HK1051069A

HK1051069A - Methods and apparatuses for stretching polymers

Info

Publication number: HK1051069A
Application number: HK03103267.7A
Authority: HK
Inventors: Y. Chan Eugene; C. Gleich Lance; S. Wellman Parris
Original assignee: 美国吉诺米克斯有限公司
Priority date: 1999-08-13
Filing date: 2000-08-11
Publication date: 2003-07-18

Description

Method and apparatus for stretching polymers

This application claims the benefit of U.S. provisional application 60/149,020 filed on 8/13 of 1999, which is incorporated herein by reference in its entirety.

1. Field of the invention

The present invention relates to the general field of polymer characterization. More particularly, the present invention relates to stretching polymers or selecting polymers on a length basis in a micromodule using a structure.

2. Background of the invention

Macromolecules are associated with the diversity and substantial function of biological systems. The ability to interpret functions, kinetics, and macromolecular interactions is dependent on an understanding of their chemical and three-dimensional architecture. These three aspects-chemical and three-dimensional architecture and dynamics-are interrelated. For example, the chemical composition of proteins, and more specifically the linear arrangement of amino acids, unambiguously determines the three-dimensional structure formed by folding of polypeptide chains after biosynthesis (Kim & Baldwin (1990) Ann. Rev. biochem. 59: 631-660), which in turn determines the interaction of proteins with other macromolecules, as well as the relative mobility of the phases that make the proteins behave correctly.

Biomacromolecules are both polymers and complexes of polymers. Macromolecules of different species are composed of monomers of different species, namely twenty amino acids in the case of proteins and four main nucleobases (nucleobaseses) in the case of nucleic acids. A great deal of information can be obtained from determining the linear or primary sequence of monomers in a polymer chain. For example, by determining the primary sequence of a nucleic acid, the primary sequence of a protein encoded by the nucleic acid can be determined in order to generate an expression profile for the determination of the mRNA expression profile, to determine the protein expression profile and to understand the genetic mutations corresponding to the disease state. In addition, the characteristic nucleobase sequence profile along a particular DNA polymer can be used to unambiguously identify DNA, such as for forensic assays. For this reason, fast, accurate and inexpensive methods for characterizing polymers, especially nucleic acids, are being developed due to the efforts of human genome project to rank human genomes.

The challenge to characterize the linear sequence of monomers in a polymer chain arises from the natural tendency of polymers to adopt an unpredictable, coiled morphology in most media. The average number of such windings depends on the interaction of the polymer with the surrounding solution, the rigidity of the polymer and the energy of the polymer interaction with itself. In most cases, this winding is quite pronounced. For example, lambda-phage DNA, theoretically 16 μm long when stretched in the B form, has a random coil diameter of about 1 μm (Smith et al (1989) Science 243: 203-206).

DNA and many other biopolymers can be modeled as uniform elastic rods with helical strands to determine their random coil properties (Austin et al (1997) Physics today 50 (2): 32-38). One correlation parameter is the correlation length, P, over which the directionality is preserved, which is given by:

P＝к/k_Bt (1) where κ is the flexural modulus of elasticity (Housearch et al (1989) Biophys.J.56: 507-516), k_BIs the Boltzmann constant, and T is the temperature (Austin et al (1997) Physics Today 50 (2): 32-38). A longer correlation length means that the polymer is more rigid and more stretchable. Under physiological conditions, P ≈ 50nm for DNA. Although larger than a 2.5nm molecule diameter, the relevant length is many orders of magnitude smaller than the actual length of a typical DNA molecule, such as a human chromosome, which is about 50mm long. From this correlation length, the overall coil size R (Austin et al (1997) Physics Today 50 (2): 32-38) can be calculated as follows:

<R²>2PL (2) where L is the equivalent length of the DNA molecule. For chromosomal DNA, R ≈ 70 μm. Clearly, it is easier to resolve information on stretched 5cm long pieces of DNA than on pieces of DNA with a 70 μm coil size.

The force necessary to stretch the polymer, e.g., DNA, is not very large. The spiral chain model makes a polymer appear to be spring-like, and the force (Fs) required to stretch it close to its full natural length can be calculated as follows (Austin et al (1997) Physics Today 50 (2): 32-38):

F_s≈k_BT/P (3) wherein all parameters are as defined above. Below Fs, the relationship between applied force and amount of extension is approximately linear; above Fs, the application of a greater force results in a small change in extension (Smi)th et al (1992) Science 258: 1122-1126; bustamante (1994) Science 265: 1599-1600). Therefore, full stretching must be accomplished by applying F_sTo achieve this. In the case of DNA, the force required to stretch it from the coil form to full length, the stretched form retains the B form, at about 0.1 pN. Such small forces can in principle be obtained from virtually any source, including shear forces, electricity and gravity.

In stretching DNA, the risk does not result from the destruction of covalent bonds, which require a force of at least 1nN (Grandbois et al (1999) Science 283: 1727-1730), but rather from hyperextension. It has been noted that when a force of 70pN is applied, the DNA adopts a super-relaxed form, so-called "S-DNA", having almost twice the length of normal B-type DNA with the same base number (Austin et al (1997) Physics Today 50 (2): 32-38). Others have reported this shift under a force of 50pN (Marko & Siggia (1995) Macromolecules 28: 8759-8770). The length of S-DNA is less consistent than that of B-DNA stretched to its natural length and is more dependent on the precise force applied (Cluzel et al (1996) Science 271: 792-794), varying linearly from 1.7 to 2.1 times the length of B-DNA with applied force. Because the precise force applied may not be known, it is desirable to avoid stretching the DNA into its S-form. Thus, with forces ranging from about 0.1pN to 25pN, of about two orders of magnitude, it is possible to stretch DNA consistently and predictably into well-stretched B-type DNA.

In addition, the force must be applied quickly enough so that the polymer does not rewind. The natural relaxation time, τ, of the polymer depends on the solvent (Marko (1998) Physical ReviewE 27: 2134-2149), as follows:

τ≈L²Pμ/k_Bt (4) wherein μ is the viscosity of the solvent and the other parameters are as defined above. This relaxation time is about 6 seconds for DNA under physiological conditions, and can be increased to 20 seconds in a solution with a viscosity of 220cp (Smith et al (1999) Science 283: 1724-1727), or by letting the DNA in a confined space by elongating P and changing the viscous resistance (B)akajin et al (1998) Phys. Rev. Let.80: 2737-2740). The relaxation time is also a function of the degree of stretching (Hatfield)&Quake (1999) Phys. Rev. Let.82: 3548-3551) the above calculated value is the lower limit of the actual relaxation time.

Regardless of the exact value of the relaxation time, the polymer must be stretched in a short time frame. In the case of flow through channels where the stretching arises from fluid strain on the polymer, a suitable time range for stretching is the inverse of the strain rate. The strain rate is defined as d ε/dt ═ dv_xA/dx where x is the flow direction and v_xIs the component of the velocity along x. Many of the strain rates and relaxation times are known as the debbola number, De ═ τ d ∈/dt, and can be used to determine whether stretch is to be maintained (Smith)&Chu (1998) Science 281: 1335-1340). If De is much greater than 1, the strain forces dominate and the polymer will remain stretched. If De is much smaller than 1, the natural relaxation process dominates and the polymer does not remain in the stretched state. Dimensionless values, such as wesenbau number in elongational flow, can be obtained from other reasonable time frames when other extension forces are included (Smith et al (1999) Science 283: 1724-1727).

Prior art techniques for stretching DNA include immobilizing at least one end of a molecule on a surface, then manipulating the other end, stretching with physical force, then immobilizing, or passing through a gel with a restricted size. Early attempts to stretch DNA for size measurement were made by Housearch et al. (1989, Biophys. J.56: 507-516). Contacting the DNA solution with the gold surface produced satisfactory binding using the Kleinschmidt method, which is widely used in electron microscopy to stretch DNA molecules on a protein monolayer, resulting in many molecules remaining coiled rather than stretched. Another attempt is to spread the DNA by "gradually" smearing it using a pipettor, but this technique is difficult to automate (PCT publication No. WO93/22463).

More complex protocols have been devised for immobilizing DNA and other polymers on one end of a surface. Typically, they involve exposing the surface to reactive groups such as hydroxyl, amine, thiol, aldehyde, ketone or carboxyl groups to modify the surface, or to add coupling structures such as avidin, streptavidin and vitamin H. Examples of such techniques can be found in PCT publication Nos. 97/06278; U.S. patent No.5,846,724 and Zimmermann & Cox (1994) nucleic acids res.22: 492-497. Typically these techniques involve the use of silanes (Bensimon et al (1994) Science 265: 2096-2098).

Once one end of the polymer is fixed, stretching can be performed because the force can be oriented perpendicular to the attachment surface. One common method is to align the polymer using a receding meniscus, which is sometimes referred to as "molecular combing". In this technique a second fluid is introduced, which is substantially immiscible with the first fluid, forming a meniscus at the interface. The primary fluid is then gradually removed and replaced by the new fluid by mechanical, thermal, electrical or chemical means or simply by evaporation. When the interface moves, the polymer is aligned perpendicular to the interface by surface tension, thus becoming stretched. The extension force of this method can be expressed as a function of polymer diameter D (for double-stranded DNA, D ═ 2.2nm) and surface tension γ (Bensimon et al (1994) Science 265: 2096-2098): f ═ γ π D.

For an air/water interface, γ is 0.07N/m, producing a force on DNA of approximately 40pN, which is clearly within the required range. If the second fluid is properly selected to prevent polymer movement, the polymer remains fixed in place for an extended period of time. In addition, adjacent polymers attached to the same surface are all aligned in the same direction. The two fluids involved, while usually solvents for the polymer, may be only part of the solvent and one may even be air. The degree of stretching depends on the modification of the surface (Bensimon, D. et al (1995) Phys. Rev. Lett.74 (23): 4754-4747), but is consistent for any given surface treatment. Variations of this technique have been used (U.S. Pat. No.5,851,769; PCT publication WO 97/06278; Bensimon et al (1994) Science 265: 2096-2098; U.S. Pat. No.5,840,862; Cox & Zimmermann (1994) Nucl. acids Res.22: 492-497). However, this technique is not easy to operate at high-throughput, since the immobilization is a rate-limiting step and further polymer modification after the immobilization is more difficult.

Another method of processing DNA immobilized at one end involves the use of optical traps (optical traps). In this technique, a laser beam ("optical tweezers") imparts momentum to a DNA molecule by emitting a quantum of light. By moving the position of the photon, i.e. moving the beam, the direction of movement of the DNA can be changed very precisely (U.S. Pat. No.5,079,169, Chu (1991) Science 253: 861-866). Thus, the DNA molecules can be stretched using optical tweezers. The advantage of this technique is the ability to vary the force used for stretching and has been used to validate the theory of surface slump (Perkins et al (1994) Science 264: 819-822). However, lasers can only hold one molecule in place at a time and must be rearranged for each subsequent molecule, making them unattractive for high-throughput analysis.

A third method of stretching DNA involves electrophoresis of DNA immobilized at one end, moving the free end of the molecule away from the immobilized end, and then attaching the immobilized end to a surface with avidin, or electrophoresis of DNA free at both ends, and then attaching both ends to a surface with avidin (Kabata et al (1993) Science 262: 1561-1563; Zimmerman & Cox (1994) Nucl. acids Res.22: 492-497). There has been no attempt to characterize the quality of stretching using this technique. Furthermore, this technique also has the drawbacks of the above-described technique (in terms of the post-fixation method).

In the case where one end of the molecule is not immobilized, DNA is also stretched by electrophoresis. As part of near-field detection for sequencing biomolecules, DNA has been extended by electrophoresis in both gels and solutions, where power is used to move the DNA into position for identification (u.s. patent 5,538,898). However, no data is given to determine the mass of the stretched high molecular polymer, and the technique is limited to about 3 megabases per analysis.

An extension of this idea involves the use of dielectrophoresis, or an alternating electric field, to stretch the DNA. Washizu and Kurosawa ((1990) IEEE Transactions on Industrial applications 26: 1165-1172) indicate that DNA can be stretched to its full length in the B-DNA format in a field having an intensity of 106V/m and a frequency of 400kHz or more. At some lower frequencies (about 10kHz), the DNA will also be fully stretched, but in a direction perpendicular to the field rather than parallel to it. This technique has been applied to the sizing of DNA by creating a gap between electrodes having a tapered width such that the DNA is aligned at a gap width equal to the length of the DNA. It was also found that this technique does not stretch single-stranded DNA because the solvent interactions from double-stranded DNA are different (Washizu et al (1995) IEEE Transactions on industry Applications 31: 447-456). One disadvantage of this technique is that the sample is prone to aggregation due to the presence of induced dipoles along the length of the DNA, and it is difficult to accurately identify the components in heterogeneous samples. In addition, these experiments must be performed in deionized water to avoid the undesirable effects of joule heating and electro-osmotic flow, thereby presenting sample preparation difficulties because most of the DNA is present in a salt solution or other solvent.

Gravity is also used to stretch DNA (U.S. Pat. No.5,707,797; Windle (1993) Nature Genetics 5: 17-21). In this technique, a drop of DNA from cell derived sodium dodecyl sulfate lysine is flowed down a slide fixed at an angle. Gravity acts to straighten the DNA, even to its over-stretched S-DNA form. The DNA is then immobilized on slides and, for example, fluorescently labeled before stretching is relatively difficult.

Church et al developed another method for polymer characterization that involved measuring the physical changes at the interface between the media bis (pool) as the polymer passed through the interface (u.s. patent 5,795,782). The method is relatively fixed. For example, the ion channel protocol used for nucleic acid characterization (Church et al (1999) Science 284: 1754-1756) can only be used for single-stranded DNA. Interfaces that can be used with a variety of polymers have yet to be developed.

Kambara et al developed a method for measuring DNA length (U.S. Pat. No.5,356,776). The method involves electrophoresis of DNA through a gel; when the DNA reaches a portion of the gel having a diameter of not more than several micrometers, it is forced to be in a straight line, where fluorescent label detection on each end of the DNA is completed. In another embodiment, one end of the DNA is immobilized in a well, stretched by electrophoresis, and the label at the other end of the molecule is detected. The use of a gel in this method forces the use of a higher voltage than in solution to move the DNA, and the end-labeling precludes most other characterizations of DNA. In addition, long DNA molecules tend to entangle in the gel. An improved method of electrophoresis, pulse-field electrophoresis (Schwartz & Koval (1989) Nature 338: 520-522), allows longer pieces of DNA to be fully stretched by a moving electric field. However, this technique takes longer due to field variations and has other drawbacks of electrophoresis.

Schwartz et al developed a gel-based and solution-based mixing method for stretching DNA ((1993) Science 262: 110-113). The DNA is placed in a free-melt agarose solution, stretched by gravity, and then held in place by a gelation process. Enzymes are also added during gelation to cleave the DNA at specific sites. This method is effective in generating restriction maps, however, predictable stretching in agarose media is difficult and the use of this technique for high-throughput methods of analyzing uncut DNA is problematic.

Other techniques for characterizing particles do not rely on stretching. For example, the method developed by Schwartz (U.S. patent 5,599,664; EP0391674) determines size and mass by applying forces to particles and measuring changes in configuration and position. In the case of polymers, this force is typically applied to the wound form. Another method for determining the size of and classifying DNA molecules (Chou et al (1999) Proc. Natl. Acad. Sci. USA 96: 11-13) involves devices that operate at the micrometer scale. The device utilizes the integrated fluorescence signal from the coiled DNA passing through the detector for analysis. Schmalzing et al ((1998) Analytical Chemistry 70: 2303-2310; (1997) Proc. Natl. Acad. Sci. U.S. 94: 10273-10278) developed microfabricated devices for DNA analysis that include small scale formats using conventional techniques such as electrophoresis and do not rely on sequencing for DNA stretching.

In order to accurately determine the linear sequence of information in a biopolymer, the biopolymer must be stretched so that individual units can be distinguished. Although many techniques have been developed for stretching biopolymers, especially DNA, they all have drawbacks such as consistency and reproducibility of stretching, ease of handling biopolymers and suitability for all types and sizes of biopolymers. Furthermore, none of these methods is suitable for rapid methods of analysis of information, such as sequencing large fragments of DNA on a reasonable timescale. Clearly, there is a need for a method and apparatus for reliably stretching polymers to more rapidly and accurately determine linear sequences of information therein to elucidate complex genetic functions and to diagnose diseases and genetic dysfunctions.

Citation of references herein shall not be construed as an admission that such references are prior art to the present invention.

3. Summary of the invention

In a first embodiment, the present invention relates to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel, the width of said tapered channel decreasing linearly from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in the direction from said first end to said second end; whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel.

This embodiment of the invention is suitable for stretching polymers, especially DNA, for further analysis.

In a second embodiment, the present invention relates to an integrated device comprising: (a) at least one polymer in the fluid sample; and (b) an elongated structure for stretching the at least one polymer, wherein the elongated structure comprises a tapered channel that decreases linearly in width from a first end to a second end, and wherein the at least one polymer, when present, moves along the tapered channel in a direction from the first end to the second end; whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel.

In a third embodiment, the present invention relates to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel, the width of said tapered channel decreasing from a first end to a second end at a rate greater than linear, and wherein said at least one polymer, when present, moves along said tapered channel in the direction from said first end to said second end; whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel.

This embodiment of the invention is also suitable for stretching polymers, especially DNA, for further analysis.

In a fourth embodiment; the present invention relates to an integrated device; it includes: (a) at least one polymer in the fluid sample; and (b) an elongated structure for stretching the at least one polymer; wherein the elongated structure comprises a tapered channel; the width of the tapered channel decreases from the first end to the second end at a rate greater than linear; and wherein the at least one polymer; when present; moving in a direction from the first end to the second end along the tapered channel; whereby said at least one polymer in the fluid sample moves along said tapered channel; shear forces are applied to the at least one polymer.

In a fifth embodiment, the present invention relates to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel, the width of said tapered channel decreasing from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in the direction from said first end to said second end; whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel, wherein the shear force creates a constant shear rate.

In a sixth embodiment, the present invention is directed to an integrated device comprising: (a) at least one polymer in the fluid sample; and (b) an elongated structure for stretching the at least one polymer, wherein the elongated structure comprises a tapered channel, the width of the tapered channel decreasing from a first end to a second end, and wherein the at least one polymer, when present, moves along the direction of the tapered channel from the first end to the second end; whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel, wherein the shear force creates a constant shear rate.

In a seventh embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a central channel for receiving a fluid and a plurality of lateral channels for receiving a fluid connected to said central channel; and wherein the at least one polymer, when present, moves in an elongation direction along the central channel.

In an eighth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising: (a) an elongated structure; (b) a transport channel leading into and out of said elongated structure for transporting said at least one polymer sample in said fluid to said elongated structure; and (c) means for moving said at least one polymer in said fluid sample, when present, within said elongate structure, wherein said elongate structure comprises a central channel for receiving a fluid and a plurality of lateral channels for receiving a fluid connected to said central channel; and wherein the device moves the at least one polymer in an elongate direction along the central channel when the at least one polymer is present.

In a ninth embodiment, the present invention relates to an integrated device for stretching DNA in a fluid sample, comprising: (a) an elongated structure; (b) means for delivering the DNA in the fluid sample to the elongated structure; and (c) means for moving said DNA, when present, in said fluid sample within said elongated structure, wherein said elongated structure comprises a central channel for receiving a fluid and a plurality of lateral channels for receiving a fluid connected to said central channel; and wherein the device moves the DNA along the central channel in an elongation direction when the DNA is present.

In a tenth embodiment, the present invention is directed to an integrated device comprising: (a) at least one polymer in the fluid sample; (b) an elongated structure for stretching said at least one polymer, wherein said elongated structure comprises a central channel for receiving a fluid and a plurality of lateral channels for receiving a fluid connected to said central channel.

In an eleventh embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel having at least one bend, and wherein said at least one polymer, when present, moves along said channel.

In a twelfth embodiment, the present invention relates to an integrated device for stretching DNA in a fluid sample, comprising: (a) an elongated structure; and (b) means for delivering said DNA in said fluid sample to said elongated structure, wherein said elongated structure comprises a channel having at least one bend, and wherein said DNA, when present, moves along said channel.

In a thirteenth embodiment, the present invention is directed to an integrated device comprising: (a) at least one polymer in the fluid sample; and (b) an elongated structure for stretching the at least one polymer, wherein the elongated structure comprises a channel having at least one bend.

In a fourteenth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample comprising an elongated structure, wherein said elongated structure comprises a tapered channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to the movement of said at least one polymer.

In a fifteenth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample comprising an elongated structure, wherein said elongated structure comprises a central channel along which said at least one polymer, when present, moves in a flow direction, and a plurality of lateral channels connected to said central channel, and wherein said central channel further comprises a plurality of movement obstructions of said at least one polymer.

In a sixteenth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample comprising an elongated structure, wherein said elongated structure comprises a channel having at least one bend along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of movement obstacles of said at least one polymer.

In a seventeenth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of pillars, at least one of said pillars having a cross-sectional shape that is not a quadrilateral polygon.

The fifteenth, sixteenth and seventeenth embodiments of the invention are suitable for stretching polymers, especially DNA, for further analysis.

In an eighteenth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to movement of said at least one polymer, said plurality of obstacles being positioned in a series of rows, each of said rows being positioned perpendicular to said flow direction, and each successive row being offset from the previous row, whereby along said flow direction at least one portion not equal to 1/2 times the length of one of said obstacles overlaps the extension of a gap formed by two adjacent obstacles in said previous row.

In a nineteenth embodiment, the present invention is directed to an integrated device comprising (a) at least one polymer in a fluid sample, each of said polymers having a diameter greater than or equal to a minimum diameter; and (b) an elongated structure for stretching said at least one polymer, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of moving obstacles to said at least one polymer, said plurality of obstacles being positioned in a series of rows, each of said rows being positioned perpendicular to said flow direction, and each adjacent pair of obstacles in each of said series of rows being separated by a distance greater than 50 times said minimum diameter.

In a twentieth embodiment, the present invention relates to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to movement of said at least one polymer, said plurality of obstacles decreasing in size along said flow direction.

In a twenty-first embodiment, the present invention relates to an integrated device for stretching DNA, comprising an elongated structure, wherein said elongated structure comprises a tapered central channel, said tapered central channel comprising a first end and a second end, and wherein said DNA, when present, moves along said tapered central channel in a direction from said first end to said second end, wherein said elongation further comprises a plurality of lateral channels connected to said tapered central channel, wherein said tapered central channel comprises at least one bend; and wherein said tapered central channel comprises a plurality of movement obstacles for said DNA.

In a twenty-second embodiment, the present invention relates to an integrated device for stretching DNA, comprising an elongated structure comprising: (a) a first tapered channel comprising a first end, a second end and a plurality of posts staggered between said first end and said second end, forming 12-15 rows, said first tapered channel decreasing in width at an angle of 26.6 °, said angle being defined at said first end relative to a constant-width channel, said first end having a width between 0.5 and 5 μm, said posts having a width equal to 1.5 μm²And are separated by a gap equal to 0.5 μm; and (b) a second conical channel connected to said first conical channel at said second end and reduced in width to between 0.5 and 5 μm, whereby a shear force producing a constant shear rate is applied to said DNA, said second conical channel, when present, having a length of between 1 and 3 mm.

In a twenty-third embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering the at least one polymer to an elongated structure comprising a tapered channel having a first end and a second end; and (b) moving said at least one polymer along said tapered passageway from said first end to said second end, whereby said tapered passageway imparts a shear force that produces a constant shear rate to said at least one polymer as said at least one polymer moves along said tapered passageway.

This embodiment of the invention comprises a method suitable for stretching polymers, especially DNA, for further analysis.

In a twenty-fourth embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering the at least one polymer to an elongated structure comprising a linearly tapered channel having a first end and a second end; and (b) moving the at least one polymer along the tapered passageway from the first end to the second end.

In a twenty-fifth embodiment, the present invention is directed to a method for stretching at least one polymer comprising the steps of: (a) delivering the at least one polymer to an elongated structure, the elongated structure comprising a tapered channel having a first end and a second end, the tapered channel narrowing from the first end to the second end at a rate greater than linear; and (b) moving the at least one polymer along the tapered passageway from the first end to the second end.

In a twenty-sixth embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering the at least one polymer to an elongated structure, the elongated structure comprising a central channel containing a fluid and a plurality of lateral channels containing a fluid connected to the central channel, the central channel comprising a first end and a second end; and (b) moving the at least one polymer along the central passage from the first end to the second end.

The methods of the twenty-fourth, twenty-fifth and twenty-sixth embodiments of the invention are suitable for stretching polymers, especially DNA, for further analysis.

In a twenty-seventh embodiment, the present invention is directed to a method for stretching at least one polymer comprising the steps of: (a) delivering the at least one polymer to an elongated structure, the elongated structure comprising a channel having at least one bend, the channel comprising a first end and a second end; and (b) moving the at least one polymer along the channel from the first end to the second end.

In a twenty-eighth embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering said at least one polymer to an elongated structure, said elongated structure comprising a channel and a plurality of movement obstructions of said at least one polymer within said channel, said central channel comprising a first end and a second end; and (b) moving said at least one polymer along said channel from said first end to said second end, wherein said plurality of movement obstacles decrease in size in a direction from said first end to said second end.

In a twenty-ninth embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering said at least one polymer to an elongated structure, said elongated structure comprising a channel and a plurality of movement obstructions of said at least one polymer within said channel, said central channel comprising a first end and a second end; and (b) moving the at least one polymer along the channel from the first end to the second end, wherein at least one of the obstacles has a polygonal cross-sectional shape that is not quadrilateral.

In a thirty-first embodiment, the present invention relates to a method for stretching at least one polymer, comprising the steps of: (a) delivering the at least one polymer to an elongated structure, the elongated structure comprising: (i) a tapered central passage having at least one bend, said tapered central passage comprising a first end and a second end; (ii) a plurality of side channels connected to said tapered central channel; and (iii) a plurality of movement barriers for said at least one polymer within said tapered central passage; and (b) moving the at least one polymer along the central passage from the first end to the second end.

In a thirty-first embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises at least one step which decreases the height, z, of said channel from a first end to a second end.

In a thirty-second embodiment, the present invention relates to an integrated device comprising an elongated structure comprising a channel, said channel comprising at least one step which decreases the height, z, of said channel from a first end to a second end, said channel comprising at least one polymer in a fluid sample, said channel being arranged such that a shear force is exerted on said at least one polymer when moving in a direction from said first end to said second end.

In a thirty-third embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure comprising: (a) a first channel, said first channel comprising a first end and a second end; and (b) a second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, along which said at least one polymer, when present, moves in a flow direction, and wherein said first channel decreases in width from said first end to said second end at a rate that is different from the rate at which said second channel decreases in width from said third end to said fourth end.

In a thirty-fourth embodiment, the present invention is directed to an integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure comprising: (a) a first channel having a width equal to 10 μm and a height equal to 1 μm, said first channel comprising a first end, a second end, and a plurality of columns staggered between said first end and said second end, forming at least 12 to 15 rows, said plurality of columns terminating at said second end, and each of said plurality of columns having a width of 1-25 μm²Cross-sectional area of (a); and (b) a second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel running from said third end to said fourth end at 1/x²Wherein x is a distance along the length of the second channel, the length of the second channel being equal to 5 μm, the second channel comprising reducing the height of the second channel at the third end to 0.25 μm²Wherein the at least one polymer, when present, moves in a flow direction along the first channel and the second channel.

In a thirty-fifth embodiment, the present invention is directed to an integrated device for selectively stretching at least one polymer in a fluid sample on a length basis, comprising an elongated structure, wherein the elongated structure comprises: (a) a first channel, said first channel comprising a first end, a second end, and a plurality of alternating posts between said first end and said second end, each post in said plurality of posts positioned no less than L from said second end; and (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel narrowing in width from said third end to said fourth end, said at least one polymer moving along the channel, when present, in the direction of flow.

In a thirty-sixth embodiment, the present invention is directed to an integrated device for stretching a plurality of polymers in a fluid sample having different lengths, comprising an elongated structure, wherein the elongated structure comprises: (a) a first channel, said first channel comprising a first end and a second end; (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end; and (c) a plurality of columns staggered in said first and second channels, said plurality of polymers, when present, moving in a flow direction along the channels.

In a thirty-seventh embodiment, the present invention relates to a method for stretching at least one polymer, comprising moving the at least one polymer along an elongated structure comprising a first channel comprising a first end and a second end; and a second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, wherein said first channel decreases in width from said first end to said second end at a different rate than said second channel decreases in width from said third end to said fourth end.

In a thirty-eighth embodiment, the present invention is directed to a method for stretching at least one polymer having a length greater than or equal to L in a fluid sample, comprising moving said at least one polymer along an elongated structure, said elongated structure comprising a first channel, said first channel comprising a first end, a second end and a plurality of alternating posts between said first end and said second end, each post in said plurality of posts is positioned L from said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end, wherein the polymer having a length greater than or equal to L is stretched and the polymer having a length less than L is not stretched.

In a thirty-ninth embodiment, the present invention relates to a method for stretching a plurality of polymers having different lengths in a fluid sample, comprising moving the plurality of polymers along an elongated structure comprising: (a) a first channel, said first channel comprising a first end and a second end; (b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end; and (c) a plurality of columns staggered in said first channel and said second channel.

In a forty-fourth embodiment, the present invention relates to a method for stretching at least one polymer comprising moving the at least one polymer along an elongated structure comprising: (a) a first channel having a width equal to 10 μm and a height equal to 1 μm, said first channel comprising a first end, a second end and a plurality of columns staggered between said first end and said second end, constituting at least 12 to 15 rows, said plurality of columns terminating at said second end, and each column in said plurality of columns having a width of 1-25 μm²Cross-sectional area of (a); and (b) a second channel comprising a third end and a fourth end, said third end connected to said first channel at said second end, said second channel from said third end to said fourth end being 1/x in width²Wherein x is the distance along the length of the second channel, the length of the second channel being equal to 5 μm, the second channel comprising reducing the height of the second channel at the third end to 0.25 μm²The steps of (a).

4. Description of the drawings

Fig. 1 shows examples of various structures that fall within the scope of the present invention.

FIG. 2(a-m) shows: (a) several embodiments of an extended structure including funnels, columns, branches, and tandem structures; (b) an enlarged example of a continuous two-funnel configuration with columns; (c) several embodiments of composite pillar arrangements and branching structures; (d) embodiments of structures comprising series and parallel structures; (e) an asymmetric branched structure; (f) structures with small combinations of obstacles defining small gaps; (g) a structure having a combination of polygons, grids, and pillars; (h) an asymmetric curved structure; (i) an enlarged view of the branched structure with the pillar; (j) a large funnel structure with struts; (k) a funnel structure having a column; (l) A funnel configuration with a linear increase in flow rate, with and without a column; and (m) a summary of some of the funnel structures encompassed by the present invention.

Fig. 3 illustrates an embodiment of a shear-stretch mode using a constant tapered channel.

Fig. 4 illustrates an embodiment of a shear-stretch mode in which the shear rate increases sharply as flow proceeds along the length of the channel.

Fig. 5 illustrates an embodiment of a shear-stretch mode using tapered channels for generating constant shear forces.

FIG. 6 illustrates an embodiment of a shear-stretch mode in which shear comes from the addition of fluid from the side channels.

FIG. 7(a) shows how shear forces are generated in a narrow channel, where the local components of the rotational and tensile forces are nearly equal; (b) showing how shear forces are generated when a fluid is added to generate forces where the tensile forces exceed the rotational forces.

Fig. 8 illustrates an embodiment of a shear-stretch mode in which shear comes from both the narrowing channel and the presence of the side channel.

FIG. 9(a) illustrates the "racetrack effect" of the fluid, with the fluid on the outside of the curve taking longer to traverse the curve than on the inside; (b) it is shown how the "racetrack effect" leads to stretching of the polymer in flexion.

Fig. 10 shows an embodiment in a meandering manner, wherein the channels are in the shape of a sine wave.

Figure 11 shows an embodiment in a meandering manner, wherein the channels are in the shape of a zigzag.

Figure 12 shows an embodiment in a meandering manner, wherein the channels are at right angles to a "snake".

Figure 13 shows how a curved channel can be used for multiplex detection as the polymer advances along the channel.

Fig. 14 shows how the polymer stretches in an embodiment with barrier zone patterns of graded barrier sizes.

FIG. 15 illustrates a coordinate frame of an elongated structure.

Fig. 16(a) illustrates an embodiment of an obstacle region pattern having circular obstacles in a square-grid arrangement; (b) embodiments of the barrier region embodiments having circular barriers in a staggered-grid arrangement are illustrated.

Fig. 17 illustrates an embodiment of a rectangular barrier densely spaced barrier regions with an enlarged aspect ratio.

Fig. 18 illustrates an embodiment of barrier regions having a dense spacing of circular barriers.

Fig. 19 illustrates an embodiment of an obstacle region embodiment having three circular obstacles of graded size.

FIG. 20 illustrates a configuration for consistent spreading, transport and stretching of DNA having different sizes.

FIG. 21 illustrates the structure of a preferred embodiment of a structure for stretching DNA, which binds to the column region at 1/x²Gradual changeWherein x is the distance along the length of the funnel, and a step that reduces the height of the channel.

FIG. 22 illustrates a schematic of a molecular size sorting apparatus in which the signal of molecules of length L or greater can be readily distinguished from the signal of molecules of length less than L.

FIG. 23 illustrates a simplified apparatus for stretching molecules of all lengths, wherein the signals from all molecules are uniformly detected.

Fig. 24 illustrates sensitive optics using confocal fluorescence illumination and detection.

FIG. 25 shows an embodiment of the overall polymer analysis system.

FIG. 26 illustrates various states of stretch of DNA at the entrance of the constant-shear channel.

FIGS. 27(a-g) illustrate that 50kb DNA is straightened out in a conical channel.

FIG. 28 illustrates DNA measured at 537kb straightened out in one channel.

FIG. 29 illustrates a bar graph showing experimentally determined DNA lengths.

FIG. 30 illustrates a histogram of experimentally determined phage lambda DNA lengths from the structure of FIG. 20(a) without and (b) with columns.

5. Detailed description of the invention

5.1 introduction to

The present invention provides structures that stretch polymers of any length, including nucleic acids comprising the entire genome, into a long, linear form for further analysis. The polymer is fed into a device and through the structure under the impetus of, inter alia, physical, electrical or chemical forces. Stretching is accomplished by, for example, applying shear forces as the polymer passes through the structure, placing obstacles in the path of the polymer, or a combination thereof. Because the force is applied continuously, the polymer can be stretched to a length equal to or greater than the active area of the device, i.e., where information about the polymer is collected as it is analyzed. For example, if the camera or laser illuminated volume is focused on the area where the stretching of the micromodule occurs, it is possible to monitor DNA molecules of infinite length, i.e. a length much greater than the video image or laser illuminated volume. Because many molecules can be stretched out continuously, extremely high throughput screening is achieved, for example, screening in excess of one molecule per second.

The stretched polymer or population of polymers is characterized. The stretched, marked polymer moves past at least one station where the marking units of the polymer interact with the station to produce an image-dependent pulse. As used herein, "moving through" refers to embodiments in which the station is stationary and the stretched polymer is in motion, the station is in motion and the stretched polymer is stationary and both the station and the stretched polymer are in motion.

Although the invention may be used to characterise any polymer, it is preferred that the polymer has predominantly, although not necessarily exclusively, a linear or single-chain arrangement. Examples of such polymers include biopolymers such as deoxyribonucleic acid, ribonucleic acid, polypeptides and oligosaccharides. The polymer may be heterogeneous in backbone composition and thus comprise any one of the possible combinations of individual monomeric units linked together, such as peptide-nucleic acids (PNAs) with amino acids linked to nucleic acids. In a preferred embodiment, the polymer is homogeneous in backbone composition and is, for example, a nucleic acid, a polypeptide or an oligosaccharide. The term "backbone" has its usual meaning in polymer chemistry. As used herein, a nucleic acid is a biopolymer comprising nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As used herein, a protein or polypeptide is a biopolymer composed of amino acids. In a most preferred embodiment, the extended substance is a double stranded DNA molecule.

As used herein, "linked" or "linking" with respect to individual units of a polymer means that the two units are linked to each other by any physicochemical means. Including any linkage known to those of ordinary skill in the art, covalent or non-covalent. Natural linkages, such as amide, ester and thioester linkages are the most common, and are those commonly found in nature to join individual units of a particular polymer. However, the individual units of the polymers extended by the structures of the present invention may be linked by synthetic or modified linkages.

A polymer is made up of a number of individual units, which are building blocks or monomers, which are directly or indirectly connected to other building blocks or monomers to form the polymer. The polymer preferably comprises at least two chemically different linked monomers. The at least two chemically different linked monomers may generate or be labeled to generate different signals. Different types of polymers are composed of different monomers. For example, DNA is a biopolymer comprising a deoxyribose phosphate backbone to which are attached purines and pyrimidines such as adenine, cytosine, guanine, thymine, 5-methylcytosine, 2-amino-purine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. RNA is a biopolymer comprising a phosphoribosyl backbone to which are attached purines and pyrimidines such as those described for DNA, but in which uracil replaces thymidine. Deoxyribonucleotides can be linked to each other by an ester bond through a 5 'or 3' hydroxyl group to form the DNA polymer. Ribonucleotides can be linked to each other by means of an ester bond through the 5 ', 3 ' or 2 ' hydroxyl group. In addition, a DNA or RNA unit having a 5 ', 3 ' or 2 ' amino group can be linked to other units of the polymer by means of an amide bond.

The polymer may be a naturally occurring or non-naturally occurring polymer. The polymers may be isolated, for example, from natural sources using biochemical purification techniques. Alternatively, the polymer may be synthesized, for example, by in vitro amplification using enzyme-catalyzed Polymerase Chain Reaction (PCR), by chemical synthesis or by recombinant techniques.

The structure of the invention is used in combination with a method for analyzing stretched polymers, which detects an image-dependent impulse signal. As used herein, an "image-dependent pulse" is a detectable physical quantity that conveys or conveys information about the structural characteristics of at least one unit of the stretch polymer, a unique label. As used herein, a unit-specific label may be an inherent property of a particular kind of measurable stretched polymer, such as a different maximum absorption of the nucleobases of naturally occurring DNA (which polymer is intrinsically labeled), or a compound with a measurable property that is specifically associated with one or more individual units of the polymer (which polymer is extrinsically labeled). The unit-specific label of the exogenously labeled polymer may be a specific fluorescent dye with which all nucleobases of a specific type, e.g. all thymidylate nucleobases in the DNA strand, are labeled. In addition, the unit-specific label of the exogenously labeled polymer may be a fluorescently labeled oligonucleotide of defined length and sequence that hybridizes to and thus "labels" the complementary sequence present in the target DNA. Unit-specific tags may further include, but are not limited to, sequence-specific primary or secondary groove binders (groove binders) and intercalators, sequence-specific DNA or peptide binding proteins, sequence-specific PNAs, and the like. The detectable physical quantity may be in any form that can be measured. For example, the detectable physical quantity may be electromagnetic radiation, chemical conductance, radioactivity, or the like. The image-dependent pulses may result from energy transfer, controlled excitation, quenching, a change in conductance (resistance), or any other physical change. In one embodiment, the image-dependent pulse is generated from fluorescence resonance energy transfer ("FRET") between the unit-specific label and the station or an environment surrounding the station. In a preferred embodiment, an image-dependent pulse is used that originates from direct excitation in a confined or localized region, or epidrilling of confocal volume or slit-based excitation. Possible analyses of the polymer include, but are not limited to: determination of polymer length, determination of polymer sequence, determination of polymer velocity, determination of the degree of identity of two polymers, determination of a profile of polymer unit-specific markers to generate a "fingerprint", and characterization of heterogeneous populations of polymers using statistical distribution of unit-specific markers within a sampled population.

There are a number of methods and products that can be used to analyze polymers, as described in PCT publication WO98/35012, which is incorporated herein by reference in its entirety.

The various methods used to analyze polymers differ in their potential sensitivity and resolution, i.e., the shortest distance between two unit-specific labels, wherein the unit-specific labels are distinguishable. Low resolution techniques can discriminate between cells having large distances between them-specific markers; high resolution techniques are able to discriminate cell-specific markers with small distances between them. The resolution of a particular technique is determined by the characteristic distance at which a particular unit-specific mark of the stretched polymer can be detected. Shorter feature distances favor better resolution. The lowest resolution techniques include monitoring light transmission and controlling excitation to a resolution of 50-100nm or more (Tan & Kopelman (1996) chem. anal. Ser. 137: 407-475). In contrast, the resolution of FRET is about the F * rster radius, the distance between the donor and acceptor where the most efficient energy transfer occurs, which is typically 2-7 nm. The distance between adjacent base pairs in a fully extended DNA molecule with B-morphology is 3.4  or 0.34 nm. In its natural state in solution, the DNA does not exist in its fully extended B-form, but as a coil having a diameter of about 10 μm. Therefore, it is difficult to resolve many units-specific tags on a coiled DNA molecule, and therefore the molecule should be stretched before analysis.

5.2 shear force as a means of stretching the Polymer

When a polymer molecule encounters a physical barrier, it will either pass through the barrier without interaction or "hook" the barrier so that portions of the chain remain on either side of the barrier. This does not mean that the polymer is bonded to the barrier or otherwise physically attached. The imbalance of the suspension around the obstacle determines the speed at which the molecules travel along the favored side. (see Austin & Volkmuth, Analysis 1993(21) 235-238.) furthermore, a localised velocity gradient is created at the obstruction as the cross-sectional area available for fluid flow is reduced. As a result, fluid flow between obstacles is faster than before and after. This creates a shear force on the approaching molecules, which acts as a tensile force on the polymer. When this effect is enlarged by the overall area with the correctly sized obstacles, the polymer stretches to pass all the obstacles in that area. In a preferred embodiment, the polymer is stretched in a linear fashion.

Once the polymer passes through the series of obstacles and enters a channel in a manner that it is sufficiently extended, it will naturally tend to return to a lower-energy, more coiled configuration, where it is analyzed in the preferred embodiment. To prevent this from happening, the channels are designed to provide a constant shear force on the polymer in a narrowing channel, keeping it in the stretched form.

A constant shear rate, or variation in average velocity with distance in a channel, is defined as S:

u/x where x is the distance along the substantially rectangular channel and u is the average fluid velocity in the direction of the x-axis, calculated from the total liquid flow (Q) and the cross-sectional area A of the channel as follows:

u＝Q/A (6)。

in one embodiment, where the channel cross-section is rectangular, the channel may be defined by a constant height H and width W, such that the cross-sectional area a is HW, then the average fluid velocity is given by:

Q/HW (7) applies the boundary condition that the liquid flow must be continuous (i.e. incompressible), then Q is constant. Therefore, u is inversely proportional to W. This relationship can be substituted into the expression originally used for S to determine the relationship between shear rate and width:

s＝u/x＝Q/H /x(1/W)＝(-Q/HW²)(dW/dx) (8)

dW/dx＝(-SH/Q)(W²) (9) integrating the expression, finding:

W＝(SHx/Q+C)^-1(10) where C is an integration constant determined by the initial width of the channel (boundary condition). This equation for channel width is used to define channels outside the pillar structure. Similar calculations can be readily performed by those skilled in the art for non-rectangular channel shapes. When no net momentum transfer occurs in the height axis, i.e., when a velocity profile has been developed in the z-axis, the shear rate generates a tensile force from the width profile. For example, in the case of Newtonian fluids, the stress tensor τ required to calculate the force_yzCan be easily expressed in terms of shear rate:

F＝∫∫-τ_yzdzdx＝∫∫-μ(du/dx)dzdx＝∫∫-μSdzdx， (11)

where μ is the solution viscosity. In these equations, x is the direction of motion, y is the width and z is the height. The surface over which the shear rate needs to be integrated is the channel wall surface, resulting in:

f ═ μ HLS (12) where L is the length of the channel wall, approximately the length of the channel in which shear is held constant.

Thus, a water channel having a height of 1 μm, a length of 1mm and a shear rate of 0.25/s generates a force of about 0.25pN, which is sufficient to stretch the DNA, as experimentally confirmed by the inventors. Notably, this result demonstrates that the constant-shear channel not only maintains the elongation of the pre-stretched DNA, but also helps to further stretch the DNA, or stretch the DNA alone.

In a preferred embodiment, two general methods for obtaining stretch are combined. The series of graded obstacles, which are columns that have been put into the structure, also generate shear forces on the passing molecules, not only ensuring the initial extension of the polymer by means of said obstacles, but also maintaining the extension after said polymer has passed through the obstacles.

5.3 Structure for stretching polymers

The structure for stretching DNA of the invention ("elongated structure") comprises two parts: a delivery zone and a polymer elongation zone. The transport zone is a wider channel leading into and out of the polymer elongation zone. The elongated region comprises at least one of four main portions: (1) a funnel; (2) a structure having a branched channel; (3) a channel having a curve or curve; and (4) obstacles defining a small gap, wherein the obstacles may be, inter alia, pillars or steps. The invention includes combinations of the four main parts and variations of the main parts themselves. The combination of two or more of the properties of the main part may result in additional designs which are suitable for elongating and stretching polymers, especially DNA, in a controlled manner. Furthermore, several of the same designs can be repeated in parallel or in series.

Examples of structures (FIG. 1) that fall within the scope of the invention include, but are not limited to:

i) having a funnel that increases non-linearly in fluid velocity;

ii) a funnel with a linear increase in fluid velocity;

iii) a funnel with obstacles defining small gaps as elongation regions of DNA;

iv) a funnel with obstacles that increase non-linearly in fluid velocity and define a small gap;

v) a funnel with obstacles that increase linearly in fluid velocity and define a small gap;

vi) a funnel with a mixed obstacle size and gap, including a gradient of obstacle sizes and gaps;

vii) a branched structure having a region of increased fluid velocity from the converging channel;

viii) a branched structure having a plurality of regions that increase fluid velocity from a plurality of converging channels;

ix) branched structures with obstacles defining small gaps;

x) a branching structure having at least one funnel as one of the branches;

xi) branched structures having a mixture of barrier sizes and gaps, including gradients of barrier sizes and gaps;

xii) obstacles with a defined small gap and curved or curvilinear structures;

xiii) structures with obstacles defining small gaps with periodicity (sinusoidal pattern, rectangular wave repetition, zigzag turns);

xiv) structures with non-quadrilateral obstacles defining small gaps;

xv) structures with a mixture of obstacles defining small gaps, such as a set of bars defining small gaps juxtaposed to a sinusoidal pattern area, or a triangular, annular or star-shaped area;

xvi) a combination of obstacles with a small gap and funnels, branched structures or curved or curvilinear structures;

xvii) structures having bends or turns in the funnel shape;

xviii) structures with curved or curvilinear barriers defining small gaps;

xix) structures with tandem DNA extension regions;

xx) structures with parallel DNA elongation regions;

xxi) a structure having a plurality of transport channels with respective elongation zones;

xxii) structures with three-dimensional geometries, which include other kinds of embodiments; and

xxiii) is the structure of a closed loop containing a stretch of DNA.

A further example of a structure falling within the scope of the present invention is illustrated in fig. 2 (a-1). These include several embodiments of extended structures including funnels, obstacles, branches and tandem structures; two funnels in series with a column; several embodiments of composite post arrangements and branching structures; an asymmetric branched structure; structures with small combinations of obstacles defining small gaps; a structure having a combination of polygonal, rod and post obstacles; an asymmetric curved structure; a branched structure having a pillar; a large funnel structure with struts; a funnel structure having a column; with a funnel structure with and without a column that increases linearly in flow rate. Fig. 2(m) is an outline of some possible funnel structures. In general, the elongated structures of the present invention may have a length of 1 μm to 2cm, preferably 1 μm to 1mm, a width of 2 μm to 1mm and a height of 0.1 μm to 10 μm.

Each of the four main parts of the functional polymer elongated and extended structure is described below.

A funnel structure. The funnel structure is a tapered channel that exerts shear forces in a regular and continuous manner as the polymer flows along the channel. The specific shear force is defined by the type of channel structure and shape. In one embodiment of the invention, the channel is a tapered channel (fig. 3) starting at a given width and continuously decreasing to a second width, creating an increased shear force in the funnel portion of said channel, defined as:

du/dx＝(-Q/H)(dW/dx)(1/W²) (13)

in one embodiment of the invention, the width decreases linearly, so dW/dx is a constant; in this embodiment, the shear, du/dx, increases as W decreases. In this embodiment, the angle of the funnel, when calculated from the extension of the straight wall, is preferably 1-75 °, most preferably 26.6 ° for DNA in a low viscosity solution such as TE (10mM TRIS, 1mM EDTA) buffer, and the pH of the solution is 8.0. For the linear funnel embodiment, the starting width is preferably 1 micron to 1cm and the tail width is preferably 1nm to 1mm, depending on the polymer under study; for DNA, the most preferred values are 50 microns and 5 microns, respectively.

The channels may also be arranged such that the width decreases at an increasing rate as fluid passes through the channels (fig. 4), with a resultant increase in shear as it passes through the channels. Such a gradual decrease provides particularly good protection against natural relaxation of the polymer, since as time passes and the molecules move along the channel, they are subjected to increased reaction forces, preventing their tendency to recoil. Furthermore, the increased force gradient allows flexibility in certain designs; any polymer that will encounter shear forces large enough to cause the polymer to extend in the tapered structure and will not encounter shear forces large enough to cause the polymer to break in the tapered structure can successfully pass through the tapered structure and extend. It is not necessary to find an ideal or limiting force for the polymer, only a valid range is required. In embodiments involving pressure-driven fluid flow (see driving force below), increased shear also provides the greatest velocity increase for a given pressure drop, since the final velocity is a function of cross-sectional area, while the pressure drop is a function of cross-sectional area and channel length. The same small cross-sectional area (and hence large velocity) can be reached at a shorter distance (and hence a smaller pressure drop). In a preferred embodiment, the width W of the funnel is in the range of 1/(ax)ⁿ+ b) reduction, where n is any real number greater than 1, a is a non-zero real number, b is a real number, andx is the distance along the length of the funnel (and is the direction of polymer flow). The potential equation for tapering of the added shear funnel includes W1/x²，W＝1/x³And so on.

In another embodiment, the channel is designed such that the shear rate is constant, resulting in a tapered channel, for example as shown in fig. 5. The constant shear rate to achieve a force sufficient to fully stretch the polymer in a stroke across the channel will vary depending on the length of the channel (see equation (12)). Thus, for a complete stretching of the polymer in a very long channel, e.g. > 1cm, a reasonable shear rate may be 0.01/s, but this value may hardly stretch the polymer in a very short channel, e.g. < 10 μm. The channel length may vary considerably, with preferred values of 10 μm to 1cm and most preferred values of 1-2 mm. In one embodiment, the channel is 1mm long and the shear rate is 0.075/s.

The shear rate of the funnel can be determined by measuring the distance between two known points of the DNA strand. For example, concatamers of lambda DNA (concatamers) are used as standard samples for measuring shear forces. The unique sequence on each concatemer is fluorescently labeled with a hybridization probe. The inter-probe distance on the concatemer is thus the length of a single lambda DNA molecule (48 kilobases). The physical distance between the probes is determined using image microscopy or time-of-flight measurements. For lambda DNA in natural solution, the physical distance is 14.1 μm. This value matches the actual measured physical distance. For example, if the measurement distance is 15.0 μm, the shear rate can be calculated from the amount of stretch experienced by the DNA in the stretched structure. The predicted shear force on the DNA, as measured by the velocity of the DNA and the size of the channel (see equation 10), is consistent with the elongation of the DNA and its inherent nonlinear stiffness.

A branched channel. A second aspect of the present invention for stretching and extending a polymer is to create a branched structure that causes a change in fluid flow rate or a change in polymer directionality (see below, structures having a bend or curve). The side channels feed more fluid into the main channel, resulting in a change in fluid velocity and thus polymer stretching. A typical arrangement of branched channels is shown in figure 6. The side channels preferably have a total cross-sectional area corresponding to about 1% to 500% of the cross-sectional area of the main channel. Most preferably, the total cross-sectional area of the side channels is about 50% of the cross-sectional area of the main channel. In one embodiment, the side channels are present in a repeating pattern that results in shear dilution at each individual entrance into the main channel, thus resulting in a close approximation of the constant-shear condition. This solution highlights the advantages and disadvantages of the side channels. One disadvantage of this polymeric elongate member is that the entire force of fluid acting on the main channel is dissipated in a relatively small area near the junction of the main channel and the side channels. Therefore, this structure cannot lead to a constant-force situation. However, an advantage of this polymer elongation assembly is that because the additional fluid moves in the same direction in the side channels as in the main channel, the force is not a pure shear force but rather has a substantial elongation flow component. Pure shear, which is applied to the polymer by means of a conical funnel, is a superposition of tensile and rotational forces, as shown in fig. 7 (a). The tensile force on the polymer accelerates its movement in the direction of the fluid flow, causing the portion of the polymer located in the elongated flow region to move faster than the portion still located in the more stagnant region, thereby stretching the polymer. The rotational force causes the polymer to rotate or "roll" in shape, which may cause the stretched portion of the polymer to overlap upon itself and rewind. In embodiments with stronger tensile forces, such as the side channel connection configuration shown in fig. 7(b), the polymer tends to accelerate away from the junction, which results in lower rotational forces and therefore better stretching.

As will be appreciated by those skilled in the art, the channel dimensions may vary and the flow rate may increase in the same area of the micromodule. In fact, the method of significantly increasing the flow rate before the constant-shear part is not only to stretch the polymer but also to direct it away from the walls of the channel. An arrangement incorporating this embodiment of the invention is shown in figure 8. In another embodiment, additional flow is introduced from only one side of the main channel, thereby positioning the polymer advancing along the main channel to one side. This positioning design can be used to ensure that the polymer is aligned across a narrow detector in a wide channel.

Having a curved or curvilinear configuration. A third aspect of the invention uses tortuosity to achieve stretching. When the fluid flow encounters a change in its course, from a small bend to a right angle alignment, the fluid outside the curve or corner will take longer to bypass the turn than the fluid inside the curve or corner (fig. 9 (a)). This so-called "racetrack effect" can help to stretch the polymer. Such a bend does not include a 'T' junction. In a rectangular cross-section of one channel, the polymer can flow across more than one fluid flow path, as the fluid proceeds at the same speed on each path, and thus it remains in configuration. In contrast, when the distance traveled by each fluid flow path deviates at a bend or corner, the polymer is locally stretched by virtue of the velocity differential. Furthermore, the polymer tends to move towards higher-speed streamlines, so even if the channel curves back to its original direction, the polymer does not completely re-wind because locally it is in the same streamlines. One possible sequence of such stretching is shown in fig. 9 (b). While this effect is not sufficient to stretch molecules of an overall length in a single turn, it can gradually stretch specific regions, and a sufficiently repetitive tortuous path can stretch the entire molecule.

A more specific tortuous form is one in which the configuration of the channels is sinusoidal in form (fig. 10). In another embodiment, the channels take the form of a zigzag shape (fig. 11), or, in a further embodiment, even a "snake" shape with only right angle corners (fig. 12), although such sharp corners tend to cause stagnation and other undesirable hydrodynamic problems. For those embodiments in which the channel has a zig-zag, tortuous shape, each bend preferably has an angle of 5-75 °; for DNA, a preferred value for each such angle is 26.6 (effectively 53.4 ° angle at the turn of the zigzag). Such a zigzag shape may be cyclic, wherein the bending angles are always the same, or may comprise different bending patterns. For the zigzag turns, the cycles can be as small as 2 μm to 1cm, with values of 20-50 μm (1000 times the correlation length) being preferred for DNA. For those embodiments in which the channels have the form of sine waves, the amplitude to period ratio is preferably 0.01 to 5. For any of these patterns, the number of cycles may be 1-500, with a preferred value of 10.

In a further embodiment, a meandering channel is used to create the possibility of multiple detections. When detectors, such as position-dependent photomultiplier tubes arranged in a 1x 256 array, are arranged along the flow direction of the channel, the curved channel may be arranged such that it repeatedly passes through the detection zone at defined locations. The stretched polymer is observed at several sites, creating duplication and error checking in the system. Such an arrangement is shown in fig. 13, where the fluid travels along channel 111, at six locations, 112-117, through detection zone 110.

An obstacle defining a small gap. A fourth aspect of the structure that helps to induce stretch is the barrier region. As described more generally above, the obstacles induce stretch by reducing the available cross-sectional area of the channel (creating local strain on the molecule), and acting as a physical barrier through which large polymer coils cannot pass. One example is the pillar configuration of a virtually stretched polymer, shown in FIG. 14.

The obstacles may vary in cross-sectional shape and cross-sectional area. The terms "cross-sectional shape" and "cross-sectional area" as used herein with respect to an obstruction, unless otherwise stated, refer to the shape of the X-Y projection and the area of the X-Y plane, respectively, of the obstruction, as shown in FIG. 15. In particular embodiments, the obstacles comprise square posts, round posts, oval posts, or posts of rectangular cross-section having any aspect ratio (including extremely long "grids"); in another embodiment, the barrier comprises a post having a cross-sectional shape such as a regular or irregular non-quadrilateral polygon. In a preferred embodiment, the cross-sectional shape is triangular. In another preferred embodiment, the shapes are modified to have a concave surface (e.g., a shallow U-shape) on the edge facing the direction of fluid ingress. In another embodiment, pillars having a cross-sectional shape in which one dimension is longer than the other preferably have an aspect ratio of 2 to 20, more preferably 2 to 5.

Each of these obstacles may be placed at any angle to the flow direction. In preferred embodiments, the obstacles are arranged to have either a plane perpendicular to the direction of flow or at a 45 ° angle to flow, using other angles that can physically direct the polymer toward the destination if preferred positioning of the polymer molecules is desired. Preferably, obstacles in which one dimension is longer than the other are placed with their longer dimension perpendicular to the flow direction. Another factor in the layout of the obstacles is the grid they are placed on. If placed on a repeating square matrix (fig. 16(a)), some fluid flow paths are barely affected by the obstructions, so that unstretched or poorly stretched polymer may be able to follow these flow lines and pass through the obstruction areas without being stretched. To prevent this, each successive column is preferably displaced, placing the next obstacle in the gap of the previous column (fig. 16(b)), forcing all the streamlines to have a bend and causing all the molecules passing through to stretch. The misalignment may also be less than 50% of the total of the repeating units, such that every other column is not in the same arrangement, as shown in this figure; for example every fourth or sixth column may have the same alignment or there may be no repeating alignment, as long as the flow lines are forced to bend around the obstacle at some point.

In addition to the permutations in the procedure, there are two other parameters related to the obstacles: the size of the channel between them, the total Y-Z cross-sectional area of the posts relative to the Y-Z cross-sectional area of the channel (fig. 15), both of which affect the preferred barrier size. The width of the channels between the obstacles should not be less than the diameter of the stretched polymer and preferably not less than about 50 times the diameter of the stretched polymer to increase the probability of the polymer passing through the channels without sticking to the obstacle areas. An example of an insufficient channel width resulting in polymer failure through the obstruction is shown in fig. 17. On the other hand, the channel is preferably not as wide as the diameter of the wound polymer, in which case the clew may pass through the barrier region without having to stretch at all. Therefore, the preferred spacing of the obstacles is highly dependent on the polymer being analyzed. In the case of long DNA having a chain diameter of 2nm and a winding diameter varying upwards from about 1 μm, the channel width is preferably between 100nm and 800nm, most preferably a value equal to 500 nm. For polymers with very small diameters, gels can be used instead of barrier regions, with pore sizes (corresponding to the channel widths of the regions) of 1nm to 1000 nm.

The total Y-Z cross-sectional area occupied by the obstacles most directly affects the velocity gradient that occurs between the obstacles and it encourages stretch. Therefore, it is preferable to have a larger ratio of the cross-sectional area of the obstacles Y-Z to the cross-sectional area of the total channel Y-Z (also known as the packing factor, which is the ratio of the total area of the pillars to the total area of the channel when expressed in percent multiplied by 100) to maximize the velocity gradient. On the other hand, if more than one polymer is trying to enter a channel at the same time, forcing too much material through a relatively small gap may cause clogging. Therefore, to balance these conflicting considerations, the packing factor is preferably between 33% and 95%. This is the ratio of the blocked area to the total area expressed as a percentage in a particular channel. For example, having a thickness of 1 μm²Column with cross-sectional area of Y-Z at 3 μm²The channel of Y-Z cross-sectional area has a filling ratio of 33% and a thickness of 20 μm²The column was at 21 μm²The packing factor in the channels was 95%. The most preferred packing factor for DNA is between 50% and 80%. An example of an obstruction that is too large resulting in an obstruction is shown in fig. 18.

To alleviate the problem of polymer blocking small channels in the column region, different channel widths are used in certain embodiments of the invention. In certain embodiments, this is accomplished by varying the size of the obstruction. In other embodiments, this is accomplished by varying the packing factor. In other embodiments, both the obstacle size and the packing factor are varied. In such embodiments, the polymer first encounters wide inter-barrier channels, and then encounters reduced width channels (fig. 19), forcing them to become progressively more extensive to pass through smaller channels. In a preferred embodiment, the channel width is in order from about 5 μm per channel to about 1 μm per channel in the flow direction. In another embodiment, the column size in the flow direction from the cross-sectional area of about 10m²To about 1 μm². In other embodiments, the barrier cross-sectional area and channel width may be varied separately to achieve similar results, i.e., the barrier size may be varied and the channel size may be held constant, or the channel size may be varied and the barrier size may be held constant. In a preferred embodiment, all obstacles have the same cross-sectional area, but the packing factor increases in the direction of flow. The cross-sectional area of the column may be from 0.1 μm²To 1mm²Varying, preferably from 0.1 μm²To 10 μm²More preferably from 1 μm²To 100 μm²Even more preferably from 1 μm²To 25 μm²(ii) a change; depending on the size of the polymer being stretched and the size of the channels used. The pre-positioning of such polymers serves to reduce the likelihood of entanglement, thus providing more predictable stretching.

Obstacles can also be made as height or z-dimensional structures, i.e. by introducing "steps" at the top and/or bottom of the channel to reduce the height. Instead of obstacles being placed across the channel, as discussed above, the overall channel may vary in height, providing the same kind of obstacles and shear forces around the obstacles when placed along the width of the channel. Furthermore, variations in height can be accomplished relatively inexpensively, as it is generally easier to control the height of an etch at the sub-micron scale using photolithography than to try to produce feature sizes at the sub-micron scale. Without being bound by any theory, a significant change in height at a particular location produces essentially the same effect as a single row of columns, or as a funnel of infinite length x. To simulate a funnel in a manner that is convenient to manufacture using standard microfabrication techniques, the height variation may be designed as several steps along the length of the channel, rather than one step at a single location. In a preferred embodiment, the mono-step configuration reduces the height of the channel by up to one fifth. In other embodiments, a configuration having at least one step reduces the channel height from about 1/2 to about 1/100. In other embodiments, the step varies in height from about 0.1 to about 0.9 μm.

And (4) combining the components. In further embodiments of the invention, three general aspects of the structure, shear induction (i.e., tapered and branched channels), tortuosity, and barrier-filling are used in combination. For example, the constant-shear tapered channel is not only good in stretching itself, but also in maintaining the stretch of the polymer that has stretched through the barrier region. The channels with tortuous profiles may also be reduced in width in a constant-shear fashion to take advantage of the effects of both. In preferred embodiments, a graded barrier region or arrangement is used to pre-stretch the polymer, followed by a precision barrier section, a tortuous form or a high shear region to complete the stretch, and a constant-shear or increased shear section to maintain the stretch until the point of detection is reached.

Applicants have found that a particularly effective structure is a combination of barrier regions upstream of the tapered channel. The barrier region serves to stretch the DNA in a random coil configuration, preferentially sending one end of the molecule to a downstream structure. Advantageously, the obstacle region is in the wide region of the channel where the flow rate is low, so that the resistance applied to the molecules folded around or otherwise held by one of the obstacles is insufficient to break the molecules. When a molecule snakes through the barrier region, one end will tend to guide the molecule and enter the tapered channel first. The molecules will then be further stretched by the shear force of the flow through the tapered channel. Without being bound by any theory, the applicant has found that the partial stretch and the end lead, which are influenced by the barrier region, in combination with the stretch in the tapered channel, are particularly effective in accomplishing DNA stretching. Comparison of experimental data from a tapered channel with an upstream pillar region with data from a tapered channel alone shows that better stretching is obtained by combining the pillar region and tapered channel under similar flow and temperature conditions (see example 2 and fig. 29(a) and (b)). The experimental data show that while the tapered channel can stretch the DNA, the structure that binds the tapered channel to the pillar region provides significantly greater average stretch and a greater proportion of DNA stretched.

In a preferred embodiment, an obstacle region, step or array structure is used to pre-stretch and align the polymer, followed by a constant or increasing shear or elongation portion to complete and maintain the stretch until the detection zone is reached. Preferably, the barrier region is matched to the tapered channel to avoid constricted flow (i.e. reduced velocity). It is therefore preferred that the pillars or steps are located at or end in the tapering portion of the channel.

In a more preferred embodiment, the channel is a two-funnel configuration, i.e. it has two regions of different tapers in series. An example of two funnel configurations is shown in fig. 20. In one embodiment, the two funnel structures further comprise a pillar region in the first conical region. In the two funnel configurations, the stretching of the polymer is completed in the second conical region (rightmost channel region in fig. 20). Pressure driven flow is the preferred driving force because of its simplicity and ease of application.

At an optimumIn an alternative embodiment, the structure has a first channel region with a constant width of about 10 μm and a height of about 1 μm, in which the barrier regions are located along the flow direction, and a second channel region, which is a funnel, is introduced in a 1/x ratio²Decreasing in width from about 10 μm to about 1 μm and decreasing in height in a single step at the entrance of the funnel from about 1 μm to about 0.25 μm (fig. 21). The ratio of the initial channel width to the final channel width is preferably greater than 10, and the funnel portion length is preferably less than one-half of the initial width. The barrier region preferably comprises at least 12-15 rows of pillars having a cross section substantially equal to 1 μm, wherein the rows have an increasing packing factor in the flow direction. In one embodiment, six rows have a packing factor that increases from 0% to 50% in the direction of flow, and the subsequent 12-15 rows have a constant packing factor of 50%, wherein the distance of the centrally adjacent row of the subsequent 12-15 rows is about 2 μm (fig. 21). In another embodiment, the rows have a continuously increasing packing factor in the flow direction, from 0% to 80%.

5.4 Structure for polymers by Length selection

As described in the above section, the column regions can be used to create a non-random arrangement of polymers and to effectively separate one end of a polymer chain from a random coil, which is an equilibrium structure of the polymers in solution. If a pillar region is placed at a distance L from the mouth of the tapered channel, which may be any shape that it is desired to maintain or produce stretch, such as straight, constant shear or high order polynomials, the resulting structure may also be used to select molecules by length. This method is illustrated in fig. 22.

Figure 22 illustrates a schematic diagram of a column region (see method of making a structure) constructed according to the method described below, positioned before a funnel region where flow is sheared or extended. Because the post fills a portion of the channel, fluid moving through the channel will experience a decrease in velocity as it moves from the post region into the straight portion of the channel. This velocity reduction produces a constricted flow, i.e. the polymer will rewind in the region of reduced fluid velocity. DNA molecules that have advanced along the channel and hooked around the column will stretch through the flow. If the length of the molecule is equal to or greater than the distance L from the column to the beginning of the tapered region, it will be released from the column region into the extensional flow region, will actually pass through the region of reduced fluid velocity without re-winding, and will remain stretched, as illustrated schematically by the DNA molecule in FIG. 22. If the molecule is shorter than L, such as DNA molecule 2 in FIG. 22, it will exit the column and remain in the constricted flow region of the channel, where it will contract rapidly into an equilibrium coil. Thus, molecules with a length greater than or equal to L will be stretched, while molecules with a length less than L will not. If a detector is placed at the exit of the funnel, as shown in FIG. 22, the signals of coiled molecules (length less than L) and extended molecules (length greater than or equal to L) will be distinguishable. For example, if the detector monitors intercalator-stained DNA, contracted molecules will produce a short, intense pulse, while fully extended molecules will produce a long, less intense signal. Thus, a structure can be made which can separate a mixed population of polymers into two clusters, i.e., those having a length less than L and those having a length equal to or greater than L, by simply setting L, the distance from the end of the column region to the mouth of the tapered region, to a length substantially the same as the length of the molecule whose signal is to be detected.

In another embodiment, it may be desirable to stretch and uniformly detect signals from molecules of all lengths in a given population. This can be done by eliminating the constricted flow region, for example by extending the post region of figure 22 into the channel, as shown in figure 23. Because the detector is located at the entrance of the channel (see fig. 22), at the end of this column region, all molecules will be stretched as they pass through the detector, and thus the signals from all molecules, regardless of their length, will be detected. In these embodiments, the flow remains constant because the area between the pillars matches the area of the channel into which the pillar regions extend.

5.5 design basis

Stretch considerations and the type of structure used. Different structures produce different types of DNA stretch and elongation. There is tethering (tethered) stretch and uniform stretch. Tethered stretching requires an uneven force distribution at one end of the molecule to produce complete elongation in the flow profile. The tethering extension is directly created using an obstruction that defines a small gap. On the other hand, uniform stretching is more complex and involves extensive simulation of the polymer dynamics. Uniform stretching is defined as the creation of uniform tension on each unit of DNA molecule. Structures designed to produce uniform stretch, including those with constant shear in the x-axis direction of the design, such as funnels with non-linear increases in flow rate.

Polymer size considerations. The structural design makes them scalable and some universal. The structures may be increased in size and the relative sizes varied to accommodate polymer molecules of different lengths. DNA of sizes from several kilobases to at least megabases is of value, although there is no upper limit on the length of polymer molecules that can be accommodated. One megabase of DNA has a length greater than 300 microns. The channel size can be made several millimeters. In this manner, a complete chromosome (50-250 megabases in size) can be processed and stretched.

The configuration of the channels on the overall microassembly. The transport channel to the DNA extension region may comprise a transport channel that is a parallel, radial, branched, interconnected, and closed loop. The transport channel in the preferred embodiment is a wide channel, i.e., 1-1000 microns, which leads to the DNA stretch and elongation region.

A method of fabricating a structure. Preferred methods of manufacturing the design structure are lithography, such as electron beam lithography, deep-uv lithography, photolithography, LIGA (headers of the german words "Lithographie", "Galvanoformung" and "aboforming", meaning lithography, electroplating and molding), and molding of elastomeric materials. With the aid of these techniques two-dimensional and three-dimensional structures are produced. Additional methods of creating three-dimensional defined channels include track-etching and molding techniques.

Other methods of creating nano-sized barriers include methods involving chemical methods such as optical deposition of colloids, localized self-assembly of polymers, and cross-linked networks of polymers. For example, a non-linear funnel with locally deposited agarose gel in the funnel may create an environment that controls stretching.

A conveying device. The structure used to stretch the polymer is not the only useful component placed in the channel. Structures designed to advantageously localize the polymer in one portion of the channel but not another are useful to ensure that the polymer is supplied to a particular spreading structure or a particular detection zone. In addition to adding fluid to a single channel as described above (see the branch channels), this can be done by forcing streamlines to approach each other. The polymer driven by the fluid flow (induced by any subsequently cited method such as pressure differential and gravity) will move primarily along the fluid flow lines (in electrophoresis for charged biopolymers, the polymer follows the electric field lines, which can likewise be altered). The irregular motion may cause portions of the chain to move toward adjacent streamlines. If the flow lines encounter a constriction or obstruction, the flow lines become closer to each other around the obstruction, resulting in a greater chance that irregular motion of the same side causes a change in the flow lines. When the streamlines return to their original spacing on the other side of the structure (if the channel returns to its original width), the velocity gradient between the streamlines tends to drag the polymer towards the faster streamlines. In this way, previously randomly distributed polymers can be turned into more regular ones. For example, in one embodiment, a large triangle at the center of the channel, one side of which faces downstream perpendicular to the channel, tends to position the polymer toward the center because the polymer originally near the wall tends to pull toward the center by virtue of the fluid moving laterally on the downstream side of the triangle. In other embodiments, other shapes are used to facilitate positioning, such as cross-shaped obstacles, wedges, and areas with misaligned obstacles that help guide a larger channel on a particular side of the channel. Although a channel with a simple bend should have a positional effect, which seems intuitive, the velocity gradient involved is in fact rather small, its individual effect being rather small.

A method of improving stretch in a structure. In a further embodiment of the invention, the effectiveness of the scissor-induced mode is increased by increasing the viscosity of the solution. The actual force generated by the constriction of the channel is proportional to the solution viscosity. In certain embodiments, the solution viscosity is increased by the addition of one or more viscosity modifying components. Glycerol (which will have a viscosity of approximately 900cP at room temperature) can be added to the aqueous solution at concentrations up to 70% (w/v) if it does not react chemically with the polymer. Sugars such as sucrose, xylose and sorbitol may also be added. Water-soluble polymers, such as polyethylene glycol, may also be added. In the case of DNA, high molecular weight polyacrylamides, polyethylene oxides or long-chain glycans, even at concentrations as low as 0.01% by weight, can increase the viscosity of aqueous solutions without altering the structure of the DNA to be characterized.

The viscosity can also be altered by adding an appropriate amount of the polymer being characterized, but which is not detected by the detection zone of the structure. For example, if FRET is performed on an exogenously labeled DNA molecule, additional DNA molecules that are not exogenously labeled may be added to the labeled polymer solution to increase viscosity. Thus, only the labeled molecules are detected and unlabeled DNA merely changes the viscosity of the solution and does not interfere with the signal of the labeled molecules.

In another embodiment, the viscosity is increased by decreasing the temperature; for example, pure water may have a viscosity that approximately doubles as it approaches freezing. In addition to increasing viscosity, lowering the temperature is used to minimize brownian motion and extend relaxation time. Stretching is considerably improved when the temperature of an aqueous buffer solution, such as a 1 XTE solution (10mM TRIS, 1mM EDTA), is changed from ambient temperature to 4 ℃.

A driving force. The driving force for moving the polymer through the structure may come from any device, including physical, electrical, thermal or chemical forces. The simplest driving force is to drive the flow by capillary action when the sample solution and the device are first in contact. Although the surface energy involved can provide high velocity in the channel, the control of the flow in this manner is limited.

Indirect and therefore limited control can be obtained by using chemical potentials. One advantage of establishing a concentration gradient is that it provides an extremely slow, steady flow rate. This is done by creating a large excess of substance on one side of the structure and consuming the diffused substance after it causes fluid to flow through the structure to the other side, controlled based on the excess concentration. The polymer flows through the structure with the fluid flow induced by means of the migrating species.

A preferred embodiment directly controls the flow of fluid. In such an embodiment, a head is formed on the inlet side of the structure to force fluid flow distally, which is either open to atmospheric pressure or maintained at a reduced pressure. The ram may come from any device that utilizes physical force, such as a syringe pump. Currently, syringe pumps dispense up to the range of 100pL/s, whereas the flow rates required in the device may be at 1pL/s, and therefore it may be necessary to establish a "bypass" with a large cross-sectional area, thus increasing the flow rate required by the device and allowing control with commercially available equipment, losing only a few volumes of sample. In another embodiment of the pressure control system, one end of the system is gradually sucked in through the structure by vacuum suction in a device where the pressure drop is less than atmospheric pressure. The pressure drop required to induce flow at the required velocity is a function of the channel geometry (particularly the minimum cross-sectional dimension) and velocity, but is typically around 10psi for a flow of 100 microns per second in a millimeter long, micron deep channel (otherwise wide for most devices). In another embodiment, a combination of a pressure head at a first end of the channel and a vacuum at a second end of the channel is used to push the polymer from the first end to the second end.

In a further embodiment, the polymer flow through the fluid is controlled by means of establishing a temperature gradient on each side of the expansion zone. Natural convection thus establishes fluid flow through the extended zone. Since it is very difficult to establish and control the temperature gradient at the micron scale of operation of these devices, this method, like the chemical potential method, is preferred for very low liquid flow rates.

In another embodiment, the flow of polymer, for charged polymers such as DNA, is controlled by establishing an electric field that acts on the charge of the polymer, but not on the surrounding fluid at all (if it is uncharged). The electric field is preferably formed by the presence of two oppositely-charged electrodes in the solution, but the overall series of electrodes can be used to create a more complex or uniform field pattern. The polymer moves along electric field lines rather than streamlines (in some cases, there is an abrupt change depending on the physical layout of the micromodules and the charge density of the solution). This can impair stretching if the surrounding solution contains oppositely-charged species that flow in opposite directions (electrokinetic flow), or surface charges on the channel walls create ionic flow along the walls (electroosmotic flow), both of which can induce fluid flow in opposite directions and create viscous forces on the polymer. However, in low conductivity solutions, where the walls are suitably coated to avoid surface charges, the reverse viscous forces have a negligible effect on the driving force of electrophoresis, allowing the polymer to travel through the structure and be stretched. Furthermore, with appropriately charged wall surfaces, the electroosmotic flow can be reversed to provide a viscous force that facilitates the electrophoretic stretching. A field strength of 1000 to 2000V/m results in a suitable polymer velocity in the range of 100 microns per second.

In the case of electrophoretic and pressure driving forces, the means for generating the driving force is typically physically separated from the extended zone. The electrodes are positioned a few millimeters to many centimeters away from the extended zone, and the power supply is located even further away. The syringe pump, while advantageously as close as possible to the expansion region to minimize the required pressure drop, tends to be placed outside the device because of its large volume. In fact, for structural flexibility, it is preferable to simply place the stretching and detection structure itself on a small micromodule, preferably no longer than 2cm in side length, and perhaps as small as a 1mm square, with the most preferred dimensions (from an operational point of view) being about 1.5cm by 1cm, and 0.2cm thick. On the substrate, various fluid flow channels are provided. In such a module, 1-160 channels can be comfortably placed on the substrate, 30-40 channels can be well balanced: repetition in the case of channel blockage or substrate cracking and only one channel at a time are in the detection field of view (typically with a 60X objective).

A substrate. The choice of substrate used is appropriate for the solution and conditions used in the analysis, including but not limited to extreme salt concentrations, acid or base concentrations, temperature, electric field, and transparency to wavelengths used for light excitation or emission. The substrate material may include those associated with the semiconductor industry, such as fused silica, quartz, silicon, or gallium arsenide, or inert polymers such as polymethylmethacrylate, polydimethylsiloxane, polytetrafluoroethylene, polycarbonate, or polyvinyl chloride. Quartz is a preferred embodiment because of its transmissive properties at a wide variety of wavelengths.

The use of quartz as a substrate for the aqueous solution means that the contact surface with the solution has a positive charge. When working with charged molecules, especially under electrophoretic conditions, it is desirable to have a neutral plane. In one embodiment, a coating is applied to the surface to eliminate the interaction that causes the charge. The coating can be applied commercially (Supelco's capillary coating, Bellafonte PA) or by using silanes with functional groups at one end. The silane end will effectively irreversibly bond to the glass and the functional group can further react to produce the desired coating. For DNA, the silane with polyethylene oxide can effectively prevent interaction between the polymer and the wall without further reaction, and the silane with acrylamide groups can participate in the polymerization reaction to produce a polyacrylamide coating that not only does not interact with DNA, but also inhibits electroosmotic flow during electrophoresis.

The channels can be constructed on the substrate by a number of techniques, many of which are derived from the semiconductor industry, depending on the substrate selected. These techniques include, but are not limited to, photolithography, reactive ion etching, wet chemical etching, electron beam writing, laser or air ablation, LIGA, and injection molding. Various of these techniques applied to polymer-processed microcomponents have been discussed in the following documents, including Harrison et al (Analytical Chemistry 1992(64)1926-1932), Seiler et al (Analytical Chemistry 1993(65)1481-1488), Woolley et al (Proceedings of the National Academy of sciences 1994 November (91)11348-11352), and Jacobsen et al (Analytical Chemistry 1995(67) 2059-2063).

Other considerations are taken into account. In a preferred embodiment of the invention, the velocity is substantially uniform in a rectangular channel at a given channel plane height. This is true when the height of the channel is significantly less than the width, so that the non-slip condition on the walls results in a viscosity-induced parabolic velocity profile, which is significant in the height axis, leaving only a small slower-flowing boundary region in the width axis. An aspect ratio (width/height) of about 10 or more is required for embodiments that approximate according to lubrication theory (Deen, Analysis of Transport Phenomena, new york: Oxford university press, 1998.275-278.) furthermore, a small height facilitates detection when using a microscope objective in an optical system a typical objective may have a depth of focus of 500nm to several microns, so while the height of the channel may be 50nm to 100 μm, the preferred embodiment has a channel height of 200nm to 1 μm, so that all material passing in the channel will be in focus and accurately observed, as long as the aspect ratio is around 10 to accommodate the polymer being analyzed.

The invention also includes embodiments in which the channels are not planar and are fabricated using three-dimensional channel fabrication techniques. In such embodiments, constant shear is induced not only from the side walls, but also from the gradient in channel height. Likewise, in a further embodiment, the combination of structures has a force acting on one axis and another force acting on another axis. In some such embodiments, the barrier region spans the width of the channel, while its height decreases in a tapered shape. In other embodiments, a serpentine, inward-spiral design in a single plane, which also decreases in channel width, is used to generate shear forces that feed in the center, enter the vertical outlet of the device through a hole in the bottom of the material, detecting the entrance near the hole. Gravity is used in certain embodiments to help create velocity differences in the fluid when the structure is in a vertical orientation. (notably, gravity alone is not sufficient to stretch the polymer or significantly cause it to flow, since the force on the 100kD polymer is just over 10 kD^-18N; any effect of gravity will be felt by the molecules through viscous forces. )

6. Examples of the embodiments

6.1 example 1: fabrication of a micromodule for stretching DNA, and

their use in a device for detecting fluorescence emitted from labeled DNA

An experimental device. The sensitive optics used for the detection are shown in FIG. 24. The device utilizes confocal fluorescence illumination and detection. Confocal illumination causes a small optical volume (on the order of femtoliters) to be illuminated. The use of a small probe volume minimizes both Rayleigh and Raman scattering. The beam from the 1mW argon ion laser passed through a laser line filter (514nm), sent to a dichroic mirror, passed through a 100x 1.2NA oil immersion objective, and then to the sample. The fluorescent label on the DNA may be one of several dyes including: cy-3, tetramethylrhodamine, rhodamine 6G, and Alexa 546. In addition, intercalator dyes such as TOTO-3 (molecular probe) may be used. The fluorescence emission from the sample is focused through a dichroic, narrow bandpass (e.g., omega optical), onto a 100 μm pinhole, through an aspheric lens, and finally onto an avalanche photodiode in photon counting mode (EG & G canada). The output signal was collected with a multi-way scalar (EG & G) and computer analyzed using Pentium III. The confocal assembly is suitable for quantitative applications including time of flight. Such applications include testing distances on DNA, detecting the order of labels, and determining the degree of stretch in DNA. The device can be used to detect single fluorescent molecules. For applications requiring an image, a device using an intensified CCD (ICCD) placed on a microscope is suitable.

And (4) manufacturing a micro assembly. On a 0.090 inch thick quartz substrate, a set of constant-shear channels with a designed shear rate of 0.085/s, guided by two rows of 2 micron spaced 1.5 micron obstacles, was created by photolithography and electron beam methods. The substrate was first cleaned by placing in an RCA solution (5 parts deionized water to 1 part 30% ammonium hydroxide/30% hydrogen peroxide, the latter two from Sigma Chemical co., st. louis, MO) heated to 80 ℃ for twenty minutes, then dried under a stream of nitrogen. Shipley S1813 photoresist diluted with R type diluent (Shipley, Newton, MA) at a 2: 1 ratio was then spin coated on the quartz surface in a spin coater at 3250rpm for 45 seconds and then cured in an oven at 90 ℃ for 0.5 hours. The rough constant-shear form is then contact printed on the surface by exposure to a mercury lamp for 12s, for example in a contact lithography machine from Carl Zeiss, germany, then rinsed in 351 developer (Shipley) diluted with deionized water at a ratio of 5: 1 for 30s, further rinsed in deionized water, and then dried under a stream of nitrogen. After a 10s UV-ozone purge, the substrate was treated with CHF3 at Reactve Ion Etch (R)IE) machine for 40 minutes. After another wash in RCA solution, a solution of polymethyl methacrylate (650MW) diluted to 3% in chlorobenzene was spin coated on the surface in a spin coater at 2000rpm for 45 seconds. The coating was cured in an oven at 180 ℃ for one hour and then a 60  chrome layer was added to the evaporator. Electron beam writing is performed to produce fine structures, such as barrier rows, followed by chrome etching in a REI machine and rinsing with deionized water. The substrate was then immersed for 90 seconds in a 2: 1v/v solution of isopropanol methyl isobutyl ketone heated to 21 ℃ to develop and then subjected to another UV-ozone purification. Then another CHF is performed in the REI machine₃Etching followed by RCA solution washing.

Coverslips (Fisher Scientific, Pittsburgh, Pa.) measuring 45mm by 50mm by 0.15mm were rinsed with deionized water and dried under nitrogen. A10: 1w/w RTV 615A: a solution of RTV 615B silicone (General Electric, Schenectady, NY) was spin coated on the coverslip in a spin coater at 4000rpm for 60 seconds and then cured at 80 ℃ for two hours. A silicone plate with wells in which the microassembly was placed on a cover glass, which was then exposed to a 30W plasma cleaner for 50 seconds to create a hydrophilic surface. The silicone plate was then removed and the coverslip rinsed in deionized water and then dried under nitrogen. The fully prepared micromodule is then carefully positioned on the cover glass.

A device for monitoring the image-dependent pulses from stretched DNA. As shown in fig. 25, the delivery system consists of a polymer supply 151 driven by a syringe pump 150 through a microassembly 152 (described above) in which the polymer is stretched and excited by a laser beam from a laser 154, which is detected by a photodetector 153 and analyzed by a computer 155, which also controls the pump 150 and detector 153.

Fluorescence emission in the stretched DNA was monitored. Coli phage T4 DNA (Sigma, St.Louis, Mo.) was labeled by adding 4040-1 at a 5: 1 (base pair: dye) ratio, incubated for one hour, and then diluted to one-50,000 in 0.5 XTBE running buffer (45mM TRIS, 32.3mM boric acid and 1.25mM EDTA, pH8.3, all from Sigma, St.Louis, Mo.).

A microliter of the sample is then pipetted onto the coverslip, which is next to the micromodule where it is introduced into the channel by capillary action. The micromodules and coverslips were mounted on the stage of a fluorescence microscope (micropht series from Nikon) equipped with a 60X plano apo lens (from Nikon or Carl Zeiss, for example). Excitation was from a mercury arc lamp with a Nikon B2A filter to ensure sufficient excitation around the 490nm peak excitation of YOYO-1. The emission above 520nm passes through the B2A filter set and is captured by a silicon-enhanced camera (C2400-08 to Hammamatsu) or a CCD camera. The image from the camera is output to a computer via an image capture card (e.g., PCI-1408 from national instruments, Austin, TX) and analyzed with image processing software, which is a custom-written program that identifies DNA on the screen based on brightness relative to the background and calculates pixels to determine polymer length.

Various DNA molecules were observed in the device (fig. 26). The constantly sheared portions of the micromodules in FIGS. 27(a-g) show stretched DNA molecules of about 190kb (63 μm). A fully stretched DNA in the micromodule is shown in FIG. 28. The molecule measures 139 microns, or 535 kb.

And (4) data. A small sample (half microliter) of T4 DNA (Sigma) stained with YOYO-1 (molecular probe) was loaded into a micromodule having a rectangular funnel section along with a column and operating under capillary action. The samples were excited with a 100WHg lamp and observed with a SIT video camera (Hammatsuc 2400-08). The video signal from the camera was sent to an image capture card in the Pentium class computer running a custom LabView software that determined the length of the DNA fragment in pixels based on its speed and the time spent in the study area. Lengths of less than 30 microns are considered debris and are automatically eliminated, which results in only ten data points being obtained in about two minutes of sample run. Using known scaling of amplification levels, DNA was found to be 50.6 μm long, between 42 and 62 μm. A bar graph is shown in figure 29. The length was slightly shorter than the expected value of 71.1 μm for the pigmented 164kbp T4 DNA, indicating that the stretch was not completely completed in the design.

6.2 example 2: extension of bacteriophage lambda DNA Using the device of the invention

Two different devices were used to obtain the data shown in fig. 30(a) and 30 (b). The data shown in fig. 30(b) was obtained using the apparatus shown in fig. 20. The apparatus used to obtain the data of fig. 30(a) has the same channel boundaries (i.e. the same size ratio of the two tapered regions of the two-funnel apparatus) as the apparatus used to obtain the data of fig. 30(b), except that no pillars are present in the structure.

A fused silica sheet (Hoya corp., San Jose, CA) was etched to have the pattern of fig. 20 using the photolithography method described above. The sheet was cut into 1cm by 2cm micro-assemblies using a dicing saw (e.g. from discocorp., Santa Clara, CA) and then a cover glass of fused silica (e.g. from Esco, Oak Ridge, NJ) was attached by means of thermal bonding.

Double-stranded lambda DNA (Promega, Madison, Wis.) having a uniform length of 48.5 kilobases (i.e., the expected extension length is 16-17 microns) was labeled by adding a similar amount of 3. mu. MTOTO-3 iodide (molecular probe, Eugene OR) intercalating dye and then diluted to approximately one 50,000 in 1 XTE buffer (10mM TRIS and 1mM EDTA, pH8.0, all from Sigma, St. Louis, Mo.). For the double stranded 48.5 kilobase DNA samples used here, the expected stretch length of lambda DNA stained with the intercalating dye was 21 μm (approximately 30% longer than the unstained DNA).

The micromodules and coverslips are placed on the microscope stage of a fluorescence microscope (e.g., the micropht series from Nikon) equipped with a 100X plano apo lens (e.g., from Nikon, Carl Zeiss) and a filter set optimized for TOTO-3 (e.g., XF-47 from Omega Optical, Brattleboro, VT). Excitation from a 633nm helium-neon laser (e.g., from Melles Griot) is focused at two points arranged on the same flow line within the microchannel. The sample is input at the entrance of the channel by means of capillary action and the flow is continued by means of a vacuum at the other end of the micromodule (generated by means of a vacuum pump, e.g. Welch vacuum, Skokie, IL). When a DNA molecule passes through a laser spot, emissions in excess of 650nm pass through the filter set and are received by a pair of confocal detectors arranged on the spot. The time of flight between the detectors is used to determine the velocity, which is used together with the dwell time in one laser spot to calculate the length of the molecule.

The results of this experiment show that the two-funnel device including the column stretches 48.5 kilobases of double stranded, dye-stained lambda DNA to a length of about 19.5 μm (FIG. 30(b)), while the two-funnel device without the column stretches DNA only to a length of about 10 μm (FIG. 30 (a)). Thus, while there is stretch of DNA in the tapered channel without pillars, on average, DNA stretches only slightly over half its full length, and very few individual molecules are fully stretched, as evidenced by the broad distribution of the histogram in fig. 30 (a). By contrast, in a structure having a pillar region in combination with a downstream tapered channel, on average, the molecules are stretched to nearly full length, and most of the molecules are within 20% of their fully stretched length expected. Thus, a two-funnel device with a column is better able to stretch DNA than the same device without a column. In addition, the apparatus is capable of stretching the polymer more uniformly and efficiently than a two-funnel configuration without a column.

7. Reference to the cited references

All references cited herein are incorporated by reference in their entirety and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

It will be apparent to those skilled in the art that many modifications and variations can be made in the present invention without departing from the spirit or scope thereof. The particular embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel that decreases linearly in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end;

whereby a shear force is applied to the at least one polymer in the fluid sample as the at least one polymer moves along the tapered channel.

2. The integrated device of claim 1, wherein the tapered channel decreases in width at an angle of 1-75 °, the angle being defined as the angle relative to a constant width channel at the first end.

3. The integrated device of claim 2, wherein the angle equals 26.6 ° and the at least one polymer comprises DNA.

4. The integrated device of claim 1, wherein the width of the first end is 1 μm to 1cm and the width of the second end is 1nm to 1 mm.

5. The integrated device of claim 4, wherein the width of the first end is equal to 50 μm, the width of the second end is equal to 5 μm, and the at least one polymer sample comprises DNA.

6. The integrated device of claim 1, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

7. The integrated apparatus of claim 6, wherein said transport device comprises a transport channel that leads in and out of said elongated structure.

8. The integrated device of claim 1, further comprising means for moving said at least one polymer, when present, within said elongated structure.

9. The integrated apparatus of claim 8, wherein said means comprises means for creating a pressure differential along said elongated structure.

10. An integrated device, comprising:

an elongated structure comprising a tapered channel that decreases linearly in width from a first end to a second end, the tapered channel comprising at least one polymer in a fluid sample, the tapered channel being configured such that a shear force is applied to the at least one polymer when the at least one polymer moves from the first end to the second end.

11. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel that decreases in width from a first end to a second end at a greater than linear rate, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end;

whereby a shear force is applied to the at least one polymer as the at least one polymer in the fluid sample moves along the tapered channel.

12. The integrated device of claim 11, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

13. The integrated apparatus of claim 12, wherein the transport device comprises a transport channel that leads in and out of the elongated structure.

14. The integrated device of claim 11, further comprising means for moving said at least one polymer in said fluid sample within said elongated structure.

15. The integrated apparatus of claim 14, wherein said means comprises means for creating a pressure differential along said elongated structure.

16. An integrated device, comprising:

an elongated structure comprising a tapered channel that decreases in width at a rate greater than linear from a first end to a second end, the tapered channel comprising at least one polymer in a fluid sample, the tapered channel being configured such that a shear force is applied to the at least one polymer when the at least one polymer moves in a direction from the first end to the second end.

17. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel that decreases in width from a first end to a second end, and wherein said at least one polymer, when present, moves along said tapered channel in a direction from said first end to said second end;

whereby a shear force is applied to the at least one polymer as the at least one polymer in the fluid sample moves along the tapered channel, wherein the shear force creates a constant shear rate.

18. The integrated device of claim 17, wherein the width of the tapered channel satisfies the equation: w ═ C (SHx/Q)^-1Where W is the width, S is the shear rate, Q is the total flow and C is a constant.

19. The integrated device of claim 18, wherein the shear rate is at 0.01s^-1-10s^-1In the meantime.

20. The integrated device of claim 19, wherein the shear rate is equal to 0.075s^-1。

21. The integrated device of claim 18, wherein the tapered channel has a length of 1mm to 2 mm.

22. The integrated device of claim 18, wherein the fluid sample is maintained at a temperature below ambient temperature.

23. The integrated device of claim 17, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

24. The integrated apparatus of claim 23, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

25. The integrated device of claim 17, further comprising means for moving said at least one polymer, when present, within said elongated structure.

26. The integrated apparatus of claim 25, wherein said means comprises means for creating a pressure differential along said elongated structure.

27. An integrated device, comprising:

an elongated structure comprising a tapered channel that decreases in width from a first end to a second end, the tapered channel comprising at least one polymer in a fluid sample, the tapered channel being configured such that a shear force is applied to the at least one polymer when the at least one polymer is moving in a direction from the first end to the second end, wherein the shear force produces a constant shear rate.

28. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a central channel for receiving a fluid and a plurality of lateral channels for receiving a fluid connection to said central channel; and

wherein the at least one polymer, when present, moves in an elongated direction along the central channel.

29. The integrated device of claim 28, wherein said plurality of side channels are oriented such that fluid injected from said plurality of side channels moves substantially along said central channel in said elongated direction.

30. The integrated device of claim 28, wherein said central channel has a central-channel cross-sectional area and said plurality of side channels have a total side-channel cross-sectional area that is between 1% and 500% of said central-channel cross-sectional area.

31. The integrated device of claim 30, wherein the total side-channel cross-sectional area is equal to 50% of the center-channel cross-sectional area.

32. The integrated device of claim 28, wherein the plurality of side channels are arranged in a repeating pattern.

33. The integrated device of claim 28, wherein the central channel is tapered.

34. The integrated device of claim 28, wherein the fluid is maintained at a temperature below ambient temperature.

35. The integrated device of claim 28, wherein at least one of said plurality of side channels is tapered.

36. The integrated device of claim 28, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

37. The integrated apparatus of claim 36, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

38. The integrated device of claim 28, further comprising means for moving said at least one polymer, when present, within said elongated structure.

39. The integrated apparatus of claim 38, wherein said means comprises means for creating a pressure differential along said elongated structure.

40. An integrated device for stretching at least one polymer in a fluid sample, comprising:

(a) an elongated structure;

(b) a transport channel leading into and out of said elongated structure, which transports said at least one polymer sample in said fluid to said elongated structure; and

(c) means for moving said at least one polymer in said fluid sample, when present, within said elongated structure;

wherein said elongated structure comprises a central channel for receiving a fluid and a plurality of side channels for receiving a fluid connected to said central channel; and

wherein, when the at least one polymer is present, the apparatus moves the at least one polymer along the central passage in an elongate direction.

41. An integrated device for stretching DNA in a fluid sample, comprising:

(a) an elongated structure;

(b) means for delivering said DNA in said fluid sample to said elongated structure; and

(c) means for moving said DNA in said fluid sample, when present, within said elongated structure;

wherein, when the DNA is present, the device moves the DNA along the central channel in an elongation direction.

42. An integrated device, comprising:

an elongated structure comprising a central channel for receiving a fluid, said central channel comprising a first end and a second end, and a plurality of lateral channels for receiving a fluid connected to said central channel, said central channel comprising at least one polymer in a fluid sample,

wherein the at least one polymer moves in a direction from the first end to the second end.

43. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel having at least one bend, and wherein said at least one polymer, when present, moves along said channel.

44. The integrated device of claim 43, wherein the channels have a sinusoidal shape.

45. The integrated device of claim 44, wherein the sinusoidal shape has an amplitude to period ratio of 0.01 to 5.

46. The integrated device of claim 43, wherein the channel comprises a plurality of linear portions connected at a non-zero angle.

47. The integrated device of claim 46, wherein each of a middle of said plurality of straight portions has the same length, and said non-zero angle is equal in magnitude and varies alternately in sign, such that said channel has a zig-zag, turning shape.

48. The integrated device of claim 47, wherein each of said non-zero angles is between 5 ° and 75 °.

49. The integrated device of claim 48, wherein each of the non-zero angles is equal in size to 26.6 ° and the at least one polymer sample comprises DNA.

50. The integrated device of claim 43, wherein the channel comprises a plurality of right angle bends such that the polymer moves in a repeating path of two 90 ° turns and two-90 ° turns.

51. The integrated device of claim 43, wherein the channels comprise a cyclic shape and have a cycle number of 1-500.

52. The integrated device of claim 51, wherein the number of cycles is 10.

53. The integrated device according to claim 51, further comprising means for detecting said at least one polymer along a detection zone, wherein said detection means is positioned such that said channel repeatedly passes through said detection zone at defined locations.

54. The integrated apparatus according to claim 43, wherein said means for transporting said at least one polymer comprises a transport channel leading into and out of said elongated structure.

55. The integrated device of claim 43, wherein the channel contains a solution that is maintained at a temperature below ambient temperature.

56. The integrated device of claim 43, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

57. The integrated apparatus of claim 56, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

58. The integrated device of claim 43, further comprising means for moving said at least one polymer, when present, within said elongated structure.

59. The integrated apparatus of claim 58, wherein the means comprises means for creating a pressure differential along the elongated structure.

60. An integrated device for stretching DNA in a fluid sample, comprising:

(a) an elongated structure; and

(b) means for delivering the DNA in the fluid sample to the elongated structure;

wherein the elongated structure comprises a channel having at least one bend, an

Wherein said DNA, when present, moves along said channel.

61. The integrated device of claim 41, further comprising means for moving said DNA, when present, within said elongated structure.

62. An integrated device, comprising:

an elongated structure comprising a channel having at least one bend, said channel comprising a first end and a second end, said channel comprising at least one polymer in a fluid sample for stretching said at least one polymer,

63. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a tapered channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to the movement of said at least one polymer.

64. The integrated device of claim 63, wherein the tapered channel decreases linearly in width from the first end to the second end.

65. The integrated device of claim 63, wherein the tapered channel decreases in width from the first end to the second end at a rate greater than linear.

66. The integrated device of claim 63, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

67. The integrated apparatus according to claim 66, wherein the transport device comprises a transport channel leading into and out of the elongated structure.

68. The integrated device of claim 63, further comprising means for moving said at least one polymer, when present, within said elongated structure.

69. The integrated apparatus according to claim 68, wherein said means comprises means for creating a pressure differential along said elongated structure.

70. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a central channel along which said at least one polymer, when present, moves in a flow direction, and a plurality of lateral channels connected to said central channel, and wherein said central channel further comprises a plurality of obstacles to the movement of said at least one polymer.

71. The integrated device of claim 70, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

72. The integrated apparatus of claim 71, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

73. The integrated device of claim 70, further comprising means for moving said at least one polymer, when present, within said elongated structure.

74. The integrated apparatus of claim 73, wherein said means comprises means for creating a pressure differential along said elongated structure.

75. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel having at least one bend along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to the movement of said at least one polymer.

76. The integrated device of claim 75, wherein the channels comprise a cyclical shape having a period number of 1-500.

77. The integrated device of claim 76, wherein the shape of the cycle is sinusoidal.

78. The integrated device of claim 76, wherein the shape of the loop is a zig-zag turn.

79. The integrated device of claim 75, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

80. The integrated apparatus of claim 79, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

81. The integrated device of claim 75, further comprising means for moving said at least one polymer, when present, within said elongated structure.

82. The integrated apparatus of claim 81, wherein the means comprises means for creating a pressure differential along the elongated structure.

83. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of pillars, at least one of said pillars having a cross-sectional shape that is a polygon other than a quadrilateral.

84. The integrated device of claim 83, wherein at least one of the plurality of posts comprises a concave edge, wherein the concave edge faces in a direction of fluid ingress.

85. The integrated device of claim 83, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

86. The integrated apparatus of claim 85, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

87. The integrated device of claim 83, further comprising means for moving said at least one polymer, when present, within said elongated structure.

88. The integrated apparatus of claim 88, wherein the means comprises means for creating a pressure differential along the elongated structure.

89. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to movement of said at least one polymer, said plurality of obstacles being positioned in a series of rows, each of said rows being positioned perpendicular to said flow direction, and each successive row being offset from a preceding row, whereby along said flow direction at least one portion that is not equal to a multiple of 1/2 of one of said obstacles overlaps the extension of a gap formed by two adjacent obstacles in said preceding row.

90. The integrated device of claim 89, wherein said channel is tapered.

91. The integrated device of claim 89, further comprising a plurality of side channels connected to said channel.

92. The integrated device of claim 89, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

93. The integrated apparatus of claim 92, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

94. The integrated device of claim 89, further comprising means for moving said at least one polymer, when present, within said elongated structure.

95. The integrated apparatus of claim 94, wherein the means comprises means for creating a pressure differential along the elongated structure.

96. An integrated device, comprising:

an elongated structure comprising a channel, the channel comprising:

(a) a first end and a second end;

(b) a plurality of obstacles to movement of at least one polymer, said plurality of obstacles being positioned in a series of rows, each said row being positioned perpendicular to said flow direction, and each adjacent pair of obstacles in each row of said series of rows being separated by a distance greater than 50 times said minimum diameter; and

(c) at least one polymer in the fluid sample, each of the polymers having a diameter greater than or equal to a minimum diameter,

97. The integrated device of claim 96, wherein the at least one polymer comprises DNA, and the distance separating each adjacent pair of obstacles in each row of the series of rows is from 100nm to 800 nm.

98. The integrated device of claim 97, wherein said distance separating each adjacent pair of obstacles in each row of said series of rows is equal to 500 nm.

99. The integrated device of claim 96, wherein each row of said series of rows has a total obstruction cross-section and a total channel width, said total obstruction cross-section being equal to a total area obstructed along said flow direction by said obstruction in each row of said series of rows, and said total channel width being equal to a total area unobstructed along said flow direction by said obstruction in said each row of said series of rows, wherein said total obstruction cross-section and said total channel width have a ratio of 0.5-20.

100. The integrated device of claim 99, wherein the at least one polymer comprises DNA and the ratio is between 2 and 4.

101. The integrated device of claim 96, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

102. The integrated apparatus of claim 101, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

103. The integrated device of claim 96, further comprising means for moving said at least one polymer, when present, within said elongated structure.

104. The integrated apparatus of claim 103, wherein the means comprises means for creating a pressure differential along the elongated structure.

105. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises a plurality of obstacles to the movement of said at least one polymer, said plurality of obstacles decreasing in cross-sectional area along said flow direction.

106. The integrated device of claim 105, wherein the channel is tapered.

107. The integrated device of claim 105, further comprising a plurality of side channels connected to said channel.

108. The integrated device of claim 105, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

109. The integrated apparatus of claim 108, wherein the transport device comprises a transport channel that leads in and out of the elongated structure.

110. The integrated device of claim 105, further comprising means for moving said at least one polymer, when present, within said elongated structure.

111. The integrated apparatus of claim 110, wherein the means comprises means for creating a pressure differential along the elongated structure.

112. An integrated device for stretching DNA, comprising an elongated structure,

wherein the elongated structure comprises a tapered central channel comprising a first end and a second end, and wherein the DNA, when present, moves along the tapered central channel in a direction from the first end to the second end,

wherein said elongated further comprises a plurality of side channels connected to said tapered central channel,

wherein the tapered central passage comprises at least one bend; and

wherein said tapered central channel comprises a plurality of obstacles to movement of said DNA.

113. The integrated device of claim 112, wherein said tapered central channel decreases linearly in width from said first end to said second end.

114. The integrated device of claim 112, wherein said tapered central channel decreases in width from said first end to said second end at a rate greater than linear.

115. The integrated device of claim 112, wherein at least one of said plurality of side channels is tapered.

116. The integrated device of claim 112, wherein said means for delivering said DNA comprises a delivery channel, said delivery channel leading into and out of said elongated structure.

117. The integrated device of claim 112, wherein said tapered central channel has a sinusoidal shape.

118. The integrated device of claim 112, wherein the tapered central channel has a zig-zag shape.

119. The integrated device of claim 112, wherein said plurality of obstacles decrease in cross-sectional area from said first end to said second end.

120. The integrated device of claim 112, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

121. The integrated apparatus of claim 120, wherein the transport device comprises a transport channel that leads in and out of the elongated structure.

122. The integrated device of claim 112, further comprising means for moving said at least one polymer, when present, within said elongated structure.

123. The integrated apparatus of claim 122, wherein the means comprises means for creating a pressure differential along the elongated structure.

124. An integrated device for stretching DNA, comprising an elongated structure comprising:

(a) a first tapered channel comprising a first end, a second end and a plurality of posts staggered between said first end and said second end, forming 12-15 rows, said first tapered channel decreasing in width at an angle of 26.6 °, said angle being defined at said first end relative to a constant-width channel, said first end having a width between 0.5 and 5 μm, said posts having a width equal to 1.5 μm²And are separated by a gap equal to 0.5 μm; and

(b) a second conical channel connected to said first conical channel at said second end and reduced in width to between 0.5 and 5 μm, whereby a shear force producing a constant shear rate is applied to said DNA, said second conical channel having a length, when present, of between 1 and 3 mm.

125. The integrated device of claim 124, wherein the width of said second tapered channel satisfies the equation: w ═ C (SHx/Q)^-1Where W is the width, S is the shear rate, Q is the total flow and C is a constant.

126. The integrated device of claim 125, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

127. The integrated apparatus of claim 126, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

128. The integrated device of claim 125, further comprising means for moving said at least one polymer, when present, within said elongated structure.

129. The integrated apparatus of claim 128, wherein the means comprises means for creating a pressure differential along the elongated structure.

130. A method for stretching at least one polymer, comprising the steps of:

moving the at least one polymer along an elongated structure comprising a tapered channel having a first end and a second end;

whereby said tapered channel causes a shear force that creates a constant shear rate applied to said at least one polymer as it moves along said tapered channel from said first end to said second end.

131. The integrated device of claim 130, wherein the width of the tapered channel satisfies the equation: w ═ C (SHx/Q)^-1Where W is the width, S is the shear rate, Q is the total flow and C is a constant.

132. The method of claim 131, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by means of capillary action.

133. The method of claim 131, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by forming a concentration gradient along said tapered passageway.

134. The method of claim 131, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by forming an indenter at said first end of said tapered passageway.

135. The method of claim 134, wherein said head is formed by connecting a syringe pump to said first end of said tapered passageway.

136. The method of claim 135, wherein said elongated structure further comprises a bypass connected to said tapered channel.

137. The method of claim 131, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by creating a pressure drop at said second end of said tapered passageway.

138. The method of claim 131, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by forming a temperature gradient along said tapered passageway.

139. The method of claim 131, wherein said at least one polymer comprises a charged polymer and said step of moving said at least one polymer along said tapered passageway is accomplished by creating an electric field along said tapered passageway.

140. The method of claim 139, wherein said electric field has a field strength between 1000 and 2000V/m.

141. The method of claim 139, wherein the electric field is formed with two oppositely charged electrodes in the solution.

142. The method of claim 139, wherein the electric field is formed with a series of electrodes in solution.

143. A method for stretching at least one polymer, comprising the steps of:

moving at least one polymer along an elongated structure comprising a linearly tapered channel having a first end and a second end,

wherein the at least one polymer moves along the channel from the first end to the second end.

144. A method for stretching at least one polymer, comprising the steps of:

moving at least one polymer along an elongated structure, said elongated structure comprising a tapered channel having a first end and a second end, said tapered channel decreasing from said first end to said second end at a rate greater than linear,

145. A method for stretching at least one polymer, comprising the steps of:

moving at least one polymer along an elongated structure, said elongated structure comprising a central fluid-containing passageway and a plurality of lateral fluid-containing passageways fluidly connected to said central passageway, said central passageway comprising a first end and a second end,

wherein the at least one polymer moves along the central channel from the first end to the second end.

146. The method of claim 145, wherein said step of moving said at least one polymer along said central passageway is accomplished by means of capillary action.

147. The method of claim 145, wherein said step of moving said at least one polymer along said central channel is accomplished by forming a concentration gradient along said central channel.

148. The method of claim 145, wherein said step of moving said at least one polymer along said central passageway is accomplished by creating an indenter at said first end of said central passageway.

149. The method of claim 148 wherein said head is formed by connecting a syringe pump to said first end of said central passage.

150. The method of claim 149, wherein said elongated structure further comprises a bypass connected to said central channel.

151. The method of claim 145, wherein said step of moving said at least one polymer along said central passageway is accomplished by creating a pressure drop at said second end of said central passageway.

152. The method of claim 145, wherein said step of moving said at least one polymer along said central channel is accomplished by creating a temperature gradient along said central channel.

153. The method of claim 145, wherein the at least one polymer comprises a charged polymer and the step of moving the at least one polymer along the central channel is accomplished by forming an electric field along the central channel.

154. The method of claim 153, wherein said electric field has a field strength between 1000 and 2000V/m.

155. The method of claim 153, wherein the electric field is formed with two oppositely charged electrodes in the solution.

156. The method of claim 153, wherein the electric field is formed with a series of electrodes in solution.

157. The method of claim 145, wherein the central passage is tapered.

158. The method of claim 145, wherein at least one of said side channels is tapered.

159. A method for stretching at least one polymer, comprising the steps of:

moving the at least one polymer along an elongated structure comprising a channel having at least one bend, the channel comprising a first end and a second end, wherein the at least one polymer is moved from the first end to the second end,

160. The method of claim 159 wherein said step of moving said at least one polymer along said channel is accomplished by means of capillary action.

161. The method of claim 159, wherein said step of moving said at least one polymer along said channel is accomplished by forming a concentration gradient along said channel.

162. The method of claim 159, wherein said step of moving said at least one polymer along said channel is accomplished by forming a ram at a first end of said channel.

163. The method of claim 162 wherein said head is formed by connecting a syringe pump to said first end of said passageway.

164. The method of claim 163, wherein said elongated structure further comprises a bypass connected to said channel.

165. The method of claim 159, wherein said step of moving said at least one polymer along said channel is accomplished by creating a pressure drop at a second end of said channel.

166. The method of claim 159, wherein said step of moving said at least one polymer along said channel is accomplished by creating a temperature gradient along said channel.

167. The method of claim 159, wherein said at least one polymer comprises a charged polymer and said step of moving said at least one polymer along said channel is accomplished by creating an electric field along said channel.

168. The method of claim 167, wherein the electric field has a field strength between 1000 and 2000V/m.

169. The method of claim 167, wherein the electric field is formed with two oppositely charged electrodes in solution.

170. The method of claim 167, wherein the electric field is formed with a series of electrodes in solution.

171. The method of claim 159, wherein the central passage is tapered.

172. The method of claim 159, wherein said elongated structure further comprises a plurality of side channels connected to said channel.

173. The method of claim 159, wherein the channel has a sinusoidal shape.

174. The method of claim 159, wherein the channel has a zigzag shape.

175. A method for stretching at least one polymer, comprising the steps of:

moving at least one polymer along an elongated structure, said elongated structure comprising a channel and a plurality of obstacles to movement of said at least one polymer within said channel, said central channel comprising a first end and a second end;

176. The method of claim 175, wherein the step of moving the at least one polymer along the channel is accomplished by capillary action.

177. The method of claim 175, wherein the step of moving the at least one polymer along the channel is accomplished by forming a concentration gradient along the channel.

178. A method as set forth in claim 175 wherein the step of moving the at least one polymer along the passageway is accomplished by forming an indenter at the first end of the passageway.

179. The method of claim 178 wherein said head is formed by connecting a syringe pump to said first end of said passageway.

180. The method of claim 179, wherein said elongated structure further comprises a bypass connected to said channel.

181. The method of claim 175, wherein the step of moving the at least one polymer along the channel is accomplished by creating a pressure drop at the second end of the channel.

182. The method of claim 175, wherein the step of moving the at least one polymer along the channel is accomplished by forming a temperature gradient along the channel.

183. The method of claim 175, wherein the at least one polymer comprises a charged polymer and the step of moving the at least one polymer along the channel is accomplished by creating an electric field along the channel.

184. The method of claim 183, wherein said electric field has a field strength between 1000 and 2000V/m.

185. The method of claim 183, wherein the electric field is formed with two oppositely charged electrodes in solution.

186. The method of claim 183, wherein the electric field is formed with a series of electrodes in solution.

187. The method of claim 175, wherein the channel is tapered.

188. The method of claim 175, wherein the elongated structure further comprises a plurality of lateral channels connected to the central channel.

189. The method of claim 175, wherein the channel comprises at least one bend.

190. The method of claim 189, wherein the channel has a sinusoidal shape.

191. The method of claim 189, wherein the channel has a zig-zag shape.

192. The method of claim 175, wherein said plurality of obstacles decrease in cross-sectional area along said passageway from said first end to said second end.

193. A method for stretching at least one polymer, comprising the steps of:

wherein at least one of the obstacles has a non-quadrilateral polygonal cross-sectional shape and wherein the at least one polymer moves along the channel from the first end to the second end.

194. The method of claim 193, wherein said step of moving said at least one polymer along said channel is accomplished by means of capillary action.

195. The method of claim 193, wherein said step of moving said at least one polymer along said channel is accomplished by forming a concentration gradient along said channel.

196. The method of claim 193, wherein said step of moving said at least one polymer along said passageway is accomplished by forming an indenter at a first end of said passageway.

197. The method of claim 196 wherein said head is formed by connecting a syringe pump to said first end of said passageway.

198. The method of claim 197, wherein the elongated structure further comprises a bypass connected to the channel.

199. The method of claim 193, wherein said step of moving said at least one polymer along said passageway is accomplished by creating a pressure drop at a second end of said passageway.

200. The method of claim 193, wherein said step of moving said at least one polymer along said channel is accomplished by creating a temperature gradient along said channel.

201. The method of claim 193, wherein said at least one polymer comprises a charged polymer and said step of moving said at least one polymer along said channel is accomplished by creating an electric field along said channel.

202. The method of claim 201, wherein said electric field has a field strength between 1000 and 2000V/m.

203. The method of claim 201, wherein the electric field is formed with two oppositely charged electrodes in solution.

204. The method of claim 201, wherein the electric field is formed with a series of electrodes in solution.

205. The method of claim 193, wherein the channel is tapered.

206. The method of claim 193, wherein said elongated structure further comprises a plurality of lateral channels connected to said central channel.

207. The method of claim 193, wherein the channel comprises at least one bend.

208. The method of claim 207, wherein the channel has a sinusoidal shape.

209. The method of claim 207, wherein the channel has a zigzag shape.

210. The method of claim 193, wherein said plurality of obstacles decrease in cross-sectional area along said passageway from said first end to said second end.

211. A method for stretching at least one polymer, comprising the steps of:

moving at least one polymer along an elongated structure, the elongated structure comprising:

(i) a tapered central passage having at least one bend, said tapered central passage comprising a first end and a second end;

(ii) a plurality of side channels connected to said tapered central channel; and

(iii) a plurality of obstructions to the movement of the at least one polymer within the tapered central passage;

212. The method of claim 211, wherein said step of moving said at least one polymer along said central channel is accomplished by means of capillary action.

213. The method of claim 211, wherein said step of moving said at least one polymer along said central channel is accomplished by forming a concentration gradient along said central channel.

214. The process of claim 211, wherein said step of moving said at least one polymer along said central passageway is accomplished by forming an indenter at said first end of said central passageway.

215. The method of claim 214 wherein said head is formed by connecting a syringe pump to said first end of said central passageway.

216. The method of claim 215, wherein said elongated structure further comprises a bypass connected to said central channel.

217. The method of claim 211, wherein said step of moving said at least one polymer along said central passageway is accomplished by creating a pressure drop at said second end of said central passageway.

218. The method of claim 211, wherein said step of moving said at least one polymer along said central channel is accomplished by forming a temperature gradient along said central channel.

219. The method of claim 211, wherein said at least one polymer comprises a charged polymer and said step of moving said at least one polymer along said central channel is accomplished by creating an electric field along said central channel.

220. The method of claim 219, wherein the electric field has a field strength between 1000 and 2000V/m.

221. The method of claim 219, wherein the electric field is formed with two oppositely charged electrodes in solution.

222. The method of claim 219, wherein the electric field is formed with a series of electrodes in solution.

223. The method of claim 211, wherein said tapered central channel has a sinusoidal shape.

224. The method of claim 211, wherein the tapered central channel has a zig-zag shape.

225. The method of claim 211, wherein said plurality of obstacles decrease in cross-sectional area from said first end to said second end.

226. The method of claim 130, 143, 144, 145, 159, 175, 193, or 211 further comprising the step of delivering the polymer to the elongated structure prior to the moving step.

227. The integrated device of claim 11 or 16, wherein said tapered channel is 1/(ax) in widthⁿ+ b) decreases in velocity from the first end to the second end, where n is a real number greater than 1, a is a real number that is non-zero, b is a real number and x is a distance along the length of the channel.

228. The integrated device of claim 227, wherein n is an integer.

229. The integrated device of claim 228, wherein n has a value of 2, 3, or 4.

230. The integrated device of claim 10, 16, 27, 42, 62, or 96, wherein the fluid sample further comprises a viscosity-modifying component.

231. The integrated device of claim 230, wherein the viscosity-modifying component is selected from the group consisting of glycerol, sucrose, xylose, sorbitol, polyethylene glycol, polyacrylamide, and polyethylene oxide.

232. The integrated device of claim 230, wherein the viscosity-modifying component comprises an aqueous buffer solution at 4 ℃.

233. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure, wherein said elongated structure comprises a channel along which said at least one polymer, when present, moves in a flow direction, and wherein said channel comprises at least one step that decreases the height, z, of said channel from a first end to a second end.

234. The integrated device of claim 233, wherein the channel has a length from 1 μ ι η to 1 mm.

235. The integrated device of claim 233, wherein the at least one step has a height of from 0.1 μ ι η to 0.9 μ ι η.

236. The integrated device of claim 233, wherein the channel decreases linearly in width from the first end to the second end.

237. The integrated device of claim 233, wherein the channel is 1/(ax) in widthⁿ+ b) decreases in velocity from the first end to the second end, where n is a real number greater than 1, a is a real number that is non-zero, b is a real number and x is a distance along the length of the channel.

238. The integrated device of claim 233, wherein the at least one step reduces the height, z, of the passageway by a factor of 2 to 100.

239. The integrated device of claim 233, further comprising means for delivering the at least one polymer in the fluid sample to the elongated structure.

240. The integrated apparatus of claim 239, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

241. The integrated device of claim 233, further comprising means for moving the at least one polymer, when present, within the elongated structure.

242. The integrated apparatus of claim 241, wherein said means comprises means for creating a pressure differential along said elongated structure.

243. An integrated device, comprising:

an elongated structure comprising a channel, said channel comprising at least one step which decreases the height, z, of said channel from a first end to a second end, said channel comprising at least one polymer in a fluid sample, said channel being arranged such that a shear force is exerted on said at least one polymer when it moves in a direction from said first end to said second end.

244. The integrated device of claim 243, wherein said channel has a length from 1 μm to 1 mm.

245. The integrated device of claim 243, wherein said at least one step has a height of from 0.1 to 0.9 μm.

246. The integrated device of claim 243, wherein said channel decreases linearly in width from said first end to said second end.

247. The integrated device of claim 243, wherein said channel is 1/(ax) in widthⁿ+ b) decreases in velocity from the first end to the second end, where n is a real number greater than 1, a is a real number that is non-zero, b is a real number and x is a distance along the length of the channel.

248. The integrated device of claim 243, wherein the fluid sample further comprises a viscosity-modifying component.

249. The integrated device of claim 243, wherein said at least one step reduces the height, z, of said passageway by a factor of 2 to 100.

250. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure comprising:

(a) a first channel, said first channel comprising a first end and a second end; and

(b) a second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end,

along the channel, the at least one polymer, when present, moves in the direction of flow, and

wherein the first channel decreases in width from the first end to the second end at a different rate than the second channel decreases in width from the third end to the fourth end.

251. The integrated device of claim 250, wherein said first channel and said second channel each independently have a length from 1 μ ι η to 1 mm.

252. The integrated device of claim 250, wherein said first channel further comprises a plurality of posts between said first end and said second end.

253. The integrated device of claim 252, wherein at least one of the plurality of posts has a non-quadrilateral, polygonal cross-sectional shape.

254. The integrated device of claim 252, wherein at least one of the plurality of posts has an elliptical cross-sectional shape, wherein a major axis of the elliptical cross-sectional shape is perpendicular to the flow direction.

255. The integrated device of claim 252, wherein at least one of the plurality of posts has a rectangular cross-sectional shape, wherein a major axis of the rectangular cross-sectional shape is perpendicular to the flow direction.

256. The integrated device of claim 252, wherein at least one of the plurality of posts comprises a concave edge, wherein the concave edge faces in a direction of fluid ingress.

257. The integrated device of claim 252, wherein each of the plurality of pillars has a height of 0.1 μm²And 10 μm²And the plurality of posts are positioned in a series of rows between 12 and 15.

258. The integrated device of claim 257, wherein the packing factor of six rows of columns increases in the direction of flow from 0% to 50%, wherein the subsequent at least 12 to 15 rows have a constant packing factor of 50%, and wherein the distance between the centers of adjacent rows of the at least 12 to 15 rows is equal to 2 μm.

259. The integrated device of claim 257, wherein the packing factor of the rows of columns increases continuously in the flow direction from 0% to 80%.

260. The integrated device of claim 257, wherein the packing factor of the column rows is constant.

261. The integrated device of claim 252, wherein the plurality of posts have a cross-sectional area along the flow direction of from 10 μm²To 1 μm²And decreases.

262. The integrated device of claim 250, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

263. The integrated apparatus of claim 262, wherein the transport device comprises a transport channel that leads into and out of the elongated structure.

264. The integrated device of claim 250, further comprising means for moving said at least one polymer, when present, within said elongated structure.

265. The integrated apparatus of claim 264, wherein the device comprises a device that creates a pressure differential along the elongated structure.

266. An integrated device for stretching at least one polymer in a fluid sample, comprising an elongated structure comprising:

(a) a first channel having a width equal to 10 μm and a height equal to 1 μm, said first channel comprising a first end, a second end, and a plurality of columns staggered between said first end and said second end, forming at least 12 to 15 rows, said plurality of columns terminating at said second end, and each column in said plurality of columns having a width of 1-25 μm²Cross-sectional area of (a); and

(b) a second channel comprising a third end and a fourth end, said third end connected to said first channel at said second end, said second channel being 1/x in width from said third end to said fourth end²Wherein x is a distance along the length of the second channel, the length of the second channel being equal to 5 μm, the second channel comprising reducing the height of the second channel at the third end to 0.25 μm²In the above-described manner, the step of (a),

wherein the at least one polymer, when present, moves in a flow direction along the first channel and the second channel.

267. The integrated device of claim 266, wherein at least one of the plurality of posts has a polygonal cross-sectional shape other than quadrilateral.

268. The integrated device of claim 266, wherein at least one of said plurality of posts has an elliptical cross-sectional shape, wherein a major axis of said elliptical cross-sectional shape is perpendicular to said direction of flow.

269. The integrated device of claim 266, wherein at least one of said plurality of posts has a rectangular cross-sectional shape, wherein the long axis of said rectangular cross-sectional shape is perpendicular to said flow direction.

270. The integrated device of claim 266, wherein at least one of said plurality of posts comprises a concave edge, wherein said concave edge faces in said flow direction.

271. The integrated device of claim 266, wherein each of said plurality of posts has a thickness equal to 1 μm²Cross-sectional area of (a).

272. The integrated device of claim 271, wherein the packing factor of six rows of columns increases in said flow direction from 0% to 50%, wherein said subsequent at least 12 to 15 rows have a constant packing factor of 50%, and wherein the distance between the centers of adjacent rows of said at least 12 to 15 rows is equal to 2 μm.

273. The integrated apparatus of claim 271, wherein the packing factor of the at least 12 to 15 rows of columns increases continuously from 0% to 80% in the flow direction.

274. The integrated device of claim 266, wherein the cross-sectional area of said plurality of posts in said flow direction is from 10 μm²To 1 μm²And decreases.

275. The integrated device of claim 266, further comprising at least one detection zone in said second channel.

276. The integrated device of claim 266, further comprising means for delivering said at least one polymer in said fluid sample to said elongated structure.

277. The integrated apparatus of claim 276, wherein the delivery device comprises a delivery channel that leads into and out of the elongated structure.

278. The integrated device of claim 266, further comprising means for moving said at least one polymer, when present, within said elongated structure.

279. The integrated apparatus of claim 278, wherein the means comprises means for creating a pressure differential along the elongated structure.

280. The integrated device of claim 266, wherein the fluid sample further comprises a viscosity-modifying component.

281. An integrated device for selectively stretching at least one polymer in a fluid sample on a length basis, comprising an elongated structure, wherein the elongated structure comprises:

(a) a first channel, said first channel comprising a first end, a second end, and a plurality of alternating posts between said first end and said second end, each post in said plurality of posts positioned no less than L from said second end; and

(b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end,

the at least one polymer, when present, moves along the channel in the direction of flow.

282. An integrated device for stretching a plurality of polymers having different lengths in a fluid sample, comprising an elongated structure, wherein the elongated structure comprises:

(a) a first channel, said first channel comprising a first end and a second end;

(b) a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end; and

(c) a plurality of columns staggered in said first channel and said second channel,

the plurality of polymers, when present, move in a flow direction along the channel.

283. The integrated device of claim 281 or 282, wherein the first channel and the second channel each independently have a length from 1 μ ι η to 1 mm.

284. The integrated device of claim 281 or 282, further comprising at least one detection zone in the second channel.

285. The integrated device of claim 281 or 282, wherein at least one of the plurality of posts has a polygonal cross-sectional shape other than quadrilateral.

286. The integrated device of claim 281 or 282, wherein at least one of the plurality of posts has an elliptical cross-sectional shape, wherein a major axis of the elliptical cross-sectional shape is perpendicular to the flow direction.

287. The integrated device of claim 281 or 282, wherein at least one of the plurality of posts has a rectangular cross-sectional shape, wherein a long axis of the rectangular cross-sectional shape is perpendicular to the flow direction.

288. The integrated device of claim 281 or 282, wherein at least one of the plurality of posts comprises a concave edge, wherein the concave edge faces the flow direction.

289. The integrated device of claim 281 or 282, wherein at least one of the plurality of postsEach has a diameter of 0.1 μm²And 10 μm²Cross-sectional area therebetween.

290. The integrated device of claim 281 or 282, wherein the second channel decreases linearly in width from the third end to the fourth end.

291. The integrated device of claim 281 or 282, wherein the second channel is 1/(ax) in widthⁿ+ b) decreases in rate from the first end to the second end, where n is a real number greater than 1, a is a real number that is non-zero, b is a real number and x is a distance along the length of the second channel.

292. The integrated device of claim 281, further comprising means for delivering the at least one polymer in the fluid sample to the elongated structure.

293. The integrated device of claim 282, further comprising means for delivering said plurality of polymers in said fluid sample to said elongated structure.

294. The integrated apparatus of claim 292 or 293, wherein the transport device comprises a transport channel leading into and out of the elongated structure.

295. The integrated device of claim 281, further comprising means for moving the at least one polymer, when present, within the elongated structure.

296. The integrated device of claim 282, further comprising means for moving said plurality of polymers, when present, within said elongated structure.

297. The integrated apparatus of claim 295 or 296, wherein the device comprises a device that creates a pressure differential along the elongated structure.

298. The integrated device of claim 233, 243, 250, 266, 281, or 282, further comprising at least one polymer in the fluid sample.

299. The method of claim 144, wherein the channel is 1/(ax) in widthⁿ+ b) decreases in velocity from the first end to the second end, where n is a real number greater than 1, a is a real number that is non-zero, b is a real number and x is a distance along the length of the channel.

300. The method of claim 130, 131, 143 or 144, wherein said channel further comprises at least one step which decreases the height, z, of said channel from said first end to said second end.

301. The method of claim 300, wherein the method further comprises delivering the at least one polymer to the elongated structure.

302. The method of claim 300, wherein said step of moving said at least one polymer along said channel is accomplished by forming a ram at a first end of said channel, by forming a vacuum at a second end of said channel, or a combination thereof.

303. A method for stretching at least one polymer, comprising:

moving the at least one polymer along an elongated structure, the elongated structure comprising a first channel, the first channel comprising a first end and a second end; and a second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, wherein said first channel decreases in width from said first end to said second end at a different rate than said second channel decreases in width from said third end to said fourth end.

304. The method of claim 303, further comprising the step of delivering the at least one polymer to the elongated structure.

305. The method of claim 303, wherein said first passageway further comprises a plurality of posts between said first end and said second end.

306. The method of claim 303, wherein the step of moving the at least one polymer along the first channel and the second channel is accomplished by forming an indenter at a first end of the first channel, by forming a vacuum at a fourth end of the second channel, or a combination thereof.

307. The method of claim 302 or 306, wherein the pressure head is formed by connecting a syringe pump to the first end of the first passageway.

308. A method for stretching at least one polymer having a length greater than or equal to L in a fluid sample, comprising:

moving said at least one polymer along an elongated structure, said elongated structure comprising a first channel, said first channel comprising a first end, a second end, and a plurality of alternating posts between said first end and said second end, each post in said plurality of posts being positioned L from said second end, and a second channel, said second channel comprising a third end and a fourth end, said third end being connected to said first channel at said second end, said second channel decreasing in width from said third end to said fourth end,

wherein the polymer having a length greater than or equal to L is stretched and the polymer having a length less than L is not stretched.

309. The method of claim 308, further comprising the step of delivering the at least one polymer to the elongated structure.

310. A method of claim 308, wherein said step of moving said at least one polymer along said first channel and said second channel is accomplished by creating an indenture at a first end of said first channel, by creating a vacuum at a fourth end of said second channel, or a combination thereof.

311. A method for stretching a plurality of polymers having different lengths in a fluid sample, comprising:

moving the plurality of polymers along an elongated structure, the elongated structure comprising:

(c) a plurality of columns staggered in said first channel and said second channel.

312. The method of claim 311, further comprising the step of delivering said plurality of polymers to said elongated structure.

313. The method of claim 311, wherein said step of moving said plurality of polymers along said first channel and said second channel is accomplished by forming an indenter at a first end of said first channel, by forming a vacuum at a fourth end of said second channel, or a combination thereof.

314. The method of claim 310 or 313, wherein the pressure head is formed by connecting a syringe pump to the first end of the first passageway.

315. A method for stretching at least one polymer, comprising:

moving the at least one polymer along an elongated structure comprising:

(a) a first channel having a width equal to 10 μm and a height equal to 1 μm, said first channel comprising a first end, a second end and a plurality of columns staggered between said first end and said second end, constituting at least 12 to 15 rows, said plurality of columns ending at said second end and each column in said plurality of columns having a width of 1-25 μm²Cross-sectional area of (a); and

(b) a second channel, said second channel comprising a third end and a fourth end, said third end connected to said first channel at said second end, said second channel 1/x in width from said third end to said fourth end²Wherein x is the distance along the length of the second channel, the length of the second channel being equal to 5 μm, the second channel comprising a step at the third end which reduces the height of the second channel to 0.25 μm²。

316. The method of claim 315, further comprising the step of delivering the polymer to the elongated structure prior to the moving step.

317. The method of claim 143 or 144, wherein said step of moving said at least one polymer along said tapered channel is accomplished by means of capillary action.

318. The method of claim 143 or 144, wherein said step of moving said at least one polymer along said tapered channel is achieved by forming a concentration gradient along said tapered channel.

319. The process of claim 143 or 144, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by forming an indenter at said first end of said tapered passageway.

320. The method of claim 319 wherein the pressure head is formed by connecting a syringe pump to the first end of the tapered passageway.

321. The method of claim 320, wherein said elongated structure further comprises a bypass connected to said tapered channel.

322. The process of claim 143 or 144, wherein said step of moving said at least one polymer along said tapered passageway is accomplished by creating a pressure drop at said second end of said tapered passageway.

323. The method of claim 143 or 144, wherein said step of moving said at least one polymer along said tapered channel is achieved by forming a temperature gradient along said tapered channel.

324. The method of claim 143 or 144, wherein said at least one polymer comprises a charged polymer and said step of moving said at least one polymer along said tapered channel is achieved by means of forming an electric field along said tapered channel.

325. The method of claim 324, wherein the electric field has a field strength between 1000 and 2000V/m.

326. The method of claim 324, wherein the electric field is formed with two oppositely charged electrodes in the solution.

327. The method of claim 324, wherein the electric field is formed with a series of electrodes in solution.

328. The integrated device of claim 1, 10, 11, 16, 17, 27, 43, 60, 62, 63, 70, 75, 83, 89, 96, or 105, further comprising at least one detection zone in said channel.

329. The integrated device of claim 28, 40, 41, 42 or 112, further comprising at least one detection zone in said central channel.

330. The integrated device of claim 124, further comprising at least one detection zone in the second channel.