JP2002522780A - Optical characterization of polymers - Google Patents

Abstract

(57) Abstract The present invention is a system for optically characterizing a polymer. Preferably, the system is used to perform a linear analysis of the polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION

The present invention relates to optical systems, methods, and products for analyzing polymers, and more particularly, optical systems, methods, and methods that utilize highly localized radiation to characterize individual units of a polymer. About the product.

[0002]

BACKGROUND OF THE INVENTION

This patent application is filed with US Provisional Application No. 60 / 096,54, filed August 13, 1998.
No. 4, and U.S. Provisional Application No. 60 / 120,414, filed February 14, 1999, which is incorporated herein by reference.

[0003] Cells have a complex microstructure that determines their function. Many of the differences associated with cell structure and function are due to the ability of cells to assemble various basic components into various compounds. Cells do this by assembling polymers from a limited set of basic components called monomers or units.
The key to unlocking the diverse functions of a polymer lies in the main sequence of monomers in the polymer, and it is important to understand cell functions such as why cells differentiate in a specific way, how cells respond to treatment with specific drugs, etc. It is essential to understand the basics.

[0004] The ability to identify the structure of a polymer by identifying the sequence of monomers is essential for understanding each active ingredient and the role that the ingredients play in cells. Create an expression map by determining the polymer sequence to determine what proteins are being expressed, understand where the mutations are occurring in the disease state, and whether certain monomers are missing Or when mutated, determines whether the polysaccharide has a better function or has lost function.

[0005] Expression maps are involved in determining mRNA expression patterns. M expressed differently
The need to identify RNA is important when trying to understand genetic programs in time and space. Various genes function or stop functioning during the life course of an organism, including the fetal, growth, and aging stages. In addition to developmental changes, there are also temporary changes to changing stimuli, such as injuries, drugs, foreign bodies, stress, and the like. Altering the expression of a particular cell population, whether for stimulation or growth,
The ability to chart at the right time allows the generation of what is called a temporal expression map. On the other hand, there are also body expression maps that contain knowledge of differentially expressed genes for different tissues and cell types. To generate an expression map, cDNA or mR
As it involves the sequencing and identification of NA, faster sequencing necessarily implies that faster expression maps are generated.

[0006] Recently, only 1% of the human genome and smaller amounts of other genomes have been sequenced. In addition, a very incomplete but unique human expression map using expressed sequence tags has been obtained (Adams et al., 1995). Current protocols for genomic sequencing are slow and involve labor intensive steps such as cloning, genomic library generation, colony collection, and sequencing. Even a partial genomic library takes several months to build. Even after the library has been established, there is a time delay in preparing the DNA for sequencing and performing the actual sequencing steps. Given the fold effects of these unfavorable facts, it is clear that even sequencing a single genome requires enormous costs, time and effort.

[0007] Generally, DNA sequencing is performed using one of two methods. The first, more widely used method is the dideoxy chain termination reaction proposed by Sanger et al. ("DNA sequencing with chain-terminating inhibitors", Proc.
tl. Acad. Sci. USA. 74: 5463-7, 1977). This method involves the enzymatic synthesis of a DNA molecule terminated with a dideoxynucleotide. Using the four ddNTPs, an individual molecule that terminates at each position of the target DNA can be synthesized. Subsequent analysis shows the length of the DNA molecules and the termination of each molecule (either A, C, G or T)
Provides information about bases. With this information, the DNA sequence can be determined. The second method is the Maxam-Gilbert sequencing method (Maxam and Gibert).
lbert, "A new method for sequencing DNA", Proc. Natl. Acad. Sci. USA. 74
: 560-4, 1977) to generate a molecular individual decomposed at a specific position of the target DNA using chemical decomposition. Depending on the cleavage specificity of the chemical reaction and the length of the fragment, D
An NA sequence is generated. Both methods are based on polyacrylamide gel electrophoresis and photographic visualization of radioactive DNA fragments. Each process takes about one to three days. The Sanger sequencing reaction can produce only 300 to 800 bases at a time.

[0008] Sanger-based methods have been proposed to improve the output of sequence information. Sanger-based methods involve multiple sequencing, capillary gel electrophoresis, and automated gel electrophoresis. Recently, the development of the Sanger Independence Law has also attracted attention.
The Sanger Independent Method obtains base information using a completely different methodology. This category comprises state-of-the-art techniques including scanning electron microscopy (STM), mass spectrometry, enzyme-linked inorganic pyrophosphate detection assay (ELIDA) sequencing, exonuclease sequencing, and sequencing by hybridization.

Recently, automated gel electrophoresis has been the most widely used method for large-scale sequencing. The automation involves converting the fluorescently labeled Sanger fragments into charge-coupled devices (C
CD) It is necessary to read in real time by a detector. Four different dideoxy chain termination reactions are performed with variously labeled primers. The reaction mixture binds and co-electrophoreses under a polyacrylamide slab. Irradiating the edge of the gel with a laser decomposes the separated DNA fragments and determines the sequence by computer. Many automated machines using various detection and labeling methods are commercially available. The most efficient of these is Applied Biosystems M
odel 377XL, which produces a real rate of up to 115,200 bases per day.

In capillary gel electrophoresis, a reaction sample is analyzed by a small-diameter gel-filled capillary. This small diameter (50 μm) capillary allows for efficient dissipation of the heat generated during electrophoresis. Therefore, while reducing the separation time to about 20 minutes per reaction,
The electric field strength (400 V / m) can be increased without excessive Joule heating. Not only does it separate bases faster, it also increases resolution over traditional gel electrophoresis. In addition, many capillaries were analyzed in parallel (Wooley and Mathies, "Ultra
-high-speed DNA sequencing using capillary electrophoresis chips, "Anal.
Chem. 67: 3676-3680, 1995), allowing amplification of the base information generated (actual rate equals 200,000 bases / day). The main drawback is that continuous loading of capillaries is not possible because new gel-filled capillaries must be prepared for each reaction. Capillary gel electrophoresis devices have recently been commercialized.

[0011] Multiplex sequencing is an efficient method using electrophoresis gels (
Church and Kieffer-Higgins, "Multiplex DNA sequencing," Science. 240: 185
-88, 1988). The Sanger reaction sample is first tagged with a unique oligomer,
Then more than 20 different samples are run on a one lane electrophoresis gel. Next, the sample is copied onto the membrane. The membrane is then sequentially probed with an oligomer that matches the tag on the Sanger reaction sample. The membrane is continuously washed and reprobed until all 20 samples have been sequenced. Although substantially fewer gels are performed, the washing and hybridization steps require as much work as running an electrophoretic gel. Actual sequencing rates are comparable to automated gel electrophoresis.

[0012] Sequencing by mass spectrometry was first introduced in the 1980's. Recent advances in this area have enabled better sequencing (Crain, MassSpectrom. Rev.
9: 505-54, 1990; Little et al., J. Am. Chem. Soc. 116: 4893-4897, 1994; Keou.
gh et al., Rapid Commun. Mass Spectrom. 7: 195-200, 1993; Smirnov et al., 1996)
. Mass spectrometry sequencing first involves the generation of individual nested DNA molecules that vary in length from base to base. Subsequent fragment analysis is performed by mass spectrometry. As an example, the 33-mer is partially digested using exonuclease (Smirnov, "Sequenci
ng oligonucleotides by exonuclease digestion and delayed extraction matr
ix-assisted laser desorption ionaization time-of-flight mass spectrometr
y, "Anal. Biochem. 238: 19-25, 1996). An individual of molecules with a common 5'-end and a 3'-end change point is generated. The reaction mixture is then analyzed. ,
It has sufficient sensitivity to distinguish mass differences between consecutive fragments, so that sequence information can be generated.

[0013] Mass spectrometry sequencing is extremely accurate, inexpensive, and fast compared to conventional methods. However, there is a great restriction that the read length is about tens of bases.
Matrix-assisted laser desorption ionization floating time measurement formula (MA
LDI-TOF mass spectrometry (Smirnov et al., "Sequencing oligonucleotides by
exonuclease digestion and delayed extraction matrix-assisted laser desor
ption ionaization time-of-flight mass spectrometry, "Anal. Biochem. 238: 1
9-25, 1996), a maximum read length of only 80-90 base pairs can be achieved.
Longer read lengths are not physically possible because long DNA fragmentation occurs at the guanidine during the analysis step. Thus, mass spectrometry sequencing is limited to the range that confirms short primer sequences and is not practically applicable to large-scale sequences.

Scanning tunneling microscope (STM) sequencing (Ferrell, "Scanning tunneling
microscopy in sequencing of DNA, "In Molecular Biology and Biotechnolog
y, RAMeyers, Ed. VCH Publishers, New York, 1997) is a method conceived when STM became commercially available. The earliest prospects that base pair information can be read directly from electron micrographs are gone. DNA molecules must be placed on a conductive surface, usually graphite (HOPG) or gold, which is highly pyrolytic. They do not have binding sites to hold strongly against the DNA being attempted to be detached by the physical and electrical forces induced by the tunnel tip. With difficulty, the DNA molecules can be stuck to the surface with static electricity. Even if the immobilization of DNA is successful, it is extremely difficult to identify base information because it requires extremely high resolution. Using current techniques, purines are distinguished from pyrimidines, but individual purines and pyrimidines cannot be identified. The ability to accomplish this separation requires an electron microscope that can distinguish between aldehyde and amine groups on purines and the presence or absence of methyl groups on pyrimidines.

[0015] Enzyme Illumination Inorganic Pyrophosphate Detection Assay (ELIDA) sequencing utilizes the detection of pyrophosphate release from DNA polymerization to determine the contiguous bases to add. Pyrophosphate released by the DNA polymerization reaction is converted to ATP by ATP sulfurylase, and ATP production is continuously monitored by firefly luminescent enzymes. To determine the specificity of a base, ATP, CTP, GTP,
And a continuous wash of TTP. If washing for ATP produces pyrophosphate, one or more adenines are incorporated. The number of bases incorporated is directly proportional to the amount of pyrophosphate produced. Enhancement of the generated sequence information can be achieved by simultaneously analyzing many ELIDA reactions in parallel.

Exonuclease sequencing involves a fluorescently labeled single-stranded DNA molecule suspended in a stream and continuously cleaved by an exonuclease. Thereafter, the individual fluorescent bases are released and pass through the single molecule detection system. The temporal sequence of the labeled nucleotide detection corresponds to the DNA sequence (Ambrose et al., "Applica
tion of single molecule detection to DNA sequencing and sizing, "Ber. Bu
nsenges. Phys. Chem. 97: 1535-1542, 1993; Davis et al., "Rapid DNA sequencing.
based on single-molecule detection, "Los Alamos Science. 20: 280-6, 1992
; Jette et al., "High-speed DNA sequencing: an approach based upon fluoresce
nce detection of single molecules, "J. Of Bio. Structure & Dynamics. 7: 3
01-9, 1989). Using an evolving exonuclease, theoretically 10,000
bp or larger fragments can be sequenced at a rate of 10 bases per second.

[0017] In sequencing by hybridization, the target DNA is probed sequentially with an oligomer assembly consisting of all possible oligomer sequences.
The sequence of the target DNA is generated using knowledge of the hybridization pattern between the oligomer and the target (Bains, "Hybridization methods for DNA
sequencing, "Genomics. 11: 294-301, 1991; Cantor et al.," Reporting on the se
quencing by hybridization workshop, "Genomics. 13: 1378-1383, 1992; Drman
ac et al., "Sequencing by hybridization," In Automated DNA Sequencing and An
alysis Techniques, J. Craig Ventor, Ed. Academic Press, London, 1994).
There are two possible ways to probe the target DNA. "Probe up"
The method involves immobilizing a target DNA on a substrate and continuously probing with a collection of oligomers. On the other hand, "probe down" requires that the aggregate of oligomers be immobilized on the substrate and hybridized with the target DNA. With the advent of "DNA chips" in which microchip synthesis techniques have been applied to DNA probes, arrays of thousands of different DNA probes can be generated in an area of 1 cm2, making the probe-down method more practical. The probe-up method requires 65,536 consecutive probes and washes for an 8-mer, which would be enormous. On the other hand, probe down hybridization produces data in seconds. With complete hybridization, 65
, 536 octamer probe will determine up to 170 bases. 65,
With 536 "mixed" 11-mers, 700 bases can be generated.

The most common limitation of most of these techniques is the short read length.
As a practical matter, a short read length means that additional gene sequence information needs to be sequenced before decoding the linear order of the target DNA. The shorter fragments are additional overlapping fragments and must be cross-linked to each other. In theory, using a read length of 500 bases, a minimum of 9 x 109 bases need to be sequenced before a linear sequence of all 3 x 109 bases in the human genome is properly sequenced. In practice, the number of bases needed to create a reliable genome is around 2 × 1010. Comparison of various techniques indicates that only the impractical exonuclease sequencing method has the theoretical capacity of long read lengths. Other methods have a theoretically short read length, and in practice have only shorter read lengths. Obviously, to reduce the number of bases that need to be sequenced, the read length must be improved.

[0019] Protein sequencing usually involves chemically induced sequential removal and identification of terminal amino acid residues, for example, by Edman degradation. Stryer, L., Biochemist
ry, WH Freeman and Co., San Francisco (1981) pp. 24-27. The Edman degradation method requires that the polypeptide have a free amino group that reacts with the isothiocyanate. The isothiocyanate is usually phenylisothiocyanate. The addition reacts intramolecularly with the closest backbone amide group of the polymer to form a five-membered ring. This addition rearranges and then the terminal amino acid residue is cleaved using a strong acid. Amino acid free phenylthiohydantoin (PTH) has been identified and the shortened polymer undergoes repeated cycles of degradation and analysis.

In addition, several new methods for carboxyl terminal sequencing of polypeptides have been published. See Inglis, AS, Anal. Biochem. 195: 183-96 (1991). Carboxyl-terminal sequencing mimics the Edman degradation method but involves continuous degradation from the opposite end of the polymer. Inglis, AS, Anal. Biochem. 19
5: 183-96 (1991). Similar to the Edman degradation method, the carboxyl-terminal sequencing method involves a chemically-induced sequential removal and identification of terminal amino acid residues.

[0021] More recently, polypeptide sequencing has been described as preparing a nested set of polymer fragments (sequence defined set) prior to mass spectrometry. Chait, BT
Et al., Science 257: 1885-94 (1992). The sequence is determined by comparing the relative mass differences between the fragments with amino acid residues of known mass. The form of nested (sequence definition) assembly of polymer fragments is a requirement for DNA sequencing, but this method differs substantially from conventional protein sequencing methods, which consist of sequential removal and identification of each residue. Although this method has the potential for practical use, it faces several problems and has not yet proven to be an effective method.

Each of the known methods for performing polymer sequencing has drawbacks. For example, most methods are slow and labor intensive. The gel-based DNA sequencing method is 300
Approximately one to three days is required to identify a sequence of ~ 800 units of polymer. Methods such as mass spectrometry and ELIDA sequencing can only be performed on very short polymers.

There is a need for new polymer sequencing methods. Heretofore, sequencing rates have limited the ability to generate a large number of physical and temporal expression maps that will undoubtedly aid in the rapid determination of complex gene function. There is also a need for improved systems and methods for analyzing polymers to increase the speed with which disease diagnosis and drug preparation can be performed.

[0024]

Summary of the Invention

The present invention relates to new systems, methods, and products for analyzing polymers, and in particular, to new systems, methods, and products useful for sequencing polymers. The present invention has a number of advantages over prior art systems and methods for sequencing polymers. Using the methods of the present invention, the entire human genome will be sequenced several orders of magnitude faster than achieved using conventional techniques. In addition to sequencing all genomes, the systems, methods, and products of the present invention are used to generate comprehensive and multiple expression maps for development and disease processes. The ability to sequence an individual's genome and generate multiple expression maps will significantly enhance the ability to determine the genetic basis of any trait expression or disease process.

According to one aspect, the present system for optically analyzing a polymer of a chain unit comprises:
Includes light source, interaction station, photodetector, and processor. The light source is configured to emit radiation of a selected wavelength. The interaction station
It is configured to receive the radiation and produce a local radiation spot from the radiation emitted from the light source. The interaction station is also configured to sequentially receive units of the polymer and is arranged to sequentially illuminate the units with a local radiation spot. The light detector is configured to detect emitted light that includes a characteristic signal resulting from the interaction of the unit with the local radiation spot. A processor is configured and arranged to analyze the polymer based on the detected emitted light.

Preferred embodiments of this aspect include one or more of the following features: The interaction station is configured to sequentially receive units selectively labeled with a radiation sensitive label, wherein the interaction is And the interaction of local radiation with the radiation sensitive label.

The radiation-sensitive label comprises a fluorophore.

[0028] The interaction station includes a waveguide configured to receive the emitted light and provide evanescent radiation accordingly.

[0029] The interaction station includes a slit having a width in the range of 1 nm to 500 nm, the slit producing a local radiation spot.

[0030] The interaction station includes microchannels having submicron widths and slits organized to produce a local radiation spot.

The slit width is in a range from 10 nm to 100 nm.

The system can include a polarizer, wherein the light source is a laser configured to emit a light beam, and the polarizer is organized to polarize the laser beam before it reaches the slit.

The polarizer may be organized to polarize the laser beam parallel to the slit width or perpendicular to the slit width.

The interaction station may include a number of slits arranged perpendicular to the microchannel organized to receive the straightened polymer.

The interaction station may include a set of electrodes configured and organized to provide an electric field for advancing the polymer unit through the microchannel.

The system may further include an alignment station configured and arranged to straighten the polymer and to supply the straightened polymer to the interaction station.

In another embodiment, a method of optically analyzing a polymer of a chain unit includes: labeling selected polymer units with a radiation-sensitive label; sequentially passing the polymer units through microchannels; Generating radiation of a selected wavelength to generate a local radiation spot; irradiating the labeled polymer units sequentially with the local radiation spot; and interacting with the local radiation spot and the label or unit. Sequentially detecting the emitted light that provides the resulting characteristic signal; and analyzing the polymer based on the detected emitted light.

In another embodiment, a product used to optically analyze a polymer of a chain unit, which is fabricated on a substrate, receives radiation, and generates a local radiation spot from the radiation. With an interaction station constructed to occur.
The interaction station is further configured to sequentially receive the units of polymer and is arranged to sequentially illuminate the units with a local radiation spot to generate a characteristic signal of the emitted light.

According to another aspect, a system for optically analyzing a polymer comprising a chain unit includes a light source, an interaction station, a photodetector, and a processor. The light source is configured to emit radiation of a selected wavelength. The interaction station is configured to receive the emitted light, is configured to sequentially receive the polymer units, and is arranged to sequentially illuminate the polymer units with evanescent radiation excited by the light emitted from the light source. The light detector is configured to detect light including a characteristic signal resulting from the interaction of the unit with the evanescent radiation. A processor is configured and arranged to analyze the polymer based on the detected emitted light.

Preferred embodiments of this aspect include one or more of the following features: The interaction station is configured to sequentially receive units selectively labeled with a radiation sensitive label, wherein the interaction is , The interaction of evanescent radiation with the radiation-sensitive label.

The synchrotron radiation sensitive label includes a fluorescent transmitter.

The interaction station includes a waveguide configured to receive the emitted light and configured to provide evanescent radiation in response to the emitted light.

The waveguide is a dielectric waveguide configured to achieve complete internal reflection of the introduced light. This waveguide is a dielectric rectangular mirror waveguide surrounded by a metal mirror layer configured to reduce the loss of introduced light. The waveguide includes a tip that includes an opening in the metal mirror layer and is organized to emit evanescent radiation. The waveguide includes a tip configured to emit evanescent radiation.

The interaction station includes a nanochannel located at the tip of the waveguide,
Knitted to receive the straightened shape of the polymer.

The interaction station includes a set of electrodes configured and arranged to provide an electric field for advancing the polymer unit through the nanochannel. These electrodes are internal electrodes.

The electrodes are external electrodes. Nanochannels are between 2 and 50 nm.

The waveguide is further configured and organized to receive radiation comprising a characteristic signal, and optically couples the received radiation to a photodetector.

The interaction station includes another waveguide configured and arranged to receive the radiation including the characteristic signal, and optically couples the received radiation with the photodetector.

[0049] The system further includes an alignment station configured and organized to straighten the polymer to provide the straightened polymer to the interaction station.

In yet another aspect, the present invention is a system for optically analyzing polymers utilizing confocal fluorescent illumination of a chain unit. The system includes: a light source configured to emit radiation; a filter configured to receive the radiation and filter at a known wavelength; a dichloic configured to receive the filtered radiation. A mirror; an interaction station configured to receive the filtered radiation and to produce a local radiation spot from the filtered radiation, wherein the interaction station is also configured to sequentially receive the polymer units. A light detector arranged to sequentially illuminate the unit at the local radiation spot; a photodetector configured to detect radiation at the local radiation spot that includes a characteristic signal resulting from the interaction of the unit; Based on the detected emitted light including the characteristic signal A processor configured and arranged to analyze the polymer accordingly.

In one embodiment, the interaction station is configured to sequentially receive, at the local radiation spot, the units selectively labeled with a radiation sensitive label that produces a characteristic signal. In another embodiment, the radiation-sensitive label comprises a fluorescent transmitter. In some embodiments, the filter is a laser line filter.

The system also includes an objective lens, which focuses the filtered light.

The proposed systems and methods for analyzing polymers are particularly useful for sequencing units within DNA, which is a major limitation of current genome-wide sequencing protocols. The need for genomic library creation, cloning, and colony collection, which make up a very long preliminary sequencing step, can be eliminated. The methods disclosed herein provide longer read lengths than achieved by the prior art, and sequence reads that are one million times faster. Planned read lengths are on the order of hundreds of thousands of nucleotides. This significantly reduces the need to find overlapping redundant sequences and can reduce the actual amount of DNA that needs to be sequenced prior to genomic rearrangement. The real time for a given number of polymer unit readings is one million times higher than with current methods due to the tremendous parallel amplifiers referred to as nanochannel plates or microchannel plates provided by the novel device claimed here. Be faster. The combination of all these elements transforms methods of polymer analysis, including sequencing, which will bring significant advances in the field of molecular and cell biology.

[0054]

[Detailed description of preferred embodiments]

Referring to FIG. 1, an interaction system for determining characteristics of an individual polymer unit includes a system controller 10, a polymer supply device 20, a microfluidic pump 25, a polymer alignment station 30, a first interaction station 4
0, and a second interaction station 50. The system controller 10 may be a general-purpose computer. Microfluidic pump 25 delivers a selected amount of polymer 27 from polymer feeder 20 to polymer alignment station 30.
Supply to A polymer alignment station 30 controlled by the system controller 10 uses the force field and mechanical obstacles to straighten and align the individual polymers and supply the first interaction station 40 with the polymers. The first interaction station 40 uses an optical system to obtain the properties of the individual polymer units passing through. The optical system includes a light source 42, an optical filter 45, and a photodetector 4.
6, and other light elements and electrical elements associated with the light source and detector. The optical system is controlled by a light controller 48.

As individual units of the polymer pass through the interaction station 40, the light sources 42 emit light toward the optical components of the interaction station 40. The optical component creates a local radiation spot that interacts directly with the polymer unit, interacts with a label selectively attached to the polymer unit, or interacts with both the polymer unit and the label. The local radiation spot comprises at least one-dimensionally localized non-radiating near-field or evanescent waves. The local radiation spot has a higher resolution than the diffraction limited resolution used in conventional optics.

In addition, the interaction station 40 has a unique organization and geometry that allows the local radiation spot to interact with one or several polymer units, or attached labels of the order of a few nanometers or less. Is used. The light detector 46 detects the light changed by the interaction, and provides a detection signal to the light controller 48.
The second interaction station 50 uses an electric or electromagnetic field, X-ray radiation, or visible or infrared light to determine the properties of the polymer passing from the first interaction station 40 through the second interaction station 50. . The controller 56 controls the operation of the second interaction station 50. Controllers 48 and 56 are both connected to system controller 10.

Referring to FIGS. 2 and 3, polymer alignment station 30 and first interaction station 40 include substrate 92, quartz wafer 60, and optional cover glass 90. Substrate 92 is made of a conductive fluid 96 (eg, agarose gel) and a non-conductive, chemically inert material, such as Teflon® or Delrin®, to facilitate the flow of the polymer under test. Process. Substrate 92 includes grooves 94A and 94B machined to receive gold wires 98A and 98B, respectively, which grooves form an electric field shape for advancing polymer molecules 39 across first interaction station 40. Has a shape selected according to The quartz wafer 60 is sealed on a substrate 92 surrounding the region 91.

Alternatively, grooves 94A and 94B and lines 98A and 98B are
It may be replaced by a metal area disposed directly on it, or by an external electrode generating an electric field. Generally, the electrodes are about 1 mm to 5 mm.
cm, preferably 2 cm apart, and typically provide a field strength of about 20 V / cm.

FIGS. 4 and 4A show a presently preferred embodiment of the alignment station 30 and the first interaction station 40. FIG. 4 is a plan view of a part of the alignment station 30A formed on the quartz wafer 60 and the first interaction station 40 (also shown in FIG. 2). Naturally, a single quartz wafer 6
0 may include hundreds to thousands of alignment stations and the first interaction station. The quartz wafer 60 includes a quartz substrate covered with a metal layer 62 (eg, aluminum, gold, silver) and has microchannels 41 formed on the substrate surface. Created through metal layer 62 are slits 36A, 36B, and 36C that form optical elements that provide a local radiation spot. The slits 36A, 36B, and 36C have a selected width in the range between 1 nm and 5000 nm, preferably in the range between 10 nm and 1000 nm, and even 10 n
A range between m and 100 nm is preferred. Slits 36A, 36B and 36C
Are arranged across a microchannel 41 having a width in the range of 1 μm to 50 μm and a length of several hundred μm. The electric field generated by the gold wires 98A and 98B draws the polymer chains 39 (such as DNA molecules) through the path slits 36A, 36B and 36C of the microchannel 41.

As shown in FIG. 4, the polymer alignment station 30 includes a number of alignment posts 32 located in a region 31. Region 31 is connected to microchannel 41 via transition region 34. The alignment column 32 has a circular cross section and a diameter of about 1 μm. The alignment posts 32 are about 1.5 μm apart and from about 5 μm to 50 μm from the microchannel 41 depending on the length of the polymer being tested.
0 μm (preferably about 10 μm to 200 μm). For example, bacteriophage T4 DNA where the polymer has about 167,000 base pairs
In this case, the alignment column 32 is arranged at a position of about 30 μm from the nanoslit 36A. Generally, the distance from the nanoslit 36A is about one-half the expected length of the polymer 39.

FIG. 4A shows that the light beam 65 emitted from the light source 42 interacts with the nanoslits 36 formed in the metal layer 62 to create a local radiation spot 67. The laser beam 65 has dimensions that are many times greater than the width of the nanoslit 36, illuminates the back of the quartz wafer 60, diffuses through the quartz wafer 60, and interacts with the nanoslit 36. It is indicated by the electric field line. The local radiation spot 67 is a non-radiating near-field, and irradiates the polymer chain unit 39 sequentially when the polymer chain 39 is sucked through the microchannel 41. The local radiation spot 67 may be understood as an evanescent wave radiated from the nanoslit 36. Since the width of the nanoslit 36 is smaller than the wavelength of the light beam 65, the radiation is in the Fresnel mode.

The optical system may include a polarizer 43 mounted between the light source 42 and the quartz wafer 60, and a notch filter 45 mounted between the quartz wafer 60 and the light detector 46. The polarizer emits a light beam 65 with an E vector parallel to the length of the nanoslit 36.
, There is near-field radiation emitted from the nanoslit 36 and no far-field radiation. If the polarizer orients the light beam 65 with an E vector perpendicular to the nanoslit 36 (longer in wavelength), there is far-field radiation from the nanoslit 36. By selectively polarizing the incident beam 65, the optical system can switch between near-field and far-field radiation.

FIG. 4B shows an optical system for determining the properties of a polymer unit labeled with a fluorophore. The optical system includes a laser source 80, an acousto-optic tunable filter 82, a polarizer 84, a notch filter 86, an intensifier and a CCD detector 88,
And a video monitor 90 connected to the video recorder VCR 92. Individual units of the polymer chain 39 are selectively labeled with a fluorescent transmitter 68 that is sensitive to a selected excitation wavelength. An acousto-optic tunable filter 82 is used to select the excitation wavelength of the light emitted from the laser source 80. Excitation beam 6
5 interacts with the nanoslits 36 (shown in FIG. 4A and noted here as regions 40) to create a non-radiating near-field 67. Gold wire 98A (of FIGS. 2 and 3)
The electric field between and B draws the polymer chain 39 at a known rate at which the interaction of the radiation 67 with each labeled unit occurs. As the fluorophore 68 moves through the pass slits 36A, 36B, and 36C (shown in FIG. 4), the emitted light 67 excites the fluorophore 68 to re-emit the fluorescent radiation 72. Notch filter 8
6 passes the fluorescence wavelength (72) of the radiation 70 and attenuates the excitation wavelength, increasing signal-to-noise resolution, as is well known in the art. A CCD detector 88 located several mm to several cm above the quartz wafer 60 detects the fluorescent radiation 72. The CCD detector 88 detects the fluorescence emission 72 as the fluorescence transmitter moves across.
Are detected separately for nanoslits 36A, 36B, and 36C. This process occurs with a number of nanoslits placed on the quartz wafer 60.

An electric field may be used to position polymer 39 near nanoslit 36. The nanoslit 36 "radiates" a non-radiative field 67 that attenuates beyond a distance of only one or two wavelengths. In order to position the fluorescence transmitter 68 within the non-radiative field 67, the polymer 39 may need to be brought closer to the metal layer 62 by being drawn closer to the nanoslit 36 (and the metal film 62). unknown. Polymer 39 is attracted close to nanoslit 36 using the dielectric force created by applying an AC electric field to metal layer 62. For example, "Trapping of DNA in Nonuniform Oscillating Electric Fields" by
Charles L. Ashbury and Ger van den Engh, Biophysical Journal Vol 74, pp
1024-1030 (1998) "Molecular Dielectrophoresis of Biopolymers" M. Washizu
, S.Suzuki, O.Kurosawa, T.Nishizaka, and T.Shinohara, in IEEE Transactio
ns on Industry Application, Vol 30, No4, pp. 835-843 (1994), and "Electr
ostatic Manipulation of DNA in Microfabricated Structures "by M. Washizu,
and O. Kurosawa, in IEEE Transactions on Industry Application, Vol 26, N
o6, pp. 1165-1172 (1990). In general, "Dielectrophoresis: The Behav
ior of Neutral Matter in Nonuniform Electric Fields "by Pohl, HA, Camb
See ridge University Press, Cambridge, UK, 1978. The non-uniform electric field attracts the polarized polymer units 39 (eg, DNA molecules) to the metal layer 62.

Referring to FIG. 5, the second interaction station 50 measures the ionic current across the nanochannel as the straightened polymer molecules pass close to the nanochannel. Blocking the detected ionic current is used to characterize the molecular length of the polymer and other properties of the polymer. The interaction station 50 receives the straightened polymer 39 from the first interaction region 40 and applies a voltage through the channel using electrodes 52 and 53 in a direction perpendicular to electrodes 54 and 55. Then, the polymer molecules are sucked through the channel 51. The electrodes 54 and 55 are connected to a controller 56
Connected to a microammeter 56A for measuring the ionic current across the nanochannel 51. Alternatively, referring to FIG. 5A, the microammeter compares the impedance (Z1) of the channel 51 without the polymer 39, replaced by the bridge 56B, with the instantaneous impedance (ZX). Polymer 3
When 9 is not present in channel 51, the voltmeter indicates 0V. As the nearly straight string 39 is extended and passes through the channel 51, its presence reduces or completely shuts off the normal ion flow from the electrode 54 to the electrode 55 in a detectable manner.

The electrodes 54 and 55 are manufactured using submicron lithography, and are connected to a bridge for detecting impedance change or a microammeter for measuring ion current. Measurement data traversing the channel is amplified, the amplified signal is filtered with a low pass filter (eg, at 64,000 samples per second), and the data is digitized with an analog to digital converter at a selected sampling rate. The system controller 10 correlates the transient decrease in ionic current with the speed of the polymer unit to determine the length of the polymer, for example, the length of a DNA or RNA molecule.

In another embodiment, the optical system includes an ultra-fast, sensitive spectrophotometer capable of detecting fluorescence from a single fluorescence transmitter. The light source 42
It is a fixed mode Nd: YAG laser that emits light of an excitation wavelength. The system uses a photodiode and a splitter that provides a reference beam to a discriminator (eg, Tennelec TC454). The discriminator converts the start pulse to a time / amplitude converter (eg, Tennel
ec 863). The main beam 65 is directed through a gray density filter that adjusts the power level. As described above, the fluorescent transmitter 68 interacting with the non-radiating near-field 67 excites the fluorescent light 72, which is spectrally filtered by an interference filter (eg, from Omega Optic) and avalanche. After being detected by a photodiode or an optical multiplexer (for example, a Hamamatsu R156UMCP microchannel optical multiplexer), it is collected by a detector 46. The signal of the microchannel optical multiplexer is amplified by an amplifier and discriminator (eg, Te)
nnelec C4534 discriminator). The signal with the appropriate time delay is provided to a time / amplitude converter (TAC). The TAC output of the time gate is counted by the multiscaler and interfaced to the system controller 10 via the VME. The system controller 10 provides, for the signal from each detector, a time lag histogram characterizing each type of fluorescent transmitter that emits fluorescence associated with the polymer 39 unit.

Different fluorophores have different fluorescence lifetimes (ie, the average time that a molecule remains excited before returning to electrical ground through emission of fluorescent photons), which typically has an exponential probability distribution. Have. Fluorescence lifetimes are useful for identifying fluorescent mediators. During fast sequencing, the system can use similar dyes with similar spectra but different lifetimes, so that only one laser source emitting the excitation wavelength and one detector detecting the fluorescence emission are used. I just need it. In another embodiment, the optical system uses a phase modulation technique to characterize the fluorescence of a single fluorophore placed next to the polymer unit and uses a phase modulation technique ranging from 10 MHz to 1 GHz (eg, a single sideband or Modulated radiation (both sidebands) is used. For example, the laser source emits an intensity-modulated light beam 65 using a sine wave having a frequency of 100 MHz. The excited fluorescent radiation 72 is detected using an optical multiplexer. The corresponding signal is a characteristic signal from the fluorescence transmitter, for example, homodyne or heterodyne detected to decode the fluorescence lifetime. (Eg Lackowicz, JR, "Gi
gaherz Frequency-Domain Fluorometry: Resolution of Complex Intensity Dec
ays, Picosecond Processes and Future Developments "Photon Migration in T
issues, Academic Press, NY, pp. 169-186, 1989; See also the other references cited therein. 6 to 7B show the fabrication of the alignment region 30, the microchannel 41 and the slits 36A, 36B and 36C shown in FIG. FIG. 6 shows a quartz wafer 60.
It is a side view of which is about 400 μm thick and both sides are polished. First thickness 300
A nm aluminum film 62 is deposited on the wafer and pretreated in hexamethyldisiloxane (HMDS) for 35 minutes. A photoresist Shipley 1813 was then spun on the wafer at 4000 rpm for 60 seconds and the wafer baked on a hot plate at 115 ° C. to cure the resist (FIG. 6A). The wafer was exposed and the photoresist was developed with 1: 1 MF312 developer and water for 60 seconds. The rough pattern of aluminum is chlorine active ion etcher PK1250
For 1.5 minutes (FIG. 6B). FIG. 6C shows an overview of the wafer with the device indicated by squares and the alignment marks indicated by Xs. All excess resist was removed using a Branson barrel etcher with a 1000 W RF power, 10 minute resist scum removal process (FIG. 6D).

Referring to FIG. 6E, the PMMA resist (4% at 950K in MIBK) is 3000
The wafer was spin coated on the wafer at 60 rpm for 60 seconds, and the wafer was baked on a hot plate at 180 ° C. for 30 minutes. A 100 ° gold layer was then deposited on the PMMA photoresist to avoid charge buildup. PMMA photoresist
It was exposed in an e-beam system to define a nanoslit. Exposed PM
The MA resist was developed in 3: 1 IPA: MIBK for 1 minute and the 100 ° gold layer was etched (FIG. 6F). Next, a nanoslit pattern was defined by etching the aluminum for 1.5 minutes using a chlorine active ion etch PK1250 (FIG. 6G). The photoresist was removed with a Branson barrel etcher at 1000 W RF power for 10 minutes (FIG. 6H). Alignment area 30
A 1 μm SiO 2 layer is deposited by plasma enhanced chemical vapor deposition (PECVD) at T = 240 ° C., 450 mTorr, R
Deposited using 15 sccm silane, 50 sccm N2O at F power 50 W (FIG. 6I). The SiO2 layer was planarized by chemical mechanical polishing (CMP).

FIGS. 7 to 7B are side views of the wafer along the nanochannel. Referring to FIG. 7, the alignment region 30 and the microchannel 41 are 1800 rpm,
Defined on the wafer with a 60 second first spin-coated photoresist Shipley 1813 and baked on a 115 ° C. hot plate for 60 seconds. The resist is 5
It was exposed in a high resolution mask aligner such as an xg-line stepper and developed in 1: 1 MF312 and water for 60 seconds. The layer of SiO 2 is made of SiO 2 shown in FIG.
CHF3 (50 sccm) + O2 (2 sccm) so as to define a two-layer pattern
) Was etched using active ion etching (RIE) (FIG. 7A).
The photoresist is RF output 1000W, 10 minutes Branson barrel etcher
Was removed. Then, a 10-100 nm SiO2 protective layer is
D (FIG. 7B). A cover glass 90 (shown in FIG. 2) may be anodically welded to the quartz wafer or attached to the chip 60 using a thin layer of RTV.

FIG. 8 shows SEM micrographs of the two alignment regions 30 and the two interaction regions 40 that have been manufactured. Each alignment region 30 includes a micro pillar 31, and each interaction region 40 includes a microchannel 41 and nanoslits 36A, 36A shown in FIG.
B, and 36C. Referring to FIGS. 9-10C, the alignment region 30 and interaction region 40 (shown in FIG. 8) that were created were tested in the following experiment.

The collimated CW laser light from the Ar: Kr ion laser was focused on the back of the wafer 60 as shown in FIG. 4A. The laser beam 65 having an excitation wavelength of 488 nm created a non-radiating near-field on the film 62 on the other side near the fluorescence transmitter 68. The microscope objective captured the 560 nm fluorescent far-field emission, which was recorded in a time-dependent manner by an optical multiplexer. The time-dependent signal then provided a pass record where the target crossed the slit with a spatial resolution approximately equal to the width of the slit.

FIG. 9 shows a 2.0 micron width (curve 94A) and a 0.1 micron width (curve 94B).
4 shows the response of the optical multiplexer for a 0.5 μm ball passing through the slit of FIG. Curves 94A and 94B represent the voltage of the optical multiplexer as a function of time.
As expected, smaller slits produce a narrower curve 94B, which is the minimum response with this device.

FIGS. 10A to 10C show the loading of fluorescent beads simultaneously passing through two nanoslits separated by 10 μm and the imposition of T4 DNA stained with yoyo-1. In FIG. 10A,
A bead with two intensity peaks passing through a first slit and then passing through a second slit is shown. FIG. 10B shows a partially unwound DN passing through a delivery channel.
A is shown. The broader peaks 96A and 96B are due to the geometry of the DNA helix. The path of the fluorescent beads is superimposed on the DNA signal. FIG. 10C shows greatly stretched DNA traveling through three slits 36A, 36B, and 36C.
Is shown. Again, the fluorescent bead signal is superimposed on the DNA signal for reference. The broader peaks 97A, 97B, and 97C are due to the DNA helix geometry.

FIG. 11 is a cross-sectional view of the quartz wafer having the waveguide 160 taken along the center axis of the waveguide. The waveguide 160 includes two waveguides 166A and 166B having a rectangular cross section made on a quartz wafer 150. The rectangular waveguides 166A and 166B may be rectangular dielectric waveguides using two types of dielectric materials having different refractive indexes, and have a refractive index (n2) larger than the refractive index (n1) of the surrounding dielectric material. ) Is confined to the core material having () (n2> n1). Alternatively, rectangular waveguides 166A and 166B may be rectangular mirror waveguides using a dielectric core material, surrounded by a metal material, or a waveguide formed by a combination of two waveguides. Wave tubes 166A and 1
66B.

A rectangular dielectric waveguide ideally achieves complete internal reflection of light propagation at angles of incidence θ1> θc. In order to confine the incoming light with perfect internal reflection, the interaction station 40 uses a triangular waveguide with a very small tip angle. Rectangular mirror waveguides typically exhibit high losses depending on the quality of the metal mirror. A rectangular mirror waveguide transmits light up to a wavelength (λ) equal to twice the height (h) of the waveguide (λ = 2 · h). Thus, these waveguides have heights designed for propagation of light in the selected wavelength range used for polymer testing. For more information, see "Fundamentals
of Photonics "by Bahaa EA Saleh and Malvin Carl Teich, John Wily & Son
s, 1991.

As shown in the perspective view of FIG. 11A, waveguides 166 A and 166 B are symmetrically arranged with respective tip portions 170 A and 170 B aligned along a target axis defining nanochannel 171. (FIG. 11B). The width of the nanochannel 171 ranges from 2 nm to 100 nm, preferably from 5 nm to 50 nm. Gold wires 198A and 198B (shown in FIGS. 2 and 3) are approximately 3 to 25 mm from nanochannel 171. Alternatively, as shown in FIG. 11C, the two waveguide arrangements may be replaced by a single waveguide with counter electrodes forming a wide channel ranging from 100 nm to 1 μm.

The triangular waveguides 166A and 166B shown in FIGS. 11 and 11A have a width of about 10 μm,
It has a length of 5000 μm, a height of more than 1 μm and is made of SiO 2. Waveguide 16
6A and 166B are separated from substrate 162 by metal layers 164A and 164B, respectively, and from cover glass 152 by metal layers 174A and 174B. (Alternatively, metal layers 164A and 174A for waveguide 166A,
Alternatively, the metal layers 164B and 174B of the waveguide 166B may be replaced by a low reflection index dielectric layer). The introduced plane wave 176 is coupled to the triangular waveguide 166A at the input surface 168A and is internally reflected at the waveguide side surfaces 172A and 173A to be directed toward the waveguide tip 170A. The waveguide chip 170A emits a wave of evanescent radiation (shown in FIG. 11B) to the nanochannel 171. In the nanochannel 171, evanescent radiation interacts with individual units of the polymer 39 to produce radiation with a characteristic signal. For example, the evanescent radiation interacts with a fluorescent transmitter located next to a particular unit of polymer 39. Triangular waveguide 166B collects radiation, including characteristic signals (eg, of fluorescent radiation), from nanochannel 171 and directs this radiation toward coupling region 168B. When the collected radiation propagates inside the waveguide 166B, the radiation is transmitted to the triangular sides 173A and 173A.
B completely reflects internally. Output surface 168B, which provides radiation 188, is optically coupled to photodetector 46 (FIG. 1). Further, radiation from the nanochannel 171 penetrates the cover glass 152 and is also radiated in the direction 189. Another external photodetector several millimeters to several centimeters above nanochannel 171 detects far-field radiation 189, as shown in FIG.

FIG. 11B shows two triangular waveguides 166 A and 1, each side surrounded by a metal layer.
FIG. 66B is a cross-sectional view of FIG.
, 172B, 173A and 173B. However, the metal layer does not
The vertices of the tip portions 170A and 170B of A and 166B are not completely covered. As described below, the metal layers of the tip portions 170A and 170B
1 or may be removed in a cutting process. The waveguide 166A carries the introduced light beam 176 to the tip 170A, substantially confining all waves within the SiO2 volume. The waveguide 166A emits an evanescent wave 177 attenuated by q-1 in the dielectric waveguide at the tip 170A. Here, q = n1, 2ω / c [(sinθ1 / sinθc) 2-1] 1/2
(Eg "Optical Waves in Layered Media" by P.Yeh, John Wiley & Sons,
1988). Thus, the evanescent wave is completely internally reflected (θ1> θc)
Therefore, it is attenuated beyond a distance of only one or two wavelengths. Evanescent radiation 177
Waves interact with the units of polymer 39 through nanochannels 171.
For example, the evanescent wave 177 interacts with a fluorescent transmitter selectively attached to a selection unit of the polymer 39. The fluorescent mediator 178 emits fluorescent radiation 179 that diffuses in all directions. Fluorescent emission 179 is collected by waveguide 166B and delivered to detector 46 (FIG. 1).

FIG. 11C is a cross-sectional view of another embodiment using a single triangular waveguide 166 and metal electrode 185. The channel 171A formed between the waveguide 166 and the metal electrode 185 is about 0.5 μm, which is significantly larger than the nanochannel 171. The triangular waveguide 166 is surrounded on all sides by a metal layer, and the waveguides 166A and 16
It is manufactured in the same manner as 6B. Here, the cross hatching pattern is the waveguide side surface 172.
And 173 shows the metal layer above. As with the waveguide 166A, the tip 170 emits an evanescent wave 177 that attenuates over a distance of only one or two wavelengths. Therefore, the polymer 39 must be drawn closer to the tip 170 than the electrode 185 to illuminate the fluorescent transmitter 178 with an evanescent wave 177.

The polymer 39 comprises an electrode 185 and a waveguide 166, ie, metal layers 164 and 174.
An AC electric field is applied to the tip 170 and is attracted closer to the tip 170 using the dielectric force generated by the DC electric field applied across the lines 98A and 98B. The AC electric field applied capacitively in relation to the DC electric field creates a non-uniform electric field in the nanochannel 171 as described above with respect to FIG. 4A.

FIG. 12 shows an optical system 100 for detecting near-field and far-field radiation emitted from a nanochannel 171. The light source 44 has 189
B emits a light beam 76 that focuses on the input side 168A of the waveguide 166A using the technique described in connection with FIG. After the interaction of the evanescent wave 176 with the polymer 39, the near-field radiation is collected by the waveguide 166B and optically coupled out of the output side 168B to the photodetector 46. Direction 89
Is collected by a lens 102, filtered by a tunable filter 104, and provided to a PMT detector 106. The light source 44, such as an LED or a laser diode,
It may be incorporated above. This organization would not require an external light source that would have to be aligned with the input side 168A. The light source is made using a direct bandgap material, such as GaN, which produces UV radiation, or Gap: N, which produces green wavelength radiation.

The quartz wafer 150 may include an integrated optical detector 46 to eliminate the need for external devices for detection and filtering. To filter out the excitation wavelength
An integrated avalanche photodiode or PIN photodiode with both in-situ filters receives the light beam 188. Various integrated optical devices are called "I
ntegrated Optoelectronics-Waveguide Optics, Photonics, Semiconductors "
by Karl Joachim Ebeling, Springer-Verlag, 1992. For example, corrugated waveguides are used as opposite couplers so that light within a narrow frequency band is reflected back, resulting in filtering. Another filter is made using two waveguides with different diffusions that are in an approximate relationship. Light from one waveguide will be coupled into the other waveguide for wavelengths with matching refractive indices.
By applying a voltage to the waveguide, the diffusion curve shifts, resulting in a change in the spectrum of the filter, providing a tunable filter.

In another embodiment, the optical system 110 is an ultrafast and sensitive spectrophotometer capable of detecting fluorescence from the single fluorescence transmitter described above.

In another embodiment, optical system 120 utilizes modulated radiation having a frequency in the range of 10 MHz to 1 GHz, as described above.

FIGS. 13 to 13B show various forms of coupling light from an external light source into a waveguide. In FIG. 13, the light source 42 emits a light beam 176, which is
Using the focusing lens 180, the input side 168A of the triangular waveguide 166A is
Focus on the top. Alternatively, referring to FIG. 13A, a prism 182 is used to couple the light beam 176 into the triangular waveguide 166A. The light beam 176 is
Diffracted by the prism 182 and pushed inside the total internal reflection. Prism 182 is disposed on the surface of SiO2 volume 166A and is organized to optically couple beam 176 across layer 184 to waveguide 66A. Referring to FIG. 13B, a diffraction grating 186 is alternatively used to couple the light beam 176 into the triangular waveguide 166A. Grating 186 is fabricated on waveguide 166A to diffract light beam 176 toward tip 170A. Alternatively, fiber optics
Light beam 176 is coupled into triangular waveguide 166A. Various methods of coupling light into waveguides are described in Fundamentals of Optics, by Clifford R. Pollok, Richard D. Irwi
n, Inc., 1995.

The waveguides 166 A and 166 B are fabricated on quartz or other insulating material to prevent current from flowing through the substrate 152. Nanochannel region (ie, 10 nm resolution)
In order to achieve the high resolution required by, the fabrication process can be either UV lithography alone or in combination with deep UV lithography, e-beam lithography, or X-ray lithography.
Line lithography is used. In a continuous waveguide, the nano-channels (or micro-channels 171A described in connection with FIG. 11C) are first defined using standard UV lithography and then defined in a single e-beam or X-ray lithography step. . In an embodiment waveguide that includes radiation slits at tips 170A and 170B, the slits (or holes) are formed at the very tip 170 of waveguides 166A and 166B.
At A and 170B, create a convex curved shape (i.e., undercut) of the photoresist and before depositing the metal, the sides 172A, 173A, 172B,
And at 173B, creating a convex curved shape of photoresist. Thus, the sides of the convex surface will be covered by the deposited metal, but the tips of the convex surface will not be covered. Alternatively, the small tips (small holes) are created by first creating a very thin wall and then lifting off or etching to create a metal film with small slits over the entire wall. When using e-beam lithography, as is known in the art, keep the thickness of the resist thin,
A hard mask made of metal is used to keep the resolution high.

FIGS. 14A to 14K are side views along the center line of the waveguides 166A and 166B.
Is created as follows. That is, the wafer is pre-treated in hexamethyldisiloxane (HMDS) for 34 minutes to improve the adhesion of the resist to the wafer (FIG. 14A). A photoresist Shipley 1830 is then spun on the wafer at 4000 rpm for 60 seconds to obtain a 1.3 μm thick resist, and the wafer is baked for 60 seconds on a 115 ° C. hot plate to cure the resist. (FIG. 14B). The photoresist is exposed in a high resolution mask aligner such as a 5xg-line stepper and baked in a pressurized NH3 oven. This inverts the positive tone of the photoresist and provides the backward-sloping shape required for the subsequent lift-off process (ie, the undercut shown in FIG. 14C). Wafers were flood exposed for 1 minute with 405 nm light in an HTG / contact aligner before
Develop for minutes. Referring to FIG. 14D, a 1000 ° aluminum layer is deposited and lift-off is performed using a Microposit 1165 resist remover or room temperature acetone (FIG. 14E). All excess resist is removed in a Branson Barrel etcher at 0.6 Torr O2, RF output 150 W using a resist scum removal process.

Referring to FIGS. 14F to 14K, a SiO 2 waveguide is created as follows: 1 μm SiO 2 is deposited by plasma enhanced chemical vapor deposition (PECVD) to form T
= 240 ° C., 450 mTorr, RF power 50 W, 15 sccm silane, 50
Deposited using sccmN2O. The SiO2 layer, as shown in FIG.
It is flattened by chemical mechanical polishing (CMP). The upper metal mask is
Photoresist Shipley 1830 was added to 400 μm to obtain a 1.3 μm thick resist.
Spin at 0 rpm for 60 seconds on the wafer to define and bake on a 115 ° C. hot plate for 60 seconds. The resist is exposed in a high resolution mask aligner such as a 5xg-line stepper and baked in a pressurized NH3 oven. This inverts the positive tone of the photoresist, as shown in FIG.
Provide the rearwardly inclined shape (ie, undercut) required for the subsequent lift-off process. The resist is 40 in the HTG / contact aligner.
Flood exposure for 1 minute with 5 nm light, then develop on Microposit 321 for 1 minute. As shown in FIG. 14J, a 1000 ° Al metal layer is deposited. Excess metal is removed by lift-off using a Microposit 1165 resist remover or acetone at room temperature.

FIGS. 15A to 15G are side views along the center line, and FIGS.
G is a side view perpendicular to the center line. PMMA resist 496K has a thickness of 200
Spin coated on the wafer at 2500 rpm to obtain nm resist and baked on a 180 ° C hot plate for 60 minutes to harden the resist. PMM
A is exposed with an e-beam system to create a pattern in the nanochannel region. The exposed PMMA resist is developed in 3: 1 IPA: MIBK for 1 minute and a 1000 ° Al metal layer is deposited as shown in FIG. 15C. After performing extra metal lift-off with acetone at room temperature, active ion etching (RIE)
In a Plasma Therm 72 etcher using CHF3 (50 sccm) + O2 (
2 sccm), RF output 200 W, 40 mTorr under the conditions shown in FIG.
The waveguide is etched to create a wall larger than μm, but without a microchannel pattern. The bottom metal is wet etched in a solution of 16: H2PO4; 1: HNO3; 1: acetic acid; 2: water; humectant, or dry etched in Cl. Excess resist was applied at RF output 1 in a Branson barrel oxygen plasma etcher.
Removed at 000 W for 15 minutes. Aluminum is 16: H2PO4; 1: H
NO3; 1: acetic acid; 2: water; removed in a wet etch of the humectant.

Deposition of the top Al layer over the waveguide is shown in FIGS. 15E to 15G and FIGS. 16D to 16G. Referring to FIGS. 15 and 16D, a photoresist Shipley 1830 was spun on the wafer at 4000 rpm for 60 seconds to obtain a 1.3 μm thick resist, and 6 seconds on a 115 ° C. hot plate to cure the resist.
Bake for 0 seconds. The resist is exposed in a high resolution mask aligner such as a 5xg-line stepper and baked in a pressurized NH3 oven. This inverts the positive tone of the photoresist and provides the backward-sloping feature (ie, undercut) required for the subsequent lift-off process. The resist is flood exposed for 1 minute with 405 nm light in an HTG / contact aligner and then developed for 1 minute in Microposit 321. As shown in FIGS. 15F and 16F, a 1000 ° Al layer is deposited. Excess metal is lifted off using Microposit 1165 resist stripper or room temperature acetone.

The metal layer Cr is deposited on the top of the device as follows. First, the mask for the nanochannel is etched, and then Shipley 1 is used to obtain a 1.3 μm thick resist.
830 resist is spin coated on the wafer at 4000 rpm for 60 seconds and baked on a hot plate at 115 ° C. for 60 seconds to cure the resist. The resist is exposed in a high resolution mask aligner, such as a 5xg-line stepper,
Fired in a pressurized NH3 oven. This process reverses the positive tone of the photoresist and provides the back-slope feature (ie, undercut) required for the subsequent lift-off process. The resist was flood exposed for 1 minute with 405 nm light in an HTG / contact aligner before Micro
Developed in posit 321 for 1 minute. A 1000 ° Cr layer was deposited and excess metal lift-off was performed in a Microposit 1165 resist remover or acetone at room temperature. PMMA 496K resist is spin-coated on the wafer at 2500 rpm to obtain a 200 nm thick resist and 180 to cure the resist.
It was baked on a hot plate at 60 ° C for 60 minutes. E to define the desired pattern
Exposed with the beam system, the wafer was developed with IPA: MIBK 3: 1 for 1 minute. A 1000 ° Cr layer was then deposited and excess metal lift-off was performed in a Microposit 1165 resist remover or acetone at room temperature.

The nanochannel 171 was created by etching the first metal layer (ie, Al layer) in a Cl-based dry etch using Cr as an etch mask. Then, S
iO2 was added to Plasma C in Plasma Therm 72 using active ion etching (RIE).
HF3 (50sccm) + O2 (2sccm), RF output 200W, 40mTo
Etching was performed under rr conditions to create walls greater than 1 μm. The bottom metal layer is
The remaining Cr was etched by a Cl-based dry etch and the remaining Cr was removed by a wet etch. Alternatively, the nanochannel 171 may be created by imaging ion beam shilling to define a gap and an opening in the tip. Individual molecules for DNA sequencing were published Feb. 11, 1998, incorporated herein by reference.
It may be selectively labeled, as described in PCT Application No. PCT / US98 / 03024, filed on the date of the Japanese Patent Application. Sequencing was performed using hybridized single-stranded DNA (sss) with fluorescently tagged test sequence oligonucleotides.
DNA). When hybridization occurs, the tagged sequence is in a fixed position on the DNA molecule. The process can use three tags, the "start" and "end" tags, which signal 3 'and 5' cues at the beginning and end of the ssDNA and sequence using the tagged oligo. By using oligonucleotide sequence spectra to observe large individuals of these tagged molecules as they pass through the microchannel, and by recording the location of the oligonucleotide labels, the system has been With no speed and accuracy, and at the level of low-concentration molecules, you will acquire molecular sequences.

FIG. 17 shows another embodiment of the present invention. Optical device 200 utilizes confocal fluorescence illumination and detection. Confocal illumination can reduce the illuminated optical volume (on the order of picoliters). Rayleigh and Raman scattering are both minimized by using small test volumes. The optical device 200 includes a light source 202, a filter 204, a dichroic mirror 206, an objective lens 208, a narrow band pass filter 210, a pinhole 212, a lens 214, and a detector 216. The light source 202 is a 1 mW argon ion laser that emits a laser beam 201 passing through a filter 204. Filter 204 is a laser line filter that provides an imaging beam at a wavelength of about 514 nm. The filtered beam 205 is reflected by the dichroic mirror 206 and is
Focus on the sample or other polymer area. The objective lens 208 is 100 × 1
. 2NA oil immersion lens.

A DNA sample is a straightened DNA molecule having one or several units tagged with a fluorescent tag. The fluorescent tag on the DNA may be one of several dyes, including Cy-3, tetramethylrhodamine, rhodamine 6G, and Alexa 546. Further, an interrupt symbol such as TOTO-3 (molecular probe) can be used.

The excited tag is attached to the dichroic mirror 206 (eg, Omega Optical
Provide fluorescent emission passing through a narrow bandpass filter 210 (made of) and focus on a 100 μm pinhole 212. The fluorescent light 213 is focused by an aspheric lens 214 onto a detector 216 that is an avalanche photodiode (eg, manufactured by EG & G, Canada) operating in a photon counting mode. The output signal from the photodiode is collected by a multi-channel scalar (from EG & G) and analyzed by a general purpose computer.

[0097] Confocal devices are suitable for quantitative applications, including flotation time. Such applications include D
Includes distance measurements on NA, detection of tagged sequences, and determination of stretched degree of DNA. Single fluorescent molecules may be detected using this device. Alternatively, the imaging device may be an enhanced CCD (ICCD, Prinston Instr.
uments).

FIG. 18 shows an alignment station 2 for aligning and stretching the polymer before reaching the interaction station 230 which interacts by light emission.
20 present 20 preferred embodiments. Alignment station 2
20 is fabricated on a quartz wafer that may be covered with a metal layer 222 (eg, aluminum, gold, silver). The alignment station 220 includes triangular microchannels 224, microposts 228, and entrance areas 230, all created on a surface. The entrance region 230 has a width of about 50 μm and communicates with the micro pillar region 228. The micro pillar region 228 includes several alignment pillars 226. The alignment column 226 has a circular cross section and a diameter of about 1 μm. Alignment micro pillar 2
Reference numeral 26 denotes 12 rows to 15 rows, which are separated at intervals of about 1.5 μm. Fine pillar region 22
8 is inclined about 26.6 °.

The microposts 226 are about 100 μm to 5,000 μm (preferably, about 1,000 μm to 3,000 μm) from the interaction station where the polymer units (eg, of DNA) interact with the light emission. Be placed. The microchannels 224 are areas where the polymer is released from the microposts 226 and where constant shear in the x-direction is applied to maintain their stretched shape. The electric field attracts the polymer to be tested through the microchannel 224.

A very effective technique for uniformly stretching a polymer (eg, of DNA) is to maintain the stretched shape, and the portion of constant shear that straightens the helix remaining in the polymer is followed by a taper To have a faulty area inside the attached microchannel 224. The preferred embodiment is a structure combining micro pillars having two regions designed to have different funnels as shown in FIG. The pressure flow is
This is preferable as a driving force because the behavior of the entire flow of the fluid can be predicted.

The rate of constant shear, or change in average velocity, with respect to the distance of the channel is defined by S: u / x = S where x is the distance below the substantially rectangular channel and u is the average fluid Velocity, calculated from total flow (Q) and channel cross-sectional area (A).

U = Q / A In one embodiment where the cross section of the channel is rectangular, the channel is defined by a constant height H and width W, so that the cross section is A = HW and the average flow velocity is It is given by: u = Q / HW Applying the boundary condition that fluid flow must be continuous, Q is constant. Therefore, u is inversely proportional to W. Substituting this relationship into the first equation of S to determine the relationship between shear rate and width: S = u / x = Q / H / x (1 / W) = (-Q / HW2) (dW / dx) dW / dx = (-SH / Q) (W2) Integrating this gives: W = (SHx / Q + C) -1 where C is the integration constant determined by the original width of the channel. (Boundary condition). This channel width equation is used to define the channel beyond the post structure.

[0103] Other embodiments are within the following claims.

[Brief description of the drawings]

FIG. 1 schematically illustrates a system for characterizing a polymer.

FIG. 2 shows an alignment station and a first interaction station used in the system of FIG.

FIG. 3 is a sectional view of the alignment station and the first interaction station shown in FIG. 2 taken along line 3-3.

FIG. 4 is a plan view of a part of the alignment station and the first interaction station shown in FIG. 2;

FIG. 4A shows the organization of a nanoslit disposed at the first interaction station shown in FIG.

FIG. 4B shows an optical system for determining the properties of a polymer unit labeled with a fluorescent transmitter.

FIG. 5

5 and 5A show a second interaction station used in the system of FIG.

FIG. 6

FIG. 6A

FIG. 6B

FIG. 6C

FIG. 6D

FIG. 6E

FIG. 6F

FIG. 6G

FIG. 6H

FIG. 6I

FIG. 7

FIG. 7A

FIGS. 6-7B illustrate the fabrication of the alignment station and the first interaction station shown in FIG.

FIG. 8 is a SEM micrograph of the fabricated alignment station and the first interaction station.

FIG. 9

FIG. 10A

FIG. 10B

FIGS. 9, 10A, 10B, and 10C show the results of test measurements of the alignment and interaction stations of FIG.

FIG. 11 is a cross-sectional view at the centerline of an optical waveguide according to another embodiment of the first interaction station.

FIG. 11A is a perspective view of the optical waveguide shown in FIG. 11;

FIG. 11B

FIGS. 11B and 11C show the interaction of a linearized polymer with evanescent radiation emanating from an optical waveguide.

FIG. 12 shows an optical system for near-field and far-field detection used in the optical waveguide of FIG.

FIG. 13

FIG. 13A

FIGS. 13, 13A and 13B show the coupling of electromagnetic radiation to the optical waveguide of FIG.

FIG. 14A

FIG. 14B

FIG. 14C

FIG. 14D

FIG. 14E

FIG. 14F

FIG. 14G

FIG. 14H

FIG. 14I

FIG. 14J

FIG. 14K

FIG. 15A

FIG. 15B

FIG. 15C

FIG. 15D

FIG. 15E

FIG. 15F

FIG. 15G

FIG. 16A

FIG. 16B

FIG. 16C

FIG. 16D

FIG. 16E

FIG. 16F

16A to 16G show the fabrication of the optical waveguide of FIG.

FIG. 17 is a schematic diagram of an optical device that utilizes confocal fluorescence illumination and detection for linear analysis of a polymer.

FIG. 18 is a plan view of another embodiment of an alignment station for polymer alignment and stretching.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/58 A 33/58 37/00 101 37/00 101 102 102 27/02 Z / / G01N 27/02 27/26 315Z 27/416 27/46 336Z (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, Z, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU , ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MG, MK, MN, MW, MX, NO, (72) Invention NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW Jonas Oh. -Tegenfeld, Alden Lane 28, Princeton, New Jersey 08540, USA (72) Robert H. Inventor. Austin, Harris Road, Princeton, New Jersey 08544, USA 135 (72) Inventor Eugene Wy.・ Chan United States, Massachusetts 02108, Boston, Beaver Place 25, Suite 4F Term (Reference) 2G042 AA01 BD12 BD18 BD20 CB03 DA08 FA11 FB05 FC07 HA07 2G043 AA03 BA16 CA09 DA05 EA01 FA01 FA06 GA02 GA03 GA04 GA06 GA07 GB01 GB12 GB19 JA03 JA03 KA01 KA02 KA03 KA07 KA09 LA02 LA03 2G045 AA35 DA12 DA13 DA14 FA12 FA15 FA25 FA27 FB05 FB07 FB12 GC15 2G060 AA05 AE40 AF06 KA05 4B063 QA13 QQ42 QR32 QR56 QS03 QS34 QS39 QX02

Claims (51)

[Claims] 1. A system for optically analyzing a polymer comprising a chain unit, comprising: a light source configured to emit radiation of a known wavelength; receiving the radiation and generating a local radiation spot from the radiation. An interaction station configured to occur, wherein the interaction station is configured to sequentially receive units of the polymer and is arranged to sequentially illuminate the units at the local radiation spot; A photodetector configured to detect radiation comprising a characteristic signal resulting from the interaction of the units; configured and arranged to analyze the polymer based on the detected radiation comprising the characteristic signal A system comprising: a processor; 2. The system of claim 1, wherein the interaction station is configured to sequentially receive, at the local radiation spot, the units selectively labeled with a radiation sensitive label that produces the characteristic signal. . 3. The system of claim 2, wherein said radiation sensitive label comprises a fluorescent transmitter. 4. The system of claim 1, wherein said interaction station includes a slit having a width in the range of 1 nm to 500 nm, said slit producing said local radiation spot. 5. The interaction station includes a microchannel and a sub-micron wide slit organized to create the local radiation spot, the microchannel communicating the polymer unit through the local radiation spot. The system of claim 1, configured to receive and advance. 6. The system of claim 1, wherein said width is in a range from 10 nm to 100 nm. 7. The light source further includes a polarizer, and the light source includes a laser configured to emit the beam of radiation, the polarizer organized to polarize the beam before reaching the slit. The system of claim 5, wherein 8. The system of claim 5, wherein said polarizer is organized to polarize said beam parallel to said width of said slit. 9. The system of claim 5, wherein the polarizer is organized to polarize the beam perpendicular to the width of the slit. 10. The system of claim 5, wherein said interaction station includes a number of said slits intersecting said microchannels arranged to receive said polymer in a straightened form. 11. The apparatus of claim 5, further comprising a set of electrodes configured and organized to provide an electric field for advancing the unit of the polymer through the microchannel.
System. 12. The system of claim 11, wherein said electrode is an internal electrode. 13. The system of claim 11, wherein said electrodes are external electrodes. 14. The system of claim 5, wherein said nanoslits are several micrometers in length. 15. An alignment station configured and arranged to straighten the polymer and provide the straightened polymer to the microchannel, wherein the alignment station has a diameter of about 1 μm and about 0 μm. 6. The system of claim 5, comprising a number of micro-pillars spaced at a distance of between 0.5 μm and 5 μm. 16. An alignment station configured and arranged to straighten the polymer and provide the straightened polymer to the microchannel, wherein the alignment station is about 5 μm to 500 μm from the slit. The system of claim 5, comprising a number of microposts disposed at a distance. 17. The system of claim 16, wherein said microposts are separated by a distance between 0.5 μm and 2.5 μm. 18. The system of claim 1, wherein the light source is a laser and the system further comprises an acousto-optically tunable filter configured to select the wavelength. 19. The system of claim 18, wherein said wavelength is an excitation wavelength of a fluorescent transmitter selectively coupled to said unit and said characteristic signal is a fluorescent wavelength emitted by said fluorescent transmitter. . 20. The system of claim 19, further comprising a notch filter arranged to send only the fluorescence wavelength to a photodetector. 21. The system of claim 1, wherein said light source is configured to emit said wavelength in the ultraviolet to infrared wavelength range. 22. The system of claim 1, wherein said photodetector comprises one of a photodiode, an avalanche photodiode, an optical multiplexer, a PIN diode, and a CCD. 23. The system of claim 1, wherein the processor is arranged to evaluate the characteristic signal that is a fluorescence lifetime. 24. The processor is arranged to evaluate the characteristic signal that is a fluorescence wavelength.
The system of claim 1. 25. The system of claim 1, wherein the processor is arranged to evaluate the characteristic signal that is an intensity of the detected radiation. 26. The system of claim 1, wherein the processor is arranged to evaluate the characteristic signal that is a time-dependent characteristic of the detected radiation. 27. A method for optically analyzing a polymer comprising chain units, comprising sequentially passing said units of said polymer through a microchannel; producing radiation of known wavelength to produce a local radiation spot Sequentially irradiating the unit of the polymer with the local radiation spot; sequentially detecting radiation at the local radiation spot to provide a characteristic signal resulting from the interaction of the units; and Analyzing the polymer based on the detected radiation,
A method that includes: 28. The method of claim 27, wherein passing the polymer through the microchannel comprises using an electric field. 29. The method of claim 27, wherein producing the local radiation spot comprises optically coupling the generated light to a nanoslit having a width of less than 1 μm. 30. Producing the local radiation spot reduces the generated light by about 1n in width.
28. The method of claim 27, comprising optically coupling to a nanoslit in the range of m to 500 nm. 31. Producing the local radiation spot comprises optically coupling the generated light into a number of nanoslits oriented longitudinally perpendicular to the microchannel. 28. The method of claim 27, comprising. 32. Producing the local radiation spot includes generating the light in the form of a laser beam and polarizing the laser beam to be oriented parallel to the width of the slit. 30. The method of claim 29. 33. Producing the local radiation spot includes generating the light in the form of a laser beam and polarizing the laser beam to be oriented perpendicular to the width of the slit. 30. The method of claim 29. 34. Further, the light emitting device is disposed at a distance of 5 to 100 μm from the local radiation spot.
28. The method of claim 27, comprising straightening the polymer with a number of microposts separated by 5 to 5 [mu] m. 35. The method further comprising labeling a selected unit of the polymer with a radiation-sensitive label, wherein the detecting is performed during a time period during which the unit passes through the microchannel. 28. The method of claim 27, comprising collecting said radiation over said characteristic signal. 36. The label of claim 35, wherein the label comprises a fluorophore, and wherein detecting comprises filtering only radiation excited by the fluorophore to provide a photodetector. Method. 37. The method of claim 27, wherein said producing comprises producing said radiation at said wavelength in the range of ultraviolet to infrared wavelengths. 38. The method of claim 27, wherein said detecting comprises using a photodiode detector, an avalanche photodiode detector, an optical multiplexer detector, a PIN diode detector, or a CCD detector. 39. The method of claim 27, wherein said polymer is a nucleic acid. 40. A product used for optically analyzing a polymer comprising a chain unit, wherein the product is organized on a substrate, receiving radiation emitted from a light source and producing a local radiation spot therefrom. An interaction station, further configured to sequentially receive units of the polymer, and arranged to sequentially illuminate the units with the local radiation spot to generate a characteristic signal of radiation. The product. 41. The article of claim 40, wherein the interaction station includes a nanoslit configured to produce the local radiation spot. 42. The article of claim 41, wherein the interaction station includes a microchannel configured to provide the polymer in a straightened state to the nanoslit. 43. The article of claim 42, wherein a width of said nanoslit is less than a wavelength of said emitted light. 44. The article of claim 42, wherein the width of said nanoslits ranges from 1 nm to 500 nm. 45. The article of claim 42, wherein the width of said nanoslits ranges from 50 nm to 100 nm. 46. The article of claim 40, further comprising a set of electrodes configured and arranged to provide an electric field for advancing said unit of said polymer through said local radiation spot. 47. The article of claim 40, wherein the width of the microchannel is less than 1 μm. 48. The method according to claim 48, wherein the distance from the local radiation spot is 0.5 μm to 5 μm.
41. The product of claim 40, further comprising an alignment station further comprising a number of microposts located at ~ 100 [mu] m. 49. The article of claim 48, wherein said microposts are located 10 μm to 200 μm from said local radiation spot. 50. The article of claim 49, wherein said microposts are separated by a distance of 0.5 μm to 5 μm. 51. The article of claim 50, wherein said microposts are separated by a distance of 1.5 μm to 2.5 μm.