CA2680558A1 - Clinical sample preparation on a microfluidic platform - Google Patents

Abstract

Genotyping clinical samples (e.g. whole blood and buccal swabs) for molecular diagnostics is typically performed in laboratories using specialized equipment by trained operators. The present invention provides for an integrated microfluidic approach wherein cell lysis, molecules of interest cleanup, molecules of interest isolation/purification and molecules of interest transport approach is demonstrated. A magnet is focussed within the microfluidic channel and facilitates the magnetic bead movement which binds the molecules of interest. Further a procedure is developed to indefinitely re-use the chips with no notable degradation in performance.

Description

CLINICAL SAMPLE PREPARATION ON A MICROFLUIDIC PLATFORM

FIELD OF THE INVENTION

The present invention pertains to the field of clinical sample preparation for use in microfluidic or other diagnostic devices.

BACKGROUND OF THE INVENTION

AII of the publications, patents and patent applications cited within this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual pubiication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.

Microfluidic technology has made great strides in the integration and validation of many of the individual analytical functionalities necessary for tests to determine the molecular characteristics of a variety of diseases for purposes of diagnosis, making treatment decisions and for clinfcal monitoring. It is envisaged that in public health monitoring such technology would play a key role. It is also envisioned to be of central importance in the diagnosis and monitoring of cancer by detecting molecular signatures that characterise the cancer in single individuals or in groups of individuais. In the developing world there is a need for diagnostic technology to monitor infections by a variety of viral, bacterial or other types of pathogens, as a public heath screening tool for detecting emerging disease, containing disease and detecting spread in the community. Microfluidic technology is an ideal candidate for point-of-care/point-of-need testing (Yager, P.; et al. Nature 442:412 (2006)). A
significant hindrance for the widespread usage of microfluidic devices is the substantial "off-chip"
clinical sample preparation, which requires considerable manual intervention by highly trained individuals, significant amounts of time and creates run-to-run variability. A major goal in the development of iab-on-a-chip devices is to create molecular diagnostic and monitoring devices that can be operated by an untrained user In a fully automated and standardized manner that does not require substantive human intervention. Such devices should be able to accept unprocessed body tissues or fluids, with. extraction of nucleic acids or proteins performerd on-chip and where needed, concentrating or enriching such nucleic acids or proteins entireiy on-chip. Such techniques should be rapid, inexpensive and Independent of the need for highly specialized diagnostic equipment that is external to the chip. Typically in conventional. protocols, it is relatively easy (and well established techniques exist) to extract DNA, RNA or protein from tissue samples or body fluids by using various chemicals, precipita4on, centrifugation and the column-based separation techniques. This conventional approach is labour intensive, time consuming and requires substantial specialized equipment and skilled operators. These purificafion protocols also introduce considerable variability when performed in different laboratories or by different individuals, with significant run-to-run variability and lack of standardizadon. Integration of sample analysis onto a microfluidic device (or "chip" or "microchip") is hindered by the complex nature of many of the samples that need to be analyzed (Lichtenberg, J. Et al. Talanta 56233 (2002)). To date, the samples Introduced to a microdevioe have been most often processed off-chip, having undergone some form of preliminary manipulations off-chip by a human operator.

In applications not involving microfluidic devices, there are several developed approaches (referred herein as the "conventional" approach) for DNA extraction leading to its purification and concentration. Phenol/chloroform and chelex methods for DNA extraction require large volume of samples making them difficult to port on a microflibidic platform (also in certain scenarios clinical sample can be sparse). Anion-exchange and silica-based purification methods are still the most popular conventional approaches for nucleic acid purification, yet they are time-consuming and may be difficult to automate. Even more problematic, these methods require the use of ethanol and chaotropic salts, both of which have the potential to contaminate the eluted nucleic acid product and tend to Inhibit many sensitive enzymatic reactions for downstream applications.

Only in extremely limited demonstrations have raw clinical samples been directly introduced to a microfluidic chip and this involves use of relatively simple bodily fluids such as urine.
One such example was the detection of viral titres in raw urine (Kaigala, G.
V.; et al.
Electrophonesis 27: 3753 (2006)). To date, there has been no demonstration of genetic amplification from whole bkad without any prior purification steps. Whole blood, plasma or serum are highly inhibitory to PCR redctions, making simple approaches to the processing of whole blood problematic. Even isolated cells in certain concentrations have an inhibitory effect on PCR. This inhibitory actlvity is evaluated by introducing cell lysate to a PCR reaction amplifying sequences from a purified nucleic acid template. The inhibitory effect becomes greater as the number of cell equivalents in the lysate is increased.

Unlike electrophoretic (electrical) means of manipulation, magnetic interactions are not generally affected by surface changes, pH, ionic conoentrations or temperature. Therefo're they represent an attractive method for manipulating blomolecules. There are several approaches for genomic clean-up within a micrnchip. One such approach is based upon immobilization of leucacytes from whole blood using a LeykosorbO-filter, followed by a wash step, lysis, and neutralization with buffers (Dadic, D.; et al. Proceedings of the 9th lntemational Conference on Miniaturized Systems for Chemistry and Life Sciences 2:811 (2005)). This requires a continuous flow, utilizing micropumps, actuators and other external interFaces; resulting in a complex implementation for microtluidic applications. Alternatively, cell lysis can be achieved with an electric AC field, with a combination of'electrokinetic and vacuum transport, in which the cell lysate is processed (Poulsen, C. R.; at al. Proceedings of.
the 9th lntemational Conference on Miniaturized Systems for Chemistry and Life Sciences 2:880 (2005)). The electric fields when used will not allow for precise separation, that is they do not result in advantageous DNA fragment separation of DNA from the rest of the lysate.
The prior art teaches the use of cell trapping using dielectrophoresis (DEP), cell lysis by electropbration and silica beads for DNA extraction, using a continuous flow approach which, due the number of processing steps required, is perhaps not practical for a portable system (Ramadan, Q.; et al. Sensors and Actuators B, 113: 944 (2006)). The additional compleadty in this approach, with use of a continuous-flow approach and the microfabrication processes involved for the DEP electrodes, is reflected in the lack of commercial use of such devices.
Lander et al. (Legendre, L. A., et al. Analytical Chemistry, 78:1444 (2006)) used solid-phase extraction (SPE) to selectively concentrate DNA and purify the clinical sample within a silica bead/sol-gel beds. A closely related demonstration is that of the SPE
technique for a disposable cyclic polyolefin chip (Bhattacharyya, A., et al. Analytical Chemistry, 78:788 (2006)) wherein the solid phase extraction was formed in situ with photo-initiated polymerization.

There are also demonstrations of the isolation of nucleic acids from bacterial cell cultures, wherein Bacillus c reus bacteria were lysed through incubation with a solution containing detergents, lysozyme and ethanol, followed by incubation with a chaotropic agent and Protease K (Wang, J., et al. Prroceedings of the 9th Intemational Conference on Miniaturized Systems for Chemistry and Life Sciences, 1:289 (2005)). DNA was then eluted on a solid phase silica membrane. SPE techniques are typically highly effeetive in the isolation of DNA
but pose several challenges during fabrication of the microchip and therefore not part of the implementation of the development directed towards portable systems.

Several demonstrations have used non-magnetic besds on a microchip far DNA
extraction.
Chung et al teach the use of immobilized beads, with.E. Coli present on the surface, as the solution is agitated to increase the probabiiity of collision of the cell lysate and the beads within bead-immobilized PMMA substrates (Chung, Y. C., et al. Lab on a Chip, 4:141 (2004)).
Hong et al. (Hong, J. W. et al. Nature Biotechnology 22:435 (2004)) teach the use of pneumatic valves, wherein mRNA purification from bacterial and mammalian cells. was demonstrated, pneumatically, by opening the valves between the lysis buffer and by flushing cell lysate over the mRNA affinity column, mRNA is retained on the column, which is then recovered after a wash step. Another closely related demonstration uses laser irradiation, with bead-based DNA extraction, leading to reahtime detection for various pathogens (Lee, J., et al. Lab on a Chip 6:886 (2006)). Hence, for effective usage substantial other external infrastructure is necessary.

Therefore the art is in need of an effective means to isolate highly pure nucleic acids free of proteins and other components in cefis or body fluids, chaotropic salts, ethanol, and other common inhibitors.

SUMMARY OF THE INVENTION

The present art has suffered from complex means of sample preparation particularly in the context of DNA isolation or concentration in a microfluidic device.

The present invention provides for a method for increasing the concentration of a molecule of interest comprising, in a first area of the microfluidic devioe allowing a clinical sample to interact with a particle capable of being attracted by a magnetic field;
applying a magnetic field to the microfluidic device such that said particles in the first area of the microfluidic device are encouraged to move to a second area of the microfluidic device;
wherein said first area of the microfluidic device and second area of said microfluidic device are in fluid communication; and wherein the particles are suspended in a fluid medium of viscosity greater than 3 centipoise and wherein 'said particle is able to preferentially capture said molecule of interest.

In a further aspect, the present invention provides for the addition of a solute to the fluid medium to Increase viscosity of said fluid medium, wherein the solute {s chosen so as not to substantially interfere with later biochemical reactions with the molecules of interest In a one embodiment, the solute is sucrose of a concentration of 20-35% w/v, in a preferred embodiment the solute is 25% sucrose wlv.

In a further aspect, the first and second areas of the mlcrofluidic device have fluidic communication by means of a microfluidic channel. In one embodiment the microfiuidic channel contains a trench, wherein said trench is of a depth and width of 15 to 25 times the diameter of the particie, more preferably 18 to 22 times the diameter of the particle, still more preferably 20 times the diameter of the particie, In an alternative embodiment the microfluidic channel is of a width of 250 to 350 times the diameter of the particle and the depth is of 35 to 65 fimes the diameter of the particie. In a preferred embodiment, the width of the microfluidic channel is 275 to 325 times the diameter of the particle and the depth is 40 to 60 times the diameter, and in an even more preferred embodiment the width of the microfluidic channel is 305 to 316 times the diameter of the particle and 53 to 57 times the diameter.

In another aspect, the present invention provides for a method of increasing translocation of the particles of the present invention by coating of the microfluidic channels within a microfiuidic device with an agent, wherein the agent is selected from the group comprised of chlorinated organopoly siloxane, linear polyacrylamide, poly-dimethylacrylamide, 3-(trimethoxysilyl)propyl methylacrylate, 3-(trimethoxysilyl)-poiyethyleneglycoi (600), bovine serum albumin, polyvinyl pyrrolydone, Triton-X-100, Brij-35 and Tween 20T"'.
IN a preferred embodiment the agent is bovine serum albumin.

The accompanying description illustrates preferred embodiments of the present invention and serves to explain the principies of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 shows a representation of the micnofluidic chip (glass-glass microchip (ith channels etched on only one of the surface, and then irreversibly bonded to the second un-pattemed glass surface) used to perform the sample puriflcation of the present invention;

FIGURE 2 shows a representation of the cross-sectional view of the micofluidic channels in a mPcrofluldic chip with "trenches ;

FIGURE 3 shows a diagram representing the various steps within the conventional (left) and microchip-based (right) DNA purification approach;

FIGURE 4 shows an electropherogram depicting the detection of beta 2 microglobulin (B2M) gene using glass-CE chip filled with commercially available molecular sieving polymer (POP6) but with puTification performed in a microchip as in Figure 1 and Figure 2 with whoie blood as the starting material;

FIGURE 5 shows an electrophenogram detecting the presence of a single nucleotide polymorphism on glass-CE chip (POP6) with whole blood as the analyte for G238C
SNP In the thiopurine methyl transferase (TPMT) gene showing a 293bp peak FIGURE 6 shows an electropherogram detecting the presence of a single nucleotide polymorphism on glass-CE chip (POP6) with'whole blood as the analyte for G460a TPMT
SNP after restriction enzyme digestion;

FIGURE 7 shows two electropherograms detecting the presence of Herpes Simplex virus on-chip ;(top) is a Herpes simplex-specific PCR performed using genital swabs (using buffer extract viral DNA from the genital swabs) and (bottom) after concentrating viral DNA using magnetic beads, wherethe low intensity PCR product signal in (top) is greatly enhanced post concentration as in (bottom).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein "bound", in the context of beads or magnetic beads, is not limited to a covalent or ionic interacfion between the nucleic acid, or other molecule of interest, with the magnetic bead contemplated by the present Invention, but includes 'hydrophobic, size-exclusion based "trapping" or other interaction wherein there is a higher concentration of molecule of interest internal to, or immediately adjacent to, the magnetic bead. It is contemplated by the present invention that the molecule of interest may be a nucleic acid, such as DNA or RNA, interacting with the bead primarily through a charge-interaction; or alternativefy a molecule of Interest Interacting with the bead through charge or other Interactions, such as an interaction between a protein and an antibody or antibody fragment with specificity for the protein. One skilled in the art would be capable of identifying potential interactions between a molecule of interest and a motecule or compound present on the bead which would result in a higher concentration of molecule of interest internal to, or immediately adjacent to, the magnetic bead.

As used herein "magnetic beads" or "beads" means a particle capable of being attracted to or repelled within a magnetic field, and of a size such that it is capable of moving through a microfluidic channel. It is contemplated that a magnetic bead may optionally include an internal cavity which is optionally accessible to the surrounding environment.
It is explicitly contemplated that "beads" or "magnetic beads" are not necessarily spherical in shape.

As used herein, "trench" means a deformation of the generafiy planar surface of a microfluidic channel of sufFicient size to allow the beads contemplated by the present invention to reside within the deformation and substantially restrict the lateral bead movement within the context of the general microfluidic channel which includes the deformation, while allowing the beads to travel along the length of the deformation or general microfluidic channel.

As used herein, "isolation/purification" means the increase of the concentration of a moEecule of interest or a decrease of molecules, compounds, or particles not of interest, in a region 25. within a microfluidic device.

The present invention, comprising an on-chip bead-based technique is applicable to a wide variety of molecular diagnostic applications and also for transport andlor manipulation of biological particles (e.g. whole cells). The efficacy of such an approach is demonstrated here in a pertinent application of human genotyping.

In addition to the DNA isolation/purification capability of the present invention, the methods and apparatus of the present invention can also be used for RNA, nucleic acid, protein or other macromolecule concentration, as wefl as for coflecting and enriching cells or specific cell populations. In molecular diagnostlc applications, template concentration is = often necessary when starting from unprocessed clinical samples. The frequency of rare cells in blood, for'example metastatic cancer cells, blood or urine-borne bacteria or viral DNA, RNA
or particles, or fetal red or white blood cells in maternal blood may be extremely low (e.g. one cell of interest among a million cells, or low viral titers with e.g. 1-10 templates per ml. of plasma or urine). In the context of microfluidics, using sub-KL volumes (low volume regimes) there may be insufficient cells or template for subsequent analysis.
Therefore, concentration and/or enrichment of the nucleic acids or proteins of interest becomes important, particulariy those which may occur in a larger sample but would otherwise be unrepresented in L
volumes. The ability to utilize sub-microliter reaction volumes for the analysis. of clinical samples is thus heavily dependent on purificafion and concentration of the analyte from, for example, whole blood, other tissues or body fluids prior to performing, for example, PCR.

The present invention contemplates the use of beads to immobilize charged biological species on the surface of beads, wherein the magnetic beads may be immobilized or transported using an appropriate magnetic field. The beads contemplated by the present invention may be either custom designed or using beads otherwise commercially available as disclosed herein. The present invention contemplates the manipulation of the magnetic beads within a microfluidic chip using a magnetic field generated either within 'the chip itself, or through the application of an external field impinging, at least in part, upon the microfluidic chip. The art has demonstrated use of an external magnetic field in combination with magnetic beads in the context of microfluidics. For example paramagnetic oligo-dT beads (Jiang, G. F., et al. Analyst 925:2176 (2000)) were used to isolate mRNA for construction of a cDNA library, but usage.was limited to mixing of the beads and sample on-chip and for using magnetic trapping (using two sets of extemal magnets) to capture, and subsequently release the beads. In another closely related demonstration, T-cell purification from human and reconstituted blood samples was performed within a chip (Furdui, V. I., et al.
Lab on a Chip 4:614 (2004)). Magnetic bead-based beds were formed and protein CD3 complexed to paramagnetic beads was introduced into the chip and captured in a field generated by an external magnet followed by off-chip PCR and subsequent electrophoresis.
Similarly, using a silicon/glass device, rare cells were captured from whole blood, with electrolytic generation of gas from Kn03 to provide pumping actuation for a device that performed the capture and purification of rare cells spiked into a 7.5 L reconstituted blood sample.
Yet, the use of magnetic beads for movement and transport of molecules of interest, as contemplated by the present invention, has not been previously disclosed in the art.

Several demonstrations using planar microfabricated coils have been reported in the literature. Deng et al. (Deng, T. et al. Applied Physics Letters 78:1775 (2001)) teach the use of magnetic beads moving towards magnetic field maxima, generated by microfabricated current carrying wires. The magnetic field maxima capture beads from solution, which are heid in the desired position and driven along complex paths. However, high currents of -10 A are necessary, resulting in high temperatures, which are detrimental to biomolecules. In another demonstration (Mirowski, E., et al. Applied Physics Ledters 84:1786 (2004)) a pattemed magnetic thin-film element (permalloy traps) is supported on a thick silicon-nitride membrane.
In the presence of an external magnetic field, the field gradients near the magnetic elements are sufficiently large to trap magnetic particles. Wirix-Speetjens et al.
(Wirix-Speetjens, R. et al.
1"EEE .TransactPons on Magnetics 40:1944 (2004)) teach an on-chip bead transport devioe based on a set of two tapered current conductors. This system is capable of guiding magnetic particles along a defined track (or channel) using a magnetic field generating current conductor which is phase shifted. For each of these demonstrations, the current conductors have rectangular cross sections and the magnetic field is maximal at the corners of the cross section. There has been no biological impiementation of such a system. Using planar coils on a microchip has the limitation that such a technique for micro-magnet fabrication typically involves compisx fabrication techniques. Furthermore, the heat generated in these devices, prevents their use for manipulation of bio-moiecuies.

The present invention provides for the substitution of liquid movement with magnetically induced movement of magnetically responsive particles within a microfluidic chip. The present invention contemplates cell lysis, magnetic particle transport and deiivery, and trapping when necessary. The resulting system is hence compatible with chip-based PCRs and electrophoretic analysis for detection. The apparatus of the present invention does not require valving, thus can easily be used as a stand-aione module or coupled to other vaived or non-valved, microfluidic systems. Hence, the method of the present invention is robust and provides minimum observable inter-run variability. ChargeSwitch technology (invitrogen) is suitable for isolating highly pure DNA and RNA from almost any source, including yeast, bacteria, plants, human or other animal tissues, blood, buccal cells, PCR
reactions, and forensic samples. Using whole blood, tests were perPo.rmed for beta 2 microgioblulin (82M) and certain SNPs.

As contempfated by the present invention, a microfluidic channel is filled with appropriate bufferliiquid medium, and an input well present on the microfluidic device, or in fluid communication therewith, is filled with the mix of beads, lysis buffer and Protease-K along with a sample of interest, which may include ceilular material such as a clinical sample, whole blood, cells, tissue, urine or any other type of biological sample known in the art.
Effective cell lysis occurs, with binding of the molecules of interest, for example but not limited to, nucleic acids such as DNA or RNA to the bead surface or by extension protein if the bead surface is appropriatety modified to bind the 'species of Interest. A
movable magnetic field maxima in the plane of, impinging on and moving in the direction of, the microfluidic channel results in movement of the beads through the microfiuidic channel into another region of the chip. It Is contemplated that the magnetic field may be external to the microfluidic device, alternatively it may be generated within the chip itself or it may be generated immediately adjacent to the chip. Means to generate magnetic fields internal to the microfluidic device are known in the art and include, but are not limited to, means for conducting an electrical current of alternating or direct polariry,.integral or adjacent to, the microfluidic chip; wherein the currerit passing through the conducting region may be increased, or decreased compared to an adjacent region on the microfluidic device. Such procedures might require microfabrication of planar coils in the vicinity where magnetic filed maxima's are required within the chip. This will allow the change in location of a magnetic field maxima on the microfluidic device. External ma,qnetic fields can be affected by means known in the art, including either a single or multiple sources of magnetic fields in proximity to the chip, to manipulate the beads within the chip. In the preferred embodiment, the source of magnet field is perpendicular to the longitudinal and lateral plane of the chip, i.e. "above" or "below" the chip.

Using the methods, protocols, buffering system and the apparatus of the present invention, a separate (i.e. off-chip) sample "clean-up" step is not necessary, though may opfionaily be included as required by the application. To ensure sufficient removal of the Protease-K (and also cell debris) that is considered inhibitory to PCR, the traditional clean-up step may be substituted by migration of the beads, using the movement of the magnetic field maxima, through a microfluidic channel (or cavity, such as a well, in the microfluidic device), containing Tween-20TM. This, results in sufficient deactivation of the Protease-K in advance of the PCR reaction; alternatively a more thorough wash mechanism could include a gravity based flow of fluid (for example, including but not limited to wash,buffer through the Tween 20T'" fiiled channel) initiated by altering the volume of fluid in each of the wetts on the micnochip. In this manner, the nucleic acid bound to the beads is migrated though the chip, thus effectively separating the nucleic acid from the cell debris and other introduced chemical constitutes (e.g. Protease -K) within the input chamber. Hence the migration of the beads, through the controlled movement of the magnetic field maxima, performs a dual function of removing nucleic acids from cell debris and removal from cellular components which would have an adverse effect on a subsequent reaction, including but not limited to, PCR.

It is contemplated that the input and output wells, as shown in Figure 1, are filled to the same level with fluid, thus minimizing potential pressure driven flows through the chip. In the preferred embodiment the movement of the beads i.s primarily by the change in location of the magnetic maxima and not due to the bulk flow of the fluid. In an altemate embodiment, a negative pressure gradient is established, relative to the directional flow of the magnetic beads, which may further assist in the removal of compounds having an adverse effect on later reactions or processes, Alternatively, the beads may remain immobilized while a fluid flow past the beads occurs. In one embodiment, PCR is performed after elution of DNA from the bead, in an elution step, using an appropriate elution buffer. It is contemplated by the present invention that the PCR step may be integrated as part of the microfluidic device. In an alternate embodiment, the wash buffer with the beads containing bound DNA
and/or RNA
may be included within the PCR reaction, without the elution step. Since the elutlon and binding of the DNA to the ChargeSwitch beads depends on the pH of the environment, the pH of the PCR buffer is sufficient to release or relax the DNA/RNA bound to the magnetic beads, allowing the target nucleic acid or other molecule to participate in the amplification reaction (PCR). Release of nucleic acids bound to beads that have concentrated them from a larger original sample provides a means to deliver a much larger amount of template to the PCR reaction well than is possible if an unconcentrated aliquot of the original sample is tested. An important aspect of this invention is the on-chip enrichment of template by immobilization on the surface of the beads for concentrated delivery to a desired region of the microfluidic device, such as a reaction well. Certain applications of the.
present invention, in particular those that include processing of the DNA and/or RNA in a manner not including the PCR reaction as a first step, may require complete elution of the bound nucleic acids.
.Figure 2 shows a cross-sectional view of one embodiment of the present invention which incorporates a trench in the microfluidic channel. It is oontemplated that the methods of the present invention may be used in conjunction with microfluidic channels In which a trench is incorporated, or otherwise.

Beads operation The nucleic acid bound to the positively charged ChargeSnritchfel surface loses its affinity for the beads as the surface becomes neutrally charged, which occurs in the presence of an elution buffer compatible with downstream processes (e.g. PCR). The ChargeSwitch@ beads have a net positive charge, thereby binding nucleic acids, when the environment surrounding the ChargeSwitch beads is below pH 6. When the pH of the environment is above 8, the net charge on the bead tends towards neutral, and the bound nucleic acid is eluted from the ChargeSwitch beads, Hence, the binding of nucleic acid to the ChargeSwitchOD
beads is controlled through the pH of the medium within which the beads are placed.
Depending of the nature of the Interaction between the molecule of interest and the bead, other means of elution of the molecule of interest from the bead may be necessary and are well known to the art. Selection of the elution means must take into account the stability of the molecule of interest as well as its effect on downstream applications or processes such as electrophoresis, PCR, labelling, or antibody detection. With respect to the ChargeSwitchOD
beads, the pH of the microfluidic device of the present invention is optimized such that the DNA will be effectively bound to the beads until delivery of the DNA to the site of PCR, or other desirous reaction point. The ChargeSwitch technology uses 100% aqueous solutions, which eliminate the possibility of PCR failure due to the presence of ethanol, phenol, chloroform, and ionic chaotropes. The ChargeSwitchO process does not require ethanol precipitation, drying, or re-suspension.

Buffering system Surfaces treated by ChargeSwitchO Technology carry a positive charge at low pH
(<pH 6.5) and are neutral at higher pH (~-pH 8.5-9.0). Therefore it is contemplated by the present invention to avoid use of a "wash bufier" to fill the microfluidic channel where bead rinsing ("Wash") is to be performed in preparation of cell lysates for PCR. As weii, use of wash buffer, which has a relatively low viscosity, can result In pressure differentials in the microfluidic device, thus potentially initiating bulk fluid "flows' within a microchip.
Therefore, in place of.a wash buffer, Tween-20TM is used, a detergent known In the art At appropriate concentrations, Tween-20Tm is viscous enough to reduce pressure driven flows in the microfluidic device thus minimizing possible contamination between the input and the output well. Viscosity may be addressed by reduction of Tween-20T14 to 1% or less, and addition of up to 25% sucrose, which is inert in the context of dQwnstream reactions, while the increased visoosity interferes with the diffusion of lysis buffer (as discussed below), proteases or cellular debris. -In a preferred embodiment the viscosity of the buffer is 4 centipoise.
Consistent results were obtained using Tween-20T"" in place of a wash buffer.

Another advantage is that any remaining proteins entangled with the bead-bound DNA are denatured, thus reducing inter-run variability of the PCR using the beads bond to DNA taken out of this purification chip. Yet another advantage is that Tween-20TM is itself a gentle detergent which assists in the cell lysis occurring in the input well of the purification well. The use of Tween-20T"" with the apparatus of the present invention results in more consistent bead movement, as compared to wash buffer, with inconsistencies likely arising from the interaction of the wash buffer with the surface of the channel. Finally, the pH of a 10%
preparation of Tween 20T"' as disclosed herein (10% Tween 20TM diluted in water) has a pH
of -5.5 which ensures that the DNA remaing hniind to the beads until they reach the output 20. well (Figure 1). As contemplated in the present invention, maintaining the bead-nucleic acid complex until beads reach the Tween-20TM containing PCR well provides a means to concentrate the DNA, thereby increasing the ability to detect templates that are otherwise present In low amounts that in unconcentrated samples may be absent, or unavailable in, the PCR reaction chamber or well, or below the limits of detection, even if using PCR
amplification.

The ability to concentrate a template as well as to remove inhibitory material using the method and apparatus of the present invention, means that microfluidic tests or analysis using certain clinical samples not otherwise be capable of being implemented on a microfluidic chip, for example but not limited to the detection of herpes simplex viruses I and 2 in cell preparations obtained from genital swabs. Often, clinical samples when" used in small volumes may not have sufficient targets for detection, even though the concentration of viral nucleic acids is sufficient for causing disease. Furthermore, when these samples are tested in small volumes this issue is further exacerbated. Hence, biomoiecule concentration in such scenarios is important. Concentration of nucleic acid from lysed cells was observed to be necessary when using genital swabs (buffer) for detecting. Herpes Simplex virus.
Although a weak signal was detected from on-chip PCR (using the buffer used to elute the viral DNA from the genital swabs) and chip-based CE detection (Figure 7, top), the signal strength is greatly enhanced with the magnetic bead-based concentration of the analyte using the apparatus and method of the present invention (Figure 7, bottom).

Operation of the external magnet for the generation of the field maximas.

In the preferred embodiment of the present invention, all other forces affecting bead movement other than the change in the focation 6f the magnetic field maxima, are minimized during this purification process (for example, but not limited to, surface effects of the glass microfluidic chip or pressure driven flows). Since the method and apparatus of the present invention contemplates use of a glass-based microfluidic device, but not excluding use of other substances in fabrication of the chip, for example PDMS, silicon or other material, surface effects may have a substantial impact. Therefore, buffer conditions in the fluid, as well as the implementation of the magnetic field, are important_ Achieving the desired magnetic field pattern, strength, maxima and gradient over the confined space of a microfluidic channel is centrally important, particularly in considering the rapid decrease of magnetic flux density with increasing distance of the source of the magnetic field form the fluidic device. As indicated previousiy, the use of Tween-20Ta in the microchannels provides a coating for the surface of the channels, thereby minimizing the potential surface effects of the glass or other material that is in contact with the beads and the other buffering system.

In a preferred embodiment, the microfluidic channels are pre-coated. Bovine Serum Albumin (BSA) In distilled water ('Emg/mt_) is injected through the microfluidic channels and wells such that all channels are filled. The solution is allowed to reside in the microfluidic device for 5 minutes, at a temperature of 18 C to 23 C. The BSA solution is removed and the channels and wells are dried with pressurized air. This pre-coating significantly enhances bead movement through the microfluidic channels. In addition to enhancing bead movement and decreasing bead clumping, pre-coating the mocrofluidic device with BSA also decreases adhesion of Polymerase Chain Reaction (PCR) enzymes to the PCR reaction chamber, while not interfering with the PCR reaction or any other desirous biochemical reactions within the microfluidic device. It is contemplated by the present invention that alternative coatings, other than BSA, may be used so long as it results in the previously described desirable characteristics. Other coatings for the chanriels contemplated by the present invention inciude hydrophobic coatings, by way of non-limiting example including Chlorinated organopoly siioxane; hydrophilic coatings, by way of non-limiting example including linear poiyacryiamide or poly-dimethylacrylamide, 3-(trimethoxysilyl)propyl methylacrylate, or 3-(trimethoxysiiyl)-polyethyieneglycol (600); or blocking agents, by way of non-limiting example Bovine serum albumin, Polyvinyl pyrrolydone, Triton-X-1 00, Brij-35 or Tween 20T'".

Bead clumping during migration can negatively affect performance of the isoiation/purification contemplated by the present invention, which is a function of bead density, bead size, bead migration velocity and the , contents of the sample for testing (i.e. nucieic acid, or other molecule of interest), does not compromise the purification of nucleic acids, protein, cells or cell constituents, but can hinder passage of the beads through the channels.
An inherent property of the beads is to clump together both before and after the i'naqnetic field has been applied, which reduces the surface area available for DNA to bind to the beads, and could result in a clogging of the channels in the microfluidic device. In one embodiment of the present invention, a 20 m deep and 20 m wide "trench" was etched within the migration channel. This trench is placed within the 500 m wide X 100 m main channel (Figure 1b).
The incorporation of the trench provides a means to focus the magnetic field (further) on the beads by restricting laterel bead movement to the physical area of the channel defined by this "trench," while retaining the ability of the beads to move in a direction that is perpendicular to both the channel direction and channel width through the trench (when the field maxima is taken away from the chip) and towards the source of the magnetic field (i.e.
into the trench) when the maxima is produced ciose to the chip (from the bottom side of the chip). This maximizes capture and enrichment of the target molecules or cells on the surface of the beads, while ensuring that clogging is minimized.

Furthermore, it is contemplated that the presence of a trench etched within the main migration channel promotes enhanced washing of the beads as they are made to migrate through the fluid-filled channel, thereby taking advantage of the two directions of freedom/motion, horizontai 'and vertical, by the beads. This embodiment of the invention further enhances the processing of bead-bound target molecules or cells by removing potential inhibitory components of the Input mixture that may have been released during the on-chip purification and/or processing. Therefore, the design of the trench is important wherein the depth of the trench must be sufficiently deep to restrict passage of a high density of beads to the smaller downstream channel. Though not necessary to practise the present invention, it is hypothesized that the trench focuses the streams of beads in the magnetic field while restricting the density of beads traversing through the trench to the downstream channel.

In an alternate embodiment of the present invention, channels of 310 m and 55 m depth are used in association with beads of 1 um size and at a concentration of 26mg/mi of solution. It has been advantageously identified,as part of the present invention that these channel dimensions result in a diminished amount of bead clumping.

ChargeSwitch beads are typically in the range of 1 m diameter, but when migrating in a magnetic field, they typically move as a loose clump, hence, a sufficiently large ratio of the trench depth to the individual bead was maintained (the preferred embodiment of the present invention has a ratio of 1:20, but the present inventiorl is not limited to this ratio). The present invention contemplates a range of trench depth to bead size ratio, that could be incorporated in a manner that is dependent on the desired outcome. Other architectures are alsa contemplated and would be obvious to one skilled in the art, dependent on the bead density used and the blomolecule of interest. In one embodiment of the present invention using ChargeSwitch@) beads, a trench approximately 20 m deep within the main migrating channel was used, A variety of other configurations would be obvious to one skiiled in the art and are contemplated as part of this invention.

In one embodiment of the present invention, focusing of the magnetic field, for an external magnetic field source, was achieved by machining the magnet in a conical shape, with tlie tip -of the magnet placed directly under the channel. It was observed that such a machined magnet performs better than, for example, a cylindrical magnet in which the magnetic field is -unfocused. The present invention contemplates the use of a variety of magnet configurations, including external magnets and Integrated magnetic coils micromachined on the chip itself, as would be obvious to one skilled in the art. When using a channel size of approximately 65 jAm depth and 310 f,.m width, absent a trench; combined with the viscous buffer contemplated by the present invention, significant isolation/purification of nucleic acids was possible in channel- lengths as short as 13 mm.

DNA purification from clinical samples.

Using the apparatus and method of the present invention, purified mono-nuclear cells (MNCs) were used as the starting material with the ChargeSwitch beads and the other components as described herein. Subsequently, extraction of DNA from whole blood was performed, with the extracted DNA tested on separate PCR chips using primers that amplify the B2M gene. To validate -the apparatus and methods of the present invention for processing of whole blood, prior to on-chip PCR, the extracted DNA (post on-chip PCR) was enzyrnaticaliy digested on the microfluidic device and tested for two single nucleotide polymporphisms (SNP) in the thiopurine s-methyltransferase gene (TPMT) gene.
Detection and size discrimination was performed using the )iTK (Vahedi G, et al;
Electrophoresis 25:
2346 (2004)) within a glass based capillary electrophoresis chip and further verified using the ABI 3100 and using agarose gels. Finally, the chip-based. PCR product was sequenced to ensure specificity of the on-chip amplification. Primer sets were chosen to ensure that only genomic DNA was being amplified in the downstream PCR reaction. Any product derived from bead-bound RNA was excluded by choice of primers (product size) and the lack of a reverse transcription step Patients and cell lines.

The purified mono-nuclear cells (MNC) used were obtained by ficoll purification of blood samples donated by patients at the Cross Cancer Institute (Edmonton, Alberta) after obtaining informed consent. For purposes of storage, cells were frozen in phosphate buffered saline (PBS) at 5000 cells/ L, the lower limit of physiological range of MNC
in normal human blood. The fresh, whole, human blood samples used in this study were obtained from healthy volunteers using a standard finger lancet (LifeScan, Milpitas, CA) that produced approximately 20-30 L of blood. Blood samples were used directly on-chip with no prior sample preparation.

EXAMPLE 1: Conventional beads-based protocol Genomic DNA (gDNA) was purified from crude clinical samples using ChargeSwitch@ (CST) magnetic beads (Invitrogen, Calsbad, CA). CST beads contain a switchable surface charge dependent on pH of the environment. In low pH conditions, the CST beads are positively charged and bind to the negatively charged backbone of nucleic acids, which were then separated by a field from the surrounding solution. The bound nucleic acids are then eluted from the CST beads by raising the pH of the solution to 8.5. Figure 3 shows an outline of the conventional bead preparation steps (left) compared to the micrnfluidic equivaient (right).

As a control, -the procedure as outlined by the vendor (Invitrogen) was exactly followed, i_e.
the DNA cleanup withiri a tube_ The clinical sample (20 }eL - in this case whole blood) is incubated in 0.5mL of a supplied lysis buffer, (L12 buffer, lnvitrogen, Calsbad, CA) and 0.1 mg Protease K(Invitrogen, Calsbad, CA) for 10 minutes, followed by addition of 0.1mL of the supplied low pH buffer (N5 buffer, Invitrogen, Caisbad, CA) and 0.5mg CST
beads. The supernatant containing proteins and other contaminants were removed with the CST beads held in a pellet by an extemal magnet. This demonstrates the immobilization of beads/nucleic complexes to remove cell components by fluid flow away from the beads. Other tests, as in Example 2, purified the'bead-bound nucleic acids by magnetic movement of beads away from cell component in the lysate. CST beads were then successively washed three times in a supplied wash buffer (W12 buffer, Invitrogen, Calsbad, CA), lysis buffer, and again in wash buffer. DNA was eluted from the beads using 450p.L supplied high pH buffer (E5 buffer, Invitrogen, Calsbad, CA). 5 pL of this elution buffer was then used as t,emplate in downstream PCR (both conventional thermal cycler PCR as well as on-chip PCR
was performed).

EXAMPLE 2: Microchip bead-based protocol Two variants of the bead based procedure were implemented, one wherein blood was used as the starting material for DNA, and,the other being where purified mono-nuclear cells were employed as the starting material. This was primarily to establish the limit of detection and to demonstrate the broader applicability of this bead-based system. In either case (MNCs or whole blood procedure) a 10 L clinical sample of thawed purified MNC cells or fresh whole human blood was mixed with 500 L of lysis buffer, 25 L low pH buffer, 0.1mg Protease K, and 0.25mg -CST beads. Microfluidic channels were filled with Tween-20T"' (Sigma Aldrich, Oakville, Canada) prior to loading 4 L of this initial cell lysate solution Into a microchip well using a pipette. The magnetic beads were then washed by migrating them through the channel, away from the ceil components in the lysate, with an external magnet.
The external magnet was machined Into a cone with a 2mm tip to improve localization of the magnetic field, with the tip used for generating the magnetic field maxima on the microfluidic device.

Magnetic beads were moved Into a microchip well at the end of the channel, which contained 4 L of Tween-20TM. The DNA carried by the beads to the Tween-20T""-containing PCR
reaction well provided the template for the subsequent PCR reaction.

EXAMPLE 3: PCR protocols After gDNA purification and recovery from the microchip, the B2M gene was amplified with on-chip and conventional PCR as a control gene to evaluate the performance of the purification with the results shown in Figure 4. Thermal cycling conditions were as follows' 5 min); 30 cycles of DNA denaturing, 95 C (30s); primer annealing, 60 C (30s);
dNTP
polymerizing, 72 C (30s), final extension, 720C (10 min). The primer set for b2m was for 1.0 forward, 5'-CCAGCAGAGAATGGAAAGTC -3' and for reverse 5'-ACTTAACTATCTTGGGCTGTGAC-3'. The reverse primer was labelled with the fluorescent dye VIC (Applied Biosystems, Foster City CA). The PCR reaction mixture contained a final concentration of 1xPCR buffer, 2.0pM MgC12, 200'nM dATP, dGTP, dCTP, and dTTP, and 0.2 M of each primer.

EXAMPLE 4: Buccal swabs for TPMT SNP genotyping.

Cells obtained by swabbing provided a non-invasive means to obtain material for genotyping.
Types of swabs include, but are not limited to, genital swabs to test for viral infect+ons,.
cervical swabs to detect cancer and buocal swabs to determine genetic characteristics. On-chip testing of cells recovered from swabs required a method to process the sample as contemplated in this invention. On-chip use of this invention has been tested using genital swabs to detect Herpes viruses 1 and 2, and buccal cells to test for genetic polymorphisms.
For both tests, purification of cellular DNA using magnetic bead processing on-chip provided an effective means of obtaining and concentrating DNA. In some cases on-chip processing using beads, as disclosed herein, proved essential for successfully performing the tests. It will be obvious that this invention also applies to a wide variety of other tests where nucleic acids must be recovered from swab samples. In one einbodiment of the invention, buccal swabs were obtained from healthy volunteers using two 15 cro sterile swabs (AMG Medical, Montreal, QC) applied to left and right buccal surface for 1 min and then incubated in 200 L
lx PBS each for 15 min. 50 L of the pooled 400 L was then used for on chip purification of gDNA. Genotyping of three signature pharmcogenetic SNP; G238C (FigUre 5) , (Figure 6), and A719G, in the thiopurine s-methyl-transferase gene that account for over 95%
of the clinical toxicity to thiopurine immunosuppressants was carried out by on-chip restriction fragment length polymorphism (RFLP). PCR products containing the SNP were amplified using specific VIC-labeled primers using the aforementioned PCR conditions.
Emmediatefy following PCR, without any purification of the PCR product, 0.25 L of b U/ L
of the specified restriction enzyme (New England Biolabs, Pickering, ON) was added and incubated for 30 min at room temperature except for the Acc1 enzyme which was incubated at 37 C for 2 hours.

EXAMPLE 5: Microchip design and fabrication The chip designs were drawn in L-Edit 0.0 (MEMS Pro 8, MEMS CAP, CA, USA) and transferred to a mask using a pattern generator (DWL 200, Heidelber+g Instruments, CA, USA). The 4 inch X 4 inch borof{oat glass jul~dOtlatO (i,a~wyvn vFril4Pi cc.,pG^y, P,^., !aA`
was cleaned in a hot Piranha (3-1 of H2SO4,:H202) and the cleaned glass was sputter-coated with 20 m of Cr and 200 m of Au. Spin coat of HPR 504 photoresist was applied at a spin speed of 500 rpm for 10 s and a spread speed of 4000 rpm for 40 s. An oven set at 100 'C
was usert to bake the wafer and the wafer was then placed in an enclosed container with a damp cleanroom wipe, light and moisture sealed for -15 hours. UV exposure (4 s, 356 nm, and intensity of (19.2 mW/cm2)) of the spin-coated substrate was performed through the chrome mask using a mask aligner (ABM Inc., CA, USA). The substrate was then chemically developed with Microposit 354 developer (Shipley Company Inc., Marlborough, MA, USA) for about 30 s. Class etch was performed using hydrofluoric acid (HF).
Subsequently, holes in the top layer of the glass (non-etched glass) are drilled using a Waterjet for accessing the channels. After sufficient cleaning the two layers of glass where then irreversibly bonded by placing in an oven at -500 'C fnr 2 hours.

EXAMPLE 6' Electrophoresis equipment and protocol Electrophoretic fragment analysis was performed within the cross-channel CE
section of the microchfp. Fragment analysis of the amplified PCR mix was performed within the microfluidic tool kit, TK (Micralyne, Edmonton, Canada). The TK provides the optical detection and high voltages needed to perform CE with confocal laser-induced fluorescence (LIF) detEction. The LIF system uses excitation at 532 nm and detection at 578 nm. A
pinch-off injection approach with 0.4 kV was applied for 60 s with a separatlon voltage of 6 kV. LIF
detection was performed at 76 mm from the channel intersection. POP6 (Applied Biosystems, Foster City, CA), a denaturing polymer, was used as the separation medium within the chip. Sizing was performed by simultaneously loading 0.3 l of a DNA ladder GeneScarOD 500 TAMRA (Applied Biosystems, Foster City, CA). Using the denaturing POP6 polymer, the polymer was first heated for 10 m at 67 C before being loaded into the .microchip, reducing the viscosity of the polymer (as well as ensuring the complete dissolution of any precipitated urea) and facilitating the loading of the polymer within the chip. In the sample loading well, the on-chip PCR product was denatured for 4 min. at 96 C
and rapidly cooled to -4 C and mixed with 1.2 i of HiDi formamide (ABI), 1 i of size standard (GS500) and 0.8 l of I X GABE (genetic analysis buffer with EDTA) to a total volume of 3 i. A mix of 1 l POP6 and 2 l of 1 X GABE is loaded in the separation wells.

EXAMPLE 7: Chip reusability procedures Between loads, the channels were soaked with 95% ethanol for -20 minutes.
After drying all residual ethanol, Millipore-purified water was run through the chip dried using a N2 gun. The chip was then placed under UV light for - 1 hour. Prior to use, the channels were loaded with Tween 20TM. A single chip can be used for -15 loads, and further usage was possible, but only after a rejuvenation procedure as described. After multiple runs, the beads were found to adhere to the walls of the microchannels, as a consequence of which they respond to the external magnetic field by changing orientation, but do not demonstrate any overall bulk movement. This hinders the overall directional induced flow of the bead population. This problem was solved by performing rejuvenation of the chip by soaking of the chip in hot Piranha (3:1 of H2S00242) solution for -15 min. This is followed by an annealing process for -12 hours at 450 C. The chips were then ready to be re-used for about 15 runs without any noticeable degradation during the clean-up procedure. After this rejuvenation procedure the beads respond as desired to the magnetic field maxima, i.e. they migrate through the channel with the same efficiency as within a new chip.

EXAMPLE 8: Optimization of bead numbers with clinical samples By binding fluorescently tagged labels to the ChargeSwitchOD beads, and running the fluorescent tagged beads through a flow cytometer, it was possible to determine the number of beads within a given volume as supplied by the manufacturer. It was observed that there were 2735 54 beads per pL of solution as determined using flow cytometry.
Using this data, the absolute number of beads introduced into the input well per unit volume was estimated. Two sets of optirnizations were performed starting with MNC, to determine the sensitivity and lower limits of detection using bead purified UNA to obtain a positive signal after the PCR. As shown in the particular example of Table 1, the number of beads introduced to the input chamber of the purification chip was kept constant, while the number of cells was varied, to determine the minimum size of clinical sample necessary to obtain a detectable signal from the. PCR reaction. The second optimization determined the minimum number of beads that were required to obtain a positive signal when the number of cells was kept constant at 50,000. As shown in the, particular example of Table 2, a minimum of -500 beads (applied onto the chip) were necessary for a positive signal when in a initial mix of Protease-K, lysis buffer a total of 50,000 cells were included. It will be obvious that the optimal numbers of cells and beads may vary depending on the instrumentation, reagents, application and cells being, analyzed, with the optimal numbers of beads determined by one skilled in the art using the apparatus, methods and procedures taught herein..

Table 1: Optimization of number of cells required to produce a positive signal using on-chip purification Cell equivalents (lysate) added to Initial input well of the Tube-based Cellslmi purification chip conventional PCR On-chip PCR
100000 367.0 +
50000 183.5 +
10000 36.7 + +
5000 18.3 + +
2500 9.2 + -1000 3.7 + +

600 +
100 0.4 - -Table 2. Optimization of the bead quantity for microchip sample purification.
On-chip bead equivalent ( L) Tube-based Initial volume of On-chip beatis In ul. (fraction of the initial mix volume) conventional PCR PCR
0.03670 + +
5 0.01$35 + +
2 0.00734 + +
1 0.00367 + -0.5 0.00183 - -5 EXAMPLE 9: Integrated bead-based isolation/purification with PCR.

The microfiuidic device is prepared as described In previous example. The channel dimensions of the microfluidic channels are 310 m In width, 55 rn in depth, with a separation length of the channel of 15 mm. No trench is present in the channels. The channels are filled with 4 L of STW buffer (25% sucrose, 1% Tween-20T^+, in 10 ChargeSwitch wash buffer). The reduction of the Tween-20TM content, compared to that used in Example 2, allows the direct transfer of the CST beads through the microfluidic channel to an on-chip well for performing a Polymerase Chain Reaction (PCR) within the microfluidic device as described previously in the art, see for example PCT/CA2007/000959.
The cells or clinical sample of interest are be added to 5004L of lysis buffer, 25 L low pH
buffer, 0.1mg Protease K, and 0.25mg CST beads, 4 L of the cell lysate solution is loaded _24 into a microchip well using a pipette. The magnetic CST beads are washed by migrating them through the. channel, away from the cell components In the lysate, with an external magnet. The extemal magnet is machined into a cone with a 2mm tip to improve localization of the magnetic field, with the tip used for generating the magnetic field maxima on the microfluidic device. Magnetic beads are moved into region of the microchip capable of performing the PCR reaction, containing the necessary PCR enzymes in an acceptable buffer, further capable of temperature cycling.

Optionally, the cells or clinical sample may be added to a well on the microfluidic device in fluid communication with at least one microfluidic channel wherein the well contains lysis buffer, low pH buffer, Protease K and CST beads such that following addition of the ceils,or clinical sample the final concentrafion is 5% low pH buffer, .2 pg/mL Protease K and .5 g/mL CST beads in lysis buffer. The magnetic CST beads are washed by migrating them through the channel, away from the cell components in the lysate, with an external magnet.
The external magnet is machined into a cone with a 2mm tip to improve locaiization of the magnetic field, with the tip used fbr generating the magnetic field maxima on the microfluidic device. Magnetic beads are moved into region of the microchip capable of performing the PCR reaction, containing the necessary PCR enzymes in an acceptable buffer, further capable of temperature cycling.

EXAMPLE 10, Use of coatings to improvie bead isolation/purification The presence or absence of i3SA coating in a microfluidic channel was assessed with respect to the CST bead clumping in a microfluidic channel without a trench.
Bead clumping refers to whether or not the magnetic beads formed a,clump such that their subsequent movement through the channei(s) of the chip was retarded. This is often a predictor of a successful downstream PCR reaction. It is hypothesized, though not necessary to practise the present invenfion, that bead clumping increases separation time which results in increased diffusion of contaminants from the input well to the sample collecting well or PCR
reaction chamber. The mock control utilized was purified water.

The average time was based on 4 different separations in each group and was started at the moment the beads were drawn into the channel from the input well under the influence of a magnetic field, and ending when the beads entered the collection well. It is important to note that there was an obvious difference in the time needed for the beads to collect under the extemal magnet in the input well when comparing the two groups, with the mock treated chips requiring more tfine (up to a minute). It was observed that a significant amount of bead clumping occurred prior to the beads leaving the input well. It was also observed that the size of the bead pellet recovered in the input well was, on average, significantly smaller in the S mock-control treated chips as compared to the BSA pre-coated chips. This was observed to result from the majority of the beads in the input well of the mock-treated chips not leaving the input'vvell at any point of the separation, making the subsequent PCR less optimal, due to having a lower concentration of DNA. .

Table 3: . Observations of pre-coating of microfluidic chips with BSA

Chip treatment Dead clumping Avg. time for Positive (pre-coating) separation PCIR
(seconds) outcomes BSA (lmg/m1) 0/20 19 20/20 Mock control 5/20 43 19/20 While particular embodiments of the present invention have been described in the foregoing, it is to be understood that other embodiments are possible within the scope of the invention and are intended to be included herein. It will be clear to any person skilled in the art that modifications of and adjustments to this invention, not shown, are possible without departing from the spirit of the invention as demonstrated through. the exemplary embodiments. The invention is therefore to be considered limited solely by the scope of the appended claims.

Claims (22)

1. A method for increasing the concentration of a molecule of interest comprising In a first area of the microfluidic device allowing a clinical sample to interact with a particle capable of being attracted by a magnetic field;

Applying a magnetic field to the microfluidic device such that said particles in the first area of the microfluidic are encouraged to move to a second area of the microfluidic device;

wherein said first area of the microfluidic device and second area of said microfluidic device are in fluid communication; and wherein the particles are suspended in a fluid medium of viscosity greater than 3 centipoise; and wherein said particle is able to preferentially capture said molecule of interest

2. The method of claim 1 wherein the viscosity of said fluid medium results from the presence of a solute which does not substantially interfere with later biochemical reactions with the molecules of interest.

3. The method of claim 2 wherein the solute is sucrose.

4. The method of claims 1, 2 or 3 wherein the particles are translocated through a microfluidic channel.

5. The method of claim 4 wherein the microfluidic channel has a deformation in the generally planar surface of the channel.

6. The method of claim 5 wherein the deformation has a width and depth of 15 to 25 times the diameter of said particle.

7. The method of claim 5 wherein the particle is of, on average, 1 micrometer diameter and the depth and width of said deformation is 25 micrometers.

8. The method of claim 5 wherein the microfluidic channels are of a width of 250 to 350 times the diameter of said particle and a depth of 35 to 65 times the diameter of said particle.

9. The method of claim 8 wherein the particle is of, on average, 1 micrometer diameter, the microfluidic channel is of 290 to 330 micrometer width and 45 to micrometers depth.

10. The method of claims 1, 2, 3, 4, 5, 6, 7, or 8 wherein the molecule of interest is a nucleic acid.

11. The method of claim 10 wherein the particle is a ChargeSwitch® bead.

12. A method for increasing the concentration of a molecule of interest comprising On a microfluidic device comprising a first and second area of the microfluidic device, with at least one microfluidic channel providing fluid communication between said first and second areas of the microfluidic device, coating said first and second areas of the microfluidic device and said at least one microfluidic channel with an agent;

In the first area of the microfluidic device allowing a clinical sample to interact with a particle capable of being attracted by a magnetic field;

Applying a magnetic field to the microfluidic device such that said particles in the first area of the microfluidic are encouraged to move to the second area of the microfluidic device;

wherein the particles are suspended in a fluid medium of viscosity greater than 3 centipoise; and wherein said particle is able to preferentially capture said molecule of interest.

13. The method of claim 12 wherein said agent is selected from the group comprised of Chlorinated organopoly siloxane, linear polyacrylamide, poly-dimethylacrylamide, 3-(trimethoxysilyl)propyl methylacrylate, 3-(trimethoxysilyl)-polyethyleneglycol (600), Bovine serum albumin, Polyvinyl pyrrolydone, Triton-X-100, Brij-35 and Tween 20.TM..

14. The method of claim 13 wherein the viscosity of said fluid medium results from the presence of a solute which does not substantially interfere with later biochemical reactions with the molecules of interest.

15. The method of claim 14 wherein the solute is sucrose.

16. The method of claim 14 wherein the microfluidic channel has a deformation in the generally planar surface of the channel.

17. The method of claim 16 wherein the deformation has a width and depth of 15 to 25 times the diameter of said particle.

18. The method of claim 17 wherein the particle is of, on average, 1 micrometer diameter and the depth and width of said deformation is 25 micrometers.

19. The method of claim 14 wherein the microfluidic channels are of a width of 250 to 350 times the diameter of said particle and a depth of 35 to 65 times the diameter of said particle.

20. The method of claim 19 wherein the particle is of, on average, 1 micrometer diameter, the microfluidic channel is of 290 to 330 micrometer width and 45 to micrometers depth.

21. The method of claims 12, 13, 14, 15, 16, 17, 18, 19 or 20 wherein the molecule of interest is a nucleic acid.

22. The method of claim 21 wherein the particle is a ChargeSwitch® bead.