JP2003532400A - Microfluidic device - Google Patents

Abstract

SUMMARY A microfluidic device for capturing non-magnetic and magnetic beads (6) is disclosed. The device has an inlet (2), an outlet (20), and a bead capture filter (4) that includes a grooved wall with a groove opening less than the diameter of the bead. The filter (4) is provided with an enlarged zone (2a) and may extend around the axis of flow (e.g. box-shaped). The device can be used in a method of sequencing by synthesis.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to microfluidic devices, and in particular, but not exclusively, to microfluidic devices for manipulating microbeads.

BACKGROUND OF THE INVENTION Microspheres, also known as beads, are routinely used as a mobile solid phase for the separation, synthesis and detection of molecules in medical diagnostics, microbiology, applied research, immunology and molecular biology. . The homogeneity of the beads and their precisely defined size ensure that each bead has the same chemical and physical properties. Beads are available in several different materials and sizes (several nanometers to millimeters in diameter). The surface chemistry of the beads can be modified with a variety of functional groups that make the beads hydrophobic, hydrophilic, fluorescent, active towards specialized ligands that bind to proteins.

In order to carry out and detect chemical reactions on the beads, the beads must be limited to a certain volume. Microfluidic devices for manipulating microspheres, to date, have predominantly included magnetic microspheres, primarily focusing on magnetically active separation. For example, P. T
elleman, U.S.A. Larsen, J .; Philip, G.M. Blankens
tein and A. Wolf's Cell sorting in microfl
uidic systems, Micro Total Analysis Sys
See tems '98, Banff Canada, Oct 13-16, 1998, pp 39-44.

Paramagnetic beads (ie, magnetic (Fe ₃ O ₄ ) cores sealed with polymer shells) are used exclusively today. This is because they can be easily separated by applying an external magnet. However, the magnetic principle is not always advantageous in Micro Total Analytical System (μ-TAS) applications. The external magnetic system complicates accurate handling,
The result is a bulky system. Incorporation of magnetic components at the wafer level is also a very complicated process.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an improved method of manipulating both non-magnetic and magnetic beads.

[0006] From a first aspect, the invention relates to a predetermined nominal size or range size of 1
A microfluidic reactor that captures the above particles
and a reaction zone having an enlarged cross-sectional area as compared to the inlet, the reaction zone including a filter means having a number of holes defined by the filter means, the hole comprising: Is smaller than the nominal size or range of sizes and is arranged to capture the particles while fluid flows from the inlet through the filter means.

In contrast to closed systems such as microtiter plates, the through-flow according to the invention (
By providing a through-flow) system, the reaction proceeds more efficiently and can be detected more accurately because there is no accumulation of by-products with the progress of the reaction. Furthermore, it will be understood by those skilled in the art that, according to the present invention, the through-flow passages of the microfluidic reactor are widened where a filter is placed to capture particles. This means that more holes can be provided for a given hole size compared to prior art arrangements. This is beneficial in reducing the tendency of the filter to become clogged and promotes a uniform flow of liquid through the filter by the liquid. This miniaturizes many of the experiments, assays, etc. currently performed in test tubes, microtiter plates, etc., and increases the possibility of utilizing available combinations of beads, etc.

Furthermore, the reagent fluid may flow through the device without interfering with the particles or their surface functional groups, so the device may be used to perform multi-step reactions in one location.

DETAILED DESCRIPTION OF THE INVENTION The enhanced flow characteristics, especially the reduced tendency of clogging, that can be achieved by the present invention require that the fluid be filtered (not required during the reaction itself). Those skilled in the art should understand that it is useful at any stage of the process. Therefore, some preferred applications of the device according to the invention include flowing the reaction fluid over the particles in a through-flow process, the device being used for “stop-flow” measurements without significant flow of the reaction fluid into the filter. It is likewise possible to be used. In such a case, the improved fluid properties described above may be achieved when the filter is loaded with particles using a fluid in which the particles are suspended and / or a washing step is performed between each step of the multi-step reaction. It is also useful when

A presently preferred application of the invention is for reactions in which the analyte is immobilized on certain beads, and the device according to the invention is used in bead-free applications (eg cell-cell separation, cell transformation). It is understood that it can also be used for sex testing and particle filtration [6,7]).

The filter means may extend laterally across the reaction zone (ie orthogonal to the direction of liquid flow). However, preferably the filter means extends around the flow axis (i.e. at least to some extent in a plane, the normal of which is not parallel to the flow axis). This is beneficial because it enhances the concentration of trapped particles in a smaller space and helps detect the reaction. This is important, for example, if the reaction is to illuminate. This is because it increases the intensity and thus facilitates the measurement of potentially low levels of light. In a particularly preferred embodiment, the filter means extends substantially completely around the flow axis so as to form a porous reaction chamber. This allows the particles to be entrapped in a compressed array while providing good porosity for the fluid to pass through. Preferably, the chamber is shaped to match the shape of the reaction monitor. Thus, in one preferred embodiment, for example, the enclosure is substantially rectangular to fit the rectangular shape of the charge coupled device. Of course, an array of such devices can be used where the arrays have different overall shapes. Also, the charge coupled device need not be rectangular (eg, if the chamber is also preferably hexagonal, then hexagonal). In another possible embodiment, the chamber is circular or partially or substantially spherical.

Confining particles in a porous reaction chamber that conforms in shape to the detector is
It is understood to be beneficial in its own right, regardless of flow characteristics. Thus, in a further aspect, the invention comprises a porous reaction chamber that captures one or more particles in the reaction chamber, and a reaction monitoring means arranged to monitor the particles captured in the reaction chamber. A reactor is provided in which the reaction chamber is arranged to substantially conform in shape to the reaction monitoring means.

The holes may be of any suitable size or shape, so long as they are smaller in at least one dimension than the size of a predetermined nominal size or range of particles. It should be understood that if the particles are not spherical, it is the smallest dimension that is chosen to represent the "size" of the particles. An important criterion is that particles do not pass and are trapped by the holes. Preferably the holes are elongated and most preferably rectangular. This is the smallest "dead space" (ie,
Low flow or no flow) promotes substantially lateral flow.

A hole is defined as an opening in a wall or between elements of a mesh, but is preferably a large number of preferably parallel parallel, separate wall elements (walls).
limited to one element). Such wall elements preferably extend perpendicular to the substrate to form pillars.

The opening according to the invention may be open on one side, but is preferably closed to prevent contamination. In a preferred embodiment, at least the reaction zone of the device is closed with a substantially transparent cover. This makes it possible to monitor reactions that generate light or change the incident light (eg fluorescence obtained by irradiating particles with laser light).

The device is preferably formed on a substantially planar substrate. Also preferred is that the outlet is provided and is preferably substantially co-linear with the inlet and the reaction zone.

The device according to the invention can be manufactured using standard lithographic processing and bulk micromachining of silicon. Preferably, strong reactive ionic corrosion (deep)
A mask processing method including reactive inonic etching) is used. Most preferably, a two-step method is used in which each surface is formed from a substantially planar substrate.

Although a wide variety of different micro-total analysis systems have been described [4], efficient standard interconnections between these devices and the macroscopic world are not yet available [5]. It is a further object to provide an improved method of forming such an interconnect, and according to a further invention disclosed herein, a method of coupling an outer tube to a channel in a microreactor. And having a guide member on the channel,
A method is provided that includes moving the tube along a guide member and coupling the end of the tube to the mouth of the channel.

The bonding is preferably accomplished by heating the mouth of the channel to partially melt the ends of the tube. In a preferred embodiment, the bond strength is enhanced by applying an adhesive around the bottom surface of the tube. In the preferred embodiment where the channels are corroded in silicon glass and the tubing is polyethylene, the adhesive is an epoxy adhesive.

Preferably, the mouth of the channel is roughened prior to connecting the tube thereto.
This increases the strength of the bond.

The device of the present invention can be used for many different purposes. A preferred use of the device is in the analysis or sequencing of nucleic acids (ie DNA, RNA or cDNA).
A particularly preferred nucleic acid sequencing application is sequencing by synthesis (s).
(equencing-by-synthesis). Any suitable method of detecting nucleotide incorporation for sequencing by synthetic reactions can be used.
That is, the use of fluorescently labeled nucleotides, colorimetric detection, the use of radioactive labels, or the use of enzyme detection systems. Most preferably, the device is used in Applicants' PyroSequencing ™ sequencing by synthesis technology, which is described in full detail in WO98 / 13523. Therefore, it is important that the analyte in question contains nucleic acids (ie DNA fragments) and the liquid contains nucleotides. Preferably, the nucleic acid fragment is single stranded. The device according to the invention is particularly useful in such a technique. Because these techniques ensure that the different nucleotides are periodically injected onto the particles (preferably microbeads such as Dynabeads ™ (available from Dynal Biotech ASA of Norway)) and the nucleotides are the same. Because it stays in position and is therefore easy to monitor in the generation of light associated with the technique.

In the context of this application, the term nucleotide is understood to include deoxy and dideoxynucleotide triphosphates (dNTPs and ddNTPs).
In addition, dNTPs and ddN, which are normally incorporated into the growing DNA strand by polymerase.
Also included are analogs of TP.

Indeed, the use of a through-flow system for such a technique is novel and inventive in its own right, and thus in a further aspect, the present invention provides a filter provided in a through-flow channel. Capturing a large number of bead-immobilized targets on the means, passing at least one nucleotide through the capture beads,
A method for synthetic sequencing is provided.

Certain preferred aspects of the invention are described below, by way of example only, with reference to the accompanying drawings.

Examples Looking at FIGS. 1a to 1c, three different schematic filters and channel arrangements can be seen. FIG. 1a shows a known filter arrangement in which a planar filter A is placed laterally over a uniform channel B. This arrangement tends to clog when beads C are packed, thus impeding the free flow of liquid into the channels.

FIG. 1b shows a filter arrangement according to the invention. In this embodiment, the inlet channel 2 is open in a zone 2a with an enlarged cross section compared to the inlet channel, over which the filter 4'is laterally arranged. For a given size or range of sizes of particles 6 (and thus the size of the holes in filter 4 '), more holes are provided than in the arrangement of Figure 1a. This means that compared to FIG.
The tendency of the filter 4'to become clogged is significantly reduced, resulting in more uniform flow characteristics.

The arrangement in FIG. 1b is an improvement over the arrangement in FIG. 1a in terms of fluid flow characteristics. However, in some situations there may be the disadvantage that the beads 6 are more spread out than in the FIG. 1a arrangement (eg if the reaction taking place on the bead surface produces a low level of light).

The arrangement shown in FIG. 1c overcomes this problem. In this arrangement, the filter 4 is likewise arranged in the zone 2a with enlarged cross section. However, in this embodiment, the filter 4 extends in all directions around the flow axis 8 to form a porous reaction chamber 10. This allows the beads 6 to be confined in a more densely packed area within the chamber 10, thereby providing an increased number of holes that facilitate further improved fluid flow compared to the arrangements of FIGS. 1a and 1b. E.g. increase the intensity of the emitted light.

A more detailed view of the preferred embodiment is shown in FIG. The microfluidic reactor is described with a substantially planar and rectangular substrate 2. The method of processing will be described in more detail below. The fluid and particles are introduced by a vertically arranged inlet tube,
2 extends vertically from the other surface. A similar outlet tube 16 is located at the other end of the device. The inlet tube 14 is in fluid communication with the inlet channel 2.
The inlet channel 2 opens into a reaction zone 2a of large cross section. Filter 4
Extend in all directions around the flow axis 8 to form a box-shaped reaction chamber 10. The remainder of the large zone 2a forms a drainage chamber 18 which collects the fluid that has passed through the filter 4. The drainage fluid flows to the outlet channel 16 which is similar in size to the inlet channel 2 and is in fluid communication with the outlet tube.

With reference to FIGS. 3, 4 a, 4 b and 5, various scanning electron micrograph (SEM) images of the embodiment depicted in FIG. 2 are seen. From these, the filter 4 is the substrate 12
It can be seen that it is composed of a series of horizontal and vertical pillars 22 extending vertically from the bottom of the enlarged zone 2a, which has been corroded. The spacing between pillars 22 defines holes that must be smaller than the beads or other particles that one wants to capture.

The device shown in FIGS. 2 to 5 is processed as follows. First, a 100 millimeter diameter, 535 μm thick p-doped silicon (100 off) wafer is used as the starting material. Photoresist (1.5 μm thick) is used as two corrosion masks. First, the front face is patterned and corroded with a first mask using Strong Reactive Ion Corrosion (DRIE) (British Surface Technology System) to introduce inlet channel 2, reaction chamber 10, filter 4, drain chamber 18 and Exit channel 2
Set 0. Gas switching in the DRIE method causes a wave-like pattern on the pillar 22 that can be observed in FIG. 170, 300 or 500 to seal the device
A Pyrex glass wafer of μm thickness is anodically bonded to the front side. The back surface is then patterned with a second mask in a second DRIE step to form fluid nodes for the inlet and outlet tubes 14,16. The silicon-glass stack is then cut into 9x5 mm chips.

External polyethylene (PE) tubing is used as the inlet and outlet tubes 14, 16 and is secured to the tip in a multi-step process, as shown schematically in FIGS. 6a-6d. In order to ensure good adhesion of the PE tube, a second DRIE step is used to roughen the silicon surface defined by the mask around the fluid opening (Fig. 6a).
). PE tube 14 with each opening on the tip during the tube immobilization process
, 16 are arranged using a guide wire 24 (FIG. 5b). Short heating of the silicon-glass stack 28 causes localized melting of the PE tubes 14, 16 onto the chip (FIG. 6c). The interface between the tip and the PE tubes 14, 16 is then covered with epoxy adhesive to provide additional strength to the assembly.

Several exemplary devices of the type shown in FIG. 2 were made of varying sizes. These sizes are shown in rows 1-8 of Table 1 below. Row 9 relates to a design similar to that of FIG. 1a and is included for comparison.

[0034]

[Table 1]

The size of the microfabricated structures was measured using a scanning electron microscope and compared to the original specifications. The uniformity of the filter pillar size within the reaction chamber and between different reaction chambers was evaluated.

The measured structure size was in good agreement with the original specifications. The microfabricated structures have been found to have a high degree of uniformity, indicating a uniform and reproducible processing method.

The melt-on method of fixing the outer PE tube to the chip has proven to be very simple and reliable. It provides interconnection to a robust, precisely positioned macroscopic world with low dead volume. Epoxy adhesives are believed to be important in providing robustness to the assembly.

The fluid behavior of the flow-through microfabricated element was investigated. To test the ability of the reaction chamber to collect the beads and simultaneously allow unhindered flow-through, streptavidin-coated beads of two different materials and sizes were used (ie, polystyrene beads with a diameter of 5.50 μm). (Bangs Laboratories, Indiana, USA) and magnetic Dynabeads with a diameter of 2.8 μm (Dynal Biotech ASA, Norway). The bead solution was pipetted under a standard light microscope with a 40 × objective. The beads were coated at a low concentration (10000 beads / mL) to allow detailed observation of the individual beads in the microfluidic device.A sample was collected at the outlet and adjusted under a microscope to It was confirmed that the beads did not pass through the filter.

To investigate the fluid behavior of the microfluidic device itself, bead-free water was first added. When a constant pressure of 3.0 kPa was applied at the inlet, the water flow rate was about 3.5 μL / min for all designs presented in Table 1. The flow rate is determined by measuring the velocity of the liquid column. Different filter sizes and number of pillars proved not to significantly affect water pressure drop.

Next, beads were filled in each reaction chamber. It was observed that it was easy to fill the chamber through the microscope. The beads were free to pass through the inlet channel, filling the reaction chamber from bottom to top. All designs successfully captured the 5.50 μm diameter beads. It was observed that no beads passed through the filter and therefore no beads were found in the filtered liquid at the outlet when examined under a microscope.

The smallest reaction chamber (design 1, 5, 6) has a volume of 0.5 nL and a diameter of 5.5
It contains about 50 beads of 0 μm. Liquid or gas flow (flow-t
There is no upper limit on the volume), which is important when working with very low sample concentrations. The minimum volume required to fill the device is 3.0 nL (volume of inlet channel and reaction chamber).

This time, when the reaction chamber was completely filled with beads, the water flow rate for each of designs 1-8 was about 2.2 μL / min (again a constant pressure of 3.0 kPa was applied at the inlet). Met. Therefore, when the reaction chamber is filled with beads, the flow rate is about 40%.
Decrease. The different size of the reaction chamber and the number of pillars do not significantly affect the flow rate. The results are shown in Table 2.

[0043]

[Table 2] Table 2. Calculated and applied pressure drop (for constant flow rate 3.5 μL / min)
At the entrance)

Analytical calculations were performed to obtain the pressure drop over the filter in the reaction chamber without the different components (eg inlet and outlet channels) and beads. To calculate the pressure drop on the channel, Poiseuille's law
Law) was used [8]. The pressure drop on the filter is calculated as

[0045]

[Formula 1] Was calculated using. Here, ΔP is pressure loss, Q _v is volumetric flow rate, and C is friction coefficient (
96) for a right-angled cross section w >> h), μ is the hydrodynamic velocity, A is the cross section of the flow path
And D _h are hydraulic diameters [9]. Equation 1 is valid when 2 <L / D _h <50 (where L is the channel length [8]). Table 2 shows the values of pressure loss calculated in the inlet and outlet channels and the filter at a flow rate of 3.5 μL / min.
For comparison, Table 2 shows the pressure applied to achieve a similar flow rate (3.5 μL / min). The theoretical and experimental values are in good agreement. The major pressure drops were in the inlet and outlet channels, even with the smallest filter designs.

There is almost no clogging of the filter, and it can be easily removed by applying a back pressure. It is concluded that designs 1-8 are much less sensitive to clogging than design 9. This is probably due to the wider filter range of designs 1-8. Design 9 is a channel with a filter consisting of only 20 pillars, compared to designs 1-8 of 70-790 pillars.

Selectivity studies were conducted on designs 7 and 8 using a mixture of 2.8 μm and 5.5 μm beads. The smaller beads easily passed the filter, while the larger beads were effectively trapped in the reaction chamber.

Air bubbles present in the sample did not affect the performance of the device. The beads can be easily removed from the reaction chamber by applying back pressure. It can be reused after removing the beads and carefully cleaning the microfabricated flow device.

The microfabricated flow reaction chamber shown here captures both non-magnetic and magnetic beads. Non-magnetic beads have a lower density that results in improved hydrodynamic behavior in μ-TAS compared to magnetic beads.

The batch assembly process of flowing microfluidic device is simple and reproducible, only 2
It only includes a single mask and two different processing techniques. These are important factors in terms of effective production cost for parallelization and economical μ-TAS. The chip area (9 x 5 mm) was chosen to facilitate practical handling, and can be made smaller if desired. A minimum reaction chamber of 0.5 nL collects about 50 beads and can be smaller if reduced number of beads or flow volume is important.

When sealing the device, Pyrex glass of different thickness (170-500 μm) was used to allow real-time optical detection of chemical reactions on the beads in the reaction chamber. The thinnest Pyrex wafers will be used for the detection of chemical reactions that generate only a few photons. Since the sample flow does not displace the beads or functional groups on its surface, a multi-step reaction can be carried out at one point in the microassembly device, facilitating optical detection.

In a preferred application of the invention, the device shown in FIG. 2 is adapted from Applicant's Pyroseq.
used in the uencing ™ sequencing-by-synthesis technique. This technique is carried out by hybridizing a sequencing primer to a template of single-stranded nucleic acid and culturing with the enzymes DNA polymerase, ATP sulfurylase and luciferase. Specific deoxyribonucleotide triphosphates (dNTPs, nucleotides) were added to the reaction. Only when complementary to the bases in the template strand is the DNA polymerase a sequencing primer DNA.
Catalyzes the incorporation of nucleotides into the chain. This technique can also be used to detect pyrophosphate (PPi) released upon nucleotide incorporation. The released PPi is converted to ATP by ATP sulfurylase and a proportional amount of detectable light is produced by luciferase. One application of this technology is the analysis of single nucleotide polymorphisms (SNPs). In this study, two primers with different 3'ends were used for direct analysis of variable positions. One SNP position in the tumor suppressor gene (exon 4, codon 72) was selected for analysis. SN
External PCR was performed to amplify the P site. The outer PCR was performed after the inner PCR that generated a -80 bp fragment. One of the internal primers was biotinylated at the 5'end for immobilization. The biotinylated internal PCR product was immobilized on beads coated with streptavidin. Single-stranded DNA was obtained by culturing the immobilized PCR product in NaOH. Immobilized DNA
The strands were resuspended in H ₂ O and the annealing buffer was added to the single-stranded template.
Then, the solution was divided into two wells, and the primer was added to each well. Primers differ at the 3'end and are 5'-GCTGCTGGGTGC
AGGGGCCACGG-3 ', and 5'-GCTGCTGGTGCAGGGG
It has a base sequence of CCACGC-3 '. Hybridization was performed, and single-stranded DNA having an annealed primer (substrate) was captured in the reaction chamber. The reagent mixture was then added. The microfluidic device was placed on top of the CCD camera and the data obtained was analyzed.

The principle of allele-specific pyro-extension in SNP used in this study is shown in FIG. A light signal is generated when the 3'end of the primer matches the template DNA (a) and a reagent mixture containing all four bases is added to the annealed template. This signal indicates that the DNA polymerase uses bases to extend the primer and the released PPi is converted to ATP to produce light. If the 3'end of the primer does not match the template DNA (b), D
NA polymerase is unable to extend the primer and no light is generated. This principle was used to analyze the SNP at codon 72 of the p53 gene. The SNP encoded at codon 72 is the amino acid proline (CCC) or arginine (CGC).
) Corresponding to either G or C residues. These two mutants were
Analysis was performed at several independent times in a device with nL reaction chamber. In FIG. 8, the total amount of light collected is plotted against the time of allele-specific pyro-extension. The extension of the matched primer gave 5 times more light than the extension of the mismatch. A snapshot of the reaction is shown in Figure 9. here,(
(a) corresponds to the case of the match in FIG. 8, and (b) corresponds to the extension of the mismatch in FIG. For match extension, light is detected inside and outside the filter chamber. Weak signal (background level) for mismatch extension
Is detected in the filter chamber. The results presented here clearly demonstrate that SNP analysis can be performed in a microfabricated flow filter chamber.

With respect to the above description of the preferred embodiments, the flow rate of the sample is also adjustable, which is important when carrying out the chemical reaction on the beads. Efficient, unlimited flow volumes of gases and liquids can be achieved with dilute molecules or biological materials (100 copies /
<11 ml) [11]. The flow-through microfluidic reaction chamber reduces the accumulation of by-products, which results in improved reaction and detection sensitivity compared to closed systems (eg, microtiter plates).

The differences in the reaction chamber area for designs 1-8 included in this study are due to the performance of the device in the bead assay (eg, bead collection, bubble sensitivity) as long as the pillars form a mechanical barrier. , Pressure loss) is not significantly affected. By analytical calculation,
It was revealed that the largest pressure drop exists in the inlet and outlet channels. Therefore, the reaction chamber and filter area can be optimized for bead size and parameters of the chemical reaction. Filter area is important for cell-based analysis. For example, when filtering cells, cell activity, stiction
ion), and it is important that the cells pass through the filter as quickly as possible so that the destruction of the cells can be reduced [12].

In order to carry out a chemical reaction on beads in a microfluidic device, it is important that the flow resistance be kept small even when the reaction chamber is filled with beads. Otherwise, it will be difficult to flow the reactants through the reaction chamber. In the device shown herein, the flow rate is reduced by 40% when the reaction chamber is filled with beads. This is about 2 μL / mi
n, and still well within the benefit of μ-TAS.

At least the preferred embodiments of the present invention can be used for solid phase DNA sequencing, automated bead introduction using micropumps, parallelization using plastic replication techniques and device fabrication.

The filter is described as being made by independent pillars,
It is also conceivable to form a filter made of walls with slits whose gap is smaller than the diameter of the beads to be filtered. It is also possible to have some slits arranged laterally along the outer circumference of the reaction chamber. These lateral slits should be narrower than the diameter of the beads to be filtered. The pillar filter design is
Minimal "dead volumes" in flow equipment
Creates a vertical filter gap that provides substantially side-to-side flow with "(i.e., almost no volume equals almost no flow). This improves the quality of chemical and biochemical analyses. It is a very attractive property because it enhances.In addition, in order to obtain the best conditions in the detection of chemical reactions (eg when using optical detection), the beads in the reaction chamber have a "point" shape. (Eg, circular or quadratic boundaries) Filters should be as close to the plane as possible, given the lowest flow resistance and the placement of beads at “points”. A circular or quadratic-shaped reaction chamber, which is made up of walls, is derived.

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[Brief description of drawings]

[Figure 1]   FIG. 1a shows a prior art filter arrangement.   1b and 1c show different embodiments of the filter arrangement according to the invention.

2 diagrammatically shows a plan view of an embodiment of the device according to the invention using the filter arrangement of FIG. 1c.

[Figure 3]   FIG. 3 shows a scanning electron microscope (SEM) photograph of one embodiment of the device according to the invention.

[Figure 4]   4a and 4b show SEM images of one embodiment of the reaction chamber according to the present invention.

[Figure 5]   FIG. 5 shows an SEM photograph of the pillar of the reaction chamber of FIG.

6a-6d show respective steps in a method of coupling a fluid connector according to the invention disclosed herein.

FIG. 7 is an allele-specific pyro extension (pyro-ex) at SNP.
Tension) is shown.

[Figure 8]   FIG. 8 shows the total integrated light versus time.

9a and 9b show snapshots of matched and mismatched pyro-extensions, respectively.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, I N, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, P L, PT, RO, RU, SD, SE, SG, SI, SK , SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Van der Wizingaart, Wote             Le             Sweden, S-104.05.stop             Kholm, Forskerbacken 17 F-term (reference) 4B029 AA07 FA12                 4B063 QA13 QA19 QQ27 QQ28 QQ42                       QQ63 QQ89 QR07 QR08 QR32                       QR42 QR50 QR55 QS34 QX02                 4G075 AA02 AA27 AA39 AA65 BA10                       BB05 BD16 DA02 EB01 EE01                       EE05 EE12 EE13 FA01 FC04

Claims (29)

[Claims] 1. A microfluidic apparatus for trapping one or more particles of a predetermined nominal size or range of sizes, the microfluidic apparatus having an inlet and an enlarged cross-sectional area compared to the inlet. A reaction zone having a plurality of holes defined by the filter means, the holes being smaller than the nominal size or range of sizes described above, while fluid flows from the inlet through the filter means. The device being arranged to capture the particles to. 2. A device according to claim 1, wherein the filter means extends laterally across the reaction zone. 3. The apparatus of claim 1, wherein the inlet defines a flow axis and the filter means extends about the flow axis. 4. The apparatus of claim 3, wherein the filter means extends substantially completely around the flow axis to form a porous reaction chamber. 5. The apparatus of claim 4, wherein the porous reaction chamber is substantially rectangular. 6. The apparatus according to claim 4, wherein the porous reaction chamber is circular or partially or substantially spherical. 7. A device according to any one of the preceding claims, wherein the holes are defined between a number of separate wall elements. 8. The device of claim 7, wherein the wall elements are substantially parallel. 9. The apparatus according to claim 1, wherein the reaction zone is covered with a transparent cover. 10. A substantially planar substrate defining an inlet and a reaction zone,
The device according to any one of the preceding paragraphs. 11. A method for reacting a fluid with a sample immobilized on a large number of particles, wherein the particles are captured on a filter means of the microfluidic reaction device according to any one of the preceding claims, and the fluid is captured. Said method comprising passing over said particles. 12. The method of claim 11, wherein the particles comprise microbeads. 13. A method according to claim 11 or 12 comprising observing trapped particles for the visible result of the reaction. 14. The method of claim 13 including using a charge coupled device to detect light emitted from the reaction. 15. The method according to claim 11, wherein the analyte comprises single-stranded DNA immobilized on microbeads. 16. A method of sequencing by synthesis according to any one of claims 11 to 15. 17. The method of claim 11, wherein the fluid comprises nucleotides. 18. The method of claim 11, wherein the particles comprise living cells. 19. A method according to any one of claims 11 to 18, comprising flowing the fluid through the particles in a through-flow process. 20. A method according to any one of claims 11 to 18, comprising flowing liquid to particles captured by the filter means which is substantially free of liquid passing through the filter means. 21. A method of coupling an outer tube to a channel in a microreactor, comprising placing a guide member in the channel, moving the tube along the guide member, and placing the end of the tube in the mouth of the channel. A method comprising conjugating. 22. The method of claim 21, including heating the mouth of the channel to partially melt the end of the tube to achieve the bond. 23. The method of claim 21, further comprising applying an adhesive around the bottom surface of the tube. 24. The method of claim 21, comprising roughening the mouth of the channel prior to joining the tubes. 25. A microfluidic device (divi) for capturing non-magnetic and magnetic beads.
c) having an inlet, an outlet, and a bead-trapping filter, the bead-trapping filter comprising a wall having a number of grooves with openings having a width less than the diameter of the beads. apparatus. 26. The microfluidic device of claim 25, wherein the wall comprises a number of pillars spaced apart, the distance between adjacent pillars being less than the diameter of the beads. 27. The method according to claim 25, wherein the pillars are arranged to form a reaction chamber, and all the flow from the inlet to the outlet passes through the reaction chamber.
The microfluidic device according to. 28. The microfluidic device according to claim 25, 26 or 27, wherein the wall is circular or square. 29. A reaction apparatus comprising a porous reaction chamber for capturing one or more particles in the reaction chamber and a reaction monitoring means arranged to monitor the particles trapped in the reaction chamber. The reactor is arranged to substantially conform in shape to the reaction monitoring means.