Flow-valve diagnostic microfluidic system

Abstract

A system for detecting concentration of a target in a solution where sample fluid is passed into a microchannel with wall coated with the receptor that reacts and crosslinks with the target to constrict the channel and slow or stop sample flow through the microchannel. Concentration of the target is determined by measuring length of the sample filled channel.

Claims

1. A fluidic device for measuring concentration of a target in solution comprising: a microchannel in a material, the microchannel having an inlet at a first end; at least the portion of the walls of the microchannel near the first end coated with a receptor, the receptor reactive with the target to form a cross-linked coating; the material at at least the first end of a deformable material, and the microchannel at at least the first end having a geometric cross-section, the cross-section and the deformable material such that the microchannel is deformable and constrictable by a progressing crosslinking between opposing wall surfaces where crosslinking between opposing wall surfaces deforms the microchannel to constrict the microchannel until closure of the microchannel to provide a concentration flow valve effect where flow distance of the solution in the microchannel until closure depends on concentration of the target; the microchannel and material having properties to show visual contrast between a portion of the microchannel filled with solution, and a portions of the microchannel not filled with solution.

2. A fluidic device for measuring concentration of a target in solution comprising: a microchannel in a material, the microchannel having an inlet at a first end; at least the portion of the walls of the microchannel near the first end coated with a receptor, the receptor reactive with the target to form a cross-linked coating; the material at at least the first end of a deformable material, and the microchannel at at least the first end having a geometric cross-section, the cross-section and the deformable material such that target-mediated crosslinking of receptors on opposing surfaces of the microchannel deform the cross-section to make capillary flow distance of solution in the microchannel dependent on target concentration; the microchannel and material having properties to show visual contrast between a portion of the microchannel filled with solution, and a portions of the microchannel not filled with solution.

3. The device of claim 2 wherein the cross-section of the microchannel at the first end is a geometric shape with at least two acute angles.

4. The device of claim 2 wherein the cross-section of the microchannel at the first end is semicircular.

5. The device of claim 2 wherein the device comprises one or more micropattern calibration markings for measuring length of the microchannel filled with solution.

6. The device of claim 2 wherein the deformable material comprises an elastomer.

7. The device of claim 2 wherein the deformable material comprises polydimethylsiloxane.

8. The device of claim 7 wherein the polydimethylsiloxane above the channel has a thickness between 0.4-1.0 mm.

9. The device of claim 2 wherein the deformable material comprises a fluoroelastomer.

10. The device of claim 2 wherein channel height is between 1 and 50 microns.

11. The device of claim 2 wherein channel height is between 5 and 20 microns.

12. The device of claim 2 wherein the channel length is between 10 and 1000 mm.

13. The device of claim 2 wherein the channel length is between 20 and 200 mm.

14. The device of claim 1 wherein receptor can react with at least two sites on the target.

15. A method of detecting the concentration of a target in a solution comprising: directing a solution containing the target into the inlet of a microchannel, at least the portion of the walls of the microchannel near the first end coated with a receptor, the receptor reactive with the target to form a cross-linked coating; the microchannel cross-section at at least the first end having a geometric cross-section and of a deformable material such that the microchannel is deformable and constrictable by product of target-mediated crosslinking of the receptor and target on adjacent surfaces; continuing flow of the solution into the microchannel as the crosslinked product of the receptor and product forms in the channel and deforms and constricts the channel to provide a concentration flow valve effect where capillary flow distance of solution in the microchannel depends on target concentration; measuring the concentration of the target in the solution by visually observing and measuring length of the portion of the microchannel filled with solution.

16. The method of claim 15 wherein the concentration is measured after the flow of the solution has stopped or is insignificant.

17. The method of claim 15 wherein the concentration is measured after a predetermined time of flow.

18. The method of claim 15 where viscosity of the sample is between 1 and 4 cP.

19. The method of claim 15 wherein receptor can react with at least two sites on the target.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Flow valve device fabrication and channel closure process. Microchannel and PDMS layer dimensions are not shown at scale. (A) Glass wafer (gray). (B) AZ50XT positive photoresist (black) spun on wafer. (C) Photolithographic patterning to form a mold with elevated feature using UV exposure and development with AZ400K developer. (D) Reflowing of photoresist at 150° C. for 5 min to yield a curved feature. (E) PDMS cured (80° C., 45 min) on mold. (F) Cured PDMS released. (G) Plasma bonding to enclose the microchannel. (H) Cross section zoom view of the open channel in (G), coated with receptors. (I) Zoom view (˜1000×) of the boxes in (H), showing receptors on the top and the bottom channel walls. (J) Cross section of a partially closed channel. (K) Zoom view (˜1000×) of the box in (J), showing receptor-target interaction leading to channel closure.

(2) FIG. 2. Flow-valve assay concept and data, with device schematic (top) and photograph (bottom) in each panel. Devices had biotinylated-BSA coated channels 17 μm tall and 58 μm wide, and a PDMS cover layer thickness of 0.5 mm. White arrows indicate direction of flow. (A) Open and empty channel, visible as no solution is in it. (B) Channel filled with solution lacking streptavidin, which travels the entire length of the channel without stopping, making the channel difficult to distinguish from the surrounding device. (C) 32 mm flow distance for 10 μg/mL streptavidin solution loaded in the channel. (D) 83 mm flow distance for 100 ng/mL streptavidin solution added to the channel.

(3) FIG. 3. Effect of solution viscosity on the flow distance of 100 ng/mL (X) and 1.0 μg/mL (◯) streptavidin solutions in biotin-modified 17 μm deep and 58 μm wide channels with a 0.5 mm PDMS top layer. Flow distance is only affected above a threshold viscosity of ˜2 cP, corresponding to 24% glycerol.

(4) FIG. 4. Effect of PDMS layer thickness on the flow distance for 1.0 μg/mL streptavidin in biotin-modified 17 μm deep and 58 μm wide channels. Flow distance decreases asymptotically as PDMS layer thickness is reduced.

(5) FIG. 5. Flow distance traveled as a function of streptavidin concentration in biotin-modified microchannels (58 μm wide) of two different heights, with a 0.5 mm thick PDMS top layer. (A) 13 μm deep channels. (B) 17 μm deep channels.

(6) FIG. 6. Plots of background subtracted, normalized fluorescence signal and peak area in 13 μm deep biotin-modified channels as a function of channel position and flow distance to probe constriction. (A-B) Unlabeled streptavidin (500 μg/mL) mixed with fluorescein sodium salt (80 ng/mL); signal (which scales with pathlength or channel height) is lowest near the reservoir, rising gradually to a constant level at about 6 mm flow distance. (C) Normalized peak area of unlabeled streptavidin versus flow distance is lowest near the reservoir, rising gradually to a constant level at about 6 mm flow distance. (D-E) Unlabeled BSA (500 μg/mL) mixed with fluorescein sodium salt (80 ng/mL); signal is essentially constant along the flow distance. (F) Normalized peak area of unlabeled BSA versus flow distance is approximately constant throughout the flow distance.

DETAILED DESCRIPTION

Example I

(7) Immediately after plasma bonding and oxidation, the microchannels of the flow valve devices were filled with biotinylated-bovine serum album (biotinylated-BSA, Thermo Scientific, Rockford, Ill., 2 mg/mL in 0.14 mM citrate buffer, pH 6.8) by capillary action. The biotinylated-BSA was allowed to adsorb to the channel walls for 15 min. After that time period, unadsorbed biotinylated-BSA was flushed from the channel using phosphate buffered saline (PBS, 10 mM, pH 7.2). Last, PBS was removed from the channel and 1 μL of streptavidin solution (New England Biolabs, Ipswich, Mass.) of known concentration in PBS was pipetted into the reservoir. Flow distance was recorded with a ruler and images were obtained with a digital camera.

(8) Results indicated that log.sub.10 [streptavidin] and flow distance share a linear relationship. Therefore, for a given device design, one is able to create a standard curve and subsequently determine the concentration of unknown samples by measuring flow distance.

(9) An aspect is a method that involves the capillary flow of target solution through a receptor-coated microchannel in a deformable material, which leads to channel constriction and flow stoppage due to target-receptor interaction. Importantly, in this “flow-valve” method, the distance of capillary flow is correlated with the target's concentration, and the ability to differentiate between filled and empty channels visually enables detectorless determination of flow distance, and hence target concentration. Polydimethylsiloxane (PDMS) devices have been fabricated and tested them with the model target-receptor system of streptavidin and biotin. Furthermore, three factors were studied that affect assay performance: solution viscosity, device material thickness and channel height. The concentration dependence of flow distance and assayed streptavidin solutions as dilute as 1 ng/mL has been measured. Finally, the mechanism of channel closure in these assays was evaluated. Notably, the “flow valve” approach should be adaptable to various target-receptor pairs, offering a very broadly applicable analysis method.

(10) Experimental Section

(11) Mold Design and Preparation.

(12) Molds were prepared using a 500 μm thickness, 10 cm diameter glass wafer (FIG. 1A) with spun on AZ50XT positive photoresist (AZ Electronic Materials, Branchburg, N.J.) of 5-20 μm thickness (FIG. 1B). Next, photolithography was used to transfer the serpentine design of the mask (FIG. 2A) onto the glass wafer by UV exposure followed by development in AZ400K developer (AZ Electronic Materials), resulting in elevated features of 50 μm width on the wafer (FIG. 1C). Reflowing of photoresist.sup.19 was then done at 150° C. for 5 min to round the edges of the elevated features in the mold (FIG. 1D).

(13) PDMS Device Fabrication.

(14) Devices were fabricated by casting PDMS against the positive relief mold. PDMS (Dow Corning, Centennial, Colo.) was prepared by mixing the base and curing agent in a 10:1 ratio, pouring it on the mold to a thickness of 0.45-1.1 mm (FIG. 1E), and heating to 80° C. for 45 min for curing. This PDMS was removed from the mold (FIG. 1F) and bonded to an unpatterned PDMS layer (thickness: 0.4-1.1 mm) after exposure to an oxygen plasma for 30 s.sup.20 to form a completed device with embedded channel (FIG. 1G). After plasma bonding, devices were stored with water in the channels to ensure that the surface remained hydrophilic.

(15) Procedure for Experimentation.

(16) Experiments were carried out on a biotin-streptavidin model system using the general protocols given here. The water-filled microchannel was first aspirated and then filled with biotinylated bovine serum albumin (b-BSA, Thermo Scientific, Rockford, Ill., 2 mg/mL in 0.14 mM citrate, pH 6.8) or a control solution of BSA (Sigma-Aldrich, St. Louis, Mo., 2 mg/mL in phosphate buffered saline) via capillary action. The b-BSA was allowed to adsorb to the PDMS channel walls for 15 min, leaving exposed biotin groups. Then, the b-BSA solution was removed and the channel was flushed with phosphate buffered saline (PBS, 10 mM, pH 7.2) to remove unadsorbed material. Finally, PBS was aspirated from the channel and a 1 μL streptavidin solution (New England Biolabs, Ipswich, Mass.) of specified concentration in PBS was pipetted into the reservoir (see FIG. 2C-D). The flow distance of streptavidin solution in the microchannel was measured with a ruler, and photographs were obtained with a digital camera. Some flow experiments were also carried out with streptavidin solutions having added glycerol (0-36%) to explore the influence of viscosity.

(17) Flow Restriction Mechanism.

(18) Fluorescein sodium salt (80 ng/mL, Spectrum, Gardena, Calif.) in PBS was mixed with unlabeled streptavidin or BSA as a control (both 500 μg/mL in PBS) and allowed to flow in 13 μm tall biotin-modified microchannels. Fluorescence signal was monitored using a CCD camera (CoolSNAP HQ2, Photometrics, Tucson, Ariz.) attached to an upright microscope (Axio Scope, A1, Zeiss, Thornwood, N.Y.). Illumination was provided by a 625 mW LED (MBLED, Thorlabs, Newton, N.J.) that passed through a filter cube (FITC-LP01-Clinical-OMF, Semrock, Rochester, N.Y.). Images were acquired using a 400 ms exposure time. Image acquisition and data analysis were performed using Image J software. The fluorescence signal from fluorescein in these images, integrated across the channel at different flow distances, was obtained. From these traces, background subtracted, normalized channel fluorescence signal peak areas (proportional to channel cross sectional areas) were obtained for flow solutions containing either streptavidin or BSA (control).

(19) Contact angles of streptavidin solution droplets of different concentrations on biotinylated PDMS substrates were measured using a contact angle goniometer (Rame-Hart, Succasunna, N.J.).

(20) Results and Discussion

(21) Experiments on a model system, biotin-streptavidin, were conducted to test the devices and enable their optimization. Studied were the effects on flow distance of channel height and shape, PDMS cover layer thickness, and solution viscosity. The mechanism through which channel closure affects flow was also probed. FIG. 2 shows a few examples of the data resulting from “flow valve” assays. Unfilled flow channels are easily seen in the photographs (e.g., FIG. 2A), and similarly under simple visual inspection. In contrast, microchannels containing liquid, as demonstrated in FIG. 2B, are no longer seen readily.

(22) Initial experiments were conducted on 35 mm long, 58 μm wide channels with a 1.1 mm thick PDMS cover layer. Channels with a height <5 μM usually became blocked by the flow of only water or during coating with b-BSA, either because of channel deformation due to capillary forces or due to surface crosslinking during BSA adsorption. In a revised device design with slightly taller microchannels (5.2 μm) and a PDMS cover layer thickness of 0.7 mm, a 1.0 mg/mL streptavidin solution traveled 10 mm, and a 0.88 mg/mL streptavidin solution traveled 15 mm, while solutions lacking streptavidin flowed the full length (35 mm) of the b-BSA coated channel. When glycerol was added to a 28% concentration, a solution with a streptavidin concentration of 60 μg/mL flowed 30 mm in a 5.2 μm tall channel with a 0.7 mm PDMS cover layer thickness. These experiments identified three assay parameters (channel height, cover layer thickness and solution viscosity) that could be altered to affect the dynamic range and limit of detection for “flow valve” experiments. Reproducible results for these initial device designs were still somewhat difficult to obtain, which was attributed to the above-noted blockage issues associated with relatively shallow channels.

(23) A curved channel cross-section (FIG. 1G) was found suitable for channel constriction, probably because pinching shut from the sides towards the center was possible with this geometry (see FIG. 1H-J). Flow experiments were done with curved and rectangular cross-section channels coated with biotin (both 58 μm wide and 5.2 μm tall with a 0.7 mm PDMS top layer thickness); 1.0 mg/mL streptavidin solution flowed only 10 mm in the curved cross-section channel before flow stopped, but the same solution flowed the full length of the rectangular cross-section channel. The results with this channel geometry are also consistent with published work showing that a curved channel is easier to close than a rectangular channel for valves actuated by external pressure..sup.21

(24) Building on these initial studies, Further characterization was made of the three factors that affected channel closure: solution viscosity, PDMS cover layer thickness and channel height. One parameter was varied while holding others constant and observed any effects on the flow distance. Also, taller (13-17 μm) channels were used to avoid some of the issues previously seen with shallower ones. When a higher streptavidin concentration (10 μg/mL) was introduced into a biotin-modified channel (FIG. 2C), more rapid cross-linking of the biotin anchored to the surface in the first few millimeters of the channel length led to faster constriction at the start of the channel and a shorter capillary flow distance traveled by the streptavidin solution. On the other hand, when a lower concentration of streptavidin solution (100 ng/mL) was loaded (FIG. 2D), slower cross-linking led to a greater capillary flow distance for the streptavidin solution before constriction in the first few millimeters of the channel stopped flow. A more in-depth discussion of this hypothesized mechanism of channel closure and flow stoppage is provided later.

(25) The effect of solution viscosity on the flow distance was studied with other variables held constant. Added glycerol adjusted the solution viscosity, and control solutions containing glycerol but lacking streptavidin flowed the entire length of the microchannels. FIG. 3 shows the effects of solution viscosity (1.0-3.2 cP, corresponding to glycerol concentrations of 0-36%).sup.22 on flow distance for 100 ng/mL and 1.0 μg/mL streptavidin. There was little effect on the flow distance for viscosities less than ˜2 cP (24% glycerol), but with further increases in viscosity, the flow distance decreased. Below 2 cP, the solution viscosity also had little effect on flow velocity (10-12 s to flow 80 mm, with or without glycerol). However, above 2 cP, the solution viscosity led to slower solution flow through the channel that increased the time for biotin-streptavidin interaction and closure of the first few millimeters of the channel. Thus, the distance solution traveled before channel constriction restricted flow was shorter for both concentrations of streptavidin. The 100 ng/mL solutions travelled a greater distance than the 1.0 μg/mL ones, in line with expectations. It was further found that added glycerol could be used to adjust the linear range for detection for a given microchannel length, although adding glycerol increased the assay complexity compared to flowing solution without viscosity adjustment. Indeed, flow experiments done in duplicate in glycerol-adjusted 3.0 cP solutions in 17 μm b-BSA coated channels yielded the following results: control solutions lacking streptavidin flowed 95 and 100 mm; solutions containing 1 ng/mL streptavidin flowed 56 mm and 67 mm; and 100 pg/mL streptavidin solutions (a factor of 10 lower concentration than this laboratory has been able to detect reliably in 1.0 cP buffer solutions) flowed 71 mm and 84 mm. It is also valuable to understand the viscosity dependence of flow distance in these devices for possible future work with viscous samples like blood.

(26) The effect of PDMS cover layer thickness on the flow distance was also studied with all other parameters held constant. FIG. 4 shows the influence of different PDMS top layer thicknesses on the flow distance for 1.0 μg/mL streptavidin in biotin-modified channels. The flow distance decreases at a slower rate as PDMS layer thickness is reduced, approaching an asymptote around 0.45 mm cover layer thickness. The shorter flow distances are attributed for thinner cover layers to the reduced force needed to deflect the cover layer and constrict the first few millimeters of the channel, resulting in more rapid constriction and hence shorter capillary flow time and distance. The leveling off observed approaching 0.45 mm thickness may occur because forces exerted in the channel by capillary flow itself become the dominant process in channel constriction at these shallower depths, leading to similar flow times and distances. From the data obtained, it was concluded that cover thickness allows control of the flow distance, and thus this parameter can adjust dynamic range or limit of detection.

(27) The correlation was explored between flow distance and streptavidin concentration for two different channel heights. A plot of flow distance for various streptavidin solution concentrations in biotin-modified 13 μm tall channels is given in FIG. 5A. The plot shows a linear relationship between the logarithm of streptavidin concentration and flow distance, along with a good R.sup.2 value of 0.95. A different set of experiments was carried out on biotin-modified 17 μm tall channels (FIG. 5B) and likewise showed a linear relationship with an improved R.sup.2 value of 0.98 and less data scatter than in the 13 μm tall channels. Also, improved assay sensitivity was observed for deeper versus shallower channels, with a 40% increase in the magnitude of the calibration curve slope. The linear relationship between logarithm of streptavidin concentration and flow distance across a broad swath of concentrations in different channel heights highlights the wide dynamic range for this method. The lowest quantified streptavidin concentration was 1.0 ng/mL, with flow distances of ˜100 mm for 13 μm tall channels and ˜130 mm for 17 μm tall channels, with potential to detect lower streptavidin concentrations using longer channels. This very low detection limit compared to the ˜0.2 μg/mL protein detection limits in paper-based assays.sup.17, 23 and excellent quantitation capability marks an important improvement in performance for simple, rapid and inexpensive assays.

(28) Several plausible explanations were evaluated and eliminated for the observed flow behaviors that do not involve channel constriction. Measured were contact angles of solutions of different streptavidin concentrations (1 ng/mL-100 μg/mL) on b-BSA coated PDMS to be 25-26°. Thus, the mechanism of flow stoppage is clearly not linked to concentration-dependent changes in surface wettability or tension. In addition, flow experiments on buffer solutions lacking streptavidin were performed in 13 μm tall b-BSA coated PDMS microchannels with 0.45 and 0.5 mm cover layers. These solutions flowed the entire channel length, indicating that the flow stoppage was not due to any pressure drop or channel constriction caused by capillary action. In addition, flow is unaffected by non-specific adsorption, as streptavidin solutions from 1 ng/mL-100 μg/mL) in 13 μm tall channels coated with BSA (lacking biotin) flowed the entire channel distance. Thus non-specific adsorption, which is a significant problem for conventional immunoassays, appears not to play a major role in the flow valve devices, showing promising potential for extension to other assay systems.

(29) Further explored was the mechanism of flow stoppage via channel closure using fluorescent imaging. After capillary flow of a solution containing streptavidin mixed with the unreactive small molecule marker fluorescein in a b-BSA-coated channel, the fluorescence in the first 10 mm of the microchannel was imaged to observe any differences due to constriction (FIG. 6A-B). Plots of normalized fluorescence signal across the channel at different flow distances demonstrate a significant, 3-fold increase in channel fluorescence (i.e., cross-sectional area) moving away from the solution introduction point until the signal plateaus at around 6 mm flow distance, as shown in FIG. 6C. Importantly, control experiments wherein streptavidin was replaced by BSA and similarly flowed with fluorescein (FIG. 6D-F) showed no appreciable change in channel cross-section over the same portion of the flow channel, clearly supporting a channel constriction mechanism that is specific to biotin-streptavidin interaction. It is hypothesized that once this initial portion of the channel is constricted to a sufficiently small aperture, flow stops. Thus, the capillary flow distance of the target solution depends on the time needed to close the first few millimeters of channel enough for flow to cease, which will be a function of target concentration. Hence, for future “flow valve” designs, only the first few millimeters of the channel need to be modified with receptor, and deeper channels after the constriction zone could also be used in designing assays without serpentine channels.

(30) Receptors must recognize at least two distinct sites on the target to crosslink channels. Streptavidin readily meets this criterion with four biotin binding sites. Polyclonal antibodies or two different monoclonal antibodies to a target would recognize different epitopes and should also cause receptor-mediated crosslinking of microchannels in response to an antigen target. Additionally, hybridization of a target nucleic acid sequence to complementary surface-attached single-stranded oligonucleotides should mediate microchannel closure. Are antigen-antibody or base pairing interactions strong enough to develop “flow valve” assays? The unbinding forces for target-receptor pairs have been studied by scanning probe microscopy, and were 200-300 pN.sup.25, 26 per biotin-streptavidin molecular pair. The measured unbinding force for a single antigen-antibody pair is 50-60 pN,.sup.27-29 which is less than biotin-streptavidin by a small factor of 3-6 that could likely be accommodated through adjusting device parameters. The unbinding force for hybridized DNA oligonucleotides, depending on the sequence and number of base pairs, ranges from 450 pN.sup.30 for 14-mer sequences to 2700 pN for 20-base-long hybridized pairs..sup.31 These published unbinding data affirm the likely feasibility of generalization of “flow valve” systems beyond biotin-streptavidin measurements to nucleic acid hybridization and antigen-antibody interactions.

(31) A key question regarding flow valve assays is the following: how can molecular-scale (˜10 nm) surface interactions translate into much larger, micrometer-dimension alterations in microchannel diameter that can affect flow? It is believed that the answer can be found in the data in FIG. 6, coupled with the posited channel constriction mechanism illustrated at the bottom of FIG. 1. At the edges of the microchannels, where the curved regions meet the flatter bottom segment (see FIG. 1), the channel height is at or near the ˜10 nm molecular scale, such that biotin-streptavidin crosslinking of the top surface to the bottom is possible. This interaction would pull the top and bottom surfaces incrementally closer, enabling similar molecular-scale crosslinking to occur moving inward toward the middle of the channel. As this interaction progresses, the edges of the channel would be constricted, while the middle would remain open (i.e., FIG. 1J-K). Importantly, the data in FIG. 6A-B are indicative of exactly this type of change in cross-sectional channel profile induced by streptavidin solution flow, strongly supporting the hypothesized mechanism. In further support of surface intermolecular interactions leading to channel constriction, some simple force calculations were made. A typical surface density of b-BSA molecules is 6×10.sup.16/m.sup.2,.sup.24 while the force needed to unbind one biotin-streptavidin molecular pair has been measured as 200-300 pN..sup.25, 26 Hence, the force per area exerted by biotin-streptavidin surface interactions would be 1.2×10.sup.7 N/m.sup.2, or 1740 psi, which is at least a factor of 100 greater than the 5-10 psi needed to completely close similarly shaped PDMS microfluidic valves..sup.21 Thus, it is concluded that molecular-scale interactions have sufficient force to induce channel constriction and that the occurrence of such interactions from the edges toward the centers of these microchannels is both plausible and consistent with the channel imaging data that was obtained.

(32) Conclusions

(33) Demonstrated is a detectorless microfluidic approach for quantifying target analytes through simple visual inspection of capillary flow distance in a microchannel. Identified and characterized are three important parameters (solution viscosity, PDMS cover layer thickness and channel height) that affect the flow distance in these assays for the biotin-streptavidin model system. In addition, found was a linear relationship between flow distance in biotin-modified channels and logarithm of streptavidin concentration over a 100,000-fold range of concentrations. Moreover, identified and studied is a plausible mechanism of channel constriction and how this leads to concentration-dependent flow distances. Importantly, streptavidin concentrations were measured as low as 1 ng/mL using these microsystems, demonstrating low detection limits, with potential for future improvement. “Flow valve” microfluidic devices show great promise for simplified, low cost, but high performance chemical analysis that could be extended to antigen and nucleic acid determinations. “Flow valve” systems are especially promising for POC testing due to their portability, and detectorless and label-free quantitation.

Example II

(34) Microdevices were constructed essentially as in Example I, except the receptors were antibodies attached to the PDMS surface by a silanization technique where the PDMS was modified with 3-glycidoxytrimethoxypropylsilane (COPS), to which antibodies were attached by reacting the COPS epoxy end groups with amine groups on antibodies. The microdevices were tested, and concentration of the target was determined by measurement of the distance of sample flow along the channel after a predetermined time.

Example III

(35) Microdevices are constructed essentially as in Example II, except the receptors are amine-modified nucleic acids that are reacted with the GOPS-silanized PDMS surface.

(36) While this invention has been described with reference to certain specific embodiments and examples, it will be recognized by those skilled in the art that many variations are possible without departing from the scope and spirit of this invention, and that the invention, as described by the claims, is intended to cover all changes and modifications of the invention which do not depart from the spirit of the invention.

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