DNA Displacement Assay Integrated into Microfluidic Channels

present results of a unique diagnostic assay based on the displacement of a sacrificial fluorescent indicator oligomer that allows for target DNA dete...
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Anal. Chem. 2004, 76, 3655-3659

DNA Displacement Assay Integrated into Microfluidic Channels Rebecca A. Zangmeister* and Michael J. Tarlov

Chemical Science and Technology Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899

This paper describes the development of a unique fluorescence-based DNA diagnostic microfluidic assay that does not require labeling of the target sequence prior to analysis. The assay is based on the displacement of a short sacrificial fluorescent-tagged indicator oligomer by a longer untagged target sequence as it is electrophoresed through a DNA-containing hydrogel plug immobilized in a microfluidic channel. The distinct advantages of this assay are the short sensing times, as a result of directed electrophoretic transport of target DNA to the sensing element, combined with the ability to detect nonlabeled target DNA. Several examples of DNA total analysis systems on microfluidic chips including cell lysis, PCR amplification, separation, and detection have been demonstrated.1-3 DNA hybridization detection elements have been integrated into microfluidic channels in two dimensions using chemical immobilization of probe oligomers directly onto the microchannel walls4 and in three dimensions using probe DNA functionalized microbeads5,6 and hydrogels.7 These examples of DNA hybridization detection require prelabeling of the target DNA for fluorescence detection. Here we present results of a unique diagnostic assay based on the displacement of a sacrificial fluorescent indicator oligomer that allows for target DNA detection without labeling prior to analysis. This assay is designed for real-time, diagnostic monitoring of nucleic acid solutions by the electrophoresis of oligomers through a sensing matrix, or hydrogel plug, formed in a microfluidic channel. This technique takes advantage of directed, electrophoretic transport of DNA oligomers to the sensing matrix, first reported using electroactive 2-D arrays, that results in complete analysis on the minute time scale.8,9 The assay is akin to molecular beacon technology10,11 in the sense that when a target hybridizes * Corresponding author: (e-mail) [email protected]. (1) Woolley, A. T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (2) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (3) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (4) Shamansky, L. M.; Davis, C. B.; Stuart, J. K.; Kuhr, W. G. Talanta 2001, 55, 909-918. (5) Fan, Z. H.; Mangru, S.; Granzow, R.; Heaney, P.; Ho, W.; Dong, Q. P.; Kumar, R. Anal. Chem. 1999, 71, 4851-4859. (6) Seong, G. H.; Zhan, W.; Crooks, R. M. Anal. Chem. 2002, 74, 3372-3377. (7) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (8) Heller, M. J.; Forster, A. H.; Tu, E. Electrophoresis 2000, 21, 157-164. 10.1021/ac035238v Not subject to U.S. Copyright. Publ. 2004 Am. Chem. Soc.

Published on Web 05/21/2004

with the probe sequence, a shorter integrated duplex is disrupted, resulting in a change in fluorescence and the signaling of the formation of the target-probe duplex. In the present study, the probe sequence is immobilized within a hydrogel sensing matrix within a microfluidic channel. A short, fluorescently tagged indicator sequence is hybridized with a portion of the probe sequence, forming a stable duplex under the conditions used for electrophoresis, prior to the assay. When a target sequence electrophoretically migrates into the hydrogel, it hybridizes with the entire probe sequence, thus displacing the indicator sequence, which is then electrophoresed out of the hydrogel and onto another hydrogel located downstream where it is detected. Because noncomplementary targets will not hybridize with the immobilized probe, the indicator sequence remains bound, and the noncomplementary targets electrophorese through the hydrogel sensing element with no effect, thus remaining silent. Therefore, this technique can be used as a real-time measure for the presents of a specific DNA sequence. The length of the indicator sequence was chosen with regard to molecular beacon literature, which reports that the probe sequence should be at least twice the length of the arm sequence;10 estimates of the ∆G of formation for the indicator-probe duplex versus the targetprobe duplex were also considered when this prototype assay was designed. The displacement assay described here is based on our ability to immobilize DNA probes in hydrogel matrixes within microfluidic channels using a procedure developed by Rehman and coworkers.7,12 The 5′ end of the oligomer is modified with an acrylic acid group that can copolymerize with acrylamide and bisacrylamide monomers during the polymerization of a polyacrylamide hydrogel. Spatial definition of the hydrogel plug is achieved by using a photoinitiator and a photomask to define the lateral dimensions of the resulting polymer. Spatially defined polyacrylamide hydrogel plugs, containing cross-linked ssDNA probes, have been successfully immobilized in poly(methyl methacrylate)/ polycarbonate (PMMA/PC) microfluidic channels using photoinitiated polymerization.7,13 The hydrogel plugs are surface grafted to the PMMA/PC microchannel walls using a polymer adhesion (9) Gurtner, C.; Tu, E.; Jamshidi, N.; Haigis, R. W.; Onofrey, T. J.; Edman, C. F.; Sosnowski, R.; Wallace, B.; Heller, M. J. Electrophoresis 2002, 23, 15431550. (10) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (11) Tyagi, S.; Bratu, D. P.; Kramer, F. R. Nat. Biotechnol. 1998, 16, 49-53. (12) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.; Kenney, M.; Boles, T. C. Nucleic Acids Res. 1999, 27, 649-655. (13) Zangmeister, R. A.; Tarlov, M. J. Langmuir 2003, 19, 6901-6904.

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Figure 1. Overview of displacement assay: (1) The first of two serial gel plugs is loaded with a fluorescent tagged 10-mer, complementary to the 10 bases nearest the 3′ end of the immobilized probe oligomer. (2) An unlabeled 20-mer target is electrophoresed through the system; the 10-mer is displaced due to the higher efficiency of hybridization associated with the 20-base pair duplex vs the 10-base pair target, 20-base pair probe duplex. (3) The displaced indicator is captured and detected in the second gel plug.

layer applied prior to hydrogel plug formation.13 The probe strands retain activity after immobilization and are able to form duplexes with target strands as they electrophorese through the hydrogel plug. The advantages of such a system are the high probe density that can be achieved in the hydrogel plug, and the enhanced mass transfer inherent to the microfluidic system. These attributes allow for low-concentration target scavenging.7 Our ability to create independent hydrogel plugs within a single microfluidic channel, each containing a different probe sequence, have been shown to be effective for selective multicolor, multitarget identification.7 The plugs are formed by photopolymerization of a solution containing 19:1 polyacrylamide/bisacrylamide and ss-DNA modified at the 5′ end with an acrylic acid group.7,12 Prior to DNA target introduction, the first plug is loaded with a fluorescent-tagged 10mer indicator oligomer that is complementary to half of the immobilized probe sequence (Figure 1, step 1). Labeled or unlabeled target oligomers of 20 bases are electrophoresed through the microchannel (Figure 1, step 2). The 20-mer complement of the immobilized probe sequence displaces the 10-mer indicator oligomer because of differences in the calculated ∆G of formation for each hybrid pair (-133.7 vs -47.8 kJ mol-1, respectively, calculated at 298 K/25 °C).14 The fluorescence of the displaced 10-mer is detected in the second capture plug, thus signaling the presence of the target in the analyte solution (Figure 1, step 3). A control, noncomplementary DNA sequence passes through the first plug without displacement of the indicator 10mer sequence. EXPERIMENTAL SECTION Disclaimer. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. (14) The estimated values of ∆G for each duplex formation were calculated using the module found online at http://ozone2.chem.wayne.edu/Hyther/ hytherm1main.html using 0.5 M NaCl, 25 °C, and the Fotin et al. correction factor.

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Materials and Chemicals. The two-component microchannel devices were made from PC (McMaster-Carr, Atlanta, GA) and UV-transparent Acrylite OP-4 (PMMA) (Cyro Industries, Mt. Arlington, NJ). Acrylamide/bisacrylamide (19:1) 40% solution (Sigma, St. Louis, MO), N,N,N′,N′-tetraethylmethylenediamine (TEMED, ∼99%, Sigma), riboflavin (Sigma), sodium hydroxide (J. T. Baker, Phillipsburg, NJ), sodium chloride (Mallinckrodt, Inc., Paris, KY), 10× TE buffer (pH 7.4, 100 mM Tris-HCl, 10 mM EDTA, Research Genetics, Huntsville, AL), and Fluoresbrite Polychromatic Red 1.0-µm beads, 2.67% solids (Polysciences, Warrington, PA) were used as received. The Acrydite-modified single-strand DNA probe, used as the probe oligomer in all experiments presented, (Acrydite-5′-AGG CCC GGG AAC GTA TTC AC-3′) was provided by Mosaic Technologies (Waltham, MA). The labeled and unlabeled 20-mer complementary targets (5′-[TAMRA] GTG AAT ACG TTC CCG GGC CT-3′ and 5′-GTG AAT ACG TTC CCG GGC CT-3′), 10-mer indicator oligomer (5′[6-FAM] GTG AAT ACG T-3′), and 20-mer noncomplementary target (5′-GAT GGT ACA TGA CAA GGT GC-3′) were purchased from Operon Technologies (Huntsville, AL). DNA solutions were made to a final concentration of 10 µM, unless otherwise indicated, using 18.2 MΩ‚cm water from a NANOpure UV system (Barnstead, Dubuque, IA). Microchannel Fabrication. A 248-nm excimer laser system (LMT-4000, Potomac Photonics Inc., Lanham, MD) was used to ablate ∼50-µm-wide and ∼95-µm-deep microchannels in polycarbonate, as described previously.7 The polycarbonate microchannel chip was thermally fused at 103 °C for 30 min with an Acrylite OP-4 lid containing ∼2.5-mm-diameter holes that provided fluid access to the microchannels.7 The Acrylite OP-4 PMMA was specifically chosen because it is transparent down to ∼300 nm, and allows for UV light to pass through for the photopolymerization. Microchannel Hydrogel Polymerization. Microchannel devices were surface modified with a polymer adhesion layer by placing in a UV/O3 cleaner for 15 min and immediately exposing to a solution of (3-methacryloxypropyl)trimethoxysilane for ∼3 h.13 The microchannels were then rinsed with 18MΩ‚cm water to remove unreacted silane prior to introduction of the monomeric polymerization solution. For polymerization, equivalent amounts of a solution containing 0.0012% (w/v) riboflavin, 10% (w/v) 19:1 acrylamide/bisacrylamide, 15 µM acrylamide-modified oligomer, 0.125% (v/v) TEMED, and 0.000 07% Fluoresbrite beads in 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) buffer were placed into each of the fluid reservoirs. Fluoresbrite beads (λex ) 591, λex ) 657) were used as visualization markers to minimize fluid flow during photopolymerization due to unequal volumes in the fluid reservoirs. A mask placed within the microscope created two hydrogel plugs ∼200 µm wide separated by ∼100 µm in the microchannel. The channel was illuminated with 340-380-nm light for 5 min to polymerize. Postpolymerization, the monomer solution was rinsed through the open channels on either side of the hydrogel plugs using pressure-driven flow. Excess monomeric species remaining between the two hydrogel plugs was removed by electrophoresis using an alkaline solution (1 M NaOH and 0.5 M NaCl, pH 14), followed by a neutral buffer solution containing 0.5 M NaCl and 1× TE. All microfluidic chips were filled with neutral buffer and refrigerated when not in use.

Figure 2. Fluorescence intensity measured of fluorescein-labeled 10-mer averaged over the first and second gel plugs while electrophoresing (A) with control buffer (top graph), a noncomplementary 20-mer target (top graph), and a complementary 20-mer target (bottom graph). Fluorescence time-lapsed images of the 10-mer indicator oligomer as it is displaced (B).

Electrophoresis. Platinum electrodes were placed in wells at either end of the microchannels and connected to a 40-V power supply (Sorensen). The magnitude of the current flowing through the microchannel was determined by measuring the voltage drop across a 100-kΩ resistor connected to the power supply in series with the microchannel. Typical currents measured at 25 V varied from 10 to 20 µA. Image Acquisition. Imaging of the microchannels was performed using a Leica DM LB fluorescence microscope equipped with a 10× objective, mercury arc lamp, and appropriate filters. Frame grabber software (Scion Image, Frederick, MD) connected to either a black and white (COHU CCD) or color (Sony 3CCD) CCD camera was used to capture fluorescence images. RESULTS AND DISCUSSION Displacement Assay of Unlabeled Target. Two hydrogel plugs containing immobilized probe oligomers (∼15 µM) of 20 bases were formed in series within a microchannel using a photomask. The first plug was used to preload the indicator oligomer, a 10-mer labeled with a fluorescein derivative (6-FAM) that is complementary to half of the 20-mer probe oligomer. The second plug was used to capture and concentrate the 10-mer indicator oligomer as it is displaced by the complementary 20mer target during the displacement assay (See illustration in Figure 1.). The fluorescence of the labeled 10-mer is observed using a fluorescence microscope to monitor the progress of the assay. Electrophoresis of a neutral buffer solution containing the 10mer indicator sequence was used to load the indicator onto the first of the two serial hydrogel plugs (e1 min). Care was taken to stop electrophoresis before the 10-mer saturated and exited the first plug to minimize the amount preloaded on the second plug. The polarity of the potential was immediately switched to reverse

the direction of electrophoresis of the 10-mer and was maintained for 3 min to remove any excess 10-mer located in the first hydrogel plug. After loading the first plug with the 10-mer indicator oligomer, a neutral buffer control solution containing either no oligomer or a noncomplementary 20-mer was electrophoresed through the system. The fluorescent signal from the labeled 10mer was monitored in both the first and second hydrogel plugs over the course of the experiment (Figure 2A, top graph). The fluorescence in the first plug stayed nearly constant over 4 min, a sufficient time scale for strand displacement, and was comparable to that of a control run using pure buffer. For both pure buffer and the same buffer containing a noncomplementary 20mer, there was no increase in fluorescence in the second plug, indicating that the slight decrease seen in the first plug is likely due to photobleaching, not strand migration (open circles in Figure 2A, top graph). Displacement of the 10-mer indicator oligomer was achieved by electrophoresing a solution containing a complementary 20mer through both plugs. A decrease in fluorescence in the first plug is observed with a concurrent increase in fluorescence in the second plug as the 10-mer is displaced by the unlabeled 20mer (Figure 2A, bottom graph and B). It is concluded that the 10-mer is captured, concentrated, and detected in the second plug. Note the concentrating effect reported previously7 of the hydrogel plug, a result of the retention and accumulation of complementary target oligomers in the probe containing hydrogel matrix, is illustrated well in this experiment by comparing the low fluorescence signal in the area between the two plugs versus that seen in either hydrogel plug at 1.75 min (Figure 2B). At 2.0 min, all of the indicator probe has been displaced by the 20-mer target, which has electrophoresed through the first plug, and is beginning to displace the 10-mer indicator off the second plug (as seen after 2.5 min in Figure 2A, bottom graph). The time scale of the assay Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Figure 4. Electrophoresis time required for complete displacement of the 10-mer indicator sequence by the 20-mer target at concentrations of 0.5, 5, and 10 µM.

Figure 3. Fluorescence images that illustrate the mechanism of the assay by using a tagged target. The 20-mer (red) target sequence displaces the 10-mer (green) indicator sequence, that is then captured on the second gel plug.

is only a few minutes and appears to be 100% complete; i.e., there is no detectable 10-mer remaining in the first plug. The stark contrast in the extent of displacement of the indicator sequence between the full complement target compared to the noncomplement sequence is clear. The selectivity achievable using this type of assay is currently being explored. We would anticipate that the selectivity achievable using this type of displacement assay would be comparable to the selectivity seen in the molecular beacon literature.10,11 The potential selectivity of this type of assay is great due to the many variables that can be explored including the design of the indicator sequence, DNA concentration, temperature, salt concentration, and electronic stringency.8,9 Displacement Assay of Labeled Target. To further illustrate that the mechanism of this assay is a displacement, a labeled target strand was used to track its position within the microchannel during electrophoresis of the target into the first hydrogel plug (Figure 3). In this example, the fluorescence images show that as a complementary TAMRA functionalized 20-mer target (red) is electrophoresed into the hydrogel containing the 10-mer indicator (green) it begins to displace the indicator strand. Over the course of the experiment (3.5 min), almost all of the 10-mer is displaced by the 20-mer before the 20-mer begins to electrophorese onto the second capture plug. The use of a tagged target demonstrates the displacement effect that is generated by the differences in ∆G of formation between the two competing duplexes. Regeneration of the System. The two hydrogel system can be reused multiple times by electrophoresing with a denaturing 3658 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

buffer (1 M NaOH/0.5 M NaCl) to remove the 10-mer indicator and 20-mer target sequences from the system. Next, the pH of the system is brought back to neutral by electrophoresing with a neutral buffer (0.5 M NaCl and 1× TE). Last, the 10-mer is reloaded onto the first hydrogel plug, as described previously, and is ready for a successive assay. On average, this process can be repeated up to six times on a single set of hydrogel plugs. It should be noted that the hydrogel plugs themselves have only a finite lifetime due to mechanical strain on the hydrogels.7 Over time, electroosmotic flow initiates along the microchannel wall/ hydrogel interface or through microscopic pathways that develop under electrophoretic conditions. Although the hydrogels can be covalently grafted to the microchannel walls with a silane surface pretreatment (as described in the Experimental Section),13 the hydrogels still have a finite lifetime (50 ( 10 min under a continuous applied field of 10 V/cm) due to repeated changes in pH and the electrophoretic movement of DNA (cross-linked and free) within the hydrogel plug. This could have further ramifications as to the limits of the length of target that can be successfully assayed using this technique. Effect of Target Concentration. Standard solutions of unlabeled 20-mer target were made at concentrations of 0.1, 0.5, 5, and 10 µM to examine the effect of target flux on the time scale of the displacement assay. Replicate measurements of the time required for full displacement of all 10-mer indicator sequences from the hydrogel plug were recorded for all but the 0.1 µM sample and are reported in Figure 4. Measurements at all concentrations were made using a single hydrogel that was regenerated using the previously described procedure. The current drop measured across the microchannel, with 25 V applied, was 9.6 ( 0.3 µA for all measurements. Reliable time measurements were unable to be made at the lowest concentration (0.1 µM) due to photobleaching of the fluorophore (6-FAM, a fluorescein derivative) over the time scale of the experiment (>15 min). As seen in Figure 4, the time for the displacement increases with decreasing concentration of target but does not appear to scale proportionally. If the complete displacement scaled proportionally, one would expect the time of displacement using a 0.5 µM target solution to be 20× longer than for a 10 µM sample.

Complete displacement of the indicator using the 0.5 µM target solution takes only 3× longer than for the 10 µM sample. We do not currently understand this discrepancy, but it may be due in part to the concentrating ability of the hydrogel plugs.7 Photobleaching of the fluorophore (6-FAM, a fluorescein derivative) precluded our ability to observe the displacement at the lowest concentration (0.1 µM). The lowest concentration of target for which complete displacement of all indicator sequences from the hydrogel plug was observed is 0.5 µM, using a 6-FAM fluorophore modification of the indicator sequence. A lower limit of detection could be attained using laser excitation. Considering its current stage of development, the displacement assay is most appropriate for use in conjunction with a preparatory step, such as PCR. CONCLUSIONS Fluorescence detection of an unlabeled target sequence was achieved through the development of a DNA displacement assay using two plugs of a hydrogel/DNA (20-mer probe) copolymer formed in series in a single microchannel device. A sacrificial fluorescent-tagged 10-mer indicator oligomer is preloaded onto the first of two serial hydrogel plugs in the microchannel prior to target introduction. Electrophoresis of a buffer solution containing the complement to the immobilized probe sequence results in displacement of the 10-mer indicator sequence. The displacement

event signals the presence of the 20-mer target in the analyte solution. No displacement is seen when a noncomplementary strand of equal length is electrophoresed through the system. The time scale of the displacement assay increases with decreasing concentration of 20-mer, and the current limit of detection for complete displacement is 0.5 µM. The displacement assay described here has potential to be further developed into a multiplex assay. Previous studies have shown that multiple targets can be detected in multiple hydrogel plugs formed in a single microfluidic channel, each containing a separate immobilized probe sequence.7 One can imagine a single hydrogel containing multiple sacrificial indicator oligomers, followed by a series of assay hydrogel plugs, each containing a different probe sequence, complementary to the sacrificial indicator oligomers. Efforts in this research direction, as well as selectivity and stringency studies, are under investigation. ACKNOWLEDGMENT R.A.Z. acknowledges the financial support of the NRC/NIST Postdoctoral Research Program.

Received for review October 20, 2003. Accepted March 25, 2004. AC035238V

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