Anal. Chem. 2005, 77, 7520-7527
Microfluidic Biosensor for the Serotype-Specific Detection of Dengue Virus RNA Natalya V. Zaytseva,† Richard A. Montagna,‡ and Antje J. Baeumner*,†
Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, and Innovative Biotechnologies International, Inc., Grand Island, New York 14072
The development of a microfluidic biosensor with fluorescence detection for the rapid, sensitive, and serotypespecific detection of Dengue virus is presented. The biosensor chip consists of poly(dimethylsiloxane) (PDMS) substrate with fabricated microchannels and a glass substrate used to seal the microchannels. These two substrates are packaged within a pressure-closed Plexiglas housing to provide a watertight reversible sealing at the PDMS-glass interface. The ability to reversibly seal the device permits easy disassembly and quick interchange of the device parts, which is ideal for developmental purposes. The biosensor employs a magnetic bead-based sandwich hybridization system in conjugation with liposome amplification for the specific detection of nucleic acids. The concentrations of the various biosensor components were optimized using a synthesized fragment of Dengue virus RNA. To evaluate the sensitivity of the assay, two detection systems, based on fluorescence measurements of intact and lysed liposomes, were analyzed. The entire analysis was complete within 20 min (including incubation time) with RNA detection limits of 0.125 nM and 50 pM for intact and lysed liposome detection systems, respectively. Subsequently, the biosensor was applied to the analysis of actual RNA obtained from Dengue virus serotypes 1-4. The resulting signals were compared to those obtained using standard electrochemiluminescence detection and shown to correspond perfectly with respect to serotype identification. Dengue virus, single-stranded RNA virus belonging to the Flavivirus genus of the Flaviviridae family, is transmitted to an individual by several species of Aedes mosquitoes and exists as four antigenically distinct serotypes (Dengue 1-4). Infection with any one of the four serotypes produces classic Dengue fever, an illness with mild febrile symptoms. Infection by more than one serotype increases the risk of developing lethal Dengue hemorrhagic fever/shock syndrome (DHF/DSS) caused by vast hemorrhage and capillary plasma leakage. Today Dengue virus is endemic in more than 100 countries in tropical and subtropical regions of the world. An estimated 50-100 million Dengue infections and 250 000-500 000 DHF/DSS with 1-30% mortality * To whom correspondence should be addressed. Tel.: +1-607-255-5433. Fax: +1-607-255-4080. E-mail:
[email protected]. † Cornell University. ‡ Innovative Biotechnologies International, Inc.
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occur annually with approximately 2.5 billion people worldwide at risk from Dengue virus infections.1,2 Dengue virus is difficult to control due to massive urbanization, overpopulation, continually increasing travel, and failure to maintain effective control programs against mosquito vectors. Moreover, no Dengue vaccine has been licensed yet. Therefore, fast and reliable diagnostic methods are necessary to ascertain and implement proper treatment of Dengue virus infections. The current techniques available for the detection of Dengue virus rely upon virus isolation3 or serological tests4-9 and typically require days to weeks to complete. In addition, the immunological assays are compromised by the cross-reactivity with other flaviviruses. Molecular-based diagnostic assays, such as the reverse transcription-polymerase chain reaction (RT-PCR), can provide quick and reliable results within a few hours. Traditional amplification methods such as nested or single-tube multiplex RT-PCR10,11 and real-time, automated TaqMan or Light Cycler techniques12-15 have been suggested for Dengue virus detection and quantification. Traditional RT-PCR, however, increases the chance of sample cross-contamination,10,11 and the real(1) Strengthening the implementation of the global strategy for dengue fever/dengue hemorrhagic fever prevention and control; World Health Organization: Geneva, Switzerland, 2000. (2) Gubler, D. J. In Dengue and dengue hemorrhagic fever; Gubler, D. J., Kuno, G, Eds.; CAB International: Cambridge, U.K., 1997; pp 1-22. (3) Henchal, E. A.; Putnak, J. R. Clin. Microbiol. Rev. 1990, 3, 376-396. (4) Innis, B. L.; Nisalak, A.; Nimmannitya, S.; Kusalerdchariya, S.; Chongswasdi, V.; Suntayakorn, S.; Puttisri, P.; Hoke, C. H. Am. J. Trop. Med. Hyg. 1989, 40 (4), 418-427. (5) Porter, K. R.; Widjaja, S.; Lohita, H. D.; Hadiwijaya, S. H.; Maroef, C. N.; Suharyono, W.; Tan, R. Clin. Diag. Lab. Immunol. 1999, 6 (5), 741-744. (6) Groen, J.; Koraka, P.; Velzing, J.; Copra, C.; Osterhaus, A. D. Clin. Diag. Lab. Immunol. 2000, 7 (6), 867-871. (7) Lam, S. K.; Ew, C. L.; Mitchell, J. L.; Cuzzubbo, A. J.; Devine, P. L. Clin. Diag. Lab. Immunol. 2000, 7 (5), 850-852. (8) Nawa, M.; Yamada, K. I.; Takasaki, T.; Akatsuka, T.; Kurane, I. Clin. Diag. Lab. Immunol. 2000, 7 (5), 774-777. (9) Balmaseda, A.; Guzman, M.; Hammond, S.; Robleto, G.; Flores, C.; Tellez, Y.; Videa, E.; Saborio, S.; Perez, L.; Sandoval, E.; Rodriguez, Y.; Harris, E. Clin. Diag. Lab. Immunol. 2003, 10 (2), 317-322. (10) Lanciotti, R. S.; Calisher, C. H.; Gubler, D. J.; Chang, G. J.; Vorndam, A. V. J. Clin. Microbiol. 1992, 30 (3), 545-551. (11) Harris, E.; Roberts, T. G.; Smith, L.; Selle, J.; Kramer, L. D.; Valle, S.; Sandoval, E.; Balmaseda, A. J. Clin. Microbiol. 1998, 36 (9), 2634-2639. (12) Laue, T.; Emmerich, P.; Schmitz, H. J. Clin. Microbiol. 1999, 37, 25432547. (13) Ito, M.; Takasaki, T.; Yamada, K.-I.; Nerome, R.; Tajima, S.; Kurane, I. J. Clin. Microbiol. 2004, 42 (12), 5935-5937. (14) Callahan, J. D.; Wu, S. J.; Dion-Schultz, A.; Mangold, B. E.; Peruski, L. F.; Watts, D. M.; Porter, K. R.; Murphy, G. R.; Suharyono, W.; King, C. C.; Hayes, C. G.; Temenak, J. J. J. Clin. Microbiol. 2004, 39 (11), 4119-4124. (15) Drosten, C.; Gottig, S.; Schilling, S.; Asper, M.; Panning, M.; Schmitz, H.; Gunther, S. J. Clin. Microbiol. 2002, 40 (7), 2323-2330. 10.1021/ac0509206 CCC: $30.25
© 2005 American Chemical Society Published on Web 10/22/2005
Figure 1. Recognition principle and detection schemes used in the biosensor. When a specific Dengue RNA is present (a), a sandwich is formed between the reporter probe, the RNA molecule, and the capture probe. The specific complexes are detected via intact liposomes with encapsulated fluorescent dye by means of fluorescence microscopy. Upon addition of the detergent solution (n-octyl β-D-glucopyranoside, OG), the liposomes release the fluorescence molecules (c) and the fluorescence signal is detected at the time when lysed liposomes pass the detection zone. No signal is detected in the presence of nonspecific RNA (b).
time probes can be expensive to synthesize. Moreover, due to the complex nature of the detection systems, which include reverse transcription and thermocycling steps, these methods present a challenge for miniaturization. In the past decade, microfluidic biosensor chips have been extensively developed for clinical and biological applications.16-19 The advances offered by miniaturization include high throughput of the analysis, low-cost fabrication, multiplex functionality, and portability. Recently our laboratory has reported the development of a microfluidic biosensor module with fluorescence detection for the rapid identification of pathogenic organisms and viruses.20 We have demonstrated the suitability of the poly(dimethylsiloxane) (PDMS) substrate for easy and rapid fabrication of microfluidic channels, which can be sealed to a glass substrate. Various applications of PDMS microfluidic channels are reviewed elsewhere.21 This elastomer has numerous advantages including elasticity, optical transparency, inertness, biocompatibility, and low price. The PDMS has the ability to seal reversibly and irreversibly to many materials and embed microelectronic and optical components. Some properties of the PDMS, such as surface hydrophobicity and incompatibility with high concentrations of organic solvents and limited application in commercial systems, can be (16) Figeys, D.; Pinto, D. Anal. Chem. 2000, 72 (9), 330A-335A. (17) Auroux, P.-A.; Koc, Y.; deMello, A.; Manz, A.; Day, P. J. R. Lab Chip 2004, 4 (6), 534-546. (18) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (19) Mitchell, P. Nat. Biotechnol. 2001, 19 (8), 717-721. (20) Zaytseva, N. V.; Goral, V. N.; Montagna, R. A.; Baeumner, A. J. Lab Chip 2005, 8 (5), 805-811. (21) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563-3576.
detrimental. However, its advantages and fabrication simplicity make this elastomer a material of choice in prototyping new, complex, and multifunctional microfluidic systems. The objectives of this study were to develop a microfluidic biosensor based on fluorescence detection and bead-based sandwich hybridization for the rapid detection of Dengue virus. This biosensor is based on earlier lateral-flow assays developed in our laboratory for the identification of Dengue virus serotypes.22,23 The advantage of the microfluidic biosensor in the long term will be integration into a micro total analysis system and thus integration of sample preparation and detection in an automated, portable, and relatively inexpensive setup. The analytical concept of the biosensor is illustrated in Figure 1. The biorecognition element of the biosensor consists of serotype-specific and generic DNA probes that are complementary to separate regions in Dengue viral RNA. The probes are designed so that the generic (reporter) probe can hybridize to the sequences found in all Dengue virus serotypes, while four serotype-specific (capture) probes can bind to the specific serotype only.24 The reporter probe is coupled to liposomes with encapsulated fluorescence dye, sulforhodamine B (SRB). The capture probes are immobilized on the surface of the paramagnetic beads via biotin-streptavidin conjugation. Viral RNA is amplified using (22) Baeumner, A. J.; Schlesinger, N. A.; Slutzki, N. S.; Romano, J.; Lee, E. M.; Montagna, R. A. Anal. Chem. 2002, 74, 1442-1448. (23) Zaytseva, N. V.; Montagna, R. A.; Lee, E. M.; Baeumner, A. J. Anal. Bioanal. Chem. 2004, 380, 46-53. (24) Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.
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the nucleic acid sequence-based amplification (NASBA) reaction. It exclusively amplifies single-stranded RNA molecules under isothermal conditions,25 which makes this reaction ideal for miniaturization. Liposomes with reporter probe and beads with capture probe hybridize with amplified target molecules; sandwich complexes subsequently are captured on the magnet and detected by fluorescence microscopy (Figure 1a). Liposomes contain a large number of fluorescent molecules26 and provide enormous signal amplification for each binding event. The fluorophore molecules are present inside the liposomes at relatively high concentrations and, therefore, undergo self-quenching. Detergent molecules (i.e., n-octyl β-D-glucopyranoside, OG) can disrupt the liposomes easily and release the fluorescent dye from the liposome’s core, which results in fluorophore dequenching and fluorescence signal increase. Thus, we evaluated the sensitivity of the assay using two different detection formats, based upon fluorescence measurements of intact and lysed liposomes, respectively (Figure 1a,c). The biosensor was optimized in terms of the amount of magnetic beads and liposomes used in the analysis, composition of the hybridization buffers, incubation times, and detection mechanism. EXPERIMENTAL SECTION Materials and Reagents. All general chemicals and buffer reagents were obtained from Sigma Co. (St. Louis, MO). Organic solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI). Lipids were obtained from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B was acquired from Molecular Probes Co. (Eugene, OR). OG was purchased from Alexis Corp. (Lausen, Switzerland). All oligonucleotides including cholesterol-tagged reporter probes were purchased from Qiagen (Valencia, CA). Silicone elastomer kit Sylgard-184 containing PDMS prepolymer and catalyst was obtained from Dow Corning Corp. (Midland, MI). Superparamagnetic beads (Dynabeads MyOne Streptavidin, 1 µM in diameter) were purchased from Dynal Biotech Inc. (Lake Success, NY). Stainless steel and Tygon tubing was supplied by Small Parts Inc. (Miami Lakes, FL). Syringes were purchased from Hamilton Co. (Reno, ND). The Cornell Nanofabrication Facility (CNF) provided clean room facilities, chemicals, and equipment for silicon template fabrication. The Plexiglas housing was constructed in the machine shop located at the School of Chemical and Biomolecular Engineering, Cornell University. Device Fabrication. A 4-in. silicone wafer with a positive surface relief was used as a mold master for the production of PDMS replicas. The silicon master’s pattern was obtained by employing standard photolithography and dry etching techniques described previously.27 Detailed protocols for the preparation of PDMS replicas are given in ref 20. Final thickness of the PDMS replica with designed topographical layout (Figure 2) was ∼170 µm. The channel network had the dimensions of ∼50 µm in depth and ∼100 and ∼500 µm in width. The microfluidic channels fabricated in PDMS were reversibly sealed by a glass coverslip (Figure 3). The slip was cut out from a microscope slide and before sealing cleansed with chromic acid (25) Compton, J. Nature 1991, 360 (6313), 91-92. (26) Locascio-Brown, L.; Plant, A. L.; Horvath, V.; Durst, R. A. Anal. Chem. 1990, 62, 2587-2593. (27) Kwakye, S.; Baeumner, A. Anal. Bioanal. Chem. 2003, 376 (7), 10621068.
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Figure 2. Typical channel layout with dimensions in micrometers. The enlarged area adjacent to the outlet hole (500 µm in width) is the detection zone; two channels, 100 µm wide, with inlets 1 and 2, are used for introduction of materials.
Figure 3. Assembly of the microfluidic device. The PDMS-glass plate structure is held together by applying a slight pressure using a housing consisting of two Plexiglas plates and four to eight screws.
cleaning solution. (Caution! The dichromate should be handled with extreme care because it is a powerful corrosive and carcinogen.) A leak-proof sealing between PDMS and glass substrates was achieved by applying slight pressure. For this purposes, two Plexiglas plates and four to eight screws were used as shown in Figure 3. To facilitate the introduction of materials, stainless steel tubing (with an outer diameter of 0.51 mm and an inner diameter of 0.25 mm) was glued into one of the Plexiglas plates at the locations lined up with the inlet and outlet holes of the PDMS film. In addition, the plate had a well for accommodation of a permanent magnet required for capturing magnetic beads in the detection zone. The rare-earth neodymium-iron-boron magnet (Grade N40, National Imports, Inc.) was placed at a distance of 270 µm from the upper wall of the microchannel. A photograph of the integrated device is shown in Figure 4. DNA Oligonucleotides. The experiments for optimizing the bead and liposome amounts and determining the limit of detection for the intact and lysed liposome detection systems were performed using a synthetic target sequence comprising the binding regions in Dengue virus serotype 3 (Table 1). NASBA-amplified RNA sequences of all four Dengue virus serotypes23 were used for the specificity analysis. The necessary biosafety level work was performed by our colleages from Advanced BioScience Laboratories. The sequences were then shipped to us for biosensor development and analysis. The DNA reporter and capture probes used have been designed earlier.24 A generic sequence was introduced into Dengue virus RNA during the NASBA reaction and, therefore regardless of the specific serotype, was present in each amplified Dengue RNA. A cholesterol-tagged (via 5′ end) generic probe complementary to this region was then used as reporter probe and was incorporated into liposomes during the liposome synthesis.28 The four serotype-specific capture probes were modified with a biotin at their 5′ end and immobilized on (28) Edwards, K. A.; Baeumner, A. J. In preparation.
Figure 4. Assembled microfluidic device used as a biosensor module. The device consists of PDMS microchannels sealed with glass substrate and is packed in Plexiglas to provide connection to the macroworld (1, 2 inlet ports; 3 outlet port; 4 magnet location). Table 1. DNA Sequences of All Oligonucleotides (Reporter, Capture, Cold Probes, Synthetic Dengue Virus Serotype 3 Sequence)a Used in the Dengue Microfluidic Biosensor name reporter probe/ generic sequence capture probes: serotype 1 serotype 2 serotype 3 serotype 4 cold probe 4 synthetic Dengue 3
DNA sequence (5′-3′) gAT gCA Agg TCg CAT ATg Ag ggg Aag CTg TAT CCT ggT ggT Aag g ATg Aag CTg Tag TCT CAC Tgg Aag g Agg gAA gCT gTA CCT CCT TgC AAA g gAg gAA gCT gTA CTC CTg gTg gAA g gAg gAA gCT gTA CTC CTg gTg gAA g TTT gTC gTA TAA CTg AgA CCC AAA CTA CgT TCC AgC gTA TAC TC AAA TCC CTT CgA CAT ggA ggA Acg TTT C
a The reporter probe was modified with cholesterol group at the 5′ end and was incorporated into the liposomes during the liposome synthesis while the capture probes were labeled with a biotin at the 5′ end and immobilized on the surface of the paramagnetic beads via biotin-streptavidin conjugation.
the streptavidin-coated superparamagnetic beads. DNA “cold probe” 4 was used to prevent the cross-reactivity of different serotypes. This is an unlabeled oligonucleotide with sequence identical to the serotype-specific capture probe 4. All probes utilized were obtained in a desalted and lyophilized form. For a better understanding of the probes, their sequences are repeated here and shown in Table 1 including their chemical modifications. Liposomes. The liposomes were prepared using a modified version29 of the reversed-phase evaporation method described by Siebert and colleagues.30 (OD532 nm ) 1.814, diluted 1:100 in osmolality adjusted phosphate-buffered saline before the OD measurements) Immobilization of Oligonucleotides on the Bead Surface. Conjugation of biotinylated capture probes to streptavidin-coated superparamagnetic beads was carried out according to the manufacturer’s protocol. Briefly, 20 µL of bead stock (10 mg/ (29) Baeumner, A. J.; Pretz, J.; Fang, S. Anal. Chem. 2004, 76 (4), 888-894. (30) Siebert, S. T. A.; Reeves, S. G.; Durst, R. A. Anal. Chim. Acta 1993, 282, 297-305.
mL) were washed two times in 2× concentrated binding and washing (B&W) buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.5) and resuspended in 18 µL of 1× concentrated B&W buffer. A 2-µL sample of DNA capture probe (300 nmol/mL) was added to the beads, and the resultant mixture was placed on a rotator for 15 min at room temperature to allow conjugation. After conjugation, the beads were washed 3 times with 1× B&W buffer and resuspended in the same buffer to a final volume of 20 µL. The beads with immobilized oligonucleotides could be stored at 4 °C for several months. Biosensor Module Operation and Assay Protocol. Fluid flow through the channel network was established by applying a positive pressure at the inlet using a syringe pump (KD Scientific Inc., Holliston, MA) and opening the outlet to atmospheric pressure. The connection between the top of the steel tubing and 500-µL Hamilton gastight syringes on the pump was made via Tygon tubing with an inner diameter of 0.5 mm. All channels were prefilled with running buffer (10% formamide, 3× SSC (1× SSC contains 15 mM sodium citrate and 150 mM sodium chloride, pH 7.0), 0.2 M sucrose, 0.2% Ficoll type 400, 0.01% Triton X-100, 10% dextran sulfate) at a slow flow rate of 1 µL/min to prevent the formation of bubbles. In a microcentrifuge tube, 1 µL of a hybridization solution (master mix) in optimal composition (60% formamide, 6× SSC, 0.15 M sucrose, 0.8% Ficoll type 400, 0.01% Triton X-100, 10% dextran sulfate), 1 µL of target sequence (DNA or RNA sample) or water (for a negative control), 1 µL of bead suspension containing 1 µg of beads, and 0.25 µL of liposomes were incubated for 15 min at room temperature in a shaker. Following the incubation, the mixture was loaded into the microfluidic channel through inlet 1 at a flow rate of 14 µL/min. The liposome-target sequence-bead complexes formed were captured by the magnet in the detection zone. After all of the beads with the specific complexes were collected on the magnet and unbound liposomes were washed away with running buffer, the fluorescence image of intact liposomes was detected as described below. For the quantification of lysed liposomes, a 30 mM OG solution was continuously injected through inlet 2 in order to lyse the liposomes completely and release the sulforhodamine B dye molecules into the microchannel. The analytical procedures required for a single biosensor assay are summarized in Table 2. The fluorescence of intact and lysed liposomes was visualized using a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany) with a setup as follows: a 10×/0.25 NA long working distance objective, the appropriate filter set (540/25 nm bandpass exciter; 620/60 nm band-pass emitter), and 100-W mercury illumination source. The images of beads in the detection zone were obtained with a digital CoolSnap CCD camera (Photometrics, Tucson, AZ) coupled to image acquisition software (Roper Scientific Inc., Tuscon, AZ). The fluorescence was quantified using Image ProExpress software (Media Cybernetics, Silver Spring, MD). RESULTS AND DISCUSSION The designed biosensor consists of PDMS substrate with embedded microchannels and a glass substrate. These two planar substrates are brought into close contact and packed in pressureclosed Plexiglas housing providing a watertight reversible sealing at the PDMS-glass interface. The reversible sealing permits easy disassembly for cleaning and interchange of the device parts. In Analytical Chemistry, Vol. 77, No. 23, December 1, 2005
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Table 2. Typical Analytical Operations Performed during Microfluidic Analysis procedure collecting beads on the magnet and washing unbound liposomes fluorescence detection of intact liposomes liposome lysis and fluorescence detection washing the channels to repeat the analysis total analysis time
time and flow rate 2 min, 14 µL/min 30 s 2 min, 0.8 µL/min 1 min, 14 µL/min 4 min for intact liposome system 6 min for lysed liposome system
Figure 5. Influence of the amount of intact liposomes (a) and beads (b) on the fluorescence signal. The amount of DNA target was kept constant at 5 fmol. Each concentration point corresponds to duplicate experiments.
addition, the sealing performed at room temperature and without the use of bonding tools/reagents allows the integration of biochemical and biological materials, delicate nanostructures, and detection systems into one biosensor chip. Otherwise, these can be easily damaged, deactivated, or change their properties under high temperature or harsh bonding conditions. Previously we have designed the experimental setup of a microbead-based sandwich nucleic acid hybridization assay in a microfluidic device and have optimized it with respect to microchannel dimensions, magnet position, and flow rates necessary for efficient capture and uniform distribution of the beads in the detection zone.20 The magnet was placed so that it could effectively capture beads from the moving fluid in a range of 1-14 µL/min and hold them in a reproducible configuration at a given flow rate. In the present study, the influence of different parameters including probe concentrations (amount of beads and liposomes), composition of the hybridization solutions, and incubation times was investigated to optimize the hybridization efficiency and maximize the sensitivity of the microfluidic biosensor, which was then applied to the detection of Dengue virus serotype 1-4 RNA and compared to standard detection technology. Optimization of Probe Concentration. The approach that is used in the microfluidic biosensor allows us to reach a lower limit of detection by optimizing different experimental conditions and theoretically is limited by the sensitivity of the microscope/ camera unit. The upper limit of detection is influenced by a variety of parameters such as the amount of liposomes and beads used in the assay and the ability of the CCD camera to record a high fluorescence signal. It was found that, due to the spatial constraints of the optical detection zone, no more than 2.5 µg of beads could 7524
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be used in the analysis. To optimize the relevant amounts of assay components, i.e., liposomes and beads, the experiments were carried out under conditions where the amount of one component was varied while the amount of the other was constant. Experiments were performed using the intact liposome detection system (i.e., liposomes were not lysed prior to detection) and hybridization buffers (master mix and running buffer) with compositions optimized previously.23 The results are summarized in Figure 5. At a constant amount of Dengue virus serotype 3 synthetic sequence (5 fmol) and bead amount of 2.5 µg (7.5 pmol of capture probe), 0.25 µL of liposomes was sufficient to label all of the captured DNA molecules (Figure 5a). The amount of capture probe was varied by the quantity of the beads while keeping the amount of target sequence (5 fmol) and reporter probe (0.25 µL of liposomes) constant. The fluorescence signal became saturated at a bead amount equal to or exceeding 1 µg (3 pmol) (Figure 5b). It should be noted that the increase in bead and liposome amounts above 1 µg and 0.25 µL, correspondingly, leads to elevated background signals, thus effectively decreasing the signalto-noise ratios. Therefore, to keep the background signals acceptably low, in all further experiments, 1 µg of magnetic beads and 0.25 µL of liposomes were used. Subsequently, a dose response curve using synthetic target DNA representing a portion of the Dengue virus 3 serotype was generated using the optimized conditions. The analyte amount was varied in the range of 0.01-25 fmol (Figure 6). The detection limit determined as three times standard deviation of the blank signal was 0.5 fmol in 4 µL of sample (0.125 nM). Comparison of Sensitivities of Intact and Lysed Liposome Detection Systems. The sensitivity of the detection system can
Figure 6. Fluorescence intensity of intact liposomes vs concentration of synthetic target DNA representing Dengue virus serotype 3. Optimized conditions of 1 µg of beads and 0.25 µL of liposomes were used. A range from 0.01 to 25 fmol of sequence was investigated in triplicates.
be improved even further with liposome lysis using a nonionic surfactant, OG. The SRB molecules encapsulated in the liposomes at high concentrations undergo self-quenching. The detergent at the critical micellar concentration causes a complete breakage of liposomes into micelles leading to a rapid release of fluorophore molecules and dequenching of fluorescence signal.31 It has been previously shown that the fluorescence signal could be significantly increased upon liposome lysis.20,32 To determine the limit of detection for the lysed liposome system, a set of additional experiments was conducted under the same hybridization conditions while injecting the detergent solution into the microfluidic device via an auxiliary channel (inlet 2, Figure 2). The limit of detection for the lysed liposome detection system was 0.2 fmol in 4 µL of sample volume (50 pM). Therefore, lysis of the liposomes provides at least a 2-fold improvement in the sensitivity of the assay. The fact that this improvement is much lower than expected can be explained by lack of a currently available detection unit to integrate the signal over the time during which the lysed liposomes pass the detection zone. Thus, most of the free fluorescence signal is lost when carrying out only a single-frame analysis. Thus, either a different fluorescence imager should be utilized or electrochemical detection can be employed, which makes the integration of the result simple, for example, by detecting Coulombs. Effect of Dextran Sulfate on Nucleic Acid Hybridization. It has been known that molecular crowding reagents, such as dextran sulfate polymer, can be used to accelerate the rate of hybridization of labeled DNA or RNA probes with nucleic acid targets.33-36 This effect is assumed to be due to the exclusion of (31) Lasch, J. Biochim. Biophys. Acta 1995, 1241, 269-292. (32) DeCory, T. R.; Durst, R. A.; Zimmerman, S. J.; Garringer, L. A.; Paluca, G.; DeCory, H. H.; Montagna, R. A. Appl. Environ. Microbiol. 2005, 71 (4), 1856-1864. (33) Ku, W. C.; Lau, W. K.; Tseng, Y. T.; Tseng, C. M.; Chiu, S. K. Biochem. Biophys. Res. Commun. 2004, 315, 30-37. (34) Storhoff, J. J.; Lucas, A. D.; Garimella, V.; Bao, Y. P.; Mueller, U. R. Nat. Biotechnol. 2004, 22 (7), 883-887. (35) Wetmur, J. G. Biopolymers 1975, 14, 2517-2524.
Figure 7. Detection of Dengue virus serotype 1 RNA in the presence (b) of dextran sulfate in the hybridization buffer (HB) and its absence (O).
probe and target molecules from the volume occupied by the polymer, which results in an increase of the local concentration of the interacting molecules. Although our hybridization buffers already contain the polymer Ficoll type 400 at an optimized concentration (i.e., at higher concentrations, the specific signal is reduced in lateral-flow assays), we investigated the addition of dextran sulfate in the microfluidic system. As can be seen in Figure 7, the addition of dextran sulfate greatly accelerates the rate of probe-target hybridization. In the presence of 10% dextran sulfate in the hybridization buffer, the maximum fluorescence signal was obtained after only 15 min of hybridization, whereas with no addition of polymer, similar fluorescence signals were only detected after as much as 1 h of hybridization. It is important that the background signal in the presence of dextran sulfate in the hybridization mixture did not increase in comparison with notarget control for the solution without any dextran sulfate (data not shown). This principle had been shown to be successful previously by Ku and colleagues.33 However, interestingly, we have not found dextran sulfate at similar concentrations to be advantageous in lateral-flow assays. We assume that this is due to the forced migration and almost two-dimensional nature of flow in the membrane-based assays versus the free flow and threedimensional interactions in a microfluidic assay. Dengue Virus RNA Assay in the Microfluidic Device. The microfluidic biosensor with optimized parameters was applied for the detection of the four Dengue virus serotypes. First, the crossreactivity pattern of Dengue virus serotypes of available strains was investigated. The amplified RNA molecule of one of the four Dengue virus serotypes hybridized with generic reporter probe coupled to liposomes and one of the specific capture probes (CP1, CP2, CP3, or CP4) immobilized on the paramagnetic beads. Thus, for the cross-reactivity analysis of one Dengue virus serotype, four successive reactions were completed. As can be seen from Figure 8a, because of the nonspecific binding of Dengue virus serotype 4 to capture probe 1, the detection of Dengue virus serotype 1 is (36) Wahl, G. M.; Stern, M.; Stark, G. R. Proc. Natl. Acad. Sci. U.S.A. 1979, 76 (8), 3683-3687.
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Figure 8. Specificity analysis of Dengue virus serotypes. All four serotypes were analyzed successively using paramagnetic beads with immobilized capture probes 1-4. The background signal (6-10 AU) obtained from the assays with no Dengue RNA was subtracted from the fluorescence signals shown. (a) No cold probe was used in the assays. (b) In the incubation mixture containing beads with immobilized CP1, 1 µM cold probe 4 was added. Table 3. Results on Decoding Blinded Samples Dengue serotype
No. of samples
No. of properly scored
percent correct
fluorescence signal
ECL values
3 4 negative 1 and 2 1 and 3 1 and 4 2 and 4 3 and 4
1 1 3 1 1 1 1 1
1 1 3 1 1 1 1 1
100 100 100 100 100 100 100 100
56 99 12; 11; 11 35 and 45 32 and 41 29 and 97 128 and 107 38 and 101
786,700 694,200 107; 43; 92 25 800 and 895 400 92 800 and 621 600 21 900 and 539 300 814 800 and 345 000 195 400 and 539 500
ambiguous whereas the detection of Dengue virus serotypes 2, 3, and 4 is very specific. In previous studies,22-24 it was found that the serotype cross-reactions could be eliminated or dramatically reduced by addition to the hybridization mixture of cold probes, which are unlabeled oligonucleotides with sequences identical to the capture probes immobilized on the test strips. The addition of 1 µM cold probe 4 into the hybridization mixture containing beads with immobilized CP1 reduced the nonspecific binding of Dengue serotype 4 to CP1 to a background signal (Figure 8b). Based on the results obtained, biosensor signals above 20 were considered positive. Identification of Coded Samples. A set of 10 coded samples was provided by our collaborators at Advanced BioScience Laboratories Inc. (ABL) and Innovative Biotechnologies International Inc. The coded samples were characterized by ABL using their standard electrochemiluminescence detection following NASBA amplification prior to shipment to our laboratory, and the code was only broken after the analyses were complete. It was found that all of the samples were decoded correctly including those containing only water (Table 3). Therefore, as shown by our blind studies, we can adequately differentiate all four serotypes of Dengue virus. The excellent results we obtained suggest that this technological approach can be exploited as a fast and reliable 7526
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diagnostic method for surveillance and proper treatment of Dengue virus infections. CONCLUSIONS The microfluidic biosensor based on the nucleic acid hybridization and fluorescence detection has been developed for the rapid, sensitive, and serotype-specific detection of Dengue virus. The assay was performed within 20 min (including incubation time) with detection limits of 0.125 nM and 50 pM for intact and lysed liposome detection systems, respectively. Comparing this to the previously developed lateral-flow assays,29,37 the sensitivity of the microfluidic assay is improved with the limit of detection 4 times lower. The addition of 10% dextrane sulfate greatly accelerated the hybridization rate without increasing the background signal. When comparing the microfluidic Dengue virus biosensor to the standard ECL detection mechanism,24 similar limits of detection are achieved. However, the potential to integrate the current module into a self-contained micro total analysis system and relatively low cost makes it a very interesting alternative to current ECL-based detection, especially in resource-limited settings of Dengue virus serotype analysis in blood samples. The (37) Hartley, H. A.; Baeumner, A. J. Anal. Bioanal. Chem. 2003, 376, 319-327.
biosensor employs a reusable microfluidic channel system that can be easily disassembled and regenerated if needed after each experiment. The developed approach based on the microfluidics and magnetic bead capture methodology can be potentially applied for the analysis of a variety of biological materials. While we have only shown the application of the microfluidic biosensor to nucleic acid detection, it can be easily envisioned to be used also for immunological and receptor-based assays by simply exchanging the reporter and capture probes with antibodies, antigens, and receptors. Several systems for RNA/DNA analysis with electrochemical detection have been developed earlier.38,39 Integrating liposome signal amplification and fluorescence detection on a single chip with microfluidic channels, we were able to achieve lower detection limits, shorter analysis time, and reduced reagent usage in comparison to the approaches used in refs 38 and 39. (38) Gabig-Ciminska, M.; Holmgren, A.; Andresen, H.; Barken, K. B.; Wumpelmann, M.; Albers, J.; Hintsche, R.; Breitenstein, A.; Neubauer, P.; Los, M.; Czyz, A.; Wegrzyn, G.; Silfversparre, G.; Jurgen, B.; Schweder, T.; Enfors, S. O. Biosens. Bioelectron. 2004, 19 (6), 537-546. (39) Gabig-Ciminska, M.; Los, M.; Holmgren, A.; Albers, J.; Czyz, A.; Hintsche, R.; Wegrzyn, G.; Enfors, S. O. Anal. Biochem. 2004, 324 (1), 84-91.
Thus, our microfluidic biosensor is a platform technology that will find numerous applications in the bioanalytical field. ACKNOWLEDGMENT The authors acknowledge financial support for this project from Innovative Biotechnologies International, Inc., from the National Institute of Allergy & Infectious Diseases (NIAID), Bethesda, MD, from the New York State CAT, Biotechnology Program at Cornell University, and from the Cooperative State Research, Education and Extension Services (NYC-123314). We thank Sutee Yoksan from Mahidol University, Thailand. and Shuenn-Jue Wu from Naval Medical Research Center, USA, for providing us with Dengue virus samples that were used in this publication. This work was performed in part at the Cornell Nanofabrication Facility (a member of the National Nanofabrication Users Network), which is supported by the National Science Foundation under Grant ECS 03-35765, its users, Cornell University, and Industrial Affiliates.
Received for review May 26, 2005. Accepted September 22, 2005. AC0509206
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