Bead-Based Electrochemical Immunoassay for Bacteriophage MS2

Apr 20, 2004 - biothreat viruses, such as smallpox.3,4 MS2 is a well-characterized virus with a known nucleic acid sequence5 and crystal structure.6...
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Anal. Chem. 2004, 76, 2700-2707

Bead-Based Electrochemical Immunoassay for Bacteriophage MS2 Jennifer H. Thomas,† Sang Kyung Kim,‡ Peter J. Hesketh,‡ H. Brian Halsall,† and William R. Heineman*,†

Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, Ohio 45221-0172, and Bioengineering Program, School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic beadbased electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-β-galactosidase conjugate to another site on the virus. β-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 × 1010 viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 × 1010 MS2 particles/mL. With recent events and the rising threat of bioterrorism, the demand for simple, specific, and sensitive detection of biothreat agents has grown.1,2 Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox.3,4 MS2 is a well-characterized * To whom correspondence should be addressed. E-mail: [email protected]. † University of Cincinnati. ‡ Georgia Institute of Technology. (1) Ember, L. R. Chem. Eng. News 2001, 79, 27-32. (2) Henderson, D. A. Science 1999, 283, 1279-1282. (3) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.; MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 1999, 71, 38463852. (4) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Anal. Chem. 2003, 75, 1924-1930.

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virus with a known nucleic acid sequence5 and crystal structure.6 MS2 RNA contsists of 3569 nucleotides containing the genetic information for the A protein, coat protein, and the replicase protein.5 The 180 copies of the coat protein and 1 copy of the A protein6 encase the RNA, forming a small viral particle with a 26nm diameter.7 Immunological detection of biothreat agents has proved successful for viruses as well as for the other biothreat classes of toxins, bacteria, and spores, but improvements are needed for more sensitive and miniaturized assays.8 Miniaturization affects portability and reduces the sample volume required for an immunoassay, and fields other than national defense would benefit from miniaturized immunoassay. For example, neonatal and geriatric patients may have very little blood to give toward diagnostics, and ultrasensitive immunoassays can aid the diagnosis of diseases in earlier stages than is currently possible.9 Paramagnetic microbeads can be used as a mobile solid phase on which an immunoassay sandwich is assembled10-12 and can easily be used in small volumes to miniaturize a method. When microbeads are dispersed throughout a solution, the distance that reagents must diffuse is minimized. With these characteristics, microbeads are good candidates for use in a fluidic system,13,14 as has been demonstrated for an electrochemical immunoassay with mouse IgG as the model analyte.12 Enzyme-labeled electrochemical immunoassay has been studied extensively over the past several decades.10-12,15-22 Electro(5) Fiers, W.; Contreras, R.; Duerinck, F.; Haegeman, G.; Iserentant, D.; Merregaert, J.; Jou, W. M.; Molemans, F.; Raeymaekers, A.; Berghe, A. V. d.; Volckaert, G.; Ysebaert, M. Nature 1974, 260, 500-507. (6) Valegard, K.; Liljas, L.; Fridborg, K.; Unge, T. Nature 1990, 345, 36-41. (7) Strauss, J. H., Jr.; Sinsheimer, R. L. J. Mol. Biol. 1963, 7, 43-54. (8) Iqbal, S. S.; Mayo, M. W.; Bruno, J. G.; Bronk, B. V.; Batt, C. A.; Chambers, J. P. Biosens. Bioelectron. 2000, 15, 549-578. (9) Herberman, R. B.; Mercer, D. W. Immunodiagnosis of Cancer, 2nd ed.; Marcel Dekker: New York, 1990. (10) Wijayawardhana, C. A.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1999, 399, 3-11. (11) Wijayawardhana, C. A.; Purushothama, S.; Cousino, M. A.; Halsall, H. B.; Heineman, W. R. J. Electroanal. Chem. 1999, 468, 2-8. (12) Heineman, W. R.; Thomas, J. H.; Wijayawardhana, C. A.; Halsall, H. B.; Ridgway, T. H.; Choi, J. W.; Oh, K. W.; Ahn, C.; Dharmatilleke, S.; Medis, P.; Henderson, T. H. Anal. Sci. 2001, 17, i281-i284. (13) Miller, M. M.; Sheehan, P. E.; Edelstein, R. L.; Tamanaha, C. R.; Zhong, L.; Bounnak, S.; Whitman, L. J.; Colton, R. J. J. Magn. Magn. Mater. 2001, 225, 138-144. (14) Andersson, H.; van der Wijngaart, W.; Enoksson, P.; Stemme, G. Sens. Actuators, B 2000, 67, 203-208. (15) Weber, S. G.; Purdy, W. C. Anal. Lett. 1979, 12, 1-9. (16) Heineman, W. R.; Halsall, H. B. Anal. Chem. 1985, 57, 1321A-1330A. 10.1021/ac035503c CCC: $27.50

© 2004 American Chemical Society Published on Web 04/20/2004

Figure 1. Schematic of the bead-based immunoassay for bacteriophage MS2. Biotinylated rabbit anti-MS2 IgG (biotin-1° Ab) is attached to the streptavidin-coated microbead. Bacteriophage MS2 is sandwiched between the biotin-1° Ab and rabbit anti-MS2 IgG labeled with β-galactosidase (conjugate). The enzyme label converts p-aminophenyl-β-D-galactopyranoside (PAPG) to p-aminophenol (PAP). PAP is oxidized through a 2-electron reaction to p-quinone imine (PQI) at an electrode with an applied potential of +290 mV vs Ag/AgCl and PQI can be reduced to PAP at -300 mV.

chemical detection can be coupled with enzyme-labeled immunoassay when the enzyme product can be oxidized or reduced. For voltammetric detection, a potential is applied to a working electrode, and the resulting current is measured. An important advantage of electrochemical detection is that the instrumentation can be miniaturized, permitting detection in small volumes or integration into a microfluidic system.22 When an interdigitated array (IDA) electrode is used, a reversible redox couple is electrochemically recycled, which can provide a signal amplification that single electrode detection cannot.23,24 This study had two objectives. The first was to develop a miniaturized electrochemical immunoassay for MS2 and detect (17) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R. Clin. Chem. 1985, 31, 1546-1549. (18) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 1559-1563. (19) Kaneki, N.; Xu, Y.; Kumari, A.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1994, 287, 253-258. (20) Wijayawardhana, C. A.; Wittstock, G.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 2000, 72, 333-338. (21) Wijayawardhana, C. A.; Wittstock, G.; Halsall, H. B.; Heineman, W. R. Electroanalysis 2000, 12, 640-644. (22) Ronkainen-Matsuno, N.; Thomas, J. H.; Halsall, H. B.; Heineman, W. R. Trends Anal. Chem. 2002, 21, 213-225. (23) Aoki, K.; Morita, M.; Niwa, O.; Tabei, H. J. Electroanal. Chem. 1988, 256, 269-282. (24) Niwa, O. Electroanal. 1995, 7, 606-613.

it with rotating disk electrode (RDE) amperometry.10 The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin-coated bead, capturing the virus, and then attaching an antibody labeled with β-galactosidase to another site on the virus, as depicted in Figure 1. β-Galactosidase converted p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI was directly proportional to the concentration of antigen in the sample. For this linear relationship to exist, the enzyme reaction rate must be constant, the virus must be saturated with enzyme-labeled antibody, and the change in current must be limited by the enzyme reaction rather than by the heterogeneous electron transfer. These situations are controlled by obtaining results from the initial velocity of the enzyme reaction, working above the enzyme-labeled antibody concentration at which the virus is saturated, and applying a potential that is positive enough to produce currents in the limiting current plateau of a hydrodynamic voltammogram. The second objective was to test in a fluidic device a more sensitive detection method that was based on our previous study of planar IDA electrode detection of an electrochemical immunoassay.25,26 The immunosandwich was prepared manually on the beads before capturing the beads in the fluidic device, where a Analytical Chemistry, Vol. 76, No. 10, May 15, 2004

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trench IDA electrode monitored the enzymatic reaction. A trench IDA has generator and collector electrodes facing each other in parallel in a plane that is perpendicular to the plane of the silicon dioxide chip. As predicted by finite element modeling (FEM), a trench IDA can improve redox cycling efficiency, enhancing the current 3-fold more than a conventional coplanar IDA electrode.26 Two significant advantages of using paramagnetic microbeads to meet both of these objectives are those of size and time. An enzyme-labeled immunoassay benefits from detection in smaller volumes since the dilution of the electroactive enzyme product depends on the detection volume. Therefore, the time necessary for a detectable concentration of PAP to be formed is inversely related to the solution volume. Detection times can be significantly less when the detection step is combined with the incubation of enzyme with enzyme substrate, and with beads positioned near the electrode surface, the enzymatic reaction can be monitored continuously, yielding detection times on the order of seconds.10 EXPERIMENTAL SECTION Materials. β-Mercaptoethanol and p-aminophenyl β-D-galactopyranoside (PAPG) were from Sigma-Aldrich (St. Louis, MO). Tween-20, NaH2PO4, Na2HPO4, KCl, MgCl2, NaN3, and NaOH were from Fisher Scientific (Fair Lawn, NJ). Bovine serum albumin (BSA) fraction V powder (98+% pure) was from Boehringer Mannheim (Indianapolis, IN). Streptavidin-coated M-280 Dynabeads, 6.7 × 108 beads mL-1, of 2.8-µm diameter were from Dynal (Great Neck, NY). Rabbit anti-MS2 IgG was a generous gift from Tetracore, Inc (Gaithersburg, MD). β-Galactosidase, the conjugation of β-galactosidase to rabbit anti-MS2 IgG, and sheep anti-rabbit IgG conjugated to β-galactosidase were from American Qualex Antibodies (San Clemente, CA). Bacteriophage MS2 (1 mg/mL) was generously given by Michael Goode (Aberdeen Proving Ground, MD). Sulfo-NHS-LC-biotin, D-Salt Dextran Desalting Column, HABA(2-(4′-hydroxyazobenzenebenzoic acid), avidin, ImmunoPure o-nitrophenyl-β-D-galactopyranoside (ONPG), and SuperBlock Blocking Buffer were from Pierce (Rockford, IL). Folin’s Reagent was from ICN Biomedicals (Irvine, CA). ELISA Immunowell plates were from Dynatech Immulon (Alexandria, VA). CuSO4‚5H2O was from Mallinckrodt (St. Louis, MO). Sodium tartrate was from J. T. Baker Chemical Co. (Phillipsburg, NJ). Na2CO3 was from Thorn Smith Laboratories (Beulah, MI). Chrompure mouse IgG, whole molecule, was from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). The reaction buffer, PBS-R, consisted of 0.044 M NaH2PO4, 0.056 M Na2HPO4, 0.1 M NaCl, 0.003 M NaN3, 1% BSA (w/v), and 0.5% (v/v) Tween-20 in OrganicPure H2O. The detection buffer, PBS-D, consisted of 0.044 M NaH2PO4, 0.056 M Na2HPO4, 0.1 M NaCl, 0.01 M MgCl2 because Mg2+ is an activator of β-galactosidase, and 0.003 M NaN3 dissolved in OrganicPure H2O. Both buffers were adjusted to pH 7.4 with concentrated NaOH. PBS-B, the biotinylation buffer, was 0.044 M KH2PO4, 0.056 K2HPO4, and 0.15 M NaCl in OrganicPure H2O, pH 7.2. Antibody Biotinylation. Biotin was attached to rabbit antiMS2 IgG27 to eventually couple the antibody to the streptavidincoated beads. In a glass vial, 24 µL of 0.018 M sulfo-NHS-LC(25) Thomas, J. H.; Kim, S. K.; Hesketh, P. J.; Halsall, H. B.; Heineman, W. R. Anal. Biochem. 2004, accepted. (26) Kim, S.-K.; Hesketh, P. J.; Li, C.; Thomas, J. H.; Halsall, H. B.; Heineman, W. R. Biosens. Bioelectron. 2003, submitted.

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biotin in OrganicPure H2O was added to 900 µL of 3.2 mg/mL rabbit anti-MS2 IgG (MW ∼150 000 Da) in PBS-B to give a mole ratio of 23:1 biotin/antibody. The vial was capped, and the mixture was incubated with gentle shaking for 30 min at room temperature. The desalting column was equilibrated with 30 mL of PBS-B before injecting the protein-biotin mixture onto the column. PBS-B was used to elute the components, and 15 mL of eluent was collected in 0.5-mL fractions. The fractions containing protein, as determined by the sample’s absorbance at 280 nm, were pooled, and the solution was concentrated to a volume of 1.25 mL with ultrafiltration. The Lowry protein assay28 was used to determine the concentration of biotinylated rabbit anti-MS2 IgG. An alkaline copper solution consisted of 1 mL of 1% CuSO4‚5H2O in H2O, 1 mL of 2% sodium tartrate in H2O, and 98 mL of 2% Na2CO3 in 0.1 M NaOH. Mouse IgG standards of 0, 25, 50, 75, and 100 µg/mL in H2O were made. In a glass test tube, 3 mL of alkaline copper solution was mixed with 500 µL of mouse IgG standards or biotin-rabbit anti-MS2 IgG solution and allowed to sit at room temperature. After 10 min, 150 µL of Folin-Ciocalteau reagent was added to each tube and mixed, and the tubes sat at room temperature for 60 min. The absorbance was read at 660 nm, and the concentration of the biotinylated antibody was determined by linear regression analysis. A HABA assay was done to determine the number of moles of biotin per mole of protein.29 The absorbance at 500 nm was measured for 60 µL of HABA and 1 mg of avidin diluted to 2 mL with PBS-B. Then 100 µL of the biotinylated antibody was added to this solution, and the absorbance was measured. The molar ratio was determined from the published algorithm.29 ELISA. For ELISA experiments, 50 µL of 100 µg/mL MS2 in OrganicPure H2O was incubated in wells of the ELISA plate at room temperature with gentle shaking overnight. Each well was washed three times with OrganicPure H2O before being filled with 200 µL of SuperBlock solution. After a 30-min incubation, the wells were washed three times with PBS-R. The wells were filled with 50 µL of varying concentrations of antibody against MS2, incubated for 60 min, and washed three times with PBS-R. The incubation and wash steps were repeated until all bioreagents were added. Following the addition of the β-galactosidase-labeled antibody, 50 µL of 9 mM ONPG in 0.1 mM β-mercaptoethanol in PBS-D was pipetted into each well and incubated for 30 min. Then 5 µL of a 1 M sodium carbonate solution was pipetted into each well to stop the enzyme reaction. The absorbance at 405 nm was read with an EIA Reader (Bio-Tek Instruments, Inc., Winooski, VT). Immunoassay Procedure with RDE Detection. In a glass test tube, 95 µL of beads in PBS-R (6.7 × 108 beads/mL) was mixed with 95 µL of 80 µg/mL biotin-rabbit anti-MS2 IgG (biotin1° Ab) to give a concentration of 40 µg/mL biotin-1° Ab. The mixture incubated for 25 min at room temperature with gentle shaking before washing three times with PBS-R to remove excess biotin-1° Ab. Wash steps have been described previously in more detail.11 The beads were resuspended in 190 µL of PBS-R and (27) Hnatowich, D. J.; Virzi, F.; Rusckowski, M. J. Nucl. Med. 1987, 28, 12491302. (28) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265-275. (29) Green, N. M. Biochem. J. 1965, 94, 23c-24c.

shaken vigorously to disperse them, and then 10 µL of bead solution was distributed into each of 18 clean test tubes (3 for each MS2 concentration). The appropriate MS2 concentration was added to each tube to give 20 µL of 0, 0.25, 0.5, 0.75, 1, and 2 µg/mL MS2, and the mixtures were incubated for 10 min at room temperature with gentle shaking. A blank consisted of 10 µL of PBS-R added to the beads instead of MS2 to determine the extent of nonspecific adsorption (NSA). Each sample was washed three times with PBS-R before being resuspended in 6.7 µL of PBS-R. Ten microliters of 250 µg/mL rabbit anti-MS2 IgG-β-galactosidase (conjugate) was added directly to the test tube, and the mixture was incubated for 30 min at room temperature with gentle shaking. Each sample was washed twice with PBS-R and four times with PBS-D. Each sample was resuspended in 20 µL of PBS-D for RDE experiments. RDE experiments were done with a polished MF-2068 3-mmdiameter gold electrode (Bioanalytical Systems, Inc.), a 0.015-in.diameter Ag/AgCl wire reference electrode (A-M Systems, Inc. (Carlsborg, WA)), and a platinum wire auxiliary electrode. Amperometric detection was done at an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm. The detection of bead-based procedures used a 30-µL drop of 5.3 mM PAPG in PBS-D initially, and the potential was applied to the electrode spinning at 3000 rpm. After a 15-s quiet time, the current was measured to establish a background slope. At 30 s, 10 µL of bead solution was added to the drop. The run terminated after 70 s. The slope between 50 and 70 s was corrected by subtracting the background slope, taken between 15 and 25 s, to give the slope of the current-time plot. Fabrication of the Trench IDA Electrode. The fabrication process for comb IDAs has been described by Kim et al.26 The 4-in.-diameter (100) Si wafers patterned with photoresist and combshaped trenches, 3 µm deep, were defined using deep reactive ion etching (RIE). Thermal oxidation was preferred to chemical vapor deposition (CVD) processes because of the resulting pinhole-free structure and conformal growth rates at the flat surface and inside the trenches. Polymer (Futerrex, PC3-6000) was spin-coated to fill the trenches and provide a flat surface for metal patterning, and oxygen RIE removed excess polymer from the wafer. A negative photoresist (Futerrex, NR7 1500PY) was exposed and developed, and exposed areas of polymer in the trenches were removed. Metal was deposited by an electron beam using a wafer holder tilted relative to the source. The shadows of the trenches prevented metal from being deposited on the bottom, so the two facing electrodes were electrically disconnected. A 10nm-thick titanium layer was deposited, followed by 100 nm of platinum, and the IDA pattern was defined by lift-off of the resist. Si3N4 was selected as a passivation layer because the sensors were cleaned with acetone and an acidic solution in the final step to guarantee the electrode performance. Si3N4 (500 nm thick) was deposited by plasma-enhanced CVD at 250 °C, patterned lithographically, and etched by RIE in SF6. The smallest final gap between electrode pairs was 2.4 µm. Fabrication of Fluidic Channel. Fabrication of the flow channels was done with stereolithography and poly(dimethyl siloxane) (PDMS) molding. Stereolithography has been applied

to the packaging of sensors and other novel fluidic devices.30 The channels were drawn in the ProE program (Parametric Technology Corporation, Needham, MA) in a wire frame model, and a 3500 Rapid Prototyping machine (3D Systems, Valencia, CA) was used to build the mold. The channel mold was filled with PDMS solution (10 parts Sylgard 184 silicone elastomer base (Dow Corning, Midland, MI) to 1 part curing agent). After degassing in a vacuum desiccator overnight, the curing process took place at 60 °C for 1 h. The PDMS was easily removed from the mold, and leak-free contact was made with the silicon nitride-coated silicon wafer using finger pressure contact. Immunoassay Procedure with IDA Electrode Detection. In a glass test tube, 120 µL of beads in PBS-R (6.7 × 108 beads/ mL) was mixed with 120 µL of 80 µg/mL biotin-1° Ab. The mixture was incubated for 25 min at room temperature with gentle shaking before being washed three times with PBS-R to remove excess biotin-1° Ab. The beads were resuspended in 240 µL PBSR, shaken vigorously to disperse them, and then distributed into seven clean test tubes at 30 µL of bead solution per tube. The appropriate MS2 concentration was added to each tube to give 60 µL of 0, 0.05, 0.1, 0.25, 0.5, 1, and 2 µg/mL MS2, and the mixtures were incubated for 10 min at room temperature with gentle shaking. Each sample was washed three times with PBS-R before being resuspended in 20 µL of PBS-R. Thirty microliters of 250 µg/mL conjugate was added directly to the test tube, and the mixture was incubated for 30 min at room temperature with gentle shaking. Each sample was washed twice with PBS-R and four times with PBS-D before being resuspended in 335 µL PBSD. IDA Electrode Detection in the PDMS Fluidic Channel. Potentials of +290 and -300 mV were applied to the two working electrodes of the IDA, and the current was measured throughout the following procedure. The channel was rinsed with PBS-D at 500 µL/min for 30 s using a model M362 ThermoOrion Sage (Beverly, MA) syringe pump to control the flow before injecting 100 µL of bead solution at 50 µL/min. Then PBS-D was injected into the channel for 100 s at 50 µL/min. At 250 s, 100 µL of 4 mM PAPG was injected at 50 µL/min. The flow was stopped at 360 s. The flow resumed at 480 s, sending PBS-D through the channel at 1 mL/min, and the beads were removed from the fluidic device. The IDA was electrochemically cleaned between each sample by cycling the potential six times between +1500 and -1200 mV at 50 mV/s in the presence of PBS-D. RESULTS AND DISCUSSION Antibody Biotinylation. It was necessary to biotinylate the rabbit anti-MS2 IgG (1° Ab) in order to attach it to the streptavidincoated bead. Sulfo-NHS-LC-biotin27,31 was used as the biotinylation agent, and the biotinylated product was determined by the Lowry28 and HABA29 assays to have a molar ratio of 0.9 mol biotin/mol antibody. Although this value is lower than that predicted for the procedure (8-14 mol biotin/mol antibody),31 it is sufficient for antibody attachment, and fewer biotin per antibody minimizes the chance of biotin’s being located in the antibody-binding site. An ELISA experiment was conducted with 100 µg/mL MS2 adsorbed to the immunowell and 1° Ab or biotin-1° Ab added at concentra(30) Tse, A. L.; Hesketh, P. J.; Gole, J. L.; Rosen, D. W. Microsyst. Technol. 2003, 9, 319-323. (31) Hermanson, G. T. Pierce Chemical Company, Rockford, IL, 1996.

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tions between 0 and 75 µg/mL. An enzyme-labeled antibody, sheep anti-rabbit IgG labeled with β-galactosidase, then bound to the 1° Ab. β-Galactosidase converted o-nitrophenyl-β-galactoside (ONPG) to o-nitrophenol (ONP), and with a path length of 0.1 cm, the absorbance at 405 nm, the wavelength at which ONP absorbs light, was monitored. Plotting absorbance vs 1° Ab concentration (µg/mL) or biotin-1° Ab concentration (µg/mL) gave y ) 0.0014x + 0.017 and y ) 0.0015x + 0.010 (data not shown), respectively. It was concluded that the biotinylation of the antibody did not affect the 1° Ab’s ability to bind MS2. Then the binding of biotin-1° Ab to the streptavidin-coated beads was examined. The biotin-1° Ab was exposed to the bead in varying concentrations and then incubated with 75 µg/mL sheep anti-rabbit IgG, which recognized biotin-rabbit anti-MS2 IgG (1° Ab), labeled with β-galactosidase. The enzyme reaction was monitored with RDE amperometry.10 Between 0 and 30 s, the background slope, or change in current with time, was established for the microdrop of PAPG solution. Once beads had been added to the drop, the enzymatic conversion of PAPG to PAP began. The anodic current increased with the increasing concentration of enzymatically generated PAP, since the current resulted from oxidation of PAP to PQI at the RDE. The slope of the current-time plot increased from 7.3 nA/s at 1 µg/mL biotin1° Ab to 70.6 nA/s with 30 µg/mL biotin-1° Ab. Beyond this concentration, the slope of the current-time plot was independent of biotin-1° Ab concentration. To ensure that biotin-1° Ab was present in excess, 40 µg/mL biotin-1° Ab was chosen as the appropriate concentration. Antibody-Enzyme Conjugate. The enzyme activity of the commercially conjugated antibody, rabbit anti-MS2 IgG labeled with β-galactosidase (conjugate), was monitored by RDE amperometry. The following kinetic parameters were determined by plotting the slope of the current-time plot vs PAPG concentration and using the nonlinear least-squares fit:32 Km ) 200 µM; Vmax ) 6.9 µM PAP/s; kcat ) 430/s. It was therefore decided to use a 4 mM solution of the enzyme substrate, since the concentration was 20 times the Km value and would not limit the enzymatic reaction. Additionally, the rapid conversion of PAP from PAPG, indicated by the kcat value, made β-galactosidase a suitable choice as the enzyme label to rapidly generate detectable PAP concentrations. Beads were saturated with 1° Ab and MS2 before being exposed to conjugate concentrations between 0 and 250 µg/mL. The slope of the current-time plot, monitored with the RDE method, increased through the entire range but deviated from linearity above 150 µg/mL conjugate. Although higher conjugate concentrations gave slightly higher reaction velocities, 150 µg/ mL conjugate was the highest practical concentration, considering the stock reagent concentration and cost. Optimization of Incubation Times. Figure 2 shows how the slope of the current-time plot (nA/s) depends on the incubation times (min) for (A) beads saturated with biotin-1° Ab incubated with MS2 and (B) beads saturated with biotin-1° Ab and MS2 incubated with conjugate. The reaction between MS2 and beads occurred immediately as 96% of the maximal velocity was reached after only 1 min incubation. In the case of the conjugate, however, the slope of the current-time plot depended on the incubation (32) Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis; VCH Publishers: New York, 1996, pp 93-119.

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Figure 2. Slope of the current-time plot vs incubation time for (A) binding of MS2 to beads saturated with 1° Ab and for (B) binding of 150 µg/mL conjugate to MS2 attached to beads saturated with 1° Ab. Data were obtained with the RDE method at +290 mV vs Ag/ AgCl at 3000 rpm.

Figure 3. RDE data for the MS2 immunoassay with the following concentrations of MS2: (A) 0, (B) 0.1, (C) 0.25, (D) 0.5, (E) 0.75, (F) 1, (G) 2.5, (H) 5, (I) 10, and (J) 25 µg/mL. The RDE had an applied potential of +290 mV vs Ag/AgCl and rotated at 3000 rpm. Initially, 30 µL of 5.3 mM PAPG in PBS-D was present, and 10 µL of beads was added at 30 s.

time. Two significant regions are seen in Figure 2B. The slope of the current-time plot increases from 10.8 nA/s at 1 min to 22.0 nA/s at 30 min. Beyond 30 min, the slope of the current-time plot appears to be independent of incubation time, and therefore, 30 min was chosen as the appropriate incubation time for conjugate and beads modified with biotin-1° Ab and MS2. The parameters for the MS2 assay were chosen to be 40 µg/ mL of the biotinylated rabbit R-MS2 IgG and 150 µg/mL rabbit R-MS2 IgG*β-galactosidase conjugate. The optimal incubation time of MS2 with the beads coated with primary antibody was 10 min, and the optimal incubation time of the antibody-enzyme conjugate with the beads coated with primary antibody and MS2 was 30 min. As the incubation time for the conjugate with the antigen increases, it is possible that nonspecific adsorption will increase; however, under these conditions, minimal NSA was observed with RDE detection. MS2 Immunoassay with RDE Detection. The MS2 assay was demonstrated with MS2 concentrations between 0 and 25 µg/ mL using the RDE procedure for detection. The RDE data are seen in Figure 3, and the results were obtained in the same manner as described in the Experimental Section. The assay was limited by the detection method rather than NSA, because a significant signal was not detected for the 0 µg/mL MS2 standard. The following three regions are seen in Figure 4, a plot of slope of the current-time plot vs MS2 concentration: (A) the enzymatic reaction was not detectable over the background, (B) the slope

Figure 5. (A) Schematic of the fluidic device and (B) a picture of the fluidic device.

Figure 4. Log-log plot of slope of current-time plot vs MS2 concentration for the MS2 immunoassay with RDE detection. There are three significant regions of the plot: (A) the enzymatic reaction was not detectable over the background, (B) the slope of the currenttime plot increased linearly with increasing MS2 concentration, and (C) the beads were saturated with MS2.

of the current-time plot increased linearly with increasing MS2 concentration, and (C) the beads were saturated with MS2, so the signal was independent of MS2 concentration. A linear range was observed for MS2 concentrations between 0.25 and 2.5 µg/ mL. A plot of this region as log slope of current vs time (nA/s) vs log MS2 concentration (µg/mL) was described by y ) (1.00 ( 0.12)x + (0.81 ( 0.01). The detection limit, determined by three times the standard deviation of the blank signal, was 200 ng/mL MS2, or 3.2 × 1010 viral particles/mL. The upper limit is defined by saturation of the beads, but the lower limit can be pushed even lower with an improved detection method, by using an antibody with a higher affinity for MS2 or by using a conjugate with higher activity. If the practical considerations of cost and reagent consumption were not important, increasing the conjugate concentration may also increase the sensitivity, since the NSA signal appeared to be independent of the conjugate concentration up to 150 µg/mL. MS2 Immunoassay with IDA Electrode Detection in the PDMS Fluidic Channel. A PDMS channel was fabricated and positioned over a platinum trench IDA electrode, Ag/AgCl reference electrode, and platinum auxiliary electrode on a silicon dioxide chip. A 1.5-mm-diameter rare earth magnet was positioned beneath the trench IDA electrode on the silicon dioxide chip to hold the beads above the electrode. The PDMS channel was 300 µm deep, 500 µm wide, and 12 mm long. The injection tubing had a 0.08-cm i.d. and was 8.4 cm long; therefore, the total volume of the fluidic device was 44 µL. Figure 5 depicts the fluidic device and its schematic. The PDMS channel was designed to avoid 90° angles to minimize areas where beads might be trapped, because bead carryover could affect reproducibility and the background signal. Following an injection of 10 000 beads, the channel was rinsed for 1 min with PBS-D at 1 mL/min. The channel was inspected under a microscope for the presence of beads, and only 10 beads, or 0.1% of injected beads, were found in the channel. Therefore, it was decided that untreated PDMS was a suitable material for bead-based experiments, and the fluidic channel configuration was not prone to significant bead carryover between samples. The IDA electrode was a trench structure with 2.4-µm gaps between electrode pairs. Electrochemistry with a trench IDA has

Figure 6. (A) Schematic of the trench IDA electrode. The schematic shows a cross section of two electrode pairs. The bead diameter was greater than the width of the trench, so the beads sat on top of the trench. The electrodes were separated by 2.4 µm. PAP was oxidized to PQI at the anode, +290 mV vs Ag/AgCl, and PQI was reduced to PAP at the cathode, -300 mV vs Ag/AgCl. (B) A picture of the sensor.

been described, and it was predicted by FEM that a trench IDA could enhance the current 3-fold, as compared to a conventional coplanar IDA electrode.26 Figure 6 shows a schematic of two electrode pairs and a picture of the sensor. The dimensions of the sensing area were 250 µm by 300 µm, which was small enough for the entire sensing area to be exposed to solution and beads in the PDMS channel. The two working electrodes were held at different potentials so that PAP was oxidized to PQI at the anode and PQI was reduced to PAP at the opposing cathode. Our previous IDA study involved a planar IDA electrode in which the electrodes were horizontally positioned in the plane of the silicon dioxide chip. In the trench IDA, however, each electrode pair is positioned perpendicular to the plane of the silicon dioxide chip, and the two electrodes in the pair face each other. Since the separation distance was less than the bead diameter (2.8 µm), the beads could sit on top of the electrode without sitting directly on and blocking access to the conducting surfaces. An amperogram for the 100 ng/mL MS2 sample is shown in Figure 7 to illustrate the procedure in terms of current vs time. Initially, potentials were applied to the two working electrodes, +290 and -300 mV vs Ag/AgCl, while PBS-D was flowing through the fluidic device. At 30 s, 100 µL of the bead sample, 1 × 106 beads/100 µL, was injected into the device, followed by 80 µL of PBS-D to remove any beads not retained by the magnet. At 250 s, 100 µL of the enzyme substrate, PAPG, was injected, and an increase in anodic current was seen when the enzyme substrate reached the beads held over the electrode. In Figure 7, this increase in anodic current was seen at ∼290 s. The flow was stopped at 360 s, a steady state was reached, and increasing anodic current was observed that was directly related to the increasing PAP concentration as the enzyme converted PAPG to PAP. At Analytical Chemistry, Vol. 76, No. 10, May 15, 2004

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Figure 7. Amperogram for the 100 ng/mL MS2 sample detected in the PDMS channel at an IDA electrode. (A) PBS-D was flowing through the channel at 500 µL/min. (B) 100 µL of bead solution was injected at 30 s at 50 µL/min. (C) 100 µL of 4 mM PAPG was injected at 250 s at 50 µL/min. (D) The flow was stopped at 360 s, and steadystate current was achieved. The working electrodes were held at +290 mV and -300 mV vs Ag/AgCl. The anodic current increased as the enzymatically generated PAP increased in concentration in proportion to the concentration of MS2 in the sample.

Figure 8. Current-vs-time plots for the following MS2 concentrations detected in the PDMS channel at an IDA electrode for the time period that the enzyme substrate was present in the fluidic device: (A) 0, (B) 0.1, (C) 0.25, (D) 0.5, (E) 1, and (F) 2 µg/mL. The working electrodes were held at +290 mV and -300 mV vs Ag/AgCl.

480 s, beads were rinsed out of the channel with PBS-D at 1 mL/ min. Within 8 min, beads were collected on top of the electrode, enzyme substrate was introduced, and enzymatic conversion of PAP was monitored, which related to the concentration of MS2 present in the sample. Figure 8 shows the current vs time plots for MS2 concentrations between 0 and 2 µg/mL for the time period that the enzyme substrate was present in the fluidic device. The upper limit of MS2 concentration was set, since beads were saturated with MS2 above 3 µg/mL, as seen with the RDE data. The lower MS2 concentrations extended down to 50 ng/mL MS2 in hopes of lower detection limits with the IDA electrode than with the RDE method. Each MS2 concentration was detected in triplicate, and the same sensor was used throughout the study. The log of the slope of current vs time (nA/s) between 400 and 450 s, which was during the time of zero flow and steady-state current, was plotted against log MS2 2706

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concentration (µg/mL). A linear range was observed from 0.1 to 2 µg/mL MS2, and the plot was described by y ) (1.18 ( 0.09)x + (0.06 ( 0.25). The limit of detection was 0.09 µg/mL MS2, or 1.5 × 1010 MS2 particles/mL, as determined by three times the standard deviation of the signal for the 0 µg/mL MS2 sample. Detection limits with the IDA electrode in the fluidic channel were lower than those obtained with the RDE method, and therefore, the linear working range for slope vs virus concentration was wider with the IDA electrode than with the RDE electrode. Although the rotation of the RDE provided enhanced mass transport, the electrochemical recycling of the PAP/PQI couple by the IDA electrode gave a detection limit approximately onehalf that of the RDE method. The two detection methods had similar levels of background noise, as determined by comparing the standard deviations: 2.8 nA for RDE (3.9% RSD) and 3.2 nA (3.1% RSD) for IDA. The RDE electrode (0.071 cm2) had an area 76 times greater than the IDA electrode (9.4 × 10-4 cm2). Normalizing the slopes of the current-time plots for the 500 ng/ mL MS2 samples with electrode areas gave 50 nA/(cm2‚s) for RDE detection vs 1750 nA/(cm2‚s) for IDA detection, which illustrates the effect of electrochemical recycling on the current. Furthermore, a signal from NSA of biomolecules was detectable with the IDA electrode, in contrast to the RDE method, so the IDA electrode detection was limited by NSA of the biomolecules rather than by instrumental capabilities. Suppressing the interference of NSA should lead to even lower detection limits with the IDA method. It was found during our previous study coupling a bead-based immunoassay with an IDA electrode that having beads packed directly on a planar IDA electrode surface was an ineffective configuration;25 however, the IDA used in this study was a trench IDA, so beads did not sit directly on the metal surface. In addition, the flow of the solution through the channel spread the beads over the diameter of the magnet, reducing the bead clump seen in a stagnant microdrop of solution. This appeared to minimize enzyme substrate depletion within the vicinity of the bead clump seen in the previous study. Having the beads as close as possible to the electrode minimized the distance that PAP needed to diffuse, which reduced the detection time, once all of the necessary reagents were present in the channel. Sources of variability connected with the previous study were minimized. Injecting the beads into the channel with a syringe pump instead of manually pipetting beads into a drop was more reproducible because the bead injection did not rely on the experimenter’s ability to position the pipet at the same point on the drop. Other sources of variability still remain as long as the immunosandwich is prepared by the manual procedure. CONCLUSION A paramagnetic bead-based electrochemical immunoassay has been developed to detect bacteriophage MS2, a biothreat agent simulant. The detection methods described in this study compare well with other MS2 immunoassays that have been recently reported. Rowe et al. have demonstrated an array biosensor that uses a sandwich immunoassay format and fluorescent labels to detect MS2 at 400 ng/mL.3 McBride et. al. reported a detection limit of 3 ng/mL MS2, using a bead-based assay and a flow cytometer for detection.4 Although the assay detected with the flow cytometer is more sensitive, it is more likely that the fluidic

channel with IDA electrode detection can be developed into a hand-held device to be used in the field. The logical next step would be to include automated bead preparation within the fluidic device. It has been reported that exposure to only 10 particles of smallpox can be lethal. Our method is far from detecting concentrations at that level, but falls nearer to the lethal range of hemorrhagic fever viruses, between 105 and 1010 particles.33 Although improvements in detection limit are needed for an early warning system, the reported method can selectively detect a virus of interest, assuming the appropriate antibody is available. (33) Burrows, W. D.; Renner, S. E. Environ. Health Perspect. 1999, 107, 975984.

ACKNOWLEDGMENT J.H.T. acknowledges the Society for Analytical Chemists of Pittsburgh for sponsoring an ACS Division of Analytical Chemistry Graduate Fellowship, allowing this work to be completed. Michael Goode provided a supply of bacteriophage MS2, and Tetracore, Inc. generously gave a sample of rabbit anti-MS2 IgG. P.J.H. and S.K.K. acknowledge the support by a grant from Motorola Inc., Science Advisory Board Associate. Received for review December 18, 2003. Accepted February 20, 2004. AC035503C

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