Electrochemical Enzyme Immunoassays on Microchip Platforms

Anal. Chem. 2001, 73, 5323-5327

Electrochemical Enzyme Immunoassays on Microchip Platforms Joseph Wang,* Alfredo Iba´n˜ez, Madhu Prakash Chatrathi, and Alberto Escarpa†

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003

A microfluidic device for conducting electrochemical enzyme immunoassays is described. The new “lab-on-achip” protocol integrates precolumn reactions of alkaline phosphatase-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibody-antigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the 4-aminophenyl phosphate substrate and a downstream amperometric detection of the liberated 4-aminophenol product. Factors influencing the reaction, separation, and detection processes were optimized, and the analytical performance was characterized. An applied field strength of 256 V/cm results in free antibody and antibody-antigen complex migration times of 125 and 340 s, respectively. A remarkably low detection limit of 2.5 × 10-16 g/mL (1.7 × 10-18 M) is obtained for the mouse IgG model analyte. Such combination of a complete integrated immunoassay, an attractive analytical performance, and the distinct miniaturization/portability advantages of electrochemical microsystems offers considerable promise for designing self-contained and disposable chips for decentralized clinical diagnostics or onsite environmental testing. Microfluidic devices have experienced phenomenal success since their introduction.1 The advantages of such analytical microsystems, including high performance, design flexibility, reagent economy, high throughput, miniaturization, and automation, have been well documented.2,3 Such “lab-on-a-chip” devices can thus dramatically change the speed and scale at which chemical analyses are performed. The power and scope of microanalytical systems can be greatly enhanced by performing highly selective biochemical reactions. In particular, on-chip immunoassays combine the analytical power of microfluidic devices with the high specificity of antibodyantigen interactions. Microchip platforms have proven themselves as highly suitable vehicles for conducting various immunoassay protocols.4-8 Koutny et al.4 reported on a microchip electrophoretic immunoassay for serum cortisol involving a competitive assay and † On leave from Departamento de Quimica Analitica, Universidad de Alcala, Alcala de Henares, Madrid, Spain. (1) (a) Manz, A.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244. (b) Jacobson, S. C.; Hergenroder, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127. (c) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Luedi, H.; Widmer, H. M. J. Chromatogr., B 1992, 593, 253. (2) Figeys, D.; Pinto, D. Anal. Chem. 2000, 71, 330A. (3) Freemantle, M. Chem. Eng. News 1999, (Feb. 22), 27.

10.1021/ac010808h CCC: $20.00 Published on Web 10/02/2001

© 2001 American Chemical Society

laser-induced fluorescence (LIF) detection. Chiem and Harrison5,6 described an effective immunoassay for serum theophylline based on on-chip separation and optical measurements of the free and bound fluorescence tracer. Kitamori’s group reported on a beadbased immunoassay with thermal lens microscopic (TLM) detection for cancer diagnosis.7 Schmalzing et al.8 demonstrated a competitive immunoassay of serum thryoxine using a fluoresceinthryoxine conjugate. While early lab-on-a-chip immunoassay studies have focused on optical (LIF and TLM) detection, there are no reports of analogous on-chip electrochemical immunoassays. Electrochemistry offers considerable promise for on-chip immunoassays and for designing self-contained and disposable chips for point-of-care diagnostics. The attractive features of electrochemical detection for microchip systems, including its high sensitivity, inherent miniaturization, low cost, low power requirements, freedom from turbidity, and high compatibility with advanced micromachining technologies, were documented and discussed earlier.9,10 In this article, we demonstrate the potential and advantages of using amperometry as a detection scheme for on-chip enzyme immunoassays. Electrochemical enzyme immunoassays, combining antigen-antibody reactions with amperometric detection of the product of the enzymatic reaction, have evolved dramatically over the past two decades.11-14 Excellent (attomole-zeptomole) detection limits have been reported by combining such enzyme immunoassays with amperometric detection to liquid chromatography (LC) or flow injection analysis (FIA). Most of these schemes have relied on the use of the alkaline phosphatase (ALP) enzyme label since it generates easily oxidizable phenolic products. As shown schematically in Figure 1, the microchip platform allows complete integration of the multiple steps of electrochemical enzyme immunoassays to create a lab-on-a-chip. The new protocol thus consists of the precolumn reaction of the ALPlabeled antibody and the antigen analyte, a rapid electrophoretic (4) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18. (5) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373. (6) Chiem, N.; Harrison, D. J. Clin. Chem. 1998, 44, 591. (7) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 1213. (8) Schmalzing, D.; Koutny, L. B.; Taylor, T. A.; Nashabeh, W.; Fuchs, M. J. Chromatogr., B 1997, 697, 175. (9) Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684. (10) Wang, J.; Tian, B.; Sahlin, E. Anal. Chem. 1999, 71, 5436. (11) Heineman, W. R.; Halsall, H. B. Anal. Chem. 1985, 57, 1321A. (12) Halsall, H. B.; Heineman, W. R.; Jenkins, S. H. Clin. Chem. 1988, 34, 1701. (13) Purushothama, S.; Kradtap, S.; Wijayawardhana, A.; Heineman, W. R.; Halsall, H. B. Analyst 2001, 126, 337. (14) Skladal, P. Electroanalysis 1997, 9, 737.

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Figure 1. Schematic of the immunochip used in this study. Key: RB, running buffer; Ab-E, enzyme-labeled antibody; Ag, antigen; S, substrate; IRC, immunoreaction chamber; RE, reference electrode; CE, counter electrode; WE, working electrode; B, unused reservoir. See text for exact dimensions and details.

separation of labeled free antibody and the labeled antigenantibody complex, postcolumn addition of the 4-aminophenyl phosphate (p-APP) substrate and its enzymatic conversion to a 4-aminophenol (p-AP), along with a downstream low-potential anodic detection of p-AP at a mass-produced screen-printed electrode detector. To our knowledge, this is the first report describing such an on-chip integration of precolumn immunoreaction with postcolumn enzymatic reaction. The combination of such a complete integrated assay, with the very attractive analytical performance, and distinct portability/disposability advantages of electrochemical microsystems, offers considerable promise for decentralized clinical or environmental testing. Operating conditions, efficiency, and benefits of the new electrochemical immunoassay biochip are reported below in connection with highly sensitive measurements of the mouse IgG model analyte. EXPERIMENTAL SECTION Reagents. Alkaline phosphatase, tris(hydroxymethyl)aminoethane (Tris) and polyoxyethylenesorbitan monolaurate (Tween 20), reagent-grade mouse IgG analyte, and anti-mouse IgG (whole molecule) conjugated to alkaline phosphatase developed in goat (3 mg/mL stock solution) were purchased from Sigma. pAminophenyl phosphate was purchased from Universal Sensors (Cork, Ireland). All chemicals were used without further purification. The 5 × 10-5 g/mL antigen working standard solution was prepared in a mixture of 10 mM phosphate buffer, 2.7 mM KCl, 120 mM NaCl, and 0.1% (w/v) sodium azide. More diluted antigen solutions were prepared daily from the working standard solution. The electrophoresis buffer (pH 8.0) consisted of 50 mM Tris buffer and 0.02% (v/v) Tween 20. The Tris buffer was proven to enhance the activity of the ALP enzyme tag.15 Tween 20 was added to minimize the adsorption of the immunoglobulins onto the walls.5 The pH of the running buffer, 8.0, was chosen in accordance with (15) (a) Simopoulos, T. T.; Jencks, W. P. Biochemistry 1994, 38, 10375. (b) Plocke, D. J.; Vallee, B. L Biochemistry 1962, 1, 1039.

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the activity of the alkaline phosphatase.16 The p-APP substrate solution (for the postcolumn reaction) consisted of 5 mM p-APP in 50 mM Tris buffer (pH 9.0) and 0.02% (v/v) Tween 20. The pH 9.0 of the postcolumn buffer was selected to meet the stability requirements of the p-APP substrate.17 Sample solutions were prepared by diluting the corresponding stock solutions in the electrophoresis buffer. All the solutions were prepared in highpurity water filtered through a Milli-Q water system (Millipore, Bedford, MA). Apparatus. The glass chip used in this study was designed at NMSU and fabricated by Micralyne (formally, Alberta Microelectronic Co., Edmonton, Canada; model MC-BF4-001). The layout is different from that employed previously10 in that it permits both pre- and postcolumn reactions (Figure 1). The chip thus consisted of “reagent” (Ab-E) and “analyte” (Ag) reservoirs, connected through 50-µm-wide channels to the immunoreaction chamber (IRC; 200 µm wide; 3.6 mm long) to a four-way injection cross. A running buffer reservoir (RB), injection-waste reservoir (each measuring 5 mm long), and a 78-mm-long separation channel were connected to the other side of the injection cross. A 77-mm postcolumn channel was joined 10 mm from the end of the separation channel to introduce p-APP substrate. All the channels were 50 µm wide and 20 µm deep. The fabrication of a Plexiglas holder, supporting the separation chip and housing the detector, reservoirs, and electrical contacts, and the amperometric detection protocols were described elsewhere.10 The detector design allows fast, convenient, and reproducible replacement of the working electrode. A laboratoryconstructed power supply, containing multiple voltage terminals, was used for applying the selected driving voltage (between 0 and +4000 V) to a given reservoir and for switching between the “reaction/injection” and “postcolumn/separation” modes. The screen-printed working electrodes were fabricated with a semiautomatic printer (model TF 100, MPM, Franklin, MA). The Acheson carbon ink Electrodag 440B (49AB90, Acheson Colloids, Ontario, CA) was used for printing electrode strips. Details of the printing process were described previously.10 Amperometric measurements were performed with an electrochemical analyzer 621A (CH Instruments, Austin, TX). Assay Protocol. The reagent reservoir (Ab-E) was filled with the 7.5 × 10-6 g/mL anti-mouse IgG solution (prepared by 400fold dilution of the 3 mg/mL stock solution). The analyte reservoir (Ag) was filled with the required concentration of mouse-IgG (diluted from a 5 × 10-5 mg/mL working standard solution). The on-chip complexation was carried out in the immunoreaction chamber after mixing the analyte and reagent streams by applying suitable voltages to the reagent and analyte reservoirs. Sample (complex and free antibody) injections were usually performed by applying a voltage of +250 V for 2 s to the analyte reservoir and subsequent application of +2000 V for 3 s to the reagent reservoir. The substrate reservoir was filled with the p-APP dissolved in its corresponding buffer. Separations were usually achieved by applying +2000 V simultaneously to both “running buffer” and “postcolumn reservoirs. All the injections/separations were performed by applying the high voltage to the required reservoir with the detection reservoir grounded while other (16) Garen, A.; Levinthal, C. Biochim. Biophys. Acta 1960, 38, 470. (17) Kreuzer, M. P.; O’Sullivan, C. K.; Guilbault, G. G. Anal. Chim. Acta 1999, 393, 95.

Figure 2. Electropherograms resulting from the postcolumn addition of 5 mM p-APP substrate in connection with (A) enzyme-labeled free antibody alone, (B) off-chip complexation of the enzyme-labeled antibody with 2.5 × 10-3 g/mL antigen, (C) on-chip complexation of the enzyme-labeled antibody with 2.5 × 10-3 g/mL antigen, and (D) enzyme-labeled free antibody (a) and complex (b) using on-chip complexation with 1.56 × 10-15 g/mL antigen. Antibody concentration, 7.5 × 10-6g/mL. The running buffer (pH 8.0) and postcolumn buffer (pH 9.0) were a 50 mM Tris with 0.02% v/v Tween 20. Separation and postcolumn voltages, 2000 V; injection voltage, (Ag) 250 V for 2 s and (Ab-E) 2000 V for 3 s. Screen-printed carbon electrode held at +0.7 V (vs Ag/AgCl wire reference electrode).

reservoirs floating. The immunochip was washed between a group of runs using 0.1 M NaOH and water. Sample injections were performed after stabilization of the baseline current; the electropherograms were recorded using an applied potential of +0.7 V (vs Ag/AgCl), a time resolution of 0.1 s, and without noise filtration. The off-chip immunoreaction (used for comparison purposes) was carried out by mixing the antigen and antibody solutions (with final concentrations of 2.5 × 10-3 and 7.5 × 10-6 g/mL, respectively). The mixing took place in a 1-mL cell at 25 °C for an incubation time of 2 h, and no efforts were made in further optimizing the conditions. Safety Considerations: The high-voltage power supply and associated open electrical connections should be handled with extreme care to avoid electrical shock. RESULTS AND DISCUSSION The on-chip electrochemical enzyme immunoassay protocol (Figure 1) relies on precolumn reactions of enzyme-labeled antibody (anti-mouse IgG) with the antigen (mouse IgG), followed by electrophoretic separation of the free antibody and antibodyantigen complex. The separation is followed by a postcolumn reaction of the enzyme tracer with the p-APP substrate and a downstream amperometric detection of the liberated p-AP product. The present protocol represents the first example of on-chip integration of a precolumn immunological reaction with a postcolumn enzymatic reaction. The ability to mix the labeled antibody with the antigen, separate the labeled antibody from the labeled antibody-antigen complex, and detect electrochemically the enzyme tracer (through the p-AP oxidation) is demonstrated in Figure 2. Electrophero-

grams were first recorded to identify the individual peaks of the new immunochip protocol. An injection of the ALP-labeled antibody (7.5 × 10-6 g/mL), coupled to the postcolumn enzymatic reaction, resulted in a well-defined “free antibody” peak at around 125 s (A). A similar injection of the labeled antibody-antigen complex (produced in an off-chip external incubation cell using a large excess of the antigen) resulted in a well-defined peak around 345 s (B). No response is observed for the free antibody, indicating the completeness of the off-chip reaction (due to the large excess of the antigen). A similar “saturation” experiment, conducted “onchip” by employing a precolumn mixing of the reagents (at the levels employed in B) also yielded a single complex peak (at a similar migration time) and no free antibody signal (C). Precapillary mixing of the antigen (1.56 × 10-15 g/mL) with the labeled antibody (7.5 × 10-6 g/mL), coupled to the postcapillary mixing of the substrate, produced an electropherogram (D) with two well-defined peaks at migration times of 125 (a) and 340 (b) s, corresponding to the migration of the labeled antibody and complex, respectively (D vs A,B). Apparently, the mobilities of the free and complexed antibodies are vastly different. These data indicate that the chip layout and protocol are adequate for forming the antibody-antigen complex, for separating it from the free antibody, and for the postcolumn enzymatic conversion. As will be illustrated in the following sections, both the complex and free antibody peaks offer convenient quantitation of extremely low levels of the target antigen. As expected for such on-chip reactions,18,19 the pre- and postcolumn mixing resulted in significant band broadening compared to the signals observed for the injection of the “pure” biomolecules (D vs A,B). The 4-aminophenol product has desirable electrochemical behavior of a low-potential, reversible redox reaction and negligible surface fouling.13 Hydrodynamic voltammograms were constructed for assessing the on-chip electrochemical behavior of p-AP by changing the potential of the screen-printed carbon detector over the +0.2 - +0.7 V range. Figure 3 shows such hydrodynamic voltammograms for the on-chip-generated p-AP in connection with postcolumn reactions of the substrate with the free enzyme (A) and the enzyme-labeled antibody (B). As expected, the p-AP product of both reactions displays a similar voltammetric profile, with oxidation starting above +0.3 V, a gradual rise in the current between +0.3 and +0.5 V, and a leveling off thereafter. All subsequent work employed a potential of +0.7 V that offered the most favorable signal/background characteristics. Note also the similarity in the E1/2 and E3/4 - E1/4 values (0.472 (A) vs 0.456 (B) and 26 (A) vs 29 (B)) mV, respectively. The influence of the applied (separation and postcolumn) voltages upon the analytical performance was examined over the 1000-2500-V range (Figure 4, A-D). The resulting electropherograms indicate that such voltages are sufficient for effective immunological and enzymatic reactions and subsequent separation of the antibody-antigen complex from the free antibody. As expected, increasing the separation and postcolumn voltages over the 1000-2500-V range decreases the migration times for the free antibody and its complex with antigen. Yet, higher field strengths shorten the contact/reaction time of the separated substrates in the postcolumn reactor and, hence, impair the efficiency of the (18) Schmalzing, D.; Nashabeh, W. Electrophoresis 1997, 18, 2184. (19) Colyer, C. L.; Mangru, S. D.; Harrison, D. J. J. Chromatogr., A 1997, 781, 271.

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Figure 3. Hydrodynamic voltammograms for the p-AP product, constructed by injecting solutions of 50 units/mL of the free ALP enzyme (A) and 7.5 × 10-6 g/mL of the enzyme-labeled antibody (B) in connection with postcolumn addition of 5 mM p-APP substrate and the absence of the antigen. Injection voltage, 1500 V for 3 s; other conditions, as in Figure 2C.

Figure 4. Influence of the separation and postcolumn voltages upon the response for the free antibody (a) and its complex with the antigen (b). Separation voltage, (A) 1000, (B) 1500, (C) 2000, and (D) 2500 V. Ab-E concentration, 6.0 × 10-6 g/mL; Ag concentration, 2 × 10-15 g/mL. Also shown in the inset is the effect of separation voltage upon the difference in migration times (b) of the complex and free antibody and upon current response (2) of the complex. Other conditions, as in Figure 2C.

enzymatic reaction. It is necessary that the postcolumn mixing is carried out in a controlled and efficient manner. The effect of the separation and postcolumn voltages upon the postcolumn mixing was examined by monitoring corresponding currents (not shown; note the similarity in length of the separation and postcolumn channels). Maximum mixing was observed when the separation and postcolumn voltages were similar; these results are consistent with the reported observations made by Harrison’s group.20 Also shown in Figure 4 (as inset) are the plots of the voltage effect (20) Fluri, K.; Fitzpatrick, G.; Chiem, N.; Harrison, D. J. Anal. Chem. 1996, 68, 4285.

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Figure 5. Calibration plots: dependence of the free antibody (A, O) and complex (B, b) peak currents upon the concentration of the IgG antigen. Also shown (as inset) is the portion of the electropherogram for 3.0 × 10-16 g/mL antigen concentration (following digital smoothing of the raw data). Other conditions, as in Figure 2C.

upon the difference in migration times and upon the peak height of the complex. The peak separation decreases rapidly up 1000 V and then more slowly. The complex peak height increases rapidly up to 1500 V and decreases slightly at higher voltages. A voltage of 2000 V was used for all subsequent work, as it provided the most favorable balance between speed, sensitivity, resolution, and isolation from the detection circuitry. The electrochemical immunoassay microsystem displays a well-defined concentration dependence. Figure 5 shows calibration data obtained for IgG concentrations up to 3.9 × 10-15 g/mL in steps of 7.8 × 10-16 g/mL. Well-defined antibody and complex peaks were observed over this range. The heights of these peaks can be used for quantitating the antigen and lead to well-defined calibration plots. The complex peak increases in a nearly linear fashion up to 3.1 × 10-15 g/mL (sensitivity of 1.2 × 10-15 nA‚ mL/g; correlation coefficient of 0.997) and then more slowly (B). Similarly, the free antibody signal decreases in a linear fashion with the IgG concentration up to 2.4 × 10-15 g/mL (sensitivity of 3.5 × 10-15 nA‚mL/g; correlation coefficient of 0.996), and levels off above 3.1 × 10-15 g/mL (A). In addition to enhanced sensitivity, the use of the antibody peak for quantitation results in significantly (nearly 3-fold) shorter analysis times (e.g., Figure 2C). Welldefined calibration plots (analogous to those of Figure 5) were obtained by using of the peak area instead of height (not shown). Extension of the dynamic working range could be readily accomplished through control of the antibody concentration, onchip dilution, or both. The highly sensitive response is coupled to a low noise level and, hence, results in extremely low detection limits. Figure 5 (inset) displays the actual complex signal obtained for an antigen concentration of 3.0 × 10-16 g/mL. A detection limit of around 2.5 × 10-16 g/mL (1.7 × 10-18 M, can be estimated based on the signal-to-noise characteristics of these data (S/N ) 3). Such detectability is slightly lower than that (1.4 × 10-14 g/mL (6.7 × 10-17 M)) reported for off-chip electrochemical enzyme immunoassay protocols.12 This may be attributed to the enhanced

enzymatic amplification at ultrasmall volumes21 and to the continuous “supply” of the substrate. Higher detection limits (in the ng/ mL to pg/mL range) are common to nonenzymatic chip-based immunoassays.4-8 Relative standard deviations of 4.1 and 3.4% were calculated for six repetitive measurements of IgG with and without, respectively, cleaning the chip between runs (conditions as in Figure 2C; not shown). The migration times were also very reproducible with RSDs of less than 1.0%. Such good precision reflects the high reproducibility of the mixing/reaction/separation/detection processes and indicates a negligible electrode fouling or enzyme/ antibody adsorption onto the channel walls. CONCLUSIONS A microfabricated device that integrates multiple steps of electrochemical enzyme immunoassays on a chip platform was successfully evaluated. Such an on-chip integration of immunological and enzymatic reactions, electrophoretic separation, and amperometric detection allows performing immunoassays more rapidly, easily, and economically. While the concept of on-chip electrochemical enzyme immunoassays has been illustrated using (21) de Frutos, M.; Paliwal, S. K.; Regnier, F. E. Anal. Chem. 1993, 65, 2159.

the IgG model analyte, it could be readily extended to a broad variety of analytes of clinical, environmental, or biotechnological significance. Such extension would require proper attention to the effect of the analyte molecular weight upon the separation. Multiple analytes may be detected simultaneously in connection with a multichannel on-chip parallel operation. The power and utility of such on-chip bioassays will be greatly enhanced by integrating additional sample processing functions (e.g., cleanup, preconcentration, dilution) into their protocol. Such sample manipulations are essential for addressing the complexity of biological fluids. The new electrochemical immunoassay biochip strategy offers considerable promise for designing self-contained and disposable chips for decentralized clinical or environmental testing. ACKNOWLEDGMENT Financial support from the National Institute of Health (NIH grant R01 14549-02) is gratefully acknowledged. A.E. acknowledges a NATO fellowship through the European Community. Received for review July 18, 2001. Accepted August 22, 2001. AC010808H

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