Anal. Chem. 2003, 75, 3316-3321
Microfabricated On-Chip-Type Electrochemical Flow Immunoassay System for the Detection of Histamine Released in Whole Blood Samples Tae-Kyu Lim, Hiroko Ohta, and Tadashi Matsunaga*
Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fcconjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/ mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chiptype electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results. The development of miniaturization of chemical and biotechnological processes is moving at a rapid pace. The advantages of such analytical microsystems are high performance, design flexibility, reagent economy, high throughput, and automation.1-7 Microfluidic devices have been used for transporting and manipulating minute amount of fluids or biological entities through microchannel manifolds, allowing integration of various chemical and biochemical processes into fast and automated monolithic microflow systems.8 The various types of microfabricated chemical and biochemical devices have been often referred to as lab-on-achip systems, which encompass miniaturized separation systems, * Author for correspondence. Phone: +81-42-388-7020. Fax: +81-42-385-7713. E-mail:
[email protected]. (1) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (2) Tsukagoshi, K.; Obata, Y.; Nakajima, R. J.Chromatogr., A 2002, 971, 255260. (3) Burns, M. A. Science 2002, 296, 1818-1819. (4) Schulte, T. H.; Bardell, R. L.; Weigl, B. H. Clin. Chim. Acta 2002, 321, 1-10. (5) Kakuta, M.; Bessoth, F. G.; Manz, A. Chem. Rec. 2001, 1, 395-405. (6) Mayer, G.; Schober, A.; Kohler, J. M. J. Biotechnol. 2001, 82, 137-159. (7) Chovan, T.; Guttman, A. Trends Biotechnol. 2002, 20, 116-122. (8) Jandik, P.; Weigl, B. H.; Kessler, N.; Cheng, J.; Morris, C. J.; Schulte, T.; Avdalovic, N. J. Chromatogr., A 2002, 954, 33-40.
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microreactors, microarrays, and combinations of any of the above.9-11 In particular, lab-on-a-chip immunoassays combine the analytical power of microfluidic devices with the high specificity of antibody-antigen interactions.12,13 Microchip platforms have proven to be highly suitable vehicles for conducting various immunoassay protocols.14-17 Although early lab-on-a-chip immunoassay studies have focused on optical detection,18,19 there are no reports of analogous on-chip electrochemical immunoassays. Electrochemistry offers considerable promise for on-chip immunoassays and for designing selfcontained and disposable chips for medical diagnosis.14 The attractive features of electrochemical detection for microchip systems include its high sensitivity, inherent miniaturization, low cost, low power requirements, and high compatibility with advanced micromachining technologies.20 We have described a new approach to the performance of miniaturized electrochemical flow immunoassay system for the detection of human chorionic gonadotrophin.21,22 In this report, an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column coated with cation-exchange resin on PMMA (poly(methyl methacrylate)) plate is described. This system detects histamine released in whole blood samples from patients suffering from allergic reactions. EXPERIMENTAL SECTION Reagents. Histamine 2-HCl and 1,4-benzoquinone were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). (9) Mao, H.; Holden, M. A.; You, M.; Cremer, P. S. Anal. Chem. 2002, 74, 5071-5075. (10) Mouradian, S. Curr. Opin. Chem. Biol. 2002, 6, 51-56. (11) Weigl, B. H.; Hedine, K. Am. Clin. Lab 2002, 21, 8-13. (12) Khandurina, J.; Guttman, A. J. Chromatogr., A 2002, 943, 159-183. (13) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emneus, J. Anal. Chem. 2002, 74, 2994-3004. (14) Wang, J.; Ibanez, A.; Chatrathi, M. P.; Escarpa, A. Anal. Chem. 2001, 73, 5323-5327. (15) Sato, K.; Yamanaka, M.; Takahashi, H.; Tokeshi, M.; Kimura, H.; Kitamori, T. Electrophoresis 2002, 23, 734-739. (16) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (17) Purushothama, S.; Kradtap, S.; Wijayawardhana, C. A.; Halsall, H. B.; Heineman, W. R. Analyst 2001, 126, 337-341. (18) Lapos, J. A.; Ewing, A. G. Anal. Chem. 2000, 72, 4598-4602. (19) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (20) Wang, J. Trends Anal. Chem. 2002, 21, 226-232. (21) Lim, T. K.; Imai, S.; Matsunaga, T. Biotechnol. Bioeng. 2002, 77, 758-763. (22) Lim, T. K.; Matsunaga, T. Biosens. Bioelectron. 2001, 16, 1063-1069. 10.1021/ac020749n CCC: $25.00
© 2003 American Chemical Society Published on Web 06/17/2003
Figure 1. Schematic diagram of the on-chip-type electrochemical flow immunoassay system. The reaction mixture consists of histamine, histamine-BSA, Fc-IgG, histamine-Fc-IgG complex, and histamine-BSA-Fc-IgG complex. The mixture is passed through the multichanneled matrix column that selectively traps free Fc-IgG and the histamine-Fc-IgG complex. The eluted species pass through the flow cell, and the current associated with the histamine-BSA-Fc-IgG antibody complex is monitored. The applied potential is 395 mV vs Ag/AgCl.
Ferrocenemonocarboxylic acid (Fc) was obtained from Sigma Chemical Co. (St.Louis, MO). Antihistamine monoclonal antibody was purchased from Biogenesis (Florida). Sulfo-NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) were purchased from Pierce (Rockford, IL). PhastGel Gradient 8-25, PhastGel IEF 3-9, and molecular markers were purchased from Amersham Biosciences Corp. (Piscataway, NJ). Nafion was purchased from Aldrich Chemical Co (Milwaukee, WI). All other chemicals used were analytical reagent or laboratory grade. Deionized distilled water was used in all procedures. Equipment. Characterization and evaluation of the on-chiptype electrochemical flow immunoassay involves a three-electrode flow-cell system equipped with a glassy carbon electrode, a platinum counter electrode, and a Ag/AgCl reference electrode (BAS Inc. Japan). A constant potential was applied with a potentiostat (model HA-151; Hokuto Denko Co., Tokyo, Japan or model LC-4C; amperometric detector, BAS Inc.). Results were recorded on a chart recorder (SP-J5C, Riken Denshi, Tokyo, Japan). Eluent flow was produced using a peristaltic pump (AliteaXV, Sweden). Samples were applied with a pipetman (10 µL). A multichanneled matrix column was fabricated using a computeraided modeling machine (CAMM-3, PNC-300) running the CAD software package from Roland DG Corp. (Tokyo, Japan). Preparation of the Ferrocene-Conjugated IgG. The conjugation of ferrocene to IgG antibody was carried out according to the following procedure. Ferrocenemonocarboxylic acid (Fc; 4 mg) was dissolved in 800 µL of a 0.15 M Na-HEPES [sodium 4-(2hydroxyethyl-9-1-piperazineethanesulfonate] buffer (pH 7.3), was reacted with 5 mM sulfo-NHS, and was incubated with 2 mM EDC (final concentration) for 15 min at 25 °C.23,24 The reaction step was terminated by the addition of β-mercaptoethanol (20 mM final concentration), and IgG (antihistamine, 500 µg/mL, 90 µL) was added and incubated for 4 h at room temperature. The concentration of IgG in the solution was determined by the BCA protein assay method 25,26 before and after conjugation. The removal of (23) Grabarek, Z.; Gergely, J. Anal. Biochem. 1990, 185, 131-135. (24) Leszyk, J.; Grabarek, Z.; Gergely, J.; Collins, H. Biochemistry 1990, 29, 299304.
free ferrocene after conjugation was carried out by dialysis for 3 days with three changes of fresh buffer. Furthermore, ferroceneconjugated IgG (Fc-IgG) prepared in this manner was homogeneous, as determined by gel filtration using a Superdex 200 column (Amersham Biosciences Corp.). Fc-IgG was stable at 4 °C for several weeks. The number of Fc introduced into IgG was measured by atomic absorption spectroscopy (Shimadzu; AA6600G, Japan), which detects iron in modified IgG. Preparation of the Histamine-BSA Conjugate. 1,4-Benzoquinone is known to react with both thiol27 and amino groups of proteins.28 Histamine was bound to BSA (bovine serum albumin) using 1,4-benzoquinone as the coupling agent. The most efficient histamine-BSA conjugates were obtained as follows: 10 mg of BSA was dissolved in 0.1 M phosphate buffer pH 4.5 (1.7 mL) to which 0.3 mL of a solution containing 30 mg 1,4-benzoquinone in 1 mL ethanol was added, and the mixture was allowed to react for 1 h at room temperature in the dark. BSA with covalently bound benzoquinone molecules was separated from nonreacted benzoquinone by dialysis. The dialysis was continued for 3 days with three changes of fresh buffer. Multichanneled Matrix Column Fabrication. Multichanneled matrix column layout was carried out on a Machining Star 25 (Toki Corporation, Tokyo) using CAD system. Layout files in the CAD graphics format were electronically transferred and compiled to function as motion control files for the CAMM (PNC300, Roland DG Corp, Tokyo). Multichannels were fabricated on the PMMA plate by milling with a End-Mill (Toki Corp. Tokyo) fitted with a 300-µm bit. All plates were thoroughly cleaned with hydrogen peroxide to remove organic materials and particles. The column chosen for ion exchange coating was first thoroughly rinsed with water and then coated by introducing a solution of 5 wt % Nafion, which was maintained for 4 h at room temperature. The column was then rinsed using 50 mM malonate (25) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85. (26) Wiechelman, K. J.; Braun, R.; Fitzpatrick, J. D. Anal. Biochem. 1988, 175, 231-237. (27) Mason, H. S.; Peterson, E. W. Biochim. Biophys. Acta 1965, 111, 134-146. (28) Byck, J. S.; Dawson, C. R. Anal. Biochem. 1968, 25, 123-135.
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Figure 2. Conjugation of ferrocene on IgG using EDC and sulfo-NHS.
buffer (pH 6.5) and stored until used. Sampling of Blood. Blood was sampled from fingertips of volunteer donors using disposable prick needles (Misawa Medical Industry Co., Tokyo, Japan). To prevent coagulation, heparin calcium solution was added to the whole blood sample. The collected samples were used within 3 h for the experiment. On-Chip-Type Electrochemical Flow Immunoassay. The on-chip-type electrochemical flow immunoassay system using a multichanneled matrix column is shown in Figure 1. Ten microliters of Fc-IgG (3 µg/mL) was mixed with 10 µL of 40 µg/mL histamine-BSA and 10 µL of whole blood sample. The mixture was incubated for 30 min at 37 °C, loaded into the 10-µL injection section, and passed into the multichanneled matrix column holder with 50 mM malonate buffer (pH 6.5) at a flow rate of 40 µL/min. The electrochemical oxidation current of the formed histamineBSA and Fc-IgG (histamine-BSA-Fc-IgG) complex in the eluent was measured using a three electrode flow cell (BAS Inc., Japan) equipped with a glassy carbon electrode, Ag/AgCl reference electrode and a platinum counter electrode. The multichanneled matrix column was regenerated by occasional elution with 50 mM malonate buffer (pH 6.5) supplemented with 0.5 M sodium chloride to remove accumulated unreacted Fc-IgG. Identification of Histamine-BSA-Fc-IgG Antibody Complex and Free Fc-IgG Antibody. Histamine-BSA-Fc-IgG complex, free Fc-IgG, and histamine-BSA were subjected to native polyacrylamide gel electrophoresis with PhastGel Gradient 8-25 using PhastGel native buffer strips. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and BSA (67 kDa) were used as molecular markers. Electrophoresis, focusing, and silver staining of the gels (Pharmacia Phastsystem) were performed according to the manufacturer’s instructions. RESULTS AND DISCUSSION Electrochemical Properties of Ferrocene-Conjugated IgG. Initially, Fc was reacted with sulfo-NHS in the presence of EDC, which formed N-succinimidyl esters of Fc carboxyls. IgG was then added for cross-linking (Figure 2). The electrochemical evaluation revealed that these conjugates demonstrated complicated electrochemical behavior. The conjugates that were purified by Superdex 200 gel filtration in the final step contained noncovalently adsorbed ferrocenemonocarboxylic acid, some of which desorbed easily from the conjugate and behaved as free electron mediators. Therefore, the free ferrocenemonocarboxylic acid was removed thoroughly. The electrochemi3318 Analytical Chemistry, Vol. 75, No. 14, July 15, 2003
Figure 3. Cyclic voltammograms of ferrocene-conjugated IgG dissolved in HEPES buffer (pH 7.3) at a scan rate of 20 mV/s using a glassy carbon electrode.
cal properties of ferrocene and Fc-IgG in Na-HEPES buffer at the graphite electrode was investigated by cyclic voltammetry. When cyclic voltammetry was carried out in the HEPES containing Fc, a redox potential appeared at 395 mV (Figure 3). Similar electrochemical behavior was observed with Fc.29 These results suggest that the Fc is electrochemically oxidized at a potential of 395 mV vs Ag/AgCl. Therefore, the applied potential of 395 mV is appropriate for detection of Fc-IgG and histamine-BSA immunocomplexes. The relationship between the number of ferrocene labels bound to IgG antibody and peak current was investigated. We have succeeded in labeling ferrocenes from 4 to 11 units per IgG while retaining 87% of the native IgG activity. The oxidative current increased with the increasing number of ferrocene on the IgG antibody, (data not shown) however, IgG activity decreased to 65% after incorporation of at least 20 ferrocene groups per molecule. The conjugates used had similar IgG concentrations, as determined by the BCA protein assay method (Pierce, Rockford, IL) before and after conjugation. The anodic current at 395 mV vs Ag/AgCl was plotted against the number of bound ferrocene labels. The number of conjugated ferrocene per IgG molecules was measured using atomic absorption spectroscopy. Multichanneled Matrix Column Structure. This system facilitates an increased surface area for more effective cation exchange. This was made possible by the design of the plate with a multichanneled column (Figure 4). The design was reported (29) Okochi, M.; Nakamura, N.; Matsunaga, T. Electrochim. Acta 1999, 44, 3795-3799.
Figure 4. Configuration of multichanneled matrix column onto PMMA plate: design layout reflecting the 2n channels of the splitter with constant cross-sectional area.
first by Regnier’s group.1 However, conventional photolithographic methods require complicated procedures and expensive instrumentation, thus increasing analysis cost. In addition, silicon is difficult to use in clinical diagnosis, since for safety reasons, it cannot be used several times. Processing for preparing matrix columns on PMMA plates was done using CAMM. This method is simple and rapid compared with other photolithographic methods, because it does not involve etching processes. Furthermore, PMMA plates are disposable and cost-effective. These channels were coated with cation-exchange functional groups. Antigen-antibody complexes and unreacted antibodies were separated by a multichanneled matrix column. Uniform distribution and collection of analytes at the column inlet and outlet, respectively, are essential for high column efficiency. Injected samples were merged at the column terminus with a system that is the reverse of the inlet distributor. This multichanneled matrix columns have 2n channels, all channels in a plane perpendicular to the separation axis are of the same width (300 µm) and height (500 µm), and the distance from the center of the fluid distributor inlet to any channel at the head of the column is equal. This means that matrix columns attended by a binary splitting inlet distributor may only have 2, 4, 8, 16, or 2n channels across the column. The number of channels is determined by the desired size of the column. This system directs the same volume of liquid to reach individual channels at the head of the column at the same time. Assuming matrix column walls, it can be calculated that a 32channel matrix column with 100 × 300-µm, rectangular channels would have a volume of 0.3 µL/cm. Thus, the volume of the total channel length of a fabricated 60-cm column would be ∼18 µL. Because elemental analysis of cation-exchange resin coating in the channels of a fully fabricated column is impossible, coating quality was evaluated chromatographically. The condition of trapping free Fc-IgG bound on the matrix column was investigated. The results showed that unreacted Fc-IgG (50 µg/mL, 10 µL) bound to the matrix column coated with cation-exchange resin at pH 6.5 with 50 mM malonate buffer (Figure 5A). On the other hand, when a cation-exchange resin noncoated matrix column was used, free Fc-IgG did not bind to the matrix column
Figure 5. Comparison of currents measured in coated (A) and noncoated (B) cation-exchange resin matrix columns. Fc-IgG, 50 µg/mL; elution buffer, 50 mM malonate buffer (pH 6.5).
and was eluted (Figure 5B). Selective elution of antigen-antibody complex is required for detecting histamine. Protein capacity of the matrix column may not be less than 50 µg/mL. A matrix column coated with cation-exchange resin was therefore used, which could separate antigen-antibody complexes and free Fc-IgG. On-Chip-Type Electrochemical Flow Immunoassay Analysis. We previously showed that antigen-antibody complexes can be successfully separated from unreacted antibody on the basis of differences in isoelectric points.21,22,30-34 Analysis of the isoelectric points of histamine-BSA, Fc-conjugated IgG antibody, and antigen-antibody complexes was carried out by isoelectric gel electrophoresis. The three species were found to have different isoelectric points, 7.0 for Fc-IgG, 5.6 for histamine-BSA, 6.0 for histamine-BSA-Fc-IgG complex, and 7.2 for histamine-FcIgG complex. This means that free Fc-IgG and histamine-FcIgG becomes positively charged when malonate buffer (pH 6.5) is used and bind to the matrix column. Free histamine-BSA and histamine-BSA-Fc-IgG were not retained on the matrix column. The antigen-antibody complexes from the competitive immunoreaction between Fc-IgG, histamine-BSA, and histamine were (30) Lim, T. K., Nakamura, N., Matsunaga, T. Denki Kagaku (presently Electrochemistry) 1995, 63, 1154-1159. (31) Lim, T. K.; Nakamura, N.; Matsunaga, T. Anal. Chim. Acta 1997, 354, 2934. (32) Lim, T. K.; Nakamura, N.; Matsunaga, T. Anal. Chim. Acta. 1998, 370, 207-214. (33) Lim, T. K.; Komoda, Y.; Nakamura, N.; Matsunaga, T. Anal. Chem. 1999, 71, 1298-1302. (34) Lim, T. K.; Nakamura, N.; Matsunaga, T. Biotechnol. Bioeng. 2000, 68, 571574.
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Figure 6. Time course of histamine-BSA-Fc-IgG complex formation. The experiments were carried out using 3 µg/mL Fc-IgG (antihistamine) and various concentrations of histamine-BSA: (9) 40, (2) 20, and (b) 10 µg/mL at 37 °C.
Figure 7. Relationship between current and histamine concentration. Fc-IgG, 3 µg/mL; histamine-BSA, 40 µg/mL.
therefore separated from unreacted Fc-IgG and histamine-FcIgG complex under the desired conditions with a cation-exchange resin-coated matrix column equilibrated at pH 6.5 with 50 mM malonate buffer. Using the electrochemical flow immunoassay equipment described in the Experimental Section, various concentrations of histamine were injected into the system with malonate buffer eluent under optimal analysis conditions. The time course for the formation of immunocomplex was examined using various concentrations of histamine-BSA and Fc-IgG in the reaction mixture. After immunocomplex formation, the reaction mixture was applied to the matrix column. The immunoreaction reached equilibrium within 20 min using 3 µg/ mL Fc-IgG (Figure 6). A typical calibration response for histamine is shown in Figure 7. The signal from this complex decreased with increasing histamine concentration. Reversible, reproducible, and sensitive responses were obtained, indicating that the liquid-phase immunological sensing system was efficient. A good relationship was observed between the signal and histamine concentration in a range of ∼200 to 2000 ng/mL. This method was faster (2 min), simpler, and more precise (
