Correspondence Anal. Chem. 1996, 68, 1276-1278
Detection of Microspotted Carcinoembryonic Antigen on a Glass Substrate by Scanning Electrochemical Microscopy Hitoshi Shiku, Tomokazu Matsue,* and Isamu Uchida
Department of Applied Chemistry, Graduate School of Engineering, Tohoku University, Sendai 980-77, Japan
Microspots of carbinoembryonic antigen (CEA) on glass substrates were characterized by scanning electrochemical microscopy (SECM). CEA was immobilized via a sandiwch method using horseradish peroxidase (HRP)labeled anti-CEA. The reduction current of the oxidized form of ferrocenylmethanol generated by the HRP reaction was monitored to view SECM images. This method detects as low as ∼104 CEA molecules in a single 20-µmradius spot.
We have investigated the SECM characterization of glass surfaces with 20-µm-radius microspots of horseradish peroxidase (HRP) and a complex of carcinoembryonic antigen (CEA) and HRP-labeled anti-CEA on the basis of detection of the enzyme reaction. CEA is known as a specific antigen for cancer. The labeled HRP catalyzes the oxidation of a reduced electron mediator (Mred) by H2O2 to yield the oxidized mediator (Mox), which can be detected by SECM. Our objective is to couple the SECM HRP
2Mred + H2O2 + 2H+ 98 2Mox + 2H2O The enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect trace amounts of biologically important substances in clinical chemistry laboratories.1,2 However, further development and refinement of ELISA continues to improve sensitivity, simplify analytical procedures, and shorten operation time. Among the various methods developed, electrochemical detection systems have been coupled with enzyme immunoassays.3-9 We report here the characterization of antigenantibody complexes on glass substrates using scanning electrochemical microscopy (SECM).10-12 SECM, a member of the scanning probe microscopies, detects localized electrochemical phenomena at a probe microelectrode. One of the notable features of SECM is the possibility of detecting molecules in an extremely small volume, as was shown Fan and Bard,13 who detected a single molecule by SECM. (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (2) Hage, D. S. Anal. Chem. 1995, 67, 455R-462R. (3) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R. Clin. Chem. 1985, 31, 1546-1549. (4) Jenkins, S. H.; Heineman, W. R.; Halsall, H. B. Anal. Biochem. 1988, 168, 292-299. (5) Niwa, O.; Xu, Y.; Halsall, H. B.; Heinemann, W. R. Anal. Chem. 1993, 65, 1559-1563. (6) Aizawa, M.; Morioka, A.; Suzuki, S.; Nagamura, Y. Anal. Biochem. 1979, 94, 22-28. (7) Robinson, G. A.; Cole, V. M.; Rattle, S. J.; Forrest, G. C. Biosensors 1986, 2, 45-57. (8) Huet, D.; Bourdillon, C. Anal. Chim. Acta 1993, 272, 205-212. (9) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369-1377. (10) Engstrom, R. C.; Pharr, C. M. Anal. Chem. 1989, 61, 1099A-1104A. (11) Bard, A. J.; Fan, F.-R. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. O.; Zhou, F. Science 1991, 254, 68-74. (12) Arca, M.; Bard, A. J.; Horrocks, B. R.; Richards, T. C.; Treichel, D. A. Analyst 1994, 119, 719-726. (13) Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871-874.
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technology with ELISA to develop a novel SECM-ELISA system. This system has several advantages: (1) The method requires no incubation time. The products of the labeled enzyme reaction are detected directly at a microelectrode tip located nearby. (2) The sample volume needed to make a spot is extremely small (several tens of picoliters), which in turn indicates that the absolute number of the analyte molecules to be detected is very small. (3) Since a huge number of the sample spots can be formed in a small area, it is possible to characterize many samples in a single SECM image. Recently, Wittstock et al.14 demonstrated the visualization of immobilized antibodies by SECM. EXPERIMENTAL SECTION Materials. Horseradish peroxidase (HRP, Funakoshi), bovine serum albumin (BSA), glutaraldehyde (GA, Wako Chemical Co.), (3-aminopropyl)triethoxysilane, n-octadecyltrichlorosilane (Tokyo Kasei), and Tween 20 (Kanto Chemical Co.) were used as received. Ferrocenylmethanol (FMA) was synthesized by the reduction of ferrocenecarboxyaldehyde (Aldrich). FMA+ was synthesized by the electrooxidation of FMA before the measurement. Carcinoembryonic antigen (CEA), anti-CEA, and HRPlabeled anti-CEA were donated by Mochida Pharmaceutical Co. CEA is a glycoprotein, and its molar masss is ∼180 000-200 000 Da.15 Preparation of HRP Immobilized Substrate. A glass slide was dipped into a 10 mM (3-aminopropyl)triethoxysilane/benzene solution for 8 h. An aqueous mixture of equal volumes of a 1600 (14) Wittstock, G.; Yu, K.-j.; Halsall, H. B.; Ridgway, T. H.; Heinemann, W. R. Anal. Chem. 1995, 67, 3578-3582. (15) Samejima, S.; Sawada, T. Rinshyoi 1993, 19, 960-961 (in Japanese). 0003-2700/96/0368-1276$12.00/0
© 1996 American Chemical Society
Figure 1. SECM images of a HRP immobilized spot on a glass substrate in a 0.5 mM H2O2/0.1 M KCl/0.1 M phosphate buffer solution (pH 7.0). Mediator, 1.0 mM FMA+ (A) and 1.0 mM FMA (B); d ) 10 µm.
units/mL HRP/phosphate buffer solution, a 5 mg/mL BSA/ phosphate buffer solution, and a 1% (v/v) GA/water solution was spotted on the silanized glass slide at intervals of 100 µm by a glass capillary (tip radius, 20 µm) controlled with a motor-driven actuator. Preparation of Antigen-Antibody Immobilized Substrate. The antigen-antibody was immobilized by a sandwich method.4 A slide glass was successively dipped into a 10 mM n-octadecyltrichlorosilane/benzene solution for 8 h and a 500 µg/mL antiCEA/phosphate buffer solution for 2 h. CEA/phosphate buffer solution was then spotted on the anti-CEA-adsorbed glass surfaces at intervals of 100 µm by the glass capillary. The optical microscopic observation demonstrated that the sizes of the spots were almost uniform and that the average radius of the spots was ∼20 µm. If we assume that the spot is hemispherical, then the volume of the spot is 17 pL. After spotting with CEA, the substrate was soaked in a 15 µg/mL HRP-labeled anti-CEA/0.1% Tween 20/ phosphate buffer solution for 20 min. Between these steps, the substrate was washed thoroughly with water. Measurement System. The microelectrode for SECM was fabricated as follows. A Pt wire (radius, 7.5 µm) was etched electrochemically in saturated NaNO3 and inserted into a soft glass capillary. The tip was fused in a small furnace at 320 °C in vacuo to coat the Pt filament with the soft glass. The tip was then polished on a turntable (Narishige, Model EG-6). The Pt disk radius, including the insulating part, was 30 µm. The Pt disk radius was determined from the steady-state voltammogram to be 2.4 µm. The measurements were carried out in a two-electrode configuration. The current was amplified with a Keithley Model 427 amplifier. Movement of the microelectrode tip was performed by means of a motor-drive XYZ stage (Chuo Seiki, M9103). The details of the SECM system were described in a previous paper.16 The measurement solution was 0.1 M KCl/0.1 M phosphate buffer (pH 7.0) containing 0.5 mM H2O2 (unless otherwise stated), with 1.0 mM FMA+ or 1.0 mM FMA as the electron mediator. The potential of the tip electrode was held at 0.05 V vs Ag/AgCl. The tip was scanned over the substrate at 9.8 µm/s. The distance (d) between the electrode and the substrate surfaces was estimated using a working curve which represents a theoretical oxidation current vs d profile.17 (16) Shiku, H.; Takeda, T.; Yamada, H.; Matsue, T.; Uchida, I. Anal. Chem. 1995, 67, 312-317. (17) Yamada, H.; Shiku, H.; Matsue, T.; Uchida, I. Bioelectrochem. Bioenerg. 1994, 33, 91-93.
RESULTS AND DISCUSSION Before applying the SECM measurements to characterize the antigen-antibody immobilized substrate, we investigated the SECM behavior of the HRP immobilized glass surfaces. Figure 1 shows SECM images of a HRP spot on a glass surface in two observation modes, feedback mode and generation mode.18 The difference in the SECM images originates from the fundamental difference in the detection mode. In the feedback mode, the substrate was immersed in a 1.0 mM FMA+/0.5 mM H2O2/0.1 M KCl/0.1 M phosphate buffer (pH 7.0) solution, and the reduction current of FMA+ was monitored to visualize SECM images. The electrode tip was scanned over the substrate, maintaining d ) 10 µm. The spot of immobilized HRP was observed clearly. In this mode, the reduction current of FMA+ increases when the microelectrode is scanned above the spot. At the tip, microelectrode FMA+ is reduced to FMA, which diffuses into the substrate and is oxidized back to FMA+ by the HRP catalytic reaction. Thus, oxidation-reduction cycles proceed between the tip and substrate. The redox cycling enhances the reduction current observed at the tip. In the generation mode, the substrate is immersed in a 1.0 mM FMA/0.5 mM H2O2/0.1 M KCl/0.1 M phosphate buffer (pH 7.0) solution. In this mode, the enzyme reaction continuously generates FMA+, which diffuses into the solution. The electrode tip detects the enzymatically generated FMA+. The redox cycling can also be operative in enhancing the response in the generation mode. The comparison of SECM images in the both modes indicates that the generation mode gives lower background currents and larger responses. Thus, the generation system is suitable for characterizing surfaces with low enzyme activities. We used the generation mode in the following measurements. However, it should be noted that the positional resolution in the feedback mode is superior to that of the generation mode, as can be seen in Figure 1. In the generation mode, the diffusion of FMA+ generated continuously from the active HRP area obscures the fine fluctuations in enzyme activity. The concentration of H2O2 in solution affected the SECM images. Figure 2 shows the reduction current profiles of the intersection, as indicated by the arrow in Figure 1B, in the presence of various concentrations of H2O2. Typically, the reduction currents decrease to 10% of the peak currents at a distance of 60 µm from the spot center. When the concentration of H2O2 is larger than 0.1 mM, the profile is almost independent (18) Pierce, D. T.; Bard, A. J. Anal. Chem. 1993, 65, 3598-3604.
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Figure 2. Reduction current profiles with the background subtracted along the arrow indicated in Figure 1B. H2O2 concentrations; 0 (+), 0.05 (4), 0.1 (0), 0.2 (2), 0.3 ((), and 0.5 mM (O).
Figure 3. SECM image of a series of antigen-antibody immobilized spots in a 1.0 mM FMA/0.5 mM H2O2/0.1 M KCl/0.1 M phosphate buffer solution (pH 7.0). CEA concentration of the solution for making spots, 2.0 (left) and 0.2 µg/mL (right); d ) 10 µm.
of the concentration. Under this circumstance, the response is controlled by the reaction between HRP and FMA. The SECM images of the substrate immobilized with antigen-antibody adducts were obtained in the 1.0 mM FMA/0.5 mM H2O2/0.1 M KCl/0.1 M phosphate buffer (pH 7.0) solution. Figure 3 shows an SECM image of the substrate with CEA/ HRP-labeled anti-CEA spots. The appearance of circular spots with large reduction currents is attributed to the HRP-catalyzed reactions at the spots, which indicates the localized presence of CEA. The reduction currents depend on the CEA concentration of the solution for making a CEA spot. In this figure, the five spots on the left were made using a 2.0 µg/mL CEA solution, and the five spots on the right were made using a 0.2 µg/mL CEA solution. Since the volume needed to make a spot was extremely small, the number of CEA molecules in a single right
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Figure 4. Variation of the reduction current at the spot center (d ) 10 µm) as a function of the CEA concentration of the solution used for making spots. Spot size, 0.8 mm radius.
spot was as small as ∼104. When CEA was spotted on untreated glass or silanized glass surfaces, no clear image was observed in the SECM measurements. The dependence of the reduction current on the CEA concentration was investigated in more detail using a substrate with 0.8mm-radius CEA spots. These spots were formed on the substrate by dropping 1.0-µL solutions with different CEA concentrations. We measured the reduction current vs d profiles at the spot centers. Figure 4 shows the variation of the response 10 µm away from the spot center as a function of the CEA concentration used to make the spot. The response increases with concentration in the range of 10-7-10-5 g/mL. No obvious response was observed at concentrations less than 20 nM. The response tends to be saturated at higher concentrations. The amount of CEA molecules in a drop of the 20 µg/mL solution corresponds roughly to that which forms a CEA monolayer (∼10-13 mol/cm2) in the spot. The extra CEA molecules would be washed out during the cleaning process. The SECM technique described above can be applied to ELISA. This method detects electrochemical phenomena occurring in extremely small volumes and needs no incubation time for a labeled enzyme reaction. Further investigations of the SECM-ELISA system and the creation of ultrasmall biological structures are now underway in our laboratory. ACKNOWLEDGMENT This research was partially supported by Grants-in-Aid for Scientific Research (Nos. 05235102 and 07241206) from the Ministry of Education, Science and Culture, Japan. Received for review August 14, 1995. Accepted January 20, 1996.X AC950824C X
Abstract published in Advance ACS Abstracts, March 1, 1996.
