Anal. Chem. 2003, 75, 4578-4584
Ultrathin Alumina Sol-Gel-Derived Films: Allowing Direct Detection of the Liver Fibrosis Markers by Capacitance Measurement Dechen Jiang, Jia Tang, Baohong Liu, Pengyuan Yang, and Jilie Kong*
Department of Chemistry, Fudan University, Shanghai 200433, P.R. China
A capacitive immunoassay based on antibody-embedded ultrathin γ-alumina sol-gel films (∼20 to 40 nm) was successfully prepared in this work. The nanofilms greatly increased the capacitance change initiated by the recognition between the immobilized antibody and the target antigen, which allowed capacitive measurements capable of directly determining the antigen more sensitive than that of thick films. Meanwhile, the inorganic films with high permittivity significantly increased the time constant (i.e., RC value) of the films, which rendered the potentiostatic step method with acceptable S/N ratio. These two advantages enabled the immunosensor to be readily employed in a multichannel capacitance analysis system. An eight-channel hIgG capacitive sol-gel-derived immunoassay based on this system was constructed to illustrate the application. Compared with the detection limits of SiO2 sol-gel-derived hIgG capacitive immunosensors or the conventional ELISA immunoassay, the immunoassay based on thin alumina gel film showed a lower detection limit of 1 ng mL-1. The novel immunoassay was employed to co-determine two liver fibrosis markers (hyaluronan and laminin) in mixed samples from ∼0.5 to 50 ng mL-1. The little derivation caused by the interfered antigen indicated that the sensitive, specific, low-cost sol-gelderived multichannel immunosensors might be a promising approach in the application of screening disease markers. Immunosensors have provoked much interest for high specificity and selectivity that can be applied in clinical analysis and environmental monitoring.1,2 The Self-assembled monolayers (SAMs) technique was often used for the functional film immobilization because it could self-assemble the target artificially to form an ordered and dense monolayer.3-5 However, the covalent immobilization process of the antibody on the thiol-modified monolayer was somewhat complex, and the procedure for linking the antibody onto acid-terminated thiol by forming an amino acid * Corresponding author. Phone: +86-21-65642405. Fax: +86-21-65641740. E-mail:
[email protected]. (1) Luppa, P. B.; Sokoll, L. J.; Chan, D. W. Clin. Chim. Acta 2001, 314, 1-26. (2) Hennion, M. C.; Barcelo, D. Anal. Chim. Acta 1998, 362, 3-34. (3) Katz, E.; Alfonta, L.; Willner, I. Sens. Actuators, B. 2001, 76, 134-141. (4) Bardea, A.; Katz, E.; Willner, I. Electroanalysis 2000, 12, 1097-1106. (5) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977-989.
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chain between them might denature the protein.6 The use of solgel chemistry to encapsulate the proteins has been reported as an alternative effective method applied to immunosensors.7-9 Compared with the SAMs technique, the sol-gel approach could not only provide a mild environment for the embedded protein and preserve the integrity and homogeneity of the protein surface microstructure,10 but also make the batch production of multichannel immunoassays possible by screening-printing or dotmatrix patterning.11 Amperometric enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and optical immunoassay have been successfully applied on the sol-gel-derived immunosensor,7-9 while both the immunoassays needed the sample labeled, which made the analysis complex. Thus, developing a direct (label-free), quick-response immunoassay employing sol-gel-derived immunosensors motivated our group to undertake this work. Recently, capacitive immunosensors have been extensively studied as a novel label-free immunoassay with high sensitivity, speedy test time, and ordinary instrumentations.12-16 Two factors were critical for the feasibility of capacitive immunosenors. First, the formed film must be insulating to allow the capacitive measurement. Second, the antibody-embedded gel film must be as thin as a nanometer one, normally prepared by SAMs technique; otherwise, the capacitance of the diffuse layer on the electrode might dominate the total capacitance of the immunosensor, which would make the capacitance change caused by the interaction of Ag-Ab not be significantly detected.15,16 The sol-gel matrix, such as SiO2, which is the one normally used to immobilize proteins,17 often formed a thick film (at a several (6) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1-36. (7) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 11711175. (8) Bronshtein, A.; Aharonson, N.; Avnir, D.; Turniansky, A.; Altstein, M. Chem. Mater. 1997, 9, 2632-2639. (9) Pulido-Tofino, P.; Barrero-Moreno, J. M.; Perez-Conde, M. C. Anal. Chim. Acta 2001, 429, 337-345. (10) Lev, O.; Tsionsky, M.; Rabinovich, L.; Glezer, V.; Sampath, S.; Pankratov, I.; Gun, J. Anal. Chem. 1995, 67, 7, A22-A30. (11) Kim, Y. D.; Park, C. B.; Clark, D. S. Biotechnol. Bioeng 2001, 73, 331-337 (12) Berggren, C.; Bjarnason, B.; Johansson, G. Electroanalysis 2001, 13, 173180. (13) Granek, V.; Rishpon, J. Environ. Sci. Technol. 2002, 36, 1574-1578. (14) Hu, S. Q.; Wu, Z. Y.; Zhou, Y. M.; Cao, Z. X.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2002, 458, 297-304. (15) Berggren, C.; Bjarnason, B.; Johansson, G. Biosens. Bioelectron. 1998, 13, 1061-1068. (16) Berggren, C.; Johansson, G. Anal. Chem. 1997, 69, 3651-3657. (17) Holt, D. B.; Gauger, P. R.; Kusterbeck, A. W.; Ligler, F. S. Biosens. Bioelectron. 2002, 17, 95-103 10.1021/ac034046x CCC: $25.00
© 2003 American Chemical Society Published on Web 07/24/2003
hundred nanometers level),18 which might be too thick for capacitive measurements. This was the reason, on one hand, that most of the reported capacitive biosensors employed the SAMs technique to prepare biocomposite films,3,15,16 and on the other hand, almost all the reported sol-gel-based immunosensors employed labeled methods,7-9 instead of the mentioned label-free capacitive measuring approach. Thus, searching a sol-gel matrix to prepare ultrathin biocomposite films allowing capacitive detection initiated the pursuit of this work. γ-Alumina sol-gel was an efficient matrix that could immobilize biological species to fabricate biosensors. In our previous work, it had been successfully used to encapsulate tyrosinase to sensitively detect phenols at a nanomoles-per-milliliter level.19,20 Compared with a SiO2 matrix, γ-alumina had an inherent high surface area, which could reduce the thickness of formed films significantly and might be suitable to fabricate the capacitive immunosensor.21 Thus, γ-alumina solgel was chosen here to prepare an antibody-embedded thin film to enable the label-free capacitive measurement. Here, the potentiostatic step, normally performed in seconds for each test, was chosen to measure the capacitance of the immunosensors. Coupled with a multichannel sampling system, the method would be a promising approach for the high throughput screening target antigens. Compared with multichannel immunoassays reported before, such as fluorescence-labeled method,22 SPR23 and ELISA systems,24 the capacitive multichannel immunoassays emerged by its relative high data-acquiring speed, easy-handling and inexpensive instrumentations. Human immunoglobulin G (hIgG), a normal target analyte for the immunoassay, was chosen as a model protein to illustrate the application of the ultrathin γ-alumina film on the sol-gel-derived capacitive immunosensors. Then, the immunoassay was used as an efficient transducer for the immobilization of the antibodies to directly detect two main liver fibrosis markers, hyaluronan (HA) and haminin (LN) antigens, simultaneously.25 The potentiostatic step was employed to measure the capacitance change caused by the interaction between the corresponding Ag-Ab. Detailed properties for the sensitive, specific, low-cost, sol-gel-derived multichannel immunosensors were reported. EXPERIMENTAL SECTION Chemicals. Purified hyaluronan-binding cartilage protein (HABP)26-28 and rabbit anti-human laminin (LN, antibody)25,29,30 were prepared and purified as in the literature. Hyluronan (H7630, 30 kDa) and laminin (90 KDa) were purchased from Sigma. (18) Goring, G. L. G.; Brennan, J. D. J. Mater. Chem. 2002, 12, 3400-3406 (19) Liu, Z. J.; Liu, B. H.; Kong, J. L.; Deng, J. Q. Anal. Chem. 2000, 72, 47074712. (20) Liu, Z. J.; Deng, J. Q.; Li, D. Anal. Chim. Acta 1999, 392, 135-141. (21) Agrafiotis, C.; Tsetsekou, A. J. Eur. Ceram. Soc. 2002, 22, 423-434. (22) Schuderer, J.; Akkoyun, A.; Brandenburg, A.; Bilitewski, U.; Wagner, E. Anal. Chem. 2000, 72, 3942-3948. (23) Berger, C. E. H.; Beumer, T. A. M.; Kooyman, R. P. H.; Greve, J. Anal. Chem. 1998, 70, 703-706. (24) Tang, T. C.; Deng, A. P.; Huang, H. J. Anal. Chem. 2002, 74, 2617-2621. (25) Korner, T.; Kropf, J.; Gressner, A. M. J. Hepatol. 1996, 25, 684-688. (26) Tengblad, A. A. Biochim. Biophys. Acta 1979, 578, 281-289. (27) Banerjee, S. D.; Toole, B. P. Dev. Biol. 1991, 146, 186-197 (28) Wyatt, H. A.; Dhawan, A.; Cheeseman, P.; Mieli-Vergani, G.; Price, J. F. Arch. Dis. Child. 2002, 86, 190-193 (29) Chen, J. G. Immunol. J. (Chinese), 1996, 12, 53-55. (30) Capra, F.; Casaril, M.; Gabrielli, G. B.; Tognellap, P.; Rizzi, A.; Dolci, L.; Colombari, R.; Mezzelani, P.; Corrocher, R.; Desandre, G. J. Hepatol. 1993, 18, 112-118.
Human immunoglobulin G (hIgG) and goat anti-hIgG were purchased from Shensys Diagnostic Technology Co. Ltd. (Shanghai, China). 1-Hexadecanthiol was obtained from Fluka. Aluminum isopropoxide (Al(i-PrO3)) and ethylsilicate (TEOS) were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). All the other chemicals were of reagent grade and used as received. Phosphate buffered saline (PBS) was used for the preparation of buffers. All water used was demineralized water. Note: HABP, instead of anti-hyaluronan (antibody), was chosen to determine hyaluronan, with the detection sensitivity ∼100 times higher than that of the latter.31-33 Thickness Measurement. X-ray fluorescent coating thickness measurement (SEA5120 energy-dispersive X-ray spectrometer, micro-XRF element monitor, MX Instrument, Seiko Instruments. Inc) was a versatile, quick, and highly sensitive measurement for the nanometer thickness layer,34,35 which was chosen to measure the thickness of biocomposite gel films on the gold electrode surface. The X-ray (Mo) voltages for the gel and Au films were 15 and 45 V, respectively, which produced a collimated beam of X-rays to excite the film on a substrate. The induced emitted X-rays associated with the sample were detected and calibrated to the standard to obtain quantitative thickness information. The measurement time was 100 s for the gel and Au films. All of the measurements were performed under vacuum. Electrochemical Measurement. Eight-channel immunosensors were designed with eight annularly set integrated gold disk electrodes (CH Instrument, U.S.A.; 2.2 mm in diameter for each electrode). In the case of the measurement for hIgG, all eight channel sensors were employed to immobilize goat anti-hIgG antibodies. For the detection of the fibrosis serum markers, four of the eight sensors were used for the immobilization of HABP; the other four were for anti-LN. A Pt plate and saturated calomel electrode (SCE) were set in the center as the counter and reference electrodes in the eight-channel system. The electrochemical measurements were carried out with a CHI1030 multichannel voltammetric analyzer (CH Instrument, U.S.A.). The potential-step measurements for eight-channel immunosensors were performed in 10 mM PBS (pH 7.0) that allowed all the current data to be sampled at a frequency of 50 kHz. Cyclic voltammetric measurements for the eight-channel system were performed simultaneously in the presence of 5 mM K3 [Fe(CN)6]/ K4[Fe(CN)6] (1:1) mixture as redox probe in 10 mM PBS containing 0.1 M KCl (pH 7.0). The impedance spectroscopy was performed using a potentiostat/galvanostat (model 273, EG&G, Princeton Applied Research) with a two-phase lock-in analyzer (model 5208). For ac impedance measurement, the frequency range was 10 Hz to 10 KHz in PBS, and 10 mV was applied as an alternating voltage. Microgravimetric Measurement. The microgravimetric analysis was performed with a quartz microbalance (QCM) analyzer (CHI420, CH Instrument, U.S.A.) and quartz crystals (7.995 MHz) sandwiched between two Au electrodes (area 0.196 cm2). The frequency changes of the crystals were used for the quantitative (31) Scott, J. E. Methodol. Biochem. Anal. 1960, 8, 145-197. (32) Ohya, T.; Kaneko, Y. Biochim. Biophys. Acta 1970, 198, 607-609. (33) Ulla, B. G. L.; Anders, T. Anal. Biochem. 1980, 109, 386-394. (34) Sugihara, K.; Tamura, K.; Sato. M.; Ohno, K. X-ray Spectrom. 1999, 28, 446-450. (35) Link, D. D.; Kingston, H. M.; Havrilla, G. J.; Colletti, L. P. Anal. Chem. 2002, 74, 1165-1170.
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analysis of the stability of the ultrathin alumina biocomposite layer on Au electrodes, and the mass change caused by the interaction of the embedded antibody and the target antigen. Preparation of Al2O3 and SiO2 Sol. The alumina sols were prepared as previously reported work,36 briefly described as the following: the mixtures of Al(i-PrO3) and distilled water with various molar ratios of Al/H2O were stirred at 80 °C for 1 h and hydrolysis at 90 °C by adding 1 M HCl. Then the vessel was opened to evaporate 2-propanol at 90 °C. Finally, the mixture was refluxed for 8 h at that temperature to obtain a stable γ-alumina sol. SiO2 sols with different molar ratios of Si/water were prepared as in the literature:7,18 the sols were prepared by mixing 4.5 mL of TEOS with water, 1.0 mL of ethanol, and 0.1 mL of 0.05 M hydrochloric acid. The mixture was stirred for ∼1 h until a clear and homogeneous solution resulted. Fabrication of Sol-Gel-Derived Thin-Film Immunosensors. Gold disk electrodes were polished carefully with alumina slurries (0.3, 0.05 um) and then cleaned through sonication in distilled water, 3:1(v/v) H2SO4/H2O2 solution, and absolute ethanol. Finally, the electrodes were dried with purified N2. The immunosensors were fabricated by dripping the mixture of the antibody (0.1 mg mL-1) and Al2O3 sol (1:1 v/v), 0.5 µL, with a microliter syringe on bare gold electrodes.18 Afterward, the immunosensors were dried at 4 °C for 3 days. Finally, they were immersed in 1-hexadecanthiol solution for 1 h to block the pinholes on the gold surface. The same procedure was employed to fabricate SiO2 sol-gel-derived capacitive immunosensors with the above-prepared SiO2 sol-gel material. All the fabrication processes were monitored with Fe2+/3+ redox couples, as reported before.37 Capacitance Measurement. The capacitance changes for the different channels of the immunosensors were evaluated in 10 mM PBS (pH ) 7.0) using potentiostatic steps. Before each measurement, the modified immunosensors were incubated in 10 mM PBS with different concentrations of antigen for 20 min at 38 °C to make the interaction between Ag and Ab reach saturation. RESULTS AND DISCUSSION. Thickness Measurement of Al2O3 and SiO2 Biocomposite Films. As mentioned above, the thickness of the prepared biocomposite films was the key factor affecting the properties of the capacitive immunosensors. Table 1 showed the thickness of Al2O3 and SiO2 gel films with different water contents. The thickness of the prepared protein-embedded Al2O3 gel films with an Al/H2O ratio of 1:100, a normal ratio where a porous transparent Al2O3 was formed,36 was ∼40 nm. Adding the water content in the sol to 1:300, the thickness of alumina biocomposite gel decreased to ∼20 nm. The films thickness was much thinner than that of the prepared SiO2 biocomposite film, that is ∼ to 370 nm, with the reported optimum Si/H2O ratio 1:8 employed in an amperometric and optical immunoassay.7,9 Upon increasing the water content in the SiO2 sol, even to a ratio of 1:100, the film thickness was not found to decrease remarkably (∼250 to 270 nm), while the high water content in the SiO2 sol would create a looser gel, which worsened the efficiency of the encapsulation (36) Yoldas, B. E. J. Mater. Sci. 1975, 10, 1856-1860. (37) Jiang, D. C.; Tang, J.; Liu, B. H.; Shen, G. L.; Yang, P. Y.; Kong, J. L. Biosens. Bioelectron. 2003, 18, 1183-1191.
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Table 1. Thickness of Al2O3-Antibody and SiO2-Antibody Gel Filmsa Al2O3 (nm)
1 2 3 4 5
SiO2 (nm)
Al/water 1:100
Al/water 1:300
Si/water 1:10
Si/water 1:100
31 ( 3 42 ( 4 37 ( 4 40 ( 4 41 ( 4
22 ( 2 19 ( 2 21 ( 2 19 ( 2 24 ( 2
370 ( 37 350 ( 35 350 ( 35 370 ( 37 370 ( 37
250 ( 25 260 ( 36 270 ( 37 250 ( 25 250 ( 25
a Each data value was obtained by five tests for the different regions of the film; the derivation was estimated as the maximum one, 10%, of the SEA 5120 Element Monitor MX Instrument38.
and prolonged the drying time for the immunosensors.39 These indicated that the ultrathin films for SiO2 gel with a thickness of
