Anal. Chem. 2006, 78, 1769-1773
Au Nanoparticle Conjugation for Impedance and Capacitance Signal Amplification in Biosensors Jianbin Wang,† James A. Profitt,‡ Michael J. Pugia,‡ and Ian I. Suni*,†
Department of Chemical Engineering and Center for Advanced Materials Processing (CAMP), 8 Clarkson Avenue, Clarkson University, Potsdam, New York 13699-5705, and Diagnostics Division, Bayer Healthcare, 1111 Bristol Street, Elkhart, Indiana 46515
Amplification of the electrochemical impedance and capacitance signals in a biosensor is demonstrated for the model fluorescein/anti-fluorescein system. Following immobilization of fluorescein onto Au through formation of a self-assembled monolayer, goat anti-fluorescein conjugated with 10-nm Au nanoparticles is introduced into the system. This results in an increase in the capacitance of ∼400 nF/cm2, whereas no change can be observed for goat anti-fluorescein without the Au nanoparticle conjugate. An even greater sensitivity is obtained by introduction of a redox probe, [Fe(CN)6]3-/4-, whereby the chargetransfer resistance (Rct) is reduced to ∼25% of its original value. This allows construction of high-sensitivity electrochemical impedance biosensors at a single low frequency, where the signal is sensitive to the interfacial Rct. This change in the electrochemical impedance signal upon binding to goat anti-fluorescein conjugated with Au nanoparticles can be attributed to the much higher electrochemical activity of Au surfaces relative to the underlying organic layer. Electrochemical impedance and capacitance biosensors have been employed to study cell growth,1,2 protein binding at surfaces,3,4 bacterial growth,5 DNA hybrization,6-9 and antibodyantigen binding.10-13 These techniques may be more sensitive than * Corresponding author. Telephone: (315)268-4471. E-mail:
[email protected]. † Clarkson University. ‡ Bayer Healthcare. (1) Luong, J. H. T.; Habibi-Rezaei, M.; Meghrous J.; Xiao C.; Male, K. B.; Kamen, A. Anal. Chem. 2001, 73, 1844. (2) Xiao, C.; Lachance, B.; Sunahara, G.; Luong, J. H. T. Anal. Chem. 2002, 74, 1333. (3) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977. (4) Desilva, M. S.; Zhang, Y.; Hesketh, P. J.; Maclay, G. J.; Gendel, S. M.; Stelter, J. R. Biosens. Bioelectron. 1995, 10, 675. (5) Gomez, R.; Bashir, R.; Sarikaya, A.; Ladisch, M. R.; Sturgis, J.; Robinson, J. P.; Geng, T.; Bhunia, A. K.; Apple, H. L.; Wereley S. Biomed. Microdevices 2001, 3, 201. (6) Bardea, A.; Patolsky, F.; Dagan, A.; Willner, I. Chem. Commun. 1999, 1, 21. (7) Xu, Y.; Cai, H.; He, P. G.; Fang, Y. Z. Electroanalysis 2004, 16, 150. (8) Xu, Y.; Jiang, Y.; Cai, H.; He, P. G.; Fang, Y. Z. Anal. Chim. Acta 2004, 516, 19. (9) Van Gerwen, P.; Laureyn, W.; Laureys, W.; Huyberechts, G.; Op De Beeck, M.; Baert, K.; Suls, J.; Sansen, W.; Jacobs, P.; Hermans, L.; Mertens, R. Sens. Actuators, B: Chem, 1998, 49, 73. (10) Dijksma, M.; Kamp, B.; Hoogvliet, J. C.; Van Bennekom, W. P. Anal. Chem. 2001, 73, 901. 10.1021/ac051113+ CCC: $33.50 Published on Web 02/08/2006
© 2006 American Chemical Society
electrochemical biosensors that employ amperometry, where the signal depends critically on the relative proximity of the active site and the electrode surface. Ideally, reagentless label-free impedance and capacitance biosensors should be developed where the signal change arises only from the biological recognition event.10,14 However, signal amplification may also be useful in many situations, such as the detection of small-molecule ligands of biomedical interest, where ligand binding may not significantly perturb the biosensor interface. For example, the biocatalyzed precipitation of an insoluble product by a redox enzyme label onto an electrode surface has been employed as an amplification route for both antigen/antibody15-17 and DNA recognition.18,19 Signal amplification of an impedance biosensor can also be accomplished by labeling the analyte with metal ions20 or proteins.21 Signal amplification of impedance biosensors by labeling the analyte with semiconductor nano/microparticles has been reported for a DNA biosensor.22 However, the use of metal nanoparticles may be advantageous due to their more electrochemically active surface. In other words, their double layer capacitance is typically greater than that of other materials. Here, using the model fluorescein/anti-fluorescein system, we study the amplification effect of analyte conjugation with Au nanoparticles for development of electrochemical impedance and capacitance biosensors. The high affinity of anti-fluorescein antibodies for fluorescein and its derivatives make this an ideal model system.23 Au has been widely used in biosensor fabrication because it is hydrophilic, biocompatible, and inert. Furthermore, Au nanoparticles can be easily fabricated by standard methods,24 and methods for preparing Au nanoparticle conjugates are also well known.25 (11) Berggren, C.; Johansson, G. Anal. Chem. 1997, 69, 3651. (12) Ruan, C.; Yang, L.; Li, Y. Anal. Chem. 2002, 74, 4814. (13) Pei, R. J.; Cheng, Z. L.; Wang, E. K.; Yang, X. R. Biosens. Bioelectron. 2001, 16, 355. (14) Wang, J.; Carmon, K. A.; Luck, L. A.; Suni, I. I. Electrochem. Solid-State Lett. 2005, 8, H61. (15) Alfonta, L.; Willner, I. Anal. Chem. 2001, 73, 5287. (16) Ruan, C.; Yang, L.; Li, Y. Anal. Chem. 2002, 74, 4814. (17) Bardea, A.; Katz, E.; Willner, I. Electroanalysis 2000, 12, 1097. (18) Alfonta, L.; Singh, A. K.; Willner, I. Anal. Chem. 2001, 73, 91. (19) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 3703. (20) Xu, Y.; Jiang, Y.; Cai, H.; He, P.; Fang, Y. Anal. Chim. Acta 2004, 516, 19. (21) Pei, R.; Cheng, Z.; Wang, E.; Yang, X. Biosens. Bioelectron. 2001, 16, 355. (22) Xu, Y.; Cai, H.; He, P. G.; Fang, Y. Z. Electroanalysis 2004, 16, 150. (23) Harmer, I. J.; Samuel, D. J. Immunol. Methods 1989, 122, 115. (24) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959.
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 1769
Figure 1. Electrode complex after fluoresceinamine immobilization. The amine on the fluoresceinamine reacts with NHSS activated carboxlic acid on the SAM, forming a stable amide bond.
EXPERIMENTAL SECTION Materials. Glass slides with a 100-nm Au film atop a 5-nm Ti adhesion layer were purchased from Evaporated Metal Films (Ithaca, NY); fluoresceinamine isomer I and 11-mercaptoundecanoic acid (11-MUA) were purchased from Aldrich; N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), trisbuffered silane (TBS), potassium dihydrogen phosphate, and dipotassium dihydrogen phosphate were purchased from Sigma; N-hydroxysulfosuccinimide sodium salt (NHSS) was purchased from Pierce; goat anti-fluoroscein conjugated with 10-nm gold particles was purchased from Ted Pella; and goat anti-fluoroscein without conjugation was purchased from Molecular Probes. All reagents were used as received. Gold Electrode and Biosensor Preparation. The gold electrode was fixed by an O-ring onto an electrochemical cell constructed from virgin Teflon. The conical electrochemical cell was designed with an electrode area of 0.28 cm2 and a cell volume of 5 mL. The electrode was cleaned electrochemically by potential scanning in a solution containing 0.1 M H2SO4 and 0.01 M KCl.26 The gold electrode was then modified by immersion for 17 h into 1 mM 11-MUA and 50 mM phosphate buffer solution (pH ) 10) to form a self-assembled monolayer (SAM) with carboxylic group termination. To immobilize fluoresceinamine, the terminal carboxylic groups were activated for 1 h in 75 mM EDC and 15 mM NHSS in 50 mM phosphate buffer solution (pH ) 6.1).27 Since the activated carboxylic group only reacts with deprotonated amine groups, the fluoresceinamine was first dissolved in DMSO and diluted 10 times in 50 mM phosphate buffer (pH ) 8). The buffer solution with 5 mM fluoresceinamine was applied to the activated electrode for 1.5 h to immobilize fluoresceinamine. All experimental procedures involving fluoresceinamine were carried out in the dark to keep fluoresceinamine active. The biosensor electrode structure is shown as Figure 1. To passivate remaining activated carboxylate groups, the electrode was then immersed into tris buffer test solution for at least 30 min prior to use. (25) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, CA, 1995. (26) Nahir, T. M.; Bowden, E. F. J. Electroanal. Chem. 1996, 410, 9. (27) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187.
1770
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006
Experimental Methods. Electrochemical impedance measurements were obtained using a standard three-electrode electrochemical cell, with a Pt counter electrode and saturated calomel reference electrode (SCE). Impedance data were obtained with two different experimental setups, one for steady state and the other for transient measurements. The frequency spectrum from 1.2 to 950 Hz at 10 steps per decade was measured at steady state with a Bioanalytical Systems (BAS) model 100B/W electrochemical workstation, with a built-in multisine ac impedance module. Time evolution of the impedance at a single frequency was measured with a Princeton Applied Research (PAR) model 263A potentiostat and a Stanford Research Systems SR830 lock-in amplifier for phase-sensitive detection. For all experiments, a 5-mV ac probe voltage was employed. The electrolyte used here during impedance measurements is tris-buffered silane (pH ) 8.0). The frequency used for timedependent impedance/capacitance measurements is 20 Hz, and the dc potential is 100 mV versus SCE. No redox probe was added to the test solution for capacitance measurement. This distinguishes them from impedance measurements, where a typical redox probe, [Fe(CN)6]3-/4-, is added to the analyte. This yields a larger current response to the ac voltage probe and increases the electron-transfer rate at the interface, so a full Nyquist plot from 1.2 to 950 Hz can be measured before and after adding antibody. For an immunosensor, where antibody-antigen interaction is detected, impedance sensors are sometimes referred to as capacitance sensors.3 At low frequency, an electrode coated with an insulating polymer/protein layer behaves largely as a capacitor, with the imaginary (capacitive) impedance considerably higher than the real impedance and the solution-phase resistance negligible. The electrode capacitance at one frequency can be obtained from the out-of-phase (imaginary) current by means of a lock-in amplifier. The capacitance is then
C ) iim/Eω
(1)
where C is capacitance, iim is the out-of-phase current, E is the amplitude of the ac probe voltage, and ω is the angular frequency. RESULTS AND DISCUSSION SAM Formation. The Au electrode onto which fluoresceinamine has been immobilized is highly stable in aqueous solution. The capacitance does not change for several hours within the dc potential range from -150 to 150 mV versus SCE. This is not surprising, since SAMs formed from long-chain alkanethiols can be stable for days. Although Berggren and Johansson have argued that the sensitivity of a capacitance biosensor can be improved by moving the binding site closer to the electrode surface with a shorter SAM,11 such a SAM would be less stable. The capacitance of the electrode modified by 11-MUA and fluoresceinamine decreased from ∼35 to ∼3 µF/cm2 at +100 mV versus SCE. The reduced capacitance of this interface provides a lower background signal than the original bare Au surface. Change in Capacitance and Impedance after Adding AntiFluorescein Conjugated with 10-nm Au Nanoparticles. About 25 µL of anti-fluorescein conjugated with 10-nm Au nanoparticles was added to the test electrolyte, resulting in a final antibody
Figure 2. Capacitance change with time at 20 Hz and +100 mV vs SCE after adding anti-fluorescein conjugated with 10-nm Au nanoparticles ([), anti-fluorescein without conjugation (b), and 10nm Au nanoparticles alone (2). Figure 4. Impedance of Au electrode modified with 11-MUA (O), 11-MUA + fluoresceinamine ([), and 1 (2) and 4 h (0) after adding anti-fluorescein conjugated with Au nanoparticles to the fluoresceinmodified gold electrode at +190 mV vs SCE. The test solution also contains 0.5 mM K3Fe(CN)3 and 0.5 mM K4Fe(CN)3 in TBS buffer.
Figure 5. Equivalent circuit employed for all data fits.
Figure 3. Capacitance change as a function of the concentration of the anti-fluorescein/Au nanoparticle conjugate.
concentration of ∼0.15 µg/mL. Figure 2 shows the capacitance change with time following this addition at a dc potential of +100 mV versus SCE. Following addition of the anti-fluorescein/Au nanoparticle conjugate, the capacitance increases gradually and then eventually reaches steady state after ∼4 h. The total capacitance increase is ∼400 nF/cm2. This capacitance increase is irreversible, since it remains after rinsing the electrode surface with buffer solution. Modest capacitance changes of ∼60 nF/cm2 (3-6%) might be expected for antibody-antigen recognition without amplification.3 However, as also illustrated in Figure 2, addition of antifluorescein alone or Au nanoparticles alone does not induce such a capacitance change. This demonstrates that the capacitance change is specifically associated with interaction of the antifluorescein/Au nanoparticle conjugate probe with the immobilized fluorescein layer. The noise in the background capacitance shown in Figure 2 is unmeasurable or, in other words, less than the resolution of the analog-to-digital converter. Thus, although the capacitance change shown in Figure 2 is modest, the signal-tonoise ratio is quite high. The capacitance change as a function of the concentration of anti-fluorescein/Au nanoparticle conjugate is illustrated in Figure 3. This shows that the current impedance biosensor exhibits a logarithmic relationship between the capacitance change and the analyte concentration over the concentration range from 0.015 to 0.75 µg/mL. By adding a redox probe, [Fe(CN)6]3-/4-, to the test solution, a larger current response can be obtained for the same ac probe voltage. In addition, the charge-transfer resistance (Rct) at the interface is dramatically reduced, allowing one to determine
equivalent circuit elements beyond the interfacial capacitance, as will be shown below. Figure 4 shows Nyquist plots of the electrochemical impedance from 1.2 to 950 Hz for the biosensor interface at +190 mV versus SCE, which is the open circuit potential in the presence of [Fe(CN)6]3-/4-. Note that in some cases the impedance of the biosensor interface is quite large, so a complete semicircle is not obtained in this frequency range. The Nyquist plot following addition of anti-fluorescein/Au nanoparticle conjugate to 0.15 µg/mL shows a continuous decline in the charge-transfer resistance, while the other Nyquist plots in Figure 4 are at steady state. Each impedance spectrum in Figure 4 takes ∼5 min to acquire. The impedance results presented in Figure 4 for the 11-MUA monolayer and immobilized fluorescein are consistently observed for different batches of these reagents. However, the results shown for binding of the anti-fluorescein/ Au nanoparticle conjugate were only consistent within one batch of this reagent, with the results varying between different batches. Since fluorescein/anti-fluorescein is studied only as a model system, this does not impact the conclusions about the value of Au nanoparticles for signal amplification in impedance spectroscopy. The simplified Randles model shown in Figure 5 was used here as the equivalent circuit for the biosensor interface modified with 11-MUA, 11-MUA + fluoresceinamine, and following addition of anti-fluorescein conjugated with Au nanoparticles. Since frequencies below 1 Hz were not investigated, mass-transfer effects are not observed, so the Warburg impedance is omitted. However, minor deviations arising from mass-transfer effects are seen at low frequencies in Figure 4. Table 1 provides the values of the different equivalent circuit elements fit to the data in Figure 4. From Table 1, the capacitance change 1 h after adding anti-fluorescein conjugated with Au nanoparticles is ∼100 nF/cm2, which is smaller to the result Analytical Chemistry, Vol. 78, No. 6, March 15, 2006
1771
Figure 6. SEM image of an Au electrode modified with 11-MUA + fluoresceinamine (A) and 4 h after addition of anti-fluorescein conjugated with 10-nm Au nanoparticles (B). Table 1. Values of the Equivalent Circuit Elements Shown in Figure 4 for the Au Electrode Modified with 11-MUA, 11-MUA + Fluoresceinamine, and after Adding Anti-Fluorescein Conjugated with Au Nanoparticles at +190 mV vs SCE
obtained directly from the capacitance measurement in Figure 2, 190 nF/cm2. The capacitance change (400 nF/cm2) after 4 h in Table 1 is closer to the result obtained directly from the capacitance measurement in Figure 2 (400 nF/cm2). However, these measurements cannot be directly compared, since they are obtained at different potentials, and in different electrolytes, since the redox probe is only present during the impedance measurements. The most striking result from Table 1 is the dramatic decrease in the Rct following introduction of the anti-fluorescein/ Au nanoparticle conjugate, eventually reaching ∼25% of its original value. This reflects the influence of the Au nanoparticles, since the redox reaction involving [Fe(CN)6]3-/4- occurs far more rapidly on the Au surface than on the insulating organic layer of fluoresceinamine/MUA. In other words, metal surfaces are expected to be far more electrochemically active than organic surfaces. The mechanism by which the redox probe, [Fe(CN)6]3-/4-, may increase the sensitivity of this impedance biosensor deserves further comment. The adsorption of Au nanoparticles atop an organic film was studied in detail using impedance spectroscopy by Li and co-workers.28 They observed that when Au nanoparticles
are bound to a SAM formed from 1,6-hexanedithiol, the chargetransfer resistance was reduced by ∼17×.28 The likely explanation is that both the Au nanoparticles and the organic film are thin enough that they do not contribute significantly to the dc resistance (Rs), so the predominant effect of Au nanoparticles atop an organic film is to dramatically increase the electron-transfer kinetics (decreasing Rct). The effect reported in the current study is smaller, likely reflecting the greater steric hindrance in this system, as well as lower binding constants for the anti-fluorescein/ Au nanoparticle conjugate. Similar effects on the impedance spectrum were reported by Lin and co-workers for Au nanoparticles deposited atop a DNA film for an amperometric biosensor.29 The reduction in Rct illustrates the sensitivity advantage of adding a redox probe, since this effectively magnifies the signal change that occurs upon analyte binding to the immobilized fluoresceinamine layer. For example, 4 h after analyte introduction, the capacitance measurements at 20 Hz in Figure 2 above yield a 15% increase in the capacitance from 2.6 to 3.0 µF/cm2. The relative change in this value is largely frequency independent. For greater contrast, comparison can be made to the results reported in Table 1, 4 h after analyte introduction, in the presence of the redox probe. For this case, the magnitude of the impedance at 1.2 Hz decreases by 35% from 39.4 to 25.7 kΩ‚cm2. In some experiments, considerably larger amplification effects were obtained upon introduction of the redox probe. For the results in Figure 4, the redox probe [Fe(CN)6]3-/4was added to the electrolyte only during the electrochemical impedance measurements, after the recognition process between fluoresceinamine and anti-fluorescein was complete. If this redox probe is present during addition of the anti-fluorescein/Au nanoparticle conjugate, no consistent impedance change is observed, suggesting that [Fe(CN)6]3-/4- might not be biocompatible. Recent results from our laboratory indicate that the galactose/ glucose receptor protein from the periplasmic space of Escherichia coli appears to denature in the presence of [Fe(CN)6]3-/4-.14
(28) Lu, M.; Li, X. H.; Yu, B. Z.; Li, H. L. J. Colloid Interface Sci. 2002, 248, 376.
(29) Lu, L.; Wang, S.; Lin, X. Anal. Chim. Acta 2004, 519, 161.
circuit element electrode surface 11-MUA fluoresceinamine 1 h after adding antifluorescein conjugated with nanoparticles 4 h after adding antifluorescein conjugated with nanoparticles
RS (Ω‚cm2)
Cd (µF/cm2)
Rct (kΩ‚cm2)
29.6 29.3 27.1
2.47 3.06 3.16
93.0 141.8 54.4
26.9
3.46
36.8
1772 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006
Scanning Electron Microscopy (SEM) Imaging of the Electrode Surface. SEM was used to image the Au electrode surface before and after analyte binding. Figure 6 shows the Au surface modified with 11-MUA + fluoresceinamine, before and after addition of anti-fluoresein conjugated with Au nanoparticles. A thin Au film is often deposited atop insulating samples to prevent sample charging and improve the image quality. However, such an Au coating was indistinguishable from the Au nanoparticles of interest, so the samples were not coated prior to obtaining the images in Figure 6. Due to the insulating organic layer between the Au nanoparticles and Au substrate, the Au nanoparticles become charged by the electron beam and appear as bright circular dots in Figure 6B. The average density of Au nanoparticles determined from SEM images is somewhat lower than expected from the results for the observed change in capacitance. For example, for Au electrode surfaces corresponding to Figure 2 results at long times, the average density of Au nanoparticles from 10 different SEM images is ∼2.0 × 109/cm2. Given the much greater capacitance of metal surfaces relative to protein and organic surfaces, and the lack of a signal change for anti-fluorescein without Au nanoparticles, the capacitance change observed in Figure 2 can be ascribed entirely to the Au nanoparticles that are introduced. Since our measured double layer capacitance of Au at +100 mV versus SCE is ∼35 µF/cm2, one expects an Au nanoparticle density of 3.7 × 109/ cm2, ∼1.9× larger than that observed experimentally. Similar to the effects of surface roughness, the capacitance of 10-nm-diameter Au nanoparticles is likely greater than that of an Au surface. The capacitance value is also altered by the adsorption and desorption of anions, which create an adsorption capacitance, increasing the effective capacitance.30 However, these effects may be too small to explain this discrepancy. If the electron beam is left on for a long enough time, all the Au nanoparticles eventually disappear from the sample image, suggesting that they are “burned off” by the electron beam during SEM imaging, since the surface is not conductive. The use of lower energy electrons could not circumvent this problem without compromising image resolution. The use of Au nanoparticles for amplification and augmentation of electrochemical detection in DNA sensors using a variety of (30) Walter, M. J.; Garland, J. E.; Pettit, C. M.; Zimmerman, D. S.; Marr, D. R.; Roy, D. J. Electroanal. Chem. 2001, 499, 48. (31) Wang, J. Anal. Chim. Acta 2003, 500, 247. (32) Fritzsche, W.; Tatton, T. A. Nanotechnology 2003, 14, R63. (33) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739. (34) Wang, L.; Xu, D.; Kawde, A. N.; Polsky, R. Anal. Chem. 2001, 73, 5576. (35) Cai, H.; Wang, Y.; He, P.; Fang, Y. Anal. Chim. Acta 2002, 469, 165.
detection schemes has been recently reviewed.31,32 However, to the best of the authors’ knowledge, the use of nanoparticle amplification schemes has only been reported once for impedancebased biosensors, through the use of semiconductor nano/ microparticles.22 As illustrated in the current report, through the addition of a redox probe, the signal change can be amplified through correct frequency choice. The use of a relatively lowfrequency allows one to sample the Rct, which declines as the interface becomes more metallic upon binding of the Au nanoparticle-conjugated analyte. This signal can likely be further amplified by deposition of Au or Ag onto the Au nanoparticle tag, as has been shown for DNA biosensors based on anodic stripping voltammetry33,34 and differential pulse voltammetry.35 CONCLUSIONS Fluoresceinamine was immobilized onto an Au surface using standard amine attachment chemistries to an underlying SAM constructed from 11-MUA. The capacitance and electrochemical impedance of this biosensor interface do not change measurably when exposed only to goat anti-fluorescein. However, when this interface is exposed to goat anti-fluorescein conjugated with 10nm Au nanoparticles, both the interfacial capacitance and electrochemical impedance changed significantly. The capacitance measured at 20 Hz and +100 mV versus SCE without the presence of any redox probe increased by ∼400 nF/cm2 upon introduction of this analyte. Even greater sensitivity can be achieved through introduction of a redox probe, [Fe(CN)6]3-/4-, which eventually reduces the Rct obtained at +190 mV SCE to ∼25% of its original value. Thus, single-frequency impedance measurements can be performed at low frequencies, which are sensitive to the Rct. For example, the magnitude of the impedance at 1.2 Hz decreases by 35% from 39.4 to 25.7 kΩ‚cm2 upon analyte introduction. However, this particular redox probe may induce denaturation of the goat anti-fluorescein, so it must be introduced after recognition of antifluorescein by fluorescein. ACKNOWLEDGMENT This research was supported by NSF grant CTS-0329698 and by the Center for Advanced Materials Processing (CAMP) at Clarkson University. Thanks to Bayer Healthcare for donation of the Bioanalytical Systems (BAS) model 100B/W electrochemical workstation and for providing chemical materials for this project. Received for review June 22, 2005. Accepted October 24, 2005. AC051113+
Analytical Chemistry, Vol. 78, No. 6, March 15, 2006
1773
