Ferrocene Catalyzed Aptamer-Based Thrombin Immunosensor

Jul 14, 2009 - ... for the detection of spiked concentrations of thrombin. ..... The unetched side of the membrane was adhered to a strip of adhesive ...
0 downloads 0 Views 3MB Size
Download PDF
Anal. Chem. 2009, 81, 6604–6611

Gold Nanoparticles Doped Conducting Polymer Nanorod Electrodes: Ferrocene Catalyzed Aptamer-Based Thrombin Immunosensor Md. Aminur Rahman,†,‡ Jung Ik Son,† Mi-Sook Won,§ and Yoon-Bo Shim*,† Department of Chemistry, Pusan National University, and Korea Basic Science Institute, Pusan 609-735, South Korea Au nanoparticles-doped conducting polymer nanorods electrodes (AuNPs/CPNEs) were prepared by coating Au nanorods (AuNRs) with a conducting polymer layer. The AuNRs were prepared through an electroless deposition method using the polycarbonate membrane (pore diameter, 50 nm, pore density, 6 × 108 pores/cm2) as a template. The AuNPs/CPNEs combining catalytic activity of ferrocene to ascorbic acid were used for the fabrication of an ultrasensitive aptamer sensor for thrombin detection. The AuNPs/3D-CPNEs were characterized employing cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Sandwiched immunoasay for r-human thrombin with NH2-functionalized-thrombin binding aptamer (Apt) immobilized on AuNPs/3D-CPNEs was studied through the electrocatalytic oxidation of ascorbic acid by the ferrocene moiety that was bound with an antithrombin antibody and attached with the Apt/3D-CPNEs probe through target binding. Various experimental parameters affecting thrombin detection were optimized, and the performance of the thrombin aptamer sensor was examined. The Apt/AuNPs/3D-CPNEs based thrombin sensor exhibited a wide dynamic range of 5-2000 ng L-1 and a low detection limit of 5 ng L-1 (0.14 pM). The selectivity and the stability of the proposed thrombin aptamer sensor were excellent, and it was tested in a real human serum sample for the detection of spiked concentrations of thrombin. The three-dimensional (3D) nanostructured materials are promising for diverse applications.1-4 Of these applications, an ultrasensitive detection could be achieved using the 3D nanoelectrode ensemble in electrochemical biosensing.3,4 The ultrasensitivity is due to the high signal-to-noise ratio, which can be easily * Corresponding author. E-mail: [email protected]. Phone: (+82) 51 510 2244. Fax: (+82) 51 514 2430. † Department of Chemistry. ‡ Present address: Department of Applied Chemistry, Konkuk University. § Korea Basic Science Institute. (1) Sapp, S. A.; Mitchell, D. T.; Martin, C. R. Chem. Mater. 1999, 11, 1183– 1185. (2) Long, J. W.; Dunn, B.; Rolison, D. R.; White, H. S. Chem. Rev. 2004, 104, 4463–4492. (3) Gasparac, R.; Taft, B. J.; Lapierre-Devlin, M. A.; Lazareck, A. D.; Xu, J. M.; Kelley, S. O. J. Am. Chem. Soc. 2004, 126, 12270–12271. (4) Roberts, M. A.; Kelley, S. O. J. Am. Chem. Soc. 2007, 129, 11356–11357.

6604

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

obtained in the case of nanoelectrodes enasemble. It has been reported that using of the 3D nanoelectrodes ensemble is advantageous in electrochemical sensing with high efficiency because of the geometry of an exposed structure.5 The 3D nanoelectrodes ensemble can be fabricated using the template method in which metals were grown within the pores of a nanoporous membrane through electroless deposition.6,7 Gold has been commonly used for fabrication of the 3D nanoelectrodes ensemble. However, the application of 3D Au nanoelectrode ensembles in biomolecular sensing is limited3-5 due to the fact that only self-assembling thiolated biomolecules can be covalently immobilized through the formation of a Au-thiol bond.4 Thus, the modification of the surface of 3D gold nanoelectrodes is needed for directly immobilizing biomolecules. For this, 3D nanoelectrodes coated with conducting polymers having functional groups are promising materials in biosensing. Previously, we have reported the conducting polymer layer composed of nanoparticles as an enzyme immobilizing substrate for the fabrications of phosphate8 and in vivo glutamate9 biosensors. However, the application of Au nanoparticles-deposited conducting polymer coated nanoelectrodes ensemble (AuNPs/3D-CPNEs) in biomolecular sensing has not yet been reported. For the application of 3D conducting polymer electrodes to the biosensor probe, we fabricated a thrombin aptamer sensor with the 3D polymer electrode by immobilizing a catalyst for the signal enhancement. Thrombin is a protein that has many effects in coagulation cascade. It is a serine protease that converts soluble fibrinogen to insoluble strands of fibrin as well as catalyzing many coagulation-related reactions. Because of its clinical importance, many aptamer sensors have been reported using optical10-12 and electrochemical methods.13-16 Thrombin detection using an (5) Lapierre-Devlin, M. A.; Asher, C. A.; Taft, B. J.; Gasparac, R.; Roberts, M. A.; Kelley, S. O. Nano Lett. 2005, 5, 1051–1055. (6) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920–1928. (7) Martin, C. R. Science 1994, 266, 1961–1966. (8) Rahman, M. A.; Park, D.-S.; Chang, S.-C.; McNeil, C. J.; Shim, Y.-B. Biosens. Bioelectron. 2006, 21, 1116–1124. (9) Rahman, M. A.; Kwon, N.-H.; Won, M.-S.; Choe, E. S.; Shim, Y.-B. Anal. Chem. 2005, 77, 4854–4860. (10) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768–11769. (11) Ho, H.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384–1387. (12) Wang, X.; Li, F.; Su, Y.; Sun, X.; Li, X.; Schluesener, H. J.; Tang, F.; Xu, S. Anal. Chem. 2004, 76, 5605–5610. (13) Hansen, J. A.; Wang, J.; Kawde, A.; Xiang, Y.; Gothelf, K. V.; Collins, G. J. Am. Chem. Soc. 2006, 128, 2228–2229. (14) Baldrich, E.; Acero, J. L.; Reekmans, G.; Laureyn, W.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 4774–4784. 10.1021/ac900285v CCC: $40.75  2009 American Chemical Society Published on Web 07/14/2009

aptamer based probe has several advantages that make it an ideal biosensing element.17 Aptamers are synthetic DNA/RNA based receptors originated from an in vitro selection process, so-called systematic evolution of ligands by exponential enrichment (SELEX).18-20 Aptamers are chemically stable in contrast to antibodies or enzymes. They often undergo conformational changes upon target binding, which offers high flexibility in design of novel biosensors with high sensitivity and selectivity. The first in vitro selected aptamer is thrombin-binding aptamer (TBA, 5′GGTTGGTGTGGTTGG-3′), which has been targeted toward a protein, thrombin21 at certain conditions that TBA folds into a quadruplex structure.22 There have been numerous reports on the aptamer based thrombin biosensors.23-30 The use of aptamer functionalized AuNPs as a catalytic label for the amplified optical detection of thrombin was reported.29 The detection limit of thrombin was determined to be 2 nM. Although, the sensitivity and the accuracy of optical aptamer sensors are comparable to electrochemical aptamer sensors, electrochemical aptamer sensors reveal certain advantages when compared to optical aptamer sensors. Because of their high sensitivity and selectivity, simple instrumentation, low production cost, and the fact that they are fast, accurate, compact, portable, and inexpensive, electrochemical methods have received particular attention. Many of the electrochemical methods are based on a change in the electrochemical response of an electroactive label. For example, the possibility to couple catalytic or biocatalytic labeling enables amplified detection of thrombin, and thus enhances the sensitivity of sensing processes. An electrochemical thrombin aptamer sensor was developed by tethering a redox-active methylene blue (MB) label to the aptamer nucleic acid.24 In the absence of a target, the immobilized aptamer is thought to relatively unfolded, thereby allowing the tethered MB to collide with the electrode and transfer an electron. In the presence of a target, electron transfer is inhibited, presumably due to a binding-induced conformational change in the aptamer that significantly increases the electron-tunneling distance. Although the method is elegant and simple, the current response was not directly proportional to the thrombin concentration but proportional to the logarithm concentration of thrombin. Though (15) Centi, S.; Tombelli, S.; Minunni, M.; Mascini, M. Anal. Chem. 2007, 79, 1466–1473. (16) Willner, I.; Zayats, M. Angew. Chem., Int. Ed. 2007, 46, 6408–6418. (17) Wang, J.; Wang, L.; Liu, X.; Liang, Z.; Song, S.; Li, W.; Li, G.; Fan, C. Adv. Mater. 2007, 19, 3943–3946. (18) Hermann, T.; Patel, D. J. Science 2000, 287, 820–825. (19) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818–822. (20) Tuerk, C.; Gold, L. Science 1990, 249, 505–510. (21) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermass., E. H.; Toole, J. J. Nature 1992, 355, 564–566. (22) Marathias, V. M.; Bolton, P. H. Biochemistry 1999, 38, 4355–4364. (23) Wang, J. J. Am. Chem. Soc. 2006, 128, 2228–2229. (24) Xiao, Y.; Lubin, A. A.; Heeger, A. A.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456–5459. (25) Bang, G. S.; Cho, S.; Kim, B. G. Biosens. Bioelectron. 2005, 21, 863–870. (26) Dittmer, W. U.; Reuter, A.; Simmel, F. C. Angew. Chem., Int. Ed. 2004, 43, 3550–3553. (27) Li, J. W.; Fang, X. H.; Tan, W. H. Biochem. Biophys. Res. Commun. 2002, 292, 31–40. (28) Hamaguchi, N.; Ellington, A. D.; Stanton, M. Anal. Biochem. 2001, 294, 126–131. (29) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768–11769. (30) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vwemass, E. H.; Toole, J. J. Nature 1992, 355, 564–566.

the sensor could be regenerated, the aptamer itself was labeled with MB, which could affect the binding affinity between the target and aptamer to a certain degree. The detection limit of thrombin was determined to be 20 nM. Another amperometric aptamer sensor based on a redox-active indicator that intercalate into double-stranded DNA has been reported.25 A nucleic acid in hairpin configuration that includes the thrombin recognition sequence was linked to a gold electrode, and methylene blue was intercalated in the duplex stem of the probe hairpin structure. The detection limit was reported to be 1.1 nM. On the other hand, ferrocene as a label has been the subject of intense investigation, as it is a molecule that exhibits excellent reversibility of its redox reaction.31 Previously, an electrochemical aptamer sensor based on bifunctionalized aptamer with a terminal electroactive ferrocene group as the reporter and the thiol function as the anchor on a gold electrode surface was reported.32 In the presence of thrombin, the conformational changes result in the folded quadruplex structure, bringing the ferrocene label to the electrode surface, thus enhancing the electron transfer. The detection limit was reported to be 0.5 nM. However, no catalytic property was examined with this ferrocene labeled thrombin aptamer sensor. It had been shown that ferrocene can electrocatalyzed the oxidation of ascorbic acid.33 Thus, we tried to develop a highly sensitive thrombin-aptamer sensor based on gold nanoparticles (AuNPs) deposited 3D nanoelectrode ensemble (AuNPs/3DCPNEs) immobilized ferrocene that electrocatalyzed the oxidation of ascorbic acid in the sample solution as a substrate. In the present study, 3D-CPNEs were fabricated using a polycarbonate membrane (pore diameter, 50 nm, pore density, 6 × 108 pores/cm2) as a template and were characterized using cyclic voltammetry (CV), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). A sandwiched immunoassay of human thrombin at the NH2-functionalized-thrombin binding aptamer (Apt)-immobilized on the AuNPs deposited 3D-CPNEs was examined through the electrocatalytic oxidation of ascorbic acid by the ferrocene moiety that was bound with an antithrombin antibody through avidin-biotin interaction. Various experimental parameters affecting the thrombin detection were optimized, and the detection limit was determined. The selectivity and the stability of this thrombin aptamer sensor were also discussed. EXPERIMENTAL SECTION Reagents. N-hydroxysuccinimide (NHS), biotin, streptavidin, ascorbic acid, 1-ethyl-3(3-(dimethylamino)-propyl) carbodiimide (EDC), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), ferricyanide, sodium bicarbonate, sodium chloride, hydrogen tetrachloroaurate (HAuCl4), glycerol, antihuman thrombin, immunoglobulin G (IgG), human serum albumin, and bovine serum albumin (produced in mouse) were purchased from Sigma Co. Track-etch polycarbonate membrane filters were obtained from Sterlitech Corporation. The thickness of the membranes were 6 µm with a nominal pore diameter of 80 (31) Van Staveren, D. R.; Metzler-Nolte, N. Chem. Rev. 2004, 104, 5931–5986. (32) Radi, A.-E.; Sanchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128, 117–124. (33) Lertanantawong, B.; O’Mullane, A. P.; Zhang, J.; Surareungchai, W.; Somasundrum, M.; Bond, A. M. Anal. Chem. 2008, 80, 6515–6525.

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

6605

nm and a pore density of 6.0 × 10-8 pores/cm-2. Tetrabutylammonium perchlorate (TBAP, electrochemical grade) was received from Fluka, purified, and then dried under vacuum at 10-5 Torr. Acetonitrile (AN, 99.8%, anhydrous, sealed under N2 gas) and dichloromethane (99.8%, anhydrous, sealed under N2 gas), sodium sulfite (98%), formaldehyde (37% wt %), potassium chloride, ferrocene carboxylic acid, and ammonium hydroxide (NH3 content 28-30%) were obtained from SigmaAldrich Co. Tin(II) chloride and trifluoroacetic acid were obtained from Aldrich Co. Human R-thrombin (specific activity, 3 725 Units/mg; concentration, 9.7 mg/mL) was obtained from Haematologic Technologies Inc. The thrombin aptamers were obtained from Bioneer Co. (South Korea), which have the following sequences: 5′-NH2-GGT TGG TGT GGT TGG-3′ and 5′-SH-GGT TGG TGT GGT TGG-3′. A terthiophene monomer bearing a carboxylic acid group, 5,2′;5′,2′′-terthiophene-3-carboxylic acid (TTCA) was synthesized according to a previous report.34 The streptavidin-ferrocene conjugate was synthesized by the EDC-NHS method.32 Briefly, 10 µM of ferrocenecarboxylic acid was reacted for 2 h with a mixed solution of 10 mM EDC and 10 mM NHS in HEPES. To this solution, 0.1 mg/mL of streptavidin was added and incubated for 2 h. Then, ferrocene was conjugated with streptavidin through the covalent bond formation between the amine groups of streptavidin and carboxylic acid groups of ferrocenecarboxylic acid. Biotinylated antithrombin was synthesized by the crosslinking method with glutaraldehyde. Gold nanoparticles (AuNP) were prepared according to a previously reported procedure.35 From the high-resolution transmission electron microscope (HRTEM), the particle size of AuNPs were about 4.0 nm. All other chemicals were of extra pure analytical grade and used without further purification. All aqueous solutions were prepared in doubly distilled water, which was obtained from a Milli-Q water purifying system (18 MΩ cm). Instruments. Cyclic voltammograms were recorded using a Kosentech model KST-P2 (South Korea) and an EG & G 273A potentiostat/galvanostat. XPS experiments were performed using a VG Scientific ESCALAB 250 XPS spectrometer with a monochromated Al KR source with charge compensation at the Korea Basic Science Institute (KBSI), Busan. SEM images were obtained using a Cambridge Stereoscan 240. Atomic force microscopic (AFM) experiments were carried out on a multimode AFM system from Digital Instrument Inc. Commercial Si3N4 tips (125 mm length, 300 kHz resonance frequency, 5-10 nm radius) were attached to a triangular cantilever made of the same material. The force constant was 200-1000 N/m. Preparation of 3D Gold Nanorods. The 2D gold nanodisks were prepared using the electroless plating procedure reported previously6-8 with slight modification. Briefly, after the membrane was wet for 2.5 h in methanol, the polycarbonate template membrane was sensitized with Sn2+ by immersing it 30 min into a solution containing 0.026 M SnCl2 and 0.07 M trifluoroacetic acid in 1:1 methanol/water as the solvent. After the membrane was rinsed with methanol for 30 min, the sensitized membrane was immersed for 10 min into a solution of 0.029 M AgNO3 in aqueous ammonia. After the membrane was washed with (34) Lee, T. Y.; Shim, Y.-B. Anal. Chem. 2001, 73, 5629–5632. (35) Shiddiky, M. J. A.; Rahman, M. A.; Shim, Y.-B. Anal. Chem. 2007, 79, 6886–6890.

6606

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

methanol to remove excess AgNO3, it was immersed into the Au plating bath (10 mL), which included 3.75 g/L HAuCl4, 40 g/L Na2SO3, 5 g/L EDTA, 30 g/L K2HPO4, and 0.6 mL of formaldehyde. The pH of the gold plating solution was controlled at about 10. Formaldehyde was used as a reducing agent that reduces the Au(I) to metallic Au. The gold electroless deposition was carried out for 24 h at 4 °C. The membrane was rinsed with water after plating and then immersed in 25% HNO3 for 12 h to remove the surface bound chemicals. The membrane was then thoroughly rinsed with water and air-dried. To prepare 3D gold nanorods, the surface gold from one face of the membrane was removed and chemical etching was carried out on the same side of the membrane, which was carried out by using a mixture of 50:50 dichloromethane and ethanol.36 Finally, 3D gold nanorods was heated at 150 °C for 10 min to improve sealing the membrane around 3D gold nanorods. Preparation of 3D Conducting Polymer Nanoelectrodes. The unetched side of the membrane was adhered to a strip of adhesive conducting copper tape. A piece of insulating tape (Nitto Co., Japan) was punched to be made a 0.03 cm2 hole and pasted onto the etched side of the membrane so that the exposed copper tape was covered completely on both sides to prevent exposure to the analyte solution. The resulting 3D gold nanoelectrodes were used to prepare 3D conducting polymer nanoeletrodes (3D-CPNEs) by electrochemical deposition of a conducting polymer film on 3D gold nanoelectrodes. The deposition was performed through electropolymerization of the 0.1 µM TTCA monomer in 0.1 M TBAP/AN by cycling the potential between 0 and 1500 mV three times at the scan rate of 100 mV/s. After electropolymerization, the 3D-CPNEs were washed with AN to remove the excess monomer. Fabrications of the Aptamer Probe and the Indirect Sandwich Catalytic Aptamer Sensor. The 3D-CPNEs were immersed for 6 h in a 0.01 M HEPES buffer solution (pH 7.0) containing 10.0 mM of EDC and 10 mM of NHS to activate the carboxylic acid groups of the 3D-CPNEs. Then, the EDC treated 3D-CPNEs were washed with an HEPES buffer solution and subsequently incubated for 2 h in a 2 µM amine group-modified thrombin binding aptamer (NH2-TBA) in the 0.01 M HEPES buffer (pH ) 7.0) at 4 °C. The NH2-TBA was immobilized onto the 3D-CPNEs through the formation of covalent bonds between carboxylic acid groups of the conducting polymer (CP) and amine groups of the aptamer. The aptamer immobilized 3D-CPNEs were then rinsed thoroughly with the HEPES buffer to remove the weakly adsorbed aptamers and subsequently incubated for 1 h in a 0.1% BSA solution for minimizing the nonspecific binding. The 3D-CPNEs/NH2-TBA probes were then incubated for 30 min in an HEPES solution containing thrombin at various concentrations. The thrombin binding probe was incubated for 30 min in an HEPES solution containing biotinylated antithrombin antibody after being rinsed. The resulting assembly was then immersed in a HEPES solution containing steptavidin-conjugated ferrocene for 30 min and subsequently washed with HEPES buffer solution. By this step, ferrocene was attached with antithrombin through streptavidin-biotin interactions. In the presence of a substrate, (36) Krishnamoorthy, K.; Zoski, C. G. Anal. Chem. 2005, 77, 5068–5071.

Figure 1. The schematic representation of the fabrication of the Apt/ 3D-CPNEs-based thrombin aptamer sensor.

ascorbic acid, the attached ferrocene electrochemically catalyzed the oxidation of ascorbic acid, thus amplified the current response. Since, thrombin was sandwiched between thrombin aptamer and antithrombin antibody attached with ferrocene, the catalytic current response was proportional to the thrombin concentration. The schematic presentation of the fabrication of an aptamer sensor probe and electrochemical catalytic detection of thrombin are presented in Figure 1. RESULTS AND DISCUSSION Characterization of 3D Conducting Polymer Nanoelectrodes. The 3D-CPNEs were prepared through electropolymerization of the 0.1 µM TTCA monomer on 3D gold nanorods in a 0.1 M TBAP/AN solution using a potential cycling method. At the first anodic scan, the CV exhibited one oxidation peak at around 1.3 V where the monomer oxidized and formed the polymer immediately on the Au nanorods (AuNRs) (figure not shown). A polymer reduction peak was observed at around 1.1 V in the reverse cathodic scan due to the reduction of immediately formed polymer at 1.3 V. The peak currents at 1.3 and 1.1 V increased as the number of potential cycles increased, indicating the formation and the growth of the PolyTTCA film on 3D Au nanorods (3D-CPNEs). The resulted 3D-CPNEs were electrochemically characterized by recording CV in a blank phosphate buffer solution (figure not shown). The Au reduction peak obtained for 3D AuNRs at about 0.75 V vs Ag/AgCl significantly decreased, indicating that the 3D gold nanorods were coated with the conducting polymer layer. The morphology of 3D-CPNEs were characterized using SEM. Parts a and b of Figure 2 show the SEM images obtained for 3DAuNRs and 3D-CPNEs, respectively. The shining extent of 3DAuNRs decreased for 3D-CPNEs, indicating the formation of a thin conducting polymer layer on 3D-AuNRs. To confirm the formation of 3D-CPNEs, SEM images were obtained after dissolving the polycarbonate membrane by dichloromethane and are shown in the insets of Figure 2a,b. From the SEM images, the diameters of the 3D-AuNRs and 3D-CPNEs were determined to be 30 and 60 nm, respectively. The 3D-CPNEs were also characterized using the XPS method. Figure 3 shows the survey spectra obtained for (a) 3D-AuNRs and (b) 3D-CPNEs. The survey spectrum for 3D-AuNRs shows two sharp peaks at 83.50 and 87.3 eV. These peaks corresponded to Au 4f7 and Au 4f5, respectively. In addition to Au 4f peaks, Au

Figure 2. (a) SEM images obtained for the (a) 3D-AuNRs and (b) 3D-CPNEs.

Figure 3. XPS analysis for the (a) 3D-AuNRs and (b) 3D-CPNEs.

4d5, Au 4d3, and Au 4p3 peaks were also observed at 334.4, 353.3, and 546.4 eV, respectively. The survey spectrum of 3D-AuNRs also shows C 1s and O 1s peaks. The C 1s and O 1s peaks came from the polycarbonate membrane. The C 1s peak observed at 284.8 eV corresponded to C-H or C-C bonds, whereas the O 1s Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

6607

Figure 4. (a) CVs recorded for Apt/3D-CPNEs (wine line) and Apt/ AuNPs/3D-CPNEs (dark cyan line) with conjugated ferrocene and for Apt/AuNPs/3D-CPNEs (green line) without conjugated ferrocene in a 0.01 M HEPRS buffer solution. (b) The electrocatalytic oxidation of ascorbic acid with a bare gold electrode (red line), Apt/3D-CPNEs (blue line), and Apt/AuNPs/3D-CPNEs (green line) with ferroceneconjugated in a 0.01 M HEPES buffer solution (pH ) 7.0).

peaks observed at 532.3 and 533.2 eV corresponded to the CdO and C-O, respectively. On the other hand, the survey spectrum of 3D-CPNEs also shows C 1s, O 1s, and Au 4f peaks. In addition, the S 2p peak at 164.4 eV was observed, while it was not observed in the 3D-AuNRs survey spectrum due to the presence of polyterthiophene film on the surface. The intensities of C 1s and O 1s peaks increased whereas the intensity of Au peaks decreased, indicating that the conducting polymer was successfully coated on the 3D-AuNRs. Redox Properties of the Conjugated Ferrocene on the 3DCPNEs. Figure 4a shows CVs recorded for the final aptamer immobilized 3D-CPNEs probe (Apt/3D-CPNEs) in the presence of antithrombin-conjugated ferrocene as an indicator attached with the aptamer probe through the target binding (wine line) as shown in Figure 1. A redox peak was observed at approximately +0.1/ +0.32 V vs. Ag/AgCl, which was not observed in the absence of conjugated ferrocene (green line). These results indicate that the redox peak came solely from conjugated ferrocene itself. The peak current was directly proportional to the scan rate between 20 and 200 mV/s, indicating that the redox reaction was involved in a surface-confined process.37 The formal potential of the redox reaction was determined to be +0.21 V. The peak separation between anodic and cathodic peak potential was determined to be +0.22 V, which indicates a qusai-reversible electron transfer process of ferrocene. In order to enhance the electron transfer process of the conjugated ferrocene, the 3D-CPNEs were modified by electrochemically depositing gold nanoparticles (AuNPs/3D(37) Murray, R. W. Chemically Modified Electrodes. In Electroanalytical Chemistry, Vol. 13; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; pp 191368.

6608

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

CPNEs). AuNPs are widely used in biotechnology38 due to their unique physical and chemical properties.39 In our previous works, we have used AuNPs for increasing the surface conductivities of conducting polymer and dendrimer bonded conducting polymer layers.40,41 The CV recorded (dark cyan line) at the AuNPs deposited 3D-CPNEs (Apt/AuNPs/3D-CPNEs) with conjugated ferrocene also showed a pair of redox peaks at +0.04/+0.32 V vs Ag/AgCl. The formal potential of the redox peak was determined to be +0.18 V, which was lower than the obtained one without AuNPs. In this case, the peak current was also proportional to the scan rate between 20 and 200 mV/s, indicating that the redox reaction was also involved in a surface-confined process. Although the formal potential of the conjugated ferrocene lowered by only 0.03 V, the redox peak currents were significantly increased. The redox peak currents of the conjugated ferrocene obtained for the Apt/AuNPs/3D-CPNEs were three or four times higher than that obtained for Apt/3D-CPNEs. This result revealed that AuNPs acted as efficient electron transfer promoters for the redox reaction of the conjugated ferrocene. The electrocatalytic behavior of the ferrocene-conjugated Apt/ AuNPs/3D-CPNEs probe toward the ascorbic acid oxidation was studied. Figure 4b shows CVs recorded for ferrocene-conjugated Apt/3D-CPNEs without (blue line) or with AuNPs (green line) on the oxidation of ascorbic acid in a 0.01 M HEPES buffer solution (pH ) 7.0). For comparison, a CV on the oxidation of ascorbic acid at a bare gold electrode (red line) was also recorded. The small oxidation peak of ascorbic acid was observed at about +0.55 V at the bare gold electrode, while it was shifted to a less positive potential from +0.55 V to +0.3 V at the ferroceneconjugated Apt/3D-CPNEs modified electrode without AuNPs. The anodic peak current of ascorbic acid was found to be 5 times higher than that obtained for a bare gold electrode. These results revealed that conjugated ferrocene at the Apt/3D-CPNEs probe had a catalytic activity toward the oxidation of ascorbic acid. On the other hand, the oxidation potential of ascorbic acid slightly lowered for the ferrocene-conjugated Apt/3D-CPNEs (+0.27 V) with AuNPs. However, when the Apt/3D-CPNEs electrode was modified using AuNPs, the anodic peak current of ascorbic acid oxidation was found to be 2 times higher than that obtained without using AuNPs. The higher oxidation current was observed due to the fact that AuNPs increased the surface conductivities of 3D-CPNEs modified electrode. Optimization of Experimental Parameters for Thrombin Detection. The experimental parameters for the detection of thrombin with the ferrocene-conjugated Apt/AuNPs/3D-CPNEs probe were optimized in terms of aptamer concentration, ferrocene concentration in the conjugation, ascorbic acid concentration, pH, and temperature, where the thrombin concentration was kept constant. The effect of aptamer concentration on the electrocatalytic oxidation of ascorbic acid was studied between 0.2 and 5 µM (Figure 5a). The catalytic oxidation current of ascorbic acid gradually increased from 0.2 to 2.0 µM of aptamer concentration immobilized. Over 2.0 µM concentration of aptamer, the catalytic (38) Alivisatos, A. P. Science 1996, 271, 933–937. (39) Mirkin, C. R.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (40) Rahman, M. A.; Noh, H. B.; Shim, Y.-B. Anal. Chem. 2008, 80, 8020–8027. (41) Singh, K.; Rahman, M. A.; Son, J. I.; Kim, K. C.; Shim, Y.-B. Biosens. Bioelectron. 2008, 23, 1595–1601.

Figure 5. The effects of the concentration of (a) ascorbic acid, (b) ferrocene in the conjugation solution, (c) ascorbic acid and (d) the pH on the electrocatalytic response for thrombin detection.

oxidation current did not increase significantly. Thus, the aptamer concentration was used as 2.0 µM in the subsequent experiments. The effect of ferrocene concentration for the conjugation was also examined between 3 and 30 µM (Figure 5b). The catalytic oxidation current gradually increased from 3 to 10 µM. At higher concentrations over 10 µM, the catalytic current did not increase significantly. Thus, the optimum concentration of ferrocene for the conjugation was selected as 10 µM. The effect of ascorbic acid concentration was studied between 1 and 20 mM (Figure 5c). The catalytic oxidation current gradually increased from 1 to 10 mM. Over 10 mM concentration of ascorbic acid, the catalytic current was not found to be significantly increased due to the saturation effect. Thus, the optimum concentration of ascorbic acid was determined as 10 mM. The effect of pH on the ascorbic acid oxidation was studied over the pH range of 4.5-8.5 in a HEPES buffer solution containing 10 mM ascorbic acid (Figure 5d). The oxidation current gradually increased from pH 4.5 to 7.5 and then decreased at pH values higher than 7.5. The first (pK1) and second (pK2) values of ascorbic acid are 4.15 and 11.50.42 The decrease in current response over pH 7.5 may be due to the increase amount of dissociated ascorbic acid at higher pH. The maximum catalytic oxidation current was observed at a pH of 7.5. Thus, the optimum pH was chosen as 7.5. The effect of temperature on the detection of thrombin was studied between 10 and 80 °C (figure not shown). The catalytic response was found to be gradually increased as the temperature was increased from 10 to 35 °C. The catalytic response was not found to be significantly changed between 35 and 45 °C. However, the current response rapidly decreased from 45 to 80 °C due to the deactivation of thrombin. On the basis of the temperature-response profile, the optimum temperature was selected as 35 °C. (42) Kurtum, G.; Vogel, W.; Andrussow, K. Pure Appl. Chem. 1960, 1, 187536 (IUPAC Technical Reports and Recommendation, Dissociation Constant of Organic Acids in Aqueous Solutions).

Interference Effect. Common proteins, such as immunoglobin G (IgG) and human serum albumin (HSA), can interfere with the thrombin detection. In order to assess the possibility of interference, the catalytic oxidation current response was measured for IgG and HSA (figure not shown). IgG and HSA did not interfere in thrombin detection. This was due to the fact that the thrombin aptamer and antithrombin antibody cannot interact with IgG and HSA, thus, the conjugated ferrocene cannot interact on the immunosensor and no catalytic current response was observed. In addition to IgG and HSA, the ferrocene-conjugated Apt/AuNPs/ 3D-CPNEs sensor did not respond to bovine serum albumin (BSA). Thus, the response of the present aptamer sensor was very selective for human R-thrombin. Calibration Plot. The electrocatalytic current responses were measured for ferrocene-conjugated Apt/3D-CPNEs and Apt/ AuNPs/3D-CPNEs based sensors by varying the thrombin concentration. Figure 6 shows the electrocatalytic responses and calibration plots obtained for Apt/3D-CPNEs (Figure 6a,b) and Apt/AuNPs/3D-CPNEs (Figure 6c,d) based aptamer sensors for thrombin detection. The inset in part c shows the electrocatalytic responses at 0, 5.0, 10, and 50 ng L-1 of thrombin concentrations. At the thrombin concentration of 0 ng L-1 (no thrombin present), no electrocatalytic response was observed. The ferrocene conjugated antithombin antibody might not be attached with the aptamer probe without thrombin, thus the electrocatalytic oxidation of ascorbic acid by the conjugated ferrocene was not observed. On the other hand, electrocatalytic responses were obtained at 5.0, 10, and 50 ng L-1 thrombin concentrations. At lower concentrations than 5.0 ng L-1 (1.0 and 2.0 ng L-1), we did not get any electrocatalytic responses. CVs recorded at these concentrations were similar to that recorded for 0 ng L-1, indicating that the aptamer probe was not able to detect thrombin at lower concentrations than 5.0 ng L-1. Under the optimized conditions, the electrocatalytic response of thrombin detection was linear in the ranges of Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

6609

Figure 7. Electrocatalytic responses obtained in spiked (5, 10, and 20 ng L-1 thrombin) serum samples. Table 1. Comparison of the Hydrodynamic Range and the Detection Limit of Thrombin with Other Aptamer Based Nanostructured Sensor Probes

Figure 6. Electrocatalytic responses and calibration plots for thrombin detection with (a, b) Apt/3D-CPNEs and (c,d) Apt/AuNPs/ 3D-CPNEs. The inset in part c shows electrocatalytic responses at 0, 5, 10, and 50 ng L-1 of thrombin concentration.

10-1000 and 5-2000 ng L-1 for Apt/3D-CPNEs and Apt/ AuNPs/3D-CPNEs based aptamer sensors, respectively. These linear dependencies yielded regression equations of Ip (µA) ) 9.15 + 0.02[C] (ng L-1) and Ip (µA) ) 33.8 + 0.05[C] (ng L-1) with the correlation coefficients of 0.998 and 0.999, respectively. The reproducibility expressed in terms of the relative standard deviation (RSD) was about 4.3% and 5.7% at a thrombin concentration of 80 ng L-1. The detection limits of thrombin were determined to be 10.0 (0.28 pM) and 5 ng L-1 (0.14 pM) for Apt/3D-CPNEs and Apt/AuNPs/3D-CPNEs, respectively. These values of detection limits were much lower than the detection limits obtained from the electrochemical thrombin detections based on a binding induced conformational change where ferrocene (0.5 nM)32 and methylene blue (∼20 nM)24 were used as labels. The detection limits in the present study were also 2-4 times lower than the most sensitive thrombin aptasensor (0.5 pM).23 The sensitivity of the thrombin aptamer sensor was found to be 2 times enhanced with AuNPs. The detection limit of thrombin was also determined for 2DAu nanodisk (2D-AuNDs) and 3D-AuNRs electrodes, where a thiolated TBA (5′-SH-GGT TGG TGT GGT TGG-3′) was used. The results are shown in Table 1. It can be seen that the Apt/ AuNPs/3D-CPNEs exhibited the highest sensitivity for the aptamer sensor-based thrombin detection. Stability of the Apt/AuNPs/3D-CPNEs Based Thrombin Aptamer Sensor. The stability of the present thrombin aptamer sensor was determined by measuring the response once a day for 1 month. After each measurement, the aptamer sensor was 6610

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

nanostructured probe

dynamic range (ng L-1)

detection limit (ng L-1)

Apt/ 2D-AuNDs Apt/3D-AuNRs Apt/3D-CPNEs Apt/AuNPs/3D-CPNEs

10-1000 5-2000 10-1000 5-2000

10 5 10 5

stored in a phosphate buffer solution at 4 °C. From these experiments, no significant decrease in thrombin detection was observed for 3 weeks. For 3 weeks, the aptamer sensor retained more than 95% of its initial response. After 3 weeks, the response was gradually decreased due to the gradual decrease in the interaction between aptamer and thrombin. These results indicate that the thrombin aptamer sensor exhibited not only high sensitivity but also long-term stability. Real Sample Analysis. The practical applicability of the proposed aptamer sensor was investigated by detecting thrombin in a human real serum sample. The Apt/AuNPs/3D-CPNEs based thrombin aptamer sensor did not detect thrombin in the serum sample. This was due to the fact that healthy human serum sample does not contain thrombin.15 However, to examine the applicability of this aptamer sensor in serum sample, we performed spike and recovery experiments. The serum sample was spiked with 5, 10, and 20 ng L-1 thrombin. Figure 7 shows the electrocatalytic responses obtained in thrombin spiked serum samples. After electrochemical measurement, the calibration method was used to determine thrombin concentration. The current response of this aptamer sensor in a serum sample was slightly lower (about 3%) than that obtained in a blank buffer solution. The thrombin concentration recovery was between 95% and 98%, which clearly indicates the potentiality of this aptamer sensor for thrombin detection in real biological samples. CONCLUSIONS Ferrocene-catalyzed ascorbic acid oxidation based an ultrasensitive thrombin aptamer sensor was fabricated by covalently immobilizing a thrombin binding aptamer onto 3D-CPNEs. 3DCPNEs were fabricated by coating a conducting polymer layer on the surface of 3D-AuNRs, which were prepared through the electroless deposition method. The sensitivity of the thrombin detection was further enhanced by depositing AuNPs on to the

3D-CPNEs before aptamer immobilization (Apt/AuNPs/3DCPNEs). The aptamer sensor was successfully characterized using SEM, XPS, and electrochemical techniques. SEM results confirmed the formation of 3D-AuNRs and 3D-CPNEs, and the diameters of the 3D-AuNRs and 3D-CPNEs were determined to be 30 and 60 nm, respectively. XPS results further confirmed that the conducting polymer was successfully coated on the 3D-AuNRs. The redox and catalytic properties of the conjugated ferrocene at Apt/AuNPs/3D-CPNEs and Apt/3D-CPNEs were characterized using a cyclic voltammetric technique. The electrocatalytic oxidation of ascorbic acid by the labeled-ferrocene increased the sensitivity of the thrombin aptamer sensor. The Apt/AuNPs/3DCPNEs based aptamer sensor exhibited a wide linear range (5-2000 ng L-1) and a very low detection limit (0.14 pM). The RSD value was determined to be 5.7% at the thrombin concentration of 80 ng L-1. The common proteins, such as IgG,

HSA, and BSA, did not interfere with the thrombin detection. The present aptasensor also exhibited a long-term stability of 3 weeks. The sensitivity of the Apt/AuNPs/3D-CPNEs sensor was 10 times higher than the most sensitive thrombin aptamer sensor reported until now and was successfully tested in a real human serum sample for the detection of spiked amounts of thrombin. ACKNOWLEDGMENT The work is supported by Korea Ministry of Environment as “The Eco-technopia 21 project” (Grant No. 091-082-078).

Received for review February 6, 2009. Accepted July 4, 2009. AC900285V

Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

6611