Carbon Nanotubes Labeled with Aptamer and Horseradish

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Carbon nanotubes labeled with aptamer and horseradish peroxidase as probe for highly sensitive protein biosensing by postelectro-polymerization of insoluble precipitates on electrodes Jing Li, Jingjing Wang, Xiang Guo, Qiong Zheng, Jing Peng, Hao Tang, and Shouzhuo Yao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00640 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 11, 2015

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Analytical Chemistry

Carbon nanotubes labeled with aptamer and horseradish peroxidase as probe for highly sensitive protein biosensing by post-electropolymerization of insoluble precipitates on electrodes Jing Li1,2,‡, Jingjing Wang1,‡, Xiang Guo1,‡, Qiong Zheng1, Jing Peng1, Hao Tang1,*, Shouzhuo Yao1,3 1

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, P. R. China 2 Department of Chemical Engineering and Textile, Shaanxi Polytechnic Institute, Xianyang 721000, P. R. China 3 State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, P.R. China ABSTRACT: Carbon nanotubes (CNTs) labeled with aptamer and horseradish peroxidase (HRP) are used as a probe to amplify the impedimetric sensing of aptamer-protein (thrombin as model) interaction. The HRP-biocatalyzed oxidation of 3,3Diaminobenzidine (DAB) in the presence of H2O2, and the post-electro-polymerization of insoluble precipitates produced on electrode supports are used as a signal amplification route for the sensing process. The thrombin is sensed by the aptamer 1 immobilized on a glass carbon electrode. The multi-walled CNT-aptamer 2-HRP probe is linked to the aptamer 1-thrombin complex through the thrombin-aptamer 2 interaction. The post-electro-polymerization of biocatalyzed precipitates of DAB on the electrode greatly increases the electron transfer resistance at the electrode-solution interface. The cyclic voltammetry and electrochemical impedance spectroscopy were employed to follow the stepwise fabrication of the aptasensor and impedimetric detection of thrombin. Thrombin concentration as low as 0.05 pM could be detected by this method. In addition, the proposed impedimetric aptasensor exhibits good sensitivity (5195 Ω decade-1), selectivity, and reproducibility. The aptasensor also has acceptable recovery for thrombin detection in complex protein sample.

Sensitive determination of proteins is very important for medical diagnostics, elucidation of disease vectors, new drug development, proteomics, and system biology 1. Modern commercial biosensing for proteins are usually based on measuring the binding event between antibody and antigen. Aptamers, as artificial oligonucleotides, can bind to a wide variety of entities including proteins. The affinity, selectivity, and specificity of aptamers are equal to or often superior to those of antibodies. Moreover, aptamers are relatively costeffective and capable of reversible denaturation 2-4. Therefore, aptamer-based biosensors have obtained numerous attentions in the past decade. Various aptasensors that utilize different signal transduction techniques have been reported. The optical 5-7 , electrochemical 8-12, and atomic force microscopy (AFM) 13 aptasensors have exhibited great promise in protein detection with high sensitivity, selectivity, and cost-effectiveness. The impedimetric biosensor, based on electrochemical impedance spectroscopy (EIS), is one type of interesting labelfree biosensors through detection of a change in the interfacial electron transfer resistance caused by biorecognition events 14, which has exhibited wide applications due to simplicity, good sensitivity, and cost-efficiency. Up to now, numerous works have reported impedimetric protein biosensors based on aptamer-protein interaction 8, 15-22. For example, a simple and straightforward method was proposed to construct impedimetric thrombin aptasensor by using gold recordable compact discs 15. A regenerative impedimetric aptasensor, based on poly(pyrrole-nitrilotriacetic acid)-aptamer film, was also fabricated for thrombin determination 16. These works are expected

to provide attractive insights for the developments of impedimetric aptasensors. Recently, proofs of concept for new and sensitive protein detection technologies combining nanomaterials and aptamers have been developed 7, 23-28. Carbon nanotubes (CNTs), as a typical one-dimension nanomaterial, have been successfully demonstrated as sensitive electrochemical biosensors due to their unique physical, chemical, and electrical properties. Aptamer-modified CNT field effect transistors were fabricated for detection of thrombin 29 and immunoglobulin E 30. Concerned the electrochemical aptasensors, amperometric aptasensors based on CNT-based nanocomposites were obtained the most attentions. A simple and efficient post-labeling strategy, based on dye-induced peeling of the aptamer molecules off single-walled CNTs (SWCNTs), was used to develop aptasensor for thrombin detection 31. The CNT-based nanocomposites such as ferrocene-aptamer-multi-walled CNTs (MWCNTs) 32, alkaline phosphatase-aptamer-SWCNTs 33, aptamer-SWCNTs 34, aptamer-Au NPs-SWCNTs 35, aptamerMWCNTs 36, and aptamer-MWCNTs-ionic liquid-chitosan 37, were prepared and utilized to fabricate amperometric aptasensors for thrombin, platelet-derived growth factor, tetracycline, and immunoglobulin E determination. The CNTs or their composites show capacity to function as a signal amplifier by facilitating the aptamer probe immobilization and improving the electrochemical properties of the transducer due to the vast surface-to bulk ratio and excellent electrocatalytic performance.

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In contrast, very few works reported the incorporation of CNTs into impedimetric aptasensors. The CNT modified screen-printed carbon electrodes were used to fabricated simple impedimetric aptasensors for thrombin 38 and lysozyme 39 determination. Porfireva and co-workers 40 found that MWCNT-electro-polymerized methylene blue composites were favorable for aptamer immobilization. This new immobilization technique significantly improved the sensitivity of thrombin detection by EIS. The detection limits of these CNTbased impedimetric aptasensors are commonly in the range of nanomole 38-40. Many protein biomarkers, however, are present at very low level during the early stage of diseases. It is highly desired that appropriate CNT-based amplification strategies are explored to develop ultrasensitive protein impedimetric aptasensors.

Figure 1. Schemes demonstrating the principle of impedimetric aptasensor for thrombin detection by signal amplification: (1) apt1 immobilization; (2) BSA blocking; (3) interaction between apt1 and thrombin; (4) signal amplification by MWCNT-apt2-HRP hybrids; (5) enzymatically biocatalyzed reaction between HPR and DAB; (6) post-electro-polymerization of the precipitates; (7) EIS measurements.

In this work, we reported the MWCNTs labeled with aptamer and horseradish peroxidase (HRP) as probe for highly sensitive protein impedimetric detection by post-electropolymerization of insoluble precipitates on electrodes. In contrast to SWCNTs, MWCNTs are significantly cheaper and now available at a reasonable low price. Moreover, the vast surface to bulk ratio may render the MWCNTs good characteristics for biomolecule (aptamer and enzyme, for example) immobilization 41, resulting in improvement of the sensing performances. Besides aptamer immobilization, the amplification strategy of protein sensing is also a major factor that determines the sensitivity of impedimetric aptasensors. It has been reported that enzymatically biocatalyzed amplification is very effective for sensitivity enhancement in antigen/antibody 42-43 and DNA biosensors 44-45. The biocatalyzed generation of electroactive species and insoluble products, or the participation of O2, H2O2 or NH3 in the enzyme-stimulated processes enables the amplified signal transduction of the antigenantibody binding or DNA hybridization events. Microgravimetric quartz crystal microbalance (QCM) analysis 46, surface plasma resonance 47-48, voltammetry 49-51, and EIS 44, 52, and photoelectrochemistry 53 have been used as transduction methods of the amplified sensing signals. HRP is one of the most frequently used enzyme labels in the enzymatically biocatalyzed reactions. In the presence of peroxide (H2O2), HRP catalyzes the oxidation of 4-chloro-l-naphthol, 3,3diaminobenzidine (DAB), and etc. forming insoluble precipitates. For example, the quantum dots-based amplified photoelectrochemical immunoassay with the integration of HRP biocatalyzed precipitation has been reported 53. Improved sensi-

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tivity is achieved through using the photoelectrochemical detection and enzymatically biocatalyzed reaction. HRPmimicking DNAzyme has also been used as electrocatalyst for amplified biosensing and the electrochemical sensors are developed for the detection of H2O2, DNA, and adenosine monophosphate 54. Here we proposed the MWCNT-aptamerHRP hybrid probe and the post-electro-polymerization of insoluble precipitates, produced by enzymatically biocatalyzed reaction between HRP and its substrate (DAB), for signal amplification of aptamer-protein binding event and development of highly sensitive protein impedimetric aptasensor (Figure 1). As a model case, we used the thrombin, which is a serine protease that plays important roles in converting soluble fibrinogen into insoluble strands of fibrin as well as catalyzing many other coagulation-related reactions. The prepared impedimetric aptasensor exhibited excellent analytical properties (high sensitivity, low detection limit, and good selectivity and reproducibility) for thrombin detection. EXPERIMENTAL SECTION Chemicals and Instruments. MWCNTs were purchased from Shenzhen Nano-Technologies Port Co., Ltd. (China), and the detail parameters are as follows: out diameter, 10-20 nm; length, 5-15 µm; purity, > 95%. 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC, hydrochloride form, >98.5%) and N-hydroxy-succinimide (NHS, >98%) were purchased from Aladdin® Reagent Database Inc. Thrombin, HRP, bovine serum albumin (BSA), and human immunoglobulin G (h-IgG) were obtained from Sigma-Aldrich. The synthetic thrombin aptamers (apt) were purchased from Sangon Biotech (Shanghai) Co., Ltd. The base sequences of the aptamers are as follows38, 55: Amine group-modified 15-mer Apt1: 5’-GGT TGG TGT GGT TGG-NH2-3’. Amine groupmodified 29-mer Apt2 with polyT(15) tail: 5’-NH2-TTT TTT TTT TTT TTT AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3’. The apt samples were stored at -20 oC for future use. DAB (tetrahydrochlodire form) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). The hydrogen peroxide (H2O2, 30%) was obtained from Changsha Antai Chemical Industry Co., Ltd. Blood serum samples were kindly provided by Hunan Normal University Hospital. Phosphate buffered saline (PBS, 0.01 M, pH 7.0) solutions were used in the experiments. All other reagents were of analytical grade. Milli-Q ultrapure water (>18 MΩ cm, Milli-pore Co., Ltd.) and fresh prepared solutions were used throughout. CHI 600C electrochemical workstation (CH Instruments Co.) and a conventional three-electrode electrolytic cell were used in the electrochemical experiments. The glass carbon (GC, diameter 3 mm) electrode modified with thrombin apt was served as the working electrode. A KCl-saturated calomel electrode (SCE) was used as the reference electrode and a platinum wire served as the counter electrode. All potential values given below are referred to the SCE electrode. Unless otherwise specified, experiments were performed at room temperature. The measurements were repeated at least three times and the means of measurements were presented with the standard deviations (SD). Preparation of MWCNT-apt, MWCNT-apt2-HRP, MWCNT-HRP, and apt2-HRP. The MWCNTs received were firstly purified by concentrated HCl and then treated by mixed acids (concentrate H2SO4+HNO3, v/v 3:1) to obtain carboxylated MWCNTs (hereafter, unless otherwise specified,


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the carboxylated MWCNTs are denoted as MWCNTs). The details were presented in Supporting Information. The thrombin apt was tethered onto the MWCNT surface by covalent binding between 5’ (or 3’)-amine group of the apt and the carboxyl group of MWCNTs using EDC and NHS as coupling reagents. The details were presented in Supporting Information. For the preparation of MWCNT-apt2-HRP hybrids, the procedures were similar with that of MWCNT-apt. In addition, MWCNT-HRP and apt2-HRP were also prepared for comparison. These details were presented in Supporting Information. Preparation of the impedimetric aptasensors. Before further use, the GC electrode was pretreated by a well-established procedure and the details were presented in Supporting Information. For preparation of the impedimetric aptasensos, the pretreated GC electrode was immersed into 100 µL of apt1 (1 µM) PBS solutions for 3 h, followed by rinse with PBS solution three times. To prevent the nonspecific adsorption in thrombin detection, the apt1 modified GC (GC+apt1) electrode was blocked by immersing into 100 µL of PBS solutions containing BSA (1 wt.%) for 1 h, followed by rinse with PBS three times. The obtained BSA blocked GC+apt1 (GC+apt1+BSA) electrode was used for impedimetric detection of thrombin. For comparisons, various electrodes including GC+MWCNT+apt1+BSA, GC+MWCNT-apt1+BSA, and GC+MWCNT-apt2+BSA were fabricated and the details were presented in Supporting Information. FTIR, AFM, SEM, and QCM characterization. The prepared MWCNT-apt2-HRP hybrids were characterized by FTIR. The morphologies of various electrodes were characterized by AFM and SEM. The produced precipitates and the formed electro-polymerized precipitates were analyzed by QCM. These details were presented in Supporting Information. Electrochemical measurements and signal amplification. The apt1 immobilization and BSA blocking for fabrication of the impedimetric aptasensors were characterized by cyclic voltammetry (CV) and EIS. In addition, the MWCNT or MWCNT-apt modification was also investigated. The electrochemical measurements were carried out in PBS (pH 7.0) solutions containing 2 mM [Fe(CN)6]3-/4- redox probe. The CV scan rate is 50 mV s-1. The impedance data were recorded at open circuit potential in the ac frequency range of 100 kHz to 0.01 Hz with an excitation signal of 5 mV. The impedance Z is expressed in term of its real (Zre) and imaginary (-Zim) components. The data of the electrochemical impedance measurements were analyzed by ZSimpWin software. For impedimetric detection of thrombin, the prepared GC+apt1+BSA electrodes were dipped into 100 µL of PBS solutions containing different concentration of thrombin. The binding reaction between the apt1 and thrombin was allowed to proceed for 1 h at room temperature. After rinsed with PBS three times, the GC+apt1+BSA+thrombin electrodes were measured by CV and EIS in PBS (pH 7.0) solutions containing 2 mM [Fe(CN)6]3-/4- redox probe. The EIS spectrum of the GC+apt1+BSA electrode was also measured as background. For comparisons, other electrodes (GC+MWCNT+apt1+BSA, GC+MWCNT-apt1+BSA, and GC+MWCNT-apt2+BSA) were also evaluated for thrombin detection. The signal amplification of the GC+apt1+BSA electrode for thrombin detection was achieved by using the MWCNT-apt2HRP probe and post-electro-polymerization of insoluble precipitates, produced by the enzymatically biocatalyzed reaction

between HRP and DAB (Figure 1). For details, 10 µL of 1.0 mg mL-1 MWCNT-apt2-HRP suspension solution was dropcoated onto the GC+apt1+BSA+thrombin electrode and allowed to react for 1 h. The obtained GC+apt1+BSA+thrombin+MWCNT-apt2-HRP electrode was rinsed with PBS three times, and then 5 µL of 1.0 mg mL-1 DAB and 5 µL of 0.01 M H2O2 were added onto the electrode surface for 5 min to allow the enzymatically biocatalyzed reaction. After rinsed with PBS three times, the insoluble precipitates on the electrode surface (the GC+apt1+BSA+thrombin+ MWCNT-apt2-HRP+precipitates) were electro-polymerized in blank PBS (pH 7.0) by CV for 5 cycles in the potential range of -0.1 to 1.0 V vs. SCE at a scan rate of 50 mV s-1. The resulted electrode was rinsed with PBS three times, and then measured by CV and EIS in PBS (pH 7.0) solutions containing redox probe. For comparisons, apt2, apt2-HRP, MWCNTHRP, and MWCNT-apt2 hybrids were also used to amplify the impedimetric signal of the thrombin bound onto the GC+apt1+BSA electrodes. To test the specificity of the assay, the BSA, h-IgG, and HRP were used as control. Moreover, the analytical applicability of the prepared GC+apt1+BSA electrode in complex sample was evaluated by standard addition method using blood serum. RESULTS AND DISCUSSION Characterization of the impedimetric aptasensor

Figure 2. Cyclic voltammograms (A) and Nyquist plots (B) of various electrodes in PBS (0.01 M, pH 7.0) containing 2.0 mM [Fe(CN)6]3-/4- redox probe. Line I, GC; line II, GC+apt1; Line III, GC+apt1+BSA; line IV, GC+apt1+BSA+thrombin. The thrombin concentration is 50 nM.



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The transduction principle for impedimetric detection of thrombin is based on measurement of Faradaic impedance in the presence of [Fe(CN)6]3-/4- redox pair, being often used to characterize the electrode interface feature in electrochemistry 56 . The electron transfer of the redox pair can be blocked by the formation of aptamer-protein complexes on the aptasensor surface, resulting in a change of electron transfer resistance (Rct) of the redox pair. The amount of the aptamer-protein complex formed on the electrode surface is related to the concentration of the target protein in the solution. Therefore, the detection of thrombin without signal amplification can be performed by measuring the Rct change (∆Rct) of the redox pair on the GC+apt1+BSA electrode before and after formation of apt1-thrombin complex using EIS. For the thrombin detection using signal amplification, it was described in the following section. Figure 2 shows the cyclic voltammograms and Nyquist plots of various electrodes in PBS containing 2.0 mM [Fe(CN)6]3-/4redox pair. The apt1 immobilization (Figure 2A, red lines), BSA blocking (Figure 2A, blue lines), and interaction between apt1 and thrombin (Figure 2A, pink lines) lead to significant decrease of redox current density and increase of Rct (the diameter of the semicircles in Figure 2B), which demonstrate the successfully preparation of the impedimetric aptasensor and formation of apt1-thrombin complex at the surface of GC+apt1+BSA electrode. The apt1-thrombin interaction can greatly block the electron transfer of [Fe(CN)6]3-/4- redox pair and the ∆Rct before and after apt1-thrombin interaction is about 5270 Ω (Figure 2B, blue and pink lines). Table 1. Comparison of several electrodes for impedimetric detection of thrombin Electrodes

∆Rca) t ±SD (Ω)

Sensitivityb) (Ω decade-1)

Detection limitb) (pM)

GC+apt1+BSA

5270±554

1185.8

5

GC+MWCNT+ap1+BS A

425±91

-

-

GC+MWCNTapt1+BSA

1704±163

440.0

5

GC+MWCNTapt2+BSA

1298±117

-

-

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Signal amplification based on MWCNT-apt2-HRP hybrids and post-electro-polymerization of enzymatically biocatalyzed precipitates

a) ∆Rct is the change of electron transfer resistance before and after the electrodes interacted with 50 nM thrombin. The loading mass of MWCNT, MWCNT-apt1, and MWCNT-apt2 were optimized (Figures S1-S3, Supporting Information) b) Obtained from the calibration curves of the electrodes for thrombin detection (Figure S4, Supporting Information).

To study the effects of MWCNT or MWCNT-apt hybrid modifications on the responses of the impedimetric aptasensors, the GC+MWCNT+apt1+BSA, GC+MWCNT-apt1+BSA, and GC+MWCNT-apt2+BSA electrodes were prepared and investigated by CV and EIS in detail and the results were shown in Figures S1-S4 (Supporting Information) and listed in Table 1. The GC+apt1+BSA electrode exhibits the largest response (∆Rct) to 50 nM thrombin and the modifications of GC electrode with MWCNT or MWCNT-apt hybrids can not enhance the response of the GC+MWCNT+apt1+BSA or GC+MWCNT-apt+BSA electrode. The detail discussions were presented in Supporting Information.

Figure 3. The cyclic voltammograms (A) and Nyquist plots (B, C) of the GC+apt1+BSA electrode for thrombin detection by signal amplification. (B) apt2 (line II), MWCNT-apt2 (line III), and MWCNT-apt2-HRP (line IV) hybrids were used for signal amplification; (C) MWCNT-apt2-HRP and electro-polymerized precipitates (line II) were used for signal amplification. Insert plot of (C): the cyclic voltammogram of the GC+apt1+BSA electrode using the MWCNT-apt2-HRP and electro-polymerized precipitates for signal amplification. The electrolyte: 0.01M PBS (pH 7.0) containing 2.0 mM [Fe(CN)6]3-/4-. The concentration of thrombin is 50 nM.


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The thrombin binding aptamers (apt1 and apt2) consist of two G-quartet conformations that selectively bind to specific and different epitopes of human α-thrombin 57. The GC+apt1+BSA electrode was prepared and then, the thrombin was bound onto the GC+apt1+BSA electrode surface by specific interaction between apt1 and thrombin. The signal amplification for impedimetric detection of thrombin was based on the specific interaction between the apt2 and thrombin on the GC+apt1+BSA electrode, which achieved by the binding of the MWCNT-apt2-HRP hybrids onto the GC+apt1+BSA+thrombin electrode, followed by the postelectro-polymerization of the precipitates produced by the enzymatically biocatalyzed reaction between HRP and DAB (shown in Figure 1). The amount of MWCNT-apt2-HRP hybrids, which bind to the GC+apt1+BSA+thrombin electrode, was dependent on the amount of thrombin sandwiched. Because the conditions of the enzymatically biocatalyzed reaction and the CV parameters for electro-polymerization of the precipitates were fixed throughout the experiments, the produced precipitates and the formed polymeric layer should be related to the amount of MWCNT-apt2-HRP, that is, related to the thrombin sandwiched. The produced precipitates and the formed polymeric layer will be different at different thrombin concentrations, resulting in different ∆Rct. It was confirmed by the EIS characterization and QCM analysis (Figure S5, Support Information) Figure 3 shows the cyclic voltammograms and Nyquist plots of the GC+apt1+BSA electrode for thrombin determination by signal amplification. The signal amplification by apt2 or MWCNT-apt2 or MWCNT-apt2-HRP hybrids all leads to the decrease of redox current density and increse of the Rct of the redox pair at the GC+apt1+BSA electrodes (Figure 3A and B). The ∆Rct after the MWCNT-apt2 (Figure 3B, blue line) binding onto the GC+apt1+BSA+thrombin electrode reaches 1.59×104 Ω, and is about one time larger than that (0.78×104 Ω) using apt2 (Figure 3B, red line) as signal amplification probe. The MWCNT-apt2 hybrids are more effective for signal amplification due to the large surface area of MWCNTs enabling it to immobilization more apt2 molecules. To further enhance the signal amplification, MWCNT-apt2HRP hybrids were prepared and used as probe. The success of functionalization of the apt2 and HRP onto MWCNTs were verified by FTIR (Figure S6, Supporting Information). The binding of MWCNT-apt2-HRP onto the GC+apt1+BSA+ thrombin electrode leads to about 1.78×104 Ω of ∆Rct (Figure 3B, pink line), being a little larger than that using MWCNTapt2 hybrids (Figure 3B, blue line). However, the post-electropolymerization of the precipitates produced by the enzymatically biocatalyzed reaction between HRP and DAB greatly improves the signal amplification. The redox peaks of the [Fe(CN)6]3-/4- redox pair almost dispear at the electropolymerized precipitate electrode (Figure 3C, insert plot). The ∆Rct resulting from post-electro-polymerized insoluble precipitates reaches 3.2×104 Ω (Figure 3C, red line), being 5 times larger than that (5270 Ω) leaded by the thrombin binding to the GC+apt1+BSA electrode (Figure 2B). The results suggest that the proposed signal amplification by MWCNT-apt2-HRP and the post-electro-polymerization of the precipitates can be used for ultrasensitively impedimetric detection of thrombin. For comparison, the Nyquist plots of post-electro-polymerized precipitates by using apt2-HRP and MWCNT-HRP were also provided (Figure S7, Supporting Information). In addition, the possible process of post-electro-

polymerization of the insoluble precipitates was described and discussed (Figures S8-S11, Supporting Information). To demonstrate the signal amplification stratage more clearly, the morphologies and thickness of various electrodes were characterized by AFM and SEM (Figure 4 and Figures S12-S15, Supporting Information). The immobilized apt1 molecules (Figure 4A) exhbit particle shape and disperse on the GC electrode surface with relative uniformity. The BSA blocking (Figure 4B), thrombin interaction with the apt1 (Figure 4C), MWCNT-apt2-HRP amplification (Figure 4D), and the electro-polymerized precipitates (Figure 4E) are clearly verified. The immobilization of apt1, BSA blocking, and apt1-thrombin interaction decrease the electroactive surface area of GC electrode, and increase the hindrance to the electrochemical reaction of redox probe, resulting in progressively increase of the redeox probe Rct (Figure 2). Upon the signal amplification by MWCNT-apt2-HRP hybrids, the electrode shows porous microstructure and the electroactive surface area further decreases (Figure 4D). But the electrode surface is almost occupied after the post-electropolymerization of the precipitates (Figure 4E), leading to greatly increase of the Rct (Figure 3C). In addition, the thickness of the modification layers increase step by step (from about 50 nm to 9 µm; Figures S12-S15, Supporting Information). The Rct of the redox probe should be affect by not only the electroactive electrode surface area but also the thickness of the modification layers.

Figure 4. The AFM (A-C) and SEM (D and E) images of the GC+apt1 (A), GC+apt1+BSA (B), GC+apt1+BSA+thrombin (C), GC+apt1+BSA+thrombin+MWCNT-apt2-HRP (D), and GC+apt1+BSA+thrombin+MWCNT-apt2HRP+electropolymerized precipitates (E) electrodes.

Analytical properties of the proposed impedimetric aptasensor for thrombin detection To test the selectivity of the impedimetric aptasensor for thrombin detection, the BSA, h-IgG, and HRP were used as control. Figure S16 (Supporting Information) shows the responses of the GC+apt1+BSA electrodes to different proteins. The ∆Rct obtained from 0.1 µM BSA, h-IgG, and HRP are very little in contrast to 50 nM thrombin, indicating that the assay has good selectivity. On the other hand, the reproducibility was investigated. Samples (50 nM thrombin) were detected by six GC+apt1+BSA electrodes, giving a relative standard deviation (RSD) of 10.5%. The results reveal that the aptasensor has acceptable reproducibility. The proposed impedimetric aptasensor shows relatively fast response and the EIS measurement can be completed in about 9 min.



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Figure 5 shows Nyquist plots and the relationship between the ∆Rct and the logarithm of thrombin concentration (logCthrombin) using the MWCNT-apt2-HRP hybrids and the post-electro-polymerization of precipitates as signal amplification. The ∆Rct is positively correlated with thrombin concentration, and the ∆Rct-logCthrombin curve shows a linear relationship in the range of 0.05 pM to 50 nM. The regression equation in the linear detection range can be obtained and expressed as ∆Rct = 81056 + 5195logCthrombin and the linear curve has the slope of 5195 Ω decade-1 (the sensitivity of the

S3 (Supporting Information). The acceptable recovery and RSD imply that the impedimetric aptasenosr has potential application in complex thrombin samples. In addition, many other protein impedimetric aptasensors can be developed based on the same signal amplification principle and therefore, found broader applications in ultrasensitive detection of proteins. The analytical performances of the proposed impedimetric aptasensor would be affected by some aspects. Besides the aptamer affinity, the performances could be further enhanced by optimization of other factors including the apt1 immobilization onto the surface of GC electrode, the immobilization of apt2 and HRP onto the surface of MWCNTs, the kinetics of aptamer-thrombin interaction, the conditions of enzymatically biocatalyzed reaction, and the electro-polymerization of the precipitates. On the other hand, the proposed impedimetric aptasensor was singly prepared and used. The reproducibility and calibration will be somewhat limited. Moreover, it will not be adaptable for the analysis of large samples. The aptasensor arrays (chip) and multi-channel technique for electrochemical measurement would improve the reproducibility, calibration, and efficiency of preparation and measurement, which are desirable for high throughput analysis of protein samples. CONCLUSIONS We have developed a highly sensitive impedimetric aptasensor for thrombin detection. The preparation of the GC+apt1+BSA electrodes is simple and just includes pretreatment of the GC electrode, apt1 immobilization by adsorption, and BSA blocking. More importantly, the interaction between apt and thrombin can be greatly amplified by using MWCNT-apt2-HRP hybrids and the post-electropolymerization of the precipitates produced by enzymatically biocatalyzed reaction between HRP and DAB, rendering ultrasensitive detection of thrombin with a detection limit of 0.05 pM. In addition, the reagents involved in signal amplification are commercially available and widely used in enzyme linked immunosensing assay kits, which make the proposed impedimetric aptasensor be cost-effective. The impedimetric aptasensor based on MWCNT-apt-HRP hybrids and post-electropolymerization of enzymatically biocatalyzed precipitates may find wide application in the routine detection of proteins with high sensitivity and low detection limit.

Figure. 5. Nyquist plots (A) and the calibration curve (B) of the prepared impedimetric aptasensor for thrombin detection using signal amplification at various thrombin concentrations in 0.01 M PBS (pH 7.0).

ASSOCIATED CONTENT

impedimetric aptasensor). The sensitivity of impedimetric aptasensors reported previously was listed in Table S1 (Supporting Information). Moreover, the detection limt is 0.05 pM (S/N>3), which is lower than that of the existing impedimetric aptasensors by ca. 4 orders 15, 38-40, and better than or comparable with those reported previously using other protocols or signal amplification methods for thrombin determination. The detection limits of some optical, impedimetric, and voltammetric aptasensors are listed in Table S2 (Supporting Information). The effect of non-specific adsorption of MWCNT-apt2-HRP on the observed detection limit of the impedimetric aptasensor was aslo disscussed (Figure S17, Supporting Information). The analytical applicablity of the proposed impedimetric aptasensor was evaluated by the standard addition method, as listed in Table

Supporting Information Details for the preparation of carboxylated MWCNTs, MWCNTapt hybrids, MWCNT-apt2-HRP, MWCNT-HRP, and apt2-HRP; pre-treatment of GC electrode, preparation of GC+MWCNT+apt1+BSA, GC+MWCNT-apt1+BSA, and GC+MWCNT-apt2+BSA electrodes; the effects of MWCNT or MWCNT-apt hybrid modification on the responses of the impedimetric aptasensors; QCM analysis of the produced precipitates and the formed electro-polymerized precipitates; FTIR spectrum of MWCNT-apt2-HRP; the Nyquist plots of the GC+apt1+BSA electrodes for thrombin detection using apt2-HRP and MWCNTHRP for signal amplification; the process of electropolymerization of the insoluble precipitates; the thickness of various electrodes; the selectivity of the GC+apt1+BSA electrode for the thrombin detection; summary of the sensitivity of the impedimetric aptasensors reported in the literatures; summary of detection limits of the impedimetric, optical, transistor, and voltammetric aptasensors reported in literatures; effect of non-specific ad-


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sorption of MWCNT-apt2-HRP on the detection limit; detection of thrombin in the human blood serum substrate using the proposed impedimetric aptasensor. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Tel. /Fax: +86-731-88872618. E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally.

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China under grants (21275050, 21145001), the Hunan Provincial Natural Science Foundation of China (13JJ1016), the Scientific Research Fund of Hunan Provincial Education Department (13A053), the Construct Program of the Key Discipline in Hunan Province, and the Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province.

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Carbon Nanotubes Labeled with Aptamer and Horseradish

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