A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

Analytical Biochemistry 441 (2013) 95–100

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A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin Xiao Xia Jiao, Jing Rong Chen, Xi Yuan Zhang, Hong Qun Luo ⇑, Nian Bing Li ⇑ Key Laboratory of Eco-environments in the Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China

a r t i c l e

i n f o

Article history: Received 11 May 2013 Received in revised form 8 July 2013 Accepted 17 July 2013 Available online 26 July 2013 Keywords: Aptasensor Chronocoulometry Gold nanoparticles Thrombin

a b s t r a c t A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH3)6]3+ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin. Ó 2013 Elsevier Inc. All rights reserved.

The sensitive and quantitative analysis of proteins is very important in disease diagnosis and biomedical research. The establishment of a reliable detection method for proteins has attracted the efforts of the scientific community [1,2]. In many immunoassay techniques, antibodies are frequently used for the recognition of proteins because of their high specificity. In recent years, aptamers, a new class of single-stranded DNA (ssDNA) or RNA oligonucleotides obtained through a method called ‘‘systematic evolution of ligands by exponential enrichment’’ from random DNA or RNA libraries [3,4], have become an alternative in analytical bioassays as protein recognition elements. Compared with antibodies, aptamers present some advantages, such as relatively easy production, excellent target versatility, specific binding, high stability in complex physical and chemical environments, long-term storage, reversible thermodynamic denaturation, and convenient regeneration [5–8]. Thrombin, which is a specific serine protease involved in the coagulation cascade, thrombosis, and hemostasis, has attracted much interest because of its superior functions such as converting soluble fibrinogen into insoluble strands of fibrin and catalyzing many other coagulation-related reactions [9,10]. In addition to its

⇑ Corresponding authors. Fax: +86 23 68253237. E-mail addresses: [email protected] (H.Q. Luo), [email protected] (N.B. Li). 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.07.023

key role in the dynamic process of thrombus formation, thrombin has a pronounced proinflammatory character, which may influence the beginning and progression of atherosclerosis [11]. Therefore, a sensitive and specific method for the detection of thrombin at low concentration is necessary. So far, various strategies and technologies using aptasensors for thrombin have been developed, such as fluorescence [12,13], electrochemiluminescence [14], quartz crystal microbalance [15], surface plasmon resonance [16], and electrochemistry [17–21]. Among these, the electrochemical method has attracted much attention in the development of aptasensors because of its simple instrumentation, high sensitivity, fast response, and low cost. To realize ultrasensitive detection, signal amplification of the electrochemical aptasensor is essential. Many methods for signal amplification have been introduced, such as rolling circle amplification [22–24], enzyme-based amplification [25–27], strand displacement amplification [28], and so on. However, because of the complicated protocols, high cost, and rigorous detection conditions, these methods for signal amplification are disadvantageous. Recently, metal nanoparticles used in signal amplification [29,30], especially gold nanoparticles (AuNP’s), have attracted great attention because of their facile synthesis, large specific surface area, high chemical stability, favorable biocompatibility, good conductivity, and high affinity of binding to amine/thiol-containing molecules [31].

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purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (China). All reagents were of analytical grade and used without further purification. All solutions were prepared with doubly distilled water (18.2 MX cm). All glassware used in the following procedure was cleaned in a bath of freshly prepared 3:1 HCl:HNO3 and then rinsed thoroughly with doubly distilled water. Apparatus

Scheme 1. Schematic diagram for the fabrication of a chronocoulometric aptasensor based on gold nanoparticle amplification for the detection of thrombin.

Cyclic voltammetry (CV), electrochemical impedance spectroscopy, and chronocoulometry (CC) were performed with a CHI 660B electrochemical workstation (Shanghai Chenhua Instrument Corp., China). Differential pulse voltammetry was performed with an Autolab PGSTAT302 electrochemical workstation (Eco Chemie BV, Utrecht, The Netherlands). A conventional three-electrode cell was employed, which involved a gold working electrode (2 mm in diameter), a platinum foil counterelectrode, and a saturated calomel reference electrode (SCE). All the potentials in this paper are given with respect to SCE. The electrolyte buffer was thoroughly purged with nitrogen before experiments. Transmission electron microscopy (TEM) images were taken using a Hitachi 600 (Hitachi Ltd., Tokyo, Japan). Preparation of gold nanoparticles

Herein, we report a novel strategy to develop a sensitive chronocoulometric aptasensor based on gold nanoparticle amplification for the detection of thrombin. The principle of the chronocoulometric aptasensor for the detection of thrombin is illustrated in Scheme 1. First, thrombin aptamers were self-assembled on the surface of a gold electrode to form a monolayer. The DNA-functionalized AuNP’s were immobilized on a thrombin aptamer-modified gold electrode by DNA hybridization. Different from the reported AuNP’s with one kind of DNA, AuNP’s labeled with two kinds of DNA were used here. Since link DNA (LDNA) was complementary to aptamers and report DNA (RDNA) was noncomplementary, the low density of LDNA on AuNP’s would be favorable to the one-to-one combination of aptamers. In the absence of thrombin, the functional gold nanoparticles (DNA– AuNP’s) localized at the electrode surface could electrostatically adsorb abundant hexaamineruthenium(III) chloride ([Ru(NH3)6]3+; RuHex) cations, which serve as the signaling molecules. After thrombin was added, as aptamers could bind tightly to target molecules to form a tertiary complex with a binding constant greater than that of the DNA duplex, the DNA–AuNP’s were replaced by the target to form the target–aptamer complex. The release of DNA–AuNP’s was accompanied by the extrication of the abundant RuHex molecules, which induced a significant decrease in chronocoulometric signal. By employing this strategy, we demonstrated that this chronocoulometric aptasensor can sensitively and selectively detect thrombin.

Gold nanoparticles were prepared by citrate reduction of HAuCl4 according to the literature [32]. Briefly, 10 ml of 38.8 mM sodium citrate was immediately added to 100 ml of 1.0 mM HAuCl4 refluxing solution under stirring, and the mixture was kept boiling for another 15 min. The solution color turned to wine red and was cooled to room temperature with continuous stirring. The size of the AuNP’s was verified by transmission electron micrograph and the diameter was 12 ± 2.0 nm. The TEM image of AuNP’s is shown in Fig. 1. Functionalization of gold nanoparticles with DNA The immobilization of LDNA and RDNA on AuNP’s was carried out as follows. Briefly, a mixture of LDNA (5 lM) and RDNA (20 lM) was activated with 4 ll of 10 mM acetate buffer (pH 5.2)

Experimental procedures Reagents All oligonucleotides were synthesized and purified by Sangon, Inc. (Shanghai, China). The sequences of the single-stranded oligonucleotides were as follows: link DNA, 50 -SH-(CH2)6-AACCAACCACAC-30 ; report DNA, 50 -SH-(CH2)6-TTTTTTTCGGCCTGTTCCGG-30 ; aptamer, 30 -TTGGTTGGTGTGGTTGGTTT-(CH2)6-SH-50 . Mercaptohexanol (MCH), RuHex, thrombin, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris) and AuCl3HCl4H2O were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Lysozyme and bovine serum albumin were

Fig.1. TEM image of AuNP’s.

Chronocoulometric aptasensor for thrombin / X.X. Jiao et al. / Anal. Biochem. 441 (2013) 95–100

and 1.5 ll of 40 mM TCEP for 1 h and then added into 100 ll of freshly prepared AuNP’s for 16 h in darkness at room temperature. Then, the DNA–AuNP conjugates were ‘‘aged’’ in salts (0.1 M NaCl, 25 mM Tris acetate, pH 7.4) for 24 h. Excess reagents were removed by centrifuging at 15,000 rpm for 30 min. The red precipitate was washed, recentrifuged, dispersed in 100 ll of 25 mM Tris acetate buffer (pH 7.4) containing 0.1 M NaCl, and stored at 4 °C.

Fabrication of the aptasensor and thrombin detection A gold electrode was polished carefully with 0.3- and 0.05-lm alumina powder and washed ultrasonically with Milli-Q water, ethanol, and Milli-Q water for 3 min each. Subsequently, the electrode was electrochemically cleaned in 0.5 M H2SO4 solution by cyclic potential scanning between 0.2 and 1.6 V until a reproducible cyclic voltammogram was obtained. Then, the electrode was washed with Milli-Q water and dried in a mild nitrogen stream. The aptamer self-assembly process was performed by treating the clean Au electrode with 25 mM Tris–HCl buffer (pH 7.4) containing 0.2 lM aptamers and 8.0 mM TCEP for 15 h. The modified electrode was then immersed in 1.0 mM MCH for 1 h to cover aptamer-unbound surface and obtain a well-aligned aptamer monolayer. After that, the modified working electrode was further immersed in a DNA–AuNP solution for 6 h at room temperature. The electrode surface was rinsed with 25 mM Tris–HCl buffer containing 0.1 M NaCl (pH 7.4) after each step of the fabrication process to remove nonspecific DNA sequences. For the thrombin detection, 10 ll of thrombin solution (a series of concentrations from 0.1  109 to 18.5  109 M) was placed onto the modified electrode for 40 min at 36 °C. Chronocoulometry was carried out in 10 mM Tris–HCl buffer (pH 7.4) containing 20 lM RuHex.

Surface density of aptamers The surface density (the number of immobilized electroactive ssDNA moles per unit area of the electrode surface) was chronocoulometrically quantified from the redox charges of RuHex [33]. When the aptamer-modified surface was exposed to a solution containing [Ru(NH3)6]3+, the redox cations bound electrostatically to the aptamers by replacing the native charge compensation ions (Na+) until an ion-exchange equilibrium was reached. Thus, the surface density of aptamers can be calculated by the measured charge acquired from the reduction of the [Ru(NH3)6]3+. The amount of electroactive oligonucleotides on the electrode surface (Uss) is calculated based on the equation:

Css ¼ ðQ ss NA =nFAÞðz=mÞ

ð1Þ

where Qss is the charge corresponding to RuHex electrostatically bound to surface-confined ssDNA,NA is Avogadro’s number,n is the number of electrons transferred in the reaction (RuHex3+ + e ? RuHex2+, n = 1), F represents the Faraday constant (coulombs per equivalent), A is the effective surface area of gold electrode (square centimeters), z is the charge of the redox molecules, and m is the number of nucleotides in the DNA. The Qss can be calculated from the equation:

Q ss ¼ Q total  Q dl

ð2Þ

where Qtotal represents the total charge flowing through the electrode, comprising both faradaic (redox) charges and nonfaradaic (capacitive) charges (Qdl).

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Results and discussion Characterization of the aptasensor In this study, cyclic voltammetry and electrochemical impedance spectroscopy were used to monitor the process of fabrication of the aptasensor in each step. As expected, the behavior of K3[Fe(CN)6]/K4[Fe(CN)6] on a bare gold electrode was reversible with a peak-to-peak separation DEp of 91 mV (Fig. 2A, curve a). After the electrode was assembled with aptamers, the peak currents decreased and the peak-to-peak separation increased (Fig. 2A, curve b), probably because of the established kinetics barrier between [Fe(CN)6]3/4 and the negatively charged phosphate backbone of the DNA. After treatment with MCH, the peak currents decreased (Fig. 2A, curve c), implying a remarkable increase in the resistance to electron transfer. When the modified electrode was immersed in DNA-functionalized AuNP’s, an increase in the DEp of the electrode was observed (Fig. 2A, curve d). It was largely attributed to the fact that the surface of AuNP’s was covered with DNA and the bulk negative charges could further repel the probes of [Fe(CN)6]4/3 anions. A consistent result is also provided in Fig. 2B. The electrontransfer resistance increased in the order of the bare gold electrode (Fig. 2B, curve a), aptamer-modified gold electrode (Fig. 2B, curve b), MCH/aptamer-modified gold electrode (Fig. 2B, curve c), and DNA–AuNP/MCH/aptamer-modified gold electrode (Fig. 2B, curve d). The increase in electron-transfer resistance indicated that the aptamers and DNA–AuNP’s were successfully immobilized on the electrode surface. Differential pulse voltammetry was employed to characterize the electrochemistry of RuHex. As shown in Fig. 3, two peaks (peaks I and II) can be observed when MCH/aptamer-modified gold electrode (curve a) or DNA–AuNP/MCH/aptamer-modified gold electrode (curve b) was immersed in 10 mM Tris–HCl buffer (pH 7.4) containing 20 lM RuHex. Peak I arose owing to the diffusion-based redox process of RuHex (RuHex diffused to the electrode). The other peak (peak II), observed at about 0.34 V, was ascribed to the surface-confined redox process of RuHex electrostatically bound to the phosphate backbone of DNA [34]. It was noted that a small peak current (peak II) was observed at the MCH/aptamer-modified electrode, and a significant enhancement of peak II can be found at the DNA–AuNP/MCH/aptamer-modified electrode. These differential pulse voltammograms provided an intuitive impression of the amplification effect of AuNP’s, implying that AuNP’s could be used to realize the thrombin detection with high sensitivity. As shown in Supplementary Fig. S1, the chronocoulometric technique was also employed to demonstrate the amplification effect of AuNP’s. Fan and co-workers [35] have demonstrated that chronocoulometry is more accurate for detecting the signal of electrostatically trapped redox markers than other electrochemical methods, and the RuHex–DNA–electrode system can be used to generate an intense signal in chronocoulometry. This is possibly because a large portion of RuHex molecules entrapped in the heterogeneous film are kinetically electroinactive during ‘‘dynamic’’ voltammetric scans, while nearly all RuHex molecules are electroactive in the ‘‘static’’ chronocoulometric measurements. Therefore, the CC technique was employed for the thrombin detection experiments.

Optimization of surface density of aptamers Electrodes with DNA self-assembly monolayers of various surface densities were researched in this work. Low-density surfaces (0.5  1012 molecules/cm2) were obtained by incubation of the electrode with 0.2 lM aptamers in the immobilization buffer.

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Fig.2. (A) Cyclic voltammograms and (B) Nyquist plots of impedance spectra of the bare gold electrode (curve a), aptamer-modified gold electrode (curve b), MCH/aptamermodified gold electrode (curve c), and DNA–AuNP/MCH/aptamer-modified gold electrode (curve d) in 5 mM [Fe(CN)6]3/4 solution. The scan rate of CV is 100 mV s1. The frequency range is from 0.1 to 105 Hz and the amplitude of the alternate voltage is 5 mV.

(DQ = QAuNP’s  Qss, Supplementary Fig. S2) occurred at the surface density of aptamers with 0.5  1012 molecules/cm2. The inset shows chronocoulometric curves for DNA–AuNP/MCH/aptamermodified gold electrodes assembled with various concentrations of thrombin aptamers. The chronocoulometric signal change decreased when the density was larger than 0.5  1012 molecules/ cm2. The reason may be that too many aptamers on the electrode could not fully hybridize with DNA–AuNP’s. However, the aptamers without DNA–AuNP hybridization could not cause the signal amplification, resulting in a decrease in sensitivity. Therefore, the optimum DNA monolayer (0.5  1012 molecules/cm2) was adopted in the following experiments. Sensitive detection of thrombin Fig.3. Differential pulse voltammograms of MCH/aptamer-modified gold electrode (curve a) and DNA–AuNP/MCH/aptamer-modified gold electrode (curve b) in 10 mM Tris–HCl buffer (pH 7.4) containing 20 lM RuHex.

Medium-density (1.3  1012 molecules/cm2) and high-density (5.8  1012 molecules/cm2) surfaces were prepared by incubation of electrodes with 1 and 5 lM aptamers in the immobilization buffer, respectively. After the preparation of aptamer self-assembly monolayers, DNA–AuNP’s were assembled successively on the aptamer self-assembly monolayers in sufficient hybridization time. As shown in Fig. 4, the largest chronocoulometric signal change

Under the optimum conditions, the analytical performance of the aptasensor was investigated by exposing the aptasensor to a series of thrombin concentrations under the same experimental conditions, and the results are shown in Fig. 5. As shown in Fig. 5A, it can be observed that the chronocoulometric signal decreased with increasing concentration of thrombin. It was clearly demonstrated that the introduction of thrombin at various concentrations to the sensing interface resulted in varying degrees of digestion of DNA–AuNP’s. The plot of the chronocoulometric signal change as a function of thrombin concentration is illustrated in Fig. 5B. The value of DQ was linearly related to the thrombin concentration range of 0.1–18.5 nM. The calibration equation was DQ = 0.0396 Cthrombin + 0.1253 (DQ in lC, Cthrombin in nM) with a correlation coefficient of 0.992. The detection limit was down to 30 pM. The results indicated that this method can successfully detect thrombin, and the amplification based on AuNP’s was not only simple but also effective for the enhancement of the sensitivity of the aptasensor. Since one gold nanoparticle could load with a few hundred DNA strands, which absorbed thousands of RuHex molecules, after the electrode was incubated with thrombin, a significant decrease in chronocoulometric signal was observed compared with only one complementary DNA strand as the leaving group. Six replicate chronocoulometric analyses showed a relative standard deviation of 5.8% for 5.0 nM thrombin, implying that the aptasensor also had good repeatability. Detection selectivity for thrombin

Fig.4. Comparison of chronocoulometric signal change for gold electrodes with various aptamer surface densities. The inset shows chronocoulometric curves for DNA–AuNP/MCH/aptamer-modified gold electrodes assembled with various concentrations of thrombin aptamers. Concentration of thrombin aptamers (from curve a to curve c): 0.2, 1.0, and 5.0 lM, respectively.

As an aptasensor, the selectivity is obviously a crucial factor to be considered. In this work, two kinds of similar proteins, including bovine serum albumin and lysozyme, were chosen to study the selectivity. As shown in Fig. 6, a significant decrease in chronocoulometric signal induced by the interaction of the aptamers with

Chronocoulometric aptasensor for thrombin / X.X. Jiao et al. / Anal. Biochem. 441 (2013) 95–100

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Fig.5. (A) Chronocoulometric curves for DNA–AuNP/MCH/aptamer-modified gold electrode in 10 mM Tris buffer (pH 7.4) containing 20 lM RuHex after interaction with various concentrations of thrombin. Concentration of thrombin (from curve a to curve g): 0, 0.1, 0.5, 5.0, 12.0, 18.5, and 25.0 nM, respectively. Redox charges of RuHex bound to DNA are obtained from chronocoulometric intercepts at t = 0. (B) Linear relationship between DQ and various concentrations of thrombin. DQ is defined as the difference in the charge of RuHex before and after interaction with thrombin. Error bars show the standard deviations of measurements taken from at least three independent experiments.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21273174, 20975083) and the Municipal Science Foundation of Chongqing City (No. CSTC–2013jjB00002). Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab. 2013.07.023. Fig.6. The change in chronocoulometric signal of DNA–AuNP/MCH/aptamer-modified gold electrode after incubation with 18.5 nM thrombin (column a), 1 lM bovine serum albumin (column b), 1 lM lysozyme (column c), and blank (column d).

thrombin (18.5 nM) was observed. The DQ of thrombin was 0.8190 lC (Fig. 6, column a), which was much higher than that of bovine serum albumin (1 lM) and lysozyme (1 lM), with DQ of 0.0950 lC (Fig. 6, column b) and 0.0840 lC (Fig. 6, column c), respectively. The DQ of the blank background was 0.0380 lC (Fig. 6, column d). The results demonstrated that the aptasensor was able to discriminate thrombin as the target with the high specificity. The reason may be the effective surface blocking by MCH and the highly specific binding between the aptamers and thrombin.

Conclusions By taking advantage of the stronger binding affinity of the aptamers toward thrombin than toward the aptamer-complementary DNA oligonucleotide, we developed a novel chronocoulometric aptasensor for thrombin determination based on gold nanoparticles as signal-amplified strategy. The developed aptasensor was demonstrated to be very sensitive and specific for the detection of thrombin. DNA-functionalized AuNP’s were proved to be an efficient signal amplifier, which caused significant chronocoulometric signal change of surface-confined RuHex. The fabrication process of the aptasensor was time-saving, easy, and simple. A 30 pM detection limit was achieved through not only signal amplification by AuNP’s, but also the control of the self-assembly process of DNA probes at the gold electrode. Therefore, it is expected to provide new possibilities for protein analysis as well as for disease diagnosis.

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