Effective covalent immobilization of quinone and aptamer onto a gold electrode via thiol addition for sensitive and selective protein biosensing
Introduction
Detecting proteins using biosensors in a cost-effective, sensitive and rapid manner is a crucial issue in fundamental research and clinical applications [1], [2], [3]. Because of their portability, inexpensiveness, simple instrumentation, high sensitivity and fast response, the electrochemical methods have attracted substantial attention in the development of aptasensors [4], [5], [6], [7], [8], [9], [10]. In general, the electrochemical aptasensors were fabricated by employing redox-labeled aptamer [4], [11], [12] or label-free impedance strategy [13], [14]. However, it is not easy to obtain the redox-labeled aptamer, because the existing methods of covalent labeling of the aptamer require complicated and labor-intensive labeling procedures. Therefore, it is still an important and active research topic for developing sensitive, convenient, and cost-effective labeling protocols for aptasensing. Among various aptasensors that utilize differernt signal transduction techniques such as electrochemistry [15], [16], optics [17], and atomic force microscopy [18], electrochemical aptasensors have exhibited great promise in protein detection with high sensitivity, selectivity, and cost-effectiveness.
The thiols and thiolated chemicals are highly concerned in surface chemistry and biosensors [19], [20]. Interestingly, the redox-active quinone has an alkenes-like electron-conjugate structure can interact with amines for proteins [21], [22] and other molecular [23], [24] immobilization or interact with thiols for proteins [25], thiolated DNA [26] and other molecular [24] immobilization by Michael addition reaction [21], [22], [23], [24], [25], [26], [27], [28]. In view of above compelling merits, we were prompted to fabricate gold surface-attached quinone and thiolated aptamer via thiol addition (a Michael addition) that is electrochemically measurable based on TTA-BQ-HDT/Au. To our knowledge, the thiol addition serves as an effective and convenient method of immobilization of quinone and thiolated aptamer onto a gold electrode, and the quinone acts as a signaling transducer for preparation of sensitive electrochemical aptasensor have hardly been investigated for biosensing applications to date.
In this work, effective covalent immobilization of quinone and aptamer onto a gold electrode via thiol addition for sensitive and selective protein (with thrombin as the model) biosensing is reported. Herein the thiol addition acts as an effective and convenient binding protocols for aptasensing, and a new method (electrochemical conversion of Michael addition complex for signal generation) for the fabrication of biosensor is presented. In addition, the reagents involved in the proposed amperometric aptasensor are commercially available and widely used in biosensing, making the proposed amperometric aptasensor cost-effective. The thus-prepared TTA-BQ-HDT/Au electrode showed well-defined redox peaks of quinone moiety, and the TTA-thrombin interaction can sensitively decrease the electrochemical signal. The prepared amperometric aptasensor was found to exhibit good analytical properties (sensitivity, low detection limit, and good selectivity and reproducibility) for thrombin detection.
Section snippets
Chemical reagents
Human α-thrombin and 6-mercaptohexanol (MCH) were purchased from Sigma. TTA (5′-SH-(CH2)6-GGTTGGTGTGGTTGG-3′) was purchased from Sangon Co., Ltd. (Shanghai, China). BQ was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). HDT was purchased from Alfa Aesar. Human blood sera were obtained from the Hospital of Hunan Normal University. 10 mM phosphate buffer solution (PBS, 10 mM NaH2PO4–Na2HPO4) containing 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2 (pH 7.0) was used for binding
Characterization of the amperometric aptasensor
CV was used to investigate the electrochemical properties of labeled BQ on aptasensor. As shown in Fig. 1, we observed a pair of well-defined redox peaks centering at ca. 0.18 V vs. SCE during potential cycling of BQ-HDT/Au in 0.1 M PBS (pH 7.4). The peak currents are linear with potential scan rate, proving a facile electrochemical and a surface-controlled redox process of the labeled (surface-confined) BQ. Additionally, in the absence of HDT, the similar redox peaks were not found at the
Conclusions
In summary, A sensitive amperometric aptasensor for thrombin detection has been developed. The preparation of TTA-BQ-HDT/Au is simple and effective, immobilization of quinone and apt by thiol addition. More importantly, the modified electrode exhibits a pair of well-defined redox peaks of quinone moiety, and the TTA-thrombin interaction can sensitively decrease the electrochemical signal, providing sensitive detection of thrombin with a detection limit of 20 fM. In addition, the reagents
Acknowledgement
This work was supported by the National Natural Science Foundation of China (21305039, 21475041, 21175042 and 21075036), the Foundation of Hunan Province (14JJ3097), the Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, the Foundation of Hunan Provincial Education Department for Young Scholar, and the Foundation of Hunan Agricultural University (12YJ05).
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