Electronic Supplementary Material

Amperometric thrombin aptasensor using a glassy carbon electrode modified with polyaniline and multiwalled carbon nanotubes tethered with a thiolated aptamer

Zhaohong Su*, 1, Xiaolin Xu1, Haitao Xu1, Yi Zhang2, Chaorong Li1, Yan Ma1, Dongcheng Song1, Qingji Xie*, 2

1 College of Science, Hunan Agricultural University, Changsha 410128, PR China

2 Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China

* To whom correspondence should be addressed, Tel.: +86 731 88865515; fax: +86 731 88865515, Email: (Z. Su); (Q. Xie)


1. Preparation of the modified electrode.

Firstly, the PANI-MWCNTs nanocomposite was prepared similarly to a previous report [1], with a slight modification. In brief, the desired amount of the acidified MWCNTs (0.5 mg mL–1) was dispersed in 1 mol L–1 aqueous HCl by sonication for 20 min. Aniline (5 mmol L–1) and ammonium peroxydisulfate (5 mmol L–1) were then added to the aqueous suspension containing acidified MWCNTs under magnetic stirring and left for 5 h at 5 0C. Then, it was centrifuged, and the supernatant solution was removed, the PANI-MWCNTs nanocomposite was washed with ethanol and water thrice, respectively, and finally dried under vacuum at room temperature. 3 μL of well dispersed PANI-MWCNTs nanocomposite (0.5 mg mL-1) was cast on GCE, dried in air to form PANI-MWCNTs/GCE. Subsequently, the PANI-MWCNTs/GCE was activated for 60 s at 0.3 V in 0.1 M PB (pH 7.4), then 10 μL of 10 μM TTA (5’-SH-(CH2)6-T10AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’) solution was placed on the activated PANI-MWCNTs/GCE for 2 h to obtain TTA-PANI-MWCNTs/GCE. After treatment with 100 μM BSA for 1 h, the unmodified region of the electrode was blocked. Finally, the resulting electrode was incubated for 2 h with thrombin at various concentrations to obtain the thrombin-TTA-PANI-MWCNTs/GCE. After each step, the electrode was rinsed thoroughly with ultrapure water and then dried with a stream of nitrogen for electrochemical tests.

In addition, we also used the electropolymerized PANI (PANIe) to prepare a TTA-PANIe/MWCNTs/GCE for comparative studies. The experimental procedures are as follows. The MWCNTs/GCE (acidified MWCNTs) was immersed into 0.1 M aqueous H2SO4 containing 20 mM aniline, and a PANIe film was electro-deposited by a multipulse potentiostatic method between -0.2 and 0.85 V with a pulse width of 0.5 s and a total time of 150 s (optimized). Afterwards, the PANIe/MWCNTs/GCE was activated for 60 s at 0.3 V in 0.1 M PB (pH 7.4), then 10 μL of 10 μM TTA solution was placed on the activated PANIe-MWCNTs/GCE for 2 h to obtain TTA-PANIe-MWCNTs/GCE.

2. Detection method.

Quantitative determinations of thrombin were performed with differential pulse voltammetry (DPV) between -0.3 and 0.3 V in 0.1 M PB (pH 7.4). The optimal conditions were as follows: amplitude of 0.05 V; pulse width of 0.05 s; sampling width of 0.0167 s; pulse period of 0.2 s.

3. Preparation of human sera samples.

For human sera samples preparation, drug-free human blood samples were collected from Hunan Agricultural University Hospital. All the blood samples were obtained through venipuncture and centrifuged at 9400 g for 10 min after standing for 2 h at room temperature [2]. The serum samples were diluted 100 times with 0.10 M pH 7.4 phosphate buffer before detection. Dilution of human serum is a commonly pretreatment procedure for protease detection in samples of high complexity [3].

Fig. S1. SEM pictures of MWCNTs (A), PANI-MWCNTs (B) and PANIe-MWCNTs (C).

Fig. S2. UV-Vis spectra of (a) PANI-MWCNTs and (b) TTA-PANI-MWCNTs in 10 mM phosphate buffer containing 100 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2 (pH 7.0).

References (Only for Supplementary Material)

[1] Qiu Y, Yu J, Fang G, Shi H, Zhou X, Bai X (2008) Synthesis of Carbon/Carbon Core/Shell Nanotubes with a High Specific Surface Area. J Phys Chem C 113: 61-68

[2] Lin Z, Pan D, Hu T, Liu Z, Su X (2015) A near-infrared fluorescent bioassay for thrombin using aptamer-modified CuInS 2 quantum dots. Microchim Acta 182: 1933-1939

[3] Ma M, Zheng X (2015) Preparation of brightly fluorescent silica nanoparticles modified with lucigenin and chitosan, and their application to an aptamer-based sandwich assay for thrombin. Microchim Acta 182: 2193-2199

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