Electrochemical Reduction Potential Shifts of Graphene Oxide Employed in Thrombin Detection

Experimental

TBA (NH2-C6-5'TTT TTT TTT TTT TTT AGT CCG TGG TTG GTG TGG TTG GGG TGA CT-3') and blocking DNA (5'-TTT TTG GGT TTT T-3') were purchased from Genotech (Korea). All chemicals including graphite powder (< 150 µm) were purchased from Sigma Aldrich. Tris-HCl buffer consist of 25 mM Tris-HCl and 300 mM and 1 M NaCl with pH 7.4. Phosphate buffer saline (PBS) consists of 100 mM phosphate and 100 mM NaCl. The binding buffer consists of 50 mM Tris-HCl, 140 mM NaCl, 1 mM MgCl2, and 0.1% bovine serum albumin (BSA). All electrochemical measurements, including CV and EIS, were carried out using CHI-A617 and Ivium Compactstat as a potentiostat interfaced with a PC. SEM images were obtained using a JEOL JSM-7001F. Raman spectroscopy was performed by alpha 300 (WITEC).

GO suspension was synthesized from graphite powder by a modified Hummers method.23 Graphite powder (3 g) was taken in an Erlenmeyer flask and stirred in ice bath. NaNO3 (1.5 g) and concentrated H2SO4 (69 mL) were added into the Erlenmeyer flask. Then KMnO4 (9 g) was added slowly and the mixture was stirred for 3 h. 120 mL of deionized (DI) water was added slowly over 30 min. DI water (200 mL) was added, and followed by slow addition of 30% H2O2 (3 mL) solution. The color of the suspension changed from brown to yellow. The suspension was centrifuged at 4500 rpm with 5% HCl solution for 2 rounds and then centrifuged at high speed for several times with distilled water.

Prior to the surface modification, gold coated glass elect-rodes were cleaned by using piranha solution (H2SO4:H2O2 = 3:1), washed with sufficient amount of deionized water, and finally dried with N2 gas. In order to create the positive and negative charged layers in sequence, the freshly cleaned gold electrodes were soaked in 10 mM MPA and 0.1% PEI solution for 12 h and 1.5 h, respectively. The positively charged electrodes were immersed in 0.5 mg/mL solution of GO for 1 h. The blocking DNA was treated on the GO-modified electrodes to prevent non-specific binding of NH2-terminated TBA. 1:1 mixture of 200 mM EDC and 50 mM NHS was exposed to the GO modified surface for 1 h to immobilize NH2-terminated TBA. Then the surface was incubated with 1% BSA solution in PBS for 1 h to block nonspecific binding of thrombin on the surface.

The modified electrodes were exposed to various concent-rations of thrombin solutions and incubated for 1.5 h. For negative control experiment, same procedure was followed without thrombin. All electrochemical measurements were carried out by using electrochemical cell consisting of the modified gold electrode as the working electrode, and Ag/AgCl and Pt wire as the reference and counter electrodes, respectively. CV measurements were performed range from −0.4 V to −1.3 V. EIS measurements were recorded between 10 kHz and 0.05 Hz at the formal potential of K4[Fe(CN)6]/K3[Fe(CN)6] (1 mM, 1:1 molar ratio) in PBS. A Randle equivalent circuit was used to fit the obtained impedance spectra, which were then represented as a Nyquist plot in the complex plane.