Electrochemical Aptasensors Based on a Direct Assay

Some papers have used the catal3 ic activity of thrombin for the determination of this protein. a-Human thrombin is a highly specific serine protease that catalyses the hydrol is of the thrombin chromogenic substrate, j8-Ala-Gly-Arg-p-nitroaniline producing p-nitroaniline. The rate of yellow colored p-nitroaniline formation can be followed by its UV absorption at 405 nm, or electrochemi-cally by the reduction of its nitro group. The electrochemical detection offers benefits in terms of sensitivity and speed. When saturated by enzyme substrate the formation rate of p-nitroaniline is proportional to the enz3mie concentration. 

Mir et al [17] first reported the detection of thrombin bound to an aptamer selective for thrombin by the quantihcation of p-nitroaniline produced by the enzymatic reaction catal ed by thrombin. 

A mixed self-assembled monolayer was used for the aptamer immobilization on the gold electrode. The aptamer-modified electrodes were then incubated for 1 h at 37°C with thrombin (18 ag/mL). Electrochemical measurements were recorded in the thin-layer cell configured to contain a total volume of 20 [xL. Thrombin chromogenic substrate (/3-Ala-Gly-Arg-p-nitroaniline) was injected into the cell and differential pulse voltammetry (DPV) measurements between -0.2 and -1V with a pulse height of—0.05 V and pulse duration of 70 ms were carried out. The DPV measurements showed that /3-Ala-Gly-Arg-p-nitroaniline substrate and the p-nitroaniline product have different redox potentials. Moreover, the DPV experiments showed a current peak at -0.45 V in the presence of the thrombin substrate. After 5 min, the peak at —0.45 V decreased and a new peak was detected at -0.70 V, indicating the formation of p-nitroaniline. The same measurements carried out on a control electrode in order to test the specificity of the assay in this experiment bovine serum albumin (BSA) substituted thrombin and in this case only the peak at 0.45 V was measured. 

The authors demonstrated a huge reduction in the assay time considering that the optical detection of p-nitroaniline needed 3 h against 5 min necessary for the electrochemical measurement. 

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