Stable thin films of human P53 antigen on gold surface for the detection of tumour associated anti-P53 autoantibodies

Abstract

An electrochemical impedimetric immunosensor was developed for the label-free detection of autoantibodies against the human P53 antigen (protein) involved in carcinogenesis. The immunosensor was based on the covalent immobilization of human P53-protein (P53ag) using amide coupling onto gold electrode pre-grafted with phenylethylamine-succinic acid (Au-PEA-SA). The non-specific binding sites on the activated Au-PEA-SA were blocked with bovine-serum albumin (BSA) to yield an Au-PEA-SA-P53ag/BSA immunosensor. The gold electrode modification steps were monitored using X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The analytical performance of Au-PEA-SA-P53ag/BSA towards the detection of anti-P53 autoantibodies showed a concentration-dependent impedimetric (RCT) response. A wide linear range (1.0 –1000 ng.mL-1) with a low detection limit of 103 pg.mL−1 and high RCT sensitivity of 0.749 kΩ.ng.mL−1.cm−2 was obtained. The Au-PEA-SA-P53ag/BSA could be regenerated (using dimethylsulfoxide/formamide) several times without the loss of covalently bound human P53ag as a sensing element. The detection signal was retained and about 92% of the original signal was recovered without damage to the immunoelectrode after the 6 regeneration cycles. The real sample detection was investigated using serum recovery method. An excellent % recovery was obtained between 84.8 and 93.7%.