Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

Abstract

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

“…The evaluated variables, the tested ranges and the values selected for further work are summarized in Table 1 . Other experimental variables not included in the Table, such as the MBs suspension volume 45 , 46 and the potential applied for the amperometric response 47 were optimized in previous works.…”

Competitive electrochemical immunosensor for the detection of unfolded p53 protein in blood as biomarker for Alzheimer’s disease

“…Moreover, this sensor allows the quantification of unfolded p53 directly in human plasma without sample dilution. Other types of p53 protein biosensors can be found in the literature, but they are mainly focused on cancer detection [44][45][46] and don't distinguish between unfolded p53 from the "total". The biosensor here developed offers excellent figures of merit (as can be seen in Table S2 in Supporting Information) and has demonstrated great features for the quantification of unfolded p53 in plasma, which is a sample far less invasive than others used in AD diagnosis such as cerebrospinal fluid.…”

Fast Screening of Whole Blood and Tumor Tissue for Bladder Cancer Biomarkers Using Stochastic Needle Sensors

“…When applying histochemistry for the assay of p53, only a qualitative assay was achieved [19]; limits of detection of 14 pmol/L and 1.29 ng/mL were obtained using an amperometric immunosensor [20] and an amperometric magnetoimmunosensor [21]. For E-cadherin, the best limit of detection obtained to date has been 0.16 ng/mL using an amperometric immunosensor [22], and for BTA, the best limit of determination was 34 U/mL (achieved using immunoassay) [23], while for the assay of hyaluronic acid using a colorimetric enzyme-coupled technique, a limit of detection of 0.3 mg/L was achieved [24].…”

“…To our knowledge, for the assay of the proposed biomarkers, the best methods recorded to date are histochemistry for the qualitative assay of p53 [19], amperometry using an immunosensor [20], and a magnetoimmunosensor [21] for the quantitative assay of p53 [20], for E-cadherin, it is amperometry using an immunosensor [22], for BTA it is an immunoassay [23], while for the assay of hyaluronic acid, the best results were obtained using a colorimetric enzyme-coupled technique [24]. None of these methods can be applied for the simultaneous assay of the four biomarkers, and these methods cannot be applied for in vivo analysis of the biomarkers in biological fluids or tumoral tissue samples.…”