Reagentless Amperometric Immunosensors Based on Direct Electrochemistry of Horseradish Peroxidase for Determination of Carcinoma Antigen-125

Abstract

A novel strategy for immunoassay and the preparation of reagentless immunosensors was proposed. This strategy was based on the immobilization of antigen and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to an antibody. A reagentless immunosensor for carcinoma antigen-125 (CA 125) determination was developed. The immunosensor was prepared by immobilizing CA 125 with titania sol-gel on a glassy carbon electrode by the vapor deposition method. The incubation of the immunosensor in phosphate buffer solution (PBS) including HRP-labeled CA 125 antibody led to the formation of a HRP-modified surface. The immobilized HRP displayed its direct electrochemistry with a rate constant of 3.04 +/- 1.21 s(-1). With a competition mechanism, a differential pulse voltammetric determination method for CA 125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the competitive binding of the CA 125 in sample solution and the immobilized CA 125 to the limited amount of HRP-labeled CA 125 antibody. Under optimal conditions, the current decrease was proportional to CA 125 concentration ranging from 2 to 14 units mL(-1) with a detection limit of 1.29 units mL(-1) at a current decrease by 10%. The CA 125 immunosensor showed good accuracy and acceptable precision and fabrication reproducibility with intraassay CVs of 8.7 and 5.5% at 8 and 14 units mL(-1) CA 125 concentrations, respectively, and interassay CV of 19.8% at 8 units mL(-1). The storage stability was acceptable in a pH 7.0 PBS at 4 degrees C for 15 days. The proposed method provided a new promising platform for clinical immunoassay.

Detection of prostate specific antigen in serum at the femto-gram per milliliter level using the intrinsic amplification of a field-effect enzymatic immuno-sensing system

“…The first approach required the measurement of the reduction peak current of HRP and used it as the signal. This is a reagentless method [22], in which no H 2 O 2 is used to initiate the activity of HRP. To carry out the detection, cyclic voltammetry or linear voltammetry (LV) was performed with electrodes, on which the Ab-PSA-Ab(HRP) structure was formed with serum solutions containing different PSA concentrations.…”

Recent Advances in Electrochemical Enzyme Immunoassays

“…The immunoreagents have been traditionally placed, either alone or mixed with other proteins such as bovine serum albumin [130 -138] [105,114] are some of the most commonly immobilization methods used in the design of electrochemical immunosensors. In addition to these conventional methods, novel strategies based on the use of gold nanoparticles monolayers [158,159], iridium oxide thin films [160], and carbon nanotubes [106], as platforms for the immunoreagent immobilization, have also been found in the literature.…”

Gold Nanoparticles Doped Three‐Dimensional Sol‐gel Matrix for Amperometric Human Chorionic Gonadotrophin Immunosensor

“…Compared with the common optical detection methods, electrochemical immunoassays and immunosensors which are useful in the case of point-ofcare testing and in vivo monitoring appear to provide a promising platform with a wide application, especially for determination of clinically important substances [2,8,13], due to the simple and fast analytical process and inexpensive and miniaturizable instrumentation [14,15].…”

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