An electrochemical immunosensor based on graphite paper electrodes for the sensitive detection of creatine kinase in actual samples

Abstract

• The electrochemical immunosensor designed very stable, low-cost and sensitive method for determination of Creatine Kinase. • CK immunosensor exhibited high analytical performance with a linear range 0.150 pg/mL and low detection limit (0.045 pg/mL). • Disposable graphite paper electrodes were firstly used to detection CK antigen for a novel biosensor system. • The optimization was performed for all fabrication steps of the electrochemical biosensor system. The enzyme creatine kinase (CK) is one of the most well-established biomarkers in cardiovascular disease. For acute myocardial infarction (AMI) diagnosis, CK has a clinical comorbidity rate of 90%. This study has developed a novel electrochemical immunosensor using disposable graphite paper electrodes (GP). The GP electrodes were modified with gold nanoparticles (AuNP) to facilitate CK detection. Afterwards, GP electrodes were covalently immobilized with 6-mercapto-1-hexanol (6-MH), resulting in self-assembled monolayers (SAMs). Then, the electrodes were formed with a 3-Glycidyloxypropyltrimethoxysilane (3-GOPE) agent. Afterwards, the electrodes were immobilized with anti-CK (antibody creatine kinase) protein. In the final immobilization step, BSA (bovine serum albumin) protein was used to block non-covalent interactions. All parameters of the proposed immunosensor were optimized, including concentrations and incubation times. Analytical characteristics such as square wave voltammetry, linear determination range, repeatability, reproducibility, and regeneration of biosensors were determined. All characterization steps were monitored by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV). Moreover, the single frequency impedance (SFI) technique interacted with anti-CK and CK antigens. Furthermore, the proposed immunosensor was characterized using scanning electron microscopy (SEM). The proposed immunosensor exhibited a wide detection range (0.1–50 pg mL −1 ), and low limit of detection (LOD), and a low limit of quantification (LOQ); 0.045 pg mL −1 and 0.171 pg mL −1 , respectively. Finally, the developed biosensor was tried in an actual blood sample, which showed it could be utilized in clinical applications.