Novel sensory surface for creatine kinase electrochemical detection

• Substrate immobilization on screen-printed electrodes.
• Electrical changes observed after the enzymatic reaction.
• Square-wave voltammetry used for analytical calibration.
• Enzyme detection on a sensory layer.

This work describes a novel concept of biosensor for quantifying enzymes, where the substrate is immobilized directly over the working area of a screen printed electrode (Au-SPE). This concept is applied here to creatine kinase isoenzyme (CK-MB), a cardiac biomarker in ischemic conditions. It acts as a phospho-transferase on creatine (Crea), requiring the presence of phosphate. So, the phosphorylated form of creatine (Pcrea) was immobilized on the Au/SPE previously aminated with cysteamine (Cys) by self-assembling monolayer technique. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) studies were used to follow the chemical modifications in the Au-SPE.Since Pcrea is an electroactive species at low potential, its consumption over the platform by the enzyme changed the electrical response of the biosensor. So, CK-MB determination has been achieved in mediator free-conditions due the redox proprieties of the Pcrea. The analytical features of the resulting biosensor were studied by square wave voltammetry (SWV). The limit of detection was 0.11 µg/mL and the slope was −0.029(±0.0035) µA×mL/µg. The interference effect of troponin T (TnT), bovine serum albumin (BSA) and myoglobin (Myo) in the performance of the sensor was tested and good selectivity was observed.The biosensor was successfully applied to biological fluids, showing good stability at room temperature and excellent sensitivity and selectivity. This new concept of biosensor is especially useful for point of care (POC) applications, due to the low cost and small size of the final device.