Optimization of polydopamine imprinted polymer for label free sensitive potentiometric determination of proteins: Application to recombinant human erythropoietin sensing in different matrices

摘要

The integration of molecular imprinted polymers (MIPs) with ion selective electrodes (ISE) has been an ideal platform for sensing of biological products. In this work, a facile MIP was constructed over the surface of PVC polymeric membrane using self-assembled dopamine (DA) as a monomer around recombinant human Erythropoietin (rhEPO) as a template. A label free passive ion flux MIP potentiometric sensor was fabricated using tetra butyl ammonium bromide as a marker ion. MIP preparation was optimized for critical factors (monomer concentration and polymerization time) that affect the thickness of the imprinted layer by central composite response surface design. The optimum condition for DA-MIP preparation was found to be 3.79 mg mL-1 DA for 18.14 h with desirability of 0.9919. A highly selective and sensitive potentiometric sensor was obtained by combining membrane surface minimization with superficial imprinting process strategy. The sensor has elicited selectivity for template molecule as confirmed by imprinting factor exceeding 11-fold the values obtained from closely related rhEPO analogues. The imprinted layer surface was characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The quantitative determination of rhEPO was established over the range of 1.00-100.00 ng mL-1 (10-9 to 10-11 M) with a limit of detection of 0.33 ng mL-1. The sensor could classify rhEPO-alpha products in a similar attitude of natural antibodies. The potentiometric sensor was able to quantitate rhEPO in human serum with a limit of detection of 0.50 ng mL-1 and the bioanalytical assay was successfully validated. The proposed MIP biosensor elicited specificity for template recognition and selectivity in biological fluids without the need of immunoaffinity purification. Such approach shall plan the direction towards a portable sensor for doping disclosure in sports cheating.