Facile imprinted polymer for label-free highly selective potentiometric sensing of proteins: case of recombinant human erythropoietin.

In the current study, a molecularly imprinted polymer (MIP)-based potentiometric sensor was fabricated for a label-free determination of recombinant human erythropoietin (rhEPO). The MIP sensor was operated under zero current conditions using tetra-butyl ammonium bromide as a marker ion. A highly ordered rhEPO surface imprinted layer was prepared using 3-aminopropyl triethoxysilane and tetraethoxysilane as a monomer and cross-linker, respectively, under mild reaction conditions. A two-fold increase in the signal output was obtained by polymeric surface minimization (0.5 mm) that allowed more pronounced molecular recognition (imprinting factor = 20.1). The proportion of cross-reactivity was examined using different interfering biomolecules. Results confirmed sensor specificity for both structurally related and unrelated proteins. An ~40% decrease in the response was obtained for rhEPO-β compared to rhEPO-α. The imprinted polymeric surface was evaluated using scanning electron microscopy and Fourier transform infrared spectroscopy. Under the optimal measurement conditions, a linear range of 10.00-1000.00 ng mL-1 (10-10 - 10-8 M) was obtained. The sensor was employed for the determination of rhEPO in different biopharmaceutical formulations. Results were validated against standard immunoassay. Spiked human serum samples were analyzed and the assay was validated. The presence of non-specific proteins did not significantly affect (~8%) the results of our assay. A concentration-dependent linear response was produced in an identical range with detection limit as low as 6.50 ng mL-1 (2.14 × 10-10 M). The facile fabricated MIP sensor offers a cost-effective, portable, and easy to use alternative for biosimilarity assessment and clinical application.