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Ultrafiltration or dialysis was used as sample pretreatment to remove 95% of lactose. Free PRP was separated using SPE, and then total and free PRP were analyzed using HPAEC-PAD (ribitol determination) and a colorimetric assay (phosphorus determination).

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.

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-α 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.