Sensitive ‘two-steps’ competitive assay for gonadotropin-releasing hormone detection via SPR biosensing and polynorepinephrine-based molecularly imprinted polymer

Abstract

The work reports an innovative bioassay for the detection of gonadorelin in urine, a gonadotropin-releasing hormone agonist widely used in fertility medicine and to treat hormonal dysfunctions. Gonadorelin is also a synthetic hormone listed by the World Anti-Doping Agency (WADA) and of interest in anti-doping controls. The main novelty relies on the development of a biocompatible, stable, and low-cost biomimetic receptor alternative to classic antibodies. Starting from norepinephrine monomer, a highly selective and sensitive molecularly imprinted polymer (MIP) was developed and optimized for optical real-time and label-free SPR biosensing. The selectivity has been addressed by testing a series of peptides, from high to low similarity, both in terms of molecular weight and primary sequence. Due to the very low molecular weight of gonadorelin (1182 Da), a ‘two-steps’ competitive assay was developed. Particular attention has been paid to the design of the competitor and its binding affinity constant towards the MIP, being a key step for the success of the competitive strategy. The SPR assay was first optimized in standard conditions and finally applied to untreated urine samples, achieving the sensitivity required by WADA guidelines. The MIP, tested in parallel with a monoclonal antibody, gave comparable results in terms of affinity constants and selectivity towards possible interfering analytes. However, the biomimetic receptor appears clearly superior in terms of sensitivity and reproducibility. This, together with its preparation simplicity, the extremely low-cost of the monomer and its reusability for hundreds of measurements, make polynorepinephrine-based MIPs powerful rivals to immune-based approaches in the near future for similar applications.