A biomimetic enzyme-linked immunosorbent assay (BELISA) for the analysis of gonadorelin by using molecularly imprinted polymer-coated microplates

Francesca Torrini,Aldo Paolicchi,Simona Scarano,Alessandro Saba,Francesca Cipolli,Maria Minunni,Andrea Bertolini,Pasquale Palladino,Laura Caponi

doi:10.1007/s00216-021-03867-7

Abstract

An original biomimetic enzyme-linked immunoassay (BELISA) to target the small peptide hormone gonadorelin is presented. This peptide has been recently listed among the substances banned in sports by the World Antidoping Agency (WADA) since its misuse by male athletes triggers testosterone increase. Hence, in response to this emerging issue in anti-doping controls, we proposed BELISA which involves the growth of a polynorepinephrine (PNE)–based molecularly imprinted polymer (MIP) directly on microwells. PNE, a polydopamine (PDA) analog, has recently displayed impressive performances when it was exploited for MIP preparation, giving even better results than PDA. Gonadorelin quantification was accomplished via a colorimetric indirect competitive bioassay involving the competition between biotinylated gonadorelin linked to the signal reporter and the unlabeled analyte. These compete for the same MIP binding sites resulting in an inverse correlation between gonadorelin concentration and the output color signal (λ = 450 nm). A detection limit of 277 pmol L−1 was achieved with very good reproducibility in standard solutions (avCV% = 4.07%) and in urine samples (avCV% = 5.24%). The selectivity of the assay resulted adequate for biological specimens and non-specific control peptides. In addition, the analytical figures of merit were successfully validated by mass spectrometry, the reference anti-doping benchtop platform for the analyte. BELISA was aimed to open real perspectives for PNE-based MIPs as alternatives to antibodies, especially when the target analyte is a poorly or non-immunogenic small molecule, such as gonadorelin.Graphical abstractBiomimetic enzyme-linked immunosorbent assay (BELISA)

Highlights

Enzyme-linked immunosorbent assay (ELISA) technology, which conventionally relies on antibody-antigen affinity reaction, is considered one of the most widespread diagnostic tools in clinical routine measurements
A fixed concentration of the competitor molecule, the biotinylated G (BG) conjugated to the signal reporter (S-horseradish peroxidase (HRP)), was involved in the assay to trigger a competitive reaction with G for the same molecularly imprinted polymer (MIP) cavities
This is the first example of a biomimetic enzyme–linked immunoassay (BELISA) test involving a molecularly imprinted polymer based on polynorepinephrine, and able to detect the analyte in buffer and untreated human urine

Summary

Introduction

Enzyme-linked immunosorbent assay (ELISA) technology, which conventionally relies on antibody-antigen affinity reaction, is considered one of the most widespread diagnostic tools in clinical routine measurements. Catecholamine-based MIPs, in particular PDA and PNE, are very attractive since their polymerization (the mechanistic processes are currently not completely known [43,44,45,46,47]) is obtained spontaneously and under mild aqueous conditions starting from the corresponding low-cost monomers (dopamine (DA) and norepinephrine (NE), respectively), and can be performed in any laboratory as they do not require specific technology (being easy to operate) or expensive devices In this context, we successfully developed an original PNE-based MIP as capturing receptor for gonadorelin detection both in buffer and synthetic urine [40]. All these pharmacological drugs are banned by the World Anti-Doping Agency (WADA)

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