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

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. Biomimetic enzyme-linked immunosorbent assay (BELISA).

Keywords: Antibody mimetics; Enzyme-linked immunosorbent assay; Gonadotropin-releasing hormone; Molecularly imprinted polymers; Polydopamine; Polynorepinephrine.

Biomimetic enzyme-linked immunosorbent assay (BELISA)

Scheme 1

Sketched illustration of the imprinting process onto a micro-welled plate. (1) The functional monomer (norepinephrine) and the template (gonadorelin) were dropped as a mixture onto the wells’ microplate, and the polymerization process occurred for 5 h at 25 °C. (2) Then, the template was washed out the polymeric matrix, and the “two-steps” competitive BELISA (3) was set up

Fig. 1

a Schematic depiction of the assay setup to select the S-HRP concentration (0.20 μg mL⁻¹); b calibration curve of the enzyme conjugated to streptavidin (S-HRP) on the MIP-coated microplate

Fig. 2

Saturation binding curve for BG where the BG concentration (0.20–13.00 μmol L⁻¹) was plotted against Abs@450nm. Each point indicates the mean response ± SD of quadruplicate measures. The inset figure reports the BG calibration scheme. An illustrative photo (at the top of the figure) reports the wells’ color development for an increasing series of BG concentrations (from left to right)

Fig. 3

MIP-based competitive inhibition BELISA: typical S-shaped curve (x-axis logarithmically transformed) for gonadorelin spiked in standard solutions over the concentration range of 8.46•10⁻⁵–8.46 μmol L⁻¹. The curve was fitted by using a 4-parameter (4PL) logistic regression

Fig. 4

Selectivity test performed on MIP-coated microplates. a Sequence, number of amino acids, and molecular weight of each peptide tested; b the competitive assay was performed with the target G (white color) and unrelated control peptides (PEP_A, gray bars, and PEP_B, gray striped bars)

Fig. 5

(a) Competitive inhibition curve obtained by performing the MIP-based BELISA on human urine samples and standard solution fortified with gonadorelin, spanning from 0.084 to 846 nmol L⁻¹

Fig. 6

Calibration curve for the LC-MS/MS analysis of gonadorelin (G) in standard solutions (concentration range: 0.084–8.46 nmol L⁻¹, black dots) and results (red and blue dots) of urine specimens spiked with G at 0.084, 0.423, and 4.23 nmol L⁻¹

Fig. 7

Bland-Altman plot displays the regression line between measurements performed by BELISA (A) and the LC-MS/MS reference method (B). The difference of each value pair (A − B) is plotted on the y-axis against the mean of each value pair on the x-axis ((A + B)/2)