RESUMO

Microcystins (MCs) are highly toxic peptides produced by cyanobacteria during algal blooms. Microcystin-leucine-arginine (MC-LR) is the most toxic and common MC variant with major effects on human and animal health upon exposure. MC-LR detection has become critical to ensure water safety, therefore robust and reliable analytical methods are needed. This work reports the development of a simple and optimized Molecularly Imprinted Nanoparticle-Based Assay (MINA) for MC-LR detection in water. Molecularly Imprinted Nanoparticles (MINs) were prepared by solid-phase polymerization on glass beads conjugated to MC-LR through (3-aminopropyl) triethoxysilane (APTES) via amide bonding. APTES-modified glass beads were obtained under optimized conditions to maximize the density of surface amino groups available for MC-LR conjugation. Two quinary mixtures of acrylic monomers differing in charge, polarity, and functionality were selected from molecular docking calculations and used to obtain MINs for MC-LR recognition using N,N'-methylene-bis-acrylamide (BIS) as the crosslinking agent. MINs were immobilized by physical adsorption onto 96-well polystyrene microplate and evaluated as per their rebinding capacity toward the analyte by using a covalent conjugate between MC-LR and the enzyme horseradish peroxidase (HRP). Experimental conditions for the MINs immobilization protocol, HRP-MC-LR concentration, and composition of the blocking solution were set to maximize the colorimetric response of the MINs compared to non-treated wells. Optimized conditions were then applied to conduct competitive MINAs with the HRP-MC-LR conjugate and the free analyte, which confirmed the preferential binding of MC-LR to the immobilized MINs for analyte concentrations ranging from 1 × 10-5 nmol L-1 to 100 nmol L-1. The best competitive MINA showed a limit of detection of 2.49 × 10-4 nmol L-1 and coefficients of variation less than 10% (n = 6), which are auspicious for the use of MINs as analytical tools for MC-LR detection below the permissible limits issued by WHO for safe water consumption (1.00 nmol L-1). This assay also proved to be selective to the analyte in cross-reactivity studies with two analogous microcystins (MC-RR and MC-YR). Analyses of lagoon and drinking water samples enriched with MC-LR revealed strong matrix effects that reduce the MINA response to the analyte, thus suggesting the need for sample pretreatment methods in future development in this subject.

Assuntos

Assuntos

RESUMO

Domoic acid (DA) is a natural amino acid and water-soluble neurotoxic biotoxin primarily produced by the microalgae Pseudo-nitzschia. DA can cause poisoning in humans and a wide variety of marine species. In this work, a molecularly imprinted nanoparticle-based assay (MINA) was developed as an alternative to enzyme-linked immunosorbent assay (ELISA) for selective detection of DA. In contrast with ELISA, MINA uses molecularly imprinted polymer nanoparticles (nanoMIPs) as plastic antibodies due to its higher stability and lower production costs. In this work, dihydrokainic acid (DKA) was used as a dummy template because this molecule is structurally similar to DA but less toxic. The developed MINA had a high linear response for DKA and DA, showing detection limits of 2.12 nmol L-1 and 4.32 nmol L-1, respectively. Additionally, q-RMN studies demonstrated that DKA - nanoMIPs were selective for DKA, since they presented the best association parameters with a high loading load capacity of 175% and an association efficiency of 18%. No cross-reactivity towards 1, 3, 5 - pentanetricarboxylic acid was observed. These results suggest that MINA could be a more robust, more sensitive, and less expensive alternative to ELISA. The assay developed with DKA - nanoMIPs has strong potential for the detection of domoic acid in real samples of red tide.

Assuntos