Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

Abstract

The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17β-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19 ± 0,06 µg L; sulfapyridine, 0.17 ± 0.07 µg L; chloramphenicol, 0.11 ± 0.03 µg L; BDE-47, 2.71 ± 1.13 µg L; 17β-estradiol, 0.94 ± 0.30 µg L and domoic acid, 1.71 ± 0.30 µg L). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was < 20% in all the cases. No sample pre-treatment was necessary, and the results could be obtained in just 1 h 30 min. The microarray shows high sample throughput capabilities, being able to measure simultaneously more than 68 samples and screen them for a significant number of chemical contaminants of interest in environmental screening programs.

Multiplexed Immunosensor Based on the Amperometric Transduction for Monitoring of Marine Pollutants in Sea Water

“…The IC 50 values reached were 1.20 ± 0.23, 1.40 ± 0.39, 1.12 ± 0.37 and 9.72 ± 0.37 µg·L −1 for Irgarol 1051, sulfapyridine, chloramphenicol and estradiol, respectively. The results obtained with the immunosensor were similar to the results obtained by ELISA and fluorescent microarray in a previous characterization of the selected immunoreagents [ 11 , 31 ]. Moreover, our group has developed other immunosensors exploring different transducing principles with similar detectability for sulfonamides and Irgarol [ 39 , 40 , 41 ].…”

“…Those parameters should be considered in order to study the matrix effect concerning the antigen–antibody recognition. In our previous work [ 31 ], the matrix effect for all the immunoreagents was tested demonstrating to be minimum in all cases, allowing the direct measure of seawater. Therefore, it was decided to test the matrix effect of seawater in the immunosensor format.…”

“…The immunoreagents used for the detection of Chloramphenicol, CA6-BSA/As226, were prepared in-house. Additionally, the antibody R69 for the detection of estradiol was kindly provided by Prof. R. Quidant (ICFO, Spain) and the corresponding coating antigen 6E2_4-BSA was prepared by the active ester method, as described in Reference [ 31 ]. Specifically, all antibodies used were produced in rabbits and all coating antigens used are Bovine Serum Albumin (BSA) conjugates as a carrier protein.…”

The benefits of carbon black, gold and magnetic nanomaterials for point-of-harvest electrochemical quantification of domoic acid

“…A fluorescence-based microarray shows similar performance to the CB-SPE biosensor although it was tested in a less demanding matrix (artificial seawater). Additionally, the microarray is less portable due to the reader size [24]. Finally, an electrochemical method using SPEs outperformed the method reported here in sensitivity by a factor 4 [17].…”

“…The protocol for magnetic bead coating was adapted from Thermofisher™ and is detailed in the supplementary material 2.2.1. Bioconjugate preparation and hapten density determination was done following a procedure adapted from [24]. Briefly, conjugation was performed using N,N ′dicyclohexylcarbodiimide and N-hydroxysuccinimide bioconjugation while matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) allowed to calculate the toxin density on the BSA conjugate which was found to be~16 DA molecules per BSA molecule (Fig.…”