Abstract
A novel flow injection microfluidic immunoassay system for continuous monitoring of saxitoxin, a lethal biotoxin, in seawater samples is presented in this article. The system consists of a preimmobilized G protein immunoaffinity column connected in line with a lab-on-chip setup. The detection of saxitoxin in seawater was carried out in two steps: an offline incubation step (competition reaction) performed between the analyte of interest (saxitoxin or Ag, as standard or seawater sample) and a tracer (an enzyme-conjugated antigen or Ag*) toward a specific polyclonal antibody. Then, the mixture was injected through a “loop” of a few μL using a six-way injection valve into a bioreactor, in line with the valve. The bioreactor consisted of a small glass column, manually filled with resin upon which G protein has been immobilized. When the mixture flowed through the bioreactor, all the antibody-antigen complex, formed during the competition step, is retained by the G protein. The tracer molecules that do not interact with the capture antibody and protein G are eluted out of the column, collected, and mixed with an enzymatic substrate directly within the microfluidic chip, via the use of two peristaltic pumps. When Ag* was present, a color change (absorbance variation, ΔAbs) of the solution is detected at a fixed wavelength (655 nm) by an optical chip docking system and registered by a computer. The amount of saxitoxin, present in the sample (or standard), that generates the variation of the intensity of the color, will be directly proportional to the concentration of the analyte in the analyzed solution. Indeed, the absorbance response increased proportionally to the enzymatic product and to the concentration of saxitoxin in the range of 3.5 × 10–7–2 × 10–5 ng ml−1 with a detection limit of 1 × 10–7 ng ml−1 (RSD% 15, S N−1 equal to 3). The immunoanalytical system has been characterized, optimized, and tested with seawater samples. This analytical approach, combined with the transportable and small-sized instrumentation, allows for easy in situ monitoring of marine water contaminations.
Highlights
The development of rapid and sensitive analytical methods for the determination of trace analytes in liquid samples is an important goal to be achieved in analytical chemistry
Over the past few years, many different analytical approaches have been explored in the area of algal toxin detection from environmental samples, including liquid chromatography coupled with mass-spectroscopy (LC-MS) (Vilariño et al, 2013), high-performance liquid chromatography (HPLC) (Van Egmond et al, 1994), enzyme-linked immunosorbent assay (ELISA) (Micheli et al, 2002; Yu et al, 2002; Petropoulos et al, 2019), and electrochemical biosensors (Zhang et al, 2018)
Most of the research has been focused on the saxitoxin (STX) and STX-related compounds, as they are found to be more common in neurotoxic paralytic shellfish toxins (PSTs) (Deeds et al, 2008)
Summary
The development of rapid and sensitive analytical methods for the determination of trace analytes in liquid samples is an important goal to be achieved in analytical chemistry. Flow analysis methods have been widespread in the field of chemical analysis since the middle of the last century when Ruzicka and Hansen first introduced the term “Flow Injection Analysis (FIA)” (1975) (Ruzicka and Hansen, 1975) This is considered the cornerstone of a new paradigm in analytical chemistry (McKelvie, 2008). Four immunological categories (immunoassay, immunosensor, lateral flow immunoassay, and radioimmunoassay) were developed for the detection of algal toxins in shellfish and water These methods for PSTs are suitable for field testing because the extraction and detection are simple. Tian et al (2020) reported an overall vision about the recent progress of optical and electrochemical biosensors developed for the detection of shellfish toxins in food and drinking water, showing the advantages of the strategy, analyte, sensing unit, method, and property for their future application in field fast screening (Tian et al, 2020). The proposed FI-IA system consists of a bioreactor in line with a lab-on-chip (LOC) microfluidic system for reagents mixing and colorimetric detection (Figure 1 and Supplementary Figure S1)
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