Microfluidic Flow Injection Immunoassay System for Algal Toxins Determination: A Case of Study

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.

Keywords: FI-IA; algal toxins; immunoanalytical system; microchip flow-chamber system; saxitoxin.

FIGURE 1

Scheme of FI-IA approach where the bioreactor is in line with the lab-on-chip cartridge and connected to a laptop. In particluar, 1b: (A) offline incubation between STX, HRP-STX, and PAb; (B) injection of the mixture in the FI-IA system.

FIGURE 2

Schematization of the microfluidic lab-on-chip system: (A) microfluidic bioreactor and components; (B) microfluidic chip detector housing (LED 655 nm and photodiode). In particular, in (A): (a) i) outlet; ii) substrate inlet; iii) sample inlet; (b) lab-on-chip components: i) top plate, ii) black PMMA herringbone etched plate, iii) microfluidic channels, and iv) clear bottom plate; (c) microfluidic mixing chamber, reaction channel, and optical paths.

FIGURE 3

(A) Detector response (variation of absorbance) at fixed dilution of the colored substrate and different flow rates; (B) detector response (in variation of absorbance) at different dilutions and fixed flow rate (0.1 ml min⁻¹).

FIGURE 4

Gaussian regression of several responses [0.0001 (A), 0.001 (B), and 0.1 µg ml⁻¹ (C)], sequentially injected in the full system at 0.1 ml·min⁻¹.

FIGURE 5

Calibration curve of HRP in the microfluidic system using 0.1 ml min⁻¹ flow rate.

FIGURE 6

Binding curve for anti-STX PAb obtained with a fixed amount of STX-HRP (600 v v⁻¹) in 20 mM phosphate buffer pH 7.4, after 2 h of incubation; flow rate, 0.15 ml min⁻¹. The analysis was carried out using only bioreactor of the FI-IA system and the solution was collected.

FIGURE 7

Selection of the STX-HRP dilution to use for the competition assay: 1:75,000 v v⁻¹ dilutions of anti-STX PAb in 20 mM phosphate buffer pH 7.4, for 2 h at room temperature; flow rate, 0.15 ml min⁻¹. The analysis was carried out using only the bioreactor of the FI-IA system.

FIGURE 8

Calibration curve, using only bioreactor, for STX determination STX-HRP (1:600 v v⁻¹) for the Anti-STX (1:75,000 v v⁻¹) in 20 mM phosphate buffer pH 7.4, flow rate 0.15 ml min⁻¹.

FIGURE 9

(A) Calibration curve for STX determination STX-HRP (1:600 v v⁻¹) for the anti-STX (1:75,000 v v⁻¹⁾ in diluted seawater (1:3 v v⁻¹ with double distilled water) using bioreactor. (B) Calibration curve for STX obtained using microfluidic FI-IA system.