Celio Lorenzo, Ottaviani Matteo, Cancelliere Rocco, Di Tinno Alessio, Panjan Peter, Sesay Adama Marie, Micheli Laura
Departement of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome, Italy.
Department of Analytical Chemistry, University of Turin, Turin, Italy.
Front Chem. 2021 Mar 4;9:626630. doi: 10.3389/fchem.2021.626630. eCollection 2021.
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 × 10-2 × 10 ng ml with a detection limit of 1 × 10 ng ml (RSD% 15, S N 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 monitoring of marine water contaminations.
本文介绍了一种新型流动注射微流控免疫分析系统，用于连续监测海水中的致命生物毒素——石房蛤毒素。该系统由一个预固定化的G蛋白免疫亲和柱与一个芯片实验室装置串联组成。海水中石房蛤毒素的检测分两步进行：离线孵育步骤（竞争反应），在目标分析物（石房蛤毒素或作为标准品或海水样品的Ag）与针对特定多克隆抗体的示踪剂（酶联抗原或Ag*）之间进行。然后，使用六通进样阀将混合物通过几微升的“定量环”注入与该阀串联的生物反应器中。生物反应器由一个小玻璃柱组成，手动填充有固定化G蛋白的树脂。当混合物流经生物反应器时，竞争步骤中形成的所有抗体 - 抗原复合物被G蛋白保留。未与捕获抗体和蛋白G相互作用的示踪剂分子被洗脱出色谱柱，收集后通过两个蠕动泵直接在微流控芯片内与酶底物混合。当存在Ag*时，通过光学芯片对接系统在固定波长（655 nm）处检测溶液的颜色变化（吸光度变化，ΔAbs）并由计算机记录。样品（或标准品）中产生颜色强度变化的石房蛤毒素量将与分析溶液中分析物的浓度成正比。实际上，在3.5×10 - 2×10 ng/ml范围内，吸光度响应与酶产物和石房蛤毒素浓度成正比增加，检测限为1×10 ng/ml（相对标准偏差%为15，信噪比等于3）。该免疫分析系统已进行表征、优化并用于海水样品测试。这种分析方法与可运输的小型仪器相结合，便于对海水污染进行监测。
