USDA, ARS, SEA, USNPRC, 950 College Station Rd, Athens, GA 30605, USA.
Food Science Center, Merieux NutriSciences (China), Shanghai 201112, China.
Toxins (Basel). 2020 Apr 26;12(5):280. doi: 10.3390/toxins12050280.
Shiga toxin-producing (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world as well as in the United States. Current detection methods have limitation to implement for rapid field-deployable detection with high volume of samples that are needed for regulatory purposes. Surface plasmon resonance imaging (SPRi) has proved to achieve rapid and label-free screening of multiple pathogens simultaneously, so it was evaluated in this work for the detection of Shiga toxins (Stx1a and Stx2a toxoids were used as the less toxic alternatives to Stx1 and Stx2, respectively). Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted one by one by programed microarrayer, on the same high-throughput biochip with 50-nm gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference (ΔR) between immobilized testing antibodies and immunoglobulin G (IgG) control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of picogram (pg)/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.
产志贺毒素（STEC）是引起世界各地以及美国爆发的胃肠道疾病的罪魁祸首。目前的检测方法在需要进行大量样本快速现场部署检测时存在局限性，无法满足监管要求。表面等离子体共振成像（SPRi）已被证明可用于同时快速、无标记地筛选多种病原体，因此本研究评估了其用于检测志贺毒素的能力（分别使用 Stx1a 和 Stx2a 类毒素作为 Stx1 和 Stx2 的毒性较低替代品）。通过程序微阵列器将多种抗体（Stx1pAb、Stx1-1mAb、Stx1-2mAb、Stx1d-3mAb、Stx1e-4mAb、Stx2pAb、Stx2-1mAb、Stx2-2mAb 和 Stx2-10mAb）逐一固定在具有 50nm 金膜的高通量生物芯片上，通过多次交联和封闭步骤提高抗体在生物芯片表面的定向性。基于固定化检测抗体与免疫球蛋白 G（IgG）对照之间的 SPRi 信号差异（ΔR）来检测志贺毒素。在所测试的抗体中，Stx1pAb 对 Stx1 类毒素的灵敏度最高，检测限（LOD）为 50ng/mL，检测时间为 20min。Stx2-1mAb 和 Stx2-2mAb 对 Stx2 类毒素均表现出较高的灵敏度。此外，金纳米粒子（GNPs）用于在三明治平台中放大单克隆抗体的 SPRi 信号。在 GNP-抗体结合物的帮助下，LOD 达到了皮克（pg）/mL 级。该结果证明，具有选定抗体的 SPRi 生物芯片具有快速、高通量和多重检测志贺毒素的潜力。
