Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA.
School of Mechanical and Materials Engineering, Washington State University, Pullman, WA, USA.
Talanta. 2019 Dec 1;205:120112. doi: 10.1016/j.talanta.2019.07.007. Epub 2019 Jul 2.
Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.
基于纸的分析器件（PADs）由于具有成本效益、简单直接等特点，被广泛应用于即时检测（POCT）中。然而，其灵敏度较差限制了其在检测低丰度生物标志物疾病方面的应用。PADs 的检测限较差主要归因于分析物浓度低，以及生物流体的复杂性，导致分析物与捕获抗体之间的相互作用不足。本研究旨在通过开发一种基于纸的阳离子等速电泳（ITP）方法来克服这些困难，该方法可同时检测两种重要的心脏蛋白标志物的皮摩尔水平：掺入人血清样本中的酸性肌钙蛋白 T（cTnT）和碱性肌钙蛋白 I（cTnI）。该方法利用 3-氨丙基三甲氧基硅烷（APTMS）处理的具有递减截面积的玻璃纤维纸，组装在 3D 打印的盒式设备上。我们的结果表明，在存在 cTnT 单克隆抗体（mAb）的情况下，荧光标记的 cTnI 和 cTnT 可以在阳离子 ITP 中有效地被富集。每个单独的靶标随后被固定有捕获 mAb 的检测区中的检测线捕获。详细分析表明，该技术能够同时在板上耗尽丰富的血浆蛋白，并通过 ~1300 倍的富集 cTnI/cTnT，其对 cTnT 的灵敏度为 0.6 pmol/L，对 cTnI 的灵敏度为 1.5 pmol/L，整个过程不到 6 分钟。结果表明，该技术具有在即时护理点对临床血清样本中的多蛋白标志物进行快速、超灵敏和经济有效的分析的潜力。
