Institute for Sports Research, School of Mechanical and Aerospace Engineering , Nanyang Technological University , Singapore , 637460.
Center for Biomimetic Sensor Science, School of Materials Science and Engineering , Nanyang Technological University , Singapore , 639798.
ACS Sens. 2019 Apr 26;4(4):865-873. doi: 10.1021/acssensors.8b01343. Epub 2019 Apr 3.
This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate releases a colored byproduct upon cleavage by salivary α-amylase. Introduction of maltose as a noncompetitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20-500 μg/mL) with a limit of detection (LOD) of 8 μg/mL (in aqueous solution). The concentrations of substrate and noncompetitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based "strip" assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific, and easy-to-implement for colorimetric detection of salivary α-amylase between 20 and 500 μg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 μg/mL. The two assay formats are benchmarked against the Phadebas test, a state of the art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses toward stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue.
本文介绍了一种用于检测唾液α-淀粉酶的比色法，α-淀粉酶是自主神经系统 (ANS) 活性的潜在生物标志物之一，可用于评估疲劳。利用α-淀粉酶切割多糖α键的能力开发了比色测定法。在提出的方法中，2-氯-4-硝基苯基-α-d-麦芽三糖苷作为底物，在唾液α-淀粉酶切割时释放出有色副产物。引入麦芽糖作为非竞争性抑制剂，可以在生理相关浓度范围内（20-500μg/mL）获得理想的线性响应，检测限（LOD）为 8μg/mL（水溶液）。随后优化了底物和非竞争性抑制剂的浓度，以用于比色检测唾液α-淀粉酶。提出了一种简便的基于纸的“条带”测定法，用于分析人唾液样本，唾液成分的干扰很小。该测定法快速、特异，易于实现 20-500μg/mL 范围内的唾液α-淀粉酶比色检测。互补的 RGB（红、绿、蓝成分）分析提供了定量检测，检测限为 11μg/mL。两种测定方法均与 Phadebas 测试进行了基准测试，Phadebas 测试是一种用于分光光度法检测α-淀粉酶的先进方法。所报道的基于纸的方法在评估可能对压力源产生的改变的 ANS 反应方面具有很高的潜力，可能在疲劳评估和监测疲劳发作方面具有应用价值。
