Taron Wichit, Jamnongkan Wassana, Phetcharaburanin Jutarop, Klanrit Poramate, Namwat Nisana, Techasen Anchalee, Sithithaworn Paiboon, Khuntikeo Narong, Boonmars Thidarut, Loilome Watcharin, Ngeontae Wittaya
Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.
Spectrochim Acta A Mol Biomol Spectrosc. 2021 Jun 5;254:119633. doi: 10.1016/j.saa.2021.119633. Epub 2021 Feb 26.
The colorimetric AuNPs-LISA is a new, powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, the diagnostic sensitivity of the colorimetric AuNPs-LISA is powerless to screen ultralow concentrations of OvAg in urine samples in cases of early stage liver fluke infection. This work, we aimed to improve the diagnostic sensitivity of the colorimetric AuNPs-LISA by developing a new fluorescence AuNPs-LISA. O-phenylenediamine (OPD) was used as the chromogenic substrate instead of the tetramethylbenzidine (TMB) of the colorimetric AuNPs-LISA. Interestingly, the fluorescence of the OPD oxidation product by the peroxidase-like activity of labelled AuNPs can be extremely enhanced by a non-ionic surfactant, especially the Triton X-100. The proposed assay exhibited a dynamic linear detection of OvAg concentration in the range of 34.18 ng mL to 273.44 ng mL with the limit of detection at 36.97 ng mL which the detection sensitivity enhancement around 1200-fold when comparing with the colorimetric AuNPs-LISA. This model exhibits high diagnosis sensitivity, specificity and accuracy, 91.28%, 91.75%, and 91.59%, respectively, compared to the traditional ELISA. The fluorescence AuNPs-LISA showed excellent potential for the diagnosis of OvAg in urine samples from endemic areas. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis.
比色金纳米颗粒-酶联免疫吸附测定法（AuNPs-LISA）是一种用于检测尿液样本中华支睾吸虫抗原（OvAg）的新型强大技术。然而，在早期肝吸虫感染病例中，比色AuNPs-LISA的诊断敏感性对于筛查尿液样本中超低浓度的OvAg无能为力。在这项工作中，我们旨在通过开发一种新的荧光AuNPs-LISA来提高比色AuNPs-LISA的诊断敏感性。使用邻苯二胺（OPD）作为显色底物，替代比色AuNPs-LISA中的四甲基联苯胺（TMB）。有趣的是，标记的金纳米颗粒的过氧化物酶样活性产生的OPD氧化产物的荧光可以被非离子表面活性剂，特别是Triton X-100极大地增强。所提出的检测方法对OvAg浓度的动态线性检测范围为34.18 ng/mL至273.44 ng/mL，检测限为36.97 ng/mL，与比色AuNPs-LISA相比，检测灵敏度提高了约1200倍。与传统酶联免疫吸附测定法（ELISA）相比，该模型分别具有91.28%、91.75%和91.59%的高诊断敏感性、特异性和准确性。荧光AuNPs-LISA在诊断流行地区尿液样本中的OvAg方面显示出优异的潜力。这将为人体华支睾吸虫病的检测、控制和消除提供一种有效的工具。
