Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Drug Test Anal. 2021 Apr;13(4):762-769. doi: 10.1002/dta.2981. Epub 2020 Nov 25.
Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM-γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM-γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 μL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples.
辛弗林（HM）是一种存在于多种植物物种中的生物碱，当诺卡屈林合酶选择性地缩合多巴胺和 4-羟基苯乙醛时，就会得到（S）-辛弗林（（S）-HM）。世界反兴奋剂机构已将 HM 列为运动中的禁用物质。因此，全球许多商业、学术和监管机构都在致力于寻找一种快速检测（S）-HM 的方法。在本研究中，开发了一种侧向流动免疫分析（LFA），其中相关的生物传感器是由针对（S）-HM 的单克隆抗体（即 MAb E8）与胶体金纳米颗粒的缀合物制成的。HM-γ-球蛋白缀合物和兔抗鼠 IgG 抗体分别置于测试区和对照区。样品中的游离（S）-HM 分子与固定在 LFA 中的 HM-γ-球蛋白缀合物竞争反应。生物传感器的可见响应与游离（S）-HM 的含量呈反比关系，这可以通过测试区中粉红色斑点强度的变化来观察到。所开发的 LFA 的检测限为 156ng/mL。各种验证方法证实，该 LFA 具有足够的灵敏度、选择性、重复性和可靠性，非常适合植物样品和含植物产品中（S）-HM 的检测。与传统的 LFA 相比，所开发的系统仅需要较小的样品体积（20μL）和简洁的样品制备时间。因此，本研究中报道的 LFA 可以作为一种快速响应试剂盒，用于检测植物样品中的（S）-HM。
