Circulating microRNA detection using electrochemical biosensor for rapid diagnosis of liver disease in dogs

IF 3.5 Q2 CHEMISTRY, ANALYTICAL
Appan Roychoudhury, Federico Diez, Richard J. Mellanby, James W. Dear and Till T. Bachmann
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Abstract

Liver disease in dogs is a major cause of morbidity and mortality. Non-invasive diagnosis of liver disease in dogs is a clinical challenge and improved tests which could done at point-of-care are highly desirable. Liver-specific circulating microRNAs have emerged as promising biomarkers for liver injury across many vertebrate species including dogs. MicroRNA-122 (miR-122), originating from the damaged hepatocytes, provides high specificity and sensitivity in detecting liver disease, compared to the traditional biomarkers. In this study, we present the development of a point-of-care compatible electrochemical biosensor for rapid, early diagnosis of liver disease in dogs by detecting miR-122 in clinical samples. Building on our prior work utilising electrochemical impedance spectroscopy (EIS) for direct and amplification-free detection of miR-122 in human drug-induced liver injury, we have used a miR-122 target-specific short probe and implemented target overhang formation during hybridisation in a flow-based sample cycling setup for enhanced detection performance and demonstrated its performance in real clinical dog samples for the first time. We determined the hybridisation performance by analysing miR-122 specificity and sensitivity achieving a limit of detection (LOD) and limit of quantification (LOQ) of 10 pM and 100 pM, respectively, and high specificity over a nearly-complementary sequence of a miR-122 precursor. Using conventional sample preparation, the developed EIS assay was used to analyse serum samples from dogs with liver disease which were identified based on an increased serum alanine aminotransferase concentration. The test successfully distinguished samples from dogs with and without liver disease in comparable performance to the gold-standard real-time polymerase chain reaction (qPCR) detection. We will further focus on developing sample-to-answer test by combining our miR-122 EIS biosensor with a compatible sample preparation to measure miR-122 from dog blood at point of care.

Abstract Image

利用电化学生物传感器检测循环微RNA,快速诊断狗的肝病
犬肝病是发病和死亡的主要原因。狗肝脏疾病的无创诊断是一项临床挑战,因此非常需要改进可在护理点进行的检测。在包括狗在内的许多脊椎动物中,肝脏特异性循环微RNA已成为肝损伤的有希望的生物标志物。与传统的生物标记物相比,源自受损肝细胞的微RNA-122(miR-122)在检测肝脏疾病方面具有很高的特异性和灵敏度。在这项研究中,我们开发了一种与护理点兼容的电化学生物传感器,通过检测临床样本中的 miR-122,对狗的肝病进行快速、早期诊断。在利用电化学阻抗谱(EIS)直接、无放大检测人类药物性肝损伤中 miR-122 的前期工作基础上,我们使用了 miR-122 靶标特异性短探针,并在基于流动的样品循环装置中杂交过程中形成靶悬垂,以提高检测性能,并首次在真实的临床狗样本中展示了其性能。我们通过分析 miR-122 的特异性和灵敏度确定了杂交性能,其检测限(LOD)和定量限(LOQ)分别为 10 pM 和 100 pM,对 miR-122 前体的几乎互补序列具有高度特异性。利用传统的样品制备方法,所开发的 EIS 检测法被用于分析根据血清丙氨酸氨基转移酶浓度升高而确定的患有肝病的狗的血清样品。该检测方法成功区分了患有和未患有肝病的狗样本,其性能与黄金标准的实时聚合酶链反应(qPCR)检测方法相当。我们将进一步致力于开发从样本到答案的测试,将我们的 miR-122 EIS 生物传感器与兼容的样本制备相结合,在医疗点测量狗血液中的 miR-122。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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CiteScore
2.30
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