Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Sweden.
Department of Chemistry, Royal Institute of Technology, KTH, Teknikringen 30, SE-100 44, Stockholm, Sweden.
Biosens Bioelectron. 2020 Mar 1;151:112002. doi: 10.1016/j.bios.2019.112002. Epub 2019 Dec 30.
Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 μg mL of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.
本文报道了一种基于等温锁式探针的简单便携的病原体检测方法，该方法与葡萄糖氧化酶（GOx）电化学读出相结合。传染病在全球范围内仍然是一个持续存在的威胁，例如反复出现的大流行。快速便携的诊断方法有望解决疾病的传播问题，并将医疗服务去中心化，以满足就地医疗需求。埃博拉病毒是一种高变异性的 RNA 病毒，在非洲最近的疫情中，死亡率高达 90%，因此需要立即进行床边诊断。所展示的检测设计包括滚环扩增（RCA）技术，该技术负责生成作为 RCA 产物（RCP）的核酸扩增子。RCP 生成在磁性珠（MB）上，随后通过链霉亲和素-生物素键连接到 GOx。结合的 GOx 对葡萄糖的酶催化作用允许对 DNA 靶标进行间接电化学测量。通过扫描电子显微镜证实了 MB 表面上生成的 RCPs，并评估了其他实验条件，例如缓冲液类型、温度、GOx 浓度、采样和测量时间，以获得最佳的电化学检测效果。因此，选择 125 μg mL 的 GOx 与 5 mM 葡萄糖在磷酸盐缓冲盐水（PBS）中，监测 1 分钟作为理想条件。最后，我们使用来自患者的埃博拉病毒临床样本评估了生物传感策略的分析性能。总的来说，这项工作为使用电化学检测进行新兴病原体的简单便携分子诊断提供了概念验证生物测定，特别是在资源有限的环境中。
