Syed Zia Ul Quasim, Samaraweera Sathya, Wang Zhuo, Schrader James Kelby, Scott Colton, Schut Joshua, Smith Dozier Johnson, Ramsey Joshua D, Krishnan Sadagopan
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078, United States.
School of Chemical Engineering, Oklahoma State University, Stillwater, Oklahoma 74078, United States.
ACS Meas Sci Au. 2024 Sep 9;4(6):689-694. doi: 10.1021/acsmeasuresciau.4c00043. eCollection 2024 Dec 18.
This article presents a colorimetric visual biosensor designed for direct application in undiluted biofluids, which holds significant promise for point-of-need applications. Unlike traditional biosensors that struggle with heavily diluted sample matrices, the presented biosensor does not require any instrumentation or trained personnel, making it highly practical. The sensor features an oligonucleotide probe covalently attached to magnetically separable magnetite (FeO) particles. This probe selectively captures a DNA mimic of the SARS-CoV-2 RNA sequence via a base-pair hybridization. The DNA mimic oligomer sequence was tested in a buffer solution, undiluted serum, and undiluted salivary biofluids. A second complementary hybridization sequence with a biotin tag was used to bind the target oligomer already hybridized to the magnetic particle-conjugated capture probe. Subsequent detection of the target oligomer was accomplished through high-affinity selective binding of streptavidin-peroxidase labels with the detection probe biotin units for visual colorimetric detection in the presence of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide. Inverse assaying of the unbound-free streptavidin-peroxidase labels left in the detection reagent solution offered a reverse trend to the target oligomer concentration, as anticipated. We obtained detection limits of 1 fM (buffer assay), 1 pM (undiluted serum assay), and 1 pM (undiluted saliva assay) and with the linear ranges of 1 fM-10 nM (buffer assay), 1 pM-1 nM (undiluted serum assay), and 1 pM-1 nM (undiluted saliva assay), respectively. The assays in different biofluids allowed for the estimation of the analytical performance and the effect of sample matrices on the detection limits and calibration sensitivity.
本文介绍了一种比色视觉生物传感器,专为直接应用于未稀释的生物流体而设计,在即时检测应用方面具有重大前景。与传统生物传感器在高稀释样本基质中遇到困难不同,该生物传感器不需要任何仪器或专业人员,具有很高的实用性。该传感器的特点是寡核苷酸探针共价连接到可磁分离的磁铁矿(FeO)颗粒上。该探针通过碱基对杂交选择性捕获SARS-CoV-2 RNA序列的DNA模拟物。在缓冲溶液、未稀释的血清和未稀释的唾液生物流体中对DNA模拟寡聚物序列进行了测试。使用带有生物素标签的第二个互补杂交序列来结合已经与磁性颗粒偶联的捕获探针杂交的目标寡聚物。随后,通过链霉亲和素-过氧化物酶标记与检测探针生物素单元的高亲和力选择性结合来完成目标寡聚物的检测,以便在存在3,3',5,5'-四甲基联苯胺和过氧化氢的情况下进行比色视觉检测。检测试剂溶液中未结合的游离链霉亲和素-过氧化物酶标记的反向测定呈现出与目标寡聚物浓度相反的趋势,正如预期的那样。我们分别获得了1 fM(缓冲液检测)、1 pM(未稀释血清检测)和1 pM(未稀释唾液检测)的检测限,线性范围分别为1 fM - 10 nM(缓冲液检测)、1 pM - 1 nM(未稀释血清检测)和1 pM - 1 nM(未稀释唾液检测)。在不同生物流体中的检测可以评估分析性能以及样品基质对检测限和校准灵敏度的影响。
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