Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, New York 13699, United States.
CSIRO-QUT Synthetic Biology Alliance, ARC Centre of Excellence in Synthetic Biology, Centre for Agriculture and the Bioeconomy, School of Biology and Environmental Science, Queensland University of Technology, Brisbane, Queensland 4001, Australia.
Anal Chem. 2022 May 24;94(20):7303-7310. doi: 10.1021/acs.analchem.2c00650. Epub 2022 May 11.
Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.
蛋白质工程的进展促使人们加大力度开发蛋白质生物传感器，以替代仪器密集型的分析和诊断方法。灵敏度、可多重检测能力和可制造性仍然是限制蛋白质生物传感器广泛应用的关键问题。在这里，我们尝试通过构建基于 PQQ-葡萄糖脱氢酶（GDH）人工别构变体的蛋白质生物传感器阵列来解决这些问题。我们证明，固定在硅胶纳米颗粒上的 GDH 蛋白可以在不损失活性的情况下沉积在玻璃纤维片上。可以使用常用电子介体吩嗪甲硫酸盐的荧光变化来监测颗粒相关的 GDH 活性。构建的环孢素 A 和 FK-506 等大环免疫抑制剂药物的生物传感器阵列显示出非常低的背景和超过 300 倍的动态范围，从而使两种分析物的检测限达到 2 pM。这使我们能够定量检测人血液、血清、尿液和唾液中的这两种药物。该阵列可以以干燥形式储存，并使用智能手机摄像头进行定量成像，证明了该方法适用于现场和即时护理应用。所开发的方法为生物传感器阵列的发展提供了一个可推广的平台，该平台与廉价且具有潜在可扩展性的制造方法兼容。
