Wells Paulina K, Smutok Oleh, Guo Zhong, Alexandrov Kirill, Katz Evgeny
Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY, 13699, USA.
CSIRO-QUT Synthetic Biology Alliance, ARC Centre of Excellence in Synthetic Biology, Centre for Agriculture and the Bioeconomy, Centre for Genomics and Personalised Health, School of Biology and Environmental Science, Queensland University of Technology, Brisbane, QLD, 4001, Australia.
Talanta. 2023 Apr 1;255:124215. doi: 10.1016/j.talanta.2022.124215. Epub 2022 Dec 29.
Protein biosensors hold a promise to transform the way we collect physiological data by enabling quantification of biomarkers outside of specialized laboratory environment. However, achieving high specificity and sensitivity in homogeneous assay format remains challenging. Here we report construction of fluorescent biosensor arrays based on artificial allosteric α-amylase-activated PQQ-dependent glucose dehydrogenase (Amy-GDH). Amy-GDH was covalently immobilized on silica nanoparticles that were then arrayed on fiberglass sheets. The activity of the biosensor was monitored using a smartphone camera via emergence of bright fluorescence (λ 365 nm) originating from reduced phenazine methosulfate upon glucose oxidation by Amy-GDH. We show that such biosensor arrays demonstrate an apparent K of 115 pM for α-amylase with a detection limit of 2 pM. Using the developed biosensor arrays, we were able to specifically and accurately quantify the concentration of α-amylase in biological fluids such as serum and saliva. We propose that the presented approach can enable construction of ultrasensitive point-of-care diagnostic arrays.
蛋白质生物传感器有望通过在专门实验室环境之外实现生物标志物的定量，来改变我们收集生理数据的方式。然而，在均相检测形式中实现高特异性和灵敏度仍然具有挑战性。在此，我们报告了基于人工变构α-淀粉酶激活的吡咯喹啉醌依赖性葡萄糖脱氢酶（Amy-GDH）构建荧光生物传感器阵列。Amy-GDH被共价固定在二氧化硅纳米颗粒上，然后将这些纳米颗粒排列在玻璃纤维片上。通过智能手机摄像头监测生物传感器的活性，该活性通过Amy-GDH氧化葡萄糖时还原的硫酸吩嗪甲酯产生的明亮荧光（λ 365 nm）来体现。我们表明，这种生物传感器阵列对α-淀粉酶的表观K为115 pM，检测限为2 pM。使用所开发的生物传感器阵列，我们能够特异性且准确地定量血清和唾液等生物流体中α-淀粉酶的浓度。我们提出，所展示的方法能够实现超灵敏即时诊断阵列的构建。
