Departamento de Ingeniería Electrónica, Eléctrica y Automática, Universitat Rovira i Virgili, Avda. Països Catalans 26, 43007, Tarragona, Spain.
Mikrochim Acta. 2020 Mar 13;187(4):230. doi: 10.1007/s00604-020-4188-9.
An interferometric reflectance spectroscopy-based biosensor for the determination of cathepsin B (Cat B) as a cancer-related enzyme has been fabricated. For this purpose, the nanoporous anodic alumina (NAA) was fabricated electrochemically. The NAA was then modified with the amino-silane coupling agent. After that, human serum albumin (HSA) was immobilized into the NAA pores by using glutaraldehyde as a cross-linking agent. Subsequently, the carboxylic group of HSA was activated with N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to attach to thionine (TH) as a photoprobe to fabricate the labeled HSA (HSA-TH). HSA-TH plays a significant role in this sensor to determine cathepsin B as a model analyte for the development of the interferometric reflectance spectroscopy-based biosensor for the measurement of protease. The attached TH adsorbed the illuminated white light on NAA modified with HSA-TH. Therefore, the intensity of the reflected light to the charge-coupled device (CCD) detector decreased in the wavelength range 450-1050 nm. In the presence of Cat B, HAS-TH cleaved into short peptide fragments and washed away by flow cell system. Since TH was removed from NAA, the intensity of the reflected light increased. The peak area has a logarithmic relationship with the concentration of Cat B in the range 0.5 to 64.0 nM. The limit of detection of the biosensor sensor was 0.08 nM. The optical sensor was used for the determination of Cat B in a human serum sample. Graphical abstract Schematic presentation of biosensor for the determination of the cathepsin B which is based on nanoporous anodic alumina modified with HSA-thionine. The principle response of the optical biosensor is based on detecting changes in the intensity of the reflected light after cleaving the immobilized HSA-thionine by cathepsin B into short peptide fragments.
基于干涉反射光谱的用于测定组织蛋白酶 B（Cat B）作为癌症相关酶的生物传感器已经被制备。为此，通过电化学制备纳米多孔阳极氧化铝（NAA）。然后，用氨基硅烷偶联剂修饰 NAA。之后，通过戊二醛作为交联剂将人血清白蛋白（HSA）固定在 NAA 孔中。随后，通过 N-乙基-N'-(3-(二甲氨基)丙基)碳二亚胺（EDC）和 N-羟基琥珀酰亚胺（NHS）激活 HSA 的羧基以连接到硫堇（TH）作为光探针来制备标记的 HSA（HSA-TH）。HSA-TH 在这个传感器中起着重要的作用，用于测定组织蛋白酶 B 作为测定蛋白酶的基于干涉反射光谱的生物传感器的模型分析物。附着的 TH 在 HSA-TH 修饰的 NAA 上吸附被照亮的白光。因此，在 450-1050nm 的波长范围内，反射光到电荷耦合器件（CCD）探测器的强度降低。在存在 Cat B 的情况下，HAS-TH 被切割成短肽片段并通过流池系统冲洗掉。由于 TH 从 NAA 中去除，反射光的强度增加。峰面积与 Cat B 在 0.5 至 64.0nM 的浓度范围内呈对数关系。生物传感器的检测限为 0.08nM。该光学传感器用于测定人血清样品中的 Cat B。
