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.
Keywords: Biosensor; Cathepsin B; Interferometric reflectance spectroscopy; Nanoporous anodic alumina.