Abstract
An electrochemically synthesised porous anodic aluminium oxide (pAAO) layer has been analysed by means of spectroscopic ellipsometry. The determined thickness of the formed pAAO layer obtained from spectroscopic ellipsometry measurements and modelling was 322.75 ± 0.12 nm. The radius of the nanopores estimated from SEM images was 39 ± 5 nm and the distance between nanopores was 107 ± 6 nm. The investigation of human serum albumin (HSA) adsorption on the pAAO coating showed that: (i) the protein concentration inside nanopores, depending on exposure time, approximately was from 200 up to 600 times higher than that determined in buffer solution; (ii) the initial phase of the adsorption process is slow (3.23 mg·cm−3·min−1) in comparison with the protein desorption rate (21.2 mg·cm−3·min−1) by means of pAAO layer washing; (iii) conventional washing with PBS solution and deionised water does not completely remove HSA molecules from pAAO pores and, therefore, the HSA concentration inside nanopores after 16 h of washing still remains almost 100 times higher than that present in PBS solution. Thus, due to such binding ability, HSA can be successfully used for the blocking of the remaining free surface, which is applied for the reduction in non-specific binding after the immobilisation of biorecognition molecules on the pAAO surface. It was determined that some desorption of HSA molecules from the pAAO layer occurred during the sensor’s surface washing step; however, HSA concentration inside the nanopores still remained rather high. These results recommend the continued application of pAAO in the development of biosensors.
Highlights
The investigation of protein adsorption/desorption to/from various nanoporous materials is an important task due to the ability to use such materials for proteins separation, purification, and in the development of biosensors, which can be applicable for the determination of various analytes that are important in biology, biochemistry, medical diagnostics, pharmacy, or food industry [1].The ability of large molecules such as proteins to adsorb within nanopores significantly depends on Coatings 2020, 10, 1018; doi:10.3390/coatings10111018 www.mdpi.com/journal/coatingsCoatings 2020, 10, 1018 hydrophobicity/hydrophility and pore sizes
Ellipsometric measurements of porous anodic aluminium oxide (pAAO)/air, pAAO/human serum albumin (HSA)(1 h incub.), pAAO/HSA(2 h incub.), pAAO/HSA(3 h incub. + 15 min wash), and data simulation for Al2 O3 and Al2 O3 /HSA were performed in order to compare the spectral shift for non-structured (Al2 O3 ) and nanostructured substrates, which were used as sensing layers for HSA protein adsorption
It was determined that pAAO/HSA structure more sensitive to deionized water because more HSA molecules were removed from the substrate by washing using deionized water, than by 0.01 M PBS, pH 7.4
Summary
The investigation of protein adsorption/desorption to/from various nanoporous materials is an important task due to the ability to use such materials for proteins separation, purification, and in the development of biosensors, which can be applicable for the determination of various analytes that are important in biology, biochemistry, medical diagnostics, pharmacy, or food industry [1]. It is highly important to assess how strongly HSA interacts with surfaces applied in the design of bioanalytical systems and how washing procedures influence the stability of the adsorbed HSA layer As it was shown in some research works, HSA-modified pAAO substrate was used as a sensing layer for the determination of cathepsin B [16]. Significant differences in the predicted and observed modulation depth of the reflectivity appear due to the effects of surface roughness on the reflectivity of the aluminium foil substrate This is a result of the RIFTS method, which uses only one s-polarisation measurement that in the case of the porous layer, produces partial transformation of s-polarised light to p-polarisation, resulting in discrepancies of reflected light amplitude. This kind of evaluation has been determined as the main aim of the present research
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