Abstract
Developing an enhanced diagnosis using biosensors is important for the treatment of patients before disease complications arise. Improving biosensors would enable the detection of various low-abundance disease biomarkers. Efficient immobilization of probes/receptors on the sensing surface is one of the efficient ways to enhance detection. Herein, we introduced the pre-alkaline sensing surface with amine functionalization for capturing gold nanoparticle (GNP) conjugated to human blood clotting factor IX (FIX), and we demonstrated the excellent performance of the strategy. We have chosen the enzyme-linked immunosorbent assay (ELISA) and the interdigitated electrode (IDE), which are widely used, to demonstrate our method. The optimal amount for silanization has been found to be 2.5%, and 15-nm-sized GNPs are ideal and characterized. The limit of FIX detection was attained with ELISA at 100 pM with the premixed GNPs and FIX, which shows 60-fold improvement in sensitivity without biofouling, as compared to the conventional ELISA. Further, FIX was detected with higher specificity in human serum at a 1:1280 dilution, which is equivalent to 120 pM FIX. These results were complemented by the analysis on IDE, where improved detection at 25 pM was achieved, and FIX was detected in human serum at the dilution of 1:640. These optimized surfaces are useful for improving the detection of different diseases on varied sensing surfaces.
Highlights
Improvement of all aspect at the forefront of medicine is mandatory to maintain healthy human life and to extend the lifespan
Efficient immobilization of protein or antibody on the PS enzyme-linked immunosorbent assay (ELISA) surface is a crucial step to improve the limit of detection
We introduced a new and easy protein immobilization strategy on the PS ELISA surface assisted by gold nanoparticle (GNP)
Summary
Improvement of all aspect at the forefront of medicine is mandatory to maintain healthy human life and to extend the lifespan. There are different diagnostics which have been demonstrated capable of genuine detection via a wide range of biomarkers [4, 5]. It has been proven that proper orientation of antibody on the immobilized plate improves the detection by over 64fold as compared to the randomly immobilized molecules [12, 13]. Different researchers are utilizing the immobilization of proteins on PS ELISA plates by chemical or physical processes, such as a photochemical reaction assisted by polyvinyl benzyl lactonoylamide and immobilization of protein in the presence of detergent [14–16]. Dixit et al [8] have immobilized the antibodies on the amine-modified PS plate to improve the limit of detection, and they demonstrated that the premixture of (3-aminopropyl)triethoxysilane (APTES) and antibody before the immobilization step improved sensitivity by 54 times compared with the conventional ELISA and reached the limit of detection of 10 pM [8, 17]
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