Abstract
Background: Saliva is a clear and complex biological fluid with pH value in the range of 6.0 – 7.0 that is produced from three main salivary glands. Recently, saliva has been recognized as a noninvasive diagnostic tool for surveillance of disease and in healthcare research and promotion since saliva is able to indicate a wide range of health and disease conditions with distinctive advantages over serum. In this study, we have fabricated an electrochemical immunosensor for sensitive detection of salivary immunoglobulin A (sIgA) based on indirect competitive format and using magnetic beads (MBs) as reaction carrier.
Methods: Firstly, carboxylic modified MBs were covalently attached to sIgA with N-(3- dimethylaminopropyl)- N’-ethylcarbodiimide and N-hydroxysulfosuccinimide. Then, the sIgA immobilized on MBs and free sIgA in solution was allowed to compete with each other for fixed amount of primary antibody. Upon the formation of immunocomplex, biotin-conjugated secondary antibody was incubated with the modified MBs following affinity binding with streptavidin-hydrogen peroxidase. For amperometric measurement, the resulting MBs were immobilized onto the surface of single-walled carbon nanotube working electrode with a strong external magnet and subsequently 3,3’,5,5’,- tetramethylbenzidine and hydrogen peroxide were introduced that functioned as electron transfer mediator and as enzyme substrate, respectively.
Results: The proposed MB-based immunosensor demonstrated a wide linear range of 5 pg/mL – 10 ng/mL and a low detection limit of 5 pg/mL with excellent reproducibility.
Conclusion: The developed immunosensor has great potential for clinical diagnostic application to monitor sIgA in field setting.
Keywords: Salivary immunoglobulin A, magnetic beads, amperometric immunosensor, indirect competitive immunoassay format, carboxylic modified MBs, antibody.
Graphical Abstract
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