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Scanning electrochemical microscopy: Glucose oxidase as an electrochemical label in sandwich format immunoassay

I. Morkvenaite-Vilkonciene, A. Kisieliute, W. Nogala, A. Popov, B. Brasiuns, M. Kamarauskas, A.  Ramanavicius, S. Linfield, A. Ramanaviciene

Electrochimica Acta, 2023


Electrochemical immunoassays can be developed in a way that allows them to become an alternative to enzyme-linked immunosorbent assays as clinical diagnostic tools. In this research, we propose an electrochemical sandwich immunoassay for the detection of human growth hormone (hGH) using scanning electrochemical microscopy (SECM) on an array of 30 μm diameter gold micro-discs. The gold micro-discs were modified with a self-assembled monolayer, which was then used to covalently immobilize monoclonal antibodies against hGH (m-anti-hGH). After the hGH was captured by the m-anti-hGH, polyclonal detection antibodies conjugated with GOx (p-anti-hGH-GOx) interacted with the captured hGH, forming sandwich type immune complexes (Au-SAM/m-anti-hGH/hGH/p-anti-hGH-GOx). The substrate generation–tip collection (SG/TC) mode of SECM was then applied for the registration of GOx activity (which is proportional to the p-anti-hGH-GOx presence) using potassium hexacyanoferrate(III) as a redox mediator. It was shown that hGH concentrations as low as 40 nM could be detected by applying SECM to the sandwich immunoassay. The flux of the redox mediator from the gold micro-discs with formed sandwich type immune complexes after the addition of glucose was calculated using a mathematical model and the generation rate of [Fe(CN)6]4- in the presence of glucose was estimated to be 28 pmol s-1 cm-2. Thus, GOx was successfully applied as an electrochemical label for the detection of hGH in a sandwich type immunoassay by SECM operating in SG/TC mode. The methodology described herein could be adapted for the electrochemical detection of many other antigens of interest.


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