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Rapid, low-cost, and accurate tests are still required for epidemiological surveillance and healthcare services to monitor and contain the spread of SARS-CoV-2. Brazilian researchers have contributed to this field's efforts by creating an electrochemical immunosensor that identifies antibodies against the virus.

The innovation is described in an article published in the journal ACS Biomaterials Science and Engineering.

In search of a novel diagnostic method, the group opted for a material frequently used in metallurgy - zinc oxide - and combined it for the first time with fluorine-doped tin oxide (FTO) glass, a conductive material used in electrodes for photovoltaics and other advanced applications.

"With this unusual combination and the addition of a biomolecule, the viral spike protein, we developed a surface capable of detecting antibodies against SARS-CoV-2. The result is displayed as an electrochemical signal captured by this surface," said chemist Wendel Alves, lead author of the article. Alves is a professor at the Center for Natural and Human Sciences, Federal University of the ABC (UFABC), Sao Paulo state.

The electrode fabricated by the researchers detected COVID-19 antibodies in serum in about five minutes with 88.7% sensitivity and 100% specificity, outperforming even the enzyme-linked immunosorbent assay (ELISA) test, the current gold-standard clinical diagnostic tool.

The research was supported by FAPESP via the National Science and Technology Institute for Bioanalysis and a Thematic Project.

According to Alves, who heads UFABC's Electrochemistry and Nanostructured Materials Laboratory, prior knowledge of chemical properties such as the isoelectric point of the virus's spike protein (S), enabled the group to develop a platform for S to bind electrostatically to zinc oxide nanorods. Zinc oxide is increasingly used to fabricate biosensors because of its versatility and unique chemical, optical and electrical properties.

Working Principle of the Immunosensor

A method for directly connecting an electronic device to a biological environment is challenging due to the inherent complexity of biosensor development. Nanomaterials enable the miniaturization of devices, enhancing their sensitivity due to their higher surface area and long-range electron conductivity.

(72,73) The ZnONRs create a favorable environment for biomolecules adsorption, maintaining their functionality and converting biological events into a stable, selective, and sensitive measurable signal.

The SARS-CoV-2 recombinant trimeric spike protein was the biomolecule used to construct the electrochemical biosensing detection platform for anti-spike antibodies as the S protein is the main target antigen component from all structural proteins of SARS-CoV-2. (74) When working at physiological pH (7.4), the IEP of S protein (∼5) has a net negative surface charge, and the ZnONR matrix with a high isoelectric point (∼9.5) takes a net positive surface charge density.

Thus, the negatively charged protein can be immobilized by electrostatic interaction on the surface of the positively charged ZnONR matrix. This principle has been widely used for the development of multiple immunosensors.

Analysis and future uses

A total of 107 blood serum samples were analyzed. They were divided into four groups: pre-pandemic (15), COVID-19 convalescents (47), vaccinated without previous positive results for the disease (25), and vaccinated after a positive result (20). The vaccine was two doses of CoronaVac administered four weeks apart. CoronaVac is produced by the Chinese company SinoVac in partnership with Butantan Institute (Sao Paulo state).

The authors of the article - researchers affiliated with UFABC and the Heart Institute (INCOR), which is run by the University of Sao Paulo's Medical School (FM-USP) - note that the device detects antibodies produced in response to both infection by the virus and vaccination, and shows excellent potential as a tool for monitoring of seroconversion and seroprevalence. Detecting the response to vaccination is important to help public health authorities assess the effectiveness of different vaccines and of immunization campaigns or programs, they stress.

The device has been validated for detecting immunity induced by CoronaVac, but the group plan to extend its use to testing for the response to Pfizer's and AstraZeneca's vaccines.

One of the advantages of the electrode they developed is its flexible architecture, which means that it can easily be customized for other diagnostic and biomedical applications using different biomolecules on the zinc oxide nanorods and other target analytes.

"The technology is a versatile biosensing platform. As developed by us, it can be modified and customized for serological detection of other diseases of public health interest," Alves said.

Electrochemical Response Studies of the ZnONRs/Spike Immunosensor

The EIS, CV, and SWV were used to characterize the electrode at each manufacturing stage (Figure 4). It shows the Faradic impedance spectra for the redox of [Fe(CN)6]3–/4– measured at each manufacturing stage. The imaginary impedance component (Z″) is displayed as a function of the real component of impedance (Z′), as shown in Figure 4A.

*with inputs from ANI

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