Flow injection analysis coupled with photoelectrochemical immunoassay for simultaneous detection of anti-SARS-CoV-2-spike and anti-SARS-CoV-2-nucleocapsid antibodies in serum samples.

A thin-layer flow cell of low internal volume (12 µL) is incorporated in a flow injection analysis (FIA) system for simultaneous and real-time photoelectrochemical (PEC) immunoassay of anti-SARS-CoV-2 spike 1 (S1) and anti-SARS-CoV-2 nucleocapsid (N) antibodies. Covalent linkage of S1 and N proteins to two separate polyethylene glycol (PEG)-covered gold nanoparticles (AuNPs)/TiO2 nanotube array (NTA) electrodes affords 10 consecutive analyses with surface regenerations in between. An indium tin oxide (ITO) allows visible light to impinge onto the two electrodes. The detection limits for anti-S1 and anti-N antibodies were estimated to be 177 and 97 ng mL-1, respectively. Such values compare well with those achieved with other reported methods and satisfy the requirement for screening convalescent patients with low antibody levels. Additionally, our method exhibits excellent intra-batch (RSD = 1.3%), inter-batch (RSD = 3.4%), intra-day (RSD = 1.0%), and inter-day (RSD = 1.6%) reproducibility. The obviation of an enzyme label and continuous analysis markedly decreased the assay cost and duration, rendering this method cost-effective. The excellent anti-fouling property of PEG enables accuracy validation by comparing our PEC immunoassays of patient sera to those of ELISA. In addition, the simultaneous detection of two antibodies holds great potential in disease diagnosis and immunity studies.

Interference-free photoelectrochemical immunoassays using carboxymethylated dextran-coated and gold-modified TiO2 nanotube arrays.

An interference-free photoelectrochemical (PEC) immunoassay was developed for cardiac troponin I (cTnI) detection. Covalent linkage of cTnI antibody to carboxymethylated (CM-) dextran pre-immobilized onto a gold nanoparticles (AuNPs)-modified TiO2 nanotube array (NTA) affords five consecutive analyte captures with surface regenerations in between. Changes in the photocurrents at this photoanode before and after cTnI captures can be well fitted with the Langmuir isotherm from 0.220 pM to 2.20 nM cTnI. Owing to the inherently high sensitivity of the PEC detection, the detection limit (2.20 pg/mL) is lower than the range attainable with the enzyme-linked immunosorbent assay (ELISA) (6.00-40.0 pg/mL). Furthermore, CM-dextran prevents species in complex biological matrices from nonspecifically adsorbing onto the sensor surface, a feature not attainable with uncoated semiconductor electrodes or those coated with non-hydrogel-based chemical modifiers. The excellent anti-fouling property of dextran hydrogel allowed us to validate the accuracy of our regenerable sensors through a comparison of PEC immunoassays of patient sera to those of ELISA.