Enhanced Enzymatically Amplified Metallization on Nanostructured Surfaces for Multiplexed Point‐of‐Care Electrical Detection of COVID‐19 Biomarkers

Abstract

Inexpensive yet sensitive and specific biomarker detection is a critical bottleneck in diagnostics, monitoring, and surveillance of infectious diseases such as COVID‐19. Multiplexed detection of several biomarkers can achieve wider diagnostic applicability, accuracy, and ease‐of‐use, while reducing cost. Current biomarker detection methods often use enzyme‐linked immunosorbent assays (ELISA) with optical detection which offers high sensitivity and specificity. However, this is complex, expensive, and limited to detecting only a single analyte at a time. Here, it is found that biomarker‐bound enzyme‐labeled probes act synergistically with nanostructured catalytic surfaces and can be used to selectively reduce a soluble silver substrate to generate highly dense and conductive, localized surface silver metallization on microelectrode arrays. This enables a sensitive and quantitative, simple, direct electronic readout of biomarker binding without the use of any intermediate optics. Furthermore, the localized and dry‐phase stable nature of the metallization enables multiplexed electronic measurement of several biomarkers from a single drop (<10 µL) of sample on a microchip.This method is applied for the multiplexed point‐of‐care (POC) quantitative detection of multiple COVID‐19 antigen‐specific antibodies. Combining a simple microchip and an inexpensive, cellphone‐interfaced, portable reader, the detection and discrimination of biomarkers of prior infection versus vaccination is demonstrated.

Inexpensive High-Throughput Multiplexed Biomarker Detection Using Enzymatic Metallization with Cellphone-Based Computer Vision

“…Additional individual COVID+ ( n = 10), healthy ( n = 10), and buffer control samples were tested using the multiplexed assay and were found to have similar antibody signatures as above ( Figure S7 ). This matches our and others’ earlier findings that SARS-CoV-2 infection results in antibodies against a larger set of viral antigens including S and N. 12 , 40 − 42 Finally uninfected vaccine-recipient serum showed only anti-S IgG but no anti-N IgG, which is as expected since the vaccine used (Pfizer mRNA vaccine) contains only S mRNA and results in an immune response directed against the S antigen. 43 This indicates the utility of this MO-BEAM platform in rapid and inexpensive vaccine response monitoring as well.…”

“…Further, the enzymatic metallization reaction itself is unexplored here for optimization as its detailed mechanism remains unknown. We have recently hypothesized 42 that the silver deposition is self-limiting as the HRP that catalyzes the silver reduction itself serves as the nucleation site for sliver deposition. This eventually blocks and inactivates the active site of the enzyme.…”

Electronic Immunoassay Using Enzymatic Metallization on Microparticles

“…It may be possible to improve this metallization by immobilizing additional catalysts, such as gold nanoparticles, on the bead surface. We have earlier shown that this technique achieves a ∼4–100 pM LOD for electronic detection of immunoassays on microelectrode arrays . With regards to the measurement method, sensitivity is highly dependent on the electric field focusing in the aperture and could be enhanced by minimizing its relevant dimensions to more closely match the bead size.…”

“…Note that these times are obtained from assay optimization performed in our prior work with enzymatic metallization. 9 2.3. Clinical Immunoassay.…”

Regulating the microenvironment with nanomaterials: Potential strategies to ameliorate COVID-19

“…Research has largely focused on managing COVID-19, such as by applying biosensing technologies 12 , 13 , 14 , developing vaccines to prevent infection 15 , 16 , repurposing molecular drugs and neutralizing antibodies (NAbs) to fight against the virus 17 , 18 , 19 , and administering symptomatic therapy to alleviate clinical symptoms ( Fig. 1 ).…”