Direct Bioelectronic Detection of SARS-CoV-2 From Saliva Using Single-Molecule Field-Effect Transistor Array

Background: Nucleic acid tests, usually performed in specialized laboratories, have become the gold standard for COVID-19 diagnostics, most commonly through reverse-transcription quantitative polymerase chain reaction (qRT-PCR). However, specimen transport and processing often result in turnaround times of several days. While some rapid point-of-care (POC) tests provide results in less than an hour, they are expensive, require sample preparation and specialized reagents, and lack the throughput needed for population surveillance. Direct viral testing, which reduces reagent requirements, is essential for improving diagnostic testing. Four antigen tests approved for detection of SARS-CoV-2 based on immunoassays to the nucleocapsid (N) protein have limited sensitivity and cannot quantify viral load.

Materials/Methods: To address these gaps, DiagnostikosTM, an in-development rapid POC platform, was designed for direct, real-time, multiplexed, quantitative bioelectronic detection of biomolecules using an all-electronic single-molecule detection device. Single-molecule field-effect transistors (smFETs) were arrayed on a complementary metal-oxide-semiconductor (CMOS) integrated circuit chip, interfaced with a planned USB-stick-form-factor reader device. Nanobodies, or robust single-domain antibodies, were immobilized on the devices for sensitive detection of viral particles and debris. Using multiple nanobodies for a single protein, and nanobodies for different proteins in one assay, improved specificity. Targets included the four major SARS-CoV-2 structural proteins: nucleocapsid (N), spike (S), membrane (M), and envelope (E). The platform required no sample preparation or specialized reagents and was designed to operate with saliva, a reliable medium for detecting SARS-CoV-2. Individual chips could be manufactured for $35. Large, dense arrays with additional nanobodies also enabled detection of multiple pathogens in a single test. In this Direct-to-Phase-2 SBIR program, several innovations were pursued: isolation of nanobodies for SARS-CoV-2 structural proteins, development of the smFET platform for antigen detection, development of large CMOS arrays of smFET devices, and verification of detection in increasingly complex samples up to clinical samples. The project was a partnership between university researchers developing the smFET technology and Quicksilver Biosciences, a venture-based start-up commercializing smFET/CMOS arrays for diagnostics.

Outcome/Impact: The intended outcome was a functional, low-cost, and scalable POC diagnostic platform capable of rapid, direct, and quantitative detection of SARS-CoV-2 from saliva without specialized reagents. With its multiplexing capability, the platform also provided potential for detection of multiple pathogens in a single test, offering broad applicability for future molecular diagnostic applications.