Ultrasensitive, inexpensive, rapid, and portable assays are needed to detect infectious diseases at early stages to improve treatment and prevent transmission, especially to vulnerable patients. Unfortunately, most ultrasensitive assays require rinsing and development steps which increase their complexity, cost, and read time. We address these issues in a novel assay that uses a combination of buoyant microbubbles and magnetic microspheres to doubly label an analyte (SARS-CoV-2 N-protein from surfactant-lysed viruses) and form buoyant-analyte-magnetic (BAM) sandwich complexes. A permanent magnet pulls down the BAM complexes, while unbound microbubbles float towards the surface and separate without rinsing. Removing the magnet releases the buoyant BAM complexes allowing them to float upwards. Under flashlight illumination, they appear as bright rising dots. A simple digital camera (or even cell phone camera) counts the BAM complexes. Remarkably, the analytical detection limit is ~50 N-protein molecules in 5 micro-liters of saliva. The assay gave positive results for all PCR-positive saliva specimens tested, with concentrations ranging from 0.7 to 2.5x105 RNA copies/microliter. We also discuss assay limitations and ways to address them in the future.

Drs. Anker and Livi are Scientific Advisors for Akadeum Life Sciences, the company that sells the buoyant microbubbles used in the BAM assay, which owns the rights to a patent describing buoyant and magnetic analyte labeling and detection (US Patent 10,724,930).

This research was supported by "Buoyant and Magnetic (BAM) Assays for On Site Sensitive Rapid Diagnostics," COVID Testing Research Seed Grant Program from Clemson University and "Rising to the Separations and Diagnostic Challenge with Buoyancy and Magnetism (BAM)," Agilent Technologies Inc., Applications and Core Technology-University Relations (ACT-UR).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The saliva collection protocols were approved by the Institutional Review Boards (IRB) of Clemson University (IRB2021-0703) and Prisma Health (Pro00100731). All participants provided their written informed consent to provide a saliva sample. Participants provided 1-2 mL of saliva collected in a 50 mL conical tube and some demographic data (age, gender, race/ethnicity) to the research study. Samples were deidentified striped of personal identifying information before being passed to the research team. Part of the samples (200 microliters) was used for a RT-PCR clinical diagnostic test and eventual sequencing for SARS-CoV2 variant identification.

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