Proximity cleavage assay (PCA): A single-step, 20-minute platform for protein detection in 1-μL biofluids

Rapid and accurate detection of protein biomarkers from tiny body fluid samples (approximately microliters) is required for both point-of-care testing (POCT) and at-home testing, but most protein biomarker assays struggle to combine small sample size, rapidity, simplicity of operation, and high sensitivity at the same time. In this paper, we proposed a proximity cleavage assay (PCA) for the highly sensitive and one-pot detection of protein biomarkers with 1-μL biological samples. Employing dual ligands labeled with DNA tags as probes, this approach triggers a cascaded signal amplification reaction upon target binding, completing the detection process within 20 min and requiring only one manual operation step of sample spiking and mixing. By real-time measurement of the fluorescence intensities in the signal amplification reaction, this method enables the detection of anti-SARS-CoV-2 spike protein antibodies and SARS-CoV-2 spike protein, with a detection limit of 7 pM for both analytes. Alternatively, a portable light-emitting diode (LED) bulb can be used to irradiate the reaction products, and then the test results can be observed with the naked eye. This allows for the detection of biomarkers without the need for expensive equipment, making PCA more suitable for POCT and at-home testing of protein biomarkers.