Quantitation of Femtomolar‐Level Protein Biomarkers Using a Simple Microbubbling Digital Assay and Bright‐Field Smartphone Imaging

Abstract Quantitating ultra‐low concentrations of protein biomarkers is critical for early disease diagnosis and treatment. However, most current point‐of‐care (POC) assays are limited in sensitivity. Herein, we introduce an ultra‐sensitive and facile microbubbling assay for the quantification of protein biomarkers with a digital‐readout method that requires only a smartphone camera. We used machine learning to develop a smartphone application for automated image analysis to facilitate accurate and robust counting. Using this method, post‐prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit (LOD) of 2.1 fm (0.060 pg mL−1), and early pregnancy detection using βhCG can be achieved with a of 0.034 mIU mL−1 (2.84 pg mL−1). This work provides the proof‐of‐principle of the microbubbling assay with a digital readout as an ultra‐sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.