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

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 to meet this clinical need. Herein, we introduce an ultra-sensitive and facile microbubbling digital assay readout method toward POC quantitation of protein biomarkers requiring only bright-field smartphone imaging. Picolitre-sized microwells together with platinum nanoparticle labels enable the discrete “visualization” of protein molecules via immobilized-microbubbling with smartphone. We also use computer vision and machine learning to develop an automated image analysis smartphone application to facilitate accurate and robust counting. Using this method, post-prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit of 2.1 fM (0.060 pg/mL), and early pregnancy detection using βhCG with a detection limit of 0.034 mIU/mL (2.84 pg/mL). The results are further validated using clinical serum samples against clinical and research assays. This work provides the proof-of-principle of the microbubbling digital readout as an ultra-sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.