Recent progress in digital immunoassay: how to achieve ultrasensitive, multiplex and clinical accessible detection?

Abstract

The detection of low-abundance proteins in physiological samples is of high importance in clinical diagnostics and pathology research. However, protein biomarkers that can be accurately quantified by the existing detection methods and applied to clinical practice are just the tip of the iceberg. Quite a few proteins with concentrations below pg mL⁻¹, which are closely related to the occurrence and progression of diseases, are not effectively utilized due to the limitations of detection technology. In addition, simultaneously measuring multiple proteins in a single reaction improves the diagnostic accuracy and reliability since most diseases will cause abnormal changes in multiple biomarkers. Therefore, increasing demands to realize ultrasensitive and multiplexed protein detection have promoted the rapid development of digital immunoassay recently. Furthermore, the high-end digital detection is also expected to spark its potential for streamlined and integrated workflows in clinical applications, extending beyond its use in fundamental research within central laboratories. In this review, we focus on elucidating the core strategies and approaches to enhance the performance of digital immunoassay across three key dimensions: sensitivity, multiplexing, and clinical accessibility. Ultimately, we highlight the promising prospects of digital protein detection in the next decade.