Digital Biosensor for Rapid and Ultrasensitive Liquid-Phase Detection of Biomarkers With Lensless Holographic Microscopy

Digital assays hold great potential for biomarker analysis in early disease diagnosis, and a key method to achieve digital detection is through the single-molecule pull-down (SiMPull) technique. However, the SiMPull assay frequently encounters challenges in field-of-view, detection throughput and ease of use. Lensless Holographic Microscopy (LHM) is a compact imaging technique that, when combined with aggregation assays, enables the detection of aggregation events of thousands of particles in a three-dimensional liquid-phase environment. This makes it a highly promising digital biosensing approach. Herein, we propose a rapid and ultrasensitive microbead aggregation assay based on LHM imaging for the digital detection of nucleic acid and protein biomarkers in solution. After an incubation period of less than one hour, the solution is directly imaged using LHM. The localization of microbeads within an extensive three-dimensional liquid mixture is determined via an automated bead locating and focusing procedure. This process eliminates the need for complex microfluidic channels and circumvents the requirement to wait for particles to settle to the sensing plane, which greatly simplifies the sensing platform and reduces the detection time. Finally, feature-based automatic classification is proposed to identify the aggregation rates, which reflect the concentration of analytes. Experiments indicate that the assay can be performed within 60 minutes and reach a limit of detection (LOD) of 10 fM and dynamic range of at least 4 orders of magnitude for nucleic acid; for interleukin-6, the LOD is 2 pg/mL, with a dynamic range of 2-3 orders of magnitude. The proposed approach is expected to enable point-of-care applications in settings where resource scarcity and protocol simplicity are of great importance.