An integrated immunofluorescent detection system for automated and sensitive protein quantification based on a microfluidic flow cytometry platform.

Rapid and sensitive protein detection methods are of benefit to clinical diagnosis, pathological mechanism research, and infection prevention. However, routine protein detection technologies, such as enzyme-linked immunosorbent assay and Western blot, suffer from low sensitivity, poor quantification and labourious operation. Herein, we developed a fully automated protein analysis system to conduct fast protein quantification at the single molecular level. It streamlines antibody-coated beads-based protein capture, fluorescent labelling with fluorophore-conjugated antibodies, and flow-cytometric optical detection in a microfluidic chip, achieving sample-in quantitative result-out protein detection within 60 min. This platform employs a combination of spiral and ultrasonic mixing, and realizes highly efficient protein capture and immunolabelling. Leveraging non-Newtonian fluids, the beads can be well-aligned at the focus of the optical module, yielding robust protein quantification results with a low coefficient of variation of 0.4 %. As a proof of concept, interleukin 6 was determined using the integrated instrument. The results showed good linear correlation (R = 0.9938) with that obtained from Roche Electrochemiluminescence Immunoassay Analyzer (ECLIA). Moreover, the single molecule immunoanalyzer achieved a lower limit of detection (LOD) of 16 fg/mL and exhibited an 8-fold increase in sensitivity compared to ECLIA. With short turnaround time and less human intervention, the developed protein quantification system could become a valuable tool for detection of disease markers and precision medicine.