Real-Time Highly-Sensitive Protein Quantification Through On-Chip Chemiluminescence

A range of experimental methods have been developed to achieve highly sensitive detection and quantification of proteins. The majority of these methods rely on fluorescence-mediated readouts and, as such, their sensitivity can be affected by factors such as photobleaching of fluorophores and background signal from the illumination source. Both of these limitations can be overcome by using chemiluminescence-based detection: in contrast to fluorescence, chemiluminescence can be generated in an excitation source free manner, which allows for a significant reduction in background noise and for the use of an optical setup that comprises only a detection element. Here, we develop a highly-sensitive protein quantification platform by combining chemiluminescent detection of proteins with microfluidic mixing and detection. We use the platform to demonstrate quantitative detection of proteins over a concentration range of five orders of magnitude and down to 10 pg mL−1, corresponding to pM concentrations. Owing to the general presence of amine groups in peptides and proteins, our demonstrated system is applicable to characterising any protein sample and it can be used to quantify unlabelled samples.