Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.
We thank the National Basic Research Program of China (2010CB732402 and 2013CB933703), the National Science Foundation of China (91313302, 21205100, 21275122, and 21075104), National Instrumentation Program (2011YQ03012412), and the National Science Foundation for Distinguished Young Scholars of China (21325522) for their financial support.
II. EXPERIMENTAL SECTION
A. Materials and reagents
B. Synthesis of surfactant E2K0660
C. Device fabrication
D. Generation, capture, and release of droplets
E. Single β-Gal molecule assay
III. RESULTS AND DISCUSSION
A. Principle of digital enzyme detection using microfluidic droplet approach
B. The choice of fluorinated oil GH-135 and surfactant E2K0660
C. Droplet generation, capture, and release
D. Optimization of the droplet diameter
E. Highly parallel observation of single-enzyme reaction kinetics in droplets
F. Poisson distribution of single molecules in droplets
G. Digital detection of β-Gal molecules
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