A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Abstract

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 b-galatosidase (b-Gal) molecules and the fluorogenic substrate fluorescein di-b-D-galactopyranoside were injected from two separated inlets to form uniform 20 lm 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 b-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 b-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 b-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. V C 2014 AIP Publishing LLC. [http://dx

Digital Assays Part II: Digital Protein and Cell Assays

“…10 Although the majority of studies worked with the well-known enzyme β-galactosidase, the basic principle could be extended to other enzymes, since a wide range of fluorogenic substrates is commercially available. When developing a digital enzyme assay, technical requirements include (1) an imaging system able to detect fluorescence at low levels, (2) blocking proteins 7 or biocompatible surfactants to prevent adsorption of proteins to the walls or liquid interfaces, [11][12][13][14] and (3) appropriate partition sizes to accumulate detectable fluorescence in a reasonable timeframe. Compared to digital PCR, the detection of proteins is more difficult because it relies on linear enzymatic amplification rather than targeted PCR; however, the time can be decreased to minutes when the assay is miniaturized to subpicoliter partitions.…”

Highly sensitive multiplex protein detection by droplet‐free digital ELISA

“…In digital ELISA, signal amplification is performed in a droplet to enable detection of signals from a single target molecule (Figure a). This requires multiple uniformly sized microdroplets, and a number of methods to produce microdroplets were proposed so far . However, microdroplet generation results in a complex test protocol, while special‐purpose micro devices incur high cost, which makes clinical applications of digital ELISA problematic.…”

“…This requires multiple uniformly sized microdroplets, and a number of methods to produce microdroplets were proposed so far. [2][3][4] However, microdroplet generation results in a complex test protocol, while special-purpose micro devices incur high cost, which makes clinical applications of digital ELISA problematic. Thus, aiming at developing digital ELISA that does not require microdroplets, we previously proposed droplet-free digital ELISA with signals amplified on beads ( Figure 1b).…”

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detection

“…Finally, this mechanism can potentially be adapted to a microfluidic droplet platform, where individual droplets enclose distinct target and reagents, with the same temperature gradient along the channel. [39][40][41][42] The droplet functions like the microbeads as a mobile object to carry the target DNA and fluorescent dye for the melting analysis. While the droplet platform can possibly simplify the DNA immobilization step, the microbeads will provide better signal-to-noise ratio for this continuousflow melting analysis in temperature-gradient channels.…”