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

National Basic Research Program of China [2010CB732402, 2013CB933703]; National Science Foundation of China [91313302, 21205100, 21275122, 21075104]; National Instrumentation Program [2011YQ03012412]; National Science Foundation for Distinguished Young Scholars of China [21325522]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 beta-galatosidase (beta-Gal) molecules and the fluorogenic substrate fluorescein di-beta-D-galactopyranoside were injected from two separated inlets to form uniform 20 mu m 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 beta-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 beta-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 beta-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. (C) 2014 AIP Publishing LLC

Cobalt/Iron(Oxides) Heterostructures for Efficient Oxygen Evolution and Benzyl Alcohol Oxidation Reactions

Design of advanced electrocatalysts for oxygen evolution reaction (OER) and the alternative reaction is of prime importance to splitting water for hydrogen generation. Herein, cobalt/iron­(oxides) heterostructures with interface engineering for regulating surface structure properties toward enhanced OER and benzyl alcohol oxidation (BAO) are demonstrated. Interface engineering triggers generation of local crystallinity and defective oxygen, enabling the material to export 50 mA cm^–2 for OER at an overpotential of 329 mV and continuous 20 h of operation without apparent decay. Further, BAO is also boosted on the heterostructures, further propelling water splitting to export 10 mA cm^–2 at a voltage of only 1.42 V. Theoretical calculation reveals that the defective sites dominated by interfaces facilitate adsorption/dissociation of intermediates during electrocatalysis. The findings in this work place Fe/Co­(oxides) heterostructures as an excellent bifunctional OER/BAO catalyst and also provide a promising interface-regulated electrocatalysis strategy for development of other advanced heterostructures toward various applications