title: 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 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.
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Guan
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zou
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Mingxia
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Jiangquan
authors.initials: J
authors.lastname: Lv
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Huali
authors.initials: H
authors.lastname: Shen
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
authors.forename: Chaoyong
authors.initials: C
authors.lastname: James Yang
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