Dataset Information

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting.

ABSTRACT: Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real-time tracking of the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker-antibody binding in biofluids with biomarker-protein binding on the cellular membrane.

SUBMITTER: Zhou X

PROVIDER: S-EPMC10316747 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting.

Zhou Xinyu X Wang Rui R Wan Zijian Z Zhang Pengfei P Wang Shaopeng S

Analytical chemistry 20230103 2

Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counti ...[more]

PMID: 36595491

Similar Datasets

Project description: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.

Dataset Information

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting.

Publications

Multiplexed Protein Detection and Parallel Binding Kinetics Analysis with Label-Free Digital Single-Molecule Counting.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets