Accelerated protein digestion and separation with picoliter volume utilizing nanofluidics

Area of Science:

• Biochemistry
• Analytical Chemistry
• Nanotechnology

Background:

• Single cell analysis offers critical insights into cellular heterogeneity.
• Analyzing the proteome at the single-cell level is challenging due to protein amplification limitations and analyte loss during sample preparation.
• Existing liquid chromatography-mass spectrometry (LC-MS) platforms face issues with analyte loss and long analysis times for single cells.

Purpose of the Study:

• To develop an integrated nanofluidic device for high-throughput single-cell proteomics.
• To overcome challenges of analyte loss and lengthy analysis durations in single-cell proteomic workflows.
• To enable precise manipulation and analysis of femtoliter to picoliter (fL-pL) scale samples.

Main Methods:

• Development of an integrated nanofluidic device for sample manipulation on an fL-pL scale.
• Incorporation of an open/close valve for precise control of nanochannel surfaces and sample aliquoting.
• On-device capabilities including tryptic digestion, chromatographic separation, and non-labeled detection.
• Achieved an injection volume of 1.0 ± 0.1 pL with the integrated valve system.

Main Results:

• The nanofluidic device successfully suppressed analyte loss and enabled high-throughput analysis.
• Demonstrated high consistency in tryptic digestion, chromatographic separation, and detection.
• Showcased ultra-fast digestion within the nanochannel, achieving comparable results to 12-hour bulk digestion in just 15 minutes.
• Successfully implemented an injection system with a precise 1.0 ± 0.1 pL volume.

Conclusions:

• An integrated nanofluidic device was successfully developed for ultra-fast, high-throughput single-cell proteomic analysis at the picoliter scale.
• The device addresses critical issues of analyte loss and analysis duration, paving the way for more efficient single-cell proteome studies.
• This technology represents a significant advancement in single-cell proteomics, enabling analysis of limited biological samples with unprecedented speed and precision.