Joule Heating-Induced Dispersion in Open Microfluidic Electrophoretic Cytometry.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Capillary and microchannel electrophoresis enable high-resolution protein separations but are difficult to parallelize for single-cell analysis.
• Open microfluidics offers a scalable approach for concurrent separations, particularly for single-cell analysis.

Purpose of the Study:

• To investigate and mitigate the impact of Joule heating on protein electrophoresis in open microfluidic devices for single-cell analysis.
• To enhance analytical sensitivity and separation resolution in electrophoretic (EP) cytometry.

Main Methods:

• Development of an open microfluidic device using microwells within a polyacrylamide gel for in situ cell lysis and protein electrophoresis.
• Numerical simulations to model Joule heating-enhanced diffusion and protein loss.
• Implementation of a buffer exchange strategy to reduce diffusive losses and band broadening.

Main Results:

• Joule heating causes significant protein loss (∼50%) from the open microfluidic device due to diffusive losses.
• Autothermal runaway Joule heating increases analyte diffusivity, reducing separation resolution.
• Buffer exchange successfully reduced diffusive losses and enabled near-complete separation of proteins with a 4 kDa mass difference in single cells.

Conclusions:

• Open microfluidics coupled with buffer exchange provides a viable platform for high-throughput single-cell protein electrophoresis.
• The developed method significantly improves separation resolution and sensitivity, crucial for detecting subtle protein differences in diseases like cancer.
• This technique holds promise for identifying unmeasurable protein isoforms implicated in cancer progression.