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Microfluidically Partitioned Dual Channels for Accurate Background Subtraction in Cellular Binding Studies by Surface
Chaowei Han1, Tianbao Dong1, Pengcheng Wang1
1Institute of Surface Analysis and Chemical Biology, University of Jinan, Jinan 250022, Shandong, P. R. China.
Analytical Chemistry
|December 1, 2022
Summary
A novel dual-channel substrate for surface plasmon resonance (SPR) microscopy improves single-cell analysis by preventing nonspecific adsorption and enabling accurate background subtraction for reliable kinetic and affinity measurements.
Area of Science:
- Biophysics
- Surface Science
- Cell Biology
Background:
- Conventional surface plasmon resonance (SPR) microscopy for single-cell analysis faces challenges with nonspecific adsorption (NSA) and inaccurate background subtraction.
- Existing methods using protein-modified substrates and cell-free references are prone to false signals, hindering accurate kinetic analysis.
Purpose of the Study:
- To develop and validate a dual-channel substrate for SPR microscopy to overcome limitations in single-cell analysis.
- To enable precise background subtraction and obtain biologically relevant kinetic and affinity parameters.
Main Methods:
- A microfluidic device was used to pattern a dual-channel substrate with one channel for cell immobilization (poly-l-lysine on PEG) and another for reference (PEG only).
- The dual-channel substrate was tested using wheat germ agglutinin (WGA) binding to HFF cells and COVID-19 S1 protein binding to ACE2 on HEK293 cells.
- SPR microscopy was employed to compare the dual-channel approach with conventional methods, assessing signal-to-noise ratios and kinetic parameter accuracy.
Main Results:
- The dual-channel substrate effectively excluded interferences from NSA and cell-diffracted waves, allowing for accurate subtraction of refractive index changes.
- Sensorgrams exhibited higher signal-to-noise ratios and shapes consistent with correct binding models, yielding accurate kinetic and affinity parameters.
- The binding kinetics of S1 protein and ACE2 on cells differed from recombinant ACE2, indicating a kinetic barrier imposed by the cell membrane.
Conclusions:
- The patterned dual-channel substrate is a significant advancement for SPR microscopy in single-cell analysis, providing accurate and reliable kinetic and affinity data.
- This method overcomes NSA-related artifacts, leading to more biologically relevant insights into molecular interactions at the single-cell level.
- The findings highlight the importance of the cell membrane in modulating binding kinetics, as demonstrated by the S1 protein-ACE2 interaction.
