Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cell Biology

Background:

• Cell-to-cell variability is crucial for adaptation, evolution, and disease development.
• Quantitative chemical analysis of single-cell lysates is challenging.
• Microsystem technology offers potential for single-cell studies.

Purpose of the Study:

• To develop a microfluidic device for quantitative single-cell chemical analysis using ELISA.
• To reliably identify and quantify intracellular proteins, secondary messengers, and metabolites at the single-cell level.
• To investigate cell-to-cell variations in molecular responses.

Main Methods:

• Integration of a microfluidic device with enzyme-linked immunosorbent assays (ELISA).
• Parallel single-cell trapping and isolation in microchambers (625-pL).
• Quantitative determination of GAPDH and cyclic adenosine monophosphate (cAMP) using sandwich and competitive ELISA, respectively.

Main Results:

• Quantified GAPDH in U937 and HEK 293 cells (1-4 attomol/cell).
• Determined the EC50 of lutropin in MLT cells (2.51 ± 0.44 ng/mL).
• Observed significant cell-to-cell variations in cAMP levels (36-536 attomol for nonstimulated, 80-1040 attomol near EC50).

Conclusions:

• The combined microfluidic-ELISA device provides a sensitive and specific method for single-cell proteomics and metabolomics.
• The approach reveals substantial heterogeneity in cellular responses.
• This technology opens new avenues for understanding cell biology and disease mechanisms.