Probing Low-Copy-Number Proteins in a Single Living Cell

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Single-cell analysis is crucial for understanding cellular heterogeneity and function.
• Measuring low-copy-number proteins in single cells presents a significant analytical challenge.
• Existing methods lack the sensitivity required for detecting low-abundance proteins at the single-cell level.

Purpose of the Study:

• To develop a novel ultrasensitive method for probing low-copy-number proteins in single cells.
• To investigate the differential expression of proteins like alkaline phosphatase and survivin in cancer versus normal cells.
• To demonstrate the application of this technique for analyzing protein dynamics in living organisms.

Main Methods:

• Developed a plasmonic immunosandwich assay (PISA) combining in vivo immunoaffinity extraction and plasmon-enhanced Raman scattering (PERS).
• Utilized microprobes with antibodies or molecularly-imprinted polymers for specific protein extraction.
• Employed Raman-active nanotags for ultrasensitive, single-molecule level detection via PERS, achieving a 9-order magnitude enhancement in Raman intensity.
• Adapted PISA using acupuncture needles for in vivo analysis.

Main Results:

• Achieved ultrasensitive detection of low-copy-number proteins at the single-molecule level.
• Demonstrated distinct expression levels of alkaline phosphatase and survivin in cancer cells compared to normal cells.
• Observed reduced survivin expression following extended cell culture passage.
• Successfully applied acupuncture needle-based PISA for in vivo protein analysis.

Conclusions:

• PISA provides an ultrasensitive platform for single-cell protein analysis, particularly for low-abundance targets.
• The technique reveals significant differences in protein expression profiles between cancer and normal cells.
• PISA is adaptable for in vivo studies, opening avenues for real-time biological monitoring.