Non-fluorescent quantification of single mRNA with transient absorption microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Nanotechnology

Background:

• Single-molecule detection is challenged by biological background noise like autofluorescence and scattering.
• Existing methods struggle in turbid biological samples such as cells and tissues.

Purpose of the Study:

• To develop a non-fluorescence method for single-molecule detection using gold nanoparticles (AuNPs) and transient absorption microscopy (TAM).
• To quantify human epidermal growth factor receptor 2 (Her2) mRNA in cancer cells and tissues at single-copy sensitivity.

Main Methods:

• Utilized gold nanoparticles (AuNPs) as orthogonal probes for non-fluorescence detection.
• Employed transient absorption microscopy (TAM) for high-sensitivity imaging and quantification.
• Validated the TAM strategy with fluorescence in situ hybridization (FISH).

Main Results:

• TAM achieved single-copy sensitivity for Her2 mRNA detection.
• Quantified average Her2 copies in SK-BR-3 and MCF-7 cells (203.19 ± 80.48 and 11.29 ± 4.47, respectively).
• Demonstrated superior signal-to-noise ratio in clinical tissues, showing higher Her2 expression in breast cancer than normal tissues.

Conclusions:

• Transient absorption microscopy with gold nanoparticles offers a novel approach for biomarker quantification in turbid biological environments.
• This method provides a new dimension for single-molecule sensitivity analysis, crucial for clinical monitoring.
• TAM shows significant potential for accurate gene expression profiling in cancer diagnostics.