Extending the dynamic range of biomarker quantification through molecular equalization.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Precision medicine demands scalable multiplexed biomarker quantification.
• Current methods have limited dynamic range (3-4 orders of magnitude) compared to the human plasma proteome (>10 orders).
• Existing techniques like sample splitting and dilution hinder reproducibility and scalability.

Purpose of the Study:

• To develop a method for simultaneous quantification of analytes across a wide concentration range.
• To overcome the dynamic range limitations of current biomarker detection methods.
• To enhance scalability and reproducibility in multiplexed assays.

Main Methods:

• A two-pronged strategy named 'EVROS' was developed to equalize analyte signal intensity.
• The EVROS strategy was applied to a proximity ligation assay.
• Simultaneous quantification of four analytes was performed.

Main Results:

• Achieved simultaneous quantification of analytes from low femtomolar to mid-nanomolar concentrations.
• Demonstrated a seven-orders-of-magnitude dynamic range in a single 5 µl undiluted human serum sample.
• Successfully applied the EVROS strategy to a proximity ligation assay.

Conclusions:

• The EVROS strategy enables sensitive and accurate detection of diverse analytes in a highly multiplexed manner.
• This approach significantly expands the dynamic range achievable in biomarker quantification.
• Offers a scalable solution for precision medicine applications requiring broad analyte detection.