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Direct single-molecule imaging for diagnostic and blood screening assays
Qiaoqiao Ruan1, Patrick J Macdonald1, Kerry M Swift1
1Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064.
Summary
Direct single-molecule imaging significantly enhances diagnostic assay sensitivity and specificity. This breakthrough improves detection limits to attomolar levels, impacting infectious disease screening and biomarker measurement.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Medical Diagnostics
Background:
- Millions of blood units are screened annually for infectious diseases like HIV and hepatitis.
- Billions of diagnostic tests measure over 400 biomarkers for various diseases globally.
- Improvements in assay performance are crucial given the high volume of diagnostic testing.
Purpose of the Study:
- To fundamentally improve assay sensitivity and specificity using direct single-molecule imaging.
- To overcome limitations of current ensemble-based measurement techniques in diagnostic assays.
- To establish a novel method for highly sensitive and accurate biomarker detection.
Main Methods:
- Utilizing regular epifluorescence microscopes for direct single-molecule imaging.
- Acquiring intensity profiles to identify and count individual fluorescent complexes on microparticles.
- Transitioning from single-molecule counting to pixel intensity averaging for a wide linear range.
Main Results:
- Achieved attomolar (10-18 M) detection sensitivity in model systems.
- Reached low femtomolar (10-16 M) sensitivity for analyte detection in human plasma.
- Demonstrated a continuous linear response from 10-18 to 10-5 M.
- Assays showed insensitivity to microparticle number and volume variations.
Conclusions:
- Direct single-molecule imaging offers fundamentally improved assay sensitivity and specificity.
- The method provides a wide linear detection range, shorter incubation times, and simpler protocols.
- This approach minimizes reagent consumption and eliminates key sources of uncertainty in standard assays.
