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Nanopore-Based Fingerprint Immunoassay Based on Rolling Circle Amplification and DNA Fragmentation
Xinqi Kang, Connie Wu1, Mohammad Amin Alibakhshi
1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School and Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts 02115, United States.
ACS Nano
|March 6, 2023
Summary
This study introduces a novel nanopore sensing strategy using DNA reporter fingerprints for sensitive biomarker detection. This method enhances sensitivity and specificity for disease diagnostics, enabling low pM quantification of analytes like HE4.
Area of Science:
- Biotechnology
- Nanotechnology
- Molecular Diagnostics
Background:
- Nanopore sequencing offers advantages for genomics but faces limitations in sensitivity and analyte specificity for diagnostics.
- Current nanopore applications struggle to detect low-concentration disease biomarkers (pM or lower) and differentiate analytes.
- Lack of unique nanopore signals hinders sensitive and quantitative biomarker detection in biological fluids.
Purpose of the Study:
- To develop a sensitive and quantitative nanopore-based strategy for disease biomarker detection.
- To overcome limitations in nanopore sensitivity and analyte signal differentiation.
- To create a unique fingerprint signature for identifying and quantifying biomarkers.
Main Methods:
- Utilized immunocapture to isolate biomarkers.
- Employed isothermal rolling circle amplification to amplify signals.
- Incorporated sequence-specific fragmentation to release multiple DNA reporter molecules.
- Analyzed DNA fragment reporters for unique nanopore signal fingerprints.
Main Results:
- Successfully developed a nanopore detection strategy generating distinctive signal fingerprints.
- Achieved sensitive quantification of human epididymis protein 4 (HE4) at low picomolar (pM) levels.
- Demonstrated biomarker detection and quantification within a few hours.
Conclusions:
- The developed method provides a sensitive and specific approach for nanopore-based biomarker detection.
- This fingerprinting strategy enables precise identification and quantification of disease analytes.
- Future integration with nanopore arrays and microfluidics promises reduced detection limits, multiplexing, and cost-effectiveness for diagnostics.
