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Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection
Katharina Schmidt1,2, Simone Hageneder1, Bernadette Lechner1,2
1Biosensor Technologies, AIT-Austrian Institute of Technology GmbH, Konrad-Lorenz-Straße 24, 3430 Tulln an der Donau, Austria.
ACS Applied Materials & Interfaces
|November 29, 2022
Summary
This study tailors rolling circle amplification (RCA) for enhanced affinity biosensors. RCA significantly improves detection limits and enables single-molecule visualization for sensitive analyte detection.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Molecular Biology
Background:
- Affinity biosensors are crucial for detecting analytes.
- Optical probing using surface plasmon fields offers sensitive detection methods.
- Rolling circle amplification (RCA) can amplify biological signals.
Purpose of the Study:
- To tailor rolling circle amplification (RCA) for affinity biosensors.
- To investigate the impact of RCA-generated single-stranded DNA (ssDNA) chain conformation on plasmonic sensor response.
- To enhance the limit of detection (LOD) for biosensing applications.
Main Methods:
- Developed a biointerface to confine RCA within the evanescent surface plasmon field.
- Utilized surface plasmon resonance (SPR) and surface plasmon-enhanced fluorescence (PEF) for optical probing.
- Employed circular padlock probes as model target analytes.
Main Results:
- Achieved a high-femtomolar limit of detection (LOD), improving from 13 pM.
- Demonstrated an enhancement factor of approximately 2 orders of magnitude.
- Enabled direct visualization and counting of individual binding events using fluorescence microscopy.
Conclusions:
- The tailored RCA approach significantly enhances biosensor sensitivity.
- The developed biointerface effectively confines RCA for maximized sensor response.
- This method provides a versatile platform for digital readout and single-molecule detection with reduced LOD.
