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Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification.
Erik L McCarthy1, Lee E Bickerstaff, Mauricio Pereira da Cunha
1Department of Chemical and Biological Engineering/LASST, 245 ESRB-Barrows, University of Maine, Orono, ME 04469, USA.
Biosensors & Bioelectronics
|June 27, 2006
Summary
A new biosensor regeneration method uses molecular padlock probes (MPP) and rolling circle amplification (RCA) for highly specific nucleic acid detection. This isothermal technique offers low false positives and is suitable for low-power, remote sensing applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Biosensor Technology
Background:
- Nucleic acid detection is crucial for diagnostics and environmental monitoring.
- Existing methods like PCR can have limitations in specificity and power consumption.
- Regenerable biosensing surfaces are needed for cost-effective and sustainable applications.
Purpose of the Study:
- To develop and characterize a novel, regenerable biosensor platform for specific nucleic acid detection.
- To evaluate the performance of molecular padlock probe (MPP) technology combined with surface-associated rolling circle amplification (RCA).
- To assess the suitability of this isothermal technique for low-power sensing applications.
Main Methods:
- Utilized molecular padlock probes (MPP) and surface-associated rolling circle amplification (RCA) for nucleic acid detection.
- Employed gold-sputtered 96-well polystyrene microplates and a fluorescent label for assay development.
- Investigated detection limits, specificity, regeneration conditions, and reproducibility of the sensing surface.
- Tested the ability to discriminate between DNA oligonucleotides from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) viruses.
Main Results:
- Achieved highly specific detection of nucleic acid sequences with low false positive rates compared to PCR.
- Demonstrated an isothermal reaction advantageous for low-power systems.
- Detected as little as 0.6 fmol of circularized MPP using a fluorimetric assay.
- Showed that sensing layers could be reused for at least four cycles with <33% decrease in response after thermal denaturation.
- Successfully discriminated between DNA oligonucleotides specific to ISA and IHN viruses.
Conclusions:
- The developed MPP and surface-associated RCA technique provides a highly selective and regenerable method for nucleic acid detection.
- This isothermal approach is suitable for developing low-power biosensors for various applications, including remote sensing.
- The surface-bound amplification product allows direct integration with existing sensing platforms like optical and acoustic wave devices.