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Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

View Article: PubMed Central - PubMed

ABSTRACT

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3′ and 5′ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10−9 mol.L−1 over a linear ranged from 20 × 10−9 to 10 × 10−7 mol.L−1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

No MeSH data available.


(a) UV-Vis spectra of full match target assemblies in comparison with mismatch target assemblies mixed with AuNPs after salt addition. Inset aggregation trend of salt-treated AuNPs in the presence full and mismatch targets during the time (40 min), data were acquired at 1 min intervals at 550 nm. (b) Mismatch analysis on acrylamide 15% (up) and agarose gel 2.5% (down). (c) Representative visual images of full match and mismatch targets mixed with AuNPs after salt addition, in solution (up) and after spotted (10 μl) on the TLC paper (down).
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f6: (a) UV-Vis spectra of full match target assemblies in comparison with mismatch target assemblies mixed with AuNPs after salt addition. Inset aggregation trend of salt-treated AuNPs in the presence full and mismatch targets during the time (40 min), data were acquired at 1 min intervals at 550 nm. (b) Mismatch analysis on acrylamide 15% (up) and agarose gel 2.5% (down). (c) Representative visual images of full match and mismatch targets mixed with AuNPs after salt addition, in solution (up) and after spotted (10 μl) on the TLC paper (down).

Mentions: In order to test the ability of the designed assay to track the mismatch targets, the CTV severe DNA sequence was modified in 4 and 8 positions (Table 1). The thiolated probes (3′ L and 5′ R) were then hybridized with a perfect match (CTV severe) or mismatch (4, 8) DNA targets and the final products were visualized using the 15% polyacrylamide or 2.5% agarose gels electrophoresis. Interestingly, as demonstrated in Fig. 6a, a clear difference between sequences could be attained on acrylamide or agarose gels after staining with ethidium bromide. A DNA smear resulted from a product of the disulfide-induced self-assembling process was observed for all sequences, which appears to occur much more easier with full targets than mismatches. In this regard, a heavy DNA smear, which was extended toward higher molecular weights was observed for full DNA targets, while moderate and minimal smears were detected when targets with 4 or 8 mismatches were used respectively (Fig. 6a). Using the proposed novel method, it can be easily distinguish among close DNA sequences even without the addition of AuNPs.


Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles
(a) UV-Vis spectra of full match target assemblies in comparison with mismatch target assemblies mixed with AuNPs after salt addition. Inset aggregation trend of salt-treated AuNPs in the presence full and mismatch targets during the time (40 min), data were acquired at 1 min intervals at 550 nm. (b) Mismatch analysis on acrylamide 15% (up) and agarose gel 2.5% (down). (c) Representative visual images of full match and mismatch targets mixed with AuNPs after salt addition, in solution (up) and after spotted (10 μl) on the TLC paper (down).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384278&req=5

f6: (a) UV-Vis spectra of full match target assemblies in comparison with mismatch target assemblies mixed with AuNPs after salt addition. Inset aggregation trend of salt-treated AuNPs in the presence full and mismatch targets during the time (40 min), data were acquired at 1 min intervals at 550 nm. (b) Mismatch analysis on acrylamide 15% (up) and agarose gel 2.5% (down). (c) Representative visual images of full match and mismatch targets mixed with AuNPs after salt addition, in solution (up) and after spotted (10 μl) on the TLC paper (down).
Mentions: In order to test the ability of the designed assay to track the mismatch targets, the CTV severe DNA sequence was modified in 4 and 8 positions (Table 1). The thiolated probes (3′ L and 5′ R) were then hybridized with a perfect match (CTV severe) or mismatch (4, 8) DNA targets and the final products were visualized using the 15% polyacrylamide or 2.5% agarose gels electrophoresis. Interestingly, as demonstrated in Fig. 6a, a clear difference between sequences could be attained on acrylamide or agarose gels after staining with ethidium bromide. A DNA smear resulted from a product of the disulfide-induced self-assembling process was observed for all sequences, which appears to occur much more easier with full targets than mismatches. In this regard, a heavy DNA smear, which was extended toward higher molecular weights was observed for full DNA targets, while moderate and minimal smears were detected when targets with 4 or 8 mismatches were used respectively (Fig. 6a). Using the proposed novel method, it can be easily distinguish among close DNA sequences even without the addition of AuNPs.

View Article: PubMed Central - PubMed

ABSTRACT

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3′ and 5′ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10−9 mol.L−1 over a linear ranged from 20 × 10−9 to 10 × 10−7 mol.L−1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

No MeSH data available.