Lapitan, LDS, Xu, Y, Guo, Y et al. (1 more author) (2017) Ultrasensitive Detection and Discrimination of Cancer-Related Single Nucleotide Polymorphisms Using Poly-Enzyme Polymer Bead Amplification. In: MDPI Proceedings. 5th International Symposium on Sensor Science, 27-29 Sep 2017, Barcelona, Spain. MDPI , p. 806.
Abstract
We report the development of a new ultrasensitive approach for label-free DNA detection using magnetic nanoparticle (MNP)-assisted rapid target capture/separation in combination with signal amplification using poly-enzyme tagged polymer nanobead. The sensor uses a MNP linked capture DNA and a biotin modified signal DNA to sandwich bind the target followed by ligation to provide high single-nucleotide polymorphism discrimination. Only the presence of a perfect match target DNA yields a covalent linkage between the capture and signal DNAs for subsequent binding to a neutravidin-modified horseradish peroxidase (HRP) enzyme via the strong biotin–neutravidin interaction. This converts each captured full match DNA target into a HRP which can convert millions of copies of a non-fluorescent substrate (amplex red) to a highly fluorescent product (resorufin) for great signal amplification. The use of polymer nanobead each tagged with thousands of copies of HRPs as the signal amplifier greatly improves the signal amplification power, leading to greatly improved sensitivity. This biosensing approach can specifically detect an unlabelled DNA target down to 10 aM with a wide dynamic range of 5 orders of magnitude (from 0.01 fM to 1000 fM). Furthermore, our approach has a high discrimination between a perfectly matched gene and its cancer-related single-base mismatch targets (SNPs): it can positively detect the perfect match DNA target even in the presence of 100-fold excess of co-existing SNPs. This sensing approach also works robustly in clinical relevant media (e.g., 10% human serum) and gives almost the same SNP discrimination ratio as that in clean buffers. Therefore, this ultrasensitive SNP biosensor appears to be well-suited for potential diagnostic applications of genetic diseases.
Metadata
Item Type: | Proceedings Paper |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Inorganic Chemistry (Leeds) |
Funding Information: | Funder Grant number Leeds Cancer Research UK centre UNSPECIFIED Leeds Biomedical and Health Research Centre PF565 Wellcome Trust 097354/Z/11/Z |
Depositing User: | Symplectic Publications |
Date Deposited: | 18 Dec 2017 11:42 |
Last Modified: | 18 Dec 2017 11:42 |
Status: | Published |
Publisher: | MDPI |
Identification Number: | 10.3390/proceedings1080806 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:125341 |