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  • e-MagnetoMethyl IP: a magnetic nanoparticle-mediated immunoprecipitation and electrochemical detection method for global DNA methylation.

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    Accepted Manuscript (AM)
    Author(s)
    Farhana, Fatema Zerin
    Umer, Muhammad
    Saeed, Ayad
    Pannu, Amandeep Singh
    Husaini, Sediqa
    Sonar, Prashant
    Firoz, Shakhawat H
    Shiddiky, Muhammad JA
    Griffith University Author(s)
    Shiddiky, Muhammad J.
    Umer, Muhammad
    Pannu, Amandeep Singh S.
    Year published
    2021
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    Abstract
    The quantification of global 5-methylcytosine (5mC) content has emerged as a promising approach for the diagnosis and prognosis of cancers. However, conventional methods for the global 5mC analysis require large quantities of DNA and may not be useful for liquid biopsy applications, where the amount of DNA available is limited. Herein, we report magnetic nanoparticles-assisted methylated DNA immunoprecipitation (e-MagnetoMethyl IP) coupled with electrochemical quantification of global DNA methylation. Carboxyl (-COOH) group-functionalized iron oxide nanoparticles (C-IONPs) synthesized by a novel starch-assisted gel formation ...
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    The quantification of global 5-methylcytosine (5mC) content has emerged as a promising approach for the diagnosis and prognosis of cancers. However, conventional methods for the global 5mC analysis require large quantities of DNA and may not be useful for liquid biopsy applications, where the amount of DNA available is limited. Herein, we report magnetic nanoparticles-assisted methylated DNA immunoprecipitation (e-MagnetoMethyl IP) coupled with electrochemical quantification of global DNA methylation. Carboxyl (-COOH) group-functionalized iron oxide nanoparticles (C-IONPs) synthesized by a novel starch-assisted gel formation method were conjugated with anti-5mC antibodies through EDC/NHS coupling (anti-5mC/C-IONPs). Anti-5mC/C-IONPs were subsequently mixed with DNA samples, in which they acted as dispersible capture agents to selectively bind 5mC residues and capture the methylated fraction of genomic DNA. The target-bound Anti-5mC/C-IONPs were magnetically separated and directly adsorbed onto the gold electrode surface using gold-DNA affinity interaction. The amount of DNA adsorbed on the electrode surface, which corresponds to the DNA methylation level in the sample, was electrochemically estimated by differential pulse voltammetric (DPV) study of an electroactive indicator [Ru(NH3)6]3+ bound to the surface-adsorbed DNA. Using a 200 ng DNA sample, the assay could successfully detect differences as low as 5% in global DNA methylation levels with high reproducibility (relative standard deviation (% RSD) = <5% for n = 3). The method could also reproducibly analyze various levels of global DNA methylation in synthetic samples as well as in cell lines. The method avoids bisulfite treatment, does not rely on enzymes for signal generation, and can detect global DNA methylation using clinically relevant quantities of sample DNA without PCR amplification. We believe that this proof-of-concept method could potentially find applications for liquid biopsy-based global DNA methylation analysis in point-of-care settings.
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    Journal Title
    Analyst
    DOI
    https://doi.org/10.1039/d1an00345c
    Funder(s)
    ARC
    Grant identifier(s)
    DP190102944
    Copyright Statement
    © 2021 Royal Society of Chemistry. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal website for access to the definitive, published version.
    Note
    This publication has been entered in Griffith Research Online as an advanced online version.
    Subject
    Analytical chemistry
    Other chemical sciences
    Publication URI
    http://hdl.handle.net/10072/404207
    Collection
    • Journal articles

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