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 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.
Subject(s)
DNA Methylation , Magnetite Nanoparticles , Electrochemical Techniques , Immunoprecipitation , Reproducibility of ResultsABSTRACT
In recent years, a new group of nanomaterials named nanozymes that exhibit enzyme-mimicking catalytic activity has emerged as a promising alternative to natural enzymes. Nanozymes can address some of the intrinsic limitations of natural enzymes such as high cost, low stability, difficulty in storage, and specific working conditions (i.e., narrow substrate, temperature and pH ranges). Thus, synthesis and applications of hybrid and stimuli-responsive advanced nanozymes could revolutionize the current practice in life sciences and biosensor applications. On the other hand, electrochemical biosensors have long been used as an efficient way for quantitative detection of analytes (biomarkers) of interest. As such, the use of nanozymes in electrochemical biosensors is particularly important to achieve low cost and stable biosensors for prognostics, diagnostics, and therapeutic monitoring of diseases. Herein, we summarize the recent advances in the synthesis and classification of common nanozymes and their application in electrochemical biosensor development. After briefly overviewing the applications of nanozymes in non-electrochemical-based biomolecular sensing systems, we thoroughly discuss the state-of-the-art advances in nanozyme-based electrochemical biosensors, including genosensors, immunosensors, cytosensors and aptasensors. The applications of nanozymes in microfluidic-based assays are also discussed separately. We also highlight the challenges of nanozyme-based electrochemical biosensors and provide some possible strategies to address these limitations. Finally, future perspectives on the development of nanozyme-based electrochemical biosensors for disease biomarker detection are presented. We envisage that standardization of nanozymes and their fabrication process may bring a paradigm shift in biomolecular sensing by fabricating highly specific, multi-enzyme mimicking nanozymes for highly sensitive, selective, and low-biofouling electrochemical biosensors.
