Md. Nazmul Islam

Electrochemical biosensing strategies for DNA methylation analysis

DNA methylation is one of the key epigenetic modifications of DNA that results from the enzymatic addition of a methyl group at the fifth carbon of the cytosine base. It plays a crucial role in cellular development, genomic stability and gene expression. Aberrant DNA methylation is responsible for the pathogenesis of many diseases including cancers. Over the past several decades, many methodologies have been developed to detect DNA methylation. These methodologies range from classical molecular biology and optical approaches, such as bisulfite sequencing, microarrays, quantitative real-time PCR, colorimetry, Raman spectroscopy to the more recent electrochemical approaches. Among these, electrochemical approaches offer sensitive, simple, specific, rapid, and cost-effective analysis of DNA methylation. Additionally, electrochemical methods are highly amenable to miniaturization and possess the potential to be multiplexed. In recent years, several reviews have provided information on the detection strategies of DNA methylation. However, to date, there is no comprehensive evaluation of electrochemical DNA methylation detection strategies. Herein, we address the recent developments of electrochemical DNA methylation detection approaches. Furthermore, we highlight the major technical and biological challenges involved in these strategies and provide suggestions for the future direction of this important field.

Biological Functions and Current Advances in Isolation and Detection Strategies for Exosome Nanovesicles

Exosomes are nanoscale (≈30-150 nm) extracellular vesicles of endocytic origin that are shed by most types of cells and circulate in bodily fluids. Exosomes carry a specific composition of proteins, lipids, RNA, and DNA and can work as cargo to transfer this information to recipient cells. Recent studies on exosomes have shown that they play an important role in various biological processes, such as intercellular signaling, coagulation, inflammation, and cellular homeostasis. These functional roles are attributed to their ability to transfer RNA, proteins, enzymes, and lipids, thereby affecting the physiological and pathological conditions in various diseases, including cancer and neurodegenerative, infectious, and autoimmune diseases (e.g., cancer initiation, progression, and metastasis). Due to these unique characteristics, exosomes are considered promising biomarkers for the diagnosis and prognosis of various diseases via noninvasive or minimally invasive procedures. Over the last decade, a plethora of methodologies have been developed for analyzing disease-specific exosomes using optical and nonoptical tools. Here, the major biological functions, significance, and potential role of exosomes as biomarkers and therapeutics are discussed. Furthermore, an overview of the most commonly used techniques for exosome analysis, highlighting the major technical challenges and limitations of existing techniques, is presented.

A PCR-free electrochemical method for messenger RNA detection in cancer tissue samples

Despite having reliable and excellent diagnostic performances, the currently available messenger RNA (mRNA) detection methods mostly use enzymatic amplification steps of the target mRNA which is generally affected by the sample manipulations, amplification bias and longer assay time. This paper reports an amplification-free electrochemical approach for the sensitive and selective detection of mRNA using a screen-printed gold electrode (SPE-Au). The target mRNA is selectively isolated by magnetic separation and adsorbed directly onto an unmodified SPE-Au. The surface-attached mRNA is then measured by differential pulse voltammetry (DPV) in the presence of [Fe(CN)6]4-/3- redox system. This method circumvents the PCR amplification steps as well as simplifies the assay construction by avoiding multiple steps involved in conventional biosensing approaches of using recognition and transduction layers. Our method has demonstrated good sensitivity (LOD = 1.0pM) and reproducibility (% RSD = <5%, for n = 3) for detecting FAM134B mRNA in two cancer cell lines and a small cohort of clinical samples (number of samples = 26) collected from patients with oesophageal cancer. The analytical performance of our method is validated with a standard qRT-PCR analysis. We believe that our PCR-free approach holds a great promise for the analysis of tumor-specific mRNA in clinical samples.

Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies

Cell-free DNA (cfDNA) refers to short fragments of acellular nucleic acids detectable in almost all body fluids, including blood, and is involved in various physiological and pathological phenomena such as immunity, coagulation, aging, and cancer. In cancer patients, a fraction of hematogenous cfDNA originates from tumors, termed circulating tumor DNA (ctDNA), and may carry the same mutations and genetic alterations as those of a primary tumor. Thus, ctDNA potentially provides an opportunity for noninvasive assessment of cancer. Recent advances in ctDNA analysis methods will potentially lead to the development of a liquid biopsy tool for the diagnosis, prognosis, therapy response monitoring, and tracking the rise of new mutant sub-clones in cancer patients. Over the past few decades, cancer-specific mutations in ctDNA have been detected using a variety of untargeted methods such as digital karyotyping, personalized analysis of rearranged ends (PARE), whole-genome sequencing of ctDNA, and targeted approaches such as conventional and digital PCR-based methods and deep sequencing-based technologies. More recently, several chip-based electrochemical sensors have been developed for the analysis of ctDNA in patient samples. This paper aims to comprehensively review the diagnostic, prognostic, and predictive potential of ctDNA as a minimally invasive liquid biopsy for cancer patients. We also present an overview of current advances in the analytical sensitivity and accuracy of ctDNA analysis methods as well as biological and technical challenges, which need to be resolved for the integration of ctDNA analysis into routine clinical practice.

Gold-loaded nanoporous ferric oxide nanocubes for electrocatalytic detection of microRNA at attomolar level

A crucial issue in microRNA (miRNA) detection is the lack of sensitive method capable of detecting the low levels of miRNA in RNA samples. Herein, we present a sensitive and specific method for the electrocatalytic detection of miR-107 using gold-loaded nanoporous superparamagnetic iron oxide nanocubes (Au-NPFe2O3NC). The target miRNA was directly adsorbed onto the gold surfaces of Au-NPFe2O3NC via gold-RNA affinity interaction. The electrocatalytic activity of Au-NPFe2O3NC was then used for the reduction of ruthenium hexaammine(III) chloride (RuHex, [Ru(NH3)6]3+) bound with target miRNA. The catalytic signal was further amplified by using the ferri/ferrocyanide [Fe(CN)6]3-/4- system. These multiple signal enhancement steps enable our assay to achieve the detection limit of 100aM which is several orders of magnitudes better than most of the conventional miRNA sensors. The method was also successfully applied to detect miR-107 from cancer cell lines and a panel of tissue samples derived from patients with oesophageal squamous cell carcinoma with excellent reproducibility (% RSD = < 5%, for n = 3) and high specificity. The analytical accuracy of the method was validated with a standard RT-qPCR method. We believe that our method has the high translational potential for screening miRNAs in clinical samples.