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2018

(105) Abu Ali Ibn Sina, Laura G. Carrascosa, Ziyu Liang, Yadveer S. Grewal, Andri Wardiana, Muhammad J. A. Shiddiky, Robert A. Gardiner, Hemamali Samaratunga, Maher K. Gandhi, Rodney J. Scott, Darren Korbie and Matt Trau (2018)

Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker

. 2018; 9: 4915.  DOI: 10.1038/s41467-018-07214-w

Publication year: 2018

Abstract

Epigenetic reprogramming in cancer genomes creates a distinct methylation landscape encompassing clustered methylation at regulatory regions separated by large intergenic tracks of hypomethylated regions. This methylation landscape that we referred to as Methylscape is displayed by most cancer types, thus may serve as a universal cancer biomarker. To-date most research has focused on the biological consequences of DNA Methylscape changes whereas its impact on DNA physicochemical properties remains unexplored. Herein, we examine the effect of levels and genomic distribution of methylcytosines on the physicochemical properties of DNA to detect the Methylscape biomarker. We find that DNA polymeric behaviour is strongly affected by differential patterning of methylcytosine, leading to fundamental differences in DNA solvation and DNA-gold affinity between cancerous and normal genomes. We exploit these Methylscape differences to develop simple, highly sensitive and selective electrochemical or colorimetric one-step assays for the detection of cancer. These assays are quick, i.e., analysis time≤10minutes, and require minimal sample preparation and small DNA inpu

(104) Shuaihua Zhang, Qian Yang, Xin Zhou, Zhi Li, Wenjin Wang, Xiaohuan Zang, Chun Wang, Muhammad J. A. Shiddiky, Adharvana Chari Murugulla, Nam-Trung Nguyen, Zhi Wang and Yusuke Yamauchi (2018)

Synthesis of nanoporous poly-melamine-formaldehyde (PMF) based on Schiff base chemistry as a highly efficient adsorbent

Analyst, 2018. Accepted Manuscript. DOI: 10.1039/c8an01623b

Publication year: 2018

Abstract

This study proposes the construction of nanoporous poly-melamine-formaldehyde (PMF) through the Schiff base condensation reaction of paraformaldehyde and melamine. The PMF nanoparticles showed a good adsorption capability to some benzene-ring-containing dyes including acid fuchsine, nigrosine, and methyl orange. Moreover, the as-prepared PMF nanoparticles were employed as the coating adsorbent for the solid-phase microextraction (SPME) of seven volatile fatty acids (VFAs) with high enrichment factors. A PMF-assisted SPME method was established for the enrichment of VFAs from environmental water samples with satisfactory recoveries (88.5%–102.0%) and acceptable precisions (relative standard deviations <10.9%). This contribution might furnish an advanced benchmark for the exploitation of new porous organic polymers as the effective adsorbents for SPME or other fields of utilization.

(103) Ripon Bhattacharjee, Sofia Moriam, Nam-Trung Nguyen and Muhammad J. A. Shiddiky (2018)

A bisulfite treatment and PCR-free global DNA methylation detection method using electrochemical enzymatic signal engagement

Biosensors and Bioelectronics, 2018, Accepted Manuscript. DOI: 10.1016/j.bios.2018.10.020

Publication year: 2018

Abstract

In this paper we report on a bisulfite treatment and PCR amplification-free method for sensitive and selective quantifying of global DNA methylation. Our method utilises a three-step strategy that involves (i) initial isolation and denaturation of global DNA using the standard isolation protocol and direct adsorption onto a bare gold electrode via gold-DNA affinity interaction, (ii) selective interrogation of methylation sites in adsorbed DNA via methylation-specific 5mC antibody, and (iii) subsequent signal enhancement using an electrochemical-enzymatic redox cycling reaction. In the redox cycling reaction, glucose oxidase (GOx) is used as an enzyme label, glucose as a substrate and ruthenium complex as a redox mediator. We initially investigated the enzymatic properties of GOx by varying glucose and ruthenium concentration to delineate the redox cyclic mechanism of our assay. Because of the fast electron transfer by ruthenium (Ru) complex and intrinsic signal amplification from GOx label, this method could detect as low as 5% methylation level in 50 ng of total DNA input. Moreover, the use of methylation-specific 5mC antibody conjugated GOx makes this assay relatively highly selective for DNA methylation analysis. The data obtained from the electrochemical response for different levels of methylation showed excellent interassay reproducibility of RSD (relative standard deviation) <5% for n = 3. We believe that this inexpensive, rapid, and sensitive assay will find high relevance as an alternative method for DNA methylation analysis both in research and clinical platforms.

(102) Ripon Bhattacharjee, Sofia Moriam, Muhammad Umer, Nam-Trung Nguyen and Muhammad J A Shiddiky (2018)

DNA methylation detection: recent developments in bisulfite free electrochemical and optical approaches

Analyst, 2018, Accepted Manuscript (Critical Review)

Publication year: 2018

Abstract

DNA methylation is one of the significant epigenetic modifications involved in mammalian development as well as the initiation and progression of various diseases like cancer. Over the past few decades, enormous research has been carried out for quantification of DNA methylation in the mammalian genome. Earlier, most of these methodologies used bisulfite treatment. However, low conversion, false reading, longer assay time and complex chemical reaction are the common limitations of this method that hinders their application to routine clinical screening. Thus, alternative to bisulfite conversion-based DNA methylation detection, numerous bisulfite-free methods have been proposed. In this regard, electrochemical biosensors have gained much attention in recent years for being highly sensitive yet cost effective, portable, and simple to operate. On the other hand, biosensors with optical readout enable direct real time detection of biological molecules and are easily adaptable to multiplexing. Incorporation of electrochemical and optical readouts to bisulfite free DNA methylation analysis is paving the way for translation of this important biomarker to standard patient care. In this review, we provide a critical overview of recent advances in the development of electrochemical and optical readout based bisulfite free DNA methylation assays.

(101) Ahmed Munaz, Muhammad J. A. Shiddiky, Nam-Trung Nguyen (2018)

Magnetophoretic separation of diamagnetic particles through parallel ferrofluid streams

Sensors and Actuators B: Chemical 275, 459-469.

https://doi.org/10.1016/j.snb.2018.07.176

Publication year: 2018

Abstract

Particle separation based on microfluidic technology offers a simple, reliable, and low-cost approach for the diagnosis of diseases. The separation concept can be extended to genetic engineering, cell transplantation, and immunology. This paper reports a simple microfluidic platform for the separation of diamagnetic particles of different sizes utilizing parallel ferrofluid streams. The ferrofluid streams with predefined concentrations of magnetic nanoparticles promote negative magnetophoresis and are able to separate a particle mixture with a subtle size variation. Numerical simulation was used to optimise the magnetic field gradient, e.g. the number and position of the external permanent magnets. The effect of flow rate ratio and the concentration distribution were analyzed by the simulation and validated by experiments. Furthermore, two-stream and three-stream ferrofluid configurations were evaluated to find the optimum separation performance. The experimental results show a maximum separation efficiency of 78% and 75% with three-stream configuration for 3.2-µm and 4.8-µm particles, respectively.

(100) Ripon Bhattacharjee, Shunsuke Tanaka, Sofia Moriam, Mostafa Kamal Masud, Jianjian Lin, Saad M. Alshehri, Tansir Ahamad, Rahul R Salunkhe, Nam-Trung Nguyen, Yusuke Yamauchi, Md Shahriar Hossain, Muhammad J A Shiddiky (2018)

Porous Nanozymes: Peroxidase-Mimetic Activity of Mesoporous Iron Oxide for Colorimetric and Electrochemical Detection of Global DNA Methylation

Journal of Materials Chemistry B, 2018, 6, 4783 – 4791

Publication year: 2018

Abstract

Nanomaterials (nanozymes) with peroxidase-mimetic activity have been widely used in biosensing platforms as low-cost, relatively stable and prevailing alternatives to natural enzymes. Herein, we report on the synthesis and application of the peroxidase-mimetic activity of mesoporous iron oxide (MIO) for the detection of global DNA methylation in colorectal cancer cell lines. The target DNA was extracted and denatured to get ssDNA followed by direct adsorption onto the surface of a bare screen-printed gold electrode (SPGE). A 5-methylcytosine antibody (5mC) functionalized nanomaterial (MIO-5mC) was then used to recognise the methylcytosine groups present on the SPGE. The MIO-5mC conjugates catalyse the TMB solution in the presence of hydrogen peroxide to give the colorimetric (i.e., naked-eye observation) and electrochemical detection of DNA methylation. The assay could successfully detect as low as 10% difference in the global DNA methylation level in synthetic samples and cell lines with good reproducibility and specificity (%RSD = <5%, for n = 3). This strategy avoids the use of natural enzyme horseradish peroxidase (HRP), traditional PCR based amplification and bisulfite treatment steps that are generally used in many conventional DNA methylation assays. We envisage that our assay could be a low-cost platform with great potential for genome-wide DNA methylation analysis in point-of-care applications.

Graphical abstract: Porous nanozymes: the peroxidase-mimetic activity of mesoporous iron oxide for the colorimetric and electrochemical detection of global DNA methylation

(99) Md. Nazmul Islam, Lena Gorgannezhad, Mostafa Kamal Masud, Shunsuke Tanaka, Md. Shahriar A. Hossain, Yusuke Yamauchi, Nam-Trung Nguyen, Muhammad J. A. Shiddiky (2018)

Novel Graphene Oxide‐loaded Iron Oxide Superparamagnetic Nanoparticles for Ultrasensitive Electrocatalytic Detection of MicroRNA

ChemElectroChem, First published: 12 June 2018

Publication year: 2018

Abstract

We report on the electrocatalytic activity of a new class of superparamagnetic nanoparticle graphene oxide‐ loaded iron oxide (GO/IO hybrid material) towards the reduction of ruthenium hexaammine(III) chloride (Ru(NH3)6]3, RuHex). Leveraging the electrocatalytic activity of GO/IO hybrid material, and signal enhancement capacity of [Ru(NH3)6]3+/[Fe(CN)6]3‐ electrocatalytic cycle, an ultrasensitive and specific electrochemical sensor was developed for the detection of cancer‐related microRNA (miRNA). Using direct affinity interaction between RNA and graphene oxide, magnetically isolated and purified target miRNA were directly adsorbed onto the GO/IO hybrid material‐ modified screen‐printed electrode. The detection was enabled by chronocoulometric (CC) readout of charge compensating [Ru(NH3)6]3+ followed by an enhancement in CC charge display via Ru(NH3)6]3+/[Fe(CN)6]3‐ system. We demonstrated an excellent LOD of 1.0 fM by accurately detecting miR‐21 in synthetic samples, and showcased its clinical utility in ovarian cancer cell lines with high sensitivity (10 cells) and good reproducibility (% RSD = < 5%, for n = 3).

(98) Ahmed Munaz, Muhammad J. A. Shiddiky, Nam-Trung Nguyen (2018)

Recent Advances and Current Challenges in Magnetophoresis Based Micro Magnetofluidics

Biomicrofluidics 12, 031501.

Publication year: 2018

Abstract

The combination of magnetism and microscale fluid flow has opened up a new era for handling and manipulation of samples in microfluidics. In particular, magnetophoresis, the migration of particles in a magnetic field, is extremely attractive for microfluidic handling due to its contactless nature, independence of ionic concentration, and lack of induced heating. The present paper focuses on recent advances and current challenges of magnetophoresis and highlights the key parameters affecting the manipulation of particles by magnetophoresis. The magnetic field is discussed according to their relative motion to the sample as stationary and dynamic fields. The migration of particles is categorized as positive and negative magnetophoresis. The applications of magnetophoresis are discussed according to the basic manipulation tasks such as mixing, separation, and trapping of particles or cells. Finally, the paper highlights the limitations of current approaches and provides the future perspective for this research area.

(97) Lena Gorgannezhad, Muhammad Umer, Mostafa Kamal Masud, Yusuke Yamauchi, Shahriar Al Hossain, Carlos Salomon, Richard Kline, Nam-Trung Nguyen, Muhammad J.A. Shiddiky (2018)

Detection of FGFR2: FAM76A Fusion Gene in Circulating Tumor RNA Based on Catalytic Signal Amplification of Graphene Oxide‐loaded Magnetic Nanoparticles

Electroanalysis 30, Available on line. 

DOI: 10.1002/elan.201800282

 

Publication year: 2018

Abstract

Circulating tumor nucleic acids (ctNAs) are promising biomarkers for minimally invasive cancer assessment. The FGFR2:FAM76A fusion gene is one of the highly promising ovarian cancer biomarkers detectable in ctNAs. Herein, we introduce a new amplification‐free electrochemical assay for the detection of FGFR2:FAM76A fusion gene in ctNAs extracted from ovarian cancer patients. The assay relies on the electrocatalytic activity of a new class of superparamagnetic graphene‐loaded iron oxide nanoparticles (GO‐NPFe2O3). After isolation and purification, the target RNA was directly adsorbed onto the GO‐NPFe2O3 surface through graphene‐RNA affinity interaction. The electrocatalytic signal was achieved by the reduction of surface‐attached ruthenium hexaammine(III) chloride which was further enhanced by using the ferricyanide redox system. Our assay depicted an excellent detection sensitivity down to 1.0 fM, high specificity and excellent reproducibility (% RSD=<5 %, for n=3). The analytical performance of our method was validated with standard qRT‐PCR analysis. We believe that this newly developed assay would be practically applicable in clinical research.

(96) Muhammad Umer, Ramanathan Vaidyanathan, Nam-Trung Nguyen, Muhammad JA Shiddiky (2018)

Circulating Tumor Microemboli: Progress in Molecular Understanding and Enrichment Technologies,

Biotechnology advances 36, 1367-1389.

 

Publication year: 2018

Abstract

Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development.

(95) Md Nazmul Islam, Sofia Moriam, Muhammad Umer, Hoang-Phuong Phan, Carlos Salomon, Richard Kline, Nam-Trung Nguyen, Muhammad J. A. Shiddiky (2018)

Naked-eye and Electrochemical Detection of Isothermally Amplified HOTAIR Long non-coding RNA,

Analyst, Just acceptedDOI: 10.1039/C7AN02109G

Publication year: 2018

Abstract

An inexpensive, simple and rapid sensor platform capable of detecting cancer-related long non-coding RNA (lncRNA) with high accuracy is of great interest in the field of molecular diagnostics. Herein, we report on the development of a new colorimetric and electrochemical assay platform for long non-coding HOX transcript antisense intergenic RNA (HOTAIR) detection. Isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) was performed to amplify HOTAIR sequences from a RNA pool extracted from a designated number of ovarian cancer cells and a small cohort of plasma samples derived from patients with ovarian cancer. During RT-RPA, biotinylated dUTPs were randomly incorporated in the amplified product. Subsequently, HOTAIR amplicons were magnetically purified and isolated followed by a horseradish peroxidase (HRP)-catalyzed colorimetric reaction in the presence of 3,3′,5,5′- tetramethylbenzidine (TMB)/ H2O2 system. We finally introduced three potential readout methods for HOTAIR detection – (i) naked-eye visualisation of the color change for a quick screening of the target, (ii) quantitative absorbance measurement by UV-vis, and (iii) amperometric quantification using the electrochemical properties of TMB. The assay has shown excellent reproducibility (% RSD = < 5%, for n = 3) and sensitivity (10 cells/ mL) while detecting HOTAIR in cancer cell lines and patient samples. Expression of HOTAIR in clinical samples was also verified with a standard RT-qPCR method. We believe that our proof of concept assay may find potential relevance for routine clinical screening of cancer- associated lncRNAs. 

(94) Lena Gorgannezhad, Muhammad Umer, Md Nazmul Islam, Nam-Trung Nguyen, Muhammad J. A. Shiddiky (2018)

Circulating Tumor DNA and Liquid Biopsy: Opportunities, Challenges, and Recent advances in Detection Technologies,

Lab on a Chip, Just accepted 

Publication year: 2018

Abstract

Cell free DNA (cfDNA) refers to short fragments of acellular nucleic acids detectable in almost all body fluids, including blood, and are involved in various physiological and pathological phenomenon such as immunity, coagulation, aging, and cancer. In cancer patients, a fraction of hematogenous cfDNA originates from tumors, termed as circulating tumor DNA (ctDNA), and may carry the same mutations and genetic alterations as those of 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 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, or 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 integration of ctDNA analysis into routine clinical practice.

(93) Kseniia Boriachek, Muhammad Umer, Md Nazmul Islam, Vinod Gopalan, Alfred K. Lam, Nam-Trung Nguyen, Muhammad J. A. Shiddiky (2018)

An Amplification-free Electrochemical Detection of Exosomal miRNA-21 in Serum Samples,

Analyst, 143, 1662-1669,  DOI: 10.1039/C7AN01843F

Publication year: 2018

Abstract

Recent evidence suggests that small non-coding RNAs or microRNA (miRNA)s encapsulated in exosomes represent an important mechanism of communication between the cells. Exosomal miRNAs play an important role in oncogenesis via  stimulating  cell to cell communication and facilitating metastasis in cancers. Despite progressive advances, current methods for exosomal miRNA detection most rely on labor intensive sequencing approaches which are often prone to amplification bias and require costly and bulky equipment. Herein, we report an electrochemical approach for detection of cancer-derived exosomal miRNAs in human serum samples by selectively isolating the target miRNA using magnetic beads pre-functionalized with capture probes and then directly absorbing the targets onto the gold electrode surface. The level of adsorbed miRNA is detected electrochemically in the presence of the [Fe(CN)6]4-/3- redox system. This method enabled an excellent detection sensitivity of 1.0 pM with a relative standard deviation (%RSD) of <5.5% in cancer cells and serum samples (n = 8) collected from patients with colorectal adenocarcinoma. We believe that our approach could be useful for the quantification of exosomal miRNA in clinical samples.

(92) Harshad Kamble, Raja Vadivelu, Matthew J. Barton, Muhammad J. A. Shiddiky, Nam-Trung Nguyen (2018)

Pneumatically Actuated Cell-stretching Array Platform to Engineer Cell Pattern in vitro,

Lab on a Chip 18, 765-774. DOI: 10.1039/C7LC01316G

Publication year: 2018

Abstract

A cellular response to mechanical stimuli is well-known phenomena known as a mechanotransduction. It is widely accepted that mechanotransduction plays an important role in cell alignment which is critical for cell homeostasis. Although many approaches have been developed in recent years to study the effect of external mechanical stimuli on cell behaviour, most of them have not explored the ability of the mechanical stimuli to engineer cell alignment to obtain patterned cell culture. This paper introduces a simple, yet effective pneumatically actuated 4×2 cell stretching array for concurrently inducing a range of cyclic normal strain onto cell cultures to achieve predefined cell alignment. We utilised ring-shaped normal strain pattern to demonstrate the growth of in vitro patterned cell culture with predefined circumferential cellular alignment. Furthermore, to ensure the compatibility of the developed cell stretching platform with general tools and existing protocols, dimensions of the developed cell-stretching platform follow the standard F bottom 96 well plate. In this study we report principle design, simulation and characterisation of the cell-stretching platform with the preliminary observations using fibroblast cells. Our experimental results of cytoskeleton reorganisation such as perpendicular cellular alignment of the cells to the direction of normal strain are consistent with those reported in the literature. After two hours of stretching, the circumferential alignment of fibroblast cells confirms the capability of the developed system to achieve a patterned cell culture. The cell-stretching platform reported is potentially a useful tool for drug screening in 2D mechanobiology experiments, tissue engineering and regenerative medicine.

(91) Shunsuke Tanaka, Yusuf Valentino Kaneti, Ripon Bhattacharjee, Md Nazmul Islam, Rina Nakahata, Nawfel Abdullah, Shin-ichi Yusa, Nam-Trung Nguyen, Muhammad J. A. Shiddiky, Md Shahriar A. Hossain, Yusuke Yamauchi (2018)

Mesoporous Iron Oxide Synthesized Using Poly(styrene-b-acrylic acid-b-ethylene glycol) Block Copolymer Micelles as Templates for Colorimetric and Electrochemical Detection of Glucose,

ACS Applied Materials & Interfaces 10, 1039-1049DOI: 10.1021/acsami.7b13835

Publication year: 2018

Abstract

Herein, we report the soft-templated preparation of mesoporous iron oxide using an asymmetric poly(styrene-b-acrylic acid-b-ethylene glycol) (PS-b-PAA-b-PEG) triblock copolymer. This polymer forms a micelle consisting of a PS core, a PAA shell and a PEG corona in aqueous solutions, which can serve as a soft-template. The mesoporous iron oxide obtained at an optimized calcination temperature of 400 °C exhibited an average pore diameter of 39 nm, with large specific surface area and pore volume of 86.9 m2 g -1 and 0.218 cm3 g -1, respectively. The as-prepared mesoporous iron oxide materials showed intrinsic peroxidase-like activities towards the catalytic oxidation of 3,3ˊ,5,5ˊ-tertamethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). This mimetic feature was further exploited to develop a simple colorimetric (naked-eye) and electrochemical assay for the detection of glucose. Both our colorimetric (naked-eye and UV-vis) and electrochemical assays estimated the glucose concentration to be in the linear range of 1.0 µM to 100 µM with a detection limit of 1.0 µM. We envisage that our integrated detection platform for H2O2 and glucose will find a wide range of applications in developing various biosensors in the field of personalized medicine, food-safety detection, environmental-pollution control, and agro-biotechnology.