Construction of AC/DC magnetic syringe device for stimulated drug release, injection and ejection of nanocarriers and testing cytotoxicity in vitro.

Iron nanoparticles are used as a targeted drug delivery system. The nanocarrier itself can be genotoxic, trigger oxidative stress or cell death. Therefore, we developed an AC/DC magnetic syringe for injecting, stimulating drug release and safe removing of the nanocarrier. Alongside we optimized the method for nanoparticles' drug release kinetics and testing cytotoxicity in vitro.•This paper presents detailed instructions for construction of AC/DC magnetic syringe device for stimulated drug release, injection and ejection of magnetic nanoparticles; nanoparticles preparation; adsorbing methylene blue on nanoparticles; determination of drug release kinetics from nanocarriers on the example of methylene blue•Gomori´s Prussian blue reaction for differentiated SH-SY5Y human neuroblastoma cell line; MTT viability assay optimized for differentiated SH-SY5Y human neuroblastoma cell line and antioxidant enzymes activities assay and lipid peroxidation methods are optimized for cell analyses cell cultivation for nanoparticles cytotoxicity testing in vitro.•Those protocols are the first step toward further testing the effect of nanoparticles in vivo, on brain tissue.

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.

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.

Gold-loaded nanoporous iron oxide nanocubes: a novel dispersible capture agent for tumor-associated autoantibody analysis in serum.

Autoantibodies are produced against tumor associated antigens (TAAs) long before the appearance of any symptoms and thus can serve as promising, non-invasive biomarkers for early diagnosis of cancer. Current conventional methods for autoantibody detection are highly invasive and mostly provide diagnosis in the later stages of cancer. Herein we report a new electrochemical method for early detection of p53 autoantibodies against colon cancer using a strategy that combines the strength of gold-loaded nanoporous iron oxide nanocube (Au@NPFe2O3NC)-based capture and purification while incorporating the inherent simplicity, inexpensive, and portable nature of the electrochemical and naked-eye colorimetric readouts. After the functionalisation of Au@NPFe2O3NC with p53 antigens, our method utilises a two-step strategy that involves (i) magnetic capture and isolation of autoantibodies using p53/Au@NPFe2O3NC as 'dispersible nanocapture agents' in serum samples and (ii) subsequent detection of autoantibodies through a peroxidase-catalyzed reaction on a commercially available disposable screen-printed electrode or naked-eye detection in an Eppendorf tube. This method has demonstrated a good sensitivity (LOD = 0.02 U mL-1) and reproducibility (relative standard deviation, %RSD = <5%, for n = 3) for detecting p53 autoantibodies in serum and has also been successfully applied to analyse a small cohort of clinical samples obtained from colorectal cancer. We believe that the highly inexpensive, rapid, sensitive, and specific nature of our assay could potentially aid in the development of an early diagnostic tool for cancer and related diseases.

Quantification of gene-specific DNA methylation in oesophageal cancer via electrochemistry.

Development of simple and inexpensive method for the analysis of gene-specific DNA methylation is important for the diagnosis and prognosis of patients with cancer. Herein, we report a relatively simple and inexpensive electrochemical method for the sensitive and selective detection of gene-specific DNA methylation in oesophageal cancer. The underlying principle of the method relies on the affinity interaction between DNA bases and unmodified gold electrode. Since the affinity trend of DNA bases towards the gold surface follows as adenine (A) > cytosine (C) > guanine (G)> thymine (T), a relatively larger amount of bisulfite-treated adenine-enriched unmethylated DNA adsorbs on the screen-printed gold electrodes (SPE-Au) in comparison to the guanine-enriched methylated sample. The methylation levels were (i.e., different level of surface attached DNA molecules due to the base dependent differential adsorption pattern) quantified by measuring saturated amount of charge-compensating [Ru(NH3)6]3+ molecules in the surface-attached DNAs by chronocoulometry as redox charge of the [Ru(NH3)6]3+ molecules quantitatively reflects the amount of the adsorbed DNA confined at the electrode surface. The assay could successfully distinguish methylated and unmethylated DNA sequences at single CpG resolution and as low as 10% differences in DNA methylation. In addition, the assay showed fairly good reproducibility (% RSD= <5%) with better sensitivity and specificity by analysing various levels of methylation in two cell lines and eight fresh tissues samples from patients with oesophageal squamous cell carcinoma. Finally, the method was validated with methylation specific-high resolution melting curve analysis and Sanger sequencing methods.

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.

Magnetic nanoparticles for "smart liposomes".

Liposomal drug delivery systems (LDDSs) are promising tools used for the treatment of diseases where highly toxic pharmacological agents are administered. Currently, destabilising LDDSs by a specific stimulus at a target site remains a major challenge. The bacterial mechanosensitive channel of large conductance (MscL) presents an excellent candidate biomolecule that could be employed as a remotely controlled pore-forming nanovalve for triggered drug release from LDDSs. In this study, we developed superparamagnetic nanoparticles for activation of the MscL nanovalves by magnetic field. Synthesised CoFe2O4 nanoparticles with the radius less than 10 nm were labelled by SH groups for attachment to MscL. Activation of MscL by magnetic field with the nanoparticles attached was examined by the patch clamp technique showing that the number of activated channels under ramp pressure increased upon application of the magnetic field. In addition, we have not observed any cytotoxicity of the nanoparticles in human cultured cells. Our study suggests the possibility of using magnetic nanoparticles as a specific trigger for activation of MscL nanovalves for drug release in LDDSs.