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The current and emerging liquid biopsy methods for analysing three major circulating biomarkers namely ctDNA, CTCs and exosomes as well as biological and technical challenges associated with these methods have been reviewed.

INTRODUCTION: Obstructive sleep apnea (OSA) is a common sleep disorder with multiple comorbidities including hypertension, diabetes, and cardiovascular disorders. Detected based on an overnight sleep study is called polysomnography (PSG); OSA still remains undiagnosed in majority of the population mainly attributed to lack of awareness. To overcome the limitations posed by PSG such as patient discomfort and overnight hospitalization, newer technologies are being explored. In addition, challenges associated with current management of OSA using continuous positive airway pressure (CPAP), etc. presents several pitfalls. AREAS COVERED: Conventional and modern detection/management techniques including PSG, CPAP, smart wearable/pillows, bio-motion sensors, etc., have both pros and cons. To fulfill the limitations in OSA diagnostics, there is an imperative need for new technology for screening of symptomatic and more importantly asymptomatic OSA patients to reduce the risk of several associated life-threatening comorbidities. In this line, molecular marker-based diagnostics have shown great promises. EXPERT OPINION: A detailed overview is presented on the OSA management and diagnostic approaches and recent advances in the molecular screening methods. The potentials of biomarker-based detection and its limitations are also portrayed and a comparison between the standard, current modern approaches, and promising futuristic technologies for OSA diagnostics and management is set forth.ABBREVIATIONS AHI: Apnea hypopnea index; AI: artificial intelligence; CAM: Cell adhesion molecules; CPAP: Continuous Positive Airway Pressure; COVID-19: Coronavirus Disease 2019; CVD: Cardiovascular disease; ELISA: Enzyme linked immunosorbent assay; HSAT: Home sleep apnea testing; IR-UWB: Impulse radio-ultra wideband; MMA: maxillomandibular advancement; PSG: Polysomnography; OSA: Obstructive sleep apnea; SOD: Superoxide dismutase; QD: Quantum dot.

Exosomes are vesicles released by healthy and cancer cells into the extracellular matrix and bodily fluid. Cancer cell-derived exosomes have attracted much attention in early-stage detection and prognostication of treatment response. Thus, detecting exosomes is of great interest to biology and medicine. However, many conventional detection methods require high-cost equipment and centralized laboratory facilities, making diagnostics inaccessible in limited-resource settings. This study reports a proof-of-concept low-cost electrochemical paper-based analytical device to quantify both the total bulk and cancer cell-derived exosomes in cell culture media. The device employs a sandwich immune assay design, where exosomes are initially captured using the electrode-bound generic antibodies (

Sugarcane leaf scald caused by the bacterium Xanthomonas albilineans is a major disease of sugarcane worldwide. Whereas erratic symptoms make phenotypic detection challenging, molecular methods require expensive instruments and labour, and longer sample-to-answer times. We report a novel method for detection of X. albilineans DNA in sugarcane xylem sap. The method involves (i) boiling lysis-based DNA extraction from sugarcane sap; (ii) magnetic purification of target sequences directly from the lysate through use of magnetic bead-bound capture probes; and (iii) DNA sandwich hybridisation platform for HRP/TMB/H2O2 reaction-based naked eye visualisation and electrochemical detection of the target. The method is sensitive (limit of detection 100 fM) and reproducible (relative standard deviation

Magnetic nanozymes with peroxidase-mimicking activity have been widely investigated for developing molecular biosensors. Herein, we report a starch-assisted method for the synthesis of a novel class of carboxyl group-functionalized iron oxide nanoparticles (C-IONPs). Scanning electron and transmission electron microscopy analysis revealed that the nanoparticles possess a spherical shape with an average size of ∼250 nm. Peroxidase-mimicking activity of C-IONPs was investigated through catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The results showed that nanoparticles follow typical Michaelis−Menten kinetics and exhibit excellent affinity toward TMB and H2O2 with estimated KM and VMax values of 0.0992 mM and 0.156 × 10−8 Ms−1 for TMB and 114 mM and 0.197 × 10−8 Ms−1 for H2O2, respectively. C-IONPs were used to develop a simple method for the direct isolation and quantification of disease-specific exosomes. This method utilized a two-step strategy that involved (a) initial isolation of bulk exosomes present in the sample media using tetraspanin biomarker (i.e., CD9)-functionalized C-IONPs and (b) subsequent electrochemical quantification of disease-specific exosomes within the captured bulk exosomes using tumor-specific markers (in this case, the ovarian cancer biomarker CA-125). In the first step, C-IONPs were used as “dispersible nanocarriers” to capture the bulk population of exosomes, and in the second step, they were used as nanozymes to generate an enzyme-catalyzed current indicative of the presence of tumor-specific exosomes. Chronoamperometric analysis showed that the method exhibits an excellent specificity for OVCAR3 cell-derived exosomes (linear dynamic range, 6.25 × 105 to 1.0 × 107 exosomes/mL; detection limit, 1.25 × 106 exosomes/mL) with a relative standard deviation of

In this article, we demonstrate that specifically engineered oxide nanoparticles (NPs) have the potential to act as theranostic materials that are able to generate or prevent oxidative stress through their oxi-redox activity in various types of malignant and nonmalignant cells. The oxi-redox activity is related to the type and presence of surface defects, which is modified with appropriate synthesis conditions. In the present work, we used MDA-MB-231 and MCF-7 human breast cancer cells and nonmalignant MCF-10A human breast cells to demonstrate how controlled oxidative stress mediated by specifically nanoengineered indium tin oxide (ITO) NPs can selectively induce cell death in the cancer cells while reducing the oxidative stress in the normal cells and supporting their proliferation. The ITO NPs are also promising nanotheranostic materials for cancer therapy and contrast agents because of their multimodal imaging capabilities. We demonstrate that the synthesized ITO NPs can selectively increase the generation of reactive oxygen species (ROS) in both breast tumor cell lines, resulting in activation of apoptosis, and can also greatly suppress the cellular proliferation in both types of tumor cells. In contrast, the ITO NPs exhibit ROS scavenging-like behavior, significantly decreasing the ROS levels in MCF-10A cells exposed to the additional ROS, hydrogen peroxide (H2O2), so that they protect the proliferation of nonmalignant MCF-10A cells from ROS damage. In addition, fluorescent microscopy images revealed that the ITO NPs emit strong fluorescence that could be used to reveal their location. Moreover, computed tomography imaging demonstrated that the ITO NPs exhibited a comparable capability toward anatomical contrast enhancement. These results suggest that the synthesized ITO NPs have the potential to be a novel selective therapeutic agent with a multimodal imaging property for anticancer treatment.

Dysregulated microRNA associated pathways contribute to the pathology of neurological disorders, hence presenting themselves as a potential candidate for motor neuron disease (MND) diagnosis. Herein, we reported an enzymatic amplification‐free approach for the electrochemical detection of exosomal microRNA (miR‐338‐3p) from preconditioned media of motor neurons obtained from amyotrophic lateral sclerosis (ALS) patients and healthy controls. Our assay utilizes a three‐step strategy that involves i) initial isolation and purification of exosomal miR‐338‐3p from patients and healthy controls using biotinylated complementary capture probe followed by heat‐release of the specific target, ii) direct adsorption of target miR‐338‐3p onto the gold‐loaded ferric oxide nanocatalyst (AuNP‐Fe2O3NC) through affinity interaction between microRNA and exposed gold surfaces within the AuNP‐Fe2O3NC, and iii) gold nanocatalyst‐induced electrocatalytic signal amplification through methylene blue‐ferricyanide redox cycling (MB/[Fe(CN)6]3−). The electrocatalytic signal is monitored by using chronocoulometry at the AuNP–Fe2O3NC‐modified screen‐printed carbon electrode (AuNP‐Fe2O3NC/SPCE). We demonstrated the detection of miR‐338‐3p as low as 100 aM in spiked buffer samples with a relative standard deviation of (%RSD)

We report a sensitive, yet low-cost biosensor based on laser induced graphene for femtomolar microRNA (miRNA) detection. Combined with the miRNA extraction and magnetic isolation process, the target miRNAs were purified for further detection using laser induced graphene sensor. The laser induced graphene was prepared by direct laser writing on commercial polyimide (PI) and patterned via a computer-aided design system as an electrode for electrochemical biosensing. We found that the laser reduction of PI resulted in nitrogen-doped porous graphene, not only improving its conductivity but also its sensitivity to nucleic acids. Preeclampsia specific miRNA hsa-miR-486-5p was magnetically purified and directly adsorbed on the surface of graphene electrode via graphene-miRNA affinity interaction. Surface attached miRNAs were then electrochemically quantified using [Fe(CN)6]3-/4- redox system. Our assay demonstrates detection of miRNA has-miR-486-5p up to concentrations as low as 10 fM with excellent reproducibility. Owing to its facile fabrication, low cost and high performance, the laser induced N-doped graphene biosensor presented here shows great potential for applications in detecting miRNA in biomedical applications.

This article reports on the peroxidase-like catalytic activity of polyoxometalates (POMs) and their potential use as natural peroxidases for developing a simple and efficient colorimetric glucose sensor. Two Keggin-type vanadium-substituted tungstosulfates, [SVW11O40]3− (SVW11) and [SV2W10O40]4− (SV2W10), were tested for their potential as natural enzyme mimetics and exhibited strong peroxidase-like catalytic activity. The catalysis reaction was found to be in accordance with Michaelis-Menten and Lineweaver-Burk kinetics models. Michaelis-Menten constant (Km) and maximum velocity (Vmax) parameters were calculated to be 0.0759 mM and 0.329×10−8 Ms−1 for SVW11, and 0.0543 mM and 2.67×10−8 Ms−1 for SV2W10, respectively, indicating a high catalytic activity and a strong affinity of POMs towards 3,3,5,5-tetramethylbenzidine (TMB). In the case of H2O2, these values were found to be 57.1 mM and 0.325×10−8 mMs−1 for SVW11, and 47.7 mM and 2.72×10−8 mMs−1 for SV2W10. The peroxidase-like catalytic activity of these POMs was used to develop colorimetric glucose sensors as a proof-of-concept model for the POM-based naked-eye detection of biomolecules. The limit of detcetion (LOD) of glucose for SVW11 and SV2W10 was 1.14 μM and 1.24 μM, respectively. Our findings propose broad-ranging potential applications of these novel POMs in biosensing and bioanalytical chemistry.

Small extracellular vesicles (sEVs) released from the extravillous trophoblast (EVT) are known to regulate uterine spiral artery remodeling during early pregnancy. The bioactivity and release of these sEVs differ under differing oxygen tensions and in aberrant pregnancy conditions. Whether the placental cell-derived sEVs released from the hypoxic placenta contribute to the pathophysiology of preeclampsia is not known. We hypothesize that, in response to low oxygen tension, the EVT packages a specific set of proteins in sEVs and that these released sEVs interact with endothelial cells to induce inflammation and increase maternal systemic blood pressure. Using a quantitative MS/MS approach, we identified 507 differentially abundant proteins within sEVs isolated from HTR-8/SVneo cells (a commonly used EVT model) cultured at 1% (hypoxia) compared with 8% (normoxia) oxygen. Among these differentially abundant proteins, 206 were up-regulated and 301 were down-regulated (P < 0.05), and they were mainly implicated in inflammation-related pathways. In vitro incubation of hypoxic sEVs with endothelial cells, significantly increased (P < 0.05) the release of GM-CSF, IL-6, IL-8, and VEGF, when compared with control (i.e. cells without sEVs) and normoxic sEVs. In vivo injection of hypoxic sEVs into pregnant rats significantly increased (P < 0.05) mean arterial pressure with increases in systolic and diastolic blood pressures. We propose that oxygen tension regulates the release and bioactivity of sEVs from EVT and that these sEVs regulate inflammation and maternal systemic blood pressure. This novel oxygen-responsive, sEVs signaling pathway, therefore, may contribute to the physiopathology of preeclampsia.

In recent years, conducting polymers are playing a significant role in the field of display devices, transistors, solar cells, sensors, and electrochromic windows due to their outstanding optoelectronic and semiconducting properties due to their conjugated backbone. One potential application that is not as widely explored using these materials is biosensing, where advantage is taken of the porosity that can be generated by the polymerization of a three-dimensional network. There are various approaches for producing conjugated microporous polymers using trifunctional or multifunctional monomers synthesized via chemical or electrochemical methods. In this work, we have used electropolymerization to synthesize conjugated polymer films on a working electrode of flexible indium tin oxide (FITO) using a trifunctional conjugated monomer 1,3,5-tri(furan-2-yl)benzene (TFB). There are several parameters that influence the formation of a porous polymer film, and the most critical ones are substrate conductivity, roughness, method of electropolymerization, and choice of an electrolyte. These porous electropolymerized films were characterized using UV–vis spectroscopy (UV–vis), X-ray photoelectron spectroscopy (XPS), surface profilometry, four-point probe conductivity measurements, and scanning electron microscopy (SEM). The polymer films that were electropolymerized using chronoamperometry rather than repetitive potential cycling demonstrated a more suitable morphology to trap DNA/RNA analytes for biosensing applications.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

This paper reports the development of fluorescent-linked immunosorbent paper-based assay for exosome detection. The paper-based device was fabricated with sandwich lamination for easy handling and was coated with exosome-specific antibody as a biosensing platform to detect exosome sample from the cell culture media. This assay employed fluorescent detection which is followed by tagging fluorophore-conjugated detecting antibody on exosome samples. The fluorescent readout was evaluated and quantified from image processing software. This assay can detect high concentration of exosome samples (~ 1010 exosome/mL). However, this assay has encountered various challenges. First, the exosome concentration prepared from cell culture media from cancer-derived ovarian and mesothelial cell lines may be insufficient to reach detectable range. Second, chemical contamination from exosome isolation kits may affect assay sensitivity. Therefore, assay optimization and minimizing chemical contamination are required which could enhance assay specificity and sensitivity.

This paper reports the development of colorimetric immunological paper-based assay for exosome detection. The paper-based device was fabricated with lamination technique for easy handling and create hydrophilic/hydrophobic region for analytical paper-based devices. Exosome-specific antibody was coated onto the paper-based devices as a biosensing platform to detect exosome sample from the cell culture media. This assay employed a colorimetric reaction which is followed by reaction between horseradish peroxidase (HRP) and 3,3’,5,5’-tetramethylbenzidine substrate (TMB). The colorimetric readout was qualitatively evaluated by naked eyes and was quantitatively assessed by image processing software. The result indicated that this assay faces many challenges. First, the exosome concentration may be inadequate to reach detectable range. Second, high background signal due to non-specific binding on the platform results in lack of sensitivity for exosome detection. Therefore, modification on the paper should promote protein binding for specific target and prevent non-specific binding to reduce the high background signal.

We present a novel amplification-free sandwich type platform assay for electrochemical detection of miRNA. The assay is based on T4 DNA polymerase mediated synthesis of the p53 binding DNA sequence at the 3' end of target miRNA. The resulting miRNA-DNA chimera is detected via an electrochemical sandwich hybridization assay where HRP-labelled p53 binds to its recognition sequence and an amperometric signal is generated by hydroquinone-mediated enzymatic reduction of H2O2. The limit of detection of our assay was estimated to be 22 fM with a linear dynamic range of 100 fM-1 nM. This new platform method of detecting miRNA shows superior performance to conventional electrochemical miRNA biosensors and has the potential for amplification-free analysis of miRNA with high specificity and sensitivity.

This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 minutes using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard qPCR machine (~10-20 minutes vs >38 minutes) for 1×105 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need SARS-CoV-2 detection and patient management.

The upregulated expression of thyrosine kinase AXL has been reported in several hematologic and solid human tumors including gastric, breast, colorectal, prostate, and ovarian cancers. Thus, AXL can potentially serve as a diagnostic and prognostic biomarker for various cancers. This paper reports the first-ever use of loop-mediated isothermal amplification (LAMP) of the AXL gene as a diagnostic method for ovarian cancer. We demonstrated simple instrumentation toward a point-of-care device to perform LAMP. This paper also reports the first-ever use of core-shell beads as a microreactor to perform LAMP as an attempt to promote environmentally friendly laboratory practices.

We report on an immunofluorescent paper-based assay for the detection of severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper-based device was fabricated by using lamination technique for easy and optimized handling. Our approach utilises a two-step strategy that involves (i) initial coating of the paper-electrode with recombinant SARS-CoV-2 nucleocapsid antigen to capture the target SARS-CoV-2 specific antibodies, and (ii) subsequent detection of SARS-CoV-2 antibodies using fluorophore-conjugated IgG antibody. The fluorescence readout was observed with fluorescence microscopy. The images were processed and quantified using a MATLAB program. The assay can selectively detect SARS-CoV-2 humanized antibodies spiked in PBS and healthy human serum samples with the relative standard deviation of approximately 6.4% (for n = 3). It has broad dynamic ranges (1 ng to 50 ng/µL in PBS and 5 to 100 ng/µL in human serum samples) for SARS-CoV-2 humanized antibodies with the detection limits of 2 ng/µL (0.025 IU/mL) and 10 ng/µL (0.125 IU/mL) in PBS and human serum samples, respectively. We believe that our assay has the potential to be used as a simple, rapid, and inexpensive paper-based diagnostic device with a portable fluorescent reader to provide point-of-care diagnosis. This assay can be used for rapid examination of a large batch of samples toward clinical screening of SARS-CoV-2 specific antibodies as a confirmed infected active case or to evaluate the immune response to a SARS-CoV-2 vaccine.

Simple Summary Genomic profiling of cancer-derived materials in circulation has become an alternative approach for tumour genotyping. The detection of tumour origin markers such as DNA methylation in bodily fluids enables cancer screening, early-stage diagnosis and evaluation of therapy response. The development of broad platform technologies that underpin many in vitro clinical diagnostic tests has brought about a paradigm shift in cancer management and diagnosis. This study developed a multifaceted technology platform based on bioengineered polymer nanobeads for efficient capture and electrochemical detection of DNA methylation in ovarian cancer patient samples. This could be a versatile diagnostic platform for detecting numerous disease biomarkers, thus allowing several disease diagnoses. Abstract DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

Early sensitive diagnosis of cancer is critical for enhancing treatment success. We previously bioengineered multifunctional core-shell structures composed of a poly-3-hydroxybutyrate (PHB) core densely coated with protein functions for uses in bioseparation and immunodiagnostic applications. Here, we report bioengineering of Escherichia coli to self-assemble PHB inclusions that codisplay a ferritin-derived iron-binding peptide and the protein A-derived antibody-binding Z domain. The iron-binding peptide mediated surface coating with a ferrofluid imparting superparamagnetic properties, while the Z domain remained accessible for binding of cancer biomarker-specific antibodies. We demonstrated that these nanobeads can specifically bind biomarkers in complex mixtures, enabling efficient magnetic separation toward enhanced electrochemical detection of cancer biomarkers such as methylated DNA and exosomes from cancer cells. Our study revealed that superparamagnetic core-shell structures can be derived from biological self-assembly systems for uses in sensitive and specific electrochemical detection of cancer biomarkers, laying the foundation for engineering advanced nanomaterials for diverse diagnostic approaches.

Metal nanoprobes have recently attracted board research interestinr their application in establishing sensing systems due to their unique optical, electrical, physical, and chemical properties. In comparison to gold and silver nanoprobes, analytical platform based on copper nanoprobes (Cu-NPs) is still in the early stages of development. In this review, we focus on single-stranded, and double-stranded DNA capped Cu-NPs sensing systems which have been designed for various analytes, including metal ions, anions, small molecules, biomolecules (DNA, RNA, and protein, etc.). In addition, the application of Cu-NPs in biological labeling or bio-imaging platforms has also been introduced and summarized.

Most of the current exosome-analysis strategies are time-consuming and largely dependent on commercial extraction kit-based preisolation step, which requires extensive sample manipulations, costly isolation kits, reagents, tedious procedures, and sophisticated equipment and is prone to bias/artifacts. Herein we introduce a simple method for direct isolation and subsequent detection of a specific population of exosomes using an engineered superparamagnetic material with multifunctional properties, namely, gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC). In this method, the Au-NPFe2O3NC were initially functionalized with a generic tetraspanin (exosomes-associated) antibody (i.e., CD63) and dispersed in sample fluids where they work as “dispersible nanocarriers” to capture the bulk population of exosomes. After magnetic collection and purification, Au-NPFe2O3NC-bound exosomes were transferred to the tissue-specific, antibody-modified, screen-printed electrode. As a proof of principle, we used a specific pl...

Porous nanomaterials with superior peroxidase mimetic activity (nanozyme) at room temperature have gained increasing attention as potential alternatives to natural peroxidase enzymes. Herein, we report the application of porous iron oxide nanoflakes (IONFs), synthesized using the combination of solvothermal method and high-temperature calcination as peroxidase nanozyme for the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of HO. The four IONF catalysts possess porous structures with a wide pore size distribution between 2–30 nm and high specific surface areas around to 200 m g. The increase of calcination temperature of the IONFs from 250 °C to 400 °C resulted in a gradual decrease in their specific surface area and Michaelis-Menten constant (K) for TMB oxidation. The optimum IONF sample showed a much lower K at 0.24 mM (towards TMB) compared to natural enzyme horseradish peroxidase (HRP) at 0.434 mM, revealing the promising potential of the as-prepared IONFs as alternatives to HRP for biosensing applications.

This work reports the fabrication of a three-dimensional (3D) zinc oxide/zinc cobaltite (ZnO/ZnCo2O4) hybrid with a hierarchical macroporous honeycomb-like structure using highly uniform cobalt glycerate spheres as a self-sacrificial template. In the proposed method, the conversion of the template cobalt glycerate nanospheres into a 3D hierarchical macroporous honeycomb-like ZnO/ZnCo2O4 hybrid is achieved via a facile room-temperature reaction with aqueous zinc nitrate solution, followed by calcination in air at 350 °C. The proposed method offers several benefits including (i) the attainment of the ZnO/ZnCo2O4 hybrid in one step without additional or separate coating steps, (ii) the achievement of a unique 3D hierarchical macroporous honeycomb-like structure with interconnecting nanosheets and macropores which are assembled from smaller mesopores, leading to higher surface area and good interparticle separation, (iii) the relatively low calcination temperature required to obtain the ZnO/ZnCo2O4 hybrid (350 °C) and (iv) potential generalization for the creation of other macroporous honeycomb-like cobalt-based oxide nanostructures (including Al–Co and Cu–Co systems). When evaluated as a sensing material for carbon monoxide (CO), the hierarchical honeycomb-like ZnO/ZnCo2O4 hybrid sensor displays a higher sensing response with enhanced selectivity and stability towards CO gas at 300 °C compared to both ZnO hierarchical spheres and ZnCo2O4 nanospheres. The enhanced sensing performance of the hierarchical honeycomb-like ZnO/ZnCo2O4 hybrid is derived from the synergistic cooperation of the formed p–n heterojunction, large surface area and hierarchical macroporous nature of the as-synthesized ZnO/ZnCo2O4 hybrid. It is expected that the proposed general method may open a new path for creating other hierarchical macroporous honeycomb-like oxide nanostructures with enhanced surface areas and improved functional performance.

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

Diketopyrrolopyrrole (DPP) is one of the most fascinating organic dyes, and has gained significant attention in the organic electronic community in recent times. Due to their outstanding semiconducting properties, DPP-based small molecules and polymers can be used as active semiconductor thin films for various high performance electronic devices. However, although several flanking groups attached to the DPP backbone have been reported in the literature for DPP molecules, there is little study about the effect of fused rings flanked on both sides of the DPP core. Recently our group reported for the first time a naphthalene-flanked DPP as a new DPP family member. In this work, we have performed electropolymerization studies on a newly synthesized naphthalene flanked DPP core with two alkyl chains, hexyl (H-DPPN) and octyl (O-DPPN), as conjugated monomers. Electrochemical studies have been carried out on glassy carbon (GC) and indium tin oxide (ITO) electrodes by using repetitive cyclic voltammetry (CV) as the deposition technique for up to 50 cycles. The concentration of H-DPPN and O-DPPN solution in dichloromethane was found to be a key parameter when optimizing conditions for obtaining an electropolymerised thin film, although other parameters such as the electrolyte, potential range, number of cycles, and the nature of the electrode all play a role in successful thin film formation. The electropolymerized thin films were characterised using UV-Vis spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photon spectroscopy (XPS) and surface profilometry. We found that each monomer produced different film morphologies on glassy carbon and ITO electrodes. We believe that electropolymerized poly(H-DPPN) and poly(O-DPPN) thin film electrodes could be used for various applications such as studying redox reactions and bio-sensing.

International audience; This work presents crystalline SiC-on-glass as a transparent, robust, and optically stable electrode for simultaneous electrochemical characterization and optical microscope imaging. Experimental results show a large potential window, as well as excellent stability and repeatability over multiple cyclic voltammetric scans in common redox biomarkers such as ruthenium hexaammine and methylene blue. The high optical transmittance and biocompatibility of SiC-on-glass were also observed, enabling cell culture, electrical stimulation, and high resolution fluorescence imaging. This new platform opens exciting opportunities in multi-functional biosensing-probes and observation.

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.

Exosomes are nano-sized extracellular vesicles that serve as a communications system between cells and have shown tremendous promise as liquid biopsy biomarkers in diagnostic, prognostic, and even therapeutic use in different human diseases. Due to the natural heterogeneity of exosomes, there is a need to separate exosomes into distinct biophysical and/or biochemical subpopulations to enable full interrogation of exosome biology and function prior to the possibility of clinical translation. Currently, there exists a multitude of different exosome isolation and characterization approaches which can, in limited capacity, separate exosomes based on biophysical and/or biochemical characteristics. While notable reviews in recent years have reviewed these approaches for bulk exosome sorting, we herein present a comprehensive overview of various conventional technologies and modern microfluidic and nanotechnological advancements towards isolation and characterization of exosome subpopulations. The benefits and limitations of these different technologies to improve their use for distinct exosome subpopulations in clinical practices are also discussed. Furthermore, an overview of the most commonly encountered technical and biological challenges for effective separation of exosome subpopulations is presented.

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) =

Long non-coding RNA HOX transcript antisense intergenic RNA (HOTAIR) is one of the promising biomarkers that has widely been used in determining the stages of many cancers, including ovarian cancer. In cancer diagnostics, the two key analytical challenges for detecting long non-coding RNA biomarkers are i) the low concentration levels (nM to fM range) in which they are found and ii) the analytical method where broad dynamic range is required (four to six orders of magnitude) due to the large variation in expression levels for different HOTAIR RNAs. To meet these challenges, we report on a biosensing platform for the visual (colorimetric) estimation and subsequent electrochemical quantification of ovarian-cancer-specific HOTAIR using a screen-printed gold electrode (SPE-Au). Our assay utilizes a two-step strategy that involves (i) magnetic isolation and purification of target HOTAIR sequences and (ii) subsequent detection of isolated sequences using a sandwich hybridization coupled with horseradish peroxidase (HRP)-catalyzed reaction of 3,3&prime, 5,5&prime, tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. The assay achieved a detection limit of 1.0 fM HOTAIR in spiked buffer samples with excellent reproducibility (% RSD &le, 5%, for n = 3). It was successfully applied to detect HOTAIR in cancer cell lines and a panel of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was validated with standard RT-qPCR. We believe that the proof of concept assay reported here may find potential use in routine clinical settings for the screening of cancer-related lncRNAs.

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.

The discovery of large transcripts of long RNAs that have limited protein coding capacity, known as long non-coding RNAs (lncRNAs) present new concepts on RNA-mediated gene regulation. Increasing evidence suggests that large intervening ncRNAs regulate key pathways in cancer genesis and metastasis. Among the most characterized lncRNAs, homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) acts as an oncogenic molecule in different cancer cells, and thus its expression level serves as a potential biomarker for diagnostic and therapeutic purposes in several human cancers, such as breast, prostate, liver and ovarian cancer. This paper reports a simple and sensitive sensor platform for the detection of HOTAIR. Extracted HOTAIR sequences from ovarian cancer cells and plasma samples derived from ovarian cancer patients were magnetically isolated and purified, followed by a sandwich hybridization event at a screen-printed gold electrode. This event was monitored by amperometry using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) system. The catalytic enhancement of the amperometric signal enabled our assay to achieve a detection limit of 1.0 fM with a good inter-assay reproducibility (relative standard deviation (%RSD) = < 5.0%, n = 3). The method was used for the analysis of specific HOTAIR in cell line and a small cohort of plasma samples derived from patients with ovarian cancer. The analytical performance of the method was also demonstrated using a standard RT-qPCR. We believe that the proof of the concept assay demonstrated here could be a cost-effective alternative platform for screening cancer-related lncRNAs in routine clinical settings.

Ovarian cancer is the most aggressive of all gynaecological malignancies and is the leading cause of cancer-associated mortality worldwide. Over the recent years, there has been a sharp increase in this mortality rate, mostly due to late diagnosis, which can be attributed to the lack of an early and specific biomarker. Under this scenario, recent interest has shifted towards ovarian cancer associated miRNAs which play strong regulatory roles in various cellular processes. miRNAs have emerged as promising non/minimally invasive cancer biomarkers for improved diagnostic, prognostic and streamlined therapeutic applications. A large number of miRNA assays have been reported that are based on nucleic acid detection-based techniques such as RT-qPCR, microarrays and RNA sequencing methods. Despite demonstrating commendable analytical performances, these laboratory-based techniques are expensive and hence not ideally suited for routine use in resource-limited settings. In recent years, considerable attention has been dedicated to the development of relatively simple, rapid and inexpensive miRNA biosensor strategies. Among these, electrochemical sensors have shown a great promise towards point-of-care diagnostics, due to their inherent advantages such as simplicity, sensitivity, amenability to high levels of multiplexing as well as low cost. In this paper, we provide an overview of the potential role of miRNAs in ovarian cancer, as well as recent advances in the development of nanotechnology-based, optical, and electrochemical biosensing-strategies for miRNA detection.