A mini review on recent progress of microfluidic systems for antibody development.

Objectives: Antibody is specific reagent that be utilized in various field of biomedical research. Monoclonal antibodies are mostly produced using two common techniques namely hybridoma and antibody engineering, which suffer from some limitations such as boring screening procedures, long production time, low efficacy and a degree of automation. To address these limitations, various microfluidics techniques have been developed for the antibody isolation and screening. Methods: This study specifically investigates nearly recent reports published in peer-reviewed journals indexed in various databases including Web of Science, Scopus, PubMed, Google Scholar, and Science Direct. Results: In this study, we identified a total of seventy papers from a pool of 130 articles. These papers focus on the application of three major groups of microfluidic platforms, namely valves, microwells, and droplets, in the development of antibodies using hybridoma method and phage display technology. We provide a summary of these applications and also discuss the key findings in this field. Additionally, we illustrate our discussion with several examples to enhance understanding. Conclusions: Microfluidics has the potential to serve as a valuable tool in streamlining complex laboratory procedures involved in antibody discovery. However, it is important to note that microfluidics is limited to laboratory settings. Further enhancements are needed to address existing challenges and to make microfluidics a reliable, accurate, and cost-effective tool for antibody discovery.

Engineered Robust Hydrophobic/Hydrophilic Nanofibrous Scaffolds with Drug-Eluting, Antioxidant, and Antimicrobial Capacity.

Multifunctional nanofibrous architectures have attracted extensive attention for biomedical applications due to their adjustable and versatile properties. Electrospun fabrics stand out as key building blocks for these structures, yet improving their mechanobiological and physicochemical performance is a challenge. Here, we introduce biodegradable engineered hydrophobic/hydrophilic scaffolds consisting of electrospun polylactide nanofibers coated with drug-eluting synthetic (poly(vinyl alcohol)) and natural (starch) polymers. The microstructure of these composite scaffolds was tailored for an increased hydrophilicity, optimized permeability, water retention capacity of up to 5.1 g/g, and enhanced mechanical properties under both dry and wet conditions. Regarding the latter, normalized tensile strengths of up to 32.4 MPa were achieved thanks to the improved fiber interactions and fiber-coating stress transfer. Curcumin was employed as a model drug, and its sustained release in a pure aqueous medium was investigated for 35 days. An in-depth study of the release kinetics revealed the outstanding water solubility and bioavailability of curcumin, owing to its complexation with the hydrophilic polymers and further delineated the role of the hydrophobic nanofibrous network in regulating its release rate. The modified curcumin endowed the composites with antioxidant activities up to 5.7 times higher than that of free curcumin as well as promising anti-inflammatory and bacteriostatic activities. The cytocompatibility and cell proliferation capability on human dermal fibroblasts also evidenced the safe use of the constructs. Finally, the fabrics present pH-responsive color-changing behavior easily distinguishable within the pH range of 5-9. Thus, these designs offer a facile and cost-effective roadmap for the fabrication of smart multifunctional biomaterials, especially for chronic wound healing.

Mercury (II) sensing using a simple turn-on fluorescent graphene oxide based aptasensor in serum and water samples.

A simple, highly sensitive, and selective fluorometric aptasensing platform based on aptamer and graphene oxide (GO) is proposed for the determination of mercury (II) ion (Hg2+). In the designed assay, two aptamer probes, a carboxy-fluorescein (FAM) labeled aptamer (aptamer A) and its complementary (aptamer B) with partial complement containing several mismatches and GO as the quencher were used. In the absence of Hg2+, both A and B aptamers were adsorbed on the surface of GO by π-π-stacking, leading to fluorescence quenching of FAM due to fluorescence resonance energy transfer (FRET). Upon exposure to Hg2+, the A and B aptamer strands bind Hg2+ and form T-Hg2+-T complexes, leading to the formation of a stable double-stranded aptamer. The double-stranded aptamer is detached from the GO surface, resulting in the recovery of FAM fluorescence. The fluorescence intensity (FI) of the developed sensor was correlated with the Hg2+ concentration under optimized experimental conditions in two wide linear ranges, even in the presence of 10 divalent cations as interferences. The linear ranges were obtained from 200.0 to 900.0 fM and 5.0 to 33.0 pM, a limit of detection (LOD) of 106.0 fM, and a limit of quantification (LOQ) of 321.3 fM. The concentration of Hg2+ was determined in five real samples containing three water and two serum samples, using spiking and standard addition methods and the results were compared with the spiked amounts and atomic absorption (AAS) as standard method respectively, with acceptable recoveries. Furthermore, in the standard addition method, to overcome the effects of matrix influence of real samples in quantitative predictions, the excitation-emission matrix (EEM) data for samples was simultaneously analyzed by multivariate curve resolution with alternating least squares (MCR-ALS) as a second-order standard addition method (SOSAM).

Smart Wearable Nanopaper Patch for Continuous Multiplexed Optical Monitoring of Sweat Parameters.

Notwithstanding the substantial progress in optical wearable sensing devices, developing wearable optical sensors for simultaneous, real-time, and continuous monitoring of multiple biomarkers is still an important, yet unmet, demand. Aiming to address this need, we introduced for the first time a smart wearable optical sensor (SWOS) platform combining a multiplexed sweat sensor sticker with its IoT-enabled readout module. We employed our SWOS system for on-body continuous, real-time, and simultaneous fluorimetric monitoring of sweat volume (physical parameter) and pH (chemical marker). Herein, a variation in moisture (5-45 µL) or pH (4.0-7.0) causes a color/fluorescence change in the copper chloride/fluorescein immobilized within a transparent chitin nanopaper (ChNP) in a selective and reversible manner. Human experiments conducted on athletic volunteers during exercise confirm that our developed SWOS platform can be efficiently exploited for smart perspiration analysis toward personalized health monitoring. Moreover, our system can be further extended for the continuous and real-time multiplexed monitoring of various biomarkers (metabolites, proteins, or drugs) of sweat or other biofluids (for example, analyzing exhaled breath by integrating onto a facemask).

Lateral Flow Assay: A Summary of Recent Progress for Improving Assay Performance.

Lateral flow tests are one of the most important types of paper-based point-of-care (POCT) diagnostic tools. It shows great potential as an implement for improving the rapid screening and management of infections in global pandemics or other potential health disorders by using minimally expert staff in locations where no sophisticated laboratory services are accessible. They can detect different types of biomarkers in various biological samples and provide the results in a little time at a low price. An important challenge regarding conventional LFAs is increasing their sensitivity and specificity. There are two main approaches to increase sensitivity and specificity, including assay improvement and target enrichment. Assay improvement comprises the assay optimization and signal amplification techniques. In this study, a summarize of various sensitivity and specificity enhancement strategies with an objective evaluation are presented, such as detection element immobilization, capillary flow rate adjusting, label evolution, sample extraction and enrichment, etc. and also the key findings in improving the LFA performance and solving their limitations are discussed along with numerous examples.

Toward waterborne protozoa detection using sensing technologies.

Drought and limited sufficient water resources will be the main challenges for humankind during the coming years. The lack of water resources for washing, bathing, and drinking increases the use of contaminated water and the risk of waterborne diseases. A considerable number of waterborne outbreaks are due to protozoan parasites that may remain active/alive in harsh environmental conditions. Therefore, a regular monitoring program of water resources using sensitive techniques is needed to decrease the risk of waterborne outbreaks. Wellorganized point-of-care (POC) systems with enough sensitivity and specificity is the holy grail of research for monitoring platforms. In this review, we comprehensively gathered and discussed rapid, selective, and easy-to-use biosensor and nanobiosensor technologies, developed for the early detection of common waterborne protozoa.

Nitrogen and sulfur co-doped carbon quantum dots fluorescence quenching assay for detection of mercury (II).

Mercury is a highly toxic and potentially bioaccumulative heavy metal ion that can cause severe health problems in humans even at very low concentrations. Thus, the development of a simple, rapid, and sensitive assay for the effective detection of mercury ions at trace levels is of great importance. Here, nitrogen and sulfur co-doped carbon quantum dots (N,S-CQD) were synthesized by a simple hydrothermal treatment of chitosan in the presence of thiourea and citric acid with a quantum yield (QY) up to 33.0 % and used as a selective fluorescent probe to detect mercury ions (Hg2+). The effect of pH, ionic strength, and time on the fluorescence intensity of N,S-CQD were investigated and optimized. The synthesized N,S-CQD showed ultrasensitive ability to detect Hg2+ ions in the water samples, also in the presence of 11 interfering metal ions, with a low detection limit (∼4 nM) over a wide linear range from ∼5-160 nM. The sensing performance of N,S-CQD probe in real sample applications was evaluated by the detection of Hg2+ in lake water samples, which confirmed its potential application in environmental analysis.

Three-layered PCL-collagen nanofibers containing melilotus officinalis extract for diabetic ulcer healing in a rat model.

Active wound dressing with physicochemical and biological characteristics is more effective in healing diabetic foot ulcer (DFU). In this study, a 3-layer electrospun nanofiber wound dressings was fabricated, while its outer, middle and inner layers of the scaffold were made of PCL, PCL/collagen and collagen nanofibers, respectively. Various amounts of Melilotus officinalis extract were also loaded in the collagen nanofibers as a biologically active compound. The diameter and morphology of the obtained nanofibers were investigated by scanning electron microscopy (SEM) and FT-IR spectroscopy to analyse the composition of prepared dressings. The efficacy of the fabricated dressings as wound healing agent was assessed in streptozotocin-induced diabetic rats. The results demonstrated that the mean diameter of nanofibers are 373 ± 179 nm, 266 ± 108 nm, 160 ± 52 nm, and 393 ± 131 nm for PCL, PCL/collagen, pure collagen, and collagen nanofibers containing 0.08 g extract, respectively. The histo-pathology and histomorphometry assessments demonstrate the herbal extract-loaded electrospun dressings (especially containing 0.08 g of the extract) are promising in improving the diabetic ulcer healing. Our results indicated that the combination of drug did not compromise the physicochemical characteristics of wound dressing, while improving its biological activities.

Multilayered Mesoporous Composite Nanostructures for Highly Sensitive Label-Free Quantification of Cardiac Troponin-I.

Cardiac troponin-I (cTnI) is a well-known biomarker for the diagnosis and control of acute myocardial infarction in clinical practice. To improve the accuracy and reliability of cTnI electrochemical immunosensors, we propose a multilayer nanostructure consisting of Fe3O4-COOH labeled anti-cTnI monoclonal antibody (Fe3O4-COOH-Ab1) and anti-cTnI polyclonal antibody (Ab2) conjugated on Au-Ag nanoparticles (NPs) decorated on a metal-organic framework (Au-Ag@ZIF-67-Ab2). In this design, Fe3O4-COOH was used for separation of cTnI in specimens and signal amplification, hierarchical porous ZIF-67 extremely enhanced the specific surface area, and Au-Ag NPs synergically promoted the conductivity and sensitivity. They were additionally employed as an immobilization platform to enhance antibody loading. Electron microscopy images indicated that Ag-Au NPs with an average diameter of 1.9 ± 0.5 nm were uniformly decorated on plate-like ZIF-67 particles (with average size of 690 nm) without any agglomeration. Several electrochemical assays were implemented to precisely evaluate the immunosensor performance. The square wave voltammetry technique exhibited the best performance with a sensitivity of 0.98 mA mL cm-2 ng-1 and a detection limit of 0.047 pg mL-1 in the linear range of 0.04 to 8 ng mL-1.

Smartphone-Based Electrochemiluminescence for Visual Simultaneous Detection of RASSF1A and SLC5A8 Tumor Suppressor Gene Methylation in Thyroid Cancer Patient Plasma.

Visual one-step simultaneous detection of low-abundance methylation is a crucial challenge in early cancer diagnosis in a simple manner. Through the design of a closed split bipolar electrochemistry system (BE), detection of promoter methylation of tumor suppressor genes in papillary thyroid cancer, RASSF1A and SLC5A8, was achieved using electrochemiluminescence. For this purpose, electrochemiluminescence of luminol loaded into the Fe3O4@UiO-66 and gold nanorod-functionalized graphite-like carbon nitride nanosheet (AuNRs@C3N4 NS), separately, on the anodic and cathodic pole bipolar electrodes (BPEs) in two different chambers of a bipolar cell were recorded on a smartphone camera. To provide the same electric potential (ΔEelec) through the BPEs to conduct simultaneous light emission, as well as to achieve higher sensitivity, anodic and cathodic poles BPEs were separately connected to ruthenium nanoparticles electrodeposited on nitrogen-doped graphene-coated Cu foam (fCu/N-GN/RuNPs) to provide a hydrogen evolution reaction (HER) and polycatechol-modified reduced graphene oxide/pencil graphite electrode (PC-rGO/PGE) to provide electrooxidation of hydrazine. Moreover, taking advantages of the strong cathodic ECL activity due to the roles of AuNRs, as well as the high density of capture probes on the UiO-66 and Fe3O4 roles in improving the signal-to-background ratio (S/B) in complicated plasma media, a sensitive visual ECL immunosensor was developed to detect two different genes as model target analytes in patient plasma samples. The ability of discrimination of methylation levels as low as 0.01% and above 90% clinical sensitivity in thyroid cancer patient plasma implies that the present strategy is able to diagnose cancer early, as well as monitor responses of patients to therapeutic agents.

Electrochemical immunosensor for determination of cardiac troponin I using two-dimensional metal-organic framework/Fe3O4-COOH nanosheet composites loaded with thionine and pCTAB/DES modified electrode.

Cardiac troponin-I (CTnI) is one of the most popular biomarkers which can be utilized for the diagnosis and control of acute myocardial infarction in clinical practice. Here, a sandwich-type electrochemical immunosensor has been established using the zinc-based metal-organic framework/Fe3O4-COOH/thionine labeled anti-CTnI monoclonal antibody (Ab1-Zn-MOF/Fe3O4-COOH/Thi) nanocomposite as signaling molecule and a polymer film of cetyltrimethylammonium bromide (pCTAB) in the presence of choline chloride-urea deep eutectic solvent (DES) and anti-CTnI polyclonal antibody (Ab2) as immobilization substance of detecting surface. The porous ultrathin layers of Zn-MOF nanosheets successfully prepare a well-defined structure for Fe3O4-COOH electrocatalyst and Thi within a certain two dimensional (2D) regions, which enhances electrochemical reduction of Thi. The Ab1-Zn-MOF/Fe3O4-COOH/Thi nanocomposites were introduced to CTnI in the specimen and on the surface of pCTAB/DES-Au-SPE quantitative determination of CTnI was achieved using differential pulse voltammetry after sandwiching the CTnI target between Ab1-nanocomposite and Ab2 which was encapsulated into the pCTAB/DES-Au-SPE. This immunosensor indicated the appropriate assay performance for CTnI with the detection range of 0.04 ng mL-1 to 50 ng mL-1 and the limit of detection of 0.0009 ng mL-1. This study provides convenient plan for sensitive detection of bioanalytes and opens a path for the establishment of user-friendly and cost-effective device.

Simultaneous Detection of SARS-CoV-2 IgG/IgM Antibodies, Using Gold Nanoparticles-Based Lateral Flow Immunoassay.

The novel coronavirus disease (COVID-19), known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), exhibits a strong human-to-human transmission infectivity and could cause acute respiratory infections. Therefore, simple and rapid serological testing is urgently needed to recognize positive cases. In this study, a point-of-care serological test based on lateral flow immunoassay (LFIA) was developed and its application for the simultaneous detection of IgM/IgG antibodies against SARS-CoV-2 was evaluated. The recombinant SARS-CoV-2 antigens were conjugated to the produced colloidal gold nanoparticles and used as the detection reagent. This test required only 10-15 minutes to achieve simultaneous qualitative detection of IgM/IgG antibodies specific to SARS-CoV-2 in 20 µL of serum or plasma samples. The clinical performance and reliability of the assay were evaluated by performing the test with 60 samples and comparing the results of these tests with those obtained via real-time polymerase chain reaction. The sensitivity and specificity of our assay were defined to be 90% and 96.6%, respectively. The presented LFIA was sufficiently sensitive and accurate to be used for the rapid diagnosis of coronavirus disease 2019 in laboratories or in patient care settings, particularly in emergency conditions, in which many samples require to be evaluated on time.

Electrochemiluminescence paper-based screen-printed electrode for HbA1c detection using two-dimensional zirconium metal-organic framework/Fe3O4 nanosheet composites decorated with Au nanoclusters.

Glycated hemoglobin (HbA1c) is one of the most popular biomarkers which can be utilized for the diagnosis and control of diabetes in clinical practice. In this study, a sandwich paper-based electrochemiluminescence (ECL) biosensor has been developed using the zirconium metal-organic framework/Fe3O4(trimethyl chitosan)/gold nanocluster (Zr-MOF/Fe3O4(TMC)/AuNCs) nanocomposite as tracing tag to label anti-HbA1c monoclonal antibody and reduced graphene oxide (rGO) as immobilization platform of sensing element. The screen-printed electrodes (SPEs) were constructed and modified by sputtering a thick layer of gold on the paper substrate, followed by electrochemical reduction of aminophenylboronic acid (APBA)-functionalized GO to rGO/APBA, respectively. Different types of surface analysis methods were applied to characterize the Zr-MOF/Fe3O4(TMC)/AuNCs nanomaterials fabricated. Finally, antibody-labeled Zr-MOF/Fe3O4(TMC)/AuNCs nanocomposites were subjected to HbA1c in the sample and on the paper-based SPE. Quantitative measurement of HbA1c was performed using ECL and cyclic voltammetry (CV) over a potential range of - 0.2 to 1.7 V vs gold reference electrode with a sweep rate of 0.2 V.s-1 in the presence of triethylamine as a co-reactant after sandwiching the HbA1c target between antibody and APBA on the sensing area. This immunosensor demonstrated the desirable assay performance for HbA1c with a wide response range from 2 to 18% and a low detection limit (0.072%). This new strategy provides an effective method for high-performance bioanalysis and opens avenues for the development of high-sensitive and user-friendly device. Graphical abstract.

Point-of-care cancer diagnostic devices: From academic research to clinical translation.

Early and timely diagnosis of cancer plays a decisive role in appropriate treatment and improves clinical outcomes, improving public health. Significant advances in biosensor technologies are leading to the development of point-of-care (POC) diagnostics, making the testing process faster, easier, cost-effective, and suitable for on-site measurements. Moreover, the incorporation of various nanomaterials into the sensing platforms has yielded POC testing (POCT) platforms with enhanced sensitivity, cost-effectiveness and simplified detection schemes. POC cancer diagnostic devices provide promising platforms for cancer biomarker detection as compared to conventional in vitro diagnostics, which are time-consuming and require sophisticated instrumentation, centralized laboratories, and experienced operators. Current innovative approaches in POC technologies, including biosensors, smartphone interfaces, and lab-on-a-chip (LOC) devices are expected to quickly transform the healthcare landscape. However, only a few cancer POC devices (e.g. lateral flow platforms) have been translated from research laboratories to clinical care, likely due to challenges include sampling procedures, low levels of sensitivity and specificity in clinical samples, system integration and signal readout requirements. In this review, we emphasize recent advances in POC diagnostic devices for cancer biomarker detection and discuss the critical challenges which must be surmounted to facilitate their translation into clinical settings.

Point-of-care biosensors in medicine: a brief overview of our achievements in this field based on the conducted research in EMRI (endocrinology and metabolism research Institute of Tehran University of medical sciences) over the past fourteen years.

The growing demand of diagnostic tools with enhanced analytical characteristics in term of sensitivity, selectivity, and low response time has encouraged researches to conduct their research towards development of point-of-care (POC) biosensors. POC diagnostic devices are powerful tools for detection, diagnosis, and monitoring of diseases at its initial stage. The above characteristics encouraged us to conduct active multidisciplinary and collaborative research oriented towards the design and development of POC sensing systems. Here, we present a brief overview of our recent achievement in the field of biomedical POC devices implemented in paper based microfluidic and screen printing electrodes and discuss the critical limitations that need to be surmounted to facilitate their translation into clinical practice in the future.

Seed-mediated Electrochemically Developed Au Nanostructures with Boosted Sensing Properties: An Implication for Non-enzymatic Glucose Detection.

A new approach has been developed to improve sensing performances of electrochemically grown Au nanostructures (AuNSs) based on the pre-seeding of the electrode. The pre-seeding modification is simply carried out by vacuum thermal deposition of 5 nm thin film of Au on the substrate followed by thermal annealing at 500 °C. The electrochemical growth of AuNSs on the pre-seeded substrates leads to impressive electrochemical responses of the electrode owing to the seeding modification. The dependence of the morphology and the electrochemical properties of the AuNSs on various deposition potentials and times have been investigated. For the positive potentials, the pre-seeding leads to the growth of porous and hole-possess networks of AuNSs on the surface. For the negative potentials, AuNSs with carved stone ball shapes are produced. The superior electrode was achieved from AuNSs developed at 0.1 V for 900 s with pre-seeding modification. The sensing properties of the superior electrode toward glucose detection show a high sensitivity of 184.9 µA mM-1 cm-2, with a remarkable detection limit of 0.32 µM and a wide range of linearity. The excellent selectivity and reproducibility of the sensors propose the current approach as a large-scale production route for non-enzymatic glucose detection.

A review on nanomaterial-based field effect transistor technology for biomarker detection.

Field effect transistor (FET) based sensors have attractive features such as small size, ease of mass production, high versatility and comparably low costs. Over the last decade, many FET type biosensors based on various nanomaterials (e.g. silicon nanowires, graphene, and transition metal dichalcogenides) have been developed to detect various classes of biomolecular targets due to their integration into portable and rapid test systems, both for use in the clinical lab and in point-of-care testing. This review (with 197 refs.) starts with an introduction into the specific features of FET biosensor technology. This is followed by a description of the essentials of methods for immobilization of recognition elements. The next section discusses the progress that has been made in FET based biosensors using semiconducting nanostructures composed of silicon, graphene, metal oxides, and transition metal dichalcogenides. A further section is devoted to microfluidic systems combined with FET biosensors. We then emphasize the biosensing applications of these diagnostic devices for analysis of clinically relevant biomarkers, specifically to sensing of neurotransmitters, metabolites, nucleic acids, proteins, cancer and cardiac biomarkers. Two tables are presented which summarize advances in applications of 1D and 2D nanomaterial-based FETs for biomarker sensing. A concluding section summarizes the current status, addresses current challenges, and gives perspective trends for the field. Graphical abstract Field effect transistor devices based on the use of 1D and 2D semiconductor nanostructures (so called nano-FETs) are making use of materials including silicon nanowires, graphene, zinc oxide, indium oxide, titanium oxide, and molybdenum disulfide that are further modified with recognition elements for biosensing application.

Biosensor design using an electroactive label-based aptamer to detect bisphenol A in serum samples.

A new and simple procedure was applied to detect bisphenol A (BPA) based on a BPA aptamer and its complementary strand (Comp. Str.). An electrode was modified with a mixture of carboxylated multiwalled carbon nanotubes and chitosan. The Comp. Str. was immobilized on a modified-glassy carbon electrode (GCE) surface via covalent binding. After the incubation of the aptamer with the electrode surface, it could interact with the Comp. Str. In the presence of BPA, its aptamer will interact with the analyte, resulting in some changes in the configuration and leading to separation from the electrode surface. Due to the attached ferrocene (Fc) group on the 50 head of the aptamer, the redox current of Fc has reduced. This aptasensor can sense the level of BPA in the linear range of 0.2-2 nM, with a limit of detection of 0.38 nM and a sensitivity of 24.51 lA/µM. The proposed aptasensor showed great reliability and selectivity. The acceptable selectivity is due to the specificity of BPA binding to its aptamer. The serum sample was used as a real sample; the aptasensor was able to effectively recover the spiked BPA amounts. It can on-site monitor the BPA in serum samples with acceptable recoveries.

Electrochemical sensors and biosensors based on the use of polyaniline and its nanocomposites: a review on recent advances.

Polyaniline and its composites with nanoparticles have been widely used in electrochemical sensor and biosensors due to their attractive properties and the option of tuning them by proper choice of materials. The review (with 191 references) describes the progress made in the recent years in polyaniline-based biosensors and their applications in clinical sensing, food quality control, and environmental monitoring. A first section summarizes the features of using polyaniline in biosensing systems. A subsequent section covers sensors for clinical applications (with subsections on the detection of cancer cells and bacteria, and sensing of glucose, uric acid, and cholesterol). Further sections discuss sensors for use in the food industry (such as for sulfite, phenolic compounds, acrylamide), and in environmental monitoring (mainly pesticides and heavy metal ions). A concluding section summarizes the current state, highlights some of the challenges currently compromising performance in biosensors and nanobiosensors, and discusses potential future directions. Graphical abstract Schematic presentation of electrochemical sensor and biosensors applications based on polyaniline/nanoparticles in various fields of human life including medicine, food industry, and environmental monitoring. The simultaneous use of suitable properties polyaniline and nanoparticles can provide the fabrication of sensing systems with high sensitivity, short response time, high signal/noise ratio, low detection limit, and wide linear range by improving conductivity and the large surface area for biomolecules immobilization.

Voltammetric immunosensor for E-cadherin promoter DNA methylation using a Fe3O4-citric acid nanocomposite and a screen-printed carbon electrode modified with poly(vinyl alcohol) and reduced graphene oxide.

Silencing of tumor suppressor genes (E-cadherin) by promoter DNA methylation may lead to the development of invasive phenotypes in epithelial tissues. The authors describe an electrochemical nanobiosensor for early detection and screening of circulating methylated DNA as a biomarker for cancers. First, the antibody against 5-methylcytosine was physically immobilized onto modified with reduced graphene oxide and polyvinylalcohol. In the next step, methylated target DNA in samples was hybridized with ssDNA probe conjugated to Fe3O4-citric acid nanocomposites and placed on the modified electrode. Then, the hexacyanoferrate redox system was added and electron transfer recorded. Cyclic voltammetry and electrochemical impedance spectroscopy showed that the modification process was well accomplished. Quantitative measurement of E-cadherin DNA promoter methylation was performed using differential pulse voltammetry. The electrochemical analysis achieved in the presence and absence of nonmethylated DNA mixed with samples indicated the high specificity and selectivity in methylation analysis using this system. With the linear range of concentration from 1 × 10-4 ng.mL-1 to 20 ng.mL-1 and the detection limit of 9 × 10-5 ng.mL-1, this method represents a promising approach for analysis of other biomarkers. Graphical abstract A label free electrochemical nanobiosensor was constructed for detection of methylated circulating cell-free DNA using screen-printed carbon electrode (SPCE) modified with reduced graphene oxide (rGO) and polyvinylalcohol (PVA).