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We report here a dual-nanopore biosensor based on modulation of surface charge density coupled with a microwell array chip for in situ monitoring of ROS secretion from single MCF-7 cells. [ABSTRACT FROM AUTHOR]

Direct measurement of DNA repair is critical for the annotation of their clinical relevance and the discovery of drugs for cancer therapy. Here we reported a "repaired and activated" DNAzyme (RADzyme) by incorporating a single methyl lesion (O6MeG, 3MeC, or 1MeA) at designated positions through systematic screening. We found that the catalytic activity of the RADzyme was remarkably suppressed and could be restored via enzyme‐mediated DNA repair. Benefit from these findings, a fluorogenic RADzyme sensor was developed for the monitoring of MGMT‐mediated repair of O6MeG lesion. Importantly, the sensor allowed the evaluation of MGMT repair activity in different cells and under drugs treatment. Furthermore, another RADzyme sensor was engineered for the monitoring of ALKBH2‐mediated repair of 3MeC lesion. This strategy provides a simple and versatile tool for the study of the basic biology of DNA repair, clinical diagnosis and therapeutic assessment. [ABSTRACT FROM AUTHOR]

CRISPR‐dCas9 systems that are precisely activated by cell‐specific information facilitate the development of smart sensors or therapeutic strategies. We report the development of an activatable dCas9 transcriptional circuit that enables sensing and silencing of mRNA in living cells using hybridization‐mediated structure switching for gRNA activation. The gRNA is designed with the spacer sequence blocked by a hairpin structure, and mRNA hybridization induces gRNA structure switching and activates the transcription of reporter RNA. An mRNA sensor developed using a light‐up RNA reporter shows high sensitivity and fast‐response imaging of survivin mRNA in cells under drug treatments and different cell lines. Furthermore, a feedback circuit is engineered by incorporating a small hairpin RNA in the reporter RNA, demonstrating a smart strategy for dynamic sensing and silencing of survivin with induced tumor cell apoptosis. This circuit illustrates a broadly applicable platform for the development of cell‐specific sensing and therapeutic strategies. [ABSTRACT FROM AUTHOR]

A novel Spinach-based fluorescent light-up biosensor utilizing the T7 in vitro transcription process to generate unmodified Spinach sequences for multiplexed microRNA detection has been developed. [ABSTRACT FROM AUTHOR]

DNAzymes are a promising platform for metal ion detection, and a few DNAzyme-based sensors have been reported to detect metal ions inside cells. However, these methods required an influx of metal ions to increase their concentrations for detection. To address this major issue, the design of a catalytic hairpin assembly (CHA) reaction to amplify the signal from photocaged Na+-specific DNAzyme to detect endogenous Na+ inside cells is reported. Upon light activation and in the presence of Na+, the NaA43 DNAzyme cleaves its substrate strand and releases a product strand, which becomes an initiator that trigger the subsequent CHA amplification reaction. This strategy allows detection of endogenous Na+ inside cells, which has been demonstrated by both fluorescent imaging of individual cells and flow cytometry of the whole cell population. This method can be generally applied to detect other endogenous metal ions and thus contribute to deeper understanding of the role of metal ions in biological systems. [ABSTRACT FROM AUTHOR]

This study develops a simple and label-free biosensor for sensitive and selective detection of microRNA (miRNA) based on the formation of the adenosine2–coralyne–adenosine2 complex mediated by miRNA-specific polyadenosine extension. [ABSTRACT FROM AUTHOR]

A novel, highly sensitive split aptamer mediated endonuclease amplification strategy for the construction of aptameric sensors is reported. [ABSTRACT FROM AUTHOR]

Abstract: A glucose biosensor was developed using ruthenium purple nanowire array (RPNWA) as the substrate layer for immobilization of glucose oxidase (GOx). The RPNWA was prepared by means of electrochemical deposition technique on the glassy carbon electrode (GCE) directly from the mixture of Fe3+and Ru(CN)6 4− solution. Scanning electron microscopy (SEM), Raman spectroscopy and electrochemical methods were employed to characterize the RPNWA. The RPNWA-coated electrode was demonstrated to exhibit high catalytic activity for H2O2 reduction at a relatively positive potential of −0.1V. Glucose biosensor was then fabricated by crosslinking GOx with chitosan on the RPNWA electrode. The RPNWA-based biosensor thus obtained was shown to afford high sensitivity to glucose in the range from 1μM to 1mM with a slope of 2.641μA/mM and a detection limit down to 0.5μM. Moreover, common redox species such as ascorbic acid (AA) and uric acid (UA) were observed to have little interference due to the positive detection potential of this working system. [Copyright &y& Elsevier]

Abstract: A novel chemiluminescence biosensor based on a supported lipid layer incorporated with ganglioside GM1 was developed for the detection of cholera toxin. The planar supported lipid membrane was prepared as biosensing interface via spontaneous spread of ganglioside-incorporated phospholipid vesicles on the octadecanethiol-coated gold surface. The specific interaction of multivalent CT by ganglioside GM1 molecules enables the biosensor to be implemented via a sandwiched format using a liposome probe functionalized with GM1 and horseradish peroxidase (HRP). Then, the presence of the target CT could be determined via the HRP-catalyzed enhanced chemiluminescence reaction. The developed strategy offers several unique advantages over conventional biosensors in that it allows for an easy construction and renewal of the sensing interface, a small background signal due to low non-specific adsorption of serum constituents on the lipid membrane, and effective immobilization of multiple biocatalytic amplifiers and recognition components via common phospholipid reagents. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1pgmL−1 to 1ngmL−1 with readily achievable detection limit of 0.8pgmL−1. [Copyright &y& Elsevier]

Abstract: A novel electrochemical immunosensor was developed based on enzymatic deposition of copper onto platinum (Pt) nanoparticle-modified electrode that inhibited the electrocatalytic reduction of protons to hydrogen in acidic medium by Pt. The method was implemented for the determination of a model target, human immunoglobulin G (hIgG), using a microtiter-based sandwiched immunoassay with alkaline phosphatase (ALP)–antibody conjugate as the detection probe. The binding of ALP on the microtiter interface due to the presence of target hIgG catalyzed the hydrolysis of a substrate ascorbic acid 2-phosphatase (AAP), producing a reductive product ascorbic acid that mediated the deposition of copper on a Pt nanoparticle-modified electrode. A negative shift of hydrogen evolution potential was thus obtained at the Pt nanoparticle-modified electrode, which could be determined using linear sweep voltammetry in 0.1M HCl. The influence of experimental variables including the concentrations of HPtCl6, Cu2+, and AAP as well as the reaction time of enzymatic copper deposition upon the potential shift was investigated. Under optimized conditions, the potential shift was observed to show linear dependency on hIgG concentration over a range from 10pgmL−1 to 1.0μgmL−1 with a readily achievable detection limit of 2.0pgmL−1. The developed method could be implemented with simple operation, high sensitivity and multiple-sample format, indicating that this technique might hold great promise in various environmental, food, and clinical applications. [Copyright &y& Elsevier]

Abstract: We report the synthesis of a novel looped enzyme–polyamidoamine nanocomposite with high enzyme loading density and long-term retention of bioactivity. The horseradish peroxidase (HRP) is first immobilized on fourth-grade (G4) poly(amidoamine) (PAMAM) dendrimer to form relatively a small enzyme–PAMAM composite, which is allowed to grow up into a larger one. The looped horseradish peroxidase-polyamidoamine (HRP-PAMAM) nanohybrid was characterized by TEM. The material obtained shows promising features as applied to the fabrication of high sensitive and long lifetime biosensors. In the presence of the hydroquinone mediator in the solution, the immobilized HRP exhibited excellent electro-catalytical response to H2O2. Under the optimal conditions, the resulting biosensor showed a linear response to H2O2 over a concentration range from 3.1×10−6 to 2.0×10−3 molL−1 with a sensitivity of 0.36ALmol−1 cm−2 and a detection limit of 8.0×10−7 molL−1. The sensitivity of the sensor response maintained over 70% of the original over 10 weeks. The catalytic activity of the looped enzyme–PAMAM nanohybrid form of HRP enzyme was obviously stabilized. As an extension, bienzyme sensor modified with glucose oxidase and HRP enzymatic PAMAM nanocomposites was constructed. The sensor exhibited improved performance and can be applied to the detection of glucose in real samples. [Copyright &y& Elsevier]

Abstract: A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin–peroxide horseradish (SA–HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in −28 site of the β-thalassemia gene with a detection limit of 0.1nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection. [Copyright &y& Elsevier]

Abstract: By grafting with poly(amidoamine) (PAMAM) dendrimer, novel carbon nanotube (CNT) nano-composites have been successfully prepared. The novel functionalized matrix with plenty amino groups circumvents the troublesome solubility problem of CNTs in solvents, especially in water, greatly expanding the scope of the application of carbon nanotubes. The GO x and HRP immobilized CNT-PAMAM based on the functional CNTs was synthesized. The bi-enzymatic CNT-PAMAM nano-composites are highly dispersible in water and show very promising applications in the fabrication of mediator-free bi-enzymatic biosensors for sensitive detection of glucose. The cooperation of nano-composite between CNT and high dense GO x and HRP results in very high sensitivity to glucose with a current response of 2200nAmM−1 and fast response (∼1s). The modified electrode exhibits a wide linear response range for glucose from 4.0μM to 1.2mM (R =0.9971, N =15), with a detection limit of 2.5μM. The negative electrode potential of −0.34V is favorable for glucose detection in real samples without interference caused by other biomolecules. [Copyright &y& Elsevier]

The ability to recognize mRNA with high efficiency in cells would greatly facilitate the elucidation of mRNA-mediated cellular cascades and their disease associations. However, most traditional electrochemical strategies targeting nucleotides are always confronted with cumbersome interface operation and washing procedures, as well as the high cost of labeling and the strict reaction conditions of tool enzymes, limiting their potential applications. To address these issues, herein we reported, for the first time, a simple label-free, isothermal, non-enzymatic, and ultrasensitive homogeneous electrochemical biosensor based on autonomous proximity-dependent surface hybridization chain reaction (HCR), for sensitive signal amplification and highly specific detection of target survivin mRNA with a detection limit of 3 fM. The target triggers hybridization chain reaction and mRNA-fueled surface hybridization of ferrocene-tagged metastable DNA hairpin probes on proximity-dependent surface hybridization, resulting in the formation of multiple long-range duplex DNA chains which are immobilized onto the gold electrodes with a substantially stable ferrocene-mediated redox current. Thus, a significant electrochemical signal increase is observed dependent on the concentration of the target RNA, with a very low detection limit. Mo-reover, this molecular biosensor also exhibits excellent specificity to distinguish even single base mismatched, with strong reliability. The developed biosensor provides a novel promising tool for ultra-sensitive and selective detection, and it has great potential to be applied in mRNA-related biochemical research and clinical cancer diagnostics in more detail. Image 1 • A novel label-free, isothermal, non-enzymatic, and ultrasensitive electrochemical biosensor was successfully designed. • HCR-programmed proximity-based surface hybridization strategy for survivin mRNA detection. • The proposed biosensor exhibits excellent specificity to distinguish even single base mismatched, with strong reliability. • High selectivity, enough sensitivity and stability were achieved in reveal true mRNA detection. [ABSTRACT FROM AUTHOR]

Graphitic carbon nitride nanosheets (CNNS) are a promising kind of graphene-like two-dimensional (2D) nanomaterials, which have great potential as biological probes for biosensing and bioimaging applications. However, the pristine CNNS are usually chemically inert, making the functional modification is difficult, which limits their practical biological applications. Therefore, development of new designs for CNNS-based biosensors to extend biological applications is interesting and also of great significance. Herein, we developed a novel CNNS-based turn-on biosensor for simple, sensitive and label-free detection of adenylate kinase (ADK) activity. The approach relies on ADK-mediated catalytic reverse transphosphorylation reaction (2ADP ⇋ ATP + AMP). In this method, quantitative analysis of ADK activity in the concentration range from 0.1 to 100 U/L with the low detection limit of 0.06 U/L. Moreover, it was successfully applied in detecting ADK in the complex biological media. We believe the developed biosensing strategy might provide a rapid, cost-efficient, highly sensitive and label-free platform for ADK-based clinical diagnosis and biomedical research. [ABSTRACT FROM AUTHOR]

Design of DNA self-assembly with reversible responsiveness to external stimuli is of great interest for diverse applications. We for the first time develop a pH-responsive, fully reversible hybridization chain reaction (HCR) assembly that allows sensitive sensing and imaging of pH in living cells. Our design relies on the triplex forming sequences that form DNA triplex with toehold regions under acidic conditions and then induce a cascade of strand displacement and DNA assembly. The HCR assembly has shown dynamic responses in physiological pH ranges with excellent reversibility and demonstrated the potential for in vitro detection and live-cell imaging of pH. Moreover, this method affords HCR assemblies with highly localized fluorescence responses, offering advantages of improving sensitivity and better selectivity. The proton-fueled, reversible HCR assembly may provide a useful approach for pH-related cell biology study and disease diagnostics. [ABSTRACT FROM AUTHOR]

Highlights: [•] We discuss nanomaterials of different types for intracellular fluorescent sensors. [•] We outline strategies for delivery for intracellular fluorescent nanosensors. [•] We review analytical strategies for intracellular fluorescent nanobiosensors. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a cytotoxicity assay was developed for profiling the cytotoxicity of cigarette smoke condensates (CSCs) base on a cellular impedance biosensor (CIB). Compared with the traditional in vitro cytotoxicity assays, this CIB-based method offered distinct advantages in real-time kinetic measurement which provided a comprehensive understanding of cellular responses for the entire duration of the experiment and prediction of the potential mechanism of action of a given treatment. The time-dependent cell response profiles provided valid evidences for optimization of cell number per well, cell quality control, and identification of the optimal time points for compound treatment and endpoint assays. According to the time dependent IC50 values, the CIB could provide dynamic information that can be used to identify maximum toxicity of cigarette smoke and reversibility of the toxic effects which are difficult to achieve by the endpoint assays. The comparative IC50 values indicated that the as-developed biosensor offered analytical results in good consistency with the commonly used NRU method. The features of the CIB-based cytotoxicity assay, such as no cell labeling, automatic detection, and easy operation, give this assay potential to become routine setting for evaluating the cytotoxicity of CSCs. [Copyright &y& Elsevier]

Abstract: Graphene (GR) was covalently functionalized with chitosan (CS) to improve its biocompatibility and hydrophilicity for the preparation of biosensors. The CS-grafted GR (CS-GR) rendered water-soluble nanocomposites that were readily decorated with palladium nanoparticles (PdNPs) using in situ reduction. Results with TEM, SEM, FTIR, Raman and XRD revealed that CS was successfully grafted without destroying the structure of GR, and PdNPs were densely decorated on CS-GR sheets with no aggregation occurring. A novel glucose biosensor was then developed through covalently immobilizing glucose oxidase (GOD) on a glassy carbon electrode modified with the PdNPs/CS-GR nanocomposite film. Due to synergistic effect of PdNPs and GR, the PdNPs/CS-GR nanocomposite film exhibited excellent electrocatalytical activity toward H2O2 and facilitated high loading of enzymes. The biosensor demonstrated high sensitivity of 31.2μAmM−1 cm−2 for glucose with a wide linear range from 1.0μM to 1.0mM as well as a low detection limit of 0.2μM (S/N=3). The low Michaelis–Menten constant (1.2mM) suggested enhanced enzyme affinity to glucose. These results indicated that PdNPs/CS-GR nanocomposites held great potential for construction of a variety of electrochemical biosensors. [Copyright &y& Elsevier]

Abstract: Based on the aptamer-conjugated core–shell near-infrared fluorescent nanoparticles (NIR-Nps) and fluorescence anisotropy measurement, the present study reported proof-of-principle for a rapid homogeneous assay approach that can detect target cancer cells without the need of the complicated separation steps in whole blood samples. Experimental investigation showed that the novel NIR-Nps have negligible background fluorescence and low inner filtration interference in complex biologic systems such as whole blood. The specific recognition characteristic of aptamer in whole blood samples was investigated by using the proposed fluorescence anisotropy method. The results showed that the fluorescent nanoparticle-tagged aptamer probes sequence could achieve specific recognition of the target cancer cells from complex mixtures including whole blood samples. And the reaction conditions for the binding between fluorescent nanoparticle-conjugated aptamer probes and target cancer cells were optimized. The present approach can exhibit sensitive and reproducible fluorescence anisotropy responses to the target cells concentration and the calibration curve showed good linearity when the target cells concentration is in the range from 4.0×103 to 7.0×105 cells/mL. Moreover, the present fluorescence anisotropy assay technique could be practically utilized for the detection of acute leukemia samples with improved capabilities and be comparable to the immunophenotyping methods clinically used. [Copyright &y& Elsevier]

Abstract: A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25ngmL−1 CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins. [Copyright &y& Elsevier]

Abstract: A new method for the determination of platelet-derived growth factor BB (PDGF-BB) was developed using an electrochemical immunosensor with an aptamer-primed, long-strand circular detection probe. Rabbit anti-human PDGF-B polyclonal antibody was immobilized on the electrode to serve as the capture antibody. The detection probe was synthesized via polymerase extension along a single-stranded circular plasmid DNA template with a primer headed by the anti-PDGF-B aptamer. In the presence of the analyte, the aptamer-primed circular probe was captured on the electrode via the formation of an antibody/PDGF-BB/aptamer sandwiched complex. The electroactivity indicator methylene blue was adsorbed on the electrode surface via the analyte-sandwiched complex with long-strand circular DNA, thus yielding a strong electrochemical signal for the quantification of PDGF-BB. This strategy allowed electrochemical detection with enormous signal amplification arising from the long-strand localized circular probe. The oxidation peak current of methylene blue in square wave voltammetric measurements showed a linear dependence on the concentration of PDGF-BB in the range from 50 to 500ng mL−1, with a detection limit as low as18 pg mL−1. [Copyright &y& Elsevier]

Abstract: A successively signal-amplified electrochemical immunoassay has been reported on the basis of the biocatalytic deposition of silver nanoparticles with their subsequent enlargement by nanoparticle-promoted catalytic precipitation of silver from the silver-enhancer solution. The immunoassay was carried out based on a heterogeneous sandwich procedure using polystyrene microwells to immobilize antibody. After all the processes comprising the formation of immunocomplex, biocatalytic deposition of silver nanoparticles and following silver enhancement were completed, the silver on polystyrene microwells was dissolved and quantified by anodic stripping voltammetry (ASV). The effect of relevant experimental conditions, including the concentration of ascorbic acid 2-phosphate (AA-p) substrate and Ag(I) ions, the biocatalytic deposition time, and of crucial importance, the silver enhancement time, were investigated and optimized. The anodic stripping peak current was proportional to the concentration of human IgG in a dynamic range of 0.1–10ngml−1 with a detection limit of 0.03ngml−1. Scanning electron microscope (SEM) was applied to characterize the silver nanoparticles before and after silver enhancement on the surface of polystyrene microplates. By coupling the highly catalytic effect of enzyme and nanoparticles to successively amplify the analytical signal, the sensitivity of immunoassay was enhanced so dramatically that this approach would be a promising strategy to achieve a lower detection limit for bioassays. [Copyright &y& Elsevier]

Abstract: CeO2/Chitosan (CHIT) composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. Such matrix combined the advantages of CeO2 and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity, showing the enhanced loading of ssDNA probe on the surface of electrode. The preparation method is quite simple and inexpensive. The hybridization detection was accomplished by using methylene blue (MB), an electroactive lable, as the indicator. The differential pulse voltammetry (DPV) was employed to record the signal response of MB and determine the amount of colorectal cancer target DNA sequence. The experimental conditions were optimized. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.59×10−11 to 1.16×10−7 molL−1 and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence. [Copyright &y& Elsevier]