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Abstract: A homogeneous chitosan–ferrocene/graphene oxide/glucose oxidase (CS–Fc/GO/GOx) nanocomposite film was successfully constructed as a novel platform for the fabrication of glucose biosensor. The morphologies and electrochemistry of the nanocomposite film were investigated by using scanning electron microscopy and electrochemical techniques including electrochemical impedance spectroscopy and cyclic voltammetry, respectively. Results demonstrated that the uniformly dispersed GO within the CS matrix could significantly improve the stability of GO and make it exhibit a positive charge, which was more favorable for the further immobilization of biomolecules, such as GOx, with higher loading. Further attaching redox mediator ferrocene group (Fc) to CS could not only effectively prevent the leakage of Fc from the matrix and retain its electrochemical activity, but also improve the electrical conductivity of CS and promote the electron-transfer between GOx and electrode. Biosensors based on this CS–Fc/GO/GOx film had advantages of fast response, excellent reproducibility, high stability, and showed a linear response to glucose in the concentration range from 0.02 to 6.78mM with a detection limit of 7.6μM at a signal-to-noise ratio of 3 and exhibited a higher sensitivity of 10μAmM−1 cm−2. The proposed strategy based on CS–Fc/GO nanocomposite for the immobilization of enzymes can be of practical relevance for the facile design of biosensors, as well as for the construction of new multifunctional bioelectrochemical systems. [Copyright &y& Elsevier]

Abstract: A novel amperometric sensor for glucose was constructed by entrapping glucose oxidase (GOD) in a new chitosan composite doped with ferrocene monocarboxylic acid-modified 3-(aminopropyl) triethoxysilane enwrapping multiwalled carbon nanotubes (FMC-AMWNTs). The approach was very simple, timesaving and effectively prevented the leakage of the ferrocene monocarboxylic acid mediator during measurements. The entrapped FMC-AMWNTs composite performed excellent redox electrochemistry and acted as an electron transfer mediator. This matrix showed a biocompatible microenvironment for retaining the native activity of the entrapped GOD and a very low mass transport barrier to the substrate. Under optimal conditions, this biosensor was able to detect glucose in the linear range of 0.01–4.2mM with a detection limit of 3.4μM (S/N=3), the value of was 6.3mM. This immobilization approach effectively improved the stability of the electron transfer mediator and is promising for construction of biosensor and bioelectronic devices. [Copyright &y& Elsevier]

Highlights • This is the first report on the peroxidase-like activity of CoOOH nanoflakes for the detection of MTase activity. • The peroxidase-like activity of CoOOH nanoflakes can be improved by adsorbing ssDNA. • The colorimetric sensor with a low detection limit of 0.069 U/mL for M.SssI MTase activity detection. • The colorimetric sensor allows detection of M.SssI MTase in cell lysates of lung cancer cells (A549). Abstract A label-free colorimetric assay for MTase activity based on the nanozyme activity of cobalt oxyhydroxide (CoOOH) nanoflakes and the difference in affinity of the nanoflakes with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) has been developed. In this strategy, ssDNA can enhance the nanozyme activity of CoOOH nanoflakes, which could be attributed to the electrostatic attraction and π-π stacking interaction between 3,3′,5,5′-tetramethylbenzidine (TMB) and ssDNA. The proposed method presents a wide linear range from 0.08 to 50 U/mL and a low detection limit (LOD) of 0.069 U/mL. Additionally, this colorimetric sensor also has been proved to detect MTase in the cell lysates of lung cancer cells (A549) with good recovery and reproducibility. Furthermore, the novel strategy can be applied for high-throughput evaluation and screening the inhibitors for M.SssI MTase by using representative anticancer drugs as model inhibitors, therefore, the colorimetric sensor can not only achieve the purpose of sensitively detecting the activity of MTase, but also has the potential in clinical diagnostics and drug discovery. [ABSTRACT FROM AUTHOR]

Highlights • Cancer-associated telomerase activity can be detected with QDs-SA biosensor. • The detection limit of telomerase can be down to 13 Hela cells and 9 A549 cells. • The QDs-SA biosensor allows intracellular telomerase tracking and imaging. • Inhibitor screening can be realized in vitro and in vivo. Abstract In order to overcome the shortcomings of traditional methods for telomerase activity detection of time-consuming and susceptibility to interferences in cell extracts, here, we develop a semiconductor quantum dots-streptavidin conjugates (QDs-SA)-based biosensor for sensitive detection of telomerase activity. In the absence of telomerase, the aggregation and thus the fluorescence quenching of QDs-SA can be observed in buffer solution due to charge shielding effect. In the presence of telomerase and biotin-dATP, QDs-SA will bind to telomerase elongation products due to the high affinity between the biotin and streptavidin, protecting the QDs-SA from aggregation and retaining their strong fluorescence. This sensor exhibited good fluorescence responses toward telomerase activity from HeLa cells in the range of 40–1000 cells with a detection limit of 13 cells or A549 cells within the linear range of 20–500 cells with a detection limit of 9 cells. Furthermore, the sensor can be used to efficiently monitor the evolution of telomerase activity in living cells, demonstrating great potential in tumor diagnosis and inhibitor drug screening. [ABSTRACT FROM AUTHOR]

Human single-stranded-selective monofunctional uracil glycosylase (hSMUG1) and alkyladenine glycosylase (hAAG) are important base-excision repair (BER) glycosylases responsible for deamination and alkylation repair. Their dysfunctional activities have strong association with various multifactorial diseases and cancers. Herein, by integrating three-dimensional (3D) DNA walker with single-molecule technique, we construct bidirectional strand displacement-driven 3D DNA walkers for single-molecule monitoring of multiple DNA glycosylases. In the presence of hSMUG1 and hAAG, the damaged U and I in bifunctional dumbbell probe are removed by APE1, activating bidirectional self-priming strand displacement amplification (SP-SDA) to produce two trigger DNAs. Trigger DNAs hybridize with Cy5/Alexa Fluor 488 signal probes and subsequently serve as the walker DNAs to induce cyclic APE1-powered cleavage of two signal probes, liberating abundant Cy5 and Alexa Fluor 488 from AuNPs. Due to high precision of intracellular BER mechanisms, high efficiency of bidirectional SP-SDA-directed 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging, this nanosensor exhibits a detection limit of 8.14 × 10−10 U/μL for hSMUG1 and 4.50 × 10−9 U/μL for hAAG. Importantly, it can measure kinetic parameters, identify potential inhibitors, discriminate cancer from normal cells, and quantify glycosylases activities in various cancer cells at single-cell level, with promising potentials in biomedical and clinical applications. • We construct bidirectional strand displacement-driven 3D DNA walkers. • This nanosensor enables single-molecule monitoring of multiple human glycosylases. • This nanosensor can measure kinetic parameters and screen potential inhibitors. • This nanosensor can discriminate cancer cells from normal cells. [ABSTRACT FROM AUTHOR]

Methionine (Met) is one of sulfur-containing essential amino acids, it plays an indispensable role in physiological processes. Its quantitative and sensitive determination is imperative. However, currently available analytical approaches for determination of Met are scarce and also exhibit many disadvantages, such as poor selectivity for Met towards other amino acids and biothiols. In this work, a label-free colorimetric biosensor based on bio-dots assembly-triggered aggregation of gold nanoparticles (AuNPs) for detecting Met is firstly developed. In the presence of Met, Met effectively prevents the bio-dots assembly-induced aggregation of AuNPs since that AuNPs are protected inside the Met layer. This colorimetric biosensor provides a simple, fast, highly sensitive and highly selective method for Met detection. It presents lower detection limit of 3.2 nM and exhibits highly selectivity toward bio-thiols surprisingly. Furthermore, this proposed biosensor has been successfully applied for the determination of Met in human serum. [ABSTRACT FROM AUTHOR]

A novel fluorescent probe, 2, 4-cyclohexanedione-1-propyl-pyrene (TAR), has been synthesized to research thioredoxin (Trx) disulfenylation and visualize the activity of Trx in cells. The turn-on fluorescence change was induced by combining the vicinal dithiol proteins with the spatially sensitive fluorescent signal of the pyrene excimer which emitted at 480 nm. Trx activity can be detected at nanomolar concentrations. Trx preference of TAR in vitro and in vivo was proved by fluorometric and confocal microscopic experiments. In addition, it could realize dynamic tracking of protein disulfenylation in situ with minimal disturbance to disulfenylated proteins and less perturbation with cellular function. We herein report the first turn-on fluorescent sensor for the selective detection of proximally disulfenylated protein sites, suitable for application in both aqueous solutions and in cells. [ABSTRACT FROM AUTHOR]

In this paper, we present a colorimetric method for the assay of DNA methyltransferase (MTase) activity based on strand displacement amplification (SDA). In our study, a well-designed hairpin DNA I (HPI) containing the sequence of 5′-CCGG-3′ is specifically recognized by CpG methyltransferase (M.SssI) and HpaII endonuclease. The methylated HPI is able to coexist with all the DNA and enzymes in the solution while the unmethylated HPI can be cleaved into single-stranded DNA (ssDNA) fragments. The amplification can be triggered by the HpaII digestion products hybridization with another hairpin structure DNA II (HPII) to form a duplex, which would be replaced by probe DNA, leading to the aggregation of gold nanoparticles (AuNPs). Simultaneously, ssDNA fragments released from the duplex, and triggered the cycle anew. Varying concentrations of M.SssI in the solution therefore would lead to differences of absorption and color changed from red to pale. A linear response was obtained when the M.SssI concentration ranging from 0.2 to 50 U mL −1 with a detection limit of 0.08 U mL −1 . In addition, the developed assay in this study can also be applied to screen the inhibitors of M.SssI. [ABSTRACT FROM AUTHOR]

DNA methylation is one important epigenetic modification that involves in regulating multiple cellular functions. DNA methyltransferases (MTases) maintain methylation patterns in genomic DNA, and their aberrant activities may lead to multiple diseases including cancers. However, the efficient sensing of multiple DNA MTases is limited by the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases. Herein, inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification, we construct a novel methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases. Taking advantage of ultrahigh specificity of MDREs toward methylated DNAs, high efficiency of bicyclic cascade amplifications and high signal-to-noise ratio of light-up RNA aptamer-fluorophore pairs, this nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77 × 10−3 U/mL for M.SssI and 7.21 × 10−5 U/mL for Dam. Furthermore, it can screen potential inhibitors and quantify M.SssI and Dam MTases activities in E. coli cells and human serum samples. Notably, this assay can be completed homogeneously within 2 h in a single tube in a label-free manner, providing a powerful platform for sensing multiple DNA MTases in biomedical research and clinical therapeutics. • A dual-color light-up RNA nanosensor is developed for label-free and ultrasensitive sensing of multiple DNA MTases. • This nanosensor relies on methylation-powered bicyclic cascade amplifications for generating two light-up aptamers. • The assay may be completed homogeneously within 2 h in single tube. • This nanosensor exhibits good specificity and high sensitivity. • This nanosensor can screen inhibitors and quantify DNA MTases activity from clinical samples. [ABSTRACT FROM AUTHOR]

The instant and on-site detection of trace aqueous nitrite ions still remains a challenge for environmental monitoring and protection. In this work, we synthesized water-soluble striking fluorescent Cu nanoclusters (Cu NCs) by a green, simple, one-pot and stabilizer-free chemical reduction method. The fluorescence (FL) of the obtained Cu NCs can be selectively and efficiently quenched by the nitrite ions through the oxidation of Cu (0) atoms to Cu (I) in the core of Cu NCs. Under optimal conditions, two excellent linear relationships between the FL quenching degree of the Cu NCs and the concentrations of nitrite ions in the range of 0.0125–125 μM and 125–5000 μM with a low detection limit of 3.6 nM were obtained. More importantly, the portable paper-based sensor has been explored for visual detection of trace nitrite ions in aqueous solution by the appearance of different degrees of blue FL under a UV lamp. The very simple and effective strategy reported here should facilitate the development of portable and reliable FL chemosensors for nitrite ions in pollution control. [ABSTRACT FROM AUTHOR]

A novel and sensitive ratiometric fluorescent assay for monitoring kinase activity based on carboxypeptidase Y (CPY) digestion by using Au nanoclusters (AuNCs) and CdSe/ZnS QDs@SiO 2 as the dual-emission nanoparticles is developed. This approach can not only sensitively monitor the kinase activity with affording built-in correction which avoids environmental interferences, but also realize visual detection. Under optimal conditions, a linear relationship between the fluorescence intensity ratio (I 415 /I 630 ) of CdSe/ZnS QDs@SiO 2 @peptide-AuNCs and the protein kinase A (PKA) concentration in a range of 0.01 to 40 U mL −1 with a detection limit of 0.004 U mL −1 (3 σ ) is obtained. The feasibility of this CdSe/ZnS QDs@SiO 2 @peptide-AuNCs-based ratiometric sensor has been further demonstrated by assessment of kinase inhibition by ellagic acid in cell lysates. The IC 50 value (inhibitor concentration producing 50% inhibition) for ellagic acid is estimated to be 0.08 μM. The detection of other enzymes can also be realized with precise design of the peptide substrate sequences. The present assay is universal and visual for kinase assay and promises potential application in kinase-related biochemical fundamental research and inhibitor screening. [ABSTRACT FROM AUTHOR]

An exonuclease III-assisted recycling amplification (ERA) coupled with fluorescent DNA-scaffolded silver nanoclusters (Ag NCs) for sensitive determination of hepatitis B virus (HBV) DNA is proposed. The sensing mechanism is based on the employment of two different molecular beacons (MBs), MB1 and MB2. In the absence of targets, the MBs self-hybridize into stable stem-loop structures with exonuclease III (Exo III) cleaving resistance, and the MB2 containing a C-rich loop can be utilized to synthesize Ag NCs with high fluorescence signal. In the presence of targets, the targets can bind with MB1 to form partly duplex structure. With the cleaving of Exo III, the liberated target DNA can initiate the cycle of MB1-target hybridization, while the generated trigger DNA can hybridize with MB2, inducing a conformational change of MB2 from hairpin to single-stranded structure. The structural change of MB2 would influence the formation of Ag NCs, thus producing weak fluorescence signal, and the released trigger DNA could activate the cycle of MB2-trigger hybridization. The present method for HBV DNA analysis exhibits a detection limit of 0.97 nM ( S / N = 3) with high specificity, and demonstrates its applicability for the HBV detection in human serum. [ABSTRACT FROM AUTHOR]

Abstract: In this contribution, we develop a novel type of multi-walled carbon nanotubes/silica nanoparticles array (MWCNTs/SiO2) composite by the galvanostatic deposition technique. In this process, MWCNTs were mixed with ammonium fluorosilicate to form a doped precursory sol solution, the electrochemically generated hydroxyl ions at negative potentials promote the hydrolysis of ammonium fluorosilicate, and the simultaneously generated hydrogen bubbles assist the formation of three-dimensional porous silica matrix, further facilitating the construction of SiO2 NPs array with uniform distribution, as confirmed by scanning electron microscopy. The formation of SiO2 NPs array provides a higher surface area and biocompatible microenvironment for retaining the native activity of the immobilized biomolecules. Further incorporation of MWCNTs into SiO2 NPs array improves the electronic conductivity and enhances the electroactive surface area of the film, making the fabricated three-dimensional porous MWCNTs/SiO2 electrode an ideal platform for further immobilization of alpha-fetoprotein (AFP), as a model protein. This method achieves ultrasensitive detection of AFP antigen with ferricyanide as a probe. The obtained results provided a linear response range from 0.1 to 30.0ngmL−1 AFP antigen with a lower detection limit of 0.018ngmL−1. This work implies that the biocompatible and controllable three-dimensional porous SiO2 NPs array possessed potential applications for biosensing. [Copyright &y& Elsevier]