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We developed a single quantum dot-based fluorescence resonance energy transfer biosensor for antibody-free detection of ten-eleven translocation 1 (TET1). This biosensor can sensitively detect TET1 in a homogeneous manner without the involvement of any specific antibodies, and it can be used for accurate measurement of TET1 activity in human neuroblastoma cells and the screening of TET1 inhibitors. [ABSTRACT FROM AUTHOR]

We demonstrate the development of a new fluorescent biosensor for sensitive DNA methylation assay by integrating single-molecule detection with endo IV-assisted signal amplification. This biosensor possesses the characteristics of good selectivity and high sensitivity with a detection limit of 7.3 × 10−17 M. It can distinguish as low as 0.01% methylation level, and can analyze genomic DNA methylation even in a single cancer cell. [ABSTRACT FROM AUTHOR]

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]

n amperometric immunosensor has been developed for sensitive determination of hepatitis B surface antigen as a model protein. A glassy carbon electrode was modified with an assembly of positively charged poly(allylamine)-branched ferrocene (PAA-Fc) and negatively charged gold nanoparticles (Au NPs). The formation of PAA-Fc effectively avoids the leakage of Fc, retains its electrochemical activity, and enhances the conductivity of the composite. The adsorption of Au NPs onto the PAA-Fc matrix provides sites for the immobilization of the antigen and a favorable micro-environment to maintain its activity. The morphologies and electrochemistry of the sensing film were investigated via scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Factors influencing the performance of the immunosensor were studied in detail. The concentration of the antigen can be quantitated (by measuring the decrease of the amperometric response resulting from the specific binding between antigen and antibody) in the range between 0.1 and 150 ng mL, with a detection limit of 40 pg mL (S/N = 3). The method is economical, efficient, and potentially attractive for clinical immunoassays. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: In this work, the magnetic core–shell Fe3O4@Au nanoparticles attached to the surface of a magnetic glassy carbon electrode (MGCE) were applied to the immobilization/adsorption of myoglobin (Mb) for fabricating Mb/Fe3O4@Au biofilm. The morphology, structure, and electrochemistry of the nanocomposite were characterized by transmission electron microscope, UV–vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry, respectively. The resultant Fe3O4@Au NPs not only have the magnetism of Fe3O4 NPs that make them easily manipulated by an external magnetic field, but also have the good conductivity and excellent biocompatibility of Au layer which can maintain the bioactivity and facilitate the direct electrochemistry of Mb in the biofilm. The modified electrode based on this Mb/Fe3O4@Au biofilm displayed good electrocatalytic activity to the reduction of H2O2 with a linear range from 1.28 to 283μM. The proposed method simplified the immobilization methodology of proteins and showed potential application for fabricating novel biosensors and bioelectronic devices. [Copyright &y& Elsevier]

Abstract: A novel experimental methodology based on the unique characteristics of chitosan-branched ferrocene (CS-Fc) and gold nanoparticles (Au NPs) was developed to design a label-free amperometric immunosensor for the sensitive detection of hepatitis B surface antigen (HBsAg) as a model protein. The controllable electrodeposition of CS-Fc solution formed a three-dimensional robust film with good biocompatibility and large surface area for the assembly of Au NPs and further immobilization of hepatitis B surface antibody (HBsAb) on an electrode. The morphologies and electrochemistry of the formed nanocomposite biofilm were investigated by using scanning electron microscopy and electrochemical techniques including cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The HBsAg concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltametric values were proportional to the HBsAg concentration in the range of 0.05–305ngmL−1 with a detection limit 0.016ng. This would provide an approach for the application of mediator in immunoassays. [Copyright &y& Elsevier]

Abstract: A novel dopamine sensor was fabricated by forming the 6-ferrocenylhexanethiol (HS(CH2)6Fc) functionalized Fe3O4@Au nanoparticles (NPs) films on the surface of a carbon paste electrode with the aid of a permanent magnet. HS(CH2)6Fc, which acted as the redox mediator, was self-assembled to Fe3O4@Au NPs via Au–S bond. Transmission electron microscopy, UV–visible absorption spectroscopy, Fourier transform infrared spectra, and cyclic voltammetry were used to characterize the properties of the Fe3O4@Au NPs/HS(CH2)6Fc nanocomposite. It is shown that the prepared ferrocene-functionalized Fe3O4@Au NPs composite shuttled electrons between analyte and electrode, increased the mediator loading, and more importantly prevented the leakage of the mediator during measurements, which resulted in the substantially enhanced stability and reproducibility of the modified electrode. The electrooxidation of dopamine could be catalyzed by Fc/Fc+ couple as a mediator and had a higher electrochemical response due to the unique performance of Fe3O4@Au NPs. The nanocomposite modified electrode exhibited fast response (3s) and the linear range was from 2.0×10−6 to 9.2×10−4 M with a detection limit of 0.64μM. This immobilization approach effectively improved the stability of the electron transfer mediator and is promising for construction of other sensors and bioelectronic devices. [Copyright &y& Elsevier]

Abstract: A kind of nanocomposite with good dispersion in water was prepared through covalent adsorption of ferrocenecarboxaldehyde on multiwalled carbon nanotubes (MWNTs) for electrical communication between glucose oxidase (GOD) and electrode. The ferrocene-modified multiwalled carbon nanotube nanocomposites (MWNTs-Fc) could be conveniently cast on electrode surfaces. With the aid of chitosan, GOD was then immobilized on the nanostructure film to form a reagentless amperometric sensor for glucose determination. FTIR spectra and cyclic voltammetry were used to characterize the nanocomposites. The presence of both ferrocene as mediator of electron transfer and MWNTs as conductor enhanced greatly the enzymatic response to the oxidation of glucose. The novel biosensor exhibited a fast response toward glucose with a detection limit of 3.0×10−6 mol/L and the linear range extended up to 3.8×10−3 mol/L. [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]

• We review enzyme-free nucleic acid amplification for DNA repair enzyme imaging. • Their sensing mechanisms and practical applications are highlighted. • The performances of different approaches are compared. • We discuss the potential challenges and future perspectives in this field. To maintain the stability and integrity of cellular genomic DNA, DNA repair enzymes catalyze the repair of DNA damage caused by exogenous and endogenous factors through various DNA repair pathways (e.g., base excision repair pathway). Abnormal DNA repair enzyme activity hampers the timely repair of DNA damage, inducing cell growth stagnation, cell death, transformation of normal cells into tumor cells and eventually the occurrence of human diseases including cancer. DNA repair enzymes have become the valuable biomarkers and potential therapeutic targets in biomedical research and clinical diagnosis. To accurately monitor DNA repair enzymes with low expression levels in cells, signal amplification strategies have been introduced to achieve reliable signal sensing and signal gain, facilitating diagnosis and treatment of malignant diseases. Conventional strategies for amplified detection of DNA repair enzymes involve multiple enzymes, washing/separation steps, and thermal cycling, and they are unable to provide spatiotemporal information of intracellular DNA repair enzymes. In recent years, some enzyme-free nucleic acid amplification approaches have been introduced for real-time imaging of intracellular DNA repair enzymes, providing new insights into the dynamic changes of DNA repair enzymes at the single-cell level. Herein, we review the recent advances in enzyme-free nucleic acid amplification-based fluorescent biosensors for the imaging of intracellular DNA repair enzymes. We highlight the mechanisms and applications of these enzyme-free nucleic acid amplification approaches, and further compare the performances of different enzyme-free nucleic acid amplification approaches for DNA repair enzyme imaging. Furthermore, we discuss the potential challenges and future perspectives in this field. [ABSTRACT FROM AUTHOR]

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease Dpn II and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons. [Display omitted] • We construct a new biosensor for label-free detection of FTO activity. • This biosensor exhibits high sensitivity and good specificity. • This biosensor can detect the FTO activity in a single MCF-7 cell. • It can screen inhibitors and measure enzyme the kinetic parameters. • It can discriminate breast cancer tissues from healthy person tissues. [ABSTRACT FROM AUTHOR]

DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 − 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors. [Display omitted] • We review single-molecule counting-based biosensors for DNA-modifying enzyme detection. • The principles and applications of these biosensors are highlighted. • The performances of different biosensors are compared. • We discuss the future challenges and perspectives in this field. [ABSTRACT FROM AUTHOR]

Abstract An electrochemiluminescence (ECL) biosensor based on Au NPs enhanced ECL signal of S 2 O 8 2−–O 2 system has been constructed for ultrasensitive detection of protein kinase activity. In the presence of ATP-s and protein kinase A (PKA), Au NPs can be captured on the thoil-phosphorylated peptides modified electrode surface, generating an enhanced ECL emission of S 2 O 8 2−–O 2 system. With increasing the PKA activity, more Au NPs can be captured on the modified electrode surface, resulting in the gradually enhanced ECL intensity of S 2 O 8 2−–O 2 system. Based on the Au NPs mediated the ECL amplification of S 2 O 8 2−–O 2 system, the activity of PKA can be detected sensitively with a limit of detection of 0.0002 U/mL, which is much lower than the most sensitive method in previous reports. The good conductivity and high catalytic ability of the Au NPs are revealed to account for the enhanced sensitivity in this ECL biosensor. The ECL biosensor has been successfully used for monitoring PKA activity in biological samples and screening of protein kinase inhibition. Graphical abstract ECL biosensor based on the Au NPs mediated ECL amplification of the S 2 O 8 2−–O 2 system has been designed for sensitive PKA activity detection and inhibitor screening. Unlabelled Image Highlights • ECL biosensor based on the Au NPs mediated ECL amplification of S 2 O 8 2−–O 2 has been designed. • Ultrasensitive detection of PKA activity and protein kinase inhibitor screening are realized. • Due to the good conductivity and catalytic activity of Au NPs, ECL signal for PKA detection is amplified. • This work presences an excellent PKA biosensor with a low detection limit down to 0.0002 U/mL. [ABSTRACT FROM AUTHOR]

The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10−17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery. We develop a single quantum dot-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues. [Display omitted] • We develop a single quantum dot-based FRET biosensor. • This biosensor can sensitively detect the METTL3/14 complex activity. • It can measure the METTL3/14 complex activity in a single cell. • This biosensor can evaluate kinetic parameters and screen inhibitors. • It can discriminate breast cancer patient tissues from healthy tissues. [ABSTRACT FROM AUTHOR]

We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA’), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (I Thi /I Fc ), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11 aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Highlights: [•] We reported a novel ECL biosensor for sensitive analysis of protein kinase activity. [•] GOx/Au NPs/DNA-biotin was used as the ECL signal amplification nanoprobe. [•] This strategy can in situ generate coreactant H2O2 to improve the luminol ECL signal. [•] Highly sensitive detection of PKA activity and inhibition is achieved. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a novel biomolecule immobilization approach has been proposed to the synthesis of multi-functional core–shell glucose oxidase–Au–polydopamine–Fe3O4 magnetic bionanoparticles (GOx–Au–PDA–Fe3O4 MBNPs) using the one-pot chemical polymerization method. Then, a high performance biosensor has been constructed by effectively attaching the proposed GOx–Au–PDA–Fe3O4 MBNPs to the surface of the magnetic glassy carbon electrode. Scanning electron microscope, energy dispersive x-ray spectrometer, UV–vis spectroscopy, and electrochemical methods were used to characterize the GOx–Au–PDA–Fe3O4 MBNPs. The resultant GOx–Au–PDA–Fe3O4 MBNPs not only have the magnetism of Fe3O4 nanoparticles which makes them easily manipulated by an external magnetic field, but also have the excellent biocompatibility of PDA to maintain the native structure of the GOx, and good conductivity of Au nanoparticles which can facilitate the direct electrochemistry of GOx in the biofilm. Hence, the present GOx–Au–PDA–Fe3O4 biofilm displays good linear amperometric response to glucose concentration ranging from 0.02 to 1.875mM. This efficient biomolecule immobilization platform is recommended for the preparation of many other MBNPs with interesting properties and application potentials in many fields, such as biosensing, biocatalysis, biofuel cells, and bioaffinity separation. [Copyright &y& Elsevier]

Abstract: A novel “smart” electrochemical affinity nanobiosensor with “on-off” switchable property was designed for the ultrasensitive determination of glucose. The sensing approach was based on the glucose-ConA-dextran competitive system induced charge evolution in the use of graphene oxide (GO) as transducer element, resulting in the enhancement of interfacial electron transfer kinetics between the redox probe and the electrode. As concanavalin A (ConA) constituent was pH-sensitive, when the ConA-DexP/GO film electrode switched in probe Fe(CN)6 3−/4− solution between pH 4.0 and 8.0, the film was cycled between the “on” and “off” states by the electrostatic attraction and repulsion of Fe(CN)6 3−/4− to and from the electrode surface. Upon introduction of glucose into the ConA-DexP/GO complex at the “off” state, glucose competed with DexP for ConA and displaced ConA from the GO platform, resulting in gradual decrease of the surface negative charge as well as the resistance of probe for electron communication on the sensor surface, and making the switching from “off” state to “on” state simultaneously. This ultrasensitive glucose nanobiosensor had a broad linearity between the decrease in electron transfer resistance (ΔR) and the glucose concentration over a range from 5.0μM to 9.0mM with a detection limit as low as 0.34μM. The proposed method showed potential application for fabricating novel biosensors and bioelectronic devices. [Copyright &y& Elsevier]

Abstract: In this study, bifunctional Fe3O4@ZrO2 magnetic core–shell nanoparticles (NPs), synthesized by a simple and effective sonochemical approach, were attached to the surface of a magnetic glassy carbon electrode (MGCE) and successfully applied to the immobilization/adsorption of myoglobin (Mb) for constructing a novel biosensor platform. With the advantages of the magnetism and the excellent biocompatibility of the Fe3O4@ZrO2 NPs, Mb could be easily immobilized on the surface of the electrode in the present of external magnetic field and well retained its bioactivity, hence dramatically facilitated direct electron transfer of Mb was demonstrated. The proposed Mb/Fe3O4@ZrO2 biofilm electrode exhibited excellent electrocatalytic behaviors towards the reduction of H2O2 with a linear range from 0.64μM to 148μM. This presented system avoids the complex synthesis for protecting Fe3O4 NPs, supplies a simple, effective and inexpensive way to immobilize protein, and is promising for construction of third-generation biosensors and other bio-magnetic induction devices. [Copyright &y& Elsevier]

A new electrochemical biosensor for sensitive detection of telomerase activity based on the hybridization chain reaction and the dual interaction of the poly-adenine (polyA) DNA with Au electrode and Ce-Ti dioxide nanorods (Ce-Ti NRs) has been constructed. Deoxynucleotide triphosphates (dNTPs) can be incorporated at the 3´-OH of TS primer by telomerase to generate the extension products with telomeric repeats (TTAGGG) n (TEP), which can hybridize with hairpin (HP) DNA and open it to form a duplex DNA with a short poly-adenine DNA single-stranded tail (s1). The duplex DNA can be recognized and selectively cleaved into single-stranded DNA fragment s1 and TEP by exonuclease III (Exo III). The released TEP can then hybridize with the HP again to initiate the s1 recycling process, which will result in the massive release of s1 fragments from HP. The s1 fragments can be immobilized on the Au electrode surface through the interaction between polyA and Au electrode on one hand. On the other hand, Ce-Ti NRs can be adsorbed on the Au electrode surface by the interaction between Ce-Ti NRs and the phosphate groups of s1, resulting in the decrease of the differential pulse voltammetry (DPV) current of the redox probe. The higher activity of telomerase, more HP can be hybridized and cleaved, more poly-adenine and Ce-Ti NRs can be immobilized on electrode surface, and remarkable decrease of the DPV signal can be observed. The biosensor exhibited good sensitivity toward telomerase activity in HeLa cells. Unlabelled Image • A novel electrochemical biosensor for sensitive detection of telomerase activity has been designed. • HCR and dual interaction of polyA DNA with Au and Ce-Ti NRs were greatly amplified the DPV signal. • Sensitive detection of telomerase activity with a low detection limit was achieved. • The method can be used for qualitative screening of telomerase inhibitor. [ABSTRACT FROM AUTHOR]