The present work investigates the capability of single-stranded DNA (ssDNA) in enhancing the intrinsic peroxidase-like activity of the g-C3N4 nanosheets (NSs). We found that ssDNA adsorbed on g-C3N4 NSs could improve the catalytic activity of the nanosheets. The maximum reaction rate of the H2O2-mediated TMB oxidation catalyzed by the ssDNA-NSs hybrid was at least 4 times faster than that obtained with unmodified NSs. The activity enhancement could be attributed to the strong interaction between TMB and ssDNA mediated by electrostatic attraction and aromatic stacking and by both the length and base composition of the ssDNA. The high catalytic activity of the ssDNA-NSs hybrid permitted sensitive colorimetric detection of exosomes if the aptamer against CD63, a surface marker of exosome, was employed in hybrid construction. The sensor recognized the differential expression of CD63 between the exosomes produced by a breast cancer cell line (MCF-7) and a control cell line (MCF-10A). Moreover, a similar trend was detected in the circulating exosomes isolated from the sera samples collected from breast cancer patients and healthy controls. Our work sheds lights on the possibility of using ssDNA to enhance the peroxidase-like activity of nanomaterials and demonstrates the high potential of the ssDNA-NSs hybrid in clinical diagnosis using liquid biopsy.

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.

Light-up RNA aptamers are valuable tools for fluorescence imaging of RNA in living cells and thus for elucidating RNA functions and dynamics. However, no light-up RNA sensor has been reported for imaging of microRNAs (miRs) in mammalian cells. We report a novel genetically encoded RNA sensor for fluorescent imaging of miRs in living tumor cells using a light-up RNA aptamer that binds to sulforhodamine and separates it from a conjugated contact quencher. On the basis of the structural switching mechanism for molecular beacon, we show that the RNA sensor activates high-contrast fluorescence from the sulforhodamine-quencher conjugate when its stem–loop responsive motif hybridizes with target miR. The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to miR target. We also realize the RNA sensor for dual-emission, ratiometric imaging by coexpression of RNA sensor with GFP, enabling quantitative studies of target miR in living cells. Our design may provide a new paradigm for developing robust, sensitive light-up RNA sensors for RNA imaging applications.

AbstractDNAzymes 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.

In recent years, many activatable fluorescent probes have been developed for hNQO1 detection. However, most of the reported fluorescent probes are susceptible to the interferences of endogenous fluorescence and have the drawback of inadequate penetration depth. Very recently, researchers have reported a two-photon excitation (TPE) fluorescent probe for hNQO1 detection. Nevertheless, this probe only exhibits a compromised signal-to-background ratio, and has not been applied to image hNQO1 in living tissues. Herein, a novel TPE fluorescent probe, trimethyl locked quinone caged Acedan (Q3CA-P), has been developed for hNQO1 detection and imaging in living cells and tissues. Q3CA-P displays over 25-fold enhancement in fluorescence intensity toward hNQO1 with a Stokes shift over 100 nm in one-photon excitation and exhibits a very low detection limit of 5.6 ng mL−1. The imaging experiments performed in tumour cells and tissue slices using Q3CA-P demonstrate that Q3CA-P could image the endogenous hNQO1 with high selectivity and sensitivity with a TPE probing depth of 120 μm. Thus, our probe may have great potential for use in cancer diagnosis and image-guided surgery.

Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better understanding their biological functions. Since there are a few fluorescent probes concerning NTR activity specifically located in mitochondria, we developed a novel fluorescent benzoindocyanine probe (BICP) for mitochondrial NTR activity monitoring and imaging via extending a benzoindole moiety into a benzoindocyanine based fluorophore (BICF) with a strong intramolecular charge transfer (ICT) effect and incorporating 4-nitrobenzyl as a fluorescence-quenching and enzyme-responsive moiety. Live cell imaging of HeLa and A549 demonstrates that the developed BICP is able to realize sensitive and selective mitochondrial NTR activity probing with high-contrast “off–on” fluorescence. These findings implied the great potential of the developed probe for monitoring mitochondrial-specific NTR activities in living cells and related applications in cell biology.

AbstractDNAzymes 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.

•Highly efficient assembly of DNA nanostructure on nanopore membrane utilizing hybridization chain reaction.•A label-free nanopore biosensor has been developed utilizing hybridization chain reaction as signal amplification strategy.•Significant improvement of sensitivity has been achieved compared to conventional amplification-free nanopore biosensor.•It may provide a new paradigm for the design of nanopore biosensor utilizing nucleic acid-based amplification strategy.A label-free nanopore biosensor for detection of DNA target is proposed utilizing hybridization chain reaction (HCR) strategy for signal amplification. The DNA target triggered HCR to form large DNA nanostructure inside the nanopore and out the nanopore membrane, which inducing the ionic current decrease effectively due to the blockage of the nanopore. The developed method achieves a desirable sensitivity of 30 fM with a wide linear dynamic range from 0.1 to 10 pM and demonstrated good application for real sample analysis. This work has great potential to be applied in the early diagnosis of gene-related diseases and provide a new paradigm for label-free nucleic acid amplification strategy in ultrasensitive nanopore biosensor.A label-free nanopore biosensor has been developed for the detection of DNA target with high sensitivity and specificity utilizing hybridization chain reaction as signal amplification strategy.Download high-res image (135KB)Download full-size image

•A silica nanoparticle-based dual-responsive ratiometric fluorescent sensor was synthesized.•The sensor is responsive to ClO− and temperature independently and sensitively.•The detection limit of sensor is 26 nM for ClO−.•The sensor can realize monitoring of ClO− in human serum and live cells.Hypochlorite (ClO−) and temperature play crucial roles in a wide range of physiological processes, and they are also implicated in various diseases, including cancer, inflammation of tissues and so on. Therefore, it is of great importance to explore a novel method to detect ClO− and temperature instantly. In this study, we developed a silica nanoparticle-based dual-responsive ratiometric fluorescent sensor (DRFS), whose correlative dual emissions can response to ClO− and temperature independently and sensitively. The detection limit of DRFS can reach to as low as 26 nM for the detection of ClO−. And further research demonstrates that DRFS possesses excellent anti-interference feature when other possible interferents exist, and has been successfully applied in ClO− detection in human serum and recognition of exogenous/endogenous ClO− in HeLa cells and macrophages by fluorescence microscopic imaging. Moreover, DRFS can also be used as a ratiometric temperature sensor, and the fluorescence intensity ratio (I576/I445) exhibits a linear temperature response in the range from 20 to 60 °C with a change ratio as large as a factor of 5. Based on the above research, the DRFS can be used as versatile fluorescence sensor in various physiological and environmental systems.Download high-res image (116KB)Download full-size imageA dual-response ratiometric fluorescent biosensor based on silica nanoparticle was synthesized. The sensor exhibited the attractive property of independent dual-response, is not only a ratiometric fluorescent sensor for sensitively and selectively detecting ClO−, but also a ratiometric fluorescence thermometers with good cycling capability towards temperature. More importantly, the detection limit of sensor for detecting ClO− can reach as low as 26 nM, and the sensor has been proved to be a an effective and outstanding fluorescent ClO− sensor in biological system (human serum) and for microscopic cell imaging.

•A label-free method for UDG detection is proposed.•UDG initiates strand displacement DNA synthesis among three designed hairpin probes.•The established strategy is robust, cost-effective, and highly sensitive.•This method can be used to screen UDG inhibitors and displays good performance for UDG detection in real samples.Uracil-DNA glycosylase (UDG) plays essential roles in base excision repair (BER) pathway by eliminating uracil from DNA to sustain the genome integrity. Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications. We develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification (SPEA). In the presence of active UDG, the uracil base in helper hairpin probe (HP) can be excised to generate an abasic site (AP site), which can be cleaved by endonuclease IV (Endo IV) with a blocked primer released. This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA, which can initiate a loop-mediated isothermal amplification (LAMP) reaction. This reaction generates a large number of long double-strand DNA replicates, which can be stained by SYBR Green (SG) I to deliver enhanced fluorescence for quantitative detection of UDG activity. A linear range from 0.001 U/mL to 1 U/mL and a detection limit down to 0.00068 U/mL are achieved. This strategy has also been demonstrated for UDG assay in complex cell lysates, implying its great potential for UDG based clinical diagnostics and therapeutics.Download high-res image (163KB)Download full-size image

A novel microfluidic paper-based analytic device (μPAD) biosensor is developed for sensitive and visualized detection of glucose. This biosensor is easily fabricated using the wax printing technique, with a hybrid nanocomplex composed of dual enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) and Cu3(PO4)2 inorganic nanocrystals incorporated in the detection zones. The hybrid nanocomplex is found to exhibit a flower-like structure, which allows co-immobilization of these two enzymes in a biocompatible environment. These nanoflowers not only preserve the activity and enhance the stability of the enzymes, but also facilitate the transport of the substrates between the two enzymes. The biosensor is demonstrated to enable rapid and sensitive quantification of glucose in the concentration range of 0.1–10 mM with a limit of detection (LOD) of 25 μM. It is also shown to be applicable to colorimetric quantitative detection of glucose in human serum and whole blood samples, implying its potential for clinical applications.

A commercially available fluorescent hydrogen sulfide (H2S) probe 7-azido-4-methylcoumarin (AzMC) was developed into its lysosome-targeted counterpart Lyso-C via a four-step synthetic approach. Lyso-C displayed fast response (within 5 min), excellent sensitivity (with a detection limit of 37 nM) and high selectivity toward H2S. More importantly, Lyso-C was successfully applied to imaging lysosomal H2S and showed potential capability to quantitatively detect H2S in living cells.

6-(Diethylamino)-2,3-dihydro-1H-xanthene-4-carbaldehyde (DDXC), a reported synthetic intermediate for near-infrared fluorescent dyes, was developed into a fluorescent pH probe for extreme acidity. The unique sensing mechanism of DDXC for pH is based on the reversible protonation of the carbonyl oxygen followed by keto–enol tautomerization. The probe displays a linear ratiometric fluorescence response (I512/I580) to H+ over the extremely acidic range of pH 2.0–4.0 with a pKa of 3.11, and features high fluorescence quantum yield (Φ = 0.60) and excellent selectivity. More importantly, the probe can be applied to ratiometric fluorescence imaging of pH changes in living cells, making it a potential molecular tool for pH-related cell biology study.

Enzyme-free, signal-amplified nucleic acid circuits utilize programmed assembly reactions between nucleic acid substrates to transduce a chemical input into an amplified detection signal. These circuits have shown great potential for developing biosensors for high-sensitivity and high-selectivity detection of varying targets including nucleic acids, small molecules and proteins in vitro and for high-contrast in situ visualization and imaging of these targets in tissues and living cells. We review the background of the enzyme-free, signal-amplified nucleic acid circuits, including their mechanism, significance, types and development. We also review current applications of these circuits for biosensors and bioimaging.

A fluorescence aptasensor is described that combines target-cycling strand displacement amplification (TCSDA) and synthesis of copper nanoclusters (CuNCs) templated with double-stranded DNA (dsDNA). Specifically, the detection scheme was applied to the determination of adenosine-5′-triphosphate (ATP) via target-induced structure switching design. The binding of an aptamer hairpin probe (AP) to ATP induces a structural switch from a hairpin shape to an open conformation. This facilitates hybridization with a primer and triggers a TCSDA reaction. This amplification step produces a large quantity of dsDNA that can directly act as a template for the synthesis of fluorescent CuNCs, thereby producing a strong red fluorescence (with excitation/emission maxima at 340/598 nm) that can be used to quantify ATP. The assay has a dynamic range that extends over 4 decades (from 0.01 nM to 100 nM) and a 5 pM detection limit. Conceivably, this detection scheme is applicable to numerous other analytes for which suitable aptamers are available.

Cobalt oxyhydroxide (CoOOH) nanoflakes, an emerging type of two-dimensional nanomaterial, show great potential for use in molecular detection. Previous assays utilizing such materials have largely been based on their outstanding fluorescence quenching ability and oxidizing power. Herein, we report the intrinsic peroxidase-like activity of cobalt oxyhydroxide (CoOOH) nanoflakes, and we show how this activity can be employed for glucose detection. We found that, in the presence of hydrogen peroxide (H2O2), the nanoflakes accelerated the conversion of peroxidase substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) into colored products. By combining the CoOOH nanoflakes with the biological enzyme glucose oxidase (GOx), we developed a colorimetric method for the detection of glucose within the concentration range 5.3–500 μM. The proposed method was applied to detect elevated blood glucose levels in diabetic patients, and the intense color change induced by elevated glucose levels was found to be readily apparent to the naked eye, proving the utility of our assay for point-of-care testing.

•This is the first fluorescent hydrazine probe based on nucleophilic aromatic substitution of hydrazine toward aryl 4-nitrobenzenesulfonate.•The probe exhibits a large Stokes shift, high selectivity and sensitivity for hydrazine with a detection limit of 0.716 ppb (22 nM).•The probe displays excellent cell permeation and low cytotoxicity and has been successfully applied to imaging hydrazine in living cells.A turn-on fluorescent probe (Naphsulf-O) for hydrazine was developed by protecting the hydroxy group of the fluorophore 6-acetyl-2-hydroxynaphthalene via O-4-nitrobenzenesulfonylation, where 4-nitrobenzene was used as a fluorescence quenching moiety as well as an electrophile. Upon nucleophilic aromatic substitution (NAS) reaction of hydrazine toward the probe, the protecting group was removed and fluorophore was released. The probe exhibits a large Stokes shift, excellent selectivity and high sensitivity for hydrazine detection in aqueous solution with a detection limit of 0.716 ppb (22 nM), which is of great importance in both environmental and biological system. Furthermore, it was successfully applied to imaging of hydrazine in living cells.

Graphitic C3N4 (g-C3N4) nanosheets provide an attractive option for bioprobes and bioimaging applications. Utilizing highly fluorescent and water-dispersible ultrathin g-C3N4 nanosheets, a highly sensitive, selective and label-free biosensor has been developed for ALP detection for the first time. The developed approach utilizes a natural substrate of ALP in biological systems and thus affords very high catalytic efficiency. This novel biosensor is demonstrated to enable quantitative analysis of ALP in a wide range from 0.1 to 1000 U L−1 with a low detection limit of 0.08 U L−1, which is among the most sensitive assays for ALP. It is expected that the developed method may provide a low-cost, convenient, rapid and highly sensitive platform for ALP-based clinical diagnostics and biomedical applications.

We developed novel activatable probe using self-assembled peptide nanowires with low affinity and toxicity to tumor cells in the absence of matrix metalloproteinase that showed activated high affinity and toxicity and provided a highly selective and efficient platform for targeted therapy and tumor imaging.

We report the development of a novel plasmon coupling enhanced Raman scattering (PCERS) method, PCERS nanobeacon, for ultrasensitive, single-step, homogeneous detection of cholera toxin (CT). This method relies on our design of the plasmonic nanoparticles, which have a bilayer phospholipid coating with embedded Raman indicators and CT-binding ligands of monosialoganglioside (GM1). This design allows a facile synthesis of the plasmonic nanoparticle via two-step self-assembly without any specific modification or chemical immobilization. The realization of tethering GM1 on the surface imparts the plasmonic nanoparticles with high affinity, excellent specificity, and multivalence for interaction with CT. The unique lipid-based bilayer coated structure also affords excellent biocompatibility and stability for the plasmonic nanoparticles. The plasmonic nanoparticles are able to show substantial enhancement of the surface-enhanced Raman scattering (SERS) signals in a single-step interaction with CT, because of their assembly into aggregates in response to the CT-sandwiched interactions. The results reveal that the developed nanobeacon provides a simple but ultrasensitive sensor for rapid detection of CT with a large signal-to-background ratio and excellent reproducibility in a wide dynamic range, implying its potential for point-of-care applications in preventive and diagnostic monitoring of cholera.

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.

High-throughput and sensitive detection of proteins are essential for clinical diagnostics and biomarker discovery. We develop a novel high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay method, which utilizes antibody-modified phospholipid bilayer coated gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized magnetic beads as the capture reagent. This method enables magnetic enrichment of the PBL-AuNPs label specific to target protein, allowing sensitive surface enhanced laser desorption ionization (SELDI)-TOF MS detection of the protein via its specific label. AuNPs act as not only the support but also the matrix for the phospholipids in SELDI TOF MS detection. Moreover, with phospholipids with varying molecular weights as the encoded MS reporters, this method allows multiplexed detection of multiple proteins. With the use of a predefined phospholipids internal standard, this method also affords excellent reproducibility in protein quantification. We have demonstrated this method using the assays of two tumor biomarkers, and the results reveal that it provides a sensitive platform for multiplexed protein detection with detection limits in the picomolar ranges. This method may provide a useful platform for high-throughput and sensitive detection of protein biomarkers for clinical diagnostics.

Small molecule probes suitable for high-resolution fluorescence imaging of enzyme activity pose a challenge in chemical biology. We developed a novel design of activity localization fluorescence (ALF) peptide probe, which enables spatially resolved, highly sensitive imaging of peptidase in live cells. The ALF probe was synthesized by a facile thiol-ene click reaction of a cysteine-appended peptide with an acryloylated fluorophore. Upon cleavage by peptidase, the probe undergoes a seven-membered intramolecular cyclization and releases the fluorophore with the excited-state intramolecular photon transfer (ESIPT) effect. A highly fluorescent, insoluble aggregate was formed around the enzyme, which facilitates high-sensitivity and high-resolution imaging. This design is demonstrated for detection of caspase-8 activation. The results show that our design allows easy, high-yield synthesis of the probe, and the probe affords high sensitivity for caspase-8 detection. Live cell imaging reveals that the probe is able to render highly localized and high-contrast fluorescence signal for caspase-8. Our design holds the potential as a generally applicable strategy for developing high-sensitivity and high-resolution imaging peptide probes in cell biology and diagnostics.

Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

A novel fluorescent nanosensor has been developed by combining super fluorescence quenching ability of graphene oxide and hybridization chain reaction amplification, which enables highly sensitive detection of base excision repair enzyme activity with a wide dynamic range from 0.0001 to 100 U mL−1 and a detection limit of 0.00006 U mL−1.

A novel fluorescent sensor using graphene oxide (GO)–peptide nanoassembly is developed for histone deacetylases (HDACs) based on deacetylation mediated cleavage of substrate peptides, which provides a simple, cost-effective platform for monitoring the activity of HDACs.

A simple and highly sensitive fluorometric method has been developed for inorganic pyrophosphatase (PPase) activity detection based on the disaggregation and aggregation of graphene quantum dots (GQDs). Copper ions can trigger the severe aggregation of GQDs with rich carboxyl groups, which results in effective fluorescence quenching. While, with the addition of pyrophosphate (PPi), the quenched fluorescence is effectively recovered owing to the strong interaction between PPi and Cu2+. Furthermore, under the catalytic hydrolysis of PPase, the complex of PPi–Cu2+–PPi is rapidly disassembled, and the fluorescence is re-quenched. This method is highly sensitive and selective for PPase detection, with a linear correlation between the fluorescence intensity and the PPase concentration in the range from 1 to 200 mU mL−1 with a detection limit down to 1 mU mL−1 (S/N = 3). Additionally, the inhibition effect of NaF on the PPase activity is also studied. Thus, the proposed method may hold a potential application in the diagnosis of PPase-related diseases and screening of PPase inhibitors, to evaluate the function and inhibition of PPase in biological systems.

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.

The design of effective tools capable of sensing lysosome pH is highly desirable for better understanding its biological functions in cellular behaviors and various diseases. Herein, a lysosome-targetable ratiometric fluorescent polymer nanoparticle pH sensor (RFPNS) was synthesized via incorporation of miniemulsion polymerization and surface modification technique. In this system, the donor: 4-ethoxy-9-allyl-1,8-naphthalimide (EANI) and the acceptor: fluorescein isothiocyanate (FITC) were covalently linked to the polymer nanoparticle to construct pH-responsive fluorescence resonance energy transfer (FRET) system. The FITC moieties on the surface of RFPNS underwent structural and spectral transformation as the presence of pH changes, resulting in ratiometric fluorescent sensing of pH. The as-prepared RFPNS displayed favorable water dispersibility, good pH-induced spectral reversibility and so on. Following the living cell uptake, the as-prepared RFPNS with good cell-membrane permeability can mainly stain in the lysosomes; and it can facilitate visualization of the intracellular lysosomal pH changes. This nanosensor platform offers a novel method for future development of ratiometric fluorescent probes for targeting other analytes, like ions, metabolites, and other biomolecules in biosamples.A lysosome-targetable ratiometric fluorescent polymer nanoparticle pH sensor (RFPNS) was synthesized via incorporation of miniemulsion polymerization and surface modification technique. The as-prepared RFPNS displayed favorable water dispersibility, good pH-induced spectral reversibility and so on. Following the living cell uptake, the as-prepared RFPNS with good cell-membrane permeability can mainly stain in the lysosomes; and it can facilitate visualization of the intracellular lysosomal pH changes.

Herein we report for the first time a hybridization chain reaction (HCR) lightened by DNA-stabilized silver nanoclusters (AgNCs) as a label-free and turn on fluorescence platform for nucleic acid assays in a homogeneous format.

G-rich DNA sequences could be promoted to form G-quadruplex structures in the presence of a water-soluble fluorogenic dye, thioflavin T (ThT), which is weakly fluorescent in the free state, but exhibits obvious fluorescence enhancement once it binds to G-quadruplex structures with high specificity. We developed a novel approach using G-quadruplex-specific fluorescence enhancement of ThT for the label-free detection of Ag+ and biothiols. This approach relies on the coordination of Ag+ with guanine, which inhibits the formation of the G-quadruplex structure and delivers a quenched fluorescence signal, and the stronger coordination of biothiols with Ag+, which releases Ag+ from guanine and restores the G-quadruplex with an activated fluorescence. This “turn-off/on” biosensor may provide a label-free, robust, yet sensitive platform for the detection of Ag+ and GSH.

Gold nanoparticles (AuNPs) have been extensively used in optical biosensing and bioimaging due to the unique optical properties. Biological applications including biosensing and cellular imaging based on optical properties of AuNPs will be reviewed in the paper. The content will focus on detection principles, advantages and challenges of these approaches as well as recent advances in this field.

Graphitic carbon nitride (g-C3N4) nanosheets, an emerging graphene-like carbon-based nanomaterial with high fluorescence and large specific surface areas, hold great potential for biosensor applications. Current g-C3N4 nanosheets based fluorescent biosensors majorly rely on single fluorescent intensity reading through fluorescence quenching interactions between the nanosheets and metal ions. Here we report for the first time the development of a novel g-C3N4 nanosheets-based ratiometric fluorescence sensing strategy for highly sensitive detection of H2O2 and glucose. With o-phenylenediamine (OPD) oxidized by H2O2 in the presence of horseradish peroxidase (HRP), the oxidization product can assemble on the g-C3N4 nanosheets through hydrogen bonding and π–π stacking, which effectively quenches the fluorescence of g-C3N4 while delivering a new emission peak. The ratiometric signal variations enable robust and sensitive detection of H2O2. On the basis of the glucose converting into H2O2 through the catalysis of glucose oxidase, the g-C3N4-based ratiometric fluorescence sensing platform is also exploited for glucose assay. The developed strategy is demonstrated to give a detection limit of 50 nM for H2O2 and 0.4 μM for glucose, at the same time, it has been successfully used for glucose levels detection in human serum. This strategy may provide a cost-efficient, robust, and high-throughput platform for detecting various species involving H2O2-generation reactions for biomedical applications.Keywords: glucose; graphitic carbon nitride nanosheets; H2O2; ratiometric;

Efficient approaches for intracellular delivery of nucleic acid reagents to achieve sensitive detection and regulation of gene and protein expressions are essential for chemistry and biology. We develop a novel electrostatic DNA nanoassembly that, for the first time, realizes hybridization chain reaction (HCR), a target-initiated alternating hybridization reaction between two hairpin probes, for signal amplification in living cells. The DNA nanoassembly has a designed structure with a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically assembled outer layer of fluorophore-labeled hairpin DNA probes. It is shown to have high efficiency for cellular delivery of DNA probes via a unique endocytosis-independent mechanism that confers a significant advantage of overcoming endosomal entrapment. Moreover, electrostatic assembly of DNA probes enables target-initialized release of the probes from the nanoassembly via HCR. This intracellular HCR offers efficient signal amplification and enables ultrasensitive fluorescence activation imaging of mRNA expression with a picomolar detection limit. The results imply that the developed nanoassembly may provide an invaluable platform in low-abundance biomarker discovery and regulation for cell biology and theranostics.

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

T7 exonuclease is reported for the first time to have high specificity in discriminating single-base mismatch and utilized for developing a target cyclic amplification biosensor strategy for sensitive SNP detection based on graphene oxide quenching of uncleaved probes.

MicroRNAs (miRNAs) are quite short single-stranded RNA molecules playing crucial roles in many biological processes and recognized as potential diagnostic biomarkers as well as targets for drug discovery in cancers. It has fueled a great need for the development of highly sensitive and selective detection methods for miRNAs. Many nucleic acid amplification technologies are demonstrated methods which have exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of miRNA. Herein, we review the basic principles of five types of nucleic acid amplification-based miRNA assay methods that have been established in recent three years. Sensitive miRNA detection based on polymerase chain reaction, rolling circle amplification, strand displacement amplification, duplex-specific nuclease signal amplification, and hybridization chain reaction techniques is discussed.

We developed a novel label-free biosensor for biomolecule detection based on the thioflavin T (ThT)-induced conformational change of guanine-rich oligonucleotides and self-assembled aptamer/GO nanosheet architecture. In the presence of target biomolecules, the aptamer sequence could specifically bind to the target and release from the surface of GO nanosheets to form a G-quadruplex conformation with ThT as an inducer, resulting in enhancement of fluorescence. The proposed biosensor exhibits a “turn-on” signal, which allows sensitive, selective and rapid detection of biomolecules.

Photoswitchable fluorescent polymeric nanoparticles (PFPNs) with controllable molecular weight, high contrast, biocompatibility, and prominent photostability are highly desirable but still scarce for rewritable printing, super-resolution bioimaging, and rewritable data storage. In this study, novel amphiphilic BODIPY-based PFPNs with considerable merits are first synthesized by a facile one-pot RAFT-mediated miniemulsion polymerization method. The polymerization is performed by adopting polymerizable BODIPY and spiropyran derivatives, together with MMA as monomer, and mediated by utilizing biocompatible PEO macro-RAFT agent as both control agent and reactive stabilizer. The amphiphilic BODIPY-based PFPNs not only exhibit reversibly photoswitchable fluorescence properties under the alternative UV and visible light illumination through induced intraparticle fluorescence resonance energy transfer (FRET) but also display controllable molecular weight with narrow polydispersity index (PDI), high contrast of fluorescence, tunable energy transfer efficiency, good biocompatibility, excellent photostability, favorable photoreversibility, etc. The as-prepared PFPNs are successfully demonstrated for rewritable fluorescence patterning and high-contrast dual-color fluorescence imaging of living cells, implying its potential for rewritable data storage and broad biological applications in cell biology and diagnostics.

Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. Here, we propose a simple, efficient, biocompatible, and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and semiquantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery, and tissue engineering.

MicroRNAs (miRNAs) play vital roles in physiologic and pathologic processes and are significant biomarkers for disease diagnostics and therapeutics. However, rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their short length, sequence homology, and low abundance. Herein, we report for the first time that WS2 nanosheet can exhibit differential affinity toward short oligonucleotide fragment versus ssDNA probe and act as an efficient quencher for adsorbed fluorescent probes. This finding is utilized to develop a new strategy for simple, sensitive, and selective detection of miRNA by combining WS2 nanosheet based fluorescence quenching with duplex-specific nuclease signal amplification (DSNSA). This assay exhibits highly sensitive and selective with a detection limit of 300 fM and even discriminate single-base difference between the miRNA family members. The result indicates that this simple and cost-effective strategy holds great potential application in biomedical research and clinical diagnostics.

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.

•The strategy carried out a visual assay for NAD+.•A strategy based on ligase-mediated inhibition on strand displacement amplification.•The colorimetric assay is simple, high sensitivity and high selectivity.•A novel platform for investigating cofactors, small molecules and DNA ligases.Existing strategies for detecting nicotinamide adenine dinucleotide (NAD+) or other cofactors are commonly cumbersome and moderate sensitive. We report a novel DNAzyme-based visual assay strategy for NAD+ based on ligase-mediated inhibition of the strand displacement amplification (SDA). In the presence of NAD+, the SDA can be inhibited by the ligase reaction of two primers, which can initiate the SDA reaction in the case of no ligation, resulting in a dramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that can quantitatively catalyze the formation of a colored product. Therefore, the quantitative analysis for NAD+ can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivity against most interferences and a detection limit as low as 50 pM. It also provides a universal platform for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases.

A novel label-free fluorescent biosensor platform has been developed for protease activity assay using peptide-templated gold nanoclusters (AuNCs). The biosensor was demonstrated with elastase as the model to have high sensitivity, excellent specificity, simplicity and rapidity.

A novel and label-free method for micrococcal nuclease (MNase) detection has been presented based on single-stranded DNA (ssDNA)-scaffolded fluorescent silver nanoclusters (AgNCs). The ssDNA was introduced as the substrate for MNase and also as the scaffold for the synthesis of the AgNCs. In the absence of MNase, the ssDNA was not digested. As a result, the fluorescent AgNCs were formed and exhibited strong fluorescence. In the presence of MNase, the DNA was digested, which prohibited the formation of the AgNCs due to the lack of the DNA scaffold, resulting in weak fluorescence. The fluorescence intensity exhibits a linear correlation to MNase concentration in the range of 0 U mL−1 to 2 × 10−4 U mL−1 with a detection limit of 8 × 10−6 U mL−1. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.

An electrochemical method for microRNA (miRNA) detection has been proposed with a dual signal amplification strategy. The method relies on polymerase extension and a two-step signal amplification using streptavidin–gold nanoparticle biocomplexes and alkaline phosphatase. The target miRNA can hybridize with the capture DNA template, which can act as a primer and be extended along the template in the presence of DNA polymerase and dNTPs. A biotin group was introduced into the duplex by the incorporation of biotin-11-dUTP. Thus, biotinylated alkaline phosphatase would bind to the duplex using streptavidin–gold nanoparticles as linkers, which resulted in an amplified electrochemical signal. The electrochemical signal exhibited a linear correlation to the logarithm of miRNA concentration ranging from 100 fM to 1 nM, with a detection limit of 99.2 fM. The specificity of the method allowed single-nucleotide differences between miRNA family members to be discriminated. The established biosensor displayed an excellent analytical performance towards miRNA detection and might present a convenient tool for biomedical research and clinical diagnostic applications.

•A free MATLAB toolbox for class modeling is described;•It includes ordinary, nonlinear and robust OCPLS algorithms.•Two functions are sufficient to tune and train an OCPLS model.One-class classifiers are widely used to solve the classification problems where control or class modeling of a target class is necessary, e.g., untargeted analysis of food adulterations and frauds, tracing the origins of a food with Protected Denomination of Origin, fault diagnosis, etc. Recently, one-class partial least squares (OCPLS) has been developed and demonstrated to be a useful technique for class modeling. For analysis of nonlinear and outlier-contaminated data, nonlinear and robust OCPLS algorithms are required.This paper describes a free MATLAB toolbox for class modeling using OCPLS classifiers. The toolbox includes ordinary, nonlinear and robust OCPLS methods. The nonlinear algorithm is based on the Gaussian radial basis function (GRBF), and the robust algorithm is based on the partial robust M-regression (PRM). The usage of the toolbox is demonstrated by analysis of a real data set.

•A homogeneous colorimetric biosensor is developed for phosphoinositide enzyme assay.•The biosensor relies on enzyme-activated assembly of phospholipid membrane-coated AuNPs.•This biosensor allows sensitive, visual detection of PI3K and PTEN with pM detection limits.Enzyme mediated phosphoinositide signaling plays important regulatory roles in diverse cellular processes and has close implication in human diseases. However, detection of phosphoinositide enzymes remains a challenge because of the difficulty in discriminating the phosphorylation patterns of phosphoinositide. Here we develop a novel enzyme-activated gold nanoparticles (AuNPs) assembly strategy as a homogeneous colorimetric biosensor for activity detection of phosphoinositide kinases and phosphatases. This strategy utilizes a biomimetic mechanism of phosphoinositide signaling, in which AuNP supported phospholipid membranes are constructed to mimic the cellular membrane substrate, and AuNPs modified with the pleckstrin homology (PH) domain of cytosolic proteins are designed for specific, multivalent recognition of phosphorylated phosphoinositides. This biomimetic strategy enables efficient enzymatic reactions of the substrate and highly selective detection of target enzyme. The biosensor is demonstrated for the detection of phosphoinositide 3-kinase (PI3K) and phosphatase with tensin homology (PTEN). The results revealed that it allows sensitive, rapid visual detection of the enzymes with pM detection limits and four-decade wide dynamic ranges, and is capable of detecting enzyme activities in complex cell lysate samples. This biosensor might provide a general biosensor platform for high-throughput detection of phosphoinositide enzymes with high sensitivity and selectivity in biomedical research and clinical diagnostics.

•A novel plasmonic ELISA strategy for the detection of syphilis.•Based on ELISA-mediated surface plasmon resonance of gold nanoparticles.•1000-Fold improvements in sensitivity over a conventional ELISA.•A potential method for clinic diagnosis and therapeutic monitoring of syphilis.In this report, we have developed a plasmonic ELISA strategy for the detection of syphilis. Plasmonic ELISA is an enzyme-linked immunoassay combined with enzyme-mediated surface plasmon resonance (SPR) of gold nanoparticles (AuNPs). Immune response of the Treponema pallidum (T. pallidum) antibodies triggers the acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine to produce abundant thiocholine. The positive charged thiol, in turn, alters the surface charge distribution the AuNPs and leads to the agglomeration of the AuNPs. The induced strong localized SPR effect of the agglomerate AuNPs can, thus, allow the quantitative assay of T. pallidum antibodies due to the remarkable color and absorption spectral response changes of the reaction system. The plasmonic ELISA exhibited a quasilinear response to the logarithmic T. pallidum antibody concentrations in the range of 1 pg/mL–10 ng/mL with a detection limit of 0.98 pg/mL. Such a low detection limit was 1000-fold improvements in sensitivity over a conventional ELISA. The results of plasmonic ELISA in syphilis assays of serum specimens from 60 patients agreed with those obtained using a conventional ELISA method. The plasmonic ELISA has characteristics (analyte specific, cost-effective, ease of automatic, low limit of detection) that provide potential for diagnosis and therapeutic monitoring of syphilis.

•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.Nanomaterial-based fluorescent probes represent a significant approach to intracellular detection with high spatiotemporal resolution. We review the properties of various nanomaterials that can be used for intracellular nanosensors in terms of the sensor design and the approaches to delivery of nanosensors based on engineering their surfaces. We also review general strategies for these nanosensors based on the transduction mechanisms of the fluorescence signal.

We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3′-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3′-phosphoryl end that served as the substrate for DNA 3′-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3′-phosphatases, the resulting HP with a 3′-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3′-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL−1 and 0.003 U mL−1, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.

A novel surface enhanced Raman scattering (SERS) based assay using a formaldehyde-selective reactive probe for sensitive detection of activity of histone demethylases (HDMs) by direct observation of by-product formaldehyde was reported.

We have developed a novel biosensor platform for colorimetric detection of active DNA methyltransferase/glycosylase based on terminal protection of the DNA-gold nanoparticle (AuNP) probes by mechanistically covalent trapping of target enzymes. This biosensor relied on covalent capture of target enzymes by activity-based DNA probes which created terminal protection of the DNA probes tethered on AuNPs from degradation by Exo I and III. This biosensor has the advantages of having highly sensitive, rapid, and convenient detection due to its use of the homogeneous assay format and strong surface plasmon absorption. Because the activity-based probes (ABPs) are mechanistically specific to target enzymes, this strategy also offers improved selectivity and can achieve the information about both abundance and activity of the enzymes. We have demonstrated this strategy using a human DNA (cytosine-5) methyltransferase (Dnmt 1) and a human 8-oxoguanine glycosylase (hOGG 1). The results reveal that the colorimetric response increases dynamically with increasing activity of the enzymes, implying a great potential of this strategy for DNA methyltransferase/glycosylase detection and molecular diagnostics and drug screening. Our strategy can also be used as a promising and convenient approach for visualized screening of ABPs for DNA modifying enzymes.

Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs’ fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.

We report the development of a novel single-step, multiplexed, homogeneous immunoassay platform for sensitive detection of protein targets based on our realization of high surface-enhanced raman spectroscopy (SERS) signal enhancement by controlled assembly of SERS nanoparticles. An essential design of this platform is the use of gold nanoparticles or nanorods codecorated with specially reduced antibody half-fragments, nonfluorescent Raman-active dyes, and passivating proteins as the SERS nanoparticles. These nanoparticles offer a facile approach to accomplish orientational immobilization of antibodies, minimized interparticle distance, multicolor Raman fingerprint coding, low fluorescence background, as well as excellent biocompatibility and stability. Through sandwiched antibody–antigen interactions, controlled assembly of SERS nanoparticles is realized with a strong SERS signal achieved via plasmonic coupling, creating an immunoassay platform for rapid, sensitive, multiplexed quantification of proteins. This platform is demonstrated for reproducible quantification of three cytokines, interferon gamma, interleukin-2, and tumor necrosis factor alpha, with large signal-to-noise ratio. It is also successfully applied to multiplexed cytokine analysis for T cell secretion studies in complicated biological samples. The developed SERS immunoassay platform may create a simple but valuable tool for facilitating accurate validation and early detection of disease biomarkers as well as for point-of-care tests in clinical diagnostics.

On the basis of the inhibition of double strand DNA (dsDNA)-templated fluorescent copper nanoparticles (CuNPs) by pyrophosphate (PPi), a novel label-free turn-on fluorescent strategy to detect alkaline phosphatase (ALP) under physiological conditions has been developed. This method relies on the strong interaction between PPi and Cu2+, which would hamper the effective formation of fluorescent CuNPs, leading to low fluorescence intensity. The ALP-catalyzed PPi hydrolysis would disable the complexation between Cu2+ and PPi, facilitating the formation of fluorescent CuNPs through the reduction by ascorbate in the presence of dsDNA templates. Thus, the fluorescence intensity was recovered, and the fluorescence enhancement was related to the concentration of ALP. This method is cost-effective and convenient without any labels or complicated operations. The present strategy exhibits a high sensitivity and the turn-on mode provides a high selectivity for the ALP assay. Additionally, the inhibition effect of phosphate on the ALP activity was also studied. The proposed method using a PPi substrate may hold a potential application in diagnosis of ALP-related diseases or evaluation of ALP functions in biological systems.

T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of λ exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5′-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by λ exonuclease. As a result, the blocked ‘HRPzyme’ sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL−1 and a wide detection range from 0.06 to 100 U mL−1. Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs.Graphical abstractHighlights► The strategy was based on the coupled reaction triggered by polynucleotide kinase. ► The strategy was a colorimetric assay visible to the naked eye. ► The strategy had obvious advantages in controlling cost and simplifying operations. ► The strategy exhibited an improved signal to noise ratio and a wide linear range. ► The strategy could be extended to high-throughput phosphorylation investigations.

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

An electrochemical method for the determination of polynucleotide kinase (PNK) activity has been proposed with a dual signal amplification strategy. The method relies on the phosphorylation-induced DNA digestion and two-step signal amplification using streptavidin–gold nanoparticle biocomplexes and alkaline phosphatase. In the presence of T4 PNK, the 5′-terminus of the immobilized PNK substrate probe was phosphorylated, and the substrate probe would be cleaved by λ exonuclease after being hybridized with the complementary detection probe biotinylated in the 3′-hydroxyl terminus. As a result, the detection probe would be released, with an electrochemical signal decrease, due to less biotinylated alkaline phosphatase loading on the electrode surface. The electrochemical signal exhibited a linear correlation to the logarithm of PNK concentration ranging from 0.01 U mL−1 to 5 U mL−1 with the detection limit of 0.01 U mL−1. The inhibiting effect of (NH4)2SO4 on the activity of PNK was also evaluated.

A label-free fluorescence aptamer-based sensor method for cocaine detection has been constructed based on polymerase aided isothermal circular strand-displacement amplification (ICSDA) and graphene oxide (GO) absorption. In this assay, a hairpin probe (HP), which consists of the aptamer sequence of cocaine, is partially complementary to the primer. When cocaine was bound with the HP, the primer could anneal with the single-stranded sequence of the opened HP and then trigger polymerase elongation, generating a double-stranded DNA (dsDNA) and displacing the cocaine. In the absence of cocaine, the HP remained intact and ICSDA would not occur. Through combining the unique properties of SYBR Green I (SG) and the preferential binding of GO to single-stranded DNA over dsDNA, a significant fluorescence enhancement was achieved. Whereas, the SG-stained HP without polymerase elongation was absorbed and quenched by GO. The proposed method is simple, convenient, and has high sensitivity and selectivity. This method displayed a good linear correlation within the cocaine concentration range from 0.2 μM to 100 μM, and the detection limit was down to 190 nM. In addition, this aptamer-based sensor method was also successfully applied for cocaine quantification in human urine samples.

A label free exonuclease III (Exo III)-aided fluorescence assay for adenosine triphosphate (ATP) was developed based on the ATP-dependent enzymatic reaction and graphene oxide (GO). This strategy relies on the principle that Exo III shows different cleavage capacity for a DNA substrate in the absence and presence of ATP and the preferential binding of GO to single-stranded DNA over double-stranded one. By combining the unique properties of SYBR Green I and GO adsorption, this sensor displays an improved sensitivity and a wide linear range within the ATP concentration from 1 nM to 200 nM with a low detection limit of 0.2 nM. The proposed method is simple, cost-effective and convenient, which might create a new methodology for developing a sensitive ATP biosensor.

The generation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA is a common type of DNA damage after exposure to reactive oxygen species or drugs. Human 8-oxoG DNA glycosylase/AP lyase (hOGG1) is a kind of base excision repair enzyme specifically used to repair the base excision of 8-oxoG. In this paper, we develop a novel, simple and sensitive strategy for the detection of hOGG1 activity based on the self-assembly of the active HRP-mimicking DNAzyme coupled with lambda exonuclease (λ exo) cleavage. We designed two DNA oligonucleotides that are fully complementary to each other. One is modified with 8-oxoG, the other contains the G-quadruplex DNAzyme sequence. The two single-stranded DNA (ssDNA) firstly hybridize to form a DNA duplex containing an 8-oxoG. In the presence of hOGG1, the formed DNA duplex is selectively cleaved at the 8-oxoG site, yielding a new DNA duplex with a recessed 5′-phosphate terminus. Upon treatment with λ exo, the 5′-phosphoryl ssDNA of the new DNA duplex is digested by λ exo, releasing the G-quadruplex DNAzyme sequence. After addition of hemin, the G-quadruplex–hemin complex is used as a peroxidase-mimicking DNAzyme, catalyzing H2O2-mediated oxidation of 2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS2−) to generate a colorimetric signal. The activity of hOGG1 is directly related to UV/Vis absorption intensity. The results revealed that the method allowed a sensitive quantitative assay of the hOGG1 concentration with a wide range from 0.05–32 U mL−1 and a low detection limit of 0.01 U mL−1.

A novel silver nanocluster is synthesized using a DNA template with short guanine at the 3′-end of a DNA scaffold to enhance the fluorescence intensity. The obtained DNA-stabilized silver nanoclusters (G-DNA-Ag NCs) are found to exhibit a strong red fluorescence at 605 nm and its fluorescence can be sensitively quenched by H2O2. This allows us to develop a new enzymatic method for fluorescent detection of cholesterol through oxidation by cholesterol oxidase which can generate H2O2. The present method is convenient without the need for complicated operations, and demonstrates desirable selectivity with no interferences from species such as ascorbic acid, glucose and urea. It shows a linear detection range for cholesterol from 0.2 to 200 μM with a detection limit of 0.15 μM.

A label-free molecular logic system is developed based on a G-quadruplex conformation change, which can be reported using a small dye molecule N-methyl mesoporphyrin IX (NMM) as the fluorescent signaling indicator. In the absence of Pb2+, the K+-stabilized G-quadruplex could bind with NMM, resulting in strong fluorescent intensity. In contrast, the fluorescence decreases greatly when Pb2+ is present. This molecular logic system provides a simple biosensor for Pb2+. The biosensor has shown high accuracy, selectivity, and sensitivity, as well as linearity within a wide concentration range with a detection limit of 5 nM, which meets the EPA standard for Pb2+ (50 nM).

The interactions between small molecules and proteins constitute a critical regulatory mechanism in many fundamental biological processes. A novel biosensing strategy has been developed for sensitive and selective detection of small molecule and protein interaction on the basis of terminal protection of small molecule-linked ssDNA-SWNT nanoassembly. The developed strategy is demonstrated using folate and its binding protein folate receptor (FR) as a model case. The results reveal the developed technique displays superb resistance to non-specific binding, very low detection limit as low as subnanomolar, and a wide dynamic range from 100 pmol/L to 500 nmol/L of FR. Thus, it may offer a simple, cost-effective, highly selective and sensitive platform for homogeneous fluorescence detection of small molecule–protein interaction and related biochemical studies.This strategy mainly relies on the binding event of a target protein to a small molecule-linked ssDNA-SWNT nanoassembly, which can efficiently protect the nanoassembly from the degradation by exonuclease. By incorporating a fluorophore in the small molecule-linked ssDNA, the small molecule–protein interaction can be readily probed by the fluorescent signals of the reaction system.

•An electrochemical biosensor for DNA detection was developed.•Dual signal amplification was achieved by combining CSDPR and HCR.•DNA was detected sensitively and selectively.•The biosensor worked well in complex biological samples.We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10 fM to 1 nM, and the detect limit was as low as 8 fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems.

Uracil-DNA glycosylase (UDG) as one of the most important base excision repair enzymes plays a crucial role in protecting the genome from endogenous DNA damage and sustaining the genome integrity. Quantitative activity analysis of UDG is a central challenge and of fundamental importance in bioanalysis. Here, we proposed a novel biosensor constituted by adsorbing a fluorophore-labeled hairpin probe onto the surface of graphene oxide (GO) as a homogeneous assay platform for sensitive UDG activity assay. Active UDG could excise the uracil base in the hairpin probe, and further hydrolysis of the leaving abasic site gave rise to high fluorescence. Thus, it provided a convenient approach for UDG activity quantification. Because of the unique ability of GO in universal fluorescence quenching, a low background fluorescence signal can be obtained for the efficient fluorescence resonant energy transfer from the fluorophore-labeled on the hairpin probe to GO sheet. A quite wide dynamic range from 0.0017 U/mL to 0.8 U/mL was achieved for UDG assay and the detection limit was estimated to be 0.0008 U/mL. The results indicated that this strategy offers a simple, cost-effective, highly sensitive and selective homogeneous detection platform for UDG activity assay related biochemical studies.Highlights► A novel biosensor for activity analysis of uracil-DNA glycosylase. ► A graphene oxide-hairpin probe nanocomposite was constructed in this strategy. ► Simple, cost-effective, highly sensitive and selective assay was achieved. ► A promising homogeneous platform for UDG assay and related biochemical studies.

A novel electrochemical biosensor was developed for activity assay of DNA methyltransferase and its inhibitor based on methylation-sensitive cleavage, which activated a primer for terminal transferase-mediated extension of biotinylated dUTP followed by sensitive detection via enzymatic amplification.

A novel phospholipid–graphene nanoassembly is developed based on self-assembly of phospholipids on nonoxidative graphene surfaces. The nanoassembly can be prepared easily through noncovalent hydrophobic interactions between the lipid tails and the graphene without destroying the electronic conjugation within the graphene sheet. This imparts the nanoassembly with desired electrical and optical properties with nonoxidative graphene. The phospholipid coating offers excellent biocompatibility, facile solubilization, and controlled surface modification for graphene, making the nanoassembly a useful platform for biofunctionalization of graphene. The nanoassembly is revealed to comprise a bilayer of phospholipids with a reduced graphene oxide sheet hosting in the hydrophobic interior, thus affording a unique planar mimic of the cellular membrane. By using a fluorescein-labeled phospholipid in this nanoassembly, a fluorescence biosensor is developed for activity assay of phospholipase D. The developed biosensor is demonstrated to have high sensitivity, wide dynamic range, and very low detection limit of 0.010 U/L. Moreover, because of its single-step homogeneous assay format it displays excellent robustness, improved assay simplicity and throughput, as well as intrinsic ability to real-time monitor the reaction kinetics.

We have developed a novel concept for enzymatic control of plasmonic coupling as a surface enhanced Raman scattering (SERS) nanosensor for DNA demethylation. This nanosensor is constructed by decorating gold nanoparticles (AuNPs) with Raman reporters and hemimethylated DNA probes. Demethylation of DNA probes initiates a degradation reaction of the probes by methylation-sensitive endonuclease Bsh 1236I and single-strand selective exonuclease I. This destabilizes AuNPs and mediates the aggregation of AuNPs, generating a strong plasmonic coupling SERS signal in response to DNA demethylation. This nanosensor has the advantages in its high signal-to-noise ratio, superb specificity, and rapid, convenient, and reproducible detection with homogeneous, single-step operation. Thus, it provides a useful platform for detecting DNA demethylation and related molecular diagnostics and drug screening. This work is the first time that enzymatic degradation of DNA substrate probes has been utilized to induce aggregation of AuNPs such that reproducible, sensitive SERS signals can be achieved from biological recognition events. This enzymatic control mechanism for plasmonic coupling may create a new paradigm for the development of SERS nanosensors.

Activity screening of histone-modifying enzymes is of paramount importance for epigenetic research as well as clinical diagnostics and therapeutics. A novel biosensing strategy has been developed for sensitive and selective detection of histone-modifying enzymes as well as their inhibitors. This strategy relies on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. This design allows a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvents the requirements to prefunctionalize the antibody and affords improved assay simplicity and throughput. Additionally, the use of antibody-based recognition of modified peptides could offer improved specificity as compared with existing techniques based on the enzyme coupled assay. We have demonstrated this strategy using a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The results reveal that the absorption peak characteristic for AuNPs decreases dynamically with increasing activity of the enzymes with concomitant visualizable color attenuation, and subnanomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a robust and convenient visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity.

This paper reported a novel homogeneous fluorescence assay strategy for probing small molecule–protein interactions based on endonucleolytic inhibition of a DNA/Fok I transducer. The transducer could cyclically cleave fluorescence-quenched probes to yield activated fluorescence signal, while protein binding to the small molecule label would prevent Fok I from approaching and cleaving the fluorescence-quenched probes. Because of the efficient signal amplification from the cyclic cleavage operation, the developed strategy could offer high sensitivity for detecting small molecule–protein interactions. This strategy was demonstrated using folate and its high-affinity or low-affinity binding proteins. The results revealed that the developed strategy was highly sensitive for detecting either high- or low-affinity small molecule–protein interactions with improved selectivity against nonspecific protein adsorption. This strategy could also be extended for assays of candidate small-molecule ligands using a competitive assay format. Moreover, this strategy only required labeling the small molecule on a DNA heteroduplex, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new method could have potential to become a universal, sensitive, and selective platform for quantitative assays of small molecule–protein interactions.

A simple, amplification-free and sensitive fluorescent biosensor for ATP detection was developed based on the ATP-dependent enzymatic reaction (ATP-DER) and the different adsorption affinity between graphene oxide (GO) and DNA structures. The proposed method was simple and convenient and also showed high sensitivity and selectivity to ATP.

A simple, selective and sensitive electrochemical biosensor has been developed to detect protease biomarker from Bacillus licheniformis, a recognized model of the biochemical warfare agent Bacillus anthracis. In this assay, the biosensor is constructed using a D-amine acid containing substrate peptide via self-assembly of cysteine residual at the C-terminal. A biotin modifier is labelled at the N-terminal of the substrate peptide. This enables sensitive electrochemical detection of the intact substrate peptide using a streptavidin-conjugated alkaline phosphatase, which catalyzes the conversion of electrochemically inactive 1-naphthyl phosphate into electrochemically active phenol. In the presence of the protease biomarker, the peptide is cleaved, and the biotin moiety is removed away from the electrode surface, which results in a decreased electrochemical signal corresponding to the concentration of the protease biomarker. This electrochemical biosensor is simple, sensitive and cost effective. The introduction of D-amino acids into the peptide substrate enables high species selectivity and eliminates the steps for enzyme isolation and purification. Under optimized conditions, the protease can be determined in the concentration range from 0.5 to 100 μg mL−1 with a detection limit to 0.16 μg mL−1.

DNA methyltransferase (MTase) is a kind of important regulatory factor in various biological processes. Current methods to investigate DNA MTase activity are still limited in the sensitivity and/or generality. Therefore, developing methods with high sensitivity and improved generality is needed. Here, we develop a new bioluminescence strategy based on methylation-resistant cleavage and protein expression in vitro to detect DNA MTase activity. In the strategy, Dam MTase was used as a model enzyme and MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR–DNA) was used as their action target. Because the completely methylated LR–DNA could be expressed as detectable luciferase, Dam MTase activity was quantified by measuring the luminescence intensity of the expressed luciferase. The assay provides a very low detection limit (0.08 U/ml) as well as a wide linear range (0.2–100 U/ml). Besides, the analysis mode has improved generality and could be extended to the detection of other DNA MTases and the corresponding inhibitor screening.

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies.Graphical abstractHighlights► We report a homogeneous label-free CYP2D6*10 genotyping technique. ► Genotyping is accomplished by ligase chain reaction. ► The technique is based on ligation-mediated strand displacement amplification. ► The signal of detection is DNAzyme-catalyzed chemiluminescence.

Based on exonuclease III (Exo III) aided amplification and graphene oxide (GO) platform for fluorescence quenching, a novel, turn-on fluorescent aptasensor for lysozyme (Lys) protein was constructed. The system contains a hairpin probe (HP) and a signal probe (SP) labeled with carboxyfluorescein (FAM) at its 5′ end. HP, which consists of the aptamer sequence of Lys, is partially complementary to SP. Lys could bind with the aptamer region of the HP and facilitate the opening of the hairpin structure of HP, exposing a single-stranded sequence to hybridize with SP. This triggered the Exo III aided amplification and caused the degradation of SP, which liberated the free fluorophore labels. Upon the addition of GO, the released fluorophore could not be adsorbed and no fluorescence quenching occured, while the intact SPs could be adsorbed on GO surface with the fluorescence substantially quenched. The results revealed that the proposed method displayed fluorescence responses in a linear correlation to the concentrations of Lys within the range from 0.125 μg/ml to 1 μg/ml and the detection limit is 0.08 μg/ml. Besides such sensitivity, the proposed strategy is also low-cost and simple due to its homogeneous and fluorescence-based detection format.Highlights► A simple, low cost and sensitive fluorescent biosensor was presented. ► Signal amplification strategy based on exonuclease III was employed. ► Fluorescence quenching was performed based on graphene oxide (GO) platform. ► Labeling of fluorophore/quencher pairs is not needed. ► This method can be expanded to detect other proteins, small molecules, DNAs.

Genotyping of single nucleotide polymorphisms (SNPs) is a central challenge in disease diagnostics and personalized medicine. A novel label-free homogeneous SNP genotyping technique is developed on the basis of ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatches is first accomplished using DNA ligase to generate a ligation product between a discriminant probe and a common probe. The ligated product then initiates two consecutive SDA reactions to produce a great abundance of aptamer sequences against hemin, which can be probed by chemiluminscence detection. The developed strategy is demonstrated using a model SNP target of cytochrome P450 monooxygenase CYP2C19*2, a molecular marker for personalized medicines. The results reveal that the developed technique displays superb selectivity in discriminating single-base mismatches, very low detection limit as low as 0.1 fM, a wide dynamic range from 1 fM to 1 nM, and a high signal-to-background ratio of 150. Due to its label-free, homogeneous, and chemiluminescence-based detection format, this technique can be greatly robust, cost-efficient, readily automated, and scalable for parallel assays of hundreds of samples. The developed genotyping strategy might provide a robust, highly sensitive, and specific genotyping platform for genetic analysis and molecular diagnostics.

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 31.2 μA mM−1 cm−2 for glucose with a wide linear range from 1.0 μM to 1.0 mM as well as a low detection limit of 0.2 μM (S/N = 3). The low Michaelis–Menten constant (1.2 mM) 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.

Quantitative analysis of interactions between small molecules and proteins is a central challenge in chemical genetics, molecular diagnostics and drug developments. Here, we developed a RNA transcription nanomachine by assembling T7 RNA polymerase on a small molecule-labeled DNA heteroduplex. The nanomachine, of which the RNA transcription activity can be quantitatively inhibited by protein binding, showed a great potential for small molecule-protein interaction assay. This finding enabled us to develop a novel homogeneous label-free strategy for assays of interactions between small molecules and their protein receptors. Three small molecule compounds and their protein receptors have been used to demonstrate the developed strategy. The results revealed that the protein-small molecule interaction assay strategy shows dynamic responses in the concentration range from 0.5 to 64 nM with a detection limit of 0.2 nM. Due to its label-free, homogeneous, and fluorescence-based detection format, besides its desirable sensitivity this technique could be greatly robust, cost-efficient and readily automated, implying that the developed small molecule-protein interaction assay strategy might create a new methodology for developing intrinsically robust, sensitive and selective platforms for homogeneous protein detection.

We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3′-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3′-phosphoryl end that served as the substrate for DNA 3′-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3′-phosphatases, the resulting HP with a 3′-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3′-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL−1 and 0.003 U mL−1, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.

A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.

A novel surface enhanced Raman scattering (SERS) based assay using a formaldehyde-selective reactive probe for sensitive detection of activity of histone demethylases (HDMs) by direct observation of by-product formaldehyde was reported.

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

We developed novel activatable probe using self-assembled peptide nanowires with low affinity and toxicity to tumor cells in the absence of matrix metalloproteinase that showed activated high affinity and toxicity and provided a highly selective and efficient platform for targeted therapy and tumor imaging.

A novel silver nanocluster is synthesized using a DNA template with short guanine at the 3′-end of a DNA scaffold to enhance the fluorescence intensity. The obtained DNA-stabilized silver nanoclusters (G-DNA-Ag NCs) are found to exhibit a strong red fluorescence at 605 nm and its fluorescence can be sensitively quenched by H2O2. This allows us to develop a new enzymatic method for fluorescent detection of cholesterol through oxidation by cholesterol oxidase which can generate H2O2. The present method is convenient without the need for complicated operations, and demonstrates desirable selectivity with no interferences from species such as ascorbic acid, glucose and urea. It shows a linear detection range for cholesterol from 0.2 to 200 μM with a detection limit of 0.15 μM.

We developed a novel label-free biosensor for biomolecule detection based on the thioflavin T (ThT)-induced conformational change of guanine-rich oligonucleotides and self-assembled aptamer/GO nanosheet architecture. In the presence of target biomolecules, the aptamer sequence could specifically bind to the target and release from the surface of GO nanosheets to form a G-quadruplex conformation with ThT as an inducer, resulting in enhancement of fluorescence. The proposed biosensor exhibits a “turn-on” signal, which allows sensitive, selective and rapid detection of biomolecules.

A novel and label-free method for micrococcal nuclease (MNase) detection has been presented based on single-stranded DNA (ssDNA)-scaffolded fluorescent silver nanoclusters (AgNCs). The ssDNA was introduced as the substrate for MNase and also as the scaffold for the synthesis of the AgNCs. In the absence of MNase, the ssDNA was not digested. As a result, the fluorescent AgNCs were formed and exhibited strong fluorescence. In the presence of MNase, the DNA was digested, which prohibited the formation of the AgNCs due to the lack of the DNA scaffold, resulting in weak fluorescence. The fluorescence intensity exhibits a linear correlation to MNase concentration in the range of 0 U mL−1 to 2 × 10−4 U mL−1 with a detection limit of 8 × 10−6 U mL−1. Given its simplicity, easy operation, sensitivity and cost-effectiveness, this method can be extended to other nuclease assays.

Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better understanding their biological functions. Since there are a few fluorescent probes concerning NTR activity specifically located in mitochondria, we developed a novel fluorescent benzoindocyanine probe (BICP) for mitochondrial NTR activity monitoring and imaging via extending a benzoindole moiety into a benzoindocyanine based fluorophore (BICF) with a strong intramolecular charge transfer (ICT) effect and incorporating 4-nitrobenzyl as a fluorescence-quenching and enzyme-responsive moiety. Live cell imaging of HeLa and A549 demonstrates that the developed BICP is able to realize sensitive and selective mitochondrial NTR activity probing with high-contrast “off–on” fluorescence. These findings implied the great potential of the developed probe for monitoring mitochondrial-specific NTR activities in living cells and related applications in cell biology.

The generation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA is a common type of DNA damage after exposure to reactive oxygen species or drugs. Human 8-oxoG DNA glycosylase/AP lyase (hOGG1) is a kind of base excision repair enzyme specifically used to repair the base excision of 8-oxoG. In this paper, we develop a novel, simple and sensitive strategy for the detection of hOGG1 activity based on the self-assembly of the active HRP-mimicking DNAzyme coupled with lambda exonuclease (λ exo) cleavage. We designed two DNA oligonucleotides that are fully complementary to each other. One is modified with 8-oxoG, the other contains the G-quadruplex DNAzyme sequence. The two single-stranded DNA (ssDNA) firstly hybridize to form a DNA duplex containing an 8-oxoG. In the presence of hOGG1, the formed DNA duplex is selectively cleaved at the 8-oxoG site, yielding a new DNA duplex with a recessed 5′-phosphate terminus. Upon treatment with λ exo, the 5′-phosphoryl ssDNA of the new DNA duplex is digested by λ exo, releasing the G-quadruplex DNAzyme sequence. After addition of hemin, the G-quadruplex–hemin complex is used as a peroxidase-mimicking DNAzyme, catalyzing H2O2-mediated oxidation of 2,2′-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS2−) to generate a colorimetric signal. The activity of hOGG1 is directly related to UV/Vis absorption intensity. The results revealed that the method allowed a sensitive quantitative assay of the hOGG1 concentration with a wide range from 0.05–32 U mL−1 and a low detection limit of 0.01 U mL−1.

An electrochemical method for microRNA (miRNA) detection has been proposed with a dual signal amplification strategy. The method relies on polymerase extension and a two-step signal amplification using streptavidin–gold nanoparticle biocomplexes and alkaline phosphatase. The target miRNA can hybridize with the capture DNA template, which can act as a primer and be extended along the template in the presence of DNA polymerase and dNTPs. A biotin group was introduced into the duplex by the incorporation of biotin-11-dUTP. Thus, biotinylated alkaline phosphatase would bind to the duplex using streptavidin–gold nanoparticles as linkers, which resulted in an amplified electrochemical signal. The electrochemical signal exhibited a linear correlation to the logarithm of miRNA concentration ranging from 100 fM to 1 nM, with a detection limit of 99.2 fM. The specificity of the method allowed single-nucleotide differences between miRNA family members to be discriminated. The established biosensor displayed an excellent analytical performance towards miRNA detection and might present a convenient tool for biomedical research and clinical diagnostic applications.

A label-free molecular logic system is developed based on a G-quadruplex conformation change, which can be reported using a small dye molecule N-methyl mesoporphyrin IX (NMM) as the fluorescent signaling indicator. In the absence of Pb2+, the K+-stabilized G-quadruplex could bind with NMM, resulting in strong fluorescent intensity. In contrast, the fluorescence decreases greatly when Pb2+ is present. This molecular logic system provides a simple biosensor for Pb2+. The biosensor has shown high accuracy, selectivity, and sensitivity, as well as linearity within a wide concentration range with a detection limit of 5 nM, which meets the EPA standard for Pb2+ (50 nM).

T7 exonuclease is reported for the first time to have high specificity in discriminating single-base mismatch and utilized for developing a target cyclic amplification biosensor strategy for sensitive SNP detection based on graphene oxide quenching of uncleaved probes.

A novel electrochemical biosensor was developed for activity assay of DNA methyltransferase and its inhibitor based on methylation-sensitive cleavage, which activated a primer for terminal transferase-mediated extension of biotinylated dUTP followed by sensitive detection via enzymatic amplification.