MicroRNAs (miRNAs) play important roles in cell differentiation, proliferation, and apoptosis and have been recognized as valuable biomarkers for clinical disease diagnosis. Here, we adopt for the first time zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier to efficiently deliver a nucleic acid probe to living cells and develop a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled substrate strand containing a segment complementary to the target miRNA-21 first form a duplex probe, and fluorescence resonance energy transfer (FRET) takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomplex degrades in acidic endosome and releases duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridizes with the complementary segment of the substrate strand and results in dissociation from the DNAzyme–substrate duplex probe after DNAzyme cleaves the substrate into two fragments, accompanied by the change in the FRET signal. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa, and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor can also be monitored through the obvious imaging color change. Taken together, the proposed method provides a powerful tool for cancer diagnosis and miRNA-associated biological study.

As an important biological small molecule, ascorbic acid (AA) plays a key role in many bioprocesses. Here, we developed a novel method to evaluate AA based on the redox reaction between CoOOH and AA. In our work, a nanosystem was constructed with fluorescent polydopamine (PDA) nanoparticles and CoOOH nanosheets, which were used as signal indicators and an oxidant respectively. This is the first time that fluorescent PDA nanoparticles were synthesized through oxidation by CoOOH nanosheets. In the absence of AA, dopamine was oxidized to quinone derivatives and further spontaneously polymerized into fluorescent PDA nanoparticles which had strong fluorescence signals. When there was AA in the reaction system, CoOOH nanosheets would be reduced to Co2+, which would prevent the synthesis of fluorescent PDA nanoparticles due to the absence of oxidant CoOOH, resulting in weak fluorescence. We hence used the fluorescence intensity of the PDA nanoparticles to detect AA concentration. The fluorescence of this sensing platform was linear with the concentration of AA in the range of 0–500 μM with a detection limit of 4.8 μM. In addition, the sensor was simple, fast, label-free, and low cost, and may be used to detect other small molecules based on the redox reaction.

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.

Blood glucose detecting has aroused considerable attention because diabetes mellitus has become a worldwide publish health problem. Herein, we construct an exceptionally simple upconverting hybrid nanocomposite, composed of DNA-templated Ag nanoparticles (DNA-AgNPs) and NaYF4:Yb/Tm@NaYF4 core–shell upconversion nanoparticles (UCNPs), for the sensing of H2O2 and glucose. In this design, UCNPs with bared surface act as the donor, and DNA-AgNPs serve as efficient quenchers. DNA-AgNPs can be directly assembled on the bared surface of UCNPs, which further decreases the distance of donor-to-acceptor. The formation of DNA-AgNPs/UCNP nanocomposite results in luminescence quenching of UCNP by DNA-AgNPs through luminescence resonance energy transfer (LRET). Upon H2O2 addition, AgNPs can be etched and transformed into Ag+, leading to inhibition of the LRET process and causing the recovery of upconversion luminescence. Based on the conversion of glucose into H2O2 by glucose oxidase, the DNA-AgNPs/UCNP nanocomposite can also be exploited for glucose sensing. Moreover, due to the non-autofluorescence offered by UCNPs, the approach developed can be applied to monitor glucose levels in human serum samples with satisfactory results.

Phosphorylation of nucleic acids with 5′-OH termini catalyzed by polynucleotide kinase (PNK) is an inevitable process and has been implicated in many important cellular events. Here, we found for the first time that there was a significant difference in the adsorbent ability of cobalt oxyhydroxide (CoOOH) nanoflakes between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which resulted in the fluorescent dye-labeled dsDNA still retaining strong fluorescence emission, while the fluorescence signal of ssDNA was significantly quenched by CoOOH nanoflakes. Based on this discovery, we developed a CoOOH nanoflake-based nanoprobe for the fluorescence sensing of T4 PNK activity and its inhibition by combining it with λ exonuclease cleavage reaction. In the presence of T4 PNK, dye-labeled dsDNA was phosphorylated and then cleaved by λ exonuclease to generate ssDNA, which could adsorb on the CoOOH nanoflakes and whose fluorescence was quenched by CoOOH nanoflakes. Due to the high quenching property of CoOOH nanoflakes as an efficient energy acceptor, a sensitive and selective sensing approach with satisfactory performance for T4 PNK sensing in a complex biological matrix has been successfully constructed and applied to the screening of inhibitors. The developed approach may potentially provide a new platform for further research, clinical diagnosis, and drug discovery of nucleotide kinase related diseases.

A novel fluorescent sensing platform has been developed for protein kinase assay based on the phosphorylation-induced formation of a cytochrome c-peptide complex.

Endonuclease IV (Endo IV), as a DNA repairing enzyme, plays a crucial role in repairing damaged DNA comprising abasic sites to maintain genomic integrity. The cleaving capability of Endo IV to apurinic/apyrimidinic sites (AP) in single-stranded DNA (ssDNA) was demonstrated. It was found that Endo IV has considerably high cleaving activity to AP sites in ssDNA compared with that in double-stranded DNA (dsDNA). The unique feature of Endo IV in cleaving AP sites in ssDNA was further applied to construct a novel dual signal amplified sensing system for highly sensitive enzyme and protein detection by a combination of exonuclease III (Exo III)-aided cyclic amplification reaction and a rolling circle replication (RCR) technique, which showed a good sensing performance with a detection limit of 0.008 U mL−1 for Endo IV and 2.5 pM for streptavidin. In addition, the developed method had considerably high specificity for Endo IV and streptavidin over other potential interferences. The developed strategy indeed provides a novel platform for protein and enzyme assays and may find a broad spectrum of applications in bioanalysis, disease diagnosis, and drug development.

•A novel "light-up" sensor for human 8-oxoguanine DNA glycosylase activity assay was developed.•The work mainly relies on target-induced formation of 5′ phosphorylated probe and autocatalytic DNAzyme-generated rolling circle amplification strategy.•The assay exhibited a low detection limit of 0.001 U/mL for hOGG1.•The strategy showed excellent specificity over other nonspecific enzymes and good analytical performance in complex biological media.Human 8-oxoguanine DNA glycosylase (hOGG1) plays a crucial role in maintaining the genomic integrity of living organisms for its capability of repairing DNA oxidative damage. The expression level of hOGG1 is closely associated with many diseases including various kinds of cancers. In this study, a novel “light-up” sensor based on target-induced formation of 5′ phosphorylated probe and autocatalytic DNAzyme-generated rolling circle amplification has been developed for highly sensitive human 8-oxoguanine DNA glycosylase (hOGG1) activity assay. The approach reaches detection limit as low as 0.001 U/mL for hOGG1 via scarcely increased background signal and dual signal amplification strategy. To the best of our knowledge, it is one of the most sensitive methods for the detection of base excision repair enzyme. Moreover, the approach shows excellent specificity over other nonspecific enzymes would interfere with the assay and holds great promise for application in real sample analysis. Hence, the proposed method provides a highly sensitive, selective, and desirable hOGG1 sensing platform.

Ascorbic acid (AA), a potent antioxidant readily scavenging reactive species, is a crucial micronutrient involved in many biochemical processes. Here, we have developed a cobalt oxyhydroxide (CoOOH)-modified upconversion nanosystem for fluorescence sensing of AA activity in human plasma. The nanosystem consists of upconversion nanoparticles (UCNPs) NaYF4:30% Yb,0.5% Tm@NaYF4, which serve as energy donors, and CoOOH nanoflakes formed on the surface of UCNPs, which act as efficient energy acceptors. The fluorescence resonance energy transfer (FRET) process from the UCNPs to the absorbance of the CoOOH nanoflakes occurs in the nanosystem. The AA-mediated specific redox reaction reduces CoOOH into Co2+, leading to the inhibition of FRET, and resulting in the recovery of upconversion emission spectra. On the basis of these features, the nanosystem can be used for sensing AA activity with sensitivity and selectivity. Moreover, due to the minimizing background interference provided by UCNPs, the nanosystem has been applied to monitoring AA levels in human plasma sample with satisfactory results. The proposed approach may potentially provide an analytical platform for research and clinical diagnosis of AA related diseases.

Blood glucose monitoring has attracted extensive attention because diabetes mellitus is a worldwide public health problem. Here, we reported an upconversion fluorescence detection method based on manganese dioxide (MnO2)-nanosheet-modified upconversion nanoparticles (UCNPs) for rapid, sensitive detection of glucose levels in human serum and whole blood. In this strategy, MnO2 nanosheets on the UCNP surface serve as a quencher. UCNP fluorescence can make a recovery by the addition of H2O2, which can reduce MnO2 to Mn2+, and the glucose can thus be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. Because of the nonautofluorescent assays offered by UCNPs, the developed method has been applied to monitor glucose levels in human serum and whole blood samples with satisfactory results. The proposed approach holds great potential for diabetes mellitus research and clinical diagnosis. Meanwhile, this nanosystem is also generalizable and can be easily expanded to the detection of various H2O2-involved analytes.Keywords: glucose; H2O2; lanthanide-doped upconversion nanoparticles; manganese dioxide;

The ability to probe low-abundance biomolecules or transport a high-load drug in target cells is essential for biology and theranostics. We develop a novel activatable theranostic approach by using a structure-switching aptamer triggered hybridization chain reaction (HCR) on the cell surface, which for the first time creates an aptamer platform enabling real-time activation and amplification for fluorescence imaging and targeting therapy. The aptamer probe is designed not to initiate HCR in its free state but trigger HCR on binding to the target cell via structure switching. The HCR not only amplifies fluorescence signals from a fluorescence-quenched probe for activatable tumor imaging but also accumulates high-load prodrugs from a drug-labeled probe and induces its uptake and conversion into cisplatin in cells for selective tumor therapy. An in vitro assay shows that this approach affords efficient signal amplification for fluorescence detection of target protein tyrosine kinase-7 (PTK7) with a detection limit of 1 pM. Live cell studies reveal that it provides high-contrast fluorescence imaging and highly sensitive detection of tumor cells, while renders high-efficiency drug delivery into tumor cells via an endocytosis pathway. The results imply the potential of the developed approach as a promising platform for early stage diagnosis and precise therapy of tumors.

We have developed a facile one-step approach to make hydrophilic and DNA-functionalizable upconversion nanoparticles (UCNPs), which are used to act as a biosensor for determining Hg2+ in complex matrices. The proposed approach is simple and exhibits low background interference, high sensitivity and rapid response.

A robust fluorescent sensing platform for ultrasensitive assay of nuclease has been established based on rolling circle amplification and exonuclease III-aided recycling amplification. Rolling circle amplification (RCA) is an isothermal DNA amplification process that can convert a short DNA primer into a long single-stranded DNA containing a large amount of tandem repeats. In this work, the substrate DNA (sDNA) of S1 nuclease was designed as the primer to generate RCA products that can hybridize with the pre-quenched TaqMan probes and form recessed 3′ terminus double-stranded DNAs. In the presence of exonuclease III (Exo III), the TaqMan probes were digested from the 3′-hydroxyltermini, releasing the fluorophore and generating enhanced fluorescence signals. Meanwhile, the RCA products with 3′ protruding ends were liberated and hybridized with other TaqMan probes, triggering another cycles and obtaining remarkablely increasing fluorescence. However, in the presence of S1 nuclease, sDNA was cleaved into mono- or short-oligonucleotides pieces, which could not cause the RCA reaction and subsequent Exo III-aided recycling amplification reaction, resulting in extremely weak fluorescence. The fluorescence intensity gradually reduced with increasing concentration of S1 nuclease. Due to the double signal amplification, the developed method was demonstrated to exhibit exceedingly excellent sensitivity with a detection limit of 5 × 10−7 U μL−1. The system was also used to establish a turn-on biosensor of ATP since ATP can prevent the S1 nuclease from digesting the sDNA. ATP can be detected in a range of 5 × 10−4 to 0.2 mM with a detection limit of 5 × 10−4 μM and good selectivity. Moreover, the sensing system was used for the real sample analysis with satisfied results.

We have developed a reliable, sensitive, label-free and turn-on sensor for H2O2 and glucose based on the cleavage of ssDNA by ˙OH and the fluorescence enhancement effect when guanine-rich (G-rich) DNA sequences are in proximity to DNA–silver nanoclusters (DNA–Ag NCs). In addition, we have also proved that ˙OH is indeed produced in the sensing system by adding antioxidants.

A facile one-step approach was proposed to make hydrophilic and DNA-functionalizable upconversion nanoparticles through ligand exchange at the liquid–liquid interface, and an ultrasensitive and selective biosensor for nuclease assay and its inhibitors assay based on FRET from the DNA-functionalizable UCNPs to graphene oxide was designed. A high sensitivity exhibited with a detectable minimum concentration of 1 × 10−4 units per mL S1 nuclease, which was more sensitive than the developed approaches.

•A label-free and turn-on biosensor of endonuclease based on silver nanoclusters.•The proposed method shows high sensitivity and selectivity.•The assay is applied for inhibitor screening.Endonuclease plays a vital role in a variety of biological processes and the assay of endonuclease activity and inhibitors is of high importance in the fields ranging from biotechnology to pharmacology. Howerer, traditional techniques usually suffer from time intensive, laborious, and cost-expensive. This work aims to develop a facile and sensitive method for endonuclease activity assay by making use of the fluorescence enhancement effect when DNA–silver nanoclusters (DNA–Ag NCs) are in proximity to guanine-rich DNA sequences. The system mainly consists of block DNA (B-DNA), G-DNA and Ag-DNA. B-DNA serves as the substrate of the endonuclease (S1 nuclease as the model enzyme). G-DNA, which is predesigned entirely complementary to B strand, contains a guanine-rich overhang sequence and hybridization part at the 5′-end. Ag–DNA involves a sequence for Ag NCs synthesis and a sequence complementary to the hybridization part of the G-DNA. In the “off” state, B-DNA plays the role as a blocker that inhibit the proximity between Ag NCs and guanine-rich DNA sequences, resulting in a low fluorescence readout. However, if S1 nuclease is introduced into the system, B-DNA was cleaved into mono- or short-oligonucleotides fragments, which could not hybridize with G-DNA. As a result, the subsequent addition of DNA–Ag NCs could bring guanine-rich DNA sequences close to the Ag NCs, accompanied by a significant fluorescence enhancement. Therefore, endonuclease activity could be successfully quantified by monitoring the variation in fluorescence intensity. In addition, this approach can also be applied for inhibitor screening of endonuclease. This label-free and turn-on fluorescent assays employing the mechanism proposed here for the detection of nuclease and inhibitors turn out to be sensitive, selective, and convenient.

We have developed an aptameric nanosensor for fluorescence activation imaging of cytochrome c (Cyt c). Fluorescence imaging tools that enable visualization of key molecular players in apoptotic signaling are essential for cell biology and clinical theranostics. Cyt c is a major mediator in cell apoptosis. However, fluorescence imaging tools allowing direct visualization of Cyt c translocation in living cells have currently not been realized. We report for the first time the realization of a nanosensor tool that enables direct fluorescence activation imaging of Cyt c released from mitochondria in cell apoptosis. This strategy relies on spatially selective cytosolic delivery of a nanosensor constructed by assembly of a fluorophore-tagged DNA aptamer on PEGylated graphene nanosheets. The cytosolic release of Cyt c is able to dissociate the aptamer from graphene and trigger an activated fluorescence signal. The nanosensor is shown to exhibit high sensitivity and selectivity, rapid response, large signal-to-background ratio for in vitro, and intracellular detection of Cyt c. It also enables real-time visualization of the Cyt c release kinetics and direct identification of the regulators for apoptosis. The developed nanosensor may provide a very valuable tool for apoptotic studies and catalyze the fundamental interrogations of Cyt c-mediated biology.

We have developed a novel fluorescence-activated DNA–MoS2 nanosheet biosensor for detecting biomolecular targets such as proteins and small molecules based on the self-assembled architecture of a DNA aptamer and a MoS2 nanosheet. The proposed design is simple to prepare and exhibits low background interference, high sensitivity and rapid response.

A novel fluorescent nanosensor has been developed for detecting biothiols including cysteine and glutathione using graphene oxide based hairpin DNA-selective fluorescence quenching and thymine–Hg(II)–thymine coordination-controlled hybridization chain reaction, which provides a simple but the most sensitive platform for biothiol assays.

A facile one-step approach was proposed to prepare hydrophilic and peptide-functionalized upconversion nanoparticles (UCNPs), which were used in the design of a biosensor for the sensitive and selective determination of human immunodeficiency virus antibodies in human serum based on FRET from the UCNPs to the graphene oxide.

The ability to detect spatial and temporal microRNA (miRNA) distribution at the single-cell level is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. We report for the first time the development of a target-primed RCA (TPRCA) strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free 3′ terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands of complementary segments. After hybridization with fluorescent detection probes, target miRNA molecules can be visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated by the free 3′ end of target miRNA, the developed strategy offers the advantage over existing ISH methods in eliminating the interference from precursor miRNA or mRNA. This strategy is demonstrated to show high sensitivity and selectivity for the detection of miR-222 expression levels in human hepatoma SMMC-7721 cells and hepatocyte L02 cells. Moreover, the developed TPRCA-based ISH strategy is successfully applied to multiplexed detection using two-color fluorescent probes for two miRNAs that are differentially expressed in the two cell lines. The results reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and clinical diagnostics.

Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.

The phosphorylation of nucleic acids with 5′-hydroxyl termini catalyzed by polynucleotide kinase (PNK) plays a critical role in numerous cellular activities, including DNA recombination, DNA replication and DNA repair, during strand damage and interruption. The evolution of polynucleotide kinase activity and inhibition has attracted considerable attention in recent years. Here, we describe a new method that combines a coupled λ exonuclease reaction and exonuclease III-aided fluorescence signal amplification for PNK activity and inhibition detection. By recycling trigger DNA and continuously liberating the fluorescence probe for signal amplification, the strategy can be used for the simple and accurate determination of polynucleotide kinase (PNK) activity. Moreover, the inhibition effect of adenosine diphosphate on T4 PNK activity has also been investigated. The strategy also performs well in complex biological samples. This method not only offers a new opportunity for DNA phosphorylation-related process evolution, but also holds a great potential to apply in detection of other end-processing enzymes and biomolecular diagnosis.

A novel electrochemical immunosensor was developed for the detection of human immunoglobulin G (IgG) by using gold nanoparticles (AuNPs) and telomerase extension reaction as dual signal amplification. The immunosensor was implemented based on a heterogeneous sandwich procedure on the gold electrode surface. Goat anti-human IgG (Ab) and telomerase primer P1 co-labelled gold nanoparticles (Ab–DNA–AuNP complexes) was used as secondary antibody for telomerase extension and binding with human IgG. After the telomerase extension reaction, the extension products then hybridized with the biotinylated probe P2, followed by binding of streptavidin-labelled alkaline phosphatase (SA–ALP). The ALP converted ascorbic acid 2-phosphate (AA-P) into ascorbic acid, which reduced the silver ions in the solution into metal silver, leading to the deposition of silver onto the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver which was proportional to the concentration of human IgG. The electrochemical immunosensor showed a dynamic range of 0.1–100 μg mL−1 with a detection limit of 0.02 μg mL−1, acceptable precision, reproducibility and stability. The real human serum sample assay results demonstrated this approach could be used for clinical diagnosis.

•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.

A novel nanocomplex displaying single-excitation and dual-emission fluorescent properties has been developed through a crown-like assembly of dye-encapsulated silica particles decorated with satellite AuNCs for live cell imaging of highly reactive oxygen species (hROS), including •OH, ClO– and ONOO–. The design of this nanocomplex is based on our new finding that the strong fluorescence of AuNCs can be sensitively and selectively quenched by these hROS. The nanocomplex is demonstrated to have excellent biocompatibility, high intracellular delivery efficiency, and stability for long-time observations. The results reveal that the nanocomplex provides a sensitive sensor for rapid imaging of hROS signaling with high selectivity and contrast.

A novel aptamer biosensor for cancer cell assay has been reported on the basis of ultrasensitive electrochemical detection. Cancer cell capturing is first accomplished via aptamer-aided recognition, and the cell–aptamer binding events then mediate an alkaline phosphatase-catalyzed silver deposition reaction which can be probed by electrochemical detection. Following biocatalytic silver deposition, an efficient amplification approach for sensitive electrochemical measurements is demonstrated, for cell detection with high sensitivity. Ramos cell are used as a model case, a typical biomarker of the acute blood cell cancer, Burkitt's lymphoma. The results reveal that the developed technique displays desirable selectivity in Ramos cell discrimination, and linear response range from 10 to 106 cells with a detection limit as low as 10 cells. Due to the simple procedures, label-free and electrochemistry based detection format, this technique is simple and cost-effective, and exhibits excellent compatibility with miniaturization technologies. The electrochemical cell detection strategy may create an intrinsically specific and sensitive platform for cancer cell assay and associated studies.

In this work, we report a simple and sensitive method for the detection of biothiols including glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), using fluorescent copper nanoclusters synthesized by a double-stranded DNA template (DNA-CuNCs) as a probe. The photoluminescence intensity of DNA-CuNCs was found to be quenched effectively with the increase in the concentration of biothiols due to the formation of nonfluorescent coordination complexes between the DNA-CuNCs and biothiols, whereas the fluorescence of DNA-CuNCs was not changed in the presence of other amino acids at a 10-fold higher concentration. Satisfactory detection limits and linear relationships for the detection of GSH, Cys and Hcy were obtained The resulting calibration plots exhibited good linear correlations in the range from 2.0 × 10−6 to 1.0 × 10−4 mol L−1 for Cys, 2.0 × 10−6 to 8.0 × 10−5 mol L−1 for GSH, and 5.0 × 10−6 to 2.0 × 10−4 mol L−1 for Hcy. The detection limits of Cys, GSH and Hcy were 2 μmol L−1, 2 μmol L−1 and 5 μmol L−1, respectively. In addition, the method was successfully applied in the detection of biothiols in human plasma samples.

A sensitive fluorescence strategy based on T7 exonuclease-assisted target recycling amplification was developed for telomerase detection in cancer cells. The novel strategy improved the fluorescence signal and sensitivity compared with the previously reported methods.

A novel fluorescence biosensing strategy for simple, rapid and sensitive selecting quadruplex-binding ligands was reported by using graphene oxide (GO) as the fluorescence quencher. Data from transmission electron microscopy (TEM), atomic force microscopy (AFM) image, Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy demonstrated that single-layered GO sheets were successfully prepared with well dispersion in aqueous solution. The fluorescein amidite (FAM)-labeled signal probe first adsorbed onto the surface of GO through π-stacking interaction between the ring structure in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. When the quadruplex-binding ligands were introduced, the signal probe folded to form intramolecular G-quadruplex structure, which led to the releasing of FAM-labeled signal probe from the surface of GO and the fluorescence intensity recovered. Three series of Chinese medicine monomers were investigated by the proposed method, and the flavonoids were demonstrated to be the potential quadruplex-binding ligands by fluorescence measurement and melting temperature analysis. The results indicated that this strategy offers a simple, rapid and sensitive method for screening G-quadruplex ligands and it could find wide applications in the discovery of new antitumor drugs.Highlights► This work developed a novel graphene oxide (GO)-based biosensing platform for simple, rapid and high-throughput selecting of quadruplex-binding ligands. ► We chose graphene oxide as the fluorescence quencher. ► Three series of traditional Chinese medicine monomers are chosen to explore the natural quadruplex-binding ligands. ► A family of planar flavonoid compounds is found to be effective G-quadruplex ligands.

A novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNA-binding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. Interestingly, we disclosed a new finding that binding of target proteins to their binding sequences in the aggregated AuNP network rendered a stable and long-period protection of DNA. Unlike conventional fluorescence assays merely based on temporal protection of DNA from Exo III digestion, the stable protection afforded a static color transition indicator for DNA−protein interactions with no time-dependent monitoring required in the assay. Therefore, it furnished the developed strategy with improved technical robustness and operational convenience. Furthermore, we introduced thioctic acid as a stable anchor for tethering DNA on AuNPs. This DNA-tethering protocol circumvented the interferences from thiol compounds in common enzymatic systems. The Exo III protection-based colorimetric biosensor was demonstrated using a model target of TATA binding protein, a key transcriptional factor involving in various transcriptional regulatory networks. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.

A facile one-step approach was proposed to prepare hydrophilic and peptide-functionalized upconversion nanoparticles (UCNPs), which were used in the design of a biosensor for the sensitive and selective determination of human immunodeficiency virus antibodies in human serum based on FRET from the UCNPs to the graphene oxide.

The phosphorylation of nucleic acids with 5′-hydroxyl termini catalyzed by polynucleotide kinase (PNK) plays a critical role in numerous cellular activities, including DNA recombination, DNA replication and DNA repair, during strand damage and interruption. The evolution of polynucleotide kinase activity and inhibition has attracted considerable attention in recent years. Here, we describe a new method that combines a coupled λ exonuclease reaction and exonuclease III-aided fluorescence signal amplification for PNK activity and inhibition detection. By recycling trigger DNA and continuously liberating the fluorescence probe for signal amplification, the strategy can be used for the simple and accurate determination of polynucleotide kinase (PNK) activity. Moreover, the inhibition effect of adenosine diphosphate on T4 PNK activity has also been investigated. The strategy also performs well in complex biological samples. This method not only offers a new opportunity for DNA phosphorylation-related process evolution, but also holds a great potential to apply in detection of other end-processing enzymes and biomolecular diagnosis.

A facile one-step approach was proposed to make hydrophilic and DNA-functionalizable upconversion nanoparticles through ligand exchange at the liquid–liquid interface, and an ultrasensitive and selective biosensor for nuclease assay and its inhibitors assay based on FRET from the DNA-functionalizable UCNPs to graphene oxide was designed. A high sensitivity exhibited with a detectable minimum concentration of 1 × 10−4 units per mL S1 nuclease, which was more sensitive than the developed approaches.

A novel fluorescent sensing platform has been developed for protein kinase assay based on the phosphorylation-induced formation of a cytochrome c-peptide complex.

A novel fluorescent nanosensor has been developed for detecting biothiols including cysteine and glutathione using graphene oxide based hairpin DNA-selective fluorescence quenching and thymine–Hg(II)–thymine coordination-controlled hybridization chain reaction, which provides a simple but the most sensitive platform for biothiol assays.

A sensitive fluorescence strategy based on T7 exonuclease-assisted target recycling amplification was developed for telomerase detection in cancer cells. The novel strategy improved the fluorescence signal and sensitivity compared with the previously reported methods.

We have developed a novel fluorescence-activated DNA–MoS2 nanosheet biosensor for detecting biomolecular targets such as proteins and small molecules based on the self-assembled architecture of a DNA aptamer and a MoS2 nanosheet. The proposed design is simple to prepare and exhibits low background interference, high sensitivity and rapid response.

In this work, we report a simple and sensitive method for the detection of biothiols including glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), using fluorescent copper nanoclusters synthesized by a double-stranded DNA template (DNA-CuNCs) as a probe. The photoluminescence intensity of DNA-CuNCs was found to be quenched effectively with the increase in the concentration of biothiols due to the formation of nonfluorescent coordination complexes between the DNA-CuNCs and biothiols, whereas the fluorescence of DNA-CuNCs was not changed in the presence of other amino acids at a 10-fold higher concentration. Satisfactory detection limits and linear relationships for the detection of GSH, Cys and Hcy were obtained The resulting calibration plots exhibited good linear correlations in the range from 2.0 × 10−6 to 1.0 × 10−4 mol L−1 for Cys, 2.0 × 10−6 to 8.0 × 10−5 mol L−1 for GSH, and 5.0 × 10−6 to 2.0 × 10−4 mol L−1 for Hcy. The detection limits of Cys, GSH and Hcy were 2 μmol L−1, 2 μmol L−1 and 5 μmol L−1, respectively. In addition, the method was successfully applied in the detection of biothiols in human plasma samples.

A novel electrochemical immunosensor was developed for the detection of human immunoglobulin G (IgG) by using gold nanoparticles (AuNPs) and telomerase extension reaction as dual signal amplification. The immunosensor was implemented based on a heterogeneous sandwich procedure on the gold electrode surface. Goat anti-human IgG (Ab) and telomerase primer P1 co-labelled gold nanoparticles (Ab–DNA–AuNP complexes) was used as secondary antibody for telomerase extension and binding with human IgG. After the telomerase extension reaction, the extension products then hybridized with the biotinylated probe P2, followed by binding of streptavidin-labelled alkaline phosphatase (SA–ALP). The ALP converted ascorbic acid 2-phosphate (AA-P) into ascorbic acid, which reduced the silver ions in the solution into metal silver, leading to the deposition of silver onto the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver which was proportional to the concentration of human IgG. The electrochemical immunosensor showed a dynamic range of 0.1–100 μg mL−1 with a detection limit of 0.02 μg mL−1, acceptable precision, reproducibility and stability. The real human serum sample assay results demonstrated this approach could be used for clinical diagnosis.