A versatile bio-logic system is proposed using a surface charge tunable fluorescent protein (H39GFP) as a generic processor. On the basis of the cell penetration of H39GFP triggered by H+ and metal ions, OR logic gates are successfully operated in living cells with transfection of functional nucleic acids (Cy5 labelled DNA and siRNA) as the readout, as well as RNA interference.

•A FRET system between CCP and NIR CdTe/CdS QDs was first found.•A near-infrared ratiometric biosensor for glucose detection was developed.•This biosensor is applicable for the directly determination of glucose in whole blood.Quantum dots (QDs) have attracted extensive attention in biomedical applications， because of their broad excitation spectra, narrow and symmetric emission peaks etc. Furthermore, near-infrared (NIR) QDs have further advantages including low autofluorescence, good tissue penetration and low phototoxicity. In this work, the electrostatic interaction and fluorescence resonance energy transfer (FRET) between NIR CdTe/CdS QDs and cationic conjugated polymer (CCP) was studied for the first time. Based on the newly discovered phenomena and the result that hydrogen peroxide (H2O2) can efficiently quench the fluorescence of NIR CdTe/CdS QDs, a novel NIR ratiometric fluorescent probe for determination of H2O2 and glucose was developed. Under the optimized conditions, the detection limit of H2O2 and glucose assay were 0.1 mM and 0.05 mM (S/N=3), with a linear range of 0.2–4 mM and 0.1–5 mM, respectively. Because of the NIR spectrum, this ratiometric probe can be also applied for the determination of glucose in whole blood samples directly, providing a valuable platform for glucose sensing in clinic diagnostic and drug screening.

DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system.Keywords: cascade reaction; DNA hydrogel; multiple-enzyme system; protein encapsulation; self-assembly

DNA-related enzymes, including nucleases and DNA modifying enzymes, play key roles in DNA metabolism. Herein, a versatile platform using supercharged green fluorescence protein (ScGFP) as a recognition and signal output element was developed for homogeneous, turn-on, rapid, and sensitive detection of various DNA-related enzymes, including endonucleases, restriction nucleases, as well as DNA methyltransferase (MTase). This platform takes advantage of the DNA length-dependent binding affinity between ScGFP and DNA for assaying the cleavage activity of nucleases. The long negatively charged DNA probe (labelled with a quencher) can tightly bind to the positively charged ScGFP, and efficiently quench the fluorescence of ScGFP through fluorescence resonance energy transfer (FRET). After nuclease cleavage or the coupling reaction of DNA MTase/nuclease, the resulting short DNA segment with the quencher with significantly less negative charges loses the ability to be absorbed on ScGFP, causing the restored fluorescence and signal on detection. This method is a simple mix-and-read platform capable of screening enzyme inhibitors. Furthermore, the multiplex detection of various enzymes was realized by using a microplate reader. In principle, our platform presents a promising method for DNA-related enzymes-targeted biochemical research.

Thrombin, a kind of serine protease, is a significant regulator of thrombosis in vivo. Herein, we proposed a label-free and sensitive fluorescence assay for probing thrombin activity based on an enhanced green fluorescent protein (EGFP) probe and gold nanoparticles (AuNPs). The EGFP probe containing a hexahistidine sequence (His-tag) and a thrombin recognition site at the N-terminal was designed. His-tag enables the probe to be adsorbed on the surface of AuNPs through high affinity of His–Au bonds, and as a consequence, fluorescence resonance energy transfer (FRET) would happen, where EGFP is the fluorescence donor and AuNPs are the acceptors. Because of the high extinction coefficient of AuNPs, the fluorescence of EGFP will be quenched. When there is thrombin, it cleaves the recognition site, and results in the leaving of the His-tag from EGFP. EGFP without the His-tag cannot be efficiently adhered on AuNPs, and its fluorescence is protected. Thus, the corresponding fluorescence signal can respond to the activity of thrombin. Using this turn-on fluorescence assay, we obtained a highly sensitive and specific detection of thrombin activity (limit of detection = 0.0025 U mL−1) with a linear range from 0.01 U mL−1 to 0.25 U mL−1 under optimized conditions. The assay can also be used to measure the inhibition of thrombin activity (where hirudin was used as an example) with an IC50 value of 1.38 nM, and this was successfully performed on real samples. Furthermore, the proposed assay has the potential to be a fluorescence-colorimetry dual detection method.

The interaction between supercharged green fluorescent protein (ScGFP) and graphene oxide (GO) as well as the resulting quenching effect of GO on ScGFP were investigated. Based on this unique quenching effect and the DNA-mediated ScGFP/GO interaction, a label-free fluorescence method has been established for homogeneously assaying the activity and inhibition of base excision repair enzyme.

Protein phosphorylation catalyzed by protein kinases plays a critical role in many intracellular processes, and detecting kinase activity is important in biochemical research and drug discovery. Herein, we developed a novel fluorescent biosensor to detect protein kinase activity based on phosphorylation-mediated assembly of semisynthetic green fluorescent protein (GFP). A chimaera S-peptide composed of the 10th β-strand of GFP (s10) and a kinase substrate peptide was synthesized. Kinase-catalyzed phosphorylation of the S-peptide can protect its s10 part against cleavage by carboxypeptidase Y (CPY). Then, the peptide can bind the truncated GFP (tGFP, GFP without s10) to assemble intact GFP and recover fluorescence. Unphosphorylated S-peptide would be degraded by CPY, and fluorescent protein assembly could not occur. Thus, the kinase-catalyzed phosphorylation can switch on the fluorescence signal. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase with a low detection limit of 0.50 mU/μL and its inhibition of H-89 with an IC50 value of 23.4 nM. The feasibility of this method has been further demonstrated by assessment of the kinase activity and inhibition in the cell lysate. Moreover, based on the reverse principle, this method was expanded to detect the activity of protein phosphatase 1. Our method, using semisynthetic GFP as a readout, is facile, sensitive, label-free, and highly versatile, thus showing great potential as a promising platform for protein kinase detection and inhibitor screening.

Nowadays, large-scale screening for enzyme discovery, engineering, and drug discovery processes require simple, fast, and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel automatic and integrated micro-enzyme assay (AIμEA) platform was proposed based on a unique microreaction system fabricated by a engineered green fluorescence protein (GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and to be released after thrombin digestion. Combined with capillary electrophoresis-laser-induced fluorescence (CE-LIF), all the procedures, including thrombin injection, online enzymatic digestion in the microreaction system, and label-free detection of the released GFP, were integrated in a single electrophoretic process. By taking advantage of the ultrahigh loading capacity of the AIμEA platform and the CE automatic programming setup, one microreaction column was sufficient for many times digestion without replacement. The novel microreaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly, the AIμEA platform was highly sensitive with a limit of detection down to 1 pM of thrombin. Moreover, the AIμEA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AIμEA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics, and drug screening.

Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu2+ sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu2+ by electron transfer. The high affinity of H39GFP with Cu2+ (Kd, 16.2 nM) leads to rapid detection of Cu2+ in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu2+ complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu2+ and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL–1) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.

Endonuclease V (EndoV) plays important roles in DNA repair. In the absence of a quantitative assay method for EndoV, we have developed a dual enzymatic amplified strategy for the detection of EndoV activity based on a nicking enzyme and a template independent polymerase. Every hydrolysis process performed on a substrate by EndoV can generate only one 3′-hydroxyl terminal to support the polymerization of polymerase, however, with the assistance of a nicking enzyme, abundant 3′-hydroxyl terminals are generated. Next, terminal deoxynucleotidyl transferase (TdT) involved in the second amplified procedure prolongs the 3′-hydroxyl terminus DNA with repeated T bases, providing a long template for the synthesis of fluorescent CuNPs. Consequently, a wide linear dynamic range of 0.02 to 10 U mL−1 is achieved with a detection limit of 0.02 U mL−1. This method exhibits several advantages such as high sensitivity and desirable selectivity, which shows great potential as a promising platform for the sensitive analysis of EndoV or other biomolecules.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays with the advantage of a short, simple synthesis. Herein, a strategy for generating polyT ssDNA-templated CuNPs with terminal deoxynucleotidyl transferase (TdT) to detect DNA-related enzyme activities is proposed. A short oligonucleotide primer triggered polymerization in a template-free way through TdT to form a long polyT, which acted as a template in the formation of fluorescent CuNPs. In comparison with short T-rich CuNPs, the fluorescence intensity of TdT-generated polyT-CuNPs was greatly improved. The proposed method can be used to develop a label-free turn-on fluorescence assay to detect TdT activity with a detectable minimum concentration of 3.75 U mL−1. Furthermore, our strategy can be used to perform versatile turn-on DNA-related enzyme assays based on enzyme-activated TdT polymerization. Alkaline phosphatase (ALP) and BamHI were selected as model targets to test this strategy. As a result, simple, low-cost, selective detection of DNA-related enzymes was achieved with a competitive limit of detection of 0.052 × 10−3 U mL−1 for ALP and 0.005 U mL−1 for BamHI. This method showed a high signal-to-background ratio of 62.2, 44.6, and 31.4 corresponding to TdT, ALP, and BamHI. This TdT polymerization-based DNA-CuNP synthesis strategy can be used as a universal and efficient biosensing platform.

•A simple and label-free fluorescence assay for thrombin and its inhibitor was developed.•Thrombin activity can be selectively and sensitively detected based on peptide-modulated aggregation behavior of unmodified quantum dots.•The quenching mechanism of unmodified quantum dots was primarily elucidated.•This method is successfully applied to thrombin activity measurement in human blood serum.Rapid and sensitive assay of thrombin and its inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. In this article, we developed a novel biosensor for the detection of thrombin and its inhibition based on the aggregation behavior of the unmodified CdTe QDs. A cationic substrate peptide of thrombin (GGLVPRGSCC-NH2, S-peptide) can attach to the surface of CdTe QDs, partly balance their surface negative charge, and induce the aggregation of QDs, which results in the fluorescence quenching of QDs. After hydrolysis of S-peptide by thrombin, two kinds of shorter peptides (P1-peptide, GGLVPR, and P2-peptide, GSCC) are produced. The uncharged P2-peptide rather than the cationic P1-peptide would bind to QDs. Hence, the CdTe QDs were kept stable in the solution with the fluorescence being maintained. The change of fluorescence intensity would sensitively respond to thrombin activity and its inhibition. Fluorescence spectroscopy, transmission electron microscopy and dynamic light scattering were performed to discuss the quenching mechanism. Under optimized conditions, this method enables measurement of thrombin in the range of 10–100 μU/mL with the detection limit of 1.5 μU/mL. Not only in buffer, but also in blood serum, such sensor exhibited extraordinarily high sensitivity and excellent specificity. In addition, the typical inhibitor of thrombin, hirudin, was also successfully assayed by this method (from 2 μU/mL to 30 μU/mL with the LOD of 0.21 μU/mL). Furthermore, the present approach could also be potentially extended to other proteases and their inhibitors detection with unmodified CdTe QDs.

•A simple method for protein kinase activity analysis based on (+)AuNPs is presented.•(+)AuNPs can fast, selectively, and sensitively recognize P-peptide from S-peptide.•(+)AuNPs are first and successfully used to detect the activity and inhibition of PKA.•The unmodified (+)AuNPs are easily synthesized.Herein, we report a fluorometric method for monitoring the activity and inhibition of protein kinase based on positively charged gold nanoparticles, (+)AuNPs. In this assay, when the cationic substrate peptide (S-peptide) is phosphorylated by protein kinase, the resulting negatively charged product peptide (P-peptide) will be adsorbed onto (+)AuNPs through electrostatic interaction, and the fluorescence of fluorescein isothiocyanate (FITC) on the peptide will be quenched by (+)AuNPs. Thus, the fluorescence of solution can respond to the activity of protein kinase. The feasibility of this (+)AuNPs-based method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU μL−1). Furthermore, the system is successfully applied to estimate the IC50 value of PKA inhibitor H-89. The fast mix-and-readout detection process as well as the simple synthesis of the unmodified (+)AuNPs makes this proposed method a promising candidate for simple and cost-effective kinase activity detection and a good potential in high-throughput screening of kinase-related drugs.

The rapid development in nanoparticle production and application during the past decade requires an easy, rapid, and predictive screening method for nanoparticles toxicity assay. In this study, the toxicological effects and the source of toxicity of copper nanoparticles (CuNPs) are investigated based on a stress-responsive bacterial biosensor array. According to the responses of the biosensing strains, it is found that CuNPs induce not only oxidative stress in E. coli, but also protein damage, DNA damage, and cell membrane damage, and ultimately cause cell growth inhibition. Through enzyme detoxification analysis, the toxicological effects of CuNPs are traced to H2O2 generation from CuNPs. Rapid copper release from CuNPs and Cu(I) production are observed. The oxidation of the released Cu(I) has a close relation to H2O2 production, as tris-(hydroxypropyltriazolylmethyl) amine, the specific Cu(I) chelator, can largely protect the cells from the toxicity of CuNPs. In addition, the TEM study shows that CuNPs can be adsorbed and incepted fast by the cells. Comparatively, copper microparticles are relatively stable in the system and practically non-toxic, which indicates the importance of toxic estimation of materials at the nanoscale. In addition, the Cu(II) ion can induce protein damage, membrane damage, and slight DNA damage only at a relatively high concentration. The current study reveals the preliminary mechanism of toxicity of CuNPs, and suggests that the stress-responsive bacterial biosensor array can be used as a simple and promising tool for rapid screening in vitro toxicity of nanoparticles and studying the primary mechanism of the toxicity.

Cystatin C (Cys C) is a significant cysteine protease inhibitor in human bodies, and is proposed as a fascinating novel marker of glomerular filtration rate for kidney injury detection. Almost all traditional methods for Cys C measurement are immunoassays. In this article, we report a simple, immune-independent (no need to rely on immunoassay) and label-free method for Cystatin C detection using BSA-stabilized Au nanoclusters (Au NCs) as a fluorescent probe. This method relies on the BSA scaffold degradation caused by the cysteine protease activity of papain and the specific inhibition of papain activity by Cys C. The fluorescence of BSA-Au NCs can be effectively quenched by papain, and restored by the coexistence of Cys C. Under optimized conditions, this method enables sensitive and selective measurement of Cys C concentration in the range of 25 ng/mL–2.0 μg/mL with the detection limit of 4.0 ng/mL, which is above 40 fold lower than that of commercial immune-based methods. SDS-PAGE, the absorption spectroscopy, transmission electron microscope, dynamic light scattering, and X-ray photoelectron spectroscopy were performed to discuss the quenching mechanism. In addition, percentage recoveries of Cys C in the spiked urine samples were ranged from 102.2% to 114.9% with the relative standard deviation ranging from 0.9–1.8%, demonstrating the applicability of the developed method in clinical samples. Furthermore, the present approach would be potentially extended to other proteases and their inhibitors detection with different protein-stabilized Au NCs.Highlights► A simple, immune-independent and label-free method for Cystatin C detection was developed. ► Cys C can be selective and sensitive detected using BSA-Au NCs as a fluorescent probe. ► The quenching mechanism of Au-NCs was primarily elucidated.

A novel colorimetric copper(II) biosensor has been developed based on the high specificity of alkyne–azide click reaction to the catalysis of copper ions and unmodified gold nanoparticles (AuNPs) as the signal reporter. The clickable DNA probe consists of two parts: an azide group-modified double-stranded DNA (dsDNA) hybrid with an elongated tail and a short alkyne-modified single-stranded DNA (ssDNA). Because of low melting temperature of the short ssDNA, these two parts are separated in the absence of Cu2+. Copper ion-induced azide–alkyne click ligation caused a structural change of probe from the separated form to entire dsDNA form. This structural change of probe can be monitored by the unmodified AuNPs via mediating their aggregation with a red-to-blue colorimetric read-out because of the differential ability of ssDNA and dsDNA to protect AuNPs against salt-induced aggregation. Under the optimum conditions, this biosensor can sensitively and specifically detect Cu2+ with a low detection limit of 250 nM and a linear range of 0.5–10 μM. The method is simple and economic without dual-labeling DNA and AuNPs modification. It is also highly selective for Cu2+ in the presence of high concentrations of other environmentally relevant metal ions because of the great specificity of the copper-caused alkyne–azide click reaction, which potentially meets the requirement of the detection in real samples.

•Present a label-free fluorescent method to sensitively detect thrombin activity.•Ni2+-NTA MNPs and EGFP is firstly implemented in thrombin activity and inhibition detection.•The Ni2+-NTA MNPs are reusable.•This method is successfully applied to thrombin activity measurement in human blood serum.Herein, a novel label-free fluorescent assay has been developed to detect the activity of thrombin and its inhibitor, based on a recombinant enhanced green fluorescence protein (EGFP) and Ni2+ ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni2+–NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni2+-NTA MNPs through Ni2+-hexahistidine interaction, and dragged out of the solution by magnetic separation. Thrombin can selectively digest EGFP accompanied by His-tag peptide sequence leaving, and the resulting EGFP cannot be captured by Ni2+-NTA MNPs and kept in supernatant. Hence the fluorescence change of supernatant can clearly represent the activity of thrombin. Under optimized conditions, such assay showed a relatively low detection limit (3.0×10−4 U mL−1), and was also used to detect the thrombin inhibitor, Hirudin, and further applied to detect thrombin activity in serum. Combined with the satisfactory reusability of Ni2+-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.

Endonuclease V (EndoV) plays important roles in DNA repair. In the absence of a quantitative assay method for EndoV, we have developed a dual enzymatic amplified strategy for the detection of EndoV activity based on a nicking enzyme and a template independent polymerase. Every hydrolysis process performed on a substrate by EndoV can generate only one 3′-hydroxyl terminal to support the polymerization of polymerase, however, with the assistance of a nicking enzyme, abundant 3′-hydroxyl terminals are generated. Next, terminal deoxynucleotidyl transferase (TdT) involved in the second amplified procedure prolongs the 3′-hydroxyl terminus DNA with repeated T bases, providing a long template for the synthesis of fluorescent CuNPs. Consequently, a wide linear dynamic range of 0.02 to 10 U mL−1 is achieved with a detection limit of 0.02 U mL−1. This method exhibits several advantages such as high sensitivity and desirable selectivity, which shows great potential as a promising platform for the sensitive analysis of EndoV or other biomolecules.

The interaction between supercharged green fluorescent protein (ScGFP) and graphene oxide (GO) as well as the resulting quenching effect of GO on ScGFP were investigated. Based on this unique quenching effect and the DNA-mediated ScGFP/GO interaction, a label-free fluorescence method has been established for homogeneously assaying the activity and inhibition of base excision repair enzyme.