DNA-templated Copper Nanoclusters Research Articles

In situ formation of copper nanoclusters for efficient analysis of β-glucosidase activity

An efficient and convenient fluorescent method has been developed to detect β-glucosidase via utilizing the in situ synthesis of double-stranded DNA templated copper nanoclusters (dsDNA-Cu NCs). The dsDNA-Cu NCs can be formed by using random dsDNA with (AT-TA) rich sequence as the template. However, with the addition of amygdalin and β-glucosidase, amygdalin was catalyzed by β-glucosidase to generate cyanide ion (CN−). The CN− had the high affinity to bind with Cu2+ to inhibit the production of fluorescent dsDNA-Cu NCs, resulting in low fluorescence intensity. This method exhibits both sensitivity and specificity in detecting β-glucosidase under the optimal conditions. In addition, the technique's detection limit is 0.00084 U mL−1. The serum samples were also used to verify this fluorescent strategy with satisfactory results. In addition, this method does not require complex preparation process of probes and fluorescent dye labelling, greatly simplifying the experimental processes. Thus, it can provide a sensitive method for the β-glucosidase related biomedical research.

Efficient Electrocatalytic Nitrate Reduction to Ammonia Based on DNA-Templated Copper Nanoclusters

In alkaline solutions, the electrocatalytic conversion of nitrates to ammonia (NH3) (NO3RR) is hindered by the sluggish hydrogenation step due to the lack of protons on the electrode surface, making it a grand challenge to synthesize NH3 at a high rate and selectivity. Herein, single-stranded deoxyribonucleic acid (ssDNA)-templated copper nanoclusters (CuNCs) were synthesized for the electrocatalytic production of NH3. Because ssDNA was involved in the optimization of the interfacial water distribution and H-bond network connectivity, the water-electrolysis-induced proton generation was enhanced on the electrode surface, which facilitated the NO3RR kinetics. The activation energy (Ea) and in situ spectroscopy studies adequately demonstrated that the NO3RR was exothermic until NH3 desorption, indicating that, in alkaline media, the NO3RR catalyzed by ssDNA-templated CuNCs followed the same reaction path as the NO3RR in acidic media. Electrocatalytic tests further verified the efficiency of ssDNA-templated CuNCs, which achieved a high NH3 yield rate of 2.62 mg h-1 cm-2 and a Faraday efficiency of 96.8% at -0.6 V vs reversible hydrogen electrode. The results of this study lay the foundation for engineering catalyst surface ligands for the electrocatalytic NO3RR.

DNA-templated copper nanocluster: A robust and universal fluorescence switch for bleomycin assay

Bleomycin (BLM) is widely utilized for cancer treatment due to the outstanding antitumor activity, but BLM with imprecisely controlled dosage may lead to lethal consequences. It is thus a profound task to accurately monitor the BLM levels in clinical settings. Herein, we propose a straightforward, convenient, and sensitive sensing method for BLM assay. Poly-T DNA-templated copper nanoclusters (CuNCs) are fabricated with strong fluorescence emission and uniform size distribution and served as fluorescence indicators for BLM. The high binding affinity of BLM for Cu2+makes it able to inhibit fluorescence signals generated from CuNCs. This is the underlying mechanism rarely explored and can be utilized for effective BLM detection. A detection limit of 0.27 μM (according to 3σ/s rule) is achieved in this work. And the precision, producibility, and practical useability are also confirmed with satisfactory results. Furthermore, the accuracy of the method is verified by high-performance liquid chromatography (HPLC). To sum up, the established strategy in this work exhibits the advantages of convenience, rapidness, low cost, and high precision. The construction of BLM biosensors is important to achieve the best therapeutic effect with minimal toxicity, which opens a new avenue for monitoring antitumor drugs in clinical settings.

DNA micelle-templated copper nanoclusters for fluorescent imaging of MUC1-positive cancer cells.

DNA micelles formed by hydrophobic, self-assembly of amphiphilic DNA monomers have enormous potential in biological imaging owing to its unique and programmable, three-dimensional nanostructure. Herein, we rationally design double-stranded DNA oligonucleotides with two cholesterols that can spontaneously form the lipid-mediated DNA micelles and generate the high fluorescence signal after the formation of DNA-templated copper nanoclusters (CuNCs). Furthermore, the DNA aptamer specific to MUC1 protein, aberrantly overexpressed on the surface of cancer cells, is attached to lipid-mediated DNA micelles to confer the selectivity towards the target cancer cells. With the well-defined DNA nanostructures, the cell membrane of MUC1-positive cancer cells are stained by CuNCs exhibiting an intense, red fluorescence signal, which are clearly distinguished from MUC1-negative cancer cells. This approach may not only expand the application scope of both DNA micells and CuNCs, especially in the area of cellular imaging, but also provides a basis for developing other types of DNA nanostructures to detect target biomarkers.

High Fructose Concentration Increases the Fluorescence Stability of DNA-Templated Copper Nanoclusters by Several Thousand Times.

DNA-templated copper nanoclusters (CuNCs) have limited applications because of their low fluorescence stability (several tens of minutes). In this study, we prepared CuNCs with improved temporal fluorescence stability by introducing fructose into the CuNC synthesis process and optimizing the reaction conditions. The inclusion of fructose increased the operating lifetime of CuNCs by approximately 5200-fold from 30 min to 108 days and improved their stability against heat, acids, and bases compared to CuNCs synthesized under original conditions. In addition, the fluorescence signal of CuNCs was maintained for a significantly longer time when stored at refrigeration (4 °C) and freezing (-20 °C) temperatures. Importantly, this method did not require the addition of substances other than fructose or any additional physicochemical treatment to maintain the fluorescence of DNA-templated CuNCs for more than several tens of days. As such, this study could serve as a basis to improve the stability of CuNCs for various applications.

Detection of Pyrophosphate and Alkaline Phosphatase Activity Based on PolyT Single Stranded DNA - Copper Nanoclusters

The determination of pyrophosphate and alkaline phosphatase activity plays a significant role in medical diagnosis. In this work, a label-free "ON-OFF-ON" fluorescence strategy is developed for the analysis of pyrophosphate and alkaline phosphatase activity. Using PolyT single strand DNA as templates to synthesize fluorescent copper nanoparticles, the coordination effect of pyrophosphoric acid on Cu2+ inhibited the generation of fluorescence. Afterwards, the addition of alkaline phosphatase into hydrolyze pyrophosphoric acid resulted in the release of Cu2+, whereby the fluorescence intensity could be recovered. Thereupon enhanced-sensitivity for alkaline phosphatase was obtained (0.1 mU/L), much better than previously reported methods. Meanwhile, it could be performed directly in homogeneous solution, which was very close to the actual activity level of alkaline phosphatase under physiological conditions. Likewise, satisfactory results were also obtained in specificity assessment, which demonstrated its potential application in clinical diagnosis. Notably, a new, sensitive, low-cost, short-time, and high-sensitivity platform for alkaline phosphatase detection was constructed, and the design of biosensor using DNA-templated Copper nanoclusters (CuNCs) was instructed in this study.

Detection of AluI Endonuclease Activity by Using Double Stranded DNA-Templated Copper Nanoclusters

Detection of AluI Endonuclease Activity by Using Double Stranded DNA-Templated Copper Nanoclusters

Integration of ion-tuned oligonucleotide structural motifs and DNA-templated copper nanoclusters as a manipulable logic device

Molecular logic gates possess great potential for multi-parameter sensing, intelligent diagnostics, and molecular computation. One remaining challenge was to construct novel new logic gates based on smart nanomaterials and functional nucleic acids. In this study, two novel new logic devices were constructed utilizing ion-tuned DNA/copper nanoclusters (CuNCs), which were dual-responsive to K+ and H+. By merely altering the arrangement of a specific sequence of the DNA stabilizer, two kinds of well-designed DNA probes permuted by G-tracts, C-tracts, and poly-T were designed as the building blocks of the logic device. Poly-T acted as the template for the formation of CuNCs. G-tracts and C-tracts enabled a K+/H+-responsive conformational switch into G-quadruplex (G4) and i-motif structures. Such a conformational conversion significantly modulated the fluorescence behaviors of CuNCs. This study discovered the unique interaction between the logic device and graphene oxide (GO). The logic devices were implemented for the analysis of K+ and microRNA.

Enhanced-assay of alkaline phosphatase based on polyAT dsDNA-templated copper nanoclusters

DNA-templated copper nanoclusters (CuNCs) have attracted extensive interest owing to their low cost, easy preparation, excellent optical properties and well biocompatibility, especially in the field of biosensor. However, exploration of DNA-templated CuNCs in biochemical application still suffer from the disadvantage of low sensitivity. Inspired by the report that dsDNA made up of poly adenine-thymine pair (polyAT) was a better template for fluorescent CuNcS, we reported herein for the first example to improve the CuNCs-based fluorescent assay platform, where alkaline phosphatase (ALP) was used as a model target. As a result, enhanced-assay for ALP was obtained (0.05 U/L), much lower than previously reported methods using CuNCs as probe. Meanwhile satisfying results were still obtained in the evaluation of the specificity and the detection of ALP in human serum, demonstrating its potential applications in clinical diagnosis. Notably, this research not only provides a novel and sensitive platform for ALP assay, but also plays an important guiding role in the design of biosensor using DNA-templated CuNCs.

Self-assembly of DNA-templated copper nanoclusters and carbon dots for ratiometric fluorometric and visual determination of arginine and acetaminophen with a logic-gate operation.

This work describes the synthesis of red-emitting copper nanoclusters (CuNCs) by using DNA as the template. DNA-templated CuNCs combined with blue-emitting carbon dots (CDs) form the self-assembled complex DNA-CuNC/CDs through electrostatic interactions. In the presence of arginine (Arg), the blue fluorescence of CDs (with excitation/emission maxima at 350/440nm) is quenched. Addition of acetaminophen (AP) induces the competitive combination of Arg and AP for the CDs. This results in the release of Arg from CDs and the recovery of blue fluorescence. On addition of both Arg and AP, the red fluorescence of CuNCs (with excitation/emission maxima at 350/670nm) undergoes only slight changes. Hence, the DNA-CuNC/CD complex can serve as a dually emitting ratiometric probe to determine both Arg and AP, with detection limits of 0.35μM and 0.26μM, respectively. The probe also enables on-site, visual determination of Arg and AP in aqueous samples, best by placing the system in cuvettes or dropping it onto filter paper strips. An "INHIBIT" logic gate was designed based on this ratiometric and visual fluorometric assay, with Arg and AP as the inputs. Graphical abstractSchematic presentation of self-assembly of DNA-templated copper nanoclusters and carbon dots to construct novel dual-emitting nanoprobes for ratiometric fluorometric and visual determination of arginine and acetaminophen in aqueous solutions and on wetting filter paper strips.

Nano-fluorescent probes based on DNA-templated copper nanoclusters for fast sensing of thiocyanate

A fast and label-free fluorescent sensor was developed to determine SCN−via inhibiting the formation of DNA-templated copper nanoclusters (CuNCs).

Facile fluorescence strategy for sensitive detection of microcystin-LR based on dsDNA-templated copper nanoclusters

We developed a simple, sensitive, low-cost and label-free method to detect microcystin-leucine-arginine (MC-LR) using double-strand DNA-templated copper nanoclusters (dsDNA-CuNCs) as fluorescent probes.

DNA-templated copper nanoclusters as a fluorescent probe for fluoride by using aluminum ions as a bridge.

A selective fluorescent on-off-on probe has have designed for the detection of fluoride (F-) ions based on DNA-templated copper nanocluster (CuNCs) and by using aluminum(III) ions as a bridge. A 40-mer polythymine acts as a template for the reduction of Cu(II) to Cu(0) by ascorbic acid. This result is the formation of redfluorescent CuNCs, with excitation/emission peaks at 340/640nm. After addition of Al3+ ions, the fluorescence of CuNCs is quenched because the interaction of Al3+ and DNA disturbs the formation of DNA-templated CuNCs. Fluorescence is restored on addition of fluoride to the system. This is due to the desorption of Al3+ from the DNA and the formation of the Al(OH)3F- complex. This system displays a fast fluorometric response to fluoride, with high selectivity over other anions. Fluorescence increases linearly in the 2 to 150μMF- concentration range, and the detection limit is 1.0μM. This probe has been successfully used for the detection of F- ions in four brands oftoothpaste. The method is rapid, cost-effective, selective, and does not require toxic solvents and reagents. Graphical abstract Schematic presentation of a method for fluorometric determination of fluoride by using DNA-templated copper nanoclusters (CuNCs) and using aluminum(III) as a bridge. The red fluorescence of the CuNCs is quenched in the presence of Al(III) ions but restored after addition of fluoride.

Enzyme-free fluorescent detection of microcystin-LR using hairpin DNA-templated copper nanoclusters as signal indicator

Microcystin-LR (MC-LR) is a potent hepatotoxin that is often associated with blooms of cyanobacteria. The analysis of trace MC-LR plays important role in environmental and health fields. Herein, we developed a low-cost and enzyme-free detection method of MC-LR by using hairpin DNA-templated copper nanoclusters (hpDNA-CuNCs) as fluorescent probe. The hpDNA-template was designed and fabricated by a MC-LR aptamer loop and a double strand stem, which can specifically recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as a template for the formation of CuNCs. The formed fluorescent sensing probe of hpDNA-CuNCs exhibits maximum emission wavelength at 575 nm. Upon the addition of target MC-LR into the hpDNA-CuNCs, we observed fluorescence was quenched considerably due to the high affinity between MC-LR and hpDNA aptamer strand loop, which indicated a conformational change of hairpin probe from the stem-loop DNA structure to single-stranded DNA. Then, the change of fluorescence intensity can be used to monitor the concentration of MC-LR from 0.005 to 1200 μg L−1 with a detection limit of 0.003 ng L−1. Compared with the previous reports, this method does not require complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. Moreover, the target MC-LR in real water samples has been detected.

A Fluorescence Strategy for Silver Ion Assay via Cation Exchange Reaction and Formation of Poly(thymine)-templated Copper Nanoclusters.

The detection of Ag+ ions in the environment and biological systems is important to both environmental monitoring and modern medicine. Herein, a novel and label-free method was developed for Ag+ detection, which utilizes a florescence strategy combining DNA-templated copper nanoclusters (Cu NCs) with cation exchange reactions. The method is primarily based on the effective detection of an Ag+-triggered cation exchange reaction and the release of free Cu2+ from CuS nanoparticles (CuS NPs), while the probe T30 serves as an effective template for the formation of fluorescence-inducing Cu NCs. Under optimal conditions, this sensing system displays high sensitivity with a 50 nM limit of detection and a range from 0 - 100 μM. In addition, the proposed method exhibits high selectivity and, therefore, was successfully applied to the analysis of real samples. Overall, these results demonstrate that our established method has advantages of design and operation simplicity, as well as cost-effectiveness.

Double stranded DNA-templated copper nanoclusters as a novel fluorescent probe for label-free detection of rutin

In this study, we developed a simple, sensitive, low-cost and label-free method to detect rutin by using double-stranded DNA-templated copper nanoclusters (dsDNA-CuNCs) as a fluorescent probe.

Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters.

A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3'-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07UmL-1 to 15UmL-1 and a detection limit of 0.06UmL-1. Graphical abstract The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.

Ultrasensitive Biosensor for Detection of Mercury(II) Ions Based on DNA-Cu Nanoclusters and Exonuclease III-assisted Signal Amplification.

This paper describes a novel method for label-free mercury(II) ion detection based on exonuclease III-induced target signal recycling amplification using double-stranded DNA templated copper nanoclusters. The synthesized DNA-Cu nanoclusters were used with exonuclease III loop amplification technology for ultra-high sensitivity detection of mercury(II) ions, which were detected by significantly decreased fluorescence intensity. Under the optimal experimental conditions, there was a clear linear relationship between Hg2+ concentration in the range of 0.04 to 8 nM and fluorescence intensity. The detection limit for Hg2+ was 4 pM. In addition, the interference of other metal ions on the mercury(II) ion detection was also studied. To confirm the application of the fluorescent sensor, it was applied to determine the concentrations of mercury(II) ions in tap water, and the results showed that the method can be used to detect mercury(II) ions in water samples successfully.

Label-free rapid and simple detection of exonuclease III Activity with DNA Templated Copper Nanoclusters

Label-free rapid and simple detection of exonuclease III Activity with DNA Templated Copper Nanoclusters

Label-free rapid and simple detection of exonuclease III Activity with DNA Templated Copper Nanoclusters.

In this study, DNA-templated copper nanoclusters (DNA-CuNCs) were used to detect exonuclease III (Exo III) activity, which is important for the diagnosis and therapy of several diseases. The results of this study showed that Exo III was affected by the concentrations of magnesium ions and sodium ions, and its optimal conditions for cleavage were 5 mM Mg2+ and less than 25 mM Na+. With a blunt-end DNA, more than 98% of DNA was digested by Exo III. As expected, with two or four cytosines in the terminal position of a 4-base overhanging DNA such as 5'-GGCC-3' and 5'-CCCC-3', there was little cleavage by Exo III compared to with a blunt-end DNA.