DNA-templated Copper Nanoparticles Research Articles

Electrochemical Detection of miRNA Combining T7 Exonuclease-Assisted Cascade Signal Amplification and DNA-Templated Copper Nanoparticles.

miRNA has been serving as an ideal biomarker for diagnosis, prognosis, and therapy of many severe diseases. In this study, we have developed an amplified electrochemical method for miRNA detection using T7 exonuclease (exo) and copper nanoparticles (CuNPs). Double-stranded DNA modified on the electrode surface is used as the template for in situ synthesis of CuNPs as excellent electrochemical signal sources. Two cycles of DNA cleavage reactions are carefully designed according to the catalytic activity of T7 exo and occur in the solution and at the electrode surface, respectively. The two cycles are integrated for cascade signal amplification. Briefly, target miRNA triggers the first cycle and its product triggers the second cycle, which destroys the template on the electrode for CuNPs synthesis. As a result, electrochemical signal is decreased and can be used to reflect the level of initial miRNA. Due to T7 exoassisted cascade signal amplification and intense electrochemical responses from CuNPs, the biosensor is developed with excellent sensitivity. A linear range from 10-16 to 10-13 M and the limit of detection as low as 4.5 × 10-17 M are achieved. Meanwhile, it shows the capability of discriminating single base mismatch and exhibits the eligibility in the analysis of miRNA extracted from cells. Therefore, it has great potential for biomedical research and disease management.

DNA-templated copper nanoparticles for voltammetric analysis of endonuclease activity.

DNA endonucleases play critical roles in medicinal chemistry, which are also commonly used in molecular biology investigation. Sensitive quantification of endonuclease activity is of great significance. In this study, a reliable electrochemical approach for endonuclease activity sensing is developed with the adoption of copper nanoparticles (CuNPs) as electrochemical reporters. Firstly, a DNA duplex is designed and modified on a gold electrode, which acts as the template for the synthesis of CuNPs. Subsequently, the formed NPs are dissolved and then electrodeposited on a glassy carbon electrode for DPV measurements. With the effect of target endonuclease, the DNA duplex is specifically recognized and cleaved. Thus, CuNPs cannot be synthesized and a declined DPV peak is obtained to reveal the level of endonuclease activity. This developed sensor has a wide linear range from 10-3 to 10 U mL-1, and the limit of detection is 10-3 U mL-1, which is extremely low. High stability and excellent reproducibility are also researched. Besides, this sensor shows good selectivity, which can successfully distinguish target endonuclease from possible interferences.

A label-free fluorometric assay for actin detection based on enzyme-responsive DNA-templated copper nanoparticles

Herein, we develop a fluorescent strategy for label-free detection of actin by the in situ formation of double-stranded DNA (dsDNA) templated copper nanoparticles (CuNPs) and the digestion capability of deoxyribonuclease I (DNase I). In this design, the introduction of actin can effectively hinder the enzymic digestion owing to the exceptional target-mediated conformational structure between actin and DNase I. Consequently, the remaining adenine-thymine-rich dsDNA can act as an efficient template for fluorescent CuNPs (λex = 340nm, λem = 585nm) with a Mega-Stokes shifting (245nm). This novel fluorescent strategy exhibits several advantages such as low-cost and environmental-friendly, because it does not require the toxic organic dyes and laborious procedures. Under optimized experimental conditions, this method realizes a turn-on selective and sensitive response for actin with a detection limit of 0.12μgmL−1 and a detection capability in complex biological media with satisfactory results.

Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy.

Smart Composite Reagent Composed of Double-Stranded DNA-Templated Copper Nanoparticle and SYBR Green I for Hydrogen Peroxide Related Biosensing.

On the basis of an interesting experimental phenomenon, a novel and smart composite reagent consisting of double-stranded DNA-templated copper nanoparticles (dsDNA-CuNPs) and DNA intercalator (SYBR Green I) was developed and exploited for hydrogen peroxide (H2O2) detection as well as oxidase-based biosensing. The study found that, within the composite reagent, the small molecule SYBR Green I was easily adsorbed on the surfaces of CuNPs, instead of intercalating into the dsDNA. So the composite reagent only exhibited the red fluorescence generated from dsDNA-CuNPs. However, when the solution of H2O2 was added into the composite reagent, the CuNPs were deconstructed and their fluorescence was quenched; meanwhile, the inhibition of SYBR Green I binding with dsDNA was eliminated. As a result, the mixed solution of the composite reagent with H2O2 exhibited green fluorescence generated from the intercalation of SYBR Green I into dsDNA. Since H2O2 is an important molecule and involved in various research fields, this developed composite reagent could be employed for many applications in biological analysis. As a proof-of-application demonstration, the sensitive detection of glucose was conducted. Moreover, the method was also extended to the detection of other biomolecules, such as cholesterol and horseradish peroxidase, which indicated the broad applications of the proposed sensing strategy in biomedical analysis.

Hairpin-shaped DNA Templated Copper Nanoparticles for Fluorescence Detection of Adenosine Triphosphate Based on Ligation-mediated Exonuclease Cleavage.

DNA-templated copper nanoparticles (CuNPs) have recently received considerable interest as functional fluorescent probes for biochemical analysis. In this work, a novel ATP-dependent ligation reactions (ATP-DLR) based ATP assay strategy was proposed by using hairpin-shaped (HS) DNA templated CuNPs as a fluorescent probe. Nick sealing by T4 DNA ligase leads to the formation of intact HS DNA, which can resist the exonuclease cleavage and be taken as the template for CuNPs formation, resulting in strong fluorescence. The proposed ATP detection is label free, sensitive and highly selective, and it has good linearity from 0.02 to 4 μM and a detection limit of 7 nM. This strategy is expected to promote the exploitation and application of DNA-templated CuNPs in biochemical and biomedical studies, and holds great promise in fluorescence detection for other ligation-related biomolecules.

A fast, highly sensitive and selective assay of iodide ions with single-stranded DNA-templated copper nanoparticles as a fluorescent probe for its application in Kunming mice samples.

The development of fast, sensitive, selective and flexible methods for the detection of iodide is highly demanded and is of great significance. In this work, single-stranded DNA-templated copper nanoparticles (ssDNA-CuNPs) generated by sodium ascorbate reduction of Cu2+ along the single-stranded DNA of poly-T were utilized as a fluorescent probe for the determination of iodide ions (I-). The detection scheme is based on the instant quenching of the fluorescence of ssDNA-CuNPs by iodide ions. I- can be quantified in the concentration range from 0.050 to 40 μM and from 40 to 80 μM, and the limit of detection is as low as 15 nM. This method provides a simple and convenient strategy for the biochemical assay of I-, which is also helpful for early diagnosis of related diseases. The establishment of a low cost and fast detection method would be particularly important in developing countries where medical supplies are lacking.

Sensitive detection of glutathione by using DNA-templated copper nanoparticles as electrochemical reporters

Herein, a new electrochemical method for the detection of glutathione (GSH) is proposed with the adoption of DNA-templated copper nanoparticles (CuNPs) as electrochemical reporters. The general sensing concept of the method relies on the strong interaction between GSH and copper ions, which would cause great hindrances to DNA-templated formation of CuNPs on the electrode surface, resulting in decreased electrochemical signals. Experimental results reveal that this method exhibits desirable selectivity and reproducibility, and allows absolute detection of as low as 0.27nM of the target, showing superior sensitivity as compared to previous electrochemical methods. This work may not only provide an effective tool for GSH detection, but also promote the practice of DNA-templated CuNPs in electrochemical biosensing applications, which may be of great attraction in areas ranging from gene analysis, clinical diagnosis and environmental monitoring.

A label-free and enzyme-free ultra-sensitive transcription factors biosensor using DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier

Detection and quantification of specific protein with ultralow concentration play a crucial role in biotechnological applications and biomedical diagnostics. In this paper, a label-free and enzyme-free amplified fluorescent biosensor has been developed for transcription factors detection based on AT-rich double-stranded DNA-templated copper nanoparticles (ds DNA/Cu NPs) and hairpin DNA cascade reaction. This strategy was demonstrated by using nuclear factor-kappa B p50 (NF-κB p50) and specific recognition sequences as a model case. In this assay, a triplex consists of double-stranded DNA containing NF-κB p50 specifically binding sequences and a special design single-stranded DNA (Trigger) which is able to activate the hairpin DNA cascade amplifier (HDCA). In the presence of NF-κB p50, the triplex became unstable since the target bound to the recognition sequences with strong affinity. The selective binding event confirmed that the Trigger was successfully released from the triplex and initiated HDCA to yield the product which could effectively template the formation of fluorescent Cu NPs. The experimental results revealed that the advanced strategy was ultra-sensitive for detecting NF-κB p50 in the concentration range from 0.1 to 1000 pM with a detection limit of 0.096 pM. In addition, the relative standard deviation was 4.08% in 3 repetitive assays of 500 pM NF-κB p50, which indicated that the reproducibility of this strategy was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, cost-effective, and simplified operations. In addition, the proposed biosensing platform is versatile. By conjugating with various specific recognition units, it could hold considerable potential to sensitive and selective detect various DNA-binding proteins and might find wide applications in biomedical fields.

H2O2-mediated fluorescence quenching of double-stranded DNA templated copper nanoparticles for label-free and sensitive detection of glucose

A label-free fluorescent sensor has been developed for glucose detection based on H2O2-mediated fluorescence quenching of ds-DNA templated Cu NPs.

Inhibition of double-stranded DNA templated copper nanoparticles as label-free fluorescent sensors for l-histidine detection

A label-free fluorescent sensing strategy was reported for l-histidine detection by the inhibition of double-stranded DNA templated copper nanoparticles.

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

In this paper, we report an improved electrochemical aptasensor based on exonuclease III and double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) assisted signal amplification. In this sensor, duplex DNA from the hybridization of ligated thrombin-binding aptamer (TBA) subunits and probe DNA can act as an effective template for the formation of CuNPs on the electrode surface, so copper ions released from acid-dissolution of CuNPs may catalyze the oxidation of ο-phenylenediamine to produce an amplified electrochemical response. In the presence of thrombin, a short duplex domain with four complementary base pairs can be stabilized by the binding of TBA subunits with thrombin, in which TBA subunit 2 can be partially digested from 3′ terminal with the cycle of exonuclease III, so the ligation of TBA subunits and the subsequent formation of CuNPs can be inhibited. By electrochemical characterization of dsDNA-templated CuNPs on the electrode surface, our aptasensor can display excellent performances for the detection of thrombin in a broad linear range from 100fM to 1nM with a low detection limit of 20.3fM, which can also specially distinguish thrombin in both PBS and serum samples. Therefore, our aptasensor might have great potential for clinical diagnosis of biomarkers in the future.

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

A simple and sensitive sensor for rapid detection of sulfide anions using DNA-templated copper nanoparticles as fluorescent probes

A simple and rapid method for the detection of S(2-) anions with high selectivity and sensitivity has been developed by using random double-strand DNA-templated formation of copper nanoparticles as novel fluorescence probes in aqueous solution.