DNA-templated Silver Nanoclusters Research Articles

Cationic-Polyelectrolyte-Modified Fluorescent DNA-Silver Nanoclusters with Enhanced Emission and Higher Stability for Rapid Bioimaging.

DNA-templated silver nanoclusters (DNA-Ag NCs) have shown great potential in various bioanalysis and bioimaging applications, owing to their facile synthesis and ultrasmall sizes and especially their programmable fluorescence emission depending on the sequences of DNA templates. However, the bioimaging applications of DNA-Ag NCs are severely limited by their poor stability in physiological environments and their poor cell permeability, resulting from the highly negatively charged DNA backbones. In this paper, cationic polyelectrolytes were used to modify fluorescent DNA-Ag NCs via electrostatic interactions between the positive polymer backbones and the negatively charged phosphate groups of the DNA strands. The fluorescence properties of a typical single-stranded-DNA (ssDNA)-templated Ag NC changed dramatically after incorporation with the cationic polyelectrolyte caused by conformational changes in the DNA, with a 3-fold fluorescence-emission enhancement of the Ag NCs observed after incorporation with a typical cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA), whereas the fluorescence of double-stranded-DNA (dsDNA)-templated Ag NCs changed little after incorporation. Moreover, the modification with PDDA greatly prolonged the stability of both ssDNA- and dsDNA-stabilized Ag NCs against nuclease digestion and enhanced the cell uptake of the DNA-Ag NCs.Rapidcell imaging was demonstrated using NIH/3T3 cells as a model system.

An enhanced silver nanocluster system for cytochrome c detection and natural drug screening targeted for cytochrome c

A label-free and turn-off fluorescence method for detecting cytochrome c (Cyt c) was developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs@tween 80) as a novel fluorescence-enhancing nanoprobe. Cyt c-induced electron transfer between Cyt c and DNA-AgNCs results in a significant fluorescence quenching effect of DNA-AgNCs. The coated tween 80 around DNA-AgNCs particles significantly causes fluorescence signal enhancement in the solution. Meanwhile, the efficiency of fluorescence quenching by Cyt c increases substantially by adding 0.1% tween 80 (final volume concentration) into the detection system. Based on these findings, a simple method with the detection limit of 0.8 nM was established for Cyt c based on tween 80 enhanced DNA-AgNCs. A good linear relationship between the fluorescent intensity and the concentration of Cyt c ranging from 0.8 nM to 20,000 nM (R2 = 0.9910) was obtained. Consequently, the method was applied to monitor the Cyt c variation in HCT-116 and BGC-823 tumor cell culture medium after being treated with doxorubicin (DOX)and two kinds of natural compounds (J5 and S6, extracted from plants Abacopteris Penangiana and Swertia punicea Hemsl, respectively). The results indicate that the level of Cyt c in cell culture medium decreases according to the presence of DOX, J5 and S6. The reliability of the result was further confirmed by using cell viability assay and flow cytometry for apoptosis analysis (FACS). From these data, it can be concluded that this groundbreaking nanoprobe with high sensitivity, great simplicity and good biocompatibility boosts a promising outlook for exploring the molecular mechanisms of apoptosis regulation and nature drugs screening.

A "turn-on" fluorometric assay for kanamycin detection by using silver nanoclusters and surface plasmon enhanced energy transfer.

A rapid method is described for the determination of the antibiotic kanamycin. It integrates a kanamycin-binding aptamer and surface plasmon enhanced energy transfer (SPEET) between DNA-templated silver nanoclusters (AgNCs) and gold nanoparticles (AuNPs). The AgNCs and AuNPs were selected as energy donor and energy acceptor, respectively. The aptamer was designed to regulate the energy transfer between AgNCs and AuNPs. The aptamer was adsorbed on the AuNPs. Upon addition of kanamycin, the aptamer-kanamycin complex is formed, and this results in the aggregation of the AuNPs in high salt concentration, the formation of a blue coloration, and in the suppression of the SPEET process. The fluorescence of the AgNCs (with excitation/emission peaks at 560/600nm) is quenched by the aptamer protected AuNPs in absence of kanamycin. The fluorescence on addition of kanamycin increases linearly in the 5 to 50nM concentration range, with a lower detection limit of 1.0 nM (at S/N = 3). The assay can be performed within 30min. It was successfully applied to the determination of kanamycin in spiked milk samples, and recoveries ranged between 90.2 and 95.4%. Conceivably, the strategy has a wide potential for screening by simply changing the aptamer. Graphical abstract Schematic presentation of the aptamer regulated surface plasmon enhance energy transfer (SPEET) between silver nanoclusters (Ag NCs) and gold nanoparticles (Au NCs) in high salt concentration buffer, and of the procedure for the detection of kanamycin.

Multifunctional G-quadruplex-based fluorescence probe coupled with DNA-templated AgNCs for simultaneous detection of multiple DNAs and MicroRNAs

A rapid, label-free, and multifunctional fluorescent probe for simultaneous detection of multiple targets was fabricated based on G-quadruplexes and DNA-templated silver nanoclusters (AgNCs). In this work, a probe with two capitated recognized regions was coupled with a locked G-quadruplex at the 5′-terminus and dark AgNC at the 3′-terminus. Upon the addition of the virus subtype H5N1 gene or microRNA-141, only the sequence of the G-quadruplex was released and bound with Thioflavin T (ThT) for a specific fluorescent response. On the contrary, with the presence of the influenza virus subtype H1N1 gene or microRNA-21, the fluorescence intensity was enhanced because of two split AgNCs approaching closely to produce a nanocluster dimer. Subsequently, with multiple target addition, fluorescence signals were produced for both G-quadruplexes and AgNCs. Moreover, this single and duplex detection for virus DNAs and microRNAs, which provides a versatile platform for different targets, was sufficiently sensitive for the expected detection limit, and still possessed unique selectivity with similar oligonucleotides. The simultaneous detection of targets in biological fluids indicated that there is great opportunity for this strategy to be further applied in biomedical research and clinical diagnosis.

A single fluorophore ratiometric nanosensor based on dual-emission DNA-templated silver nanoclusters for ultrasensitive and selective Pb2+ detection

Lead ion (Pb2+) is known as a major non-degradable environmental pollutant and is quite harmful to human health. In our investigation, a novel ratiometric fluorescent nanosensor with DNA-AgNCs as single fluorophore has been successfully constructed for ultrasensitive and specific detection of Pb2+. The single-strand DNA templated silver nanoclusters present green emission, which can be converted into red emission after approaching a specific DNA segment by forming duplex with its complementary DNA strands (ds-DNA-AgNCs). The ds-DNA-AgNCs contains rA cleavage site of Pb2+-dependent DNAzyme configuration. In the presence of Pb2+, the specific DNA segment would be released from the ds-DNA-AgNCs, resulting in discoloration of DNA-AgNCs from red to green. Since Pb2+ can successively cleave the rA site, the signal change from red to green emission is amplified. Thus, benefiting from the Pb2+-dependent DNAzyme and the charming properties of DNA-AgNCs, the proposed platform exhibits a good linear relationship from 0.001 nM to 10 nM with a detection limit of 1.0 pM for Pb2+ determination, which is lower than most of the reported Pb2+ biosensors. Moreover, this sensitive ratiometric fluorescent probe exhibits excellent selectivity toward Pb2+ detection in real samples, such as lake water, tap water, and human serum samples, illustrating the huge potential applications for complicated samples in the future.

A Turn-On Detection of DNA Sequences by Means of Fluorescence of DNA-Templated Silver Nanoclusters via Unique Interactions of a Hydrated Ionic Liquid.

Nucleic acid stability and structure, which are crucial to the properties of fluorescent DNA-templated silver nanoclusters (DNA-Ag NCs), significantly change in ionic liquids. In this work, our purpose was to study DNA-Ag NCs in a buffer containing the hydrated ionic liquid of choline dihydrogen phosphate (choline dhp) to improve fluorescence for application in DNA detection. Due to the stabilisation of an i-motif structure by the choline cation, a unique fluorescence emission—that was not seen in an aqueous buffer—was observed in choline dhp and remained stable for more than 30 days. A DNA-Ag NCs probe was designed to have greater fluorescence intensity in choline dhp in the presence of a target DNA. A turn-on sensing platform in choline dhp was built for the detection of the BRCA1 gene, which is related to familial breast and ovarian cancers. This platform showed better sensitivity and selectivity in distinguishing a target sequence from a mutant sequence in choline dhp than in the aqueous buffer. Our study provides new evidence regarding the effects of structure on properties of fluorescent DNA-Ag NCs and expands the applications of fluorescent DNA-Ag NCs in an ionic liquid because of improved sensitivity and selectivity.

Detecting the adulteration of antihypertensive health food using G-insertion enhanced fluorescent DNA-AgNCs

In this work, a new phenomenon was found that the insertion of guanine (G) bases into the cytosine (C)-rich DNA could endow enhanced fluorescence intensity of DNA-templated silver nanoclusters (DNA-AgNCs). This could be another important finding after Yeh’s report that DNA-AgNCs can be greatly enhanced when placed in proximity to G-rich bases and, a supplementary proof for the understanding of G&C roles in the synthesis of DNA-AgNCs. A sequence (C6G5G6) was selected for the synthesis of DNA-AgNCs of quantum yield 28%. The AgNCs were employed as a visual and facile probe for the sensitive detection of captopril in antihypertensive health food. This detection strategy showed good linear response to captopril with applicability in complex matrices of real samples.

DNA Templated Metal Nanoclusters: From Emergent Properties to Unique Applications.

Metal nanoclusters containing a few to several hundred atoms with sizes ranging from sub-nanometer to ∼2 nm occupy an intermediate size regime that bridges larger plasmonic nanoparticles and smaller metal complexes. With strong quantum confinement, metal nanoclusters exhibit molecule-like properties. This Account focuses on noble metal nanoclusters that are synthesized within a single stranded DNA template. Compared to other ligand protected metal nanoclusters, DNA-templated metal nanoclusters manifest intriguing physical and chemical properties that are heavily influenced by the design of DNA templates. For example, DNA-templated silver nanoclusters can show bright fluorescence, tunable emission colors, and enhanced stability by tuning the sequence of the encapsulating DNA template. DNA-templated gold nanoclusters can also serve as excellent cocatalysts, which are integratable with other biocatalysts such as enzymes. In this Account, DNA-templated silver and gold nanoclusters are selected as paradigm systems to showcase their emergent properties and unique applications. We first discuss the DNA-templated silver nanoclusters with a focus on the creation of a complementary palette of emission colors, which has potential applications for multiplex assays. The importance of the DNA template toward enhanced stability of silver nanoclusters is also demonstrated. We then introduce a special class of activable fluorescence probes that are based on the fluorescence turn-on phenomena of DNA-templated silver nanoclusters, which are named nanocluster beacons (NCBs). NCBs have distinct advantages over molecular beacons for nucleic acid detection, and their emission mechanisms are also discussed in detail. We then discuss a universal method of creating novel DNA-silver nanocluster aptamers for protein detection with high specificity. The remainder of the Account is devoted to the DNA-templated gold nanoclusters. We demonstrate that DNA-gold nanoclusters can serve as enhancers for enzymatic reduction of oxygen, which is one of the most important reactions in biofuel cells. Although DNA-templated metal nanoclusters are still in their infancy, we anticipate they will emerge as a new type of functional nanomaterial with wide applications in biology and energy science. Future research will focus on the synthesis of size selected DNA-metal nanoclusters with atomic monodispersity, structural determination of different sized DNA-metal nanoclusters, and establishment of structure-property correlations. Some long-standing mysteries, such as the origin of fluorescence and mechanism for emission color tunability, constitute the central questions regarding the photophysical properties of DNA-metal nanoclusters. On the application side, more studies are required to understand the interaction between nanocluster and biological systems. In the foreseeable future, one can expect that new biosensors, catalysts, and functional devices will be invented based on the intriguing properties of well-designed DNA-metal nanoclusters and their composites. Overall, DNA-metal nanoclusters can add additional spotlights into the highly vibrant field of ligand protected, quantum sized metal nanoclusters.

A Label-Free Detection of NdeI Endonuclease Activity by Using DNA-Templated Silver Nanoclusters.

Restriction endonucleases play an important role in genetic recombination, molecular cloning, clinical diagnosis, and pharmacological application in drug studies. In the present study, DNA-templated Ag+ nanoclusters (DNA-AgNCs) were used to detect NdeI endonuclease activity due to their facile synthesis, outstanding optical properties, and good biocompatibility. This study monitored fluorescence intensity of DNA-AgNCs to detect NdeI endonuclease activity and its inhibition in the presence of 5-fluorouracil under optimal conditions.

DNA-templated fluorescent silver nanoclusters for sensitive detection of pathogenic bacteria based on MNP-DNAzyme-AChE complex

The rapid and sensitive detection of bacteria is of great importance for the field of food safety, water quality and clinical diagnosis. Herein, we present a novel DNA-templated fluorescent silver nanoclusters (DNA-AgNCs) based sensing system integrated with magnetic nanoparticles (MNP)-DNAzyme-AChE complex for detection of pathogenic bacterial using Escherichia coli (E. coli) as a target bacteria. The platform takes advantage of a DNA-AgNCs based enzyme-responsive fluorescent signal element and a DNAzyme recognition element, providing an ultrasensitive detection limit of 60 cfu mL−1, and good specificity to the target bacteria. Moreover, the platform could be developed into a potentially versatile system by simply altering corresponding DNAzyme as the objects recognition probes.

Molecular beacon-templated silver nanoclusters as a fluorescent probe for determination of bleomycin via DNA scission

The authors describe a molecular beacon-based fluorescent probe for the determination of the cancer drug bleomycin (BLM). The probe was tagged with DNA-templated silver nanoclusters (DNA-AgNCs) and guanine-rich sequences (GRSs) at two terminals serving as signal reporter with a loop. In the absence of the BLM-iron(II) complex [BLM-Fe(II)], the probe has a hairpin shape and displays strong fluorescence because the AgNCs are close to the GRSs. In the presence of the BLM-Fe(II) complex, it will selectively cleave the probe at the 5'-GC-3' scission site of the loop. This displaces the AgNCs away from the GRSs and causes a decrease in fluorescence, best measured at excitation/emission wavelengths of 565/623nm. This effect enables BLM to be detected with a detection limit as low as 33 pM, which was 1-3 orders of magnitude more sensitive than most of the previous reports. The probe was applied for the determination of BLM in spiked human serum samples, and excellent performance was achieved. In our perception, the method described here represents a promising tool for highly sensitive and specific analysis of BLM during cancer treatment. Graphical abstract Schematic of a highly sensitive fluorometric assay forbleomycin. It is based on molecular beacon-templated silver nanoclusters and DNA scission.

Lighting Up Fluorescent Silver Clusters via Target-Catalyzed Hairpin Assembly for Amplified Biosensing.

Isothermal enzyme-free nucleic acid circuits have been developed for carrying out diverse functions ranging from dictate biocomputing to amplified biosensing. Catalytic hairpin assembly (CHA), the catalyzed cross-opening of two hairpin substrates by an initiator, has attracted increasing attention because of its facile design and high amplification capacity. The complex labeling and frequent photobleaching of a conventional fluorescent CHA biosensor still remains a challenge that needs to be solved. Herein, we constructed a new label-free and enzyme-free isothermal CHA lighting up AgNCs strategy for amplified nucleic acid assay by integrating the interfacially and spatially sensitive feature of DNA-templated fluorescent silver nanoclusters (DNA-AgNCs) and the high signal amplification capability of the CHA circuit. In this strategy, one polyguanine-grafted hairpin and the other AgNCs-capturing hairpin were engineered as assembly constitutes, which were kinetically impeded from cross-hybridizations without target. However, in the presence of target, the CHA-catalyzed assembly of two functional hairpins was successively progressed and concomitantly accompanied by an efficient accommodation of AgNCs to the polyguanine-elongated dsDNA product, leading to highly efficient AgNCs-lighting up and to the generation of an amplified fluorescence signal. As a simple mix-and-detect strategy, the isothermal enzyme-free CHA-mediated lighting up AgNCs (CHA-AgNCs) system provided a facile visualization way for amplified detection of DNA with a detection limit of 20 pM, which was comparable to or even better than some enzyme-involved amplification methods. The homogeneous CHA-AgNCs system can be used as a general sensing platform and be easily adapted for analyzing other biologically important analytes, for example, microRNA (miRNA), by introducing the sensing module consisting of an auxiliary hairpin through an easy-to-integrate procedure. By taking advantage of the signal amplification features of CHA and the robust AgNCs-lighting up procedure, we anticipate that the CHA-lighting up AgNCs system can provide an important tool for biomedicine and bioimaging applications and thus should hold great promise in clinical diagnoses and treatment fields.

The role of spacer sequence in modulating turn-on fluorescence of DNA-templated silver nanoclusters.

Guanine activation of fluorescence in DNA templated silver nanoclusters (AgNCs) is an interesting physical phenomenon which has yet to be fully understood to date. While the individual role of cytosine and guanine has been established, there is still a knowledge gap on how the AgNC–DNA system switches from dark to bright state. Here, we present evidence on the universal role of the DNA spacer sequence in physically separating two Ag+-binding cytosine sites to maintain the dark state while holding them together for structural re-organization by the guanine-rich strand to activate the bright state. The extent of turn-on signal could be modulated by adjusting the spacer length and composition. The ATATA spacer sequence was found to have negligible dark state fluorescence and a turn-on effect of 2440-fold, which was almost five times of the highest factor reported to date.

Fluorescent DNA-Templated Silver Nanoclusters from Silver(I)-Mediated Base Pairs.

Silver nanoclusters (AgNCs) stabilized by double-stranded DNA (dsDNA) are of special interest because the duplex structures provide rigidity and chirality that can be transferred to the metal clusters. This work reports fluorescent AgNCs obtained from dsDNA templates containing artificial ligand-derived nucleobases. They are compared with fluorescent AgNCs obtained from DNA templates containing C:C mismatches (C, cytosine). Towards this end, the new metal-mediated Im-AgI -C base pair composed of imidazole (Im) and cytosine was introduced into dsDNA and reduced to form AgNCs. The clusters were characterized by UV/Vis, fluorescence and circular dichroism spectroscopy. The AgNCs form chiral aggregates with dsDNA. Their optical properties are highly sequence-dependent. As a result, a simple method to detect a cytosine insertion into a run of cytosine residues in a duplex is proposed using the human CDH1 gene as an example.

Ultrasensitive and non-labeling fluorescence assay for biothiols using enhanced silver nanoclusters

A label-free, ultra-sensitive and turn-off fluorescence method for detecting cysteine (Cys) has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. The method is based on the specific interaction between Cys and DNA-AgNCs via robust Ag-S bonds and the fluorescence quenching ability of Cys to DNA-AgNCs. Using this method for Cys assay, it was found that the change of fluorescent intensity has a good linear relationship with Cys concentration in the range from 0.1 nM to 100 nM (R2 = 0.991). The detection limit of Cys was 0.05 nM. Furthermore, the method was successfully used for the detection of Cys in human serums, the result of which was confirmed using clinical colorimetric assay. In summary, our study shows that this simple, sensitive and rapid detection method can be hopefully used for theranostics.

The detection of a mismatched DNA by using hairpin DNA-templated silver nanoclusters

Fluorescence assays have been developed to detect biomolecules using DNA-templated silver nanoclusters (DNA-AgNCs) utilizing their unique physical and optical properties. This study was designed to detect single-mismatched DNA by the hybridization of target DNA to template DNA either before or after DNA-AgNCs synthesis. The results showed that the detection specificity of a single-mismatched DNA was clearly enhanced when the target DNA (cDNA) was hybridized to template DNA prior to DNA-AgNCs synthesis compared with cDNA hybridization subsequent to DNA-AgNCs synthesis.

How G-quadruplex topology and loop sequences affect optical properties of DNA-templated silver nanoclusters

In the study of the fabrication of DNA-templated silver nanoclusters (DNA-Ag NCs), how templates affect the fluorescence of the nanoclusters remains unclear, and it has been a challenge to understand the correlation between the properties of the DNA template and the Ag NCs. In this respect, based on the rational design of a series of structurally defined intramolecular G-quadruplexes, we prepared G-quadruplex-templated Ag NCs with a defined G-tetrad-to-silver ratio of 1:2. We evaluated the effect of G-quadruplex topology and loop sequences on the fluorescence of DNA-Ag NCs using circular dichroism, and extinction and emission spectroscopy. G-quadruplex templates with an anti-parallel topology were found to produce Ag NCs with stronger fluorescence compared with parallel and hybrid configurations. Loop bases adjacent to G-tetrads have a more significant impact on the fluorescence of Ag NCs compared with those in the middle of the loop, with adenine largely exhibiting an enhancement effect and thymine being detrimental. Generally, G-quadruplexes having an anti-parallel topology with adenine in the loop adjacent to the G-tetrad would be good templates for producing highly fluorescent Ag NCs. This is the first study to focus on the correlation between G-quadruplex topology/sequence and the optical properties of Ag NCs. We hope that the results of this study will facilitate a more in-depth understanding of correlation between G-quadruplex templates and Ag NCs, and help to understand and utilize their unique attributes.

Novel dual fluorescence temperature-sensitive chameleon DNA-templated silver nanocluster pair for intracellular thermometry

For the first time, we are reporting a novel type of dual fluorescence temperature-sensitive DNA-templated silver nanocluster (AgNC) pair, which contains two pieces of single-stranded AgNC in proximity through hybridization. Both the chameleon AgNC pairs, A-NCP and B-NCP, possess two bright fluorescence peaks that achieve sensitive variations corresponding to temperature change from 15 to 45 °C. With the increase in temperature, one of the fluorescence emissions of A-NCP (A-FL570) increases, while the other (A-FL640) decreases. However, both the emissions of B-NCP (B-FL685 and B-FL620) decrease simultaneously. Therefore, A-NCP shows a remarkable fluorescence color variation from orange to yellow, while the fluorescence color of B-NCP changes from orange to colorless, with increase in temperature. Moreover, the temperature responding linear range of A-NCP can be regulated by adjusting the structures and sequences of assistant DNA templates. It is assumed that the two single-stranded segmental AgNCs are integrated together as they are assembled into AgNC pairs, leading to a dramatic variation in fluorescence properties. The temperature-sensitive phenomenon is due to the dehybridization-induced separation of two pieces of segmental AgNC, caused by temperature increase. The temperature-sensitive AgNC pairs have been successful in indicating the temperature of living cells, showing the potential for a new application of silver nanocluster as a nanothermometer with adjustable response range, bringing novel insight into the regulatory mechanism of AgNC fluorescence variation.

One-Step Synthesis of DNA Templated Water-Soluble Au-Ag Bimetallic Nanoclusters for Ratiometric Fluorescence Detection of DNA.

DNA strands have been used as templates to form different sized silver nanoclusters. Although DNA templated silver nanoclusters (NCs) probes which show outstanding fluorescence properties have been widely applied in chemical sensing and cellular imaging, synthesis of DNA template Au-Ag bimetallic nanoclusters remains a challenge. A facile one-step synthesis method was thus developed in this study to form Au-Ag NCs using C4-ATAT-C4 as template. C4-ATAT-C4 acted as a stabilizer for preventing aggregation of the Au-Ag NCs. The obtained Au-Ag NCs stabilized by C4-ATAT-C4 showed bright fluorescence and high stability. A series of experiments showed that temperature, citrate-citric acid buffer, sequence and concentration of DNA played an important role in the synthesis of fluorescent Au-Ag NCs. In addition, a ratiometric fluorescence probe was designed through combining the nucleotide sequence (C4-ATAT-C4) and hybridization sequence in the presence of the double-strand-chelating dye Super SYBR Green (SG). The combination of prepared Au-Ag NCs, Super SG, and perfectly matched DNA emitted fluorescence at 500 and 590 nm, respectively, when the composite was excited at 290 nm. A gastric cancer gene was also selectively detected by our developed ratiometric fluorescence probe.

Cu2+ modulated DNA-templated silver nanoclusters as a turn-on fluorescence probe for the detection of quinolones

In this contribution, DNA-templated silver nanoclusters (DNA-AgNCs) were successfully utilized as novel fluorescent probes for the detection of quinolones for the first time.