DNA-templated Silver Nanoclusters Research Articles

An approach toward SNP detection by modulating the fluorescence of DNA-templated silver nanoclusters

A rapid, homogeneous, and in-situ labelable format designed to detect single nucleotide polymorphism (SNP) is presented. Fluorescent silver nanoclusters produced following hydride-mediated reduction of Ag+ bound to a partial double-stranded oligodeoxynucleotide were employed as probes for SNPs. The sensing mechanism is based on the fluorescence enhancement of silver nanoclusters through an irreversible cluster transfer upon hybridization. The key element of our design is modulating the “turn on” mechanism by introducing a competitor that can be displaced in response to a target DNA. In a controlled model system, the fluorescence intensity of silver nanoclusters is approximately 3-fold enhanced upon hybridization with a perfectly matched target DNA, and single-base mismatch detection is achieved within 5min, regardless of the position of mismatches. Human aldehyde dehydrogenase 2 (ALDH2), which is responsible for the oxidation of aldehydes to carboxylic acids, is then utilized as a target for SNP. Upon addition of a perfectly matched sequence, dramatically enhanced fluorescence intensity is observed (ca. 48-fold), and prompt SNP genotyping is accomplished.

DNA-Templated Silver Nanoclusters that Fluoresce upon Hybridization

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences, termed enhancer sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that “lights up” upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. In addition, we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. We have exploited this color shift to directly detect single nucleotide polymorphisms (SNPs). This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

DNA-templated silver nanoclusters as label-free fluorescent probes for detection of bleomycin

Noble metal nanoclusters, which have molecule-like properties and offer the missing understanding link between metal atoms and nanoparticles, have become a hot research topic due to their amazing properties. Herein, we develop a label-free, low-cost and sensitive fluorescence turn-off strategy for the detection of bleomycin (BLM) with strong fluorescent silver nanoclusters, wherein BLM coordinated with Fe2+ could highly oxidize the core of fluorescent silver nanoclusters. Under the optimal conditions, this strategy could be applied to the detection of BLM in the range of 100 to 400 nM with a detection limit of 54 nM.

Correlation of photobleaching, oxidation and metal induced fluorescence quenching of DNA-templated silver nanoclusters

Few-atom noble metal nanoclusters have attracted a lot of interest due to their potential applications in biosensor development, imaging and catalysis. DNA-templated silver nanoclusters (AgNCs) are of particular interest as different emission colors can be obtained by changing the DNA sequence. A popular analytical application is fluorescence quenching by Hg(2+), where d(10)-d(10) metallophilic interaction has often been proposed for associating Hg(2+) with nanoclusters. However, it cannot explain the lack of response to other d(10) ions such as Zn(2+) and Cd(2+). In our effort to elucidate the quenching mechanism, we studied a total of eight AgNCs prepared by different hairpin DNA sequences; they showed different sensitivity to Hg(2+), and DNA with a larger cytosine loop size produced more sensitive AgNCs. In all the cases, samples strongly quenched by Hg(2+) were also more easily photobleached. Light of shorter wavelengths bleached AgNCs more potently, and photobleached samples can be recovered by NaBH4. Strong fluorescence quenching was also observed with high redox potential metal ions such as Ag(+), Au(3+), Cu(2+) and Hg(2+), but not with low redox potential ions. Such metal induced quenching cannot be recovered by NaBH4. Electronic absorption and mass spectrometry studies offered further insights into the oxidation reaction. Our results correlate many important experimental observations and will fuel the further growth of this field.

An anti-galvanic replacement reaction of DNA templated silver nanoclusters monitored by the light-scattering technique

An anti-galvanic replacement reaction (AGRR) of copper(II) ions reduced by ultra-small ssDNA-templated silver nanoclusters forming Ag-Cu alloy nanoparticles was observed. The reaction is against the classic galvanic theory and was monitored sensitively by the light-scattering technique.

Fluorescent silver nanoclusters as DNA probes

Fluorescent silver nanoclusters (few atoms, quantum sized) have attracted much attention as promising substitutes for conventional fluorophores. Due to their unique environmental sensitivities, new fluorescent probes have been developed based on silver nanoclusters for the sensitive and specific detection of DNA. In this review we present the recent discoveries of activatable and color-switchable properties of DNA-templated silver nanoclusters and discuss the strategies to use these new properties in DNA sensing.

Base-Stacking-Determined Fluorescence Emission of DNA Abasic Site-Templated Silver Nanoclusters

DNA-templated silver nanoclusters (Ag NCs) are emerging sets of fluorophores that are widely applicable because of high brightness, good photostability, and visible to near-infrared emissions tunable using the DNA sequence and length to change the NC size. We find that fluorescent Ag NCs can be size-selectively grown at DNA abasic sites (AP site) using a constrained duplex environment opposed by a cytosine and flanked by two guanines. The size of the AP site-grown Ag NCs is not affected by the increasing Ag(+) concentration. A Job's plot analysis shows that Ag(2) NCs are the species responsible for the observed emissions. Although varying the DNA sequence one base away from the AP site (i.e., the Ag NC growth site) does not alter the size of the fluorescent Ag NCs, the emissions of the formed Ag NCs are still gradually red shifted as the sequence changes from thymine (T) to cytosine (C), adenine (A), and guanine (G). Furthermore, this emission shift is strongly dependent on the base-stacking direction of the 3'-side sequence of the 5'-G stack exactly flanking the AP site, which exhibits a larger emission alteration than altering the 5'-side sequence of the 3'-G stack flanking the AP site on the other side of the site. The excited-state lifetimes of the Ag NCs are inversely proportional to the singlet energies (ΔE(0,0)) of the Ag NCs relative to their ground state and of the vertical ionization potentials of the guanines directly flanking the AP site as determined by the base stacking. All of these results support the conclusion that the Ag NC excited state becomes more stable by interacting with a guanine base because of the larger electronic dipole moment that can be modified by the stacked sequences. Additionally, the size of the formed Ag NCs seems to be dependent on the consecutive AP site number. Thus, the AP site design in this work provides an easy way to shed light on the role of DNA base stacking in the optical properties of Ag NCs.

Emission Modulation of DNA-Templated Fluorescent Silver Nanoclusters by Divalent Magnesium Ion

DNA-templated fluorescent silver nanoclusters (Ag NCs) composed of several or tens of atoms are gaining much interest because of their unique properties and convenient emission tunability by DNA sequence and length. However, the modulation by other factors other than DNA is also dependent on the special DNA secondary structure formation such as i-motif or G-quadruplex that is stimulated by pH or K+. One main observation considered in this work is emission modulation of Ag NCs by divalent Mg2+ during or after the clusters' creation. Tuning the emitting intensities and band positions can be realized by Mg2+ addition for the examined single-stranded DNA (ss-DNA) templates, which is dependent on the addition moment of Mg2+, while only intensity modulation should be achieved for the used double-stranded DNA (ds-DNA). Despite of this discrepancy, Mg2+ addition always induces a lifetime-varied emission state of Ag NCs. The modulated emission still follows the common nature of the previously used DNA sequence- and length-dependent emitting. Efficient screening the negative charges of DNA backbone upon addition of the divalent ion is responsible for the modulation by adaptively accommodating the formed Ag NCs. This strategy could be more advantageous over the emission modulation by DNA sequence and length because a desired emitting could be achieved only by alteration of the electrolyte conditions during or after the Ag NCs' creation.

Fenton’s reagent-tuned DNA-templated fluorescent silver nanoclusters as a versatile fluorescence probe and logic device

A label-free strategy based on the Fenton reaction with DNA-templated silver nanoclusters (DNA-Ag NCs) as a probe is demonstrated for the sequential detection of Cu(2+), ascorbic acid (AA) and H(2)O(2). Cu(2+) causes a structural change of the DNA template in DNA-Ag NCs to resist the environmental quenching and emit stronger fluorescence. The addition of AA in the presence of Cu(2+) results in a further fluorescence increase of the DNA-Ag NCs. Interestingly, an even higher fluorescence enhancement is recorded by introducing Cu(2+) into the DNA-Ag NCs-AA probing system. The fluorescence turn-on probe offers detection limits of 3 nM for Cu(2+) and 7 nM for AA. Thereafter, the addition of H(2)O(2) generates hydroxyl radicals from the Fenton reaction, which induces cleavage of the DNA template, leading to fluorescence quenching of the DNA-Ag NCs. This facilitates H(2)O(2) detection. Moreover, based on the DNA-templated fluorescent silver nanoclusters and Fenton reaction, a multiple logic gate system, including AND and a three-input logic gate, is constructed, with Cu(2+), AA and H(2)O(2) as inputs, and the fluorescence intensity of the DNA-Ag NCs probe as output.

A DNA-templated fluorescent silver nanocluster with enhanced stability

We report the discovery of a DNA sequence that templates a highly stable fluorescent silver nanocluster. In contrast to other DNA templated silver nanoclusters that have a relatively short shelf-life, the fluorescent species templated in this new DNA sequence retains significant fluorescence for at least a year. Moreover, this new silver nanocluster possesses low cellular toxicity and enhanced thermal, oxidative, and chemical stability.

A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase

A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators.

DNA-templated silver nanoclusters–graphene oxide nanohybrid materials: a platform for label-free and sensitive fluorescence turn-on detection of multiple nucleic acid targets

In this study, we develop an efficient method for multiple DNA detection by exploring silver nanoclusters (AgNCs)-graphene oxide (GO) nanohybrid materials. Because of the extraordinarily high quenching efficiency of GO, the ssDNA-AgNCs probe exhibits minimal background fluorescence, while strong emission is observed when it forms a double helix with the specific target DNA, leading to a high signal-to-background ratio. Therefore the AgNCs-GO nanohybrid materials can be successfully applied for DNA detection. The system described here exhibits not only high sensitivity with a detection limit of 1 nM, but also an excellent differentiation ability for single-base mismatched sequences. In addition, by exploring AgNCs as signal reporters and GO as the nanoquencher, this approach avoids labeling the probe DNA or target DNA, which offers the advantages of simplicity and cost efficiency. Moreover, the large planar surface of GO allows adsorption of different DNA-AgNCs probes, each with a distinct emission, leading to a multicolor sensor for the detection of multiple DNA targets in the same solution.

DNA-templated fluorescent silver nanoclusters

In this review, we discuss the synthesis and applications of DNA-templated fluorescent silver nanoclusters in aqueous solution. Various oligonucleotide sequences or conformations have been utilized to synthesize silver nanoclusters with excellent fluorescence properties. The range of applications has expanded greatly, from live cell staining and the detection of metal ions and small biomolecules to the detection of DNA or proteins.

Ag K-Edge EXAFS Analysis of DNA-Templated Fluorescent Silver Nanoclusters: Insight into the Structural Origins of Emission Tuning by DNA Sequence Variations

DNA-templated silver nanoclusters are promising biological fluorescence probes due to their useful fluorescence properties, including tunability of emission wavelength through DNA template sequence variations. Ag K-edge EXAFS analysis of DNA-templated silver nanoclusters has been used to obtain insight into silver nanocluster bonding, size, and structural correlations to fluorescence. The results indicate the presence of small silver nanoclusters (<30 silver atoms) containing Ag-Ag bonds and Ag-N/O ligations to DNA. The DNA sequence used leads to differences in silver-DNA ligation as well as silver nanocluster size. The results support a model in which cooperative effects of both Ag-DNA ligation and variations in cluster size lead to the tuning of the fluorescence emission of DNA-templated silver nanoclusters.

Lighting‐Up Single‐Walled Carbon Nanotubes with Silver Nanoclusters

Light at the end of the tunnel: A new noncovalent strategy to light up single-walled carbon nanotubes (SWNTs) by functionalizing them with DNA-templated silver nanoclusters is reported (see scheme). The resulting nanohybrids combine the unique properties of both nanomaterials and could serve as a convenient and efficient platform for fluorescence visualization of SWNTs. Such nanohybrids could also have promising applications in novel electrical materials, biological probes, and fluorescent nanosensors.

Control of synthesis and optical properties of DNA templated silver nanoclusters by varying DNA length and sequence

We have used a simple method to prepare five fluorescent Ag nanoclusters (NCs) through the NaBH4-mediated reduction of Ag+ ions in the presence of various DNA scaffolds. The emission intensities and wavelengths (536–644 nm) of the as-prepared DNA–Ag NCs were dependent on the sequence and length of the DNA scaffold. Electrospray ionization mass spectrometry of the DNA–Ag NCs revealed that different numbers of Ag atoms (2–6 atoms) were present per DNA scaffold, depending on the number and position of the cytosine bases. Using the oligonucleotide 5′-CCC(TTCC)2TT(CCAA)2CCC-3′ (DNATAr2) as the scaffold, we obtained DNATAr2–Ag NCs exhibiting a quantum yield (Φf) of 61% at 608 nm; these NCs were stable in the presence of the tested thiols, Cl− ions and DNase I. Because of their strong fluorescence and stability, the DNATAr2–Ag NCs were highly selective and sensitive for the detection of Hg2+ ions [linear range: 2.5–50 nM; limit of detection (signal-to-noise ratio = 3): 0.9 nM]. We validated the practicality of this probe through analyses of several water samples spiked with Hg2+ ions (10 nM); the recoveries were 98–118%.

Modulating DNA-templated silver nanoclusters for fluorescence turn-on detection of thiol compounds

Based on the fact that the fluorescence response pattern of a silver nanocluster to a specific analyte is highly dependent on the nature of the DNA template, we develop a novel fluorescence turn-on assay for thiol compounds with high specificity and sensitivity by modulating DNA-templated silver nanoclusters.

A DNA−Silver Nanocluster Probe That Fluoresces upon Hybridization

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that "lights up" upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Photoinduced-Electron Transfer between Guanine Bases and Silver Nanoclusters Enables Increased Shelf Life

We studied the interactions between DNA-templated fluorescent silver nanoclusters and nearby guanine bases. Fluorescence quenching by guanine due to photoinduced charge transfer has been reported for many widely used organic dyes. In contrast, we found that for red- and infrared-emitting fluorescent Ag nanoclusters (NCs), formed on DNA templates, interactions with nearby guanine bases tended to protect these NCs against oxidation, making them brighter and more stable in aqueous solution. Nanoclusters formed in the absence of guanine-rich DNA changed from a red-emitting reduced NC into a green-emitting oxidized species in a few hours in air-saturated solutions. In contrast, when guanine bases were brought close to the NCs, through DNA hybridization, guanine served as an electron donor and reducing agent, which prevented the Ag NCs from being quickly oxidized in air-saturated solutions, increasing the stability from hours to days. In addition, hybridization with guanine-rich DNA could be used to reduce the already oxidized NCs back to the red-emitting reduced ones. Single-stranded DNA templates were also designed with a nanocluster formation sequence and a guanine-rich sequence at each end. Similarly, we found that the guanine-rich tail helped stabilize the fluorescence of the red-emitting NC fluorophores, in comparison to single-stranded templates with only cluster formation sequences but no guanine-rich tails. Using this strategy, we have designed a DNA sequence that produces a highly emissive Ag NC fluorophore with an extended shelf life, which should prove useful in a variety of biological applications, including fluorescence imaging and biosensing.