Upconversion emission of fluorescent silver nanoclusters and in situ selective DNA biosensing

Due to their good photostability, high quantum yield and low toxicity, fluorescent silver nanoclusters (Ag NCs) have received much attention as novel fluorophores for sensing applications. In this work, we investigate the upconversion emission of Ag NCs templated by single- and double-stranded DNAs. DNA-templated Ag NCs exhibit upconversion emission at wavelengths identical to those observed for the corresponding Stokes emission. Consequently, the Ag NCs' upconversion behavior can be easily tuned by the used DNA sequences. In addition, the Ag NCs are more stable under such an NIR excitation with the upconversion mode relative to the Stokes mode. As a proof-of-concept application, DNA nucleobase recognition with the in situ formed Ag NCs is realized using the Ag NCs' upconversion emission. We expect that the Ag NCs' upconversion emission is more advantageous than the previously used rare-earth materials, at least with respect to easy modulation of the emission energies by DNA sequences, and could find wide application in sensor design.     

Ag nanoclusters as probes for turn-on fluorescence recognition of TpG dinucleotide with a high selectivity

CpG dinucleotide in DNA has a great tendency to mutate to TpG dinucleotide and this transition can cause some serious diseases. In this work, fluorescent Ag nanoclusters (Ag NCs) were employed as useful inorganic fluorophores for the potential of selectively discriminating TpG dinucleotide from CpG dinucleotide. Opposite the base Y of interest in YpG dinucleotide (Y = C or T), a bulge site was introduced so as to make the base Y to be unpaired and ready for Ag⁺ binding. Such that the unpaired Y and context base pairs can provide a specific space suitable for creating fluorescent Ag NCs. We found that in comparison with CpG dinucleotide, TpG dinucleotide is much more efficient in growing fluorescent Ag NCs. Therefore, mutation of CpG dinucleotide to TpG can be identified by a turn-on fluorescence response and a high selectivity. More interestingly, Ag NCs exhibit a better performance in the TpG recognition over the other dinucleotides (Y = A and G) than the previously used organic fluorophores. Additionally, the effectiveness of the bulge site design in discriminating these dinucleotides was evidenced by control DNAs having the abasic site structure. We expect that a practical method for TpG dinucleotide recognition with a high selectivity can be developed using the bulge site-grown fluorescent Ag NCs as novel probes.     

日光辐射光还原合成左旋多巴胺功能化的荧光发射的银纳米簇和铁离子检测

以左旋多巴胺(L-3,4-dihydroxyphenylalanine,DOPA)为稳定剂,采用日光辐射光还原法,合成了强荧光发射的银纳米簇(silver nanoclusters,Ag NCs)。透射电镜分析表明,所合成的Ag NCs表现亚纳米非晶态结构。Ag NCs在可见-近红外波长范围内(400~750 nm)有明显光吸收带,最大荧光激发和发射峰分别为550和630 nm,荧光量子产率为2.3%(相对于罗丹明B)。Ag NCs的荧光强度与合成时的日光辐射时间、DOPA浓度以及pH值等因素有关。进一步优化了合成Ag NCs的条件。基于荧光猝灭原理,所合成的DOPA功能化的Ag NCs能选择性地灵敏响应Fe3+。修饰在Ag NCs表面的配体DOPA能够选择性地结合Fe3+,导致Ag NCs显著聚集,伴随荧光猝灭。Ag NCs具有的较高量子产率和红荧光发射特性,有利于提高Fe3+的分析灵敏度。     

日光辐射光还原合成左旋多巴胺功能化的荧光发射的银纳米簇和铁离子检测

以左旋多巴胺（L-3,4-dihydroxyphenylalanine,DOPA）为稳定剂,采用日光辐射光还原法,合成了强荧光发射的银纳米簇（silvernanoclusters,AgNCs）。透射电镜分析表明,所合成的AgNCs表现亚纳米非晶态结构。AgNCs在可见-近红外波长范围内（400~750nm）有明显光吸收带,最大荧光激发和发射峰分别为550和630nm,荧光量子产率为2.3%（相对于罗丹明B）。AgNCs的荧光强度与合成时的日光辐射时间、DOPA浓度以及pH值等因素有关。进一步优化了合成AgNCs的条件。基于荧光猝灭原理,所合成的DOPA功能化的AgNCs能选择性地灵敏响应Fe³⁺。修饰在AgNCs表面的配体DOPA能够选择性地结合Fe³⁺,导致AgNCs显著聚集,伴随荧光猝灭。AgNCs具有的较高量子产率和红荧光发射特性,有利于提高Fe³⁺的分析灵敏度。     

Characterization and application to the detection of single-stranded DNA binding protein of fluorescent DNA-templated copper/silver nanoclusters

A simple strategy for the preparation of strongly fluorescent and stable DNA-Cu/Ag NCs from reduction of AgNO(3) and Cu(NO(3))(2) by NaBH(4) in the presence of DNA having a sequence 5'-CCCTTAATCCCC-3' has been demonstrated. Fluorescence, absorption, X-ray photoelectron spectroscopy (XPS), and electrospray ionization-mass spectrometry (ESI-MS) measurements have been applied to the characterization of the DNA-Cu/Ag NCs. The ESI-MS data reveal that each DNA-Cu/Ag NC contained 2 Ag and 1 Cu atoms. The interactions among DNA with the Ag and Cu atoms are further supported by the data of low-temperature fluorescence. In the presence of Cu(2+) ions, the reaction time is 1.5 h, which is much shorter than that (120 h) for the preparation of Ag-DNA NCs that are prepared in a mixture of AgNO(3), NaBH(4) and DNA without containing Cu(2+) ions. Relative to the DNA-Ag NCs, the DNA-Cu/Ag NCs have greater fluorescence (quantum yield 51.2% vs. 11.5%). The DNA-Cu/Ag NCs are highly sensitive and selective for the detection of single-stranded DNA binding protein (SSB), with a linear range 1-50 nM and a limit of detection 0.2 nM at a signal-to-ratio of 3.     

A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition

In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5′-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1–2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.     

Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide

Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD⁺), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5′ end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3′ end. Only in the presence of NAD⁺, the probe was linked at 5′ and 3′ ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM–500 nM with a detection limit of as low as 1 pM NAD⁺, and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD⁺ level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD⁺, taking advantage of specificity, sensitivity, cost-efficiency, and simplicity.

The Relationship between DNA Sequences and Oligonucleotide‐Templated Silver Nanoclusters and Their Fluorescence Properties

A novel approach was developed to study the relationship between DNA sequences and DNA‐templated silver nanoclusters (DNA‐Ag NCs) in detail by using an ordinary DNA strand as an example. Three kinds of Ag NCs are formed by using the DNA strand as a scaffold. By dividing the DNA template into several parts according to their different affinities to Ag⁺, it was found that the fluorescence properties of DNA‐Ag NCs are related to not only the sequences but also to the position of different parts in the template, which provides a more efficient approach to obtain DNA‐Ag NCs with required photoluminescence properties and may ultimately contribute to the targeted synthesis of DNA‐Ag NCs.     

Label-free fluorescent molecular beacon based on a small fluorescent molecule non-covalently bound to the intentional gap site in the stem moiety

A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K₁₁ > 10⁶). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required.     

Structural, dynamical, and electronic transport properties of modified DNA duplexes containing size-expanded nucleobases

Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.     

Synthesis of aptamer-functionalized Ag nanoclusters for MCF-7 breast cancer cells imaging

Aptamer-functionalized silver nanoclusters(Ag NCs) have been attracting a lot of interest as label-free probes which have been successfully applied to both cell imaging and molecular detection.MUCl aptamer is an ssDNA aptamer that specifically binds to MUCl mucin which is a large transmembrane glycoprotein whose expression level increases at least 10-fold in primary and metastatic breast cancers.Using C4A4C3-linker-MUCl as template,the Ag NCs were synthesized through one-pot process.The fluorescence intensity of Ag NCs was found to be closely related to the length and type(poly adenine or thymine) of the linker,the optimum linker being-AAAAA-.Using the C4A4C3-A5-MUC1 as the scaffold,the synthesized Ag NCs emitted fluorescence with high quantum yield(QY) of 66.5%.Based on the specific interaction between the MUCl aptamer and MUCl mucin,the C4A4C3-A5-MUC1-stabilized Ag NCs could recognize and differentiate the MCF-7 breast cancer cells from MDA-MB-231 breast cancer and A549 human lung cancer cells.     

Cytidine-stabilized gold nanocluster as a fluorescence turn-on and turn-off probe for dual functional detection of Ag and Hg

In this study, we have developed a label-free, dual functional detection strategy for highly selective and sensitive determination of aqueous Ag⁺ and Hg²⁺ by using cytidine stabilized Au NCs and AuAg NCs as fluorescent turn-on and turn off probes, respectively. The Au NCs and AuAg NCs showed a remarkably rapid response and high selectivity for Ag⁺ and Hg²⁺ over other metal ions, and relevant detection limit of Ag⁺ and Hg²⁺ is ca. 10 nM and 30 nM, respectively. Importantly, the fluorescence enhanced Au NCs by doping Ag⁺ can be conveniently reusable for the detection of Hg²⁺ based on the corresponding fluorescence quenching. The sensing mechanism was based on the high-affinity metallophilic Hg²⁺–Ag⁺ interaction, which effectively quenched the fluorescence of AuAg NCs. Furthermore, these fluorescent nanoprobes could be readily applied to Ag⁺ and Hg²⁺ detection in environmental water samples, indicating their possibility to be utilized as a convenient, dual functional, rapid response, and label-free fluorescence sensor for related environmental and health monitoring.     

The determination of copper ions based on sensitized chemiluminescence of silver nanoclusters

We report on the first application of novel, water-soluble and fluorescent silver nanoclusters (Ag NCs) in a chemiluminescent (CL) detection system. A method has been developed for the determination of copper(II) ion that is based on the fact that the weak CL resulting from the redox reaction between Ce(IV) ion and sulfite ion is strongly enhanced by the Ag NCs and that the main CL signals now originate from Ag NCs. UV-visible spectra, CL spectra and fluorescent (FL) spectra were acquired to investigate the enhanced CL mechanism. It is proposed that the electronic energy of the excited state intermediate SO₂* that originates from the CL reaction is transferred to Ag NCs to form an electronically excited NC whose emission is observed. In addition, it is found that copper(II) is capable of inhibiting the CL of the nanoclusters system, but not if other common metal ions are present. The detection of copper(II) is achieved indirectly by measuring the CL intensity of Ag NCs. Under the optimized experimental conditions, a linear relationship does exist between the intensity of CL and the concentrations of copper(II) in the range of 0.2?nM to 0.1?m??. The detection limit is 0.12?nM. The method is applied to the determination of copper(II) ion in tap water with satisfactory results.

Synthesis and properties of transparent luminescent nanocomposites with surface functionalized semiconductor nanocrystals

5-Amino-1,10-phenanthroline (Aphen) was used as an organic ligand to functionalize CdS nanocrystals (NCs) by a ligand-exchange process. The functional Aphen-CdS NCs have strong luminescent emission at 552 nm and good dispersibility in the polar organic monomers. The Aphen-CdS NCs were dispersed in polymeric monomers to prepare a series of transparent luminescent nanocomposites with excellent thermal stability via in-situ bulk polymerization. The fluorescent properties of the Aphen-CdS NCs were well retained in the polymer matrix. It was found that when the methacrylic acid (MAA) and glycidyl methacrylate (GMA) as the comonomers were introduced into the polymer matrix, the emission peaks of the resultant nanocomposites had a blue shift and the fluorescent intensities also increased due to the interaction between NCs and the polymer matrices. The transparent NCs/polymer nanocomposites with tunable fluorescent emission can be potentially used for the fabrication of optoelectronic devices.     

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.     

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.     

Luminescent Noble Metal Nanoclusters as an Emerging Optical Probe for Sensor Development

In the past few years, highly luminescent noble metal nanoclusters (e.g., Au and Ag NCs or Au/Ag NCs in short) have emerged as a class of promising optical probes for the construction of high‐performance optical sensors because of their ultrasmall size (<2 nm), strong luminescence, good photostability, excellent biocompatibility, and unique metal‐core@ligand‐shell structure. In this Focus Review, we briefly summarize the common syntheses for water‐soluble highly‐luminescent thiolate‐ and protein‐protected Au/Ag NCs and their interesting luminescence properties, highlight recent progress in their use as optical sensors with an emphasis on the mechanisms underlying their selectivity, and finally discuss approaches to improving their sensitivity. The scope of the works surveyed is confined to highly luminescent thiolate‐ and protein‐protected Au/Ag NCs.     

Luminescent silver nanoclusters for efficient detection of adenosine triphosphate in a wide range of pH values

In this work, polymethacrylic acid (PMAA)-templated silver nanoclusters (Ag NCs) were developed as the fluorescent probe for the efficient and sensitive detection of adenosine triphosphate (ATP) in a wide range of pH values. The fluorescence intensity of the Ag NCs could keep stable with pH values ranging from 2.5 to 9.3. The detection of ATP was based on the quenching of the fluorescent Ag NCs in the presence of ATP. The fluorescence quenching of the Ag NCs with increasing ATP concentration was studied at pH 2.5, 4.5, 7.0 and 8.5 which involved a wide pH environment in body fluids. The limit of detection (LOD) for ATP was as low as 0.1 mmol/L in an acidic environment with pH of 2.5 and all the linear correlation coefficients were satisfactory under wide-span pH values from 2.5 to 8.5. In addition, the sensitive determination of ATP was also achieved by adding copper ions (Cu²⁺). The high selectivity and rapid detection process proved that the fluorescent probe had great potential to detect ATP in biological samples under different pH conditions.     

Chemical ligation as a method for the assembly of double-stranded nucleic acids: Modifications and local structure studies

A new procedure was developed as an alternative to the enzymatic assembly of natural and modified double-stranded DNAs using chemical reagent (chemical ligation). BrCN was suggested as an efficient coupling reagent, which induces superfast reactions in DNA duplexes. The physicochemical properties and the structure of new types of DNA duplexes, which are the substrates for chemical ligation, with breaks in phosphodiester chains, including concatemers, were studied. Chemical ligation was applied to prepare biologically active 17–200 base-pair double-stranded DNAs and DNA-RNA block-copolymers, to incorporate various modifications into DNA duplexes including pyrophosphate and phosphoramidate unnatural internucleotide bonds. The unique possibilities of this approach were demonstrated in the development of methods for circularization of oligodeoxy ribonucleotides and assembly of branched DNAs. The structural-kinetic concept of chemicalligation was created and the relationship between the reactivity of interacting groups and sequence-dependent local conformation of the ligation site in B-DNA was established. The lesser efficiency of chemical ligation of RNA fragments in comparison to that of DNA analogs was demonstrated and rationalized. This approach was used as a sensitive monitor of a stable double helix formation and third-strand binding to a DNA duplex.Translated from Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1889–1911, August, 1996.     

Luminescent silver nanoclusters for efficient detection of adenosine triphosphate in a wide range of pH values

In this work, polymethacrylic acid (PMAA)-templated silver nanoclusters (Ag NCs) were developed as the fluorescent probe for the efficient and sensitive detection of adenosine triphosphate (ATP) in a wide range of pH values. The fluorescence intensity of the Ag NCs could keep stable with pH values ranging from 2.5 to 9.3. The detection of ATP was based on the quenching of the fluorescent Ag NCs in the presence of ATP. The fluorescence quenching of the Ag NCs with increasing ATP concentration was studied at pH 2.5, 4.5, 7.0 and 8.5 which involved a wide pH environment in body fluids. The limit of detection (LOD) for ATP was as low as 0.1 mmol/L in an acidic environment with pH of 2.5 and all the linear correlation coefficients were satisfactory under wide-span pH values from 2.5 to 8.5. In addition, the sensitive determination of ATP was also achieved by adding copper ions (Cu²⁺). The high selectivity and rapid detection process proved that the fluorescent probe had great potential to detect ATP in biological samples under different pH conditions.