DNA-stabilized Silver Nanoclusters Research Articles

Fluorescent aptasensor based on DNA-AgNCs emitting in the visible red wavelength range for detection of kanamycin in milk

Fluorescence sensors detect veterinary drug residues in milk, which are usually interfered with by the complex milk matrix. The specific DNA template can produce DNA stabilized silver nanoclusters (DNA-AgNCs) emitting in the visible red wavelength range. This will reduce the fluorescence interference of milk by avoiding the fluorescence signal generated by the inherent component of milk in the relatively short wavelength range. Furthermore, the red fluorescence has relatively high penetration, which can also reduce the impact of interfering substances. Here, on the basis of fluorescence resonance energy transfer (FRET), we designed a fluorescent aptasensor using DNA-AgNCs and gold nanoparticles (AuNPs) to detect kanamycin (KAN) residue in milk. Due to electrostatic interaction, DNA adsorbs on the AuNPs. The decrease in the distance between DNA-AgNCs and AuNPs generates FRET, which causes the fluorescence of DNA-AgNCs to be quenched. When KAN is added, the aptamer and KAN can specifically bind with high affinity, resulting in a weakening of the electrostatic interaction between DNA-AgNCs and AuNPs. This results in a decrease in FRET between DNA-AgNCs and AuNPs, and recovery of the fluorescent signal intensity of DNA-AgNCs. The designed sensor has satisfactory specificity and sensitivity for the detection of KAN residues. The applied strategy showed the limit of KAN detection to be as low as 22.6 nM. Moreover, this work provides a way for reducing the interference of the milk background fluorescence signal in the fluorescence sensor and has broad application prospects.

A Novel Fluorescence Aptasensor Based on Magnetic Beads/Gold Nanoparticles/DNA-Stabilized Silver Nanoclusters for Detection of Salmonella Typhimurium.

Salmonella Typhimurium (S. Typhimurium) is a globally distributed foodborne pathogen, which can lead to outbreaks of foodborne infectious diseases. It is essential to guarantee food safety by timely and correct detection of S. Typhimurium. In this investigation, an original fluorescence aptasensor was constructed to detect S. Typhimurium rapidly and sensitively. Through the coupling of magnetic beads, aptamer, and gold nanoparticles (AuNPs), a fluorescence quenching system with a “sandwich structure” was established. The aptamer acted as a link, and its specific binding to S. Typhimurium could release AuNPs from the system. Meanwhile, fluorescent DNA-stabilized silver nanoclusters (DNA-AgNCs) were synthesized. The fluorescence intensity changes caused by the fluorescence resonance energy transfer between DNA-AgNCs and AuNPs were utilized to detect S. Typhimurium. The purposed aptasensor exhibited high selectivity and sensitivity with a linear response to S. Typhimurium, ranging from 3.7 × 102 to 3.7 × 105 cfu/mL. The limit of detection (LOD) was estimated to be 98 cfu/mL within 2 h 10 min. In addition, this method showed excellent application for detection of S. Typhimurium in artificially contaminated milk, with LOD reaching 3.4 × 102 cfu/mL. Therefore, the developed fluorescence aptasensor has great potential to identify S. Typhimurium in foodstuffs.

Probing emission of a DNA-stabilized silver nanocluster from the sub-nanosecond to millisecond timescale in a single measurement.

A method for measuring emission over a range of sub-nanosecond to millisecond timescales is presented and demonstrated for a DNA-stabilized silver nanocluster (DNA-AgNC) displaying dual emission. This approach allows one to disentangle the temporal evolution of the two spectrally overlapping signals and to determine both the nano- and microsecond decay times of the two emission components, together with the time they take to reach the steady-state equilibrium. Addition of a second near-infrared laser, synchronized with a fixed delay, enables simultaneous characterization of optically activated delayed fluorescence (OADF). For this particular DNA-AgNC, we demonstrate that the microsecond decay times of the luminescent state and the OADF-responsible state are similar, indicating that the OADF process starts from the luminescent state.

The effect of inosine on the spectroscopic properties and crystal structure of a NIR-emitting DNA-stabilized silver nanocluster.

The effect of replacing guanosines with inosines in the two stabilizing strands (5′-CACCTAGCGA-3′) of the NIR emissive DNA-Ag16NC was investigated. The spectroscopic behavior of the inosine mutants is position-dependent: when the guanosine in position 7 was exchanged, the nanosecond fluorescence decay time shortened, while having the inosine in position 9 made the decay time longer. Thanks to structural information gained from single crystal X-ray diffraction measurements, it was possible to propose a mechanistic origin for the observed changes.

A novel fluorescent platform of DNA-stabilized silver nanoclusters based on exonuclease III amplification-assisted detection of Salmonella Typhimurium

A novel fluorescent platform of DNA-stabilized silver nanoclusters (DNA-AgNCs) has been developed based on exonuclease III (Exo III) amplification-assisted for simple and sensitive detection of Salmonella Typhimurium (S. Typhimurium). The platform was designed by using magnetic beads, aptamer, its complementary DNA, hairpin probe (HP), Exo III, AgNO3, and NaBH4. The functionalized HP contained a cytosine-rich oligonucleotide loop (C-rich loop), which served as an effective template for the chemical reduction of Ag+ with NaBH4 to synthesize DNA-AgNCs. In the presence of S. Typhimurium, the C-rich loop was converted into an open form of ssDNA by the recycle digestion of Exo III, leading to a corresponding decrease in fluorescence intensity. Based on the fluorescence changes of the formed DNA-AgNCs, the sensitive detection of S. Typhimurium was achieved. Under the optimal conditions, a wide linear relationship was observed in the concentration of S. Typhimurium ranging from 4.6 × 102 to 4.6 × 107 cfu mL−1 with the limit of detection (LOD) being 82 cfu mL−1. The method showed good selectivity for detecting S. Typhimurium. In addition, the platform could be used for the detection of S. Typhimurium in milk samples. The LOD reached 6.6 × 102 cfu mL−1 with a good linear range, indicating that the method had excellent practicability in complex food samples.

Silver nanocluster-lightened catalytic hairpin assembly for enzyme-free and label-free mRNA detection

Quantitative mRNA expression level is very important for differential diagnosis and clinical diagnosis. In this work, an enzyme-free and label-free fluorescence assay based on DNA silver nanocluster-lightened catalytic hairpin assembly has been developed for detection of mRNA. The strategy employed two hairpin probes, their tails are reasonably designed to contain DNA-stabilized silver nanoclusters capture sequence and guanine-rich sequence. In the presence of the target mRNA, it can trigger the CHA cycle reaction and generated assembled products, thereby capture the prepared dark DNA/AgNCs, bring AgNCs and guanine-rich sequence in close proximity, which transform the dark green-emitting species into a bright red fluorescent DNA/AgNCs and lights up the CHA assembly products. The lightened DNA/AgNCs specifically to target mRNA, affording sensitivity for detection. In the range of 0.1–100 nM, the fluorescence intensity has a linear relationship with the logarithm of concentration of surviving mRNA with a detection limit is estimated about 45 pM. These results suggested that this developed strategy provided a useful platform for sensitive and cost-effective detection of mRNA and has shown great potential in the detection of disease-related biomarkers and early clinical theragnostic.

Single-Molecule Detection of DNA-Stabilized Silver Nanoclusters Emitting at the NIR I/II Border.

The near-infrared (NIR) I and II regions are known for having good light transparency of tissue and less scatter compared to the visible region of the electromagnetic spectrum. However, the number of bright fluorophores in these regions is limited. Here we present a detailed spectroscopic characterization of a DNA-stabilized silver nanocluster (DNA-AgNC) that emits at around 960 nm in solution. The DNA-AgNC converts to blue-shifted emitters over time. Embedding these DNA-AgNCs in poly(vinyl alcohol) (PVA) shows that they are bright and photostable enough to be detected at the single-molecule level. Photon antibunching experiments were performed to confirm single emitter behavior. Our findings highlight that the screening and exploration of DNA-AgNCs in the NIR II region might yield promising bright, photostable emitters that could help develop bioimaging applications with unprecedented signal-to-background ratios and single-molecule sensitivity.

The effect of deuterium on the photophysical properties of DNA-stabilized silver nanoclusters.

We investigated the effect of using D2O versus H2O as solvent on the spectroscopic properties of two NIR emissive DNA-stabilized silver nanoclusters (DNA–AgNCs). The two DNA–AgNCs were chosen because they emit in the same energy range as the third overtone of the O–H stretch. Opposite effects on the ns-lived decay were observed for the two DNA–AgNCs. Surprisingly, for one DNA–AgNC, D2O shortened the ns decay time and enhanced the amount of µs-lived emission. We hypothesize that the observed effects originate from the differences in the hydrogen bonding strength and vibrational frequencies in the two diverse solvents. For the other DNA–AgNC, D2O lengthened the ns decay time and made the fluorescence quantum yield approach unity at 5 °C.

Studies on the interactions of Ag(i) with DNA and their implication on the DNA-templated synthesis of silver nanoclusters and on the interaction with complementary DNA and RNA sequences.

Silver nanoclusters (AgNCs) prepared by the reduction of silver ions in the presence of DNA oligonucleotides have attracted great interest as potential diagnostic tools for their tunable and high fluorescent properties. In this work, three DNA sequences that consist of a 12-nucleotide long probe sequence at the 5′-end linked to the complementary sequence to three miRNAs are studied. First, the interaction of these sequences with Ag(i) was characterized by means of circular dichroism spectroscopy. By applying multivariate methods to the analysis of spectroscopic data, two complexes with different Ag(i) : DNA ratios were resolved. Secondly, the impact of several experimental variables, such as temperature, borohydride concentration and reaction time, on the formation of AgNCs templated by these three sequences was studied. Finally, the fluorescence properties of the duplexes formed by DNA probes with complementary DNA or miRNA sequences were studied. The results presented here highlight the role of the secondary structure adopted by the DNA probe on the fluorescence properties of DNA-stabilized AgNCs which, in turn, affect the development of methods for miRNA detection.

Observation of microsecond luminescence while studying two DNA-stabilized silver nanoclusters emitting in the 800-900 nm range.

We investigated two DNA-stabilized silver nanoclusters (DNA-AgNCs) that show multiple absorption features in the visible region, and emit around 811 nm (DNA811-AgNC) and 841 nm (DNA841-AgNC). Both DNA-AgNCs have large Stokes shifts and can be efficiently excited with red light. A comparison with the commercially available Atto740 yielded fluorescence quantum yields in the same order of magnitude, but a higher photon output above 800 nm since both DNA-AgNCs are more red-shifted. The study of both DNA-AgNCs also revealed previously unobserved photophysical behavior for this class of emitters. The fluorescence quantum yield and decay time of DNA841-AgNC can be increased upon consecutive heating/cooling cycles. DNA811-AgNC has an additional absorption band around 470 nm, which is parallel in orientation to the lowest energy transition at 640 nm. Furthermore, we observed for the first time a DNA-AgNC population (as part of the DNA811-AgNC sample) with green and near-infrared emissive states with nanosecond and microsecond decay times, respectively. A similar dual emissive DNA-AgNC stabilized by a different 10-base DNA strand is also reported in the manuscript. These two examples highlight the need to investigate the presence of red-shifted microsecond emission for this class of emitters.

Correction to "Aptamer-Based Fluorescent Biosensing of Adenosine Triphosphate and Cytochrome c via Aggregation-Induced Emission Enhancement on Novel Label-Free DNA-Capped Silver Nanoclusters/Graphene Oxide Nanohybrids".

Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA–AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and targ...

Fluorescent silver nanoclusters stabilized in guanine-enhanced DNA hybridization for recognizing different small biological molecules

Fluorescent DNA-stabilized Ag nanoclusters (DNA/Ag NCs) plays significant roles in biochemical detections due to their high fluorescence quantum yield, high stability and good biocompatibility. In this paper, two guanine-enhanced fluorescence DNA-stabilized silver nanoclusters were designed to study the effect of guanine coating degree on silver nanoclusters fluorescence and their interactions with small biological molecules. The results indicated that the degree of guanine coating had a slight influence on the fluorescence of the two DNA/Ag NCs, however, the two DNA/Ag NCs had different fluorescence response when interacting with different small biological molecules. The fluorescence of the DNA/Ag NCs wrapped in the guanine-rich strand could be quenched by small biomolecules such as ascorbic acid, dopamine and cysteine, while the fluorescence of the DNA/Ag NCs approached to the guanine-rich strand could only be quenched by dopamine and cysteine. Based on this interesting phenomenon, an analytical method for recognition of ascorbic acid, dopamine and cysteine from other small biological molecules can be established by measuring the fluorescence change of DNA/Ag NCs and a method for selective detection of ascorbic acid in the absence of dopamine and cysteines can be achieved by combining the two DNA/Ag NCs probe.

Enzyme-free amplified detection of miRNA based on target-catalyzed hairpin assembly and DNA-stabilized fluorescent silver nanoclusters.

MicroRNAs (miRNAs) have been shown to be promising biomarkers for disease diagnostics and therapeutics. However, the rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their characters of small size, vulnerability to degradation, low abundance, and sequence similarity. Herein, we describe an enzyme-free amplification platform, consisting of a catalytic hairpin assembly (CHA) and DNA-templated silver nanoclusters (DNA/AgNCs), for miRNA analysis. In this work, two DNA hairpins (H1 and H2) were first designed for target miR-21-induced CHA, and then the fluorescence of DNA/AgNCs was quenched by BHQ1 to construct an activatable probe (AP). In the presence of target miR-21, hairpin H1 was opened by miR-21 through a hybridization reaction, and hairpin H2 was then opened by H1. During this process, miR-21 was released from H1 and participated in the next round of hybridization, triggering the CHA cycle reaction. The obtained H1-H2 products with sticky ends could react with the AP, forcing BHQ1 away from the DNA/AgNCs and thus causing the fluorescence recovery of the DNA/AgNCs. The assay for miR-21 detection demonstrated an excellent linear response to concentrations varying from 200 pM to 20 nM with the detection limit of 200 pM. The simple and cost-effective strategy holds great potential for application in biomedical research and clinical diagnostics.

Unusually large fluorescence quantum yield for a near-infrared emitting DNA-stabilized silver nanocluster

A near-infrared emitting DNA-stabilized silver nanocluster (DNA-AgNC) with an unusually high fluorescence quantum yield is presented. The steady-state and time-resolved fluorescence properties of the DNA-AgNC were characterized, together with its ability to generate optically activated delayed fluorescence (OADF) and upconversion fluorescence (UCF).

The effect of pH and ionic strength on the fluorescence properties of a red emissive DNA-stabilized silver nanocluster

DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of promising fluorophores for imaging and sensing applications. All aspects of their spectroscopic properties are not yet fully characterized, leaving this field still with a number of fundamental studies to be addressed. In this work, we studied the spectroscopic properties of red-emitting DNA-AgNCs at different pH (5 to 9) and ionic strength μ (0.005 to 0.5). The photophysical properties of high performance liquid chromatography (HPLC) purified DNA-AgNCs proved to be constant over a large range of pH and μ, with absorption, emission and fluorescence decay times only being affected at very high pH and μ values. Non-purified DNA-AgNCs were also unaffected by pH and/or μ variations, but significant differences can be observed between the rotational correlation times of purified and non-purified DNA-AgNCs.

Aptamer-Based Fluorescent Biosensing of Adenosine Triphosphate and Cytochrome c via Aggregation-Induced Emission Enhancement on Novel Label-Free DNA-Capped Silver Nanoclusters/Graphene Oxide Nanohybrids.

Four fluorescent DNA-stabilized fluorescent silver nanoclusters (DNA-AgNCs) were designed and synthesized with differences in lengths of cytosine-rich DNA strand (as the stabilizing agent) and target-specific strand DNA aptamers for adenosine triphosphate (ATP) and cytochrome c (Cyt c). After their nanohybrid formation with graphene oxide (GO), it was unexpectedly found that, depending on the composition of the base and length of the strand DNA aptamer, the fluorescence intensity of three of the nanohybrids significantly enhanced. Our experimental observations and quantum mechanical calculations provided an insight into the mechanisms underlying the behavior of DNA-AgNCs/GO nanohybrids. The enhanced fluorescence was found to be attributed to the aggregation-induced emission enhancement (AIE) characteristic of the DNA-AgNCs adsorbed on the GO surface, as confirmed evidently by both fluorescence and transmission electron microscopies. The AIE is a result of hardness and oxidation properties of GO, which lead to enhanced argenophilic interaction and thus to increased Ag(I)-DNA complex shell aggregation. Consequently, two of the DNA-AgNCs/GO nanohybrids were successfully extended to construct highly selective, sensitive, label-free, and simple aptasensors for biosensing of ATP (LOD = 0.42 nM) and Cyt c (LOD = 2.3 nM) in lysed Escherichia coli DH5 α cells and mouse embryonic stem cells, respectively. These fundamental findings are expected to significantly influence the designing and engineering of new AgNCs/GO-based AIE biosensors.

Detection of tiopronin in body fluids and pharmaceutical products using red-emissive DNA-stabilized silver nanoclusters as a fluorescent probe.

Tiopronin is a widely used drug for treatment of cystinuria, rheumatoid arthritis and hepatic disorders. It is also an antidote to heavy metal poisoning and a radioprotective agent. A method is described for rapid and sensitive determination of tiopronin using DNA-stabilized silver nanoclusters (DNA-AgNCs) as a fluorescent probe. Tiopronin can selectively bind to DNA-AgNCs to form a stable Ag-S bond upon which the red photoluminescence (best measured at excitation/emission wavelengths of 590/640nm) is quenched. The finding is used to design an assay that has a linear response in the 1-150nM tiopronin concentration range and a 270 pM limit of detection. Compared with previously reported methods, the present approach is more rapid, highly sensitive and selective. It has been successfully applied in the detection of tiopronin in spiked urine and serum, and in pharmaceutical products (tablets and injections). Graphical abstract An ultrasensitive and reliable method for tiopronin assay is developed using red-emissive silver nanoclusters as a fluorescent probe. It has been successfully applied in the determination of tiopronin in biological fluids and pharmaceutical products.

DNA-programming multicolor silver nanoclusters for sensitively simultaneous detection of two HIV DNAs

A novel DNA-stabilized silver nanoclusters (AgNCs)-based label-free fluorescent platform for simultaneously detecting two human immunodeficiency virus oligonucleotides (HIV DNAs) was developed. The sensing platform was established based on fluorescence enhancement of guanine (G)-rich and the phenomenon in the process of two silver nanoclusters (AgNCs) forming a nanoclusters dimer. The probe (AgNCs/G) utilized for HIV-1 detection adopted an effective conformation based on enhancement effect of G-rich sequence (at 500 nm ex / 565 nm em) while the probe (AgNCs/AgNCs) for HIV-2 generated fluorescence signals (at 580 nm ex / 630 nm em) with bright fluorescence only in nanoclusters dimer. The nanoprobe shows high selectivity for multiplexed analysis of target DNA with a detection limit of 11 pM, respectively. Moreover, this is the first time to use the affectivity of fluorescent AgNCs and two HIV DNAs simultaneous detection integrated into a novel method, which shows a great promise in biomedical research and early clinical diagnosis.

Disentangling optically activated delayed fluorescence and upconversion fluorescence in DNA stabilized silver nanoclusters.

Optically activated delayed fluorescence (OADF) is a powerful tool for generating background-free, anti-Stokes fluorescence microscopy modalities. Recent findings, using DNA-stabilized silver nanoclusters (DNA-AgNCs), indicate that OADF is usually accompanied by a dark state-mediated consecutive photon absorption process leading to upconversion fluorescence (UCF). In this study, we disentangle the OADF and UCF process by means of wavelength-dependent NIR excitation spectroscopy. We demonstrate that, by appropriate choice of secondary NIR excitation wavelength, the dark state population can be preferentially depleted through OADF, minimizing the UCF contribution. These findings show that dark state depletion by OADF might enable background-free STED-like nanoscopy.

Novel ELISA based on fluorescent quenching of DNA-stabilized silver nanoclusters for detecting E. coli O157:H7

Escherichia coli O157:H7 (E. coli O157:H7) is a potential threat to human health; thus, a rapid and sensitive method for detecting it is necessary. We designed a single-stranded DNA that contained an appended block and anchoring block. The appended block acted as a scaffold to prepare fluorescent Ag nanoclusters (AgNCs). The anchoring block contained Poly A, which bound with the surface of gold nanoparticles to quench the fluorescence of AgNCs. An interesting ELISA approach for detecting E. coli O157:H7 was established via fluorescent quenching of DNA-stabilized AgNCs by using a sandwich complex. The changes in fluorescence intensity of AgNCs were used to quantitatively detect E. coli O157:H7. The sensitivity for detecting E. coli O157:H7 reached 1.905 × 103 CFU/mL with a good linear range. Compared with conventional ELISA, the sensitivity of this technique increased by 30-fold. Moreover, this method demonstrated specificity and reproducibility and could be used in food samples.