DNA-templated Silver Nanoclusters Research Articles

A Ratiometric Fluorescence Detection Method for Berberine Using Triplex-Containing DNA-Templated Silver Nanoclusters

Berberine (BBR), as a natural isoquinoline alkaloid, has demonstrated various pharmacological activities, and is widely applied in the treatment of diseases. The quantitative analysis of BBR is important for pharmacological studies and clinical applications. In this work, utilizing the specific interaction between BBR and triplex DNA, a sensitive and selective fluorescent detecting method was established with DNA-templated silver nanoclusters (DNA-AgNCs). After binding with the triplex structure in the template of DNA-AgNCs, BBR quenched the fluorescence of DNA-AgNCs and formed BBR-triplex complex with yellow–green fluorescence. The ratiometric fluorescence signal showed a linear relationship with BBR concentration in a range from 10 nM to 1000 nM, with a detection limit of 10 nM. Our method exhibited excellent sensitivity and selectivity, and was further applied in BBR detection in real samples.

Rapid enzyme-free detection of miRNA-21 in human ovarian cancerous cells using a fluorescent nanobiosensor designed based on hairpin DNA-templated silver nanoclusters

BackgroundCancer is known as one of the main non-communicable diseases and the leading cause of death in the new era. Early diagnosis of cancer requires the identification of special biomarkers. Currently, microRNAs (miRNAs) have attracted the attention of researchers as useful biomarkers for cancer early detection. Hence, various methods have been recently developed for detecting and monitoring miRNAs. Among all miRNAs, detection of miRNA-21 (miR-21) is important because it is abnormally overexpressed in most cancers. Here, a new biosensor based on silver nanoclusters (AgNCs) is introduced for detecting miR-21. ResultsAs a fluorescent probe, a rationally designed hairpin sequence containing a poly-cytosine motif was used to facilitate the formation of AgNCs. A guanine-rich sequence was also employed to enhance the sensing signal. It was found that in the absence of miR-21, adding a guanine-rich sequence to the detecting probe caused only a slight change in the fluorescence emission intensity of AgNCs. While in the presence of miR-21, the emission signal enhanced. A direct correlation was observed between the increase in the fluorescence of AgNCs and the concentration of miR-21. The performance of the proposed biosensor was characterized thoroughly and confirmed. The biosensor detected miR-21 in an applicable linear range from 9 pM to 1.55 nM (LOD: 2 pM). SignificanceThe designed biosensor was successfully applied for detecting miR-21 in human plasma samples and also in human normal and lung and ovarian cancer cells. This biosensing strategy can be used as a model for detecting other miRNAs. The designed nanobiosensor can measure miR-21 without using any enzymes, with fewer experimental steps, and at a low cost compared to the reported biosensors in this field.

Multimodal lab-on-a-tube detection of AFB1 based on fluorescence and nanozyme activity of DNA templated silver nanocluster: Enhanced sensitivity through G-quadruplex formation

Aflatoxin B1 (AFB1) is the potent carcinogenic toxin secreted by fungi with widespread distribution around the world as the food contaminant. In the present study, a novel strategy has been developed for sensitive detection of AFB1 based on multiplex aptamer mediated probe and ExoIII enzyme activity. Inspired by generated fluorescence signal by DNA templated silver nanocluster (AgNCs), a specific aptasensor was designed to provide efficient interaction with AFB1 and subsequent amplify the released heterostructure included G-quadruplex after Exo-assisted activity for AgNCs fabrication. This strategy triggered repeated recognition-sensing cycle, leading to output colorimetric and fluorometric response signal. The integration of G-quadruplex in released probe, amplified the intrinsic low quantum yield and fluorescence of synthesized AgNCs and also enhanced its nanozyme activity on TMB and H2O2 substrate for colorimetric detection in single tube reaction. The developed AFB1 sensing assay showed LOD of 2.5 pM in colorimetric mode and had more sensitive LOD of 8.5 fM in fluorescence analysis. Smartphone assisted assay was also applied successfully as competitive detection approach. The proposed strategy was applicable in real sample and showed reliable results. Overall, this platform takes advantages of sensitive fluorometric analysis and nanozyme assisted colorimetric method which make it promising in practical application.

Programmable DNA Templates for Silver Nanoclusters Synthesis To Develop On-Demand FRET Aptasensor.

DNA-templated silver nanoclusters (AgNCs-DNA) can be synthesized via a one-pot method bypassing the tedious process of biomolecular labeling. Appending an aptamer to DNA templates results in dual-functionalized DNA strands that can be utilized for synthesizing aptamer-modified AgNCs, thereby enabling the development of label-free fluorescence aptasensors. However, a major challenge lies in the necessity to redesign the dual-functionalized DNA strand for each specific target, thus increasing the complexity and hindering widespread application of these aptasensors. To overcome this challenge, we designed six DNA strands (DNA1-DNA6) that incorporate the templates for AgNCs synthesis and A4-linker for further aptamer coupling. Among all the synthesized AgNCs-DNA samples, it was found that both AgNCs-DNA1 and AgNCs-DNA2 stood out for their excellent long-term stability. After capturing the T4-linker that connected with aptamer1 specific for aflatoxin B1 (AFB1), however, we found that only AgNCs-DNA1/aptamer1 maintained excellent long-term stability. This finding highlighted the potential of AgNCs-DNA1 as a versatile label-free fluorescence probe for the development of on-demand fluorescence aptasensors. To emphasize its benefits in aptasensing applications, we utilized AgNCs-DNA1/aptamer1 as the fluorescence probe and MoS2 nanosheets as the quencher to develop a FRET aptasensor for AFB1 detection. This aptasensor demonstrated remarkable sensitivity, enabling the detection of AFB1 within a wide concentration range of 0.03-120 ng/mL, with a limit of detection as low as 3.6 pg/mL (S/N = 3). The versatility of the aptasensor has been validated through the recognition of diverse targets, employing aptamer2 specific for ochratoxin A and aptamer3 specific for zearalenone, thereby showcasing its extensive applicability for on-demand detection. The universal applicability of this aptasensor holds great promise for future applications in diverse fields including food safety, environmental monitoring, and clinical diagnosis.

Leveraging Concentration Imbalance-Driven DNA Circuit as an Operational Amplifier to Enhance the Sensitivity of Hepatitis B Virus DNA Detection with Hybridization-Responsive DNA-Templated Silver Nanoclusters

Leveraging Concentration Imbalance-Driven DNA Circuit as an Operational Amplifier to Enhance the Sensitivity of Hepatitis B Virus DNA Detection with Hybridization-Responsive DNA-Templated Silver Nanoclusters

Plasmon-emitter coupling in cytosine-rich hairpin DNA-templated silver nanoclusters: Thermal reversibility, white light emission, and dynamics inside live cells.

Bio-templated luminescent noble metal nanoclusters (NCs) have attracted great attention for their intriguing physicochemical properties. Continuous efforts are being made to prepare NCs with high fluorescence quantum yield (QY), good biocompatibility, and tunable emission properties for their widespread practical applications as new-generation environment-friendly photoluminescent materials in materials chemistry and biological systems. Herein, we explored the unique photophysical properties of silver nanoclusters (AgNCs) templated by cytosine-rich customized hairpin DNA. Our results indicate that a 36-nucleotide containing hairpin DNA with 20 cytosine (C20) in the loop can encapsulate photostable red-emitting AgNCs with an absolute QY of ∼24%. The luminescent properties in these DNA-templated AgNCs were found to be linked to the coupling between the surface plasmon and the emitter. These AgNCs exhibited excellent thermal sensitivity and were employed to produce high-quality white light emission with an impressive color rendering index of 90 in the presence of dansyl chloride. In addition, the as-prepared luminescent AgNCs possessing excellent biocompatibility can effectively mark the nuclear region of HeLa cells and can be employed as a luminescent probe to monitor the cellular dynamics at a single molecular resolution.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization-mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag13 (in the intact DNA host) to Ag10 (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

A Combined Quantum Mechanics and Molecular Mechanics Approach for Simulating the Optical Properties of DNA-Stabilized Silver Nanoclusters.

DNA-stabilized silver nanoclusters have emerged as an intriguing type of nanomaterial due to their unique optical and electronic properties, with potential applications in areas such as biosensing and imaging. The development of efficient methods for modeling these properties is paramount for furthering the understanding and utilization of these clusters. In this study, a hybrid quantum mechanical and molecular mechanical approach for modeling the optical properties of a DNA-templated silver nanocluster is evaluated. The influence of different parameters, including ligand fragmentation, damping, embedding potential, basis set, and density functional, is investigated. The results demonstrate that the most important parameter is the type of atomic properties used to represent the ligands, with isotropic dipole-dipole polarizabilities outperforming the rest. This underscores the importance of an appropriate representation of the ligands, particularly through the selection of the properties used to represent them. Moreover, the results are compared to experimental data, showing that the applied methodology is reliable and effective for the modeling of DNA-stabilized silver nanoclusters. These findings offer valuable insights that may guide future computational efforts to explore and harness the potential of these novel systems.

Turn-on fluorescent nanoprobe for ATP detection based on DNA-templated silver nanoclusters.

A turn-on fluorescence nanoprobe was constructed for the determination of adenosine 5'-triphosphate (ATP) based on DNA-templated silver nanoclusters (DNA-AgNCs). The significant enhancement fluorescence intensity of DNA-AgNCs in the presence of ATP is due to the high special binding affinity between ATP and the aptamer, resulting in the environment of DNA-AgNCs with darkish fluorescence lying at one terminus of DNA slightly altering owing to the change of ATP aptamer conformation. A good linear range runs from 9 to 24 mM with a satisfactory detection limit of 3 μM. Furthermore, the proposed nanoprobe exhibited good performance for ATP detection in diluted fetal bovine serum.

An electrochemiluminescence microsensor based on DNA-silver nanoclusters amplification for detecting cellular adenosine triphosphate.

Adenosine triphosphate (ATP), as the primary energy source, plays vital roles in many cellular events. Developing an efficient assay is crucial to rapidly evaluate the level of cellular ATP. A portable and integrated electrochemiluminescence (ECL) microsensor array based on a closed bipolar electrode (BPE) was presented. In the BPE unit, the ECL chemicals and oxidation/reduction were separated from the sensing chamber. The ATP aptamer was assembled with single-stranded DNA (ssDNA) in the sensing chamber. ATP capture made the aptamer disassemble from the ssDNA and facilitated DNA-templated silver nanocluster (Ag NC) generation by the target-rolling circle amplification (RCA) reaction. The guanine-rich padlock sequence produced tandem periodic cytosine-rich sequences by the RCA, inducing Ag NC generation in the cytosine-rich region of the produced DNA strands through Ag+ reduction. The in situ Ag NC generation enhanced the circuit conductivity of the BPE and promoted the ECL reaction of [Ru(bpy)2dppz]2+/tripropylamine in the anodic reservoir. On this ECL microsensor, a good linear relationship of ATP was achieved ranging from 30 to 1000 nM. The ATP content in HepG2 cells was selectively and sensitively determined without complex pretreatment. The ATP amount of 25 cells could be successfully detected when a sub-microliter sample was loaded.

Demonstrating the Synthesis and Antibacterial Properties of Nanostructured Silver.

Investigating and understanding novel antibacterial agents is a necessary task as there is a constant increase in the number of multidrug-resistant bacterial species. The use of nanotechnology to combat drug-resistant bacteria is an important research area. The laboratory experiment described herein demonstrates that changes in the nanostructure of a material lead to significantly different antibacterial efficacies. Silver has been known to be an effective antibacterial agent throughout history, but its therapeutic uses are limited when present as either the bulk material or cations in solution. Silver nanoparticles (AgNPs) and DNA-templated silver nanoclusters (DNA-AgNCs) are both nanostructured silver materials that show vastly different antibacterial activities when incubated with E. coli in liquid culture. This work aims to provide students with hands-on experience in the synthesis and characterization of nanomaterials and basic microbiology skills; moreover, it is applicable to undergraduate and graduate curricula.

Highly Sensitive Fluorescence Detection of Three Organophosphorus Pesticides Based on Highly Bright DNA-Templated Silver Nanoclusters

It is still challenging to achieve simultaneous and sensitive detection of multiple organophosphorus pesticides (OPs). Herein, we optimized the ssDNA templates for the synthesis of silver nanoclusters (Ag NCs). For the first time, we found that the fluorescence intensity of T base-extended DNA-templated Ag NCs was over three times higher than the original C-riched DNA-templated Ag NCs. Moreover, a "turn-off" fluorescence sensor based on the brightest DNA-Ag NCs was constructed for the sensitive detection of dimethoate, ethion and phorate. Under strong alkaline conditions, the P-S bonds in three pesticides were broken, and the corresponding hydrolysates were obtained. The sulfhydryl groups in the hydrolyzed products formed Ag-S bonds with the silver atoms on the surface of Ag NCs, which resulted in the aggregation of Ag NCs, following the fluorescence quenching. The fluorescence sensor showed that the linear ranges were 0.1-4 ng/mL for dimethoate with a limit of detection (LOD) of 0.05 ng/mL, 0.3-2 µg/mL for ethion with a LOD of 30 ng/mL, and 0.03-0.25 µg/mL for phorate with a LOD of 3 ng/mL. Moreover, the developed method was successfully applied to the detection of dimethoate, ethion and phorate in lake water samples, indicating a potential application in OP detection.

Excited-State Dynamics in a DNA-Stabilized Ag16 Cluster with Near-Infrared Emission.

Due to desirable optical properties, such as efficient luminescence and large Stokes shift, DNA-templated silver nanoclusters (DNA-AgNCs) have received significant attention over the past decade. Nevertheless, the excited-state dynamics of these systems are poorly understood, as studies of the processes ultimately leading to a fluorescent state are scarce. Here we investigate the early time relaxation dynamics of a 16-atom silver cluster (DNA-Ag16NC) featuring NIR emission in combination with an unusually large Stokes shift of over 5000 cm-1. We follow the photoinduced dynamics of DNA-Ag16NC on time ranges from tens of femtoseconds to nanoseconds using a combination of ultrafast optical spectroscopies, and extract a kinetic model to clarify the physical picture of the photoinduced dynamics. We expect the obtained model to contribute to guiding research efforts toward elucidating the electronic structure and dynamics of these novel objects and their potential applications in fluorescence-based labeling, imaging, and sensing.

A fluorescent aptasensor based on ZIF-8 @PdNCs and DNA-AgNCs for tobramycin detection in milk

A novel ratiometric fluorescent aptasensor was constructed utilizing ZIF-8-encapsulated palladium nanoclusters (ZIF-8 @PdNCs) as reference signals that emit blue fluorescence, and DNA-templated silver nanoclusters (DNA-AgNCs) containing aptamer fragments as response signals that emit yellow fluorescence. In the absence of tobramycin (TOB), the aptamers of DNA-AgNCs bind to ZIF-8 @PdNCs via π-π stacking, resulting in the quenching of the fluorescent signals of DNA-AgNCs. The presence of TOB can weaken the π-π stacking interaction between ZIF-8 @PdNCs and single-stranded DNA (ssDNA) aptamers, which can lead to the desorption of DNA-AgNCs from the surface of ZIF-8 @PdNCs. As a result, the fluorescence signals of DNA-AgNCs are restored. The developed fluorescent sensor exhibits a linear range between 50 nM and 500 nM, with a limit of detection determined to be 15.3 nM. Exposure to UV light triggers a visible change in fluorescent color from blue to yellow as the concentration of TOB increases. To build a miniaturized and portable device for detecting TOB concentration, we employed 3D printing technology to create an RGB (Red Green Blue) instrument recognition framework with ultraviolet lamp beads and RGB recognition components. The RGB standard curve and programming in Python can be used to directly read the TOB concentration.

Fluorescence Enhancement Method for Aptamer-Templated Silver Nanoclusters and Its Application in the Construction of a β-Amyloid Oligomer Sensor.

DNA-templated silver nanoclusters (DNA-AgNCs) have attracted significant attention due to their unique fluorescence properties. However, so far, the relatively low quantum yields of the DNA-AgNCs and the complex design of DNA-AgNC-based sensors have limited their application in biosensing or bioimaging. Herein, we report a novel fluorescence enhancement method. The β-Amyloid Oligomer (AβO) aptamer (AptAβO) with A10/T10 at its 3' end can be directly used as the template to fabricate the AgNCs. When the AgNCs were hybridized with the complementary strand that has 12 bases suspended at its 3' terminal, being the same or complementary to the A/T at the 3' end of the AptAβO, and two-base mismatches in the complementary region of the aptamer excluded A10/T10, a dramatic fluorescence enhancement (maximum: ∼500-fold; maximum quantum yield: 31.5%) can be realized. The fluorescence enhancement should result from the aggregation-induced emission of the AgNCs, which can be attributed to forming the reticular structure of the hybridized product. To some extent, the method developed in this work is extendable. The fluorescence enhancement was also realized from the thrombin aptamer-templated AgNCs through designing the aptamer and the corresponding complementary strand according to the method. Based on the fluorescence enhancement of the AptAβO-templated AgNCs, an "on-off" fluorescence sensor was constructed for the sensitive and selective detection of AβO. This work provides a rational strategy to realize fluorescence enhancement for the aptamer-templated AgNCs and design an aptamer-based fluorescence sensor.

Dual-channel fluorescence detection of antibiotic resistance genes based on DNA-templated silver nanoclusters

The aqueous environment is an ideal site for the generation and transmission of antibiotic resistance genes (ARGs), and has become a sink for multiple ARGs. Detection of multiple ARGs in one-pot by a simple method is essential to control the spread of antibiotic resistance. Herein, we developed a novel fluorescence sensing strategy based on chameleon DNA-templated silver nanoclusters (AgNCs) to achieve simultaneous detection of two ARGs (tet-A and sul-1). A DNA fluorescent probe with AgNCs stabilized at both termini and another DNA probe carried enhancer sequences were designed. The hybridization of the target ARGs and probes can form an infinitely extended linear DNA structure containing multi-branched AgNCs beacons, and the chameleon AgNCs approach the fluorescence enhancer sequence, thereby realizing the transduction and amplification of green and red fluorescence signals. Through this strategy, we successfully achieved highly specific detection of two ARGs with the LOD of 0.45 nM for tet-A and 0.32 nM for sul-1. In addition, the strategy still had good applicability in the detection of actual samples containing complex components. In this study, fluorescent DNA-AgNCs were applied to the rapid, enzyme-free and reliable detection of ARGs for the first time. The excellent performance of the simultaneous detection of two ARGs displayed that this method can be used to simultaneously analyze different types of ARGs, indicating its great potential in rapid screening and quantitative detection of ARGs in various environmental medias.

Sensitive Fluorescent Determination of Nucleic Acids by a Cytosine-Rich Loop and Silver Nanoclusters in the Hybridization Chain Reaction (HCR)

Deep understanding of the effect of DNA sequence and structure on the fluorescent properties of DNA-templated silver nanoclusters (DNA–Ag NCs) affords alternative strategies for developing and exploiting DNA biosensors. DNA secondary structures (e.g., DNA loop) are essential for generating highly emissive DNA–Ag NCs. In this study, a cytosine-rich DNA loop was used as a facile template for the formation of silver nanoclusters. The fluorescence of silver nanocluster encapsulated within a cytosine-rich DNA loop was turned on by switching the size of the loop from large to small. Target nucleic acid served as the trigger for the hybridization chain reaction of hairpin probe 1 (H1) containing an inactivated cytosine-rich DNA loop with a large size at the ends and hairpin probe 2 (H2). Long-nicked double helices were obtained coupled with the opening of the large-size cytosine-rich DNA loop in H1 and switching to an activated cytosine-rich DNA loop with a small size. As a result, an increase in the fluorescent signal was observed. A sensitive detection limit of 10 pM and a linear detection range from 10 pM to 400 nM were obtained for nucleic acid determination. This facile assay provides a new approach capable of switching the fluorescence emission of DNA–Ag NCs for nucleic acid biosensing.

DNA-Templated Silver Nanoclusters as Dual-Mode Sensitive Probes for Self-Powered Biosensor Fueled by Glucose.

Nanomaterials have been extensively explored in developing sensors due to their unique properties, contributing to the development of reliable sensor designs with improved sensitivity and specificity. Herein, we propose the construction of a fluorescent/electrochemical dual-mode self-powered biosensor for advanced biosensing using DNA-templated silver nanoclusters (AgNCs@DNA). AgNC@DNA, due to its small size, exhibits advantageous characteristics as an optical probe. We investigated the sensing efficacy of AgNCs@DNA as a fluorescent probe for glucose detection. Fluorescence emitted by AgNCs@DNA served as the readout signal as a response to more H2O2 being generated by glucose oxidase for increasing glucose levels. The second readout signal of this dual-mode biosensor was utilized via the electrochemical route, where AgNCs served as charge mediators between the glucose oxidase (GOx) enzyme and carbon working electrode during the oxidation process of glucose catalyzed by GOx. The developed biosensor features low-level limits of detection (LODs), ~23 μM for optical and ~29 μM for electrochemical readout, which are much lower than the typical glucose concentrations found in body fluids, including blood, urine, tears, and sweat. The low LODs, simultaneous utilization of different readout strategies, and self-powered design demonstrated in this study open new prospects for developing next-generation biosensor devices.

Detection of cancer-associated miRNA using a fluorescence switch of AgNC@NA and guanine-rich overhang sequences.

DNA-templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single-stranded (dC)12 when adjacent G-rich sequences (dGN , with N = 3-15) were added. The 'red' emission of AgNC@dC12 with λMAX = 660 nm dramatically changed upon the addition of a G-rich overhang with NG = 15. The pattern of the emission-excitation matrix (EEM) suggested the emergence of two new emissive states at λMAX = 575 nm and λMAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA-based switch that brings AgNC and the G overhang near one another, turning 'ON' the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR-371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR-371 molecule.

Primer Exchange Reaction Coupled with DNA-Templated Silver Nanoclusters for Label-Free and Sensitive Detection of MicroRNA.

MicroRNAs (MiRNAs) play pivotal roles in regulating gene expression, and serve as crucial biomarkers for diagnosis of a variety of disease. However, label-free and sensitive miRNA detection remains a huge challenge due to the low abundance. Herein, we developed an approach through integrating primer exchange reaction (PER) with DNA-templated silver nanoclusters (AgNCs) for label-free and sensitive miRNA detection. In this method, PER was used to amplify miRNA signals and produce single-strand DNA (ssDNA) sequences. The produced ssDNA sequences mediated DNA-templated AgNCs based signal generation by unfolding the designed hairpin probe (HP). The generated AgNCs signal was correlated with the dosage of target miRNA. Eventually, the established approach exhibited a low detection of limit of 47 fM with a great dynamic range of more than five orders of magnitude. In addition, the method was also utilized to detect the miRNA-31 expression in collected clinical samples from pancreatitis patients and demonstrated that miRNA-31 was upregulated in patients, showing a great promising of the method in clinical application.