DNA Silver Nanoclusters Research Articles

A highly cost-effective and sensitive biosensing platform: Fuel-propelled DNA nanomachines modulate fluorescence switching of DNA silver nanoclusters

It is of great significance to develop a low-cost, simple but highly sensitive fluorescence sensing platform for low-abundance molecule detection. However, traditional organic fluorophore labeling often requires expensive and sophisticated synthesis, making it difficult to use in resource-limited areas. Besides, the traditional “one-to-one” detection mechanism presents an obstacle to sensitive detection of low-level molecules, because one target only triggers one signal. To tackle these predicaments, in this work, a brightly red-emissive three-stranded DNA stabilized silver nanocluster (Ag NC) has been successfully synthesized. However, the fluorescence emission of Ag NCs can be darkened when it is in a loose single-stranded DNA. Building on this finding, we elaborately engineer a highly cost-effective and sensitive biosensing platform, which exploits a target-triggered and fuel-driven DNA nanomachine to precisely manipulate the structural transformation of Ag NCs from the rigid three-stranded DNA structures to the flexible single-stranded DNA, accordingly achieving the fluorescence switching for amplified sensing of low-abundance molecules. It is worth noting that nucleases and complex secondary structures are not involved in the entire system, offering a simple and flexible design. Furthermore, the novel and red-emissive DNA Ag NCs are employed as signal reporting, avoiding expensive and time-consuming organic fluorophore labeling. Experiment results demonstrate that this newly developed biosensing platform has a high signal-to-noise (∼15-folds), low limit of detection (1.59 pM), and good specificity to H5N1 DNA. Importantly, it can be used for H5N1 DNA detection in complex biological samples with a satisfactory recovery.

Upgraded and Light-Up Biosensing Platform: Entropy-Driven Catalysis Circuit Manipulates the Configuration Transformation of Novel DNA Silver Nanoclusters on the Graphene Oxide Surface.

To tackle the predicament of the traditional turn-off mechanism, exploring an activated turn-on system remains an intriguing and crucial objective in biosensing fields. Herein, a dark DNA Ag nanocluster (NC) with hairpin-structured DNA containing a six-base cytosine loop (6C loop) as a template is atypically synthesized. Intriguingly, the dark DNA Ag NCs can be lit to display strong red-emission nanoclusters. Building upon these exciting findings, an unprecedented and upgraded turn-on biosensing system [entropy-driven catalysis circuit (EDCC)-Ag NCs/graphene oxide (GO)] has been created, which employs an EDCC to precisely manipulate the conformational transition of DNA Ag NCs on the GO surface from adsorption to desorption. Benefiting from the effective quenching of GO and signal amplification capability of the EDCC, the newly developed EDCC-Ag NCs/GO biosensing system displays a high signal-to-background (S/B) ratio (26-fold) and sensitivity (limit of detection as low as 0.4 pM). Meanwhile, it has good specificity, excellent stability, and reliability in both buffer and biological samples. To the best of our knowledge, it is the first example that adopts an EDCC to precisely modulate the configuration transformation of DNA Ag NCs on the GO surface to obtain a biosensor with low background, strong fluorescence, high contrast, and sensitivity. This exciting finding may provide a new route to fabricate a novel turn-on biosensor based on hairpin-templated DNA Ag NCs in the optical imaging and bioanalytical fields.

An enzyme-free and label-free ratiometric fluorescence signal amplification biosensor based on DNA-silver nanoclusters and catalytic hairpin assembly for tetracycline detection

Sensitive and accurate in antibiotic detection is crucial for food security, environmental conservation, public health, and environmental protection. In this study, we developed an enzyme-free and label-free ratiometric fluorescence biosensor for highly sensitive detection of tetracycline (TET). This biosensor utilized a combination of target-triggered catalytic hairpin assembly (CHA) and DNA-silver nanocluster (DNA-AgNCs) dual-color fluorescent signal probes. Upon TET binding to the aptamer, it initiated the leakage of CHA initiator, resulting in the formation of numerous double-stranded DNA complexes. This process exposed the foothold sequence of hairpin-structured DNA-AgNCs (H3-AgNCs) that exhibit yellow fluorescence. Subsequently, the conformational changes in H3-AgNCs significantly reduced the yellow fluorescence intensity (FI) and reveal the auxiliary DNA-AgNCs (a-DNA-AgNCs) linkage sequence. The binding of a-DNA-AgNCs brought two dark AgNCs close, forming nanocluster dimers emitting red fluorescence. The results demonstrate that the variation in ratiometric signal is linear with TET concentration between 0.1 and 50 ng/mL, with a detection limit as low as 0.086 ng/mL. Additionally, this approach exhibited good recovery results with spiked milk samples. This enzyme-free and label-free combined signal amplification method with proportional signal output based on DNA-AgNCs provides valuable empirical reference for creating low-cost, stable, and highly-sensitive biosensors.

Silver nanocluster-based aptasensor for the label-free and enzyme-free detection of ochratoxin A

Mycotoxin contamination in cereal is a global concern, threatening food safety and human health, necessitating the development of rapid on-site methods. Here, a label- and enzyme-free biosensor was developed based on aptamer-regulated DNA-silver nanoclusters (AgNCs) for rapid detection of ochratoxin A (OTA). A novel DNA-templated AgNCs emitting strong red fluorescence was designed and synthesized in this study. The partial sequence of the DNA template was selected from the complementary OTA aptamer (Apt-OTA) sequence, which can quench fluorescence from the AgNCs via hybridization in the absence of OTA. In the presence of OTA, the high OTA-Aptamer affinity prevented the Apt-OTA from quenching the AgNCs, resulting in “turn on” of the fluorescence. This biosensor eliminated the use of costly reagents, complex pretreatments, and sophisticated equipment, which could realize the point-of-care testing (POCT) of OTA with a limit of detection (LOD) of 1.3 nM and a detection time of 45 min.

Detection of pyrimidine-rich DNA sequences based on the formation of parallel and antiparallel triplex DNA and fluorescent silver nanoclusters

In this work, the use of DNA-stabilized fluorescent silver nanoclusters for the detection of target pyrimidine-rich DNA sequences by formation of parallel and antiparallel triplex structures is studied by molecular fluorescence spectroscopy. In the case of parallel triplexes, the probe DNA fragments are Watson-Crick stabilized hairpins, and whereas in the case of antiparallel triplexes, the probe fragments are reverse-Hoogsteen clamps. In all cases, the formation of the triplex structures has been assessed by means of polyacrylamide gel electrophoresis, circular dichroism, and molecular fluorescence spectroscopies, as well as multivariate data analysis methods. The results have shown that it is possible the detection of pyrimidine-rich sequences with an acceptable selectivity by using the approach based on the formation of antiparallel triplex structures.

Construction of an electrochemical-fluorescent dual-mode sensor with a dual-mode signal AgNC probe synthesized from cytosine-rich DNA for OTA detection.

Herein, we have used DNA-silver nanocluster (DNA-AgNC) signal probes with both electrochemical and fluorescent signals for the first time to construct an electrochemical-fluorescent dual-mode sensor. The sensor has an easy-to-prepare dual-signal property combined with the magnetic separation technique for dual-mode detection of ochratoxin A (OTA). In the absence of OTA, the DNA strand used to synthesize AgNCs was not available in the system after magnetic separation. DNA-AgNCs probes could not be synthesized in the system, resulting in low fluorescence and electrochemical signals. In the presence of OTA, it led to the shedding of sulfhydryl-modified and cytosine-rich DNA (C-DNA). DNA-AgNCs probes with high fluorescence and electrochemical signals were formed by adding AgNO3 and NaBH4 to the supernatant after magnetic separation. Dual-mode detection of OTA was achieved by the signal response of fluorescence and electrochemistry. The detection ranges were 2.5 × 10-4-50 ng mL-1 and 2.5 × 10-4-25 ng mL-1 in the fluorescence mode and electrochemical mode with detection limits of 0.11 pg mL-1 and 0.025 pg mL-1, respectively. Meanwhile, the dual-mode sensor displayed better specificity, repeatability and reproducibility than conventional electrochemical and fluorescent single-mode sensors. The results of the spiked peanut and wheat flour detection showed that the fluorescence and electrochemical modes of the sensor exhibited satisfactory average recoveries.

Bacterial DNA analysis based on target aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction

We have constructed a simple, enzyme-free and label-free detection method for bacterial DNA. First, bacterial DNA (T) is designed as a trigger to make the designed metastable hairpin probes P1, P2, P3 (carrying DNA silver nanoclusters sequences) autonomously cross-open to achieve catalytic self-assembly, inducing a three-way DNA junction formation through the catalyzed hairpin self-assembly (CHA) mode. Then, G-rich sequences are introduced to form DNA silver nanoclusters G quadruplex (DNA-AgNCs-G quadruplex), and a strong fluorescence signal can be collected under the action of silver nitrate and sodium borohydride solutions. Through the fluorescent reporter of DNA silver nanoclusters (DNA-AgNCs) and the amplification of CHA, we successfully detected target DNA at concentrations as low as 19.95 fM. Compared with the constructed acyclic system, the sensitivity of this cyclic system is improved about three orders of magnitude. Moreover, this recycling system achieves good selectivity for mismatched target DNA and excellent recovery rate in practical samples. Therefore, bacterial DNA analysis based on target-aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction is successfully developed, promising its great application in biological sensing.

Multi pathogenic microorganisms determination using DNA composites-encapsulated DNA silver nanocluster/graphene oxide-based system through rolling cycle amplification.

A multi pathogenic microorganisms determination method is reported using DNA composites encapsulated DNA silver nanocluster (AgNCs)/graphene oxide (GO)-based system through rolling cycle (RCA) amplification. Firstly, two different RCA-based DNA composites are assembled, coupled with thousands of DNA-stabilized AgNCs probe and ssDNA aptamer specific for two pathogen bacteria targets. GO was then introduced into the system to capture ssDNA aptamer of DNA composites and as a selective fluorescence quencher of DNA/AgNCs. Upon recognizing the target bacteria, ssDNA aptamer part would combine with bacteria and release from the surface of GO. Thus, DNA/AgNCs of RCA-based DNA composites can generate strong fluorescence signal. With the fluorescent report of RCADNA-AgNCs/530 and RCADNA-AgNCs/625, the assay successfully detect Escherichia coli and Staphylococcus aureus atconcentrations as low as 38CFU/mL, and a highly selective and efficient sensing platform was achieved. Therefore, this RCA/DNA-AgNCs/GO-based platform shows excellent application in multi pathogenic microorganisms determination and potential clinic therapy.

A novel fluorescence sensor for streptomycin detection based on hairpin DNA-AgNCs and core-shell gold-palladium nanoparticles

A novel strategy for streptomycin (STR) detection in milk was described. It was realized based on guanine (G) base-regulated hairpin DNA silver nanoclusters (DNA-AgNCs) and core-shell gold-palladium nanoparticles (Au@PdNPs) according to the principle of fluorescence resonance energy transfer (FRET). It differs from most strategies to light up the DNA-AgNCs with weak fluorescence emission due to the proximity of the G bases sequences. Different numbers of G bases were added to the hairpin loop to obtain a redshift of the emission spectrum and increase the fluorescence intensity of the DNA-AgNCs. In addition, the Au@PdNPs were prepared by seed growth and deposition. The Au@PdNPs composite material improved the quenching ability of the palladium nanoparticles (PdNPs) in the long wavelength range and significantly expanded the ultraviolet absorption range of gold nanoparticles (AuNPs). These characteristics ensure that the Au@PdNPs are excellent quenching material for fluorescent sensors. The developed sensor showed a good detection performance for STR in the range of 50–1250 nM with a limit of detection of 18.7 nM. In addition, this strategy was successfully applied for STR detection in milk, indicating its suitability for STR detection in animal-derived foods.

Inhibition of Bacteria In Vitro and In Vivo by Self-Assembled DNA-Silver Nanocluster Structures.

Antimicrobial nanomaterials hold great promise for bacteria-infected wound healing. However, it remains a challenge to balance antimicrobial efficacy and biocompatibility for these artificial antimicrobials. Here we employed biocompatible genetic molecule DNA as a building material to fabricate antimicrobial materials, including self-assembled Y-shaped DNA-silver nanocluster composite (Y-Ag) and Y-Ag hydrogel (Y-Ag-gel). We demonstrate that macroscopic and microcosmic DNA-Ag composites can effectively inhibit bacterial growth but do not affect cell proliferation in vitro. In particular, Y-Ag spray can speed up the process of wound healing in vivo. Considering the efficacy and advantages of DNA-based materials, our findings provide a promising route to fabricate a novel wound dressing such as spray and hydrogel for therapeutic wound healing.

Label-free and dual-mode biosensor for HPV DNA based on DNA/silver nanoclusters and G-quadruplex/hemin DNAzyme

Specific and cost-effective methodologies for human papillomavirus (HPV) gene detection are significant for clinical diagnosis and cancer control. Herein, a label-free and fluorimetric/colorimetric dual-mode sensing strategy was developed for the quantitative determination of HPV DNA based on the integration of fluorescent DNA-silver nanoclusters (DNA/AgNCs) and G-quadruplex/hemin DNAzyme. The fluorimetric sensing strategy was based on the phenomena that the fluorescence enhancement of DNA/AgNCs obtained in proximity of guanine-rich DNA sequences and the photoinduced electron transfer (PET) effect between the electron donor (DNA/AgNCs) and electron receptor (G-quadruplex/hemin). The colorimetric sensing strategy was relied on the peroxidase-like activity of G-quadruplex/hemin DNAzyme. By integrating DNA/AgNCs and DNAzyme, this dual-mode strategy could produce two independent signals to improve the analytical diversity and accuracy. Under optimized conditions, the fluorimetry and colorimetry of the strategy displayed a linear range of 0.01–4 and 0.02–4 nM, with the low detection limit of 2.3 and 5.2 pM, respectively. Additionally, this dual-mode strategy has been successfully applied to HPV DNA analysis in different real samples with excellent results. Moreover, the sensing platform could be developed for different HPVs DNA assay by only adjusting the recognition sequence, which provided a universal strategy for various kinds of virus analysis.

A "turn-on" DNA-scaffolded silver-nanocluster probe for detection of tumor-related mRNA.

A novel and "turn-on" DNA-silver nanocluster probe (DNA/AgNCs) was developed that can detect the level of breast tumor-related mRNA markers: cyclin D1 mRNA. The fluorescence of DNA/AgNCs probe rendered a "turn-on" response based on the secondary structure changes induced by the target strand. In the presence of target chain, the fluorescence enhances in a concentration-dependent manner, which enables the quantitative detection of analytes. By this method, a detection limit of 2.1nM and a linear range of 15-125nM were achieved. In addition, the probe displays excellent discrimination between a perfectly complementary target and single-base mismatch targets, providing an alternative approach for quick and specific recognition and quantification of mRNA or DNA.

Aptamer-Functionalized DNA-Silver Nanocluster Nanofilm for Visual Detection and Elimination of Bacteria.

As a new type of nanomaterial, DNA-templated silver nanoclusters (DNA-AgNCs) have been widely studied because of their fluorescence and antibacterial properties. In this study, we combined the DNA-AgNCs with aptamers of bacteria to achieve a novel approach for the visual detection and effective elimination of bacteria. The aptamers of Staphylococcus aureus (S. aureus) were linked to G-rich sequences to achieve fluorescence enhancement when approaching the DNA-AgNCs. The capture of aptamers not only realized the visual monitoring of bacteria but also promoted the antibacterial effects. Additionally, a fluorescent nanofilm with excellent selectivity and antibacterial activity in the detection and elimination of S. aureus was developed based on the DNA-AgNCs. These aptamer-functionalized DNA-AgNCs show significant potential for many applications in food packaging and biomedical engineering.

DNA-templated silver nanoclusters as an efficient catalyst for reduction of nitrobenzene derivatives: a systematic study

Nitrobenzene compounds are highly toxic pollutants with good stability, and they have a major negative impact on both human health and the ecological environment. Herein, it was found for the first time that fluorescent DNA-silver nanoclusters (DNA-AgNCs) can catalyze the reduction of toxic and harmful nitro compounds into less toxic amino compounds with excellent tolerance to high temperature and organic solvents. In this study, the reduction of p-nitrophenol (4-NP) as a model was systematically investigated, followed by expending the substrate to disclose the versatility of this reaction. This report not only expanded the conditions for utilizing catalytic reduction conditions of DNA-AgNCs as an efficient catalyst in the control of hazardous chemicals but also widened the substrate range of DNA-AgNCs reduction, providing a new angle for the application of noble metal nanoclusters.

Fluorometric detection of cancer marker FEN1 based on double-flapped dumbbell DNA nanoprobe functionalized with silver nanoclusters

Flap endonuclease 1 (FEN1), a ubiquitous enzyme involved in DNA repair and replication, is overexpressed in highly proliferative cancer cells. FEN1 has been recognized as a promising diagnostic marker of cancers; however, very few analytical techniques have been developed for the convenient detection of FEN1. To realize the simplified quantification of FEN1, we developed a FEN1-responsive fluorescent nanoprobe based on DNA-silver nanoclusters (DNA-AgNCs). The nanoprobe was rationally designed with a double-flapped dumbbell conformation, where its 5′ flap was produced with DNA-AgNCs, and the 3′ flap was elongated by a guanine-rich enhancer sequence (GRS). Rigidified by the DNA scaffold, DNA-AgNCs and the GRS are in close proximity, resulting in high fluorescence because of the GRS-induced activation of DNA-AgNCs. Upon the addition of FEN1, the 5’ flap of the nanoprobe is cleaved due to the structure-specific endonuclease activity of FEN1. This cleavage released the DNA-AgNCs from the nanoprobe, broke the proximity between DNA-AgNCs and the GRS, and caused decreased fluorescence. This nanoprobe can be applied in the sensitive detection of FEN1 with a detection limit of 40 fM, and it showed high specificity for the monitoring of FEN1 in clinical samples. As the first attempt to develop biosensors targeting FEN1 based on DNA-AgNCs, this work provided a potent platform for monitoring FEN1 and screening FEN1 inhibitors.

Base amount-dependent fluorescence enhancement for the assay of vascular endothelial growth factor 165 in human serum using hairpin DNA-silver nanoclusters and oxidized carbon nanoparticles.

A base amount-dependent fluorescence enhancement-based strategy is put forward to determine vascular endothelial growth factor 165 (VEGF165) in human serum by the use of hairpin DNA-silver nanoclusters (hDNA-AgNCs) and oxidized carbon nanoparticles (CNPs). The hDNA-AgNCs aptasensing probe consists of AgNCs-contained hairpin loop (that generates afluorescence signal), hairpin stem (that makes the structure stable), and the terminal aptamer 1 (that recognizesthe target together with aptamer 2). It has been demonstrated that the fluorescence intensity of hDNA-AgNCs is ~ 3-fold stronger than that of single-stranded DNA-AgNCs (ssDNA-AgNCs), and hDNA-AgNCs have a strong dependence of fluorescence enhancement on the base amount in hairpin stem and loop. Upon the addition of oxidized CNPs, the terminal aptamer 1 of hDNA-AgNCs can adsorb onto the surface of oxidized CNPs via π-π stacking, and the fluorescence of hDNA-AgNCs (with excitation/emission maxima at 490/567nm) is quenched via fluorescence resonance energy transfer (FRET). When aptamer 2 and VEGF165 are subsequently added, aptamer 1, VEGF165, and aptamer 2 reassemble into anintact tertiary structure, and the fluorescence is recovered because hDNA-AgNCs are far away from the surface of oxidized CNPs and the FRET efficiency decreases. Under the optimized conditions, the aptasensing probe can selectively assay VEGF165 with a detectionlimit of 14pM. The results provide a label-free and sensitive method to monitor VEGF165 in human serum. Schematic representation of the strong dependence of fluorescence enhancement on base amount in stem and loop of hairpin DNA-silver nanoclusters. The probe can be used to assay vascular endothelial growth factor 165 (VEGF165) and give a judgment whether human serum VEGF165 is at a normal or abnormal level for clinical diagnosis.

Specific and visual assay of iodide ion in human urine via redox pretreatment using ratiometric fluorescent test paper printed with dimer DNA silver nanoclusters and carbon dots

The fluorescence-based assay of iodide ion (I−) has been extensively studied by the use of different sensing probes and techniques, but it remains a tricky task to eliminate the interference of chloride ion (Cl−) for the analysis of low-level I− in complex genuine samples. Herein, we develop a redox pretreatment strategy for specific separating I− from human urine. Simultaneously, a novel ratiometric fluorescent probe is constructed by a simple mixing of dimer DNA silver nanoclusters (dDNA-AgNCs) and carbon dots (CDs) with the ratio of 5:1 in fluorescent intensity, and used for visual assay of I−. After addition of I−, the fluorescence of orange dDNA-AgNCs can be quenched by I− as the result of I−-induced oxidative etching and aggregation of dDNA-AgNCs, while blue CDs as the stable internal standard are unresponsive to I−. With the increase of I−, the fluorescence intensity ratio (I577/I446) of binary-color probe gradually decreased, which leads to color variation from salmon pink to lighter salmon pink to lilac to light steel blue to final deep sky blue (under a UV lamp) with a sensitive detection limit of 19.8 nM. The assay for I− can also be convenient to implement for visual monitoring of I− by observing color change of test paper printed with the ratiometric probe, responding to 50 nM that is about 1 order of magnitude lower than the median urinary I− concentration defined by the World Health Organization (WHO) for school-age children. The sensitive test paper can provide an advanced platform for colorimetric and visual monitoring of I− in human urine.

A versatile turn-on fluorometric biosensing profile based on split aptamers-involved assembly of nanocluster beacon sandwich

As an important member of the aptamer family, split aptamers are used to enhance the sensitivity of aptasensors by forming a sandwich structure with target molecules. Here, we explored whether it is feasible to construct a fluorescent tunable biosensor by tethering a nanocluster beacon (NCB) pair, which is composed of DNA-silver nanoclusters (DNA-AgNCs) and a G-rich enhancer sequence (GRS), at each end of split aptamers. The addition of the target generates the formation of nanocluster beacon sandwich (NCBsandwich), resulting in fluorescence enhancement because of the proximity of DNA-AgNCs and GRS. This strategy is applied to build versatile aptasensors for the sensitive analysis of small molecules including adenosine, cocaine, and 17β-estradiol with detection limits of 40 pM, 35 pM, and 15 pM respectively. We have also shown that the NCB pair can be properly lengthened to bypass steric hindrance, allowing this strategy to be used to probe macromolecules such as thrombin. Evidence has also revealed that the high sensitivity in this system relies on the advanced properties of the NCB. The analysis of targets in human serum was also accomplished to illustrate the practical specificity and reliability of this method. This is the first attempt to incorporate NCBs into the design of a split aptamer-based biosensor.

Micro RNA Sensing with Green Emitting Silver Nanoclusters.

Micro RNA (miR) are regulatory non-coding RNA molecules, which contain a small number of nucleotides ~18–28 nt. There are many various miR sequences found in plants and animals that perform important functions in developmental, metabolic, and disease processes. miRs can bind to complementary sequences within mRNA molecules thus silencing mRNA. Other functions include cardiovascular and neural development, stem cell differentiation, apoptosis, and tumors. In tumors, some miRs can function as oncogenes, others as tumor suppressors. Levels of certain miR molecules reflect cellular events, both normal and pathological. Therefore, miR molecules can be used as biomarkers for disease diagnosis and prognosis. One of these promising molecules is miR-21, which can serve as a biomarker with high potential for early diagnosis of various types of cancer. Here, we present a novel design of miR detection and demonstrate its efficacy on miR-21. The design employs emissive properties of DNA-silver nanoclusters (DNA/AgNC). The detection probe is designed as a hairpin DNA structure with one side of the stem complimentary to miR molecule. The binding of target miR-21 opens the hairpin structure, dramatically modulating emissive properties of AgNC hosted by the C12 loop of the hairpin. “Red” fluorescence of the DNA/AgNC probe is diminished in the presence of the target miR. At the same time, “green” fluorescence is activated and its intensity increases several-fold. The increase in intensity of “green” fluorescence is strong enough to detect the presence of miR-21. The intensity change follows the concentration dependence of the target miR present in a sample, which provides the basis of developing a new, simple probe for miR detection. The detection strategy is specific, as demonstrated using the response of the DNA/AgNC probe towards the scrambled miR-21 sequence and miR-25 molecule. Additionally, the design reported here is very sensitive with an estimated detection limit at ~1 picomole of miR-21.

Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters.

A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectly-matched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample. Graphical abstractSchematic representation of microRNA detection by integrating hairpin assisted cascade isothermal amplification with light-up DNA Ag nanoclusters. With microRNA, G-abundant overhang DNA sequences from amplification reaction hybridize with dark green Ag nanoclusters to produce a concentration-dependent bright red fluorescence.