Aptamer functionalized and reduced graphene oxide hybridized porous polymers SPE coupled with LC-MS for adsorption and detection of human α-thrombin.

In this study, reduced graphene oxide (rGO) hybridized high internal phase emulsions were developed and polymerized as porous carriers for aptamer (5'/5AmMC6/-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3') modification to enrich human α-thrombin from serum. The structure and properties of the materials were confirmed by scanning electron microscope (SEM), Fourier transform infrared spectroscope (FT-IR), and X-ray photoelectron spectra (XPS). The adsorption ability and selectivity were studied and the thrombin was detected with liquid chromatography-mass spectrometry (LC-MS). The adsorption of thrombin onto the sorbent was achieved within 30 min and the desorption was realized using 5.0 mL of acetonitrile/water (80/20, v/v). The thrombin was quantified by LC-MS according to its characteristic peptide sequence of ELLESYIDGR.

A versatile biomolecular detection platform based on photo-induced enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) as one of the effective tools for sensitive and selective detection of biomolecules has attracted tremendous attention. Here, we construct a versatile biomolecular detection platform based on photo-induced enhanced Raman spectroscopy (PIERS) effect for ultrasensitive detection of multiple analytes. In our PIERS sensor, we exploit the molecular recognition capacity of aptamers and the high affinity of aptamers with analyte to trigger TiO2@AgNP substrates binding with Raman tag-labeled gold nanoparticles probes via analyte, thus forming sandwich complexes. Additionally, combining plasmonic nanoparticles with photo-activated substrates allows PIERS sensor to achieve increased sensitivity beyond the normal SERS effect upon ultraviolet irradiation. Accordingly, the PIERS can be implemented for analysis of multiple analytes by designing different analyte aptamers, and we further demonstrate that the constructed PIERS sensor can serve as a versatile detection platform for sensitively analyzing various biomolecules including small molecules (adenosine triphosphate (ATP), limit of detection (LOD) of 0.1 nM), a biomarker (thrombin, LOD of 50 pM), and a drug (cocaine, LOD of 5 nM). Therefore, this versatile biomolecular detection platform based on PIERS effect for ultrasensitive detection of multiple analytes holds great promise to be a practical tool.

Electrochemical sandwich-type thrombin aptasensor based on dual signal amplification strategy of silver nanowires and hollow Au-CeO2.

Signal amplification is crucial in electrochemical biosensor to obtain low detection limits. In this work, a highly sensitive sandwich-type thrombin aptasensor is constructed, based on dual signal amplification of uniform silver nanowires (AgNWs) and hollow Au-CeO2 nanocomposites. AgNWs are decorated on the ITO surface to immobilize amino functionalized thrombin capture apemeter 1 (Apt1). And Au nanoparticles (AuNPs) grown directly on the surface of hollow CeO2 microstructure are used to immobilize sulfydryl functionalized thrombin reporter apemeter 2 (Apt2). Thus, sandwich-type apatasensor has been successfully designed, due to the specific recognition between thrombin and the two kinds of aptamers. One of the signal amplifications is from the good conductivity of uniform AgNWs. Moreover, uniform AgNWs together with hollow Au-CeO2 exhibit the good catalytic performance for the reduction of H2O2, further resulting in significant electrochemical signal amplification. Because the electrochemical signal amplification is closely related to the thrombin concentration, differential pulse voltammetry is used to specifically detect thrombin. Under the optimized conditions, the proposed method has a good linear response ranged from 0.5 pM to 30 nM with a low detection limit of 0.25 pM (S/N = 3) for thrombin. The proposed thrombin aptasensor displays good selectivity, reproducibility and stability, providing a good platform for the ultrasensitive detection of thrombin.

Purification and characterization of a thrombin-like enzyme isolated from Vipera lebetina venom: its interaction with platelet receptor.

: Snake venoms contain various molecules that can be used as tools in the diagnosis and in the treatment of hemostatic disorders. This study reports the isolation and functional characterization of a new thrombin-like enzyme and its role in the modulation of platelet aggregation and coagulation. The molecule was purified by gel filtration, anion exchange chromatography and reverse-phase-HPLC on C8 column; its molecular weight was determined. Natural and synthetic substrates were used to evaluate its enzymatic activities. The fibrinogenolytic activity was tested electrophoretically and by reverse-phase-HPLC on C18 column. Otherwise, the effect on blood coagulation and deficient plasma factors were also evaluated. The mechanism by which a thrombin-like enzyme VLCV (thrombin-like enzyme)-induced platelet aggregation was explored in presence of ticlopidin, clopidogrel and aspirin. VLCV (45 kDa) isolated from Vipera lebetina as a thrombin-like enzyme seems to be able to modulate platelet function. This enzyme showed an amidolytic activity by hydrolyzing the chromogenic-specific substrate of thrombin and the α-chain of fibrinogen. It is also able to clot human plasma and the deficient human plasma in factor X, suggesting that it is involved in the intrinsic and common pathways. The aggregating effect of VLCV is more sensitive to ticlopidine than to the clopidogrel suggesting the involvement of ADP/P2Y12/PI3K pathway. VLCV seems to be able to promote human platelet aggregation suggesting an interaction between P2Y12 and PAR1. Due to its ability to replace the missing factor X and its proaggregating activity, VLCV could be used as molecular tool to better understand the hemostasis mechanism.

A coumarin-appended cyclometalated iridium(III) complex for visible light driven photoelectrochemical bioanalysis.

In this study, a coumarin-appended cyclometalated iridium (III) complex was prepared and demonstrated to be an efficient photoelectrochemical (PEC) active species with stable and reproducible cathodic photocurrent illuminated by visible light. A gold nanoparticles (AuNPs)-based PEC probe was assembled using the as-prepared iridium (III) complex as signal reporter. Integrating aptamer/protein proximity binding-triggered strand displacement and catalytic hairpin assembly (CHA) amplification strategy, an enzyme-free and sensitive PEC assay was developed. Benefiting from superior photon-to-current conversion character of the iridium (III) complex and effective amplification strategy, the proposed assay exhibited enhanced sensitivity for thrombin detection with a detection limit of 23 fM. It also showed a high specificity in serum samples. This study further demonstrated that cyclometalated iridium (III) complexes could be adopted as favorable photoactive material for bioanalysis by improving their ability of absorbance in the visible region.

Thrombin Aptamer-Modified Metal-Organic Framework Nanoparticles: Functional Nanostructures for Sensing Thrombin and the Triggered Controlled Release of Anti-Blood Clotting Drugs.

This paper features the synthesis of thrombin-responsive, nucleic acid-gated, UiO-68 metal-organic framework nanoparticles (NMOFs) loaded with the drug Apixaban or rhodamine 6G as a drug model. Apixaban acts as an inhibitor of blood clots formation. The loads in the NMOFs are locked by duplex nucleic acids that are composed of anchor nucleic acids linked to the NMOFs that are hybridized with the anti-thrombin aptamer. In the presence of thrombin, the duplex gating units are separated through the formation of thrombin-aptamer complexes. The unlocking of the NMOFs releases the drug (or the drug model). The release of the drug is controlled by the concentration of thrombin. The Apixaban-loaded NMOFs revealed improved inhibition, as compared to free Apixaban, toward blood clot formation. This is reflected by their longer time intervals for inducing clot formation and the decreased doses of the drug required to affect clots formation. The beneficial effects of the Apixaban-loaded NMOFs are attributed to the slow-release mechanism induced by the NMOFs carriers, where the inhibition of factor Xa in the blood clotting cycle retards the formation of thrombin, which slows down the release of the drug.

Mixed monolayer decorated SPR sensing surface for thrombin detection.

The development of surface plasmon resonance (SPR) based immunosensor for thrombin detection was aimed. For this purpose, 3,3' Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DSP):6-mercapto-1-hexanol (MCH) mixed self-assembled monolayers (mSAMs) were formed on gold surfaces for immobilization of anti-thrombin antibody. The performance of the immunosensor was determined against the target protein thrombin at various concentrations using flow cell coupled SPR. The linear detection range of the immunosensor was 30.0-100.0 nM with an R2 value of 0,992. Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined to be 6.0 nM and 30.0 nM, respectively. The selectivity of the immunosensor was tested against a non-target model protein, human serum albumin (HSA) and the obtained ΔRU value was found to be below the ΔRU value corresponding to the LOQ concentration for thrombin. The immunosensor's capability to detect thrombin in diluted complex serum matrix was also tested and the obtained ΔRU value (159 ±â€¯16) was compared with ΔRU value obtained for thrombin detection in PBS solution (137 ±â€¯19). Based on the results, it was shown that DSP:MCH interface is a promising immobilization platform for binding biological recognition elements for the development of biosensors.

Dependent signal quenching and enhancing triggered by bipedal DNA walker for ultrasensitive photoelectrochemical biosensor.

Herein, by utilizing bipedal DNA walker as booster to adjust the distance of quencher ferrocene (Fc) and sensitizer methylene blue (MB) to photoactive material perylene-3,4,9,10-tetracarboxylic acid (PTCA), a novel "on-off-super on" photoelectrochemical (PEC) biosensor was proposed for ultrasensitive detection of thrombin (TB). Firstly, the PTCA matrix on electrode could provide a high initial PEC signal assisted by depAu. Upon the Fc labeled on hairpin DNA 1 (H1-Fc) proximate to PTCA, the PEC signal could obviously decrease to reduce the background signal. Interestingly, the target TB-related bipedal DNA walker implemented the opening of H1-Fc for the departure of Fc toward PTCA, which achieved the recovery of PEC signal and exposed the prelocked toehold domain for the hybridization with hairpin DNA 2 labeled with MB (H2-MB), thereby making the MB approach to PTCA for achieving the "super on" signal. As a result, this proposed strategy showed a wide linear range from 0.5 fM to 100 nM with a low detection limit down to 0.17 fM for TB detection, providing an efficient and available avenue for sensitive detection of biomolecules in bioanalysis and disease diagnosis.

An aptamer-based hook-effect-recognizable three-line lateral flow biosensor for rapid detection of thrombin.

In this paper, a three-line LFB was successfully developed by adding a thrombin line to a conventional two-line LFB for the detection of thrombin in a wide range of human serum. We introduced a thrombin line between the test line and the control line. The concentration of thrombin in the sample was quantitatively related to the signal formation on the three lines of the LFB. We can make use of signal on three lines to quantitative determinate the thrombin by data processing. The detection range of thrombin concentrations measured in 10 min was 1 nM to 100 µM and the LOD was 0.85 nM. Our approach paves way for rapid and sensitive thrombin detection and a superior device for testing in a wide range of physiological concentrations, which also can be used in other hook-effect-limited aptamers or antibodies based sandwich LFBs, and has a high accuracy even within the range of the hook-effect.

Microfluidic paper-based photoelectrochemical sensing platform with electron-transfer tunneling distance regulation strategy for thrombin detection.

This work reports a microfluidic paper-based photoelectrochemical (µ-PEC) sensing platform for thrombin (TB) detection with electron-transfer tunneling distance regulation (ETTDR) and aptamer target-triggering nicking enzyme signaling amplification (NESA) dual strategies. Specifically, paper-based TiO2 nanosheets (PTNs) were prepared with an efficient hydrothermal process, serving as the direct pathway for the charge carriers transfer. When CeO2-labeled hairpin DNA 3 (HP3) was closely located at the PTNs, the CeO2-PTNs heterostructure was formed, which could great facilitate the photogenerated carries separation of CeO2. In addition, with the aid of aptamer target-triggering NESA strategy, the input TB could be transducted to numerous output target of DNA (tDNA), achieving the goal of desirable signal amplification. In the presence of TB, the output tDNA could be further hybridized with HP3 and unfold its hairpin loop, which forced the CeO2 away from the surface of PTNs and vanished the CeO2-PTNs heterostructure, resulting in the obviously reducing of photocurrent signal. The as-designed sensing platform exhibited a linear range from 0.02 pM to 100 pM with a detection limit of 6.7 fM. Importantly, this µ-PEC sensing platform could not only realize the highly efficient TB detection, but also pave a luciferous way for the detection of other protein in bioanalysis.

Label-free and reagentless capacitive aptasensor for thrombin.

This investigation develops a label-free and reagentless aptasensor, based on a capacitive transducer with simple face-to-face electrode pairs. The electrode pairs of the transducer are composed of a gold electrode and an indium tin oxide film with micrometer separation with a double-side polyethylene terephthalate tape. Aptamers and 1-dodecanethiol are modified to form a self-assembled monolayer (SAM) on the gold electrode surfaces, and function as bio-recognition elements and preventers of non-specific protein binding, respectively. Electrochemical characterization results indicate that the SAM also forms an effective insulating layer, which is sufficient for capacitive sensing. The feasibility of the capacitive biosensor is validated using thrombin as a model analyte. The ultra-small value changes of capacitance originating from thrombin binding with the aptamers modified on the biosensor were measured with a home-made capacitance measuring circuit based on switched capacitor (SC) technology. The developed biosensor has detection limits of 1 pM and 10 pM of thrombin in phosphate buffered saline and mimic serum solution, respectively. The linear range for thrombin detection in human serum solution is from 10 pM to 1 µM, with a regression coefficient of 0.98. Additionally, the proposed aptasensor does not have significant levels of non-specific binding of bovine serum albumin and human serum albumin. Accordingly, the combination of SC and SAM bringing capacitive transduction at the forefront of ultrasensitive label-free and reagentless biosensing devices, particularly for point-of-care clinical analysis, which adopts small numbers of biological samples with low analyte concentrations.

Design of an aptamer-based magnetic adsorbent and biosensor systems for selective and sensitive separation and detection of thrombin.

An aptasensor was designed for sensitive detection of thrombin using in biological fluids by integrating a magnetic aptamer-microbeads. To achieve this goal, the surface of gold plated QCM crystals was coated with L-cysteine and a thrombin binding DNA aptamer was immobilized on the L-cysteine coated QCM crystals surface via glutaraldehyde coupling. The binding interactions of thrombin to QCM crystals were characterized. Magnetic poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate-vinylene carbonate), Mp(HEMA-EGDMA-VC) microbeads were synthesized and thrombin binding aptamer (TBA) was immobilized. The Mp(HEMA-EGDMA-VC)-TBA microbeads were effectively adsorbed thrombin from serum in a relatively short contact time (ca. 5.0 min), and the eluted protein from Mp(HEMA-EGDMA-VC)-TBA was transferred to the QCM aptasensor that showed a specific detection of thrombin from serum. The detection limit of thrombin using aptasensor was 1.00 nmol L-1. The calculation dissociation constant of the aptasensor was 68.5 nmol L-1. The selectivity of the aptasensor system was tested with three different proteins (i.e., elastin, immunoglobulin G (IgG) and human serum albumin (HSA)) and showed high specificity to thrombin. The aptasensor was regenerated by washing with NaOH solution, and repeatedly used until 20 cycles without a change in the performance.

Ce(III, IV)-MOF electrocatalyst as signal-amplifying tag for sensitive electrochemical aptasensing.

Metal-organic frameworks (MOFs) as a new class of porous materials have attracted increasing attention in the field of biomimetic catalysis. This study firstly reports a mixed valence state Ce-MOF possessing intrinsic catalytic activity towards thionine (Thi), and its application in constructing an amplified electrochemical aptasensor for thrombin detection. As noticed, the novel catalytic process combines the advantages of 3D infinite extension of the Ce(III, IV)-MOF skeleton containing large amounts of catalytic sites and spontaneous recycling of the Ce(III)/Ce(IV) for electrochemical reduction of Thi, thereby presenting amplified electrochemical signals. To further improve the aptasensor performance, the high selectivity of proximity binding-induced DNA strand displacement and high efficiency of exonuclease III-assisted recycling amplification were incorporated into the assay. The aptasensor was employed to detect thrombin in complex serum samples, which shows high sensitivity, specificity, stability and reproducibility. This work offers an opportunity to develop MOF-based electrocatalyst as signal-amplifying tag for versatile bioassays and catalytic applications.

QCM-D surpassing clinical standard for the dose administration of new oral anticoagulant in the patient of coagulation disorders.

The study focuses the dose administration of dabigatran to avoid the deaths due to hemorrhagic complications and thromboembolic stroke in clinics worldwide. To target the issue, a novel emerging acoustic technology, namely ''Quartz Crystal Microbalance with Dissipation'' (QCM-D) has been applied, while the acoustic assays namely ''activated Partial Thromboplastin Time'' (aPTT) and ''Prothrombinase complex-induced Clotting Test'' (PiCT) have been compared with the standard methods in parallel. Both techniques have been applied to 300 samples, including 220 plasma samples of patients suffering coagulation disorders and 80 plasma samples of non-patients. In comparison, the coagulation times of the acoustic aPTT and PiCT yielded an excellent correlation with the standard methods with in analytical standard deviation limits. Finally, the acoustic aPTT assay is the ''gold standard'' for a dose administration of the new oral anticoagulant, where the Δf/ΔΓ ratio of the acoustic assay demonstrates that dabigatran with FEIBA 50 combination could be a safe remedy to avoid the deaths in clinics.

Electrochemical aptasensor for thrombin using co-catalysis of hemin/G-quadruplex DNAzyme and octahedral Cu2O-Au nanocomposites for signal amplification.

In this work, novel octahedral Cu2O-Au nanocomposites were synthesized and first applied in an electrochemical aptasensor to detect thrombin (TB) with the aid of a DNAzyme for signal amplification. The octahedral Cu2O-Au nanocomposites have not only simultaneously served as signal amplifying molecules but have also been utilized as an ideal loading platform to immobilize a large number of electroactive substances and recognition probes. Gold nanoparticles (AuNPs) were grown directly on the surface of the octahedral Cu2O nanocrystals, and the Cu2O-Au nanocomposites obtained had the advantages of large surface areas and excellent biocompatibilities. The hemin/G-quadruplex, which was formed by intercalating hemin into the amino terminated thrombin binding aptamer (NH2-TBA), and the electroactive toluidine blue (Tb) were immobilized onto the Cu2O-Au nanocomposite surfaces through a stable Au-N bond. AuNPs, Cu2O and hemin/G-quadruplex co-catalyse the H2O2 in the working buffer to promote the electron transfer of Tb as a multiple signal amplification strategy in order to improve the performance of the electrochemical aptasensor. Under optimal conditions, the designed aptasensor exhibited sensitive detection of TB from 100 fM to 20nM with a lower detection limit of 23fM. This proposed aptasensor exhibited good sensitivity, high specificity and acceptable reproducibility and could be widely applied in bioassay analysis.

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

A novel silver nanocluster in situ synthesized as versatile probe for electrochemiluminescence and electrochemical detection of thrombin by multiple signal amplification strategy.

In this work, a novel silver nanoclusters (AgNCs) were in situ synthesized and used as versatile electrochemiluminescence (ECL) and electrochemical (EL) signal probes for thrombin detection by using DNAzyme-assisted target recycling and hybridization chain reaction (HCR) multiple amplification strategy. The presence of target thrombin firstly opened the hairpin DNA, followed by DNAzyme-catalytic recycling cleavage of excess substrates, which could generate large number of substrate fragments (s1). Then these s1 fragments were captured by SH-DNA on the Au nanoparticle-modified electrode, which further triggered the subsequent HCR of the hairpin DNA probes (H1 and H2) to form the long dsDNA. The numerous AgNCs were thus in situ synthesized by incubation the dsDNA template (with cytosine-rich loop)-modified electrode in solution with AgNO3 and sodium borohydride. By integrating the DNAzyme recycling and HCR dual amplification strategy, the amount of AgNCs is dramatically enhanced, leading to substantially amplified ECL and electrochemical signals for sensitive thrombin detection. Importantly, this design introduces the novel AgNCs into versatile ECL and EL bioassays by multiple amplification strategy, thus it is promising to provide a highly sensitive platform for various target biomolecules.

Ultrasensitive electrochemical aptasensor for the detection of thrombin based on dual signal amplification strategy of Au@GS and DNA-CoPd NPs conjugates.

In this work, an ultrasensitive electrochemical aptasensor for the detection of thrombin was developed based on Au nanoparticles decorated graphene sheet (Au@GS) and CoPd binary nanoparticles (CoPd NPs). A sulfydryl-labeled thrombin capture probe (Apt1) and a biotin-labeled thrombin reporter probe (Apt2) were designed to achieve a sandwich-type strategy. Au@GS was used as a sensing platform for the facile immobilization of Apt1 through Au-S bond, forming a sensing interface for thrombin. The specific recognition of thrombin induced the attachment of Apt2-CoPd NPs to the electrode. The labeled CoPd NPs showed good catalytic properties toward the reduction of H2O2, resulting in an amperometric signal. The amperometric response was correlated to the thrombin concentration in sample solutions. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) confirmed the successful fabrication of the aptasensor. A linear response to thrombin in the range of 0.01-2.00 ng mL(-1) with a low detection limit (5 pg mL(-1)) was achieved. The proposed aptasensor showed good selectivity, good reproducibility and acceptable stability. This proposed strategy may find many potential applications in the detection of other biomolecules.

Label-free cascade amplification strategy for sensitive visual detection of thrombin based on target-triggered hybridization chain reaction-mediated in situ generation of DNAzymes and Pt nanochains.

A new magnetic bead-based cascade amplification strategy for highly sensitive visual detection of proteins (thrombin as a model analyte) was developed by coupling target-triggered hybridization chain reaction (HCR) with the synergistic catalysis of DNA concatemer-mediated hemin/G-quadruplex DNAzymes and Pt nanozymes. Initially, the biotinylated primer DNA (P-DNA) was complementary with aptamer to form dsDNA which was further linked to streptavidin-coated magnetic bead (MB), thereby fabricating the expected MB-based aptasensor. In the presence of target TB, the aptamer was taken away from the aptasensor, and the free P-DNA immediately triggered HCR to spontaneously form DNA concatemer-directed nanochains with numerous DNAzymes and Pt nanoclusters (PtNCs) to achieve cascades signal amplification. The dual peroxidase mimetics catalyzed the H2O2-mediated oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) into the colored TMB oxides (oxTMB), causing intensified color change of the chromogenic solution for the highly sensitive naked-eye detection of as low as 100.0 pM TB. In this strategy, the employment of magnetic separation and exonuclease III (Exo III)-assisted digestion of residual dsDNA minimized the background noise and avoided the false positive results, greatly improving the detection accuracy and sensitivity with a low limit of detection (LOD=15.0 pM). The proposed visual platform has promise for detecting various types of proteins with careful DNA sequence designs.

Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system.

We report here an ultrasensitive strategy based on the recognition-induced conformational alteration of aptamer and fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequence in proximity to silver nanoclusters for adenosine triphosphate (ATP), adenosine (A) and thrombin (TB) detection. Herein, we designed two tailored DNA sequences noted as complementary DNA (abbreviated as c-DNA) and signal probe DNA (abbreviated as s-DNA), respectively. c-DNA is designed as a special structure consisting of a sequence complementary to aptamer at the 3'-end and a guanine-rich DNA sequence at the 5'-end; s-DNA contains a cytosine-rich sequence responsible for Ag NCs templated synthesis at the 3'-end and a link sequence (part of aptamer) complementary to partial of the c-DNA at the 5'-end. In the presence of target, the aptamer associated with the target, resulting in the formation of duplex DNA (dsDNA), the DNA-Ag NCs thereafter could close to the guanine-rich sequence, leading to enhanced fluorescence signal readout. The widespread application of the sensing system is achieved success in the detection of three biomolecules. ATP, adenosine and thrombin in the range of 0.5-8.0 µM, 0.5-7.0 µM and 50-900 nM could be linearly detected with the detection limits of 91.6 nM, 103.4 nM and 8.4 nM, respectively. This label-free and turn-on fluorescent sensing system employing the mechanism proposed here turns out to be sensitive, selective, and convenient for the detection of biomolecules without washing and separation steps.