RESUMO
To address the limitations of typical hairpin-structural molecular beacons, exploring the ability of a quasi-molecular beacon (qMB) to create label-free fluorescence biosensors is intriguing and remains a challenge. Herein, we propose the first example of modular qMB with the feature of a stimulation-responsive conformation switch to develop an aggregated Ag nanocluster (aAgNC) in a bifurcated DNA scaffold for fluorescently sensing a specific initiator (I*). This qMB was well designed to program four functional modules: I*-recognizable element adopting metastable stem-loop bihairpin structure and two DNA splits (exposed C3GT4 and locked C4AC4T) of aAgNC template that is separated by a tunable hairpin spacer for the customized combination of selective recognition and signaling readout. When presenting I* in an assay route, the specific hybridization induces the directional disassembly of the bihairpin unit, on which the qMB is configurationally switched to liberate the locked split. Thus, the bifurcated parent template pair of C3GT4/C4AC4T is proximal, affording in situ nucleation and clustering of emissive aAgNC. By collecting the fluorescence signal, the quantitative detection of I* is achieved. Benefiting from the ingenious programming of qMB, the recognizing and signaling integration actuates the construction of a facile and convenient fluorescent biosensor featuring rapid reaction kinetics, a wide linear range, high sensitivity, and specificity. This would provide a new paradigm to exploit versatile qMB-based biosensing platforms via stimulation-responsive conformation switches for developing various DNA-scaffolded Ag clusters.
Assuntos
Técnicas Biossensoriais , DNA , DNA/química , Hibridização de Ácido Nucleico , Corantes , Conformação MolecularRESUMO
Exploring the ability of four-stranded DNA nanorings (fsDNRs) to host multiple nanosilver clusters (NAgCs) for cooperatively amplifiable fluorescence biosensing to a specific initiator (tI*) is fascinating. By designing three DNA single strands and three analogous stem-loop hairpins, we developed a functional fsDNR through sequential cross-opening and overlapped hybridization. Note that a substrate strand (SS) was programmed with six modules: two severed splits (sT and sT') of NAgCs template, two sequestered segments by a middle unpaired spacer, and a partition for tI*-recognizable displacement, while sT and sT' were also tethered in two ends of three hairpins. At first, a triple dsDNA complex with stimulus-responsiveness was formed to guide the specific binding to tI*, while the exposed toehold of the SS activated the forward cascade hybridization of three hairpins, until the ring closure in the tailored self-assembly pathway for forming the fsDNR. The resulting four duplexes forced each pair of sT/sT' to be merged as the parent template in four nicks, guiding the preferential synthesis of four clusters in the shared fsDNR, thereby cooperatively amplifying the green fluorescence signal for sensitive assay of tI*. Meanwhile, the topological conformation of fsDNR can be stabilized by the as-formed cluster adducts to rivet the pair of two splits in the nicks. Benefitting from the self-enhanced effect of multiple emitters, this label-free fluorescent sensing strategy features simplicity, rapidity, and high on-off contrast, without involving complicated nucleic acid amplifiers.
Assuntos
Técnicas Biossensoriais , DNA , Técnicas Biossensoriais/métodos , DNA/química , Prata/química , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Fluorescência , Espectrometria de Fluorescência , Nanotubos/químicaRESUMO
Long-term continuous imaging of endogenous HClO burst is of great importance for the elucidation of various physiological or pathological processes. However, most of the currently reported HClO probes have failed to achieve this goal due to their insufficient photobleaching resistance under a laser source. Herein, a highly stable ratiometric probe, HFTC-HClO 1, which is capable of continuously monitoring endogenous HClO burst over a long period of time, has been judiciously developed. Briefly, the de novo development of HFTC-HClO 1 mainly involved three main steps: (1) novel coumarins (HFTC 1-5) were designed and synthesized; (2) the most stable scaffold, HFTC 3, was selected through dye screening and cell imaging validation; and (3) based on HFTC 3, three candidate HClO probes were constructed, and HFTC-HClO 1 was finally selected due to its superior sensing properties toward HClO. Furthermore, HFTC-HClO 1 can quantitatively measure HClO levels in various real samples with excellent recovery (>90.4%), and the use of HFTC-HClO 1-coated test strips for qualitative analysis of HClO in real samples was also achieved. In addition, the application of HFTC-HClO 1 for long-term continuous monitoring of intracellular HClO burst was successfully demonstrated. Significantly, HFTC-HClO 1 was able to visualize HClO generated in the rheumatoid arthritis mouse model.
Assuntos
Corantes Fluorescentes , Ácido Hipocloroso , Camundongos , Animais , Ácido Hipocloroso/análise , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , CumarínicosRESUMO
Exploring the cooperative amplification of peroxidase-like metal nanocomposites and cycled hairpin assembly is intriguing for sensitive bioanalysis. Herein, we report the first design of a unique electrochemical biosensor based on mimicking Au@FeCo nanozymes and bicycled hairpin assembly (BHA) for synergistic signal amplification. By loading the enzyme-like FeCo alloy in Au nanoparticles (AuNPs), the as-synthesized Au@FeCo hybrids display great improvement of electronic conductivity and active surface area and excellent mimic catalase activity to H2O2 decomposition into â¢OH radicals. The immobilization of Au@FeCo in an electrode sensing interface is stabilized via the resulting electrodeposition in HAuCl4 while efficiently accelerating the electron transfer of electroactive ferrocene (Fc). Upon the immobilization of a helping hairpin (HH) via Au-S bonds, a specific DNA trigger (T*) is introduced to activate BHA operation through competitive strand displacement reactions among recognizing hairpin (RH), signaling hairpin (SH), and HH. T* and RH are rationally released to catalyze two cycles, in which the transient depletion of dsDNA intermediates rapidly drives the progressive hairpin assemblies to output more products SH·HH. Thus, the efficient amplification of Au@FeCo mimic catalase activity combined with BHA leads to a significantly increased current signal of Fc dependent on miRNA-21 analogous to T*, thereby directing the creation of a highly sensitive electrochemical biosensor having applicable potential in actual samples.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Ouro/química , Técnicas Eletroquímicas/métodos , Peróxido de Hidrogênio , Catalase , Nanopartículas Metálicas/química , DNA/química , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
The reaction kinetics and yield of traditional DNA assembly with a low local concentration in homogeneous solution remain challenging. Exploring confined catalytic DNA assembly (CCDA) is intriguing to boost the reaction rate and efficacy for creating rapid and sensitive biosensing platforms. A rolling circle amplification (RCA) product containing multiple tandem repeats is a natural scaffold capable of guiding the periodic assembly of customized functional probes at precise sites. Here, we present a RCA-confined CCDA strategy to speed up amplifiable conversion for ratiometric fluorescent sensing of a sequence-specific inducer (I*) by using string green-/red-Ag clusters (sgAgCs and srAgCs) as two counterbalance emitters. Upon recognition of I*, CCDA events are operated by two toehold-mediated strand displacements and localized in repetitive units, thereby releasing I* for recycled signal amplification in the as-grown RCA concatemer. The local concentration of reactive species is increased to facilitate rapider dsDNA complex assembly and more efficient input-output conversion, on which the clustering template sequences of sgAgCs and srAgCs are blocked and opened, enabling srAgCs synthesis but opposite to sgAgCs. Thus, the fluorescence emission of srAgCs goes up, while sgAgCs go down. With the resultant ratio featuring inherent built-in correction, rapid, sensitive, and accurate quantification of I* at the picomolar level is achieved. Benefiting from efficient RCA confinement to enhance reaction kinetics and conversion yield, this CCDA-based strategy provides a new paradigm for developing simple and diverse biosensing methodologies.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , DNA/genética , Espectrometria de Fluorescência/métodos , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
The DNA frame structure as a natural shell to stably shield the sequence-templated Ag nanocluster core (csAgNC) is intriguing yet challenging for applicable fluorescence biosensing, for which the elaborate programming of a cluster scaffold inside a DNA-based cage to guide csAgNC nucleation might be crucial. Herein, we report the first design of a symmetric tetrahedral DNA nanocage (TDC) that was self-assembled in a one-pot process using a C-rich csAgNC template strand and four single strands. Inside the as-constructed soft TDC architecture, the template sequence was logically bridged from one side to another, not in the same face, thereby guiding the in situ synthesis of emissive csAgNC. Because of the strong electron-repulsive capability of the negatively charged TDC, the as-formed csAgNC displayed significantly improved fluorescence stability and superb spectral behavior. By incorporating the recognizable modules of targeted microRNAs (miRNAs) in one vertex of the TDC, an updated TDC (uTDC) biosensing platform was established via the photoinduced electron transfer effect between the emissive csAgNC reporter and hemin/G-quadruplex (hG4) conjugate. Because of the target-interrupted csAgNC switching in three states with the spatial proximity and separation to hG4, an "on-off-on" fluorescing signal response was executed, thus achieving a wide linear range to miRNAs and a limit of detection down to picomoles. Without complicated chemical modifications, this simpler and more cost-effective strategy offered accurate cell imaging of miRNAs, further suggesting possible therapeutic applications.
RESUMO
Engineering of multivariate biosensing and imaging platforms involved in disease plays a vital role in effectively discerning cancer cells from normal cells and facilitating reliable targeted therapy. Multiple biomarkers such as mucin 1 (MUC1) and nucleolin are typically overexpressed in breast cancer cells compared to normal human breast epithelium cells. Motivated by this knowledge, a dual-responsive DNA tetrahedron nanomachine (drDT-NM) is constructed through immobilizing two recognition modules, MUC1 aptamer (MA) and a hairpin H1* encoding nucleolin-specific G-rich AS1411 aptamer, in two separate vertexes of a functional DT architecture tethering two localized pendants (PM and PN). When drDT-NM identifiably binds bivariate MUC1 and nucleolin, two independent hybridization chain reactions (HCRM and HCRN) as amplification modules are initiated with two sets of four functional hairpin reactants. Among them, one hairpin for HCRM is dually ended by fluorescein and quencher BHQ1 to sense MUC1. The responsiveness of nucleolin is executed by operating HCRN utilizing another two hairpins programmed with two pairs of AS1411 splits. In the shared HCRN duplex products, the parent AS1411 aptamers are cooperatively merged and folded into G-quadruplex concatemers to embed Zn-protoporphyrin IX (ZnPPIX/G4) for fluorescence signaling readout, thereby achieving a highly sensitive intracellular assay and discernible cell imaging. The tandem ZnPPIX/G4 unities also act as imaging agents and therapeutic cargos for efficient photodynamic therapy of cancer cells. Based on drDT-NM to guide bispecific HCR amplifiers for adaptive bivariate detection, we present a paradigm of exquisitely integrating modular DNA nanostructures with nonenzymatic nucleic acid amplification, thus creating a versatile biosensing platform as a promising candidate for accurate assay, discernible cell imaging, and targeted therapy.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Quadruplex G , Humanos , Hibridização de Ácido Nucleico/métodos , DNA/genética , DNA/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodosRESUMO
Rhodamines have emerged as a useful class of dye for bioimaging. However, intrinsic issues such as short emission wavelengths and small Stokes shifts limit their widespread applications in living systems. By taking advantage of the homoadamantane-fused tetrahydroquinoxaline (HFT) moiety as an electron donor, we developed a new class of asymmetric NIR rhodamine dyes, NNR1-7. These new dyes retained ideal photophysical properties from the classical rhodamine scaffold and showed large Stokes shifts (>80 nm) with improved chemo/photostability. We found that NNR1-7 specifically target cellular mitochondria with superior photobleaching resistance and improved tolerance for cell fixation compared to commercial mitochondria trackers. Based on NNR4, a novel NIR pH sensor (NNR4M) was also constructed and successfully applied for real-time monitoring of variations in lysosomal pH. We envision this design strategy would find broad applications in the development of highly stable NIR dyes with a large Stokes shift.
Assuntos
Elétrons , Corantes Fluorescentes , Rodaminas/química , Corantes Fluorescentes/química , LisossomosRESUMO
Ratiometric assays of label-free dual-signaling reporters with enzyme-free amplification are intriguing yet challenging. Herein, yellow- and red-silver nanocluster (yH-AgNC and rH-AgNC) acting as bicolor ratiometric emitters are guided to site-specifically cluster in two template signaling hairpins (yH and rH), respectively, and originally, both of them are almost non-fluorescent. The predesigned complement tethered in yH is recognizable to a DNA trigger (TOC) related to SARS-CoV-2. With the help of an enhancer strand (G15E) tethering G-rich bases (G15) and a linker strand (LS), a switchable DNA construct is assembled via their complementary hybridizing with yH and rH, in which the harbored yH-AgNC close to G15 is lighted-up. Upon introducing TOC, its affinity ligating with yH is further implemented to unfold rH and induce the DNA construct switching into closed conformation, causing TOC-repeatable recycling amplification through competitive strand displacement. Consequently, the harbored rH-AgNC is also placed adjacent to G15 for turning on its red fluorescence, while the yH-AgNC is retainable. As demonstrated, the intensity ratio dependent on varying TOC is reliable with high sensitivity down to 0.27 pM. By lighting-up dual-cluster emitters using one G15 enhancer, it would be promising to exploit a simpler ratiometric biosensing format for bioassays or clinical theranostics.
Assuntos
Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , COVID-19/diagnóstico , DNA , Fluorescência , Humanos , SARS-CoV-2 , Prata , Espectrometria de FluorescênciaRESUMO
Proximity-localized catalytic hairpin assembly (plCHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (T*). Using template-integrated green Ag nanoclusters (igAgNCs) as emitters, herein, we report the first design of a T*-activated plCHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of T*. To this end, the T*-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of igAgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input T*, the 3WJA-localized plCHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and T* is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting igAgNCs, outputting T*-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the plCHA operation, achieving rapider reaction kinetics via T*-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various plCHA-based circuits capable of modulating the fluorescence emission of igAgNCs for applicable biosensing and bioanalysis.
Assuntos
Técnicas Biossensoriais , DNA/química , Hibridização de Ácido Nucleico , Catálise , Espectrometria de Fluorescência , Limite de DetecçãoRESUMO
Exploring the replication of hybridization chain reaction HCR (rHCR) for reciprocal amplification is intriguing in biosensing and bioanalysis. Herein, we develop a rHCR-based fluorescence platform that is manipulated by the combination of a specific DNA trigger (T) and a T-analogous amplicon (T*), thereby concatenating multi green-emissive Ag nanoclusters (mgAgNCs) for amplifiable signal readout. Four well-designed hairpins (H1 recognizing T, H2, H3, and H4) with sequential complements are executed to operate rHCR. The termini of H1/H3 are merged to hybridize an inhibiting strand (I). The parent scaffold for mgAgNCs is separated into two splits (C4AC4T and C3GT4) that are individually overhung in H2/H4. The presence of T activates the first HCR amplifier through cross-hybridization of four reactive hairpins for forming HCR duplexes. The next invasion of a complex (T*·I) drives I to hybridize the tandem repeats in H1/H3, so that the displaced T* functions as T to catalyze the second amplifier rHCR for feeding back more hairpin assemblies with rapid reaction kinetics. In the shared rHCR polymers, the parent scaffolds (C4AC4TC3GT4) in H2/H4 are collectively concatenated for the preferential clustering of mgAgNCs adducts, which cooperatively emit enormous T-responsive fluorescence signal. Because of the localization of T in HCR products, an alternative amplicon T* is introduced to drive rHCR progress via DNA strand displacement, generating more nucleating sites of emitters. Thus, the rational combination of nonenzymatic rHCR and label-free fluorescent concatemers would create a reciprocal signal amplification, achieving a simplified, rapid, and highly sensitive assay down to femtomolar concentrations.
Assuntos
Técnicas Biossensoriais , Hibridização de Ácido Nucleico , DNA/genética , DNA/análise , Espectrometria de Fluorescência , Limite de DetecçãoRESUMO
It is intriguing to modulate the fluorescence emission of DNA-scaffolded silver nanoclusters (AgNCs) via confined strand displacement and transient concatenate ligation for amplifiable biosensing of a DNA segment related to SARS-CoV-2 (s2DNA). Herein, three stem-loop structural hairpins for signaling, recognizing, and assisting are designed to assemble a variant three-way DNA device (3WDD) with the aid of two linkers, in which orange-emitting AgNC (oAgNC) is stably clustered and populated in the closed loop of a hairpin reporter. The presence of s2DNA initiates the toehold-mediated strand displacement that is confined in this 3WDD for repeatable recycling amplification, outputting numerous hybrid DNA-duplex conformers that are implemented for a transient "head-tail-head" tandem ligation one by one. As a result, the oAgNC-hosted hairpin loops are quickly opened in loose coil motifs, bringing a significant fluorescence decay of multiple clusters dependent on s2DNA. Demonstrations and understanding of the tunable spectral performance of a hairpin loop-wrapped AgNC via switching 3WDD conformation would be highly beneficial to open a new avenue for applicable biosensing, bioanalysis, or clinical diagnostics.
Assuntos
Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , DNA/química , DNA/genética , Humanos , Nanopartículas Metálicas/química , SARS-CoV-2 , Prata/química , Espectrometria de FluorescênciaRESUMO
Exploring the ratiometric fluorescence biosensing of DNA-templated biemissive silver nanoclusters (AgNCs) is significant in bioanalysis, yet the design of a stimuli-responsive DNA device is a challenge. Herein, using the anti-digoxin antibody (anti-Dig) with two identical binding sites as a model, a tweezer-like DNA architecture is assembled to populate fluorescent green- and red-AgNCs (g-AgNCs and r-AgNCs), aiming to produce a ratio signal via specific recognition of anti-Dig with two haptens (DigH). To this end, four DNA probes are programmed, including a reporter strand (RS) dually ended with a g-/r-AgNC template sequence, an enhancer strand (ES) tethering two same G-rich tails (G18), a capture strand (CS) labeled with DigH at two ends, and a help strand (HS). Initially, both g-AgNCs and r-AgNCs wrapped in the intact RS are nonfluorescent, whereas the base pairing between RS, ES, CS, and HS resulted in the construction of DNA mechanical tweezers with two symmetric arms hinged by a rigid "fulcrum", in which g-AgNCs are lighted up due to G18 proximity ("green-on"), and r-AgNCs away from G18 are still dark ("red-off"). When two DigHs in proximity recognize and bind anti-Dig, the conformation switch of these tweezers resultantly occurs, taking g-AgNCs away from G18 for "green-off" and bringing r-AgNCs close to G18 for "red-on". As such, the ratiometric fluorescence of r-AgNCs versus g-AgNCs is generated in response to anti-Dig, achieving reliable quantization with a limit of detection at the picomolar level. Based on the fast stimulated switch of unique DNA tweezers, our ratiometric strategy of dual-emitting AgNCs would provide a new avenue for a variety of bioassays.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Anticorpos , DNA , Fluorescência , Prata , Espectrometria de FluorescênciaRESUMO
Designing antibody-powered DNA nanodevice switches is crucial and fascinating to perform a variety of functions in response to specific antibodies as regulatory inputs, achieving highly sensitive detection by integration with simple amplified methods. In this work, we report a unique DNA-based conformational switch, powered by a targeted anti-digoxin mouse monoclonal antibody (anti-Dig) as a model, to rationally initiate the hybridization chain reaction (HCR) for enzyme-free signal amplification. As a proof-of-concept, both a fluorophore Cy3-labeled reporter hairpin (RH) in the 3' terminus and a single-stranded helper DNA (HS) were individually hybridized with a recognition single-stranded DNA (RS) modified with Dig hapten, while the unpaired loop of RH was hybridized with the exposed 3'-toehold of HS, isothermally self-assembling an intermediate metastable DNA structure. The introduction of target anti-Dig drove the concurrent conjugation with two tethered Dig haptens, powering the directional switch of this DNA structure into a stable conformation. In this case, the unlocked 3'-stem of RH was implemented to unfold the 5'-stem of the BHQ-2-labeled quench hairpin (QH), rationally initiating the HCR between them by the overlapping complementary hybridization. As a result, numerous pairs of Cy3 and BHQ-2 in the formed long double helix were located in spatial proximity. In response to this, the significant quenching of the fluorescence intensity of Cy3 by BHQ-2 was dependent on the variable concentration of anti-Dig, achieving a highly sensitive quantification down to the picomolar level based on a simplified protocol integrated with enzyme-free amplification.
Assuntos
Técnicas Biossensoriais , DNA , Animais , DNA/genética , DNA de Cadeia Simples/genética , Corantes Fluorescentes , Imunoensaio , Limite de Detecção , Camundongos , Hibridização de Ácido NucleicoRESUMO
Dark or weak-emissive DNA-harbored silver nanoclusters (AgNCs) can be remarkably lighted up when approaching to guanine bases. The resultant bright AgNCs acting as a fluorescent reporter are fascinating in biosensing. To explore the applicable guanine-enhanced emission of AgNCs for biosensing microRNA-155 (miR-155) as a model, here we designed a unique stem-loop hairpin beacon (HB) encoding with an miR-155-recognizable sequence and a AgNCs-nucleable template, as well as a hairpin helper tethering a partially locked guanine-rich (15-nt) tail (G15H), while two identical cytosine-rich segments were inserted in HB and G15H to merge for folding/unfolding of i-motif at changed pHs. Initially, the intact clusters populated in HB (HB/AgNCs) were almost nonfluorescent in a buffer (pH 7.0). Then, miR-155 was introduced to trigger a repeated hairpin assembly of HB and G15H by competitive strand displacement reactions at decreased pH 5.0 within 10 min, consequently generating numerous duplex DNA constructs (DDCs). With the resultant template of pH-responsive i-motifs incorporated in DDCs, their folding at pH 5.0 brought the proximity of unlocked G15 overhang to the clusters in a crowded environment, remarkably lighting up the red-emitting fluorescence of HB/AgNCs (λem = 628 ± 5 nm) for amplified signal readout. About 3.5-fold enhancement of quantum yield was achievable using different variants of i-motif length and G15 position. Simply by adding OH-, the configuration fluctuation of i-motifs was implemented for switchable fluorescence biosensing to variable miR-155. Based on a one-step amplification and signaling scheme, this subtle strategy was rapid, low-cost, and specific for miR-155 with high sensitivity down to 67 pM.
Assuntos
Técnicas Biossensoriais , DNA/química , Guanina/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Prata/química , Fluorescência , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7RESUMO
A programmed dual-functional DNA tweezer (DFDT) as a signaling molecule is reported for the simultaneous and recognizable fluorescence detection of microRNA 21 (miRNA 21) and mucin 1 (MUC1). This unique DFDT is assembled from two Au-NP-attached central strands (C1 and C2) and an arm strand (A) dually ended by fluorophores Cy3 and Cy5, which are spatially separated from Au NP in the originally opened state. Through the competitive affinity interaction between targets and their complementary and aptamer sequences tethered in two recognition strands (R1 and R2), miRNA 21 and MUC1 are respectively converted into two dependently displaced fuel strands (F1 and F2). The next hybridization with two pairs of unpaired segments overhung in open DFDT leads to its conformational closure, resulting in the approach of Cy3 and Cy5 to Au NP. On the basis of the nanometal surface energy transfer scheme, the fluorescence emission of Cy3 or Cy5 is cooperatively quenched by Au NPs attached in C1 and C2. The significant variation of fluorescence intensity enables one-step, cost-effective, and specific quantization of miRNA 21 and MUC1 with high sensitivity down to 32 fM and 2.6 fg·mL-1 (8.5 pM), respectively. The novel DFDT-based assay route of multiplex analytes is promising and has the potential for rapid and reliable diagnosis and treatment of cancer-related diseases.
Assuntos
Técnicas Biossensoriais/métodos , DNA/química , MicroRNAs/análise , Mucina-1/análise , Espectrometria de Fluorescência , Fatores de TempoRESUMO
An electrochemical biosensor is described for highly sensitive determination of tDNA, an Alzheimer's disease (AD)-related biomarker. Electroactive molybdophosphate anions were precipitated in-situ on a glassy carbon electrode (GCE) via catalytic hydrolysis by alkaline phosphatase (ALP). This is followed by recycling amplification of tDNA. Four DNA strands (referred to as S1, S2, S3 and S4) were designed to assemble X-shape DNA (X-DNA) building blocks. These were further extended into four directions under the action of DNA polymerase. The resultant two X-DNA motifs were polymerize. Simultaneously, ALP is encapsulated into a hydrogels network to obtain a porous material of type ALP@DNAhg. The GCE was modified with reduced graphene oxide functionalized with gold nanoparticles (Au@rGO). If ALP@DNAhg are captured via strand displacement, tDNA recycling assembly for signal amplification is initiated. This results in the immobilization of large amounts of ALP. On introduction of pyrophosphate and molybdate (MoO42-), ALP will catalyze the hydrolysis of pyrophosphate to produce phosphate. It will react with molybdate to form redox active phosphomolybdate anions (PMo12O403-). Its amperometrical signal depends on the concentration of tDNA in the 1.0 × 10-2 to 1.0 × 104 pM concentration range, and the detection limit is 3.4 × 10-3 pM. Graphical abstract Schematic presentation of (a) preparation of alkaline phosphatase-encapsulated DNA hydrogel (ALP@DNAhg). (b) fabrication of the biosensor for target DNA (tDNA) based on ALP@DNAhg to catalyze in situ precipitation of electroactive molybdophosphate anion (PMo12O403-) and tDNA recycling amplification, achieving tDNA-dependent electrochemical signal readout (X-DNA: X-shape DNA building block. TdT: terminal deoxynucleotidyl transferase. dATP: deoxyadenosine triphosphate. dTTP: deoxythymidine triphosphate. X-DNA-pAn and X-DNA-pTn: X-DNA motifs with poly-A and poly-T tails. ALP: alkaline phosphatase. ALP@DNAhg: ALP-encapsulated DNA hydrogels. Au@rGO: gold nanoparticles-functionalized reduced graphene oxide. GCE: glass carbon electrode. HP1, 2: hairpin DNA 1, 2. MCH: 6-mercaptohexanol. tDNA: target DNA. CV: cyclic voltammetry).
Assuntos
Doença de Alzheimer/diagnóstico , DNA/análise , Técnicas Eletroquímicas/métodos , Fosfatase Alcalina/metabolismo , Doença de Alzheimer/genética , Biomarcadores , Catálise , Eletrodos , Humanos , Hidrogéis , Molibdênio , Ácidos FosfóricosRESUMO
A cascade nucleic acid amplification strategy is presented for fluorometric aptamer based determination of the model protein carcinoembryonic antigen (CEA). Amplification is accomplished by combining catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). In this assay, a specially designed single-stranded DNA containing the aptamer sequence (AS) specific for CEA is hybridized with an inhibitor strand (IS) to form a double-stranded DNA (IS@AS). In the presence of CEA, it will recognize and bind to the AS strand which causes the release of IS. By introducing two DNA hairpins (H1 and H2; these containing complementary sequences) CHA will be activated via the hybridization reactions of H1 and H2. This is accompanying by the formation of a double-stranded DNA (H1-H2) and the release of CEA@AS. The liberated CEA@AS further drives successive recycling of the CHA, thereby generating further copies of H1-H2. The resultant H1-H2 hybrids act as primers and trigger HCR with the help of other two DNA hairpins (H3 and H4) containing G-rich toehold at the 5'-terminus and 3'-terminus of H3 and H4, respectively. The fluorescent probe N-methyl mesoporphyrin IX (NMM) is finally intercalated into the G-rich domains of the long DNA nanostructures due to formation of G-quadruplex structures. This generates a fluorescent signal (best measured at excitation/emission wavelengths of 399/610 nm) that increases with the concentration of target (CEA). This aptamer-based fluorescence assay is highly sensitive and has a linear range that covers the 1 pg·mL-1 to 2 ng·mL-1 CEA concentration range, with a 0.3 pg·mL-1 detection limit. Graphical abstract By integrating catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for effective signal enhancement, a novel cascade amplification strategy is presented to develop a sensitive and selective fluorescent method for the assay of the model protein carcinoembryonic antigen (CEA).
Assuntos
Aptâmeros de Nucleotídeos/genética , Antígeno Carcinoembrionário/análise , Corantes Fluorescentes/química , Fluorometria/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Fluorometria/normas , Quadruplex G , Hibridização de Ácido NucleicoRESUMO
Metal nanoclusters (NCs) as a new type of electrochemiluminescence (ECL) nanomaterials have attracted great attention, but their applications are limited due to relatively low luminescent efficiency and a complex preparation process. Herein, an ultrasensitive ECL biosensor for the detection of Cyclin-D1 (CCND1) was designed by utilizing in situ electrogenerated silver nanoclusters (AgNCs) as ECL emitters and Fe3O4-CeO2 nanocomposites as a coreaction accelerator. The ECL luminous efficiency of AgNCs on the electrode could be significantly enhanced with the use of the Fe3O4-CeO2 for accelerating the reduction of S2O82- to generate the strong oxidizing intermediate radical SO4â¢-. As a result, the assay for CCND1 detection achieved excellent sensitivity with a linear range from 50 fg/mL to 50 ng/mL and limit of detection down to 28 fg/mL. Impressively, the efficiency of Traditional Chinese Medicines (TCM), sophorae, toward MCF-7 cells was successfully investigated due to the overexpression of CCND1 in relation to the growth and metastasis of MCF-7 human breast cancer cells. In general, the proposed strategy provided an effective method for anticancer drug screening and expanded the application of metal NCs in ultrasensitive biodetection.
Assuntos
Ciclina D1/análise , Medições Luminescentes/métodos , Nanocompostos/química , Prata/química , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Técnicas Biossensoriais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cério/química , Ciclina D1/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Eletrodos , Galvanoplastia , Feminino , Óxido Ferroso-Férrico/química , Humanos , Limite de Detecção , Células MCF-7 , Teoria Quântica , Regulação para Cima/efeitos dos fármacosRESUMO
In this work, a simple and sensitive electrochemical aptasensor for protein (thrombin - TB used as the model) was developed by using cubic Cu2O nanocages (Cu2O-NCs) loaded with Au nanoparticles (AuNPs@Cu2O-NCs) as non-enzymatic electrocatalysts and robust redox probes. Through the specific sandwich-type reaction between TB and TB aptamers (TBA), the formed AuNPs@Cu2O-NCs bound with NH2-TBA were captured onto the electrode surface modified with SH-TBA. Based on the inherent redox activity of AuNPs@Cu2O-NCs with cubic nanostructures, a detectable electrochemical signal was generated which was dependent on the analyte concentration. Meanwhile, AuNPs@Cu2O-NCs showed an efficient electrocatalytic capability in the reduction of H2O2, resulting in a significant enhancement of the response signal. Thus, the simplification of the proposed strategy and the improvement of analytical performances were easily achieved with a sub-picomolar sensitivity (the limit of detection was 0.066 pmol L-1). The applicability of the simple and sensitive aptasensor was successfully demonstrated by assaying TB in human serum samples. This non-enzymatic detection platform would be potential and promising in clinical diagnostics and protein analysis techniques.