RESUMO
Sensitive, reliable, and specific detection of microRNAs (miRNAs) is a key objective for disease diagnosis and prognosis. Here, a ratiometric fluorescent/electrochemiluminescent (FL/ECL) sensor was designed for the dual-mode detection of miRNA-122, a hepatocellular carcinoma biomarker. The strong ECL emission was achieved from imine-linked covalent organic framework (COF-LZU1) accelerator enriched Ru(bpy)32+ molecules (Ru@COF-LZU1), which was applied as a delimited reaction micro-reactor to enhance ECL emission. Impressively, to construct an efficient sensing platform, self-feedback circuit was grafted at the vertex of DNA tetrahedral scaffold (DTS), which could provide a solution-phase-like environment and transform miRNA-122 into abundant single-stranded DNAs on the disposable electrode. Simultaneously, the carboxyfluorescein (FAM) tagged DNA segment was cleaved and released into the reaction solution, bringing in the recovery of FL response (FL on). Finally, the introduction of glucose oxidase (GOD) could generate H2O2 by in situ catalyzing GOD to glucose, resulting in the decrease of ECL signal (ECL off). Relying on FL/ECL ratio value, miRNA-122 was quantified with high sensitivity, well selectivity, stability and favorable practicability, suggesting that the proposed biosensor hold great potential for clinical diagnosis.
Assuntos
Técnicas Biossensoriais , Estruturas Metalorgânicas , MicroRNAs , Técnicas Biossensoriais/métodos , MicroRNAs/análise , Humanos , Estruturas Metalorgânicas/química , Medições Luminescentes , Técnicas Eletroquímicas/métodos , Rutênio/química , Limite de Detecção , Glucose Oxidase/química , DNA/química , Peróxido de Hidrogênio/química , Neoplasias Hepáticas/diagnóstico , Carcinoma Hepatocelular/diagnósticoRESUMO
In situ monitoring of intracellular microRNAs (miRNAs) often encounters the challenges of surrounding complexity, coexistence of precursor miRNAs (pre-miRNAs) and the degradation of biological enzyme in living cells. Here, we designed a novel probe encapsulated DNA tetrahedral molecular sieve (DTMS) to realize the size-selective detection of intracellular miRNA 21 that can avoid the interference of pre-miRNAs. In such strategy, quencher (BHQ-1) labeled probe DNA (S6-BHQ 1) was introduced into the inner cavity of fluorophore (FAM) labeled DNA tetrahedral scaffolds (DTS) to prepare DTMS, making the FAM and BHQ-1 closely proximate, and resulting the sensor in a "signal-off" state. In the presence of miRNA 21, strand displacement reaction happened to form more stable DNA double-stranded structure, accompanied by the release of S6-BHQ 1 from the inner cavity of DTMS, making the sensor in a "signal-on" state. The DTMS based sensing platform can then realized the size-selective detection of miRNA 21 with a detection limit of 3.6 pM. Relying on the mechanical rigidity of DTS and the encapsulation of DNA probe using DTMS, such proposed method achieved preferable reproducibility and storage stability. Moreover, this sensing system exhibited good performance for monitoring the change of intracellular miRNA 21 level during the treatment with miRNA-related drugs, demonstrating great potential for biological studies and accurate disease diagnosis.
Assuntos
DNA , Corantes Fluorescentes , MicroRNAs , MicroRNAs/análise , Humanos , DNA/química , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodos , Limite de Detecção , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Técnicas Biossensoriais/métodos , Tamanho da PartículaRESUMO
Size selectivity is crucial in highly accurate preparation of biosensors. Herein, we described an innovative electrochemiluminescence (ECL) sensing platform based on the confined DNA tetrahedral molecular sieve (DTMS) for size-selective recognition of nucleic acids and small biological molecule. Firstly, DNA template (T) was encapsulated into the inner cavity of DNA tetrahedral scaffold (DTS) and hybridized with quencher (Fc) labeled probe DNA to prepare DTMS, accordingly inducing Ru(bpy)32+ and Fc closely proximate, resulting the sensor in a "signal-off" state. Afterwards, target molecules entered the cavity of DTMS to realize the size-selective molecular recognition while prohibiting large molecules outside of the DTMS, resulting the sensor in a "signal-on" state due to the release of Fc. The rigid framework structure of DTS and the anchor of DNA probe inside the DTS effectively avoided the nuclease degradation of DNA probe, and nonspecific protein adsorption, making the sensor possess potential application prospect for size-selective molecular recognition in diagnostic analysis with high accuracy and specificity.
Assuntos
Técnicas Biossensoriais , Medições Luminescentes , Medições Luminescentes/métodos , Fotometria , Técnicas Biossensoriais/métodos , DNA , Sondas de DNA , Técnicas Eletroquímicas/métodosRESUMO
A two-step resonance energy transfer (RET)-based fluorescence/electrochemiluminescence (FL/ECL) biosensor was developed for ratiometric measurement and annihilation of Staphylococcus aureus (S. aureus). Using coupled dual-recognition-triggered target conversion with the catalytic hairpin assembly (CHA) technique, the monitoring of S. aureus was obtained at the single-cell level.
Assuntos
Técnicas Biossensoriais , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Transferência de Energia , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Medições Luminescentes/métodos , Limite de DetecçãoRESUMO
In this paper, a photoelectrochemical (PEC)-surface-enhanced Raman scattering (SERS) dual-mode biosensor is constructed coupled with a dual-recognition binding-induced DNA walker with a carbon nitride nanosheet (C3N4)/MXene-gold nanoparticles (C/M-Au NPs) accelerator, which is reliable and capable for sensitive and accurate detection of Staphylococcus aureus (S. aureus). Initially, a photoactive heterostructure is formed by combining C3N4 and MXene via a simple electrostatic self-assembly as they possess well-matched band-edge energy levels. Subsequently, in situ growth of gold nanoparticles on the formed surface results in better PEC performance and SERS activity, because of the synergistic effects of surface plasmon resonance and Schottky barrier. Furthermore, a three-dimensional, bipedal, and dual-recognition binding-induced DNA walker is introduced with the formation of Pb2+-dependent DNAzyme. In the presence of S. aureus, a significant quantity of intermediate DNA (I-DNA) is generated, which can open the hairpin structure of Methylene Blue-tagged hairpin DNA (H-MB) on the electrode surface, thereby enabling the switch of signals for the quantitative determination of S. aureus. The constructed PEC-SERS dual-mode biosensor that can be mutually verified under one reaction effectively addresses the problem of the low detection accuracy of traditional sensors. Experimental results revealed that the effective combination of PEC and SERS is achieved for amplification detection of S. aureus with a detection range of 5-108 CFU/mL (PEC) and 10-108 CFU/mL (SERS), and a detection of limit of 0.70 CFU/mL (PEC) and 1.35 CFU/mL (SERS), respectively. Therefore, this study offers a novel and effective dual-mode sensing strategy, which has important implications for bioanalysis and health monitoring.
Assuntos
Nanopartículas Metálicas , Infecções Estafilocócicas , Humanos , Ouro , Staphylococcus aureus , DNARESUMO
Sensitive and accurate determination of estriol level is vastly significant for the fetal growth and development. Herein, we constructed a dual-mode ratiometric biosensor for estriol assay combining the competitive immunoreaction, proximity hybridization with a two-step resonance energy transfer (RET) strategy. Estriol antibody and goat anti-rabbit antibody labeled DNA probes (Ab1-DNA1-Pt NPs and Ab2-DNA2) both hybridized with silver nanoclusters labeled DNA strands (H1-Ag NCs). Thus, the formed proximity hybridization enabled the occurrence of fluorescence RET (FL-RET, as the primary RET) between Ag NCs (donor) and Pt NPs (acceptor), quenching FL intensity of Ag NCs (FL off). When target estriol existed, the competitive reaction of Ab1-DNA1-Pt NPs with estriol and Ab2-DNA2 avoided the proximity hybridization. Then, the estriol-dependent H1-Ag NCs quenched electrochemiluminescence (ECL) emission of CdS quantum dots (CdS QDs, ECL off), generating ECL-RET (as the second RET). Consequently, according to the reverse changes of FL and ECL responses, this sensor realized the quantification of estriol from 1 to 100 ng/mL. Moreover, satisfactory results were achieved while testing estriol in pregnancy serum specimens, suggesting that the system is promising for potential application in samples analysis.
Assuntos
Anticorpos , Bioensaio , Feminino , Gravidez , Animais , Coelhos , Hibridização de Ácido Nucleico , Estriol , Transferência Ressonante de Energia de Fluorescência , CabrasRESUMO
Pathogenic bacteria have a significant impact on food safety. Herein, an innovative dual-mode ratiometric aptasensor was constructed for ultrasensitive and accurate detection of Staphylococcus aureus (S. aureus) based on recycling of DNAzyme activation on gold nanoparticles-functionalized MXene nanomaterials (MXene@Au NPs). Electrochemiluminescent (ECL) emitter-labeled probe DNA (probe 2-Ru) containing the blocked DNAzyme was partly hybridized with aptamer and then captured by electrochemical (EC) indicator-labeled probe DNA (probe 1-MB) on electrode surface. When S. aureus presented, the conformation vibration of probe 2-Ru activated the blocked DNAzymes, leading to recycling cleavage of probe 1-MB and ECL tag close to electrode surface. Based on the reverse change tendencies of ECL and EC signals, aptasensor achieved S. aureus quantification from 5 to 108 CFU/mL. Moreover, the self-calibration characteristic of the aptasensor with dual-mode ratiometric readout ensured the reliable measurement of S. aureus in real samples. This work showed useful insight into sensing foodborne pathogenic bacteria.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Nanopartículas Metálicas , Técnicas Eletroquímicas , Ouro/química , Staphylococcus aureus/genética , Nanopartículas Metálicas/química , Aptâmeros de Nucleotídeos/química , Limite de DetecçãoRESUMO
An amplified DNA logic sensor was constructed for the identification of multiple biomarkers, in which the inputs of targets triggered the disassembly of a V-shaped probe (VSP) structure by a strand displacement reaction, leading to the synthesis of silver nanoclusters (AgNCs) for electrocatalytic reduction of H2O2. The sensing platform achieved sensitive detection of methylated DNA and microRNA 122 with detection limits down to 3.4 and 4.1 fM, respectively, and can be used for the assay of clinical serum samples from healthy volunteers and liver injury patients with satisfactory results. The DNA logic sensor exhibited the advantages of convenience, low cost, and versatility without the involvement of electroactive label modification, which is helpful for disease diagnosis as well as the fundamental investigation of interfacial electrochemistry and molecular biology.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Humanos , Prata/química , Peróxido de Hidrogênio , Nanopartículas Metálicas/química , DNA/química , Biomarcadores , MicroRNAs/genéticaRESUMO
We described a two-step förster resonance energy transfer (FRET) system for ratiometric Staphylococcus aureus (S. aureus) detection based on a dual-recognition proximity binding-induced toehold strand displacement reactions (TSDR). Ru(bpy)32+ and platinum nanoparticles (Pt NPs) labeled DNA (Ru-S3 and Pt NPs-S4) hybridized to enable the occurrence of the primary FRET using Ru(bpy)32+ as the energy donor and Pt NPs as the energy acceptor. TSDR happened by integrating vancomycin hydrochloride labeled S1 (Van-S1) and gold nanoclusters labeled S2-aptamer (Au NCs-S2-aptamer) with S. aureus. The single DNA segments of Van-S1 bond to the terminal toehold of Ru-S3, displacing Pt-S4, inducing the secondary FRET using Au NCs as the energy donor and Ru(bpy)32+ as the energy acceptor. This two-step FRET system efficiently improved the reaction efficiency of S. aureus with a detection limit of 1.0 CFU/mL. Furthermore, satisfactory results obtained while detecting S. aureus in food samples, indicating a great potential for food analysis.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Transferência Ressonante de Energia de Fluorescência , Nanopartículas Metálicas/química , Staphylococcus aureus/genética , Platina , Ouro/química , Bactérias/genética , DNA/análise , Limite de DetecçãoRESUMO
the accurate, reliable and specific analysis of foodborne pathogenic bacteria is vital for human health and safety. Staphylococcus aureus (S. aureus), as a common bacterium, is regularly found in food, water, and other biological samples. Herein, a signal-off electrochemical DNA sensor (E-DNA sensor) was designed for the sensitive detection ofS. aureusamplified withthecombination of a dna walker and pb2+-specific dnazyme. In this work, vancomycin functionalized gold nanoclusters (Van@Au NCs) and an aptamer strand as identification units were modified at the termini of two proximity probes. upon the addition of targetS. aureus, a dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pba dual-recognition binding-induced dna walker was driven by the formation of pb2+-dependent dnazyme, achieving the conversion of oneS. aureus to many intermediate dna (t) strands. then, the released t strands hybridized with methylene blue-tagged hairpin dna (h-mb) on the electrode. consequently, the conformational alteration of t strands reduced the electron transfer efficiency of mb to the electrodeinterface (signal-off). therefore, sensitive analysis of S. aureus was readily acquired within a range of 10-107 CFU/mL and a low detection limit at 1 CFU/mL. Undoubtedly, dual recognition by aptamer and vancomycin in an integrated scheme brought about a good recognition performance of S. aureus in complex samples, as well as an efficient annihilation of harmful pathogenic bacteria during the experiment.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA Catalítico , Infecções Estafilocócicas , Aptâmeros de Nucleotídeos/química , DNA/química , DNA Catalítico/química , Técnicas Eletroquímicas , Ouro/química , Humanos , Chumbo , Limite de Detecção , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/química , Vancomicina/químicaRESUMO
Pathogenic bacteria infections pose a major threat to human health which can be found in contaminated food and infected humans. Herein, an electrochemical sensor was developed for pathogenic bacteria assay using a dual amplification strategy of polymethylene blue nanoparticles (pMB NPs) and dumbbell hybridization chain reaction (DHCR). The strong binding ability of aptamer to targets endowed outstanding performance in identifying Staphylococcus aureus (S. aureus) among other typical bacteria. The released T strands were hybridized with capture DNA on electrode surface which triggered DHCR in the presence of two dumbbell-shaped helper DNA, leading to the formation of extended and tight dsDNA polymers. In combination with pMB NPs (redox indicators), S. aureus was quantitatively detected in a range of 10-108 CFU/mL and the detection limit reached 1 CFU/mL. Moreover, this sensor was successfully applied for S. aureus detection in human serum and foods, demonstrating the reliability in practical applications.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Nanopartículas Metálicas , Nanopartículas , Técnicas Eletroquímicas , Ouro , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Staphylococcus aureus/genéticaRESUMO
An in situ quenching electrochemiluminescence (ECL) biosensor sensitized with the aptamer recognition-induced multi-DNA release was designed for pathogenic bacterial detection. Benefitting from the high binding ability of the aptamer to targets and large enrichment capacity of magnetic bead separation, the proposed sensing system not only exhibited outstanding identification to Staphylococcus aureus (S. aureus) among various bacteria, but also released abundant signal transduction DNAs. One S. aureus initiated the dissociation of four kinds of DNA sequences, achieving a one-to-multiple amplification effect. These multi-DNA strands were further hybridized with capture DNA, which were assembled to an electrode modified with Ru(bpy)32+-conjugated silica nanoparticles (RuSi NPs). Then, glucose oxidase (GOD) was introduced via the functional conjugation of GOD-multi-DNA, leading to the presence of H2O2 by in situ catalysis of GOD on glucose. Relying on the ECL quenching of H2O2 in the Ru(bpy)32+ system, S. aureus was quantified with a linear range from 10 to 107 CFU/mL. In addition, the negative results of non-target bacteria and good recovery efficiency in real samples revealed the system's remarkable selectivity and potential application in infectious food tests.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA , Peróxido de Hidrogênio , Medições Luminescentes , Staphylococcus aureusRESUMO
The sensitive detection of telomerase activity is of great significance for the early diagnosis and treatment of cancer. Here, an innovative electrochemiluminescence resonance energy transfer (ECL-RET) sensor was explored to reliably detect telomerase activity based on proximity binding-triggered multipedal DNA walker. In this system, CdS quantum dots (CdS QDs) and silver nanoclusters (Ag NCs) were applied as ECL donor and acceptor, respectively. By ingeniously introducing a repetitive bases sequence (TTAGGG) along the telomerase primer, multiple same DNA "legs" were formed, leading to the activation of proximity binding-triggered multipedal DNA walker. Unlike the traditional unipedal DNA walker, one walking step of multipedal DNA walker concurrently initiated the responsivity of multiple signals, resulting in the shortening of the walking time and improvement of the signal amplification efficiency. Thus, under optimal conditions, the designed ECL-RET sensor exhibited a wide dynamic correlation of HeLa cells' telomerase activity from 1 × 102 to 1 × 106 cells/mL and a low detection limit of 16 cells/mL. Moreover, this sensor realized the general and reliable analysis of telomerase activity in different cell lines. Due to the outstanding application potential in real samples, it is believed that the ECL-RET sensing system provides a new approach for the application of telomerase activity assays in cancer diagnostics.
Assuntos
Técnicas Biossensoriais , Pontos Quânticos , Telomerase , DNA , Técnicas Eletroquímicas , Células HeLa , Humanos , Limite de Detecção , Medições LuminescentesRESUMO
Carbon dots (CDs)-based biosensors have attracted considerable interest in reliable and sensitive detection of microRNA (miRNA) because of their merits of ultra-small size, excellent biosafety and tunable emission, whereas complicated labeling procedure and expensive bioenzyme associated with current strategies significantly limit their practical application. Herein, we developed a label-free and enzyme-free fluorescence strategy based on strand displaced amplification (SDA) for highly sensitive detection of miRNA using sulfydryl-functionalized CDs (CDs-SH) as probe. CDs-SH displayed excellent response to G-quadruplex DNA against other DNAs based on based on the catalytic oxidation of -SH into -S-S- by hemin/G-quadruplex. Further, CDs-SH were employed to detect miRNA, using miRNA-21 as target model, which triggered the SDA reaction of P1 and P2 to generate hemin/G-quadruplex, subsequently making CDs-SH transform from dot to aggresome along with the quenched fluorescence. Therefore, label-free, enzyme-free, and highly sensitive analysis of miRNA-21 was readily acquired with a limit of detection at 0.03 pM. This proposed biosensor couples the advantages of CDs and label-free/enzyme-free strategy, and thus has a significant potential to be used in early and accurate diagnosis of cancer.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , MicroRNAs , Carbono , Catálise , Hemina , Limite de DetecçãoRESUMO
The detection of a small number of exosomes provides the possibility for early cancer diagnosis and prognosis. Here, a multi-signal amplified electrochemical sensing platform was explored for the ultrasensitive detection of tumor exosomes relying on catalytic hairpin assembly-triggered DNA walker, entropy beacon-based DNA assembly and Ag@C core-shell nanocomposites. In this work, the utilization of Ag@C nanocomposites as electrode interface effectively enhanced functional active sites and electron transfer capability. By designing a target-assisted entropy beacon-based DNA assembly, single exosome initiated the release of multiple special DNA sequences, which could be separated conveniently by magnet and then hybridize with the blocking DNA to liberate swing arm. DNA walker was activated with the assistance of catalytic hairpin assembly, introducing extensive electroactive methylene blue (MB) to electrode surface. Thus, the detection of exosomes was transferred into the measurement of the MB current, with a good liner range from 100 to 75 000 particles/µL. Furthermore, this constructed sensing system displayed acceptable reproducibility, long-term stability, favorable selectivity, and highlighting application potential in real samples.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Exossomos , Nanocompostos , Neoplasias , DNA , Técnicas Eletroquímicas , Humanos , Reprodutibilidade dos Testes , PrataRESUMO
A critical challenge for improving the detection performance of sensors is building a favorable sensing interface. Herein, an innovative electrochemical biosensing system relying on hierarchical mesoporous NiO@N-doped C microspheres coupled with catalytic hairpin assembly was developed for DNA analysis. In this strategy, the utilization of NiO@N-doped C microspheres and multiwalled carbon nanotubes as electrode materials effectively enhanced the interfacial electron transfer and improved the surface active sites for subsequent reactions. By designing a target-assisted catalytic hairpin assembly, single target DNA could initiate the introduction of multiple signal probes labeled with ferrocene (Fc) onto a working electrode surface. Because the change in the Fc signal is dependent on the amount of target DNA, the resulting electrochemical sensing platform is highly sensitive. Under optimized reaction conditions, this testing platform has a wide linear range for target DNA detection from 100 aM to 100 pM with a detection limit of 45 aM. Furthermore, the platform displayed excellent selectivity, acceptable reproducibility, and long-term stability, highlighting the application potential of this sensing system.
RESUMO
A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of probe DNA and auxiliary DNA, methylated target DNA triggered the exonuclease III (Exo III) digestion of auxiliary DNA from 3'-terminus, resulting in the conformational change of probe DNA with an electroactive methylene blue (MB) tag at 5'-terminus. Consequently, the MB tag in the probe DNA was close to the electrode surface for electron transfer, generating an increased current signal. Because of the target recycling of methylated DNA, significant signal amplification was obtained. Moreover, bisulfite conversion conferred an efficient approach for the universal analysis of any CpG sites without the restriction of specific DNA sequence. As a result, the target DNA with different methylation statuses were clearly recognized, and the fully methylated DNA was quantified in a wide range from 10â¯fM to 100 pM, with a detection limit of 4â¯fM. The present work realized the assay of methylated target DNA in serum samples with satisfactory results, illustrating the application performance of the system in complex sample matrix.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Metilação de DNA/genética , Técnicas Eletroquímicas , Sondas de DNA/química , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Conformação de Ácido NucleicoRESUMO
An aptamer-binding DNA walking machine triggered by the recognition of aptamers to exosomes was firstly reported for sensitive electrochemiluminescence (ECL) detection of exosomes.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Exossomos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Técnicas Eletroquímicas/métodos , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Tetraspanina 30/metabolismoRESUMO
A highly sensitive and selective biosensing system was designed to analyze DNA methylation using a dual-signal readout technique in combination with the signal amplification of supersandwich DNA structure. Through the ingenious design of target-triggered cascade of hybridization chain reaction, one target DNA could initiate the formation of supersandwich structure with multiple signal probes. As a result, one-to-multiple amplification effect was achieved, which conferred high sensitivity to target molecular recognition. Based on probe 1 labeled with ferrocene and probe 2 modified with methylene blue, the target DNA was clearly recognized by two electrochemical signals at independent potentials, which was helpful for the acquisition of more accurate detection results. Taking advantage of bisulfite conversion, the methylation status of cytosine (C) was changed to nucleic acid sequence status, which facilitated the hybridization-based detection without enzymatic reaction. Consequently, the methylated DNA was detected at the femtomolar level with satisfactory analytical parameters. The proposed system was effectively used to assess methylated DNA in human blood serum samples, illuminating the possibility of the sensing platform for applications in disease diagnosis and biochemistry research.
Assuntos
Técnicas Biossensoriais , Metilação de DNA , Técnicas Eletroquímicas , DNA/metabolismo , Dissulfetos/química , Ouro/química , Humanos , Nanopartículas Metálicas/química , Molibdênio/química , Nanocompostos/química , Hibridização de Ácido NucleicoRESUMO
The cytosines in cluster-nucleation sequences play a vital role in the formation of silver nanoclusters (Ag NCs). Here, an innovative electrochemiluminescence (ECL) resonance energy transfer (RET) sensing system was developed using CdS quantum dots (QDs) as ECL donor and Ag NCs as ECL acceptor. Modulation of the number of cytosines in the cluster-nucleation sequences allowed tuning of Ag NCs absorption bands to match with the ECL emission spectrum of CdS QDs, yielding effective ECL-RET. The sensitivity of detection was improved by dual-target recycling amplification based on duplex-specific nuclease (DSN) and catalytic hairpin assembly. In the presence of target microRNA-21 (miRNA-21), DSN selectively cleaved the complementary DNA section (S1), resulting in the release of the transduction section (S2) and the reuse of miRNA-21 in the next recycling amplification. Interaction of the stem-loop structure of the DNA1 segment (H1) on CdS QDs-modified electrode with S2 led to the opening of the hairpin structure of H1 and the formation of H1:S2 duplex. Then, hairpin DNA2 encapsulated Ag NCs hybridized with the remaining single-stranded DNA segment of H1, and the S2 strand was replaced. Finally, the dissociated S2 participated in subsequent reaction cycles, introducing Ag NCs to the electrode surface and leading to ECL signal quenching of the CdS QDs. The proposed sensor showed excellent performance in detecting miRNA-21 at a wide linear range from 1 fM to 100 pM. The practical application ability of the strategy was tested in HeLa cells with acceptable results, suggesting that the detection platform is a promising approach for disease diagnosis and molecular biology research.