RESUMEN
The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.
RESUMEN
Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg2+-stabilized DNA duplex (Hg2+-dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg2+ in Hg2+-dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios.
Asunto(s)
Técnicas Biosensibles , ADN Catalítico , Técnicas Electroquímicas , Mercurio , Metalotioneína , ADN Catalítico/química , ADN Catalítico/metabolismo , Metalotioneína/química , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Mercurio/análisis , Mercurio/química , Humanos , Límite de Detección , G-Cuádruplex , Electrodos , Hemina/química , Catálisis , ADN/químicaRESUMEN
Sensitive monitoring of luteinizing hormone (LH), a glycoprotein that regulates the synthesis of regulatory steroid hormones, can facilitate the diagnosis of various reproductive diseases. In this work, a new and highly catalytic Sulfur-doped and bimetal-coordinated CoFe(CN)5NO (denoted as S-CoFe(CN)5NO) nanoparticles are synthesized. Such material is further used to construct high performance sensing interface and coupled with primer exchange reaction (PER) and hybridization chain reaction (HCR) amplification cascades for sensitive electrochemical aptamer-based LH assay. Target LH molecules bind aptamer sequences in DNA duplex probes to liberate ssDNA strands, which initiate subsequent PER/HCR amplification cascades for the capture of many ferrocene (Fc)-tagged DNAs on sensing interface. S-CoFe(CN)5NO subsequently leads to catalytic oxidation of these Fc tags for yielding substantially magnified currents for realizing ultrasensitive assay of LH with the detection limit of 0.69 pM in range from 5 pM to 10 nM. Owing to the high specificity of aptamer, such sensor has high selectivity and can achieve low levels of LH assay in diluted serum samples. With the successful demonstration for detecting trace LH, such sensor can be easily extended as a universal aptamer-based electrochemical sensing method for monitoring various target analytes in the biomedical and biological fields.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Electroquímicas , Límite de Detección , Hormona Luteinizante , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Humanos , Técnicas Electroquímicas/métodos , Hormona Luteinizante/sangre , Hormona Luteinizante/química , Catálisis , Azufre/química , Nanopartículas del Metal/química , Cobalto/química , Hibridación de Ácido Nucleico , Nanopartículas/química , Compuestos Ferrosos/químicaRESUMEN
The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.
Asunto(s)
Aptámeros de Nucleótidos , MicroARNs , Neoplasias , MicroARNs/genética , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/química , Límite de Detección , ADN/química , Sondas de ADN/química , Colorantes Fluorescentes/química , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMEN
The aberrant expression of microRNAs (miRs) in cells is closely linked to the initiation and progression of various diseases. Sensitive monitoring of their level is hence vital for biomedical research and disease diagnosis. Herein, a highly sensitive and non-label fluorescence sensor based on multiple recycling signal amplification cascades is constructed for the detection of miR-21 in human sera. The presence of miR-21 initiates the primer-fueled target recycling process for the generation of many primer/hairpin templates for the subsequent auto-cycling primer extension (APE) amplification cycles, which result in the formation of lots of long-stem hairpins. The enzyme-based cleavage of such hairpins via polymerization/excision cycles further leads to the generation of abundant G-quadruplex strands, which associate with the thioflavin T (ThT) dye to emit remarkably magnified fluorescence for detecting miR-21 in the range of 1 pM-100 nM with a 0.32 pM detection limit without labeling the probes. Besides, the proposed assay can selectively discriminate miR-21 against other control molecules and realize the sensing of low levels of miR-21 in diluted sera. With features of high sensitivity via the triplex signal amplification cycles and simplicity in a non-label homogeneous manner, our miR sensing protocol can be a robust means for detecting various nucleic acids for the early diagnosis of diseases.
Asunto(s)
Técnicas Biosensibles , G-Cuádruplex , MicroARNs , Humanos , MicroARNs/genética , Límite de Detección , Espectrometría de Fluorescencia/métodos , Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
Sensitively monitoring the concentration change of lipocalin 1 (LCN1) can provide data support for accurately diagnosing diabetic retinopathy and efficacy of treatment. Using a new aptamer triplex switch (ATS) probe and catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual signal amplifications, a highly sensitive electrochemical and enzyme-free biosensor for detecting LCN1 is reported. The ATS probes recognize and bind LCN1 to change its structure and release ssDNA sequences, which trigger the capture of methylene blue (MB)-tagged DNA on the sensor electrode via the CHA and HCR reactions. These MB tags are then subjected to electrochemical measurements to obtain highly amplified current outputs to sensitively detect LCN1 from 5 pM to 1 nM with a detection limit of 0.85 pM. This sensor also exhibits high interfering protein discrimination capability and can be employed to monitor low levels of LCN1 in diluted tear samples. Our established sensor significantly outperforms current major LCN1 detection methods based on immunoassays and thus holds promising potential for evaluating the severity of diabetic retinopathy and monitoring treatment response.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Retinopatía Diabética , Humanos , Lipocalina 1 , Técnicas Electroquímicas/métodos , ADN/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Azul de Metileno/química , Límite de DetecciónRESUMEN
The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg2+-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed.
Asunto(s)
Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , ADN Catalítico/química , Sondas de ADN , Colorantes , Catálisis , Técnicas Biosensibles/métodos , Límite de DetecciónRESUMEN
We report here a highly sensitive fluorescent thrombin biomarker sensing method by integrating the DNA walker and CRISPR/Cas12a system. The presence of thrombin causes the localization of DNA moving arms on AuNP tracks via their proximity bindings with the dye-labeled probes immobilized on AuNPs. With the assistance of the primer and DNA polymerase, the arm sequences move continuously on the AuNP tracks to generate many CRISPR/Cas12a-responsive dsDNAs, which push the dye to move away from AuNPs to restore its fluorescence. Moreover, the dsDNAs can be recognized and cut by the CRISPR/Cas12a to trigger its trans-cleavage activity for cyclically cleaving the fluorescently quenched signal probes on the AuNP tracks, which liberates the dye from AuNPs to further enhance the fluorescence restoration to achieve highly sensitive thrombin assay with detection limit of 29.5 fM. Selectively detecting thrombin against other interference proteins and in diluted serums by such sensing method has also been verified, making it an attractive approach for monitoring other protein biomarkers at low levels for the diagnosis of diseases.
Asunto(s)
Técnicas Biosensibles , Nanopartículas del Metal , Sistemas CRISPR-Cas , Oro , Trombina , ADN/genéticaRESUMEN
Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.
Asunto(s)
Aptámeros de Nucleótidos , Neoplasias , ADN/genética , ADN/metabolismo , ADN Ligasas , Sondas de ADN , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , HumanosRESUMEN
Accurate and ultrasensitive detection of biomarkers is significance for the diagnosis of diseases at early stage. For this purpose, we herein develop a bidirectional dynamic DNA nano-device for amplified fluorescent detection of tumor marker of mucin 1 (MUC1). The nano-device is constructed by immobilizing two sets of DNA cascade catalytic probes on two opposite directions of a single-stranded DNA tracker to limit probe reactants to a compact space. Once target MUC1 binds to the aptamer sequence, the initiator DNA locked in the duplex DNA substrate can be released to induce DNA-initiated cascade hybridization reactions (DCHRs) simultaneously in two opposite directions along the tracker DNA, accompanying the displacement of two quencher labeled-DNA intermediate initiators to facilitate successively execution of DCHRs on the DNA nano-devices, which results in the separation of fluorophore (FAM) and quencher (Dabycl) to produce substantially recovered fluorescent signals for rapid and sensitive detection of MUC1 with a detection limit down to 0.18 pM. In addition, this strategy also exhibits high selectivity against other interfering proteins and potential application capacity in real serum samples, indicating its promising application prospects in disease diagnosis and treatment.
Asunto(s)
ADN , Mucina-1 , Oligonucleótidos , Biomarcadores de Tumor , Colorantes FluorescentesRESUMEN
Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.
Asunto(s)
Técnicas Biosensibles , FN-kappa B , Técnicas Biosensibles/métodos , Sistemas CRISPR-Cas , ADN/química , Células HeLa , Humanos , Hidrogeles , FN-kappa B/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN , Factores de Transcripción/genéticaRESUMEN
Single nucleotide polymorphisms (SNPs) are main causes of differences in human phenotypes and drug resistance, especially the development of genetic diseases. Monitoring SNPs is of significant benefit to drug design and disease diagnosis. In this work, on the basis of tripartite DNAzyme junction formation induced by flap endonuclease 1 (FEN1) invader assay-triggered catalytic hairpin assembly (CHA), we describe an amplified and highly sensitive fluorescent strategy for detecting SNP of K-ras gene with substantial discrimination capability. The mutant DNA (MtDNA) of K-ras gene hybridizes with the sensing probe to inhibit the enzymatic activity of FEN1 to trigger subsequent CHA of three hairpins for the formation of tripartite DNAzyme junctions. And, the fluorescently quenched signal probes are efficiently and cyclically cleaved by the DNAzymes to restore largely magnified fluorescence for detecting MtDNA target sequence at 4.23 fM. Besides, the existence of low levels of MtDNA strands in diluted human serums and high concentrations of wild DNA solutions can be differentiated by such a method, showing its high potential for monitoring various SNPs for biological research and disease diagnosis.
Asunto(s)
Técnicas Biosensibles , ADN Catalítico , Técnicas Biosensibles/métodos , Catálisis , ADN Catalítico/genética , ADN Mitocondrial , Humanos , Límite de Detección , Polimorfismo de Nucleótido SimpleRESUMEN
Simple, selective and sensitive monitoring of antibiotic residues in food is essential for food safety and human health because of its side effects upon inappropriate usage. Here, with a new label- and enzyme-free significant signal enhancement approach by the coupling of catalytic hairpin assembly (CHA) with nonlinear hybridization chain reaction (nHCR), we developed a fluorescence aptamer sensor for the detection of trace kanamycin in milk samples with high selectivity and sensitivity. The binding of the target kanamycin to the aptamer probe could initiate the CHA between two hairpins for the formation of partial DNA duplexes, which further triggered the nHCR of other three hairpins to yield branched DNA complexes with a multitude of active G-quadruplex structures. The subsequent intercalation of the organic dye, thioflavin T, into G-quadruplex structures resulted in significantly enhanced fluorescence responses for realizing sensitive sensing of kanamycin in the dynamic range of 0.1-300 nM with a detection limit of 46.1 pM. Besides, this strategy could also achieve the monitoring of kanamycin selectively in spiked milk samples. With features of high sensitivity and simplicity in a non-enzyme/label fashion, our signal amplification strategy has high potentials for establishing sensitive and convenient means to monitor various antibiotics.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Antibacterianos , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , ADN/genética , Humanos , Kanamicina , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido NucleicoRESUMEN
MicroRNAs (miRNA) are closely associated with the development of diseases in life processes, and the sensitive detection of microRNA biomarkers is beneficial to disease diagnosis and treatment at early stage. Herein, by coupling the target sequence recycling with the primer exchange reaction (PER), a highly sensitive and non-label approach is constructed to detect miRNA-122, the biomarker for drug-induced liver injury. The target sequence cyclically displaces two hairpins on the DNA track via toehold-mediated strand displacement reactions with the assistance of the fuel DNA. The released hairpins further bind the primer to trigger the polymerase-aided PER process for the yield of plenty of G-quadruplex sequences, which then combine with the thioflavin T to drastically enhance its fluorescence for sensing miRNA-122 with a low 49.4 fM detection limit. Highly specific discrimination of the target miRNA-122 can also be realized with the proposed method. Because of the non-label format, high selectivity and sensitivity, such a method can be a convenient and universal means for sensing various biomarkers for disease diagnosis at the early stages.
Asunto(s)
Técnicas Biosensibles , G-Cuádruplex , MicroARNs , Biomarcadores , ADN , Límite de Detección , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Accurate and sensitive monitoring of the abused antibiotics is vital because excessive antibiotics in human body can cause toxicity to kidney or lead to potential loss of hearing. In this work, we described a label-free and highly sensitive fluorescent aptasensing platform for detecting kanamycin in milk samples based on the synchronization signal amplification of primer exchange reaction (PER) and metal-ion dependent DNAzyme. The target kanamycin binds the aptamer sequence hybridized on a hairpin template and initiates PER for autonomous synthesis of Mg2+-dependent DNAzyme sequences with aid of Bst-DNA polymerase at isothermal conditions. Such a synthesis process can be repeated many times to produce lots of DNAzymes to cyclically cleave the rA site in the signal hairpin substrates under the assistance of Mg2+ cofactor to liberate numerous free G-quadruplex fragments. The organic dye thioflavin T (ThT) further associates with these G-quadruplex fragments to yield substantially intensified fluorescence for sensitive detection of kanamycin with a low detection limit of 0.36 nM. In addition, the developed aptamer sensing method also shows a good selectivity for kanamycin against other interfering antibiotics, and can realize the monitoring of kanamycin added in milk samples, highlighting its potential for sensitive monitoring of trace amount of kanamycin for food safety applications.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , Animales , ADN Catalítico/genética , Humanos , Kanamicina , Límite de Detección , LecheRESUMEN
The abnormal variation of the mucin 1 (MUC1) protein level is associated with the development of multiple cancers, and the monitoring of trace MUC1 can be useful for early disease diagnosis. Here, on the basis of the synchronization of DNA-fueled sequence recycling and dual rolling circle amplification (RCA), the establishment of a non-label and highly sensitive fluorescent aptamer-based detection strategy for the MUC1 protein biomarker is described. The target MUC1 binds the aptamer hairpin probe and causes its structure switching to release an ssDNA tail to trigger the recycling of the complex via two toehold-mediated strand displacement reactions under assistance of a fuel DNA. Such a recycling amplification leads to the formation of a partial dsDNA duplex with two primers at both ends, which cooperatively bind the circular DNA ring template to start the dual RCA to produce many G-quadruplex sequences. The protoporphyrin IX dye further associates with the G-quadruplex structures to show a dramatically elevated fluorescent signal for sensitively detecting MUC1 with a low detection limit of 0.5 pM. The established aptamer-based detecting strategy is also highly selective and can realize assay of MUC1 in diluted human serums, highlighting its potential for the detection of different protein biomarkers at low contents.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , G-Cuádruplex , Biomarcadores , ADN , Cartilla de ADN , Humanos , Límite de Detección , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The sensitive and quantitative analysis of mucin 1 (MUC1) is very important for the prevention and early diagnosis of cancers. In the present work, based on the mechanism of the four-way DNA branch migration cascades, we constructed a simple and effective signal amplification strategy for aptamer-based sensitive detection of MUC1. The specific binding of MUC1 to the aptamer sequence in the hairpin probe unfolds and switches its structure, triggering the formation of the DNA Holliday junction structure for cascaded branch migrations with the assistance of two fuel DNA duplexes. Importantly, a target analogue DNA complex can be generated in such processes for recycling the branch migration reactions for the production of substantial amounts of G-quadruplexes, which can bind the thioflavin T dye to show significantly intensified fluorescence for detecting MUC1 with a low detection limit of 2.8 nM without the involvement of any labels or enzymes. In addition, this detection strategy could be successfully applied to monitor the target MUC1 in diluted human serums with a high selectivity and acceptable accuracy to demonstrate its potential application for real samples with the advantages of simplicity and signal amplification capability.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , G-Cuádruplex , ADN/genética , Humanos , Límite de Detección , Mucina-1/genéticaRESUMEN
Monitoring circulating tumor cells (CTCs) in human blood can offer useful information for convenient metastasis diagnosis, prognosis, and treatment of cancers. However, it remains a substantial challenge to detect CTCs because of their particular scarcity in complex peripheral blood. Herein, we describe an in situ-generated multivalent aptamer network-modified electrode interface for efficiently capturing and sensitively detecting CTCs in whole blood by electrochemistry. Such an interface was fabricated via rolling circle amplification extension of the electrode-immobilized primer/circular DNA complexes for the yield of long ssDNA strands with many repeated aptamer segments, which could achieve efficient capture of rare CTCs in a multivalent cooperative manner. The antibody and horseradish peroxidase-functionalized gold nanoparticles further specifically associated with the surface-bound CTCs and generated electrocatalytically amplified current outputs for highly sensitive detection of CTCs with an attractive detection limit of five cells. Also, the multivalent aptamer network interface could successfully distinguish the target cells from other control cells and achieve CTC detection in whole blood, demonstrating its promising potential for monitoring different rare CTCs in human blood.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Electroquímicas , Células Neoplásicas Circulantes/patología , Humanos , Células MCF-7 , Células Tumorales CultivadasRESUMEN
MicroRNAs (miRNAs) are important biomarkers because their abnormal expressions or mutations can usually indicate the development of cancers or other diseases. However, their high family sequence homology and low abundance pose a major challenge for the analysis of miRNAs. In the present work, we developed a dual strand extension recycling signal amplification strategy for non-label detection of miRNA-155 with high sensitivity on the basis of DNA polymerase and the G-quadruplex/thioflavin T (ThT) complexes. The specific miRNA-155 target sequences could initiate two strand extension-based independent recycling cycles under the function of the DNA polymerase, resulting in the production of many active G-quadruplex segments. The dye of ThT further bound the G-quadruplexes to induce greatly enhanced fluorescence for ultra-sensitively detecting miRNA-155 sequences at the low concentration of 35 fM in the dynamic range from 0.1 pM to 100 pM. The proposed detection strategy used the completely unmodified and synthetic DNA as probes and could be performed in homogeneous solutions. This method with a high selectivity could also be used for diluted serum samples. The demonstration of our new method for miRNA detection thus offers it with high potentials for miRNA biomarker analyses with the purpose of clinical diagnostics and biomedical applications.
Asunto(s)
MicroARNs/sangre , MicroARNs/genética , Técnicas de Amplificación de Ácido Nucleico , HumanosRESUMEN
Detecting molecular biomarkers in high sensitivity plays an important role in the diagnosis of various diseases at the early stage. Here, by combining the target-induced polymerization nicking reaction (TIPNR) with the loop-mediated isothermal amplification (LAMP), we describe an ultrasensitive and label-free aptamer-based sensing method for detecting low levels of proteins in human serum by using thrombin as the model target analyte. The target thrombin binds and causes spontaneous assembly of two distinct aptamer probes to form the templates for the polymerization nicking reaction recycling amplification to produce many forward inner primer sequences. Subsequently, downstream LAMP reactions are initiated by these sequences for the generation of tremendous DNA hairpins with various lengths via automated cyclic strand displacement reactions. The SYBR Green I organic dye further binds the many hairpins to show drastically amplified fluorescence for ultrasensitive detection of thrombin down to 3.6 fM in the linear range from 0.01 pM to 10 nM. Such a sensing method based on aptamers has high discrimination capability for the target molecules against other non-specific proteins and is applicable for diluted serum samples. With the successful demonstration of the substantial signal amplification ability and simplicity feature of this assay approach, highly sensitive and convenient detection of other disease biomarkers with this method can be envisioned in the near future.