RESUMEN
Long noncoding RNAs (lncRNAs) act as the dynamic regulatory molecules that control the expression of genes and affect numerous biological processes, and their dysregulation is associated with tumor progression. Herein, we develop a fluorescent light-up aptasensor to simultaneously measure multiple lncRNAs in living cells and breast tissue samples based on the DNAzyme-mediated cleavage reaction and transcription-driven synthesis of light-up aptamers. When target lncRNAs are present, they can be recognized by template probes to form the active DNAzyme structures, initiating the T4 PNK-catalyzed dephosphorylation-triggered extension reaction to generate double-strand DNAs with the T7 promoter sequences. The corresponding T7 promoters can initiate the transcription amplification catalyzed by the T7 RNA polymerase to generate abundant Broccoli aptamers and malachite green aptamers, which can bind DFHBI-1T and MG to generate strong fluorescence signals. Taking advantage of the good selectivity of DNAzyme-mediated cleavage of lncRNAs, high amplification efficiency of T7 transcription-driven amplification reaction, and bright fluorescence of the RNA aptamer-fluorophore complex, this method exhibits high sensitivity with a detection limit of 21.4 aM for lncRNA HOTAIR and 18.47 aM for lncRNA MALAT1, and it can accurately measure multiple lncRNAs in both tumor cell lines and breast tissue samples, providing a powerful paradigm for biomedical research and early clinic diagnostics.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN Catalítico , Colorantes Fluorescentes , ARN Largo no Codificante , ADN Catalítico/química , ADN Catalítico/metabolismo , ARN Largo no Codificante/análisis , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Humanos , Aptámeros de Nucleótidos/química , Colorantes Fluorescentes/química , Límite de Detección , FluorescenciaRESUMEN
Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.
Asunto(s)
ARN Interferente Pequeño , Humanos , ARN Interferente Pequeño/genética , Catálisis , Hibridación de Ácido Nucleico , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Cinética , ARN de Interacción con PiwiRESUMEN
Telomerase is an important biomarker for early diagnosis of cancers, but current telomerase assays usually rely on measuring the extension products of telomerase substrates, which increases the assay complexity. More evidence indicates that human telomerase RNA (hTR), as a core component of telomerase, is positively correlated with the telomerase activity. Herein, we demonstrate the development of a duplex-specific nuclease (DSN)-propelled 3D quantum dot (QD) nanoassembly with two-step Föster resonance energy transfer (FRET) for the one-step sensing of hTR in breast cancer cells and tissues. This assay involves only one hairpin probe modified with a Cy5 at the sixth base from the 5'-biotin end and a BHQ2 at the 3'-terminus, which integrates three functions of target recognition, target recycling amplification, and signal readout. The anchoring of the hairpin probe on the 605QD surface results in the formation of a 3D 605QD-Cy5-probe-BHQ2 nanoassembly in which two-step FRET occurs among the 605QD, Cy5, and BHQ2 quencher. Notably, the formation of 605QD-Cy5-probe-BHQ2 nanoassembly facilitates the reduction of background signal and the increase of signal-to-background ratio due to its dense, highly oriented nucleic acid shell-induced steric hindrance effect. This assay can achieve one-step and rapid detection of hTR with a detection limit of 2.10 fM, which is the simplest and most rapid hTR assay reported so far. Moreover, this assay can efficiently distinguish single-base mismatched sequences, and it can discriminate the hTR level between breast cancer patients and healthy donors with a high accuracy of 100%, with great prospects for early diagnosis of cancers.
Asunto(s)
Neoplasias de la Mama , Transferencia Resonante de Energía de Fluorescencia , Puntos Cuánticos , ARN , Telomerasa , Humanos , Telomerasa/metabolismo , Telomerasa/análisis , Puntos Cuánticos/química , ARN/metabolismo , ARN/análisis , Femenino , Carbocianinas/química , Técnicas Biosensibles/métodosRESUMEN
We present a dimensional regulating charge transfer strategy to achieve an enhanced electrochemiluminescence (ECL) by constructing a one-dimensional pyrene-based covalent organic framework (1D-COF). The dual-chain-like edge architecture in 1D-COF facilitates the stabilization of aromatic backbones, the enhancement of electronic conjugations, and the decrease of energy loss. The 1D-COF generates enhanced anodic (92.5-fold) and cathodic (3.2-fold) signals with tripropylamine (TPrA) and K2S2O8 as the anodic and cathodic coreactants, respectively, compared with 2D-COF. The anodic and cathodic ECL efficiencies of 1D-COF are 2.08- and 3.08-fold higher than those of 2D-COF, respectively. According to density functional theory (DFT), the rotational barrier energy (ΔE) of 1D-COF enhances sharply with the increase of dihedral angle, suggesting that the architecture in 1D-COF restrains the intramolecular spin of aromatic chains, which facilitates the decrease of nonradiative transitions and the enhancement of ECL. Furthermore, 1D-COF can be used to construct an ECL biosensor for sensitive detection of dopamine.
RESUMEN
Aberrant long noncoding RNA (lncRNA) expression is linked to varied pathological processes and malignant tumors, and lncRNA can serve as potential disease biomarkers. Herein, we demonstrate the autonomous enzymatic synthesis of functional nucleic acids for sensitive measurement of lncRNA in human lung tissues on the basis of multiple primer generation-mediated rolling circle amplification (mPG-RCA). This assay involves two padlock probes that act as both a detection probe for recognizing target lncRNA and a domain for producing complementary DNAzyme. Two padlock probes can hybridize with target lncRNA at different sites, followed by ligation to form a circular template with the aid of RNA ligase. The circular template can initiate mPG-RCA to generate abundant Mg2+-dependent DNAzymes that can specifically cleave signal probes to induce the recovery of Cy3 fluorescence. The inherent characteristics of ligase-based ligation reaction and DNAzymes endow this assay with excellent specificity, and the introduction of multiple padlock probes endows this assay with high sensitivity. This strategy can rapidly and sensitively measure lncRNA with a wide linear range of 1 fM - 1 nM and a detection limit of 678 aM within 1.5 h, and it shows distinct advantages of simplicity and immobilization-free without the need of precise temperature control and tedious procedures of nanomaterial preparation. Moreover, it enables accurate measurement of lncRNA level in normal cells and malignant tumor cells as well as differentiation of lncRNA expressions in tissues of non-small cell lung cancer (NSCLC) patients and normal individuals, with promising applications in biomedical studies and disease diagnosis.
Asunto(s)
ADN Catalítico , Pulmón , Técnicas de Amplificación de Ácido Nucleico , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ADN Catalítico/química , ADN Catalítico/metabolismo , Pulmón/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Límite de DetecciónRESUMEN
Mycotoxin contamination in food products may cause serious health hazards and economic losses. The effective control and accurate detection of mycotoxins have become a global concern. Even though a variety of methods have been developed for mycotoxin detection, most conventional methods suffer from complicated operation procedures, low sensitivity, high cost, and long assay time. Therefore, the development of simple and sensitive methods for mycotoxin assay is highly needed. The introduction of nucleic acid signal amplification technology (NASAT) into aptasensors significantly improves the sensitivity and facilitates the detection of mycotoxins. Herein, we give a comprehensive review of the recent advances in NASAT-based aptasensors for assaying mycotoxins and summarize the principles, features, and applications of NASAT-based aptasensors. Moreover, we highlight the challenges and prospects in the field, including the simultaneous detection of multiple mycotoxins and the development of portable devices for field detection.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Micotoxinas , Técnicas de Amplificación de Ácido Nucleico , Micotoxinas/análisis , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Contaminación de Alimentos/análisis , Ácidos Nucleicos/análisisRESUMEN
Precisely controlling the architecture and spatial arrangement of plasmonic heterostructures offers unique opportunities to tailor the catalytic property, whereas the lack of a wet-chemistry synthetic approach to fabricating nanostructures with high-index facets limits their practical applications. Herein, we describe a universal synthetic strategy to construct Au/Rh freestanding superstructures (SSs) through the selective growth of ordered Rh nanoarrays on high-index-faceted Au nanobipyramids (NBPs). This synthetic strategy works on various metal nanocrystal substrates and can yield diverse Au/Rh and Pd/Rh SSs. Especially, the obtained Au NBP/Rh SSs exhibit high photocatalytic activity toward N2 fixation as a result of the spatially separated architecture, local electric field enhancement, and the antenna-reactor mechanism. Both theoretical and experimental results reveal that the Au NBPs can function as nanoantennas for light-harvesting to generate hot charge carriers for driving N2 fixation, while the Rh nanoarrays can serve as the active sites for N2 adsorption and activation to synergistically promote the overall catalytic activity in the Au NBP/Rh SSs. This work offers new avenues to rationally designing and constructing spatially separated plasmonic photocatalysts for high-efficiency catalytic applications.
RESUMEN
BACKGROUND: 5-hydroxymethylcytosine (5hmC) as an epigenetic modification can regulate gene expression, and its abnormal level is related with various tumor invasiveness and poor prognosis. Nevertheless, the current methods for 5hmC assay usually involve expensive instruments/antibodies, radioactive risk, high background, laborious bisulfite treatment procedures, and non-specific/long amplification time. RESULTS: We develop a glycosylation-mediated fluorescent biosensor based on helicase-dependent amplification (HDA) for label-free detection of site-specific 5hmC in cancer cells with zero background signal. The glycosylated 5hmC-DNA (5ghmC) catalyzed by ß-glucosyltransferase (ß-GT) can be cleaved by AbaSI restriction endonuclease to generate two dsDNA fragments with sticky ends. The resultant dsDNA fragments are complementary to the biotinylated probes and ligated by DNA ligases, followed by being captured by magnetic beads. After magnetic separation, the eluted ligation products act as the templates to initiate HDA reaction, generating abundant double-stranded DNA (dsDNA) products within 20 min. The dsDNA products are measured in a label-free manner with SYBR Green I as an indicator. This biosensor can measure 5hmC with a detection limit of 2.75 fM and a wide linear range from 1 × 10-14 to 1 × 10-8 M, and it can discriminate as low as 0.001% 5hmC level in complex mixture. Moreover, this biosensor can measure site-specific 5hmC in cancer cells, and distinguish tumor cells from normal cells. SIGNIFICANCE: This biosensor can achieve a zero-background signal without the need of either 5hmC specific antibody or bisulfite treatment, and it holds potential applications in biological research and disease diagnosis.
Asunto(s)
5-Metilcitosina/análogos & derivados , Técnicas Biosensibles , Neoplasias , Sulfitos , Glicosilación , ADN/genética , 5-Metilcitosina/metabolismoRESUMEN
DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 - 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors.
Asunto(s)
Técnicas Biosensibles , ADN , BiomarcadoresRESUMEN
Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.
Asunto(s)
Nanopartículas del Metal , Telomerasa , Humanos , Telomerasa/metabolismo , Oro/química , Nanopartículas del Metal/química , ADN/química , Células HeLa , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismoRESUMEN
O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/µL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.
Asunto(s)
Carbocianinas , ADN Catalítico , Guanina/análogos & derivados , Puntos Cuánticos , Humanos , ADN Catalítico/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , ADN/química , DesmetilaciónRESUMEN
Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of DNA damage, and it has become not only an oxidative stress biomarker but also a new epigenetic-like biomarker. However, few approaches are available for the locus-specific detection of 8-oxoG because of the low abundance of 8-oxoG damage in DNA and the limited sensitivity of existing assays. Herein, we demonstrate the elongation and ligation-mediated differential coding for label-free and locus-specific analysis of 8-oxoG in DNA. This assay is very simple without the involvement of any specific labeled probes, complicated steps, and large sample consumption. The utilization of Bsu DNA polymerase can specifically initiate a single-base extension reaction to incorporate dATP into the opposite position of 8-oxoG, endowing this assay with excellent selectivity. The introduction of cascade amplification reaction significantly enhances the sensitivity. The proposed method can monitor 8-oxoG with a limit of detection of 8.21 × 10-19 M (0.82 aM), and it can identify as low as 0.001% 8-oxoG damage from a complex mixture with excessive undamaged DNAs. This method can be further applied to measure 8-oxoG levels in the genomic DNA of human cells under diverse oxidative stress, holding prospect potential in the dynamic monitoring of critical 8-oxoG sites, early clinical diagnosis, and gene damage-related biomedical research.
Asunto(s)
ADN Polimerasa Dirigida por ADN , ADN , Guanina/análogos & derivados , Humanos , ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Daño del ADN , Biomarcadores , Reparación del ADNRESUMEN
Accurate and sensitive analysis of circulating tumor cells (CTCs) in human blood provides a non-invasive approach for the evaluation of cancer metastasis and early cancer diagnosis. Herein, we demonstrate the controllable assembly of a quantum dot (QD)-based aptasensor guided by CRISPR/Cas12a for direct measurement of CTCs in human blood. We introduce a magnetic bead@activator/recognizer duplex core-shell structure to construct a multifunctional platform for the capture and direct detection of CTCs in human blood, without the need for additional CTC release and re-identification steps. Notably, the introduction of magnetic separation ensures that only a target-induced free activator can initiate the downstream catalysis, efficiently avoiding the undesired catalysis triggered by inappropriate recognition of the activator/recognizer duplex structure by crRNAs. This aptasensor achieves high CTC-capture efficiency (82.72%) and sensitive detection of CTCs with a limit of detection of 2 cells mL-1 in human blood, holding great promise for the liquid biopsy of cancers.
Asunto(s)
Células Neoplásicas Circulantes , Puntos Cuánticos , Humanos , Células Neoplásicas Circulantes/patología , Puntos Cuánticos/química , Sistemas CRISPR-Cas/genética , Biopsia LíquidaRESUMEN
Herein, we develop a dual-mode biosensor for photoelectrochemical and colorimetric detection of organophosphate pesticides (OPPs) based on ultrathin-FeOOH-coated MnO2 (MO@FHO) nanozyme. In this biosensor, OPPs can inhibit the alkaline phosphatase (ALP) activity and hinder the dephosphorylation of l-ascorbic acid-2-phosphate, preventing the decomposition of MO@FHO nanozyme and inducing both a photoelectrochemical (PEC) signal and the colorimetric change. The MO@FHO nanozyme not only possesses an enhanced catalase-like activity to degrade H2O2 for the generation of an improved cathodic photocurrent, but also exhibits an excellent oxidase-like activity to oxidize 3,3,5,5-tetramethylbenzidine with high catalytic efficiency. This biosensor displays a detection limit of 50 pmol/L for the PEC mode and a detection limit of 0.8 nmol/L for the colorimetric mode. Moreover, this biosensor exhibits excellent performance in complex biological matrices, and the smartphone-based visual sensing platform facilitates rapid and sensitive detection of OPPs, holding promising applications in food safety monitoring, and on-site detection.
Asunto(s)
Técnicas Biosensibles , Insecticidas , Plaguicidas , Catalasa , Compuestos Organofosforados , Colorimetría , Peróxido de Hidrógeno , Compuestos de Manganeso , ÓxidosRESUMEN
The reported organic electrochemiluminescence (ECL) luminophors for the detection of various markers often suffer from intermolecular π-π stacking-induced luminophore quenching. Herein, we demonstrate one-pot synthesis of a new aggregation-induced electrochemiluminescence (AIECL) emitter (i.e., TPE@SiO2/rGO composite) for sensitive measurement of microcystin-leucine arginine (MC-LR). The TPE@SiO2/rGO composite is constructed by embedding the silica-encapsuled 1,1,2,2-tetra(4-carboxylphenyl)ethylene (TPE) in the reduced graphene oxide. In comparison with the monomer TPE, this composite exhibit high luminescence efficiency and strong ECL emission, because the AIECL phenomenon triggered by the spatial confinement effect in the SiO2 cage induces the restriction of the internal motion and vibration of molecules. Notably, this composite has distinct advantages of easy preparation, simple functionalization, and stable luminescence. Especially, the TPE@SiO2/rGO-based ECL-RET system exhibits a high quenching efficiency (ΦET) of 69.7%. When target MC-LR is present, it triggers DNA strand displacement reaction (SDR), inducing the quenching of the ECL signal of TPE@SiO2/rGO composite due to ECL resonance energy transfer between TPE@SiO2/rGO composite and methylene blue (MB). The proposed biosensor enables highly sensitive, low-cost, and robust measurement of MC-LR with a large dynamic range of 7 orders of magnitude and a detection limit of 3.78 fg/mL, and it displays excellent detection performance in complex biological matrices, holding potential applications in food safety and water monitoring.
Asunto(s)
Técnicas Biosensibles , Toxinas Marinas , Microcistinas , Dióxido de Silicio , Estilbenos , Vibración , Transferencia de Energía , Mediciones Luminiscentes , Técnicas Electroquímicas , Límite de DetecciónRESUMEN
Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3'-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10-10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , ADN , ADN de Cadena Simple/genética , ARN , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genéticaRESUMEN
We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10-8 U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.
Asunto(s)
Técnicas Biosensibles , Neoplasias , Humanos , Endonucleasas de ADN Solapado , Sistemas CRISPR-Cas/genética , Colorantes , Descubrimiento de DrogasRESUMEN
The development of highly sensitive and selective analytical approaches for monitoring enzymatic activity is critical for disease diagnosis and biomedical research. Herein, we develop an exogenous co-reactant-free electrochemiluminescence (ECL) biosensor for the ratiometric measurement of α-glucosidase (α-Glu) based on a zeolitic imidazolate framework (ZIF-67)-regulated pyrene-based hydrogen-bonded organic framework (HOF-101). Target α-Glu can hydrolyze maltose to α-d-glucose, which can subsequently react with GOx to produce gluconic acid. The resultant gluconic acid can dissolve ZIF-67, leading to the recovery of the HOF-101 cathodic ECL signal and the decrease of the luminol anodic ECL signal. The long-range ordered structure of HOF-101 can speed up charge transfer, resulting in a stable and strong cathodic ECL signal. Moreover, ZIF-67 can not only efficiently quench the ECL signal of HOF-101 due to ECL resonance energy transfer between HOF-101 and ZIF-67 as well as the steric hindrance effect of ZIF-67 but also enhance the anodic ECL emission of luminol in dissolved O2 system because of its ordered and porous crystalline structure and the atomically dispersed Co2+. Notably, HOF-101 possesses a higher ECL efficiency (32.22%) compared with the Ru(bpy)32+ standard. Importantly, this ratiometric ECL biosensor shows high sensitivity (a detection limit of 0.19 U L-1) and a broad linear range (0.2-50 U L-1). This biosensor can efficiently eliminate systematic errors and enhance detection reliability without the involvement of exogenous co-reactants, and it displays good assay performance in human serum samples, holding great promise in biomedical research studies.
Asunto(s)
Técnicas Biosensibles , Gluconatos , alfa-Glucosidasas , Humanos , Mediciones Luminiscentes/métodos , Reproducibilidad de los Resultados , Luminol , Técnicas Biosensibles/métodosRESUMEN
N6-Methyladenine (6mdA) and N4-methylcytosine (4mdC) are the two most dominant DNA modifications in both prokaryotes and eukaryotes, but standard hybridization-based techniques cannot be applied for the 6mdA/4mdC assay. Herein, we demonstrate the silver-coordinated Watson-Crick pairing-driven three-dimensional (3D) DNA walker for locus-specific detection of genomic 6mdA/4mdC at the single-molecule level. 6mdA-DNA and 4mdC-DNA can selectively hybridize with the binding probes (BP1 and BP2) to form 6mdA-DNA-BP1 and 4mdC-DNA-BP2 duplexes. The 6mdA-C/4mdC-A mismatches cannot be stabilized by AgI, and thus, 18-nt BP1/BP2 cannot be extended by the catalysis of KF exonuclease. Through toehold-mediated strand displacement (TMSD), the signal probe (SP1/SP2) functionalized on the gold nanoparticles (AuNPs) can competitively bind to BP1/BP2 in 6mdA-DNA-BP1/4mdC-DNA-BP2 duplex to obtain SP1-18-nt BP1 and SP2-18-nt BP2 duplexes. The resulting DNA duplexes can act as the substrates of lambda exonuclease, leading to the cleavage of SP1/SP2 and the release of Cy3/Cy5 and 18-nt BP1/BP2. The released 18-nt BP1/BP2 can subsequently serve as the walker DNA, moving along the surface of the AuNP to activate dynamic 3D DNA walking and releasing abundant Cy3/Cy5. The released Cy3/Cy5 can be quantified by single-molecule imaging. This nanosensor exhibits high sensitivity with a limit of detection (LOD) of 9.80 × 10-15 M for 6mdA-DNA and 9.97 × 10-15 M for 4mdC-DNA. It can discriminate 6mdA-/4mdC-DNA from unmodified genomic DNAs, distinguish 0.01% 6mdA-/4mdC-DNA from excess unmethylated DNAs, and quantify 6mdA-/4mdC-DNA at specific sites in genomic DNAs of liver cancer cells and Escherichia coli plasmid cloning vector, providing a new platform for locus-specific analysis of 6mdA/4mdC in genomic DNAs.
Asunto(s)
Adenina/análogos & derivados , Carbocianinas , Citosina/análogos & derivados , Nanopartículas del Metal , Plata , Oro , Nanopartículas del Metal/química , ADN , Genómica , ExonucleasasRESUMEN
We construct a single quantum dot-based nanosensor for piRNA detection based on ligation-mediated multi-cycle signal amplification. This nanosensor is homogenous, selective, and sensitive with a detection limit of 0.104 fM. Moreover, it can detect the endogenous piRNA level in different cell lines, and discriminate cancer tissues from normal tissues.