RESUMO
Nanozyme-based chemodynamic therapy (CDT) for fighting bacterial infections faces several major obstacles including low hydrogen peroxide (H2 O2 ) level, over-expressed glutathione (GSH) in infected sites, and inevitable damage to healthy tissue with abundant nonlocalized nanozymes. Herein, a smart ultrasmall Fe3 O4 -decorated polydopamine (PDA/Fe3 O4 ) hybrid nanozyme is demonstrated that continuously converts oxygen into highly toxic hydroxyl radical (â¢OH) via GSH-depleted cascade redox reactions for CDT-mediated bacterial elimination and intensive wound disinfection. In this system, photonic hyperthermia of PDA/Fe3 O4 nanozymes can not only directly damage bacteria, but also improve the horseradish peroxidase-like activity of Fe3 O4 decorated for CDT. Surprisingly, through photothermal-enhanced cascade catalytic reactions, PDA/Fe3 O4 nanozymes can consume endogenous GSH for disrupting cellular redox homeostasis and simultaneously provide abundant H2 O2 for improving â¢OH generation, ultimately enhancing the antibacterial performance of CDT. Such PDA/Fe3 O4 can bind with bacterial cells, and reveals excellent antibacterial property against both Staphylococcus aureus and Escherichia coli. Most interestingly, PDA/Fe3 O4 nanozymes can be strongly retained in infected sites by an external magnet for localized long-term in vivo CDT and show minimal toxicity to healthy tissues and organs. This work presents an effective strategy to magnetically retain the therapeutic nanozymes in infected sites for highly efficient CDT with good biosafety.
Assuntos
Radical Hidroxila , Oxigênio , Desinfecção , Glutationa , Peróxido de Hidrogênio , Indóis , Oxirredução , PolímerosRESUMO
We successfully constructed a new class of nanoflares based on ultra-thin silica-coated gold nanoparticles (Au@SiO2) with the covalent binding of nucleic acids, which demonstrated more resistance to biothiols than that exhibited in the traditional Au-S binding strategy, for imaging the target miRNA-21 with high fidelity in living cells.
Assuntos
Nanopartículas Metálicas , MicroRNAs , Ouro , Dióxido de Silício , Nanopartículas Metálicas/toxicidade , Diagnóstico por Imagem , MicroRNAs/genéticaRESUMO
As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (ΔfimA and ΔfimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, ΔfimA, and ΔfimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation. KEY POINTS: ⢠Reduce the number of fimbriae. ⢠Berberine targeting fimA. ⢠Effective biofilm inhibitor.
Assuntos
Berberina , Salmonella typhimurium , Berberina/farmacologia , Biofilmes , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Humanos , Salmonella typhimurium/genéticaRESUMO
Herein, a simple all-in-one biosensor based on a DNA three-way junction has been constructed for in situ simultaneous detection of multiple miRNAs by competitive strand displacement. In our design, three oligonucleotides (Y1, Y2 and Y3) of a Y-type scaffold were extended at their 5' ends by introducing three single-stranded recognition sequences with quenchers (BHQ1, BHQ2 and BHQ2), respectively. Subsequently, three reporter sequences labeled with different fluorophores (FAM, Cy3 and Cy5) were bound to the corresponding recognition sequences to form a multicolour DNA biosensor that gives self-quenched fluorescence. The biosensor can effectively enter into exosomes and then hybridize to the complementary miRNA targets to form longer duplexes and release the reporter sequences, thus activating the readable fluorescence signals for the simultaneous detection of multiple miRNAs in exosomes. As a proof of principle, miR-21, miR-27a and miR-375 were chosen as model targets because of their high expressions in breast cancer cells (MCF-7). Fluorescence signals of MCF-7 exosomes after being treated with the biosensor exhibited positive correlations to their concentrations and the limits of detection were determined to be 0.116 µg mL-1, 0.125 µg mL-1 and 0.287 µg mL-1 for exosomes by detecting three exosomal miRNAs (miR-21, miR-27a and miR-375), respectively. In contrast, there were no obvious correlations between fluorescence intensities and control MCF-10A exosome concentrations. Importantly, by testing multiple exosomal miRNAs using the biosensor in clinical serum samples, breast cancer patients can be effectively differentiated from healthy donors. Consequently, the developed biosensor demonstrates high potential as a routine bioassay for the multiplex quantification of exosomal miRNAs in clinical diagnosis.
Assuntos
Técnicas Biossensoriais/métodos , Neoplasias da Mama/diagnóstico , Exossomos/metabolismo , MicroRNAs/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Feminino , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Células MCF-7RESUMO
MicroRNAs (miRNAs) have been shown to be promising biomarkers for disease diagnostics and therapeutics. However, the rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their characters of small size, vulnerability to degradation, low abundance, and sequence similarity. Herein, we describe an enzyme-free amplification platform, consisting of a catalytic hairpin assembly (CHA) and DNA-templated silver nanoclusters (DNA/AgNCs), for miRNA analysis. In this work, two DNA hairpins (H1 and H2) were first designed for target miR-21-induced CHA, and then the fluorescence of DNA/AgNCs was quenched by BHQ1 to construct an activatable probe (AP). In the presence of target miR-21, hairpin H1 was opened by miR-21 through a hybridization reaction, and hairpin H2 was then opened by H1. During this process, miR-21 was released from H1 and participated in the next round of hybridization, triggering the CHA cycle reaction. The obtained H1-H2 products with sticky ends could react with the AP, forcing BHQ1 away from the DNA/AgNCs and thus causing the fluorescence recovery of the DNA/AgNCs. The assay for miR-21 detection demonstrated an excellent linear response to concentrations varying from 200 pM to 20 nM with the detection limit of 200 pM. The simple and cost-effective strategy holds great potential for application in biomedical research and clinical diagnostics.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Catálise , DNA/genética , Limite de Detecção , MicroRNAs/genética , Prata , Espectrometria de FluorescênciaRESUMO
A sandwich-type surface-enhanced Raman scattering (SERS) sensor using dual aptamers and gold-enhanced Raman signal probes has been successfully constructed for the detection of tumor-derived extracellular vesicles. The simple and sensitive sensor has the capability to detect tumor extracellular vesicles in 10-fold diluted human serum samples.
Assuntos
Vesículas Extracelulares , Nanopartículas Metálicas , Neoplasias , Ouro , Humanos , Neoplasias/diagnóstico , Análise Espectral RamanRESUMO
A fluorescence method based on functionalized magnetic nanoparticles (FMNPs) and hybridization chain reaction (HCR) is developed for the enzyme-free amplified determination of thrombin. In the proposed design, aptamer against thrombin was hybridized with the capture DNA-modified magnetic nanoparticles to yield the FMNPs. In the presence of thrombin, aptamers are released due to the specific and high-affinity binding between thrombin and its aptamer. The exposed capture DNA subsequently hybridized with the partial sequence of helper DNA, and the vacant sequence of helper DNA further hybridized with HCR products which is pre-formed by the alternate hybridization of single-stranded DNAs (H1 and H2). The immobilized HCR products were then labeled with YOYO-1 for fluorescence measurement. Fluorescence signal intensity of labeled YOYO-1 was measured at an emission wavelength of 519 nm (excitation under 488 nm) and used for calibration. By taking advantage of HCR amplification, this direct assay strategy showed a linear response in the 20- to 200-pM concentration range, and the limit of detection is 9.2 pM which is about 3-orders of magnitude lower than the serum thrombin concentration (10 nM) that triggers blood clotting. This developed method can efficiently differentiate the target protein from a protein matrix, and it is verified by determination of thrombin in spiked serum samples with recoveries in the range of 94.5-103.3%. Graphical abstract A fluorometry method for thrombin detection using magnetic nanoparticles and enzyme-free hybridization chain reaction.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Fluorometria , Nanopartículas de Magnetita/química , Hibridização de Ácido Nucleico , Trombina/análise , HumanosRESUMO
Tumor exosomes (Exo) are presumed to expedite both the growth and metastasis of tumors by actively participating in nearly all aspects of cancer development. Tumor-derived Exos are thus proposed as a resource for diagnostic biomarkers in bodily fluids. However, most Exo assays require large samples and are time-consuming, complicated, and costly, and thus unsuited for practical applications. Herein, we show an ultrasensitive assay that can directly visualize and quantify tumor Exos in plasma microsamples (1 µL) at the single-vesicle level. The assay uses the specific binding of activatable aptamer probes (AAP) to target Exos captured by Exo-specific antibodies on the surface of a flow cell to produce activated fluorescence. Furthermore, the bound AAP triggers in situ assembly of a DNA nanodevice with enhanced fluorescence that improves the Exo-detection sensitivity. By identifying tyrosine-protein-kinase-like 7 (PTK7), a total-internal-reflection-fluorescence (TIRF) assay for PTK7-Exo distinguishes target tumors from control subjects. This assay is also informative in monitoring tumor progression and early responses to therapy. The developed assay can be readily adapted for diagnosis and monitoring of other disease-associated Exo biomarkers.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Exossomos/patologia , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Desenho de Equipamento , Exossomos/química , Fluorescência , Humanos , Masculino , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Neoplasias/sangue , Neoplasias/química , Imagem Óptica/instrumentação , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/sangueRESUMO
Exosomes are cell-derived small extracellular vesicles that are naturally secreted by all types of cells and widely distributed in various biofluids. They carry a variety of key bioactive molecules (e.g., nucleic acids, proteins, growth factors, cytokines) from their parent cells and convey them to neighboring or even distant cells through circulation. In recent years, tumor-derived exosomes have attracted great interest from investigators because they actively participate in nearly all aspects of tumor development and facilitate both tumor growth and metastasis through exosome-mediated intercellular communication. The vesicular contents are increasingly considered potential biomarkers for tumor diagnoses and prognosis. With the progress made in isolation and analytical technologies, the functions of exosomes and their contents in tumor development are also becoming clearer. In this review article we describe the recent developments in exosome isolation techniques and analysis of exosomal contents. We also address their applications in cancer detection and therapy.
Assuntos
Exossomos/metabolismo , Neoplasias/diagnóstico , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Comunicação Celular , Portadores de Fármacos/química , Exossomos/química , Humanos , MicroRNAs/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteínas/metabolismo , RNA Mensageiro/metabolismoRESUMO
Intracellular pH (pHi) is an important parameter associated with cellular behaviors and pathological conditions. Sensing pHi and monitoring its changes are essential but challenging due to the lack of high-sensitive probes. Herein, a ratiometric fluorescent probe with ultra pH-sensitivity is developed based on hairpin-contained i-motif strand (I-strand, labeled with Rhodamine Green and BHQ2 at two termini) and complementary strand (C-strand, labeled with Rhodamine Red at its 5'-end). At neutral pH, both I-strand and C-strand hybridize into a rigid duplex (I-C), which holds the Rhodamine Red and the BHQ2 in close proximity. As a result, the fluorescence emission (F597 nm) of the Rhodamine Red is strongly suppressed, while the Rhodamine Green (F542 nm) is in a "signal on" state. However, the slightly acidic pH enforced the I-strand to form an intramolecular i-motif and initiated the dehybridization of I-C duplex, leading to Rhodamine Red in a "signal on" state and a decreased fluorescence of Rhodamine Green. The ratio (F542 nm/F597 nm) can be used as a signal for pH sensing. Due to the rational internal hairpin design of I-C duplex probe, almost 70-fold change in the ratio was observed in the physiological pH range (6.50-7.40). This probe possesses efficient stability, fast response, and reversible pH measurement capabilities. Furthermore, intracellular application of the ratiometric probe was demonstrated on the example of SMMC-7721 cells. With different recognition elements in engineering of i-motif based platforms, the design might hold great potential to become a versatile strategy for intracellular pH sensing.
Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Corantes Fluorescentes/química , Rodaminas/química , Linhagem Celular Tumoral , Citoplasma/química , Humanos , Concentração de Íons de Hidrogênio , Conformação de Ácido Nucleico , Imagem Óptica/métodos , Espectrometria de Fluorescência/métodosRESUMO
We herein report an efficient hybridization chain reaction (HCR)- and DNAzyme-based enzyme-free signal amplification for the detection of specific exosomal miRNAs in the culture medium of cancer cells and serum samples from cancer patients via the target-triggered self-assembly of the polymer DNAzyme nanostructure.
Assuntos
DNA Catalítico/química , Exossomos/genética , MicroRNAs/análise , Nanoestruturas , Neoplasias/diagnóstico , Hibridização de Ácido Nucleico , Técnicas Biossensoriais , Células HeLa , Humanos , Neoplasias/genética , PolímerosRESUMO
An enzyme-free and ultrasensitive fluorescence assay for the detection of nucleic acids was successfully established by a hybridization-triggered DNAzyme cascade (HTDC). This simple and cost-effective sensor has good sensitivity, selectivity and the capability for detection of target DNA from complex fluids.
Assuntos
Bioensaio/métodos , DNA Catalítico/química , DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , DNA/química , Fluorescência , Corantes Fluorescentes/química , Hibridização de Ácido Nucleico , Zinco/químicaRESUMO
A novel and adjustable lamp based on competitive interaction among dsDNA-SYBR Green I (SGI), ion quencher, and analyte was designed for bioanalysis. The "filament" and switch of the lamp could be customized by employing different dsDNA and ion quencher. The poly(AT/TA) dsDNA was successfully screened as the most effective filament of the lamp. Two common ions, Hg2+ and Fe3+, were selected as the model switch, and the corresponding ligand molecules cysteine (Cys) and pyrophosphate ions (PPi) were selected as the targets. When the fluorescence-quenched dsDNA/SGI-ion complex was introduced into a target-containing system, ions could be bound by competitive molecules and separate from the complex, thereby lighting the lamp. However, no light was observed if the biomolecule could not snatch the metal ions from the complex. Under the optimal conditions, sensitive and selective detection of Cys and PPi was achieved by the lamp, with practical applications in fetal bovine serum and human urine. This ion quencher regulated lamp for fluorescent bioassays is simple in design, fast in operation, and is more convenient than other methods. Significantly, as many molecules could form stable complexes with metal ions selectively, this ion quencher operated lamp has potential for the detection of a wide spectrum of analytes. Graphical abstract A novel and adjustable lamp on the basis of competitive interaction among dsDNA-SYBR Green I, ions quencher and analyte was designed for bioanalysis. The filament and switch of lamp could be customized by employing different dsDNA and ions quencher.
Assuntos
Corantes Fluorescentes/química , Ferro/química , Mercúrio/química , Benzotiazóis , Bioensaio/métodos , Cisteína/análise , DNA/química , Diaminas , Fluorescência , Íons , Compostos Orgânicos/química , QuinolinasRESUMO
Presented herein is a simple, robust, and label-free homogeneous electrochemical sensing platform constructed for the detection of protein kinase activity and inhibition by integration of carboxypeptidase Y (CPY)-assisted peptide cleavage reaction and vertically ordered mesoporous silica films (MSFs). In this sensing platform, the substrate peptide composed of kinase-specific recognized sequence and multiple positively charged arginine (R) residues was ingeniously designed. In the presence of protein kinase, the substrate peptide was phosphorylated and then immediately resisted CPY cleavage. The phosphorylated peptide could be effectively adsorbed on the negatively charged surface of MSFs modified indium-tin oxide (ITO) electrode (MSFs/ITO) by noncovalent electrostatic attraction. The adsorbed peptide was subsequently used as a hamper to prevent the diffusion of electroactive probe (FcMeOH) to the electrode surface through the vertically aligned nanopores, resulting in a detectable reduction of electrochemical signal. As demonstrated for the feasibility and universality of the sensing platform, both protein kinase A (PKA) and casein kinase II (CK2) were selected as the models, and the detection limits were determined to be 0.083 and 0.095 UmL-1, respectively. This sensing platform had the merits of simplicity, easy manipulation, and improved phosphorylation and cleavage efficiency, which benefited from homogeneous solution reactions without sophisticated modification or immobilization procedures. In addition, given the key role of inhibition and protein kinase activity detection in cell lysates, this proposed sensing platform showed great potential in kinase-related bioanalysis and clinical biomedicine.
Assuntos
Catepsina A/metabolismo , Técnicas Eletroquímicas/métodos , Proteínas Quinases/metabolismo , Dióxido de Silício/química , Catepsina A/química , Células HeLa , Humanos , Membranas Artificiais , Inibidores de Proteínas Quinases , Proteínas Quinases/químicaRESUMO
Measuring the levels of Fe3+ in human body has attracted considerable attention for health monitoring as it plays an essential role in many physiological processes. In this work, we reported a selective fluorescent nanoprobe for Fe3+ detection in biological samples based on ultrabright N/P codoped carbon dots. By employing adenosine 5'-triphosphate (ATP) as the carbon, nitrogen, and phosphorus source, the N/P codoped carbon dots could be simply prepared through hydrothermal treatment. The obtained carbon dots exhibited high quantum yields up to 43.2%, as well as excellent photostability, low toxicity, and water solubility. Because of the Fe-O-P bonds formed between Fe3+ and the N/P codoped carbon dots, this nanoprobe showed high selectivity toward Fe3+ against various potential interfering substances in the presence of EDTA. The fluorescence quenching of as-fabricated carbon dots was observed with the increasing Fe3+ concentration, and the calibration curve displayed a wide linear region over the range of 1-150 µM with a detection limit of 0.33 µM. The satisfactory accuracy was further confirmed with the river samples and ferrous sulfate tablets, respectively. With the above outstanding properties, these N/P codoped carbon dots were successfully applied for direct detection of Fe3+ in biological samples including human blood serum and living cells. As compared to the most reported carbon dots-based Fe3+ sensors, this nanoprobe showed high fluorescence, good accuracy, and excellent selectivity, which presents the potential practical application for diagnosis of Fe3+ related disease.
Assuntos
Carbono/química , Compostos Férricos/sangue , Corantes Fluorescentes/química , Nanopartículas/química , Pontos Quânticos/química , Células HeLa , Humanos , SolubilidadeRESUMO
Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.
Assuntos
Citoplasma/química , Pontos Quânticos/química , Carbono/química , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/química , Cloroquina/farmacologia , Dexametasona/química , Dexametasona/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Microscopia Confocal , Pontos Quânticos/toxicidade , Espécies Reativas de Oxigênio/análise , Compostos de Sulfidrila/análiseRESUMO
Herein, a simple, facile, and label-free electrochemiluminescence (ECL) aptasensor platform was constructed by integration of aptamer-gated systems and vertically ordered mesoporous silica films (MSFs) grown in suit of indium-tin oxide (ITO) electrode. In this aptasensor platform, aptamer could be effectively adsorbed on the surface of aminated MSFs by noncovalent electrostatic attraction and then employed as an ideal gate material to control the blocking and releasing of luminescence reagents (Ru(bipy)32+) entrapped within the pores of MSFs. In the presence of target, the specific aptamer-target binding could trigger the uncapping the pores, releasing the Ru(bipy)32+ with detectable reduced of ECL signal. The feasibility and universality of this design was validated by employing three aptamers that bind to lysozyme, adenosine, and K+ as gate materials, and the detection limits were determined to be 0.06 nM, 0.75 nM, and 0.5 µM, respectively. This ECL aptasensor, based on the simple competitive procedure, was simple design, undemanding, and fast in operation. In addition, no other chemical modification of the aptamer was required, suggesting that this ECL aptasensor could be applied to many other target detections just by altering the aptamer sequence.
RESUMO
Amino acid-dithiocarbamate (amino acid-DTC) was developed as both the reductant and ligand stabilizer for biomimetic synthesis of gold nanoparticles (AuNPs), which served as an excellent surface-enhanced Raman scattering (SERS) contrast nanoprobe for cell imaging. Glycine (Gly), glutamic acid (Glu), and histidine (His) with different isoelectric points were chosen as representative amino acid candidates to synthesize corresponding amino acid-DTC compounds through mixing with carbon disulfide (CS2), respectively. The pyrogenic decomposition of amino acid-DTC initiated the reduction synthesis of AuNPs, and the strong coordinating dithiocarbamate group of amino acid-DTC served as a stabilizer that grafted onto the surface of the AuNPs, which rendered the as-prepared nanoparticles a negative surface charge and high colloidal stability. MTT cell viability assay demonstrated that the biomimetic AuNPs possessed neglectful toxicity to the human hepatoma cell, which guaranteed them good biocompatibility for biomedical application. Meanwhile, the biomimetic AuNPs showed a strong SERS effect with an enhancement factor of 9.8 × 10(5) for the sensing of Rhodamine 6G, and two distinct Raman peaks located at 1363 and 1509 cm(-1) could be clearly observed in the cell-imaging experiments. Therefore, biomimetic AuNPs can be explored as an excellent SERS contrast nanoprobe for biomedical imaging, and the amino acid-DTC mediated synthesis of the AuNPs has a great potential in bio-engineering and biomedical imaging applications.
Assuntos
Aminoácidos/metabolismo , Carcinoma Hepatocelular/patologia , Ouro/química , Neoplasias Hepáticas/patologia , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Aminoácidos/química , Biomimética , Linhagem Celular Tumoral , Sobrevivência Celular , Diagnóstico por Imagem , Humanos , Tamanho da Partícula , Propriedades de Superfície , TiocarbamatosRESUMO
Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.
Assuntos
Enzimas/química , Ácidos Nucleicos/química , Colorimetria , Metilação de DNA , Proteínas/análiseRESUMO
Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probe's oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.