RESUMEN
Sensitive detection of resistance mutation T790 M is of great significance for early diagnosis and prognostic monitoring of non-small-cell lung cancer (NSCLC). In this paper, we showed a highly sensitive detection strategy for T790 M using a three-level characteristic current signal pattern in an α-hemolysin nanopore. A probe was designed that formed a C-T mismatched base pair with wild-type/P and a T-T mismatched with the T790M/P. The T790M/P produced a unique three-level characteristic current signal in the presence of mercury ions(II): first, T790M-Hg2+-P entering the vestibule of α-HL under the transmembrane potential and overhang of probe occupying the ß-barrel, then probe unzipping from the T790M/P, T790 M temporally residing inside the nanocavity due to the interaction with Hg(II), and finally T790 M passing through the ß-barrel. The blocking current distribution was concentrated with a small relative standard deviation of about 3%, and the signal peaks of T790 M and wild-type can be completely separated with a high separation resolution of more than 2.5, which achieved the highly sensitive detection of T790 M down to 0.001 pM (confidence level P 95%) with a linear range from 0.001 pM to 1 nM in human serum samples. This highly sensitive recognition strategy enables the detection of low abundance T790 M and provides a method for prognostic monitoring in NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Mercurio , Nanoporos , Humanos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Timina , Proteínas Hemolisinas/genética , Receptores ErbB/genética , Mutación , Inhibidores de Proteínas QuinasasRESUMEN
Multiplex detection can enhance diagnostic precision and improve diagnostic efficiency, providing important assistance for epidemiological investigation and epidemic prevention. There is a great need for multi-detection sensing platforms to accurately diagnose diseases. Herein, we reported a µPAD-based chemiluminescence (CL) assay for ultrasensitive multiplex detection of AIV biomarkers, based on three DNAzyme/Lum/PEI/CaCO3. Three time-resolved CL signals were sequentially generated with detection limits of 0.32, 0.34, and 0.29 pM for H1N1, H7N9, and H5N1, respectively, and with excellent selectivity against interfering DNA. The recovery test in human serum displayed satisfactory analysis capabilities for complex biological samples. The µPAD-based CL assay achieved multiplex detection within 70 s, with a high time resolution of 20 s. The proposed strategy has the advantages of low cost, high sensitivity, good selectivity, and wide time resolution, the µPAD-based CL assay has shown great potential in the early and accurate diagnosis of diseases.
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Biomarcadores , Mediciones Luminiscentes , Mediciones Luminiscentes/métodos , Humanos , Biomarcadores/sangre , Biomarcadores/análisis , Papel , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H7N9 del Virus de la Influenza A/aislamiento & purificación , Subtipo H7N9 del Virus de la Influenza A/genética , Animales , Gripe Aviar/diagnóstico , Gripe Aviar/virología , ADN Catalítico/química , ADN Catalítico/metabolismo , Aves/virología , Límite de Detección , Gripe Humana/diagnóstico , Gripe Humana/virología , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
With the aim of detecting low frequency of drug resistant mutation T790M against wild-type sequences, we reported a two-dimensional signal analysis strategy by combining a three locked nucleic acids (LNAs)-modified probe (LP15-3t) and an α-HL nanopore. The specific hybridization of the LP15-3t probe with the T790M generated unique long two-level signals, including characteristic blocking current and characteristic dwell time. Due to the significant dwell time difference (114.2-fold) and the blocking current difference ranging from 81% to 96%, this two-dimensional signal analysis strategy can simultaneously distinguish T790M sequences with a sensitivity of 0.0001% against wild-type sequences. The LOD of T790M was 0.1 pM. This high discrimination capability would have great potential in the detection of rare mutation sequences and the early monitoring of clinical outcome of NSCLC patients with TKI drug resistance.
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Resistencia a Antineoplásicos/genética , Receptores ErbB/sangre , Nanoporos , Oligonucleótidos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Análisis Discriminante , Receptores ErbB/genética , Humanos , Límite de Detección , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Células Madre Neoplásicas/metabolismo , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
An enzyme-free and label-free visual sensing strategy was developed for sensitively detecting thrombin using a plasmonic nanoplatform. Both the thrombin-triggered catalytic hairpin assembly (CHA) amplification reaction and G-quadruplex/hemin DNAzyme-controlled plasmonic signal readout were engineered on an electrospun nanofibrous membrane. Owing to its large specific surface area and porous structure, the nanofibrous membrane enhanced the loading capacity of B-H2 and the interface interaction efficiency. This plasmonic nanoplatform was used to perform the sensitive and naked-eye detection of thrombin as low as 1.0 pM in human serum samples. This visual strategy can discriminate thrombin from other co-existing proteins very well. Moreover, the visual sensing platform exhibited excellent reusability and long-term stability. The proposed enzyme-free and label-free plasmonic nanoplatform is low-cost, easy to operate and highly sensitive, and has potential applications in the point-of-care detection of protein biomarkers.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , G-Cuádruplex , Trombina/análisis , ADN Catalítico/química , Hemina/química , Humanos , Límite de Detección , Nanofibras/químicaRESUMEN
A rapid room-temperature DNA nanowires preparation strategy on the basis of self-replicating catalyzed hairpin assembly (SRCHA) was reported. In this system, three hairpin probes (P1, P2, and P3) were well-designed and partially hybridize to each other, and two split trigger DNA sequences were integrated into P1 and P3, respectively. When the SRCHA was initiated by the trigger DNA, a series of DNA assembly steps based on the toehold-mediated DNA strand displacement were activated, and the Y shaped DNA (P1-P2-P3) was formed. In that case, the two split trigger DNA sequences will come into close-enough proximity to form the trigger DNA replicas, which can initiate the additional SRCHA reaction cycles for DNA nanowire preparation, and eventually a rapid room-temperature DNA nanowires preparation strategy without need of fuel strands was successfully developed. Furthermore, the prepared DNA nanowires have been used to develop a rapid and signal amplified sensing platform for sensitive adenosine triphosphate (ATP) detection.
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Adenosina Trifosfato/análisis , Técnicas Biosensibles , ADN/química , Nanocables/química , TemperaturaRESUMEN
Identification of single-base mismatches has found wide applications in disease diagnosis, pharmacogenetics, and population genetics. However, there is still a great challenge in the simultaneous discrimination of single-base mismatch and full match. Combined with a nanopore electrochemical sensor, a shared-stem structure of molecular beacon was designed that did not need the labeling, but achieved an enhanced signal-to-background ratio of â¼104, high thermodynamic stability to bind with target sequences, and a fast hybridization rate. Fully matched and single-base mismatched sequences were simultaneously discriminated at the single-molecule level, which is expected to have potential applications ranging from the quick detection, early clinical diagnostics to point-of-care research.
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Disparidad de Par Base , Técnicas Biosensibles/métodos , Estudios de Factibilidad , Modelos Moleculares , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Relación Señal-Ruido , Termodinámica , Factores de TiempoRESUMEN
A plasmonic nanoplatform to perform an enzyme-free, naked-eye, and trace discrimination of single-base mutation from fully matched sequence is reported. The nanoplatform showed great potential to enhance catalytic hairpin assembly (CHA) amplification efficiency and biocatalytic activity of hemin/G-quadruplex (DNAzyme). When human immunodeficiency virus (HIV) DNA biomarker was used as the model analyst, a naked-eye detection with high selectivity and high sensitivity down to 10-17 M in whole serum was achieved by observing red-to-blue color change. Single-base mismatch and two-base mismatch were detected at the low concentrations of 10-11 and 10-8 M, respectively. The naked-eye detection based on the enzyme-free plasmonic nanoplatform is expected to have potential applications ranging from quick detection and early diagnostics to point-of-care research.
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Disparidad de Par Base/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Color , ADN Catalítico , ADN Viral/sangre , G-Cuádruplex , VIH/genética , Hemina , Humanos , Nanotecnología/métodos , Sensibilidad y EspecificidadRESUMEN
Demethyleneberberine (DMB) is an essential metabolite of Berberine (BBR) in vivo. Recent reports have revealed multiple novel therapeutic applications of BBR. However, the pharmacological activities of DMB remain to be elucidated. This study aimed to demonstrate the hepatoprotective and anti-fibrotic effects of DMB both in vitro and in vivo. Here we showed that DMB protects against thioacetamide (TAA)-induced hepatic fibrosis in mice and exhibits a higher safety profile as compared to BBR. Flow cytometry and Western blotting analysis showed that DMB is able to suppress the activation of hepatic stellate cells (HSCs) and induce cell apoptosis through the nuclear factor-κB (NF-κB) cascade. Immunohistochemical (IHC) and quantitative polymerase chain reaction (qPCR) analysis indicated that DMB also has inhibitory effects on collagen synthesis and is able to increase collagen degradation by blocking the transforming growth factor ß 1 (TGF-ß1)-Smad signaling and reducing the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs). These findings indicate that DMB has the potential to attenuate hepatic fibrosis via suppressing HSC activation.
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Berberina/análogos & derivados , FN-kappa B/metabolismo , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Berberina/farmacología , Línea Celular , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos ICR , Ratas , Proteínas Smad/metabolismo , Tioacetamida/toxicidad , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Enzyme-free signal amplification has enabled sensitive in vitro detection of biomolecules such as proteins and nucleic acids. However, monitoring targets of interest in live cells via enzyme-free amplification is still challenging, especially for analytes with low concentrations. To the best of our knowledge, this paper reports the first attempt to perform mRNA imaging inside live cells, using a nonenzymatic hairpin DNA cascade reaction for high signal gain, termed a hairpin DNA cascade amplifier (HDCA). In conventional nucleic acid probes, such as linear hybridization probes, mRNA target signaling occurs in an equivalent reaction ratio (1:1), whereas, in HDCA, one mRNA target is able to yield multiple signal outputs (1:m), thus achieving the goal of signal amplification for low-expression mRNA targets. Moreover, the recycled mRNA target in the HDCA serves as a catalyst for the assembly of multiple DNA duplexes, generating the fluorescent signal of reduced MnSOD mRNA expression, thus indicating amplified intracellular imaging. This programmable cascade reaction presents a simple and modular amplification mechanism for intracellular biomarkers of interest, providing a significant boost to the search for clues leading to the accurate identification and effective treatment of cancers.
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ADN/química , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , ARN Mensajero/química , Línea Celular Tumoral , HumanosRESUMEN
Excessive alcohol consumption induces oxidative stress and lipid accumulation in the liver. Mitochondria have long been recognized as the key target for alcoholic liver disease (ALD). Recently, the artificial mitochondria-targeted antioxidant MitoQ has been used to treat ALD effectively in mice. Here, we introduce the natural mitochondria-targeted antioxidant demethyleneberberine (DMB), which has been found in Chinese herb Cortex Phellodendri chinensis. The protective effect of DMB on ALD was evaluated with HepG2 cells and acutely/chronically ethanol-fed mice, mimicking two common patterns of drinking in human. The results showed that DMB, which is composed of a potential antioxidant structure, could penetrate the membrane of mitochondria and accumulate in mitochondria either in vitro or in vivo. Consequently, the acute drinking-caused oxidative stress and mitochondrial dysfunction were significantly ameliorated by DMB. Moreover, we also found that DMB suppressed CYP2E1, hypoxia inducible factor α, and inducible nitric oxide synthase, which contributed to oxidative stress and restored sirtuin 1/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ coactivator-1α pathway-associated fatty acid oxidation in chronic ethanol-fed mice, which in turn ameliorated lipid peroxidation and macrosteatosis in the liver. Taking these findings together, DMB could serve as a novel and potential therapy for ALD in human beings.
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Antioxidantes/farmacología , Berberina/análogos & derivados , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Antioxidantes/uso terapéutico , Berberina/farmacología , Berberina/uso terapéutico , Citocromo P-450 CYP2E1/metabolismo , Modelos Animales de Enfermedad , Etanol/efectos adversos , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Hepatopatías Alcohólicas/tratamiento farmacológico , Hepatopatías Alcohólicas/etiología , Masculino , Ratones , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Oxidación-Reducción/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal/efectos de los fármacos , Sirtuina 1/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.
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Computadores Moleculares , Ácidos Nucleicos/química , Animales , Secuencia de Bases , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Nanotecnología/instrumentación , Conformación de Ácido NucleicoRESUMEN
Lanthanide doped multicolor luminescent materials have attracted extensive attention due to their advanced anti-counterfeiting properties. However, designing a simple, hard-to-copy and multicolor anti-counterfeiting strategy based on upconversion nanoparticles (UCNPs) remains a huge challenge. Herein, a strategy to modulate luminescence color by altering the mediating action of Tm3+ was proposed. As a proof of concept, the mediating action of Tm3+ was explored in NaYbF4:30%Er,1%Tm@NaYF4 by changing the doping ratio of Yb3+/Er3+/Tm3+, and red, yellow and blue luminescence was successfully obtained. Then, NaYbF4:x%Er,1%Tm@NaYF4 (x = 2, 10, 30, 50, 99), NaYbF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) and NaYbF4:1%Tm@NaYF4:x%Er@NaYF4 (x = 2, 10, 30, 50, 100) were synthesized to further identify that the mediating action of Tm3+ was related to the doping ratio and distance between dopant ions. In addition, the luminescence color of NaYbF4:30%Er,1%Tm@NaYF4 changed from red to yellow with the increase of excitation power density. Based on the above, NaYbF4:Er,Tm@NaYF4 UCNPs show excellent performance in anti-counterfeiting of paintings, thus revealing their great potential in advanced anti-counterfeiting applications.
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Dental caries is one of the most prevalent and biofilm-associated oral diseases in humans. Streptococcus mutans, with a high ability to form biofilms by adhering to hard surfaces, has been established as an important etiological agent for dental caries. Therefore, it is crucial to find a way to prevent the formation of cariogenic biofilm. Here, we report an electrospun fibrous membrane that could inhibit the adhesion and biofilm formation of S. mutans. Also, the polystyrene (PS)/polyvinyl pyrrolidone (PVP) electrospun fibrous membrane altered the 3D biofilm architecture and decreased water-insoluble extracellular polysaccharide production. Notably, the anti-adhesion mechanism which laid in Coulomb repulsion between the negatively charged PS/PVP electrospun fibrous membrane and S. mutans was detected by zeta potential. Furthermore, metagenomics sequencing analysis and CCK-8 assay indicated that PS/PVP electrospun fibrous membrane was microbiome-friendly and displayed no influence on the cell viability of human gingival epithelial cells and human oral keratinocytes. Moreover, an in vitro simulation experiment demonstrated that PS/PVP electrospun fibrous membrane could decrease colony-forming unit counts of S. mutans effectively, and PS/PVP electrospun fibrous membrane carrying calcium fluoride displayed better anti-adhesion ability than that of PS/PVP electrospun fibrous membrane alone. Collectively, this research showed that the PS/PVP electrospun fibrous membrane has potential applications in controlling and preventing dental caries.
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Polystyrene electrospun optical fibrous membrane (EOF) was fabricated using a one-step electrospinning technique, functionalized with glucose oxidases (GOD/EOF), and used as a quick and highly sensitive optical biosensor. Because of the doped iridium complex, the fibrous membrane emitted yellow luminescence (562 nm) when excited at 405 nm. Its luminescence was significantly enhanced with the presence of extremely low concentration glucose. The detection limit was of 1.0 × 10(-10) M (S/N = 3), superior to that of reported glucose biosensor with 1.2 × 10(-10) M. A linear range between the relative intensity increase and the logarithm of glucose concentration was exhibited from 3.0 × 10(-10) M to 1.3 × 10(-4) M, which was much wider than reported results. Notably, the response time was less than 1 s. These high sensitivity and fast response were attributed to the high surface-area-to-volume of the porous fibrous membrane, the efficient GOD biocatalyst reaction on the fibers surface, as well as the fast electron or energy transfer between dissolved oxygen and the optical fibrous membrane.
Asunto(s)
Técnicas Biosensibles , Glucemia/análisis , Tecnología de Fibra Óptica , Iridio/química , Compuestos Organometálicos/química , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Humanos , Mediciones Luminiscentes , Estructura Molecular , Tamaño de la Partícula , Poliestirenos/química , Propiedades de SuperficieRESUMEN
Ruthenium nanoparticle tris(2,2'-bipyridyl)ruthenium(II) bis(hexafluorophosphate) (Ru(bpy)3(PF6)2, RuNP) was self-assembled on polystyrene (PS) electrospun microfibers. The formation of RuNP is attributed to the sulfonated PS (SPS) microfibers' high adsorptive capability of 94% for Ru(bpy)3(2+), as well as the strong interaction between the Ru(bpy)3(2+) and ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6). The RuNP/SPS microfibers exhibited an enhanced electrochemiluminescence (ECL) emission, 2.3 times higher than that from Ru(bpy)3(2+)/SPS microfibers and 6.6 times higher than that from Ru(bpy)3(2+)/SPS continuous thin films. It is worthy of note that, as a result of the hydrophobic nature of the RuNP, the transfer of water-insoluble α-naphthol is accelerated, and thus the α-naphthol ECL quenching efficiency is enhanced. An ECL sensor based on the RuNP/SPS microfibers was fabricated and used to detect low concentrations of α-naphthol. The detection limit was of 1.0 nM (S/N > 3), and the linear response ranged from 0 to 18 µM. This sensor has been successfully applied to measure the α-naphthol content in pesticide carbaryl samples. Our work provides a very simple and cost-effective method to fabricate RuNP on polymer microfibers with great potential in the field of chemo/biosensors.
RESUMEN
The term hydrogel describes a type of soft and wet material formed by cross-linked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the chemical properties of their constituent polymer(s). However, a group of hydrogels, called "smart hydrogels," changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this Review, DNA-based hydrogels are discussed in terms of their stimulus response, as well as their applications.
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ADN/química , Hidrogeles/química , Aptámeros de Nucleótidos/química , Materiales Biocompatibles/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Portadores de Fármacos/química , Nanopartículas del Metal/químicaRESUMEN
Developing functionalized nanomaterials with strong chemiluminescence (CL) properties is highly significant for ultrasensitive bioanalysis. Here, we report chitosan (CS), luminol, and Co2+-functionalized flower-like gold nanoparticles (Co2+/CS/Lum/AuNFs) with strong CL for the label-free sensing of the HCV core protein (HCVcp). The Co2+/CS/Lum/AuNFs exhibited a greatly enhanced CL emission at around 425 nm, which is 50 times stronger than that of CS/Lum/AuNFs, and is superior to other commonly reported CL nanomaterials. The HCVcp aptamer (HCVcp-apt) further functionalized the surface of the Co2+/CS/Lum/AuNFs through electrostatic interactions blocked the Co2+ catalytic site, depressing the CL. Owing to the high affinity of HCVcp for the HCVcp-apt, the presence of HCVcp predominated its binding and effectively separated the HCVcp-apt from the surface of the Co2+/CS/Lum/AuNFs, so that the CL intensity was significantly enhanced. As the results showed, the HCVcp-apt/Co2+/CS/Lum/AuNFs were successfully used to detect the HCVcp in human serum samples with a linear range from 0.50 ng mL-1 to 1.00 µg mL-1, a detection limit of 0.16 ng mL-1 and an excellent selectivity over other analogs. The strategy is universal for the development of the ultrasensitive detection of other proteins in the field of early disease diagnostics.
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Quitosano , Hepatitis C , Nanopartículas del Metal , Humanos , Nanopartículas del Metal/química , Oro/química , Luminiscencia , Luminol/química , Quitosano/química , Hepatitis C/diagnósticoRESUMEN
Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.
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Técnicas Biosensibles , Nanoporos , Ácidos Nucleicos , Escherichia coli/genética , NanotecnologíaRESUMEN
A simple and cost-effective method was reported to synthesize small size (6 nm) gold nanoparticles (AuNPs) on polyacrylonitrile (PAN) electrospun nanofibers (AuNPs/PAN). The formation of AuNPs is attributed to the in situ reduction of Au(III) to Au(0) by 4-(dimethylamino)benzaldehyde doped in the PAN nanofibers. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) confirmed that the AuNPs/PAN nanofibers showed good conductivity. The AuNPs/PAN nanofibers were used to immobilize tris(2,2'-bipyridyl)ruthenium(II) ions (Ru(bpy)(3)(2+)) to form an electrochemiluminescence (ECL) sensor. The AuNPs on the PAN nanofibers exhibited an excellent catalytic effect on the ECL of Ru(bpy)(3)(2+) which could be employed to detect low concentrations of phenolic compounds. The linear response range of the ECL sensor to hydroquinone is 0.55-37 µM with limit of detection of 80 nM (S/N = 3). This sensor has been successfully applied to determine the hydroquinone content in photographic developer samples. Our work provides a very simple and cost-effective method to synthesize AuNPs on polymer nanofibers which shows great potential in the field of electrocatalysis and chemo/biosensors.
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The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.