RESUMEN
Many interactions between small molecules and particles occur in solutions. They are surrounded by other molecules that do not react, for example, biological processes in water, chemical reactions in gas or liquid solutions, and environmental reactions in air and water. However, predicting the rate of such diffusive interactions remains challenging, due to the random motion of molecules in solutions, as exampled by the famous Brownian motion of pollen particles. In this report, we experimentally confirmed that a disruptive rate equation we have published before can predict the association rate of typical adsorption at interfaces, which has a surprising fraction order of 4/3 that has not been considered before. This could be an important step toward a generalized method to predict the adsorption rate of many reactions.
Asunto(s)
ADN , Compuestos de Quinolinio , Adsorción , ADN/química , Compuestos de Quinolinio/química , Ácidos Nucleicos Inmovilizados/química , BenzoxazolesRESUMEN
It is known that the abnormal expression of specific cellular miRNAs is closely related to cell apoptosis, and so monitoring the level change of these miRNAs can in principle be used to evaluate the process of apoptosis stimulated by drugs. Towards this goal, here we construct an ultrasensitive electrochemiluminescence (ECL) nanoplatform via the target miRNA-triggered immobilization of spherical nucleic acid enzymes (SNAzymes) onto tetrahedral DNA nanostructures on the electrode surface, which catalyzes the luminol-H2O2 reaction to output an ECL signal. This enables the sensitive and specific detection of two apoptosis-related miRNAs, miR-21 and miR-133a, with a detection limit of 33 aM. Furthermore, we employed the developed ECL nanoplatform to monitor the levels of these two miRNAs inside cancer cells stimulated by DOX, showing that the level of miR-21 decreases, while that of miR-133a increases in the early apoptotic cells. This difference highlights the distinct roles of the two target miRNAs, where miR-21 promotes the early apoptosis of cancer cells, whereas miR-133a suppresses it, providing new insight into cell physiological processes.
Asunto(s)
Apoptosis , Técnicas Electroquímicas , Límite de Detección , Mediciones Luminiscentes , Luminol , MicroARNs , MicroARNs/análisis , Humanos , Apoptosis/efectos de los fármacos , Mediciones Luminiscentes/métodos , Técnicas Electroquímicas/métodos , Luminol/química , Peróxido de Hidrógeno/química , Técnicas Biosensibles/métodos , Doxorrubicina/farmacología , Doxorrubicina/química , Nanoestructuras/química , ADN/química , ADN/genética , Electrodos , Células HeLa , Línea Celular Tumoral , Enzimas Inmovilizadas/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genéticaRESUMEN
Large DNA molecules (>20 kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.
Gel-Stacks technology provides multiple non-covalent molecular presentation modes, coupled with an unusually facile reagent delivery system designed for large-scale analytes, enhancing live imaging and manipulation. Enhanced further by modeling and software, Gel-Stacks technology becomes adaptable to a broad range of experimental applications.
Asunto(s)
ADN , ADN/química , Microscopía Fluorescente/métodos , Hidrogeles/química , Ácidos Nucleicos Inmovilizados/química , Resinas Acrílicas/química , Espermidina/química , Imagen Individual de Molécula/métodosRESUMEN
A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤103 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from Thermococcus kodakaraensis (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.
Asunto(s)
Enzimas Inmovilizadas , Dióxido de Silicio , ADN , ADN Polimerasa Dirigida por ADN/química , Enzimas Inmovilizadas/genética , Ácidos Nucleicos Inmovilizados/química , Reacción en Cadena de la Polimerasa/métodos , Dióxido de Silicio/químicaRESUMEN
The therapeutic use of small interfering RNAs (siRNAs) as gene regulation agents has been limited by their poor stability and delivery. Although arranging siRNAs into a spherical nucleic acid (SNA) architecture to form siRNA-SNAs increases their stability and uptake, prototypical siRNA-SNAs consist of a hybridized architecture that causes guide strand dissociation from passenger strands, which limits the delivery of active siRNA duplexes. In this study, a new SNA design that directly attaches both siRNA strands to the SNA core through a single hairpin-shaped molecule to prevent guide strand dissociation is introduced and investigated. This hairpin-like architecture increases the number of siRNA duplexes that can be loaded onto an SNA by 4-fold compared to the original hybridized siRNA-SNA architecture. As a result, the hairpin-like siRNA-SNAs exhibit a 6-fold longer half-life in serum and decreased cytotoxicity. In addition, the hairpin-like siRNA-SNA produces more durable gene knockdown than the hybridized siRNA-SNA. This study shows how the chemistry used to immobilize siRNA on nanoparticles can markedly enhance biological function, and it establishes the hairpin-like architecture as a next-generation SNA construct that will be useful in life science and medical research.
Asunto(s)
Ácidos Nucleicos Inmovilizados/farmacología , ARN Interferente Pequeño/farmacología , Línea Celular Tumoral , Silenciador del Gen/efectos de los fármacos , Oro/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/toxicidad , Secuencias Invertidas Repetidas , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Hibridación de Ácido Nucleico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/toxicidadRESUMEN
A simple fluorescence and electrochemical dual-channel biosensor based on bifunctional Zr(IV)-based metal-organic framework (Zr-MOF) was proposed to detect Ochratoxin A (OTA). The bifunctional Zr-MOF, with photoluminescence properties and enormous electroactive ligands, was exploited to load OTA-specific aptamers for designing signal probes, greatly simplifying the probe-fabrication process and improving sensing reliability. Upon specific recognition of aptamer toward OTA, the anchored probe was released from the sensing interface into the reaction solution. In this circumstance, the increased amount of the signal probe in reaction solution led to an enhanced fluorescence response, while the decreased amount of the signal probe on the sensing interface resulted in a diminished electrochemical response. According to the dual-channel signal change with increasing OTA concentration, the visual fluorescence strategy was established for intuitive OTA detection, and meanwhile, sensitive electrochemical assay with a detection limit of 0.024 pg/mL was also achieved with the help of one-step electrodeposition as a sensing platform. Moreover, the proposed dual-channel assay has been successfully applied to determine OTA levels in corn samples with rapid response, superior accuracy, and high anti-interference capability, providing a promising method for food safety monitoring.
Asunto(s)
Técnicas Biosensibles/métodos , Estructuras Metalorgánicas/química , Ocratoxinas/análisis , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/instrumentación , ADN/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Contaminación de Alimentos/análisis , Oro/química , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Ocratoxinas/química , Zea mays/química , Circonio/químicaRESUMEN
The simple and sensitive detection of protein is of great significance in biological research and medical diagnosis. However, the commonly-used methods, such as enzyme-linked immunosorbent assay (ELISA), usually rely on signal tags labeled on the antibody, which limits the sensitivity and stability. Herein, we have designed and constructed a colorimetric immunosensor in this work for the analysis of protein by taking advantage of 2D metal-organic framework (2D-MOF) nanomaterials as enzyme mimics. The nanomaterial shows a strong peroxidase mimetic activity, and good selectivity after it is modified with a specific aptamer. Therefore, taking carcinoembryonic antigen (CEA) as an example, this immunosensor achieves a good detection performance with a linear range from 1 pg mL-1 to 1000 ng mL-1 and a limit of detection (LOD) of 0.742 pg mL-1. Moreover, the sensor can successfully distinguish the human serum of colorectal cancer patients from healthy people, which suggests that this sensor has great potential in clinical applications. More importantly, the mass production, low cost, stability and ease of transport of the MOFs nanomaterials, as well as the ability for visual detection will make this sensor suitable for point-of-care (POC) testing in remote or resource-poor areas.
Asunto(s)
Antígeno Carcinoembrionario/sangre , Colorimetría/métodos , Inmunoensayo/métodos , Estructuras Metalorgánicas/química , Nanoestructuras/química , Anticuerpos Inmovilizados/inmunología , Aptámeros de Nucleótidos/química , Bencidinas/química , Biomarcadores/sangre , Antígeno Carcinoembrionario/inmunología , Catálisis , Compuestos Cromogénicos/química , Neoplasias Colorrectales/sangre , Humanos , Ácidos Nucleicos Inmovilizados/química , Límite de DetecciónRESUMEN
Single-molecule Förster resonance energy transfer (smFRET) is widely utilized to investigate the structural heterogeneity and dynamics of biomolecules. However, it has been difficult to simultaneously achieve a wide observation time window, a high structure resolution, and a high time resolution with the current smFRET methods. Herein, we introduce a new method utilizing two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) and surface immobilization techniques. This method, scanning 2D FLCS, enables us to examine the structural heterogeneity and dynamics of immobilized biomolecules on a time scale from microsecond to subsecond by slowly scanning the sample stage at the rate of â¼1 µm/s. Application to the DNA Holliday junction (HJ) complex under various [Mg2+] conditions demonstrates that scanning 2D FLCS enables tracking reaction kinetics from 25 µs to 30 ms with a time resolution as high as 1 µs. Furthermore, the high structure resolution of scanning 2D FLCS allows us to unveil the ensemble nature of each isomer state and the heterogeneity of the dynamics of the HJ.
Asunto(s)
ADN Cruciforme , ADN/química , Espectrometría de Fluorescencia/métodos , Colorantes Fluorescentes/química , Ácidos Nucleicos Inmovilizados/química , Isomerismo , Cinética , Rodaminas/química , Factores de TiempoRESUMEN
Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , ADN/química , Ácidos Nucleicos Inmovilizados/química , SARS-CoV-2/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Humanos , Límite de Detección , Microscopía/métodos , Óptica y Fotónica/instrumentación , Óptica y Fotónica/métodos , SARS-CoV-2/química , Saliva/virología , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Rapid and sensitive detection of enteropathogenic Escherichia coli (EPEC) in fluids with complex background is an important task for safety quality control in the field of medicine, environment, and food. In this study, a gold foil paper-based aptasensor was developed for the detection of enteropathogenic EPEC O26:K60 with surface-enhanced Raman spectroscopy (SERS) and magnetic separation technology mediated by Fe3O4@Au composite. The gold foil paper was firstly modified with thiolated capture probe and SERS tag. The thiolated aptamer probe for EPEC was immobilized onto a Fe3O4@Au composite. In the presence of EPEC, highly specific recognition between the aptamer probe and EPEC made the Fe3O4@Au composite partially dissociated from the gold foil paper. This led to a decreased Raman intensity response, which showed an obvious negative linear correlation with increasing concentration of EPEC over a wide concentration range from 10 to 107 CFU/mL under an excitation wavelength of 633 nm. The detection limit was about 2.86 CFU/mL in a buffer solution and a licorice extractum and the detection time was only 2.5 h. The results demonstrate that the gold foil paper-based aptasensor can be an excellent biosensing platform that offers a reliable, rapid, and sensitive alternative for EPEC detection.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Escherichia coli Enteropatógena/aislamiento & purificación , Oro/química , Papel , Ácidos Borónicos/química , Escherichia coli Enteropatógena/química , Glycyrrhiza/microbiología , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Fenómenos Magnéticos , Nanopartículas de Magnetita/química , Nanocompuestos/química , Espectrometría Raman , Compuestos de Sulfhidrilo/químicaRESUMEN
A simple nanoplatform based on molybdenum disulfide (MoS2) nanosheets, a fluorescence quencher (signal off), and a hybridization chain reaction (HCR) signal amplification (signal on) used for the enzyme-free, label-free, and low-background signal quantification of microRNA-21 in plasma exosome is reported. According to the sequence of microRNA-21, carboxy-fluorescein (FAM)-labeled hybridization probe 1 (FAM-H1) and hybridization probes 2 (FAM-H2) were designed with excitation maxima at 488 nm and emission maxima at 518 nm. MoS2 nanosheets could adsorb FAM-H1 and FAM-H2 and quenched their fluorescence signals to reduce the background signal. However, HCR was triggered when microRNA-21 was present. Consequently, HCR products containing a large number of FAM fluorophores can emit a strong fluorescence at 518 nm and could realize the detection of microRNA-21 as low as 6 pmol/L and had a wide linear relation of 0.01-25 nmol/L. This assay has the ability of single-base mismatch recognition and could identify microRNA-21 with high specificity. Most importantly, this approach was successfully applied to the detection of plasma exosomal microRNA-21 in patients with lung cancer, and it is proposed that other targets can also be detected by changing the FAM-H1 and FAM-H2 corresponding to the target sequence. Thus, a novel, hands-on strategy for liquid biopsy was proposed and has a potential application value in the early diagnosis of lung cancer.
Asunto(s)
Exosomas/química , Neoplasias Pulmonares/sangre , MicroARNs/sangre , Sondas de ADN/química , Sondas de ADN/genética , Disulfuros/química , Colorantes Fluorescentes/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Límite de Detección , Neoplasias Pulmonares/diagnóstico , MicroARNs/genética , Molibdeno/química , Nanoestructuras/química , Hibridación de Ácido Nucleico , Espectrometría de FluorescenciaRESUMEN
A reliable and sensitive detection approach for SARS-CoV 2 is essential for timely infection diagnosis and transmission prevention. Here, a two-dimensional (2D) metal-organic framework (MOF)-based photoelectrochemical (PEC) aptasensor with high sensitivity and stability for SARS-CoV 2 spike glycoprotein (S protein) detection was developed. The PEC aptasensor was constructed by a plasmon-enhanced photoactive material (namely, Au NPs/Yb-TCPP) with a specific DNA aptamer against S protein. The Au NPs/Yb-TCPP fabricated by in situ growth of Au NPs on the surface of 2D Yb-TCPP nanosheets showed a high electron-hole (e-h) separation efficiency due to the enhancement effect of plasmon, resulting in excellent photoelectric performance. The modified DNA aptamer on the surface of Au NPs/Yb-TCPP can bind with S protein with high selectivity, thus decreasing the photocurrent of the system due to the high steric hindrance and low conductivity of the S protein. The established PEC aptasensor demonstrated a highly sensitive detection for S protein with a linear response range of 0.5-8 µg/mL with a detection limit of 72 ng/mL. This work presented a promising way for the detection of SARS-CoV 2, which may conduce to the impetus of clinic diagnostics.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Estructuras Metalorgánicas/química , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/análisis , Secuencia de Bases , Técnicas Biosensibles/instrumentación , COVID-19/diagnóstico , ADN/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Oro/química , Oro/efectos de la radiación , Humanos , Ácidos Nucleicos Inmovilizados/química , Luz , Límite de Detección , Nanopartículas del Metal/química , Nanopartículas del Metal/efectos de la radiación , Faringe/virología , Procesos Fotoquímicos , Porfirinas/química , Glicoproteína de la Espiga del Coronavirus/química , Iterbio/químicaRESUMEN
Detection and characterization of DNA damage plays a critical role in genotoxicity testing, drug screening, and environmental health. We developed a fully integrated origami paper-based analytical device (oPAD) for measuring DNA damage. This simple device allows on-paper cell lysis, DNA extraction, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and signal readout with simple operation steps, enabling rapid (within 30 min) and high throughput assessment of multiple DNA damages induced by exogenous chemical agents.
Asunto(s)
Daño del ADN , ADN/análisis , Papel , Animales , Línea Celular , ADN/química , ADN Nucleotidilexotransferasa/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Ácidos Nucleicos Inmovilizados/análisis , Ácidos Nucleicos Inmovilizados/química , Etiquetado Corte-Fin in Situ , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Pez CebraRESUMEN
Plasmonic nanosensors for label-free detection of DNA require excellent sensing resolution, which is crucial when monitoring short DNA sequences, as these induce tiny peak shifts, compared to large biomolecules. We report a versatile and simple strategy for plasmonic sensor signal enhancement by assembling multiple (four) plasmonic sensors in series. This approach provided a fourfold signal enhancement, increased signal-to-noise ratio, and improved sensitivity for DNA detection. The response of multiple sensors based on AuNSpheres was also compared with AuNRods, the latter showing better sensing resolution. The amplification system based on AuNR was integrated into a microfluidic sequential injection platform and applied to the monitoring of DNA, specifically from environmental invasive species-zebra mussels. DNA from zebra mussels was log concentration-dependent from 1 to 1 × 106 pM, reaching a detection limit of 2.0 pM. In situ tests were also successfully applied to real samples, within less than 45 min, using DNA extracted from zebra mussel meat. The plasmonic nanosensors' signal can be used as a binary output (yes/no) to assess the presence of those invasive species. Even though these genosensors were applied to the monitoring of DNA in environmental samples, they potentially offer advantage in a wide range of fields, such as disease diagnostics.
Asunto(s)
ADN/análisis , Técnicas Analíticas Microfluídicas/métodos , Resonancia por Plasmón de Superficie/métodos , Animales , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Dreissena/química , Oro/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Nanosferas/química , Nanotubos/química , Hibridación de Ácido Nucleico , Alimentos Marinos/análisisRESUMEN
HKUST-1 MOFs and its derivative HKUST-CuO were coupled with TiO2 nanoparticles to form the heterogeneous composites of HKUST-1/TiO2 and HKUST-CuO/TiO2 based on their well-suitable bandgap energies (Eg). Compared with mono-component HKUST-1 or HKUST-CuO, the prepared composites displayed photoelectrochemical (PEC) response due to the synergistic effect from their heterogeneous structure. Higher photocurrent response was obtained on HKUST-CuO/TiO2-modified ITO electrode (HKUST-CuO/TiO2/ITO), which could be attributed to the hollow structure with a thin shell of HKUST-CuO greatly enhancing visible spectra harvesting. The CuO component in HKUST-CuO not only could accelerate electron transfer on the heterojunction interface but also effectively separate the photo-generated charge carriers (e-1/h+). Based on the excellent PEC performance of prepared photoactive composite material, under visible-light excitation (λ ≥ 420 nm) and with a working potential of 0 V (vs. Ag/AgCl), the S1 (probe DNA)/HKUST-CuO/TiO2/ITO PEC platform was successfully fabricated for colitoxin DNA detection without using ascorbic acid (AA) as an electron donor. Compared with the analysis results on S1/HKUST-1/TiO2/ITO electrode, S1/HKUST-CuO/TiO2/ITO displayed a wider linear response range from 1.0 × 10-6 to 4.0 × 10-1 nM with a lower detection limit of 3.73 × 10-7 nM (S/N = 3), the linear regression equation was ΔI (10-6 A) =0.5549-0.1858 log (CS2/M), which confirmed the HKUST-CuO could improve sensitivity because of its prominent PEC property. The relative standard deviation (RSD) of the PEC sensor for target DNA detection of 2.0 × 10-4 nM was 7.4%. The proposed DNA biosensor also possessed good specificity and stability. Hence, this reported work was a promising strategy for molecular diagnosis in the bio-analysis field. (A) Schematic illustration of the preparation process of the proposed PEC biosensors for colitoxin DNA detection. (B) The preparation process of HKUST-1 and HKUST-CuO.
Asunto(s)
Técnicas Biosensibles/métodos , Cobre/química , ADN/análisis , Técnicas Electroquímicas/métodos , Estructuras Metalorgánicas/química , Titanio/química , Toxinas Bacterianas/genética , Técnicas Biosensibles/instrumentación , Cobre/efectos de la radiación , Sondas de ADN/química , Técnicas Electroquímicas/instrumentación , Electrodos , Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácidos Nucleicos Inmovilizados/química , Luz , Límite de Detección , Nanopartículas del Metal/química , Nanopartículas del Metal/efectos de la radiación , Estructuras Metalorgánicas/efectos de la radiación , Procesos Fotoquímicos , Titanio/efectos de la radiaciónRESUMEN
Accurate and sensitive fluorescence imaging of intracellular miRNA is essential for understanding the mechanism underlying some physiological and pathological events, as well as the prevention and diagnosis of diseases. Herein, a highly sensitive ratiometric fluorescent nanoprobe for intracellular miRNA imaging was fabricated by integrating a Ru-SiO2@polydopamine (Ru-SiO2@PDA) nanoplatform with a near-infrared light (NIR)-assisted DNA strand displacement signal amplification strategy. The Ru-SiO2@PDA spheres have excellent biosafety, high photothermal effect, and unique photophysical properties that can both emit a stable red fluorescence and well quench the fluorophores getting closer to them. So, when the fuel DNA and carboxyfluorescein (FAM)-labeled signal DNA are co-assembled on their outer surfaces, the FAM's green fluorescence is quenched, and a low ratiometric signal is obtained. However, in the presence of miRNA, the target displaces the signal DNA from the capture DNA, releasing the signal DNA far away from the Ru-SiO2@PDA. Then, the green fluorescence recovers and leads to an enhanced Igreen/Ired value. Under NIR light irradiation, the Ru-SiO2@PDA increases the local temperature around the probe and triggers the release of fuel DNA, which thus recycles the target miRNA and effectively amplifies the ratiometric signal. Using A549 cells as a model, the nanoprobe realizes the highly sensitive ratiometric fluorescence imaging of miRNA let-7a, as well as its in vivo up- and down-regulation expressions. It provides a facile tool for highly sensitive and accurate intracellular miRNA detection through one-step incubation and may pave a new avenue for single-cell analysis.
Asunto(s)
Colorantes Fluorescentes/química , MicroARNs/análisis , Nanopartículas/química , Células A549 , ADN/química , ADN/genética , Regulación hacia Abajo , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Indoles/química , Rayos Infrarrojos , Límite de Detección , MicroARNs/genética , MicroARNs/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Hibridación de Ácido Nucleico , Polímeros/química , Rutenio/química , Dióxido de Silicio/química , Regulación hacia ArribaRESUMEN
Liposomal spherical nucleic acids (LSNAs) modified with polyethylene glycol (PEG) units are studied in an attempt to understand how the circulation time and biodistribution of the constructs can be manipulated. Specifically, the effect of (1) PEG molecular weight, (2) PEG shell stability, and (3) PEG modification method (PEG in both the core and shell versus PEG in the shell only) on LSNA blood circulation, biodistribution, and in vivo cell internalization in a syngeneic, orthotopic triple-negative breast cancer mouse model is studied. Generally, high PEG molecular weight extends blood circulation lifetime, and a more lipophilic anchor stabilizes the PEG shell and improves circulation and tumor accumulation but at the cost of cell uptake efficiency. The PEGylation strategy has a minor effect on in vitro properties of LSNAs but significantly alters in vivo cell uptake. For example, surface-only PEG in one design contributed to higher in vivo cell internalization than its counterpart with PEG both in the shell and core. Taken together, this work provides guidelines for designing LSNAs that exhibit maximal in vivo cancer cell uptake characteristics in the context of a breast cancer model.
Asunto(s)
Ácidos Nucleicos Inmovilizados/metabolismo , Liposomas/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Polietilenglicoles/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/farmacocinética , Liposomas/química , Liposomas/farmacocinética , Ratones Endogámicos BALB C , Peso Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacocinética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacocinética , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Distribución TisularRESUMEN
Timely and rapid detection of biomarkers is extremely important for the diagnosis and treatment of diseases. However, going to the hospital to test biomarkers is the most common way. People need to spend a lot of money and time on various tests for potential disease detection. To make the detection more convenient and affordable, we propose a paper-based aptasensor platform in this work. This device is based on a cellulose paper, on which a three-electrode system and microfluidic channels are fabricated. Meanwhile, novel nanomaterials consisting of amino redox graphene/thionine/streptavidin-modified gold nanoparticles/chitosan are synthesized and modified on the working electrode of the device. Through the biotin-streptavidin system, the aptamer whose 5'end is modified with biotin can be firmly immobilized on the electrode. The detection principle is that the current generated by the nanomaterials decreases proportionally to the concentration of targets owing to the combination of the biomarker and its aptamer. 17ß-Estradiol (17ß-E2), as one of the widely used diagnostic biomarkers of various clinical conditions, is adopted for verifying the performance of the platform. The experimental results demonstrated that this device enables the determination of 17ß-E2 in a wide linear range of concentrations of 10 pg mL-1 to 100 ng mL-1 and the limit of detection is 10 pg mL-1 (S/N = 3). Moreover, it enables the detection of targets in clinical serum samples, demonstrating its potential to be a disposable and convenient integrated platform for detecting various biomarkers.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Estradiol/sangre , Ácidos Nucleicos Inmovilizados/química , Papel , Biomarcadores/sangre , Biomarcadores/química , Técnicas Biosensibles/instrumentación , Biotina/química , Quitosano/química , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Estradiol/química , Oro/química , Grafito/química , Humanos , Límite de Detección , Nanopartículas del Metal/química , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Fenotiazinas/química , Estreptavidina/químicaRESUMEN
Nucleic acid amplification tests (NAATs) are powerful tools for the Japanese encephalitis virus (JEV). We demonstrated highly sensitive, specific, and rapid detection of JEV by colorimetric reverse-transcription loop-mediated isothermal amplification (cRT-LAMP). Under optimized conditions, the RT-LAMP assay results showed that the limit of detection was approximately equivalent to 1 RNA genome copy/µL with an assay time of 30 min. The assay was highly specific to JEV when tested with other mosquito-borne virus panels (Zika virus and dengue virus types 2-4). The ability to detect JEV directly from crude human sample matrices (serum and urine) demonstrated the suitability of our JEV RT-LAMP for widespread clinical application. The JEV RT-LAMP provides combination of rapid colorimetric determination of true-positive JEV RT-LAMP amplicons with our recently developed JEV-nanobarcodes, measured at absorbance wavelenght of 530 (A530) and 650 (A650), which have a limit of detection of 23.3 ng/µL. The AuNP:polyA10-JEV RT-LAMP nanobarcodes exhibited superior capability for stabilizing the true-positive JEV RT-LAMP amplicons against salt-induced AuNP aggregation, which improved the evaluation of true/false positive signals in the assay. These advances enable to expand the use of RT-LAMP for point-of-care tests, which will greatly bolster JEV clinical programs. The JEV RT-LAMP nanobarcode assay targeting the envelope (E) gene and MgSO4 induced AuNP aggregation, indicated by an instant pink-to-violet colorimetric read-out.
Asunto(s)
Colorimetría/métodos , Virus de la Encefalitis Japonesa (Especie)/química , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , Animales , Secuencia de Bases , Sangre/virología , Oro/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Nanopartículas del Metal/química , Poli A/química , ARN Viral/sangre , ARN Viral/orina , Porcinos , Orina/virologíaRESUMEN
The efficient recognition of circulating tumor cells (CTCs) with an aptamer probe confers numerous benefits; however, the stability and binding affinity of aptamers are significantly hampered in real biological sample matrices. Inspired by the efficient preying mechanism by multiplex tubing feet and endoskeletons of sea urchins, we engineered a superefficient biomimetic single-CTC recognition platform by conjugating dual-multivalent-aptamers (DMAs) Sgc8 and SYL3C onto AuNPs to form a sea urchin-like nanoprobe (sea urchin-DMA-AuNPs). Aptamers Sgc8 and SYL3C selectively bind with the biomarker proteins PTK7 and EpCAM expressed on the surface of CTCs. CTCs were captured with 100% efficiency, followed by sorting on a specially designed multifunctional microfluidic configuration, integrating a single-CTC separation unit and a hydrodynamic filtrating purification unit. After sorting, background-free analysis of biomarker proteins in single CTCs was undertaken with inductively coupled plasma mass spectrometry by measuring the amount of 197Au isotope in sea urchin-DMA-AuNPs. With respect to a single-aptamer nanoprobe/-interface, the dual-aptamer nanoprobe improves the binding efficiency by more than 200% (Kd < 0.35 nM). The microchip facilitates the recognition of single CTCs with a sorting separation rate of 93.6% at a flow rate of 60 µL min-1, and it exhibits 73.8 ± 5.0% measurement efficiency for single CTCs. The present strategy ensures the manipulation and detection of a single CTC in 100 µL of whole blood within 1 h.