RESUMEN
Point-of-care testing (POCT) technologies facilitate onsite detection of pathogens in minutes to hours. Among various POCT approaches, pressure-based sensors that utilize gas-generating reactions, particularly those catalyzed by nanozymes (e.g., platinum nanoparticles, PtNPs, or platinum-coated gold nanoparticles, and Au@PtNPs) have been shown to provide rapid and sensitive detection capabilities. The current study introduces Au-Pt alloy-coated gold nanoparticles (Au@AuPtNPs), an innovative nanozyme with enhanced catalytic activity and relatively high stability. For pathogen detection, Au@AuPtNPs are modified with H1 or H2 hairpin DNAs that can be triggered to undergo a hybridization chain reaction (HCR) that leads to their aggregation upon recognition by an initiator strand (Ini) with H1-/H2-complementary aptamers tethered to magnetic beads (MBs). Pathogen binding to the aptamer exposes Ini, which then binds Au@AuPtNPs and initiates a HCR, resulting in Au@AuPtNP aggregation on MBs. These Au@AuPtNP aggregates exhibit strong catalysis of O2 from the H2O2 substrate, which is measured by a pressure meter, enabling detection of Escherichia coli (E. coli) O157:H7 at concentrations as low as 3 CFU/mL with high specificity. Additionally, E. coli O157:H7 could also be detected in simulated water and tea samples. This method eliminates the need for costly, labor- and training-intensive instruments, supporting its further testing and validation for deployment as a rapid-response POCT application in the detection of bacterial contaminants.
Asunto(s)
Escherichia coli O157 , Oro , Nanopartículas del Metal , Platino (Metal) , Escherichia coli O157/aislamiento & purificación , Nanopartículas del Metal/química , Oro/química , Platino (Metal)/química , Catálisis , Técnicas Biosensibles/métodos , Sistemas de Atención de Punto , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/análisis , Aptámeros de Nucleótidos/químicaRESUMEN
Tethering oligonucleotide aptamers to a DNA tetrahedron structure can enhance the recognition of SARS-CoV-2 spike protein to effectively overcome challenges with its detection in current diagnostic assays. Building on this framework, we have developed a unique portable detection method for COVID-19 that provides exceptional sensitivity and selectivity via pressure meter readout. This innovative assay streamlines the detection process, providing a rapid, sensitive, cost-effective, and user-friendly diagnostic tool. This point-of-care test exhibits high sensitivity and specificity, achieving an impressive detection limit of 0.1 pg mL-1 for the spike protein. The effectiveness of this method was validated using pseudoviruses and oropharyngeal swab samples, and its utility for environmental monitoring is demonstrated by testing sewage samples. With a wide linear range and strong potential for clinical or home application, our assay represents a major innovation in point-of-care diagnostics and provides a vital contribution to the current toolkit for controlling the impacts of COVID-19.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , OligonucleótidosRESUMEN
A visual and quantitative ELISA-like method for Escherichia coli O157:H7 is developed based on highly catalytic and stable Au@AuPt nanoparticles. The proposed enhanced ELISA method can visually detect 100 CFU mL-1 O157:H7 with high specificity and without the need for strict low-temperature reagent storage, thereby increasing the utility. Moreover, it is applicable to spiked tap water and milk tea samples without additional treatments.
Asunto(s)
Escherichia coli O157 , Nanopartículas , Ensayo de Inmunoadsorción Enzimática , Microbiología de AlimentosRESUMEN
DNA nanotechnology plays an increasingly important role in the biomedical field; however, its application in the design of organic nanomaterials is underexplored. Herein, we report the use of DNA nanotechnology to transport a NIR-II-emitting nanofluorophore across the blood-brain barrier (BBB), facilitating non-invasive imaging of brain tumors. Specifically, the DNA block copolymer, PS-b-DNA, is synthesized through a solid-phase click reaction. We demonstrate that its self-assembled structure shows exceptional cluster effects, among which BBB-crossing is the most notable. Therefore, PS-b-DNA is utilized as an amphiphilic matrix to fabricate a NIR-II nanofluorephore, which is applied in inâ vivo bioimaging. Accordingly, the NIR-II fluorescence signal of the DNA-based nanofluorophore localized at a glioblastoma is 3.8-fold higher than the NIR-II fluorescence signal of the PEG-based counterpart. The notably increased imaging resolution will significantly benefit the further diagnosis and therapy of brain tumors.
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Barrera Hematoencefálica/metabolismo , Colorantes/metabolismo , ADN/química , ADN/metabolismo , Rayos Infrarrojos , Transporte Biológico , Línea Celular , Humanos , Imagen MolecularRESUMEN
A new DNA nanoprobe based on a Y-shape and pyrene-modified DNA self-assembly is developed to sensitively and specifically detect microRNA through a pyrene excimer-monomer switch. Exhibiting the capability of self-delivery and resistance to nuclease degradation, the nanoprobe has been successfully applied for microRNA imaging in live cells.
Asunto(s)
Sondas de ADN/química , MicroARNs/metabolismo , Nanoestructuras/química , Línea Celular Tumoral , Sondas de ADN/metabolismo , Colorantes Fluorescentes/química , Humanos , MicroARNs/química , Microscopía Confocal , Pirenos/químicaRESUMEN
Hydrogels are polymeric three-dimensional network structures with high water content. Due to their superior biocompatibility and low toxicity, hydrogels play a significant role in the biomedical fields. Hydrogels are categorized by the composition from natural polymers to synthetic polymers. To meet the complicated situation in the biomedical applications, suitable host-guest supramolecular interactions are rationally selected. This review will have an introduction of hydrogel classification based on the formulation molecules, and then a discussion over the rational design of the intelligent hydrogel to the environmental stimuli such as temperature, irradiation, pH, and targeted biomolecules. Further, the applications of rationally designed smart hydrogels in the biomedical field will be presented, such as tissue repair, drug delivery, and cancer therapy. Finally, the perspectives and the challenges of smart hydrogels will be outlined.
RESUMEN
In this study, catalase-linked immunosorbent pressure assay with a gas-generation reaction was established for quantitative detection of disease biomarker C-reactive protein (CRP) by a portable pressuremeter. The pressure-based detection system recognizes, transduces, and amplifies the target signal to a convenient target-correlated pressure signal reading in a closed chamber. Biotin molecules were modified on the surface of catalase in order to incorporate catalase into the pressure immunoassay by the streptavidin-biotin interaction. To improve the assay performance, the modification ratios of biotin molecules to catalase, and the concentrations of capture and detection antibodies were further optimized. The catalase-linked immunosorbent pressure assay allows portable and quantitation analysis of CRP with a limit of detection of 1.8 nM, which can satisfy the clinical needs for determining the risk of cardiovascular disease. The catalase-linked immunosorbent pressure assay also shows superior specificity and good accuracy. Compared to the previously reported assay catalyzed by PtNP nanozyme, catalase is not easily deactivated during storage and operation. With the merits of enzymatic efficiency, biocompatibility, low non-specific adsorption and facile modification, catalase can be reasonably used for reproducible, stable, simple quantitative detection of disease markers using a portable pressure-based assay in resource-limited settings.
Asunto(s)
Proteína C-Reactiva/análisis , Catalasa/química , Biotina/química , Catálisis , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Límite de Detección , Nanopartículas del Metal/química , Oxígeno/química , Platino (Metal)/química , Presión , Sensibilidad y EspecificidadRESUMEN
A new DNA-based fluorescent probe, which is a hybrid molecule of an i-motif forming sequence (IFS) and mono-functionalized tetraphenylethene (TPE), has been synthesized and investigated. A distinct pH-responsive aggregation-induced emission (AIE) effect has been observed from this hybrid molecule, i.e. the fluorescence of TPE will be turned on when the IFS part folds up under acidic conditions. According to the fact that a hybrid molecule with a relatively rigid structure shows a more obvious AIE effect, and whose nano-sized aggregates are formed at a high concentration, we assume that the solubility of the hybrid molecule in water will be reduced due to IFS folding, resulting in aggregation and the resultant AIE effect. Finally, due to its excellent pH responsiveness, this DNA-based probe employing an AIEgen has been applied in monitoring intracellular pH.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Estilbenos/química , Técnicas Biosensibles , ADN/síntesis química , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Espectrometría de Fluorescencia , Estilbenos/síntesis químicaRESUMEN
Oligonucleotide-stabilized silver nanoclusters (AgNCs) show promising applications in bioimaging and bio-/chemo-sensing. However, their unsatisfactory photostability limits their practical applications. In this work, fluorescent AgNCs were synthesized in situ in a DNA hydrogel, consisting of cross-linked enzymatically amplified polymeric DNAs with cytosine-rich sequences in the presence of Ag+. The fluorescence property of the resultant AgNCs was optimized by a rational design of the DNA sequences to cover a broad spectrum with comparable green and red emissions. Under the protection of the DNA hydrogel, the AgNCs showed significantly improved photostability in an ambient oxygen environment, as well as low cytotoxicity even at a high concentration. Therefore, these properties show the rolling-circle-amplification-stabilized AgNCs to be a promising possible fluorescent probe for the detection of reactive oxygen/nitrogen species (ROS/RNS) in live cells because red-emitting species are susceptible to oxidation and consequently convert to green-emitting species. Finally, the as-prepared AgNCs were demonstrated to be a sensitive and specific probe for cellular imaging and the monitoring of ROS/RNS levels, which broadens the applications of AgNCs and provides a new tool for related biological investigations.
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Nanoestructuras , ADN , Colorantes Fluorescentes , Hidrogeles , Radical Hidroxilo , Nanopartículas del Metal , PlataRESUMEN
Functional nucleic acids, which can target cancer cells and realize stimuli-responsive drug delivery in tumor microenvironment, have been widely applied for anticancer chemotherapy. At present, high cost, unsatisfactory biostability, and complicated fabrication process are the main limits for the development of DNA-based drug-delivery nanocarriers. Here, a doxorubicin (Dox)-delivery nanoparticle for tumor-targeting chemotherapy is developed taking advantage of rolling circle amplification (RCA) technique, by which a high quantity of functional DNAs can be efficiently collected. Furthermore, Mg2+, a major electrolyte in human body showing superior biocompatibility, can sufficiently condense the very long sequence of an RCA product and better preserve its functions. The resultant DNA nanoparticle exhibits a high biostability, making it a safe and ideal nanomaterial for in vivo application. Through cellular and in vivo experiments, we thoroughly demonstrate that this kind of Mg2+-stabilized multifunctional DNA nanoparticles can successfully realize tumor-targeted Dox delivery. Overall, exploiting RCA technique and Mg2+ condensation, this new strategy can fabricate nanoparticles with a nontoxic composition through a simple fabrication process and provides a good way to preserve and promote DNA functions, which will show a broad application potential in the biomedical field.
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Nanopartículas , ADN , Doxorrubicina , Sistemas de Liberación de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Magnesio , NeoplasiasRESUMEN
DNA condensation is a facile method to construct DNA nanostructure with a high biostability and low cost, which is mainly used in DNA separation and gene transfection. The recent emerging condensed DNA nanostructures from the rolling circle amplification (RCA), i.e., the complexes between RCA products and magnesium pyrophosphate (RCA-MgPPi), have quickly become attractive biomedical materials with broad application potential because they combine the advantages of the designable and high-throughput isothermal amplification technique and the high stability of DNA condensation structures. However, we find that only approximately 10% of RCA products can be condensed after an RCA reaction, which limits the practical application of the RCA-MgPPi nanostructures. Therefore, in this paper, we investigate how to control the condensation efficiency of RCA-synthesized DNAs in depth. The very long RCA products, which show high charge densities, can be efficiently condensed by an excessive amount of Mg2+ to form RCA-MgPPi nanostructures at a yield approaching 100%. Additionally, the new condensation approach is general and is not limited to the RCA products, which can be applied to other polymeric DNAs. These RCA-MgPPi nanoparticles exhibit a high biostability and low toxicity, in addition, which can be efficiently functionalized with foreign components to create hierarchical properties. Finally, as a proof of concept, based on RCA-MgPPi nanostructures, a ratiometric fluorescence sensor system has been constructed and demonstrated to be an efficient lysosomal pH tracker.
RESUMEN
Recently, smart DNA hydrogels, which are generally formed by the self-assembly of oligonucleotides or through the cross-linking of oligonucleotide-polymer hybrids, have attracted tremendous attention. However, the difficulties of fabricating DNA hydrogels limit their practical applications. We report herein a novel method for producing pH-responsive hydrogels by rolling circle amplification (RCA). In this method, pH-sensitive cross-linking sites were introduced into the polymeric DNA chains during DNA synthesis. As the DNA sequence can be precisely defined by its template, the properties of such hydrogels can be finely tuned in a very facile way through template design. We have investigated the process of hydrogel formation and pH-responsiveness to provide rationales for functional hydrogel design based on the RCA reaction.
Asunto(s)
ADN/síntesis química , Hidrogeles/química , Oligonucleótidos/química , Dicroismo Circular/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo/métodos , Técnicas de Amplificación de Ácido Nucleico , Transición de Fase , Reología/métodosRESUMEN
A rolling-circle-amplification method was developed to produce DNA hydrogels with horseradish-peroxidase-like catalytic capability. The catalytic hydrogel exhibits highly improved stability at elevated temperatures or during a long-term storage. Integrated with glucose oxidase, the complex hydrogel can be applied to the sensitive and reliable detection of glucose.
Asunto(s)
ADN/biosíntesis , Peroxidasa de Rábano Silvestre/metabolismo , Hidrogeles/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Biocatálisis , ADN/química , Peroxidasa de Rábano Silvestre/química , Hidrogeles/químicaRESUMEN
Due to uranium's increasing exploitation in nuclear energy and its toxicity to human health, it is of great significance to detect uranium contamination. In particular, development of a rapid, sensitive and portable method is important for personal health care for those who frequently come into contact with uranium ore mining or who investigate leaks at nuclear power plants. The most stable form of uranium in water is uranyl ion (UO2(2+)). In this work, a UO2(2+) responsive smart hydrogel was designed and synthesized for rapid, portable, sensitive detection of UO2(2+). A UO2(2+) dependent DNAzyme complex composed of substrate strand and enzyme strand was utilized to crosslink DNA-grafted polyacrylamide chains to form a DNA hydrogel. Colorimetric analysis was achieved by encapsulating gold nanoparticles (AuNPs) in the DNAzyme-crosslinked hydrogel to indicate the concentration of UO2(2+). Without UO2(2+), the enzyme strand is not active. The presence of UO2(2+) in the sample activates the enzyme strand and triggers the cleavage of the substrate strand from the enzyme strand, thereby decreasing the density of crosslinkers and destabilizing the hydrogel, which then releases the encapsulated AuNPs. As low as 100nM UO2(2+) was visually detected by the naked eye. The target-responsive hydrogel was also demonstrated to be applicable in natural water spiked with UO2(2+). Furthermore, to avoid the visual errors caused by naked eye observation, a previously developed volumetric bar-chart chip (V-Chip) was used to quantitatively detect UO2(2+) concentrations in water by encapsulating Au-Pt nanoparticles in the hydrogel. The UO2(2+) concentrations were visually quantified from the travelling distance of ink-bar on the V-Chip. The method can be used for portable and quantitative detection of uranium in field applications without skilled operators and sophisticated instruments.
Asunto(s)
Técnicas Biosensibles/métodos , ADN Catalítico/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Lagos/análisis , Uranio/análisis , Contaminantes Radiactivos del Agua/análisis , Resinas Acrílicas/química , Secuencia de Bases , Colorimetría/métodos , Oro/química , Dispositivos Laboratorio en un Chip , Límite de Detección , Nanopartículas del Metal/químicaRESUMEN
Targeted drug delivery is important in cancer therapy to decrease the systemic toxicity resulting from nonspecific drug distribution and to enhance drug delivery efficiency. We have developed an aptamer-based DNA dendritic nanostructure as a multifunctional vehicle for targeted cancer cell imaging and drug delivery. The multifunctional DNA dendrimer is constructed from functional Y-shaped building blocks with predesigned base-pairing hybridization including fluorophores, targeting DNA aptamers and intercalated anticancer drugs. With controllable step-by-step self-assembly, the programmable DNA dendrimer has several appealing features, including facile modular design, excellent biostability and biocompatibility, high selectivity, strong binding affinity, good cell internalization efficiency, and high drug loading capacity. Due to the unique structural features of DNA dendrimers, multiple copies of aptamers can be incorporated into each dendrimer, generating a multivalent aptamer-tethered nanostructure with enhanced binding affinity. A model chemotherapeutic anticancer drug, doxorubicin, was delivered via these aptamer-based DNA dendrimers and exerted a potent toxicity for target cancer cells (human T cell acute lymphoblastic leukemia cell line) with low side effects for the non-target cells (human Burkitt's lymphoma cell line). This controllable aptamer-based DNA dendrimer is a promising candidate for biomedical applications.
Asunto(s)
Aptámeros de Nucleótidos/química , Dendrímeros/química , Diagnóstico por Imagen/métodos , Sistemas de Liberación de Medicamentos/métodos , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Electroforesis en Gel de Agar , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Nanoestructuras/química , Fracciones Subcelulares/metabolismoRESUMEN
A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core-shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Micotoxinas/análisis , Ocratoxinas/análisis , Aptámeros de Nucleótidos/síntesis química , Técnicas Biosensibles/instrumentación , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis químicaRESUMEN
Based on the protective properties of polydopamine nanospheres for DNA probes against nuclease digestion, we have developed a DNase I-assisted target recycling signal amplification method for highly sensitive and selective detection of miRNA.
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Técnicas Biosensibles/métodos , Sondas de ADN/química , Desoxirribonucleasa I/metabolismo , Indoles/química , Límite de Detección , MicroARNs/metabolismo , Nanosferas/química , Polímeros/química , Línea Celular Tumoral , Sondas de ADN/genética , Portadores de Fármacos/química , Humanos , MicroARNs/genética , Modelos Moleculares , Técnicas de Amplificación de Ácido Nucleico , Conformación de Ácido NucleicoRESUMEN
Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb(2+) in biological and environmental samples is of great importance. In this work, a Pb(2+)-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb(2+). Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb(2+) can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold-platinum core-shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.
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Reactivos de Enlaces Cruzados/química , ADN Catalítico/química , ADN Catalítico/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Plomo/análisis , Plomo/metabolismo , Colorimetría , Oro/química , Nanopartículas del Metal/química , Técnicas Analíticas Microfluídicas , Platino (Metal)/químicaRESUMEN
Based on the protective properties of carbon nanoparticles for aptamers against the digestion of nuclease, we have developed a nuclease-assisted target recycling signal amplification method for highly sensitive detection of biomolecules, such as ATP and kanamycin. The high binding specificity between aptamers and targets leads to excellent selectivity of the assay.