RESUMEN
The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.
Asunto(s)
Aptámeros de Nucleótidos , Becaplermina , Técnicas Biosensibles , Hibridación de Ácido Nucleico , Poli A , Técnicas Biosensibles/métodos , Humanos , Aptámeros de Nucleótidos/química , Becaplermina/sangre , Poli A/química , Oro/química , Sondas de ADN/químicaRESUMEN
Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in â¼5.9- and â¼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.
Asunto(s)
Enzimas Inmovilizadas , Glucosa Oxidasa , Enzimas Inmovilizadas/química , Peroxidasa de Rábano Silvestre/química , Glucosa Oxidasa/química , ADN/químicaRESUMEN
Intracellular enzyme cascades are essential for various biological processes, and mimicking their functions in artificial systems has attracted significant research attention. However, achieving convenient and efficient spatial organization of enzymes on interfaces remains a critical challenge. In this work, we designed a simple single-DNA scaffold using triblock polyA single-stranded DNA for the arrangement of coupled enzymes. The scaffold was assembled onto a gold electrode through the affinity of polyA-Au, and two enzymes (glucose oxidase and horseradish peroxidase) were captured through hybridization. The molecular distance between the enzymes was regulated by changing the length of the polyA fragment. As a proof of concept, a glucose biosensor was constructed based on the enzyme cascade amplification. The biosensor exhibited excellent detection capability for glucose in human serum samples with a limit of detection of 1.6 µM. Additionally, a trienzyme cascade reaction was successfully activated, demonstrating the potential scalability of our approach for multienzyme reactions. This study provides a promising platform for the development of easy-to-operate, highly efficient, and versatile enzyme cascade systems using DNA scaffolds.
RESUMEN
The development of electrochemical DNA biosensors has been limited by their reliability and reproducibility due to many interfering factors such as electrode properties, DNA surface densities, and complex biological samples. In this work, we developed a nanobalance polyA hairpin probe (polyA-HP), which was effectively assembled onto the gold electrode surface through the affinity between the central polyA fragment and the Au surface. One flanking probe of the polyA-HP captured the target sequence together with a MB-labeled signal probe, and the other flanking probe captured a reference probe simultaneously. The MB signal related to the amount of target was normalized by the reference Fc signal; thus, the signal-to-noise (S/N) was as high as 2000, and the reproducibility was remarkably improved to 2.77%, even facing deliberately changed experiment conditions. By designing a hairpin structure at the terminal of the polyA-HP, the selectivity and specificity were dramatically improved for the analysis of mismatched sequences. The analysis performance of biological samples was dramatically improved after normalization, which is critical for its practicability. Our novel biosensor is a universal single-molecule platform for ratiometric biosensors with excellent performance in real samples, indicating great potential for next-generation high-precision electrochemical sensors.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Reproducibilidad de los Resultados , ADN/análisis , Oro/química , Límite de DetecciónRESUMEN
Designing an ocular drugs delivery system that can permeate the outer blood-retinal barrier (oBRB) is crucial for the microinvasive or noninvasive treatment of ocular fundus diseases. However, due to the lack of a nanocarrier that can maintain structure and composition at the oBRB, only intravitreal injection at the eyeball can deliver therapeutics directly to the ocular fundus via paracellular and intercellular routes, despite the intraocular operations risks. Here, we demonstrated tetrahedral framework nucleic acids (tFNAs) can penetrate the oBRB and deliver therapeutic nucleic acids to the retina of the rat eye in vivo following subconjunctival injection. We also discovered that tFNAs were transported via a paracellular route across the intercellular tight junctions at the oBRB. The histology analysis for ocular layers indicated that individual and aptamer/doxorubicin-loaded tFNAs penetrated all layers of the posterior segment of the eyeball to reach the innermost retina and persisted for over 3 days with minimal systemic biodistribution. We expect that the programmability and penetrability of tFNAs will provide a promising method for drug delivery across oBRB and long-term sustenance at the target site via periocular administration to various tissues.
Asunto(s)
Barrera Hematorretinal , Ácidos Nucleicos , Ratas , Animales , Distribución Tisular , Retina , Sistemas de Liberación de Medicamentos/métodosRESUMEN
Cell lysates from a laboratory strain of Escherichia coli can be exploited for seamless DNA cloning in vitro, which is named the seamless ligation cloning extract (SLiCE) cloning method. The SLiCE method can incorporate DNA fragments into a vector to achieve conventional DNA cloning and is more cost-effective than commercially seamless DNA cloning kits. In this study, we found that the SLiCE extracts could easily be prepared with different methods, such as 3% Triton X-100 lysis buffer, 3% SDS lysis buffer, or freeze-thaw cycles. At high E. coli transformation efficiency, the SLiCE extracts prepared using different simple and ultra-low cost methods did not affect the DNA cloning efficiency. These results further revealed that the SLiCE cloning method can be efficiently used for seamless DNA cloning in vitro.
Asunto(s)
ADN , Escherichia coli , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , ADN/genética , Laboratorios , Vectores Genéticos , PlásmidosRESUMEN
A DNA origami nanocaliper is employed as a shape-resolved nanomechanical device, with pH-responsive triplex DNA integrated into the two arms. The shape transition of the nanocaliper results in a subtle difference depending on the local pH that is visible via TEM imaging, demonstrating the potential of these nanocalipers to act as a universal platform for pH sensing at the nanoscale.
Asunto(s)
Nanoestructuras , Nanotecnología , ADN , Concentración de Iones de Hidrógeno , Nanotecnología/métodos , Conformación de Ácido NucleicoRESUMEN
The precise arrangement of single entity is a crucial objective of nanoscience and holds great promise in various fields such as biology and material science. In this work, we develop a "DNA framework-mediated geometric renormalization" (DFMGR) strategy to reassemble gold nanoparticles into specific geometric shapes on a 2-dimensional (2D) fluidic membrane interface. Cholesterol-modified AuNPs are randomly anchored on the supported lipid bilayer (SLB) via the cholesterol-lipid interaction. We demonstrate that AuNPs are laterally mobile on SLB and could be further rearranged into a specific geometric shape by DNA framework containing algebraically topological DNA arms. Using scanning electron microscope (SEM) imaging approach, simple geometric shapes, such as points assembled by monomers, line segments assembled by dimers, triangles assembled by trimers are visually presented. Interestingly, we found that the statistic angle (58.77°) and side length (12.21â nm) of triangles obtained from SEM images were both agreed well with the theoretical angle of 60° and side length of 12.58â nm. And the relative error of the angle calculated was as low as 0.33 %. These results indicated that the DFMGR strategy showed precise regulation ability for the AuNPs renormalization. We believe that DNA framework-mediated geometric renormalization strategy would be a powerful means for regulating ligand-receptor interactions in biosystems and for nanoparticle assembling in material science.
RESUMEN
This review focuses on emerging applications of surface-enhanced Raman spectroscopy (SERS) optical nanosensors for miRNA analysis, in which the key enhancement factors of the SERS signal, i.e. SERS-active substrates, SERS nanoprobes and nano-assembly strategy, are emphasized. This article includes many nanomaterials for miRNA analysis by the SERS technique. We summarize these reported nanomaterials mainly according to their function in the miRNA assay biosensor. We also briefly summarize the research progress of these nanomaterials in SERS detection of intracellular miRNA. Finally, we discussed the prospect and limitations of SERS nanosensors for analyzing miRNA.
Asunto(s)
MicroARNs/análisis , Nanoestructuras/química , Animales , Humanos , Tamaño de la Partícula , Espectrometría Raman , Propiedades de SuperficieRESUMEN
Developing ultrasensitive probes holds great significance for simultaneous detection of multiplexed cancer-associated nucleic acids. Bimetallic nanoparticles containing silver may be exploited as nanoprobes for disease detection, which can produce stable and strong surface-enhanced Raman scattering (SERS) signals. However, it remains extremely challenging that such SERS nanoprobes are directly synthesized. Herein gold-silver nanosnowmen, grown via a DNA-mediated approach and attached to thiol-containing Raman dyes, are successfully synthesized. Stable SERS-enhanced gold substrates are also prepared and used as the enriching containers, where the capture DNAs are tethered to sense the target genes jointly enhanced by the SERS nanoprobes in a sandwich hybridization assay. This means detection of the target gene can obtain a limit of detection close to 0.839 fM. Such double-enhanced SERS nanosensors are further employed to simultaneously detect the three types of prostate carcinoma-related genes with high sensitivity and specificity, which meanwhile exhibit robust capacity of resisting disturbance in practical samples. Simultaneous and multiplexed detection of cancer-related genes may provide further biomedical applications with new opportunity.
Asunto(s)
Espectrometría Raman , ADN , Oro , Nanopartículas del Metal , PlataRESUMEN
A variety of nucleic acid amplification techniques have been integrated into different detection methods to promote the development of sensitive and convenient analysis of nucleic acids. However, it is still in urgent need to develop amplified nucleic acid biosensors for the analysis of susceptible gene and even distinguishing single-base mismatched DNA in complex biological samples. Benefiting from the achieved detection strategies, here we boost isothermal nucleic acid amplification by resorting to enzyme amplification, and combine this two-stage amplification method with surface-enhanced Raman spectroscopy (SERS) to develop a signal-on nucleic acid detection platform. Due to the high cleavage efficiency of Exonuclease III (Exo III), a large amount of trigger DNA are produced to initiate multiple hybridization chain reaction circles. The product structure tagged with Tamra is then anchored onto the plasmonic SERS substrate and meanwhile enriched. It is demonstrated that this detection platform is sensitive toward the myocardial infarction disease related gene. A detection limit of 1 fM for the gene analysis in a linear relationship in the wide range from 1 fM to 10nM is achieved, better than most of previous counterparts. Meanwhile, our developed detection platform exhibits a high selectivity for the target gene over mismatched analogues. Our strategy provides a robust tool for signal amplification of gene detection even in blood samples.