RESUMEN
Protein acetylation, a fundamental post-translational modification, plays a critical role in the regulation of gene expression and cellular processes. Monitoring histone deacetylases (HDACs) is important for understanding epigenetic dynamics and advancing the early diagnosis of malignancies. Here, we leverage the dynamic characteristics of DNA-peptide interactions in biomimetic nanochannels to develop a HDAC detection method. In specific, the catalysis of peptide deacetylation by HDACs triggers alterations in the charge states of the nanochannel surface to accommodate DNA molecules. Then, the interaction between DNA and peptides shifts the nanochannel surface charge from positive to negative, leading to a reversal of the ion current rectification (ICR). By calculation of the ICR ratio, quantitative detection of HDACs can be efficiently achieved using the nanochannel-based method in an enzyme-free and label-free manner. Our experimental results demonstrate that HDACs can be detected by using this method within a concentration range of 0.5-500 nM. The innate simplicity and efficiency of this strategy may render it a valuable tool for advancing both fundamental research and clinical applications in the realm of epigenetics and personalized medicine.
Asunto(s)
Biomimética , Histona Desacetilasas , Histona Desacetilasas/metabolismo , ADN/metabolismo , Péptidos/metabolismo , Epigénesis Genética , Acetilación , Inhibidores de Histona DesacetilasasRESUMEN
Bacteria inherently possess the capability of quorum sensing in response to the environment. In this work, we have proposed a strategy to confer bacteria with the ability to recognize targets with quorum-sensing behavior. Meanwhile, we have successfully achieved artificial control over the target-triggered aggregation of Escherichia coli (E. coli) by modifying the bacteria surface in a new way. Furthermore, by making use of green fluorescent protein (GFP) expressed by E. coli as the output signal, the aggregation of modified E. coli can be observed with the naked eye. Therefore, via the detection of the target, MUC1, an ovarian cancer biomarker, a simple and conveniently operated method to diagnose ovarian cancer is developed in this work. Experimental results show that the developed low-background and enzyme-free amplification method enables the highly sensitive detection of MUC1, achieving a remarkable limit of detection (LOD) of 5.47 fM and a linear detection range spanning from 1 pM to 50 nM and 50 nM to 100 nM, respectively. Clinical samples from healthy donors and patients can give distant assay results, showing great potential for clinical applications of this method.
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Escherichia coli , Mucina-1 , Neoplasias Ováricas , Neoplasias Ováricas/diagnóstico , Escherichia coli/aislamiento & purificación , Humanos , Femenino , Mucina-1/análisis , Mucina-1/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Límite de Detección , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Percepción de Quorum , Técnicas Biosensibles/métodosRESUMEN
Precise profiling of the cytokine panel consisting of different levels of cytokines can provide personalized information about several diseases at certain stages. In this study, we have designed and fabricated an "all-in-one" diagnostic tool kit to bioassay multiple inflammatory cytokines ranging from picograms per milliliter to µg/mL in a small cytokine panel. Taking advantage of the kit fabricated by the DNA-encoded assembly of nanocatalysts in dynamic regulation and signal amplification, we have demonstrated the multiplex, visual, and quantitative detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) with limits of detection of 1.6 ng/mL (61.54 pM), 20 pg/mL (1.57 pM), and 4 pg/mL (0.19 pM), respectively. This diagnostic tool kit can work well with commercial kits for detecting serum cytokines from breast cancer patients treated with immunotherapies. Furthermore, a small cytokine panel composed of CRP, PCT, and IL-6 is revealed to be significantly heterogeneous in each patient and highly dynamic for different treatment courses, showing promise as a panel of quantitative biomarker candidates for individual treatments. So, our work may provide a versatile diagnostic tool kit for the visual detection of clinical biomarkers with an adjustable broad detection range.
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Neoplasias de la Mama , Citocinas , Humanos , Femenino , Interleucina-6 , Neoplasias de la Mama/diagnóstico , Proteína C-Reactiva , Biomarcadores , Polipéptido alfa Relacionado con CalcitoninaRESUMEN
In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.
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Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/genética , ADN Ribosómico , Edición Génica/métodos , Hidrazonas/farmacologíaRESUMEN
Replicating phosphorylation-responsive ionic gates via artificial fluidic systems is essential for biomolecular detection and cellular communication research. However, current approaches to governing the gates primarily rely on volume exclusion or surface charge modulation. To overcome this limitation and enhance ion transport controllability, we introduce graphene oxide (GO) into nanochannel systems, simultaneously regulating the volume exclusion and wettability. Moreover, inspired by (cAMP)-dependent protein kinase A (PKA)-regulated L-type Ca2+ channels, we employ peptides for phosphorylation which preserves them as nanoadhesives to coat nanochannels with GO. The coating boosts steric hindrance and diminishes wettability, creating a substantial ion conduction barrier, which represents a significant advancement in achieving precise ion transport regulation in abiotic nanochannels. Leveraging the mechanism, we also fabricated a sensitive biosensor for PKA activity detection and inhibition exploration. The combined regulation of volume exclusion and wettability offers an appealing strategy for controlled nanofluidic manipulation with promising biomedical applications in diagnosis and drug discovery.
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Biomimética , Fosforilación , Humectabilidad , Transporte IónicoRESUMEN
Micro/nanocarriers hold great potential in bioanalysis for molecular recognition and signal amplification but are frequently hampered by harsh synthesis conditions and time-consuming labeling processes. Herein, we demonstrate that Escherichia coli (Ec) can be engineered as an efficient biocarrier for electrochemical immunoassay, which can load ultrahigh amounts of redox indicators and simultaneously be decorated with detection antibodies via a facile polydopamine (PDA)-mediated coating approach. Compared with conventional carrier materials, the entire preparation of the Ec biocarrier is simple, highly sustainable, and reproducible. Moreover, immune recognition and electrochemical transduction are performed independently, which eliminates the accumulation of biological interference on the electrode and simplifies electrode fabrication. Using human epidermal growth factor receptor 2 (HER2) as the model target, the proposed immunosensor exhibits excellent analytical performance with a low detection limit of 35 pg/mL. The successful design and deployment of Ec biocarrier may provide new guidance for developing biohybrids in biosensing applications.
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Técnicas Biosensibles , Humanos , Inmunoensayo , Límite de Detección , Escherichia coli , Preparaciones de Acción RetardadaRESUMEN
The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/H2O2 substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.
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Técnicas Biosensibles , COVID-19 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Hemina/química , Peróxido de Hidrógeno , Amplificación de Genes , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Biosensibles/métodos , Límite de DetecciónRESUMEN
Uniform covalent organic framework nanoparticles (COF NPs) with a well-defined pore structure may provide a robust platform for scaffolding enzymes. Herein, bipyridine-based spherical COF NPs have been successfully prepared in this work through the Schiff base condensation reaction. Moreover, they are functionalized by metal modification and are further used for biosensor fabrication. Experimental results reveal that the metal-modified COF NPs also display impressive peroxidase-like catalytic activities, while they can load enzymes, such as glucose oxidase (GOx) and sarcosine oxidase (SOx), to develop a cascade catalysis system for design of various kinds of biosensors with very well performance. For example, the optimized GOx@Fe-COFs can achieve a sensitive detection of glucose with a low limit of detection (LOD) of 12.8 µM. Meanwhile, the enzymes also exhibit a commendable preservation of 80% enzymatic activity over a span of 14 days under ambient conditions. This work may pave the way for advancing cascade catalysis and the analysis of different kinds of biological molecules based on COF NPs.
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Técnicas Biosensibles , Nanopartículas del Metal , Estructuras Metalorgánicas , Estructuras Metalorgánicas/química , Glucosa/análisis , Nanopartículas del Metal/química , Peroxidasas , Glucosa Oxidasa/química , Catálisis , Técnicas Biosensibles/métodosRESUMEN
The persistent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still infecting hundreds of thousands of people every day. Enriching the kits for SARS-CoV-2 detection and developing the drugs for patient treatments are still urgently needed for combating the spreading virus, especially after the emergence of various mutants. Herein, an electrochemical biosensor has been fabricated in this work for the detection of SARS-CoV-2 via its papain-like cysteine protease (PLpro) and the screening of protease inhibitor against SARS-CoV-2 by using our designed chimeric peptide-DNA (pDNA) nanoprobes. Utilizing this biosensor, the sensitive and specific detection of SARS-CoV-2 PLpro can be conducted in complex real environments including blood and saliva. Five positive and five negative patient throat swab samples have also been tested to verify the practical application capability of the biosensor. Moreover, we have obtained a detection limit of 27.18 fM and a linear detection range from 1 pg mL-1 to 10 µg mL-1 (I = 1.63 + 4.44 lgC). Meanwhile, rapid inhibitor screening against SARS-CoV-2 PLpro can be also obtained. Therefore, this electrochemical biosensor has the great potential for COVID-19 combating and drug development.
RESUMEN
Bioinspired nanochannels have emerged as a powerful tool for bioengineering and biomedical research due to their robust mechanical and controllable chemical properties. Inspired by inward-rectifier potassium (K+) channels, herein, the charged peptide assembly has been introduced into a nano-confined space for the modulation of ion current rectification (ICR). Peptide-responsive reaction-triggered sequence changes can contribute to polarity conversion of the surface charge; therefore, ICR reversal (ICRR) is generated. Compared with other responsive elements, natural charged peptides show the merit of controllable charge polarity. By electrochemically monitoring the ICRR as an output signal, one can utilize the peptide assembly-mediated ICRR to construct an ionic sensory platform. In addition, a logic gate has been established to demonstrate the availability of an ionic sensory platform for inhibitor screening. As peptide nanoassemblies may also have various structures and functions due to their diverse properties, the ionic modulation system can provide alternatives for the assay of peptide-associated biotargets with biomedical applications.
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Péptidos , Transporte Iónico , IonesRESUMEN
Breast cancer is very heterogeneous and the most frequently diagnosed cancer worldwide, and precise therapy targeting specific subtypes may improve the survival rates of breast cancer patients. In this study, we designed a biomimetic vesicle by camouflaging catalytic DNA machinery with a breast cancer cell membrane, which enabled the molecular classification of circulating exosomes for subtype-based diagnosis through homotypic recognition. In addition, the vesicles specifically targeted and fused with breast cancer exosomes with phenotypic homology and manipulated the DNA machinery to amplify electrochemical signaling using exosomal RNA as an endogenous trigger. The biomimetic vesicles prepared with MCF-7 cancer cell-derived membranes were shown to recognize estrogen receptor-positive breast cancer exosomes and exhibited a low detection limit of 557 particles mL-1 with microRNA-375 used as the endogenous biomarker. Furthermore, the biomimetic vesicles prepared with MDA-MB-231 cancer cell-derived membranes displayed satisfactory performance in a homotypic analysis of triple-negative breast cancer exosomes with a potential therapeutic target, PD-L1 mRNA, used as the endogenous biomarker. Most importantly, cross-validation experiments confirmed the high accuracy and selectivity of this homotypic recognition-driven analysis for molecular subtyping of breast cancer. When applied to clinical samples of breast cancer patients, the vesicles demonstrated feasibility and reliability for evaluating the molecular features of cancer cell-derived exosomes and enabled stage-specific monitoring of breast cancer patients because the electrochemical signals showed a positive correlation with disease progression. Therefore, this work may provide new ideas for the precise diagnosis and personalized treatment of breast cancer patients throughout the whole disease process.
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Neoplasias de la Mama , Exosomas , MicroARNs , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , MicroARNs/metabolismo , Reproducibilidad de los Resultados , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Capture and analysis of circulating tumor cells (CTCs) from complex matrixes is pivotal for the prediction of cancer metastasis and personalized treatment of cancer. Herein, we propose a strategy for CTC capture by design and fabrication of a polyvalent aptamer network on an electrode surface, which can be further used for the sensitive analysis of CTCs. In our design, the polyvalent aptamer network, which is constructed via a rolling circle amplification reaction, can significantly enhance the cell-binding abilities. Meanwhile, tetrahedral DNA structures previously assembled on the electrode surface will promote the spatial orientation and reduce the steric hindrance effect of the cell capture, thus improving the cell capture efficiency. Importantly, a detectable electrochemical signal can be obtained without additional signal probes by means of target-induced allostery of the DNA hairpin structures. Further studies reveal that the electrochemical response is proportional to the logarithm of the CTC abundance ranging from 102 to 5 × 104 cell mL-1 with a low limit of detection of 23 cell mL-1. Moreover, the proposed capture strategy exhibits excellent stability and anti-interference in human whole blood, indicating its promising potential in clinical diagnosis.
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Aptámeros de Nucleótidos , Células Neoplásicas Circulantes , Aptámeros de Nucleótidos/química , Línea Celular Tumoral , ADN , Electrodos , Humanos , Células Neoplásicas Circulantes/patologíaRESUMEN
The functionalization of covalent organic frameworks (COFs) with biomacromolecules can extend their functions, which is the premise of their application in biomedical research. However, strategies to functionalize COFs with biomacromolecules, which can ensure the stability in complex medium and minimize the undesired effects, are still lacking. In this work, we have proposed a strategy to functionalize COFs with DNA by covalently linking DNA to the functional group on the COF surface through Cu(I)-catalyzed azide/alkyne cycloaddition (CuAAC) reaction. The as-prepared DNA-functionalized COFs (DNA-COFs) can exhibit good hybridization ability and cargo loading ability; thus, we have designed a DNA-COF-based nanoprobe and then fabricated an electrochemical biosensor for the detection of exosomes. In this design, the functionalization with DNA enables COFs to recognize and capture exosomes, and the encapsulation of a large number of methylene blue (MB) in COFs facilitates signal amplification, which can enhance the sensitivity of the biosensor. Moreover, by simply replacing the oligonucleotide sequences, the strategy proposed here can generally be used to build different DNA-COFs with diverse functions for broader biomedical applications.
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Técnicas Biosensibles , Exosomas , Estructuras Metalorgánicas , Reacción de Cicloadición , ADN , Estructuras Metalorgánicas/químicaRESUMEN
Although covalent organic frameworks (COFs) have received extensive attention for biomedical research due to their unique properties, their application is still hindered by the challenges of incorporating COFs with functional biomolecules. Since peptides have shown advantages in biomedical applications, herein, we propose the functionalization of COFs with peptides by a polymer-assisted surface modification strategy. Furthermore, a method based on the peptide-functionalized COFs for protein detection has also been developed to demonstrate their application potential. With the help of the polymers, peptides and horseradish peroxidase are attached onto COFs with a high surface density, and the developed method has achieved simple and sensitive detection of the secreted protein acidic and rich in cysteine. We speculate that the facile method proposed in this work to prepare peptide-functionalized COFs can not only benefit protein detection but also promote more biomedical applications of COFs.
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Estructuras Metalorgánicas , Estructuras Metalorgánicas/química , Polímeros/química , Osteonectina , Porosidad , PéptidosRESUMEN
Analyzing single-cell phenotypes is increasingly required in biomedical studies, for non-genetic understanding of cellular activities and the biological significance of rare cell subpopulations. However, as compared to the genotypic analysis, single-cell phenotype analysis is technically more challenging. Herein, a tractable method that allows quantitative phenotyping of single cell is developed in this work, termed as the aptamer-mounted nest-PCR (Apt-nPCR). In specific, only two rounds of PCR reactions are required to complete the analysis, where aptamers (short oligonucleotides that bind to specific target molecules) are used as the recognition elements to bind antigens and also as the templates of nPCR for multiplexed and quantitative detection. So, quantitative information of these target antigens can be revealed by quantitative PCR analysis of these aptamers, which can thus be used to interpret cell phenotypes in a quantitative-to-qualitative way. By addressing two technical issues that are involved in single-cell phenotype analysisâmultiplexed detection plus high sensitivity, we have shown the availability of this method for single-cell phenotyping. Therefore, the Apt-nPCR method may represent a tractable method to facilitate the single-cell phenotype analysis, which can be used as a complementary method against these single-cell genotyping methods in our daily research.
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Aptámeros de Nucleótidos , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The ongoing outbreak of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted that new diagnosis technologies are crucial for controlling the spread of the disease. Especially in the resources-limit region, conveniently operated detection methods such as "naked-eye" detection are urgently required that no instrument is needed. Herein, we have designed a novel and facile strategy to fabricate covalent organic framework (COF) capsules, which can be utilized to establish a new colorimetric assay for naked-eye detection of SARS-CoV-2 RNA. Specifically, we employ the digestible ZIF-90 as the sacrificial template to prepare the hollow COF capsules for horseradish peroxidase (HRP) encapsulation. The fabricated COF capsules can provide an appropriate microenvironment for the enzyme molecules, which may improve the conformational freedom of enzymes, enhance the mass transfer, and endow the enzyme with high environmental resistance. With such design, the proposed assay exhibits outstanding analytical performance for the detection of SARS-CoV-2 RNA in the linear range from 5 pM to 50 nM with a detection limit of 0.28 pM which can go parallel to qTR-PCR analysis. Our method also possesses excellent selectivity and reproducibility. Moreover, this method can also be served to analyze the clinical samples, and can successfully differentiate COVID-19 patients from healthy people, suggesting the promising potential in clinical diagnosis.
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Breast cancer is one of the most common malignant diseases among women worldwide, and the existence of breast cancer stem cells is closely associated with poor outcomes. Herein, we report an electrochemical phenotyping method to characterize the stemlike phenotype in breast cancer, offering a low-cost but robust choice other than the highly expensive and experience-dependent flow cytometry. Specially, after immune-magnetic beads-assisted enrichment, an in situ programmable DNA circuit is designed using capture probes to bring in the toeholds for DNA assembly and effector probes to accelerate the removal of background signals. The electrochemical phenotyping method could sensitively determine breast cancer stem cells in a wide linear range and exhibit desirable accuracy and reliability. The method can not only monitor the phenotypic transition of breast cancer cells and the drug-reversed effect but also determinate stemlike phenotype in the mice bearing breast cancer xenograft tumor. Overall, the electrochemical phenotyping method may provide promising technical support for precise management of breast tumors.
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Neoplasias de la Mama/clasificación , Técnicas Electroquímicas , Células Madre Neoplásicas/fisiología , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , ADN , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias ExperimentalesRESUMEN
To improve long-term graft patient outcomes and develop more effective antirejection therapies, noninvasive monitoring of acute cellular rejection (ACR) after organ transplantation is urgently needed. As a biomarker of ACR, Granzyme B (GrB) is expected to be applied in the noninvasive monitoring of ACR. Herein, we have developed a method for detecting the GrB activity based on the target-initiated great change in electrochemical steric hindrance by designing a nanoprobe. The nanoprobe is prepared by conjugating a specific peptide, which is responsive to GrB cleavage activity, to gold nanoparticles (AuNPs). Meanwhile, a piece of DNA sequence with G-quadruplex (G4) is attached at the distal end of the peptide. Upon exposure to GrB, the peptide substrate is cleaved to eliminate the steric hindrance between inter-nanoprobes as well as nanoprobe and DNA tetrahedron (TDN), allowing the released DNA strand to hybridize with TDN, giving sensitive signal output. The method can also be used to detect GrB activity in complex biological settings, so it has a great potential for monitoring GrB activity in the blood or urine of graft patients.
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Oro , Rechazo de Injerto/diagnóstico , Nanopartículas del Metal , Trasplante de Órganos , Granzimas , HumanosRESUMEN
Phenotypic plasticity is an emerging paradigm for providing biological and clinical insights into cancer initiation, progression, and resistance to therapy. However, it is a great challenge to track phenotypic information on live cells with high levels of sensitivity, specificity, and simplicity, when a specific cancer-cell subset is being targeted. In this work, we have successfully achieved cascade assembly of nanoparticles on the surface of specific cancer cells by designing a dual-aptamer-weaved molecular AND logic system. Taking advantage of spatial addressability, precise controllability, and targeting recognition of the nanostructure assemblies, we can precisely label the target-cell subset in a large population of similar cells and rapidly obtain phenotypic information in response to the surface changes of captured cancer cells. Without sophisticated instruments, we can know the phenotypic information on HepG2 cells in whole blood with a high level of sensitivity and rapid naked-eye tracking of on-cell phenotype changes of HepG2 cells undergoing epithelial-mesenchymal transition.
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Aptámeros de Nucleótidos , Nanoestructuras , Tecnología de Seguimiento Ocular , Células Hep G2 , Humanos , FenotipoRESUMEN
Metal-organic framework (MOF) thin films with flexible nature and prominent qualities have opened doors to new technological applications in different fields. Herein, we propose an electrochemical biosensor for the dual detection of Staphylococcus aureus based on the electrodeposition of Cu metal-organic framework (Cu-MOF) thin films. The promising sensing layer with features of good electronic conductivity and enhanced electron-transfer property can not only identify S. aureus through specific micrococcal nucleases in the supernatant but also detect the pathogen directly via aptamer recognition. The dual analysis design ensures the accuracy of this method for S. aureus detection in the range of 7-7 × 106 cfu/mL with the limits of detection of 1.9 and 5.2 cfu/mL. Moreover, the analytical method validation confirmed that the biosensor could efficiently work in complex biological samples, showing good selectivity and specificity and great potential for clinical diagnosis. More importantly, the current proposed strategy is simple and easy to implement without the need for extra signaling elements, which is convenient for timely clinical detection.