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OBJECTIVES: It has been reported that serum Clara cell secreted protein 16 (CC16) is a potential biomarker for lung injury diseases, but currently, there is no other method that is faster, more accurate, or more sensitive being applied in clinical practice apart from ELISA. The current study was designed to established a magnetic nanoparticles chemiluminescence immunoassay (MNPs-CLIA) for highly sensitive automated detection of serum Clara cell secretory protein 16 (CC16), and validated its diagnostic performance for lung disease. METHODS: The study included the expression of CC16 recombinant protein, the preparation and screening of its monoclonal antibody (MAb), as well as the construction, optimization and analytical evaluation of the MNPs-CLIA method. The clinical application value of this method was investigated by detecting CC16 level in 296 serum samples. RESULTS: The linear range of the MNPs-CLIA assay system was 0.2-50â¯ng/mL, and the limit of detection was 0.037â¯ng/mL. Performance parameters such as specificity, recovery rate, and precision can meet the industry standards of in vitro diagnostic reagents. The established method reveals consistent results with ELISA (R2=0.9962) currently used clinically, and it also exhibits satisfactory diagnostic efficacy of silicosis, chronic obstructive pulmonary disease (COPD), and pulmonary sarcoidosis, with areas under the curve (AUC) of 0.9748, 0.8428 and 0.9128, respectively. CONCLUSIONS: Our established MNPs-CLIA method has the advantages of automation, high throughput, rapidity, and simplicity, and can be promoted for widely popularized in clinical applications. MNPs-CLIA detection of serum CC16 has efficient diagnostic potentiality for predicting and diagnosing lung diseases.
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BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.
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Antígeno B7-H1 , Inmunoterapia , Polietileneimina , ARN Interferente Pequeño , Animales , Polietileneimina/química , ARN Interferente Pequeño/química , Antígeno B7-H1/metabolismo , Ratones , Inmunoterapia/métodos , Línea Celular Tumoral , Femenino , Humanos , Ratones Endogámicos BALB C , Liposomas/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Neoplasias/inmunología , Ratones Endogámicos C57BL , Silenciador del GenRESUMEN
Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics.
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Técnicas Biosensibles , Nanoestructuras , Hibridación Fluorescente in Situ , ADN/química , Diagnóstico por Imagen , Nanoestructuras/química , Sondas de ADN/genética , Sondas de ADN/química , Técnicas Biosensibles/métodosRESUMEN
RNA In situ imaging through DNA self-assembly is advantaged in illustrating its structures and functions with high-resolution, while the limited reaction efficiency and time-consuming operation hinder its clinical application. Here, we first proposed a new strand displacement reaction (SDR) model (Cas12a thrusting SDR, CtSDR), in which Cas12a could overcome the inherent reaction limitation and dramatically enhance efficiency through energy replenishment and by-product consumption. The target-initiated CtSDR amplification was established for RNA analysis, with order of magnitude lower limit of detection (LOD) than the Cas13a system. The CtSDR-based RNA in situ imaging strategy was developed to monitor intra-cellular microRNA expression change and delineate the landscape of oncogenic RNA in 66 clinic tissue samples, possessing a clear advantage over classic in situ hybridization (ISH) in terms of operation time (1 h versus 14 h) while showing comparable sensitivity and specificity. This work presents a promising approach to developing advanced molecular diagnostic tools.
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Técnicas Biosensibles , ARN , ARN/genética , Sistemas CRISPR-Cas , ADN/genética , ADN/química , Sensibilidad y Especificidad , Hibridación in Situ , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas Biosensibles/métodosRESUMEN
Nowadays, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have constituted a new challenge for anti-infective treatment. Precise identification and rapid clinical diagnostics of MRSA from other methicillin-sensitive strains entail assays with robust diagnostic efficiency and simple operation steps. Sensitive detection of MecA gene is promising to indicate MRSA infection, but it is challenged by the lack of isothermal and simple strategies. A visual assay based on isothermal rolling circular amplification and G-quadruplex/hemin (G4/hemin) DNAzyme proximity assembly was proposed for the immediate, efficient, and cost-effective detection of MecA in simple operation steps and in a single tube. The presence of MecA specifically drove the formation of circular templates, which further triggered isothermal amplification. The amplified product offered abundant binding sites for DNA-grafted hemin probes to form a novel proximity-assembled G4/hemin DNAzyme structure for colorimetric changing diagnosis. This tandem-repeated novel DNAzyme possessed higher catalytic activity and a lower background signal than traditional G4/hemin DNAzyme, ensuring sensitive discrimination of MRSA (limit of detection: 9.6 pM). Assay stability and antimatrix interference capability enable clinical application, which shows compared diagnostic ability with classic methods (100% sensitivity and 100% specificity) but possesses more simplified procedures and shorter turnaround time (<6 h). This colorimetric strategy in a nonsite-specific and hypersensitive manner holds foreseeable prospects in clinical diagnostic and research applications.
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Técnicas Biosensibles , ADN Catalítico , G-Cuádruplex , Staphylococcus aureus Resistente a Meticilina , ADN Catalítico/química , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Hemina/química , ADN , Técnicas Biosensibles/métodosRESUMEN
Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA' performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients' samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.
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Accurate cancer diagnosis and effective drug therapy entail sensitive and dynamic monitoring of intracellular key enzymes, since their expression level is closely related to disease progression. Simultaneous monitoring of correlated enzymes is promising to help unveiling mystery of cytobiological events during tumor progression and drug response, while is challenged by lacking of a robust and simple simultaneous detection strategy. In order to construct a simple and smart strategy which is complex design-avoided and doesn't need other auxiliary enzyme, here we develop an AND-gate strategy for simultaneously monitoring correlated enzymes which both are upregulated in cancer cells (telomerase and apurinic/apyrimidinic endonuclease 1). An innovative AND-gate DNA nanoprobe has been designed to avoid mutual interference and background noise, guaranteeing an enhanced fluorescent signal output upon catalyzation of dual enzymes. This AND-gate strategy achieves sensitive detection of two enzymes in an individual manner in test tube, through which the diagnostic potential of bladder cancer has been validated by telomerase detection in clinical urine sample. The AND-gate strategy enables specific intracellular imaging of dual enzymes in different cancer cell lines. Importantly, in contrast to traditional single-targeting strategies, AND-gate imaging of dual enzymes significantly improves cancer cell selectivity. Moreover, this strategy dynamically monitors enzymatic activity changes during chemoresistance induced by chemotherapeutic treatment. This simple and smart strategy has foreseeable prospect in the fields of disease diagnosis, drug prognosis evaluation, and precise fluorescence-guided surgery.
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Técnicas Biosensibles , Telomerasa , Técnicas Biosensibles/métodos , ADN/metabolismo , Endonucleasas/metabolismo , Telomerasa/metabolismoRESUMEN
A multifunctional catalytic nanomaterial (Co-MOF@AuNP@ABEI) composed of cobalt-doped metal-organic frameworks (Co-MOF), gold nanoparticles (AuNP), and N-(4-aminobutyl)-N-(ethylisoluminol) (ABEI) is reported. Co-MOF@AuNP@ABEI exhibits high synergistic and zero-distance catalytic properties, which are beneficial to the improvement of the detection sensitivity of an electrochemiluminescent (ECL) biosensor. After coupling with the ECL system and 3D magnetic walking nanomachine amplification strategy, the Co-MOF@AuNP@ABEI can achieve an ultrasensitive ECL assay of Burkholderia pseudomallei with the limit of detection (LOD) of 60.3 aM, which is 2 and 4 orders of magnitude lower than individual ECL system without the nanomachine (4.97 fM) and individual walking nanomachine (340 fM), and superior to the pathogenic bacteria analyses in the previous report. Moreover, the LOD of the proposed ECL detection system for the determination of B. pseudomallei in serum sample was as low as 9.0 CFU mL-1. The relative standard deviations (RSD) of ECL intensity for the detection of five B. pseudomallei-spiked serum samples were 4.02%, 0.84%, 0.84%, 1.55%, and 0.21%, respectively. The recoveries of the ECL biosensor for the detection of B. pseudomallei DNA-spiked serum samples were 93.63 ~ 107.83%. Therefore, this work demonstrated that the developed multifunctional catalytic nanomaterial with synergistic and zero-distance catalytic properties can be used as excellent ECL signal reporter to improve the detection sensitivity of ECL biosensor.
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Técnicas Biosensibles , Burkholderia pseudomallei , Luminol/análogos & derivados , Nanopartículas del Metal , Estructuras Metalorgánicas , Cobalto , Técnicas Electroquímicas , Oro , Mediciones Luminiscentes , Luminol/químicaRESUMEN
CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.
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Edición Génica , Neoplasias , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias/genética , Neoplasias/terapia , ARN , ARN Mensajero/genéticaRESUMEN
The cytochrome c oxidase subunit III (COX III) gene is a powerful biomarker for the early diagnosis of acute kidney injury. However, current methods for COX III gene detection are usually laborious and time-consuming, with limited sensitivity. Herein, we report a novel self-electrochemiluminescence (ECL) biosensor for highly sensitive detection of the COX III gene based on CRISPR/Cas12a and nanoemitters of luminol-loaded multicomponent metal-metalloid PdCuBP alloy mesoporous nanoclusters. The nanoemitter with excellent self-ECL in neutral media exhibited a high specific surface area for binding luminol and outstanding oxidase-like catalytic activity toward dissolved O2. Meanwhile, the CRISPR/Cas12a system, as a target-trigger, was employed to specifically recognize the COX III gene and efficiently cleave the interfacial quencher of dopamine-labeled hairpin DNA. As a result, the ECL biosensor showed superior analytical performance for COX III gene detection without exogenous coreactant. Benefiting from the high-efficiency ECL emission of the nanoemitter and Cas12a-mediated interfacial cleavage of the quencher, the developed ECL biosensor exhibited high sensitivity to COX III with a low detection limit of 0.18 pM. The established ECL biosensing method possessed excellent practical performance in urine samples. Meaningfully, the proposed strategy presents promising prospects for nucleic acid detection in the field of clinical diagnostics.
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Lesión Renal Aguda , Técnicas Biosensibles , Técnicas Biosensibles/métodos , Sistemas CRISPR-Cas/genética , Técnicas Electroquímicas/métodos , Complejo IV de Transporte de Electrones , Femenino , Humanos , Límite de Detección , Mediciones Luminiscentes/métodos , Luminol , MasculinoRESUMEN
A novel cytosensor was constructed for the ultrasensitive detection and nondestructive release of circulating tumor cells (CTCs) by combining Au nanoparticles-loaded two-dimensional bimetallic PdMo (2D Au@PdMo) nanozymes and electrochemical reductive desorption. The 2D Au@PdMo nanozymes possessed high-efficiency peroxidase-like activity and were assembled with an aptamer composed of a thiol-modified epithelial specific cell adhesion molecule (EpCAM) to strengthen CTCs adhesion. Moreover, the electrode surface was decorated with highly fractal Au nanostructures (HFAuNSs) composites due to the similarity in fractal nanostructure with the CTCs membrane to enhance the CTCs anchoring efficiency and release capability. The captured CTCs could be further efficiently dissociated and nondestructively released from the modified electrodes upon electrochemical reductive desorption. The designed cytosensor showed an excellent analytical performance, with a wide linear range from 2 to 1 × 105 cells mL-1 and low limit of detection (LOD) of 2 cells mL-1 (S/N = 3) at the working potential in the range -0.6 to 0.2 V. A satisfactory CTCs release reaching a range of 93.7-97.4% with acceptable RSD from 3.55 to 6.41% and good cell viability was obtained. Thus, the developed cytosensor might provide a potential alternative to perform CTC-based liquid biopsies, with promising applications in early diagnosis of tumors. Preparation and mechanism of desorption of the cytosensor based on 2D Au@PdMo nanozymes and electrochemical reductive desorption for the detection and release of CTCs. A Preparation procedure of the Apt/Au@PbMo bioconjugates. B Fabrication process of the sandwich-type cytosensor. C Electrochemical signal produced by the Au@PdMo nanozymes. D Mechanism of electrochemical reductive desorption for CTCs release.
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Aptámeros de Nucleótidos/metabolismo , Separación Celular/métodos , Nanopartículas del Metal/química , Células Neoplásicas Circulantes/química , Aptámeros de Nucleótidos/química , Catálisis , Línea Celular Tumoral , Técnicas Electroquímicas/métodos , Molécula de Adhesión Celular Epitelial/metabolismo , Oro/química , Humanos , Límite de Detección , Molibdeno/química , Paladio/químicaRESUMEN
BACKGROUND: Retinol binding protein 4 (RBP4) has been regarded as an important serological biomarker for type 2 diabetes mellitus (T2DM). Hence, the construction of a highly sensitive detection method for RBP4 is the key to early prevention and multidisciplinary intervention of T2DM. In this work, a dual-quenched electrochemiluminescence (ECL) immunosensor has been fabricated for ultrasensitive detection of RBP4 by combining zeolitic imidazolate framework-67/AuPt-supported luminol (luminol@AuPt/ZIF-67) with MnO2 nanosheets-grown on carbon nanotubes (MnO2@CNTs). RESULTS: AuPt/ZIF-67 hybrids with high-efficiency peroxidase-like activity could provide multipoint binding sites for luminol and antibodies and significantly boost the amplified initial signal of the ECL immunosensor. Upon glutathione/H2O2 coreactants system, MnO2@CNTs composites could quench the initial signal by inhibiting mimic peroxidase activity of luminol@AuPt/ZIF-67. Moreover, the absorption spectrum of the MnO2@CNTs composites completely overlaps with the emission spectrum of luminol, which can further reduce initial signal by ECL resonance energy transfer (ECL-RET). CONCLUSIONS: Benefiting from the above-mentioned properties, the designed immunoassay sensitivity exhibited excellent sensitivity and relative stability for RBP4 detection range from 0.0001 to 100 ng mL-1 with a low detection limit of 43 fg mL-1. Therefore, our ECL immunosensor provides an alternative assaying strategy for early diagnosis of T2DM.
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Inmunoensayo/métodos , Luminol/química , Estructuras Metalorgánicas/química , Nanocompuestos/química , Proteínas Plasmáticas de Unión al Retinol/análisis , Técnicas Electroquímicas , Oro/química , Humanos , Límite de Detección , Mediciones Luminiscentes , Compuestos de Manganeso/química , Nanotubos de Carbono/química , Óxidos/química , Platino (Metal)/química , Reproducibilidad de los ResultadosRESUMEN
Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.
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Proteínas Proto-Oncogénicas B-raf , Neoplasias de la Tiroides , Biopsia con Aguja Fina , Análisis Mutacional de ADN , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Recombinasas/genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genéticaRESUMEN
Rolling circle amplification (RCA) is an efficient enzymatic isothermal reaction that using circular probe as a template to generate long tandem single-stranded DNA or RNA products under the initiation of short DNA or RNA primers. As a simplified derivative of natural rolling circle replication which synthesizes copies of circular nucleic acids molecules such as plasmids, RCA amplifies the circular template rapidly without thermal cycling and finds various applications in molecular biology. Compared with other amplification strategies, RCA has many obvious advantages. Firstly, because of the strict complementarity required in ligation of a padlock probe, it endows the RCA reaction with high specificity and can even be utilized to distinguish single base mismatches. Secondly, through the introduction of multiple primers, exponential amplification can be achieved easily and leads to a good sensitivity. Thirdly, RCA products can be customized by manipulating circular templates to generate functional nucleic acids such as aptamer, DNAzymes and restriction enzyme sites. Moreover, the RCA has good biocompatibility and is especially suitable for in situ detection. Therefore, RCA has attracted considerable attention as an efficient and potential tool for highly sensitive detection of biomarkers. Herein, we comprehensively introduce the fundamental principles of RCA technology, summarize it from three aspects including initiation mode, amplification mode and signal output mode, and discuss the recent application of RCA-based biosensor in this review.
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Técnicas Biosensibles , ADN Catalítico , ADN/genética , Cartilla de ADN , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The cancer diagnosis with single level of biomarkers suffers from limitation of insufficient accuracy. Hence, developing sensitive, rapid and adaptative analytical strategies for double-level biomarkers are essential for improving the accuracy of clinical cancer diagnosis at early stage. Herein, a DNA biosensor was established based on the catalytic hairpin assembly-mediated Y-junction nicking enzyme assisted signal amplification (CHA-YNEASA) circuits, where the two circuits were concatenated by molecular beacon (MB). In absence of target, both the CHA and YNEASA circuits were effectively hindered because of MB's outstanding ability to control signal background. In presence of target, the initiated CHA circuits made enzyme recognition sequences in close proximity to the assisted sequences to open MB, leading to further trigger the YNEASA circuits. Due to the unique design of dual signal amplification strategies, CHA-YNEASA circuits significantly shorten the reaction time, and improve signal-to-background ratio as well as facilitate the analysis process. It was demonstrated that a high sensitivity with limit of detection (LOD) of 0.9 pM for p53 gene detection was obtained just within 23 min by the proposed DNA biosensor. Moreover, mismatched p53 gene at nucleic acid level was effectively discriminated and strong anti-interference capability was achieved. Noticeably, the DNA biosensor was adaptative for designing a cytosensor at cell level using hairpin DNA, containing MUC1 aptamer and initiation strand of CHA-YNEASA circuits, as switch based on modularity principle. The cytosensor is able to measure MUC1 positive breast cancer cells (MCF-7) with the LOD as low as 100 cells/mL. Excellent specificity for MUC1 negative cells, and good anti-interference capability in 10% fetal bovine serum (FBS) were observed by the cytosensor. Therefore, the proposed DNA biosensor is a sensitive, rapid, adaptative platform for detection of double-level biomarkers, offering novel strategy applied for clinical cancer diagnosis.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles , ADN de Neoplasias/química , Técnicas Electroquímicas , Técnicas de Amplificación de Ácido Nucleico , Proteína p53 Supresora de Tumor/análisis , Humanos , Células MCF-7 , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and â¼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (â¼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.
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Proteínas Argonautas/genética , Ácidos Nucleicos Libres de Células/genética , Neoplasias/genética , Ácidos Nucleicos de Péptidos/genética , Alelos , Humanos , Mutación/genética , Neoplasias/diagnóstico , Neoplasias/patología , Ácidos Nucleicos de Péptidos/aislamiento & purificación , Thermus thermophilus/genéticaRESUMEN
A label-free and enzyme-free colorimetric sensor for rapid detection of Pb2+ is reported, which is based on the strategy of DNAzyme-mediated RNA cleavage combined with an annealing-accelerated DNA hybridization chain reaction (HCR). As a trigger DNA, the substrate strand (STM) of DNAzyme can initiate HCR effectively. However, when it is cleaved by DNAzyme in the presence of Pb2+, the separation of DNA functional domains leads to a serious decrease in HCR efficiency. As a result, the difference in Pb2+ concentration converts into the difference of DNA assembly, which eventually leads to the color change of colloidal gold nanoparticles (AuNPs). In this work, a DNA strand (cGR5) completely complementary to the catalytic strand (GR5) of DNAzyme is used to improve the dissociation of STM to enhance the HCR efficiency. In addition, the simple operation of DNA annealing is first used to accelerate the HCR process, enabling the Pb2+ detection to be completed in about 30 min. As advantages of high sensitivity, good selectivity, strong anti-interference ability, and good practical performance are achieved, it is anticipated that the cheap and simple colorimetric sensor will be helpful for on-site detection of environmental and food samples.
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Colorimetría , ADN Catalítico/metabolismo , Plomo/análisis , Hibridación de Ácido Nucleico , Técnicas Biosensibles , ADN/química , ADN/metabolismo , ADN Catalítico/química , División del ARNRESUMEN
In this work, we report a low-cost and easy operation biosensor platform capable of detection of various analytes with high sensitivity and good selectivity. By ingeniously assigning the specific aptamer into a primer-template integrated DNA template, and using monolayer graphene oxide as a reversible and nonspecific inhibitor, the simple biosensor platform is set up. Without a target, the DNA template is constrained by the graphene oxide sheet and results in low signal. In the presence of a target, the constrained DNA template is released from the graphene oxide surface via a target-induced aptamer conformational change, and further amplified through the improved strand displacement amplification reaction. Therefore, the target detection is simply converted to DNA detection, and a correlation between target concentration and fluorescence signal can be set up. As a result, dozens-fold signal enhancement, high sensitivity, good selectivity, and potential practicability are achieved in target detection. More importantly, the proposed biosensor platform is versatile, meaning that it can greatly facilitate the detection of a variety of analytes. Due to the low cost and easy availability of sensing materials, and the elimination of tedious detection operations, we believe that this simple and universal biosensor platform can find wide applications in biological assay and environment monitoring.
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Técnicas Biosensibles/métodos , ADN de Cadena Simple/química , Grafito/química , Animales , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Benzotiazoles , Bovinos , ADN de Cadena Simple/análisis , ADN de Cadena Simple/genética , Diaminas , Humanos , Plomo/análisis , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Compuestos Orgánicos/química , Estanques/análisis , Quinolinas , Trombina/análisisRESUMEN
A novel, Ω-shaped fiber-optic localized surface plasmon resonance (FOLSPR) biosensor was designed for sensitive real-time and label-free bacterial detection. The designed Ω-shaped fiber-optic probe exhibits an outstanding sensitivity, due to the effect of unique geometry on performance. The results show that refractive index (RI) sensitivity of the Ω-shaped fiber-optic probe is 14 times and 2.5 times higher than those of the straight-shaped and the U-shaped FOLSPR, respectively. In addition, the reason for the geometry and the bending radius effects on RI sensitivity was discussed by investigating the relationship between RI sensitivity and the bending area. The results show that RI sensitivity was enhanced with the increase of bending area, and the best RI sensitivity obtained by Ω-shaped FOLSPR was 64.582 (a.u.)/RIU. Combined with this newly designed Ω-shaped FOLSPR biosensor, a real-time, label-free, sensitive, and highly selective bacterial detection method was established. In this work, the aptamers immobilized on the surface of FOLSPR could specifically capture Salmonella Typhimurium, resulting in an intense change of the absorption peak. In line with this principle, the FOLSPR biosensor achieved high detection sensitivity for Salmonella Typhimurium down to 128 CFU/mL within a linear range from 5 × 102 to 1 × 108 CFU/mL and showed good selectivity for Salmonella Typhimurium detection compared to other bacteria. Furthermore, the FOLSPR biosensor was successfully applied to the detection of Salmonella Typhimurium in a chicken sample with the recoveries of 85-123%. With these characteristics, the novel biosensor is a potential alternative tool in food analysis and environmental monitoring.