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The need to contrive interventions to curb the rise in cancer incidence and mortality is critical for improving patients' prognoses. Adoptive cell therapy is challenged with quality large-scale production, heightening its production cost. Several cancer types have been associated with the expression of highly-immunogenic CTAG1 and CTAG2 antigens, which share common epitopes. Targeting two antigens on the same cancer could improve the antitumor response of TCR-T cells. In this study, we exploited an efficient way to generate large-fold quality TCR-T cells and also demonstrated that the common epitopes of CTAG1 and CTAG2 antigens provide an avenue for improved cancer-killing via dual-antigen-epitope targeting. Our study revealed that xeno/sera-free medium could expand TCR-T cells to over 500-fold, posing as a better replacement for FBS-supplemented media. Human AB serum was also shown to be a good alternative in the absence of xeno/sera-free media. Furthermore, TCR-T cells stimulated with beads-coated T-activator showed a better effector function than soluble T-activator stimulated TCR-T cells. Additionally, TCR-T cells that target multiple antigens in the same cancer yield better anticancer activity than those targeting a single antigen. This showed that targeting multiple antigens with a common epitope may enhance the antitumor response efficacy of T cell therapies.
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Antígenos de Neoplasias , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T , Antígenos de Neoplasias/imunologia , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Epitopos de Linfócito T/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Camundongos , Linhagem Celular Tumoral , Linfócitos T/imunologia , Epitopos/imunologiaRESUMO
Molecularly imprinted polymers (MIPs) are tailor-made functional composites which selectively recognize and bind the target molecule of interest. MIP composites are products of the massively cross-linked polymer matrices, generated via polymerization, with bio-inspired recognition cavities that are morphologically similar in size, shape and spatial patterns to the target conformation. These features have enabled researchers to expand the field of molecular recognition, more specifically for target with peculiar requirements. Nevertheless, MIPs alone are characterized with weak sensitivity. Besides, nanoparticles (NPs) are remarkably sensitive but also suffer from poor selectivity. Intriguingly, the combination of the two results in a highly sensitive and selective MIP composite. For instance, the conjugation of different functional NPs with MIPs can generate new flexible target capture tools, either a dynamic sensor or a novel drug delivery system. In this regard, although the technology is considered an established and feasible approach, it is still perceived as a burgeoning technology for various fields, which makes it unceasingly worthy reviewing. Therefore, in this review, we attempt to give an update on various custom-made biosensors based on MIPs in combination with various NPs for the detection of mycotoxins, the toxic secondary metabolites of fungi. We first summarize the classification, prevalence, and toxicological characteristics of common mycotoxins. Next, we provide an overview of MIP composites and their characterization, and then segment the role of NPs with respect to common types of MIP-based sensors. At last, conclusions and outlook are discussed.
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Técnicas Biossensoriais , Impressão Molecular , Micotoxinas , Nanopartículas , Polímeros Molecularmente Impressos , Impressão Molecular/métodos , Técnicas Biossensoriais/métodosRESUMO
BACKGROUND: The transient receptor potential vanilloid receptor 2 (TRPV2) has been found to participate in the pathogenesis of various types of cancers, however, its role(s) in the tumorigenesis of ESCC remain poorly understood. METHODS: Western blotting and immunohistochemistry were performed to determine the expression profiles of TRPV2 in the ESCC patient tissues. A series of in vitro and in vivo experiments were conducted to reveal the role of TRPV2 in the tumorigenesis of ESCC. RESULTS: Our study first uncovered that the activation of TRPV2 by recurrent acute thermal stress (54 °C) or O1821 (20 µM) promoted cancerous behaviours in ESCC cells. The pro-angiogenic capacity of the ESCC cells was found to be enhanced profoundly and both tumour formation and metastasis that originated from the cells were substantially promoted in nude mouse models upon the activation of TRPV2. These effects were inhibited significantly by tranilast (120 µM) and abolished by TRPV2 knockout. Conversely, overexpression of TRPV2 could switch the cells to tumorigenesis upon activation of TRPV2. Mechanistically, the driving role of TRPV2 in the progression of ESCC is mainly regulated by the HSP70/27 and PI3K/Akt/mTOR signalling pathways. CONCLUSIONS: We revealed that TRPV2-PI3K/Akt/mTOR is a novel and promising target for the prevention and treatment of ESCC.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Canais de Cátion TRPV , Animais , Canais de Cálcio , Carcinogênese/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Humanos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
The advancement in developing sensitive, rapid, and specific sensing tools is crucial in diagnostics and biotechnological applications. Although various isothermal amplification approaches exist for the detection and identification of nucleic acids, post-amplicon analysis is still based on traditional methods such as gel electrophoresis, colorimetry, turbidity, which could be non-specific and inconvenient. Thus, this review will first elaborate various isothermal amplification techniques (principle, merits, and demerits) and their potentials when combined with lateral flow approach for point-of-care nucleic acid diagnostics. Different methods for monitoring carryover contamination resulting from amplification product contamination will be discussed. Then, we will present recent advances in diagnostics with both target pre-amplification and CRISPR-Cas systems, which exhibit collateral cleavage of target nucleic acid and a reporter single strand nucleic acid within the vicinity. When the reporter is fluorophore-labeled, it provides a detectable signal by fluorescence or lateral flow biosensors. Lastly, we will discuss how CRISPR-Cas system based diagnostics could be more effective, affordable and portable for on-site detection.
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Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/genética , Sistemas CRISPR-Cas/genética , HumanosRESUMO
MicroRNAs (miRNAs), a kind of single-stranded small RNA molecule, play significant roles in the physiological and pathological processes of human beings. Currently, miRNAs have been demonstrated as important biomarkers critically related to many diseases and life nature, including several cancers and cell senescence. It is valuable to establish sensitive assays for monitoring the levels of intracellular up-regulated/down-regulated miRNA expression, which would contribute to the early prediction of the tumor risk and cardiovascular disease. Here, an oriented gold nanocross (AuNC)-decorated gold nanorod (AuNR) probe with "OFF-enhanced ON" fluorescence switching was developed based on fluorescence resonance energy transfer and surface enhanced fluorescence (FRET-SEF) principle. The nanoprobe was used to specifically detect miRNA in vitro, which gave two linear responses represented by the equation F = 1830.32 log C + 6349.27, R2 = 0.9901, and F = 244.41 log C + 1916.10, R2 = 0.9984, respectively, along with a detection limit of 0.5 aM and 0.03 fM, respectively. Furthermore, our nanoprobe was used to dynamically monitor the expression of intracellular up-regulated miRNA-34a from the HepG2 and H9C2 cells stimulated by AFB1 and TGF-ß1, and the experimental results showed that the new probe not only could be used to quantitively evaluate miRNA oncogene in vitro, but also enabled tracking and imaging of miRNAs in living cells.
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Ouro/química , MicroRNAs/análise , Nanoconjugados/química , Nanotubos/química , Animais , Linhagem Celular Tumoral , DNA de Cadeia Simples/química , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Limite de Detecção , Nanoconjugados/toxicidade , Nanotubos/toxicidade , RatosRESUMO
In this study, the flavour-enhancing properties of the Maillard reaction products (MRPs) for different systems consisted of different peptides (sunflower, SFP; corn, CP and soyabean SP) with, xylose and cysteine were investigated. Maillard systems from peptides of sunflower, corn and soyabean with xylose and cysteine were designated as PXC, MCP and MSP, respectively. The Maillard systems were prepared at pH of 7.4 using temperature of 120C for 2 h. Results showed that all systems were significantly different in all sensory attributes. The highest scores for mouthfulness and continuity were observed for MCP with the lowest peptides distribution between 1000 and 5000 Da, known as Maillard peptide. This revealed that the MCP with the lowest Maillard peptide content had the strongest "Kokumi" effect compared to the other MRPsand demonstrated that "kokumi effect" of MRPs was contributed by not only the "Maillard peptide" defined by the molecular weight (1000-5000 Da). Results on sensory evaluation after fractionation of PXC followed by enzymatic hydrolysis showed no significant differences between PXC, P-PXC and their hydrolysates. This observation therefore confirmed that the presence of other contributors attributed to the "Kokumi" effect rather than the Maillard peptide. It can be deduced that the unhydrolyzed crosslinking products might have contributed to the "Kokumi" effect of MRPs. The structures of four probable crosslinking compounds were proposed and the findings have provided new insights in the sensory characteristics of xylose, cysteine and sunflower peptide MRPs.
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BACKGROUND: Cancer involves uncontrolled cell growth due to genetic mutations. Tumors can form when CDK6, a gene essential for controlling cell growth, isn't working correctly. Researchers are investigating drugs that inhibit CDK6; some of them appear promising. Nevertheless, CDK6 is advantageous and harmful to cancer because it controls other cellular processes. By inhibiting CDK6 and CDK4, CDK4/6 inhibitors offer a novel therapeutic strategy that stops cell proliferation. The study investigates the function of CDK6 in cancer, the difficulties in targeting CDK6, and possible remedies. OBJECTIVE: Scientists have developed drugs designed to block CDK6 and prevent it from altering other proteins. These drugs, also known as CDK6 inhibitors, help treat cancer. Finding the best drugs for CDK6 is still tricky, though. The drugs' selectivity, potency, and cost are some difficulties. These factors depend on CDK6's structure and interactions with other proteins. The structure of CDK6 and how it influences its function and regulation are explained in this review. It also describes CDK6's function in cancer and its interaction with other molecules and proteins, which is crucial for cell division. Moreover, this review describes how CDK6 interacts with the drugs that block it and what the current and future treatments that target CDK6 are. CONCLUSION: This review presents the structure, current research, and overview of CDK6. It also reviews the role of CDK6 in cancer, function, and regulation. Additionally, it explores its role in cancer signaling networks and its interaction with CDK6 inhibitors. Lastly, it discusses the current status and prospects of therapies targeting CDK6.
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BACKGROUND: The Chinese chaste tree Vitexnegundo (VN) is a popular herb in South and Southeast Asia that has several health benefits, including the ability to inhibit tumor growth and induce apoptosis in multiple tumors. Literature revealed scanty research on breast cancer, with little focus on the molecular mechanism of the disease and an emphasis on targets, biological networks, and active components. Exploring natural compounds as possible therapeutic options is an old but still promising approach for drug discovery and development. This study used a thorough computational and statistical method to screen potential drug candidates. METHODS: The active ingredients and targets of VN were identified using SwissADME, SwissTargetPrediction, STITCH, IMPPAT database, KNapSAcK database, and literature. The OMIM and GeneCards databases were searched for possible targets related to breast cancer. The PASS online server was used to check the probability of active metabolite (Pa) against breast cancer. To build protein-protein interactions (PPI) networking, the intersection of disease and drug targets was uploaded to the STITCH database. Cytoscape software was used to analyze the topology parameters of networking to identify hub targets. Gene Ontology (GO) was analyzed using Metascape and ShinyGO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using the David database and SR plot, and the site of expression and protein domain were studied using FunRich. We employed AutoDockvina, Discovery Studio, and UCSF ChimeraX software and auxiliary tools for molecular docking and analysis. Zincpharmer was used for pharmacophore mapping. ADMET analysis was conducted using ADMETsar, Swiss ADME, ADMETLab servers, and mypresto using GROMACS for molecular dynamics simulation (MDS). RESULTS: A total of 65 targets and 21 active ingredients were identified. Further investigation was conducted on 20 hub targets selected through PPI networking construction. The enrichment analysis results indicated that the key factors were P, amyloid-beta response, cellular response to amyloid- beta, Pos. reg. of G2/M transition of the mitotic cell cycle, and response to a toxic substance. The molecular docking, pharmacophore mapping, and MD simulation results indicated that apigenin, kaempferol, and luteolin positively interacted with CDK1 and CDK6 proteins. CONCLUSION: This study is the first to use network pharmacology, molecular docking, pharmacophore mapping, and MD simulation to identify the active ingredients, molecular targets, and critical biological pathways responsible for VN anti-breast cancer. The study provides a theoretical basis for further research in this area.
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Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.
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Neoplasias , Linfócitos T , Apresentação de Antígeno , Anticorpos , Citometria de Fluxo , Receptores de Antígenos de Linfócitos T , Neoplasias/terapiaRESUMO
Stunted linear growth continues to be a public health problem that overwhelms the entire world and, particularly, developing countries. Despite several interventions designed and implemented to reduce stunting, the rate of 33.1% is still high for the proposed target of 19% in 2024. This study investigated the prevalence and associated factors of stunting among children aged 6-23 months from poor households in Rwanda. A cross-sectional study was conducted among 817 mother-child dyads (two individuals from one home) living in low-income families in five districts with a high prevalence of stunting. Descriptive statistics were used to determine the prevalence of stunting. In addition, we used bivariate analysis and a multivariate logistic regression model to measure the strength of the association between childhood stunting and exposure variables. The prevalence of stunting was 34.1%. Children from households without a vegetable garden (AOR = 2.165, p-value < 0.01), children aged 19-23 months (AOR = 4.410, p-value = 0.01), and children aged 13-18 months (AOR = 2.788, p-value = 0.08) showed increased likelihood of stunting. On the other hand, children whose mothers were not exposed to physical violence (AOR = 0.145, p-value < 0.001), those whose fathers were working (AOR = 0.036, p-value = 0.001), those whose parents were both working (AOR = 0.208, p-value = 0.029), and children whose mothers demonstrated good hand washing practice (AOR = 0.181, p-value < 0.001) were less likely to be stunted. Our findings underscore the importance of integrating the promotion of handwashing practices, owning vegetable gardens, and intimate partner violence prevention in the interventions to fight child stunting.
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Transtornos do Crescimento , Feminino , Humanos , Lactente , Criança , Prevalência , Estudos Transversais , Ruanda , Fatores de Risco , Transtornos do Crescimento/epidemiologiaRESUMO
Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the 13C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.
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Técnicas Biossensoriais , Infecções por Helicobacter , Helicobacter pylori , Úlcera Péptica , Neoplasias Gástricas , Humanos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Substâncias Redutoras , Sistemas CRISPR-Cas , Detecção Precoce de Câncer , Úlcera Péptica/diagnóstico , Úlcera Péptica/genética , Genótipo , Citotoxinas/genética , Infecções por Helicobacter/diagnóstico , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismoRESUMO
Conventional and routine diagnostics such as polymerase chain reaction (PCR) and serological tests are less sensitive, costly, and require sample pretreatment procedures. CRISPR/Cas systems that inherently assist bacteria and archaea in destroying invading phage genetic materials via an RNA-mediated interference strategy have been reconstituted in vitro and harnessed for nucleic and non-nucleic acid diagnostics. CRISPR/Cas-based diagnostics (CRISPR-Dx) are cost-effective, possess excellent sensitivity (attomolar) and specificity (single base distinction), exhibit fast turnaround response, and support nucleic acid extraction-free workflow. However, CRISPR-Dx still needs to address various challenges to translate the laboratory work into end-user tailored solutions. In this perspective, we review the relevant progress of CRISPR/Cas systems-based diagnostics, focusing on the comprehensive customization and applications of leading and trending CRISPR/Cas systems as platform technologies for fluorescence, colorimetric, and electrical signal detection. The impact of the CRISPR game-changing technology on the COVID-19 pandemic is highlighted. We also demonstrate the role of CRISPR/Cas systems for carryover contamination prevention. The advancements in signal amplification strategies using engineered crRNAs, novel reporters, nanoparticles, artificial genetic circuits, microfluidics, and smartphones are also covered. Furthermore, we critically discuss the translation of CRISPR-Dx's basic research into end-user diagnostics for commercialization success in the near future. Finally, we discuss the complex challenges and alternative solutions to harness the CRISPR/Cas potential in detail.
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Técnicas Biossensoriais , COVID-19 , COVID-19/diagnóstico , Sistemas CRISPR-Cas/genética , Humanos , Pandemias , SARS-CoV-2/genéticaRESUMO
Plant polysaccharides, as prebiotics, fat substitutes, stabilizers, thickeners, gelling agents, thickeners and emulsifiers, have been immensely studied for improving the texture, taste and stability of fermented foods. However, their biological activities in fermented foods are not yet properly addressed in the literature. This review summarizes the classification, chemical structure, extraction and purification methods of plant polysaccharides, investigates their functionalities in fermented foods, especially the biological activities and health benefits. This review may provide references for the development of innovative fermented foods containing plant polysaccharides that are beneficial to health.
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Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a progressive autosomal dominant neurodegenerative disease caused by abnormal CAG repeats in the exon 10 of ATXN3. The accumulation of the mutant ataxin-3 proteins carrying expanded polyglutamine (polyQ) leads to selective degeneration of neurons. Since the pathogenesis of SCA3 has not been fully elucidated, and no effective therapies have been identified, it is crucial to investigate the pathogenesis and seek new therapeutic strategies of SCA3. Induced pluripotent stem cells (iPSCs) can be used as the ideal cell model for the molecular pathogenesis of polyQ diseases. Abnormal CAG expansions mediated by CRISPR/Cas9 genome engineering technologies have shown promising potential for the treatment of polyQ diseases, including SCA3. In this study, SCA3-iPSCs can be corrected by the replacement of the abnormal CAG expansions (74 CAG) with normal repeats (17 CAG) using CRISPR/Cas9-mediated homologous recombination (HR) strategy. Besides, corrected SCA3-iPSCs retained pluripotent and normal karyotype, which can be differentiated into a neural stem cell (NSCs) and neuronal cells, and maintained electrophysiological characteristics. The expression of differentiation markers and electrophysiological characteristics were similar among the neuronal differentiation from normal control iPSCs (Ctrl-iPSCs), SCA3-iPSCs, and isogenic control SCA3-iPSCs. Furthermore, this study proved that the phenotypic abnormalities in SCA3 neurons, including aggregated IC2-polyQ protein, decreased mitochondrial membrane potential (MMP) and glutathione expressions, increased reactive oxygen species (ROS), intracellular Ca2+ concentrations, and lipid peroxidase malondialdehyde (MDA) levels, all were rescued in the corrected SCA3-NCs. For the first time, this study demonstrated the feasibility of CRISPR/Cas9-mediated HR strategy to precisely repair SCA3-iPSCs, and reverse the corresponding abnormal disease phenotypes. In addition, the importance of genetic control using CRISPR/Cas9-mediated iPSCs for disease modeling. Our work may contribute to providing a potential ideal model for molecular mechanism research and autologous stem cell therapy of SCA3 or other polyQ diseases, and offer a good gene therapy strategy for future treatment.
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Células-Tronco Pluripotentes Induzidas , Doença de Machado-Joseph , Doenças Neurodegenerativas , Sistemas CRISPR-Cas , Humanos , Doença de Machado-Joseph/genética , FenótipoRESUMO
Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR-Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10-15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.
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Vírus da Febre Suína Africana/isolamento & purificação , Técnicas Biossensoriais , Sistemas CRISPR-Cas , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples , Fluorescência , Reação em Cadeia da Polimerase , SuínosRESUMO
Lipopolysaccharide (LPS), a characteristic component of the outer membrane of Gram-negative bacteria, can be used as an effective biomarker to detect bacterial contamination. Here, we reported a 293/hTLR4A-MD2-CD14 cell-based fluorescent biosensor to detect and identify LPS, which is carried out in a 96-well microplate which is nondestructive, user-friendly, and highly efficient. The promoter sequence of the critical signaling pathway gene ZC3H12A (encoding MCPIP1 protein) and enhanced green fluorescence protein (EGFP) were combined to construct a recombinant plasmid, which was transferred into 293/hTLR4A-MD2-CD14 cells through lipid-mediated, DNA-transfection way. LPS was able to bind to TLR4 and coreceptors-induced signaling pathway could result in green fluorescent protein expression. Results show that stable transfected 293/hTLR4A-MD2-CD14 cells with LPS treatment could be directly and continually observed under a high content screening imaging system. The novel cell-based biosensor detects LPS at low concentration, along with the detection limit of 0.075 µg/mL. The cell-based biosensor was evaluated by differentiating Gram-negative and Gram-positive bacteria and detecting LPS in fruit juices as well. This proposed fluorescent biosensor has potential in sensing LPS optically in foodstuff and biological products, as well as bacteria identification, contributing to the control of foodborne diseases and ensurance of public food safety with its high throughput detection way.
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Bactérias/isolamento & purificação , Técnicas Biossensoriais/métodos , Sucos de Frutas e Vegetais/microbiologia , Proteínas de Fluorescência Verde/metabolismo , Lipopolissacarídeos/análise , Bactérias/química , Bactérias/metabolismo , Contaminação de Alimentos/análise , Sucos de Frutas e Vegetais/análise , Proteínas de Fluorescência Verde/genética , Humanos , Lipopolissacarídeos/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Herein, a novel class-specific artificial receptor-based on molecularly imprinted polymer (MIP)-coated quantum dots (QDs@MIP) was synthesized, characterized, and used for the detection and quantification of the bacterial quorum signaling molecules N-acyl-homoserine lactones (AHLs), a class of autoinducers from Gram-negative bacteria. The QDs@MIP was prepared by surface imprinting technique under controlled conditions using CdSe/ZnS QDs as the signal transducing material. The synthesis of the QDs@MIP was characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, and fluorescence spectroscopy. After template elution, the obtained cavities sensitively and selectively recognized the target AHLs of interest. The fluorescence intensity of the QDs@MIP was significantly quenched compared to the control non-imprinted polymer (QDs@NIP) upon exposure to different AHL concentrations. It also had a good linearity in the range from 2 to 18â¯nM along with a detection limit of 0.66, 0.54, 0.88, 0.72 and 0.68â¯nM for DMHF, C4-HSL, C6-HSL, C8-HSL and N-3oxo-C6-HSL, respectively. Most interestingly, the proposed sensor exhibited high sensitivity, good stability and fast response (30â¯s) towards the target molecules due to successful formation of surface imprints. The practicability of the developed sensor in real samples was further confirmed through the analysis of bacterial supernatant samples with satisfactory recoveries ranging from 89 to 103%. According to these results, the as-prepared QDs@MIP can be used as a new potential supporting technique for the rapid and real-time detection of bacterial pathogens in food safety and healthcare facilities.