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Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.
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CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.
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Alelos , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Humanos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , Animales , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Polimorfismo de Nucleótido Simple , Mutación , ADN/metabolismo , ADN/genética , Células HEK293RESUMEN
RNA helicases are involved in the innate immune response against pathogens, including bacteria and viruses; however, their mechanism in the human airway epithelial cells is still not fully understood. Here, we demonstrated that DEAH (Asp-Glu-Ala-His) box polypeptide 35 (DHX35), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family, boosts antiviral innate immunity in human airway epithelial cells. DHX35 knockdown attenuated the production of interferon-ß (IFN-ß), IL6, and CXCL10, whereas DHX35 overexpression increased their production. Upon stimulation, DHX35 was constitutively expressed, but it translocated from the nucleus into the cytosol, where it recognized cytosolic poly(I:C) and poly(dA:dT) via its HELICc domain. Mitochondrial antiviral signaling protein (MAVS) acted as an adaptor for DHX35 and interacted with the HELICc domain of DHX35 using amino acids 360-510. Interestingly, DHX35 interacted with retinoic acid-inducible gene 1 (RIG-I), enhanced the binding affinity of RIG-I with poly(I:C) and poly(dA:dT), and formed a signalsome with MAVS to activate interferon regulatory factor 3 (IRF3), NF-κB-p65, and MAPK signaling pathways. These results indicate that DHX35 not only acted as a cytosolic nucleic acid sensor but also synergized with RIG-I to enhance antiviral immunity in human airway epithelial cells. Our results demonstrate a novel molecular mechanism for DHX35 in RIG-I-mediated innate immunity and provide a novel candidate for drug and vaccine design to control viral infections in the human airway.
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Proteína 58 DEAD Box , ARN Helicasas DEAD-box , Inmunidad Innata , Receptores Inmunológicos , Humanos , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/inmunología , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/inmunología , Receptores Inmunológicos/metabolismo , Poli I-C/inmunología , Poli I-C/farmacología , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Células HEK293RESUMEN
Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target.
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Apoptosis , Neoplasias Colorrectales , Proteínas Inhibidoras de la Apoptosis , Survivin , Ubiquitina Tiolesterasa , Ubiquitinación , Humanos , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Survivin/metabolismo , Survivin/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
BACKGROUND: Cleft palate repair surgery may result in severe pain in the immediate postoperative period. The aim of this study is to compare the effects of different doses of nalbuphine for postoperative analgesia in children with cleft palate. METHODS: From November 2019 to June 2021, 90 children (45 males and 45 females, age 9-20 months old, ASA class I-II) were selected for palatoplasty. They were randomly divided into three groups: the control group (Group C), the N1 group (postoperative analgesia with 0.05 mg/kg/h nalbuphine) and the N2 group (postoperative analgesia with 0.075 mg/kg/h nalbuphine). Each group had 30 cases. Nalbuphine was not continuously infused in Group C but was continuously infused in Groups N1 and N2 at rates of 0.05 mg/kg/h and 0.075 mg/kg/h, respectively, for 24 h for postoperative analgesia. The FLACC analgesia score and Ramsay Sedation score were recorded at 10 min (T1), 30 min (T2), 2 h (T3), 12 h (T4) and 24 h (T5) after the operation. Adverse reactions such as nausea, vomiting and respiratory depression were observed and recorded. RESULTS: Compared with those in Group C, the FLACC scores in the N1 and N2 groups decreased significantly at T1-T5 (p < 0.05); the Ramsay Sedation score in the N1 group was significantly higher at T3 and T4 (p < 0.05), and that in the N2 group was significantly higher at T1-T5 (p < 0.05). Compared with that in the N1 group, the FLACC score in the N2 group was not significantly different, and the Ramsay Sedation score increased significantly at T5 (p < 0.05). CONCLUSION: Using 0.05 mg/kg/h Nalbuphine continuously for 24 h for postoperative analgesia in children with cleft palate has a better effect and fewer adverse reactions. TRIAL REGISTRATION: This study was registered at ChiCTR1900027385 (11/11/2019).
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Analgesia , Fisura del Paladar , Nalbufina , Masculino , Niño , Femenino , Humanos , Lactante , Analgésicos Opioides , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/inducido químicamente , Fisura del Paladar/cirugíaRESUMEN
A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.
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Bencidinas , Colorimetría , Oro , Peróxido de Hidrógeno , Límite de Detección , Platino (Metal) , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Colorimetría/métodos , Oro/química , Platino (Metal)/química , Porosidad , Bencidinas/química , Peróxido de Hidrógeno/química , Aptámeros de Nucleótidos/química , Nanopartículas del Metal/química , Vancomicina/química , Técnicas Biosensibles/métodos , Catálisis , HumanosRESUMEN
Many organic reactions rely on CO2 sources to generate important structural units and valuable chemicals. In this study, we compared the effects of cannabidiol (CBD) and cannabidiolic acid (CBDA) on the supercritical CO2 (scCO2)-induced de/carboxylation reaction. The results showed that CBD was directly carboxylated in the ortho-position to form CBDA with up to 62% conversion. Meanwhile, CBDA decarboxylation occurred on hemp plant material via varying composition. Mechanistic studies revealed that CBD carboxylation was influenced not only by the physical properties of scCO2, but also by the vegetable matrix.
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Cannabidiol , Cannabinoides , Cannabis , Dióxido de Carbono , Cannabidiol/química , Cannabis/química , Cannabinoides/química , Dióxido de Carbono/química , Estructura Molecular , DescarboxilaciónRESUMEN
OBJECTIVES: To evaluate the efficacy and safety of marking ground glass nodules (GGNs) with pulmonary nodules localization needle (PNLN) prior to video-assisted thoracoscopic surgery (VATS). MATERIALS AND METHODS: From June 2020 to February 2021, all patients with GGNs who received CT-guided localization using PNLN before VATS were enrolled. Clinical and imaging data were retrospectively analyzed. RESULTS: A total of 352 consecutive patients with 395 GGNs were included in the study. The mean diameter of GGNs was 0.95 ± 0.48 cm, and the shortest distance from nodules to the pleura was 1.73 ± 0.96 cm. All 395 GGNs were marked using PNLNs. The time required for marking was 7.8 ± 2.2 min. The marking success rate was 99.0% (391/395). The marking failure of four nodules was all due to the unsatisfactory position of PNLNs. No marker dislocation occurred. Marking-related complications included pneumothorax in 63 cases (17.9%), hemorrhage in 34 cases (9.7%), and hemoptysis in 6 cases (1.7%). All the complications were minor and did not need special treatment. Localization and VATS were performed on the same day in 95 cases and on different days in 257 cases. All GGNs were successfully removed by VATS. No patient converted to thoracotomy. Histopathological examination revealed 74 (18.7%) benign nodules and 321 (81.3%) malignant nodules. CONCLUSIONS: It is safe and reliable to perform preoperative localization of GGNs using PNLNs, which can effectively guide VATS to remove GGNs. KEY POINTS: ⢠Preoperative localization of GGNs could effectively guide VATS to remove GGNs. ⢠PNLN was based on the marking principle of hook-wire, through the improvement of its material, specially designed to mark pulmonary nodules. ⢠The application of PNLN to mark GGNs had high success rate, good patient tolerance, and no dislocation. Meanwhile, VATS could be performed 2 to 3 days after marking GGNs with PNLN.
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Neoplasias Pulmonares , Nódulos Pulmonares Múltiples , Nódulo Pulmonar Solitario , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/cirugía , Nódulos Pulmonares Múltiples/diagnóstico por imagen , Nódulos Pulmonares Múltiples/cirugía , Estudios Retrospectivos , Nódulo Pulmonar Solitario/diagnóstico por imagen , Nódulo Pulmonar Solitario/cirugía , Cirugía Torácica Asistida por Video/métodosRESUMEN
A facile and general method for palladium-catalyzed stereoselective bisthiolation of terminal alkynes with allyl phenyl sulfides has been developed. The scope and versatility of the reaction have been demonstrated, and a broad range of substrates bearing electron-donating and -withdrawing groups on the aromatic rings were all compatible with this reaction, providing the desired (Z)-1,2-dithio-1-alkenes in moderate to good yields. Preliminary mechanistic studies demonstrated that the sulfur source of the desired products may be successively incorporated into alkynes via C-S bond cleavage of two molecules of allyl phenyl sulfides and ruled out the possibility of vinyl sulfides, alkynyl sulfides, and disulfide intermediates being involved in this reaction.
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Alquinos , Paladio , Alquenos/química , Alquinos/química , Catálisis , Paladio/química , Sulfuros/químicaRESUMEN
Photoacoustic imaging techniques with gold nanoparticles as contrast agents have received a great deal of attention. The photoacoustic response of gold nanoparticles strongly depends on the far-field optical properties, which essentially depend on the dielectric constant of the material. The dielectric constant of gold not only varies with wavelength but is also affected by temperature. However, the effect of the temperature dependence of the dielectric constant on gold nanoparticles' photoacoustic response has not been fully investigated. In this work, the Drude-Lorentz model and Mie theory are used to calculate the dielectric constant and absorption efficiency of gold nanospheres in aqueous solution, respectively. Then, the finite element method is used to simulate the heat transfer process of gold nanospheres and surrounding water. Finally, the one-dimensional velocity-stress equation is solved by the finite-difference time-domain method to obtain the photoacoustic response of gold nanospheres. The results show that under the irradiation of a high-fluence nanosecond pulse laser, ignoring the temperature dependence of the dielectric constant will lead to large errors in the photothermal response and the nonlinear photoacoustic signals (it can even exceed 20% and 30%). The relative error of the photothermal and photoacoustic response caused by ignoring the temperature-dependent dielectric constant is determined from both the temperature dependence of absorption efficiency and the maximum temperature increase of gold nanospheres. This work provides a new perspective for the photothermal and photoacoustic effects of gold nanospheres, which is meaningful for the development of high-resolution photoacoustic detectors and nano/microscale temperature measurement techniques.
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Owing to the small number of patients with tyrosine hydroxylase (TH) deficiency, no genotype-phenotype correlations have yet been identified. To investigate the genotype-phenotype correlation of R233H mutation in TH deficiency, we analyzed the clinical manifestations and treatment responses of four patients with the R233H homozygous mutation. Thirty-eight additional patients, available from the literature, known to be homozygous or heterozygous for the R233H mutation, were combined with the four cases from our hospital. Data for a total of 42 patients were retrieved. Our four patients showed clinical presentation consistent with Type A TH deficiency, and responded well to levodopa therapy, with an improvement in clinical symptoms within 1-2 weeks. In the 42 patients, 20 of 42 patients (48%) were homozygous and 22 (52%) were heterozygous for the R233H mutation. Of the 20 patients who were homozygous for the R233H mutation, a majority (80%) suffered from Type A TH deficiency. Of the 8 patients that were heterozygous for the R233H/the mutation located downstream of exon 11, 7 patients (86%) suffered from Type B TH deficiency. Of the 7 patients who were heterozygous for the R233H/nonsense mutation, 6 (86%) suffered from Type B TH deficiency. Genotype-phenotype correlation of R233H mutation was observed in TH deficiency. The homozygous R233H mutation frequently manifests as Type A TH deficiency, whereas R233H/nonsense mutation or any mutation located downstream of exon 11 manifests as Type B TH deficiency.
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Trastornos Distónicos/congénito , Niño , Preescolar , Trastornos Distónicos/genética , Trastornos Distónicos/fisiopatología , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Masculino , FenotipoRESUMEN
Cyclic polymers attract attention because of their endless structure and unique properties, which differ from the linear analogs. However, the synthesis of cyclic polymers is difficult and prohibits their functions and applications. In this study, we reported chiral cyclic PdII -catalysts that initiate a living ring-expansion polymerization of isocyanides, yielding a single-handed cyclic-helical poly(phenyl isocyanide), with predictable molecular weight (Mn ) and low dispersity (Mw /Mn ), in good yield. Using this method, cyclic bottlebrush polymers were prepared via the grafting-onto strategy. The cyclic topology was confirmed using various spectroscopic data and atomic force microscope observation. Moreover, the cyclic polymer brushes, comprising of a one-handed helical backbone, showed interesting photoluminescence and circularly-polarized luminescence.
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Cyclic GMP-AMP synthase (cGAS) recognizes cytosolic DNA and catalyzes the formation of cyclic GMP-AMP, which upon activation triggers the induction of stimulator of interferon genes (STING), leading to type I interferons production; these events then promote the cross-priming of dendritic cells and the initiation of a tumor-specific CD8+ T cell response. However, cancer cells in the tumor microenvironment cannot trigger intrinsic cGAS-STING signaling, regardless of the expression of cGAS and STING. This dysfunctional cGAS-STING signaling enables cancer cells to evade immune surveillance, thereby promoting tumorigenesis. Here, we review recent advances in the current understanding of the activation of cGAS-STING signaling and immunotherapies based on this pathway and focus on the mechanisms for the inactivation of this pathway in tumor cells to promote the development of cancer immunotherapy. The discovery of inherent resistance and the selection of appropriate combination therapies are of great significance for tumor treatment development.
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Proteínas de la Membrana/inmunología , Neoplasias/terapia , Nucleotidiltransferasas/inmunología , Escape del Tumor , Animales , Humanos , Inmunoterapia/métodos , Neoplasias/inmunología , Transducción de Señal , Microambiente TumoralRESUMEN
An approach for the synthesis of quinolizinone with potential bioactivity has been developed via palladium-catalytic dearomative cyclocarbonylation of allyl alcohol. Diverse quinolizinone compounds could be attained with good efficiencies. A feasible reaction pathway could be a successive procedure of allylation, dearomatization, CO insertion and the Heck reaction.
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For the ill-posed inverse problem of LII-based nanoparticle size measurement, recovered primary particle size distribution (PPSD) is sensitive to the uncertainty of LII model parameters. In the absence of reliable prior knowledge, the thermal accommodation coefficient (TAC) and fractal-dependent shielding factor are often required to be inferred simultaneously with the PPSD. In the simplified LII model for low fluence regime, TAC and fractal-dependent shielding factor are combined to define a new fractal-dependent TAC. The present study theoretically verified the feasibility of inferring PPSD and fractal-dependent TAC from the normalized LII signals. Moreover, the inversion is independent of prior knowledge of most full LII model parameters, which is attributed to low laser fluence, normalized signal, and fractal-dependent TAC.
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A new technique is developed to retrieve the fractal dimension and size distribution of soot aggregates simultaneously from the relative intensities of multi-wavelength angular-resolved light scattering. Compared with other techniques, the main advantage of this method is its independence of knowing complex refractive index, number density of aggregate, fractal prefactor and primary particle diameter. The forward light scattering procedure of soot aggregate is described by Rayleigh-Debye-Gans polydisperse fractal aggregate (RDG-PFA) scattering theory, and the retrieval process is performed by using the covariance matrix adaption-evolution strategy algorithm (CMA-ES). Three different measurement models, i.e. absolute scattering and transmittance, absolute scattering, relative scattering (RS), are investigated in present research. Numerical experiments have been performed to test the feasibility of the CMA-ES algorithm. Combined with the multi-wavelength RDG-PFA strategy, the retrieval accuracy of soot aggregate size distribution is proved to be more effectively by using the RS model. Satisfactory results under 10% Gaussian measurement noise have demonstrated the feasibility of the proposed method.
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The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-H2O2) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H2O2 through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range (3 × 102 to 3 × 107 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 104 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.
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Cronobacter/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Oro , Fórmulas Infantiles/microbiología , Nanopartículas del Metal , Cronobacter/genética , Cronobacter sakazakii/genética , ADN Bacteriano/aislamiento & purificación , Microbiología de Alimentos , Inocuidad de los Alimentos , Oro/química , Humanos , Peróxido de Hidrógeno , Hierro , Nanopartículas del Metal/química , PolvosRESUMEN
We applied urease-induced silver metallization on the surface of gold nanorods (AuNR) to improve colorimetric ELISA for the rapid and sensitive detection of Salmonella enterica Choleraesuis. To this end, we introduced a biotin-streptavidin system as a bridge to determine the correlation between urease and S. enterica Choleraesuis concentrations. The captured urease can catalyze the hydrolysis of urea into carbon dioxide and ammonia, and the generated ammonia can then induce the deposition of silver shell on the surface of AuNR in the presence of silver nitrate and glucose. With the decreased aspect ratio (length divided by width) of AuNR, a multicolor change of AuNR solution and a significant blue shift in the longitudinal localized surface plasmon resonance absorption peak (Δλmax) of AuNR were obtained at the target concentrations of 1.21 × 101 to 1.21 × 108 cfu/mL. Consequently, the detection limits of our proposed colorimetric ELISA were as low as 1.21 × 102 cfu/mL for qualitative detection with naked eyes, and 1.21 × 101 cfu/mL for quantitative detection, in which changes in Δλmax of AuNR were recorded with a microplate reader. These values were at least 2 to 3 orders of magnitude lower than those obtained with conventional horseradish peroxidase-based ELISA. The analytical performance of our developed colorimetric ELISA in terms of selectivity, accuracy, reliability, and practicability were investigated by analyzing S. enterica Choleraesuis-spiked pasteurized whole milk samples.
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Colorimetría/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Oro/química , Leche/microbiología , Nanotubos/química , Salmonella enterica/aislamiento & purificación , Ureasa/química , Animales , Colorimetría/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Microbiología de Alimentos/métodosRESUMEN
The rapid and sensitive detection of foodborne pathogens is one of the most important issues in food safety control. In this work, we developed a novel fluorescence immunoassay method for the sensitive detection of Salmonella choleraesuis. The method uses the fluorescent signals of histone-ds-poly(AT)-templated copper nanoparticles (His-pAT CuNP) as signal transducers and glucose oxidase as an alternative for horseradish peroxidase for the generation of hydrogen peroxide (H2O2) through the catalysis of glucose. The H2O2 is then further converted into hydroxyl radical (·OH) by Fenton reagents. Owing to the ultrahigh sensitivity of His-pAT CuNP synthesis toward ·OH, the proposed fluorescence immunoassay method exhibited excellent sensitivity for S. choleraesuis, with a limit of detection of 8.04 × 101 cfu/mL, which is 3 orders of magnitude lower than that of the tetramethylbenzidine-based traditional immunoassay. The reliability of the proposed method was evaluated by using spiked milk samples with S. choleraesuis concentration ranging from 8.8 × 101 to 8.8 × 104 cfu/mL. The average recoveries for the intra- and inter-assay ranged from 73.52 to 96.59% and from 66.99 to 98.24% with a coefficient of variation from 6.85 to 31.26% and 5.46 to 17.99%, respectively. These results indicated that the proposed fluorescence immunoassay possesses a great potential for ultra-sensitive detection of foodborne pathogens in food safety control.