RESUMO
Aptamers are good affinity receptors for bio-assays, while colorimetric method is suitable for point-of-care sensing via direct visualization. But previously aptamers often need complex re-engineering for colorimetric measurement at the cost of affinity and performance. Here isoquinoline alkaloids are found to own unique light-activated oxidative capacity, which can be specifically triggered by unmodified aptamers. This feature is universal for two alkaloids to efficiently oxidize four chromogenic substrates with obvious color changes. Based on a dye-displacement process, we have developed a novel light-activated aptamer system for the colorimetric assay of estradiol. It shows a good sensitivity with a detection limit of 326 nM, and this homogeneous assay is reliable to avoid artifacts in previous heterogeneous scheme. Besides, it is proven to be a universal design to assay other two targets. Significantly, they do not employ any aptamers re-engineering but only simply use their parental aptamers. Therefore, this light-activated oxidative capacity of isoquinoline alkaloid can serve as an ideal tool for colorimetric assay of various targets based on aptamer's specific recognition.
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A novel colorimetric aptasensor assay based on the excellent magnetic responsiveness and oxidase-like activity of Fe3O4@MIL-100(Fe) was developed. Fe3O4@MIL-100(Fe) absorbed with aptamer and blocked by BSA served as capture probe for selective isolation and enrichment of Listeria monocytogenes one of the most common and dangerous foodborne pathogenic bacteria. The aptamer absorbed on Fe3O4@MIL-100(Fe) was further used as signal probe that specifically binds with target bacteria conjugation of capture probe for colorimetric detection of Listeria monocytogenes, taking advantages of its oxidase-like activity. The linear range of the detection of Listeria monocytogenes was from 102 to 107 CFU mL-1, with the limit of detection as low as 14 CFU mL-1. The approach also showed good feasibility for detection of Listeria monocytogenes in milk and meat samples. The spiked recoveries were in the range 81-114% with relative standard deviations ranging from 1.28 to 5.19%. Thus, this work provides an efficient, convenient, and practical tool for selective isolation and colorimetric detection of Listeria monocytogenes in food.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Colorimetria , Microbiologia de Alimentos , Limite de Detecção , Listeria monocytogenes , Leite , Listeria monocytogenes/isolamento & purificação , Colorimetria/métodos , Aptâmeros de Nucleotídeos/química , Leite/microbiologia , Leite/química , Técnicas Biossensoriais/métodos , Animais , Contaminação de Alimentos/análise , Oxirredutases/química , Carne/microbiologia , Nanopartículas de Magnetita/químicaRESUMO
Hydroxyl radical (·OH) scavenging capacity (HOSC) estimation is essential for evaluating antioxidants, natural extracts, or drugs against clinical diseases. While nanozymes offer advantages in related applications, they still face limitations in activity and selectivity. In response, this work showcases the fabrication of laminarin-modulated osmium (laminarin-Os) nanoclusters (1.45 ± 0.05 nm), functioning as peroxidase-like nanozymes within a colorimetric assay tailored for rational HOSC estimation. This study validates both the characterization and remarkable stability of laminarin-Os. By leveraging the abundant surface negative charges of laminarin-Os and the surface hydroxyls of laminarin, oxidation reactions are facilitated, augmenting laminarin-Os's affinity for 3,3',5,5'-tetramethylbenzidine (TMB) (KM = 0.04 mM). This enables the laminarin-Os-based colorimetric assay to respond to ·OH more effectively than citrate-, albumin-, or other polysaccharides-based Os. In addition, experimental results also validate the selective peroxidase-like behavior of laminarin-Os under acidic conditions. Antioxidants like ascorbic acid, glutathione, tannic acid, and cysteine inhibit absorbance at 652 nm in the colorimetric platform using laminarin-Os's peroxidase-like activity. Compared with commercial kits, this assay demonstrates superior sensitivity (e.g., responds to ascorbic acid 0.01-0.075 mM, glutathione 1-15 µg/mL, tannic acid 0.5-5 µM, and monoammonium glycyrrhizinate cysteine 1.06-10.63 µM) and HOSC testing for glutathione, tannic acid, and monoammonium glycyrrhizinate cysteine. Overall, this study introduces a novel Os nanozyme with exceptional TMB affinity and ·OH selectivity, paving the way for HOSC estimation in biomedical research, pharmaceutical analysis, drug quality control, and beyond.
Assuntos
Benzidinas , Sequestradores de Radicais Livres , Glucanos , Radical Hidroxila , Osmio , Benzidinas/química , Colorimetria/métodos , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Glucanos/química , Radical Hidroxila/química , Radical Hidroxila/análise , Osmio/química , Oxirredução , Peroxidase/química , Peroxidase/metabolismoRESUMO
A novel Fe-MoOx nanozyme, engineered with enhanced peroxidase (POD)-like activity through strategic doping and the creation of oxygen vacancies, is introduced to catalyze the oxidation of TMB with high efficiency. Furthermore, Fe-MoOx is responsive to single electron transfer (SET) and hydrogen atom transfer (HAT) mechanisms related to antioxidants and can serve as a desirable nanozyme for total antioxidant capacity (TAC) determination. The TAC colorimetric platform can reach a low LOD of 0.512 µM in solution and 24.316 µM in the smartphone-mediated RGB hydrogel (AA as the standard). As proof of concept, the practical application in real samples was explored. The work paves a promising avenue to design diverse nanozymes for visual on-site inspection of food quality.
Assuntos
Antioxidantes , Colorimetria , Oxirredução , Antioxidantes/química , Antioxidantes/análise , Antioxidantes/metabolismo , Colorimetria/métodos , Catálise , Molibdênio/química , Limite de Detecção , Ferro/química , Benzidinas/química , Smartphone , Hidrogéis/química , Transporte de Elétrons , Técnicas Biossensoriais/métodos , Óxidos/químicaRESUMO
It is critical to develop quick, accurate, and efficient sterilization for detecting Escherichia coli O157:H7 in order to prevent infections and outbreaks of foodborne illnesses. Herein, we established a colorimetric biosensor with sterilizing properties using copper selenide nanoparticles to detect E. coli O157:H7. The sample was mixed with magnetic nanoprobes and nanozyme probes to form a sandwich structure, and then the unbound nanozyme probes were collected by magnetic separation. Finally, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-hydrogen peroxide (H2O2) reporting system was added for signal amplification. The change from colorless to green can be seen with the naked eye. Under the optimal conditions, the detection range of E. coli O157:H7 was 102-106 CFU/mL, and the detection limit was 0.35 × 102 CFU/mL. The total detection time was 80 minutes, which can be successfully applied to milk and mineral water. In addition, the colorimetric sensor can kill the target bacteria by irradiating it under a 980-nm laser for 5 minutes. In conclusion, this sensor is a promising tool for rapidly detecting foodborne pathogens and promptly eliminating bacteria. IMPORTANCE: Escherichia coli O157:H7 is a major threat to public health. At present, the detection methods for E. coli O157:H7 mainly include traditional bacterial culture, immunology (enzyme-linked immune-sorbent assay) and molecular biology techniques (polymerase chain reaction). These methods have the limitations of professional operation, waste of time and energy, and high cost. Therefore, we have developed a simple, fast, bactericidal colorimetric biosensor to detect E. coli. O157:H7. The entire process was completed in 80 minutes. The method has been successfully applied to milk and mineral water samples with satisfactory results, proving that the method is an effective method for real-time detection and inactivation of bacteria.
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Técnicas Biossensoriais , Colorimetria , Escherichia coli O157 , Microbiologia de Alimentos , Escherichia coli O157/isolamento & purificação , Colorimetria/métodos , Técnicas Biossensoriais/métodos , Microbiologia de Alimentos/métodos , Cobre , Leite/microbiologia , Animais , Nanopartículas/química , Peróxido de Hidrogênio/farmacologiaRESUMO
A colorimetric analysis platform has been successfully developed based on FeCo-NC dual-atom nanozyme (FeCo-NC DAzyme) for the detection of organophosphorus pesticides (OPPs). The FeCo-NC DAzyme exhibited exceptional oxidase-like activity (OXD), enabling the catalysis of colorless TMB to form blue oxidized TMB (oxTMB) without the need for H2O2 involvement. By combining acid phosphatase (ACP) hydrolase with FeCo-NC DAzyme, a "FeCo-NC DAzyme + TMB + ACP + SAP" colorimetric system was constructed, which facilitated the rapid detection of malathion. The chromogenic system was applied to detect malathion using a smartphone-based app and an auxiliary imaging interferogram device for colorimetric measurements, which have a linear range of 0.05-4.0 µM and a limit of detection (LOD) as low as 15 nM in real samples, comparable to UV-Vis and HPLC-DAD detection methods. Overall, these findings present a novel approach for convenient, rapid, and on-site monitoring of OPPs.
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Colorimetria , Limite de Detecção , Praguicidas , Smartphone , Colorimetria/métodos , Praguicidas/análise , Compostos Organofosforados/análise , Compostos Organofosforados/química , Malation/análise , Malation/química , Oxirredutases/química , Ferro/química , Fosfatase Ácida/análise , Fosfatase Ácida/química , BenzidinasRESUMO
The study aimed to develop a rapid and sensitive colorimetric platform based on the Emerson reaction to visualize and determine total aflatoxins (AFs) in peanut oil. This method offers the advantage of fast screening for AFs (AFB1, AFB2, AFG1, and AFG2), eliminating the need for specific antibodies. The proposed approach combined colorimetric detection with magnetic dummy imprinted solid-phase extraction and purification, enhancing sensitivity and selectivity. The oxidizer aided the colorless AFs in reacting with 4-aminoantipyrine, producing green condensates. Thus, a dual-mode approach was developed for AFs detection, employing both UV-vis colorimetric and smartphone-based colorimetry. Both methods showed a good linear relationship with the concentration of AFs. Notably, the smartphone-based method demonstrated a detection range of 0.5-57 µg/kg, with a detection limit as low as 0.21 µg/kg. The suggested colorimetric methods present a promising potential for onsite detection and fast screening of AFs in actual samples.
Assuntos
Aflatoxinas , Colorimetria , Contaminação de Alimentos , Óleo de Amendoim , Smartphone , Extração em Fase Sólida , Colorimetria/métodos , Extração em Fase Sólida/métodos , Extração em Fase Sólida/instrumentação , Aflatoxinas/análise , Aflatoxinas/isolamento & purificação , Óleo de Amendoim/química , Contaminação de Alimentos/análise , Limite de Detecção , Impressão MolecularRESUMO
A microscale colorimetric assay was designed and implemented for the simultaneous determination of clinical chemistry tests measuring six parameters, including glucose (GLU), total protein (TP), human serum albumin (HSA), uric acid (UA), total cholesterol (TC), and triglycerides (TGs) in plasma samples. The test kit was fabricated using chromogenic reagents, comprising specific enzymes and binding dyes. Multiple colors that appeared on the reaction well when it was exposed to each analyte were captured by a smartphone and processed by the homemade Check6 application, which was designed as a colorimetric analyzer and simultaneously generated a report that assessed test results against gender-dependent reference ranges. Six blood checkup parameters for four plasma samples were conducted within 12 min on one capture picture. The assay achieved wide working concentration ranges of 10.45-600 mg dL-1 GLU, 1.39-10.0 g dL-1 TP, 1.85-8.0 g dL-1 HSA, 0.86-40.0 mg dL-1 UA, 11.28-600 mg dL-1 TC, and 11.93-400 mg dL-1 TGs. The smartphone-based assay was accurate with recoveries of 93-108% GLU, 93-107% TP, 92-107% HSA, 93-107% UA, 92-107% TC, and 99-113% TGs. The coefficient of variation for intra-assay and inter-assay precision ranged from 3.2-5.2% GLU, 4.6-5.3% TP, 4.3-5.3% HSA, 2.8-6.6% UA, 2.7-6.5% TC, and 1.1-3.9% TGs. This assay demonstrated remarkable accuracy in quantifying the concentration-dependent color intensity of the plasma, even in the presence of other suspected interferences commonly present in serum. The results of the proposed method correlated well with results determined by the microplate spectrophotometer (R2 > 0.95). Measurement of these six clinical chemistry parameters in plasma using a microscale colorimetric test kit coupled with the Check6 smartphone application showed potential for real-time point-of-care analysis, providing cost-effective and rapid assays for health checkup testing.
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Colorimetria , Smartphone , Humanos , Colorimetria/métodos , Colorimetria/instrumentação , Feminino , Masculino , Glicemia/análise , Sistemas Automatizados de Assistência Junto ao Leito , Colesterol/sangue , Ácido Úrico/sangue , Triglicerídeos/sangue , Testes Imediatos , Albumina Sérica Humana/análiseRESUMO
To realize the reutilization of waste Myrica rubra in the analytical field, we synthesized Myrica rubra-based N-doped carbon dots (MN-CDs) and further anchored them onto the surface of Fe3S4 to fabricate Fe3S4@MN-CD nanocomposites. The as-fabricated nanocomposites possessed higher peroxidase-mimetic activity than its two precursors, resulting from the synergistic effect between them, and could catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) into deep blue oxTMB with a strong 652-nm absorption. Under optimized conditions (initial solution pH, 3.5; incubation temperature, 35 â; Fe3S4@MN-CD concentration, 50 µg mL-1, and 652-nm absorption), Fe3S4@MN-CDs were employed for colorimetric assay of p-aminophenol (p-AP) with wide linear range (LR, 2.9-100 µM), low detection limit (LOD, 0.87 µM), and satisfactory recoveries (86.3-105%) in environmental waters. Encouragingly, this colorimetric assay provided the relative accuracy of 97.0-99.4% as compared with conventional HPLC-UV detection. A portable smartphone-based colorimetric application was developed by combining the Fe3S4@MN-CD-based visually chromogenic reaction with a "Thing Identify" APP software. Besides, we engineered an image-capturing device feasible for field use, in which the internal-compact sealing prevented external light source from entering photography chamber, thereby reducing light interference, and also the bottom light source enhanced the intensity of blue imaging. This colorimetric platform exhibited satisfactory LR (1-500 µM), low LOD (0.3 µM), and fortification recoveries (86.6-99.6%). In the chromogenic reaction catalyzed by Fe3S4@MN-CDs, ·O2- played a key role in concomitant with the participation of â¢OH and h+. Both the colorimetric assay and smartphone-based intelligent sensing show great promising in on-site monitoring of p-AP under field conditions.
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Aminofenóis , Carbono , Colorimetria , Limite de Detecção , Pontos Quânticos , Smartphone , Poluentes Químicos da Água , Colorimetria/métodos , Aminofenóis/química , Aminofenóis/análise , Carbono/química , Poluentes Químicos da Água/análise , Pontos Quânticos/química , Materiais Biomiméticos/química , Benzidinas/química , Peroxidase/químicaRESUMO
Wax printing is the most widely used method for fabricating microfluidic paper-based analytical devices (µPADs), but it still suffers from disadvantages like discontinuation of wax printers and need for additional equipment for heating treatment. To address these issues, this work initially describes a new class of wax printing approach for high-precision, batch fabrication of µPADs using a household 3D printer. It only involves a one patterning step of printing polyethylene wax into rice paper body. Under optimized parameters, a fabrication resolution, namely the minimum hydrophilic channel width, down to ~189 ± 30 µm could be achieved. In addition, the analytical applicability of such polyethylene wax-patterned µPADs was demonstrated well with enhanced colorimetric detection of dopamine as a model analyte by combining metal-organic framework (MOF) based nanoenzymes (ZIF-67) with a smartphone (for portable quantitative readout). The developed nanosensor could linearly detect dopamine over a concentration range from 10 to 1000 µM, with a detection limit of ca. 2.75 µM (3σ). The recovery results for analyzing several real samples (i.e., pig feed, chicken feed, pork and human serum) were between 91.82 and 102.79%, further validating its good detection accuracy for potential practical applications in food safety and medical diagnosis.
Assuntos
Dopamina , Limite de Detecção , Papel , Impressão Tridimensional , Dopamina/análise , Dopamina/sangue , Animais , Humanos , Estruturas Metalorgânicas/química , Colorimetria/métodos , Colorimetria/instrumentação , Suínos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Dispositivos Lab-On-A-Chip , Galinhas , Ração Animal/análise , Desenho de EquipamentoRESUMO
BACKGROUND: Human rhinovirus 3C protease (HRV-3Cpro) plays a crucial role in viral proliferation, establishing it as a prime target for antiviral therapy. However, research on identifying HRV-3Cpro inhibitors is still limited. OBJECTIVE: This study had two primary objectives: first, to validate the efficacy of an end-point colorimetric assay, previously developed by our team, for identifying potential inhibitors of HRV-3Cpro; and second, to discover phytochemicals in medicinal plants that inhibit the enzyme's activity. METHODS: Rupintrivir, a well-known inhibitor of HRV-3Cpro, was used to validate the colorimetric assay. Following this, we conducted a two-step in silico screening of 2532 phytochemicals, which led to the identification of eight active compounds: apigenin, carnosol, chlorogenic acid, kaempferol, luteolin, quercetin, rosmarinic acid, and rutin. We subsequently evaluated these candidates in vitro. To further investigate the inhibitory potential of the most promising candidates, namely, carnosol and rosmarinic acid, molecular docking studies were performed to analyze their binding interactions with HRV-3Cpro. RESULTS: The colorimetric assay we previously developed is effective in identifying compounds that selectively inhibit HRV-3Cpro. Carnosol and rosmarinic acid emerged as potent inhibitors, inhibiting HRV-3Cpro activity in vitro by over 55%. Our analysis indicated that carnosol and rosmarinic acid exert their inhibitory effects through a competitive mechanism. Molecular docking confirmed their competitive binding to the enzyme's active site. CONCLUSION: Carnosol and rosmarinic acid warrant additional investigation for their potential in the development of common cold treatment. By highlighting these compounds as effective HRV-3Cpro inhibitors, our study presents a promising approach for discovering phytochemical inhibitors against proteases from similar pathogens.
Assuntos
Proteases Virais 3C , Antivirais , Resfriado Comum , Simulação de Acoplamento Molecular , Compostos Fitoquímicos , Rhinovirus , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Proteases Virais 3C/antagonistas & inibidores , Rhinovirus/enzimologia , Rhinovirus/efeitos dos fármacos , Humanos , Antivirais/farmacologia , Antivirais/química , Resfriado Comum/tratamento farmacológico , Resfriado Comum/virologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores de Proteases/farmacologia , Inibidores de Proteases/químicaRESUMO
Oxidized albumin is considered a short-term biomarker of oxidative stress and its measurement in blood contributes to evaluate the impact of diseases, drugs, dialytic treatments, physical activity, environmental contaminants etc. on the red-ox balance of humans as well as of other mammalians. Nevertheless, the most common methods for quantifying the oxidized and reduced albumins are costly and time-consuming. Furthermore, there is a dearth of information regarding the proper ways to store human serum or plasma samples in order to prevent inaccurate quantification of these various albumin forms. This paper explores these aspects and proposes a few spectrophotometric assay procedures which make the quantitation of oxidized and reduced albumin very fast, precise and un-expensive in various mammals.
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Oxirredução , Albumina Sérica , Animais , Humanos , Biomarcadores/sangue , Mamíferos/sangue , Estresse Oxidativo , Albumina Sérica/análise , EspectrofotometriaRESUMO
An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.
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Colorimetria , Muramidase , Saliva , Muramidase/análise , Muramidase/química , Muramidase/metabolismo , Colorimetria/métodos , Humanos , Saliva/química , Saliva/enzimologia , Limite de Detecção , Peptídeos/química , Aptâmeros de Nucleotídeos/química , Proteínas/análise , Técnicas Biossensoriais/métodos , Histidina/análise , Histidina/químicaRESUMO
Phytosterols (PSs), a class of naturally occurring bioactive lipid compounds, have been found to possess a significant cholesterol-lowering effect. In developing countries, the consumption of rapeseed oil is the primary pathway of PS intake for the general population. However, developing low-cost, real-time, and high-throughput screening techniques for PSs remains a challenge. Here, a Cu-based nanocomposite CuOx@C was synthesized via a simple method of the calcination of HKUST-1 and systematically characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The CuOx@C demonstrated excellent peroxidase-like (POD-like) activity, functioning as a peroxidase mimic to facilitate the catalysis of 3,3',5,5'-tetramethylbenzidine (TMB) into its oxidized form (oxTMB), thereby initiating a discernible color response. On the basis of this discovery, a CuOx@C-based colorimetric method for detecting total sterols in rapeseed was successfully constructed via cascade reactions. After optimizing the conditions, the high-throughput screening of total sterols in rapeseed could be completed in only 21 min, which significantly facilitated the sensing of PSs. A linear range of 0.6-6 mg/g was achieved for the detection of total sterols in rapeseed samples, thereby satisfying the requirements for detection. In addition, due to the high stability of CuOx@C and the specificity of cholesterol oxidase, the developed method had excellent stability and selectivity toward PSs, indicating that this work has huge prospects for commercial application. This innovative work overcomes the limitation of the instrumental method and provides a portable and reliable tool for total sterols detection. It can also facilitate the development of oilseeds with a high content of PSs.
Assuntos
Benzidinas , Colorimetria , Cobre , Fitosteróis , Colorimetria/métodos , Fitosteróis/análise , Fitosteróis/química , Cobre/química , Benzidinas/química , Estruturas Metalorgânicas/química , Limite de Detecção , Catálise , Nanocompostos/química , OxirreduçãoRESUMO
Nanoceria have demonstrated a wide array of catalytic activity similar to natural enzymes, holding considerable significance in the colorimetric detection of alkaline phosphatase (ALP), which is a biomarker of various biological disorders. However, the issues of physiological stability and formation of protein corona, which are strongly related to their surface chemistry, limit their practical application. In this work, CeO2 nanoparticles characterized by enhanced dimensional uniformity and specific surface area were synthesized, followed by encapsulation with various polymers to further increase catalytic activity and physiological stability. Notably, the CeO2 nanoparticles encapsulated within each polymer exhibited improved catalytic characteristics, with PAA-capped CeO2 exhibiting the highest performance. We further demonstrated that the PAA-CeO2 obtained with enhanced catalytic activity was attributed to an increase in surface negative charge. PAA-CeO2 enabled the quantitative assessment of AA activity within a wide concentration range of 10 to 60 µM, with a detection limit of 0.111 µM. Similarly, it allowed for the evaluation of alkaline phosphatase activity throughout a broad range of 10 to 80 U/L, with a detection limit of 0.12 U/L. These detection limits provided adequate sensitivity for the practical detection of ALP in human serum.
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Osteoarthritis occurs in any joints, and identification in its earlier stages helps to treat the disease and increase the recovery rate. The radiography method and imaging techniques are traditionally used to identify osteoarthritis. But these methods are expensive, and with the complicated steps. Researchers are working toward developing a highly sensitive biosensor in identifying the osteoarthritis biomarker. This research was focused on developing a C-terminal telopeptide of type II collagen (CTX-II) colorimetric sensor with gold nanoparticle (AuNP) for diagnosing osteoarthritis. Anti-CTX-II was conjugated with AuNP and then added with CTX-II and sodium chloride for the color change. In the presence of CTX-II, antibody releases from AuNP then binds with CTX-II, and the color of AuNP changed to purple. Without the CTX-II, AuNP remains its red color (dispersed). This easier colorimetric assay detected the CTX-II as low as 2 ng/mL on linear regression [y = 0.0131x - 0.0051; R2 = 0.9205]. Furthermore, control performances with the relevant proteins osteopontin, IL-6, and nonimmune antibody failed to change the color confirming the specific identification of CTX-II.
Assuntos
Colorimetria , Ouro , Nanopartículas Metálicas , Osteoartrite , Ouro/química , Nanopartículas Metálicas/química , Osteoartrite/metabolismo , Humanos , Colágeno Tipo II , Cloreto de Sódio/química , Técnicas BiossensoriaisRESUMO
Bloodstream infections (BSIs) caused by multidrug-resistant bacteria are a critical life-threatening challenge which necessitates the urgency to trigger life-saving treatment in a timely manner. This study aimed to evaluate the time required for rapid detection of carbapenemase-producing Enterobacterales (CPE) directly from blood culture bottles to optimize empirical treatment of BSI, especially in pediatric and infant patients, using a cost-effective method. This study included 419 Gram-negative bacteria, of which Klebsiella pneumoniae and Escherichia coli were the most common CPE causing BSI in pediatric and neonatal patients. Phenotypic and genotypic resistance of the selected isolates (45 K. pneumoniae and 9 E. coli) were determined by VITEK-2 Compact system and PCR, respectively. BACT/ALERT bottles were spiked with isolates. Finally, colorimetric RESIST-BC assay and Vitek-2 compact system were evaluated for the rapid detection of carbapenem-resistant bacteria directly from positive blood culture bottles. All selected isolates were phenotypically resistant to carbapenems. PCR showed that blaNDM and blaOXA-48 were present in all isolates, blaVIM was present in 44.4%, while blaKPC and blaIMP were entirely absent. The RESIST-BC kit showed good agreement with PCR for blaNDM and blaOXA-48, demonstrating high sensitivity and specificity, but not with blaVIM. These findings point out that RESIST-BC assay demonstrated an exceptionally short detection time for CPE, completing all cases within the first hour after the blood culture bottles flagged positive. It is also superior in providing a clue for clinicians on antibiotic combinations that can be administered, depending on the type of ß-lactamases detected, promptly and efficiently, with low expenses.
Assuntos
Antibacterianos , Proteínas de Bactérias , Hemocultura , beta-Lactamases , Humanos , beta-Lactamases/genética , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/genética , Lactente , Criança , Bacteriemia/microbiologia , Bacteriemia/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/sangue , Reação em Cadeia da Polimerase/métodos , Recém-NascidoRESUMO
Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.
Assuntos
Biocatálise , Éteres , Silanos , Espectrofotometria , Hidrólise , Espectrofotometria/métodos , Silanos/química , Cinética , Éteres/química , Éteres/metabolismo , Animais , Catepsinas/metabolismo , Catepsinas/químicaRESUMO
Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.
Assuntos
Colorimetria , Enzimas Imobilizadas , Fluorometria , Peroxidase do Rábano Silvestre , Urato Oxidase , Ácido Úrico , Ácido Úrico/sangue , Ácido Úrico/química , Ácido Úrico/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Urato Oxidase/química , Urato Oxidase/metabolismo , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Tamanho da Partícula , Humanos , Suspensões , Oxazinas/químicaRESUMO
Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.