RESUMEN
Tyrosinase (TYR) emerges as a key enzyme that exerts a regulatory influence on the synthesis of melanin, thereby assuming the role of a critical biomarker for the detection of melanoma. Detecting the authentic concentration of TYR in the skin remains a primary challenge. Distinguished from ex vivo detection methods, this study introduces a novel sensor platform that integrates a microneedle (MN) biosensor with surface-enhanced Raman spectroscopy (SERS) technology for the in situ detection of TYR in human skin. The platform utilized dopamine (DA)-functionalized gold nanoparticles (Au NPs) as the capturing substrate and 4-mercaptophenylboronic acid (4-MPBA)-modified silver nanoparticles (Ag NPs) acting as the SERS probe. Here, the Au NPs were functionalized with mercaptosuccinic acid (MSA) for DA capture. In the presence of TYR, DA immobilized on the MN is preferentially oxidized to dopamine quinone (DQ), a process that results in a decreased density of SERS probes on the platform. TYR concentration was detected through variations in the signal intensity emitted by the phenylboronic acid. The detection system was able to evaluate TYR concentrations within a linear range of 0.05 U/mL to 200 U/mL and showed robust anti-interference capabilities. The proposed platform, integrating MN-based in situ sensing, SERS technology, and TYR responsiveness, holds significant importance for diagnosing cutaneous melanoma.
Asunto(s)
Técnicas Biosensibles , Detección Precoz del Cáncer , Melanoma , Monofenol Monooxigenasa , Espectrometría Raman , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/metabolismo , Piel/enzimología , Animales , Ratones , Melanoma/diagnóstico , Melanoma/enzimología , Nanopartículas del Metal/química , Oro/química , Agujas/normas , Ensayo de Inmunoadsorción Enzimática , Plata/química , Sensibilidad y Especificidad , Detección Precoz del Cáncer/instrumentación , Detección Precoz del Cáncer/métodosRESUMEN
Sensitive and convenient strategy of tyrosinase (TYR) and its inhibitor atrazine is in pressing demand for essential research as well as pragmatic application. In this work, an exquisite label-free fluorometric assay with high sensitivity, convenience and efficiency was described for detecting TYR and the herbicide atrazine on the basis of fluorescent nitrogen-doped carbon dots (CDs). The CDs were prepared via one-pot hydrothermal reaction starting from citric acid and diethylenetriamine. TYR catalyzed the oxidation of dopamine to dopaquinone derivative which could quench the fluorescence of CDs through a fluorescence resonance energy transfer (FRET) process. Thus, a sensitive and selective quantitative evaluation of TYR can be constructed on the basis of the relationship between the fluorescence of CDs and TYR activity. Atrazine, a typical inhibitor of TYR, inhibited the catalytic activity of TYR, leading to the reduced dopaquinone and the fluorescence was retained. The strategy covered a broad linear range of 0.1-150 U/mL and 4.0-80.0 nM for TYR and atrazine respectively with a low detection limit of 0.02 U/mL and 2.4 nM/mL. It is also demonstrated that the assay can be applied to detect TYR and atrazine in spiked complex real samples, which provides infinite potential in application of disease monitoring along with environmental analysis.
Asunto(s)
Atrazina , Dihidroxifenilalanina/análogos & derivados , Puntos Cuánticos , Monofenol Monooxigenasa/análisis , Carbono , Atrazina/análisis , Benzoquinonas , Colorantes Fluorescentes , NitrógenoRESUMEN
OBJECTIVE: To characterize the chemical profile of methanolic crude extract and its fractions (Ethyl acetate, n-butanol and aqueous) using liquid chromatography-mass spectrometry (LC-MS) analysis, to evaluate their biological and pharmacological properties: antioxidant (1, 1-diphenyl-2-pycrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic) (ABTS), galvinoxyle free radical scavenging, reducing power, phenanthroline and ß carotene-linoleic acid bleaching assays), enzymes inhibitory ability against several enzymes [acetyl-cholinesterase (AChE), buthyrylcholinesterase (BChE), urease and tyrosinase]. METHODS: Secondary metabolites were extracted from Tamarix africana air-dried powdered leaves by maceration, the crude extract was fractionated using different solvents with different polarities (Ethyl acetate, n-butanol and aqueous). The amount of polyphenols, flavonoids and tannins (hydrolysable and condensed) were determined using colorimetric assays. A variety of biochemical tests were carried out to assess antioxidant and oxygen radical scavenging properties using DPPH, ABTS, galvinoxyle free radical scavenging, reducing power, phenanthroline and ß carotene-linoleic acid bleaching methods. Neuroprotective effect was examined against acetylcholinesterase and buthy-rylcholinesterase enzymes. The anti-urease and anti-tyrosinase activities were performed against urease and tyrosinase enzymes respectively. The extract's components were identified using LC-MS and compared to reference substances. RESULTS: The results indicated that Tamarix africana extracts presented a powerful antioxidant activity in all assays and exhibited a potent inhibitory effect against AChE and BChE as well as urease and tyrosinase enzymes. LC-MS analysis identified amount of eight phenolic compounds were revealed in this analysis; Apigenin, Diosmin, Quercetin, Quercetine-3-glycoside, Apigenin 7-O glycoside, Rutin, Neohesperidin and Wogonin in methanolic extract and its different fractions of Tamarix africana from leaves. CONCLUSIONS: Based on these findings, it is reasonable to assume that Tamarix africana could be considered as a potential candidate for pharmaceutical, cosmetics, and food industries to create innovative health-promoting drugs.
Asunto(s)
Antioxidantes , Monofenol Monooxigenasa , Humanos , Antioxidantes/farmacología , Antioxidantes/química , Monofenol Monooxigenasa/análisis , Extractos Vegetales/farmacología , Extractos Vegetales/química , Acetilcolinesterasa/análisis , Acetilcolinesterasa/metabolismo , Ureasa/análisis , Ureasa/metabolismo , 1-Butanol/análisis , Apigenina/análisis , Ácido Linoleico/análisis , Fenantrolinas/análisis , beta Caroteno/análisis , Hojas de la Planta/química , Flavonoides/farmacología , Radicales Libres , Glicósidos/análisisRESUMEN
myrsine coriacea (Sw.) R. Br. ex Roem. & Schult. (Primulaceae) conhecida popularmente como capororoquinha ou capororoca, é amplamente distribuída nas regiões sul e sudeste do Brasil. As espécies desse gênero apresentam um potencial antioxidante e anti-inflamatório, que pode ser acessado na busca de novos ativos para o tratamento de desordens pigmentares da pele. Desta forma, este trabalho teve como objetivos avaliar o potencial antitirosinase e antioxidante de extratos e frações de M. coriacea e identificar os possíveis compostos responsáveis por essas atividades. Foram realizados ensaios para avaliar o potencial antioxidante das amostras através do método do DPPH, enquanto a capacidade hipopigmentante das amostras foi avaliado pela inibição da enzima tirosinase. Como complemento, foram determinados os teores de compostos fenólicos totais e flavonoides através dos métodos colorimétricos empregando o reagente Folin-Ciocalteau e AlCl3. Adicionalmente, os extratos de M. coriacea tiveram avaliados seus potenciais citotóxicos utilizando diferentes linhagens tumorais humanas. O perfil fitoquímico de M. coriacea foi analisado por cromatografia a gás acoplada com espectrometria de massas (CG-EM) e cromatografia em camada delgada (CCD) com padrões. Nessas análises foram identificados 34 compostos, sendo o ácido palmítico e o palmitato de etila os compostos majoritários nas amostras de M. coriacea. O extrato bruto das folhas apresentou o maior teor de fenólicos totais, enquanto a fração de acetato de etila das folhas teve o maior teor de flavonoides. Contudo, o extrato bruto dos frutos apresentou a melhor atividade antioxidante de todas as amostras analisadas, apresentando também a melhor atividade antitirosinase. Dentre os compostos anotados, mandenol, ácido -linoleico e o linolenato de etila foram os compostos considerados como possíveis inibidores da tirosinase, com boa interação molecular com a enzima nas análises de ancoragem molecular in silico. Das amostras analisadas com relação a inibição de crescimento frente as células tumorais, a amostra da fração de clorofórmio das folhas foi a que apresentou potencial antitumoral frente as células de adenocarcinoma de cólon (HCT116)
myrsine coriacea (Sw.) R. Br. ex Roem. & Schult. (Primulaceae) popularly known as capororoquinha or capororoca, is widely distributed in southern and southeastern Brazil. Myrsine species have an antioxidant and anti-inflammatory potential, which can be accessed in the search for new actives for the treatment of skin pigmentation disorders. Thus, this work aimed to evaluate the antityrosinase and antioxidant potential from extracts and fractions of M. coriacea and to identify the probable compounds responsible for these activities. Assays were performed to evaluate the antioxidant potential of the samples using the DPPH method, while the hypopigmentation capacity of the samples was evaluated by the tyrosinase inhibition. As a complement, the amounts of total phenolic compounds and flavonoids were determined through colorimetric methods using the Folin-Ciocalteau reagent and AlCl3. Additionally, M. coriacea extracts had their cytotoxic potential evaluated using different human tumor cell lines. M. coriacea phytochemical profile was obtained by gas chromatography coupled with mass spectrometry (GC-MS) and thin layer chromatography (TLC) with standards. In these analyses, 34 compounds were identified, with palmitic acid and ethyl palmitate as the major compounds in M. coriacea samples. The leaf crude extract presented the highest total phenolics contents, while the leaf ethyl acetate fraction had the highest flavonoid amounts. However, the fruit crude extract showed the best antioxidant and antityrosinase activities of all analyzed samples. Among the annotated compounds, mandenol, -linoleic acid and ethyl linolenate were the compounds considered as putative tyrosinase inhibitors, presenting good molecular interaction with the enzyme active site in the in silico molecular docking analysis. The leaf chloroform fraction was the only sample that showed an antitumor potential against colon adenocarcinoma cells (HCT116)
Asunto(s)
Monofenol Monooxigenasa/análisis , Primulaceae/metabolismo , Myrsine/clasificación , Frutas/clasificación , Antioxidantes/análisis , Espectrometría de Masas/métodos , Pigmentación de la Piel/inmunología , Cromatografía en Capa Delgada/métodos , Hipopigmentación/patologíaRESUMEN
This study aimed to investigate the chemical composition of essential oils isolated from Acca sellowiana (feijoa) leaves and stems and elaborate on their relevance as natural anti-aging, coupled with molecular-docking studies. The isolated oils were analysed using gas chromatography-mass spectrometry analysis and investigated for inhibitory effects against acetylcholinesterase, ß-secretase, collagenase, elastase and tyrosinase. Molecular-modelling study was performed using MOE-Dock program to evaluate binding interactions of major components with the above-mentioned targets. The leaf oil revealed the predominance of caryophyllene oxide (24.3 %), linalool (7.9 %), and spathulenol (6.6 %), while the stem oil was presented by caryophyllene oxide (38.1 %), α-zingiberene (10.1 %) and humulene oxideâ II (6.0 %). The stem oil expressed superior inhibitory activities against acetylcholinesterase (IC50 =0.15±0.01â µg/mL), ß-secretase (IC50 =3.99±0.23â µg/mL), collagenase (IC50 =408.10±20.80â µg/mL), elastase (IC50 =0.17±0.01â µg/mL) and tyrosinase (IC50 =8.45±0.40â µg/mL). The valuable binding interactions and docking scores were observed for caryophyllene oxide and α-zingiberene with acetylcholinesterase. Besides, α-zingibirene followed by linalool and τ-cadinol revealed tight fitting with collagenase and elastase. Additionally, linalool, spathulenol and τ-cadinol showed the best binding energy to tyrosinase. This study provides valuable scientific data on A.â sellowiana as potential candidates for the development of natural antiaging formulations. The current study provided scientific evidence for the potential use of feijoa essential oils in antiaging formulations and as an adjuvant for the prophylaxis against Alzheimer disease.
Asunto(s)
Feijoa , Aceites Volátiles , Acetilcolinesterasa , Monoterpenos Acíclicos , Secretasas de la Proteína Precursora del Amiloide , Feijoa/química , Simulación del Acoplamiento Molecular , Sesquiterpenos Monocíclicos , Monofenol Monooxigenasa/análisis , Aceites Volátiles/química , Óxidos , Elastasa Pancreática/análisis , Hojas de la Planta/química , Sesquiterpenos Policíclicos , Sesquiterpenos , TerpenosRESUMEN
Seven pairs of enantiomeric phenylpropanoids (1a/1b-7a/7b), including eight undescribed phenylpropanoids (1a, 2a/2b, 3a, 5a, 6a/6b, and 7b) were found in the fruits of Crataegus pinnatifida Bge. The enantiomers were separated using chiral column and elucidated through various spectroscopic methods and quantum chemical ECD calculations. The tyrosinase inhibitory activity of enantiomers was tested in vitro. Among them, enantiomers 1a/1b displayed moderate tyrosinase inhibitory activities. The interaction patterns were further studied by molecular docking, and the results indicated compounds 1a/1b may bind in the active site near the copper atom and interacted with the hydrophobic areas in the active pocket. PRACTICAL APPLICATIONS: Melanins exist widely in organisms and will be produced more against environmental stresses. Without th enzymatic action of tyrosinase, melanin biosynthesis would be blocked. Thus, tyrosinase inhibitors have been developed to modulate melanin formation. In this study, the enantiomeric phenylpropanoid isolated from the fruits of C. pinnatifida Bge displayed moderate tyrosinase inhibitory activities. This work may provide foundations for further research on tyrosinase inhibitor from natural sources.
Asunto(s)
Crataegus , Cobre , Crataegus/química , Frutas/química , Melaninas/análisis , Simulación del Acoplamiento Molecular , Estructura Molecular , Monofenol Monooxigenasa/análisisRESUMEN
Bee pollen, which is known as a "full-nutrient food", has outstanding anti-tyrosinase activity. However, the chemical components contributing to this activity remain unknown. To comprehensively elucidate the chemical components of bee pollen inhibiting tyrosinase, we performed the anti-tyrosinase activity evaluation of bee pollen extract (BPE) of eight species, metabolomic analysis of chemical composition, multivariate statistical analysis and correlation analysis. The results revealed that the anti-tyrosinase activity of eight BPEs was significantly different (p < 0.05), with IC50 value ranging from 10.08 to 408.81 µg/mL. A total of 725 metabolites were detected from these BPEs, and 40 differential metabolites were identified, all of which were phenolamides. All these phenolamides were positively correlated with the anti-tyrosinase activity, among which 26 phenolamides (21 spermidine derivatives and five spermine derivatives) showed particularly high correlations (r > 0.7). This is the first report to reveal the main contributor to the anti-tyrosinase activity of bee pollen.
Asunto(s)
Metabolómica , Polen , Animales , Antioxidantes/química , Abejas , Monofenol Monooxigenasa/análisis , Extractos Vegetales/química , Polen/químicaRESUMEN
Ginkgo biloba L. (Ginkgoaceae) is one of the best-selling products, popular in nutritional properties and health benefits. In the present study, the total phenolic compounds and flavonoid content of the ethanolic extract from G. biloba tea were also evaluated. Furthermore, the antioxidant activity was determined using DPPH assay and tyrosinase inhibitory activity was also determined with L-DOPA as a substrate. The extract showed the total phenolic compound and flavonoid content were 14.13 mg GE g-1 extract and 71.33 mg rutin equivalence g-1 DW, respectively. Taking into account the results of the DPPH, the antioxidant property at the concentration of 500 µg ml-1 was 95.29% that is similar to that of the BHT, ascorbic acid, and gallic acid used as positive controls. The inhibitory capacity of the sample against tyrosinase is lower than that of positive controls at all concentrations. The results of inhibition in terms of IC50 confirm the inhibition patterns. On the other hand, the statistical similarity of the anti-DOPA auto-oxidation (IC50) of G. biloba leaf extract and kojic acid was found (456.27 and 418.5 µg ml-1) but was lower than that of ascorbic acid (IC50 989.61 µg ml-1). A relationship was observed between the potential of antioxidant activity, tyrosinase inhibition, and anti-DOPA auto-oxidation with concentration levels of the extracts. The results of phytochemical analysis revealed the presence of tannins, flavonoids, terpenoids, and reducing sugars.
Asunto(s)
Antioxidantes , Monofenol Monooxigenasa , Antioxidantes/análisis , Antioxidantes/farmacología , Ginkgo biloba/química , Monofenol Monooxigenasa/análisis , Extractos Vegetales/química , TéRESUMEN
We report a chemiluminescent probe (CLPT1) that permits the paired detection of tyrosinase (Tyr) and biological thiols. Tyr only leads to a poor chemiluminescence response, a finding ascribed to the formation of a stable o-benzoquinone intermediate. The addition of glutathione (GSH), or ascorbate to the o-benzoquinone intermediate results in thiol conjugation or reduction to this intermediate, respectively. This produces a strong chemiluminescence response. Thiol co-dependence was demonstrated in live cells using the cell permeable analogue, CLPT3. The present chemiluminescence-based strategy allows the concurrent detection of tyrosinase activity and biological thiols.
Asunto(s)
Colorantes Fluorescentes/química , Monofenol Monooxigenasa/análisis , Compuestos de Sulfhidrilo/análisis , Ácido Ascórbico/química , Benzoquinonas/química , Técnicas Biosensibles , Permeabilidad de la Membrana Celular , Glutatión/química , Humanos , Mediciones Luminiscentes , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
An amperometric biosensor based on tyrosinase, immobilized onto a carbon black paste electrode using glutaraldehyde and BSA was constructed to detect competitive inhibitors. Three inhibitors were used in this study: benzoic acid, sodium azide, and kojic acid, and the obtained values for fifty percent of inhibition (IC50) were 119 µM, 1480 µM, and 30 µM, respectively. The type of inhibition can also be determined from the curve of the degree of inhibition by considering the shift of the inhibition curves. Amperometric experiments were performed with a biosensor polarized at the potential -0.15 V vs. Ag/AgCl and using 0.1 M phosphate buffer (pH 6.8) as an electrolyte. Under optimized conditions, the proposed biosensor showed a linear amperometric response toward catechol detection from 0.5 µM to 38 µM with a detection limit of 0.35 µM (S/N = 3), and its sensitivity was 66.5 mA M-1 cm-2. Moreover, the biosensor exhibited a good storage stability. Conversely, a novel graphical plot for the determination of reversible competitive inhibition was represented for free tyrosinase. The graph consisted of plotting the half-time reaction (t1/2) as a function of the inhibitor concentration at various substrate concentrations. This innovative method relevance was demonstrated in the case of kojic acid using a colorimetric bioassay relying on tyrosinase inhibition. The results showed that the t1/2 provides an extended linear range of tyrosinase inhibitors.
Asunto(s)
Técnicas Biosensibles , Monofenol Monooxigenasa/análisis , Catecoles , Electroquímica , Electrodos , Inhibidores Enzimáticos/análisis , Enzimas Inmovilizadas , Concentración de Iones de Hidrógeno , Cinética , Monofenol Monooxigenasa/antagonistas & inhibidoresRESUMEN
A coumarin-based two-photon (TP) fluorescent off-on probe has been developed for detecting tyrosinase activity. High selectivity, sensitivity and biocompatibility enable the probes to successfully image tyrosinase activity in live cells and tissues using TP microscopy.
Asunto(s)
Cumarinas/química , Colorantes Fluorescentes/química , Monofenol Monooxigenasa/análisis , Animales , Línea Celular Tumoral , Cumarinas/metabolismo , Diseño de Fármacos , Colorantes Fluorescentes/metabolismo , Humanos , Límite de Detección , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Monofenol Monooxigenasa/metabolismo , Fotones , RatasRESUMEN
A self-correcting fluorescent assay of tyrosinase (TYR) was developed by utilization of Fe-MIL-88B-NH2 as a peroxidase-like nanozyme and a capture probe. Fe-MIL-88B-NH2 nanozyme was selected as an electron donor, and the oxidization product (dopamine-o-quinone) acts as an energy acceptor. First, TYR catalyzes the oxidation of tyramine hydrochloride to dopamine and then to dopamine-o-quinone. Second, Fe-MIL-88B-NH2 with intrinsic peroxidase-like activity decomposes H2O2 to produce ·OH radicals, which further accelerate the oxidation of dopamine to dopamine-o-quinone. Excessive H2O2 and ·OH radicals reduce the interferences from ascorbic acid at the same time providing a self-correcting ability. Dopamine-o-quinone reacts with -NH2 groups on the ligand of Fe-MIL-88B-NH2 through Michael reaction which results in fluorescence quenching. Under 365-nm excitation, the fluorescence emission intensity at 452 nm gradually decreased with increasing TYR concentration varying from 0 to 10 U mL-1. The linear range is from 1 to 5 U mL-1 and the detection limit is 0.05679 U mL-1. This self-correcting fluorescent assay of tyrosinase exhibits good sensitivity and selectivity which is also successfully applied for tyrosinase inhibitor detection. Schematic representation of fluorescent assay for tyrosinase determination based on Fe-MIL-88B-NH2 nanozyme. A self-correcting fluorescent assay for tyrosinase was developed based on the Fe-MIL-88B-NH2 nanozyme.
Asunto(s)
Pruebas de Enzimas/métodos , Estructuras Metalorgánicas/química , Monofenol Monooxigenasa/análisis , Catálisis , Dopamina/análisis , Dopamina/química , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Peróxido de Hidrógeno/química , Hierro/química , Límite de Detección , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/química , Oxidación-Reducción , Pironas/análisis , Pironas/química , Espectrometría de Fluorescencia/métodos , Tiramina/químicaRESUMEN
Innate immunity in arthropods is achieved largely through melanization which is in turn the result of the prophenoloxidase (ProPO) activation cascade; a series of biochemical reactions triggered by the immune identification of pathogen-recognition proteins (PRPs). Within this activation cascade, inactive proPO is cleaved to form the reactive enzyme phenoloxidase (PO). Methods of detecting PO are used to assess an arthropod's ability to respond to immune challenges. These detection assays have been described for some arthropods, especially those of commercial value, but none are available for Euastacus, a genus within the superfamily Parastacoidea. This study is the first step in developing a standardized protocol for the detection and quantification of PO activity in wild or captive Murray crayfish Euastacus armatus. Hemolymph extracts from 49 crayfish were assessed for PO activity using an assay measuring the conversion of l-dopa (3,4-dihydroxy-l-phenylalanine) into dopachrome. Short periods (up to 15 min) out of water did not cause any measurable change in PO activity. Phenoloxidase activity was detected in captive (n = 24, stressed) and wild (n = 25, healthy) crayfish with captive crayfish showing lower levels of PO possibly indicating immunosuppression. The proven protocol is the first of its kind to propose a standardized methodology for the detection and quantification of PO activity in Murray crayfish hemolymph as a means of determining stress.
Asunto(s)
Acuicultura/métodos , Proteínas de Artrópodos/análisis , Astacoidea/enzimología , Monofenol Monooxigenasa/análisis , Animales , Femenino , MasculinoRESUMEN
Artificial enzymes with modulated enzyme-mimicking activities of natural systems represent a challenge in catalytic applications. Here, we show the creation of artificial Cu metalloenzymes based on the generation of Cu nanoparticles in an enzyme matrix. Different enzymes were used, and the structural differences between the enzymes especially influenced the controlled the size of the nanoparticles and the environment that surrounds them. Herein, we demonstrated that the oxidase-like catalytic activity of these copper nanozymes was rationally modulated by enzyme used as a scaffold, with a special role in the nanoparticle size and their environment. In this sense, these nanocopper hybrids have confirmed the ability to mimic a unique enzymatic activity completely different from the natural activity of the enzyme used as a scaffold, such as tyrosinase-like activity or as Fenton catalyst, which has extremely higher stability than natural mushroom tyrosinase. More interestingly, the oxidoreductase-like activity of nanocopper hybrids was cooperatively modulated with the synergistic effect between the enzyme and the nanoparticles improving the catalase activity (no peroxidase activity). Additionally, a novel dual (metallic and enzymatic activity) of the nanozyme made the highly improved catechol-like activity interesting for the design of 3,4-dihydroxy-l-phenylalanine (l-DOPA) biosensor for detection of tyrosinase. These hybrids also showed cytotoxic activity against different tumor cells, interesting in biocatalytic tumor therapy.
Asunto(s)
Materiales Biomiméticos/uso terapéutico , Técnicas Biosensibles , Cobre/uso terapéutico , Nanopartículas/uso terapéutico , Neoplasias/terapia , Bacterias/enzimología , Biocatálisis , Materiales Biomiméticos/química , Técnicas Biosensibles/métodos , Cobre/química , Terapia Enzimática/métodos , Hongos/enzimología , Humanos , Modelos Moleculares , Monofenol Monooxigenasa/análisis , Nanopartículas/química , Oxidorreductasas/química , Oxidorreductasas/uso terapéutico , Conformación ProteicaRESUMEN
Tyrosinase (TYR) expression and activity determine the rate and yield of melanin production. Studies have shown that TYR is a potential biomarker for melanoma and highly sensitive detection of TYR benefits early diagnosis of melanoma-related diseases. In this study, we developed a method that combines surface-enhanced Raman scattering (SERS) and sandwich-type immunity for sensitive detection of TYR, in which 4-mercaptobenzonitrile (4 MB) embedded between the Au core and Au shell (Au4MB @ Au) core-shell structure was employed as a SERS probe for quantitative detection of TYR while the magnetic bead serves as a capture substrate. Our results demonstrated that under magnetic separation, the specific SERS signal obtained is highly correlated with TYR concentrations. Furthermore, the combination of magnetic beads and Au4MB @ Au core-shell structure significantly improved the sensitivity of the sensing platform, resulting in detection limits of 0.45 ng mL-1. More importantly, the detection and analysis of TYR concentration in human serum samples showed good accuracy and an excellent recovery rate. Accuracy of the system was investigated from % recovery of spiked TYR standard solutions and found to be in the range of 90-104%, which further verified the feasibility and reliability of our method applied in a complex environment. We anticipate this SERS-based immunoassay method to be applied to TYR detection in the clinical setting and to be extended to other promising related fields.
Asunto(s)
Oro , Nanopartículas del Metal , Monofenol Monooxigenasa/análisis , Humanos , Fenómenos Magnéticos , Monofenol Monooxigenasa/sangre , Reproducibilidad de los Resultados , Espectrometría RamanAsunto(s)
Butanoles/efectos adversos , Pigmentación de la Piel/efectos de los fármacos , Piel/patología , Vitíligo/inducido químicamente , Administración Cutánea , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/análisis , Antígenos CD/metabolismo , Butanoles/administración & dosificación , Cadherinas/análisis , Cadherinas/metabolismo , Estudios de Casos y Controles , Femenino , Glutamato-Cisteína Ligasa/análisis , Glutamato-Cisteína Ligasa/metabolismo , Humanos , Inmunohistoquímica , Masculino , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Persona de Mediana Edad , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Piel/citología , Piel/efectos de los fármacos , Vitíligo/patología , Adulto JovenRESUMEN
A fluorescence resonance energy transfer (FRET)-based in situ fluorescence signal amplification strategy is described for the determination of tyrosinase (TYR). In this assay, a dual-templated copper nanocluster (CuNCs) stabilized by bovine serum albumin (BSA) and glycylglycine (Gly-Gly) was used as an energy donor. Metyrosine was employed as a TYR substrate because its enzyme catalytic product (methyldopa) was able to function as a monomer molecule to form fluorescent polymethyldopa (PMeDP) with the assistance of BSA/Gly-Gly CuNCs. In this process, PMeDP can combine with BSA/Gly-Gly CuNCs without extra modification and then acts as an energy receptor, which leads to a remarkable FRET from BSA/Gly-Gly CuNCs to PMeDP. Interestingly, the fluorescence intensity of PMeDP was strengthened greatly in the FRET-based sensor compared to the separate excitation, which provided good sensitivity for TYR sensing. Illuminated under a UV light source, the fluorescence signal change is observed from dark violet to bright green. Therefore, the present sensing system affords a reliable ratiometric assay for TYR determination. Also, the ratio of fluorescence intensity between PMeDP (λem at 505 nm, F505) and BSA/Gly-Gly CuNCs (λem at 415 nm, F415) was used for quantitative determination of TYR. The sensing system was easily operated in aqueous media with an exciting detection limit of 44.0 U L-1. This sensing strategy has been applied to the screening of inhibitors. Graphical abstract Schematic representation of the strategy for the determination of tyrosinase.
Asunto(s)
Cobre , Fluorescencia , Nanopartículas del Metal/química , Monofenol Monooxigenasa/análisis , Transferencia Resonante de Energía de Fluorescencia , Límite de Detección , Metildopa , alfa-MetiltirosinaRESUMEN
Tyrosinase is the key enzyme in melanin biosynthesis and inherently involves both monophenolase activity and diphenolase activity. A continuous fluorometric assay method was developed for the first time to directly monitor the real monophenolase activity without the interference of diphenolase reactions through exclusively quenching the native fluorescence of DOPA by borate. Complexation with borate at pH 8.0 allowed for selective quantitation of tyrosine in a binary mixture of tyrosine and DOPA at 335 nm. The time course for consumption of tyrosine was established to measure the initial velocity by recording the tyrosine fluorescence intensity at discrete intervals. The assay worked in the monophenolase activity range from 0.13 to 2.01 U mL-1 with the limit of detection (LOD) of 0.10 U mL-1. The assay method exhibited a promising prospect in application in kinetics of monophenolase and high throughput screening for monophenolase inhibitors.
Asunto(s)
Boratos/metabolismo , Fluorescencia , Fluorometría , Levodopa/metabolismo , Monofenol Monooxigenasa/análisis , Tirosina/metabolismo , Boratos/química , Levodopa/química , Estructura Molecular , Monofenol Monooxigenasa/metabolismo , Tirosina/químicaAsunto(s)
Monofenol Monooxigenasa/análisis , Tumor Rabdoide/diagnóstico , Teratoma/diagnóstico , Preescolar , Metilación de ADN , Femenino , Humanos , Inmunohistoquímica , Lactante , Masculino , Monofenol Monooxigenasa/metabolismo , Tumor Rabdoide/metabolismo , Sensibilidad y Especificidad , Teratoma/metabolismoRESUMEN
Analyte-responsive chromo-fluorogenic reactions under accessible conditions are important for designing small-molecule spectroscopic probes. We describe a series of newly constructed motifs based on the chromo-fluorogenic reaction between catechol derivatives (typically hydroxytyrosol, dopamine, and levodopa) and naphthoresorcin (NR) in aqueous solution under ambient conditions. The weakly absorptive and fluorogenic catechols/NR was converted to products having visible absorption and bright fluorescence within several minutes. The chromo-fluorophores produced from this reaction had a maximum absorbance at 458 nm and emission at 480 nm with high fluorescence quantum yields (30-84%). Inspired by the tyrosinase-catalyzed hydroxylation of monophenols to catechols, the tyrosinase-enabled chromo-fluorogenic reaction was verified by using monophenol (typically tyrosol) as the substrate. In this regard, a dual-readout tyrosinase activity assay was developed by virtue of the in situ "turn-on" optical signals. Furthermore, a test of tyrosinase inhibition, by using a common inhibitor kojic acid, demonstrated the potential of the chromo-fluorogenic reaction for developing other tyrosinase related assays and signal transduction.