RESUMEN
Kynurenic acid (KYNA) is a product of the tryptophan (TRP) metabolism via the kynurenine pathway (KP). This pathway is activated in neurodegenerative disorders, such as Alzheimer´s disease (AD). KYNA is primarily produced by astrocytes and is considered neuroprotective. Thus, altered KYNA levels may suggest an inflammatory response. Very recently, significant increases in KYNA levels were reported in cerebrospinal fluid (CSF) from AD patients compared with normal controls. In this study, we assessed the accuracy of KYNA in CSF for the classification of patients with AD, cognitively healthy controls, and patients with a variety of other neurodegenerative diseases, including frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP). Averaged KYNA concentration in CSF was higher in patients with AD when compared with healthy subjects and with all the other differentially diagnosed groups. There were no significant differences in KYNA levels in CSF between any other neurodegenerative groups and controls. These results suggest a specific increase in KYNA concentration in CSF from AD patients not seen in other neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/líquido cefalorraquídeo , Ácido Quinurénico/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Ácido Quinurénico/metabolismo , Masculino , Persona de Mediana Edad , Triptófano/líquido cefalorraquídeo , Triptófano/metabolismoRESUMEN
The entrapment of enzymes within biomimetic silica nanoparticles offers unique and simple immobilization protocols that merge the stability of proteins confined in solid phases with the high loading and reduced diffusion limitations inherent to nano-sized structures. Herein, we report on the biomimetic silica entrapment of chemically derivatized horseradish peroxidase for amperometric sensing applications. Scanning electron microscopy shows evidence of the formation of enzyme-modified nanospheres using poly(ethylenimine) as a template for silicic acid condensation. When these nanospheres are directly deposited on graphite electrodes, chemically modified anionic peroxidase shows direct electron transfer at 0 mV vs Ag|AgCl. Microgravimetric measurements as well as SEM images demonstrate that negatively charged peroxidase is also entrapped when silica precipitates at gold electrodes are modified with a self-assembled monolayer of poly(ethylenimine). Electrostatic interactions may play a crucial role for efficient enzyme entrapment and silica condensation at the PEI template monolayer. The in-situ biomimetically synthesized peroxidase nanospheres are catalytically active, enabling direct bioelectrocatalysis at 0 mV vs Ag|AgCl with long-term stability.
Asunto(s)
Biomimética , Enzimas/química , Enzimas/metabolismo , Nanopartículas/química , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Biocatálisis , Electroquímica , Electrodos , Transporte de Electrón , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Oro/química , Grafito/química , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Modelos Moleculares , Muramidasa/química , Muramidasa/metabolismo , Nanocompuestos/química , Nanotubos de Carbono/química , Peroxidasas/química , Peroxidasas/metabolismo , Conformación Proteica , Propiedades de SuperficieRESUMEN
Discriminating wines according to their denomination of origin using cost-effective techniques is something that attracts the attention of different industrial sectors. In search of simplicity, direct UV-visible spectrophotometric techniques and different multivariate statistical techniques are used with admissible results to characterize wine produced in specific regions. However, most of the reported classification methods do not exploit all of the statistical relations in the investigated dataset and are inherently affected by the presence of outliers. The aim of this paper is to test novel classification methods such as support vector machines as a means of improving the classification rate when UV-visible spectrophotometric methods are used to discriminate wines. The advantages of such a discrimination tool are demonstrated when classification rates are compared for a large number of Spanish red and white wines and classification rates above 96% are achieved. The proposed methodology also enables the selection of the most relevant wavelengths for sample discrimination. The proposed methodology also enables the selection of the most relevant wavelengths for sample discrimination.
Asunto(s)
Espectrofotometría Ultravioleta/métodos , Vino/clasificación , Análisis de Varianza , EspañaRESUMEN
This work is aimed towards the generation of enzyme arrays on electrochemically active surfaces by taking advantage of the DNA-directed immobilization (DDI) technique. To this end, two different types of horseradish peroxidase (HRP)-DNA conjugates were prepared, either by covalent coupling with a bifunctional cross-linker or by the reconstitution of apo-HRP, that is, HRP lacking its prosthetic heme (protoporphyrin IX) group, with a covalently DNA-modified heme cofactor. Both conjugates were characterized in bulk and also subsequent to their immobilization on gold electrodes through specific DNA hybridization. Electrochemical measurements by using the phenolic mediator ortho-phenylendiamine indicated that, due to the high degree of conformational orientation, the apparent Michaelis-Menten constants of the reconstituted HRP conjugate were lower than those of the covalent conjugate. Due to the reversible nature of DDI, both conjugates could be readily removed from the electrode surface by simple washing and, subsequently, the electrodes could be reloaded with fresh enzymes, thereby restoring the initial amperometric-response activity. Moreover, the specific DNA hybridization allowed us to direct the two conjugates to distinct sites on a microelectrode array. Therefore, the self-assembly and regeneration capabilities of this approach should open the door to the generation of arrays of redox-enzyme devices for the screening of enzymes and their effectors.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/química , Enzimas Inmovilizadas/metabolismo , Microelectrodos , Técnicas Biosensibles/instrumentación , Cromatografía Liquida , Pruebas Enzimáticas Clínicas , Sondas de ADN , Electroquímica , Oro/química , Hemo/química , Hemo/metabolismo , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Cinética , Oxidación-Reducción , Fenoles/química , Fenilendiaminas/química , Protoporfirinas/química , Protoporfirinas/metabolismo , Espectrofotometría UltravioletaRESUMEN
A multianalyte flow electrochemical cell (MAFEC) for bioanalysis is constructed, characterised and used for simultaneous carbohydrate analysis incorporating mediated amperometric enzyme electrodes. Although multidetection schemes can be addressed with microfabricated systems, it is demonstrated that a "meso" analytical device of low cost can give answers to traditional simultaneous multianalysis problems, being robust, and easy to construct and operate. The cell operates as a radial flow thin-layer device and can achieve mass transport controlled response for fast electrochemical reactions. When appropriate enzymatic electrodes are used the response becomes kinetically limited, but still shows a better than 5% R.S.D. for response to different sugars analysed. All the enzymatic sensors are mediated with different osmium compounds appropriate for each enzyme's mechanism (NAD or PQQ dehydrogenases) in some cases combining multienzyme sensors. All sensors were optimised so that different sugars do not produce interferences to other sensors. Matrix interferences were kept low by operating all sensors at or below 150 mV versus Ag/AgCl. The integrated system was used for the simultaneous detection of fructose, sucrose, glucose, galactose, and lactose, fully characterising the system for these analytes (sensitivity, dynamic range). Cross referenced calibration curves were used for signal treatment and interpretation and it was possible to analyse real juice and milk samples with results agreeing with the standard enzymatic methods for the same analyses with a sampling frequency of more than 100 h(-1).
Asunto(s)
Técnicas Biosensibles/instrumentación , Carbohidratos/análisis , Electroquímica/instrumentación , Análisis de Inyección de Flujo/instrumentación , Glucosa 1-Deshidrogenasa/química , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Biosensibles/métodos , Carbohidratos/química , Catálisis , Mezclas Complejas/análisis , Electroquímica/métodos , Enzimas Inmovilizadas/química , Diseño de Equipo , Análisis de Falla de Equipo , Análisis de Inyección de Flujo/métodos , Técnicas Analíticas Microfluídicas/métodos , Integración de SistemasRESUMEN
The modification of enzymes with multiple single-stranded oligonucleotides opens up a new concept for the development of DNA sensors with enhanced sensitivity. This work describes the generation of reporter sequences labeled with an enzyme for the demonstration of their ability to specifically hybridize and to permit signal amplification by successive hybridization steps. The synthetic pathway for the labeling of GOx with oligonucleotide sequences is based on the oxidation of the glycosidic residues of the enzyme and their covalent binding with 5'-end amine-modified oligonucleotides. Spectrophotometric characterization of these functionalized sequences results in an average number of three linked oligonucleotides per enzyme molecule. Their specificity is demonstrated in both a direct and a sandwich-type hybridization assay. The transduction of the enzyme-linked DNA sensors is based on self-assembled multilayers, including a chemically modified anionic horseradish peroxidase electrochemically connected to a water-soluble cationic poly[(vinylpyridine)Os(bpy)(2)Cl] redox polymer in an electrostatic ordered assembly. The sensing layer is constructed by the covalent binding of the DNA probe over the redox polymer through the 3'-phosphate group, enabling the capture of the target sequence. Upon addition of glucose, hybridization results in the production of H(2)O(2), which readily diffuses to the electrocatalytic assembly, giving rise to a cathodic current at 100 mV vs Ag/AgCl. Hybridization is always performed at room temperature, and after 30 min of incubation, an amperometric response is obtained that is proportional to DNA concentration. The simultaneous sandwich assay enables the quantification of a free-label 44-mer oligonucleotide at 1 nM concentration. Signal amplification is realized by a new hybridization step over the free sequences, giving rise to a dendritic architecture that accumulates enzyme molecules per hybridization event.