RESUMEN
In this paper we report on the development of a label-free low-volume (12.5 microL), high-throughput microplate calorimetric biosensor for fast ascorbic acid quantification in food and pharmaceutical products. The sensor is based on microplate differential calorimetry (MiDiCal) technology in which the heat generation, due to the exothermic reaction between ascorbic acid and ascorbate oxidase, is differentially monitored between two neighboring wells of an IC-built wafer. A severe discrepancy is found between expected and observed sensor readings. To investigate the underlying mechanisms of these findings a mathematical model, taking into account the biochemical reactions and diffusion properties of oxygen, ascorbic acid, and ascorbate oxidase, is developed. This model shows that oxygen depletion in the microliter reaction volumes, immediately after injection of sample (ascorbic acid) into the well, causes the enzymatic reaction to slow down. Calibration experiments show that the sensor's signal is linearly correlated to the area under the output versus time profile for the ascorbic acid concentration range from 2.4 to 350 mM with a limit of detection of 0.8 mM. Validation experiments on fruit juice samples, food supplements, and a pain reliever supplemented with ascorbic acid reveal that the designed method correlates well with HPLC reference measurements. The main advantages of the presented biosensor are the low analysis cost due to the low amounts of enzyme and reagents required and the possibility to integrate the device in fully automated laboratory analysis systems for high-throughput screening and analysis.
Asunto(s)
Ácido Ascórbico/análisis , Técnicas Biosensibles/métodos , Ascorbato Oxidasa/metabolismo , Ácido Ascórbico/metabolismo , Calorimetría , Análisis de los Alimentos , Oxígeno/metabolismo , Preparaciones Farmacéuticas/análisisRESUMEN
A versatile method for localized (1H) NMR spectroscopy is presented. The method intrinsically combines B0-based spatial localization with the possibility of water suppression and spectral editing. With this sequence it is feasible to localize not only single spectra but also phase-encoded images and spectroscopic images. The technique essentially integrates the "Hahn spin-echo" with the "stimulated echo" sequence and is therefore called ACE (acquiring combined echoes). It realizes water-suppressed three-dimensional localization in a single shot and can be used for localized shimming. Studies in which the new method is applied to phantoms with metabolites diluted at low concentrations are presented. Discrimination between lactate and alanine, employing an adapted spectral editing method with complete inversion, combined with simultaneous water suppression and localization of a 0.06-cc volume is shown. The suppression of signals from outside the selected volume is greater than or equal to 24,000. Also, the method is demonstrated by in vivo experiments at 6.3 T. Localized water-suppressed 1H spectra are obtained completely noninvasively, leaving scalp and fur intact, from well-defined volumes of 0.15 cc in the brain of a living rat. Water-suppressed spectroscopic imaging over a localized volume with "body" coil excitation and noninvasive surface coil detection yielded spectra from voxels as small as 25 microliters in the in vivo rat brain.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética , Animales , Encéfalo/anatomía & histología , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Modelos Estructurales , Ratas , Ratas EndogámicasRESUMEN
Hydrogen-1 magnetic resonance (MR) spectroscopic images of patients with intracranial tumors were obtained. Metabolite maps of N-acetyl aspartate, choline, lactate, and creatine concentrations were reconstructed with a nominal spatial resolution of 7 mm and a section thickness of 25 mm. The metabolite maps showed variations in metabolite concentrations across the tumor. In one patient, it was observed that choline concentration was increased in one part of the tumor but decreased in another part. In another patient, the concentration of N-acetyl aspartate was extremely low in one part of the tumor but only slightly increased in another part of the tumor. Lactate was observed in all patients. In one patient, a combined measurement made with positron emission tomography (PET) and MR spectroscopic imaging was performed. This demonstrated that increased lactate concentration measured with H-1 MR spectroscopic imaging corresponded topographically with increased glucose uptake measured with fluorine-18 fluoro-2-deoxyglucose PET. Combined MR spectroscopic and PET measurements provide an opportunity to investigate, in greater detail than before, glucose uptake and catabolism by intracranial tumors.
Asunto(s)
Astrocitoma/diagnóstico , Neoplasias Encefálicas/diagnóstico , Glioma/diagnóstico , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Oligodendroglioma/diagnóstico , Tomografía Computarizada de Emisión , Adulto , Encéfalo/patología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
In vivo 31P NMR spectra of rat submandibular glands were obtained. The glands were exposed, leaving the neurovascular system intact, and placed in a solenoidal coil. Resonances of nine phosphate metabolites were identified in the spectra and metabolite concentrations were estimated from the corresponding integrals ([ATP] = 3.4 +/- 0.7 mM). Tissue pH, as deduced from the chemical shift of the inorganic phosphate resonance, was 7.26 (+/- 0.07). T1 relaxation times of ATP, phosphocreatine, inorganic phosphate, and phosphomonoester 31P spin systems were examined. The effect of hypoxia was followed as a function of time. 31P NMR spectra of the glands have also been obtained noninvasively by the use of a surface coil, adapted to the dimensions of the glands, and depth pulses.