RESUMEN
The use of rod-like and vesicle-like mesoporous SiO(2) particles for fabricating high performance glucose biosensors is reported. The distinctively high surface areas of mesoporous structures of SiO(2) rendered the adsorption of glucose oxidase (GOx) feasible. Both morphologies of SiO(2) enhanced the sensitivities of glucose biosensors, but by a factor of 36 for vesicle-like SiO(2) and 18 for rod-like SiO(2), respectively. The greater enhancement of vesicle-like SiO(2) can be accounted for by its higher specific surface area (509 m(2)g(-1)) and larger total pore volume (1.49 cm(3)g(-1)). Interestingly, the current responses of GOx immobilized in interior channels of the mesoporous SiO(2) were enhanced much more than those of simple mixtures of GOx and the mesoporous SiO(2). This suggests that the enhancement of current responses arise not only from the high surface area of SiO(2) for high enzyme loading, but also from the improved enzyme activity upon its adsorption on mesoporous SiO(2). Also compared were the performances of glucose biosensors with GOx immobilized on mesoporous SiO(2) by physical adsorption and by covalent binding to 3-aminopropyltrimethoxysilane (APTMS) modified SiO(2) using glutaraldehyde as the cross-linker. The covalent binding approach resulted in higher enzyme loading but lower current sensitivity than with the physical adsorption.
Asunto(s)
Técnicas Biosensibles , Enzimas Inmovilizadas/química , Glucosa Oxidasa/química , Glucosa/análisis , Dióxido de Silicio/química , Adsorción , Electrodos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , TermodinámicaRESUMEN
A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG-horseradish peroxidase conjugate (Gt anti-M IgG-HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3',5,5'-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H(2)O(2)) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H(2)O(2) in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H(2)O(2)-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 microM and 100 microM.
Asunto(s)
Pruebas de Química Clínica/métodos , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/análisis , Animales , Anticuerpos Antiidiotipos/química , Glucemia , Glucosa Oxidasa/metabolismo , Peroxidasa de Rábano Silvestre/química , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
A lateral-flow, enzyme-based, bar-code assay for creatinine employing the concept of combination of diffusion and kinetics controlled has been developed. Unlike the traditional bar-code version of immunochromatographic assay, which depends on the stepwise capture of colorimetric tracer-labeled antibody-antigen complex by the immobilized antibody on each successive line, the principle of our proposed assay is based on the delay in TMB release and its diffusion in combination with horseradish peroxidase kinetics. Hydrogen peroxide (H(2)O(2)) produced from enzymatic reactions acts as a limiting factor, which controls the rate of conversion of TMB to blue color complex. The assay takes advantage of giving ladder bar result therefore without the need of any reading device. Depending on the amount of enzymes used, the assay can be one (9 min) or two steps (19 min). The strip assay semiquantitatively measures creatinine concentrations ranging from 0 to 400 microM. Thirty urine samples and thirty serum samples were tested, and the assay showed 90.0% and 86.7% agreement compared with conventional Jaffé method, respectively. This assay provides a tool for quick identification of creatinine for patients without the requirement of any instrument.