Ingeniería de alimentos

 

Detección de Lactobacillus plantarum 299V usando biosensores basados en microcantilevers con microscopía de fuerza dinámica

 

Detection of Lactobacillus plantarum 299V using microcantilever-based biosensor with dynamic force microscopy

 

A.G. Mendoza-Madrigal¹, J.J. Chanona-Pérez¹*, J.V. Méndez-Méndez², E. Palacios-González³, G. Calderón-Domínguez¹ y H. Hernández-Sánchez¹

 

¹ Departamento de Ingeniería Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Plan de Ayala y Carpio s/n, Col. Santo Tomás, C.P. 11340, México, D.F. * Autor para la correspondencia. E-mail: jorge_chanona@hotmail.com.

² Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, Luis Enrique Erro s/n, Unidad Profesional Adolfo López Mateos, Col. Zacatenco, C.P. 07738, México, D.F.

³ Laboratorio de Microscopía de Ultra Alta Resolución, Instituto Mexicano del Petroleo, Eje Central Lázaro Cárdenas 1552, Col. San Bartolo Atepehuacan, C.P. 07730, México.

 

Recibido 19 de junio de 2013
Aceptado 1 de julio de 2013

 

Abstract

The aim of this study was the detection of active L. plantarum 299v (probiotic microorganism) growth using microcantilever based biosensors in air and in dynamic mode by atomic force microscopy. Commercial cantilevers were cleaned with Piranha solution in order to eliminate contaminants and were functionalized with silylating solution; afterwards the cantilevers were coated by an agarose layer using trie capillaries technique. An atomic force microscope in tapping mode was required to evaluate the resonance frequency shift of commercial cantilevers inoculated with L. plantarum 299v. Humidity and temperature were controlled inside an atmospheric hood during; the biodetection. The resonance frequency curves were seen to be narrower with higher Q factor values (~219). The resulti showed that the resonance frequency shifted by approximately 5.2 ± 0.8 kHz on the inoculated cantilevers throughout the growth kinetics. From the resonance frequency curves and known mechanical properties of the cantilevers, the biosensor sensitivity was determined to be 383 ± 3 pg/Hz and the biosensor can detect ~400 bacteria. In addition, L. plantarum growth on ihe cantilever's surface was confirmed by scanning electron microscopy. The results showed that it is possible to construct a microbiotensor by using commercial cantilevers and atomic force microscopy. These sensors can be used as a platform for the detection of micro organisms asto ciated with /un ctlonal foods.

Keywords: biosensors, cantilever, atomic force microscopy, foods.

 

Resumen

El objetivo de este estudio fue la detección del crecimiento activo de L. plantarum 299v (microorganismo probiótico) utilizando microcantilevers como biosensores en modo dinámico en microscopía de fuerza atómica (MFA). Cantilevers comerciales se limpiaron con solución Piraña y fueron funcionalizados con solución sililada; posteriormente se recubrieron con agarosa utilizando la técnica de capilares. Un MFA en modo intermitente se utilizó para evaluar el cambio de la frecuencia de resonancia de cantilevers comerciales inoculados con L. plantarum 299v. La humedad y la temperatura se controlaron dentro de una campana atmosférica durante la biodetección. Las curvas de frecuencia de resonancia mostraron valores altos de factor Q (~219). Los resultados mostraron que el desplazamiento de la frecuencia de resonancia cambia 5.2 ± 0.8 kHz en cantilevers inoculados a lo largo de la cinética de crecimiento. A partir de las curvas de frecuencia de resonancia y las propiedades mecánicas de los cantilevers, la sensibilidad del biosensor fue 383 ±3 pg/Hz y el biosensor puede detectar ~400 bacterias. El crecimiento de L. plantarum en los cantilevers se confirmó por microscopía electrónica de barrido. Los resultados mostraron que es posible la construcción de microbiosensores mediante el uso de cantilevers y MFA como plataforma para detección de microorganismos asociados a alimentos funcionales.

Palabras clave: biosensores, cantilever, microscopía de fuerza atómica, alimentos.

 

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