El trabajo presentado en este documento fue apoyado por becas de la Universidad de Auburn y AUDFS USAF CRADA 07-277-60MDG-01. Las opiniones expresadas en este artículo son las de los autores y no reflejan la política o posición de la Fuerza Aérea de Estados Unidos, el Departamento de Defensa, ni del Gobierno de EE.UU. oficial.

1. Preparando el escenario <ol><li> Obtener la cepa de tipo S. aureus ATCC 12600, S. aureus ATCC 27690 y Bacillus subtilis ATCC 6051. Cepas resistentes a la meticilina de S. aureus - MRSA1, MRSA 2, MRSA 5, 13 MRSA, MRSA 26, 34 MRSA, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; El fago lítico 12600. </li><li> Obtener PBP 2a anticuerpos conjugados con perlas de látex. </li><li> Preparar medio NZY tal como se describe 21. </li><li> Obtener solución tamponada con fosfato salino (PBS). </li><li> Preparar agua desionizada como subfase de Langmuir-Blodgett (LB) deposición monocapa. </li></ol> 2. Propagación del bacteriófago y Titulación <ol><li> Incubar 5 ml de cultivo de una noche de S. aureus ATCC 12600 (3,6 x 10 8 unidades formadoras de colonias (UFC) / ml) con 500 l de fago (3 x 10 9 &lt;/ Sup&gt; PFU / ml) en 500 ml de medio NZY en 2 L de Flack en un agitador-incubador a 37 ° C durante la noche. </li><li> Centrifugar cultivo a 4424 xg durante 10 min a 4 ° C. </li><li> Sobrenadante se volvió a centrifugar a 11.325 xg durante 10 min a 4 ° C. </li><li> Filtrar el sobrenadante a través de un filtro Millipore de 0,22 micras. </li><li> Centrifugar el filtrado a 75.395 xg durante 1,5 horas. </li><li> Disolver el precipitado en 100 l de agua destilada. </li><li> Preparar diluciones de 10 veces de la suspensión de fagos en medio NZY. </li><li> Placa 1 ml de cultivo de una noche (ON) de S. aureus ATCC 12600 en cada una de 2 placas con agar NZY para asegurarse de cubrir toda la superficie. Retire el exceso de la cultura y deje que la superficie se seque durante 30 minutos. </li><li> Detectar una muestra (10 l) de la dilución apropiada del fago sobre la superficie de la placa y se incuba a 37 ° C durante 18-24 horas. </li><li> Examinar la formación de placas y calcular el título de fagos. Los títulos de bacteriófagos (T) se calcula sobre la base del número de pLaques (N) formadas por 10 l de volumen (V) de suspensión de fago y el factor de dilución (F). El título se calculó por la fórmula:. T = N / (vxf) Por ejemplo, para el número de placas 75 a la dilución 10 -7 y el volumen de 10 l (10 x 10 -3 ml), el título es igual 75 / (10 x 10 x 10 -3 -7) = 7,5 x 10 10 unidades formadoras de placa (UFP) por ml de suspensión. </li></ol> 3. Phage Oro-inmovilizada <ol><li> Piezas de cuarzo recubiertos de oro Limpio (60 mm 2) de grabado con plasma en atmósfera de argón durante 10 min y después se esterilizan durante 6 horas bajo luz UV en un gabinete estéril. </li><li> Añadir 50 microlitros de 1 x 10 9 UFP / ml de suspensión de fagos a la superficie de oro de cada pieza en una placa de Petri estéril, y luego incubar durante la noche en una cámara húmeda a temperatura ambiente. </li><li> Retire y título de la suspensión de fago restante. </li><li> Lavar piezas con fago unido 5 veces con PBS para eliminar no unidofagos. </li></ol> 4. Las pruebas de infectividad fago inmovilizado sobre una superficie seca <ol><li> Difundir una cultura de O / N de S. Aureus ATCC 12600 en una placa de agar NZY y dejar secar. </li><li> Coloque una pieza de oro con el fago inmovilizado en la placa frontal hacia abajo. </li><li> Observe una zona de lisis después de 12 h de incubación a 37 ° C que indica la infectividad. </li><li> Utilice piezas cubiertos de oro sin fagos en los experimentos de control. </li></ol> 5. Pruebas de actividad lítica de fagos libres y ligados en el líquido <ol><li> Añadir una cultura ON del S. aureus ATCC 12600 a 10 ml de NZY en cada uno de cuatro matraces de 300 ml brazo lateral (6 x 10 6 UFC / ml en cada matraz). </li><li> Añadir en dos matraces de 2 x 10 6 PFU / ml de fagos libres. </li><li> Utilice las otras dos frascos como controles. </li><li> Vigilar la evolución temporal de la S. lisis celular aureus para 570 min por medición de la densidad óptica (DO 600) a intervalos de 30 min para todos los matraces.</li><li> Tome una medición final a las 24 horas. </li><li> Repita los pasos 5.1 a 5.5, pero el uso de piezas de oro con fagos unidos en vez de fagos libres, y piezas de oro sin fago en frascos de control. </li></ol> 6. La pérdida de los fagos unidos Durante la Incubación <ol><li> Añadir 50 l de 1 x 10 9 PFU / ml de fago sobre la superficie de la pieza de oro estéril en una placa de Petri que se coloca en una cámara húmeda durante la noche a temperatura ambiente. </li><li> Extraiga la suspensión con el fago no unido de la superficie de oro y el título. </li><li> Lave la pieza de oro con fagos unidos 5 veces con PBS y colóquelos en 1 ml de solución NZY 15 ml tubo de agitador-incubador a 37 º C durante 8 horas. </li><li> Determinar el título del fago individual como se describe en 2. </li></ol> 7. Phage Spheroids Preparación <ol><li> La combinación de 400 l de suspensión de fago 12600 (10 11 UFP / ml) con un volumen igual de cloroformo espectrofotométrico grado en 1 ml frascoa temperatura ambiente. </li><li> Suavemente vórtex con la suspensión de fago-cloroformo 5-6 veces (intervalos de 5 segundos) más de un minuto de duración. </li><li> Con la suspensión de estabilizar durante 30 segundos y luego pipeta de la fase superior que contiene esferoides se pipeta para la preparación monocapa. </li><li> Tómese unos microlitros de la suspensión esferoides para la transmisión y microscopía electrónica de barrido. </li><li> Utilizar una suspensión esferoide restante para la fabricación de biosensores. </li></ol> 8. Preparación biosensor <ol><li> Limpiar sensores QCM-D por plasma grabado en argón durante 10 min PDC-32G limpiador plasma Harrick. </li><li> Enjuague sensor con hexano para eliminar las posibles impurezas orgánicas. </li><li> Preparar y limpiar un canal de la balanza película como se describe en el 22. </li><li> Rellene través LB con una solución subfase y limpiarlo con una barrera a través y estabilizar el canal a 20 ± 0,1 ° C </li><li> Extienda 300 l alícuota de fago 12600 en Suspensiòn acuosaen (10 11 UFP / ml) en subfase LB. </li><li> Prepare monocapa fago sobre una solución subfase LB al permitir 300 l alícuota de fago 12600 suspensión acuosa (10 11 PFU / ml) a correr por una varilla de vidrio mojable inclinado que está parcialmente sumergido en la subfase de 22,23. </li><li> Estabilizar la monocapa preparada durante 10 min, y luego comprimir a una velocidad de 30 mm / min (45 cm 2 / min), hasta que una presión constante se alcanza 19 N / m. </li><li> Realizar una deposición de película vertical sobre posicionado perpendicular sensor de QCM-D a una velocidad de 4,5 mm / min por inmersión sucesiva sensor de dentro y fuera de la monocapa siete veces. La microscopía electrónica de depositado monocapas de fagos antes de la exposición a muestras de bacterias mostró que las capas estaban continua, homogénea y uniforme (datos no mostrados). </li><li> Repetir los pasos 4.5 a 4.8 con una suspensión de esferoide fago que se prepara en 3. </li><li> Utilice biosensores fagos fabricados por MRSA detección electrónicamicroscopía, y elipsometría. </li></ol> 9. Prueba biosensor, la Discriminación de la meticilina resistente y bacterias estafilococos sensibles <ol><li> En este protocolo, se preparan biosensores con fago no modificado y modificado y esferoides de fagos. Una microbalanza de cristal de cuarzo con cuatro cámaras de flujo de sensor se utiliza para monitorizar la unión de las bacterias a los fagos inmovilizada en la superficie del sensor QCM. PBP 2a anticuerpos conjugados con perlas de látex se utilizan para discriminar entre resistente a la meticilina (SARM) y bacterias estafilococos sensibles. Todas las mediciones se llevan a cabo en el modo de flujo (50 l / min) a 5 MHz. </li><li> Establecer una línea de base de la frecuencia de resonancia del sensor QCM en agua (~ 30 min). </li><li> Dibuje una suspensión de células vivas sensibles a meticilina de estafilococos bacterianas en el agua (10 9 UFC / ml) a través de la celda de ensayo. </li><li> Un seguimiento continuo de los cambios en la frecuencia de resonancia de sensores y la disipación de la energía para el primer armónico, using el software Q-Soft. </li><li> Cuando los cambios en la frecuencia de resonancia de sensores y la disipación alcanzaron niveles de saturación, añadir la suspensión de PBP de anticuerpos conjugados con perlas de látex (6,77 x10 10 perlas / ml) para el flujo durante el monitoreo continuo de la frecuencia y la disipación. </li><li> Repita los pasos de 09.02 a 09.05 para las células bacterianas por estafilococos resistentes a la meticilina (MRSA). </li><li> Definir un cambio de frecuencia a través de la variación de la masa debido a las bacterias de unión por la ecuación de Sauerbrey 24,25 Df = xn-Delta M / C, donde C es la sensibilidad masa constante (C = 17,7 ng x cm x -2 -1 Hz a 5 MHz ), m es la masa y n es el número de armónicos (n = 1). </li><li> Definir un cambio de disipación de energía (Δ D) a partir de la grabación de la disminución exponencial de oscilación (frecuencia y amplitud de amortiguación, lo que permitió la cuantificación de la energía disipada y se almacena durante un período de oscilación,E se disipó y E almacenada, respectivamente: Δ D = E disipada / 2 πE almacenada. Δ D se mide en unidades de disipación (DU), uno DU = 10 -6 unidades relativas). </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Plating+Methods.">Aseptic Laboratory Techniques: Plating Methods.</a> J. Vis. Exp. (63), e3064 (2012).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Es bien sabido que los fagos se pueden utilizar como sondas de biosensores para patógenos bacterianos 28. Se demuestra en este trabajo que fago junto con anticuerpos PBP 2a puede ser utilizada para resolver el viejo problema: las cepas resistentes y sensibles a antibióticos discriminación rápidos. Se encontró sin embargo esos fagos de estafilococos no modificados normales no son adecuados para la detección de bacterias con dispositivos QCM, a pesar de que las bacterias se un...

Cepas resistentes a la meticilina de Staphylococcus aureus se han sugerido como un factor en las infecciones esenciales y los brotes nosocomiales 4-8. Las formas más comunes de reconocimiento de la resistencia a la meticilina, tales como la prueba de difusión en disco oxacilina agar pantalla, o de microdilución en caldo, se basan en las condiciones de cultivo a medida para mejorar la expresión de la resistencia. Las alteraciones incluyen la utilización de oxacilina, incubación a 30 o 35 ° C en lugar de 37 ° C, y la inclusión de NaCl al medio de crecimiento. Por otra parte, para la detección correcta de estos tipos de técnicas, en lugar de 16 a 18 horas se requiere un largo período de incubación de 24 h. Técnicas rápidas con nivel apropiado (&gt; 96%) de la sensibilidad para la identificación de la resistencia a la meticilina incluyen técnicas de microdilución automatizados tales como la tarjeta Vitek GPS-SA, el sistema Rapid ATB Staph, y el sistema Rapid Panel de Microscan que producen resultados después de 3-11 hr 9-11. El Crystal MRSA sistema de identificación es un método rápido que se basa en el reconocimiento de crecimiento de S. aureus en presencia de 2% de NaCl y 4 mg de oxacilina por litro con un sensor de fluorescencia sensible al oxígeno. Sensibilidades reclamados varían entre el 91 y el 100% después de 4 horas de incubación 12-14. Estos métodos fenotípicos están limitados en sus exactitudes por el impacto de las cepas prevalentes que expresan resistencia heterogénea. Por lo tanto, los mejores métodos aceptados para el reconocimiento de la resistencia a la meticilina es PCR o hibridación de ADN del gen mecA 15. Sin embargo, esta técnica requiere ADN purificado y es extremadamente sensible a varias mezclas (impurezas), que incluyen restos de células de 16. Además, estas técnicas necesitan mucho tiempo para llevar a cabo. Estrategias para el reconocimiento del producto del gen mecA, proteína PBP 2a, se podrían utilizar para determinar la resistencia y pueden ser más fiable en comparación con las técnicas de ensayo estándar 17. Staphylococcus aureus incluso los que tienen resistencia a la meticilina 1,2,18. En este trabajo hemos propuesto una nueva técnica en el reconocimiento específico y la detección de MRSA, tales como el reconocimiento de las bacterias, junto con la conformación de MRSA en tiempo real. Para este propósito específico de una S. bacteriófago aureus con un amplio espectro de huéspedes (incluyendo cepas de SARM) en combinación con el anticuerpo monoclonal contra la proteína (PBP 2a) se han utilizado. PBP 2a es una proteína de la pared celular y que es la causa de la resistividad antibiótico de MRSA. Sin embargo anticuerpo PBP 2a no es específico para S. aureus, ya que algunas otras bacterias tienen proteínas de unión a los antibióticos con secuencia similar a PBP 2a 19,20. En consecuencia, en este trabajo, S. bacteriófago aureus y anticuerpos contra la proteína PBP 2a se han utilizado. Para ser capaz de desarrollar un biosensor que específicamentecamente detectar e identificar MRSA un dispositivo con una acción en dos etapas se ha utilizado. El paso inicial utilizó una S. aureus monocapa bacteriófago como un sensor de la sonda, mientras que el segundo paso emplea PBP 2a anticuerpos específicos. Por lo tanto, el primer paso será reconocer S. bacteria Staphylococcus, como el otro serán sensibles a la proteína de unión a los antibióticos. Cuando las señales recibidas de los dos pasos son positivos, que indica la detección específica de MRSA.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

biosensor for detection of antibiotic resistant staphylococcus bacteria

Un bacteriófago lítico estructuralmente transformado que tiene una amplia gama de huéspedes de cepas de Staphylococcus aureus y una proteína de unión a penicilina (PBP 2a) de anticuerpos conjugados con perlas de látex se han utilizado para crear un biosensor diseñado para la discriminación de resistente a la meticilina (SARM) y sensible a la meticilina (SASM) S . especies aureus 1,2. Los fagos líticos se han convertido en esferoides de fagos por el contacto con la interfase agua-cloroformo. Fagos monocapas esferoide se han movido en una superficie biosensor de Langmuir-Blodgett (LB) Técnica 3. Los biosensores creados han sido examinados por una microbalanza de cristal de cuarzo con seguimiento de disipación (QCM-D) para evaluar las interacciones bacteria-fago. Interacciones bacteria-esferoide llevaron a la reducción de la frecuencia de resonancia y un aumento de la disipación de la energía, tanto para las cepas de SARM y SASM. Después de la unión bacteriana, estos sensores han sido expuestos más al anticuerpo de proteína de unión a penicilina perla de látexs. Sensores analizados con MRSA respondieron a PBP 2a cuentas de anticuerpos, aunque sensores inspeccionados con SASM dieron ninguna respuesta. Esta distinción experimental determina una discriminación inequívoca entre resistente a la meticilina y S. sensible cepas de Staphylococcus. Igualmente consolidados y no consolidados bacteriófagos suprimen el crecimiento de bacterias en las superficies y en suspensiones de agua. Una vez que los fagos líticos se transforman en esferoides, que conservan su fuerte actividad lítica y muestran una alta capacidad de captura bacteriana. Los esferoides de fago y los fagos se pueden utilizar para la prueba y la esterilización de los microorganismos resistentes a los antibióticos. Otras aplicaciones pueden incluir el uso en la terapia de bacteriófagos y las superficies antimicrobianas.

El fago demostró actividad lítica contra todas las cepas ensayadas de S. aureus, incluyendo cepas de MRSA, tal como se indica por la prueba de la mancha fago. Tamaños de placa generalmente variaron de 5 a 15 mm. No se encontró actividad contra otros-cultivos de ensayo (Tabla 1). Un crecimiento normal de S. aureus ATCC 12600 en un medio NZY en un agitador-incubador a 37 ° C se muestra en la Figura 1A (una curva marcada por círculos vac...

Watch this Scientific Journal Video about Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria at JoVE.com

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Biosensores fagos líticos y los granos de anticuerpos son capaces de discriminar entre resistente a la meticilina (MRSA) y la bacteria estafilococo sensibles. Los fagos se inmovilizaron mediante un método de Langmuir-Blodgett sobre una superficie de un sensor de microbalanza de cristal de cuarzo y trabajaron como amplias sondas estafilococo gama. Perlas de anticuerpos reconocen SARM.

Biosensor para la detección de antibióticos Las bacterias estafilococo resistentes

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

23.9K vistas.  Auburn University. Biosensores fagos líticos y los granos de anticuerpos son capaces de discriminar entre resistente a la meticilina (MRSA) y la bacteria estafilococo sensibles. Los fagos se inmovilizaron mediante un método de Langmuir-Blodgett sobre una superficie de un sensor de microbalanza de cristal de cuarzo y trabajaron como amplias sondas estafilococo gama. Perlas de anticuerpos reconocen SARM.

Video: Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

La fabricación de nanotubos de carbono de alta frecuencia Nanoelectrónica biosensor para la detección in High Solutions fuerza iónica

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Investigación

Bioingeniería

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Detección óptica de<em&gt; E. coli</em&gt; Bacterias por mesoporosos Silicon Biosensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Pruebas de susceptibilidad a antibióticos inducidas por el estrés en un chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

De alto rendimiento de identificación de bacterias polímeros repelentes para dispositivos médicos

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

El uso de un análisis de biosensores conductimétricos β-lactamase-base para detectar las interacciones biomoleculares

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desarrollo de un Biosensor de ADN electroquímico para la detección de un patógeno de transmisión alimentaria

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Estrategia de análisis bioaformático y bioinformática basada en biosensores para la validación global de interacciones entre fármacos y proteínas

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Interfaces de hidrogel fotodegradables para detección, selección y aislamiento de bacterias

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

Pick any colony from the MRS agar plate containing electroporated L.reuteri cells and inoculate into a 96 well microplate containing filter sterilized MRS broth per well and an appropriate selection antibiotic. Incubate aerobically for 24 hours at 37 degrees Celsius without shaking. The next day, L.reuteri will precipitate out of the media in the stationary phase.Resuspend the culture by pipetting. Add 200 microliters of culture into a flat, clear, bottom 96 well plate. Transfer the plate to a plate reader and measure the optical density and fluorescence of M cherry two and other relevant reporters.Colony morphology noticeably changes in the presence of oxygen. Under low oxygen conditions colonies appear white, opaque, smooth and round. Under partial or leaky conditions, colonies appear undulate, and umbanet.Whereas under atmospheric oxygen levels colonies always appear low bait, round and dry. In wild Type L.rueteri, growth and reporter protein expression vary between colonies making accurate deductions difficult. Plasmid curing, and re-transformation can result in more stable expression but it is an uncommon and unique occurrence reliant on more stable mutant phenotypes spontaneously occurring.

Medición de la proteína reportera fluorescente resistente a los ácidos mCherry2

<meta charset="utf8"></head><body>Nanocompuestos antibacterianos de óxido de grafeno/cobre para bacterias resistentes

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

<meta charset="utf8"></head><body>Autor destacado: Nanocompuestos metálicos para eliminar bacterias resistentes a los antibióticos

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Fabricación de nanocompuestos antibacterianos de óxido de grafeno/cobre

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...