Este trabajo fue apoyado por la Fundación de Ciencias de Israel (subvención N º 1118/08 y la concesión N º 1146/12) y el Fondo de Investigación Conmemorativo Kroll Minna. ES agradece el apoyo financiero del Instituto de Nanotecnología Russell Berrie.

1. Preparación de oxidado poroso de SiO 2 <ol><li> Obleas de Si Etch (de un solo lado pulido en el &lt;100&gt; cara y fuertemente dopado, de tipo p, 0,0008 Ω · cm) en una solución de HF acuoso y etanol absoluto durante 30 seg 03:01 (v / v) a una corriente constante densidad de 385 mA / cm 2. Tenga en cuenta que la IC es un líquido altamente corrosivo y debe manejarse con sumo cuidado. </li><li> Enjuague la superficie del Si (psi) película porosa resultante con etanol absoluto varias veces; secar las películas bajo un gas nitrógeno seco. </li><li> Oxidar las muestras PSi recién grabados-en un horno tubular a 800 º C durante 1 hora en el aire ambiente (colocar la muestra en el horno a temperatura ambiente, caliente el horno a 800 ° C, deje en el horno durante 1 hora, apague la horno y retirar las muestras del horno sólo a temperatura ambiente). </li></ol> 2. Biofuncionalización de PSIO 2 Andamios <ol><li> Incubar una PSi oxidado (PSiO 2) de la muestra durante 1 hora en mercaptopropil (95% (MPTS) solución de trimetoxisilano-3) (108 mM en tolueno). </li><li> Enjuague la muestra tratada con silano PSIO 2 con tolueno, metanol y acetona; seco bajo un gas nitrógeno seco. </li><li> Incubar la muestra modificada con silano PSIO 2 durante 30 min en 1 ml de solución de biotina 100 mM de PEO-yodoacetilo. </li><li> Enjuague la muestra tratada con biotina PSIO 2 con 0,1 M de tampón fosfato salino (PBS) varias veces. </li><li> Incubar la muestra modificada biotina-PSIO 2 durante 30 min en 1 ml de 100 / g solución ml de estreptavidina (SA). </li><li> Enjuague el PSIO 2 muestra tratada con SA con solución 0,1 M PBS varias veces. </li><li> Incubar la modificado-SA PSIO 2 muestra resultante durante 30 min en 1 ml de 100 mg / ml de E. biotinilado anticuerpo monoclonal coli (Inmunoglobulina G, IgG) o solución con 100 g / ml de IgG biotinilado-conejo (como modelo para un anticuerpo monoclonal). </li></ol> 3. Marcaje fluorescente y microscopía de fluorescencia <ol><li> Incubar las superficies modificadas-IgG con 15 g / ml de fluoresceína (FITC) marcado contra IgG de conejo durante 40 minutos y con 15 g / ml de fluoresceína (FITC) etiquetados de IgG anti-ratón como control. </li><li> Enjuague las muestras modificadas con solución 0,1 M PBS varias veces. </li><li> Examine las muestras bajo un microscopio de fluorescencia. </li></ol> 4. Cultura de las bacterias <ol><li> Cultivar E. bacterias de E. coli K12 en un tubo de 10 ml con 5 ml de medio Luria Bertani (LB) (composición del medio en 1 L de agua desionizada: 5 g de NaCl, 5 g de extracto de levadura, y 10 g de triptona). Se incuban las bacterias agitación durante la noche a 37 ° C. </li><li> Monitorear la concentración de bacterias mediante la lectura de la densidad óptica (DO) a una longitud de onda de 600 nm. Después de un crecimiento durante la noche en medio LB, leer la OD 600 utilizando un espectrofotómetro para determinar la concentración bacteriana. El número de células es gravectly proporcional a las OD 600 mediciones (1 OD 600 = 10 8 células / ml). </li></ol> 5. Las bacterias de detección <ol><li> Coloque el PSIO modificado-IgG 2 y ordenada PSIO 2 (como control) muestras en una celda de flujo de plexiglás a medida. Fijar la celda de flujo para asegurar que la reflectividad de la muestra se mide en el mismo lugar durante todas las mediciones. </li><li> Incubar las muestras con E. coli K12 suspensión (10 4 células / ml) durante 30 min a temperatura ambiente. A continuación, retirar la suspensión de bacterias mediante el lavado de la célula con 0,85% w / v de solución salina durante 30 min. </li><li> Monitorear los cambios en los datos de reflectividad de todo el experimento. Todas las mediciones ópticas necesitan ser llevado a cabo en un medio acuoso circundante. Los espectros deben ser recogida utilizando un espectrómetro de CCD y se analizó mediante la aplicación de transformada rápida de Fourier (FFT), como se describió previamente 25,26. </li><li> Confirmar la presencia de las bacterias en lasuperficie del biosensor, mediante la observación de las muestras bajo un microscopio de luz en posición vertical inmediatamente después del experimento biosensores. </li></ol>

<ol>
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Los autores declaran no tener intereses financieros en competencia.

Un inmunosensor óptica-etiqueta libre, basado en un PSIO 2 nanoestructura (una película delgada de Fabry-Perot) se fabrica, y su aplicabilidad potencial como un biosensor para la detección de bacterias se confirma. Modificaciones y solución de problemas Una de las principales preocupaciones cuando se diseña un inmunosensor es la susceptibilidad de los anticuerpos a someterse a cambios de conformación no deseados durante la deposición y el patrón s...

Temprana y precisa identificación de bacterias patógenas es extremadamente importante para la alimentación y la seguridad del agua, monitoreo ambiental, y el punto de atención de diagnóstico 1. Como las técnicas tradicionales de microbiología son lentos, laboriosos, y carecen de la capacidad para detectar microorganismos en &quot;tiempo real&quot; o fuera del entorno de laboratorio, los biosensores están evolucionando para responder a estos desafíos 2-5. En los últimos años, Si poroso (PSI) se ha convertido en una plataforma prometedora para el diseño de sensores y biosensores 6-20. Durante la última década numerosos estudios sobre los sensores y biosensores ópticos basados ​​en la ISP fueron publicados 21,22. La capa PSi nanoestructurado se fabrica típicamente por grabado electroquímico anódica a partir de un solo cristal de la oblea de Si. Los nanomateriales PSi resultantes exhiben muchas características ventajosas, como la gran superficie y volumen libre, tamaños de poro que se pueden controlar y opti sintonizablepropiedades cal 10,16. Las propiedades ópticas de la capa PSi, como fotoluminiscencia 8,11 y la luz blanca a base de reflectancia interferometría 7,19, están fuertemente influenciadas por las condiciones ambientales. Captura de los huéspedes analitos moléculas / objetivo dentro de los resultados capa porosa en un cambio en el índice de refracción promedio de la película, observado como una modulación en el espectro de fotoluminiscencia o como un cambio de longitud de onda en el espectro de reflectividad 10. Aunque la gran innovación en tecnología de biosensor óptico PSi, sólo hay pocos informes sobre plataformas basadas en psi para la detección de bacterias 6,8,20,23-29. Además, la mayoría de estos estudios de prueba de concepto han demostrado &quot;indirecta&quot; de detección de bacterias. Por lo tanto, se requiere la lisis generalmente antes de las células para extraer los fragmentos de proteína / ADN dirigidos, característicos de las bacterias estudiadas 29. Nuestro enfoque es capturar directamente las bacterias dianacélulas sobre el biosensor psi. En consecuencia, los anticuerpos monoclonales, que son específicos para las bacterias diana, se inmovilizan sobre la superficie porosa. La unión de células de las bacterias, a través de interacciones anticuerpo-antígeno, sobre la superficie del biosensor inducir cambios en la amplitud (intensidad) de la espectro de reflectividad 24-26. En este trabajo, nos informe sobre la construcción de un biosensor basado en PSi óptica y demostrar su aplicación como una plataforma de biosensores sin etiqueta para la detección de Escherichia coli (E. coli) K12 (utilizado como modelo de microorganismos). El monitoreado señal óptica es la luz reflejada desde el nanoestructura PSi debido a Fabry-Perot de interferencia de película delgada (Figura 1A). Los cambios en la luz de amplitud / intensidad se correlacionan a la inmovilización específica de las células de las bacterias diana sobre la superficie del biosensor, lo que permite la rápida detección y cuantificación de las bacterias.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Si wafer</td>
			<td>Siltronix Corp.</td>
			<td></td>
			<td>Highly-B-doped, p-type, 0.0008 &#937;-cm resistivity, &#60;100&#62; oriented</td>
		</tr>
		<tr>
			<td>Aqueous HF (48%)</td>
			<td>Merck</td>
			<td>101513</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck</td>
			<td>818760</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>Saline 0.85% w/v</td>
			<td></td>
			<td></td>
			<td>prepared by dissolving 0.85 g NaCl in 100 ml Milli-Q water (18.2 M&#937;)</td>
		</tr>
		<tr>
			<td>95% (3-Mercaptopropyl)trimethoxysilane (MPTS)</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>175617</td>
			<td></td>
		</tr>
		<tr>
			<td>PEO-iodoacetyl biotin</td>
			<td>Sigma Aldrich Chemicals</td>
			<td>B2059</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin (SA)</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-000-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein (DTAF)-streptavidin</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>016-010-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>011-060-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>111-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently tagged anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>115-095-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated E. coli antibody</td>
			<td>Jackson ImmunoResearch Labs Inc.</td>
			<td>1007</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli (K-12)</td>
			<td></td>
			<td></td>
			<td>was generously supplied by Prof. Sima Yaron, Technion</td>
		</tr>
	</tbody>
</table>

optical detection of e. coli bacteria by mesoporous silicon biosensors

<html> Un biosensor óptico libre de etiquetas basado en un nanoestructurado Si poroso está diseñado para la captura rápida y la detección de bacterias de Escherichia coli K12, como un modelo de microorganismo. El biosensor se basa en la unión directa de las células de las bacterias diana sobre su superficie, mientras que no se requiere ningún tratamiento previo (por ejemplo, mediante la lisis celular) de la muestra estudiada. Una película delgada de Si mesoporoso se utiliza como el elemento transductor óptico del biosensor. Bajo iluminación de luz blanca, la capa porosa muestra patrones de franjas de Fabry-Perot bien resueltas en su espectro de reflectividad. La aplicación de una transformada rápida de Fourier (FFT) a los resultados de datos de reflectividad en un solo pico. Los cambios en la intensidad del pico de FFT son monitoreados. Por lo tanto, bacterias diana capturan sobre la superficie del biosensor, a través de interacciones anticuerpo-antígeno, induce cambios mensurables en la intensidad de los picos de FFT, lo que permite una observación &quot;tiempo real&quot; de unión bacterias. NT &quot;&gt; La película mesoporosa de Si, fabricado por un proceso de anodización electroquímica, se conjuga con anticuerpos monoclonales, específicos para las bacterias diana. La inmovilización, actividad inmunológica y especificidad de los anticuerpos son confirmados por experimentos de marcaje fluorescente. Una vez que el biosensor está expuesto a la bacterias diana, las células son capturados directamente sobre la superficie porosa de Si-anticuerpo modificado. Estos eventos de captura específicas resultan en cambios de intensidad en el espectro de interferencia óptica de película delgada del biosensor. Se demuestra que estos biosensores pueden detectar concentraciones de bacterias relativamente bajas (detección límite de 10 4 células / ml) en menos de una hora.

PSi oxidado (PSIO 2) las películas se prepararon como se describe en la sección de texto del protocolo. Figura 1B muestra una alta resolución de micrografía electrónica de barrido de la película PSi resultante después de la oxidación térmica. La capa PSIO 2 se caracteriza por poros cilíndricos bien definidas con un diámetro en el intervalo de 30-80 nm. El anticuerpo monoclonal (IgG) se injertan moléculas sobre las superficies PSIO 2</su...

Watch this Scientific Journal Video about Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors at JoVE.com

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Se introduce un biosensor óptico libre de marca para la detección de bacterias rápidos. El biosensor se basa en una Si poroso nanoestructurado, que está diseñado para capturar directamente las células de las bacterias diana sobre su superficie. Utilizamos anticuerpos monoclonales, inmovilizados sobre el transductor porosa, como las sondas de captura. Nuestros estudios demuestran la aplicabilidad de estos biosensores para la detección de concentraciones bacterianas bajas dentro de minutos sin procesamiento de la muestra antes (tales como la lisis celular).

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 ...

optical-detection-of-e-coli-bacteria-by-mesoporous-silicon-biosensors

Research

JoVE Journal

Bioengineering

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

16.8K vistas.  Technion - Israel Institute of Technology.  Se introduce un biosensor óptico libre de marca para la detección de bacterias rápidos. El biosensor se basa en una Si poroso nanoestructurado, que está diseñado para capturar directamente las células de las bacterias diana sobre su superficie. Utilizamos anticuerpos monoclonales, inmovilizados sobre el transductor porosa, como las sondas de captura. Nuestros estudios demuestran la aplicabilidad de estos biosensores para la detección de concentraciones bacterianas...

Video: Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricación de ADN electroquímico biosensores para la detección Reagentless de ácidos nucleicos, proteínas y moléculas pequeñas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Investigación

Bioingeniería

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox P Enfoque de recombinación para la detección de la fusión celular En Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

Colocación de sondas biológicas de biosensores ópticos de silicio mediante agentes de acoplamiento de silano

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

El análisis de la internalización celular de nanopartículas y bacterias en un Multi-espectral de Citometría de Flujo de imágenes

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

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) ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

El uso de biosensores ópticos sin etiqueta para Detectar la modulación de los canales de potasio por la proteína G-receptores acoplados

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Nanomanipulación de Soltero moléculas de ARN por Pinzas Ópticas

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Individual Detección Molecule-Label gratis con Microtoroid ópticos Resonadores

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor detección por fluorescencia de la gotita de microfluídica el uso de fibras ópticas

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.