This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1. Configuración experimental y la preparación de muestras <ol><li> Fabrique microtoroids utilizando la litografía, el grabado, y el derretimiento de procedimiento que el descrito anteriormente 6. Fabricar microtoroids en la parte superior de una oblea de silicio (chip) que típicamente tienen un diámetro mayor de de 80-100 micras, y un diámetro menor de 2 micras. </li><li> Relájese más o menos un metro de la fibra óptica monomodo (125 revestimiento m, 4,3 m de diámetro modo de campo) de su carrete de fibra. </li><li> En el centro de la porción desenrollada de la fibra óptica, tira de un pequeño segmento (2,5 cm) del recubrimiento de polímero alrededor de la fibra óptica utilizando pelacables. Nota: Esta es la parte de la fibra óptica que se utilizará para evanescente de luz par en el microtoroid. </li><li> Limpie la fibra despojado con alcohol isopropanol y un trapo que no suelte pelusa. </li><li> Mantenga esta parte de la fibra en su lugar usando un soporte de fibra hecha de abrazaderas magnéticas. </li><li> Delgado la fibra despojado a ~ 500 nm por melting y tirando usando un soplete de hidrógeno y dos motores paso a paso que se mueven en direcciones opuestas a 60 m / min. Coloque la fibra óptica dentro de la porción superior de la llama, que debe ser ~ 10 mm de altura. Deje de tirar la fibra cuando la transmisión a través de la fibra se detiene fluctuante, que puede ser monitoreada, ya sea visualmente (mirando la luz que se dispersa lateralmente desde el abrir y cerrar de fibra de encendido y apagado) o mediante la conexión de la fibra a un fotodiodo que está unido a un osciloscopio . </li><li> Cleave un extremo de la fibra óptica y la inserta en un adaptador de fibra desnuda. Coloca este extremo de la fibra en la entrada del fotorreceptor. </li><li> Fibra acoplar el otro extremo del carrete de fibra para el láser mediante el uso de un acoplador de fibra óptica. </li><li> Coloque el chip microtoroid en la parte superior de un soporte de muestra (acero inoxidable, 37,8 mm x 6,4 mm x 3,2 mm) utilizando epoxi o cinta de doble cara. </li><li> Montar el soporte de la muestra en la parte superior de una etapa de posicionamiento que comprende de un 3-eje nano-posicionamiento (nanocube) etapa (ver lista de equipo) en la parte superior de un micrómetro de 3 ejes. Realizar todos los experimentos sobre una mesa óptica neumática aislado para minimizar las vibraciones. </li><li> Posición aproximada del chip muestra utilizando el micrómetro de 3 ejes. </li><li> Alinear la que contiene microtoroid-chip paralelo a la fibra óptica usando el nano-posicionador. Nota: Alinear el microtoroid dentro de una distancia de una longitud de onda de la luz de entrada (~ 633 nm). Para la visualización de este proceso de usar dos columnas de imagen (tubo con lente objetivo y la cámara, ver la lista de equipo) colocado en la parte superior y en el lado del chip. </li><li> Optimizar la polarización de la luz láser dirigido a través de la fibra óptica que utiliza un controlador de polarización en línea (ver lista de equipo) con un mando para ajustar la polarización. Nota: la polarización óptimo se consigue cuando el dip medido en la transmisión de la fibra óptica aparece el más estrecho. Observar esta inmersión en un osciloscopio (consulte el paso 2.2 para más detalles). </li><li> Construiruna cámara de muestra por epoxying un cubreobjetos de vidrio sobre la etapa de la muestra usando un portaobjetos de vidrio como un espaciador. Nota: Un recinto de plexiglás que cubre toda la configuración puede ser útil para minimizar las corrientes de aire. Una pequeña abertura debe dejarse para permitir la solución para ser bombeada en el uso de tubos. </li><li> Térmicamente equilibre suspensiones de partículas o soluciones acuosas de una sola molécula de ≥ 1 hora en un baño de agua RT (~ 500 ml). Nota: Las muestras se diluyeron a la concentración deseada en tubos de microcentrífuga utilizando los buffers asociados especificados por el fabricante, por ejemplo, PBS o HEPES. Si no se detectan eventos de unión, aumentar la concentración de sal del tampón. </li><li> Partícula Vortex contiene soluciones (1 ml) brevemente durante ~ 2 seg. </li><li> Inyectar las soluciones de partículas que contiene en la cámara de muestra a 1 ml / min utilizando una bomba de jeringa 1 ml. </li><li> Después de la cámara de la muestra ha llenado, apague la bomba de jeringa. </li><li> Espere 30 segundos antes de la grabación de datos para reducir al mínimolos efectos de las vibraciones mecánicas que resultan de flujo de fluido que puede afectar a la medición. </li></ol> 2. Frecuencia de bloqueo <ol><li> Re-acoplar el toroide a la fibra óptica moviendo el soporte de la muestra con el nanopositioner, debido a que el acoplamiento se verá perturbada debido a la inyección de fluido. </li><li> Ubicar la longitud de onda de resonancia de la microtoroid mediante el escaneo de láser de entrada controlada por ordenador a través de una variedad de longitudes de onda. Realice este paso mediante el envío de una señal de tensión de forma de onda triangular de un elemento piezoeléctrico dentro del controlador de láser que regula la longitud de onda del láser. Realizar experimentos usando la luz visible (635 nm ± 2,5 nm), ya que es baja absorción de la luz en el agua en esta longitud de onda. </li><li> Medir la transmisión de la luz a través de la fibra óptica conectando la salida de la fibra óptica en una fotorreceptor auto-equilibrado. Enchufe la salida del fotorreceptor en un osciloscopio mediante un cable BNC. Observar on el osciloscopio que en la longitud de onda de resonancia de la microtoroid, transmisión a través de la fibra óptica gotas. </li><li> Conecte la salida del fotorreceptor a la entrada principal de la frecuencia de bloqueo de controlador de realimentación (ver lista de equipo) a través de un cable. </li><li> Ejecute el controlador de realimentación de frecuencia de bloqueo en el modo de bloqueo automático usando el pico más alto de bloqueo con una frecuencia de oscilación de 2 kHz y una amplitud de onda de oscilación de 19 fm. Empíricamente establecer la configuración proporcional-integral-derivados en la ventana del software usando reglas de ajuste de Ziegler-Nichols 14. Nota: Estos valores sólo deben fijarse una vez al comienzo de todos los experimentos. </li><li> Auto-bloqueo de la longitud de onda del láser a la longitud de onda de resonancia de la microtoroid. Realice este paso después de llenar la cámara de muestra. Nota: Si el cambio de longitud de onda es demasiado grande, entonces el controlador de realimentación perderá bloqueo y cambia automáticamente a modo de exploración con el fin de localizar la ubicación de longitud de onda de resonancia. Este occurs de longitud de onda desplaza más grande que aproximadamente una anchura de línea (al menos 600 fm para todos los sistemas investigados aquí). </li><li> Registre la salida del controlador de retroalimentación a 20 kHz usando una tarjeta de adquisición de datos de 24 bits. Exportar los datos como un archivo de texto a través del software de adquisición de datos. </li></ol> 3. Procesamiento de Datos y Análisis <ol><li> Transformada de Fourier de los datos en MATLAB. </li><li> Paso Bajo los datos utilizando un filtro de &quot;ladrillo-pared&quot; con un corte a 1 kHz para eliminar la frecuencia de oscilación impuesto de 2 kHz (ver Código Suplementario Captura de pantalla 1 archivo). </li><li> Muesca Computacionalmente filtrar los datos utilizando un tamaño de ventana de 16 Hz. Nota: Esto se hace para eliminar las fuentes conocidas de ruido, en este caso, 60 Hz ruido en la línea electrónica y sus armónicos, además de 100 Hz (viniendo desde el controlador de láser) y sus armónicos (ver Código Suplementario Captura de pantalla 1 archivo). </li><li> Fourier transformada inversa de los datos de nuevo en el dominio del tiempo. </li><li> Filtro de mediana de los datos utilizando una ventanatamaño de 1.001 muestras (ver Código Suplementario de pantalla 2 archivos). </li><li> Busque cambios de paso en la longitud de onda de resonancia utilizando el algoritmo paso a la búsqueda de Kerssemakers et al. 15. </li><li> Generar histogramas de la amplitud de cada etapa de unión. </li><li> Calcular el tamaño de partícula medio de la ecuación. (1) (ver Discusión). </li></ol>

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The authors declare that they have no competing financial interests.

Como una partícula se une, la longitud de onda de resonancia (λ) del toroide aumenta. Si una partícula desenlaza, la longitud de onda de resonancia disminuye correspondientemente (un evento de bajada). El diámetro de partícula (d) se puede determinar a través de histogramas de la amplitud de cada paso de longitud de onda. La altura de cada paso de longitud de onda varía debido a las variaciones de tamaño de la partícula atado y debido a la ubicación en la microtoroid donde la partícula se un...

Experimentos de detección de moléculas individuales son útiles para reducir la cantidad de analito utilizado en biosensores, para la detección precoz de la enfermedad, y para examinar las propiedades fundamentales de moléculas 3. Tales experimentos se realizan normalmente usando etiquetas, sin embargo, las etiquetas no siempre son posibles de obtener para una proteína particular, aumentar el costo, puede perturbar los eventos están estudiando, y puede ser un inconveniente, sobre todo por el tiempo real en el sitio experimentos o punto-de- diagnósticos de atención. El estándar de oro actual de biosensor libre de etiquetas es resonancia de plasmón superficial 4, sin embargo los sistemas comercial resonancia de plasmón superficial tienen típicamente un límite inferior de detección típica del orden de nM. Recientemente, resonadores ópticos han emergido como una tecnología prometedora para la etiqueta libre sola molécula biodetección 5. Resonadores ópticos trabajo basado en los de largo plazo (ns) confinamiento de la luz 6,7. La luz es evanescentejunto a estos dispositivos normalmente a través de una fibra óptica. Cuando la longitud de onda de la luz que pasa a través de la fibra coincide con la longitud de onda de resonancia del resonador, de manera eficiente la luz se acopla al resonador. Esta luz acoplada refleja totalmente internamente dentro de la cavidad del resonador generar un campo evanescente en la proximidad de la circunferencia del resonador. Como partículas entran en el campo evanescente y se unen al resonador, la longitud de onda de resonancia de los cambios de resonador en proporción al volumen de la partícula 8. En términos de capacidad de detección, resonadores de microesferas anterior se han utilizado para detectar solo la influenza A partículas de virus (100 nM) 9,10. Recientemente, resonadores ópticos mejorada plasmonically en microesferas se han utilizado para detectar el suero de albúmina bovina sola moléculas de oligonucleótidos 11 y 8-mer 12, sin embargo, este enfoque limita el área de captura de partículas de 0,3 micras por 2 devicio. Grandes biosensores zona de captura son ideales para maximizar la posibilidad de detección de partículas. Las tecnologías actuales basados ​​en soluciones de etiqueta libre de biosensores con grandes (&gt; 100 m 2) zonas de captura se han limitado a la detección de partículas de poliestireno ≥ 25 nm. Hemos desarrollado un sistema biosensor etiqueta gratuito basado en la tecnología resonador óptico conocido como Frecuencia Bloqueo óptico Whispering Evanescent Resonador (FLOR) 13 (Figura 1) que es capaz de detectar de resolución temporal de las moléculas individuales en solución. FLOR utiliza la larga vida útil de fotones de resonadores ópticos microtoroid combinados con una frecuencia de bloqueo de control de retroalimentación, detección equilibrada, y filtrado de cómputo para detectar partículas pequeñas a moléculas de proteínas individuales. El uso de la frecuencia de bloqueo permite que el sistema para rastrear siempre la resonancia desplazamiento de la microtoroid como partículas se unen, sin la necesidad de barrer o escanear la longitud de onda láser sobregrandes rangos. Los principios de la FLOR pueden utilizarse para mejorar las capacidades de detección de otras técnicas que incluyen la mejora plasmónica. En lo que sigue, se describen los procedimientos para realizar FLOR.

<table border="0" cellpadding="0" cellspacing="0" width="396">
	<tbody>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Tunable diode laser</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Laser controller</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300-LN</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Frequency locking feedback controller</td>
			<td class="xl63" style="width:99pt" width="132">Toptica Photonics</td>
			<td class="xl63" style="width:99pt" width="132">Digilock 110</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Auto-balanced photoreceiver</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">Model 2007</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">In-line polarization controller</td>
			<td class="xl63" style="width:99pt" width="132">General Photonics</td>
			<td class="xl63" style="width:99pt" width="132">PLC-003-S-90</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">24-bit data acquisition card</td>
			<td class="xl63" style="width:99pt" width="132">National Instruments</td>
			<td class="xl63" style="width:99pt" width="132">NI-PCI-4461</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">Recombinant human interleukin-2</td>
			<td class="xl63" style="width:99pt" width="132">Pierce Biotechnology</td>
			<td class="xl63" style="width:99pt" width="132">R201520</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">20 nm polystyrene beads</td>
			<td class="xl63" style="width:99pt" width="132">Thermo Scientific</td>
			<td class="xl63" style="width:99pt" width="132">3020A</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">NanoCube XYZ Piezo Stage</td>
			<td class="xl63" style="width:99pt" width="132">Physik Instrumente</td>
			<td class="xl63" style="width:99pt" width="132">P-611.3</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Optical table</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">VH3660W-OPT</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Objective lens for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl64">1-60228</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Imaging column (adaptor tube)</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl65">1-60228</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">High-Res CCD camera for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Edmund Industrial Optics</td>
			<td class="xl64">NT39244</td>
		</tr>
	</tbody>
</table>

label-free single molecule detection using microtoroid optical resonators

La detección de pequeñas concentraciones de moléculas hasta el límite de una sola molécula tiene impacto en áreas como la detección precoz de la enfermedad, y los estudios fundamentales sobre el comportamiento de las moléculas. Técnicas individuales de detección molécula comúnmente utilizan etiquetas como etiquetas fluorescentes o puntos cuánticos, sin embargo, las etiquetas no siempre están disponibles, aumentar el costo y la complejidad, y pueden perturbar los eventos están estudiando. Resonadores ópticos han surgido como un medio prometedor para detectar moléculas individuales sin el uso de etiquetas. Actualmente la partícula más pequeña detectada por un sistema resonador óptico no mejorada plasmonically desnudo en solución es un poliestireno esfera 25 nm 1. Hemos desarrollado una técnica conocida como frecuencia de bloqueo óptico Whispering Evanescent Resonador (FLOR) que puede superar este límite y lograr sola detección de moléculas de etiqueta libre en solución acuosa 2. Como fuerza de la señal escalas con el volumen de partículas, nuestro trabajo representa un&gt; improveme 100xnt en la relación señal a ruido (SNR) sobre el estado actual de la técnica. Aquí los procedimientos detrás FLOR se presentan en un esfuerzo por aumentar su uso en el campo.

Eventos de unión de partículas se ven claramente como cambios de paso-como en la longitud de onda de resonancia de la microtoroid lo largo del tiempo (Figura 2). Las alturas de estos pasos se muestran como un histograma en la Figura 2B. Figuras 2-4 muestran trazas representativas de la unión de los exosomas (nanovesículas), perlas de sílice 5 nm y solteras humanos interleucina-2 moléculas, respectivamente. El hecho de que los eventos escalonadas e...

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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 ...

label-free-single-molecule-detection-using-microtoroid-optical

Research

JoVE Journal

Bioengineering

9.1K vistas.  California Institute of Technology. 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. 

Video: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexado de una sola molécula de mediciones de la Fuerza proteólisis utilizando pinzas magnéticas

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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 ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sin contacto, libre de marca Seguimiento de células y matriz extracelular utilizando espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

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 ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Un biochip electroquímico basado en microfluidos para el análisis de hibridación ADN-Label libre

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Una técnica-Label libre para el Imaging espacio-temporal de las secreciones sola célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bilayers Individual Molécula microscopía de fluorescencia en planar compatibles

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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 ...