Name: label-free single molecule detection using microtoroid optical resonators
Uploaded: 2015-12-29T14:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

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><tbody><tr><td>Tunable diode laser</td><td>Newport</td><td>TLB-6300</td><td></td></tr><tr><td>Laser controller</td><td>Newport</td><td>TLB-6300-LN</td><td></td></tr><tr><td>Frequency locking feedback controller</td><td>Toptica Photonics</td><td>Digilock 110</td><td></td></tr><tr><td>Auto-balanced photoreceiver</td><td>Newport</td><td>Model 2007</td><td></td></tr><tr><td>In-line polarization controller</td><td>General Photonics</td><td>PLC-003-S-90</td><td></td></tr><tr><td>24-bit data acquisition card</td><td>National Instruments</td><td>NI-PCI-4461</td><td></td></tr><tr><td>Recombinant human interleukin-2</td><td>Pierce Biotechnology</td><td>R201520</td><td></td></tr><tr><td>20 nm polystyrene beads</td><td>Thermo Scientific</td><td>3020A</td><td></td></tr><tr><td>NanoCube XYZ Piezo Stage</td><td>Physik Instrumente</td><td>P-611.3</td><td></td></tr><tr><td>Optical table</td><td>Newport</td><td>VH3660W-OPT</td><td></td></tr><tr><td>Objective lens for imaging column</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>Imaging column (adaptor tube)</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>High-Res CCD camera for imaging column</td><td>Edmund Industrial Optics</td><td>NT39244</td><td></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 ...

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Bioengineering

9.2K 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

Fabrication of Silica Ultra High Quality Factor Microresonators

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the quality factor (Q) of the cavity, can be in excess of 500ns for cavities with Q factors above 100 million. As a result of their low material losses, silica microcavities have demonstrated some of the longest photon lifetimes to date1-2. Since a portion of the circulating light extends outside the resonator, these devices can also be used to probe the surroundings. This interaction has enabled numerous experiments in biology, such as single molecule biodetection and antibody-antigen kinetics, as well as discoveries in other fields, such as development of ultra-low-threshold microlasers, characterization of thin films, and cavity quantum electrodynamics studies.3-7
The two primary silica resonant cavity geometries are the microsphere and the microtoroid. Both devices rely on a carbon dioxide laser reflow step to achieve their ultra-high-Q factors (Q&gt;100 million).1-2,8-9 However, there are several notable differences between the two structures. Silica microspheres are free-standing, supported by a single optical fiber, whereas silica microtoroids can be fabricated on a silicon wafer in large arrays using a combination of lithography and etching steps. These differences influence which device is optimal for a given experiment.
Here, we present detailed fabrication protocols for both types of resonant cavities. While the fabrication of microsphere resonant cavities is fairly straightforward, the fabrication of microtoroid resonant cavities requires additional specialized equipment and facilities (cleanroom). Therefore, this additional requirement may also influence which device is selected for a given experiment.
Introduction
An optical resonator efficiently confines light at specific wavelengths, known as the resonant wavelengths of the device. 1-2 The common figure of merit for these optical resonators is the quality factor or Q. This term describes the photon lifetime (&tau;o) within the resonator, which is directly related to the resonator's optical losses. Therefore, an optical resonator with a high Q factor has low optical losses, long photon lifetimes, and very low photon decay rates (1/&tau;o). As a result of the long photon lifetimes, it is possible to build-up extremely large circulating optical field intensities in these devices. This very unique property has allowed these devices to be used as laser sources and integrated biosensors.10
A unique sub-class of resonators is the whispering gallery mode optical microcavity. In these devices, the light is confined in circular orbits at the periphery. Therefore, the field is not completely confined within the device, but evanesces into the environment. Whispering gallery mode optical cavities have demonstrated some of the highest quality factors of any optical resonant cavity to date.9,11 Therefore, these devices are used throughout science and engineering, including in fundamental physics studies and in telecommunications as well as in biodetection experiments. 3-7,12
Optical microcavities can be fabricated from a wide range of materials and in a wide variety of geometries. A few examples include silica and silicon microtoroids, silicon, silicon nitride, and silica microdisks, micropillars, and silica and polymer microrings.13-17 The range in quality factor (Q) varies as dramatically as the geometry. Although both geometry and high Q are important considerations in any field, in many applications, there is far greater leverage in boosting device performance through Q enhancement. Among the numerous options detailed previously, the silica microsphere and the silica microtoroid resonator have achieved some of the highest Q factors to date.1,9 Additionally, as a result of the extremely low optical loss of silica from the visible through the near-IR, both microspheres and microtoroids are able to maintain their Q factors over a wide range of testing wavelengths.18 Finally, because silica is inherently biocompatible, it is routinely used in biodetection experiments.
In addition to high material absorption, there are several other potential loss mechanisms, including surface roughness, radiation loss, and contamination loss.2 Through an optimization of the device size, it is possible to eliminate radiation losses, which arise from poor optical field confinement within the device. Similarly, by storing a device in an appropriately clean environment, contamination of the surface can be minimized. Therefore, in addition to material loss, surface scattering is the primary loss mechanism of concern.2,8
In silica devices, surface scattering is minimized by using a laser reflow technique, which melts the silica through surface tension induced reflow. While spherical optical resonators have been studied for many years, it is only with recent advances in fabrication technologies that researchers been able to fabricate high quality silica optical toroidal microresonators (Q&gt;100 million) on a silicon substrate, thus paving the way for integration with microfluidics.1
The present series of protocols details how to fabricate both silica microsphere and microtoroid resonant cavities. While silica microsphere resonant cavities are well-established, microtoroid resonant cavities were only recently invented.1 As many of the fundamental methods used to fabricate the microsphere are also used in the more complex microtoroid fabrication procedure, by including both in a single protocol it will enable researchers to more easily trouble-shoot their experiments.

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.

La fabricación de sílice de alta calidad Ultra microresonators Factor

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the ...

Investigación

Ingeniería

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Trampas ópticas de las nanopartículas

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

Síntesis y funcionamiento de los núcleos fluorescentes microcavidades de Percepción refractométrico

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

Implementación de un interferómetro de referencia para Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Fabricación y Pruebas de microfluidos optomecánicos Osciladores

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems 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) ...

Bioingeniería

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and&#160;limits the&#160;resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

Fabricación y operación de una banda transportadora nano-óptica

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological ...

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 ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Uso de pinzas ópticas duales y microfluídica para estudios de moléculas individuales

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Bioquímica

Optical Biosensing

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence,&#160;or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Types of Optical Biosensing; Surface Plasmon Resonance SPR

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence, or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Biosensores ópticos

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable ...