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. Versuchsaufbau und die Probenvorbereitung <ol><li> Herzustellen Mikrotoroide Verwendung der Lithographie, Ätzen und Schmelzvorgang, wie zuvor. 6 beschrieben Herzustellen Mikrotoroide auf einem Silizium-Wafer (Chip), die typischerweise einen Außendurchmesser von 80-100 um und einem kleineren Durchmesser von 2 um. </li><li> Entspannen etwa einen Meter von single-mode optische Faser (125 um Verkleidungen, 4,3 um Modenfelddurchmesser) von ihrer Faserspule. </li><li> In der Mitte des abgewickelten Abschnittes der optischen Faser, Streifen ein kleines Segment (2,5 cm) der Polymerbeschichtung auf der optischen Faser mit Abisolierzange. Anmerkung: Dies ist der Teil der optischen Faser, die evaneszent Licht in Mikrotoroid verwendet wird. </li><li> Reinigen Sie die abisolierten Faser mit Isopropanol Alkohol und einem fusselfreien wischen. </li><li> Halten Sie diese Abschnitt der Faser an Ort und Stelle mit einem Faserhalter des Haftmagneten gemacht. </li><li> Thin das abgestreifte Faser bis ~ 500 nm von Melting und das Ziehen unter Verwendung eines Wasserstoffbrenner und zwei Schrittmotoren gegenläufig in 60 &amp; mgr; m / min. Positionieren der optischen Faser im Inneren des oberen Teils der Flamme, die sein sollte ~ 10 mm hoch. Stoppen Ziehen der Faser, wenn die Übertragung durch die Faser stoppt schwankt, das entweder visuell überwacht werden kann (durch die Beobachtung der Licht, das seitlich aus der Faser Aufblinken gestreut wird und aus) oder durch Verbinden der Faser mit einer Fotodiode, die mit einem Oszilloskop verbunden ist . </li><li> Spalten einem Ende der optischen Faser und fügen diesen in einer nackten Faser-Adapter. Dieses Ende der Faser in den Eingang des Fotoempfängers. </li><li> Faser Paar das andere Ende der Faserspule zu dem Laser unter Verwendung eines faseroptischen Kopplers. </li><li> Legen Sie die Mikrotoroid Chip auf einem Probenhalter (Edelstahl, 37,8 mm x 6,4 mm x 3,2 mm) unter Verwendung von entweder Epoxy oder doppelseitiges Klebeband. </li><li> Montieren Sie den Probenhalter auf einem Positionierungstisch, der aus einem 3-Achsen-Nanopositionierung umfasst (nanocube) Bühne (siehe Geräteliste) auf einem 3-Achsen-Mikrometer. Führen Sie alle Experimente an einem pneumatisch getrennt optischen Tisch, um Vibrationen zu minimieren. </li><li> Grobposition der Probenchip unter Verwendung des 3-Achsen-Mikrometer. </li><li> Richten Sie die Mikrotoroid haltigen Chips parallel zu der optischen Faser unter Verwendung der Nano-Positionierer. Hinweis: Richten Sie die Mikrotoroid in einem Abstand von einer Wellenlänge des Eingangslichts (~ 633 nm). Zur Visualisierung dieses Prozesses verwenden zwei Bildsäulen (Rohr mit Objektiv und Kamera siehe Ausstattungsliste) auf der Oberseite und auf der Seite des Chips angeordnet. </li><li> Optimieren Sie die Polarisation des durch die optische Faser unter Verwendung eines in-line-Polarisationssteuerung gerichtete Laserlicht (siehe Geräteliste) mit einem Drehknopf, um die Polarisation einzustellen. Anmerkung: Die optimale Polarisation erreicht wird, wenn die gemessene Tauch bei der Übertragung von der optischen Faser wird der engste. Beachten Sie diese Dip auf einem Oszilloskop (siehe Schritt 2.2 für weitere Einzelheiten). </li><li> Konstrukteine Probenkammer durch epoxying einem Deckglas über dem Probentisch mit einem Objektträger aus Glas als Abstandshalter. Hinweis: Ein Plexiglas-Gehäuse, die den gesamten Setup kann nützlich zur Minimierung von Luftströmungen zu sein. Eine kleine Öffnung gelassen werden sollten, um die Lösung zu ermöglichen, sich mit der Rohrleitung gepumpt werden. </li><li> Thermisch äquilibrieren Partikelsuspensionen oder Einzelmolekül wässrigen Lösungen zur ≥ 1 Stunde in einem Wasserbad RT (~ 500 ml). Anmerkung: Die Proben werden auf die gewünschte Konzentration in Mikrozentrifugenröhrchen unter Verwendung der vom Hersteller, beispielsweise PBS oder HEPES spezifizierten assoziierten Puffer verdünnt. Wenn keine Bindungsereignisse detektiert werden, erhöht die Salzkonzentration des Puffers. </li><li> Vortex Partikelhaltigen Lösungen (1 ml) kurzzeitig für ca. 2 Sek. </li><li> Injizieren Partikel enthaltenden Lösungen in die Probenkammer bei 1 ml / min unter Verwendung einer 1 ml-Spritzenpumpe. </li><li> Nachdem die Probenkammer gefüllt hat, schalten Sie die Spritzenpumpe. </li><li> Warten Sie 30 Sekunden vor Aufnahmedaten zu minimierendie Auswirkungen von mechanischen Schwingungen von Flüssigkeitsströmung, die die Messung beeinflussen können, resultieren. </li></ol> 2. Frequenz Locking <ol><li> Erneute Koppeln des Toroids zu der optischen Faser durch Bewegung des Probenhalters mit dem Positionierer, denn die Kupplung aufgrund der Einspritzung des Fluids gestört werden. </li><li> Lokalisieren die Resonanzwellenlänge des Mikrotoroid durch Abtasten des rechnergesteuerten Eingangslaser durch eine Vielzahl von Wellenlängen. Dieser Schritt durch Senden einer Dreieckspannung Signal an ein piezoelektrisches Element innerhalb des Lasercontroller, der die Wellenlänge des Lasers regelt. Experimente durchführen unter Verwendung von sichtbarem Licht (635 nm ± 2,5 nm), da es eine geringe Absorption von Licht in Wasser bei dieser Wellenlänge. </li><li> Messung der Lichtdurchlässigkeit durch die optische Faser durch Einstecken des Ausgangs der optischen Faser in einem Auto-ausgeglichenen Photoempfänger. Stecker des Ausgangssignals des Photoempfängers in ein Oszilloskop mit einem BNC-Kabel. Beachten Sie on das Oszilloskop, das an der Resonanzwellenlänge des Mikrotoroid, Übertragung durch die optische Faser fällt. </li><li> Befestigen Sie den Ausgang des Photoempfängers auf den Haupteingang des Frequenzverriegelungsrückkopplungsregler (siehe Geräteliste) über ein Kabel. </li><li> Führen Sie das Frequenzverriegelungsrückkopplungssteuerung im Autolock-Modus mit top-of-Spitzensperr mit einer Zitterfrequenz von 2 kHz und einer Amplitude von Wellenlängenschwingung von 19 fm. Empirisch setzen die Proportional-Integral-Differential-Einstellungen in der Software-Fenster unter Verwendung von Ziegler-Nichols-Regeln 14. Anmerkung: Diese Werte sind nur einmal zu Beginn aller Experimente eingestellt werden. </li><li> Auto-Lock, die Wellenlänge des Lasers auf die Resonanzwellenlänge des Mikrotoroid. Führen Sie diesen Schritt nach dem Füllen der Probenkammer. Hinweis: Wenn die Wellenlängenverschiebung zu groß ist, dann wird die Rückkopplungssteuerung wird Schloss verlieren und automatisch auf Scan-Modus, um die Resonanzwellenlänge Standort lokalisieren zu wechseln. Diese ocKöter für Wellenlängenverschiebungen größer als etwa eine Linienbreite (mindestens 600 fm für alle Systeme hier untersucht). </li><li> Aufzeichnen des Ausgangs des Rückkopplungsreglers auf 20 kHz unter Verwendung eines 24-Bit-Datenerfassungskarte. Exportieren Sie die Daten als Textdatei über Datenerfassungssoftware. </li></ol> 3. Datenverarbeitung und Analyse- <ol><li> Fourier-Transformation der Daten in MATLAB. </li><li> Low übergeben Sie die Daten mit Hilfe eines &quot;Backsteinmauer&quot; Filter mit einer Grenz bei 1 kHz, um die auferlegten Zitterfrequenz von 2 kHz zu entfernen (siehe Zusatzcodedatei Screenshot 1). </li><li> Rechnerisch Notch-Filter, die Daten mit Hilfe einer Fenstergröße von 16 Hz. Hinweis: Dies geschieht, um bekannten Quellen von Rauschen zu entfernen, in diesem Fall, elektronische Leitungsrauschen 60 Hz und deren Oberwellen, sowie 100 Hz (aus dem Lasertreiber) und ihre Oberschwingungen (siehe Zusatzcodedatei Screenshot 1). </li><li> Inverse Fourier-Transformation die Daten zurück in den Zeitbereich. </li><li> Medianfilter die Daten unter Verwendung eines FenstersGröße von 1.001 Proben (siehe Zusatzcodedatei Screenshot 2). </li><li> Finde Schritt Veränderungen in der Resonanzwellenlänge mit der Schritt-Suchalgorithmus von Kerssemakers et al. 15. </li><li> Erzeugen Histogramme der Amplitude von jedem Bindungsschritt. </li><li> Berechnen Partikelgröße unter Verwendung von Gl. (1) (siehe Diskussion). </li></ol>

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The authors declare that they have no competing financial interests.

Als ein Teilchen gebunden ist, die Resonanzwellenlänge (λ) der Toroid zunimmt. Wenn ein Teilchen tatsächlich freigegeben wird, die Resonanzwellenlänge nimmt entsprechend ab (ein Untersetzungsereignis). Der Partikeldurchmesser (d) kann durch Histogramme der Amplitude jeder Wellenlänge Schritt bestimmt werden. Die Höhe jeder Wellenlänge Schritt variiert je nach Größenvariationen des gebundenen Teilchen und durch die Lage auf der Mikrotoroid wo das Teilchen bindet. Die maximale Änderung der Reso...

Einzelmoleküldetektion Experimente sind nützlich für die Verringerung der Menge an Analyt in Biosensoren verwendet wird, für die Früherkennung von Krankheiten und zur Untersuchung der grundlegenden Eigenschaften von Molekülen 3. Solche Experimente werden normalerweise mit den Etiketten sind jedoch Etiketten nicht immer möglich, für ein bestimmtes Protein zu erhalten, erhöhen die Kosten, können stören die Ereignisse untersucht und kann unbequem sein, insbesondere für die Echtzeit-Vor-Ort-Versuche oder Point-of- Care-Diagnostik. Der aktuelle Goldstandard für die markierungsfreien Biosensor ist die Oberflächenplasmonresonanz 4, aber die kommerziellen Oberflächenplasmonresonanz-Systeme haben in der Regel eine typische untere Nachweisgrenze in der Größenordnung von nM. In jüngster Zeit haben optische Resonatoren als vielversprechende Technologie für markierungsfreie Einzelmolekül Biodetektion 5 entstanden. Optischen Resonatoren Arbeit auf der Grundlage der langfristigen (ns) Führung von Licht 6,7. Licht ist evaneszenttypischerweise über eine optische Faser in diese Geräte gekoppelt sind. Wenn die Wellenlänge des Lichts geht durch die Faser entspricht der Resonanzwellenlänge des Resonators, Licht wirksam koppelt an den Resonator. Dieses gekoppelte Licht vollständig intern reflektiert innerhalb des Resonators Hohlraum Erzeugung eines evaneszenten Feldes in der Nähe des Umfangs des Resonators. Als Partikel in das evaneszente Feld und binden an den Resonator, der Resonanzwellenlänge des Resonators ändert sich proportional zu dem Volumen des Teilchens 8. In Bezug auf die Erfassungsfähigkeit, haben Mikrokugelresonatoren früher verwendet worden, um einzelne Influenza-A-Virus-Partikel zu erkennen (100 nm) 9,10. Kurzem plasmonically verstärkte Mikrokugel optischen Resonatoren sind verwendet worden, um einzelne Rinderserumalbuminmoleküle detektieren 11 und 8-mer Oligonukleotiden 12, aber dieser Ansatz begrenzt die Partikelfangbereich auf 0,3 &amp; mgr; m 2 pro deLaster. Größere Capture-Bereich Biosensoren sind ideal für die Maximierung der Wahrscheinlichkeit des Partikeldetektion. Aktuelle lösungsbasierten markierungsfreien Biosensor-Technologien mit großen (&gt; 100 &amp; mgr; m 2) Capture-Bereiche wurden auf die Erfassung Polystyrol-Partikel ≥ 25 nm beschränkt. Wir haben einen markierungsfreien Biosensor-System auf Basis von optischen Resonator-Technologie als Frequenzverriegelung Optical Whispering Evanescent Resonator (BLUME) 13 (Abbildung 1) bekannt, die in der Lage, zeitaufgelöste Detektion von Einzelmolekülen in Lösung entwickelt. FLOWER verwendet die lange Lebensdauer der Photonen Mikrotoroid optischen Resonatoren in Kombination mit Frequenzsperr Regelung, ausgeglichen Erkennung und Rechen Filterung, um kleine Partikel bis hin zu einzelnen Proteinmoleküle zu erkennen. Die Verwendung von Frequenzverriegelung kann das System immer verfolgen die Verschiebung Resonanz des Mikrotoroid als Teilchen zu binden, ohne die Notwendigkeit zu kehren oder Scannen der Laserwellenlänge übergroße Bereiche. Die Grundsätze der FLOWER verwendet werden, um die Erkennungsfähigkeiten von anderen Techniken, einschließlich plasmonischer Steigerung zu erhöhen. Im Folgenden werden die Verfahren für die Durchführung FLOWER beschrieben.

<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

Nachweis kleiner Konzentrationen von Molekülen bis zur Einzelmolekül Grenze hat Auswirkungen auf Bereiche wie die Früherkennung von Krankheiten und grundlegenden Studien über das Verhalten von Molekülen. Einzelmoleküldetektionsverfahren verwenden üblicherweise Markierungen, wie Fluoreszenzmarkierungen oder Quantenpunkte sind jedoch nicht immer verfügbar Etiketten, erhöhen die Kosten und die Komplexität und kann stören die Ereignisse untersucht. Optischen Resonatoren als ein vielversprechendes Mittel zur Einzelmoleküle ohne Einsatz von Etiketten erfassen taucht. Derzeit kleinste durch eine nicht plasmonically verstärkte blanke optische Resonatorsystem in Lösung nachgewiesen Teilchen ein 25 nm Polystyrolkugel 1. Wir haben eine Technik, die als Frequenzverriegelung Optical Whispering Evanescent Resonator (Blume), die in wässriger Lösung 2 dieses Limit übertreffen können und erreichen markierungsfreie Einzelmoleküldetektion bekannt entwickelt. Als Signalstärke Waage mit Partikelvolumen, unsere Arbeit für eine&gt; 100x improvement im Signal-Rausch-Verhältnis (SNR) über den aktuellen Stand der Technik. Hier die Verfahren hinter FLOWER werden in dem Bemühen, ihre Nutzung auf dem Gebiet erhöhen vorgestellt.

Partikelbindungsereignisse eindeutig als stufenartigen Änderungen in der Resonanzwellenlänge des Mikrotoroid über die Zeit (2A) ersichtlich. Die Höhen dieser Schritte sind als Histogramm in Figur 2B gezeigt. Die Abbildungen 2-4 zeigen repräsentative Spuren aus der Bindung von Exosomen (nanovesicles), 5 nm Silikonkügelchen, einzelne menschliche Interleukin-2-Moleküle sind. Die Tatsache, dass die stufenartigen Ereignissen Skala mit Teilchengröße zeigt, daß das V...

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.

Markierungsfreie Einzelmoleküldetektion Mit Mikrotoroid optischen Resonatoren

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

9.2K Aufrufe.  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. 

Serie: 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.

Herstellung von Silica Ultra High Quality Factor Mikroresonatoren

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

Forschung

Ingenieurwesen

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.

Optical Trapping von Nanopartikeln

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.

Synthese und Betrieb von Leuchtstofflampen-core Mikrokavitäten für Refraktometrische Sensing

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.

Implementierung eines Referenz-Interferometer für 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.

Herstellung und Prüfung von Mikrofluidik-Optomechanische Oszillatoren

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.

Anbringen biologischen Sonden zur Silica Optische Biosensoren Mit 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) ...

Biotechnik

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.

Herstellung und Betrieb eines Nano-Optical Fließband

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.

Mehrfarbenfluoreszenzdetektion für Droplet Mikrofluidik Verwendung von 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 ...

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.

Verwendung von zwei optischen Pinzetten und Mikrofluidik für Einzelmolekülstudien

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

Biochemie

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.

Optische Biosensoren

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