Name: Label-free Single Molecule Detection Using Microtoroid Optical Resonators (Video) | JoVE Journal
Uploaded: 2015-12-29T14:00:00.000+00:00
Duration: 8 min 53 s
Description: Learn label-free single molecule detection using microtoroid optical resonators and frequency locking technology. This method enables studying protein folding and binding kinetics without altering target molecules.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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

1. Experimental Setup and Sample Preparation
<ol>
	<li>Fabricate microtoroids using the lithography, etching, and melting procedure as described previously6. Fabricate microtoroids on top of a silicon wafer (chip) that typically have a major diameter of 80-100 &#181;m, and a minor diameter of 2 &#181;m.</li>
	<li>Unwind roughly a meter of single-mode optical fiber (125 &#181;m cladding, 4.3 &#181;m mode field diameter) from its fiber spool.</li>
	<li>In the middle of the unwound portion of the optical fiber, strip a small segment (2.5 cm) of the polymer coating around the optical fiber using wire strippers. Note: This is the portion of the optical fiber that will be used to evanescently couple light into the microtoroid.</li>
	<li>Clean the stripped fiber with isopropanol alcohol and a lint free wipe.</li>
	<li>Hold this portion of the fiber in place using a fiber holder made of magnetic clamps.</li>
	<li>Thin the stripped fiber to ~500 nm by melting and pulling using a hydrogen torch and two stepper motors moving in opposite directions at 60 &#181;m/min. Position the optical fiber inside the top portion of the flame, which should be ~10 mm tall. Stop pulling the fiber when the transmission through the fiber stops fluctuating, which can be monitored either visually (by watching the light that is scattered laterally from the fiber blink on and off) or by connecting the fiber to a photodiode which is attached to an oscilloscope.</li>
	<li>Cleave one end of the optical fiber and insert it into a bare fiber adaptor. Place this end of the fiber into the input of the photoreceiver.</li>
	<li>Fiber couple the other end of the fiber spool to the laser by using a fiber optic coupler.</li>
	<li>Place the microtoroid chip on top of a sample holder (stainless steel, 37.8 mm x 6.4 mm x 3.2 mm) using either epoxy or double sided tape.</li>
	<li>Mount the sample holder on top of a positioning stage that comprises of a 3-axis nano-positioning (nanocube) stage (see equipment list) on top of a 3-axis micrometer. Perform all experiments on a pneumatically isolated optical table to minimize vibrations.</li>
	<li>Coarse position the sample chip using the 3-axis micrometer.</li>
	<li>Align the microtoroid-containing chip parallel to the optical fiber using the nano-positioner. Note: Align the microtoroid within a distance of one wavelength of the input light (~633 nm). For visualization of this process use two imaging columns (tube with objective lens and camera, see equipment list) positioned on the top and on the side of the chip.</li>
	<li>Optimize the polarization of the laser light directed through the optical fiber using an in-line polarization controller (see equipment list) with a knob to adjust the polarization. Note: Optimum polarization is achieved when the measured dip in the transmission of the optical fiber appears the narrowest. Observe this dip on an oscilloscope (see step 2.2 for more details).</li>
	<li>Construct a sample chamber by epoxying a glass coverslip over the sample stage using a glass microscope slide as a spacer. Note: A Plexiglas enclosure covering the entire setup may be useful for minimizing air currents. A small opening should be left to allow for the solution to be pumped in using tubing.</li>
	<li>Thermally equilibrate particle suspensions or single-molecule aqueous solutions for &#8805; 1 hr in a RT water bath (~500 ml). Note: Samples are diluted to the desired concentration in microcentrifuge tubes using the associated buffers specified from the manufacturer, e.g., PBS or HEPES. If no binding events are detected, increase the salt concentration of the buffer.</li>
	<li>Vortex particle containing solutions (1 ml) briefly for ~2 sec.</li>
	<li>Inject particle containing solutions into the sample chamber at 1 ml/min using a 1 ml syringe pump.</li>
	<li>After the sample chamber has filled, turn off the syringe pump.</li>
	<li>Wait 30 sec before recording data to minimize the effects of mechanical vibrations resulting from fluid flow that can affect the measurement.</li>
</ol>
2. Frequency Locking
<ol>
	<li>Re-couple the toroid to the optical fiber by moving the sample holder with the nanopositioner, because the coupling will be disturbed due to the injection of fluid.</li>
	<li>Locate the resonance wavelength of the microtoroid by scanning the computer-controlled input laser through a variety of wavelengths. Perform this step by sending a triangular waveform voltage signal to a piezoelectric element within the laser controller that regulates the wavelength of the laser. Perform experiments using visible light (635 nm &#177; 2.5 nm) as there is low absorption of light in water at this wavelength.</li>
	<li>Measure the light transmission through the optical fiber by plugging the output of the optical fiber into an auto-balanced photoreceiver. Plug the output of the photoreceiver into an oscilloscope using a BNC cable. Observe on the oscilloscope that at the resonance wavelength of the microtoroid, transmission through the optical fiber drops.</li>
	<li>Attach the output of the photoreceiver to the main input of the frequency locking feedback controller (see equipment list) via a cable.</li>
	<li>Run the frequency locking feedback controller in autolock mode using top-of-peak locking with a dither frequency of 2 kHz and an amplitude of wavelength oscillation of 19 fm. Empirically set the proportional-integral-derivative settings in the software window using Ziegler-Nichols tuning rules14. Note: These values need only be set once at the beginning of all experiments.</li>
	<li>Auto-lock the wavelength of the laser to the resonance wavelength of the microtoroid. Perform this step after filling the sample chamber. Note: If the wavelength shift is too large, then the feedback controller will lose lock and automatically switch to scanning mode in order to locate the resonance wavelength location. This occurs for wavelength shifts larger than approximately one linewidth (at least 600 fm for all systems investigated here).</li>
	<li>Record the output of the feedback controller at 20 kHz using a 24-bit data acquisition card. Export the data as a text file via data acquisition software.</li>
</ol>
3. Data Processing &#38; Analysis
<ol>
	<li>Fourier transform the data in MATLAB.</li>
	<li>Low pass the data using a &#34;brick-wall&#34; filter with a cutoff at 1 kHz to remove the imposed dither frequency of 2 kHz (see Supplemental Code File Screenshot 1).</li>
	<li>Computationally notch filter the data using a window size of 16 Hz. Note: This is done to remove known sources of noise, in this case, 60 Hz electronic line noise and its harmonics, as well 100 Hz (coming from the laser driver) and its harmonics (see Supplemental Code File Screenshot 1).</li>
	<li>Inverse Fourier transform the data back into the time domain.</li>
	<li>Median filter the data using a window size of 1,001 samples (see Supplemental Code File Screenshot 2).</li>
	<li>Locate step changes in the resonance wavelength using the step-finding algorithm of Kerssemakers et al.15.</li>
	<li>Generate histograms of the amplitude of each binding step.</li>
	<li>Calculate particle size using Eq. (1) (see Discussion).</li>
</ol>

<ol><li>Lu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+sensitivity+nanoparticle+detection+using+optical+microcavities%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">High sensitivity nanoparticle detection using optical microcavities.</a> Proc. Natl. Acad. Sci. U. S. A. 108, 5976-5979 (2011).</li>
<li>Su, J., Goldberg, A. F. G., Stoltz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+detection+of+single+nanoparticles+and+biological+molecules+using+microtoroid+optical+resonators%5BTitle%5D)+AND+%22Light%3A+Science+and+Applications%22%5BJournal%5D)">Label-free detection of single nanoparticles and biological molecules using microtoroid optical resonators.</a> Light: Science and Applications. , (2016).</li>
<li>Knight, A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKnight%2C+A+%22Single+molecule+biology%22">Single molecule biology</a>. , Elsevier/Academic. (2009).</li>
<li>Jonsson, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+biospecific+interaction+analysis+using+surface+plasmon+resonance+and+a+sensor+chip+technology%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.</a> BioTechniques. 11, 620-627 (1991).</li>
<li>Vollmer, F., Arnold, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whispering-gallery-mode+biosensing%3A+label-free+detection+down+to+single+molecules%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Whispering-gallery-mode biosensing: label-free detection down to single molecules.</a> Nat. Methods. 5, 591-596 (2008).</li>
<li>Armani, D. K., Kippenberg, T. J., Spillane, S. M., Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultra-high-Q+toroid+microcavity+on+a+chip%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultra-high-Q toroid microcavity on a chip.</a> Nature. 421, 925-928 (2003).</li>
<li>Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+microcavities%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Optical microcavities.</a> Nature. 424, 839-846 (2003).</li>
<li>Arnold, S., Khoshsima, M., Teraoka, I., Holler, S., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shift+of+whispering-gallery+modes+in+microspheres+by+protein+adsorption%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Shift of whispering-gallery modes in microspheres by protein adsorption.</a> Opt. Lett. 28, 272-274 (2003).</li>
<li>Vollmer, F., Arnold, S., Keng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+virus+detection+from+the+reactive+shift+of+a+whispering-gallery+mode%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Single virus detection from the reactive shift of a whispering-gallery mode.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 20701-20704 (2008).</li>
<li>He, L., Ozdemir, S. K., Zhu, J., Kim, W., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+single+viruses+and+nanoparticles+using+whispering+gallery+microlasers%5BTitle%5D)+AND+%22Nat+Nano%22%5BJournal%5D)">Detecting single viruses and nanoparticles using whispering gallery microlasers.</a> Nat Nano. 6, 428-432 (2011).</li>
<li>Dantham, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-free+detection+of+single+protein+using+a+nanoplasmonic-photonic+hybrid+microcavity%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Label-free detection of single protein using a nanoplasmonic-photonic hybrid microcavity.</a> Nano Lett. 13, 3347-3351 (2013).</li>
<li>Baaske, M. D., Foreman, M. R., Vollmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+nucleic+acid+interactions+monitored+on+a+label-free+microcavity+biosensor+platform%5BTitle%5D)+AND+%22Nat+Nanotechnol%22%5BJournal%5D)">Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform.</a> Nat Nanotechnol. 9, 933-939 (2014).</li>
<li>Su, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Label-Free+Single+Exosome+Detection+Using+Frequency-Locked+Microtoroid+Optical+Resonators%5BTitle%5D)+AND+%22ACS+Photonics%22%5BJournal%5D)">Label-Free Single Exosome Detection Using Frequency-Locked Microtoroid Optical Resonators.</a> ACS Photonics. (9), 1241-1245 (2015).</li>
<li>Åström, K. J., Murray, R. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3a%C3%85str%C3%B6m%2C+KJ+%22Feedback+systems+%3A+an+introduction+for+scientists+and+engineers%22">Feedback systems : an introduction for scientists and engineers</a>. , Princeton University Press. (2008).</li>
<li>Kerssemakers, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+dynamics+of+microtubules+at+molecular+resolution%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Assembly dynamics of microtubules at molecular resolution.</a> Nature. 442, 709-712 (2006).</li>
<li>Su, T. -T. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Label-free+detection+of+single+biological+molecules+using+microtoroid+optical+resonators%22">Label-free detection of single biological molecules using microtoroid optical resonators</a>. , California Institute of Technology. (2014).</li>
</ol>

The authors declare that they have no competing financial interests.

As a particle binds, the resonance wavelength (λ) of the toroid increases. If a particle unbinds, the resonance wavelength correspondingly decreases (a step-down event). The particle diameter (d) may be determined through histograms of the amplitude of each wavelength step. The height of each wavelength step varies due to size variations of the bound particle and due to the location on the microtoroid where the particle binds. The maximum change in resonance wavelength (step height) occurs when particle...

Single molecule detection experiments are useful for reducing the amount of analyte used in biosensors, for early detection of disease, and for examining the fundamental properties of molecules3. Such experiments are typically performed using labels, however, labels are not always possible to obtain for a particular protein, increase cost, can perturb the events being studied, and can be inconvenient, particularly for real time on-site experiments or point-of-care diagnostics.
The current gold standard for label-free biosensing is surface plasmon resonance4, however the commercial surface plasmon resonance systems typically have a typical lower limit of detection on the order of nM. Recently, optical resonators have emerged as a promising technology for label-free single molecule biodetection5. Optical resonators work based on the long-term (ns) confinement of light6,7. Light is evanescently coupled into these devices typically via an optical fiber. When the wavelength of the light going through the fiber matches the resonance wavelength of the resonator, light efficiently couples to the resonator. This coupled light totally internally reflects within the resonator&#39;s cavity generating an evanescent field in the vicinity of the circumference of the resonator. As particles enter the evanescent field and bind to the resonator, the resonance wavelength of the resonator changes in proportion to the volume of the particle8.
In terms of detection capability, microsphere resonators have earlier been used to detect single influenza A virus particles (100 nm)9,10. Recently, plasmonically-enhanced microsphere optical resonators have been used to detect single bovine serum albumin molecules11 and 8-mer oligonucleotides12, however this approach limits the particle capture area to 0.3 &#181;m2 per device. Larger capture area biosensors are ideal for maximizing the chance of particle detection. Current solution-based label-free biosensing technologies with large (&#62; 100 &#181;m2) capture areas have been limited to detecting polystyrene particles &#8805; 25 nm.
We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER)13 (Figure 1) that is capable of time-resolved detection of single molecules in solution. FLOWER uses the long photon lifetime of microtoroid optical resonators combined with frequency locking feedback control, balanced detection, and computational filtering to detect small particles down to single protein molecules. The use of frequency locking allows the system to always track the shifting resonance of the microtoroid as particles bind, without the need to sweep or scan the laser wavelength over large ranges. The principles of FLOWER may be used to enhance the detection capabilities of other techniques including plasmonic enhancement. In what follows, the procedures for performing FLOWER are described.

<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

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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

Particle binding events are clearly seen as step-like changes in the resonance wavelength of the microtoroid over time (Figure 2A). The heights of these steps are shown as a histogram in Figure 2B. Figures 2-4 show representative traces from the binding of exosomes (nanovesicles), 5 nm silica beads, and single human interleukin-2 molecules, respectively. The fact that the step-like events scale with particle size shows that the technique has been performed correctly. Thi...

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.

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

The overall goal of this procedure is to detect single molecules and ultra small nanoparticles without the use of labels using a technique based on optical resonator technology and frequency locking. This method can help answer key questions in biochemical fields such as protein folding and binding kinetics. The main advantage of this technique is that the target molecule does not need to be labeled in order to detect it.Set up for this technique involves coupling a micro OID chip to an optical fiber. First, obtain a micro OID chip. This is an approximately 6.5 millimeter by 5.5 millimeter silicon chip with a micro OID fabricated on it.As in this image, the micro OID has a major diameter of 80 to 100 micrometers and a minor diameter of two micrometers. For now, put the chip aside and prepare the fiber in order to couple the chip. Work with single mode fiber on a spool and unwind roughly a meter of the fiber.Get wire strippers and roughly in the middle of the unwind fiber. Use them to strip a 2.5 centimeter segment of the polymer coating from the fiber. This stripped region is the coupling region of the fiber.After stripping the fiber, use a lint-free wipe and isopropanol alcohol to clean the stripped region. Next, use a fiber holder with magnetic clamps to hold the cleaned portion in place. For the next step, have stepper motors arranged to pull the fiber in opposite directions.Position the fiber holder to allow attaching the stepper motors to the fiber on either side of the stripped region. In addition, attach a laser to one end of the fiber to serve as a light source. Start the stepper motors moving in opposite directions to stretch the fiber and use a hydrogen torch to melt the stripped portion of the fiber.Constantly monitor the light scattered laterally from the fiber. Blinking indicates the light transmission through the fiber fluctuates and more thinning is needed. When blinking ceases, stop heating and pulling the fiber, which will have been thin to about 500 nanometers.After the fiber has been thinned, detach the light source and remove the fiber and its magnetic clamp holder from the stubborn motors. Next, move the fiber to a pneumatically isolated bench equipped with the positioning stage. Place the fiber in its magnetic clamp holder on a support block in front of the sample chip.The positioning stage is a three axis nano positioning stage on top of a three axis micrometer. In addition to the nano positioning stage, there are top and side view imaging columns to help with alignment. Continue working with the fiber by ensuring its stripped portion is near the positioning stage.Prepare to introduce the fibers free end into the measurement system. After cleaving the free end, insert it into a bare fiber adapter. Use the adapter to connect the fiber to the input of an auto balanced photo receiver connected to an oscilloscope.Now focus on the other end of the fiber. First, attach this end to an optical coupler. Then couple the fiber to the output of an inline polarizer, which has input from a 50 50 beam splitter and ATT tuneable diode laser.Here is a schematic of the connections made to this point. Note that the second output of the beam splitter passes through an inline polarizer and is used as the reference for the auto balanced photo receiver. The next step is to prepare to mount the micro OID chip using a stainless steel sample holder.Employ double-sided tape to secure the chip to the sample holder. Then place the micro chip on top of the sample holder. Now mount the sample holder with the chip on top of the nano positioning stage.Here the holder and chip are in place on the positioning stage with the three Xs micrometer. Coarsely position, the sample chip with respect to the fiber. Here are the chip and fiber after the course positioning has been completed.Next, further adjust the positioner and use the imaging columns to align the chip parallel to the optical fiber With a micro OID with a laser wavelength of the fiber, this image is of a fiber and microt. After the fine positioning step, the circular sticker near the center of the field of view is a positioning aid. Now move on to search for the resonance wavelength of the micro.Generate a triangular waveform voltage signal to regulate the laser wavelength at about 635 nanometers plus or minus 2.5 nanometers. Now scan the wavelengths to search for resonance. Observe the transmission through the fiber on the oscilloscope.Note that at the resonance wavelength, the transmission drops. At this point work to adjust the polarization of the laser light. Adjust the inline polarization controllers to optimize the polarization of the laser light in the optical fiber.View the output of the photo receiver with the oscilloscope and adjust polarization until the measured transmission dip appears narrowest. After optimizing the polarization, construct a sample chamber. On the sample stage.The chamber consists of a glass cover slip epoxy to a piece of microscope.Slide. The slide acts as a spacer allowing the cover slip to overhang the chip and fiber. Arrange a one milliliter syringe pump and tubing to introduce experiment samples into the chamber at one milliliter per minute.Next, obtain a one milliliter room temperature equilibrated sample to load into the pump. In this case, it is a solution with five nanometer silica beads. Once loaded, observe the sample chamber and inject the sample Stopping when the chamber is filled, sample injection has stopped in this chamber, which is filled with a solution containing five nanometer silica beads.After waiting for 30 seconds to minimize the effect of vibration, search for the resonance wavelength of the micro. Again, observe the dip and transmission through the fiber at resonance using the oscilloscope. This is a schematic of the experimental setup at this point, including the proportional integral derivative or PID controller.The integrator and dither. Run the controller in auto lock mode with top of peak locking. Set the dither frequency to two kilohertz and set the wavelength oscillation amplitude to 19 femto meters.After empirically finding the PID settings auto lock the wavelength of the laser to the resonance wavelength of the micro oid. Collect data by recording the output of the feedback controller. Here are representative traces of resonance shift in femto meters versus time in seconds produced with a protocol using three different binding particles in order of size.The particles are exosomes or nano vesicles. Five nanometer silica beads and human interleukin two molecules. Note the different scales of both the vertical and horizontal AEs in each dataset.Observing different scales in the data for different particle sizes is an indication the technique has been performed correctly. When a particle binds to the oid, the resonance wavelength of the OID increases causing a step up in the trace. When a particle unbinds, the resonance wavelength decreases leading to a step down.The height of each wavelength step is determined by the size of the particle and its position on the micro oid. The timescales are determined by the particle OID dynamics Once mastered, this technique can be performed in about three hours if it is done correctly. While attempting this procedure, it is important to remember to keep everything as clean and as dust free as possible.

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

Research

JoVE Journal

Bioengineering

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.

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

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.

Fabrication of Silica Ultra High Quality Factor Microresonators

This video demonstrates localized surface plasmon resonance imaging to detect protein secretions from a single cell. Using a microfluidic setup and a peptide-bound plasmonic nanostructure chip, changes in reflected light intensity at the resonance angle reveal interactions with secreted proteins from a single cell.

Localized Surface Plasmon Resonance Imaging to Detect Protein Secretions from a Single Cell

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.

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

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.

Implementation of a Reference Interferometer for Nanodetection

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.

Fabrication and Testing of Microfluidic Optomechanical Oscillators

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.

Fabrication and Operation of a Nano-Optical Conveyor Belt

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 Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

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.

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

This article traces the life cycle of a silicon nitride membrane resonator, from design through fabrication to characterization.

Fabrication and Characterization of High-Q Silicon Nitride Membrane Resonators

Learn label-free single molecule detection using microtoroid optical resonators and frequency locking technology. This method enables studying protein folding and binding kinetics without altering target molecules.

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Explore More Videos