Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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.