Schilke Lab

Optical Waveguide Lightmode Spectroscopy (OWLS)

We have acquired an OWLS 210 system from MicroVacuum, Inc. in Budapest, Hungary.

This instrument allows in situ, real-time analysis of adsorption of proteins, DNA, viruses and other biomolecules to surfaces. The sensitivity is such that OWLS can detect protein adsorption at 1% of a monolayer. Adsorption experiments can be run in various buffers or other liquids.

The OWLS instrument is located in Dr. Joe McGuire’s laboratory in Gleeson Hall 300. Contact Dr. Karl Schilke

<!–
Nix.decode(“”);
//–>
(schilkek@onid.orst.edu)<!–
Nix.decode(“”);
//–>

for more information or to schedule instrument time.

View the OWLS Google Calendar

Theory


Overview of OWLS operation	OWLS instrumentation setup

OWLS operates by “incoupling” of a HeNe (632.8 nm) laser through the bottom of the chip (right) and into a waveguide embossed in the surface of the chip. This incoupling occurs only at two distinct angles (with a very sharp peak in the intensity vs. angle plot). Total internal reflection occurs, but an evanescent wave extends ~100-200nm into the medium above the waveguide. Changes in the refractive index at the interface are sampled by this evanescent wave, and slightly change the angle, α at which the reflected light leaving the sensor reaches a maximum. From the angle and other parameters, the refractive index and thickness of the overlayer can be calculated. From the film thickness and RI, the total adsorbed amount can be estimated.

Some videos are also available for your viewing pleasure:

http://www.youtube.com/watch?v=SFyLYcp40LU (MicroVacuum product demonstration video)

Documentation

Manuals for the system are available here:

Sensors

Δ
OWLS sensor chip (click for details)

Sensors are made by depositing a thin layer of Si_0.25Ti_0.75O₂, by a sol-gel process. As the gel solidifies, it is embossed with a ~20nm waveguide (2400 lines/mm = 416.6 nm spacing), then heated to solidify the surface.

The waveguide can be coated with SiO₂ or other pure materials. This overlayer is ~10-20 nm thick, so does not interfere substantially with the optical sensing mechanism. We have purchased a number of SiO2-coated Δ and uncoated sensor chips Δ.

Sample Injection Valve

As shown at right, the HPLC-style injector port has two positions, “Load” and “Inject”. Click the diagram for an elarged view.

When set to “Load”, the flowing liquid bypasses the sample loop. A blunt needle can be used to fill up the loop. Note that an additional volume of sample is required to flush out any residual buffer — the extra liquid emerges from the waste ports.

When the injector valve is quickly turned to “Inject”, the loop is switched into the flowpath, and the sample is transported into the OWLS flow cell.

Sample Flow Loop

We have modified the original sample injection loop to hold a substantially larger volume of fluid (a few mL instead of 100 µL). We made this change to allow continuous flow of a solution of protein of known volume for the duration of an adsorption cycle, rather than the suggested method of injection and stopped flow (with an uncontrolled concentration). The results were substantially improved in terms of repeatability and noise.

The loop should be replaced before each set of experiments. Currently we are using 0.03″ I.D. Tygon tubing (which is available at ChemStores) for the loop instead of PEEK.

Each turn of the loop will contain XXX uL of fluid. This allows calculation of the total loop volume based on the number of turns. The additional volume from the connecting tubing is approximately equivalent to XXX turns.

****THIS PORTION WILL BE UPDATED ONCE A “FINAL” FLOW LOOP SCHEME HAS BEEN DEVELOPED. CURRENTLY THE FLOW LOOP IS 7FT LONG (214 CM) AND CONTAINS ROUGHLY 2.7 ML OF FLUID. (I KNOW THE MATH DOESN’T EQUATE TO THAT, I’M WORKING ON IT) ~MATT****

Protocols

How to check the slope of measurements

Select a region in both NTM and NTE windows. The button with the black rectangular region in the tool box must be selected to select regions.
Click “SHOW.”
Click “USE SELECT” button in the bottom left hand corner. This will display the SLOPE and DEVIATION values in the Measurement Properties/Data Window in the bottom right.

Calibrating n_f and d_f Variables

Place a completely dry sensor into flow cell with the dot in the TOP LEFT corner (portrait orientation). The grating will be facing down.
Do not hook up to syringe pump. The sensor should first be measured under air.
Start measurements.
When the slope for NTM and NTE are on the 10^-8 order of magnitude…
1. Select regions in NTM and NTE windows.
2. Click “SHOW.”
3. Be sure the n_c value is the refractive index for air.
4. Click “USE SELECT” under the n_f and d_f windows. This will input the calculated n_f (refractive index) and d_f (sensor thickness). These values will be used to later calculate the adsorbed layer thickness.

Loading Sensor

Load sensor into flow cell with the dot in the TOP LEFT corner (portrait orientation). The grating will be facing down. With top properly aligned, screw top on. Hook up to syringe pump that is NOT flowing. Check to make sure the fittings are tightly screwed in place.
Syringe Pump Rate: 100 µL/min
Check for leaks in the flow cell by holding the flow cell over the waste beaker, turning on the syringe pump, and watching for leaks.
Place flow cell in chamber block.

Equilibrating Flow Environment

Once sensor is properly loaded and CHECKED FOR LEAKS (see above) start the syringe pump.
Change the n_c value to the refractive index of the fluid flowing over device.
1. Click “BASE”
2. Keep the N-ref the same (it is set to the value of air).
3. Click “GET”
4. Select from the drop down menu for proper value.
5. Input the proper temperature at which the experiments are run.
Start measurements on user interface: click the green triangle PLAY button.
Wait until the slope of a selected region in both NTM and NTE windows is in the 10^-8 order of magnitude. See above for how to check the slope.
When the slope reaches this order of magnitude the sensor surface is at (approximate) equilibrium with the flowing fluid.
Proceed with experiment: see “How to inject samples” and “Switching syringe pumps.”

Changing the interface to display dA and Mass

dA is the thickness of the adsorbed layer. This is an average value?
Mass is the adsorbed mass shown in ng/cm^2.
Click “SHOW.”
Unselect “Peak Window” and “Data Window.”
Select dA and Mass from drop down menus under “Window 3” and “Window 4”.
NOTE: There are many options of what can be displayed in windows 1-4. Play around with them and have fun.

How to inject samples

Clean glass syringe by drawing up and expelling water 2 or so times.
Draw up approximately 200 µL of sample into the syringe.
Remove red cap and placeholder needle from the injection port.
NOTE: Only blunt end needles may be used. Beveled (pointy) needles will puncture the inerts of the injection port and require a very expensive repair.
The level on the injection port should be set at “LOAD.”
Insert the syringe into the injection port. You will feel some resistance when you reach the o-ring. Gently push past this point until the needle cannot be advanced farther.
Inject approximately 150 µL of the sample into the loading chamber. Visually check to make sure liquid exits the waste tube as you inject. Once visualized, the injection port is fully loaded and will not have air bubbles.
It is important to leave a portion of the sample in the syringe to maintain fluid continuity (keeping no air in the system).
Switch the level from “LOAD” to “INJECT.”
Keep the syringe in place until the sample flows through the flow cell.
To remove the syringe…
1. Swtich the level from “INJECT” to “LOAD.”
2. Pull out syringe.

Cleaning the sensor

Clean sensor with HCl before and after an experiment.
Clean before the experiment but after attaining n_f and d_f under air.
1. Inject HCl into the system via the injection port. Follow with a buffer injection.
2. Repeat 2-3 times.
Clean after the experiment is complete while the syringe pump is still flowing.
1. Inject HCl into the system via the injeciton port 2-3 times.
2. Follow with water cleaning: see “Cleaning the injecitn port/cartridge.”

Cleaning the injection port/cartridge

The injection cartridge must be cleaned after the experiment is over.
Fill the glass syringe with pure water.
Run water through the system (with the syringe pump flowing to the flow cell).
Repeat 2-3 times.
The injection port should be cleaned after an experiment.
Attach the port-syringe attachment to the injection port.
Attach an appropriately sized syringe filled with water to the end.
Inject while the level is set to “LOAD.”
Repeat 2-3 times.

References

Key references for understanding and using OWLS include:

General theory and background

Székács et al. Applied Optics (2009) 48(4), B151-158: Good introduction to data collection and interpretation, surface modification of sensors, biosensing.

:A PowerPoint review of OWLS theory Δ.

Biomaterials and Biointerfaces Lab

OWLS