The overall goal of this procedure is to coat the surface of the silico microsphere optical resonator with biotin through a covalent attachment process without degrading the sensitivity of the device. This is accomplished by a three step process, beginning with hydroxylation using oxygen plasma or piha etch hydroxylation is followed by emanation through vapor deposition of a slan coupling agent. Then biotin is carried out using NHS Ester chemistry to link the valeric side chain of biotin to the primary means on the resonator surface.
Ultimately, this method creates an optical biosensor that is both highly sensitive and specific for a molecule of interest, and could be modified further through fluorescent labeling of a mean terminated silica, as well as through the Ava tin biotin complexation method to create a biosensor platform for a wide array of potential targets. The main advantage of this technique over existing methods of surface functionalization, such as physical absorption, is that our probe molecule is covalently attached, which makes our sensors more robust and reliable. This means that they can be stored for longer periods of time and still retain their use, and they remain highly sensitive after functionalization.
Generally, individuals new to this method will struggle because of the difficulty in handling the micros size devices throughout the functionalization process. Poor handling technique is the easiest way to degrade the optical performance of the sensor and to generate contamination on the sensor surface Using scissors. Cut a one quarter inch thick piece of cardboard into a one inch by one inch square.
Tape this onto a standard glass slide and attach a one inch section of rolled scotch tape to the cardboard. This creates a platform on which the microsphere can be elevated and isolated from the surrounding environment and prevents damage to its surface using scissors. Cut a three inch section of optical fiber from the spool.
Next, use a no nick fiber stripper to strip the protective polymeric coating from the last 0.5 inches on the end of the cut piece of fiber, leaving just the silica core, clean the surface of any remaining polymer with a Kim wipe dampened with methanol by gently wiping the fiber using a bare fiber cleaver trim the stripped end so that only about one millimeter of stripped fiber remains on the end of the fiber. Place the stripped end of the optical fiber into the path of a carbon dioxide laser taking care of vertically. Align the fiber such that its stripped end is facing down.
Turn on the carbon dioxide laser and direct it to the surface of the stripped end of the optical fiber. The laser will melt the stripped end of the fiber into a sphere using approximately 3.5%power in two seconds. Using tweezers, carefully grasp the stem of the microsphere.
Attach the stem to the tape roll on the microsphere housing. The glass slide may be stored in a Petri dish, allowing for safe storage of the microsphere in a 60 milliliter polypropylene vial with a hinged cap. Prepare a piranha solution by adding three milliliters of hydrogen peroxide to the vial, followed by seven milliliters of fuming sulfuric acid.
Transfer the glass slide holding at least one microsphere to the vial. The microsphere should be in contact with the liquid, but the liquid should not touch the cardboard. Adjust the volume of the solution if needed.
After the spheres have been immersed in the piranha solution for 15 minutes, gently remove the glass slide from the vial and insert it into another plastic vial containing deionized distilled water. Place the vial on the tilt tray for five minutes at angled five degrees and tilt speed five RPM. Gently remove the glass slide from the vial and place it in an oven at 80 degrees Celsius for 10 minutes.
To dry the surface, place the glass slide containing at least one microsphere into a vacuum.Desiccate. Open the slan coupling agent bottle, such as A-P-T-M-S and place the open bottle in the desiccate. Replace the lid on the vacuum desiccate and attach the outlet port to an aspirator or house vacuum line.
Turn on the house, vacuum or the waterline and evacuate the vacuum desiccate. Once a vacuum seal is formed between the lid and the base, begin timing the reaction. This will deposit the sine coupling agent onto the surface as a thin film.
After 15 minutes, turn off the vacuum and slowly open the port to let air into the desiccate. For this coupling agent, 15 minutes is sufficient to form a uniform monolayer on the surface for other coupling agents. The time may need to be adjusted approximately one hour before attaching the biotin, prepare a 10 millimolar solution of en hydroxyl oxide biotin in anhydrous dimethyl sulfoxide in a 60 milliliter polypropylene vial with a hinged cap.
The most difficult part of this protocol is microsphere handling and avoiding contamination of the sphere surface in order to ensure success. In addition to creating the slide apparatus to hold the spheres, the biotin solution is sonicate beforehand to break up the particles that remain on the surface. Sonicate the solution for one hour to fully dissolve the NHS biotin powder in the solvent.
Transfer the glass slide containing the microsphere into another plastic vial with the microsphere at the bottom and the stem at the top of the vial. Using a plastic pipette transfer an appropriate volume of the NHS biotin solution down the side of the vial behind the glass slide so that the solution does not touch the microsphere as it is being added to the vial. Add enough solution to cover the microsphere surface.
Place the vial now containing the MICROSPHERE and NHS biotin and DMSO solution onto a rocking incubator for 30 minutes at room temperature with the speed and angle of tilt at five RPM and five degrees respectively. Gently remove the glass slide from the vial and gently slide it into another vial filled with deionized distilled water. Place the vial on the tilt tray for another 10 minutes at the same speed and tilt angle.
As before, repeat this step twice with fresh water each time. This helps remove excess DMSO from the surface and removes any physically absorbed biotin that did not actually graft to the surface. Remove the glass slide from the vial and place it in an oven at 80 degrees Celsius for 10 minutes or until all water droplets are removed from the surface.
Proceed to perform fluorescent labeling of a mean terminated silica as discussed in the protocol to fluorescently. Label the biotin terminated silica. First, prepare a 10 microgram per milliliter solution of Texas Red Aden in phosphate buffered saline.
Add the solution to a 60 milliliter polypropylene vial with a hinged cap and gently slide the glass slide containing a biotin terminated microsphere into the solution so that the microsphere is just covered by the solution. Then wrap the vial and foil following a 30 minute reaction. Time at room temperature in the dark excess fluoro four is removed through two 10 minute rinses of the microsphere in PBS buffer.
As before, fill a 60 milliliter polypropylene vial with the buffer and gently transfer the glass slide into the solution. Take care of that. The solution only covers the microsphere and not the cardboard housing.
Cover the vial with aluminum foil and place the vial on a tilt tray set to rocket five degrees and five RPM for 10 minutes. Gently remove the glass slide from the vial and gently slide it into another vial filled with deionized distilled water and covered with aluminum foil. Place the vial on the tilt tray for another 10 minutes at the same speed and tilt angle.
Repeat this step twice with fresh water each time. This helps remove excess dye from the surface. As a final step, generally remove the glass slide from the vial and dry in an oven set at 80 degrees Celsius for 10 minutes before imaging.
If the surface functionalization is done correctly, it should result in a uniformly dense coverage of biotin molecules on the surface as probe by fluorescent microscopy. Additionally, the surface should remain defect and contaminant free after functionalization in order to maintain their high sensitivities. During detection experiments as visualized by optical microscopy shown here are examples of correctly functionalized microspheres.
These images show that there is no surface damage or contamination due to functionalization, and that the microspheres show a uniform consistent coverage of either am mean groups or biotin groups. On the surface, if the microspheres have not been functionalized correctly, the optical microsphere images will exhibit surface contamination, clumping or non-uniform coverage and obvious defects in the surface like surface cracks shown here is a common example of surface contamination resulting from clumping of reagents on the surface. After watching this video, you should have a good understanding of how to functionalize the surface of any silica optical resonator with biotin.
Since this is a general method for silica devices, you should be able to apply it broadly to many different types of silica sensor platforms. Through the developments of this technique, researchers in the field of bio photonics will be able to more easily explore new methods and new probe sensor combinations for creating highly sensitive and specific biosensor platforms.
