You will use a custom-built laser-induced fluorescence microscope with microchip holder and power supplies. This instrument is interfaced with a computer for data acquisition. Do not touch anything on this setup until either the instructor or TA has briefed you on its usage. A micropipettor with disposable tips and a vacuum hose in the drawer below the computer are available for filling and emptying your device. Don’t use the micropipettor until you have been instructed on its operation.
Caution: This instrument uses high voltages (up to 5 kV) and laser radiation. You should never put anything reflective or any part of your body in the path of the laser. The high-voltage power supplies have a current limit set for your protection, but you should also take appropriate precautions. Specifically, you must verify that all wires are in the appropriate reservoirs before you turn on the power supplies, and you must always ensure that the power supplies are off before you touch any wires.
Chemicals and Materials
Amino acids have been labeled with fluorescein isothiocyanate (FITC) and diluted for you. You will be given a set of microcentrifuge tubes containing the following solutions: borate buffer, borate buffer with added hydroxypropyl cellulose (to reduce electroosmotic flow), fluorescein, FITC-Gly, FITC-Asp, FITC-Phe, and an unknown containing one or more of the amino acids. Note: fluorescent compounds are light and temperature sensitive, so you need to store them refrigerated between lab periods.
You will be given a PMMA and a PDMS/glass microdevice. Contaminants from skin will hinder device operation, so make sure you wear gloves when you handle the microdevices at all stages of use.
You will use the two different types of microchip devices to carry out rapid separations of fluorescently labeled amino acids and identify the peaks in the mixture. You will also calculate the separation efficiency and compare the performance of the two systems.
- You will first learn how to work with microliter-scale volumes and fill microfluidic devices. Use a water bottle or the micropipettor to transfer a small volume (<10 μL) of water into one of the three reservoirs at the top of a PMMA microdevice. The channels should all fill gradually via capillary action. You may want to place the vacuum hose over the various reservoirs to help in the filling process. If all the channels fill, pipet any liquid from the reservoirs, and then use the vacuum hose to dry the channels, and then try filling a PDMS/glass microchip using the same procedure. Be careful that you don’t separate the PDMS from the glass when you put the pipette tip into the reservoir. You may also find it difficult to use vacuum with the PDMS/glass devices; if so, devices can be cleaned by separating the PDMS sheet from the glass, carefully rinsing, drying, and then reassembling the PDMS/glass stack. If all your channels do not fill in either device, the instructor or TA can help you try to assess why, by inspecting the microchip with the microscope.
- A device that filled with water is ready to be loaded with buffer solution; start with your PMMA microchip. You should use the same procedure that worked for filling with water. Once all the channels are full of buffer, fill all four buffer reservoirs to be level with the top of the device.
- Turn on the detection system as described in the Operating Instructions.
- Make sure that buffer in all reservoirs is still level with the top of the device. Then, pipet out the contents of the top left (sample) reservoir, and add ~5 μL of sample. Place the device on the chip holder on the microscope stage, making sure that the injection intersection is just to the right of the left edge of the circular opening in the chip holder. Secure the device in position with 2-3 pieces of tape. Verify that the high-voltage power supplies are off, and then carefully insert the four electrodes into their respective reservoirs.
- Follow the Operating Instructions for initial alignment of the microchip in the optical detection system, focusing the laser in the injection channel near the intersection region.
- Check that the electrodes are still in position, and verify that the inject switch is in the “inject” position. Then, turn on the high-voltage power supplies, following the Operating Instructions for trial injection and fine alignment. If you have any questions about whether or not you are doing this properly, check with the instructor or TA first. After a time, the fluorescent sample will flow through the injection channel, and the PMT will detect the fluorescence signal. Align the pinhole and adjust the fine focus, then turn off the high voltage and the power supplies.
- Move the detection spot ~1 cm down the separation channel and adjust the Z-position the distance you optimized above. Then, carry out an injection/separation, following the Operating Instructions. Be sure to hit the “record” button on the VI at the same time you switch the box from “inject” to “run”.
- Obtain as many replicate separations as you desire. You should also explore the effects on separation of parameters such as injection time and detection position. When you are ready to switch samples, follow the procedures in the Operating Instructions.
- Carry out separations of the amino acid mixture in both a PMMA and a PDMS/glass device, using the same separation/injection conditions, so you can compare separation performance.
- Identify which peak corresponds to which analyte in your unknown.
- At the end of each lab period, flush your chips with water, vacuum the channels dry, and follow the shutdown procedures for the instrument in the Operating Instructions.
Some things to include in your report (along with whatever other information is needed)
List any instrumental settings that differed from the recommended ones for the experiment.
Describe the approach you used to identify the peaks in your sample.
Results and Discussion
Discuss (and be quantitative about) the reproducibility of peak migration times and theoretical plate counts.
How did the injection time and separation distance affect the number of theoretical plates and peak height? Did the number of theoretical plates scale as expected with separation distance?
Show the electropherograms you used to identify the peaks, and identify the amino acid peaks on an electropherogram of the sample. Also, explain why the analytes migrate in the order observed.
Discuss any strengths and weaknesses of microchip capillary electrophoresis.
Compare the separation performance in PMMA vs. PDMS/glass microchips.
Compare the ease of use of PMMA vs. PDMS/glass microchips.
Address all other questions and issues raised in the lab procedure.
Discuss any other conclusions you can draw from your experiments. Talk about what you would do differently, if you had to do the experiment over again, or what you would do additionally, if you had more time.
Show representative calculations for theoretical plate count. Show other calculations only as they aid your discussion.