On-chip Electrophoresis Devices: Experimental

26 March 2007

• Do filter ALL solutions immediately before putting them in chip reservoirs.  Syringe filters work great.
• Do check the mercury bulb alignment on an epifluorescent microscope before starting a serious experiment.
• Doom: Differing heights of the liquid columns in chip reservoirs can cause significant pressure driven flows.  These can lead to high dispersion and low resolution.  You can check for these by, say, starting an electrokinetic focusing/injection and then suddenly deactivating the applied potentials.  Once the field is off, pressure gradients will show themselves as drifts in the (e.g., fluorescent stream) material lines.
• Do save a data set whenever possible (as in all experiments). For quantitative imaging, also remember to take a flatfield image often (in fact, whenever it is convenient and perhaps more often). An accidental bump of the microscope stage can void hours of quantitative imaging data (since your background illumination pattern is now different).
• Don't assume that the dye fluorescence remains constant in your reservoirs over time when running long experiments with large applied potentials (in the hundreds of volts and higher).  Most fluorescent dyes are broken down electrochemically; see for example [5].
• Do experiment with and optimize your electrokinetic sample injection schemes. A good injection can significantly increase the resolution of the electrophoretic injection, and properly balance the demands of signal to noise ratio and resolution [6]. As a first step, instead of simply injecting sample from one channel to another, pinch the injection by introducing "focusing streams" on both sides of the injected stream [7].  See Fig. 1a.  This also applies to injections in "double T" channels. Note that the method of gated injection can also give clean injections [8], especially if you are hungry for signal and want a relatively large sample plug. If you choose the pinching method, it is possible to insert a step of flow reversal before the injection in the main channel for further optimization, by reversing the flow of analyte for a very short time (Fig. 1b) [6].           

Figure 1: Optimization of On-Chip electrophoretic injection [6]. (a) is the pinching step, (b) the flow reversal step, and (c) the dispensing in the separation channel. The same procedure is applicable to a double T channel geometry.

Here we provide a step-by-step description of a typical On-Chip electrophoretic injection experiment:

1. Make sure your setup is protected from external light to limit noise. 
2. Rinse the channels with buffer for >15 minutes. If you want to inject sample from the North channel (as shown on Fig. 1), fill the West, South and East wells and apply vacuum at the North well.
3. Remove the vacuum from the North well and rinse the well (not the channel) with DI water. Empty it before filling it with your sample. (This rinsing is critical if the background buffer of the sample is to be significantly different than that in the other wells).
4. Place platinum electrodes (e.g., wires) in the wells. Make sure they don't touch each other or another surrounding metallic part like the stage (to avoid short circuits). Ideally, place the wire in the well but well away from the channel inlet.(This keeps the region where electrolysis occurs away from the channel inlet.)Also, make sure there are no unwanted "liquid bridges" connecting two wells above or below the chip.
5. Take a background image. Taking a background image at this point will capture any stray fluorescence that may be visible from the well, although the channel should still be empty of sample.
6. Inject liquid from North to South before starting any injection. After starting this flow, you can start your flows from East and West to the South (as per your pinching scheme).
7. Initiate the rest of your electric field scheme. A simple scheme is suggested in Table 1, but you can customize it depending on your setup and interests.
8. After optimizing your scheme, re-focus your microscope and camera on the region of interest within the chip and take another background image prior to injection (this background will be locally free of sample). This image will help you determine background signal due to autofluorescence of solution, channel wall, etc.
9. Inject and image away!
10. After acquiring your data, don't move the chip or the stage. Instead, first rinse all of your channels with DI and then fill all channels with your fluorescent sample solution, focus your microscope and camera on the region of interest of the channel, and take a "flat field"/background image.  A "flat field" image is one where the entire region of interest is filled with fluorescent dye. This image will help you correct for non-uniformities in illumination, non-uniform channel depths, etc. This step can also be performed before actually taking data.
11. Rinse the channels with DI water and go back to step 1 or store properly.           

Injection Step	Duration (s)	En/Es	Ee/Es	Ew/Es
Pinching	20	0.42	0.29	0.29
Flow Reversal	0.2	2.32	0.66	0.66
Dispensing	100	1.00	0.43	2.43

Table 1: Electric field scheme for optimized sample injection, assuming all streams have the same conductivity [6]. Ei is the value of the electric field in the channel i

References

5. Jain, R., Sharma, N., Bhargava, M., J. Sci. Ind. Res., 2003, 62(12), 1138-1144.
6. Bharadwaj, R., Santiago, J. G., and Mohammadi, B., Electrophoresis, 2002, 23(16), 2729-2744.
7. Jacobson, S.C., Hergenroder, R., Koutny, L.B., Warmack, R.J., Ramsey, J. M., Anal. Chem., 1994, 66(7), 1107-1113.
8. Jacobson, S. C., Koutny, L. B., Hergenroder, R., Moore Jr., A. W., Ramsey, J. M., Anal. Chem., 1994, 66(20), 3472-3476.