Biochemical Interactions in Imprinted Plastic Microfluid Devices
Objective: To study biochemical interactions with plastic surfaces in imprinted plastic micro-analytical devices.
Problem: Micro-analytical technology is gaining commercialization momentum due to the greatly reduced cost of using plastics. One current disadvantage of using plastic substrates is that many of the basic properties of plastics necessary for their successful application in bioanalytical separations have not been characterized. This lack of fundamental information has limited the broad use of plastics in electrophoretic and chromatographic separations in microanalytical systems.
Approach: Use of plastic substrates required for applications in bioanalytical micro-separations was studied using a simple, low-cost method for device fabrication in plastics, a low-temperature imprinting techniques. Several characteristics of imprinted plastic microchannels are currently under investigation including parameters affecting the electro-osmotic (EO) flow and characteristics of protein absorption. EO flow is the bulk fluid flow that occurs in microchannels under the influence of an electric field and is strongly influenced by surface charge. Since the EO flow rate is directly related to the native charge on the walls of the plastic micro-channels, changes in EO flow in the presence of protein are indicative of wall adsorption. This information is useful in choosing the appropriate plastic substrates for bioanalytical separations.
Results/Future Plans: EO flow rates were measured in three different plastic substrates, acrylic, polystyrene, and copolyester, and found to be 0.07, 0.05, and 0.15 cm s-1 (electric field = 300 V cm-1), respectively. The direction of the EO flow indicated that the total surface charge in each of the plastic channels was negatively charged. The non-specific adsorption on the walls of the acrylic and polystyrene channels was significant, and caused reductions in EO flow by more than 50 %.
Figure 1 shows the results of an immunoassay separation performed
in the microanalytical device. Fluorescent peaks were monitored using a photomultiplier
tube mounted on a fluorescence microscope viewing the microchannel. The peaks
are associated with a fluorescently-labeled morphine derivative used as the
label in a competitive homogeneous immunoassay. Morphine antibody was mixed
with sample morphine and a known quantity of fluorescently-labeled morphine.
Electrokinetic injection and separation using an electric field resulted in
two resolved peaks with one peak representing labeled morphine bound to antibody,
and the other, unbound labeled morphine. The seven sequential injections shown
in Figure 1 (each showing two separate peaks) should have been identical.
However, wall adsorption effects caused irreproducible measurements, as well
as shifts in retention times caused by flow rate fluctuations. The reproducibility
of sequential injections could be improved by pre-priming the device with
several injections of the sample. A successful immunoassay for morphine was
demonstrated in an imprinted plastic micro-channel device with electrophoretic
separation of components. After priming the system with four test injections,
the analytical data showed two well-resolved peaks with retention times of
14.0 s and 31.0 s. For three injections of sample, the relative standard deviation
for peak height for the two peaks were 1.3 % and 5.1 %. Future work
will include the characterization of other commercially-available plastics,
and the evaluation of cell adsorption and lysis in these substrates
Publications:
Martynova, L., Locascio, L.E., Gaitan, M., Kramer, G.W., Christensen, R.G., and MacCrehan, W.A., "Fabrication of Plastic Microfluid Channels by Imprinting Methods," Anal. Chem., 69, 4783-4789 (1997).