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43469-AC7
Molecularly Imprinted Polymer Thin Films and Microarrays
The objective of this research is to develop a new imprinting chemistry and to fabricate molecularly imprinted polymer (MIP) thin films and microarrays. The new imprinting chemistry differs from the conventional method where the binding sites are formed by way of a photochemically initiated crosslinking process. During the first year of the project, we focused on studying the binding interactions between the functional monomer, PFPA-COOH, with the template molecule ethyladenine-9-acetate (EA9A). We carried out NMR titration experiments and determined the binding constants. Much of the work was centered around improving the quality of the MIP films, testing the reproducibility of the procedure. A variety of polymers were tested for their interactions to analyte molecules to select the matrix material that minimized the non-specific binding.
Because the MIP films were thin, with the thickness on the order of ~100 nm, it is challenging to accurately measure the amount of the analyte molecules before and after rebinding. We therefore used radioactive ligands and measured the analyte concentration either by scintillation counting or a phosphor storage screen. Standard calibration curves and detection limited were obtained for both methods, thus establishing the procedure for subsequent batch and competitive binding studies.
In the second year of this project, we focused our efforts on optimizing the imprinting chemistry. Batch binding and competitive binding studies were carried out to characterize the performance of MIP films. Initial studies on creating MIP arrays were conducted. The following summarizes the findings of these studies. Some results of these studies are presented as graphs in attached Nugget slide.
We first subjected the MIP films to batch rebinding studies, varying the concentration of radiolabelled adenine and measured the amount of adenine rebound in the MIP films. The MIP films showed consistent higher binding for adenine than the non-imprinted polymer films, ie polystyrene in our case. However, for two other control samples, ie, polymers prepared without adenine as the template, the binding was high. This was likely due to the non-specific adsorption of 14C-adenine to these films by H-bonding, dipole interactions or hydrophobic interactions. Also, we clearly observed a difference in film morphology between MIP films and the control samples. The films prepared without the template molecule were noticeably more fragile and had many pinholes. This could contribute to the higher amount of 14C-adenine adhere to the polymer matrix. Similar results were also obtained for MIP films prepared with caffeine or theophylline as the template molecule. In addition, the results were not reproducible, which was the result of the poor film quality.
The imprinted films were then subjected to competitive binding studies. The competitors chosen were hypothanthine and o-methylguanine, both structurally related to adenine. The experiments were carried out by treating EA9A-imprinted polymer films to a mixture of 14C-adenine and hypothanthine or 14C-adenine and o-methylguanine at various competitor concentrations, and the uptake of 14C-adenine was measured. As the concentration of the competitor increased, more competitor would be taken by the MIP films, resulting in a decrease in the amount of 14C-adenine in the MIP films. This was observed when o-methylguanine was used as the competitor. With hypothanthine, however, the trend was less obvious. Control samples were prepared and were treated under the same conditions as MIPs and the uptake of 14C-adenine was measured at various competitor concentrations. Similar to the batch binding studies, PS showed the least amount of non-specific adsorption whereas the control samples containing either PFPA-acid or PFPA-ester showed higher non-specific binding.
Both the batch binding studies and the competitive binding studies indicated that the MIP films were not as selective or specific towards the template molecules. Therefore the next logic step was to use MIP arrays. The design was to prepare several MIP films, each against a template that is structurally related to the target molecule so that each MIP would have different affinity for a particular analyte. An array made from these MIPs would give a specific recognition pattern for the analyte molecule. This arrangement does not require each MIP to be very selective, but rather the array of MIPs to display a recognition pattern which can be unique and highly specific for the analyte. In the initial studies, we prepared MIP films against EA9A, theophylline, and caffeine. Each MIP was then tested against radioligands of 14C-adenine, 14C-theophylline, and 14C-caffeine, respectively. Preliminary results showed that each MIP had different binding affinity towards different ligands. We will continue the studies on MIP arrays, carry out data analysis, optimize imprinting formulation, and demonstrate reproducibility of the approach.
