Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).

Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 µM, 44 ± 3 µM, 17 ± 2 µM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface.

Quantification and confocal imaging of protein specific molecularly imprinted polymers.

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.

Voltammetric behaviour and trace determination of copper at a mercury-free screen-printed carbon electrode.

Screen-printed carbon electrodes (SPCEs), without chemical modification, have been investigated as disposable sensors for the measurement of trace levels of Cu(2+). Cyclic voltammetry was employed to elucidate the electrochemical behaviour of Cu(2+) at these electrodes in a variety of supporting electrolytes. For all of the electrolytes studied the anodic peaks, obtained on the reverse scans, showed that the Cu(2+) had been deposited as a thin layer on the surface of the SPCE. The anodic peak of greatest magnitude was obtained in 0.1 M malonic acid. The possibility of determining Cu(2+) at trace levels using this medium was examined by differential pulse anodic stripping voltammetry (DPASV). The effect of Bi(3+), Cd(2+), Fe(3+), Hg(2)(2+), Pb(2+), Sb(3+) and Zn(2+) on the Cu stripping peak was examined and under the conditions employed, only Hg(2)(2+) was found to significantly effect the response gained. The sensors were evaluated by carrying out Cu(2+) determinations on spiked and unspiked serum and water samples. The mean recovery was found in all cases to be >90% and the performance characteristics indicated the method holds promise for trace Cu(2+) levels by employment of Hg-free SPCEs using DPASV.