Padró-Cortés, Lorena R.

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    Towards the design of highly selective molecularly imprinted hydrogels for biosensor applications
    (2009) Padró-Cortés, Lorena R.; Torres-Lugo, Madeline; College of Engineering; Rinaldi, Carlos; Lopez Garriga, Juan; Juan, Eduardo; Department of Chemical Engineering; Aponte, Maria
    Robust molecular recognition is essential to enhance the accuracy of diagnostic devices aiming for a better efficacy in the clinical treatment of patients. The molecular imprinting technique has gained attention to generate novel biosensor and clinical diagnostic devices with high sensitivity and specificity, which demonstrated affinities compared to their natural counterparts. A method for the rational design of biomimetic sensors based on molecularly imprinted polymer for the detection of hydrocortisone is described and applied. The thermodynamic of the association between hydrocortisone and the functional monomer, methacrylic acid (MAA) was investigated by nuclear magnetic resonance (NMR) spectroscopy. Dissociation constants for the complex formation between hydrocortisone and a functional monomer analogue, acetic acid, as a function of solvent nature were estimated by NMR titration. The results demonstrate lower affinity using ethanol as solvent (K=0.5814 0.0.1163 M). However, the formation of adduct was confirmed, which suggested creation of MAA-Hydrocortisone at the pre-polymeric mixture. The stoichiometry of the complex formation between hydrocortisone and an acetic acid on each solvent was evaluated by the Job method of continuous variation. Dimethyl sulfoxide and ethanol were selected as porogens to assess solvent effect. The dissociation constants obtained for ethanol-d6 reflected a greater proximity of interaction between solution adducts compared to dimethyl sulfoxide-d6. It is consequently associated by the dielectric constant of the solvents. Job plot results suggested a complex mole ratio of 1.5:1 and 1:2 for dimethyl sulfoxide-d6 and ethanol-d6, respectively. The collective analysis of NMR titration and Job plot method indicated the extent of shift displacement is proportional to the proximity to the interaction site that is not apparently associated with its stoichiometric capabilities of complex formation. To evaluate synthesis condition, in situ free radical copolymerization was monitored by ATR-FTIR spectroscopy with methacrylic acid (MAA) as the functional monomer and tetra(ethylene glycol) dimethacrylate (TEGDMA) as the crosslinking agent in different solvents. The synthesis was performed in presence and absence of the template molecule. The combined set of analysis allowed a better understanding of the recognition events giving rise to the imprinting effect during MIP synthesis and to ligand-MIP binding events. In situ polymerization results demonstrated a delay of the auto-acceleration during the imprinting process. In essence, the propagation kinetic was reduced by the decrease of monomer mobility, which suggested the functional monomer-template complexation already confirmed by the NMR spectroscopic studies. Consequently, the information was to be applied for the design of thin films MIP. It can be for seen that using this information, the collapse-swelling transition of MIPs could be programmed to promote binding capabilities and enhance template diffusion. To these aims, hydrocortisone imprinted polymers were synthesized in aqueous media. The feasibility of hydrogel-based MIPs was evaluated by measuring equilibrium swelling, structural parameters (e.g. mesh size, ) and template permeation as function of pH and copolymer composition. The MIP characterization results demonstrated an increase of template permeation directly influenced by mesh size at pH equal to 5.5 at 37°C and ionic strength of 0.1M. A different behavior was shown for the characterization at pH equal to 6.0 at 37°C and ionic strength of 0.1M. A reduction was observed on the permeability coefficient for MIP with a MAA/EGDMA ratio of 17:1. It suggested the influence of MIP-ligand binding on hydrocortisone transport through the polymeric network synthesized by molecular imprinting technique based on the permeation reduction. In order to confirm this hypothesis, the binding constant (b) was estimated from the slope (15.17309 M-1) of linear regression of the Langmuir isotherm and the saturation capacity (qs = 0.09128) from its intercept (1.385). In addition, the hydrocortisone rebinding to 17:1 MIP was confirmed with the electroconductivity results of MIP 17:1, which demonstrated higher sensitivity toward hydrocortisone in comparison to non-MIP. Overall, this work reported the evaluation of the principal factors affecting the stabilization of functional monomer-template complex before and during the imprinting process, which provides essential information for the rational design of molecularly imprinted polymers. In addition, the sensitivity of imprinted gels confirmed the presence of binding sites.