Molecularly imprinted polymers (MIPs) are synthetic receptors that specifically recognise target molecules. MIPs have found applications for example as antibody mimics in immunoassays or as recognition elements in biosensors [1]. They are produced by polymerising interacting and cross-linking monomers in the presence of the target molecule that acts as a molecular template. Although the technique has been used preferentially with small targets of the size of an amino acid or a small peptide, proteins can also serve as templates. However, due to their size and the possibility of denaturation under the conditions normally used in molecular imprinting, specifically adapted protocols and materials have to be developed.

A possible approach is the synthesis of the MIP in the form of soluble, nanometer-sized microgels. In that case, statistically, the majority of the binding sites created in the particle will be accessible from the solution. We describe here a new strategy for the synthesis of such materials by generating a three-dimensional image of the region at and around the substrate binding site of a model protein, the enzyme trypsin. The polymerisation is conducted with a specific monomer carrying the trypsin-inhibitor benzamidine. The growing polymer chain is thus confined to the substrate binding site of the enzyme, while the imprinted network is obtained by adding hydrophilic co-monomers. All imprinted polymers exhibit a much higher affinity towards the enzyme than the chemically identical non-imprinted control polymers (NIPs). We were able to demonstrate competitive inhibition of trypsin by the MIP with an inhibition constant of K_i= 79 nM, almost three orders of magnitude was stronger than that of free 4-aminobenzamidine (K_i=18.4 µM) [2].

Another approach is to create imprints of the protein in a thin film deposited on a surface. Here we describe the preparation of a MIP for cytochrome c. We use, for the first time, chemical force spectroscopy for the direct detection of molecularly imprinted binding sites. In order to obtain accessible surface binding sites, we chemically immobilised the template protein onto a flat surface before imprinting. The MIP was then cast on that surface and was bonded onto a glass support. After removal of the template protein, the MIP was tested for selective binding of cytochrome c by fluorescence measurements with FITC-labelled cytochrome c. Other proteins and a non-imprinted polymer surface were used as controls. At the nanometric scale, AFM and molecular force spectroscopy were employed to directly reveal molecularly imprinted binding sites. For the latter, AFM tips were modified with cyt c and used to evaluate the affinity of the MIP for the target protein. Control experiments with other proteins were performed to confirm the existence of specific binding sites [3].

As an extension of this approach, the MIPs can be synthesised in the form of surface-bound nanofilaments with a high aspect ratio. This results in an increase in surface area and thus the number of accessible binding sites, which is particularly important for the use of the MIP in a biosensor. This can be achieved by nanomoulding the polymer on a porous starting material like anodised alumina. The target protein was first coupled to the alumina, and the polymer was then cast on that surface. After chemical dissolution of the alumina, polymer nanofilaments were obtained with surface molecular imprints. The number and size of the nanostructures could be fine-tuned by adjusting the morphology of the initial template surface. The molecular imprinting effect was demonstrated using the FITC-labelled protein. By patterning the MIP surface using projection photolithography, arrays of nanostructured dots were obtained that can be used in biochips [4].

In order to increase the sensitivity of detection with MIP-based sensing systems, we have explored the possibility of using nanocomposites of MIPs and metal nanostructures. We have found that Raman spectroscopy and surface-enhanced Raman scattering (SERS) measurements were possible on single MIP nanostructures [5].

[1] K. Haupt (2003) Imprinted polymers - Tailor-made mimics of antibodies and receptors. Chem. Commun. 2, 171-178.

[2] A. Cutivet, C. Schembri, J. Kovensky, K. Haupt (2009) Molecularly imprinted microgels as enzyme inhibitors. J. Am. Chem. Soc. (in press, doi: 10.1021/ja901600e).

[3] K. El Kirat, M. Bartkowski, K. Haupt (2009) Probing the recognition specificity of a protein molecularly imprinted polymer using force spectroscopy. Biosens. Bioelectron. 24, 2618–2624.

[4] A. V. Linares, F. Vandevelde, J. Pantigny, A. Falcimaigne-Cordin, K. Haupt (2009) Polymer films composed of surface-bound nanofilaments with a high aspect ratio molecularly imprinted with small molecules and proteins. Adv. Funct. Mater.19, 1-5.

[5] M. Bompart, L. A. Gheber, Y. De Wilde, K. Haupt (2009) Direct detection of analyte binding to single molecularly imprinted polymer particles by confocal Raman spectroscopy.  Biosens. Bioelectron.(in press, doi:10.1016/j.bios.2009.01.020).

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1. Micro and nanofabrication of molecularly imprinted polymer synthetic receptors for sensing applications
2. Biochips and Acoustic Biosensor Arrays Based on Molecularly Imprinted Polymers
3. Piezoelectric Microgravimetry for Determination of Some Biologically Important Compounds