100% coverage 60% 0.05 mM MFI / a.u. c /mM 0.05 mM

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Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) was accomplished by a hybrid nanofilm composed of a propidium-terminated self-assembled monolayer (Prop-SAM) which binds AChE via its peripheral anionic site (PAS) and an ultra-thin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate-modified derivative of 3,4-propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop-SAM nanofilm covered electrode was detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine was dependent on the AChE concentration from ca. 0.04 µM to 0.4 µM. An imprinting factor of 9.9 was obtained for the hybrid MIP, which is among the best values reported for protein imprinting.The dissociation constant characterizing the strength of the MIP-AChE binding was 4.2 ×10 −7 M indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer was the effective suppression of the cross-reactivity towards competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by (i) the "shapespecific" MIP filter, (ii) the propidium-SAM, (iii) signal generation only by the AChE bound to the nanofilm show promise for assessing AChE activity levels in CSF.

Here we introduce microelectrospotting as a new approach for preparation of protein-selective molecularly imprinted polymer microarrays on bare gold SPR imaging chips. During electrospotting both the gold chip and the spotting tip are electrically connected to a potentiostat as working and counter electrodes, respectively. The spotting pin encloses the monomer-template protein cocktail that upon contacting the gold surface is in-situ electropolymerized resulting in surface confined polymer spots of ca. 500 µm diameter. By repeating this procedure at preprogrammed locations for various composition monomer-template mixtures microarrays of nanometer-thin surface-imprinted films are generated in a controlled manner. We show that the removal and rebinding kinetics of the template and various potential interferents to such microarrays can be monitored in real-time and multiplexed manner by SPR imaging. The proof of principle for microelectrospotting of electrically insulating surface-imprinted films is made by using scopoletin as monomer and ferritin as protein template. It is shown that microelectrospotting in combination with SPR imaging can offer a versatile platform for label-free and enhanced throughput optimization of the molecularly imprinted polymers for protein recognition and for their analytical application.

A novel strategy to prepare a surface confined molecularly imprinted polymer (MIP) film directly on a transducer surface for protein sensing is achieved by combining interaction with a natural binding receptor and binding to a fully synthetic MIP. A thiolated oligoethyleneglycol (OEG)/mannose conjugate is first self‐assembled on the transducer surface. Then the carbohydrate binding protein, concanavalin A (ConA), is “vectorially” immobilized as a submonolayer on the underlying mannose modified surface. Afterwards, an ultrathin polyscopoletin film with the thickness comparable to that of the protein is electrodeposited on the top. This architecture ensures that the target is confined to the film surface. The resulting functional material shows an approximately 20‐fold higher affinity than that obtained from the mannose self‐assembled monolayer. This result shows a synergism between multivalent binding of the natural sugar ligand and the non‐covalent interactions of the target within the MIP cavities. Recognition capability of the film is characterized by a real‐time measurement using quartz crystal microbalance. In comparison to the non‐imprinted film, the imprinted film reveals 8.6 times higher binding capacity towards ConA. High discrimination towards the target protein's homologues shows size and shape specificity of the imprint.

Nanoparticles modified with either 6-amino-1-hydroxy-2,1-benzoxaborolane (3-aminobenzoboroxole) or 3-aminophenylboronic acid were prepared by nucleophilic substitution of a styrene-co-DVB-co-vinylbenzylchloride latex (25 nm). Isothermal titration calorimetry (ITC) was used as a label-free detection method for the analysis of the binding between monosaccharides and these two differently derivatized nanoparticle systems at pH 7.4. Because ITC reveals, thermodynamical parameters such as the changes in enthalpy ΔH, free energy ΔG, and entropy ΔS, possible explanations for the higher binding constants can be derived in terms of entropy and enthalpy changes. In case of the modified nanoparticles, the free energy of binding is dominated by the entropy term. This shows that interfacial effects, besides the intrinsic affinity, lead to a higher binding constant compared with the free ligand. The highest binding constant was found for fructose binding to the benzoboroxole modified nanoparticles: Its value of 1150 M(-1) is twice as high as for the free benzoboroxole and five times as high as with phenylboronic acid or 3-aminophenylboronic acid. In contrast to the binding of fructose to free boronic acids, which is an enthalpically driven process, the binding of fructose to the modified nanoparticles is dominated by the positive entropy term.

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