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Electrochemical synthesis and signal generation dominate among the almost 1200 papers published annually on protein imprinted polymers. Such polymers can be easily prepared directly on the electrode surface and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture the molecularly imprinted polymer (MIP) layer represents only one "separation plate", thus the selectivity does not reach the values of "bulk" measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an "overall apparent" signal which may include non-specific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.

Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration.

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.

Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either “evolution in the test tube” of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the “biological” degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.

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.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of −184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).