Vectorially Imprinted Hybrid Nanofilm for Acetylcholinesterase Recognition

Abstract

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.

Electrosynthesized molecularly imprinted polymers for protein recognition

“…Similar methodology was implemented by Bosserdt et al to induce oriented adsorption of cytochrome c by coating the electrode with an anionic self-assembled monolayer (SAM) which attracted the positively charged lysine residues of the protein neighboring its heme group [6]. Recently the enzyme acetylcholinesterase (AChE) has been "vectorially" bound via its peripheral anionic site to a propidium terminated SAM prior the deposition of an ultrathin polymer film of a ProDOT derivative [52] (Figure 3C). …”

“…The precise control over electrosynthesis enables the fine tuning of the polymer layer thickness, which is particularly important for the surface imprinting of proteins. Electrosynthesis can be used straightforwardly to create several nanometer thick polymer films [52,53] and with the aid of sacrificial materials, micro and nanostructures with surface confined binding sites [5,[54][55][56]. It must be noted that beside direct electrooxidation of suitable monomers the versatility of electrochemical synthesis enables surface confined polymer deposition also by electrochemically generating the "active" initiator [57] or electrochemically changing the local pH in the close vicinity of the electrode [58].…”

Electrosynthesized molecularly imprinted polyscopoletin nanofilms for human serum albumin detection

“…The thickness of the polymer films was determined by mechanically removing them from the gold over a rectangular area by using AFM in contact mode then remapping the surface in tapping mode [19]. Representative depth profiles taken across the indented area reveal a polymer film thickness close to 3 nm for the non-imprinted and 12-14 nm for the ferritinimprinted film ( Figure 5).…”

“…[15] While high affinity MIPs were reported for a number of targets [16,17], the selectivity of protein-MIPs is often enhanced by incorporating in the MIPs compounds known to interact with the target, e.g. substrates or inhibitors of an enzyme target [18,19], aptamers [20] and various nanomaterials [16,21]. Alternatively, rational design of monomers tuned for the specific target and semi-covalent imprinting was also shown to give MIPs with high affinity towards protein templates [22].…”

Enzymes as Tools in MIP-Sensors

“…Some ten percent of MIP papers describe artificial receptors for proteins [7,[10][11][12][13], including enzymes [13][14][15][16][17][18][19][20][21]. Molecularly imprinted polymers have been mostly developed for binding of targets, thus mimicking the function of antibodies.…”