Electrosynthesized molecularly imprinted polymers for protein recognition

Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of non-specific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing

MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing

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

Vectorially Imprinted Hybrid Nanofilm for Acetylcholinesterase Recognition

Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is 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 ultrathin 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 is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10−6 to 0.4 × 10−6m. An imprinting factor of 9.9 is 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 is 4.2 × 10−7m indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer is the effective suppression of the cross-reactivity toward competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by i) the “shape-specific” MIP filter, ii) the propidium-SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid

Novel Moessbauer experiment in a rotating system and the extra-energy shift between emission and absorption lines

We present the results of a novel Mossbauer experiment in a rotating system, implemented recently in Istanbul University, which yields the coefficient k=0.69+/-0.02 within the frame of the expression for the relative energy shift between emission and absorption lines dE/E=ku2/c2. This result turned out to be in a quantitative agreement with an experiment achieved earlier on the subject matter (A.L. Kholmetskii et al. 2009 Phys. Scr. 79 065007), and once again strongly pointed to the inequality k>0.5, revealed originally in (A.L. Kholmetskii et al. 2008 Phys. Scr. 77, 035302 (2008)) via the re-analysis of Kundig experiment (W. Kundig. Phys. Rev. 129, 2371 (1963)). A possible explanation of the deviation of the coefficient k from the relativistic prediction k=0.5 is discussed.Comment: 21 pages, 8 figures, 3 table

Temperament Systems Influence Emotion Induction but not Makam Recognition Performance in Turkish Makam Music

We tested how induced emotions and Turkish makam recognition are influenced by participation in an ear training classes, and if either is influenced by the temperament system employed. The ear training class was attended by 19 music students and was based on the Hicaz makam presented as a between-subjects factor in either unfamiliar Turkish Original Temperament (OT, pitches unequally divided into 24 intervals) or familiar Western Equal Temperament (ET, pitches equally divided into 12 intervals). Before the and after the class, participants listened to 20 music excerpts from five different Turkish makams (in both OT and ET versions). Emotion-induction was assessed via GEMS-25, and participants were also asked to identify the makam that was present in the excerpt. The unfamiliar original temperament was experienced as less vital and more uneasy before the ear training class, and recognition of the Hicaz makam increased after ear training classes (independent of the temperament system employed). Results suggest that unfamiliar temperament systems are experienced as less vital and more uneasy. Furthermore, being exposed to this temperament system for just one hour does not seem to be enough to change participants’ mental representations of it or their emotional responses to it

Molecularly imprinted polymer-based electrochemical sensors for biopolymers

Electrochemical synthesis and signal generation dominate among the almost 1200 articles 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 nonspecific 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

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C

The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines