Jekaterina Reut

Molecularly imprinted polymer film interfaced with Surface Acoustic Wave technology as a sensing platform for label-free protein detection.

Molecularly imprinted polymer (MIP)-based synthetic receptors integrated with Surface Acoustic Wave (SAW) sensing platform were applied for the first time for label-free protein detection. The ultrathin polymeric films with surface imprints of immunoglobulin G (IgG-MIP) were fabricated onto the multiplexed SAW chips using an electrosynthesis approach. The films were characterized by analyzing the binding kinetics recorded by SAW system. It was revealed that the capability of IgG-MIP to specifically recognize the target protein was greatly influenced by the polymer film thickness that could be easily optimized by the amount of the electrical charge consumed during the electrodeposition. The thickness-optimized IgG-MIPs demonstrated imprinting factors towards IgG in the range of 2.8-4, while their recognition efficiencies were about 4 and 10 times lower toward the interfering proteins, IgA and HSA, respectively. Additionally, IgG-MIP preserved its capability to recognize selectively the template after up to four regeneration cycles. The presented approach of the facile integration of the protein-MIP sensing layer with SAW technology allowed observing the real-time binding events of the target protein at relevant sensitivity levels and can be potentially suitable for cost effective fabrication of a biosensor for analysis of biological samples in multiplexed manner.

Molecularly Imprinted Polymer Integrated with a Surface Acoustic Wave Technique for Detection of Sulfamethizole

The synergistic effect of combining molecular imprinting and surface acoustic wave (SAW) technologies for the selective and label-free detection of sulfamethizole as a model antibiotic in aqueous environment was demonstrated. A molecularly imprinted polymer (MIP) for sulfamethizole (SMZ) selective recognition was prepared in the form of a homogeneous thin film on the sensing surfaces of SAW chip by oxidative electropolymerization of m-phenylenediamine (mPD) in the presence of SMZ, acting as a template. Special attention was paid to the rational selection of the functional monomer using computational and spectroscopic approaches. SMZ template incorporation and its subsequent release from the polymer was supported by IR microscopic measurements. Precise control of the thicknesses of the SMZ-MIP and respective nonimprinted reference films (NIP) was achieved by correlating the electrical charge dosage during electrodeposition with spectroscopic ellipsometry measurements in order to ensure accurate interpretation of label-free responses originating from the MIP modified sensor. The fabricated SMZ-MIP films were characterized in terms of their binding affinity and selectivity toward the target by analyzing the binding kinetics recorded using the SAW system. The SMZ-MIPs had SMZ binding capacity approximately more than eight times higher than the respective NIP and were able to discriminate among structurally similar molecules, i.e., sulfanilamide and sulfadimethoxine. The presented approach for the facile integration of a sulfonamide antibiotic-sensing layer with SAW technology allowed observing the real-time binding events of the target molecule at nanomolar concentration levels and could be potentially suitable for cost-effective fabrication of a multianalyte chemosensor for analysis of hazardous pollutants in an aqueous environment.

Electrochemical functionalization of gold and silicon surfaces by a maleimide group as a biosensor for immunological application.

In the present study we investigated the preparation of biofunctionalized surfaces using the direct electrochemical grafting of maleimidophenyl molecules with subsequent covalent immobilization of specific peptide to detect target antibody, thereby extending the application of the biosensing systems towards immunodiagnostics. Para-maleimidophenyl (p-MP) functional groups were electrochemically grafted on gold and silicon surfaces from solutions of the corresponding diazonium salt. A specially synthesized peptide modified with cysteine (Cys-peptide) was then immobilized on the p-MP grafted substrates by cross-linking between the maleimide groups and the sulfhydryl group of the cysteine residues. Accordingly, the Cys-peptide worked as an antigen that was able to bind specifically the target antibody (anti-GST antibody), while it was non-sensitive to a negative contrast antibody (i.e. anti-Flag β). The immobilization of both specific and non-specific antibodies on the Cys-peptide-modified surfaces was monitored by infrared spectroscopic ellipsometry, a quartz crystal microbalance integrated in flow injection analysis system and potentiometric response. The results obtained clearly demonstrated that the direct modification of a surface with maleimidophenyl provides a very simple and reliable way of preparing biofunctionalized surfaces suitable for the construction of immunological biosensors.

Influence of the Para-Substitutent of Benzene Diazonium Salts and the Solvent on the Film Growth During Electrochemical Reduction

Abstract In this work the nature of the substitution process in the electrochemical deposition of different 4-substituted benzene layers on Au and the influence of the solvent were studied. We monitored the deposition from the corresponding isolated salt diazonium salts and via the “in solution diazotation” process where the diazonium salt is created just before the deposition process by means of amine and nitrite. Infrared spectroscopic ellipsometry measurements proved the presence of the various substituted benzene groups on the surfaces. Quantitative studies using electrochemical quartz crystal microbalance technique showed a decrease in the deposited organic layer thickness and the faradaic efficiency of the electrochemical process with the size of the 4-substituent. As consequence nature of the substituent in the 4-position was the dominating factor for thickness of the deposited benzene layer and the faradaic efficiency. Additionally, there was also a strong increase in the thickness of the deposited benzene layer by changing the solvent from aqueous sulphuric acid to acetonitrile pointing to a stabilization of the radical intermediates during reduction of the diazonium salt by acetonitrile. This in turn led to faradaic efficiencies of about 80%–90% for the grafting process in acetonitrile and only 15% in the aqueous acidic solution.

A computational approach to study functional monomer-protein molecular interactions to optimize protein molecular imprinting

Molecular imprinting has become a promising approach for synthesis of polymeric materials having binding sites with a predetermined selectivity for a given analyte, the so‐called molecularly imprinted polymers (MIPs), which can be used as artificial receptors in various application fields. Realization of binding sites in a MIP involves the formation of prepolymerization complexes between a template molecule and monomers, their subsequent polymerization, and the removal of the template. It is believed that the strength of the monomer‐template interactions in the prepolymerization mixture influences directly on the quality of the binding sites in a MIP and consequently on its performance. In this study, a computational approach allowing the rational selection of an appropriate monomer for building a MIP capable of selectively rebinding macromolecular analytes has been developed. Molecular docking combined with quantum chemical calculations was used for modeling and comparing molecular interactions among a model macromolecular template, immunoglobulin G (IgG), and 1 of 3 electropolymerizable functional monomers: m‐phenylenediamine (mPD), dopamine, and 3,4‐ethylenedioxythiophene, as well as to predict the probable arrangement of multiple monomers around the protein. It was revealed that mPD was arranged more uniformly around IgG participating in multiple H‐bond interactions with its polar residues and, therefore, could be considered as more advantageous for synthesis of a MIP for IgG recognition (IgG‐MIP). These theoretical predictions were verified by the experimental results and found to be in good agreement showing higher binding affinity of the mPD‐based IgG‐MIP toward IgG as compared with the IgG‐MIPs generated from the other 2 monomers.

Hybrid molecularly imprinted polymer for amoxicillin detection

The potential adverse effects of the environmental presence of antibiotics on the ecosystem demands the development of new methods suitable for accurate detection of these micropollutants in various aquatic media. An analytical method exploiting the synergistic effect of a label-free sensing platform combined with a molecularly imprinted polymer (MIP) as robust recognition element could represent an efficient tool for the real-time monitoring of antibiotics. In this work, a hybrid organic-inorganic MIP film (AMO-MIP) selective towards amoxicillin (AMO) was synthesized and integrated with a surface plasmon resonance (SPR) sensor. The film was prepared by sol-gel using methacrylamide (MAAM) as organic functional monomer, tetraethoxysilane (TEOS) as inorganic precursor, and vinyltrimethoxysilane (VTMOS) as coupling agent. The AMO-MIP film characterized with the SPR system demonstrated about 16 times higher binding capacity to AMO than corresponding reference non-imprinted polymer (NIP). AMO-MIP-modified SPR sensors could detect AMO with LoD down to 73 pM and discriminate AMO among structurally similar molecules both in buffer and in tap water. Good reproducibility was achieved for several rebinding-regeneration cycles. The sensor could be stored at room temperature for up to 6 months without losing stability.