Protein Imprinting Research Articles

Boronate affinity controllable oriented surface imprinting of glycans in organic phase using intact glycoproteins as semi-templates

Glycoproteins are a large family of proteins which have great biological and clinical importance including molecular recognition, signal transduction, cell adhesion and immune response. Many glycoproteins are important biomarkers for disease diagnosis and as therapeutic targets. However, glycoproteins are present in very low abundance in the body and coexist alongside interfering species present at much higher abundance. So, specific recognition of glycoproteins is of great significance. Although antibodies are commonly used to solve this issue, they are associated with some apparent drawbacks, such as, difficult to prepare, poor storage stability, high cost and requiring harsh conditions for target release. Therefore, novel alternatives are highly desirable. Molecularly imprinted polymers (MIPs), which are synthesized through polymerization in the presence of a template, exhibit specific binding towards the template molecules. A series of molecular imprinting approaches have been developed to produce glycoprotein-specific MIPs. Boronic acids can covalently bind cis-diol groups of cis-diol-containing compounds like glycoproteins and glycopeptides, to form stable five- or six-membered cyclic esters at relatively high pH values. The esters dissociate reversibly when the pH of the environment is made acidic. In recent years, our group has developed a series of boronate affinity molecular imprinting techniques for specific recognition of glycoproteins, especially boronate affinity controllable-oriented surface imprinting method. Based on silane hydrolysis in alcohol phase, this method permits getting the desired thickness of the imprinting layer according to the size of the template molecules. The method allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans, and monosaccharides, enabling promising applications. Recently, we can even achieve precision imprinting of glycopeptides for the facile preparation of glycan-specific MIPs. However, the molecular imprinting of intact proteins in organic phase often results in the denature of the template proteins while imprinting approaches using glycans as the templates require relatively tedious glycan preparation procedure. Herein, we developed a new approach for imprinting in organic phase using an intact target glycoprotein as a semi-template. This method allowed for the imprinting of only the glycans on the target glycoprotein, without worrying about possible denature of the target protein in organic phase. Alkaline phosphatase (ALP), which is a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was used as the target. ALP-specific molecularly imprinted magnetic nanoparticles (MI-MNPs) were prepared using this method. By using boronic acid-functionalized MNPs as the nanocores, the target molecules were immobilized onto the nanocores via boronate affinity. Then, a thin-layer of silica was controllably formed to cover the templates to an appropriate thickness through polycondensation of tetraethyl orthosilicate (TEOS) in ethanol. After that, the templates are removed facilely by washing with an acidic solution to disrupt the boronate affinity interaction. The prepared MIPs exhibited good specificity towards ALP, and this characteristic was preserved even in a complicated real sample. The developed approach exhibited high imprinting efficiency. It overcame the limitations of previous imprinting approaches. Also, the MI-MIPs synthesized in this work were simple to prepare, stable, cost-efficient and easy to collect. Besides, the target release just needed gentle conditions. It can be a promising means to produce glycoprotein-specific MIPs for important applications such as affinity separation and disease diagnosis.

Molecular imprinting for protein recognition: Current status, challenges and applications

Molecular recognition plays a critical role in numerous living systems. Over the past several decades, considerable attempts have been made to create synthetic recognition systems with high specifity and selectivity for a certain molecue by utilizing natural receptors such as antibodies, receptors, enzymes and aptamers. Despite their high selectivity and broad range of applications, these biomolecules always suffer from some drawbacks, for example, instablity in harsh condition, high production cost. In response to these limitations, the alternative molecular imprinting technique has attracted a number of researchers sustain interests, shows a promising potential in the creation of synthetic recognition systems. Molecular imprinting, a synthetic Lock to Key technique that uses molecular templates to create selective binding sites in cross-linked polymers, has become one of the most efficient methods for preparation of selective recognition materials. To date, a wide range sizes of template including inorganic ions, drugs, nucleic acids, peptides, proteins, viruses, and whole cells have been extensively applied in the synthesis of various specific molecularly imprinted polymers. Among them, the majority of the publications and successful applications in molecular imprinting are aimed at recognition and separation of small molecules. Molecular imprinting of biomacromolecules, especially proteins, still remains great challenges. Due to its large size, environmentally instability, chemical and structural complexity, traditional imprinting methods were limited and not suitable for proteins. Since 2011, protein imprinting has acheived some significant breakthroughs in imprinting methods and applications. On one hand, some novel and special protein imprinting methods have been developed, such as nanomaterial-based imprinting, boronate affinity-based surface imprinting, epitope imprinting, solid-phase synthesis and post-imprinting modification. These newly emerging methods effectively solved some key issues in protein imprinting and were applied for synthesis of MIPs with special properties that were not well demonstrated before. In this paper, we have analyzed the merits and problems of these methods in detail. On the other hand, based on the novel protein imprinting materials with special properties, some new advanced applications, such as disease diagnosis, proteomics and bioimaging, have well been demonstrated and showed great potentials. In addition, the future challenges and opportunities in protein imprinting were also discussed. Despite the tremendous interest in protein imprinting, related research studies were still behind compared to the progress made in molecular imprinting of small molecules. Particularly, the design of universal protein imprinting materials with desirabe properties still remains great challenges. As to future development, developing facile and versatile protein imprinting methods is still of significant importance. Meanwhile, as a multidisciplinary technology, protein imprinting should develop rapidly along with the advances in polymer technology, nanotechnology, analytical chemistry, biotechnology and so on. We forsee that, in a long period of time, creating new protein imprinting strategies, developing desirable protein imprinting materials with special properties based on borrowing and integration of related technologies/strategies, and thereby enable potential important applications are still the main direction of protein imprinting.

Highly efficient fluorescent QDs sensor for specific detection of protein through double recognition of hybrid aptamer-molecular imprinted polymers

This work demonstrates a strategy of double recognition hybrid aptamer–molecular imprinted polymer/quantum dots (apt–MIP/QDs) fluorescent sensor for specific recognition of target protein. We selected vinyl modified Mn–doped ZnS QDs as signal source and support. Instead of relying on single source of weak interaction between protein and small molecule monomer, thiol terminated aptamers were used as specific functional monomers to obtain apt–MIP on the surface of QDs. The double recognition was based on aptamer and imprinted cavity to overcome the challenges of protein imprinting. The synergistic effect of the aptamer and MAA contributed for highly specific recognition ability of the apt–MIP/QDs sensor toward Cyt C. The imprinting factor and the selectivity factor values of the hybrid apt–MIP/QDs sensor were 3.17 and 8.33, respectively. This composite sensor showed an excellent linear correlation for Cyt C in the concentration range of 0.20–2.00 μM. The detection limit for Cyt C was 0.054 μM A sensitive, rapid and convenient recognition system for Cyt C in biological samples was developed. The recoveries were between 94.2% and 102.5% for urine and serum samples.

Epitope imprinting of iron binding protein of Neisseria meningitidis bacteria through multiple monomers imprinting approach.

Epitope imprinting is a promising technique for fabrication of novel diagnostic tools. In this study, an epitope imprinted methodology for recognition of target epitope sequence as well as targeted protein infused by bacterial infection in blood samples of patients suffering from brain fever is developed. Template sequence chosen is a ferric iron binding fbp A protein present in Neisseria meningitidis bacteria. To orient the imprinting template peptide sequence on gold surface of electrochemical quartz crystal microbalance (EQCM), thiol chemistry was utilized to form the self-assembled monolayer on EQCM electrode. Here, synergistic effects induced by various noncovalent interactions extended by multiple monomers (3-sulfopropyl methacrylate potassium-salt and benzyl methacrylate) were used in fabricating the imprinting polymeric matrix with additional firmness provided by N,N-methylene-bis-acrylamide as cross-linker and azo-isobutyronitrile as initiator. Extraction of template molecule was carried out with phosphate buffer solution. After extraction of epitope molecules from the polymeric film, epitope molecularly imprinted polymeric films were fabricated on EQCM electrode surface. Nonimprinted polymers were also synthesized in the similar manner without epitope molecule. Detection limit of epitope molecularly imprinted polymers and imprinting factor (epitope molecularly imprinted polymers/nonimprinted polymers) was calculated 1.39ngmL-1 and 12.27 respectively showing high binding capacity and specific recognition behavior toward template molecule. Simplicity of present method would put forward a fast, facile, cost-effective diagnostic tool for mass health care.

Combined Layer/Particle Approaches in Surface Molecular Imprinting of Proteins: Signal Enhancement and Competition.

Herein we report novel approaches to the molecular imprinting of proteins utilizing templates sizing around 10 nm and some 100 nm. The first step comprised synthesizing nanoparticles of molecularly imprinted polymers (MIP) towards bovine serum albumin (BSA) and characterizing them according to size and binding capacity. In a second step, they were utilized as templates. Quartz crystal microbalances (QCM) coated with MIP thin films based on BSA MIP nanoparticles lead to a two-fold increase in sensor responses, compared with the case of directly using the protein as the template. This also established that individual BSA molecules exhibit different “epitopes” for molecular imprinting on their outer surfaces. In light of this knowledge, a possible MIP-based biomimetic assay format was tested by exposing QCM coated with BSA MIP thin films to mixtures of BSA and imprinted and non-imprinted polymer (NIP) nanoparticles. At high protein concentrations (1000 ppm) measurements revealed aggregation behavior, i.e., BSA binding MIP NP onto the MIP surface. This increased sensor responses by more than 30% during proof of concept measurements. At lower a BSA concentration (500 ppm), thin films and particles revealed competitive behavior.

Bacteria assisted protein imprinting in sol-gel derived films.

A hierarchical imprinting strategy was used to create protein imprints in a silicate film with a high binding capacity as well as selectivity toward the imprint protein and little specificity towards other proteins. In the first part of this work, rod-shaped bacteria were used as templates to create imprints in silica films of various thicknesses to open up the silica framework and increase the surface area exposed to solution. In the second part, the protein (e.g., cytochrome c (CYC) or green fluorescent protein (GFP)) was covalently attached to the surface of Bacillus subtilis and this protein-bacteria complex served as the imprint moiety. Atomic force microscopy and scanning electron microscopy were used to image the micron-size rod-shaped bacteria imprints formed on the silica surface. Fluorescence microscopy, which was used to follow the fabrication process with GFP as the representative protein, clearly demonstrated protein imprinting, protein removal and protein rebinding as well as protein specificity. Visible absorption spectroscopy using CYC as the imprint protein demonstrated relatively fast uptake kinetics and also good specificity against other proteins including bovine serum albumin (BSA), horseradish peroxidase (HRP), glucose oxidase (GOD), and lysozyme (LYZ). Collectively this work demonstrates a new surface bio-imprinting approach that generates recognition sites for proteins and provides a viable means to increase the binding capacity of such imprinted thin films.

Selective binding of matrix metalloproteases MMP-9 and MMP-12 to inhibitor-assisted thermolysin-imprinted beads.

Protein-imprinted polymers have been synthesized to recognize and specifically bind selected proteins. However, protein imprinting requires substantial amounts of pure protein to efficiently obtain imprinted polymers for large scale applications, e.g. protein purification by affinity chromatography. In the absence of large quantities of a pure protein of interest, an alternative strategy was developed. In this case study, neutral metalloprotease thermolysin was selected as a commercially available surrogate for imprinting polymer beads. Phosphoramidon-assisted thermolysin-imprinted beads were synthesized. During rebinding experiments, it was shown that these beads specifically bind to thermolysin. In addition, it was shown that these beads also bind in CHO cell culture supernatant to the matrix metalloprotease-9 and -12 (MMP-9, -12). Therefore, these beads can be applied as a selective sorbent for the rare metalloproteases MMP-9 and MMP-12 to remove these proteases from CHO cell culture supernatants. The high selectivity of thermolysin-imprinted beads can be extended to other proteases of the family of metalloproteases, and is not limited to thermolysin. This innovative approach is suitable to address the challenges in the field of protease purification and isolation from biotechnologically relevant media.

Dispersive solid-phase imprinting of proteins for the production of plastic antibodies

We describe a novel dispersive solid-phase imprinting technique for the production of nano-sized molecularly imprinted polymers (nanoMIPs) as plastic antibodies. The template was immobilized on in-house synthesized magnetic microspheres instead of conventional glass beads. As a result, high-affinity and template-free MIPs were produced in higher yields.

Composite imprinted macroporous hydrogels for haemoglobin purification from cell homogenate

Purification of haemoglobin (Hb) has been studied for many years due to its ability to act as an oxygen carrier and its possible use in urgent clinical treatment. In this study, different types of chromatography columns were developed for Hb purification. Two of them showed satisfactory results as affinity chromatography columns and were thus studied more extensively. The affinity adsorbents were prepared by molecular imprinting techniques. In the first case, Pickering emulsion polymerization was used to prepare affinity adsorbents based on molecular imprinting technology. The imprinted particles were immobilized via covalent bonds on the surface of cryogel, a macroporous hydrogel produced by free radical polymerization under sub-zero temperature. In the second case, the affinity sites for Hb were formed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The dynamic binding capacity of the composite cryogel with the immobilized particles and the directly imprinted acrylamide cryogel was found to be 5.2 mg/g and 3.6 mg/g, respectively. The affinity columns showed high selectivity towards Hb in spite of the presence of serum albumin as well as other interfering substances in non-clarified cell homogenates. The maximum capacity in batch mode, the fluid flow and other physical and chemical properties of these columns were investigated.

Epitope recognition of peptide-imprinted polymers for Regenerating protein 1 (REG1)

Abstract Molecularly imprinted polymers (MIPs) were developed to replace natural antibodies with a cost-effective and durable synthetic material. Molecular imprinting of proteins conventionally utilizes the whole protein as the template, which is complex (as many different epitopes may be imprinted) and expensive. In this work, seven peptides (13–18 amino acids) were synthesized and used as templates for the imprinting and recognition of Regenerating Protein 1 (REG1). REG1 is involved in the proliferation and differentiation of diverse cell types, and was recently described as a urinary biomarker for pancreatic ductal adenocarcinoma (PDAC). Peptide-imprinted poly(ethylene- co -vinyl alcohol)s (PIPs), containing four different mole fractions of ethylene were cast on screen-printed electrodes to find the optimum composition for both the sensing and the extraction of REG1 in an E. coli culture medium. Peptides with fewer than 16 amino acids and two or three aromatic and hydrophobic groups have a higher affinity for MIPs of poly(ethylene- co -vinyl alcohol) (EVAL) with 27 mol% of ethylene, while those with four aromatic and hydrophobic groups have a higher affinity for MIPs with EVALs that contain 32 mol% of ethylene. The peptide / EVAL combination that maximized both imprinting effectiveness and response to REG1B was the sequence NEDRETWVDADLY imprinted into 32 mol% EVAL. This EVAL composition and template peptide were then modified by incorporation of magnetic nanoparticles, thus extending applications for PIPs to include extraction of REG1 protein from E. coli culture medium.

Quantum dots based imprinting fluorescent nanosensor for the selective and sensitive detection of phycocyanin: A general imprinting strategy toward proteins

Molecular imprinting as a versatile technology has gained popularity in various fields for diverse species; however, protein imprinting still remains challenging due to some inherent problems but is potentially rewarding work. Herein, we reported a facile strategy to imprint phycocyanin (PC), as a template, using quantum dots (QDs) co-capped by thioglycollic acid and glutathione directly as functional monomer via dopamine self-polymerization, which effectively simplified the imprinting process and afforded easy accessibility to recognition sites. The resulting QDs based imprinting nanosensor showed significant fluorescence decrease of QDs within less than 16s upon binding PC owing to the ultrathin imprinting layer (ca. 3nm), leading to a high detectability down to 0.075μM. An excellent linearity was found within the wide range of 0.8–8.0μM (R2=0.9935). Good recognition selectivity toward PC was displayed over other possible competing molecules, with a high imprinting factor of 7.3. Seawater and lake water samples spiked with PC were also analyzed, presenting satisfactory recoveries ranging from 90.8–110.1% with precisions below 4.6%, indicating the practicality of the imprinting nanosensor for accurately rapid nanoscale monitoring of algae blooms. By using another protein, bovine haemoglobin (BHb), as a convincing support model, the present study can provide a simple and general imprinting strategy toward various concerned proteins through rationally utilizing diverse functionalization and interactions.

Self-cleaned electrochemical protein imprinting biosensor basing on a thermo-responsive memory hydrogel

Herein, the self-cleaned electrochemical protein imprinting biosensor basing on a thermo-responsive memory hydrogel was constructed on a glassy carbon electrode (GCE) with a free radical polymerization method. Combining the advantages of thermo-responsive molecular imprinted polymers and electrochemistry, the resulted biosensor presents a novel self-cleaned ability for bovine serum albumin (BSA) in aqueous media. As a temperature controlled gate, the hydrogel film undergoes the adsorption and desorption of BSA basing on a reversible structure change with the external temperature stimuli. In particular, these processes have been revealed by the response of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of electroactive [Fe(CN)6]3-/4-. The results have been supported by the evidences of scanning electron microscopy (SEM) and contact angles measurements. Under the optimal conditions, a wide detection range from 0.02μmolL−1 to 10μmolL−1 with a detection limit of 0.012 μmolL−1 (S/N = 3) was obtained for BSA. This proposed BSA sensor also possesses high selectivity, excellent stability, acceptable recovery and good reproducibility in its practical applications.

Protein-Imprinted Polymers: The Shape of Things to Come?

The potential to develop materials with antibody-like molecular recognition properties has helped sustain interest in protein-imprinted polymers over the past several decades. Unfortunately, despite persistent research, the field of noncovalent protein imprinting has seen limited success in terms of achieving materials with high selectivity and high affinity. In this Perspective, important yet sometimes overlooked aspects of the imprinting and binding processes are reviewed to help understand why there has been limited success. In particular, the imprinting and binding processes are viewed through the scope of free radical polymerization and hydrogel swelling theories to underscore the complexity of the synthesis and behavior of protein-imprinted polymers. Additionally, we review the metrics of success commonly used in protein imprinting literature (i.e., adsorption capacity, imprinting factor, and selectivity factor) and consider the relevance of each to the characterization of an imprinted polymer's recognition characteristics. Throughout, common shortcomings are highlighted, and experiments that could help verify or disprove the efficacy of noncovalent protein imprinting are discussed.

Immobilization of BSA on ionic liquid functionalized magnetic Fe3O4 nanoparticles for use in surface imprinting strategy

Combining template immobilization with surface imprinting technology is an effective strategy to overcome the difficulties associated with macromolecular template removal and to achieve high specific recognition ability. In this work, ionic liquid functionalized Fe3O4 nanoparticles were prepared via a simple two-step modification process and were used as substrate to immobilize bovine serum albumin (BSA). The zeta potential study revealed immobilization of BSA on the nanoparticles through multiple interactions, and the immobilization capacity was about nine times higher compared with that of bare Fe3O4. Subsequently, dopamine was utilized as functional monomer to prepare BSA surface imprinted nanoparticles. Fourier transform infrared spectroscopy, thermo-gravimetric analysis and transmission electron microscopy verified the successful preparation of BSA imprinted nanoparticles with core-shell structure. The influence of imprinted layer thickness on recognition ability of imprinted nanoparticles was investigated, and the results suggested that 20nm was an optimum thickness to achieve the best recognition ability. The adsorption isotherm studies showed that the imprinted nanoparticles had a significantly higher adsorption capacity and stronger binding affinity than the non-imprinted ones. Furthermore, the selective as well as the competitive adsorption studies revealed higher selectivity and recognition ability of the imprinted nanoparticles for BSA. Therefore, the proposed strategy is an effective way to obtain protein imprinted polymers with high adsorption capacity and good recognition ability, thus would be beneficial for the further development and application of protein imprinting technology.

The selectivity of protein-imprinted gels and its relation to protein properties: A computer simulation study.

Polymer-based protein recognition systems have enormous potential within clinical and diagnostic fields due to their reusability, biocompatibility, ease of manufacturing, and potential specificity. Imprinted polymer matrices have been extensively studied and applied as a simple technique for creating artificial polymer-based recognition gels for a target molecule. Although this technique has been proven effective when targeting small molecules (such as drugs), imprinting of proteins have so far resulted in materials with limited selectivity due to the large molecular size of the protein and aqueous environment. Using coarse-grained molecular simulation, we investigate the relation between protein makeup, polymer properties, and the selectivity of imprinted gels. Nonspecific binding that results in poor selectivity is shown to be strongly dependent on surface chemistry of the template and competitor proteins as well as on polymer chemistry. Residence time distributions of proteins diffusing within the gels provide a transparent picture of the relation between polymer constitution, protein properties, and the nonspecific interactions with the imprinted gel. The pronounced effect of protein surface chemistry on imprinted gel specificity is demonstrated.

One-step post-imprint modification achieve dual-function of glycoprotein fluorescent sensor by "Click Chemistry"

A novel molecular imprinting fluorescent sensor synthesized avoiding complicated design and synthesis of a fluorescent functional monomer, and without the extra cost of additional fluorescence materials (quantum dots, carbon dots and fluorescein), only by a click reaction making the common protein imprinting polymers upgraded to a fluorescent sensing material which can be applied to the fast detection of glycoproteins. In this study, a common protein imprinted polymer (protein-IP) which containing a disulfide bond in the imprinted cavities was first synthesized, then the disulfide linkage was cleaved by reduction, the exposed thiol groups were used for the click reaction to introduce 4-vinyl phenylboronic acid to improve the imprinted recognition for glycoproteins and convert recognition behavior into fluorescent signal simultaneously in one-step site-specific post-imprinting modifications (PIMs). Good linear relationship was obtained in the range of 0.1–10mgmL−1 by fluorescence assay, comparison of the adsorption capacity of the MIPs before and after modification proved that the method has no negative impact on the imprinting effect and exhibit good imprinting effect for glycoproteins of different molecular size and weight.

Surface-initiated synthesis of bulk-imprinted magnetic polymers for protein recognition.

Bulk imprinting of proteins was used combined with a grafting approach onto maghemite nanoparticles to develop a faster and simpler polymerization method for the synthesis of magnetic protein imprinted polymers with very high adsorption capacities and very strong affinity constants.

Guide to the Preparation of Molecularly Imprinted Polymer Nanoparticles for Protein Recognition by Solid-Phase Synthesis.

Molecularly imprinted polymers (MIPs) are synthetic antibody mimics possessing specific cavities designed for a target molecule. Nowadays, molecular imprinting of proteins still remains a challenge as the generation of selective imprinted cavities is extremely difficult, due to their flexible structure and the presence of a multitude of functional sites. To overcome this difficulty, we propose a solid-phase synthesis strategy to prepare MIPs specific for any protein that can be immobilized in an oriented way on a solid support. Trypsin and kallikrein were used as model proteins. The solid-phase support consists of glass beads functionalized with two affinity ligands of the enzymes, the competitive inhibitor p-aminobenzamidine to orient the enzymes via their active site, or a Cu2+chelate to orient via the surface histidine residues of the enzyme. Thermoresponsive molecularly imprinted polymer nanoparticles (MIP-NPs) are then synthesized around the immobilized enzyme. The MIP-NPs are released by a simple temperature change, resulting in protein-free polymers endowed with improved binding site homogeneity since all binding sites have the same orientation. The MIP-NPs exhibit apparent dissociation constants between 0.02 and 2nM toward their target proteins, which is comparable to those of natural antibodies. Moreover, these water-compatible polymers, targeting different domains of the enzyme, can also function as protective agents (armor), hence preventing the target proteins from denaturation by heat or pH.

Smart naturally plastic antibody based on poly(α-cyclodextrin) polymer for β-amyloid-42 soluble oligomer detection

This work introduces new developments into the conventional protocol of protein imprinting. It uses as principle that when the polymeric matrix is made from native building blocks in nature (amino acids, sugars, lipids or nucleic acids), conformational changes of the template protein during the polymer synthesis may be avoided or limited. Thus, the polymeric film around the protein is build-up with biomolecules that co-exist with the protein in nature. This approach is named herein as BIOPLAST, in an analogy to a Plastic material made from biological building blocks.This concept was tested herein by employing a sugar monomer (α-Cyclodextrin, α-Cd) to target a peptide biomarker (β-Amyloid-42, Aβ-42) that is relevant in Alzheimer disease. The BIOPLAST-based biosensor consisted of a screen-printed electrode (SPE) having an Au-working electrode area coated with a polyaniline (PANI) film and subsequently modified by the plastic antibody (MCd/PANI/Au-SPE). The plastic antibody was assembled by electropolymerizing αCd in the presence of Aβ-42 and the peptide molecules entrapped in the polymer film were removed by acidic action. Analysis by Atomic Force Microscopy, and Fourier Transformed Infrared and Raman spectroscopies confirmed the surface modification.The ability of the BIOPLAST film to rebind Aβ-42 was followed by electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), and compared to a control film without plastic antibody (NCd/PANI/Au-SPE). The MCd/PANI/Au-SPE devices displayed linear responses and limit of detection to Aβ-42 in EIS and SWV assays, down to 0.25ng/mL and 0.20ng/mL respectively. The dissociation constant between the BIOPLAST and Aβ-42 were 0.2±0.5ng/mL (1.8±0.5 for the control material). MCd/PANI/Au-SPE materials assembled on screen-printed electrodes showed great promising features, regarding its possible application in point-of-care.

Polyethyleneimine assisted-two-step polymerization to develop surface imprinted cryogels for lysozyme purification

Surface imprinting strategy is one of the promising approaches to synthesize plastic antibodies while overcoming the problems in the protein imprinting research. In this study, we focused our attentions on developing two-step polymerization to imprint on the bare surface employing polyethyleneimine (PEI) assisted-coordination of template molecules, lysozyme. For this aim, we firstly synthesized poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA) cryogels as a bare structure. Then, we immobilized PEI onto the cryogels through the addition reaction between GMA and PEI molecules. After that, we determined the amount of free amine (NH2) groups of PEI molecules, subsequently immobilized methacrylate functionalities onto the half of them and another half was used to chelate Cu(II) ions as a mediator between template, lysozyme and PEI groups. After the characterization of the materials developed by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and the micro-computed tomography (μCT), we optimized the lysozyme adsorption conditions from aqueous solution. Before performing lysozyme purification from chicken egg white, we evaluated the effects of pH, interaction time, the initial lysozyme concentration, temperature and ionic strength on the lysozyme adsorption. Moreover, the selectivity of surface imprinted cryogels was examined against cytochrome c and bovine serum albumin (BSA) as the competitors. Finally, the mathematical modeling, which was applied to describe the adsorption process, showed that the experimental data is very well-fitted to the Langmuir adsorption isotherm.