Protein Imprinting Research Articles

Zwitterionic polymer chain-assisted lysozyme imprinted core-shell carbon microspheres with enhanced recognition and selectivity

Constructing imprinting materials with high recognition and selectivity for protein is an always challenge in protein imprinting technology (PIT). In this work, upon the participating of a zwitterionic polymer chain (Poly (1-vinyl-3-sulfopropylimidazolium), PVSP), a lysozyme imprinted core-shell carbon microsphere (CFC-PVSP@MIPs) was prepared by combining template immobilization method and surface imprinting technology. The carboxyl-functionalized carbon microspheres as substrate provided the CFC-PVSP@MIPs satisfactory adsorption capacity (68.1 mg g−1), while the dopamine as a functional monomer and crosslinker allowed the imprinted microspheres to have a thin imprinted shell, thus endowing them a fast adsorption equilibrium rate (120 min). In addition, PVSP could be tightly bound to the imprinted layer through non-covalent interaction, which not only simplified the preparation process of CFC-PVSP@MIPs, but also reduced the non-specific adsorption of imprinted material on proteins. Therefore, the resulting CFC-PVSP@MIPs exhibited a more superior recognition ability towards lysozyme with imprinting factor value of 3.10, compared with the PVSP-free imprinted microsphere (imprinting factor value 1.93). Furthermore, benefiting from the characteristics of zwitterionic groups, CFC-PVSP@MIPs also revealed stronger selectivity in competitive adsorption studies of binary protein mixture samples. Consequently, the proposed strategy would be a promising and convenient way to obtain protein imprinted material with high recognition ability, thus would be conducive to further development and application of PIT.

Identification of biomarkers for early diagnosis of Parkinson's disease by multi-omics joint analysis.

ObjectiveThe purpose of this study is to identify the biomarkers for early diagnosis of Parkinson's disease (PD) by multi-omics joint analysis, so as to identify the biomarkers for early diagnosis of PD, and to help clinicians make early diagnosis and treatment.MethodsIn this study, mice are taken as the study subjects. The model of PD mice is established, and then lymphocyte, striatum, substantia nigra protein and proteolysis are extracted. After that, the experiments of protein imprinting and 418O labeling are carried out. Mass Spectrometry (MS) analysis technology is mainly used to study proteomics and to analyze the quantitative and qualitative situation of differential proteins in striatum, substantia nigra protein and lymphocyte. By this method, biomarkers for early diagnosis of PD are analyzed and identified.ResultsThe biomarkers of Parkinson's early onset are related to the same quantitative differential expression of lymphocyte, striatum, substantia nigra protein, lymphocyte and substantia nigra.ConclusionThis experimental method can analyze and identify the biomarkers of early diagnosis of PD, help to explore the pathophysiology and pathogenesis of PD, effectively help clinicians make timely diagnosis in advance, and improve the prevention and treatment effect of the disease.

3D ZnIn2S4 nanosheets decorated ZnCdS dodecahedral cages as multifunctional signal amplification matrix combined with electroactive/photoactive materials for dual mode electrochemical – photoelectrochemical detection of bovine hemoglobin

The construction of dual mode sensor has gained tremendous attention due to its high accuracy and sensitivity compared with a single-response system. Herein, a novel dual mode sensing platform based on a 3-dimensional (3D) ZnCdS/ZnIn2S4 double-shelled dodecahedral cages (DSDCs) is fabricated as the electrochemical (EC) - photoelectrochemical (PEC) multifunctional signal amplification matrix for the highly selective detection of bovine hemoglobin (BHb). To achieve simple and fast detection of BHb, Au@Cu2O and SnO2/SnS2 are acted as EC - PEC signal indicators, respectively. More interestingly, the electroactive Au@Cu2O and photoactive SnO2/SnS2 are assembled on the 3D ZnCdS/ZnIn2S4 DSDCs, which could effectively increase the electron transfer process, consequently amplifying the readout of the dual mode responses. Besides, polydopamine (PDA) is used as a monomer for protein imprinting. Under the optimized conditions, the dual mode sensor exhibits a wide linear concentration range from 10−19 mg mL−1 to 10−1 mg mL−1 with a low detection limit 6.5 × 10−20 mg mL−1. Furthermore, the excellent selectivity, stability and acceptable reproducibility of the designed sensor will offer an alternation for the detection of other biomacromolecules in clinic diagnosis field.

Preparation of BSA surface imprinted manganese dioxide-loaded tubular carbon fibers with excellent specific rebinding to target protein

Along with the wide development of protein imprinted polymers, the researchers still face many challenges, such as difficult template elution, slow adsorption rate and low adsorption capacity. In order to promote the progress of protein separation and purification, the surface imprinted manganese dioxide-loaded tubular carbon fibers (FTCFs@MnO2@MIPs) are prepared in this work. FTCFs@MnO2@MIPs are based on tubular carbon fibers (TCFs) coated with flaky MnO2. Dopamine (DA) and bovine serum albumin (BSA) are utilized as functional monomers and templates. The MnO2 nanosheets are grown and loaded on the surface of carboxyl-modified tubular carbon fibers (CMTCFs) to form a MnO2 shell, which provides more imprinting sites for protein imprinting. Meanwhile, this shell enhances the interaction between the imprinting sites and BSA. The content of MnO2 loaded on the surface of CMTCFs is 9.42%. The obtained materials are systematically characterized and the adsorption performances of FTCFs@MnO2@MIPs for BSA are investigated. The adsorption process of FTCFs@MnO2@MIPs exhibits significant self-driven characteristics. The adsorption capacity reaches 816.44 mg/g in 60 min and the imprinting factor (IF) is 3.31. FTCFs@MnO2@MIPs can selectively separate BSA from the mixed proteins and fetal bovine serum. Excellent reusability and practical application ability make MnO2-loaded tubular carbon fibers (FTCFs@MnO2) become a promising carrier in the field of protein imprinting.

Epitope Imprinting of Outer Membrane Protein of Neisseria Meningitidis

Epitope Imprinting of Outer Membrane Protein of Neisseria Meningitidis

Strategies for Molecular Imprinting and the Evolution of MIP Nanoparticles as Plastic Antibodies-Synthesis and Applications.

Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in solving many of the practical problems traditionally associated with molecularly imprinted polymers (MIPs), such as difficulties with imprinting of proteins, poor compatibility with aqueous environments, template leakage, and the presence of heterogeneous populations of binding sites in the polymers that contribute to high levels of non-specific binding. This success is closely related to the technology-driven shift in MIP research from traditional bulk polymer formats into the nanomaterial domain. The aim of this article is to throw light on recent developments in this field and to present a critical discussion of the current state of molecular imprinting and its potential in real world applications.

Probing Peptide Sequences on Their Ability to Generate Affinity Sites in Molecularly Imprinted Polymers.

An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

Peptide-Cross-Linked Protein-Imprinted Polymers: Easy Template Removal and Excellent Imprinting Effect

Molecular imprinting of proteins remains a huge challenge because of two major obstacles: difficulty in template removal and low imprinting efficiency. Herein, we propose a new strategy to simultan...

Effect of hepatitis B virus X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B patients complicated with hepatocellular carcinoma

Objective To investigate the effect of hepatitis B virus (HBV) X gene integration on expression of zinc finger protein ZBTB20 in chronic hepatitis B (CHB) patients complicated with hepatocellular carcinoma (HCC). Methods Eighteen CHB patients complicated with HCC who underwent surgical treatment in Taizhou Enze Medical Center Enze Hospital and Taizhou Central Hospital from July 2015 to June 2017 were enrolled. Samples of carcinoma tissue, para-carcinoma tissue and corresponding normal liver tissue were collected from each case. DNA was extracted from three kinds of tissue samples. HBV-Alu-polymerase chain reaction (PCR) was used to amplify the integrated HBVX fragments and their bilateral flanking sequences in human genomic DNA. The integrated HBV fragments were determined by PCR products sequencing. Protein was extracted from three kinds of tissue samples.The level of expression of ZBTB20 was detected by protein imprinting. Statistical analysis was performed using t test, analysis of variance, and χ2 test. Results Among the 18 CHB patients complicated with HCC, integration of HBVX gene was found in 13 carcinoma tissue samples, 16 para-carcinoma tissue samples and 9 corresponding normal liver tissue samples. The difference was statistically significant (χ2=6.353, P=0.037). Of the 18 patients, the protein expressions of ZBTB20 in carcinoma tissue, para-carcinoma tissue and corresponding normal tissue were (50.14±11.25)%, (40.71±7.17)% and (39.06±5.17)%, respectively, which was statistically different (F=9.420, P<0.01). HBVX gene integration was detected at ZBTB20 locus in five patients. The expression levels of ZBTB20 in patients with HBVX gene integration at this locus in carcinoma tissue, para-carcinoma tissue and corresponding, normal liver tissue were all significantly lower than those in patients without HBVX gene integration (carcinoma tissue (37.37±10.30)% vs (55.06±7.06)%, para-carcinoma tissue (32.06±2.61)% vs (44.04±5.24)%, corresponding normal tissue (34.66±5.59)% vs (40.76±4.04)%, t=4.205, 4.821 and 2.589, respectively, all P<0.05). Conclusions Incidence of HBVX integration in para-carcinoma tissue is higher than that in carcinoma tissue in CHB patients complicated with HCC.The expression level of ZBTB20 in carcinoma tissue is higher than that in para-carcinoma tissue. Integration of HBVX gene at ZBTB20 locus may decreases the expression of ZBTB20. Key words: Hepatitis B virus; Carcinoma, hepatocellular; Gene integration; Zinc finger protein ZBTB20

Carbon Nanotube Facilitated Interface Formation for Enhanced Protein Sensing in Electrosynthesized Molecular Imprinting.

Molecular imprinting is a sensing technique that uses synthetic ligands to form high-affinity binding sites complementary to the specific analyte of interest. It has been well developed for recognition of small compounds, however, the efficacy of molecular imprinting remains challenging in protein sensing. The attributing factors are the fragility of protein molecules and their complex surface chemistry that complicate the selection of functional monomers in the imprint synthesis. In this work, we theoretically and experimentally investigated the mechanism underlying the ultrasensitive performance of a carbon-nanotube originated electrosynthesized protein imprinting sensor. By computational screening of 14 phenolic compounds and 11 conventional functional monomers in the eletrosynthetic imprinting, it was found the possible formation of interface, and its involvement in the high affinity of the imprinting was suggested. By artificially enriching the functional compounds with high binding energy to optimize interface composition in the electrosynthetic protein imprinting, the protein sensing performance is correspondingly increased in the electrochemical nanosensor. To the best of our knowledge, it is the first proposal of interface formation for the protein imprinting design, and the hypothesis is proved in concept.

MiR-223-3p Alleviates Vascular Endothelial Injury by Targeting IL6ST in Kawasaki Disease

Background: Kawasaki disease (KD) is a self-limiting illness with acute systematic vascular inflammation. It causes pathological changes in mostly medium and small-sized arteries, especially the arteria coronaria, which adds the risk of developing coronary heart disease in adults.Materials and methods: We detected the miR-223-3p expression in 30 KD patients combined with 12 normal controls using miRNA microarrays and RT-PCR. A KD mouse model was constructed using Candida albicans water insoluble substance (CAWS). We also checked the miR-223-3p's expression using qRT-PCR. The Luciferase reporting system was implemented to validate the correlation between miR-223-3p and Interleukin-6 receptor subunit beta (IL-6ST). TNF-α was used to stimulate human coronary artery endothelial cells (HCAECs), and miR-223-3p activator or inhibitor and KD serum were used to treat HCAECs. A Western blotting automatic quantitative analysis protein imprinting system was used to test the expression of signal transducer and the activator of transcription 3 (STAT3), phosphorylated-signal transducer and the activator of transcription 3 (pSTAT3) and NF-κB p65.Results: Clinical trials found that miR-223-3p expressions were markedly different (more than 2-fold) between the acute KD group and the control group. E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) levels were also significantly higher (about 2-fold) in KD especially with coronary artery lesions. MiR-223-3p could alleviate vascular endothelial damage in KD mice, and IL-6 (Interleukin-6), E-selectin and ICAM-1 were simultaneously negative. The values of IL-6, E-selectin, and ICAM-1 mRNA expressions decreased, while the value of IL-6ST was increased between the agonist treated mice and KD mice. The RT-qPCR consequences displayed that miR-223-3p explored the highest expression on the third day in both the KD mice as well as the agonist group. MiR-223-3p can directly combine with IL-6ST 3' untranslatable regions (UTR) and held back the IL-6's expression. Overexpression of miR-223 down regulated IL6ST expression and decreased the expression of p-STAT3 and NF-κB p65, while the miR-223 inhibitor could reverse the above process.Conclusion: MiR-223-3p is an important regulatory factor of vascular endothelial damage in KD and could possibly become a potential target of KD treatment in the future.

Ni2+-BSA Directional Coordination-Assisted Magnetic Molecularly Imprinted Microspheres with Enhanced Specific Rebinding to Target Proteins.

Protein imprinting technology is of interest in drug delivery, biosensing, solid-phase extraction, and so forth. However, the efficient recognition and separation of proteins have remained challenging to date. Toward this, under the assistance of Ni2+-bovine serum albumin (BSA) directional coordination strategy, magnetic BSA-imprinted materials had been synthesized via dopamine self-polymerization on hollow Fe3O4@mSiO2 microspheres (mSiO2 referred as mesoporous silica). The well-defined imprinted microspheres possessed more satisfactory adsorption capacity (266.99 mg/g), enhanced imprinting factor (5.45), and fast adsorption saturation kinetics (40 min) for BSA, superior to many previous reports. Benefiting from the coordinate interaction between Ni2+ and BSA, these fabricated microspheres exhibited excellent specificity not only in individual and competitive protein rebinding samples but also in bovine serum. Combined with the directional coordination method, the magnetic-imprinted composite materials to selectively capture target proteins could provide promising potential in applications.

Design and preparation of self-driven BSA surface imprinted tubular carbon nanofibers and their specific adsorption performance

Increasing mass transfer rate and adsorption amount are always challenges in the field of surface protein imprinting. In order to promote the development of protein purification, surface imprinted tubular carbon nanofibers (SIPTCFs) with self-driven property are first developed in this paper. The carrier is tubular carbon nanofiber obtained by hypercrosslinking reaction, carbonization and carboxyl functionalization. Bovine serum albumin (BSA) and polydopamine (PDA) are used as template and imprinted polymer layer, respectively. The resulted materials contain cavity and porous tube wall, which make the osmotic pressure difference between inside and outside of SIPTCFs during the adsorption process. Thereby it endows the SIPTCFs self-driven specific adsorption performance. The SIPTCFs have high specific surface area, reasonable pore structure and self-driven properties. They are the main reasons for accelerating the adsorption process and increasing the adsorption amount. The adsorption amount of SIPTCFs to BSA can reach 541.99 mg/g within 60 min. In addition, SIPTCFs can recognize the target protein BSA with high selectivity from the mixed proteins of HSA, Lyz, Cyt C and Bhb as well as fetal bovine serum (FBS). Because of the excellent reusability and separation ability for actual samples, SIPTCFs will be intensely promising materials for protein separation and purification.

Effect of branched chain amino acid on apoptosis of neurons after focal cerebral ischemia in rats

Objective To investigate the regulatory effect of branched chain amino acids(BCAA) on the expression of apoptosis related proteins after cerebral ischemia reperfusion injury and the protective effects of BCAA on ischemic brain injury in rats. Methods 40 male SD rats were randomly divided into normal diet group(n=20)and branched chain amino acid (BCAA) group(n=20)according to the random number table, and each group was randomly divided into control group (n=6), sham operation group (n=6) and model group (n=8) which used suture method to make ischemia reperfusion model. After modeling, modified Neurological Severity Scores (mNSS) was used to access the neurological impairment degree of 2, 6, 24, 48 and 72 h in each group. The expression of apoptosis related proteins (Cleaved, Bax/Bcl-2) after 72 h was detected by the method of immune protein imprinting (Caspase3) and compared between normal diet group and BCAA group. Results Compared with the normal diet rats, the mNSS of BCAA diet rats after modeling at 2, 6, 24, 48, 72 h decreased(11.35±2.78 vs. 7.15±2.41, P=0.019; 9.35±1.75 vs. 5.82±1.17, P=0.002; 6.11±1.16 vs. 4.39±1.46, P=0.048; 5.87±1.32 vs. 3.55±1.94, P=0.036; 4.98±2.24 vs. 2.09±1.33, P=0.022). The expression of cleaved caspase3 protein and the ratio of Bax/Bcl-2 decreased in BCAA group. Conclusion BCAA can alleviate the apoptosis of rats after ischemia and reperfusion, reduce the damage of nerve function, and has a positive protective effect on ischemic brain injury. Key words: Branched chain amino acid; Cerebral ischemia reperfusion; Apoptosis

Whole Protein Imprinting over Magnetic Nanoparticles Using Photopolymerization

Photopolymerization using UV-light at 365 nm was used for the first time to imprint whole proteins. Combined with a grafting approach onto functionalized magnetic nanoparticles, a new and faster way to synthesize magnetic protein imprinted polymers with outstanding adsorption capacities was developed.

Dopamine-based molecularly imprinted polymers for the recognition of biomacromolecules

Compared to natural antibodies or receptors, molecularly imprinted polymers (MIPs) have shown advantages of easy preparation, low cost, high stability and reusability. MIPs have been widely used in the fields of separation, chemical sensing, drug delivery and biocatalysis. The development of new imprinting methods such as surface imprinting, epitope imprinting, post-imprinting modification have promoted the synthesis and application of MIPs against biomacromolecules. Despite of the success, it remains a challenge to prepare MIPs with high affinity and specificity to the target proteins because the options of water-soluble monomers are very limited and the protein’s conformation is liable to change during the polymerization reaction. The majority of protein imprinting studies employ the five most common monomers: acrylamide (AAm), methacrylic acid (MAA), aminophenylboronic acid (APBA), acrylic acid (AA), and N-isopropylacrylamide (NiPAAm) and the two frequently used crosslinkers, methylenebisacrylamide (MBA) and ethylene glycol dimethacrylate (EGDMA) for preparation of protein imprinted polymers. In recent years, dopamine (DA) has been frequently used for surface modification as it can self-polymerize in alkaline medium at room temperature and generate polydopamine (pDA) films that can adhere onto almost all inorganic and organic surfaces with good biocompatibility. Thus, DA emerged as an appropriate monomer as well as a crosslinker for various imprinting reactions. In this review, we highlighted the recent advances in design and fabrication of DA-based MIPs for various biomacromolecules, including proteins, glycoproteins, bacteria or virus, with focus on protein imprinted polymers. First, we briefly introduced the DA self-polymerization reaction and the reported mechanisms for pDA formation. It is generally accepted that the initial driving force for the formation of pDA is the oxidation of DA to DA-indole by dissolved oxygen in an alkaline solution. Then, we summarized the recent work that employs common protein such as human/bovine serum albumin (HSA/BSA), lysozyme (Lyz), hemoglobin, trypsin and immunoglobulin (IgG) as the imprinting templates. These MIPs were mainly used for separation and purification of the template proteins. We also introduced the combination of protein imprinting with quartz crystal microbalance (QCM), cyclic voltammetry (CV), surface plasmon resonance (SPR) and mass spectroscopy (MS) for recognition and detection of the target proteins. In the following part, we summarized the strategies developed for imprinting of glycoproteins, most of which took advantage of boron affinity reaction or lectin-sugar interaction to improve the affinity and selectivity of the resultant MIPs. Then, we introduced recent work on imprinting of microorganisms such as viruses and bacteria, in which surface imprinting technique and potential analysis or chemiluminescence (ECL) technique are combined to construct biosensors that can selectively detect target species with high sensitivity. In addition, we also introduced the synthesis of a bio-inspired virus imprinted polymer and its application for prevention of viral infections. The techniques which were used for characterizing the properties of the MIP materials, e.g., adsorption capacity, imprinting factor and selectivity, as well as the proposed theoretical model were also mentioned in the review. Finally, we discussed current challenges and possible solutions for using DA as a monomer to prepare MIPs for biomacromolecules. We believe that the interactions between the intermediate products during dopamine polymerization and the template proteins deserve further investigation, as it may provide more possibilities to modulate the binding properties of the obtained MIPs.

Near-infrared light-responsive electrochemical protein imprinting biosensor based on a shape memory conducting hydrogel

Herein, a near-infrared (NIR) light-responsive imprinting biosensor with excellent conductivity and tensibility was designed by incorporation graphene oxide/polyaniline (GO/PANI) with thermo-responsive poly(N-isopropylacrylamide) hydrogel (PNiPAAm) on a glassy carbon electrode (GCE). In this design, polyaniline (PANI) nanofiber modifies with graphene oxide (GO) acts as a physically cross-linker, providing hydrogen bond interaction with amide groups in the PNiPAAm. It is functioned to enhance the strength of imprinted hydrogel networks. Thus, the imprinted hydrogel biosensor incorporation of GO/PANI nanocomposite exhibited excellent mechanical performance and NIR light-responsive characteristic. As a NIR radiation trigger gate, the obtained biosensor not only has a function of IR-based cleaning toward bovine serum albumin (BSA) in the absence of elution solution, but also a function of proteins releasing and uptaking, basing on reversibly conformational changes with 808 nm NIR light. Simultaneously, the responsive processes were electronically monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) and verified by scanning electron microscopy (SEM). Moreover, the proposed GO/PANI nanocomposite imprinted biosensor exhibited excellent characteristics along with high conductivity and tensibility, long-term stability, and wide linear range, performing great promising for preparing remote NIR light-driven extraction protein smart devices.

Controlled preparation of molecularly imprinted polymer microspheres with surface uncrosslinked glycoprotein binding cavities

Molecularly imprinted polymers (MIPs) are synthetic receptors with tailor-made recognition sites for target molecules. Their high affinity and selectivity, excellent stability, easy preparation, and low cost make them promising substitutes to biological receptors (e.g., antibody and enzyme) in many applications where molecular recognition is important. Despite significant progress made in the imprinting of small templates, the imprinting of biomacromolecules (e.g., proteins) remains a big challenge because of their large sizes, complexed structures, and conformational variability. These inherent characteristics of biomacromolecules lead to many significant problems for the resulting macromolecularly imprinted polymers (mMIPs) such as laborious removal of large templates and their slow access to the binding sites. Recent years have witnessed much efforts being devoted to the development of mMIPs due to their great potential in proteome analysis, clinical diagnostics, and biomedicine. So far, some useful strategies have been developed for the imprinting of proteins, mainly including the bulk polymerization method, epitope imprinting strategy, and surface imprinting approach. Among them, the surface imprinting approach has been most widely used because it can readily lead to mMIPs with higher efficiency for removing large templates and more rapid template binding kinetics. Nevertheless, the presently developed mMIPs normally have crosslinked template binding sites, whose rigid structures might have negative influence on the removal of large templates and template binding kinetics. In this sense, the development of mMIPs with more flexible biomacromolecular binding cavities (e.g., uncrosslinked ones) should be useful for solving the above problems. To our knowledge, however, only rather limited numbers of publications relating to mMIPs with uncrosslinked binding sites have been disclosed, which are all based on the use of self-assembled monolayer strategy. The development of versatile new approaches for preparing mMIPs with uncrosslinked binding sites and good molecular imprinting effect is still highly desirable. We demonstrate a facile and efficient new approach for the controlled preparation of MIP microspheres with surface uncrosslinked glycoprotein binding sites. It involves the first one-pot synthesis of uniform “living” polymer microspheres with both surface-bound epoxy groups and alkyl halide groups (i.e., atom transfer radical polymerization (ATRP)-initiating groups) via atom transfer radical precipitation polymerization, their surface modification with 3-aminophenylboronic acid (APBA) for introducing surface phenylboronic acid moieties and surface immobilization of a glycoprotein (ovalbumin (OVA)), subsequent use of the “living” polymer microspheres with surface-immobilized OVA as the ATRP initiator for the controlled grafting of poly( N -isopropylacrylamide) (PNIPAAm) brushes, and final removal of OVA. A series of MIP microspheres with surface uncrosslinked OVA binding sites were readily obtained following the above procedure by just changing the polymerization time for grafting PNIPAAm brushes, and their morphologies, chemical structures, surface hydrophilicity, water dispersion stability, and template binding properties were characterized in detail. The experimental results demonstrated that the above approach could effectively provide MIPs with excellent recognition ability toward OVA in the aqueous medium. The surface hydrophilicity and water dispersion stability of MIP microspheres were largely improved due to their surface-grafting of hydrophilic polymer brushes. Moreover, the chain length of PNIPAAm brushes showed significant influence on the template binding properties of MIP microspheres, and the best template binding capacity and specific binding were achieved for MIPs only when the thickness of PNIPAAm layers was close to the total length of the diameter of OVA plus the length of the surface-attached phenylboronic acid unit. Furthermore, the optimal MIP also exhibited good selectivity toward OVA over other proteins. The strategy presented here paves a new way for controlled and efficient preparation of glycolprotein-imprinted polymer microspheres with good molecular recognition capability.

New protocol for optimisation of polymer composition for imprinting of peptides and proteins.

We present here a novel screening tool for optimisation of polymerisation mixtures used in imprinting of peptides and proteins. To facilitate rapid synthesis and screening of a combinatorial library of polymers the solid-phase synthesis method developed by Piletsky and co-workers was scaled down to 50 mg of template-immobilised solid phase, allowing a single well of a 96-well microplate to function as an individual reaction vessel. In this way, 32 different polymer compositions containing N-isopropylacrylamide, acrylic acid, N-(3-aminopropyl)methacrylamide hydrochloride, and N-tert-butylacrylamide, were tested in imprinting of three peptides and three proteins. Utilising filtration microplates has allowed the elution and washing steps to be performed in a similar manner to the large-scale synthesis, whilst incorporation of a fluorescent monomer (N-fluoresceinylacrylamide) made it possible to analyse the binding of synthesised polymer nanoparticles to the solid phase with immobilised templates under different washing conditions. The experiment has proven that the variations in monomer compositions had an effect on the yield and affinity of synthesised molecularly imprinted polymers for the peptides, but not for the proteins. Imprinting in this way presents an ideal method for performing small-scale syntheses for testing polymerisation mixtures, as information regarding the molecularly imprinted polymers affinity can be assessed as part of the elution process, without a need for time-consuming analysis such as quartz crystal microbalance or surface plasmon resonance.

Preparation of lysozyme-imprinted nanoparticles on polydopamine-modified titanium dioxide using ionic liquid as a stabilizer

Molecular imprinting of proteins has evolved into an efficient approach for protein recognition and separation. However, maintaining the structural stability of proteins during the preparation process of molecularly imprinted polymers (MIPs) remains challenging. Ionic liquids (ILs), being capable of maintaining the stability of proteins, might enable effective imprinting and accurate recognition of proteins. In this study, lysozyme (Lyz)-imprinted titanium dioxide (TiO2) nanoparticles, TiO2@Lyz-MIPs, have been successfully prepared for selective recognition and separation of Lyz. This was achieved by the free radical polymerization of hydroxyethyl acrylate (HEA) and poly(ethylene glycol) dimethacrylate (PEGDMA) on polydopamine (PDA)-modified TiO2 nanoparticles using an IL, choline dihydrogen phosphate (chol dhp), as the stabilizer of Lyz. It was found that both PDA modification of TiO2 and the use of chol dhp as stabilizer improved the adsorption capacity of TiO2@Lyz-MIPs toward Lyz. When the concentration of HEA was 7 mg mL−1, the ratio of monomer to crosslinker was 20 : 1, and the concentration of chol dhp was 12.5 mg mL−1, the highest imprinting factor of 4.40 was achieved. TiO2@Lyz-MIPs exhibited relatively high adsorption capacity with the maximum adsorption capacity up to 120 mg g−1, which was more than four times higher than that of the non-imprinted polymers (NIPs) counterpart, TiO2@Lyz-NIPs. The adsorption rate of Lyz by TiO2@Lyz-MIPs was also much higher than that of TiO2@Lyz-NIPs. TiO2@Lyz-MIPs could successfully separate Lyz from diluted egg white, a complex mixture of proteins. Findings from this study indicate that effective recognition cavities toward Lyz were formed on the surface of Lyz-imprinted TiO2 nanoparticles prepared using IL as the template stabilizer. This approach may facilitate the development of MIPs for efficient protein recognition and separation.