Epitope Imprinting Approach Research Articles

Detection of depression marker ASS1 in urine by gold nanoparticles based dual epitope-peptides imprinted sensor

Trace detection of argininosuccinate synthetase 1 (ASS1), a depression marker, in urine samples is difficult to achieve. In this work, a dual-epitope-peptides imprinted sensor for ASS1 detection in urine was constructed based on the high selectivity and sensitivity of the “epitope imprinting approach”. First, two cysteine-modified epitope-peptides were immobilized onto gold nanoparticles (AuNPs) deposited on a flexible electrode (ITO-PET) by gold-sulfur bonds (Au–S), then a controlled electropolymerization of dopamine was carried out to imprint the epitope peptides. After removing epitope-peptides, the dual-epitope-peptides imprinted sensor (MIP/AuNPs/ITO-PET) which with multiple binding sites for ASS1 was obtained. Compared with single epitope-peptide, dual-epitope-peptides imprinted sensor had higher sensitivity, which presented a linear range from 0.15 to 6000 pg ml−1 with a low limit of detection (LOD = 0.106 pg mL−1, S/N = 3). It had good reproducibility (RSD = 1.74%), repeatability (RSD = 3.60%), stability (RSD = 2.98%), and good selectivity, and the sensor had good recovery (92.4%–99.0%) in urine samples. This is the first highly sensitive and selective electrochemical assay for the depression marker ASS1 in urine, which is expected to provide help for the non-invasive and objective diagnosis of depression.

Core-Shell Magnetic Imprinted Polymers for the Recognition of FLAG-Tagpeptide

FLAG® tag (DYKDDDDK) is a small epitope peptide employed for the purification of recombinant proteins such as immunoglobulins, cytokines, and gene regulatory proteins. It provides superior purity and recoveries of fused target proteins when compared to the commonly used His-tag. Nevertheless, the immunoaffinity-based adsorbents required for their isolation are far more expensive than the ligand-based affinity resin used in combination with the His-tag. In order to overcome this limitation we report herein the development of molecularly imprinted polymers (MIPs) selective to the FLAG® tag. The polymers were prepared by the epitope imprinting approach using a four amino acids peptide, DYKD, including part of the FLAG® sequence as template molecule. Different kinds of magnetic polymers were synthesised in aqueous and organic media also using different sizes of magnetite core nanoparticles. The synthesised polymers were used as solid phase extraction materials with excellent recoveries and high specificity for both peptides. The magnetic properties of the polymers confer a new, effective, simple, and fast method in the purification using FLAG® tag.

Bovine serum albumin detection by using molecularly imprinted surface plasmon resonance sensors

Molecular imprinted polymers (MIP) have key-lock pattern binding properties specific to the size and shape of target molecules. In this study, we have prepared detection platforms based on a molecularly imprinted surface plasmon resonance (SPR) sensor that can detect bovine serum albumin (BSA) sensitively, selectively, quickly, and in real time. The polymeric film prepared on the SPR sensor surface by molecular imprinting method was obtained by selecting the N-methacryloyl-(L)-glutamic acid molecule as a suitable functional monomer using ultraviolet polymerization. Three different imprinting methods, such as epitope, bulk, and surface imprinting methods, were used to examine the imprinting efficiency. Real-time measurements were performed with BSA imprinted SPR sensor provide linearity in the concentration range from 0.10 to 7.50 nM and indicate a detection limit value of 0.015 nM. Furthermore, we performed the selectivity experiments, where transferrin and hemoglobin were chosen as competitor agents. Overall, the SPR sensor prepared by the epitope imprinting approach has been found to be highly selective and sensitive for bovine serum albumin. To statistically assess the reusability of the sensor, intraday experiments were tested three times with five replicates. The RSD% value less than

Solid-phase synthesis of imprinted nanoparticles as artificial antibodies against the C-terminus of the cannabinoid CB1 receptor: exploring a viable alternative for bioanalysis

The production of artificial anti-CB1 antibodies in nanoparticle format is described using the solid-phase imprinting approach. Instead of whole protein imprinting, a linear C-terminus sequence of the receptor comprising 15 amino acids (458-KVTMSVSTDTSAEAL-472) has been used as template, in accordance with the epitope imprinting approach. This sequence is located intracellularly, and it is involved in coupling to Gi/o proteins, being responsible for CB1 receptor desensitisation and internalisation. Developed molecularly imprinted materials were found to be in the nanometre scale, with a particle size of 126.4 ± 10.5 nm at pH 3 (25 ºC) and spherical shape. It was also observed that the size was sensible to temperature changes being reduced to 106.3 ± 15.2 nm at 35 °C. Lower critical solution temperature of this polymer was found to be ≈ 33.4 °C. The affinity and selectivity of the artificial antibody were assessed through dot blot and Western blot experiments. For the latter, recombinant fusion proteins GST-CB1414-472 and GST-CB1414-442 were produced to work respectively as target and negative control proteins. The control protein did not carry the target epitope for being devoid of last 30 amino acids at the C-terminus. The results demonstrated that the anti-CB1 material recognised selectively the target protein, thanks to the presence of the 15-amino acid sequence selected as epitope, which revealed that binding occurred at the C-terminus of the receptor itself. The methodology presented may pave the way for the development of novel imprinted nanomaterials for other proteins included in the superfamily of the G-protein-coupled receptors (GPCR).Graphical abstract

“Out of Pocket” Protein Binding—A Dilemma of Epitope Imprinted Polymers Revealed for Human Hemoglobin

The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.

Bovine serum albumin detection by using molecularly imprinted surface plasmon resonance sensors.

Molecular imprinted polymers (MIP) have key-lock pattern binding properties specific to the size and shape of target molecules. In this study, we have prepared detection platforms based on a molecularly imprinted surface plasmon resonance (SPR) sensor that can detect bovine serum albumin (BSA) sensitively, selectively, quickly, and in real time. The polymeric film prepared on the SPR sensor surface by molecular imprinting method was obtained by selecting the N-methacryloyl-(L)-glutamic acid molecule as a suitable functional monomer using ultraviolet polymerization. Three different imprinting methods, such as epitope, bulk, and surface imprinting methods, were used to examine the imprinting efficiency. Real-time measurements were performed with BSA imprinted SPR sensor provide linearity in the concentration range from 0.10 to 7.50 nM and indicate a detection limit value of 0.015 nM. Furthermore, we performed the selectivity experiments, where transferrin and hemoglobin were chosen as competitor agents. Overall, the SPR sensor prepared by the epitope imprinting approach has been found to be highly selective and sensitive for bovine serum albumin. To statistically assess the reusability of the sensor, intraday experiments were tested three times with five replicates. The RSD% value less than <1.3 indicates high reproducibility for both sensor production and reproducibility of the method. Validation studies were carried out via enzyme-linked immunosorbent analysis technique (ELISA) in order to demonstrate the applicability of the BSA imprinted SPR sensor. Due to their features such as reusability, fast response time, and ease of use, these SPR sensors, which could be used as an alternative to albumin monitoring approaches, can also be adapted to detect and monitor other proteins in real time.

Hierarchically Imprinted Polymer for Peptide Tag Recognition Based on an Oriented Surface Epitope Approach.

FLAG tag (DYKDDDDK) is a short peptide commonly used for the purification of recombinant proteins. The high price of the affinity columns and their limited reusability are a shortcoming for their widespread use in biotechnology applications. Molecularly imprinted polymers (MIPs) can circumvent some of the limitations of bioaffinity columns for such applications, including long-term stability, reusability, and cost. We report herein the synthesis of MIPs selective to the FLAG tag by hierarchical imprinting. Using the epitope imprinting approach, a 5-amino acid peptide DYKDC was selected as a template and was covalently immobilized on the surface of microporous silica beads, previously functionalized with different aminosilanes, namely, 3-(2-aminoethylamino)propyldimethoxymethylsilane, AEAPMS, and N-(2-aminoethyl)-2,2,4-trimethyl-1-aza-2-silacyclopentane, AETAZS. We investigated the effect of the type of silane on the production of homogeneous silane-grafted layers with the highest extent of silanol condensation as possible using 29Si CP/MAS NMR. We observed that the right orientation of the imprinted cavities can substantially improve analyte recoveries from the MIP. After template and silica removal, the DYKDC-MIPs were used as sorbents for solid-phase extraction (molecularly imprinted solid-phase extraction) of the FLAG peptide, showing that the polymer prepared with AETAZS-bound silica beads contained binding sites more selective to the tag (RMIP-AZA = 87.4% vs RNIP-AZA = 4.1%, n = 3, RSD ≤ 4.2%) than those prepared using AEAPMS (RMIP-DM = 73.4% vs RNIP-DM = 23.2%, n = 3, RSD ≤ 4.0%) as a functionalization agent. An extensive computational molecular modeling study was also conducted, shedding some light on the interaction mechanism between the FLAG peptide and the imprinted template in the binding cavities.

Metal chelation dual-template epitope imprinting polymer via distillation-precipitation polymerization for recognition of porcine serum albumin

A novel dual-template epitope imprinting polymer coated on magnetic carbon nanotubes (MCNTs@D-EMIP) was successfully prepared for specific recognition of porcine serum albumin (PSA) via dual-template epitope imprinting, metal chelation imprinting and distillation-precipitation polymerization (DPP). C-terminal peptides and N-terminal peptides of PSA were selected as templates simultaneously, and zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were used as functional monomer and cross-linker, respectively. The epitope templates were immobilized by metal chelation and six-membered ring formed with zinc acrylate. Finally, MCNTs@D-EMIP was synthesized by DPP in only 30 min, which was much shorter than those of other polymerization methods. The prepared MCNTs@D-EMIP displayed specific recognition ability toward PSA and its adsorption amount and imprinting factor were 45.05 mg g−1 and 4.50, which were much higher than those of single template epitope imprinting polymers. Besides, high-performance liquid chromatography (HPLC) analysis of PSA in porcine blood serum real sample indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@D-EMIP had potential to be applied in bio-separation area. In addition, the results of cross-reactivity experiment proved that this strategy had generality to prepare dual-template epitope imprinting polymer for recognition of target protein. In summary, this study provided an efficient protocol to recognize target protein in complex sample via dual-template epitope imprinting approach, metal chelation imprinting and distillation-precipitation polymerization.

Preparation of Molecularly Imprinted Mesoporous Materials for Highly Enhancing Adsorption Performance of Cytochrome C.

Molecularly imprinted mesoporous materials (MIMs) were synthesized to improve the adsorption performance of Cytochrome c (Cyt c) by using an imidazolium-based amphiphilic ionic liquid 1-octadecyl-3-methylimidazolium chloride (C18MIMCl) as surfactant in aqueous solution via the epitope imprinting approach. The surface-exposed C-terminus nonapeptide of Cyt c (residues 96–104, AYLKKATNE) was utilized as the imprinted template. The nitrogen adsorption-desorption, thermo-gravimetric analysis, and transmission electron microscopy verified the successful preparation of MIMs with ordered mesoporous structure. The adsorption isotherm studies showed that the obtained MIMs exhibited superior adsorption capacity toward Cyt c of 86.47 mg·g−1 because of the high specific surface areas of 824 m2·g−1, and the appropriate pore size promoted the mass transfer of Cyt c, causing a rapid adsorption equilibrium within 20 min. Furthermore, these MIMs still remained excellent selectivity and recognition ability according to the selective as well as the competitive adsorption studies, suggesting that the molecularly imprinted mesoporous materials is expected to be used in the field of highly efficient separation and enrichment of proteins.

Epitope Imprinting Approach to Monitor Diseases

Epitope sequences are unique combination of amino acids sequence positioned on exposed domains of proteins. Molecular imprinting is a promising technique for creating molecular receptors with recognition and binding sites that are chemically and sterically complementary in shape, size and functionality to the predetermined target molecules in synthetic polymer. This approach creates template-shaped cavities in polymer matrices with memory of template molecules to be used in molecular recognition. Imprinting whole protein denatures the tertiary and quaternary structures of protein in the polymer matrix and complexity and flexibility of its structure cannot be sustained in the polymer matrix. Epitope approach offers a way out of such snags. The epitope-imprinted film revealed high selectivity over the target protein and allow tolerance for even a single amino acid mismatch between the epitope and target protein. MIP sensors are ideal candidates for replacing biosensors as well as natural receptors in many sensing applications such as ELISA. In spite of advantages and burgeoning research in the field of MIPs, imprinting fraternity has not yet achieved commercial success. Substitution of antibodies used in diagnostic tools with synthetic analogues will cut down cost as well as time period for sample analysis. MIP sensing layers have proven to be highly economical and they have shown almost parallel feat as bio-sensing elements (antibody/antigen/enzyme) incorporated in ELISA. Rapid and accurate determination of disease biomarker proteins is vital for clinical diagnosis and medical abnormalities. Hence MIP-sensors of certain proteins will be useful in early diagnosis of diseased state.

Preparation of High-Efficiency Cytochrome c-Imprinted Polymer on the Surface of Magnetic Carbon Nanotubes by Epitope Approach via Metal Chelation and Six-Membered Ring.

A novel epitope molecularly imprinted polymer on the surface of magnetic carbon nanotubes (MCNTs@EMIP) was successfully fabricated to specifically recognize target protein cytochrome c (Cyt C) with high performance. The peptides sequences corresponding to the surface-exposed C-terminus domains of Cyt C was selected as epitope template molecule, and commercially available zinc acrylate and ethylene glycol dimethacrylate (EGDMA) were employed as functional monomer and cross-linker, respectively, to synthesize MIP via free radical polymerization. The epitope was immobilized via metal chelation and six-membered ring formed between the functional monomer and the hydroxyl and amino groups of the epitope. The resulting MCNTs@EMIP exhibited specific recognition ability toward target Cyt C including more satisfactory imprinting factor (about 11.7) than that of other reported imprinting methods. In addition, the MCNTs@EMIP demonstrated a high adsorption amount (about 780.0 mg g(-1)) and excellent selectivity. Besides, the magnetic property of the support material made the processes easy and highly efficient by assistance of an external magnetic field. High-performance liquid chromatography analysis of Cyt C in bovine blood real sample and protein mixture indicated that the specificity was not affected by other competitive proteins, which forcefully stated that the MCNTs@EMIP had potential to be applied in bioseparation area. In brief, this study provided a new protocol to detect target protein in complex sample via epitope imprinting approach and surface imprinting strategy.

Self-oriented nanoparticles for site-selective immunoglobulin G recognition via epitope imprinting approach

Molecular imprinting is a polymerization technique that provides synthetic analogs for template molecules. Molecularly imprinted polymers (MIPs) have gained much attention due to their unique properties such as selectivity and specificity for target molecules. In this study, we focused on the development of polymeric materials with molecular recognition ability, so molecular imprinting was combined with miniemulsion polymerization to synthesize self-orienting nanoparticles through the use of an epitope imprinting approach. Thus, l-lysine imprinted nanoparticles (LMIP) were synthesized via miniemulsion polymerization technique. Immunoglobulin G (IgG) was then bound to the cavities that specifically formed for l-lysine molecules that are typically found at the C-terminus of the Fc region of antibody molecules. The resulting nanoparticles makes it possible to minimize the nonspecific interaction between monomer and template molecules. In addition, the orientation of the entire IgG molecule was controlled, and random imprinting of the IgG was prevented. The optimum conditions were determined for IgG recognition using the imprinted nanoparticles. The selectivity of the nanoparticles against IgG molecules was also evaluated using albumin and hemoglobin as competitor molecules. In order to show the self-orientation capability of imprinted nanoparticles, human serum albumin (HSA) adsorption onto both the plain nanoparticles and immobilized nanoparticles by anti-human serum albumin antibody (anti-HSA antibody) was also carried out. Due to anti-HSA antibody immobilization on the imprinted nanoparticles, the adsorption capability of nanoparticles against HSA molecules vigorously enhanced. It is proved that the oriented immobilization of antibodies was appropriately succeeded.

An epitope imprinting method on the surface of magnetic nanoparticles for specific recognition of bovine serum album.

In this study, we reported an epitope imprinting method on the surface of core-shell magnetic nanoparticles (NPs) for the recognition of target bovine serum album (BSA). The epitope was selected as the template molecule from the nonapeptide of surface-exposed C-terminus of BSA. The core-shell magnetic epitope molecularly imprinted polymers (Fe3O4@EMIPs) exhibited a specific capture activity for the corresponding target protein, BSA. The magnetic NPs made it easy to separate the imprinted material from solution by an external magnetic field, and the thin imprinted layer presented fast kinetics for the rebinding of the target protein. Moreover, the Fe3O4@EMIPs could separate BSA from the bovine blood sample. The epitope imprinting approach combined with magnetic NPs provided an easy and fast method for the specific recognition of BSA.

Specific recognition of tyrosine-phosphorylated peptides by epitope imprinting of phenylphosphonic acid

Protein phosphorylation on tyrosine residues is an important cell signaling mechanism that regulates embryo development, cell growth, differentiation and migration. Here, we developed an epitope imprinting approach to selectively enrich tyrosine-phosphorylated (pTyr) peptides using molecularly imprinted polymers (MIPs). An artificial receptor based on MIPs was prepared by precipitation polymerization using phenylphosphonic acid (PPA) as a template, Ti4+-immobilized ethylene glycol methacrylate phosphate as functional monomer, poly(ethylene glycol) diacrylate and N,N′-methylenebisacrylamide as crosslinkers, and deionized water as a porogen. The performance of MIPs was first evaluated by rebinding PPA, and then a competition experiment was conducted to assess the selectivity and specificity for PPA when mixed with benzoic acid, a structural analog. The imprinting factor of the MIPs was up to 2.04, compared with just 0.24 for benzoic acid. The selectivity and capacity of the MIPs for pTyr peptides from a mixture of peptides were considerably higher than that of commercial TiO2. And after six rebinding cycles, there were still 95% adsorption efficiency retained. Finally, MIPs were used for pTyr enrichment from a complex sample containing pTyr peptide and tryptic digestion of β-casein, where it demonstrated a clear preference for the pTyr peptide over ones containing phosphorylated serine.

Immunoglobulin G recognition with Fab fragments imprinted monolithic cryogels: Evaluation of the effects of metal-ion assisted-coordination of template molecule

In this study, we applied the epitope imprinting approach to prepare molecularly imprinted monolithic cryogels for immunoglobulin G (IgG) recognition. In this respect, we imprinted Fab fragments of IgG molecules instead of intact protein molecules via two different non-covalent interactions. In the first approach, we directly coordinated Fab fragments with N-methacryloyl-L-histidine (MAH), polymerizable derivative of L-histidine, but for the second, we used cupric ions [Cu(II)] as mediator between MAH and Fab fragments. The monolithic cryogels were characterized by Fourier transform infrared (FTIR) spectroscopy, swelling test, and scanning electron microscopy. Then, the monolithic cryogels were used for Fab fragment adsorption from aqueous solution while evaluating the factors such as pH and Fab fragment concentration affecting on adsorption process in continuous set-up. After that, monolithic cryogels were used for IgG adsorption by varying pH, IgG concentration, flowrate, and temperature in appropriate ranges. Maximum IgG adsorption capacities were determined as 32.4mg/g and 49.0mg/g for directly coordinated cryogel (MIPDirect) and Cu(II) assisted cryogel (MIPCu(II) assisted), respectively. Non-imprinted monolithic cryogels were also prepared for control purposes. In addition to Fab fragments and IgG molecules, albumin and Fc fragment of IgG molecules were used as competitor biomolecules in order to investigate the selectivity gained by imprinting process. Relative selectivity constants were calculated as 1.47, 2.64 and 3.89 for MIPDirect and 2.90, 8.98, and 11.51 for MIPCu(II) assisted for Fab/IgG, Fab/Fc, and Fab/albumin as biomolecule pairs, respectively. The desorption efficiency and reusability of MIPCu(II) assisted cryogel were better than that of MIPDirect. The results reported here showed that the metal ion assistance improved the selectivity features of the imprinted cryogels and allowed to study under milder conditions with enhanced adsorptive properties.