Tag-Specific Affinity Purification of Recombinant Proteins by Using Molecularly Imprinted Polymers.

Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified ( R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

Bioinspired recognition elements for mycotoxin sensors.

Mycotoxins are low molecular weight molecules produced as secondary metabolites by filamentous fungi that can be found as natural contaminants in many foods and feeds. These toxins have been shown to have adverse effects on both human and animal health, and are the cause of significant economic losses worldwide. Sensors for mycotoxin analysis have traditionally applied elements of biological origin for the selective recognition purposes. However, since the 1970s there has been an exponential growth in the use of genetically engineered or synthetic biomimetic recognition elements that allow some of the limitations associated with the use of natural receptors for the analyses of these toxins to be circumvented. This review provides an overview of recent advances in the application of bioinspired recognition elements, including recombinant antibodies, peptides, aptamers, and molecularly imprinted polymers, to the development of sensors for mycotoxins based on different transduction elements. Graphical abstract Novel analytical methods based on bioinspired recognition elements, such as recombinant antibodies, peptides, aptamers, and molecularly imprinted polymers, can improve the detection of mycotoxins and provide better tools than their natural counterparts to ensure food safety.

Molecularly imprinted polymer beads for clean-up and preconcentration of ß-lactamase-resistant penicillins in milk.

This work describes the development and application of class-selective molecularly imprinted polymers (MIPs) for the analysis of beta-lactamase-resistant penicillins, namely cloxacillin (CLOXA), oxacillin (OXA), and dicloxacillin (DICLOXA), in milk samples. Our method is based on molecularly imprinted solid-phase extraction (MISPE) coupled to high-performance liquid chromatography (HPLC) with diode-array detection (DAD). 2-Biphenylylpenicillin (2BPEN), a surrogate with a close resemblance to beta-lactamase-resistant penicillins in terms of size, shape, hydrophobicity, and functionality, was synthesized and used as the template for the polymer synthesis. A MIP library was prepared and screened to select the optimum functional monomer, N-(2-aminoethyl)methacrylamide, and cross-linker, trimethylolpropane trimethacrylate, that provided the best recognition for the target antibiotics. For the MISPE application, the MIPs were prepared in the form of microspheres, using porous silica beads (40-75 µm) as sacrificial scaffolds. The developed MISPE method enables efficient extraction from aqueous samples and analysis of the antimicrobials, when followed by a selective washing with 2 mL acetonitrile-water (20:80 v/v) and elution with 1 mL 0.05 mol L(-1) tetrabutylammonium in methanol. The analytical method was validated according to EU guideline 2002/657/EC. The limits of quantification (S/N = 10) were in the 5.3-6.3 µg kg(-1) range, well below the maximum residue limits (MRLs) currently established. Inter-day mean recoveries were in the range 99-102 % with RSDs below 9 %, improving on the performance of previously reported MISPE methods for the analysis of CLOXA, OXA, or DICLOXA in milk samples.

Molecularly imprinted polymers for cleanup and selective extraction of curcuminoids in medicinal herbal extracts.

This paper describes the synthesis of novel molecularly imprinted polymers (MIPs), prepared by a noncovalent imprinting approach, for cleanup and preconcentration of curcumin (CUR) and bisdemethoxycurcumin (BDMC) from medicinal herbal extracts and further analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Two molecular mimics, a mixture of reduced BDMCs and 4-(4-hydroxyphenyl)-2-butanone (HPB), have been synthesized and applied as templates for MIP synthesis. The polymers were prepared using N-(2-aminoethyl) methacrylamide (EAMA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as the cross-linker (in a 1:5 molar ratio), and a mixture of acetonitrile/dimethylsulfoxide (90%, v/v) as porogen. MIPs prepared using a mixture of reduced BDMCs as template showed higher selectivity for CUR and BDMC than those obtained with HPB, with imprinting factors of 3.5 and 2.7 for CUR and BDMC, respectively, using H2O/acetonitrile (65:35, v/v) as mobile phase. The adsorption isotherms for CUR in the MIP and the nonimprinted polymer (NIP) were fitted to the Freundlich isotherm model, and the calculated average binding affinities for CUR were (17 ± 2) and (8 ± 1) mM(-1) for the MIP and the NIP, respectively. The polymers were packed into solid-phase extraction (SPE) cartridges, and the optimized molecularly imprinted solid-phase extraction (MISPE-HPLC) with fluorescence detection (FLD) method allowed the extraction of both curcuminoids from aqueous samples (50 mM NH4Ac, pH 8.8) followed by a selective washing with acetonitrile/NH4Ac, 50 mM at pH 8.8 (30:70%, v/v), and elution with 3 × 1 mL of MeOH. Good recoveries and precision ranging between 87 and 92%, with relative standard deviation (RSD) of <5.3% (n = 3), were obtained after the preconcentration of 10-mL solutions containing both CUR and BDMC at concentrations in the range of 0-500 µg L(-1). The optimized method has been applied to the analysis of both curcuminoids in medicinal herbal extracts.

Luminescent core-shell imprinted nanoparticles engineered for targeted Förster resonance energy transfer-based sensing.

Red-luminescent 200 nm silica nanoparticles have been designed and prepared as a versatile platform for developing FRET (Förster resonance energy transfer) biomimetic assays. Ru(phen)3²âº dye molecules embedded off-center in the silica core provide the long-lived donor emission, and a near-infrared labeled analyte serves as fluorescent acceptor (the measured R0 of this D-A pair is 4.3 nm). A thin surface-grafted molecularly imprinted polymer (MIP) shell intervenes as selective enrofloxacin-binding element. These nanoparticles have been tested for photochemical detection of enrofloxacin by using a competitive scheme that can be readily performed in MeCN-HEPES (pH 7.5) 7:3 (v/v) mixtures and allows for the antibiotic detection in the µM range (LOD = 2 µM) without optimization of the assay. Given the well-known difficulties of coupling the target-binding-to-MIP and the transducing events, the novel photochemical approach tuned up here will be valuable in future developments of MIP-based assays and optosensors that capitalize also on the advantages of nanomaterials for (bio)analysis.

Surface-imprinted nanofilaments for europium-amplified luminescent detection of fluoroquinolone antibiotics.

The development and characterization of novel, molecularly imprinted polymer nanofilament-based optical sensors for the analysis of enrofloxacin, an antibiotic widely used for human and veterinary applications, is reported. The polymers were prepared by nanomolding in porous alumina by using enrofloxacin as the template. The antibiotic was covalently immobilized on to the pore walls of the alumina by using different spacers, and the prepolymerization mixture was cast in the pores and the polymer synthesized anchored onto a glass support through UV polymerization. Various parameters affecting polymer selectivity were evaluated to achieve optimal recognition, namely, the spacer arm length and the binding solvent. The results of morphological characterization, binding kinetics, and selectivity of the optimized polymer material for ENR and its derivatives are reported. For sensing purposes, the nanofilaments were incubated in solutions of the target molecule in acetonitrile/HEPES buffer (100 mM, pH 7.5, 50:50, v/v) for 20 min followed by incubation in a 10 mM solution of europium(III) ions to generate a europium(III)-enrofloxacin complex on the polymer surface. The detection event was based on the luminescence of the rare-earth ion (λexc=340 nm; λem=612 nm) that results from energy transfer from the antibiotic excited state to the metal-ion emitting excited state. The limit of detection of the enrofloxacin antibiotic was found to be 0.58 µM.

Fluorescent ion-imprinted polymers for selective Cu(II) optosensing.

This paper describes the synthesis and characterization of a fluorescent ion-imprinted polymer (IIP) for selective determination of copper ions in aqueous samples. The IIP has been prepared using a novel functional monomer, 4-[(E)-2-(4'-methyl-2,2'-bipyridin-4-yl)vinyl]phenyl methacrylate (abbreviated as BSOMe) that has been spectroscopically characterized in methanolic solution, in the absence and in the presence of several metal ions, including Cd(II), Cu(II), Hg(II), Ni(II), Pb(II), and Zn(II). The stability constant (2.04 × 10(8) mol(-2) l(2)) and stoichiometry (L(2)M) of the BSOMe complex with Cu(II) were extracted thereof. Cu(II)-IIPs were prepared by radical polymerization using stoichiometric amounts of the fluorescent monomer and the template metal ion. The resulting cross-linked network did not show any leaching of the immobilized ligand allowing determination of Cu(II) in aqueous samples by fluorescence quenching measurements. Several parameters affecting optosensor performance have been optimized, including sample pH, ionic strength, or polymer regeneration for online analysis of water samples. The synthesized Cu(II)-IIP exhibits a detection limit of 0.04 µmol l(-1) for the determination of Cu(II) in water samples with a reproducibility of 3%, exhibiting an excellent selectivity towards the template ion over other metal ions with the same charge and close ionic radius. The IIP-based optosensor has been repeatedly used and regenerated for more than 50 cycles without a significant decrease in the luminescent properties and binding affinity of the sensing phase.

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.

Accelerated development procedure for molecularly imprinted polymers using membrane filterplates.

A novel technique for the synthesis and testing of large numbers of molecularly imprinted polymers is described requiring much less time than the commonly used miniMIP approach. Instead of vials, the polymers are synthesized on the surface of microfiltration membranes in multiwell filterplates. The thin polymeric films enable accelerated template removal. The MIP development procedure is thereby shortened to two days. Performance of the system was demonstrated by creating a combinatorial library of MIPs selective for cimetidine, an antiulcer drug. The polymer composition has been optimized. An experimental design combined with a multivariate analysis (i.e., response surface modeling) was used to minimize the number of experiments in the optimization process. The highest imprinting factor was obtained using a MAA/EDMA/template molar ratio of 3.5:19.5:1.

Effect of the template and functional monomer on the textural properties of molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) for zearalenone analysis have been synthesized using the template mimics cyclododecyl 2,4-dihydroxybenzoate (CDHB), resorcinol and resorcylic acid. The MIPs are photochemically prepared from 2-(diethylamino)ethyl methacrylate (2-DAEM), 4-vinylpyridine (VIPY), 2-hydroxyethyl methacrylate (HEMA) or 1-allylpiperazine (1-ALPP) as the functional monomers, trimethylolpropane trimethacrylate (TRIM) as cross-linker, azobis(isobutyronitrile) as initiator and acetonitrile as porogen. Non-imprinted polymers have been also synthesized for reference purposes. The textural properties of the novel polymers (BET areas, pore volumes and pore size distributions) have been determined from nitrogen adsorption-desorption isotherms. These parameters have shown to be strongly dependent on the presence of the template and the monomer nature. Scanning electron microscopy and solvent uptake experiments support these findings. Microporosity contributes less than 7% to the total pore volume for all the polymers prepared. Interestingly, a 3.5 nm pore opening is observed for all the polymers and additional pore apertures in the 20-40 nm region for VIPY-, HEMA- and 2-DAEM-based MIPs whereas a much wider opening size distribution has been measured for the 1-ALPP-based MIP. Molecular modeling and, particularly, (1)H NMR experiments demonstrate the strong (2:1) complex formed between 1-ALPP and the diphenolic CDHB (K(11)=4.7 x 10(4)M(-1) and K(12) = 2.6 x 10(2)M(-1) in acetonitrile) that make the corresponding MIP the most suitable for zearalenone recognition in real samples.

Tailoring molecularly imprinted polymer beads for alternariol recognition and analysis by a screening with mycotoxin surrogates.

Molecularly imprinted porous polymer microspheres have been prepared for selective binding of alternariol (AOH), a phenolic mycotoxin produced by Alternaria fungi. In order to lead the synthesis of recognition materials, four original AOH surrogates have been designed, prepared and characterized. They bear different number of phenol groups in various positions and different degree of O-methylation on the dibenzo[b,d]pyran-6-one skeleton. A comprehensive library of mixtures of basic, acidic or neutral monomers, with divinylbenzene or ethyleneglycol dimethacrylate as cross-linkers, were polymerized at a small scale in the presence of the four molecular mimics of the toxin molecule. This polymer screening has allowed selection of the optimal composition of the microbeads (N-(2-aminoethyl)methacrylamide, EAMA, and ethylene glycol dimethacrylate). The latter are able to bind AOH in water-acetonitrile (80:20, v/v) with an affinity constant of 109±10mM(-1) and a total number of binding sites of 35±2µmolg(-1), being alternariol monomethylether the only competitor species. Moreover, (1)H NMR titrations have unveiled a 1:2 surrogate-to-EAMA stoichiometry, the exact interaction sites and a binding constant of 1.5×10(4)M(-2). A molecularly imprinted solid phase extraction (MISPE) method has been optimized for selective isolation of the mycotoxin from aqueous samples upon a discriminating wash with 3mL of acetonitrile/water (20:80, v/v) followed by determination by HPLC with fluorescence detection. The method has been applied, in combination to ultrasound-assisted extraction, to the analysis of AOH in tomato samples fortified with the mycotoxin at five concentration levels (33-110µgkg(-1)), with recoveries in the range of 81-103% (RSD n=6). To the best of our knowledge, this is the first imprinted material capable of molecularly recognizing this widespread food contaminant.

Experimental mixture design as a tool for the synthesis of antimicrobial selective molecularly imprinted monodisperse microbeads.

The effect of the cross-linker on the shape and size of molecular imprinted polymer (MIP) beads prepared by precipitation polymerization has been evaluated using a chemometric approach. Molecularly imprinted microspheres for the selective recognition of fluoroquinolone antimicrobials were prepared in a one-step precipitation polymerization procedure using enrofloxacin (ENR) as the template molecule, methacrylic acid as functional monomer, 2-hydroxyethyl methacrylate as hydrophilic comonomer, and acetonitrile as the porogen. The type and amount of cross-linker, namely ethylene glycol dimethacrylate, divinylbenzene or trimethylolpropane trimethacrylate, to obtain monodispersed MIP spherical beads in the micrometer range was optimized using a simplex lattice design. Particle size and morphology were assessed by scanning electron microscopy, dynamic light scattering, and nitrogen adsorption measurements. Electron paramagnetic resonance spectroscopy in conjunction with a nitroxide as spin probe revealed information about the microviscosity and polarity of the binding sites in imprinted and nonimprinted polymer beads.

Direct extraction of penicillin G and derivatives from aqueous samples using a stoichiometrically imprinted polymer.

A molecularly imprinted polymer (MIP) prepared using penicillin G procaine salt as the template (PENGp) and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic species in the template molecule has been applied to the development of a molecularly imprinted solid-phase extraction (MISPE) procedure for the selective preconcentration of beta-lactam antibiotics (BLAs) from environmental water samples. Various parameters affecting the extraction efficiency of the polymer have been evaluated to achieve the selective preconcentration of the antibiotics from aqueous samples and to reduce nonspecific interactions. This resulted in an MISPE-HPLC method allowing the direct extraction of the analytes from the sample matrix with a selective wash using just 10% (v/v) organic solvent. On the basis of UV detection only, the method showed good recoveries and precision, ranging between 93% and 100% (RSD 3.8-8.9%, n = 3) for tap water and between 90% and 100% (RSD 4.2-9.1%, n = 3) for river water fortified with 30 or 60 microg L-1 (50 mL samples) penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin, and dicloxacillin, suggesting that this MIP can be successfully applied to the direct preconcentration of BLAs in environmental water samples.

Molecularly imprinted polymers as antibody mimics in automated on-line fluorescent competitive assays.

An automated molecularly imprinted sorbent based assay (MIA) for the rapid and sensitive analysis of penicillin-type beta-lactam antibiotics (BLAs) has been developed and optimized. The polymers were prepared using penicillin G procaine salt as template (PENGp) and a stoichiometric quantity of a urea-based functional monomer to target the single oxyanionic species in the template molecule. Highly fluorescent competitors (emission quantum yields of 0.4-0.95), molecularly engineered to contain pyrene labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers, have been tested as analyte analogues in the competitive assay. Pyrenemethylacetamido penicillanic acid (PAAP) was the tagged antibiotic providing for the highest selectivity when competing with PenG for the specific binding sites in the molecularly imprinted polymer (MIP). Upon desorption from the MIP, the emission signal generated by the PAAP was related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 1.81 x 10(-6) M PENG, and the detection limit was 1.97 x 10(-7) M. The sensor showed a dynamic range (normalized signal in the 20 to 80% range) from 6.80 x 10(-7) to 7.21 x 10(-6) M (20-80% binding inhibition) PENG in acetonitrile:HEPES buffer 0.1 M at pH 7.5 (40:60, v/v) solutions. Competitive binding studies demonstrated various degrees of cross-reactivity with penicillin-type beta-lactam antibiotics such as ampicillin (71%), oxacillin (66%), penicillin V (56%), amoxicillin (13%), and nafcillin (46%) and a lower response to other isoxazolyl penicillins such as cloxacillin (27%) and dicloxacillin (16%). The total analysis time was 14 min per determination, and the MIP reactor could be reused for more than 150 cycles without significant loss of recognition. The automatic MIA has been successfully applied to the direct analysis of penicillin G in spiked urine samples with excellent recoveries (mean value 92%). Results displayed by comparative analysis of the optimized MIA with a chromatographic procedure for penicillin G showed excellent agreement between both methods.

Molecularly imprinted polymers applied to the clean-up of zearalenone and alpha-zearalenol from cereal and swine feed sample extracts.

A molecularly imprinted polymer prepared using 1-allylpiperazine (1-ALPP) as the functional monomer, trimethyltrimethacrylate (TRIM) as the crosslinker and the zearalenone (ZON)-mimicking template cyclododecanyl-2,4-dihydroxybenzoate (CDHB) has been applied to the clean-up and preconcentration of this mycotoxin (zearalenone) and a related metabolite, alpha-zearalenol (alpha-ZOL), from cereal and swine feed sample extracts. The extraction of ZON and alpha-ZOL from the food samples was accomplished using pressurized liquid extraction (PLE) with MeOH/ACN (50:50, v/v) as the extraction solvent, at 50 degrees C and 1500 psi. The extracted samples were cleaned up and preconcentrated through the MIP cartridge and analyzed using HPLC with fluorescence detection (lambda (exc)=271/ lambda (em)=452 nm). The stationary phase was a polar endcapped C18 column, and ACN/MeOH/water 10/55/35 (v/v/v, 15 mM ammonium acetate) at a flow rate of 1.0 mL min(-1) was used as the mobile phase. The method was applied to the analysis of ZON and alpha-ZOL in wheat, corn, barley, rye, rice and swine feed samples fortified with 50, 100 and 400 ng g(-1) of both mycotoxins, and it gave recoveries of between 85 and 97% (RSD 2.1-6.7%, n=3) and 87-97% (RSD 2.3-5.6%, n=3) for alpha-ZOL and ZON, respectively. The method was validated using a corn reference material for ZON.

Molecular engineering of fluorescent penicillins for molecularly imprinted polymer assays.

The interaction of seven novel fluorescent labeled beta-lactams with a library of six polymer materials molecularly imprinted (MI) with penicillin G (PenG) has been evaluated using both radioactive and fluorescence competitive assays. The highly fluorescent competitors (emission quantum yields of 0.4-0.95) have been molecularly engineered to contain pyrene or dansyl labels while keeping intact the 6-aminopenicillanic acid moiety for efficient recognition by the cross-linked polymers. Pyrenemethylacetamidopenicillanic acid (PAAP) is the tagged antibiotic that provides the highest selectivity when competing with PenG for the specific binding sites in a MI polymer prepared with methacrylic acid and trimethylolpropane trimethacrylate (10:15 molar ratio) in acetonitrile in the presence of PenG. Molecular modeling shows that recognition of the fluorescent analogues of PenG by the MI material is due to a combination of size and shape selectivity and demonstrates how critical the choice of label and tether chain is. PAAP has been applied to the development of a fluorescence competitive assay for PenG analysis with a dynamic range of 3-890 muM in 99:1 acetonitrile-water solution. Competitive binding studies demonstrate various degrees of cross-reactivity for some antibiotics derived from 6-aminopenicillanic acid, particularly amoxicillin, ampicillin, and penicillin V (but not oxacillin, cloxacillin, dicloxacillin, or nafcillin). Other antibiotics, such as chloramphenicol, tetracycline, or cephapirin, do not compete with PAAP for binding to the imprinted polymer. The MI assay has successfully been tested for PenG analysis in a pharmaceutical formulation.

Continuous solid-phase extraction and preconcentration of bisphenol A in aqueous samples using molecularly imprinted columns.

A bisphenol A (BPA) molecularly imprinted polymer, the composition of which was optimised using a chemometric approach, has been applied to the selective preconcentration of the template from aqueous samples. The selectivity of the polymer toward BPA and related compounds was evaluated chromatographically. The BPA-imprinted polymer was packed in a column and used for continuous on-column solid-phase extraction (MISPE) of aqueous samples followed by subsequent analysis by HPLC with fluorescence detection of the eluted fractions. The composition of the washing solvent applied in the MISPE procedure was optimised to favour the specific interactions of the MIP with BPA and to remove the non-selectively bound matrix components. The MISPE method has proven to be effective for selective preconcentration of BPA in aqueous samples (recoveries >84% obtained in the eluate for 10-100 mL sample volumes) enabling detection and quantification limits of 1.0 and 3.3 ng mL(-1), respectively (based on 25 mL sample size). Analytical recoveries were between 92 and 101% for river water samples spiked with known amounts of BPA (30, 60, and 80 ng mL(-1)); relative standard deviations (RSD) were lower than 5.0%.