Affinitive separation and on-line identification of antitumor components from Peganum nigellastrum by coupling a chromatographic column of target analogue imprinted polymer with mass spectrometry

A coupled LC-MS (liquid-phase chromatography and mass spectrometry) system consisting of a combination of a column of molecularly imprinted polymer (MIP) and a MS detector was used for affinitive separation and on-line identification of the antitumor components, harmine and harmaline, from the methanol extract of Peganum nigellastrum seeds. Three molecularly imprinted polymers were synthesized with porogens bearing different hydrogen bonding capacities with harman, the structural analogue of harmaline, and harmine as the template. The affinity and selectivity of the anti-harman MIPs for the targets, harmine and harmaline, were investigated chromatographically, and the influences of the porogens and sample loads on the retention of the target compounds were also discussed. In addition, the target binding capacities of the MIPs were evaluated by frontal chromatography. When the MIPs were further used in a LC-MS system to separate the extract of herb, it was observed that imprinting with different porogens would cause the MIPs to exhibit different tendencies to adsorb the matrix components from the herb. Though the MIP prepared with a porogen of less hydrogen bonding capacity possessed higher selectivity and stronger affinity for the targets, matrix components in the herb extract interfered with the chromatographic performance more seriously when it was used as the LC solid phase in the LC-MS system for selective extraction of harmaline and harmine from the crude herb extract. Positively, the MIPs were stable and reproducible in the separation test, and the imprinting columns could efficiently separate the antitumor components from the herb extract after the sample was simply pretreated. The work in this paper would be helpful for the further extraction and identification of certain pharmacophoric compounds in herbs by a LC-MS system using MIPs as the HPLC solid phase.     

Molecularly imprinted polymeric fibers for solid-phase microextraction

Solid-phase microextraction (SPME) is widely used in analytical laboratories for the analysis of organic compounds, thanks to its simplicity and versatility. However, the current commercially available fibers are based on nonselective sorbents, making difficult in some cases the final determination of target compounds by chromatographic techniques. Molecularly imprinted polymers (MIPs) are stable polymers with selective molecular recognition abilities, provided by the template used during their synthesis. In the present work, a simple polymerization strategy allowing the obtainment of molecularly imprinted polymeric fibers to be used in SPME is proposed. Such a strategy is based on the direct synthesis of molecularly imprinted polymeric fibers (monoliths) using silica capillaries as molds, with silica being etched away after polymerization. The system propazine:methacrylic acid was used as a model for the preparation of molecularly imprinted fibers, and its ability to selectively rebind triazines was evaluated. Variables affecting polymer morphology (i.e., polymerization time, fiber thickness) and binding-elution of target analytes (i.e., solvents, time, temperature) were studied in detail. The imprinted fiber showing the best performance in terms of selectivity and affinity for triazines was successfully applied to the extraction of target analytes from environmental and food samples.     

Novel critical point drying (CPD) based preparation and transmission electron microscopy (TEM) imaging of protein specific molecularly imprinted polymers (HydroMIPs)

We report the transmission electron microscopy (TEM) imaging of a hydrogel based molecularly imprinted polymer (HydroMIP) specific to the template molecule bovine haemoglobin (BHb). A novel critical point drying based sample preparation technique was employed to prepare the molecularly imprinted polymer (MIP) samples in a manner that would facilitate the use of TEM to image the imprinted cavities, and provide an appropriate degree of both magnification and resolution to image polymer architecture in the <10 nm range. For the first time, polymer structure has been detailed that tentatively suggests molecularly imprinted cavities, ranging from 5 to 50 nm in size, that correlate (in terms of size) with the protein molecule employed as the imprinting template. The modified critical point drying sample preparation technique used may potentially play a key role in the imaging of all molecularly imprinted polymers, particularly those prepared in the aqueous phase.     

Electrosynthesis of molecularly imprinted polypyrrole for the antibiotic levofloxacin

The development of an electrosynthesized imprinted polypyrrole (PPY) film onto a platinum sheet as sorbent phase for a fluoroquinolone antibiotic (levofloxacin) is described. Experimental conditions for the electropolymerization of PPY in the presence of the template were optimized. The molecularly imprinted polymer (MIP) film was characterized by X-Ray Photoelectron Spectroscopy (XPS) to verify the template entrapment in the polymeric matrix. After being subject to washing procedures, MIP was analyzed by XPS and a very satisfactory template removal was estimated being equal to 83%. The effectiveness of washing protocol was assessed also by UV-vis and High Performance Liquid Chromatography (HPLC) analysis of corresponding washing solutions. Rebinding experiments were performed by exposing the imprinted PPY film to levofloxacin solutions, subsequently analyzed by HPLC. The effect of solvent and time of exposure was investigated. The imprinting effect was verified by comparing recognition abilities of both MIP and not imprinted polymer (a polymer prepared in the same conditions but in the absence of the template).     

Rational design of a polymer specific for microcystin-LR using a computational approach

A computational approach for the design of a molecularly imprinted polymer (MIP) specific for Cyanobacterial toxin microcystin-LR is presented. By using molecular modeling software, a virtual library of functional monomers was designed and screened against the target toxin, employed as a template. The monomers giving the highest binding energy were selected and used in a simulated annealing (molecular dynamics) process to investigate their interaction with the template. The stoichiometric ratio observed from the simulated annealing study was used in MIP preparation for microcystin-LR. The monomers were copolymerized with a cross-linker in the presence of the template. A control (blank) polymer was prepared under the same conditions but in the absence of template. A competitive assay with microcystin-horseradish peroxidase conjugate was optimized and used to evaluate the affinity and cross-reactivity of the polymer. The performance of the artificial receptor was compared to the performance of monoclonal and polyclonal antibodies raised against the toxin. The results indicate that imprinted polymer has affinity and sensitivity comparable to those of polyclonal antibodies (the detection limit for microcystin-LR using the MIP-based assay was found to be 0.1 microg L-1), while superior chemical and thermal stabilities were obtained. Moreover, cross-reactivity to other toxin analogues was very low for the imprinted polymer, in contrast to the results achieved for antibodies. It is anticipated that the polymer designed could be used in assays, sensors, and solid-phase extraction.     

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.     

Synthesis and evaluation of a molecularly imprinted polymer for selective on-line solid-phase extraction of 4-nitrophenol from environmental water

A molecularly imprinted polymer (MIP) able to bind 4-nitrophenol (4-NP) was prepared using noncovalent molecular imprinting methods and evaluated as a selective sorbent in molecularly imprinted solid-phase extraction (MISPE) on-line coupled to a reversed-phase HPLC. It has been shown that the conditions chosen for washing the MIP and for eluting the analyte in the MISPE process are extremely important for ensuring good selectivity and recovery. River water samples, spiked with the 11 Environmental Protection Agency phenolic compounds at microgram per liter levels, were preconcentrated on-line using this MIP, and 4-NP was selectively extracted. The humic acid interference was simultaneously reduced considerably. The MIP was also compared with a commercially available highly cross-linked polymer (LiChrolut EN) and the former yielded cleaner extracts.     

In-tube moleculary imprinted polymer solid-phase microextraction for the selective determination of propranolol

A molecularly imprinted polymer (MIP) material was synthesized for use as an in-tube solid-phase microextraction (SPME) adsorbent. The inherent selectivity and chemical and physical robustness of the MIP material was demonstrated as an effective stationary-phase material for in-tube SPME. An automated and on-line MIP SPME extraction method was developed for propranolol determination in biological fluids. This simplified the sample preparation process and the chromatographic separation of several beta-blocker compounds. The method developed for propranolol showed improved selectivity in comparison to alternative in-tube stationary-phase materials, overcoming the limitations of existing SPME coating materials. Preconcentration of the sample by the MIP adsorbent increased the sensitivity, yielding a limit of detection of 0.32 microg/mL by UV detection. Excellent method reproducibility (RSD < 5.0%) and column reusability (> 500 injections) were observed over a fairly wide linear dynamic range (0.5-100 microg/mL) in serum samples. To our knowledge, this is the first report on the automated application of a MIP material for in-tube SPME. The method was inexpensive, simple to set up, and simplified the choice of SPME adsorbent for in-tube extraction. The approach can potentially be extended to other MIPs for the determination of a wide range of chemically significant analytes.     

单一结合位点分子印迹聚合物的合成及性能

采用分子自组装印迹技术在光引发条件下制备了以(S)-布洛芬为模板分子,α-甲基丙烯酸为功能单体的分子印迹聚合物。通过红外对聚合物的结构进行了表征。透射电镜结果表明,交联剂用量对印迹聚合物的形貌特征具有显著的影响。同时结合Scatchard分析研究了印迹聚合物的吸附性能及选择性识别能力,表明印迹聚合物特异性吸附容量为41μmol/g,印迹指数为2.28,对(S)-布洛芬形成单一结合位点,且表现出明显的吸附选择性。     

Extraction and purification of penicillin G from fermentation broth by water-compatible molecularly imprinted polymers

Highly selective molecularly imprinted polymer (MIP) was synthesized by using methacrylic acid as functional monomer, trimethylolpropane trimethacrylate as cross-linker, chloroform as porogen and penicillin G potassium as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the selective extraction of penicillin G from the fermentation broth samples. Various parameters affecting the extraction efficiency of the MIP particles such as; effects of pH, wash and eluent solutions were evaluated. Molecular recognition properties and selectivity of these MIPs were estimated and the obtained results revealed high affinity for the target antibiotic. Equilibrium binding experiments were done to assess the performance of the MIP relative to non imprinted polymer (NIP). After optimizing the extraction parameters in molecularly imprinted solid-phase extraction (MISPE), successful imprinting was confirmed by comparison of the recoveries from the fermentation broth, ranging between 24–26% (RSD 4.1–4.5%, n = 4) for the NIPs and 83–88% (RSD 3.1–3.4%, n = 4) for the MIPs.     

Composite QDs@MIP nanospheres for specific recognition and direct fluorescent quantification of pesticides in aqueous media

Quantum dots (QDs)-based molecularly imprinted polymer (MIP) composite nanospheres were successfully prepared via a facile and versatile ultrasonication-assisted encapsulation method. Unlike the hydrogen-bond-based MIPs, these so-prepared QDs-MIP composite nanospheres, relying on the interaction including van der Waals forces and hydrophobic forces, demonstrated excellent selectivity in aqueous media. Their small particle sizes and carboxyl-enriched polymer matrixes give rise to their good dispersibility and stability in aqueous solution, and faster adsorption and desorption kinetics, which further make them extensively applicable for chemical/biological sensors in aqueous media. Based on the fluorescence quenching via template analytes (diazinon) rebinding into the recognition cavities in the polymer matrixes, the QDs-MIP nanospheres were successfully applied to the direct fluorescence quantification of diazinon, independent of extracting templates from the MIP nanospheres, as well as further complicated and time-consuming assays. This novel method can selectively and sensitively detect down to 50 ng/mL of diazinon in water, and a linear relationship has been obtained covering the concentration range of 50-600 ng/mL. The present studies provide a new and general strategy to fabricate other multifunctional (luminescent and magnetic) inorganic-organic MIP nanocomposites with highly selective recognition ability in aqueous media and are pretty desirable for biomedical/chemical sensing applications.     

Data on the structure and recognition properties of the template-selective binding sites in semi-IPN-based molecularly imprinted polymer membranes

Data on the structure and recognition properties of the template-selective binding sites in molecularly imprinted polymer membranes are presented. Porous molecularly imprinted polymer membranes based on semi-interpenetrating polymer networks (semi-IPN) were synthesized using the method of molecular imprinting in a combination with the method of computational modeling. Methacrylic acid, itaconic acid, and acrylamide were identified as optimal functional monomers for a model template — atrazine. Optimal ratios between atrazine and functional monomers as well as their binding energies were determined using the method of computational modeling and compared with the experimental data on the adsorbtion capability of porous molecularly imprinted polymer membranes. The factors influencing quality of the template-binding sites in MIP membranes (binding energy template-functional monomer and the number of functional groups taking part in the recognition of the template molecule) were revealed. The computational atrazine-selective membranes were capable of highly-selective and effective adsorbtion of atrazine from its 10^− 9–10^− 4 M aqueous solutions, and were characterized by high stability during prolonged storage. The apparent structure of the synthetic mimics of biological receptors to triazine herbicides was compared with the structure of their natural counterparts.     

Synthesis of molecularly imprinted polymer as a sorbent for solid phase extraction of citalopram from human serum and urine

This paper describes a new method for the determination of citalopram in biological fluids using molecularly imprinted solid-phase extraction as the sample cleanup technique combined with high performance liquid chromatography. The molecularly imprinted polymers were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker, chloroform as porogen and citalopram hydrobromide as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of citalopram from human serum and urine. Effective parameters on citalopram retention were studied. The optimal conditions for molecularly imprinted solid-phase extraction consisted of conditioning with 1 mL methanol and 1 mL of deionized water at neutral pH, loading of citalopram sample (50 μg L(-1)) at pH 9.0, washing using 1 mL acetone and elution with 3 × 1 mL of 10 % (v/v) acetic acid in methanol. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of citalopram. Results from the HPLC analyses showed that the calibration curve of citalopram using MIP from human serum and urine is linear in the ranges of 1-100 and 2-120 μg L(-1) with good precisions (2.5 and 1.5 % for 10.0 μg L(-1)), and recoveries (between 82-86 and 83-85 %), respectively.     

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.     

Frontal affinity chromatography combined on-line with mass spectrometry: a tool for the binding study of different epidermal growth factor receptor inhibitors

Frontal affinity chromatography (FAC) is a simple but powerful method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of retardation of the elution front. By combination of FAC with a PE-Mariner electrospray ionization mass spectrometry, a very efficient and straightforward procedure was developed herein for analyzing the binding properties of different inhibitors of the epidermal growth factor receptor (EGFR). In this study, a polyclonal antibody prepared with a known anti-EGFR inhibitor coupled with bovine serum albumin was adopted as the stationary phase in the FAC system. Using the antibody to mimic the receptor, other different anti-EGFR inhibitors as well as the small-molecule half-antigen itself were recognized directly from the crude extract of herb, which afforded us a novel promising approach for the efficient screening of lead compounds or drug candidates from natural resources.     

农药对硫磷分子印迹聚合物合成及亲和性评价

对硫磷为模板分子,甲基丙烯酸(MAA)为功能单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,偶氮二异丁腈(AIBN)为引发剂,热引发沉淀聚合法合成对硫磷分子印迹聚合物(MIP)。通过计算机模拟和紫外分析阐述模板与功能单体的分子间作用;通过电镜观察和平衡吸附试验讨论引发剂用量与聚合物性质关系;通过吸附试验Scatchard分析测得最大吸附量为3.92μmol/g,平衡解离常数为91.7μmol/L,且具有较好的吸附特异性。该分子印迹聚合物性能优良,有望应用于环境中对硫磷的富集和检测。     

Removal and destruction of endocrine disrupting contaminants by adsorption with molecularly imprinted polymers followed by simultaneous extraction and phototreatment

This study presents a method to regenerate molecularly imprinted polymers (MIPs) used for the selective removal of endocrine disrupting compounds from aqueous effluents. Regeneration was based on solvent extraction under UV irradiation to regenerate the polymer and the solvent while destroying the contaminants. Acetone was selected as the best solvent for irradiation of estrone (E1), 17beta-estradiol (E2) and ethinylestradiol (EE2) using either UVC (254 nm) or UV-vis. A MIP synthesized with E2 as template was then tested for the extraction of this compound from a 2 microg/L loaded aqueous solution. E2 was recovered by 73+/-11% and 46+/-13% from the MIPs and a non-imprinted control polymer synthesized under the same conditions, respectively, after a single step elution with acetone. The irradiated polymers and acetone were reused for an additional extraction-regeneration cycle and showed no capacity decrease.     

Polymeric receptors for the recognition of folic acid and related compounds via substructure imprinting

A range of 2-acrylamidopyridines, showing subtle differences in solution binding toward carboxylic acids, has been investigated as functional monomers in molecular imprinting. Imprinting of N-Z-L-glutamic acid with one such monomer is shown to be effective in the creation of a molecularly imprinted polymer (MIP) with recognition properties for its template and also for larger molecules containing glutamic acid residues. In comparison to a MIP prepared via a more "traditional" approach, the new polymeric receptors exhibit reduced nonspecific binding. The new receptors are compared with previously reported urea-based receptors targeting the glutamic acid residue and receptors targeting the pteridine substructure of folic acid.     

Patterning Nanostructured,Synthetic, Polymeric Receptors by Simultaneous Projection Photolithography,Nanomolding, and Molecular Imprinting

Microscope projection photolithography is combined with nanomolding and molecular imprinting for the fast microfabrication of molecularly imprinted polymer (MIP) arrays in the form of micrometric islands of nanofilaments. Dot diameters from 70–90 μm are easily obtained using a 10× objective and a photomask carrying the desired pattern. The dots are composed of parallel nanofilaments of a high aspect ratio, 150 nm in diameter and several micrometers in length, which are obtained through a nanomolding procedure on porous alumina. The arrays are molecularly imprinted with the small molecule fluorescein or with the protein myoglobin. The fluorescein MIP arrays are able to specifically recognize their target, as demonstrated by fluorescence microscopy. A four‐fold increase in binding capacity and imprinting factor (IF = 13) is obtained compared to non‐nanostructured porous dots. Imprinting of the nanofilament arrays with the protein myoglobin as the template is also possible and allows for a high imprinting factor of 4.3. Such nanostructured microarrays of synthetic receptors obtained by projection photolithography have great potential in biosensor and biochip development.     

Nanosphere molecularly imprinted polymers doped with gold nanoparticles for high selectivity molecular sensors

We report the first attempt of using molecularly imprinted polymers (MIPs) in the shape of nanoparticles that were doped with gold nanoparticles (AuNPs) for surface enhanced Raman scattering (SERS)-based sensing of molecular species.Specifically,AuNPs doped molecularly imprinted nano-spheres (AuNPs@nanoMIPs) were synthesized by one-pot precipitation polymerization using Sudan Ⅳ as the template for the SERS sensing.The AuNPs@nanoMIPs were characterized by various modes of scanning transmission electron microscopy (STEM) that showed the exact location of the AuNPs inside the MIP particles.The effects of Au concentration and solution stirring on the shape and the polydispersity of the particles were studied.Significant enhancement of the Raman signals was observed only when the MIP particles were doped with the AuNPs.The SERS signal improved significantly with increase in the Au concentration inside the AuNPs@nanoMIPs.Selectivity measurements of the Sudan Ⅳ imprinted AuNPs@nanoMIPs carried out with different Sudan derivatives showed high selectivity of the AuNPs-doped MIP particles.