Preparation of a monolithic cation-exchange material with hydrophilic external layers by two-step reversible addition-fragmentation chain transfer polymerization.

In recent years, the efficient analysis of biological samples has become more important due to the advances of life science and pharmaceutical research and practice. Because biological sample pretreatment is the bottleneck for fast process, material development for efficient sample process in the high-performance liquid chromatography analysis is highly desirable. In this research, a cation-exchange restricted access monolithic column was synthesized by a reversible addition-fragmentation chain transfer polymerization method. Utilizing the controlled/living property of the reversible addition-fragmentation chain transfer method, a monolithic column of cross-linked poly(sulfopropyl methacrylate) was prepared first and then linear poly(glycerol mono-methacrylate) was immobilized covalently on the surface of the polymer. The monolithic material has both functionalities of cation-exchange and protein exclusion. Protein recovery of 94.6% was obtained after grafting of poly(glycerol mono-methacrylate) while the cation-exchange property of the column is still retained. In the study, the relation between the synthetic conditions and properties of the materials was studied. The synthesis conditions including the porogen, monomer concentration, and ratio of monomers/initiator/reversible addition-fragmentation chain transfer agent were optimized. The study provided a method for the preparation of restricted access monolithic columns: a bifunctional material by reversible addition-fragmentation chain transfer polymerization method.

Synthesis of tetracycline-imprinted polymer microspheres by reversible addition-fragmentation chain-transfer precipitation polymerization using polyethylene glycol as a coporogen.

Tetracycline (TC)-imprinted microspheres have been synthesized by reversible addition-fragmentation chain-transfer precipitation polymerization using PEG as a coporogen. In the synthesis, methacrylic acid and ethylene dimethacrylate were used as the functional monomer and cross-linker, respectively. 2,2'-Azobisisobutyronitrile was the initiator, and cumyl dithiobenzoate was the chain-transfer reagent. Although monodispersed microspheres were obtained using acetonitrile as porogen, the particles cannot be used in the column extraction because of the high backpressure. To increase the porosity of the material, PEG was introduced as a coporogen. The influence of the molecular weight and concentration of PEG on the morphology, binding affinity, and porosity of the molecularly imprinted polymers (MIPs) have been studied. The results demonstrated that PEG as a macroporogen increased the porosity of the polymers. Meanwhile, the column backpressure was reduced using the MIPs with higher porosity. The binding affinity of the MIPs was increased when a low concentration of PEG was employed, while it was decreased when the ratio of PEG 12,000/monomers was >0.8%. Under the optimized conditions, TC-imprinted microspheres with good selectivity and size uniformity have been obtained, which facilitates its application in the column extraction for TC determinations.

Preparation of clenbuterol imprinted monolithic polymer with hydrophilic outer layers by reversible addition-fragmentation chain transfer radical polymerization and its application in the clenbuterol determination from human serum by on-line solid-phase extraction/HPLC analysis.

A novel imprinted monolithic material with the ability of protein exclusion was developed for the selective extraction of clenbuterol (CLE) from biological samples by direct injection in the HPLC analysis. The material has an imprinted inner structure and hydrophilic outer layer. The reversible addition-fragmentation chain transfer (RAFT) polymerization was employed in the material preparation by a two-step procedure. In the first step, clenbuterol imprinted monolithic polymer was synthesized by combining the molecular imprinting and the RAFT polymerization techniques. The resulting monolithic polymer has a RAFT chain transfer agent (trithioester groups) in its structure, which was used to graft poly(glycerol mono-methacrylate) [pGMMA] in the second step by post-RAFT polymerization. The hydrophilic pGMMA layers grafted on the surface of the imprinted monolith created barriers for protein diffusion. More than 90% of bovine serum albumin can be excluded from the pGMMA coated monolithic column. Meanwhile the clenbuterol was retained selectively with a large retention factor. The result indicated that the column, denoted as RA-MIM, has both the merits of a molecularly imprinted polymer and restricted access material. By using RA-MIM as the solid-phase extraction pre-column, an on-line column-switching HPLC method for the determination of clenbuterol in human serum has been established and validated. The recoveries of clenbuterol from the serum were 87.3-96.9% in the spiked level 2-1000 ng mL(-1). Both good linearity (R = 0.999) and acceptable reproducibility (RSD < 7.0%) were obtained. The limit of detection and the limit of quantitation were 0.7 ng mL(-1) and 2.0 ng mL(-1) respectively, which is sensitive in terms of UV detection. The results have demonstrated that the RAFT polymerization can be used to synthesize bi-functional monolithic columns by using its living reaction property. The resulting RA-MIM in this research can be used for efficient clenbuterol determination by HPLC from biological samples.

Preparation of bi-layer-polymer coated silica using atom transfer radical polymerization and its application as restricted access phase for HPLC separation of hydrophobic molecules in biological fluids.

A novel restricted access material was prepared by surface initiated atom transfer radical polymerization. The bi-layer-polymer structures were created on the surface of silica layer-by-layer. The inner layer was composed of poly(styrene-co-divinylbenzene), which was grafted first for binding small molecules based on hydrophobic and π-π interactions. The poly(styrene-co-divinylbenzene) bonded silica has good selectivity for aromatic hydrocarbons. It also has hydrophobicity and column efficiency similar to a C(18) bonded silica. The material has shown good ability of protein exclusion after grafting hydrophilic polymer on the external surface while its hydrophobicity and selectivity do not have obvious change. It demonstrated that the material is still qualified for hydrophobic extraction. In the study, the relations between the polymer structures and chromatographic properties of the materials were investigated. The synthetic conditions were optimized. The results have shown that the material prepared in the study has application potential in the HPLC analysis of hydrophobic molecules from biological samples by direct injection. It demonstrated that atom transfer radical polymerization can be used as a method in the preparation of restricted access material.

Monolithic poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) column with restricted access layers prepared via reversible addition-fragmentation chain transfer polymerization.

The "living"/controlled radical polymerization has provided an opportunity in making a more homogeneous polymer, which is favorable for polymer-based monolithic column fabrication. To study its application in the preparation of separation material, a capillary poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) monolithic column has been synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The correlation between the synthetic conditions and the polymer structures and separation performance was studied. The result indicated RAFT-mediated reaction provides condition for creating polymers with narrower pore size distribution and higher column efficiency compared with traditional polymerization. The "living" property of the RAFT polymerization was further utilized to graft hydrophilic polymer on the surface of poly(ethylhexyl methacrylate-co-ethylene dimethacrylate). The hydrophilic chain modified monolithic column has both abilities of protein exclusion and small hydrophobic compound retention. The result indicated that RAFT polymerization can be used for making multifunctional material. The restricted access monolithic material synthesized by this method can be used in biological sample analysis with HPLC direct injection.

Sulfonated calix[4]arene functionalized SiO2@TiO2 for recognition of lysine methylation.

Lysine methylations are common protein post-translational modifications (PTMs), that play significant roles in regulating gene activities. Studies of their functions and connections with diseases have important values. However, due to the small variations from their native structures and very low component proportions, it is very difficult to extract methylated peptides from biological mixtures. In this research, a new material that utilizes sulfonated calix[4]arene (SC4A) as the recognition unit and silica coated with TiO2 as carrier, denoted as SiO2@TiO2@SC4A, was synthesized. The equilibrium binding experiments demonstrated that SiO2@TiO2@SC4A can identify lysine and arginine methylation and peptides with these methylated residues. The maximum isotherm binding capacities are 70.0, 55.9, 31.4 and 24.8 µmol g-1 for Lys(Me3), Lys(Me)2, Lys(Me) and Lys, respectively. It demonstrated that the higher the degree of methylation, the stronger the interactions. In addition, the analyses of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) demonstrated that peptides with methylated lysine or arginine can be selectively extracted from spiked histone trypsin digestion. The recoveries for the spiked GGAK(Me)R, GGAKR(Me)2 and GGAK(Me)3R are 83%, 78%, and 84% respectively. The experiments from the nuclear extracts of HeLa cells also illustrated that SiO2@TiO2@SC4A holds a potential in the enrichment and identification of lysine methylations.

Novel restricted access chiral stationary phase synthesized via atom transfer radical polymerization for the analysis of chiral drugs in biological matrices.

A new restricted access chiral stationary phase was designed and synthesized via atom transfer radical polymerization (ATRP). Surface modified bi-functional silica with chiral selector in the inner layer and hydrophilic external layer was prepared by taking advantage of the controlled/living property of the ATRP method. ATRP initiator bound porous silica was synthesized to perform surface initiated polymerization. The chiral stationary phase was then synthesized by grafting poly(glycidyl methacrylate) (pGMA) on the surface of the silica through "grafting from" polymerization followed by beta-cyclodextrin (beta-CD) immobilization. On the surface of this beta-CD immobilized material, the external pGMA layer was synthesized via second round ATRP using the initiator on the material. The hydrophilic structure was formed after the hydrolysis, which created a diffusion barrier for proteins. The new chiral restricted access material (chiral-CD-RAM) was characterized and its abilities for chiral separation and protein exclusion were evaluated. The result demonstrates that enantio-separations can be achieved for several drugs in HPLC using chiral-CD-RAM as stationary phase. Meanwhile, good protein recovery has been obtained. It indicated that this chiral-CD-RAM can be used for determination of certain chiral drugs in biological samples with direct injection in the HPLC analysis.

Enrofloxacin-imprinted monolithic columns synthesized using reversible addition-fragmentation chain transfer polymerization.

Molecularly imprinted monolithic columns for selective separation of enrofloxacin were prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT)-mediated radical polymerization. Different ratios of initiation system were used in the synthesis. The structures of the monoliths were characterized to study the relationship between the synthetic conditions and morphology of the monolithic material. The separation performance of the monoliths was evaluated by liquid chromatography. Under optimized synthetic conditions, a monolithic molecularly imprinted polymer (MIP) with high selectivity and improved column efficiency was obtained. The research has shown that RAFT polymerization provides more adjustable conditions for making monolithic materials with different morphologies. The results also demonstrated that homogeneous macro-pore size distribution and large specific surface area are the key factors providing good separation ability and column efficiency for MIP monolithic structures.

Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer.

High-performance liquid chromatographic separation is performed to extract active components from the traditional Chinese medicine Polygonum cuspidatum using a trans-resveratrol imprinted polymer. Good separation and purification of trans-resveratrol and emodin from the Polygonum cuspidatum extract are achieved after condition optimization. The extraction recoveries are 83% and 99% for trans-resveratol and emodin, respectively. The results show that the molecularly imprinted polymer can be used as a selective extraction material for the extraction and purification of trans-resveratrol and emodin from Polygonum cuspidatum.

Preparation and application of a restricted access material with hybrid poly(glycerol mono-methacrylate) and cross-linked bovine serum albumin as hydrophilic out layers for directly on-line high performance liquid chromatography analysis of enrofloxacin and gatifloxacin in milk samples.

A novel restricted access material (RAM) was prepared through combination of hydrophilic polymer poly(glycerol mono-methacrylate) and cross-linking bovine serum albumin (BSA) for direct biological analysis. In the material preparation, the poly(styrene-co-divinylbenzene) and poly(glycerol mono-methacrylate) were grafted on silica successively by atom transfer radical polymerization. Then the BSA was adsorbed on the material and cross-linked through an in-column process. The BSA recoveries of the resulted RAM were higher than 99.3%. Small molecules such as alkylbenzene and quinolone antibiotics could be retained with a reversed phase mechanism. The column packed with RAM had good long term stability and could last at least five months without significantly changed in its efficiencies. An on-line SPE/HPLC method for the analysis of enrofloxacin and gatifloxacin in milk samples was established by using the resulted RAM as the solid-phase extraction material. The detection and quantitation limits of two antibiotics were 8.22 and 27.4 ng mL-1, respectively. In the method validation, least recovery 88.5% with relative standard deviation of less than 3.9% was obtained. It demonstrated that the method is reliable and the material can be used for the direct analysis of veterinary drug residues in biological samples.

Preparation of an epitope-imprinted polymer with antibody-like selectivity for beta2-microglobulin and application in serum sample analysis with a facile method of on-line solid-phase extraction coupling with high performance liquid chromatography.

Molecularly imprinted polymers (MIPs) for protein recognition have great application potential in the biological analysis. However, preparation of protein imprinted polymer is still facing challenge. Beta2-microglobulin (ß2m) is a protein biomarker that can be used in diagnosis of different diseases. In this research, a novel MIP with ability of ß2m recognition has been developed by epitope and surface-confined imprinting approaches. A peptide with sequence of MIQRTPKIQ was selected as template. A strategy of combination of hierarchical imprinting and template immobilization was employed in the ß2m-MIP synthesis. Imprinted binding sites with open-entrance have been created that have good accessibility for ß2m and facilitated fast reversible binding kinetics. The experimental results demonstrated that the MIP has good selectivity. It can differentiate the template from peptide with different sequence and distinguish the ß2m from other proteins with similar size and pI values. After binding property study of the ß2m-MIP, a method of ß2m determination in serum was established in which ß2m was on-line extracted by MIP and analyzed by HPLC process. The recoveries for spiked serum was ≥83% with RSD <1.1%, indicating that the method has good accuracy and precisions. The LOD and LOQ were 0.058 and 0.195mgL-1 respectively, which meet the requirements of the ß2m analysis. The successful application of the ß2m-MIP demonstrated that ß2m has reversible binding on the MIP with a kinetics that can meet the requirements of the HPLC analysis. It also indicated that the ß2m-MIP has good mechanical strength and reusability that can be applied reliably in the practical analysis. As a synthetic antibody, ß2m-MIP is advantageous compared to the biological molecules.

An epitope imprinted polymer with affinity for kininogen fragments prepared by metal coordination interaction for cancer biomarker analysis.

Development of synthetic antibodies for early-stage cancer diagnosis is a pursued goal in materials research. Molecular imprinting has shown advantages for this purpose, whereas preparation of molecularly imprinted polymer (MIP) for peptide/protein recognition is still a challenge. In the present study, a new MIP as an artificial antibody for biomarker analysis was synthesized by epitope and surface-confined imprinting approaches. The target peptides (K-1944 and K-2209) were the amino acids 440-456 and 438-456 fragments of high molecular weight kininogen that have sensitivity and specificity for the diagnosis of gastric, colorectal and liver cancers. For molecular imprinting, a heptapeptide, as an epitope for recognition, was selected as a template and immobilized on silica. Metal coordination between Cu(ii) and template residues (His and Asp) was employed to create the binding sites. 4-Vinylpyridine was used as both the monomer and coordinating ligand. After imprinting polymerization and silica removal, spherical MIP (DQGHGHQ-MIP) with recognition ability was obtained successfully. The MIP could distinguish the template from one amino acid mismatched peptide. It also has surface-confined binding sites with good affinity for epitope-containing larger molecules. The MALDI-TOF analysis demonstrated that K-1944 and K-2209 could be selectively extracted from spiked human serum by the MIP. The solid phase extraction by DQGHGHQ-MIP coupled with HPLC was performed and 71-88% recoveries for K-1944 and K-2209 in spiked serum were obtained. The results demonstrated that DQGHGHQ-MIP could be used as an artificial antibody in the target peptide analysis with good extraction and sample clean-up performance.

A molecularly imprinted polymer as an antibody mimic with affinity for lysine acetylated peptides.

Lysine acetylation is a widespread protein post-translational modification (PTM) that plays a central role in diverse physiological processes. The study on the scope and pattern of lysine acetylation is an important subject in the proteomic research. However, identification of lysine acetylation from biological sources is a great challenge due to the low abundance in the massive background of unmodified proteins and the dynamic fashion of the modifications. In this research, a novel molecularly imprinted polymer (MIP) with high affinity for peptides containing acetylated lysine (Kac) has been synthesized by the combination of an epitope and surface-confined imprinting strategy. A dipeptide: KacA, containing acetylated lysine and alanine residues, was used as the template and immobilized on the sacrificial silica support. After hierarchical imprinting and removal of silica, the surface-confined cavities were created on the resulting KacA-MIP material. The equilibrium binding and HPLC experiments demonstrated that the KacA-MIP has good selectivity and epitope affinity. It can differentiate Lys-acetylated peptides (Kac-peptides) from their native structures and has higher affinity for Kac-peptides with different sequences. The selectivity of the MIP was also proved by its ability in the extraction of Kac-peptides from spiked histone digest and by its enrichment performance in the whole cell lysates. The study developed a method of MIP preparation with affinity for PTM peptide based on recognition of peptide side chains. It also indicated that the MIP has potential to be used as an antibody mimic in the PTM analysis.

Crystallization and preliminary X-ray analysis of anti-cancer agent 3-(9-acridinylamino)-5-(hydroxymethyl)aniline complexed with the DNA hexamer d(CGTACG)2.

3-(9-Acridinylamino)-5-(hydroxymethyl)aniline (AHMA) is an anti-cancer agent with significant efficacy against murine leukemia and solid tumors. As a DNA topoisomerase inhibitor, AHMA is proposed to form a ternary complex with DNA and topoisomerase and bind to DNA in an intercalative manner. In order to understand the interactions between AHMA and DNA and study the structure-function relationship of amsacrine analogue, the AHMA-d(CGTACG)(2) complex was crystallized using the sitting-drop vapor-diffusion method. The native crystals diffract to 2.9-A resolution and belong to space group P3(1)21 or P3(2)21 with unit-cell parameters a=b=57.52, c=122.17 A when analyzed using Cu Kalpha radiation. Patterson map indicates that in the crystal, the directions of the DNA base stacking are nearly perpendicular to the c-axis of the crystal unit cell.

Molecularly imprinted solid-phase extraction of (-)-ephedrine from Chinese Ephedra.

Method of molecularly imprinted solid phase extraction (MISPE) of (-)-ephedrine from Chinese Ephedra has been developed in the research. The molecularly imprinted polymer (MIP) with good selectivity and affinity for (-)-ephedrine was synthesized with (-)-ephedrine as the template, methacrylic acid as the functional monomer. The washing and elution conditions in MISPE were selected and optimized for efficient analyte extraction and sample clean-up. A clean analytical HPLC base line of ephedra extract was obtained after MISPE, which indicated that the sample pre-treatment was efficient. Good recovery and precision were obtained in the assessment for the MISPE-HPLC procedure, which demonstrated it is a reliable method and can be used for the determination of (-)-ephedrine in herbal ephedra.

An artificial receptor synthesized by surface-confined imprinting for the recognition of acetylation on histone H4 K16.

A novel artificial receptor has been synthesized using surface-confined imprinting for the recognition of lysine acetylation in histone H4. The material has high recognition fidelity and epitope affinity. It was demonstrated that acetylated Lys plays a role in binding site creation and peptide imprinting can be performed in phosphate buffer.

Synthesis and application of molecularly imprinted polymer on selective solid-phase extraction for the determination of monosulfuron residue in soil.

A novel sample clean-up procedure using molecularly imprinted polymer as the solid-phase extraction material for the determination of monosulfuron residue in soil samples has been developed. The molecularly imprinted polymer (MIP) was synthesized by non-covalent method with monosulfuron as the template. The selectivity and affinity of the MIP was evaluated by equilibrium adsorption and HPLC experiments, which demonstrated that the MIP has specific affinity for the template. The template-MIP interaction was studied by investigating the influence of different mobile phases on the retention of the template, which provided basic knowledge for the selection of the washing and elution solutions in the molecularly imprinted solid-phase extraction (MISPE) process. The study indicated that polar organic solvents with hydrogen bonding abilities have stronger eluting strength for the monosulfuron. After the MISPE procedure, a clean baseline was obtained in the HPLC quantification analysis. The recoveries of the method using the combination of MISPE and HPLC were above 93% and the R.S.D. was less than 3.2% in the soil sample determinations. Low detection limit (0.08 microg g(-1), when defined as 3 times of the noise) was also obtained in the method evaluation study.

Determination of melamine and cyromazine in milk by high performance liquid chromatography coupled with online solid-phase extraction using a novel cation-exchange restricted access material synthesized by surface initiated atom transfer radical polymerization.

A novel strong-cation-exchange restricted access material has been synthesized by atom transfer radical polymerization (ATRP). In the synthesis, poly(3-sulfopropyl methacrylate-co-ethylene dimethacrylate), [p(SPM/EDMA)] was grafted on the silica by surface-initiated ATRP first. The poly(glycerol mono-methacrylate) [pGMMA] was then immobilized on the external surface, which created a chemical diffusion barrier for protein exclusion. The resulting Sil-g-p(SPM/EDMA)-g-pGMMA has both functions of protein exclusion and cation exchange, exhibiting the property of cation-exchange restricted access material. The application of Sil-g-p(SPM/EDMA)-g-pGMMA has been studied by the determination of melamine and cyromazine in bovine milk using the online solid-phase extraction/HPLC method. In the process, the Sil-g-p(SPM/EDMA)-g-pGMMA was used for the sample pre-treatment and a HILIC column was employed as the analytical column. The method has shown good accuracy, precision and low limits of detections. The result demonstrated that the Sil-g-p(SPM/EDMA)-g-pGMMA can be used for the cation extraction from biological samples by direct HPLC injection.

Study of the molecularly imprinted polymers for selective binding of the mono-substituted sulfonylurea herbicides.

A new molecularly imprinted polymer (MIP) was synthesized for the selective extraction of mono-substituted sulfonylurea herbicides, with monosulfuron as the template and acrylamide as the functional monomer. The recognition property and affinity of the MIP for monosulfuron and its analog, monosulfuron-ester, were evaluated by equilibrium adsorption and a chromatographic study. Computer modeling, including simulated annealing and semi-empirical quantum calculation, was employed to study the recognition mechanism. The computer modeling demonstrated that monosulfuron can form multiple hydrogen bonds with methacrylic acid and acrylamide, whereas monosulfuron-ester cannot form a stable complex with these two functional monomers, which aligns with the results of the rebinding experiment. The selectivity study further demonstrated that binding sites in the MIP interact with the hydrogen in the acylamino group of mono-substituted sulfonylurea. A comparison experiment also showed that monosulfuron-imprinted MIP offers better selectivity for monosulfuron-ester than the commercial C18 high-performance liquid chromatography stationary phase material.

Preparation of a bifunctional pyrazosulfuron-ethyl imprinted polymer with hydrophilic external layers by reversible addition-fragmentation chain transfer polymerization and its application in the sulfonylurea residue analysis.

A new bifunctional pyrazosulfuron-ethyl imprinted polymer was synthesized by the combination of molecular imprinting technology and living radical polymerization. In the synthesis, the pyrazosulfuron-ethyl imprinted polymer was obtained by the reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization followed by grafting poly(glyceryl monomethacrylate) (pGMMA) by the post-RAFT polymerization. In this research, we used polyethylene glycol (PEG) as the polymeric porogen in order to increase the porosity of the material which is a new porogen application in the precipitation polymerization. The imprinted polymer has selectivity for the template and ability of humic acids exclusion which has shown the merits of molecularly imprinted polymers and restricted access materials. An online solid-phase extraction/HPLC method for the analysis of three sulfonylurea residues in soil samples has been developed and validated. The recovery of 81-99% in the spiked levels of 40-200 µg kg(-1) was obtained and the limit of detection (LOD) and limit of quantification (LOQ) were less than 4.8 and 15.9 µg kg(-1) respectively. The results demonstrated that this bifunctional material can be used for the efficient pyrazosulfuron-ethyl extraction in the sulfonylurea residue analysis from environmental samples.