Synthesis of molecularly imprinted polymers via ring-opening metathesis polymerization for solid-phase extraction of bisphenol A.

The use of molecularly imprinted polymers (MIPs) prepared by ring-opening metathesis polymerization (ROMP) for bisphenol A (BPA) was reported in this article. The resulting MIPs have high imprinting and adsorption capacities, and can be used for separation and determination of BPA in environmental water samples. The successful application of ROMP in the molecular imprinting field is described here.

A new competitive enzyme-linked immunosorbent assay (ELISA) for determination of estrogenic bisphenols.

Bisphenol A and other hydroxylated diphenylalkanes (generally known as bisphenols) have been identified as potential estrogenic substances. In this paper, a specific polyclonal antibody was produced against these compounds by immunization of rabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serum albumin (BHPVA-BSA). The polyclonal antibody showed specific recognition of the bisphenol structure, while the cross reactions of other common phenolic compounds such as phenol, hydroquinol and p-hydroxybenzoic acid were all lower than 1%.

[Recent progress in the development of analytical methods for insulin-like growth factor-I].

Different methods for the pretreatment of insulin-like growth factor-I (IGF-I) and the derivatization of IGF-I are introduced. Detection methods such as immunoassay, chromatography, chromatography-mass spectrometry and surface plasma resonance (SPR) and so on are also reviewed.

Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction.

We describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products.

Thermosensitive and salt-sensitive molecularly imprinted hydrogel for bovine serum albumin.

A novel stimuli-responsive protein imprinted polymer for selective recognition of bovine serum albumin is presented. N-[3-(Dimethylamino)propyl]-methacrylamide, which is positively charged in neutral solution and is able to self-assemble onto the template protein through electrostatic interaction, was chosen as the functional monomer. Polymerization was carried out in the presence of N-isopropylacrylamide as an assistant monomer, which resulted in a stimuli-responsive protein imprinted polymer.

Protein-responsive imprinted polymers with specific shrinking and rebinding.

Stimuli-responsive protein imprinted polymers were obtained via a combination of molecular imprinting and reversible stimuli-responsive polymer using lysozyme or cytochrome c as template, N-isopropylacrylamide (NIPA) as major monomer, methacrylic acid (MAA) and acrylamide (AAm) as functional co-monomers, and N,N-methylenebisacrylamide (MBAAm) as crosslinker. The molecularly imprinted polymers (MIPs) can respond not only to external stimuli such as temperature and salt concentration, but also to the corresponding template protein with significant specific volume shrinking. This specific shrinking behavior was attributed to the synergistic effect of multiple-site weak interactions (electrostatic force, hydrogen bonding and hydrophobic interaction) and the cavity effect.

Polyethylene glycol diacrylate-based supermacroporous monolithic cryogel as high-performance liquid chromatography stationary phase for protein and polymeric nanoparticle separation.

A supermacroporous monolithic cryogel was directly prepared by in situ cryo-copolymerization in a stainless steel cartridge (70mmx5.0mm I.D.

Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay.

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure.

Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins.

The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins.

Multiple binding modes for dicationic Hoechst 33258 to DNA.

The binding of dicationic Hoechst 33258 (ligand) to DNA was characterized by means of the fluorescence spectra, fluorescence intensity titration, time-resolved fluorescence decay, light scattering, circular dichroism, and fluorescence thermal denaturation measurements, and two binding modes were distinguished by the experimental results. Type 1 binding has the stoichiometry of one ligand to more than 12 base pairs, and it is defined as quasi-minor groove binding which has the typical prolonged fluorescence lifetime of about 4.4 ns.

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection.

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence.

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity.

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3' (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the "wrong" genotype.

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein.

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP.

Universal molecular beacon-based tracer system for real-time polymerase chain reaction.

DNA diagnostic has been moving from expensive, low-throughput, multistep methods to inexpensive, higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays. In this method, we describe a universal molecular beacon (U-MB) as the fluorescent tracer in the real-time PCR technique.

[Analysis of recombinant erythropoietin and its tryptic digest by capillary electrophoresis and capillary electrophoresis-electrospray ionization-mass spectrometry using capillary coated with 6,6-ionene].

The microheterogeneity of recombinant human erythropoietin (rhEPO) was analyzed by capillary electrophoresis (CE) using a capillary coated with 6,6-ionene. The applicability of a volatile electrolyte for fast analysis of tryptic fragments of rhEPO with online CE-electrospray ionization-mass spectrometry (ESI-MS) was investigated, resulting in a reproducible separation of eleven rhEPO tryptic fragments within 22 min under the following conditions: running buffer 300 mmol/L acetic acid-ammonium acetate (HAc-NH4Ac), pH 4.80, separation voltage -15 kV and capillary temperature 25 degrees C.

Ionene-dynamically coated capillary for analysis of urinary and recombinant human erythropoietin by capillary electrophoresis and online electrospray ionization mass spectrometry.

In this article, a series of ionene polymers were synthesized and used to coat fused-silica capillaries for the separation of recombinant and urinary human erythropoietin (rhEPO and uEPO) standards by CE. The influence of the charge density of coatings on the separation of rhEPO and uEPO glycoforms was investigated. Then, we further studied the method for fast separation and detection of rhEPO and uEPO standards by CE-ESI-MS.

An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products.

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column.

Analysis of isoflavone daidzein in Puerariae radix with micelle-mediated extraction and preconcentration.

Nonionic surfactant oligo(ethylene glycol) monoalkyl ether (Genapol X-080) was employed as an alternative and effective solvent for the extraction of daidzein from Puerariae radix for the first time. Optimum experimental conditions were established. With 5% Genapol X-080 (w/v), liquid/solid ratio of 25:1 (mL/g), and ultrasonic-assisted extraction for 45 min, the extraction percentage of daidzein reached the highest value.

Reducing power: the measure of antioxidant activities of reductant compounds?

Electrochemical assay has been employed recently to study the activity of antioxidants; however, there is controversy as to whether reducing power fully characterizes the antioxidant activity. This study provides some essential further evidence on this point based on the reported data and mechanisms underlying the antioxidant functions as well as the anodic oxidation of phenolic antioxidants, indicating that further consideration and investigation should be made before reducing power is used as the absolute measure of antioxidant activity.

Study on the recognition of templates and their analogues on molecularly imprinted polymer using computational and conformational analysis approaches.

A simplified computational model was proposed to simulate the synthesis of molecularly imprinted polymers (MIP), removal of template and recognition of the template and its analogues by MIP. The MIPs with nicotinamide and iso-nicotinamide as templates were prepared using methacrylic acid as functional monomer. Based on our computational model, the interaction energies between the monomer and the template or its analogues were calculated, which were well correlated with the retention factors and imprinting factors obtained on HPLC columns packed with the corresponding MIP particles.

Layer-by-layer self-assembly of multilayer films containing DNA and Eu3+: their characteristics and interactions with small molecules.

Thin films of alternating DNA and rare earth ion Eu3+ layers from dilute aqueous solutions were fabricated onto quartz substrates and silicon wafers through the layer-by-layer (LbL) self-assembly technique. UV-visible spectroscopy shows that a uniform layer of DNA can be fully adsorbed onto each alternate Eu3+ layer. Microscopic FTIR spectra show Eu3+ interacts with both the phosphate groups and nitrogenous bases of DNA, and the formation of [DNA/Eu]n films induces a change of the conformation of the DNA secondary structure to a certain extent.

Synthesis of an enzyme-like imprinted polymer with the substrate as the template, and its catalytic properties under aqueous conditions.

Transition state analogues (TSAs) have long been regarded as ideal templates for the preparation of catalytically active synthetic imprinted polymers. In the current work, however, a new type of molecularly imprinted polymer (MIP) was synthesized with the substrate (homovanillic acid, HVA) as the template and hemin introduced as the catalytic center, with the use of plural functional monomers to prepare the active sites. The MIP successfully mimicked natural peroxidase, suggesting that it may not be imperative to employ a TSA as the template when preparing enzyme-like imprinted polymers and that the imprinted polymer matrix provided an advantageous microenvironment around the catalytic center (hemin), essentially similar to that supplied by apo-proteins in natural enzymes.

Sulfamethoxazole-imprinted polymer for selective determination of sulfamethoxazole in tablets.

A sulfamethoxazole (SMO)-imprinted polymer (MIP) was prepared in acetonitrile using the mixture of acrylamide and 4-vinylpyridine as functional monomers. The molecular recognition properties of the polymer was evaluated in both acetonitrile and aqueous acetonitrile mobile phases. SMO contents in two kinds of tablets were determined satisfactorily using the MIP packed HPLC column with aqueous mobile phase.

Studies of interaction between poly(allylamine hydrochloride) and double helix DNA by spectral methods.

DNA interaction with cationic polyelectrolytes promises to be a versatile and effective synthetic transfection agent. This paper presents the study on interaction between a simple artificial cationic polymer, poly(allylamine hydrochloride) (PAA), and herring sperm DNA (hsDNA) using several spectroscopic methods, including light scattering, microscopic FTIR-, CD-spectroscopy and so on. The results show that PAA interacts with DNA through both the phosphate groups and the nitrogenous bases of DNA.