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Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)--DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE--DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE--DNA adducts as low as 180 fM in the presence of I billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methytation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotexical and epigenetic analysis. 10.1021/ac102416f

Abstract: A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was applied for simultaneous determination of stachyose, sucrose and raffinose in Chinese artichoke (Stachys Sieboldii Miq.). Carbohydrates were separated on a Bondapak NH2 column using a ternary solvent mixture of methanol–acetonitrile–water (55:25:20, v/v/v) as mobile phase. Two different crafts (method 1 and method 2) for extraction and purification of stachyose in Chinese artichoke tubers were detailed and evaluated. Method 1 can meet the purpose of quantification and method 2 is appropriate for the purpose of purification. The content of stachyose in dry tubers was determined to be 236.0mg/g (from method 1) and the purity of the extracted stachyose flour was calculated to be 87.34% (from method 2), respectively. The analytical method fulfills all the standard requirements of linearity, accuracy and precision. Therefore, it is suitable for purification and routine quantification of stachyose in Chinese artichoke. [Copyright &y& Elsevier]

This study reports the utilization of thiol-induced chemical etching of aggregation-induced emission (AIE)-active Au nanoclusters (NCs) for the facile, sensitive, and selective detection of cysteine. The AIE-active Au NCs were formed in an acidic solution containing excess Au(i)-thiolate complexes. At an acidic pH (2.0), the emission of these Au NCs was enhanced by cysteine at a concentratioin below 1 mM. However, the emission was quenched by cysteine at a high concentration, e.g., 500 mM, via the thiol-induced etching of gold, although the process occurred very slowly. Interestingly, in the absence of cysteine, increasing the solution pH enhanced the emission, while the presence of cysteine remarkably accelerated the etching-induced quenching process. The complete quenching of the emission by excess cysteine at pH 2.0 and the enhancement of the emission by the increasing pH in the absence of cysteine indicated that aurophilicity might not be involved in the AIE of the Au NCs prepared using glutathione (GSH) both as the reducing and protecting reagent. On the other hand, the etching process involved the penetration of cysteine molecules through the Au(i)-thiolate complexes, which could assemble or disassemble around the embedded Au NCs in response to the solution pH to get access to the innermost Au(0) cores. Therefore, a facile, sensitive, and selective method for the detection of cysteine was established. This method exhibited an extremely wide linear range as wide as nine orders of magnitude above the cysteine concentration, including two linear regions of the relative emission intensity of the Au NCs versus the logarithm of cysteine concentration, from 10 pM to 150 μM (correlation coefficient, 0.99851) and from 150 μM to 2 mM (correlation coefficient, 0.99866). An ultra-low limit of detection of 6.3 pM (S/N = 3) was also achieved. The developed method showed superior selectivity for cysteine relative to the 19 other natural amino acids and GSH. The method was applied for the analysis of human serum samples spiked with cysteine with satisfactory results. This study demonstrates the potential of the thiol-induced chemical etching approach as a powerful tool for studying luminescent metal NCs.

A capillary monolithic bioreactor of snake venom phosphodiesterase (SVP) was constructed to generate different single-nucleotide mass ladders of oligodeoxynucleotides for mass spectrometry (MS)-based sequencing by immobilization. The immobilization of SVP in the porous silica monolith significantly enhances its stability for prolonged and repeated applications. The constructed capillary bioreactor has the advantages of handling (sub)microliter DNA samples and having good permeability. Benefiting from its good permeability, DNA solutions can be directly injected into the sequential digestion bioreactor simply by hand pushing or a low-pressure microinjection pump. Moreover, the immobilization of SVP facilitates the elimination or repression of the metal adducts of oligodeoxynucleotides, improving the analytical performance of MS sequencing. By the application of capillary bioreactor of immobilized SVP, the sequence-specific modification of single-stranded oligodeoxynucleotide induced by a ubiquitous pollutant acrolein (Acr) was identified, demonstrating its promising applications in identification of sequence-specific damage, which may further our understanding of DNA damage caused mutagenesis.

An on-line SPE-HPLC method, using a monolithic poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-EDMA)) based weak cation-exchange (WCX) column, was developed for simultaneous determination of alpha1-adrenergic receptor antagonists in human plasma. The monolithic WCX column was prepared by an in-situ polymerization protocol and modified stepwise with ethylenediamine and chloroacetic acid. On connecting this column to an injection valve, an on-line SPE protocol could be established for removal of matrices (mainly proteins and lipids) and preconcentration of four alpha1-adrenergic receptor antagonists in human plasma. This method was validated and then used for determination of terazosin, alfuzosin, prazosin, and doxazosin in clinical plasma samples. For all analytes, each calibration curve was found to be linear over a range of 0.005-5 microg/mL (R>0.997), and the limit of detection for each analyte was 0.5 ng/mL. Recovery (>80%) and precision (RSD<15%) for inter- and intra-day assay were tested at three concentration levels of each analyte and showed acceptable results for quantitative assay. Real samples from hypertensive patients were monitored and results were in agreement with those of the conventional liquid-liquid extraction-HPLC method. Furthermore, due to its good permeability and biocompatibility, the monolithic WCX sorbent could be reused more than 300 times. The proposed method was especially appropriate for multi-analyte monitoring in plasma samples.

An ester-bonded monolithic silica column for reversed-phase high performance liquid chromatography was synthesized by reacting stearic acid with gamma-glycidoxypropyltrimethoxysilane and coupling the intermediate product onto monolithic silica column. Characterization of the prepared column was carried out with Fourier transformation infrared spectrometry and elemental analysis. The chromatographic properties of the packing were evaluated by using benzene, biphenyl, and anthracene as samples and methanol-water as binary mobile phase. The column back-pressure and separation effect were also evaluated for fast separation. The results show that the stationary phase has satisfactory chromatographic properties and resists hydrolysis between pH = 2 - 8, and it can be used for the fast-separation of solutes efficiently.