Influence of the presence of surfactants and humic acid in waters on the indophenol-type reaction method for ammonium determination

This work has evaluated the influence of humic acid and/or surfactants in the quantification of ammonium in waters with the indophenol-type reaction method. Thymol has been employed with the colorimetric method for sample ammonium concentrations between 0.25 and 1 mg L⁻¹. In addition, SPE/diffuse reflectance method has been used for quantification of ammonium at low concentrations (between 0.025 and 0.25 mg L⁻¹). Matrix effect owing to humic substances were observed with the colorimetric method when the concentration was equal or higher than 25 mg L⁻¹. Lower concentrations of humic compounds produced matrix effects with the more sensitive SPE/diffuse reflectance method. Generalized H-point standard addition method (GHPSAM) was employed for evaluating the effect of humic acids and for eliminating the bias error produced by humic acids. Real water samples of different types were analyzed and accurate results for ammonium concentration were obtained with both procedures in presence or absence of humic acids. Cationic and anionic surfactants inhibited the derivatization reaction at percentages up to 0.001% and 0.5%, respectively, and non-ionic surfactants at percentages of 5.5%     

Electrophoresis studies on the contaminating glycosaminoglycans in commercially available hyaluronic acid products

Hyaluronic acid (HA) samples showing inhibition effect on digestion with testicular hyaluronidase (HAase) were found from 16 commercially available HA products, which were supplied from 11 different manufacturers. Most of these HA samples (six samples) were derived from the rooster comb, and one sample was derived from the human umbilical cord. HA oligosaccharides produced by exhaustive digestion of these HA samples with testicular HAases were monitored by capillary electrophoresis, and we found that a few HA samples gave no oligosaccharide products. Detailed analysis of HA samples by cellulose acetate membrane electrophoresis revealed that the HA samples were not digested with HAase because of the presence of a small amount of dermatan sulfate (DS). Analysis of disaccharide units of these HA samples produced by digestion with chondroitinase ABC supported the observations. And the content of DS in the sample was estimated to be ca. 8%. In contrast, these HA samples were easily digested with bacterial hyaluronate lyases from Streptomyces hyalurolyticus and Streptococcus dysgalactiae and gave endproducts of unsaturated disaccharide or unsaturated tetra- or hexasaccharides. The results suggested that the inhibitory effect of DS on HAase is specific to endo-type hydrolase (i.e. testicular HAase). In addition, pharmaceutical preparations of HA derived from rooster comb were easily digested with testicular HAase. These findings will be useful information for clinical or cosmetic use of HA preparations in terms of their half-life.     

Impact factors and thermodynamic characteristics of aquatic humic acid loaded onto kaolin

The adsorption of humic acid (HA) on kaolin particles was studied at various conditions of initial solution pH, ionic strength and solid-to-liquid ratio. The resulting affinity of interactions between humic acid and kaolin was attributed to the surface coordination of HA in ambient suspensions of mineral particles and the strong electrostatic force at low pH. Addition of inorganic salt can also influence the adsorption behavior by affecting the HA molecular structure, the clay particle zeta potential and so on. Equilibrium data were well fitted by the Freundlich model and implied the occurrence of multilayer adsorption in the process. In addition, the enthalpy dependent of system temperature was 79.17 kJ/mol, which proved that the mechanism of HA adsorption onto kaolin was comprehensive, including electrostatic attraction, ligand complexation and hydrogen bonding.     

Identification and quantification of polar naphthalene derivatives in contaminated groundwater of a former gas plant site by liquid chromatography-electrospray ionization tandem mass spectrometry

A liquid chromatography (LC) method followed by electrospray ionization (ESI) and tandem mass spectrometry (MS-MS) was developed for the quantification of acidic naphthalene derivatives in the concentration range 0.1 to 100 microg/l without excessive sample preparation. For optimal sensitivity the LC-MS-MS measurements were performed recording mass fragmentation by collision induced dissociation in the multiple reaction mode. The collision energy was optimized for every analyte. The matrix effects of the sample were investigated by spiking standards of 1-naphthoic acid with humic acid (HA) and with calcium chloride. While HA decreased the signal intensity an increase was observed in the presence of calcium chloride. For the investigated groundwater samples of a tar oil contaminated site a complete separation of the analytes from the sample matrix by reversed-phase separation could be obtained. The absence of matrix effects on quantification results was confirmed by comparison of results based on external calibration with those based on standard addition of the analytes to a groundwater sample. In four groundwater samples of the contaminated site naphthalene derivatives like 1-naphthoic acid, 2-naphthoic acid, 1-naphthylacetic acid, 2-naphthylacetic acid, 1-hydroxy-2-naphthoic acid, 2-hydroxy-3-naphthoic acid, and naphthyl-2-methylenesuccinic acid have been detected.     

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.     

Influence of extractant on quality and trace elements content of peat humic acids

Among several extractants used to isolate humic acids (HA) from terrestrial environments, sodium hydroxide (NaOH) and sodium pyrophosphate (Na₄P₂O₇) are the most utilized. In order to evaluate the influence of these different extractant solutions on the HA quality and on their trace elements content, HA were isolated from five Sphagnum-peat samples using three different solutions: (a) 0.5 M NaOH; (b) 0.1 M Na₄P₂O₇; (c) 0.5 M NaOH + 0.1 M Na₄P₂O₇. The obtained HA have been analyzed with respect to ash content, elemental composition, main atomic ratios and characterized by FT-IR and total luminescence (TL) spectroscopies. In addition, both raw peat and HA have been analyzed using X-ray fluorescence in order to determine the Br, Cu, Fe, Ni, Pb and Zn contents.

Results showed that HA extracted with NaOH and NaOH + Na₄P₂O₇ are quite similar with respect to ash, elemental contents and spectroscopic characteristics, while Na₄P₂O₇ solution, which in general reduces the extraction yield, seems to affect the nature of HA, featuring a more complex and aromatic character. With respect to the contents in the corresponding raw peat samples, the HA fractions were richer in Br, Cu and Ni, regardless of the extractant used, and poorer in Fe, Pb and Zn. Further, Br, Cu, Ni and Zn were more concentrated in HA extracted with Na₄P₂O₇ than in those extracted with NaOH and NaOH + Na₄P₂O₇, probably because of the greater affinity of these elements for these more aromatic humic molecules.     

Enhanced radiation-induced cell killing by Herbimycin A pre-treatment

Herbimycin A (HA), as in Geldanamycin, binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an integral role in cancer cell growth and survival, because it maintains the stability of several key proteins by its chaperone's activity. It is known that some of the proteins associated with radiation responses are functionally stabilized by Hsp90. In this study, we investigated the effect of HA on radiosensitivity in human cancer cells and the mechanism related to the sensitization. In order to gain a mechanistic insight of this sensitization, we examined repair of DNA double-strand breaks (DSBs) in irradiated human cancer cells pre-treated with HA, as unrepaired DSBs are thought to be the main cause of radiation-induced cell death. Cellular radiosensitivity was determined by clonogenic assay, and the DSB rejoining kinetics was examined by constant field gel electrophoresis. SQ-5, a lung squamous carcinoma cell line, showed synergistic increase in radiosensitivity when cells were pre-treated with HA. In addition, HA significantly inhibited repair of radiation-induced DSBs. These results suggest that the combination of HA and ionizing radiation may be a useful therapeutic strategy for treating certain cancer cells.     

Studies of structural alterations of humic acids from conifer bark residue during composting by pyrolysis-gas chromatography/mass spectrometry using tetramethylammonium hydroxide (TMAH-py-GC/MS)

Pyrolysis-gas chromatography/mass spectrometry using tetramethylammonium hydroxide (TMAH-py-GC/MS) was used to characterize the humic acids (HAs) produced during the composting of conifer bark. The syringyl to guaiacyl ratios of HAs during composting were estimated from the peak area ratios for 3,4,5-trimethoxy to 3,4-dimethoxy benzene derivatives (0.11–0.50), which are characteristic of conifer species. The peak areas for nitrogen-containing and fatty acid pyrolysate compounds increased significantly during composting, indicating the degree of humification during composting. HA samples from the bark composts contained higher levels of diterpene resin acids, monoterpenes and sesquiterpenes. To investigate the species of terpenes, the HA was extracted with ethanol, and the components of the extract classified into α-HA and hymatomelanic acid (HMA) fractions, respectively. The peak areas for terpenes in the α-HA fraction were much smaller than those in the original HA, indicating that the majority of terpenes were extracted into the HMA fraction. If terpenes were to bind to HMA via unspecified interactions, no peaks would be apparent for the direct injection of HMA in ethanol into the GC/MS without pyrolysis. A comparison of the total ion chromatograms of HMA for the TMAH-py-GC/MS and GC/MS revealed that terpenes in the HA had been incorporated into polymeric structures of HMA. These results indicate that terpenes are transferred from the raw bark and incorporated into HA fractions during the composting processes.     

Properties of different natural organic matter influence the adsorption and aggregation behavior of TiO2 nanoparticles

Natural organic matter (NOM) has considerable influence on the aggregation and stability of titanium dioxide nanoparticles (TiO₂ NPs). However, the effect of chemical properties of NOM on the interface interaction and the aggregation process is still not clear. In this study, we investigated the effects of two typical types of NOM (fulvic acid (FA) and humic acid (HA)) on its adsorption onto TiO₂ nanoparticles and their aggregation behavior in aqueous phase. Nuclear Magnetic Resonance (NMR) was used to study their functional groups, indicating that HA has a stronger hydrophobicity than FA. The presence of HA or FA lowered the critical coagulation concentration (CCC) of TiO₂ NPs, while FA showed a more significant effect. HA promotes the aggregation of TiO₂ NPs when ionic strength (IS) > CCC, which was likely due to the bridging effect. Contact angle measurements indicate that HA has higher hydrophobic properties than FA, and it is easier to transfer from water to the surface of TiO₂ NPs. Transmission Electron Microscopy (TEM) was applied to investigate the aggregate formation and colloid interface morphology of NOM-coated-TiO₂ NPs and NOM entanglement. Different structures of HA and FA result in various behavior and their interface interaction mechanisms including IS-induced entanglement and NOM/IS bridging.     

Electrospray ionization mass spectrometry of terrestrial humic substances and their size fractions

Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the average molecular mass of terrestrial humic substances, such as humic (HA) and fulvic (FA) acids from a soil, and humic acid from a lignite (NDL). Their ESI mass spectra, by direct infusion, gave average molecular masses comparable to those previously obtained for aquatic humic materials. The soil HA and FA were further separated in size-fractions by preparative high performance size exclusion chromatography (HPSEC) and analyzed with ESI-MS by both direct infusion and a further on-line analytical HPSEC. Unexpectedly, their average molecular mass was only slightly less than for the bulk sample and, despite different nominal molecular size, did not substantially vary among size-fractions. The values increased significantly (up to around 1200 Da) after on-line analytical HPSEC for the HA bulk sample, at both pH 8 and 4, and for the HA size-fractions when pH was reduced from 8 to 4. It was noticed that HA size-fractions at pH 8 were separated by on-line HPSEC in further peaks showing average masses which progressively increased with elution volume. Furthermore, when the HA and NDL bulk samples were sequentially ultracentrifuged at increasing rotational speed, their supernatants showed mass values which were larger than bulk samples and increased with rotational speed. These variations in mass values indicate that the electrospray ionization is dependent on the composition of the humic molecular mixtures and increases when their heterogeneity is progressively reduced. It is suggested that the dominance of hydrophobic compounds in humic supramolecular associations may inhibit the electrospray ionization of hydrophilic components. Our results show that ESI-MS is reasonably applicable to humic substances only after an extensive reduction of their chemical complexity.     

Effect of pH and Ionic Strength on the Mechanism of Association of Rodenticides with Natural Organic Components of Soil

The mechanisms of association of hydrophobic organic contaminants with natural organic matter (for example humic acid, HA) in soil and sediment are a major objective of environmental and geochemical research. This paper discusses a general model for studying the process of association between a series of rodenticides and humic acid, by use of a C₁₈ stationary phase. An approach based on extended Langmuir distribution isotherms was used to study the effect of bulk solvent pH and ionic strength (adjusted by addition of sodium cation) on the mechanism of HA–rodenticide binding. The results demonstrated that: (i) HA can be adsorbed on the surface of the C₁₈ phase; (ii) the rodenticides can be associated with HA adsorbed on the C₁₈ surface; and (iii) ionic strength and bulk solvent pH both modify the conformation of HA and thus its mechanism of association with the rodenticide molecules.     

Mass spectrometry of humic substances of different origin including those from Antarctica A comparative study

Mass spectra of humic acids (HA) from different sampling sites (Antarctica, Brazil, Czech Republic, Mexico and USA) and origin (plant, soil, peat, and coal derived) were obtained by laser desorption/ionization time of flight mass spectrometry (LDI-TOF MS). Optimisation of the experimental conditions are given as the optimal value of the laser energy at 20–30% higher than the threshold. Under these conditions, reproducible mass spectra of HA samples were obtained. In the mass spectra the majority of the peaks are observed in the m/z region 100–1000 Da. Mass spectra fingerprints of HA were analyzed and, in spite of the differences in their origin, a number of common features and profiles (patterns of peaks) were observed in most of the samples. Very similar structural groups (patterns) of the peaks are present in the m/z range 717–918 Da for HA samples of quite different origins, countries or continents.

The tandem LDI-TOF MS and multivariate statistical tools allowed us to extract and elucidate underlying information contained in the mass spectra of the HA samples under study. Applying principal components and cluster analysis, it was, e.g. demonstrated that most of the Antarctica HA samples show distinguishable differences when compared with humic acids from other continents and of different origin.     

Kinetic study of uranium speciation in model solutions and in natural waters using Competitive Ligand Exchange Method

Kinetic speciation of uranium in model solutions containing uranium and humic acid (HA) and in natural waters has been investigated by Competitive Ligand Exchange Method (CLEM). In alkaline freshwaters, most of uranium species were uranium-carbonate species, which were labile in the CLEM experiment. The uranium speciation of every sample was characterized either as “labile” or “non-labile” uranium complexes depending on the dissociation rate coefficients of the complexes. The results showed that as the U(VI)/HA ratio was decreased, the dissociation rate coefficients decreased and the labile fraction decreased as well. When the U(VI)/HA ratio was 0.1, the labile fraction of the U(VI)-HA increased with increasing pH; however, there was no pH effect on the dissociation of U(VI)-HA complexes at lower U(VI)/HA ratios. Chelex-100 had some limitations in its use for the study of dissociation of U(VI)-HA complex at very low U(VI)/HA ratios. By developing an analytical method and procedure for quantitative determination of kinetic parameters for the dissociation of uranium-HA complexes in model solutions and natural waters, this work has made a substantial contribution to analytical chemistry.     

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit

DNA extracted from forensic samples can be degraded and also contain co‐extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpF?STR^® SGM Plus^® kit. The co‐amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX‐labelled primers. Co‐amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.     

一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片

为了实现对核酸的高灵敏度检测,构建了一种新型的液滴式数字聚合酶链式反应(dd PCR)芯片.该芯片由产生液滴的聚二甲基硅氧烷(PDMS)模块和储存液滴的玻璃腔室构成.实验结果表明,该芯片可以在25 min内产生2×106个直径为20μm的微液滴(体积4.187 p L).利用该芯片定量检测了表皮生长因子受体(EGFR)基因第19号外显子,在DNA浓度为106~101copies/μL范围内呈现良好的线性关系(R2=0.9998);在浓度为106copies/μL的19号外显子野生型DNA中检测105~100copies/μL的突变型DNA,其检测敏感度可达到0.0001%.该方法在同一芯片上实现了液滴产生、核酸扩增和荧光信号读取的功能,在核酸绝对定量及痕量突变基因的检测中具有潜在应用前景.     

胡敏酸的结构特征及其吸附行为

使用0.5mol/L NaOH和0.1mol/L Na_4P_2O_7溶液分别从土壤中提取胡敏酸,并且对其进行了元素分析、红外光谱、固态13C核磁共振的定性、定量研究。结果表明,两种胡敏酸的性质很相似,但也存在一些微小差异,Na_4P_2O_7提取的胡敏酸比NaOH提取的具有芳香度较大、聚合度较高、极性官能团含量较多的特点。测定了菲在6个胡敏酸上的吸附等温线,Freundlich模型很好的拟合了所有吸附等温线,相关系数r均在0.992以上。有机碳分配系数K_(oc)与胡敏酸中极性碳(POC)之间存在明显的线性相关关系,并受到提取剂类型的影响。     

An improved liquid chromatography–tandem mass spectrometry method for the quantification of 4-aminobiphenyl DNA adducts in urinary bladder cells and tissues

Exposure to 4-aminobiphenyl (4-ABP), an environmental and tobacco smoke carcinogen that targets the bladder urothelium, leads to DNA adduct formation and cancer development [1]. Two major analytical challenges in DNA adduct analysis of human samples have been limited sample availability and the need to reach detection limits approaching the part-per-billion threshold. By operating at nano-flow rates and incorporating a capillary analytical column in addition to an online sample enrichment step, we have developed a sensitive and quantitative HPLC–MS/MS method appropriate for the analysis of such samples. This assay for the deoxyguanosine adduct of 4-ABP (dG-C8-4-ABP) gave mass detection limits of 20 amol in 1.25 μg of DNA (5 adducts in 10⁹ nucleosides) with a linear range of 70 amol to 70 fmol. 4-ABP-exposed human bladder cells and rat bladder tissue were analyzed in triplicate, and higher dose concentrations led to increased numbers of detected adducts. It was subsequently established that sample requirements could be further reduced to 1 μg digestions and the equivalent of 250 ng DNA per injection for the detection of low levels of dG-C8-4-ABP in a matrix of exfoliated human urothelial cell DNA. This method is appropriate for the characterization and quantification of DNA adducts in human samples and can lead to a greater understanding of their role in carcinogenesis and also facilitate evaluation of chemopreventive agents.     

土壤中腐殖酸与稀土离子作用的傅里叶变换红外光谱

采用NaOH碱提法提取和纯化了黑土中胡敏酸（humic acid,HA)用傅里叶变换红外光谱（FTIR）研究了HA与La^3 、Ce^3 、Cd^3 和Y^3 4种稀土离子作用前后的光谱特征。结果表明,HA与稀土离子作用后的红外光谱中归属于COO^-1600cm^-1、1400cm^-1处的振动吸收峰增强,表明腐殖酸与稀土离子之间发生了配合作用。     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.