【作者】 陈贝贝；

【导师】 胡斌；

【作者基本信息】 武汉大学 ， 分析化学， 2010， 博士

【摘要】 生命体中的微量元素对于正常生物功能的维持起着重要作用。元素的生物活性、可利用性以及毒性不仅受体内元素总量的影响,更与其所存在的化学形态密切相关,因此,生物样品中痕量元素及其形态分析具有十分重要的意义。然而,生物样品往往存在样品量有限、基质复杂、元素及其各形态的含量低、元素形态在分析过程中可能发生转变等问题。正因如此,生命体系中痕量元素及其形态分析成为分析化学中亟待解决的一个富有挑战性的课题,也是目前分析科学研究的热点之一电感耦合等离子体质谱(ICP-MS)具有灵敏度高、线性范围宽、分析速度快、多元素同时测定及同位素分析等优点,已成为痕量元素分析中一种主要的检测手段。将高选择性色谱分离技术与ICP-MS1关用是目前元素形态分析中最有效也是应用最广泛的分析方法。但生物样品中某些痕量元素及其形态含量非常低,接近或低于ICP-MS或色谱-ICP-MS的检出限水平；复杂的生物基体往往也会影响ICP-MS或色谱-ICP-MS测定结果的准确性。因此,研究/探索/发展合适的样品前处理技术对目标分析物进行富集,同时与基体分离,进一步改善方法的分析性能,是目前分析化学中的一个重要研究方向。另外,基于元素与生物分子间的相互作用,发展元素标记ICP-MS及其联用技术新方法用于生物分析不仅会拓宽ICP-MS技术的应用领域,对于进一步研究元素在生命体内的代谢、毒性及生物活性也具有重要价值。本论文的研究目的是：发展高效液相色谱(HPLC)-ICP-MS联用技术用于生物样品中元素形态分析的新方法；探讨新型样品前处理技术,并与HPLC-ICP-MS联用在生物样品中元素形态分析中的应用；建立芯片微型化样品前处理技术,拓展ICP-MS技术在细胞分析及蛋白质定量中的新应用。本论文的主要研究内容包括：(1)将两性离子胆汁酸衍生物3-[3-(胆酰胺基丙基)二甲氨基]丙磺酸盐(CHAPS)作为C18柱动态改性试剂,建立了HPLC-UV/ICP-MS在线联用直接分析人血清中Al形态的新方法。以Al-Cit和Al-Tf分别作为小分子络合铝和大分子蛋白铝的代表分析物,详细考察了CHAPS改性C18柱分离不同Al形态的色谱条件。在最优条件下,大分子蛋白铝和小分子络合铝在4 min内即可实现分离,方法测定Al-Cit和Al-Tf的检出限分别为0.83和0.74μg L-1。将所建立的方法用于健康人和慢性渗析病人血清中Al-Tf和Al-Cit的分析,结果显示Al-Tf约占人血清中总Al的87.4-91.1%,Al-Cit仅占总Al的7.1-15.2%。本方法简便、快速、无需复杂样品前处理,有效地提高了分析效率且避免了A1形态在分析过程中的转变。(2)以烷基咪唑类离子液体作为HPLC流动相的添加剂,建立了离子液体改性反相(RP)-HPLC-ICP-MS联用技术用于生物样品中硒形态分析的新方法。以亚硒酸盐(Se(Ⅳ))、硒酸盐(Se(Ⅵ))、硒胱氨酸(SeCys2)、硒蛋氨酸(SeMet)、甲基硒代半胱氨酸(MeSeCys)和硒代乙硫氨酸(SeEt)为目标分析物,采用C18柱作为固定相,详细考察了烷基咪唑类离子液体作为流动相添加剂,其种类、浓度和比例等因素对六种目标硒形态分离的影响。在优化的流动相组成为0.4%(v/v)[BMMIM]BF4和0.4%(v/v) [BMIM]Cl的水溶液时,六种目标硒形态在8 min内实现分离。采用标准样品SELM-1验证本方法的准确性,测定值与参考值吻合良好。最后,将所建立的方法应用于富硒酵母和富硒苜蓿草中硒的形态分析。结果表明,富硒酵母和富硒苜蓿草中存在的主要水溶态硒是SeMet和SeCys2,并通过SeCys2加标实验发现在活的酵母细胞中,添加的SeCys2在多步酶的作用下会转变成SeMet。本方法通过选择合适的离子液体作为HPLC流动相添加剂以调整目标分析物的保留行为,具有操作简便和进样量少的优点,在生命体系的元素形态分析中有很大的应用潜力。(3)通过乳液聚合一步合成法制备了新型聚苯乙烯磺酸钠包裹的Fe3O4磁性纳米粒子(Fe3O4@PSS)。通过改变样品溶液的pH值调节硒氨基酸的存在状态,使之与Fe3O4@PSS磁性纳米粒子上的磺酸基发生阳离子交换吸附,建立了磁固相萃取(MSPE)-HPLC-ICP-MS用于富硒酵母中硒氨基酸形态分析的新方法。实验中以SeCys2、SeMet、MeSeCys、γ-谷酰基-甲基硒代半胱氨酸(GluMeSeCys)和SeEt为目标分析物,对MSPE萃取硒氨基酸的影响因素进行了详细考察。在优化的条件下,方法对五种硒氨基酸的检出限(LODs,3σ)为21.5-89.9 ng L-1,相对标准偏差(RSDs, n=7)为6.7-12%,富集倍数在10-92倍之间,线性范围达3个数量级(R2>0.99)。采用标准样品SELM-1验证本方法的准确性,测定值与参考值吻合良好。最后,将所建立的方法成功用于富硒酵母细胞中硒氨基酸的形态分析,加标回收率在78.8-106%之间。与其他方法相比,本方法具有吸附／解吸动力学快、富集倍数高、检出限低、可进行批操作、廉价等优点。(4)建立了新型混合涂层改性的中空纤维膜管内固相微萃取(in-tube HF-SPME)与HPLC-ICP-MS在线联用用于生物样品中砷形态分析的新方法。实验中将自制的γ-巯丙基三甲氧基硅烷(γMPTS)／N-(p-氨乙基)-γ-氨丙基三甲氧基硅烷(AAPTS)混合溶胶以及部分磺化聚苯乙烯(PSP)先后涂覆于中空纤维膜内表面,并对所得改性材料进行了红外和扫描电镜表征。以亚砷酸钠(As(Ⅲ))、砷酸钠(As(V))、单甲基砷酸(MMA)、二甲基砷酸(DMA)、砷胆碱(AsB)和砷甜菜碱(AsC)为目标分析物,详细考察了影响in-tubeHF-SPME的一系列因素。在最优条件下,该方法的样品通量为6.5 h-1,检出限在0.017-0.053μgL-1范围内,富集倍数为4-19倍。为了验证方法的准确性,将本方法用于标准样品狗鱼(DORM-2)和人尿(CRM No.18)中砷的形态分析,测定值与标准值吻合良好。将本方法用于健康人尿液中砷形态的直接分析,加标回收率在92.2-107%之间。本方法具有较好的富集能力和优良的样品基质净化功能,可直接用于生命流体(如尿液)中砷的形态分析,避免了生命流体直接引入RP-HPLC引起的蛋白质在反相柱上的不可逆吸附等问题。(5)以磁性纳米粒子自组装堆积方式制备了芯片磁固相填充柱,实现了磁固相萃取技术和细胞样品引入技术在芯片上的整合,首次建立了芯片磁固相萃取-电热蒸发(ETV)-ICP-MS新方法,并将其应用于细胞中痕量重金属元素镉(Cd)、汞(Hg)和铅(Pb)的分析。在外加磁场作用下将自制的yMPTS改性纳米磁硅球填充于芯片主通道,对芯片磁固相填充柱的形成机理、影响因素以及影响芯片磁固相萃取的一系列因素进行了详细考察。在优化的实验条件下,方法对Cd、Hg和Pb的检出限达到0.72、0.82和1.16 ngL-1,富集倍数分别为45.0、41.6和48.7倍。将该方法用于HepG2肝癌细胞中痕量Cd、Hg和Pb的测定时,细胞经计数后直接引入微流控芯片进行在线破壁、混合、萃取和洗脱,将洗脱液引入ETV-ICP-MS进行测定。在仅消耗约5000个细胞的情况下,得出了单个HepG2肝癌细胞中Cd、Hg和Pb的含量在fg/亚fg级。本方法为金属组学的研究提供了新的思路。(6)基于PbS纳米粒子标记,提出了芯片磁固相免疫与ETV-ICP-MS联用高灵敏检测人血清中癌胚抗原(CEA)的新方法。设计并制备了用于免疫分析的微流控芯片装置,将亚氨基二乙酸(IDA)修饰的磁性纳米粒子填充于芯片主通道作为载体固定一抗,在芯片上完成一抗-抗原-PbS纳米粒子标记的二抗之间的夹心免疫反应。用甲酸洗脱标记在夹心免疫复合物上的PbS,通过ETV-ICP-MS定量测定Pb的浓度从而实现对CEA的定量分析。实验对一系列影响免疫反应的参数进行了考察,确定了最优分析条件。在此条件下,方法测定CEA的检出限为0.058μg L-1,相对标准偏差(RSDs,n=7)为6.7%,线性范围为0.2-50μg L-1,适用于正常人体内的CEA含量的分析。最后,将本方法应用于人血清中CEA的测定,结果与临床化学发光免疫法所得的测定值吻合良好,说明本方法在临床诊断和治疗癌症过程中具有一定的应用前景。(7)将吡咯烷二硫代氨基甲酸盐(APDC)作为浊点萃取(CPE)中的化学螯合剂以及ETV-ICP-MS测定中的化学改性剂,建立了基于非离子表面活性剂Triton X-114的CPE与低温ETV-ICP-MS联用用于环境水体中无机硒形态分析的新方法。当温度高于TritonX-114的浊点温度时,在pH值2.5-3.5的范围内,Se(Ⅳ)能与APDC生成疏水性螯合物被萃取到富胶束相中,与水相中的Se(VI)实现分离；待测物以Se(IV)-APDC螯合物的形式在ETV中蒸发有效地降低了Se的蒸发温度,延长了石墨管的使用寿命。将Se(VI)还原为Se(IV),测定样品溶液中总Se的含量,通过总硒与Se(Ⅳ)的含量差减得到Se(VI)的含量。本文对影响CPE分离富集和ETV-ICP-MS检测的诸因素进行了考察。在最优的条件下,本法测定Se(Ⅳ)的检出限为8.0 ng L-1,富集倍数为39倍,相对标准偏差(RSDs, n=7)为3.93%。为了验证方法的准确性,将本方法用于环境标准水样GSB07-1253-2000中Se的分析,测定值与参考值吻合良好。最后,将建立的方法用于环境水样中痕量无机硒的价态分析,加标回收率在82-102%之间。更多还原

【Abstract】 Trace elements play important roles in the organisms. The bioactivity, bioavailability and toxicity of the elements are not only dependent on their total amount, but also highly dependent on their existing species. Therefore, trace/ultra-trace elements and their speciation analysis in biological systems are of great significance. In bioanalysis, the sample volume is always very limited, the sample matrices is always complex, and elements or their species always occur in extremely low concentration. For these reasons, trace/ultra-trace elements and their speciation analysis in biological system have become a hot and challenging topic in analytical chemistry.Inductivity coupled plasma mass spectrometry (ICP-MS) is one of the most powerful methods for trace/ultra-trace elements analysis due to its outstanding advantages including high sensitivity, high sample throughput, wide linear dynamic range, multi-element detection and isotope analysis ability. Hyphenated techniques by combining high efficient separation techniques with ICP-MS is one of the most effective and widely-used methods for elemental speciation. Sample pretreatment techniques aiming to preconcentrate the analytes and separate them from matrix can effectively improve the analytical performance. Therefore, exploring novel miniaturized, high sensitive and effective ICP-MS based hyphenated techniques is highly demanded for trace/ultra-trace elements and their speciation analysis in biological systems. Biomarcromolecules analysis by elemental labeling ICP-MS is one of the newest advances of ICP-MS, developing elemental labelling ICP-MS methods based on the interaction between the elements and the bio-molecules is extremely significance for proteomics and metallomics.The aim of this dissertation is to develop high performance liquid chromatography (HPLC)-ICP-MS hyphenated new methods for elemental speciation in biological samples; to investigate novel sample pretreatment techniques for elemental speciation in biological samples; and to develop chip-based sample pretreatment techniques for electrothermal vaporization (ETV)-ICP-MS determination of ultra-trace elements in cells and quantification of protein in biological samples. The major contents of this dissertation are described as follows:(1) A C18 column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was used for direct injection HPLC-UV/ICP-MS speciation of aluminum (Al) in non-spiking human serum. Small molecule Al-complex compounds of Al-citrate (Al-Cit) and large molecule Al-protein compounds of Al-transferrin (Al-Tf) were chosen as the model species and their retention behaviors on CHAPS modified C18 column were studied with UV and on-line ICP-MS detection in details. Under the optimal conditions, large-molecule Al-protein compounds and small molecule Al-complex compounds could be separated within 4 min. The detection limits of the method (LODs,3σ) were 0.74 and 0.83 ng mL-1 with the relative standard deviations (RSDs, n=7) of 2.8% and 3.0% for Al-Tf and Al-Cit, respectively. The developed method was applied to the speciation of Al in real healthy human serum and chronic hemodialysis patient serum, the experimental results show that the most of Al exists as Al-protein, and the small amount of Al exists as small molecule Al-complex compounds in human serum. Compared with the reported methods in the literature, this method has several attractive features such as simplicity, rapidness, no sample preparation required, and it provides a new strategy for studying trace amount elemental speciation in human body liquids.(2) A new method based on room-temperature ionic liquids (RTILs) improved reversed-phase (RP)-HPLC-ICP-MS for selenium speciation has been developed. The different parameters affecting the retention behaviors of six target selenium species especially the effect of RTILs as mobile phase additives have been studied, it was found that the mobile phase consisting of 0.4%(v/v) 1-Butyl-3-methylimidazolium chloride ([BMIM]Cl),0.4% (v/v) 1-Butyl-2,3-dimethylimidazolium tetrafluroborate ([BMMIM]BF4) and 99.2%(v/v) water has effectively improved the peak profile and six target selenium species including Na2Se03 (Se(IV)), Na2Se04 (Se(VI)), L-Selenocystine (SeCys2), DL-Selenomethionine (SeMet), Se-methylseleno-L-Cysteine (MeSeCys), Seleno-D, L-Ethionine (SeEt) were separated within 8 min. The developed method was validated and successfully applied to the speciation of selenium in Se-enriched yeasts and clover. For fresh Se-enriched yeast cells, it was found that the spiked SeCys2 in living yeast cells could transform to SeMet. Compared with other RP- ion-pair (IP)-HPLC-ICP-MS approaches for selenium speciation, the proposed method possessed the advantages including ability to regulate the retention time of the target selenium species by selecting the suitable RTILs and adjusting their concentration, simplicity, rapidness and low injection volume, thus providing wide potential applications for elemental speciation in biological systems.(3) A new sorbent of poly[styrene-co-(sodium styrene sulfonate)-co-divinylbenzene] coated Fe3O4 magnetic nanoparticles (Fe3O4@PSS MNPs) has been synthesized by one step emulsion polymerization. Based on the cation exchange interaction between the sulfonate group on the Fe3O4@PSS MNPs and the target seleno-amino acids, a new method of magnetic solid phase extraction (MSPE)-HPLC-ICP-MS for seleno-amino acids speciation in Se-enriched yeast cells has been developed. Taking SeCys2, MeSeCys, L-y-glutamyl-Se-methyl-L-selenocysteine (GluMeSeCys), SeMet and SeEt as target selenium species, a series of factors that influence the MSPE were investigated in details. The conditions for subsequent HPLC-ICP-MS determination were optimized as well. Under the optimal conditions, the LODs (3σ) for target seleno-amino acids were in the range of 21.5-89.9 ng L-1 with the RSDs (n=7) ranging in 6.7-12%, the enrichment factors were varied from 10 to 92-fold, and the linear ranges were over three orders of magnitudes (R2>0.99). To validate the accuracy of this method, certified reference materials SELM-1 was analyzed, and the determined values were in good agreement with the certified values. The proposed method was also successfully applied for the target seleno-amino acids speciation in Se-enriched yeast cells, and the recoveries for the spiked samples were in the range of 78.8-106%. The proposed MSPE-HPLC-ICP-MS method is characterized with low cost, fast separation, high enrichment factor and low LODs.(4) A novel method based on in-tube hollow fiber-solid phase microextraction (in-tube HF-SPME) on-line coupled with RP-IP-HPLC-ICP-MS was developed for arsenic speciation in biological samples. Partial sulfonated poly(styrene) (PSP) and mixed-sol of 3- mercapto propyltrimethoxysilane (yMPTS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AAPTS) were prepared and coated on the inner surface of polypropylene HF. The prepared yMPTS-AAPTS/PSP coated HF was characterized by FT-IR spectroscopy and scanning electron microscope (SEM). With arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC) as target arsenic species, a series of factors that influence the in-tube HF-SPME were investigated in details, such as pH value, sample volume and flow rate, elution conditions and interference of co-existing ions. Under the optimal conditions, the sample frequency was 6.5 h-1, the LODs (3σ) for target arsenic species were in the range of 0.017-0.053μg L-1 with RSDs (n=7) ranging in 1.5-5.2%, and the enrichment factors were varied from 4 to 19-fold. To validate the accuracy of this method, certified reference materials DORM-2 (dogfish) and CRM No.18 (human urine) were analyzed, and the determined values were in good agreement with the certified values. The proposed method was also successfully applied for arsenic speciation in healthy human urine samples, and the recoveries for the spiked samples were in the range of 92.6-107%. The proposed in-tube HF-SPME-HPLC-ICP-MS method is low cost, time- and labor-saving, and suitable for simultaneously organic and inorganic arsenic speciation in biological samples.(5) Quantitative analysis of trace levels of heavy metals in human cells is critical to environmental and toxicological research. A new strategy for determining ultratrace levels of cadmium (Cd), mercury (Hg), and lead (Pb) in cultured cells was described. It involves the integration of cell sample introduction and magnetic solid phase microextraction (MSPME) on a microfluidic chip, combined with ETV-ICP-MS detection.γ-mercaptopropyl-trimethoxysilane (yMPTS) modified silica-coated magnetic nanoparticles were synthesized and employed as the extraction material for microextraction of Cd, Pb, and Hg. Under an external magnetic field, these magnetic nanoparticles were self-assembled in microchannels to form a solid phase packed column. The formation mechanism of the on-chip magnetic solid phase packed column and the main factors influencing the packing and analytical performance were investigated. Under the optimized conditions, MSPME enabled enrichment of Cd, Hg, and Pb by a factor of>40, and resulted in improved LODs (3σ) for Cd (0.72 ng L-1), Hg (0.86 ng L-1), and Pb (1.12 ng L-1) using ETV-ICP-MS. Quantitative analysis of trace Cd, Hg and Pb in HepG2 cells were achieved by applying the microfluidic system that integrated cell rupture, mixing, magnetic extraction and elution into one device, followed by ultrasensitive detection of ETV-ICP-MS, which required only microliter of samples. Analysis of approximately 5000 cells revealed that the average amounts of Cd, Hg and Pb in a single HepG2 cell were on the order of a few femtograms, and that the cellular levels increased with increasing concentrations of these metals in cell cultures. The concentrations of Cd, Hg, and Pb detectable in HepG2 cells were several orders of magnitude lower than their IC50 values, suggesting that the technique is potentially useful for measuring these heavy metals in studies of chronic metal toxicity.(6) A sensitive and selective method of magnetic immunoassay on microfluidic chip based on a sandwich-type immunoreaction with PbS nanoparticle (NPs) labels combined with ETV-ICP-MS was proposed for the determination of carcinoembryonic antigen (CEA). For this purpose, a microfluidic chip for magnetic immunoassay was designed and fabricated, the prepared iminodiacetic acid (IDA) modified silica coated magnetic nanoparticles (MNPs) was packed in its central microchannel to form a solid phase column by self-assembly under the magnetic field. After a complete immunoreaction among primary antibody, CEA and secondary antibody labeled with PbS NPs on magnetic solid phase packed-column, the concentration of CEA was quantified by ETV-ICP-MS determination of Pb released from captured PbS NPs with an acid-dissolution step. By optimizing a series conditions for immunoassay including blocking, incubation and elution, the established method presented a LOD (3σ) of 0.058μg L-1 for CEA based on the Pb signal, with the RSD (n=7) of 6.7%. The response of the method for CEA was linear over a dynamic range from 0.2 to 50μg L-1, and the enrichment factor of 2-folds (from 60μL sample to 30μL elution) was obtained. The proposed method was successfully applied for CEA determination in real human serum, and the analytical results are in good agreement with that obtained by the currently used clinical analytical method of chemiluminescent immunoassay. The developed method represents various advantages such as fast speed, sensitivity, selectivity, low sample/reagents consumption, versatility, and can be easily extended to other biological and medical assays.(7) A new method based on cloud point extraction (CPE)-ETV-ICP-MS has been proposed for the speciation of inorganic selenium in environmental waters. When the temperature of the system is higher than the cloud point temperature (CPT) of the selected surfactant Triton X-114, the complex of Se(IV) with ammonium pyrrolidine dithiocarbamate (APDC) can be extracted into the surfactant-rich phase, whereas the Se(VI) remains in aqueous solutions. Thus, an in situ separation of Se(IV) and Se(VI) and the preconcentration of Se(IV) could be realized. The surfactant-rich phase was diluted to 200μL with methanol and 10μL of them was introduced into the ETV-ICP-MS for subsequently determination. For Se(VI) determination, Se(VI) was reduced to Se(IV) prior to CPE, and its assay was based on subtracting Se(IV) from total selenium. The main factors affecting the CPE and the vaporization behavior of the analyte were investigated in details. Under the optimized experimental conditions, the LOD for Se(IV) was 8.0 ng L-1 with an enhancement factor of 39 when 10 mL of sample solution was preconcentrated to 0.2 mL. The RSD (n=7) was found to be 3.9%. The proposed method was validated and applied to the speciation of inorganic selenium in different environmental water samples with the recoveries for the spiked samples in the range of 82-102%.更多还原