【作者】 卢玉栋；

【导师】 陈礼辉；

【作者基本信息】 福建农林大学 ， 木材科学与技术， 2014， 博士

【摘要】 恶性肿瘤是导致人们死亡的第一位疾病,诊断是攻克肿瘤的关键一步,表面增强拉曼光谱技术已被广泛应用于肿瘤诊断的研究中,但SERS活性基底的制备是获得SERS信号的前提,以竹浆为原料,开发新型的碱水体系为溶剂,并以均相法制备取代位置可控、均一性好的纤维素衍生物,以纤维素衍生物为模板制备SERS用纳米银,并将SERS基底应用于鼻咽癌的早期诊断中,对于鼻咽癌的早期准确诊断具有重要的理论和实际意义。论文系统地研究了碱水溶剂体系对竹浆的溶解情况,开发出竹浆溶解的新的溶剂体系,在低温-7℃下,竹浆粕在NaOH/尿素、NaOH/硫脲、NaOH/羟乙基脲等二元体系中具有较好的溶解率,NaOH/硫酸钠对竹浆粕没有溶解作用；二元体系溶剂的最佳组成分别为,NaOH7.6%/尿素11.4%、NaOH7.6%/硫脲8%、NaOH7.6%／羟乙基脲6%,对竹浆粕的溶解度分别为70.50%、79.64%、84.66%；三元体系的对竹浆粕溶解的最佳组成为NaOH7.6%／尿素11.4%／羟乙基脲3.5%、NaOH7.6%/N素11.4%/硫脲5%、NaOH7.6%／尿素11.4%/硫酸钠2.5%、NaOH7.6%/羟乙基脲6%／硫酸钠2%、NaOH7.6%／羟乙基脲6%／尿素4%、NaOH7.6%/羟乙基脲6%/硫脲4.5%,对竹浆粕的溶解度分别为85.88%、83.06%、75.95%、88.58%、92.07%、93.08%；羟乙基脲的氢键供体数量(为3)与氢键受体数量(为2)均比硫脲、尿素的氢键供体数量(均为2)与氢键受体数量(均为1)来得多,拓扑分子极性表面积(TPSA)为75.4,介于硫脲(84.1)与比尿素的拓扑分子极性表面积(69.1)之间,而疏水参数计算参考值(-1.7)均比尿素(-1.4)与硫脲(-0.8)来得低,羟乙基脲比尿素、硫脲更容易形成氢键,在NaOH协同作用下,很容易与纤维素大分子中的羟基形成分子间氢键能有效地破坏聚多糖分子间和分子内氢键,从而使纤维素溶解于该溶剂体系中,NaOH/羟乙基脲溶剂体系再生后的竹浆为纤维素Ⅱ型。NaOH/羟乙基脲水溶液为溶剂,在均相体系中制备了羟乙基尿素、甲基纤维素、羧甲基纤维素、羟丙基甲基纤维素,并利用IR、核磁共振等仪器对制备的纤维素衍生物进行了表征,测定了各纤维素衍生物的取代度。利用CMC为模板制备的纳米银,对罗丹明具有很好的拉曼增强效果,其增强效果受温度、模板浓度、还原剂浓度及时间等反应条件的影响；模板法制备纳米银的最佳实验条件为：CMC的质量浓度0.15%,反应温度75℃,柠檬酸三钠的质量浓度为1.2%,采用25mmol·L-1硝酸银溶液作为银源,不调节模板溶液的pH值,反应时间1.25h。CMC水溶液粘度随着质量分数的增大而增大,曲线向上弯曲呈二次函数型的非线性增大。CMC水溶液质量分数低于0.7%时,随着溶液浓度的增加其粘度呈线性增加,此时溶液表现近似为牛顿流体；当质量分数超过0.7%时,溶液表现为非牛顿流体；CMC溶液浓度在一定范围内,溶液中极性亲水基团随CMC质量分数增大而增大,当CMC溶液浓度大到一定程度后,溶液中可利用的亲水基团反而减少。CMC质量分数为0.70%-0.80%时,CMC溶液显示出比较独特的性质。利用HPMC为模板合成纳米银,探讨了温度、时间、模板浓度等反应条件对制备纳米银增强罗丹明效果的影响；结果表明以HPMC为模板制备的纳米银是一种很好的SERS基底,对罗丹明的增强效果受纳米银制备条件的影响很大,SERS用纳米银的最佳制备条件为：将50mL10mmol·L-1AgNO3溶液滴入到50mL质量浓度为0.1%的HPMC溶液中,其中含质量浓度为0.1%柠檬酸三钠,保持温度为75℃反应150min时制备的纳米银溶胶。以HPMC为模板合成的纳米银主要是由平均粒径60-70nm的类球型颗粒组成,该溶胶常温避光下保存,保存期超过12个月,各项性能指标稳定。没有用HPMC作模板反应得到的纳米银溶液,保存期不超过15天；粘度实验表明HPMC溶液属于非牛顿流体,随着溶液质量浓度的增加,溶液中的分子易形成分子间氢键使得溶液粘度增加；在一定浓度范围内,HPMC溶液中极性亲水基团随HPMC质量浓度增大而增大。利用MC为模板成功合成了SERS基底用的纳米银,探讨了温度、时间、MC浓度等反应条件对制备纳米银增强罗丹明效果的影响；结果表明以MC为模板制备的纳米银是一种很好的SERS基底,对罗丹明的增强效果受纳米银制备条件的影响很大,当模板浓度为0.2%,银氨溶液浓度10mmol·L-1,温度75℃,还原剂为柠檬酸三钠,且浓度为0.2%,反应时间120min时,制备的纳米银具有很好SERS增强效果；以MC为模板合成的纳米银主要是由平均粒径80nm的类球型、棒状等颗粒组成,该溶胶常温避光下保存,保存期超过12个月,各项性能指标稳定；MC水溶液浓度较低时,随着溶液浓度的增加其粘度呈线性增加,此时溶液表现近似为牛顿流体。以HEC为模板合成的纳米银对罗丹明6G具有很好的拉曼增强效果,纳米银对罗丹明6G的增强效果受反应因素的影响较大,对罗丹明6G增强效果最好的纳米银的制备条件为：以50mL质量分数0.075%的HEC与50mL15mmol·L-1的硝酸银在质量分数0.1%二水合柠檬酸三钠中于75℃水浴条件下反应150min,该条件下制备的纳米银主要为粒径大小介于60～70nm的球形；该溶胶常温避光下保存,保存期超过12个月,各项性能指标稳定；HEC水溶液质量分数低于0.275%时,溶液表现近似为牛顿流体；当质量分数超过0.275%时,溶液的粘度与浓度之间的线性关系遭到破坏,曲线向上弯曲呈非线性增大,且粘度与浓度呈二次函数关系,此时溶液表现为非牛顿流体。以纤维素模板法合成的纳米银溶胶为基底,对血浆具有很好的增强作用,血浆样品的拉曼信号获得极大的增强,同时很好地抑制了生物分子中荧光的干扰；鼻咽癌患者与正常健康人血浆的表面增强拉曼光谱上存在明显差异,正常人血浆的表面增强拉曼光谱在495,591,636,812和1134cm-1处的5个吸收谱峰的强度比相对应的鼻咽癌患者血浆的表面增强拉曼光谱的谱峰强；而在725,1338,1447,1572和1653cm-1处的5个吸收谱峰位置,鼻咽癌患者血浆的表面增强拉曼光谱比相对应的正常健康人血浆的表面增强拉曼光谱的强度大；通过PCA分析后,以纤维素模板法制备纳米银溶胶为基底检测血浆样品的表面增强拉曼光谱的方法,对鼻咽癌的诊断灵敏度与特异性分别达到90.9%和97.5%；经PCA-LDA分析后,以纤维素模板法制备纳米银溶胶为基底检测血浆样品的表面增强拉曼光谱的方法,对鼻咽癌的诊断灵敏度与特异性分别为93.65%和98.72%；通过表面增强拉曼光谱的谱峰强度比较表明,鼻咽癌患者血浆与正常健康人血浆在生化成分的结构与含量上存在明显差异,鼻咽癌病人血液中核酸、苯基丙氨酸、胶原、磷脂、腺嘌呤等成分的含量高于正常健康人,氨基酸与糖类等成分的含量相对较低；以纤维素模板法合成的纳米银溶胶为基底,利用表面增强拉曼光谱技术对血浆样品进行测试,并结合PCA-LDA分析方法对鼻咽癌进行诊断,可发展为一种无损检测与筛查鼻咽癌的临床诊断工具。更多还原

【Abstract】 Malignant tumor is a leading cause of death in patients and diagnosis is a key step to conquer cancer. Surface enhanced Raman spectroscopy technology has been widely used in diagnosis of tumor. SERS active substrate is the premise of SERS signal. The homogeneous synthesis of cellulose derivatives in alkaline solvent, with bamboo pulp as raw material, was investigated in this work. The SERS ability of silver nanoparticles prepared by using cellulose derivatives as template was studied. And the silver nanoparticles have been used as SERS substrates application in early diagnosis of nasopharyngeal carcinoma, which has important theoretical and practical significance upon rapid and accurate diagnosis of nasopharyngeal carcinoma.The solubility of bamboo pulp in alkaline solvent was investigated in this work. The new solvent system for bamboo pulp was developed. Bamboo pulp can be dissolved in NaOH/thiourea, NaOH/hydroxyethyl urea and NaOH/urea aqueous solution under-7℃. NaOH/Na2SO4aqueous solution can not dissolve bamboo pulp. The optimum composition of binary system was NaOH7.6%/thiourea8%, NaOH7.6%/hydroxyethyl urea6%and NaOH7.6%/urea11.4%aqueous solution, respectively. The solubility of bamboo pulp in NaOH7.6%/urea11.4%, NaOH7.6%/thiourea8%and NaOH7.6%/hydroxyethyl urea6%aqueous solution under-7℃was70.50%,79.64%,84.66%, respectively. The optimum composition of three component system was NaOH7.6%/urea11.4%/hydroxyethyl urea3.5%, NaOH7.6%/urea11.4%/thiourea5%, NaOH7.6%/urea11.4%/Na2SO42.5%, NaOH7.6%/hydroxyethyl urea6%/Na2SO42%, NaOH7.6%/hydroxyethyl urea6%/urea4%, NaOH7.6%/hydroxyethyl urea6%/thiourea4.5%, in which the solubility of bamboo pulp was85.88%,83.06%,75.95%,88.58%,92.07%,93.08%, respectively. Both hydrogen bond donor (3) and hydrogen bond acceptor (2) of hydroxyethyl urea were more than that of thiourea (hydrogen bond donor was2and hydrogen bond acceptor was1) and urea (hydrogen bond donor was2and hydrogen bond acceptor was1). Topological molecular polar surface area of hydroxyethyl urea was75.4, which was between84.1(the TPSA of thiourea) and69.1(the TPSA of urea). Calculation values of hydrophobic parameter (CVHP) of hydroxyethyl urea was-1.7, which was lower than that of thiourea (-0.8) and urea (-1.4). It was able to be understood that the intermolecular hydrogen bonds of bamboo pulp were destroied by NaOH, urea, thiourea or hydroxyethyl urea. Hydrogen bonds of bamboo pulp can be destroyed by hydroxyethyl urea easier than others. Cellulose was dissolved completely in NaOH/hydroxyethyl urea and that cellulose I changed to cellulose II during regeneration.Cellulose derivatives (hydroxypropyl methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and methyl cellulose) were homogeneous synthesized in NaOH7.6%/hydroxyethyl urea6%aqueous solution. The cellulose derivatives were characterized by FTIR and Nuclear magnetic resonance. And the substituting degrees of cellulose derivatives were determined.The silver nanoparticles prepared with carboxymethyl cellulose (CMC) as a template have more remarkable enhancements in the SERS spectrum of R6G, which can be used as a good Ag-based SERS substrates in the analytical environment for routine measurements. The applicability of these silver nanoparticles as optical enhancers for SERS strongly relies on the preparing conditions of temperature, concentration of CMC, concentration of sodium citrate and duration time. The optimum conditions for preparing Ag-based SERS substrates were as follows:0.15%CMC,25mM silver nitrate at75℃, then reducing in1.2%sodium citrate at1.25h. The silver nanoparticles prepared with CMC as a template can be stored for more than12months away from light at room temperature. However, the silver nanoparticles prepared without CMC as a template can not stored for more than15days. Viscosity experiments indicated the CMC solution was a non-newtonian fluid when the concentration of CMC was more than0.7%, the viscosity increased with the concentration of CMC.Silver nanoparticles were prepared with hydroxypropyl methyl cellulose (HPMC) as a template. Effects of HPMC concentration, silver nitrate solution concentration, reaction duration, temperature and reducing agent on silver nanoparticles were discussed. The SERS enhancement of these silver nanoparticles was tested by using Rhodamine6G (R6G) as a probe molecule. The results show that the preparing conditions play a crucial role in the performance of nanoparticles. The silver nanoparticles prepared with HPMC as a template have been shown to provide strong enhancements in the SERS spectrum of R6G. The optimum conditions for preparing Ag-based SERS substrates were as follows:0.1%HPMC,10mM silver nitrate at75℃, then reducing in0.1%reducing agent at150min. TEM studies reveals that particles are mostly near-spherical in shape with an average size of60-70nm. The silver nanoparticles prepared with HPMC as a template can be stored for more than12months away from light at room temperature. However, the silver nanoparticles prepared without HPMC as a template can not stored for more than15days. Viscosity experiments indicated the HPMC solution was a non-newtonian fluidwhen the concentration of HPMC was more than0.4%, the viscosity increased with the concentration of HPMC.Methyl cellulose (MC) was used as a template to prepare silver nanoparticles. Effects of methyl cellulose concentration, silver ammonia solution concentration, reaction duration, reducing agent on the performance of silver nanoparticles were discussed. The results show that the reducing agent and MC concentration play a crucial role in the performance of silver nanoparticles. The silver nanoparticles prepared with MC as a template have been shown to provide strong enhancements in the SERS spectrum of R6G. The optimum conditions for preparing Ag-based SERS substrates were as follows:10mM silver ammonia at75℃, then reducing in0.2%reducing agent at120min. TEM studies reveals that particles are mostly spherical, rod in shape with an average size of80nm. The silver nanoparticles prepared with MC as a template can be stored for more than12months away from light at room temperature. Viscosity experiments indicated the MC solution was a nearly-newtonian fluid, the viscosity increased with the concentration of MC.Silver nanoparticles prepared with hydroxyethyl cellulose (HEC) as a template have more remarkable enhancements in the SERS spectrum of R6G, which can be used as a good Ag-based SERS substrates. The applicability of these silver nanoparticles as optical enhancers for SERS strongly relies on the preparing conditions of temperature, concentration of HEC, concentration of sodium citrate and duration time. The optimum conditions for preparing Ag-based SERS substrates were50ml0.075%HEC,50ml15mM of silver nitrate,0.1%tri-sodium citrate,75℃,150min. TEM studies reveals that particles are mostly near-spherical in shape with an average size of60-70nm. The silver nanoparticles prepared with HEC as a template can be stored for more than12months away from light at room temperature. Viscosity experiments indicated the HEC solution was a nearly-newtonian fluid when the concentration of HEC was less than0.275%, a non-newtonian fluid when the concentration of HEC was more than0.275%, the viscosity increased with the concentration of HEC.Raman signals of blood plasma samples were greatly enhanced by using the silver nanoparticles prepared with cellulose derivatives as templates as SERS substrates. Moreover, the intensity of the fluorescence background of blood plasma samples was decreased and the signals of blood plasma samples were reduced. There were significant differences in the Raman spectra of blood plasma from healthy subjects and nasopharyngeal cancer subjects. SERS peaks at495,591,636,725,812,1134,1338,1447,1572and1653cm-1can be observed in both normal and nasopharyngeal cancers blood plasmas. The normalized intensities of SERS peaks at495,591,636,812and1134cm-1were more intense for normal plasma than for nasopharyngeal cancers plasma, while SERS peaks at725,1338,1447,1572and1653cm-1were greater in nasopharyngeal cancer plasma sample.Using silver nanoparticle based SERS spectroscopy combined with PCA multivariate analysis we were able to differentiate the blood plasma of nasopharyngeal cancer patients from that of healthy subjects with high diagnostic sensitivity (90.9%) and specificity (97.5%). LDA based on the PCA generated features differentiated the nasopharyngeal cancer SERS spectra from normal SERS spectra with high sensitivity (93.65%) and specificity (98.72%). Raman signals of the measured SERS spectra suggested interesting cancer specific biomolecular differences, including an increase in the relative amounts of nucleic acid, collagen, phospholipids and phenylalanine and a decrease in the percentage of amino acids and saccharide contents in the blood plasma of nasopharyngeal cancer patients as compared to that of healthy subjects. Using the nanoparticles prepared with cellulose derivatives as templates as SERS substrates, SERS spectroscopy of combined with PCA-LDA analysis method was developed for blood plasma analysis into a clinical tool for non-invasive detection and screening of nasopharyngeal cancers.更多还原