Preparation Of SRES Substrate Using Cellulose As A Template And Its Applications In The Diagnosis Of Nasopharyngeal Cancer

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.