基于金纳米杆/PMMA基底SERS特性的高灵敏肿瘤标志物无标记检测

高品质贵金属纳米结构基底的制备是应用表面增强拉曼散射(SERS)技术进行高灵敏生物检测的关键。采用改进的Langmuir-Blodgett方法,通过在金纳米杆(Au NRs)溶胶注入乙醇,使得Au NRs迁移至溶胶与甲苯的交界面,并用聚甲基丙烯酸甲酯(PMMA)固定交界面处的Au NRs,形成大面积分布、均匀致密排列的二维畴状Au NRs/PMMA纳米结构薄膜基底。然后,采用等离子体清洗技术处理制备的基底,使得金纳米杆(Au NRs)的表面裸露,以增强基底的SERS特性。实验表明,Au NRs/PMMA基底具有优良的SERS特性,在785 nm波长的激光照射下,增强因子可以达到5.49×106。此外,利用制备的Au NRs/PMMA基底,开展前列腺癌症肿瘤标志物--前列腺特异性抗原(PSA)的高灵敏无标记定量检测研究。在PSA的无标记检测过程中,首先对PSA标准溶液和新生牛血清进行SERS光谱的直接检测,得到PSA分别位于823, 1 080, 1 385, 1 586和1 640 cm-1处的主要的拉曼特征峰;其次,通过对PSA标准溶液、临床男性血清样本及女性血清样本的SERS光谱进行测量和分析,筛选出在PSA的SERS光谱中与血清中PSA含量相关的拉曼特征峰,它们是分别位于649,680以及1 640 cm-1处的拉曼特征峰。进一步,通过对与PSA同属糖蛋白的肿瘤标志物甲胎蛋白(AFP)以及与PSA同源的人腺体激肽释放酶2(hK2)进行SERS光谱检测和分析,发现位于1 640 cm-1处的拉曼特征峰对于PSA具有高的特异性,将其作为临床血清样本中PSA无标记定量检测的具有特异性的拉曼特征峰,并以此为依据,对不同PSA浓度的标准溶液进行检测,得到位于1 640 cm-1处的拉曼特征峰强度与PSA样本溶液中PSA的浓度相关的剂量-响应曲线。最后,开展临床血清样本的应用检测。结果表明,基于Au NRs/PMMA基底的SERS检测结果与化学发光免疫分析(CLIA)方法的检测结果一致,且具有比CLIA更高的检测灵敏度,最低检测极限为0.06 ng·mL-1,且无标记检测范围为0.1 mg·mL-1～0.1 ng·mL-1。因此,基于Au NRs/PMMA SERS基底的高灵敏肿瘤标志物无标记检测具有重要应用前景。     

铜蒸气激光照射对血管平滑肌细胞凋亡的诱导作用及对其增殖的影响

目的：研究510.6nm铜蒸气激光照射对体外培养的血管平滑肌细胞（VSMC）凋亡的诱导作用，以及对增殖细胞核抗原（PCNA）表达的影响，探讨铜蒸气激光照射在经皮冠状动脉成形术（PTCA）后再狭窄（RS）的防治作用。方法：贴块法培养兔VSMC，510.6nm铜蒸气激光照射后透射电镜观察凋亡细胞形态学改变，TUNEL法计数凋亡细胞，免疫组化染色法计数照光对PCNA阳性表达率的影响。结果：激光照射后，VSMC凋亡率较未照光组增加12.8倍，而PCNA表达阳性率降低27．9％倍；电镜下观察细胞呈典型的凋亡形态学改变。结论：铜蒸气激光照射可以诱导VSMC凋亡，而且抑制其增殖，在RS的防治中具有一定的作用。     

用于检测癌胚抗原CEA的丝网印刷免疫传感器

采用溶胶-凝胶技术将电子媒介体亚甲基蓝和辣根过氧化物酶(HRP)标记的癌胚抗原(CEA)固定在一次性丝网印刷碳电极表面,制备了CEA免疫传感器。该免疫传感器在含CEA样品的溶液中培育后,抗原-抗体免疫结合物的形成会阻碍HRP活性中心与亚甲基蓝之间的电子传递,使HRP对H2O2电催化氧化的效率降低。循环伏安和计时电流法用于研究免疫电极的电化学特性,在优化的条件下催化效率的降低与CEA质量浓度分别在1.0~6.0μg/L和6.0~138μg/L范围内成线性关系,检出限为0.4μg/L,测定的组内和组间相对标准偏差分别为7.4%和11.2%。该测定无须分离、洗涤步骤,分析时间短,操作方便,检测成本低,具有实际应用价值。     

重氮化法合成磺胺间甲嘧啶免疫抗原的研究

采用重氮化法将磺胺间甲嘧啶(SM1)与牛血清蛋白(BSA)偶联制备合成抗原BSA-SM1作免疫抗原;用紫外扫描法和聚丙烯酰胺电泳法对其进行鉴定,并测得偶联的结合比。通过紫外扫描和聚丙烯酰胺电泳法的测定证明偶联成功,偶联的结合比为9∶1。用BSA-SDM免疫BALB/C小鼠,间接ELISA测定多抗上清(pAb)效价,结果表明,获得了高效价的pAb,为SM1残留免疫学检测方法的建立奠定了基础。     

抗肿瘤双特异免疫导向治疗制剂CAtin的表达及活性分析

结合生物信息学方法与已知癌胚抗原(Carcinoembryonic antigen, CEA)特异性单链抗体(Single chain Fv fragment, scFv)核苷酸序列, 经分子设计和密码子优化后, 通过化学方法合成CEA二硫键稳定性单链抗体(Disulfide stabilized single chain Fv fragments, scdsFv)基因片段. 将凋亡素基因(Apoptin)通过一段柔性连接肽(Linker)连接在CEA scdsFv基因下游, 并克隆入大肠杆菌表达载体质粒pET28a, 转化BL21感受态菌后经异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导, 表达融合蛋白CAtin. SDS-PAGE和Western-Blot分析表明, 目的蛋白得到良好表达, 经条件优化后表达量最高可达44.1 mg/L. 融合蛋白经分步洗涤法和谷胱甘肽对表达的目的蛋白进行初步纯化和复性后, 利用人肝癌细胞(HCC)对所制备融合蛋白进行亲和力测定、细胞结合活性测定和特异性细胞杀伤活性分析. 结果显示, 所制备融合蛋白不仅能够有效地与上述肿瘤细胞结合, 并对其具有明显的杀伤活性, 表明成功制备了具有特异性识别和特异性杀伤活性的双特异抗肿瘤免疫导向制剂.     

Antibody-Antigen Reactions in Porous Sol-Gel Matrices

Porous silica gels, synthesized via the acid hydrolysis and basic condensation of TMOS, have been used for the encapsulation of antigens. The pores of the matrix are large enough to allow the diffusion of antibodies through the gel. Antigen-antibody specific fixation occurs within the sol-gel matrix. It can be detected via the so-called enzyme linked immunosorbent assays (ELISA). Antigen-antibody associations occurring in the gel are optically detected via the reaction of a peroxidase conjugate with ortho-phenylenediamine leading to the formation of a yellow coloration. Immunoassays have been performed using the hydatid cyst fluid as the source of antigens and sera from human patients as the source of antibodies. Specific fixation appears to be as good in the sol-gel matrix as in antigen solutions.     

用于乙肝表面抗原检测的压电免疫传感器的研制

研制了一种用于乙肝表面抗原（ＨＢｓＡｇ）检测的新型传感器———石英压电免疫传感器。采用聚乙烯亚胺粘附和戊二醛交联法在金电极上固定抗体蛋白，对固定化过程进行了监测。ＨＢｓＡｇ含量的检测范围为１～４０ｍｇ／Ｌ。使用过的电极用０２ｍｏｌ／Ｌ乙醇胺（ｐＨ＝８）解吸，可重复使用。将此传感器用于实际血清样品的检测，与酶联免疫吸附法所得结果吻合。     

Traditional uses,phytochemistry, pharmacology and other potential applications of Vitellaria paradoxa Gaertn. (Sapotaceae): A review

Vitellaria paradoxa Gaertn. is a multipurpose medicinal plant of the family Sapotaceae, and it has been widely used usually in the clinical traditional medicine as remedy for a wide range of diseases for several decades. In addition, the plant has also found applications in confectionery, cosmetics and soaps, and pharmaceuticals both locally and internationally. V. paradoxa, which has been identified with >150 phytoconstituents, is rich in oleanane-type triterpene acids and glycosides, such as paradoxosides A-E, tieghemelin A, parkiosides A-C, bassic acid, as well as flavonoids such as quercetin and catechin-type compounds. The extracts and the active constituents of V. paradoxa have been investigated for various pharmacological activities, including but not limited to anticancer, melanogenesis-inhibitory, antibacterial, anti-diabetic, antioxidant, anti-inflammatory, anti-diarrhoeal, and antifungal activities. Additionally, V. paradoxa has also been utilized in nanoparticles (NPs) synthesis. These NPs among other things have shown significant antinociceptive and antiedematogenic activities as well as environmental friendly adsorptive properties for the removal of pollutants from pharmaceutical effluents. Overall, this review comprehensively examines the traditional uses, phytochemistry, pharmacology, toxicology, clinical studies, and nanoparticles synthesized from V. paradoxa and their applications.     

Amplification immunoassay for the determination of Hepatitis B surface antigen

A sensitive sandwich immunoassay for the determination of Hepatitis B surface antigen (HBs) was developed, using a cascade system of Limulus amebocyte lysate as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to anti-HBs antibody. Anti-HBs antibody was adsorbed to polystyrene beads. First, HBs were reacted to solid phase anti-HBs antibody (a-HBs). After the reaction, the beads were rinsed, and were then reacted with a-HBs-LPS. Then, LPS activity specifically bound to the beads was measured. HBs could be measured in the range of 10(-10)-10(-12) g/mL.     

Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface

A novel electrochemical immunosensor for the determination of carcinoma antigen 125 (CA125) was developed by means of immobilizing CA125 antibody (anti-CA125) on gold nanoparticles (Au) and thionine (Thi)-modified carbon paste interface. To avoid the leak of hydrophilic gold nanoparticles and thionine from carbon paste interface, the Au-Thi-modified carbon paste electrodes (CPEs) were first treated in the mixture solution containing 10% HNO₃ and 2.5% K₂Cr₂O₇ for 1.5 min at +1.5 V to make the carbon surface with -COOH groups, which can react with -NH₂ groups on the thionine molecule, in the meantime, gold nanoparticles were absorbed on the thionine surface. Subsequently, CA125 antibodies were assembled onto the surface of gold nanoparticles. The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy (FTIR) and UV-vis absorption spectroscopy. The performance and factors influencing the performance of the immunosensor were studied in detail. A direct electrochemical immunoassay format was employed to detect CA125 antigen based on the current change before and after the antigen-antibody reaction. The current change was proportional to CA125 concentration ranging from 10 to 30 U/ml with a detection limit of 1.8 U/ml (at 3δ). The immunosensors were used to analyze CA125 in human serum specimens. Analytical results of clinical samples show that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.