基于金银合金薄膜的波长检测型表面等离子体共振传感器

从实验和理论两方面详细研究了金银合金膜表面等离子体共振(SPR)传感器在可见光波段的敏感特性.实验方面,通过在玻璃基底上溅射50 nm厚的金银合金薄膜制备了一种新型的SPR传感芯片,并且自行搭建了基于Kretschmann结构的波长检测型SPR传感器测试平台.利用不同浓度的氯化钠(NaCl)水溶液和浓度为10μmol·L-1的牛血清蛋白(BSA)水溶液分别作为折射率样品和分子吸附样品,研究了传感器的折射率灵敏度和吸附灵敏度,并与金膜和银膜SPR传感器进行了对比研究.结果表明,对于折射率灵敏度的测试,金银合金膜SPR传感器大幅高于金膜SPR传感器,略低于银膜SPR传感器;而对于吸附敏感的研究,金银合金膜SPR传感器的灵敏度与银膜SPR传感器相近,是金膜SPR传感器的3倍.理论方面,利用菲涅尔公式和等效折射率计算公式仿真计算了这三种薄膜结构的SPR传感器的灵敏度和精确度,结果指出金银合金膜SPR传感器的灵敏度与银膜SPR传感器接近,是常规金膜SPR传感器的2.31倍,而半高峰宽仅为金膜和银膜SPR传感器的1.36倍.在稳定性方面,金银合金膜SPR传感器与金膜SPR传感器均具有良好的化学稳定性,而银膜SPR传感器较易氧化,使用寿命低,不常被采用.综上,金银合金膜在改善传感器灵敏度的同时,不会降低精度,是一种高灵敏、低成本、良好稳定性的SPR传感器敏感材料.     

基于金银合金薄膜的波长检测型表面等离子体共振传感器

从实验和理论两方面详细研究了金银合金膜表面等离子体共振（SPR）传感器在可见光波段的敏感特性. 实验方面,通过在玻璃基底上溅射50 nm厚的金银合金薄膜制备了一种新型的SPR传感芯片,并且自行搭建了基于Kretschmann 结构的波长检测型SPR传感器测试平台. 利用不同浓度的氯化钠（NaCl）水溶液和浓度为10 μmol·L^-1的牛血清蛋白（BSA）水溶液分别作为折射率样品和分子吸附样品,研究了传感器的折射率灵敏度和吸附灵敏度,并与金膜和银膜SPR传感器进行了对比研究. 结果表明,对于折射率灵敏度的测试,金银合金膜SPR传感器大幅高于金膜SPR传感器,略低于银膜SPR传感器;而对于吸附敏感的研究,金银合金膜SPR传感器的灵敏度与银膜SPR传感器相近,是金膜SPR传感器的3倍. 理论方面,利用菲涅尔公式和等效折射率计算公式仿真计算了这三种薄膜结构的SPR传感器的灵敏度和精确度,结果指出金银合金膜SPR传感器的灵敏度与银膜SPR传感器接近,是常规金膜SPR传感器的2.31倍,而半高峰宽仅为金膜和银膜SPR传感器的1.36 倍. 在稳定性方面,金银合金膜SPR传感器与金膜SPR传感器均具有良好的化学稳定性,而银膜SPR传感器较易氧化,使用寿命低,不常被采用. 综上,金银合金膜在改善传感器灵敏度的同时,不会降低精度,是一种高灵敏、低成本、良好稳定性的SPR传感器敏感材料.     

小型波长检测型表面等离子体共振分析仪的设计与研制

小型波长检测型表面等离子体共振(SPR)分析仪采用固定光的入射角,监测共振波长变化的测量模式,可进行多波长同时测量.设计并组装了SPR分析仪中的微型光纤光谱仪,采用光导纤维输入被测光,固定全息衍射光栅分光,线阵CCD光电传感器检测,可检测的波长范围是500～900 nm,分辨率0.23 nm.自行编制了控制传感器数据采...     

表面等离子体共振传感技术在高分子研究中的应用

介绍了表面等离子体共振 (SPR)的基本原理和SPR传感器技术的应用现状 ,综述了SPR传感器在高分子科学研究中的应用和发展前景。     

表面等离子体共振免疫传感器在蛋白质检测中的应用及其研究进展

表面等离子体共振(SPR)技术是20世纪90年代发展起来的一种新型技术,应用SPR原理可检测生物传感芯片上配位体与分析物之间的相互作用情况,在生命科学、医疗检测、药物筛选、食品检测及环境监测等领域具有广泛的应用需求.SPR技术可与免疫传感器结合,利用抗原抗体的特异性反应可用于各种蛋白质抗原的检测.本文重点总结了SPR免疫传感器在食品及医疗领域蛋白质检测的应用,综述了近年来SPR免疫传感技术在这该领域的研究热点及进展.     

基于金属纳米粒子增敏的SPR生物传感器检测吲哚乙酸

本实验建立了表面等离子体共振(SPR)生物传感器检测3-吲哚乙酸(IAA)的方法。制备了两种SPR生物传感器检测IAA:传统模式的SPR生物传感器1和Au/Ag合金纳米粒子增敏的SPR生物传感器2。结果发现:传感器1在IAA浓度范围为175~350μg/L时,浓度与其波数位移值呈线性关系,检出限为25μg/L(S/N=3);传感器2在IAA浓度范围为17.5~250μg/L时,浓度与其波数位移值呈线性关系,检出限为2.2μg/L(S/N=3)。说明基于Au/Ag合金纳米粒子的传感器2比传感器1有较高的灵敏度和较低的检出限。加标回收实验测得加标回收率范围为96%~100.2%,平均值为98.4%。本实验制备的SPR生物传感器具有较好的精密度、稳定性、重现性和特异性。     

基于色相算法的表面等离子体共振成像传感器对苯并芘的敏感特性

报道了一种用于原位探测水中苯并芘的彩色表面等离子体共振成像(SPRI)传感器,该传感器既能提供直观的图像信息,又能借助色相算法定量分析待测物质的浓度及其吸附/脱附过程。首先利用自制的波长-图像同步检测型SPR传感器测试了裸金薄膜芯片在不同入射角下的共振波长和共振图像,然后利用色相算法建立了SPR共振波长与图像色相的依赖关系,基于该依赖关系获得了SPR传感器最佳色相灵敏度对应的起始共振波长约为650 nm;另一方面,制备了聚四氟乙烯涂覆的SPR传感芯片,基于聚四氟乙烯膜对水中苯并芘的可逆富集作用实现了苯并芘的原位快速探测。实验取得以下4个结果:(1)在20-100 nmol?L-1浓度范围内彩色SPR图像的平均色相值随着苯并芘浓度的升高线性减小;(2)对100 nmol?L-1的苯并芘的响应和恢复时间分别约为7和5 s;(3)由于聚四氟乙烯膜的厚度大于SPR消逝场穿透深度,检测结果不受溶液折射率影响;(4)在聚四氟乙烯敏感膜厚度较小且不均匀的情况下,传感器容许获取敏感膜的不同厚度区域对苯并芘的色相灵敏度。实验结果有力地证明了这种彩色SPR图像传感器在生化物质检测中具有良好的应用前景。     

用于微生物体标记的CdTe半导体纳米晶超声波水相合成

利用超声波法制备高质量CdTe半导体纳米晶体, 采用该方法不仅降低了实验成本, 简化了实验流程, 而且还可制备出量子产率达到50%的CdTe纳米晶体, 具有较好的光谱学性质, 可用于莱姆病伯氏螺旋体(微生物)的荧光标记, 有利于提高对该病的检测效率.     

声光可调滤光片表面等离子体子共振传感器的研究

自行设计并组装了一套以声光可调滤光片(AOTF)为波长选择系统的波长检测型表面等离子体子共振(SPR)传感器装置,介绍了AOTF的特点和性能.研究了传感器对乙醇、葡萄糖和蔗糖的响应特性,三者的线性范围分别为5%～60%(体积分数),0.028～0.280和0.014～0.140mol/L.并对葡萄糖和蔗糖的混合溶液进行了分析.实际样品的测定结果与国家标准方法的测定值相符合     

蒽醌类药物与人血清白蛋白相互作用的研究

介绍了自行组装的波长检测型表面等离子体子共振(SPR)传感装置的原理和构造。应用此SPR传感器研究了三种蒽醌类药物与人血清白蛋白的相互作用,并分别计算了它们作用的动力学常数、热力学常数及结合百分率。结果表明,这些蒽醌类药物与人血清白蛋白都有不同程度的结合。     

Differentiation of Borrelia burgdorferi sensu lato species by temperature gradient gel electrophoresis

Borrelia burgdorferi sensu lato is a multispecies complex of pathogenic spirochetes, causing Lyme borreliosis. Due to clinical, epidemiological, and taxonomical implications, there is a need for identification of isolated Borrelia strains. In the present study, we have optimized TGGE for B. burgdorferi sensu lato species differentiation and the results were compared with two reference methods, namely PFGE and restriction of 5S-23S intergenic space region PCR product. A differentiation of B. garinii, B. afzelii, and B. burgdorferi senso stricto species with TGGE was possible and intraspecies variation was detected. Results compared between TGGE, PFGE, and restriction of 5S-23S intergenic space region PCR product showed no difference in specificity of species identification.     

Chemoenzymatic Synthesis of a Glycolipid Library and Elucidation of the Antigenic Epitope for Construction of a Vaccine Against Lyme Disease

Lyme disease (LD) is the most common tick‐borne disease in Europe, North America, and Asia. The etiologic agents of LD are spirochetes of the group Borrelia burgdorferi sensu lato, which possess a lipid content of 25–30 % of the dry weight. The major glycolipid cholesteryl 6‐O‐acyl‐β‐D ‐galactopyranoside (ACGal), present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii, is a specific and highly prevalent antigen frequently recognized by antibodies in late‐stage LD. Here we report a convenient route for the chemical synthesis of ACGal by employing a combination of chemical synthesis steps with enzymatic transformations. This synthesized molecule was compared with bacterial extracts by immunoblots with patient sera, confirming the preserved antigenicity. Next, a glycolipid library derived from the native molecules with variations in the fatty acyl moiety and derivatives in which the cholesterol has been replaced was designed and synthesized. The chemical structures were confirmed by 1D and 2D NMR spectroscopy and mass spectrometry. The native and synthetic glycolipids were utilized in immunoblots to determine the epitope recognized by antibodies in patient sera. By this method we could demonstrate that galactose, cholesterol, and a fatty acid with a minimal chain length of four carbon atoms comprises the essential structure for recognition by antibodies. Finally, this finding allowed the synthesis of a functionalized ACGal with an ω‐mercapto group at the fatty acid and a facile protection and deprotection strategy. This antigenic hapten can be conjugated to a carrier protein to effect immunization against Lyme disease.     

The effect of UV irradiation on infection of mice with Borrelia burgdorferi

These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi. Immunity against the Lyme spirochete B. burgdorferi was studied in a murine model of UV-induced immune suppression. Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR. Low-passage Borrelia were injected intradermally at the base of the tail following irradiation. At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies. Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation. The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice. Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti-interleukin (IL)-10 after UV irradiation. In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls. From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice. Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.     

Isolation of an outer membrane protein complex from Borrelia burgdorferi by n-butanol extraction and high-performance ion-exchange chromatography

Borrelia burgdorferi, the causative agent of Lyme disease, expresses two major membrane proteins, designated outer surface proteins A and B, which are of antigenic relevance, especially in the chronic phase of Lyme disease. Both proteins exhibit strain-related molecular weight variation. A method is described for obtaining these proteins from the bacterial membrane, without the use of detergents, by a combination of n-butanol extraction and cation-exchange chromatography on a Mono S fast protein liquid chromatographic column. This method yields up to five times larger amounts of the proteins in aqueous solution than previously described protocols, which applied ionic or non-ionic detergents. A comparison of extracts obtained by this method from different Borrelia burgdorferi strains is reported.     

Comprehensive detection of immunorelevant Borrelia garinii antigens by two-dimensional electrophoresis

Lyme disease is caused by spirochetes of the Borrelia burgdofferi complex and has been reported in many temperate parts of the Northern Hemisphere. The B. burgdorferi complex consists of at least five different species and five genotypes with different pathogenicity. Serodiagnosis was achieved by detection of antigens on one-dimensional (1-D) immunoblots. A systematic and comprehensive approach to elucidate antigens has been started here by the combination of two-dimensional electrophoresis (2-DE) immunoblotting with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Antigens in the proteome of B. garinii BITS were analyzed for their reactivity with sera from patients in early stage (erythema migrans) and late manifestations (neuroborreliosis late, arthritis and acrodermatitis chronica athrophicans) of borreliosis. A strategy to handle the enormous amount of data was developed and 65 antigens were detected, of which 20 were identified. These comprise the known antigens from 1-D immunoblots used routinely in serodiagnosis and additionally the two new antigens, GAPDH and the ABC transporter oligopeptide permease. Several disease-stage unique proteins were detected and some of them identified. The genetic variability between B. garinii strains BITS and 20047, B. afzelii, and B. burgdofferi, sensu stricto, seen on the 2-DE patterns underlines the necessity of the search for additional antigens to improve the serodiagnosis and development of vaccines to be used outside of Northern America. A 2-DE database of B. garinii was built up and is available on the World Wide Web (http://www.mpiib-berlin.mpg.de/2D-PAGE).     

Characterization of the P13 membrane protein of Borrelia burgdorferi by mass spectrometry

Borrelia burgdorferi sensu lato is a tick-borne pathogen that causes Lyme disease. The characterization of membrane proteins from this and other pathogens may yield a better understanding of the mechanisms of infection and information useful for vaccine design. Characterization of the highly hydrophobic Borrelia outer membrane component P13 from a mutant (OspA- OspB- OspC- and OspD-) strain was undertaken by use of a combination of mass spectrometric methods. In a previous investigation, an electrospray ionization (ESI) mass spectrum of the intact protein provided an average molecular weight that was 20 Da lower than the predicted molecular weight. The mass deviation could be explained by a modification of the N-terminus of the protein such as pyroglutamylation (-17 Da) in combination with the experimental error of measurement, however more information was required. New structural information for this membrane protein was provided by peptide mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) and sequencing with ESI-quadrupole-TOF tandem MS.     

Synthesis of two glycolipid antigens of the causative agent of Lyme disease

As a prelude to development of a human vaccine against Lyme disease, the first chemical synthesis of glycolipid antigens of Borrelia burkholderi is reported. First, cholesteryl β-d-galactopyranoside was synthesized and was converted to partially protected congeners having the HO-6 group of the galactose moiety unprotected. Treatment of these intermediates with palmitic and oleic acid, respectively, under dehydrative conditions followed by removal of the protecting groups afforded cholesteryl 6-O-palmitoyl/oleoyl-β-d-galactopyranosides that were identical to the glycolipids isolated from B. burkholderi.     

Structural determinants of inhibitor selectivity in prokaryotic IMP dehydrogenases

The protozoan parasite Cryptosporidium parvum is a major cause of gastrointestinal disease; no effective drug therapy exists to treat this infection. Curiously, C. parvum IMPDH (CpIMPDH) is most closely related to prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH gene via horizontal transfer. We previously identified inhibitors of CpIMPDH that do not inhibit human IMPDHs. Here, we show that these compounds also inhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, and Streptococcus pyogenes, but not from Escherichia coli. Residues Ala165 and Tyr358 comprise a structural motif that defines susceptible enzymes. Importantly, a second-generation CpIMPDH inhibitor has bacteriocidal activity on H. pylori but not E. coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.     

The largest protein observed to fold by two-state kinetic mechanism does not obey contact-order correlation

To probe the folding-energy landscape for a very large protein, we used Borrelia burgdorferi VlsE as a model. VlsE is a single-domain, predominantly alpha-helical protein with 341 residues. Remarkably, time-resolved folding and unfolding processes for VlsE follow two-state behavior. VlsE is by far the largest protein characterized that folds by a two-state kinetic mechanism. Thus, the common rule of thumb, that proteins larger than 110 residues fold by complex, multistate kinetic mechanisms, must be used with caution. In contrast with smaller helical proteins, the folding speed in water for VlsE is slow (5 +/- 2 s-1, pH 7, 20 degrees C) and does not agree (by 4 orders of magnitude in different directions) with the speeds predicted on the basis of native-state contact order and the topomer-search model. It is therefore questionable if the barrier height for folding is defined by gross topology for large two-state folders.