量子点在生物医学中的应用

半导体量子点是无机纳米结晶,构成于硒化镉核心和硫化锌外壳.这种荧光标记物的发射光强是常用有机荧光染料的20倍,稳定性是其100倍.量子点的发射波长取决于核心粒子的大小,而每一种单色量子点的发射波长窄而对称.这些光学特性使量子点在医学诊断、药物的高速筛选以及基因和蛋白质的高通量分析方面具有广泛的应用前景.基于量子点的稳定性和生物相容性,有可能通过标记不同颜色的量子点到不同的分子,观察它们在活细胞内的运动.     

用壳聚糖亲和磁性微球纯化血浆凝血酶的研究

通过化学共沉淀法合成纳米粒子Fe3O4磁核,以壳聚糖为包裹材料包被自制的磁核,采用乳化交联法制备了具有核-壳结构的磁性高分子微球-壳聚糖磁性微球,并偶联肝素配基得到了一种新型亲和磁性微球,应用SEM、FT-IR、XRD等对微球的粒径、形貌、结构和磁响应性进行了表征.考察了该亲和磁性微球对凝血酶的分离纯化性能,并与传统的DEAE离子交换色谱法进行了比较.结果表明,所得亲和磁性微球具有较窄的粒径分布、形状规整,粒径在50nm左右.对凝血酶一步吸附纯化获得了比活为1879.71U/mg的酶,得率85%,纯化倍数11.057,而传统柱层析法得率为72%,纯化倍数仅为5.33.制备了壳聚糖亲和磁性微球,并将磁分离技术应用于凝血酶的分离纯化,得到了较好的效果,这将对于凝血酶的纯化及生产具有一定参考价值.     

调磁对磁性微球在大鼠体内代谢速度影响的研究

应用交变磁场引发分子振荡产生退磁效应的方法,研究了磁性微球在大鼠体内的代谢过程,结果表明,在300LX调磁条件下,能明显加速大鼠体内磁性微粒的代谢速度.可防止磁性微粒在体内蓄积过长造成对机体的损害.     

高灵敏荧光探针用于肿瘤 Ramos 细胞的快速检测

本文以聚苯乙烯纳米微球为载体,基于适体特异性识别和 DNA 杂交原理,组装了一种 DNA-CdTe 量子点纳米线,制备了具有较高荧光强度的复合型荧光探针,并成功用于 Ramos 细胞的荧光成像。该探针可以用于特异性识别肿瘤细胞,在荧光成像中信号强灵敏度高,为肿瘤细胞的检测提供一种新方法。     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

基于万古霉素建立荧光酶联免疫吸附法检测金黄色葡萄球菌

以猪IgG作为捕获抗体固定金黄色葡萄球菌,修饰有万古霉素的量子点荧光微球作为"检测抗体",建立荧光酶联免疫吸附法检测金黄色葡萄球菌。文中制备了平均粒径为100 nm的量子点荧光微球并与万古霉素偶联;摸索了反应最佳盐离子浓度为0.01 mol/L,反应最佳pH为6.0。在该实验条件下,金黄色葡萄球菌的检测灵敏度为104 CFU/m L,与其他致病菌无交叉反应。以上结果表明,该方法可用于快速检测金黄色葡萄球菌,为金黄色葡萄球菌的临床监控和食品检测提供参考。     

编码悬浮微块片上多组分并行免疫检测的方法

采用热压印光刻技术制备了一种多金属构成、带数字标识图形的悬浮微块,其中的镍层与金层可分别实现微块的磁控靶向与生物探针的联接。借助微块表面的数字微通孔标识符号,实现了微块的生物探针编码;用异硫氰酸荧光素荧光标记编码的悬浮微块,通过悬浮微块的多组分并行免疫荧光检测,实现了微块的生物探针解码及生物分子的定量检测。这种编码的地址数取决于微块表面的微通孔数,理论上可以成千上万。因此,表面经过生物探针修饰的悬浮微块是建立生物分子编码库的理想途径,可作为基于高通量悬浮阵列技术的免疫分析平台。     

一种免疫生物传感器对单核细胞增生李斯特菌快速检测方法的初步研究

应用F0F1-ATP酶旋转分子马达和免疫技术相结合,建立免疫生物传感器快速检测技术。首先pH变化敏感荧光物质F1300标记到色素体(chrom atophore)的内表面,然后在F0F1-ATP酶上连接β亚基抗体-生物素-链亲和素-生物素-单核细胞增生李斯特菌多抗复合体,得到可以捕获单核细胞增生李斯特菌的免疫生物传感器。传感器上负载菌量不同,酶活性不同,酶活变化以pH敏感的荧光探针来感应,最后通过荧光扫描仪检测不同菌量负载下的荧光信号。结果表明,该方法对单核细胞增生李斯特菌标准菌株(ATCC 15313)的检测时间为4.5 h,检出浓度为100 CFU/孔。     

纤维素微球色谱介质制备、孔结构调控及聚合物改性

作为色谱技术的核心,高效色谱介质的研制与开发是提高生物大分子纯化效率的重要手段之一.纤维素微球是最早应用于生物分离的色谱介质之一.这主要是因为纤维素的基本结构为多糖化合物,表面含有大量羟基,易于修饰功能配基,且非特异性吸附弱,特别适合不稳定生物大分子的分离纯化.本文综述了纤维素微球色谱介质的研究新进展.首先简要介绍了纤...     

以双歧杆菌为载体实现量子点在体靶向肿瘤的可行性研究

应用双歧双歧杆菌作为量子点输送载体,为小动物在体生物肿瘤成像提供依据. 采用电穿孔方法,将
二棕榈酰磷脂酰胆碱（DPPC）包被的硫硒镉水溶性量子点转入细菌内,得到“量子点-细菌”复合探针,在
“量子点-细菌”表面通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺 (EDC)活化法进一步偶联叶酸分子,制得
“量子点-细菌-叶酸”复合纳米生物探针. 将纳米生物探针经尾静脉注入Lewis肺癌小鼠体内,采用冰冻组
织切片,考察探针在小鼠体内脏器及肿瘤的分布情况. 结果表明,在肿瘤部位检测到较强的量子点光致发光
信号,而在肺、肝、脾等脏器中只检测到微弱的量子点发光信号. “量子点-细菌-叶酸”复合纳米生物探
针小动物在体肿瘤靶向成像是可行的.     

Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum

A specific and sensitive immunoassay based on magnetic microbead separation for schistosomiasis japonica screening is presented in this article. So far as we know, this is the first time that magnetic microbead-based enzyme-linked immunoassay (MEIA) has been used for the determination of Schistosoma japonicum (Sj) antibody in human serum. Fluorescein isothiocyanate (FITC)-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that the MEIA method was more sensitive and more precise than traditional SEA-ELISA (enzyme-linked immunosorbent assay). In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect hemagglutination assay (IHA), dipstick dye immunoassay (DDIA), and MEIA. IHA and DDIA were then compared with MEIA, and a lower false negative rate (0.97%) was obtained.     

基于磁性颗粒“在位”PCR和通用标签技术的新型高通量SNP分型方法

单核苷酸多态性(single nucleotide polymorphism,SNP)在对复杂疾病遗传易感性以及基于群体基因识别等方面的研究中起着非常重要的作用,尤其是对复杂疾病遗传易感性的研究,需要对大量样本进行分型.为了满足这种要求,亟待需要发展一种操作简单、成本较低、适于自动化和高通量的分型技术.利用磁性颗粒"在位"固相PCR(insituMPs-PCR)扩增的靶序列,通过与野生、突变标签探针以及双色荧光(Cy3,Cy5)通用检测子杂交实现对样本的分型.应用该方法,对96个样本的亚甲基四氢叶酸还原酶(MTHFR)基因C677T位点的多态性进行了检测,其野生型和突变型样本的正错配信号比大于4.5,杂合型正错配信号比接近1,分型结果与测序结果一致.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Padlock Probes and Magnetic Nanobead-Based Readout

Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.     

Direct detection of the binding of avidin and lactoferrin fluorescent probes to heparinized surfaces

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.     

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

Micromechanical measurement of membrane receptor binding for label-free drug discovery

A potential novel binding assay based on binding-driven micromechanical motion is described. A membrane preparation containing 5-HT(3AS) receptors was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (5-hydroxytryptamine, which is also called serotonin) or the antagonist MDL-72222, but not to other similar molecules. Control experiments show that cantilevers modified by membrane preparations that do not contain 5-HT(3AS) receptors do not respond to serotonin or MDL-72222. K(d) values obtained for serotonin and MDL-72222 are identical to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label-free, drug screening applications.     

综述——新型纳米生物材料的研发：稀土上转换荧光纳米材料的制备与生物应用

近几年,稀土上转换荧光纳米材料作为新型的荧光探针受到研究者的广泛关注,其优势在于光化学稳定性好、发射谱带窄、荧光寿命长、Stokes位移大等．同时,它利用近红外激光器作为激发光源,组织穿透能力好、对生物组织的损伤小、几乎没有背景荧光,使其应用于生物活体荧光成像成为可能．本文主要综述了最近稀土上转换荧光纳米材料在制备与生物应用方面的研究进展．