应用量子点及磁微粒技术快速检测阴道毛滴虫抗原

目的 以阴道毛滴虫(trichomonas vaginalis,Tv)检测为切入点,构建以量子点和磁微粒技术为基础的夹心法快速检测抗原技术,为同步多项抗原检测作准备.方法 针对Tv特异性黏附蛋白AP33制备其抗体,以碳二亚胺交联法,分别连接抗体.量子点及抗体-磁微粒加入待测抗原AP33结合后,在荧光显微镜下观测结果.结果 确立了本检测系统的最优条件,对AP33的检测与儿种阴道常见病原菌无交叉反应,检测限为0.05υg/ml.结论 成功构建了量子点磁微粒检测技术,对Tv抗原检测,特异度为90%,准确率为88%.     

精子蛋白17抗体免疫磁性颗粒活性分析及细胞成像研究

目的:制备精子蛋白17(Sp17)抗体免疫磁性纳米颗粒, 以期用于卵巢癌的磁共振成像靶向诊断.方法:在偶联剂EDC/NHS作用下, 将壳聚糖修饰磁性氧化铁纳米颗粒与抗人Sp17抗体结合, 制备抗Sp17抗体免疫磁性纳米颗粒(IMNPs).透射电镜观察颗粒的形貌特征, 非变性聚丙烯酰胺凝胶电泳评价抗体与磁性颗粒的结合效果, ELISA检测免疫磁性颗粒的免疫活性.将IMNPs与转染人Sp17的卵巢癌HO-8910细胞共孵育, 进行体外磁共振成像检测.结果:成功实现了抗体与磁性纳米颗粒的偶联, 并且IMNPs保持良好的抗体活性.磁共振显示该颗粒成功靶向Sp17阳性的细胞, 没有发生明显的非特异吸附.结论:该免疫磁性纳米颗粒特异性良好, 可进一步用作卵巢癌的靶向治疗研究.     

建立寄生虫纳米生物传感器技术的初步应用

〔目的〕采用生物分子技术更快更准确地检测食品中寄生虫,提高检测效率。〔方法〕应用纳米金标记磁珠分离电化学检测DNA方法。以纳米量子点为DNA标记,夹心法为目标捕获方法,通过磁珠分离富集途径,利用电化学方法检测纳米金标记在碳电极表面的还原信号,建立纳米生物传感器检测技术。〔结果〕目标DNA样品出现明显信号;非碱基配对样品中无信号。在无磁性的电极上检测也无信号,说明Au67没有非特异性吸附。〔结论〕纳米生物技术是纳米技术与生物技术相结合的分子水平的检测技术。它可以从复杂的样品中快速分离、纯化、浓集出目的物。它在寄生虫检测中的应用,具有广阔的发展前景,是一种值得推广的先进的检测技术。     

Poor kidney allograft survival associated with positive B cell – Only flow cytometry cross matches: A ten year single center study

The presence of donor specific antibody (DSA) to class 1 or class 2 HLA as detected respectively in T cell or B cell – only flow cytometry cross matches (FCXMs) are risk factors for renal allograft survival, though the comparative risk of these XMs has not been definitively established. Allograft survival and FCXM data in 624 microcytotoxicity (CDC) XM negative kidney transplants were evaluated. Short and long term allograft survival was significantly less in recipients with T⁻ B⁺ FCXMs (1 year, 74%, 10 year, 58%) compared to T⁺ B⁺ FCXMs (1 year, 84%, 10 year, 68%) and to T⁻ B⁻ FCXM (1 year, 90%, 10 year, 85%). Risk factors were positive FCXM, deceased donor (DD) transplantation and donor age, but not race, gender, recipient age or previous transplant. Recipients with T⁻ B⁺ and T⁺ B⁺ FCXMs were at 4.5 and 2.5 fold greater risk, respectively, of DD allograft failure compared to patients with T⁻ B⁻ FCXMs. The quantitative value of FCXM did not correlate with the duration of graft survival. We conclude that patients with DSA to class 2 HLA have a greater risk of early and late allograft failure compared to patients with DSA to class 1 HLA.     

成年大鼠纹状体神经干细胞体外提纯及检测

目的建立成年大鼠纹状体区神经干细胞的体外提纯方法,并进行纯度检测。方法将成年大鼠的纹状体组织制成单细胞悬液,通过磁性分离系统从细胞悬液中分离出神经干细胞,并以流式细胞仪检测其纯度。结果提纯而来的细胞经干细胞特异性Nestin荧光染色后在荧光显微镜下可见细胞呈圆形且发出绿色荧光,经流式细胞仪检测Nestin荧光染色阳性率87.5 % ±2.5 %。结论利用磁性分离技术可成功将成年大鼠纹状体组织中的神经干细胞提纯。     

Liquid-phase hybridization and capture of hepatitis B virus DNA with magnetic beads and fluorescence detection of PCR product

The polymerase chain reaction (PCR) exceeds all hitherto known detection limits. This sensitivity could lead to false positive results. Every manipulation increases the risk of contamination via, for example, aerosols. Most protocols for the extraction of template nucleic acids are complicated and possible centrifugation steps do not reduce the risk of aerosols. In addition, most of the methods for analysis are time-consuming and cannot be applied to different template materials. An alternative extraction method has been developed. The fast chemical denaturation of template by guanidine thiocyanate was followed by liquid hybridization to biotinylated oligonucleotides. The template nucleic acid could be washed after binding to streptavidin-coated paramagnetic beads to reduce influence on the enzymatic amplification steps. PCR of hepatitis B virus deoxyribonucleic acid was used to demonstrate how easy, versatile, and time-saving this method is without centrifugation. The level of extracted nucleic acids was quantitated and the properties for sensitive extraction were evaluated. After PCR an additional step was developed which used fluorescent staining to detect positive amplifications. This is useful to identify positive results in predominantly negative samples.     

Hydrostatic pressure induces apoptosis of chondrocytes cultured in alginate beads.

The purpose of this study was to investigate the influence of hydrostatic pressure (HP) on apoptosis and expression of heat-shock protein 70 (HSP70) in chondrocytes cultured in alginate beads. Chondrocytes were isolated from the articular cartilage of rabbit joints and seeded in alginate beads. The beads in Group A were cultured for less than 24 h after being embedded with the chondrocytes, while those in Group B were cultured for 2 weeks. Both groups were exposed to HP of 10 or 50 MPa for 12 or 24 h. The beads in Groups A and B that were not exposed to HP were regarded as controls. Apoptotic cells induced by exposure to HP were quantified using the TUNEL method. Immunohistochemical analysis for HSP70 and in situ TUNEL analysis were also performed. Apoptotic chondrocytes were not observed in the control cells under atmospheric pressure, whereas apoptosis was observed in the beads in Group A, and the number of apoptotic cells increased as the duration and magnitude of HP increased. On the other hand, we observed no significant population of apoptotic cells in the beads in Group B. Chondrocytes expressing HSP70 were not TUNEL positive in the histological analysis. Excessively strong HP could evoke apoptosis when the extracellular matrix did not accumulate around the chondrocytes. HSP70 expression was related to occurrence of apoptosis that resulted from HP. These findings suggest a mechanism for the pathogenesis of cartilage degeneration in osteoarthritis.     

免疫磁性微珠的研制及其在免疫检测中的应用

应用材料科学和免疫学的新技术，我们在国内首次研制成功免疫磁性微珠（IMB）。在对一些临床常用血清指标以及两种激素标准品的免疫学定量检测中，自制IMB与瑞士Serono公司同类产品所获的结果十分接近（P＜0.01）,而且与放射免疫法的结果也有可比性(P＜O.01）。自制IMB在用于免疫学检测时的特异性、精密度、重复性、准确性均符合质量控制要求。     

Polylysine-Immobilized Chitosan Beads as Adsorbents for Bilirubin

Hyperbilirubinemia generally relates to an elevated bilirubin level in the blood and is usually an indication of a disease of the blood, liver, or biliary tract. Hemoperfusion using synthetic resins as sorbents has been one of the ways to reduce bilirubin. In this study, chitosan, a natural polysaccharide having structural characteristics similar to glycosaminoglycans and which is nontoxic and biocompatible, has been used for bilirubin binding. Several layers of poly-L-lysine have been coated covalently onto chitosan beads, using N2 plasma and carbodiimide treatments. Such surface-modified chitosan beads exhibited high binding affinities for bilirubin (1.13 +/- 0.18 mg/g beads) in aqueous phosphate buffer solutions at 4 degrees C in relation to activated charcoal (0.74 +/- 0.2 mg/g). The polylysine-coated resins have been reported to have an improved binding affinity for bilirubin over cholestyramine. It seems that the surface-immobilized polylysine has an increased bilirubin binding affinity and is highly stable. The binding capacity is proportional to the amount of polylysine bonded to the chitosan beads. The hemolytic potential of all modified beads is compatible with polystyrene control tubes. Studies were also performed against albumin as proof of specificity toward bilirubin binding. The albumin-coated beads have shown the highest blood compatibility and selectivity over the other modified beads. However, it appears that polylysine-modified chitosan may be an excellent sorbent system for hemoperfusion due to its high binding affinity, capacity, and blood compatibility. Further studies are needed to determine its behavior under clinical conditions.     

Quantitative characterization of magnetic separators: Comparison of systems with and without integrated microfluidic mixers

We present two new types of microfluidic passive magnetic bead separator systems as well as methods for performing quantitative characterizations of them. Both systems consist of a microfluidic channel with long rectangular magnetic elements of permalloy that are placed by the sides of the channel and magnetized by an external magnetic field. In one of the systems, a staggered herringbone microfluidic mixer is integrated in the channel. The characterization of the systems includes magnetic measurements of the capture-and-release efficiencies, estimates of distributions of captured beads in a channel from micrographs, and simulations and analytical models of bead trajectories, capture efficiencies, and capture distributions. We show that the efficiencies of both systems compare favorably to those in the literature. For the studied geometries, the mixer is demonstrated to increase the bead capture-and-release efficiency for a fixed flow rate by up to a factor of two. Moreover, high capture efficiencies can be achieved in the system with mixer at up to ten times higher flow rates than in the system without mixer.