基于上转磷光颗粒的AFP免疫层析定量检测

目的：建立了上转磷光免疫层析快速定量检测AFP的方法,用于原发性肝癌的诊断。方法：利用上转磷光标记的双抗夹心免疫层析技术检测血清中AFP的含量。将AFP单克隆抗体分别标记上转磷光颗粒并包被硝酸纤维膜,制成免疫层析检测试纸。评价该试纸的灵敏度、特异性和精密度。并通过临床50例血清样本,探讨上转磷光技术与化学发光检测方法的一致性。结果：该方法能在20min内完成,检测灵敏度达5ng/ml。浓度范围从5-1000ng/ml作标准曲线,呈现较好的线性。通过三组平行实验探讨其精密度,变异系数都在10%以内。应用于其它肿瘤标志物如CEA、CA199、AFU和NSE的检测,无交叉反应。与化学发光相比,决定系数R2=0.9692,一致性较好。结论：该方法简便、快速、廉价,可以实现定量,尤其适合基层门诊和体检现场使用。     

用磷光探针测定肿瘤细胞

用磷光探针测定肿瘤细胞马贵斌（山西大学化学系,太原０３０００６）关键词磷光探针,肿瘤细胞磷光是三重态蜕变到基态的辐射过程。它需要改变电子的自旋,因此是一种“禁止”跃迁。这就使磷光成为一种不可几的过程,所以在液态溶液中很少观察到磷光。但是在低温下（例如...     

磷光分析法在生命科学中的应用新进展

本文介绍了近年来磷光分析法在氨基酸,DNA和蛋白质研究中的应用,以及磷光分析法与其它分析技术相结合的研究进展。     

五个氨基酸磷光性质的研究

研究了5个氨基酸的磷光特性、光谱、寿命和最小检测量.以色氨酸(Trp)的磷光最强,在λ_ex/λ_em=290/438 nm,最小检测量为1 ng,酪氨酸(Tyr)其次(284/390 nm)为Trp的1/10,苯丙氨酸(Phe)在276/386 nm,脯氨酸(Pro)在308/454 nm,组氨酸(His)在320/466 nm,其磷光只有Trp的1%.Phe与Trp磷光寿命最长,在7 s左右;His最短,只有0.49 s;Tyr 2.8 s,Pro 1.34 s.另外研究氨基酸在甲醇,乙醇,丙醇,丁醇中及pH对氨基酸磷光性质的影响.还计算了Stokes位移能量及激化态pK^*_a值.     

磷光过渡金属配合物用于生物成像及癌症治疗的研究进展

顺铂及其衍生物在抗肿瘤方面取得了很大成功,但是传统的铂类抗癌药物的毒副作用和耐药性限制了这类化合物在临床上的进 一步开发。近年来,非铂类化合物,如具有 d6 电子结构的磷光过渡金属钌 ( II )、铱 ( III ) 和铼 ( I ) 配合物,由于其丰富的光物理和 光化学性质、氧化还原性质、多样的几何构型和水溶性好等优势吸引了越来越多的关注。综述上述 3 种金属配合物在生物成像及抗肿 瘤方面的研究进展。     

解偶联蛋白与阿尔茨海默病

解偶联蛋白(uncoupling protein,UCP)是线粒体内膜上的质子转运蛋白,被激活时能引发质子漏,质子经UCP渗漏回基质,使氧化磷酸化部分解耦联,降低线粒体膜电位,减少过量活性氧的产生。另外,UCP对ATP合成、钙离子稳态、能量代谢等也有调节作用。阿尔茨海默病是一种中枢神经系统退行性疾病,由多种因素共同作用引起,其中活性氧的作用越来越引起重视。UCP,特别是UCP2在中枢神经系统疾病方面的保护作用日益引起关注,有望成为阿尔茨海默病治疗的靶向目标。     

介绍一种新的半干式免疫转渍法

蛋白质免疫转渍法(Western blotting)是近十年发展起来的一种生化新技术。其原理是用SDS聚丙烯酰胺凝胶电泳分离待分析的蛋白质样品,然后用转渍装置把蛋白质转移到硝酸纤维素膜(NC膜)上。再利用免疫检测法检出要分析的蛋白质区带。过去报道的大多是利用液相夹心式电转渍仪完成转渍的,这     

禁食及胰岛素水平对大鼠解偶联蛋白-1、2、3基因表达的影响

 为探讨禁食和胰岛素对解偶联蛋白 - 1、2、3基因 (UCP1 ,2 ,3)表达的影响 ,应用 RT- PCR方法观察了在不同禁食时间和应用胰岛素条件下大鼠白色脂肪组织、棕色脂肪组织和骨骼肌中 UCP1 ,2 ,3m RNA水平的变化 .UCP1基因只在大鼠棕色脂肪组织中表达 .UCP2 ,3基因在三种组织中均有表达 ,在白色脂肪组织中以 UCP2表达为主 ;在骨骼肌中以 UCP3表达为主 .过夜禁食使棕色脂肪组织 UCP1 ,3m RNA水平明显下降 (P<0 .0 1 ) ;UCP2 m RNA水平在三种组织中均呈上升反应 ,以白色脂肪组织中表现最为明显 (P<0 .0 5) ;而对白色脂肪组织和骨骼肌中 UCP3基因表达无明显影响 .禁食时间延长至 48h,除棕色脂肪组织中 UCP2 ,3基因有明显下降外 ,各组织中UCPs基因表达基本调节至正常或高于对照组水平 .胰岛素对 UCPs基因表达水平有一定的上调作用 ,这一作用对棕色脂肪组织 UCPs各基因及骨骼肌中 UCP3基因表现得尤为明显 (P<0 .0 5) .大鼠 UCPs基因表达有一定的组织特异性 ;禁食时间对三种组织中 UCPs各成员基因表达的影响有时相上的区别 ;胰岛素可以调 UCPs各成员基因的表达 .结果反映了 UCPs各成员在能量代谢调节上的不同作用 ,这为理解膳食 -产热与体重调节的关系 ,及其能量代谢平衡与疾病关系提供了实验依据     

瘦蛋白调控小型哺乳动物产热的分子机制

瘦蛋白(leptin)是由脂肪细胞分泌的、肥胖基因编码的蛋白类激素,其在哺乳动物的体重调节和适应性产热中有重要作用。解偶联蛋白(uncoupling protein,UCP)是动物体内具有解偶联作用的一类蛋白质。UCP-1是分布于褐色脂肪组织(brown adipose tissue,BAT)线粒体内膜上的标志蛋白,其在冷适应和食物资源缺乏等条件下的产热作用已得到印证。UCP-2和UCP-3的生物学功能仍存在争论。瘦蛋白可以通过诱导UCP的表达来促进产热．瘦蛋白诱导UCP表达的机制有二：(1)瘦蛋白通过与下丘脑神经肽Y的拮抗作用诱导UCP的表达;(2)瘦蛋白以交感神经系统为媒介诱导UCP的表达。瘦蛋白诱导UCP-1的表达是调节BAT产热的主要途径。了解瘦蛋白与UCP在产热中的作用对于我们了解动物产热的分子机制和其对低温、营养缺乏等条件下的生理生态适应机制具有重要意义。     

真鲷肝脏解偶联蛋白2(UCP2)基因及其功能的探讨

从真鲷(Pagrus major)肝脏通过简并引物PCR克隆解偶联蛋白2(UCP2)cDNA部分序列。该片段长674bp,编码224个氨基酸残基。推测的此部分氨基酸序列包含线粒体载体蛋白的特征结构,并与其它脊椎动物UCP2氨基酸序列同源性在72．8％以上。对变温动物色类UCP2组织表达调控研究表明：与哺乳类UCP2基因不同,真鲷UCP2基因在肝脏大量表达,而在腹腔肠系膜脂肪组织则仅有痕迹量表达,两者表达水平相差20倍以上。饲料中添加10％绿鳕油或48h饥饿对真鲷肝脏UCP2基因的表达水平均无显著影响,表明UCP2基因在脂肪含量高的鱼类肝脏表达十分稳定,为维持其基本功能所必需。真鲷肝脏和腹腔肠系膜脂肪组织UCP2基因表达水平的强烈反差,与鱼类这两种贮脂器官完全不同的氧化活性相一致[动物学报49(1)：110—117,2003]。     

Rapid detection of severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies by up‐converting phosphor technology‐based lateral‐flow assay

In this study, an up‐converting phosphor technology‐based lateral‐flow (UPT‐LF) assay was developed to detect severe fever with thrombocytopenia syndrome virus (SFTSV) total antibodies rapidly and specifically. SFTSV recombinant N protein (SFTSV‐rNP) was coated on analytical membrane for sample capture, up‐converting phosphor (UCP) particles were used as the reporter, the luminescence emitted by UCP particles was converted to a measurable signal by a biosensor. The performance of UPT‐LF assay was evaluated by testing 302 field serum samples by ELISA (enzyme‐linked immunosorbent assay), Western blotting and UPT‐LF assay. UPT‐LF assay exhibited a lower detection limit than ELISA, and a satisfied level of agreement was exhibited by Kappa statistics (Kappa coefficient = 0.938). Considering Western blotting as the reference for comparison, the sensitivity and specificity of UPT‐LF assay could reach 98.31% and 100%. UPT‐LF assay showed no specific reaction with hantavirus total serum antibodies, which avoids the misdiagnosis of SFTSV from hantavirus that could cause similar clinical symptoms. UPT‐LF assay was able to achieve acceptable results within 15 min and needed only 10 μL sample for each test. As a whole, UPT‐LF assay is a candidate method for on‐site surveillance of SFTSV total antibodies owing to its excellent sensitivity, specificity, stability, easy operation and for being less time consuming.     

Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay

Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied using two sequence-specific oligonucleotides. One of the oligonucleotides probes was covalently bound to the UPT particle (reporter) for direct labeling and detection, whereas the second oligonucleotide probe contained biotin for capture by avidin during LF. The whole procedure of hybridization, UPT-LF detection, and analysis required a minimum time of 20 min. Moreover, aiming at minimal equipment demands, the hybridization conditions were chosen such that the entire assay could be performed at ambient temperature. During lateral flow, only targets hybridized to both capture and detection oligonucleotide were trapped and detected at an avidin capture line on the LF strip. Analysis (IR scanning) of the strips was performed in an adapted microtiter plate reader provided with a 980-nm IR laser for excitation of the phosphor particles (a portable reader was also available). Visible luminescence was measured and presented as relative fluorescence units (RFU) allowing convenient quantitation of the phosphor signal. With the assay described here as little as 0.1 fmol of a specific single-stranded nucleic acid target was detected in a background of 10 microg fish sperm DNA.     

Up-converting phosphor reporters for nucleic acid microarrays

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.     

Dual labeling of a binding protein allows for specific fluorescence detection of native protein

Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light. Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence. Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin. A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels. The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm. Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed. In a "sandwich" assay format, 10(3) org/mL E. coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium. Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10%. In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva. For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs. All assays were complete in 10 min. The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies.     

Upconverting phosphor reporters in immunochromatographic assays

Immunochromatographic assays have become popular diagnostic tools in a variety of settings because they are sensitive, fast, and easy to use. Here, we describe the use of a novel reporter, upconverting phosphors (UCP), in this assay format. UCP are submicron-sized, inorganic crystals that are excited with infrared light and that emit photons in the visible range depending on the ion composition of the crystal. Using human chorionic gonadotropin (hCG) as a model analyte to describe the properties of phosphors in immunochromatographic assays, a detection limit of 10 pg hCG in a 100-microl sample has been achieved on a regular basis, with occasional detection of 1 pg hCG. This represents at least a 10-fold improvement over conventional reporter systems such as colloidal gold or colored latex beads. Quantitation of analytes is possible over at least 3 orders of magnitude. Furthermore, an example is given of how UCP can be used for analyte multiplexing using a two-plexed wick for the detection of mouse IgG and ovalbumin. Thus, UCP lateral flow assays can be used for applications that are currently limited by assay sensitivity, and they can increase the probability of a diagnosis by verifying the presence of several analytes in the same sample.     

Feasibility of a Lateral Flow Test for Neurocysticercosis Using Novel Up-Converting Nanomaterials and a Lightweight Strip Analyzer

Neurocysticercosis is a frequent parasitic infection of the human brain, occurring in most of the world, and requires imaging of the brain to diagnose. To determine the burden of disease and to simplify diagnosis, a field-friendly rapid lateral flow (LF) based antibody screening test was developed. The assay utilizes novel nano-sized up-converting phosphor (UCP) reporter particles in combination with a portable lightweight analyzer and detects antibodies in serum samples reactive with bacterial-expressed recombinant (r) T24H, a marker for detecting neurocysticercosis cases. Three sequential flow steps allow enrichment of antibodies on the Test (T) line and consecutive binding of protein-A coated UCP reporter particles. Antibody binding was determined by measuring 550 nm emission after excitation of the UCP label with a 980 nm infrared (IR) diode. Clinical sensitivity and specificity of the assay to detect cases of human neurocysticercosis with 2 or more viable brain cysts were 96% and 98%, respectively, using a sample set comprised of sera from 63 confirmed cases and 170 healthy parasite-naïve non-endemic controls. In conclusion: Proof-of-principle, of a rapid UCP-LF screening assay for neurocysticercosis was demonstrated. The assay utilized bacterial-expressed rT24H as a potential alternative for baculovirus-expressed rT24H. Performance of the UCP-LF assay was excellent, although further studies need to confirm that bacterial expressed antigen can entirely replace previously used baculovirus antigen. In addition, the increasing availability of commercial sources for UCP reporter materials as well as the accessibility of affordable semi-handheld scanners may allow UCP-based bioanalytical systems for point-of-care to evolve at an even faster pace.     

A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen

An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA–ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP–LF test indicates a better performance than the ELISA. The UCP–LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings.     

Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.     

解偶联蛋白UCP1启动子荧光素酶报告基因载体的构建

目的:构建解偶联蛋白UCP1启动子荧光素酶报告基因载体,为寻找调控UCP1表达的小分子化合物提供有效工具。方法:从小鼠基因组DNA中PCR扩增小鼠UCP1启动子上游2000 bp序列,并将该序列连接到荧光素酶报告基因载体p GL3-basic中,构建p GL3-UCP1启动子。测序正确后,提取质粒,然后将上述载体与p RL-TK载体共转染至HEK293细胞、小鼠白色脂肪前体细胞和小鼠棕色脂肪前体细胞,48 h后裂解细胞检测荧光素酶的活性。结果:通过PCR成功扩增获得了目的片段,并将其克隆至p GL3-basic中。与细胞内源UCP1表达水平相似,荧光素酶报告系统表明构建的p GL3-UCP1在棕色脂肪细胞中启动子活性最高,在白色脂肪细胞中活性较低,在HEK293细胞中基本没有活性。同时β3肾上腺素受体激动剂CL 316,243同样能够上调p GL3-UCP1的启动子活性。结论:成功构建了小鼠UCP1启动子荧光素酶报告基因载体,并证明在棕色脂肪细胞中,该启动子具有很强的启动子活性,而在白色脂肪和HEK293细胞中,启动子活性很低。该启动子报告系统有望为寻找激活UCP1的小分子化合物提供重要平台。