压电石英晶体免疫传感器微阵列检测人免疫球蛋白的实验研究

目的以AT切型、基频10MHz的镀金石英晶体作为换能器,将抗人免疫球蛋白抗体固定在石英晶体电极表面,用2×5检测池阵列构建一种新型压电石英晶体免疫传感器。方法分别进行标准品及人血清免疫球蛋白的检测,研究了检测温度、时间、特异性等指标对检测的影响。结果该免疫传感器可在15 min内完成检测且无非特异性反应;在检测范围内传感器的响应特性良好,压电晶体频率偏移值与免疫球蛋白浓度呈良好的线性关系,将此免疫传感器用于人血清标本的测定,结果与免疫比浊法符合(r分别为0.94、0.94、0.94、0.92和0.91)。结论此传感器微阵列具有灵敏度高、特异性好,检测通量高,不需标记,操作简便,能实时在线检测和重复使用等优点,可用于临床实验诊断,具有较高的临床推广应用价值。     

适配子型压电传感器的实验研究

目的建立适配子型压电传感器阵列检测人免疫球蛋白E,对适配子型压电石英传感器的各项参数进行初步探索。方法用生物素-亲和素法将针对人IgE的适配子探针固定在压电传感器的金膜表面,通过压电传感器对适配子与IgE的反应进行实时监测,观察不同浓度IgE的反应趋势及所引起的频率变化,获得IgE检测标准曲线;通过检测BSA、IgG、IgE非特异信号,并与特异性信号相比较对适配子检测的特异性进行检验。结果亲和素法固定适配子对IgE进行检测,最低检出限0.055mg/L,线性范围0.1～2.5mg/L,非特异性信号响应小,不超过同浓度特异性信号的5%。结论构建的适配子压电传感器可检测ng级物质且非特异性信号小,显示该平台在临床检测中有较好的应用前景。     

压电蛋白芯片分析仪的研制

目的：研制一种可以快速检测血液样品中的乙肝表面抗原、HIV抗体和人禽流感H5N1抗体的便携式分析设备。方法：系统以STC89C52微处理器为控制核心,采用嵌入式系统技术,通过对石英晶体阵列谐振频率的检测处理．利用石英晶体微天平对表面质量负载的高度敏感性及抗体和其对应抗原闻的特异识别进行快速检测。结果：该压电蛋白芯片分析仪整机灵敏度高、特异性好、便于携带,可实现多参数同步检测。结论：压电蛋白分析仪的研制突破了传统的体外诊断试剂方法,达到了临床使用要求。     

适配子压电石英芯片再生性能研究

目的探索适配子压电石英芯片最佳再生试剂,对固定好的适配子型芯片保存性能进行检测。方法用生物素-亲和素法将针对人IgE的适配子探针固定在压电传感器的金膜表面,分别用HCl、NaOH、EDTA、尿素、甲酰胺5种试剂对适配子芯片进行再生实验,选取较好再生试剂;将固定好的适配子芯片保存在PBS缓冲液（0.1%叠氮钠）中,对芯片的保存时间进行测定。结果用EDTA对适配子芯片进行再生效果最好,重复检测5次后芯片响应信号仍在第1次检测的〉90%,此外HCl也可对适配子芯片重复利用3次;固定好的适配子芯片在加入防腐剂的结合缓冲液中可长期保存,保存21d后信号响应无明显下降。结论与抗体压电传感器相比较,适配子压电传感器有一定优势,适配子芯片成本更低,再生能力强,固定好的芯片保存时间长,体现出适配子检测的优越性。     

压电石英晶体传感器阵列检测尿微量白蛋白的实验研究

目的探讨并建立压电石英晶体免疫传感器检测尿液微量白蛋白的方法。方法采用葡萄球菌A蛋白(SPA)法固定石英晶体微阵列上单克隆抗白蛋白抗体,并用石英晶体微天平(QCM)检测样品反应频率漂移量;优化传感器检测条件并运用回收实验,天内、天间重复试验来验证其准确度与精密度,并将免疫比浊法作为参比方法进行方法学比较。结果该传感器平台的非特异性反应低(FS<20Hz),线性范围广(0.01~40mg/L),灵敏度达到5μg/L,准确度高(CV<10%),精密度试验总体CV<10%,并且具有检测时间短(<10 min)等优点,与传统的免疫比浊法具有可比性。结论采用压电石英晶体免疫传感器法检测尿液微量白蛋白,是一种可行的检测方法,可应用于临床检测并为诊断早期肾损伤提供帮助。     

心肌肌红蛋白压电免疫芯片检测方法的建立

目的研制一种新型、快速地用于检测心肌肌红蛋白的压电免疫芯片。方法设计四通道芯片微阵列,采用双功能基团交联剂Sulfo-LC-SPDP将心肌肌红蛋白单克隆抗体巯基化,固定于石英晶体的金膜电极表面制备生物敏感膜,构建压电免疫芯片。结果芯片用于检测心肌肌红蛋白的灵敏度达50ng/mL,与酶联荧光分析法具有较好的相关性,可用于半定量检测心肌肌红蛋白。结论构建的压电免疫芯片具有灵敏度高、特异性好等特点,具有一定的临床应用价值。     

DNA生物传感器在“基因诊断”中的实验研究

为了建立石英晶体DNA传感器检测乙肝病毒基因的方法,在石英晶体金表面固定单链DNA,构成压电晶体HBV DNA生物传感器,与待测样品进行杂交反应.结果DNA传感器能准确的诊断血清中的HBV病毒基因.石英晶体HBV DNA生物传感器具有操作省时、简单,能用于临床实验、诊断、检测.     

压电免疫传感器研究进展

压电免疫传感器操作简易、特异性强、灵敏度高,将发展成为一种有效的在线检测技术,在各个领域里中有广阔的应用前景。为此,文中介绍了压电免疫传感器的原理、基本组成、目前国内外常用的生物识别元件的固定化技术、对抗原或抗体的常用检测方法,以及压电免疫传感器在食品安全检测、医学诊断与环境监测中的应用研究进展,并对压电传感器的特点及发展趋势作出了综合评述。     

压电免疫传感器再生能力的试验研究

目的采用SPA法在镀金电极表面进行抗体固定,探索压电免疫传感器再生使用的最佳条件.方法对压电免疫传感器的检测时间、特异性,及再生试验的最佳试剂、时间进行实验研究.结果构建的压电免疫传感器检测时间为15min,无交叉反应;NaOH溶液再生10次后,频率响应下降为原有的75%.结论所构建的压电免疫传感器检测时间短,特异性好,可用于临床标本的检测,NaOH溶液用于石英晶体再生的效果较好,极大地降低了使用成本.     

尿β2微球蛋白压电传感器阵列的构建与临床应用

目的探讨并建立压电石英晶体免疫传感器检测尿液微量β2微球蛋白的方法。方法采用葡萄球菌A蛋白（SPA）法固定石英晶体微阵列上单克隆抗β2微球蛋白抗体,并用石英晶体微天平（QCM）检测样品反应频率漂移量;优化传感器检测条件并运用回收实验,天内、天间重复试验来验证其准确度与精密度,并将免疫比浊法作为参比方法与建立的传感器方法进行方法学比较。结果该传感器平台的非特异性反应低（FS〈20Hz）,线性范围广（0.01～40.00mg/L）,灵敏度达到3μg/L,准确度高（CV〈10%）,精密度试验总体CV〈10%,并且具有检测时间短（〈10min）等优点,与传统的免疫比浊法具有可比性。结论采用压电石英晶体免疫传感器法检测尿液微量β2微球蛋白是一种可行的检测方法,可应用于临床检测并为诊断早期肾损伤提供帮助。     

咳嗽变异性哮喘儿童检测血清总IgE含量及过敏原特异性IgE的临床意义

[目的]通过对132例咳嗽变异性哮喘患儿和56例其他慢性咳嗽疾病患儿(对照组)的血清总IgE含量及过敏原特异性IgE的检测,探讨血清总IgE含量及过敏原特异性IgE检测在临床诊治及预防咳嗽变异性哮喘的应用价值。[方法]用美国贝克曼化学发光DX1800检测总IgE含量,使用德国欧蒙印迹法检测20种过敏原特异性IgE。[结果]CVA总IgE阳性率87.1%,特异性IgE阳性率78.8%。对照组总IgE阳性率8.9%,特异性IgE阳性率7.1%。CVA吸入性过敏原阳性率最高,主要是屋尘螨/粉尘螨、霉菌组合、屋尘,分别是55.3%、43.2%、38.95%;食入性过敏原阳性率较高的主要是海(水)产品,其中以虾最高,为17.4%。[结论]说明CVA是特异性IgE介导的反应性疾病,过敏原是其主要致病因素,检测血清总IgE及过敏原特异性IgE对疑是咳嗽变异性哮喘疾病的儿童可起到帮助诊断作用,还可对治疗和预防起到指导意义。     

Enzyme immunoassays for total and allergen specific IgE in population studies.

OBJECTIVE--Extensive IgE serology in occupational or environmental health studies is often hampered by a lack of technical facilities and finance. The use in population studies of relatively simple and inexpensive enzyme immunoassays (EIAs) was therefore evaluated for the assessment of total serum immunoglobulin E (IgE), and of specific IgE reactions with various common (house dust mites, grass and birch pollen, and cat) or occupational (fungal alpha-amylase and rat urinary protein) allergens. METHODS--Total IgE was measured with a sandwich EIA, calibrated with commercially available IgE standards. Reproducibility was studied by testing pooled normal human serum samples in each of a large series of test plates. A panel of 156 children's serum samples with known IgE values was used to compare the assay with other total IgE assays. A previously developed EIA for anti-yeast IgE was adapted for the measurement of IgE reacting with various common and occupational allergens. Binding of IgE to microwells coated with commercially available allergen extracts, or allergen preparations from our own laboratory, was measured with a monoclonal anti-human IgE antibody and subsequent incubations with biotinylated rabbit anti-mouse Ig and avidin-peroxidase. Panels of serum samples from school children (n = 116), bakery workers (n = 126), and laboratory animal workers (n = 52) were used to study sensitivity and specificity, with reference to skin prick tests as the standard, and to compare the EIAs with commercially available test kits. RESULTS--The detection limit of the EIA for total IgE was 0.5-1 kU/l for undiluted serum samples, and the coefficient of variation between assays was less than 15% at serum concentrations between 1 and 150 kU/l. Results obtained with the panel of 156 children's serum samples were strongly correlated (r2 = 0.86) with IgE concentrations measured previously by radioimmunoassay. The results of the EIA for various occupational allergens correlated very well, both qualitatively and quantitatively, with the results of commercial test kits. Sensitivity and specificity of the EIA results as a predictor of skin prick test reactivity towards common allergens (house dust mite, grass pollen, birch pollen, and cat) were remarkably high (> 80%-90%) in the series of 116 children's serum samples. In a population of bakery workers the specificity of the EIAs was also very high (> 90%). The sensitivity was notably lower (30%-70%) in this adult population, which is, however, in agreement with results reported for conventional IgE tests. CONCLUSION--As the costs were estimated to be at least five to 10-fold lower than those of commercial test kits, the EIAs for total and specific IgE may be very useful tools in epidemiological studies of atopic respiratory or other disorders.     

基于离子液体复合膜的葡萄糖生物传感器

目的建立一种电化学葡萄糖生物传感器,进而评价其在血糖检测的应用前景。方法将葡萄糖氧化酶（GOD）分散在N,N-二甲基甲酰胺（DMF）-离子液体（[BMIM]PF6）复合膜中滴于二茂铁（Fc）修饰的丝网印刷碳电极（SPCE）上,制备一种新型三电极葡萄糖电化学生物传感器。用扫描电子显微镜（SEM）、循环伏安法（CV）和计时电流法表征生物传感器的制备以及其电化学特性。结果该生物传感器有良好的稳定性和灵敏性,响应电流与葡萄糖浓度在0.5～16mM之间有良好的线性相关,检出限为0.1mM。结论制备的电化学葡萄糖生物传感器具有良好性能,可用于血糖检测。     

链置换扩增压电DNA传感器检测铜绿假单胞菌的临床应用研究

目的探讨链置换扩增(SDA)压电DNA传感器检测铜绿假单胞菌的临床应用.方法应用SDA压电DNA传感器芯片技术和特异性寡核苷酸探针,对临床标本33份进行铜绿假单胞菌检测,并以荧光定量PCR作参照,与常规培养进行对比分析.结果在33份临床标本中,SDA压电DNA传感器检出17份铜绿假单胞菌阳性,与荧光定量PCR结果完全一致,而这两种方法与常规分离培养略有差异.结论 SDA压电DNA传感器芯片技术能直接检测铜绿假单胞菌基因组DNA,具有准确、快速、灵敏的优点.     

基于双链特异性核酸酶触发滚环扩增的microRNA-21太赫兹超材料传感方法的构建

目的:构建一种太赫兹(THz)超材料传感方法用于microRNA-21(miRNA-21)的信号放大检测。方法:首先构建THz超材料传感方法,并用聚丙烯酰胺凝胶电泳及zeta电位验证方法的可行性;对传感器检测条件进行优化之后,对不同浓度的miRNA-21以及其他不同的miRNAs进行检测,并与其他microRNA检测方法进行比较;最后,对该传感器的回收率进行了评价。结果:在最优实验条件下,通过双链特异性核酸酶(DSN)循环识别与滚环扩增(RCA)的双重信号放大策略,该THz超材料传感器对靶标miRNA-21的响应范围为10 fmol/L至10 nmol/L,检测限为8.49 fmol/L。并且该传感器具有较好的特异性,具备了从多种miRNAs中识别靶标miRNA-21的能力,并且在商业化人血清样本中的回收率可达94.33%到115.33%。结论:该THz传感器可以实现靶标miRNA-21的高灵敏、高特异性检测,具备了在miRNA相关疾病无标记诊断与早期预警的潜力。     

Associations between HLA and immune responses in individuals with chronic schistosomiasis japonica

The association of an HLA specificity with low or high immune responsiveness to Schistosoma japonicum antigen (Sj) was demonstrated among individuals who had previously been exposed to S. japonicum infection. The frequency of HLA-Aw24 specificity among low responders in the IgG antibody response determined by the enzyme-linked immunosorbent assay (ELISA) was higher than that among high responders. Significant association between HLA-B7 and high responsiveness was observed in the IgE antibody response. The frequency of HLA-Bw52-Dw12 was also found to increase among individuals with higher levels of total serum IgE. These results suggest that antibody responses to Sj are regulated by an immune response (Ir) gene(s) linked to the major histocompatibility complex (MHC), although a possibility of the presence of non-MHC-linked Ir genes is not excluded. Serum IgE levels may also be controlled by a gene(s) linked to the MHC.