CN111650371A - 基于纳米酶的膀胱癌bta检测试纸条 - Google Patents
基于纳米酶的膀胱癌bta检测试纸条 Download PDFInfo
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Abstract
本发明涉及一种基于纳米酶的膀胱癌BTA检测试纸条,试纸条同时利用了纳米酶的磁性和过氧化物酶活性,具体如下:一次纳米酶富集后用起到了样品浓缩的作用;层析结束后,利用纳米酶的过氧化物酶活性,加入显色液进行信号放大,起到了灵敏度提高的作用。
Description
技术领域
本发明属于医药生物技术领域,具体的,涉及一种基于纳米酶的膀胱癌BTA检测试纸条。
背景技术
磁性纳米颗粒具有良好的生物相容性,其既具有纳米材料所特有的性质,如粒径小、比表面积大、偶 联容量高,又具有磁响应性及超顺磁性,可以在恒定磁场下聚集和定位、在交变磁场下吸收电磁波产热, 利用这些特性,磁性纳米颗粒被广泛应用于磁共振对比剂、磁靶向药物载体、细胞与生物分子分离、生物 传感与检测以及磁感应肿瘤热疗等生物学领域。
磁性免疫层析(Magnetic ImmunoChromatographic Test,MICT)作为一种新一代单人份快速定量检测技 术逐渐发展起来,它是以超顺磁性纳米颗粒代替传统的标记物(胶体金,乳胶颗粒等)来进行免疫层析,最 后通过磁信号阅读仪读取结合在检测线处磁颗粒的磁场强度,从而对所检样品进行定性定量判断。
纳米酶BTA检测试纸条的工作原理,是将能特异识别BTA的抗体固定在硝酸纤维素膜(NC膜)上 作为检测线(T线),将能特异识别BTA的另一抗体(鼠源或兔源)固定在纳米酶上得到纳米酶探针,将 羊抗鼠或羊抗兔(视纳米酶上的抗体而定)抗体固定在NC膜上作为质控线(C线)。
当待测样品(如尿液)与纳米酶探针混合后,纳米酶探针上的抗体分子与待测样品中的BTA发生抗 原抗体反应而形成纳米酶BTA复合物,然后利用纳米酶的磁性,将待测样本中的BTA分子进行富集,加 入到96孔板的小孔中,接下来将纳米酶BTA检测试纸条插入,液体在吸水垫的作用下层析,当纳米酶BTA 复合物层析到NC膜上的T线位置时,BTA与T线上的抗BTA抗体再一次发生抗原抗体反应而被捕获, 由于纳米酶自身具有棕色因而能够在T线的位置显示条带,条带的深浅与待测液体中BTA的多少呈正相 关。
无论待测样品中是否有BTA分子,当纳米酶探针层析到NC膜上的C线位置时,此处固定的羊抗鼠 或羊抗兔抗体都能与纳米酶探针上的鼠源或兔源抗体结合将其捕获,从而显示条带,以指示纳米酶探针和 NC膜上的抗体分子均正常有效,达到了质控的目的。
发明内容
本发明首先涉及一种基于纳米酶的磁性和过氧化物酶活性的BTA抗原检测试纸条,所述的试纸条上 装有检测线,质控线。
本发明还涉及一种基于纳米酶的磁性和过氧化物酶活性的BTA抗原检测试纸条的制备方法,所述的 方法包括如下步骤:
(1)包被膜的制备:用包被缓冲液将BTA的抗体、二抗分别稀释,使用定量喷膜装置按前后顺序将 二者均匀喷印于硝酸纤维素膜上,分别形成检测线(T线)抗体带与质控线(C线)抗体带;
(2)磁颗粒探针垫的制备:使用喷膜仪将处理好的磁颗粒探针均匀喷涂于玻璃纤维垫上;
(3)样品垫的处理:将样品垫浸入样品垫处理液处理、烘干。
(4)试纸条的组装及切割:将包被膜、磁颗粒探针垫、样品垫、吸水垫以相互交错的形式依次粘贴 在背衬(底板)上,覆膜并组装成试纸板。
所述的BTA抗原(膀胱癌抗原,即人补体因子H相关蛋白)氨基酸序列如SEQ IDNO.1所示:
SEQ ID NO.1:
MWLLVSVILISRISSVGGEATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRI TCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCR STDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGP PPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRW TAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR
本发明的有益效果在于:纳米酶BTA(膀胱癌抗原)检测试纸条同时利用了纳米酶的磁性和过氧化物 酶活性,具体如下:
一次尿液50mL,纳米酶富集后用500μL缓冲液重悬,起到了核酸浓缩100倍的作用;
层析结束后,利用纳米酶的过氧化物酶活性,加入显色液进行信号放大,起到了灵敏度5倍提高的作 用。
此为纳米酶首次应用于尿液检测,尿液检测有样本量大的特点,因此两者的综合效果可实现灵敏度500 倍的提高。
附图说明
图1、检测尿液中目标物质成分的纳米酶检测试纸条结构示意图。
具体实施方式
实施例1,检测BTA抗原的纳米酶试纸条的制备
检测线,质控线的位置如图1所示,各个位置上的预装检测物如下:
1、包被膜的制备:用包被缓冲液(0.02M磷酸盐缓冲液,pH7.2)将BTA的抗体、市售羊抗鼠抗体(二 抗)分别稀释为0.5mg/ml与1mg/ml,使用定量喷膜装置以1μl/cm的量按前后顺序将二者以0.8cm的间隔 均匀喷印于3.5cm宽度的硝酸纤维素膜上,分别形成检测线(T线)抗体带与质控线(C线)抗体带。室温晾干 30min后于封闭液(含有0.5%BSA的0.02MPBS,pH7.2)中浸泡10min后于25-35℃烘干8小时,加入干燥 剂封存备用。
2、磁颗粒探针垫的制备:使用喷膜仪的专用喷头将处理好的磁颗粒探针以50μl/cm的量均匀喷涂于 0.8cm宽度的玻璃纤维垫上,过夜冷冻干燥,加入干燥剂封存备用。
3、样品垫的处理:将1.8cm宽度的样品垫(亲水性的玻璃纤维)浸入样品垫处理液(1-5%Casein(酪蛋白), 0.1-1%PVA(聚乙烯醇),0.01-0.2%Tween 20,0.02M PBS,pH7.2)处理1小时,取出后于25-35℃烘干8小 时。
4、试纸条的组装及切割:将3.5cm包被膜、0.8cm磁颗粒探针垫、1.8cm样品垫、2.5cm吸水垫以相 互交错2mm的形式依次粘贴在背衬(底板)上,覆盖一层透明塑料密封膜,组装成试纸板;使用切条机将组 装好的试纸板切成0.5cm宽的成品试纸条;将切割好的试纸条置于塑料低卡上的卡槽内,盖上上盖,使用 压卡机将上下两片塑料卡压紧,加入干燥剂室温封存备用。
BTA(膀胱癌抗原,即人补体因子H相关蛋白)氨基酸序列如SEQ ID NO.1所示:
SEQ ID NO.1:
MWLLVSVILISRISSVGGEATFCDFPKINHGILYDEEKYKPFSQVPTGEVFYYSCEYNFVSPSKSFWTRI TCTEEGWSPTPKCLRLCFFPFVENGHSESSGQTHLEGDTVQIICNTGYRLQNNENNISCVERGWSTPPKCR STDTSCVNPPTVQNAHILSRQMSKYPSGERVRYECRSPYEMFGDEEVMCLNGNWTEPPQCKDSTGKCGP PPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCRNGQWSEPPKCLHPCVISREIMENYNIALRW TAKQKLYLRTGESAEFVCKRGYRLSSRSHTLRTTCWDGKLEYPTCAKR
实施例2,检测BTA抗原
1、加样检测
微量移液器取50μl含有梯度浓度的BTA样品溶液(浓度依次是100ng/ml、10ng/ml、1ng/ml、0ng/ml) 加入检测卡上的样品垫上,再加入50μl层析缓冲液(1%Tween 20,0.5%Triton X-100,1%NP-40, 0.05%NaN3,20mM PBS,pH 7.2),等待反应进行15min。由于磁颗粒上偶联有BTA的另一个配对单抗, 它与样品溶液中的BTA结合后形成的复合物继续先前移动时,会与检测线(T线)处的BTA的另一抗体结合 形成磁颗粒的聚集,而没有结合的BTA的磁颗粒抗体探针会继续移动到质控线(C线)处通过与其二抗作用 形成磁颗粒的聚集。
2、酶催化放大检测信号
在检测线T线和质控线C线处加入50μl的显色溶液(过氧化氢H2O2,浓度为530mM;过氧化物酶 的生色底物二氨基联苯胺(3,3’-diaminobenzidine,DAB)浓度为816mM),反应5min。
由于磁颗粒的过氧化物模拟酶活性,会在磁颗粒聚集的T线和C线处生成大量棕褐色的不溶性沉淀物, 从而将检测信号放大,其检测灵敏度是传统胶体金试纸条检测法的500倍以上。
以上仅是本发明的具体应用范例。应理解,这些实施例仅用于说明本专利而不用于限制本发明的范围。
SEQUENCE LISTING
<110> 深圳市第二人民医院
中国科学院生物物理研究所
<120> 基于纳米酶的膀胱癌BTA检测试纸条
<130> CP11902124C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 330
<212> PRT
<213> 序列
<400> 1
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Claims (5)
1.一种基于纳米酶的磁性和过氧化物酶活性的BTA抗原检测试纸条的制备方法,其特征在于,所述的方法包括如下步骤:
(1)制备包被抗体的膜;
(2)磁颗粒探针垫的制备:使用喷膜仪将处理好的磁颗粒探针均匀喷涂于玻璃纤维垫上;
(3)样品垫的处理:将样品垫浸入样品垫处理液处理、烘干;
(4)试纸条的组装及切割:将包被膜、磁颗粒探针垫、样品垫、吸水垫以相互交错的形式依次粘贴在背衬(底板)上,覆膜并组装成试纸板。
2.根据权利要求1所述的方法,其特征在于,包被膜的制备方法为:用包被缓冲液将BTA的抗体、二抗分别稀释,使用定量喷膜装置按前后顺序将二者均匀喷印于硝酸纤维素膜上,分别形成检测线(T线)抗体带与质控线(C线)抗体带。
3.根据权利要求1或2所述的方法,其特征在于,所述的BTA抗原的氨基酸序列如SEQ IDNO.1所示。
4.权利要求1-3任一所述的方法制备获得的检测试纸条。
5.权利要求4所述的检测试纸条在制备检测疾病的医疗器械中的应用,所述的疾病为BTA蛋白高表达的疾病,优选的,所述的疾病是膀胱癌。
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