CN110372789A - CDs/SiO2-SFTSV单克隆抗体缀合物及其制备方法和应用 - Google Patents
CDs/SiO2-SFTSV单克隆抗体缀合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了CDs/SiO2‑SFTSV单克隆抗体缀合物的制备及其在免疫层析试纸条上的应用,所述CDs/SiO2‑SFTSV单克隆抗体缀合物以CDs/SiO2荧光纳米球接连SFTSV单克隆抗体获得。本发明还公开了基于CDs/SiO2荧光纳米球的免疫层析试纸条的制备方法。本发明制备的基于CDs/SiO2荧光纳米球的免疫层析试纸条具有简单、快速、灵敏度高、特异性强、价廉、样品所需量少等优点,其灵敏度与常规的仪器分析一致,适合现场筛选,其中的CDs/SiO2球具有高荧光强度和高稳定性,相比传统发的胶体金免疫层析试纸条,能大大提高检测极限。总之,这种快速检测的荧光试纸条能很好的应用于现场流行病检测。
Description
技术领域
本发明属于生物化学分析检测技术领域,涉及CDs/SiO2-SFTSV单克隆抗体缀合物及其制备方法和应用,具体涉及基于CDs/SiO2荧光纳米球的免疫试纸条及其制备方法和应用,特别涉及对SFTSV核蛋白检测的应用。
背景技术
发热伴血小板减少综合征布尼亚病毒(severe fever with thrombocytopeniasyndrome bunyavirus,SFTSV),简称“新布尼亚病毒”。是发热伴血小板减少综合征(俗称蜱咬病,一种以严重发热伴血小板减少为主要特征的新型传染性疾病)的致病原。临床表现以发热伴血小板减少为主要特征,少数患者病情较重且发展迅速,可因多脏器功能衰竭而死亡。这一病情主要发生在丘陵、山区地带,患者以从事农业生产的成年农民为主,部分患者被蜱虫叮咬。急性期病人血液可能有传染性。
目前,有关SFTSV的检测方法非常少,免疫层析试纸条由于其即时检测,方法简单,检测时间段,成本低等优点而成为倍受青睐的检测手段。而金胶免疫层析技术的信号依赖于金纳米粒子的局部表面等离子体共振效应,其灵敏度不高。而一些传统这些荧光信号物可能遭受光漂白(有机荧光团),低发射强度(上转换磷光体)和复杂基质(上转换磷光体,QD)中的胶体不稳定性等影响,这降低了测定分析的灵敏度和稳定性。
发明内容
发明目的:鉴于CDs/SiO2球的高荧光强度和高稳定性,以及免疫层析试纸的便携、可视化、低成本等优点,本发明解决了现有的针对SFTSV核蛋白检测技术的繁琐、低检测极限等劣势。
本发明所要解决的技术问题是提供了CDs/SiO2-SFTSV单克隆抗体缀合物。
本发明还要解决的技术问题是提供了CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法及其应用。
本发明最后要解决的技术问题是提供了一种成本低廉、高灵敏度、低检测极限的基于荧光碳球的免疫试纸条,实现了对SFTSV核蛋白抗原即时检测。
技术方案:为了解决上述技术问题,本发明提供了CDs/SiO2-SFTSV单克隆抗体缀合物,所述CDs/SiO2-SFTSV单克隆抗体缀合物以CDs/SiO2荧光纳米球接连SFTSV单克隆抗体获得。
本发明内容还包括所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,包括以下步骤:
1)硅烷化CDs的制备;
2)CDs/SiO2荧光纳米球的制备及洗涤纯化:所述CDs/SiO2荧光纳米球的制备方法如下:将十六烷基溴化胺和NaOH溶液加入到水中并搅拌,然后在强搅拌下继续加入TEOS和CDs的混合液并反应,通过离心收集沉淀物;
3)CDs/SiO2-SFTSV单克隆抗体缀合物的制备。
其中,所述步骤1)的硅烷化CDs的制备方法如下:一水合柠檬酸在高温的真空干燥箱中干燥,然后与氮气脱气的N-β-(氨基乙基)-γ-氨基丙基三甲氧基硅烷在高温下反应,形成CDs溶液,随后高速离心,弃去离心管底部大颗粒即得。
具体的,所述步骤2)CDs/SiO2荧光纳米球的制备如下:通过共水解方法制备CDs/SiO2荧光纳米球,在99mL水中,加入十六烷基溴化胺(CTAB,终浓度为0.02mg/ml~20mg/ml)和NaOH(终浓度0.010mg/ml~6mg/ml)加入到并在80℃下搅拌10min~2h,然后在强搅拌下继续加入TEOS(占水体积的百分比,终浓度0.01%~12%)和CDs(占水体积的百分比,终浓度0.001%~5%)的混合液并反应1~5h。通过离心收集沉淀物,用乙醇和H2O洗涤数次。
其中,所述步骤2)的洗涤纯化步骤如下:将离心收集沉淀物分散在HCl和乙醇的混合液中并搅拌以萃取残余物有机模板,并重复萃取,将产物洗涤至中性,最后将获得的氨基封端的CDs/SiO2荧光纳米球分散在H2O中。
其中,所述步骤3)的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法如下:先将丁二酸苷与氨基封端的CDs/SiO2荧光纳米球反应将其羧基化,得到CDs/SiO2-COOH;然后利用EDC/NHS将CDs/SiO2表面的羧基活化,加入SFTSV抗体,室温孵育得到CDs/SiO2-SFTSV抗体缀合物。
本发明内容还包括基于荧光碳球的免疫试纸条,所述基于荧光碳球的免疫试纸条条包括将所述的CDs/SiO2-SFTSV单克隆抗体缀合物与SFTSV通过抗原-抗体之间的相互作用制备的荧光信号标记物。
其中,所述基于CDs/SiO2荧光纳米球的免疫试纸条还包括依次粘连在黑色底板上的样品垫、包被有检测T线和对照C线的硝基纤维素膜和吸水垫;所述的硝基纤维素膜T线上包被有SFTSV单克隆抗体;所述的硝基纤维素膜C线上包被有羊抗鼠多克隆抗体。
其中,所述T线的SFTSV单克隆抗体的浓度为0.04~0.07mg/cm2。
其中,所述C线的羊抗鼠多克隆抗体的浓度为0.04~0.07mg/cm2。
本发明内容还包括所述的基于CDs/SiO2荧光纳米球的免疫试纸条的制备方法,所述制备方法包括以下步骤:
1)硅烷化CDs的制备;
2)CDs/SiO2荧光纳米球的制备及洗涤纯化;
3)CDs/SiO2-SFTSV单克隆抗体缀合物的制备;
4)T线上包被有SFTSV单克隆抗体、C线上包被有羊抗鼠多克隆抗体的硝基纤维素膜的制备;
5)将黑色底板、样品垫、包被有检测T线和对照C线的硝基纤维素膜、吸水垫组合,底板上的硝基纤维素膜的一端粘贴吸水垫,硝基纤维素膜与吸水垫之间相互重叠1~2mm,在硝基纤维素膜的另一端粘贴样品垫,即得基于CDs/SiO2荧光纳米球的免疫试纸条。
本发明的检测原理:检测过程中,先将微量的SFTSV核蛋白抗原与CDs/SiO2-SFTSV抗体缀合物结合形成复合物,然后滴加到样品垫上。以硝酸纤维素膜为载体,利用了微孔膜的毛细血管作用,滴加在样品垫的液体慢慢向另一端渗移,通过抗原抗体结合,先被硝基纤维素膜上T线捕获,形成双抗体夹心复合物,未被捕获的继续向另一端渗移被硝基纤维素膜上C线捕获,因此在T线和C线上有CDs/SiO2的固定并积累,在紫外灯的照射下显现出荧光。结果:T线和C线显现出荧光为阳性,即存在SFTSV核蛋白抗原;T线无荧光和C线显现出荧光为阴性,即不存在SFTSV核蛋白抗原或者SFTSV核蛋白抗原含量极少;当C线不显现荧光,T线不论有无荧光时,表明结果无效。通过CDs/SiO2特有的荧光性质示踪判断SFTSV核蛋白抗原的有无。
有益效果:相比于现有技术,本发明具备以下优点:本发明制备的基于CDs/SiO2荧光纳米球的免疫层析试纸条具有简单、快速、灵敏度高、特异性强、价廉、样品所需量少等优点,其灵敏度与常规的仪器分析一致,适合现场筛选,其中的CDs/SiO2球具有高荧光强度和高稳定性,生物相容性好,表面易修饰,无论其在水相或者固态下都有很强的荧光。相比传统发的胶体金免疫层析试纸条,能大大提高检测极限。总之,这种快速检测的荧光试纸条能很好的应用于现场流行病检测。
附图说明
图1为本发明制备的基于CDs/SiO2荧光纳米球的免疫层析试纸条原理及结构示意图;
图2为CDs/SiO2荧光纳米球的TEM图片;
图3为CDs/SiO2荧光纳米球的共聚焦图片;
图4为CDs/SiO2荧光纳米球及CDs/SiO2荧光纳米球羧基化之后在水溶液中的ZeTa电位;
图5为(A)免疫层析试纸条的结果和(B)SFTSV抗原存在(左)和不存在(右)下免疫层析试纸条在紫外灯下阳性结果及阴性结果的图片;
图6为不同硝基纤维素膜制备出的试纸条对不同浓度SFTSV抗原的检测结果在紫外灯下的图片;
图7为Sartorius CN95硝基纤维素膜制备的试纸条对不同抗原检测的特异性实验在紫外灯下的图片。
具体实施方式
下面结合附图对本发明的技术方案进行进一步说明。
本发明是基于CDs/SiO2球荧光免疫层析试纸条实现对SFTSV核蛋白检测,上述具体应用为:以CDs/SiO2球为荧光信号物,SFTSV为检测抗原,制备检测SFTSV的荧光免疫层析试纸条。
该测SFTSV的荧光免疫层析试纸条,以CDs/SiO2球为荧光信号物,羧基化之后通过EDC/NHS活化接连SFTSV单克隆抗体形成CDs/SiO2-SFTSV抗体缀合物,以SFTSV为抗原,与CDs/SiO2-SFTSV抗体缀合物结合形成荧光纳米球;滴加在样品垫上,液体慢慢向另一端渗移,通过抗原-抗体结合,先后被硝基纤维素膜上T线与C线捕获,如图1。
主要试剂和耗材如下:N-β-(氨基乙基)-γ-氨基丙基三甲氧基硅烷(AEAPTMS),一水合柠檬酸,氢氧化钠(NaOH),十六烷基三甲基溴化铵(CTAB),原硅酸四乙酯(TEOS),丁二酸酐,盐酸(HCl),无水乙醇和N,N-二甲基甲酰胺(DMF)、N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐(EDC)、N-羟基磺基琥珀酰亚胺钠盐(Sulfo-NHS)、牛血清白蛋白(BSA)、SFTSV核衣壳蛋白抗原(江苏省疾病预防控制中心)、SFTSV单克隆抗体购自(江苏省疾病预防控制中心)。羊抗鼠多克隆抗体IgG(上海杰一生物科技有限公司),样品垫,NC膜(pall vivid90、Sartorius CN95和pall vivid 170),吸水垫、聚氯乙烯(PVC)基板、离心管、移液枪及枪头、直尺、签字笔、棉纤粗笔头及紫外灯。
实施例1硅烷化CDs的制备
1)制备前反应物的准备
一水合柠檬酸中的H2O分子对合成出的硅烷化CDs有很大的干扰,因而实验反应之前将一水合柠檬酸在高温的真空干箱中干燥过夜进行脱水处理。
2)硅烷化CDs的制备
将15mL的AEAPTMS倒入50mL的三颈烧瓶中并用通氮气15min。然后将溶液加热至230℃,并在剧烈搅拌下快速加入1g脱水的柠檬酸一水合物。5min后,停止加热,自然冷却得到CDs溶液。
3)硅烷化CDs的纯化
将得到的CDs溶液10000rpm离心10min除去反应生产的大颗粒以进行纯化得到CDs。
实施例2 CDs/SiO2荧光纳米球的制备
1)CDs/SiO2荧光纳米球的制备
在99mL水中,加入0.2g CTAB和60mg NaOH,并在80℃下搅拌1h,其中CTAB、NaOH终浓度分别为2mg/ml、0.6mg/ml;然后在强搅拌下继续加入1.2mLTEOS和0.35mL实施例1制备的CDs的混合液并反应3h,通过离心收集沉淀物,用乙醇和H2O洗涤数次得到反应产物。其中TEOS和CDs占水的最终体积比分别为1.2%、0.35%。
2)CDs/SiO2荧光纳米球的洗涤纯化
将反应产物分散在15mL HCl和120mL乙醇的混合液中并在60℃下搅拌6h以萃取残余物有机模板。并重复萃取一次,将产物洗涤至中性,最后将获得的氨基封端的CDs/SiO2球体分散在H2O中得到氨基封端的CDs/SiO2球体溶液。CDs/SiO2荧光纳米球的TEM表征图如图2,直径为147nm,具有介孔结构;其共聚焦图片如图3。
实施例3 CDs/SiO2-SFTSV单克隆抗体缀合物的制备
1)CDs/SiO2荧光纳米球的羧基化
将实施例2制备的氨基封端的CDs/SiO2球体溶液离心分散在DMF中,CDs/SiO2球体在DMF溶液中的浓度为2mg/mL。然后加入丁二酸酐,丁二酸酐在DMF溶液中的浓度为5mg/mL,将CD/SiO2球体羧化并搅拌4小时。将羧基封端的CD/SiO2球用乙醇和水洗涤数次,并分散在2-(N-吗啉代)乙磺酸缓冲盐水(MES,0.01M,pH=6.0)中以供进一步使用。CDs/SiO2荧光纳米球及CDs/SiO2荧光纳米球羧基化之后在水溶液中的ZeTa电位如图4。
2)CDs/SiO2-SFTSV单克隆抗体缀合物的制备
通过经典的碳二亚胺偶联反应,用抗SFTSV抗体(mAb)功能化羧基封端的CDs/SiO2球。加入EDC和Sulfo-NHS活化MES(0.01M,pH=6.0)中的CD/SiO2球,EDC、Sulfo-NHS和CD/SiO2球在MES溶液中的浓度分别为4mg/mL、2mg/mL和1mg/mL,并反应15min。用水洗涤活化的颗粒并再分散于PBS缓冲液(0.01M,pH=7.4)中,使CDs/SiO2球在PBS中的浓度为1mg/mL。然后加入终浓度为80ug/mL的SFTSV单克隆抗体,并在25℃温育2h。通过离心收获标记CDs/SiO2的mAb,并再分散于PBS缓冲液(PBS,0.01M,pH=7.4,含有2%BSA)中以形成CDs/SiO2-SFTSV单克隆抗体缀合物存储液(CDs/SiO2,1mg/mL),保存在4℃,备用。
实施例4 SFTSV核蛋白抗原的荧光免疫层析试纸条的制备
1)样品垫的制备
将样品垫裁剪成8mm的长条,备用。
2)硝基纤维素膜的制备
T线上包被有SFTSV单克隆抗体、C线上包被有羊抗鼠多克隆抗体的硝基纤维素膜的制备;将稀释的SFTSV单克隆抗体(2mg/mL)均匀的划在硝基纤维素膜上,得到T线;将稀释好的羊抗鼠多克隆抗体(2mg/mL)均匀的划在硝基纤维素膜上,得到C线。T线和C线的抗体含量为0.05mg/cm2,37℃烘箱干燥2h,备用。
3)荧光免疫层析试纸条的制备
将黑色底板、硝基纤维素膜、样品垫和吸水垫组合。将划线的硝基纤维素膜粘贴在黑色底板的中部,然后在其两端分别粘贴样品垫和吸水垫,硝基纤维素膜和样品垫之间及硝基纤维素膜和吸水垫之间相互重叠1~2mm。用4%BSA封闭液封闭硝基纤维素膜上非特异性吸附位点。干燥后利用裁剪机器将组装好的黑色底板裁剪为宽3mm的长条,即得到所述的检测SFTSV核蛋白的荧光免疫试纸条。4℃冰箱储存,备用。
实施例5 SFTSV核蛋白抗原的荧光免疫层析试纸条的应用
1)试纸条的测试
取实施例3制备的CDs/SiO2-SFTSV单克隆抗体缀合物20uL与50uL SFTSV壳蛋白抗原在室温下孵育10min,然后在平台上将混合液滴加到实施例4制备的试纸条的样品垫上,在毛细管作用下,流体向前层析,20min后在紫外灯下观察实验结果,如图5所示。
2)结果诊断
检测过程中,微量的SFTSV核蛋白抗原与CDs/SiO2-SFTSV抗体缀合物结合形成荧光纳米球,然后滴加到样品垫上。在毛细管作用下,流体向前层析,通过抗原抗体结合,先被硝基纤维素膜上T线捕获,形成双抗体夹心荧光纳米球,未被捕获的继续向另一端渗移被硝基纤维素膜上C线捕获,因此在T线和C线上有CDs/SiO2的固定并积累,在紫外灯的照射下显现出荧光。结果:T线和C线显现出荧光为阳性,即存在SFTSV核蛋白抗原;T线无荧光和C线显现出荧光为阴性,即不存在SFTSV核蛋白抗原或者SFTSV核蛋白抗原含量极少;当C线不显现荧光,T线不论有无荧光时,表明结果无效。通过CDs/SiO2特有的荧光性质示踪判断SFTSV核蛋白抗原的有无。
实施例6不同型号的硝基纤维素膜制备荧光免疫层析试纸条
选用三种不同型号的硝基纤维素膜,分别为pall vivid 90、Sartorius CN95和pall vivid 170,按照实施例4,制备SFTSV核蛋白抗原的荧光免疫层析试纸条。
实施例7SFTSV核蛋白抗原的荧光免疫层析试纸条灵敏度测试
1)不同浓度SFTSV核蛋白抗原溶液的配制:用PBS缓冲液(0.01M,pH=7.4)配制0、1pg/mL、10pg/mL、100pg/mL、1ng/mL、10ng/mL、100ng/mL、1ug/mL的SFTSV核蛋白抗原溶液。
2)将实施例6制备的三种型号的试纸条平放在台面上,按照实施例5,将实施例6制备的不同型号的硝基纤维素膜荧光免疫层析试纸条进行应用。取实施例3制备的CDs/SiO2-SFTSV单克隆抗体缀合物20uL与50uL待测SFTSV壳蛋白抗原在室温下孵育10min,然后将混合液滴加到试纸条的样品垫上,在毛细管作用下,流体向前层析,30min后在紫外灯下观察实验结果。参见图6的检测结果,由于不同NC膜型号的差异,不同种类的NC膜对材料的非特异性吸附不同,只有Sartorius CN95型号的硝基纤维素膜,在待测SFTSV壳蛋白抗原浓度为0ng/mL时,出现明显的C线和忽略不计的T线。即使对于另外几种型号的硝基纤维素膜,在待测SFTSV壳蛋白抗原浓度为1pg/mL时在显现的亮度高于0ng/mL时的亮度,然而在实际的应用过程中仍是不可取的。Sartorius CN95型号的硝基纤维素膜制备的试纸条,在待测SFTSV壳蛋白抗原浓度为10ng/mL及以上时,由于抗原浓度大,T线能捕获较多的荧光信号物,因此在紫外灯下的颜色较亮。在待测SFTSV壳蛋白抗原浓度为1ng/mL、100pg/mL、10pg/mL、1pg/mL时,由于抗原浓度小,T线能捕获荧光信号物较少,因此在紫外灯下的颜色较弱。在待测SFTSV壳蛋白抗原浓度为1pg/mL时亮度略微高于0ng/mL时的亮度。肉眼在紫外灯下可观察最低的SFTSV壳蛋白抗原浓度为1pg/mL,可观察较亮的SFTSV壳蛋白抗原浓度为10ng/mL及以上。
实施例8 SFTSV核蛋白抗原的荧光免疫层析试纸条特异性测试
(1)溶液的配制:用PBS缓冲液(0.01M,pH=7.4)配制0ug/mLSFTSV壳蛋白抗原溶液、1ug/mLCEA溶液、1ug/mL AFP溶液、1ug/mL CA125溶液、1ug/mL HCG溶液、1ug/mL的SFTSV核蛋白抗原溶液。
(2)将实施例6中Sartorius CN95型号的硝基纤维素膜制备的试纸条平放在台面上,取实施例3制备的CDs/SiO2-SFTSV单克隆抗体缀合物20uL与50uL待测溶液在室温下孵育10min,然后将混合液滴加到试纸条的样品垫上,在毛细管作用下,流体向前层析,30min后在紫外灯下观察实验结果。参见图7的检测结果,当溶液中含有SFTSV核蛋白抗原溶液,结果成阳性;当溶液中为其他的检测抗原或者是PBS缓冲液,结果为阴性。该实验重复3次,均为此结果,表明实验结果的可靠性。
Claims (10)
1. CDs/SiO2-SFTSV单克隆抗体缀合物,其特征在于,所述CDs/SiO2-SFTSV单克隆抗体缀合物以CDs/SiO2荧光纳米球接连SFTSV单克隆抗体获得。
2.权利要求1所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,其特征在于,包括以下步骤:
1)硅烷化CDs的制备;
2)CDs/SiO2荧光纳米球的制备及洗涤纯化:所述CDs/SiO2荧光纳米球的制备方法如下:将CTAB和NaOH溶液加入到水中并搅拌,然后在强搅拌下继续加入TEOS和CDs的混合液并反应,通过离心收集沉淀物;
3)CDs/SiO2-SFTSV单克隆抗体缀合物的制备。
3.根据权利要求2所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,其特征在于,所述步骤1)的硅烷化CDs的制备方法如下:一水合柠檬酸在高温的真空干燥箱中干燥,然后与氮气脱气的N-β-(氨基乙基)-γ-氨基丙基三甲氧基硅烷在高温下反应,形成CDs溶液,随后高速离心,弃去离心管底部大颗粒即得。
4.根据权利要求2所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,其特征在于,所述步骤2)的CTAB终浓度为0.02 mg/ml~20 mg/ml,NaOH 终浓度为0.010 mg/ml~6 mg/ml,TEOS终浓度为0.01%~12%,CDs终浓度为0.001%~5%。
5.根据权利要求2所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,其特征在于,所述步骤2)的洗涤纯化步骤如下:将离心收集沉淀物分散在HCl和乙醇的混合液中并搅拌以萃取残余物有机模板,并重复萃取,将产物洗涤至中性,最后将获得的氨基封端的CDs/SiO2荧光纳米球分散在H2O中。
6.根据权利要求2所述的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法,其特征在于,所述步骤3)的CDs/SiO2-SFTSV单克隆抗体缀合物的制备方法如下:先将丁二酸苷与氨基封端的CDs/SiO2荧光纳米球反应将其羧基化,得到CDs/SiO2-COOH;然后利用EDC/NHS将CDs/SiO2表面的羧基活化,加入SFTSV抗体,室温孵育得到CDs/SiO2-SFTSV抗体缀合物。
7.基于CDs/SiO2荧光纳米球免疫层析试纸条,其特征在于,所述基于CDs/SiO2荧光纳米球的免疫试纸条包括将权利要求1所述的CDs/SiO2-SFTSV单克隆抗体缀合物与SFTSV通过抗原-抗体之间的相互作用制备的荧光信号标记物。
8.根据权利要求6所述的基于CDs/SiO2荧光纳米球免疫试纸条,其特征在于,基于CDs/SiO2荧光纳米球的免疫试纸条还包括依次粘连在黑色底板上的样品垫、包被有检测T线和对照C线的硝基纤维素膜和吸水垫;所述的硝基纤维素膜T线上包被有SFTSV单克隆抗体;所述的硝基纤维素膜C线上包被有羊抗鼠多克隆抗体。
9.根据权利要求7所述的基于CDs/SiO2荧光纳米球的免疫试纸条,其特征在于,所述T线的SFTSV单克隆抗体的浓度为0.04~0.07 mg/cm2,所述C线的羊抗鼠多克隆抗体的浓度为0.04~0.07 mg/cm2。
10.权利要求7~9任一项所述的基于CDs/SiO2荧光纳米球免疫试纸条的制备方法,其特征在于,所述制备方法包括以下步骤:
1)硅烷化CDs的制备;
2)CDs/SiO2荧光纳米球的制备及洗涤纯化;
3)CDs/SiO2-SFTSV单克隆抗体缀合物的制备;
4)T线上包被有SFTSV单克隆抗体、C线上包被有羊抗鼠多克隆抗体的硝基纤维素膜的制备;
5)将黑色底板、样品垫、包被有检测T线和对照C线的硝基纤维素膜、吸水垫组合,底板上的硝基纤维素膜的一端粘贴吸水垫,硝基纤维素膜与吸水垫之间相互重叠1~2 mm,在硝基纤维素膜的另一端粘贴样品垫,即得基于CDs/SiO2荧光纳米球的免疫试纸条。
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