CN117330749A - 一种邻苯二胺复合材料、检测试剂和检测非洲猪瘟的方法 - Google Patents
一种邻苯二胺复合材料、检测试剂和检测非洲猪瘟的方法 Download PDFInfo
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Abstract
本发明提供了一种邻苯二胺复合材料、检测试剂和检测非洲猪瘟的方法,属于抗体检测技术领域,本发明的邻苯二胺复合材料,采用如下方法制备而成:将聚醚酰亚胺和邻苯二胺溶于脱氧超纯水中搅拌至少10 min;然后再加入五水合四氯化锡搅拌至少25min;本发明的检测试剂,采用如下方法制备而成:取邻苯二胺溶液加入到水中,再加入病毒的蛋白质生物标志物并在4℃孵育2 h~2.5 h;再加入标准蛋白质溶液并孵育1.5h~2h;最后离心、洗涤、冷冻干燥制得检测试剂。本发明的邻苯二胺复合材料为纳米线,稳定性好,本发明的检测试剂具有高的灵敏度和准确度,本发明检测非洲猪瘟的方法操作简单。
Description
技术领域
本发明属于抗体检测技术领域,尤其涉及酶联免疫吸附实验,具体为一种邻苯二胺复合材料、检测试剂和检测非洲猪瘟的方法。
背景技术
酶联免疫吸附实验(ELISA)是一种常用的免疫检测方法,具有以下优点:高特异性,灵敏度高,操作简单,样本量少。然而,由于天然酶的固有缺陷,传统ELISA在低丰度疾病标志物中的应用面临巨大挑战,特别是通过稀释降低其他组合物影响的操作将进一步降低疾病检测中的分析物浓度。因此,开发灵敏度、准确性和稳定性极其优异的免疫分析法具有重要意义。邻苯二胺与3,3',5,5'-四甲基联苯胺(TMB)一样用作过氧化氢的显色底物,常用于过氧化氢的检测或与过氧化氢联合用于酶联免疫测定。与TMB相比,邻苯二胺具有更强的信号读出能力和水溶性,不需要使用停止液,但其稳定性远低于TMB。在空气中,邻苯二胺会以非常快的速度被氧氧化,这导致邻苯二胺在ELISA中很难检测到低丰度的检测底物。
发明内容
为解决上述问题,本发明的目的在于提供一种邻苯二胺复合材料、检测试剂和检测非洲猪瘟的方法,本发明的邻苯二胺复合材料由邻苯二胺、聚醚酰亚胺、Sn4+合成,在氧气条件不会被氧化,具有极高的稳定性;同时,本邻苯二胺复合材料能够与蛋白质生物标志物形成性质稳定的检测试剂,其具有较高的检测精度。
为实现上述目标,本发明的技术方案如下:
一种邻苯二胺复合材料,采用包括如下步骤的方法制备而成:将聚醚酰亚胺和邻苯二胺溶于超纯水中搅拌至少10 min;然后再加入五水合四氯化锡搅拌至少25min;其中,聚醚酰亚胺和邻苯二胺的质量比是35~40:50~55,五水合四氯化锡和邻苯二胺的质量比为12:13。
一种检测试剂,采用包括如下步骤的方法制备而成:
取上述邻苯二胺复合材料溶于水中形成溶液,再向邻苯二胺复合材料的溶液中加入某一病毒的蛋白质生物标志物并在4℃孵育2 h~2.5 h;再加入标准蛋白质溶液并孵育1.5h~2h;最后,将上述混合溶液在3℃~5℃下离心,用超纯水洗涤,干燥得到邻苯二胺纳米材料,即该病毒的检测试剂。
作为本发明的一种具体实施方式,所述蛋白质生物标志物、标准蛋白质溶液的体积比为0.8~1:1~1.2。
一种检测非洲猪瘟的方法,包括如下步骤:
S1、以非洲猪瘟的蛋白质生物标志物制备上述检测试剂作为非洲猪瘟的检测试剂;
S2、利用固定于固相上的非洲猪瘟捕获抗体吸附待测溶液中的抗原,以将抗原固定于固相上;
S3、利用步骤S2中被固相固定的抗原吸附非洲猪瘟的检测试剂中的抗体,以将非洲猪瘟的检测试剂固定在固相上;用超纯水洗涤固相,去除未粘合的非洲猪瘟检测试剂等余下试剂。
S4、利用可溶金属盐溶液浸泡步骤S3中被固相固定的非洲猪瘟的检测试剂,在此溶液中,被固相固定的检测试剂中的邻苯二胺复合材料被氧化形成荧光材料;可溶金属盐溶液中金属离子的配位能力大于Sn4+、氧化电位高于邻苯二胺的氧化电位;
S5、通过测定含荧光材料的溶液的荧光强度确定荧光材料的浓度,进而确定非洲猪瘟抗原的浓度。
作为本发明的一种具体实施方式,可溶金属盐溶液中金属离子可以为Hg2+、Au3+、Pt4+等。
作为本发明的一种具体实施方式,可溶金属盐溶液的溶剂为二甲基亚砜。
有益效果(1)制备的邻苯二胺复合材料为纳米线,具有极高的稳定性。
(2)本发明的检测试剂具有很高的灵敏度和准确度,采用荧光免疫分析法对非洲猪瘟抗原的响应范围为100 fg/mL ~10 pg/mL,检测限为16.29 fg/mL。
(3)该检测方法操作简单,检测方法简便。
附图说明
图1是本发明实施例1中制备的邻苯二胺复合材料的TEM图像;
图2是在没有聚醚酰亚胺的情况下制备的邻苯二胺复合材料的TEM图像;
图3是邻苯二胺的氧化产物在不同类型溶剂中的荧光强度图;
图4是实施例2的检测试剂的荧光激发光谱和发射光谱以及在不同浓度Au3+的二甲基亚砜中的紫外吸收光谱图;
图5是实施例2的检测试剂在不同Au3+浓度的二甲基亚砜溶剂中的荧光强度图;
图6是实施例2的检测试剂分解时荧光强度随时间的变化图;
图7是邻苯二胺和实施例2的检测试剂在静置过程中的颜色变化图;
图8是不同浓度非洲猪瘟抗原的荧光发射光谱图;
图9是526 nm处荧光强度与非洲猪瘟抗原浓度的线性相关曲线图。
具体实施方式
下面将结合实例对本发明的具体实施方式进行清楚、完整地描述,显然,所描述的实例仅仅是本发明一部分实施例,而不是全部的实施例。
实施例1(制备邻苯二胺复合材料)
先将0.2 mL聚醚酰亚胺 (MW: 10000,由上海麦克林生化科技有限公司获得)和324 mg 邻苯二胺溶于30 mL超纯水中搅拌10 min,然后取5 mL (70 mg/mL)加入五水合四氯化锡,搅拌30 min,得到邻苯二胺复合材料。最后,将上述混合溶液在4℃下离心分离,用超纯水洗涤三次,冷冻干燥备用。图1是此邻苯二胺复合材料的TEM图,由图可知,其为线性。
对比例1(制备不含聚醚酰亚胺的邻苯二胺复合材料)
将324 mg 邻苯二胺溶于30 mL超纯水中搅拌10 min,然后取5 mL (70 mg/mL)加入五水合四氯化锡,搅拌30 min,得到不含聚醚酰亚胺的邻苯二胺复合材料。由图2可知,在没有聚醚酰亚胺的情况下,邻苯二胺与Sn4+配合,通过范德华力凝聚成大尺寸的非晶态凝聚体。
实施例2(制备非洲猪瘟检测试剂)
将64mg实施例1制备的邻苯二胺复合材料溶解于25 mL 超纯水中,再加入1 mL 非洲猪瘟的蛋白质生物标志物 (0.1 mg/ mL), 4℃孵育2.5 h,再加入1 mL 标准蛋白质溶液(10 mg/ mL)孵育2h,然后,将上述混合溶液在4℃下离心分离,用超纯水洗涤三次,在零下40℃冷冻干燥得到非洲猪瘟检测试剂。
实施例3(使用非洲猪瘟检测盒检测非洲猪瘟抗原浓度)
取含1μg/mL非洲猪瘟捕获抗体的包被溶液( 50μL)于96孔板中4℃孵育过夜,此时非洲猪瘟捕获抗体固定于96孔板(固相)上,用磷酸缓冲盐溶液洗涤3次后,洗去多余的未固定于固相上的非洲猪瘟捕获抗体,再使用标准蛋白质溶液 (50μL,10 mg/ mL)孵育30min,然后,用磷酸缓冲盐溶液洗涤两到三次去除多余的标准蛋白质溶液。
向96孔板中加入不同浓度的非洲猪瘟抗原并孵育30min,待检溶液中的非洲猪瘟抗原与固相上的非洲猪瘟捕获抗体结合从而被固相固定,随后进行洗涤。
加入实施例2制备的检测试剂50μL(1 mg/ mL),在 37℃孵育30min,待检溶液中的非洲猪瘟抗原与检测试剂中非洲猪瘟抗体吸附,使得检测试剂经抗原被固相固定。然后,用超纯水洗涤三次,去除未粘合的非洲猪瘟检测试剂。
最后加入200μL含100 ppm Au3+的二甲基亚砜溶液,采集不同非洲猪瘟抗原浓度的荧光光谱确定非洲猪瘟抗原的浓度。
测试例1(优化反应溶剂的单因素实验)
邻苯二胺可被氧化成2,3-二氨基吩嗪,并在较长时间内发出荧光。在不同溶剂中,2,3-二氨基吩嗪具有不同的最大发射波长和不同的荧光强度。将0.1M的邻苯二胺分别溶解于0.1M四组不同的反应溶剂中,溶剂分别为:二甲基亚砜(DMSO)、超纯水、无水乙醇、乙腈。然后在室温下反应15分钟后,测量荧光强度。测试结果如图3所示,通过对比实验结果数据可以清楚地看到,2,3-二氨基吩嗪在二甲基亚砜中的荧光最强;并且根据图4的数据可以得知在二甲基亚砜中,其最大激发波长为434nm,最大发射波长为526nm。
测试例2(优化Au3+浓度的单因素实验)
取六份20μL(1 mg/ mL)的实施例2制备的非洲猪瘟检测试剂分别溶于含有200μLAu3+(AuCl3)浓度分别为0.1ppm、1ppm、10ppm、100ppm、1000ppm、10000ppm的二甲基亚砜溶液中,溶解15min后,测定各溶液的荧光强度。实验结果如图5所示,过高或过低的Au3+浓度都会引起荧光强度的降低,最佳的Au3+浓度为100ppm。这可以归结为Au3+浓度在1000ppm以上时,由于Au3+离子吸收光谱与2,3-二氨基吩嗪激发光谱重叠,导致金离子吸收2,3-二氨基吩嗪激发光,影响待测物浓度。而低于100 ppm时,Au3+浓度缺乏氧化能力,无法将邻苯二胺全部氧化为2,3-二氨基吩嗪。
测试例3(优化反应时间的单因素实验)
取实施例2制备的非洲猪瘟检测试剂溶于200μL Au3+浓度分别为100ppm的二甲基亚砜溶液中,然后分别测定不同时刻该溶液的荧光强度。结果如图6所示,7min后荧光强度趋于平稳。
测试例4(抗氧化性能测试)
分别将邻苯二胺、实施例2的检测试剂(两者邻苯二胺摩尔量相等)溶解于等质量的水中,比较其放置0h、12h、24h后的颜色变化。如图7所示,图中,邻苯二胺标记为A,实施例2的检测试剂标记为B,实施例2的检测试剂的颜色在24小时内无明显变化,而邻苯二胺溶液的颜色在12h小时后明显变深,由无色变为淡黄色,24小时后彻底氧化,溶液整体呈现黄色,这表明实施例2的检测试剂对邻苯二胺的抗氧化能力有积极的影响。
测试例5(实际检出限测试)
为考察所设计的荧光酶联免疫吸附试验的分析性能,在优化条件下(反应时间7min、Au3+浓度为100ppm、溶剂为二甲基亚砜)采集不同非洲猪瘟抗原浓度(浓度范围为100fg/mL ~ 100pg/mL)的荧光光谱。如图8所示,随着非洲猪瘟抗原浓度的升高,荧光光谱逐渐升高,呈现正反馈。如图9所示,在100fg/mL ~ 10pg/mL范围内,荧光光谱最大值(526nm)呈良好的线性关系,对应的线性校准曲线为Y=9.33X+4.06 (R2=0.9771),检出限(LOD)为16.29 fg/mL (3σ/S计算)。
此外,猪血清样品中非洲猪瘟抗原的检测与上述程序类似,只是用稀释的猪血清样品代替纯非洲猪瘟抗原。对于实际样品检测,将血清样品稀释到合适的浓度后,用上述方法进行检测。
为了验证本研究提出的临床非洲猪瘟抗原检测方法的准确性,我们测量了五份血清样本中的非洲猪瘟抗原水平,并在此基础上与医院所获得的结果进行比较。首先按上述方法将血清稀释至线性范围,然后用非洲猪瘟检测试剂直接检测。如表1所示,回收率为85%~ 123%,说明我们提出的方法适用于非洲猪瘟抗原的临床分析。
表 1 非洲猪瘟抗原的检测结果
表 2 与其他不同材料检测非洲猪瘟的检测低限的对比
从表2可以看出,即使现有技术中存在大量的采用ELISA作为检测材料的方法,但是这些方法的检测限仍然较高,难以应用于微量非洲猪瘟病毒抗原的检测。基于上述情况,我们搭建了一个高灵敏度、高选择性的传感平台,并通过检测非洲猪瘟病毒抗原验证了传感系统的实用性。
本发明在上文已优选实施例公开,但是本领域的技术人员应理解的是,这些实施例仅用于描述本发明,而不应理解为限制本发明的范围。在不脱离本发明原理的前提下,还能进一步改进,这些改进也应包含在本发明的保护范围之内。
Claims (5)
1.一种邻苯二胺复合材料,其特征在于,采用包括如下步骤的方法制备而成:
S1、将聚醚酰亚胺和邻苯二胺溶于超纯水中搅拌至少10 min;
S2、向步骤S1的溶液中加入五水合四氯化锡搅拌至少25min;
其中,聚醚酰亚胺与邻苯二胺的质量比为35~40:50~55,五水合四氯化锡与邻苯二胺的质量比为12:13。
2.一种检测试剂,其特征在于,采用包括如下步骤的方法制备而成:
B1、取权利要求1的邻苯二胺复合材料配置成水溶液;
B2、向步骤B1配置的水溶液中加入某一病毒的蛋白质生物标志物并在 4℃条件下孵育2 h~2.5 h;再加入标准蛋白质溶液并孵育1.5h~2h;
B3、将步骤B2孵育后的溶液在3℃~5℃下离心、洗涤、干燥得到病毒的检测试剂。
3.一种检测非洲猪瘟的方法,其特征在于,包括如下步骤:
S1、以非洲猪瘟的蛋白质生物标志物制备如权利要求2所述的检测试剂作为非洲猪瘟的检测试剂;
S2、利用固定于固相上的非洲猪瘟捕获抗体吸附待测溶液中的抗原,以将抗原固定于固相上;
S3、利用步骤S2中被固相固定的抗原吸附非洲猪瘟的检测试剂中的抗体,以将非洲猪瘟的检测试剂固定在固相上;
S4、利用可溶金属盐溶液浸泡步骤S3中被固相固定的非洲猪瘟的检测试剂,以生成荧光材料,可溶金属盐溶液中金属离子的配位能力大于Sn4+、氧化电位高于邻苯二胺的氧化电位;
S5、通过测定荧光材料荧光强度的从而确定荧光材料的浓度,进而确定非洲猪瘟抗原的浓度。
4.根据权利要求3所述的检测非洲猪瘟的方法,其特征在于,所述可溶金属盐溶液中金属离子为Hg2+、Au3+ 或Pt4+。
5.根据权利要求3所述的检测非洲猪瘟的方法,其特征在于,所述可溶金属盐溶液的溶剂为二甲基亚砜。
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