CN114236118A - 一种在硝酸纤维素膜上固载荧光染料的方法 - Google Patents
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Abstract
本发明公开了一种在硝酸纤维素膜上固载荧光染料的方法。所述荧光染料和固载液分别由以下重量百分比的组分组成:1、荧光染料为罗丹明B和罗丹明6G。2、固载液分别为5%BSA、5%γ‑聚赖氨酸和5%树枝状聚赖氨酸(PAMAM),余量为含1%吐温‑20和5%蔗糖的0.01M,pH为7.4的PBS缓冲溶液。本发明提供了一种在硝酸纤维素膜上固载荧光染料的方法,通过添加固载液使得荧光染料能高效附着于硝酸纤维素膜上,并且较高含量的蔗糖可以保护抗体。本发明所提供的固载液具有附着能力好,荧光效果好的优点。
Description
技术领域
本发明属于医学检验领域,具体地说,涉及一种在硝酸纤维素膜上固载荧光染料的方法。
背景技术
目前,在免疫层析试纸条的制备中,传统的荧光免疫层析法都是用荧光物质去标记抗体,再通过固载于硝酸纤维素膜上的捕获抗体结合产生荧光,通过对膜上检测线和质控线上荧光强度的分析实现对目标物质的检测。在目前的免疫层析试纸条的制备中,都是采用这种荧光物质标记抗体的方法。这种传统免疫层析法存在一些缺陷如(1)用于荧光标记的抗体较为昂贵,增加成本。(2)某些荧光物质有毒性,直接标记抗体会对抗体的活性产生抑制作用。这些缺陷会导致整个试纸条的检测灵敏度和稳定性降低。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种在硝酸纤维素膜上固载荧光染料的方法,此方法避免了用荧光染料直接标记抗体,而是将荧光染料固载于硝酸纤维素膜上,用胶体金标记抗体,通过胶体金对荧光的猝灭作用,实现对目标物的高灵敏检测。在硝酸纤维素膜上添加的BSA对膜起到一定封闭作用,较高含量的蔗糖能保护膜上的抗体,进而提高测试的灵敏性和延长膜的有效使用期限。
为实现上述发明目的,本发明采用了以下技术方案。
一种用于硝酸纤维素膜上固载荧光染料的方法,所述固载液由以下重量百分数的组分组成:
优选地,所述缓冲液为磷酸盐缓冲溶液。
优选地,所述缓冲溶液的浓度为10mM。
优选地,所述缓冲溶液的pH值为7.4。
优选地,所述的固载液包括如下按重量分数计的各组分:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
优选地,所述的固载液包括如下按重量分数计的各组分:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
优选地,所述的固载液包括如下按重量分数计的各组分:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
优选地,所述的固载液包括如下按重量分数计的各组分:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液
优选地,所述的固载液包括如下按重量分数计的各组分:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
本发明还提供一种用于免疫层析试纸条的固载液的制备方法,包括如下步骤:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的BSA、蔗糖、罗丹明B、吐温-20溶于基础液中,混匀并调节pH为7.4。
(3)用勾线笔将该固载液涂布于硝酸纤维素膜的1/3和2/3处。
相比传统荧光免疫层析技术,本发明的有益效果在于:
1、本发明避免了荧光染料与蛋白直接结合,使得荧光染料对蛋白不会产生失活的影响,并用胶体金对荧光染料的猝灭作用进行检测,胶体金标记抗体操作较为简便,并且用于胶体金配对的抗体成本较低。
2、本发明中的固载液中加入了较高含量的蔗糖,能够进一步提高抗体的活性,保护抗体免于失活,从而进一步提高该免疫层析试纸条的灵敏性和延长试纸条的有效期。
附图说明
为了更清楚的说明本发明的具体实施方式,下面将对具体实施方式所需要使用的附图简单的介绍。
图1是本发明中六种实施例中的试纸条的结构示意图。从左至右依次为样品垫、结合垫、硝酸纤维素膜和吸水纸。
图2为本发明中六种实施例在暗箱式紫外分析仪下固载的效果图。
具体实施方式
下面,结合附图及具体实施方式,对本发明做进一步描述。
实施例1:
一种用于硝酸纤维素膜上固载荧光染料的方法,所述固载液由以下重量百分数的组分组成:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的BSA、蔗糖、罗丹明6G、吐温-20溶于基础液中,混匀并调节pH为7.4。
实施例2:
一种用于硝酸纤维素膜上固载荧光染料的方法,所述固载液由以下重量百分数的组分组成:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的γ-聚谷氨酸、蔗糖、罗丹明6G、吐温-20溶于基础液中,混匀并调节pH为7.4。
实施例3:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的PAMAM、蔗糖、罗丹明6G、吐温-20溶于基础液中,混匀并调节pH为7.4。
实施例4:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的BSA、蔗糖、罗丹明B、吐温-20溶于基础液中,混匀并调节pH为7.4。
实施例5:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的γ-聚谷氨酸、蔗糖、罗丹明B、吐温-20溶于基础液中,混匀并调节pH为7.4。
实施例6:
所述缓冲液为浓度10mM、pH值7.4的磷酸盐缓冲溶液。
所述固载液由以下方法制备而成:
(1)用超纯水配置缓冲溶液作为基础液。
(2)称取配方量的PAMAM、蔗糖、罗丹明B、吐温-20溶于基础液中,混匀并调节pH为7.4。
将上述六例固载液用勾线笔涂布于硝酸纤维素膜的1/3和2/3处。
验证实施例:
将六例配置好的溶液放置在4℃条件下避光保存,待溶液反应一段时间后将反应后的六种溶液以条带的形式均匀涂布在硝酸纤维素膜的1/3和2/3处,在37℃的干燥箱中干燥,然后,与样品垫、金标垫、吸水垫一起组装成试纸条,然后在样品垫上滴加PBS空白样,层析完成后,在ZF-20D暗箱式紫外分析仪中开通光源,激发试纸条上的荧光,观察其在暗场中观察其在纸基上的固载情况。
从附图2可以观察出,采用实施例1-3固载液制备的试纸条,其固载效果比实施例4-6固载液的试纸条要差一些,而在实施例4-6固载液中,又以实施例6的固载效果最好,相比前面五例,其荧光强度和固载效果要更好。
综上所述,本发明的固载液具有操作简便、成本低、固载更牢固、荧光强度高等优点,又因为加入了较高质量百分比的蔗糖,对抗体也能保护,保质期也更长。
对于本领域的技术人员来说,可根据以上描述的技术方案,做出其它的改变和变形,而所有的这些改变以及变形都应该属于本发明专利要求的保护范围之内。
Claims (7)
7.权利要求1-6任一项所述的用于硝酸纤维素膜上固载荧光染料的方法,其特征在于,包括以下步骤:
1)以超纯水作为配置PBS的基础液,将各组分按重量百分比加入该基础液中溶解并混匀备用;
2)将硝酸纤维素膜剪裁为0.3×2.5cm的长度备用,并将样品垫、结合垫和吸水纸同样剪裁好备用。
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