CN113943572B - 一种用于真菌检测的荧光素碳点染色试剂、染色方法和应用 - Google Patents
一种用于真菌检测的荧光素碳点染色试剂、染色方法和应用 Download PDFInfo
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Abstract
本发明公开了一种用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.01%~0.1%、水99.90%~99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂1%~2%、柠檬酸10%~15%、乙二胺8%~12%、水73%~78%。该试剂含有荧光素碳点材料,所述荧光素碳点材料荧光稳定,荧光强度高,对真菌的成像效果好。本发明还公开了采用上述荧光素碳点染色试剂染色的方法,该方法简单易行,耗时短。以及上述荧光素碳点染色试剂在采用荧光染色法进行真菌检测方面的应用。
Description
技术领域
本发明属于荧光检测技术领域,具体涉及一种用于真菌检测的荧光素碳点染色试剂、染色方法和应用。
背景技术
近年来,随着广谱抗生素、抗肿瘤化疗、免疫抑制剂等药物的广泛应用,条件致病性真菌导致的真菌感染发病率呈明显上升趋势,而真菌感染如果得不到有效控制,可发展为危及生命的系统性疾病和并发症。因此,病原真菌的准确快速检测对疾病的诊断和治疗有着重大的意义。
目前临床上常用的诊断方法有镜检法、微生物培养法、生化法、分子生物学诊断法等。其中微生物培养方法耗时过长,分子生物学的诊断方法虽然准确,但所需成本,对仪器设备以及操作人员的要求均较高。临床的镜检法,以湿片法为主,即用10%的氢氧化钾(KOH)滴加在样本表面,加热后在光学显微镜下观察,这种方法常有各种杂质导致视野背景较杂乱,非常依赖检测人员的经验与技术水平且灵敏度、特异性及准确率低。
后续发展起来的荧光染色法,通过荧光试剂对病原微生物的染色进行形态学的观察,更加高效直观的得出诊断结论。目前应用在临床检测中的荧光试剂有钙氟白(CFW),荧光增白剂28,异硫氰酸荧光素(FITC),罗丹明等。常用的荧光增白剂可以与真菌中的壳多糖和纤维素特异性结合,在荧光显微镜下,真菌孢子及菌丝发出黄绿色或淡蓝色荧光,与暗背景形成鲜明对比。但是这些试剂虽具有较好的染色效果但其光稳定性较差,可以与大部分真菌结合没有选择性,需要另外人工判断菌属,淬灭时间短,能使背景组织发出明亮的荧光,干扰真菌与背景的判断。所以目前荧光染色法一般需要联合多种背景染料以去除背景干扰,利用促溶剂分解待测样品角质层等。现在市面上已经有成熟的商业化检测试剂,这些荧光染色液的成分多为荧光增白剂、促溶剂、复染剂、保湿剂等,但荧光染色法中荧光染料的仍存在较易猝灭、稳定性低和生物相容性差的问题。因此,有必要提供一种高抗猝灭能力、高灵敏度且不需要联用其他复杂试剂的真菌检测方法,以解决现有技术中的不足。
现有技术一中的荧光染色液主要包括以下成分:
荧光染色液的组成:分为甲液(荧光增白剂溶液);乙液(甘油、二甲基亚砜);丙液(伊文思蓝溶液)。
其使用荧光显微镜,将载玻片放置在水平位置上,直接向样本上滴几滴增强真菌染色液。染色液淹没整个样品为准,持续染色两分钟,盖上盖玻片,吸去多余染色液,置于荧光显微镜下紫外激发下观察。
其存在以下缺点:
(1)荧光增白剂较不稳定,易于光漂白,易猝灭,光亮时间短,在酸环境或强碱环境下,失去受激发射荧光能力。
(2)荧光增白剂的生物相容性较差,有一定的生物毒性。
(3)试剂配方复杂,配制繁琐,性能不稳定。
(4)组分中含有二甲基亚砜(DMSO),生物相容性差,比如公开号为CN 106467923B的专利申请一种增强真菌染色液及配制方法应用。
现有技术二中的荧光染色液主要包括以下成分:
荧光染色液的组成:凝集素、荧光素、核酸染料、缓冲溶液、抗猝灭剂、抑菌剂。
使用方法,将样品放置于载玻片上,加热固定样品。取荧光染色液与样本进行覆盖30min,27℃孵育,在380nm到600nm激发下进行镜检。
其存在以下缺点:
(1)染色液组分过多,不易储存;
(2)样品制备过程中需要加热与孵育,操作繁琐,比如公开号为CN111019999A的专利申请一种微生物荧光染色液及其应用。
碳点(carbon dots,CDs)是近些年来新兴起的一种具有强荧光、高稳定性和低毒性的新型碳纳米荧光材料,相比于传统的荧光量子点和有机荧光染料分子,碳点由于其原材料来源广泛,环保,具有良好的水溶性以及化学惰性,抗光漂白能力强,光稳定性高,表面易于官能团化,毒性低,生物相容性好制备方法简单等优势,在微生物的标记成像、残留农药检测、药物分析、食品安全检测、生物传感等方面显示出较大的潜在应用价值。特别是在生化分析检测领域,碳点可以通过在碳核表面修饰的多种亲和基团来靶向微生物,达到检测诊断的目的,因此,碳点成为取代传统荧光染料的理想选择。
目前,现有技术中公开的大多数用荧光增白剂、罗丹明、异硫氰酸荧光素等商用或药用化学分子作为染色液,进行真菌形态学识别。关于碳点的研究仅限于染色识别细胞微生物,没有用于染色识别真菌微生物的相关研究。
发明内容
本发明的目的在于提供一种用于真菌检测的荧光素碳点染色试剂,该试剂含有荧光素碳点材料,所述荧光素碳点材料荧光稳定,荧光强度高,对真菌的成像效果好。
本发明的目的还在于提供采用上述荧光素碳点染色试剂染色的方法,该方法简单易行,耗时短。
本发明的最后一个目的在于提供上述荧光素碳点染色试剂在采用荧光染色法进行真菌检测方面的应用。
本发明的上述第一个目的是通过以下技术方案来实现的:一种用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.01%~0.1%、水99.90%~99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂1%~2%、柠檬酸10%~15%、乙二胺8%~12%、水73%~78%。
所述的荧光增白剂为荧光增白剂33#、荧光增白剂113#、荧光增白剂71#和荧光增白剂VBL中的一种或多种。
本发明中的荧光增白剂都是可以直接购买的荧光增白剂药品。
以荧光增白剂33#为例,所述荧光增白剂33#的分子式为:
所述荧光增白剂33#的CAS为:61902-19-0,优选购自麦克林F823852-100g。
荧光增白剂,本身是一种化学大分子,在激发光的照射下,会被破坏和猝灭,原本能发光,变得不发光。本申请中通过在荧光增白剂中加入其他三种试剂乙二胺、柠檬酸和水,然后经过水热合成,制成碳点材料,柠檬酸和乙二胺是最为集中碳点的碳核,然后荧光增白剂负载在该碳核上。
因此,本发明提供了一种以荧光增白剂、柠檬酸、乙二胺为原料通过水热反应法合成的碳点(carbon dots,CDs),柠檬酸和乙二胺是最为集中碳点的碳核,然后荧光增白剂负载在碳核上;该碳点材料在表面负载了荧光增白剂基团,提供了真菌亲和能力,碳核提供了荧光的高稳定性与抗猝灭性,在蓝光激发下呈现黄绿色荧光,在紫外激发下呈现蓝色荧光,具有良好的抗光漂白作用。所述荧光素碳点染色试剂组成原料安全,能够对真菌进行快速高效的染色,且不需要与其他复杂试剂相结合,使得真菌呈现黄绿色或蓝色,结合形态学实现对真菌进行染色,将检测结果可视化,更为直接、合理的对微生物种类进行判断。
本发明荧光素碳点材料的制备原料有:乙二胺、荧光增白剂、柠檬酸和水。
本发明所述荧光素碳点材料优选通过以下方法制备获得:选取荧光增白剂、柠檬酸、乙二胺和水,采用水热法一步反应制备而成,反应产物经纯化冷冻干燥成粉后,得到荧光素碳点材料。
采用水热法一步反应是在温度为160~200℃条件下进行水热反应5~8小时。
更佳的,将荧光增白剂、柠檬酸、乙二胺溶解于水中,加入到聚四氟乙烯反应釜,在180℃烘箱下反应8小时(180℃测试8小时表明条件最优)。
本发明的荧光素碳点染色试剂为一个溶液体系,其制备方法为以水为溶剂,将荧光素碳点材料分散于水中,不需要加入其它成分,可稳定共存于同一体系,在生产、销售、使用过程中,更加方便快捷。
因此,本发明提出的荧光素碳点材料,表面富含羟基、羧基、苯磺酸钠等,这些基团的存在使得碳点表面呈负电势。而在生理条件下,由于静电吸引作用,可以与真菌细胞壁内呈正电的几丁质结合。将合成的碳点直接与真菌体混合后,即可使真菌菌体标记上荧光,在荧光显微镜下成像。该方法过程简单易行,耗时短,所得碳点荧光稳定,荧光强度高,对细菌的成像效果好。本发明以此为基础,制备所得的荧光素碳点材料具备发展成一种标记皮肤真菌的通用荧光染料的潜力。
本发明的上述第二个目的是通过以下技术方案来实现的:一种用于真菌检测的荧光素碳点染色试剂进行染色的方法,包括以下步骤:
(1)制片:取洁净载玻片,并将待测样本的稀释液移至载玻片中间区域,制片完成;
(2)染色:将荧光素碳点染色试剂滴加到待测样本的稀释液中进行覆盖并染色2min~10min,孵育温度为25~37℃,自然风干或加热风干;
(3)封片:取盖玻片直接加盖染色完成的载玻片;
(4)镜检:取样片载玻片于荧光显微镜下以360nm~420nm的波段进行观测,查看待测样本中真菌的形态及荧光强度。
本发明的上述最后一个目的是通过以下技术方案来实现的:上述荧光素碳点染色试剂在采用荧光染色法进行真菌检测方面的应用。
与现有技术相比,本发明具有以下优点:
(1)本发明中的荧光素碳点材料制备简单,通过水热法可一步制备;
(2)本发明获得的荧光素碳点染色试剂具有良好的光稳定性,可抗淬灭,生物相容性高;
(3)本发明中的荧光素碳点染色试剂,可用来进行真菌染色的溶液,组分单一,无需其他组分即可达到理想效果,具有一定的经济性;
(4)本发明中的荧光素碳点染色试剂,荧光染色在短时间内能很好地区分死活真菌细胞,对光照保护没有严格要求;
(5)因此,本发明中的荧光素碳点染色试剂,可对真菌染色的荧光染色液,以碳点溶液作为染色液,与传统的荧光增白剂相比,具有良好的光漂白性,及抗淬灭效果,生物毒性小,其中碳点由柠檬酸,乙二胺和荧光增白剂制备而成;偶联方式为碳点与真菌的静电结合;用于皮肤真菌染色的碳点溶液激发波长为360nm~420nm(更佳为380nm);与其它商业用试剂相比,本发明制备的具有染色效果的碳点溶液组分单一,不需要混合其它试剂成分使用,稳定性好的优点。
附图说明
图1是实施例1中#33荧光增白剂的分子式的结构图;
图2是实施例1中荧光素碳点的荧光光谱;
图3是实施例1中荧光素碳点与荧光增白剂33#的抗猝灭能力对比,其中左图是本申请中荧光素碳点,右图为市面上的普通荧光增白剂33#;
图4是实施例1中荧光素碳点检测酿酒酵母的结果图(亮度部分实际对应蓝色);
图5是实施例2中荧光素碳点检测毛霉菌的效果图(亮度部分实际对应蓝色);
图6是实施例3中荧光素碳点检测毛霉菌的效果图(亮度部分实际对应蓝色)。
图7是实施例4中荧光素碳点检测酵母菌的效果图(亮度部分实际对应蓝色);
图8是实施例5中荧光素碳点检测毛霉菌的效果图(亮度部分实际对应蓝色)。
具体实施方式
下面结合具体实施例进一步说明本发明的应用方法。下述实施例和附图仅用于示例性说明,不能理解为对本发明的限制。除非特别说明,下述实施例中使用的试剂原料为常规市购或商业途径获得的原料,使用的实验仪器均为实验室常规仪器,除非特别说明,下述实施例中使用的方法和设备为本领域常规使用的方法和设备。
实施例1
本实施例提供的用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.01%、水99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂33#1.42%、柠檬酸14.24%、乙二胺10.25%、水74.09%。
其中:
荧光增白剂33#的分子式为:
荧光素碳点材料的制备方法如下:
按用量关系,选取荧光增白剂33#1.42%、柠檬酸14.24%、乙二胺10.25%、水74.09%,在离心管内混匀并超声10min,然后倒入聚四氟乙烯内衬的反应釜中,在180℃烘箱内,水热反应8h。
反应结束后,碳点溶液在1000Da透析膜中透析纯化12h,所得纯化液在冷冻干燥机中冻干成粉,得到均一、稳定性高、生物相容性高的荧光素碳点材料。
该碳点材料水溶液的荧光光谱如图2,证明材料在蓝光至黄绿光波段有发射能力。
与市面上普遍使用的荧光增白剂33#试剂做抗猝灭稳定性对比,如图3所示,120min内碳点材料荧光强度没有明显降低,市面上普遍使用的荧光增白剂33#在20min内荧光强度快速下降,发生了猝灭现象,证明了本发明提出的荧光素碳点的性能优势。
本实施例中的荧光素碳点染色试剂因为在荧光增白剂33#中加入了柠檬酸、乙二胺,并溶于水,高温高压下将化学分子合成为碳点纳米材料,改变了材料的性质,提高了抗光漂白能力。
采用该荧光素碳点染色试剂进行染色的方法,包括以下步骤:
(1)制片:取洁净载玻片,并将待测的微生物真菌移至载玻片中间区域,制片完成;
(2)染色:将荧光染色液滴加一滴到样本稀释液进行覆盖并染色5min,孵育温度为自然温度,自然风干或加热风干均可;
(3)封片:取盖玻片直接加盖染色完成的载玻片;
(4)镜检:取样片载玻片于荧光显微镜下以380nm的波段进行观测,查看微生物样本中微生物的形态及荧光强度。
荧光素碳点检测酿酒酵母的结果图如图4所示,从图4中可以看到酵母真菌被成功染色,荧光明亮,与暗背景形成明显对比,利于对真菌的观察识别,证明了荧光素碳点的真菌检测能力。
实施例2
本实施例提供的用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.05%、水99.95%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂33#1.12%、柠檬酸11.21%、乙二胺10.94%、水76.73%。
检测过程同实施例1。
荧光素碳点检测毛霉菌的效果图如图5所示,从图5中可以看到毛霉菌真菌被成功染色,且此类丝状真菌的纵隔部分也被成功染色,发出明亮的荧光,利于对真菌的观察识别,证明了荧光素碳点的真菌检测能力。
实施例3
本实施例提供的用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.05%、水99.95%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂33#1.50%、柠檬酸12.54%、乙二胺11.50%、水74.46%。
检测过程其它同实施例1。
荧光素碳点染色毛霉菌的结果图如图6所示,从图6中可以看到在一定范围下,调整荧光素碳点溶液的浓度以及配比,不影响其对毛霉菌真菌的染色能力,证明了荧光素碳点的真菌检测性能。
实施例4
本实施例提供的用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.01%、水99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂71#1.42%、柠檬酸14.24%、乙二胺10.25%、水74.09%。
检测过程同实施例1。
荧光素碳点检测酵母真菌的效果图如图7所示,从图7中可以看到酵母真菌被成功染色,且显示出明显的真菌细胞的形状,利于对真菌的观察识别。荧光增白剂71#与荧光增白剂33#有着相似的化学结构,染色证明了类似荧光增白剂来源的荧光素碳点同样具有真菌检测能力。
实施例5
本实施例提供的用于真菌检测的荧光素碳点染色试剂,以质量百分含量计,包括:荧光素碳点材料0.01%、水99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂113#1.42%、柠檬酸14.24%、乙二胺10.25%、水74.09%。
检测过程同实施例1。
荧光素碳点检测毛霉菌真菌的效果图如图8所示,从图8中可以看到毛霉菌被成功染色,且显示出毛霉菌的纵隔形状,利于对真菌的观察识别,证明了类似荧光增白剂来源的荧光素碳点同样具有真菌检测能力。
以上所述仅是本发明的非限定实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进,这些都视为本发明的保护范围。
Claims (3)
1.一种用于真菌检测的荧光素碳点染色试剂,其特征是以质量百分含量计,包括:荧光素碳点材料 0.01%~0.1%、水99.90%~99.99%;所述荧光素碳点材料由以下质量百分含量的原料制成:荧光增白剂1%~2%、柠檬酸 10%~15%、乙二胺8%~12%、水73%~78%;
所述的荧光增白剂为荧光增白剂33#;
所述荧光素碳点材料通过以下方法制备获得:选取荧光增白剂、柠檬酸、乙二胺和水,采用水热法一步反应制备而成,反应产物经纯化冷冻干燥成粉后,得到荧光素碳点材料;
采用水热法一步反应是在温度为160~200℃条件下进行水热反应5~8小时。
2.采用权利要求1所述用于真菌检测的荧光素碳点染色试剂进行染色的方法,其特征是包括以下步骤:
(1)制片:取洁净载玻片,并将待测样本的稀释液移至载玻片中间区域,制片完成;
(2)染色:将荧光素碳点染色试剂滴加到待测样本的稀释液中进行覆盖并染色2min~10min,孵育温度为25~37℃,自然风干或加热风干;
(3)封片:取盖玻片直接加盖染色完成的载玻片;
(4)镜检:取样片载玻片于荧光显微镜下以360nm~420nm的波段进行观测,查看待测样本中真菌的形态及荧光强度。
3.权利要求1所述荧光素碳点染色试剂在采用荧光染色法进行真菌检测方面的应用。
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