CN115109079B - 聚氨酯降解酶特异性荧光探针、其制备方法及应用 - Google Patents
聚氨酯降解酶特异性荧光探针、其制备方法及应用 Download PDFInfo
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Abstract
本发明涉及聚氨酯降解酶特异性荧光探针、其制备方法及应用。利用4,4’‑亚甲基双(异氰酸苯酯)与1,4’‑丁二醇合成聚氨酯骨架结构后,将荧光分子与3‑氨基丙酸反应得到带羧基的荧光分子,所述带羧基的荧光分子与合成的所述聚氨酯骨架结构上的羟基发生酯化反应,得到所述聚氨酯降解酶特异性荧光探针。本发明设计的探针对参与聚氨酯降解的酯酶具有高度选择特异性,可以有效避免其他非特异性酶的干扰。探针被菌株分泌的酯酶水解后具有荧光增强效应,利用菌株对荧光探针水解效果不同导致的荧光差异为筛选指标,进行液滴微流控分选,挑选出荧光强度高的菌株。
Description
技术领域
本发明属于微生物和微流控技术领域,具体涉及利用人工合成的聚氨酯降解酶特异性荧光探针、其制备方法及应用。
背景技术
聚氨酯(Polyurethane,PU)是由多异氰酸酯、多元醇和扩链剂三种组分缩合而成的含有氨基甲酸酯键重复单元结构的塑料聚合物。是目前所有塑料制品中使用范围最广、用量最高的塑料之一。由于聚氨酯塑料大量使用带来的塑料白色污染问题日益严重,因此,寻找高效聚氨酯降解菌株具有重要应用价值。
PU具有高度疏水性且自然条件下难以降解,要直接以此为底物着手筛选到能高效降解PU塑料的微生物或酶非常困难。目前大多数研究都是以Impranil DLN作为结构模拟物来筛选PU塑料的降解菌。Impranil DLN被称为水性聚酯型聚氨酯分散体,呈乳白色液体状,是一种大小为0.1~0.2 μm的纳米颗粒。由于Impranil DLN不透明的性质,因此可以制成乳白色,不透明的平板,通过其被水解而形成的水解圈进而筛选降解微生物。然而由于DLN结构中的氨基甲酸乙酯键也是常见蛋白酶和脂肪酶的位点,因此使用DLN进行聚氨酯降解菌株筛选的过程中常常会出现假阳性的菌株。此外,DLN平板筛选法只能人工将菌落点在平板上,该筛选方式通量小、效率低无法应用于大规模的筛选。
微流控超高通量荧光激活液滴分选平台(Fluorescence-Activated DropletSorting,FADS)是近些年来新兴的一种单细胞分选平台,该平台可对单细胞进行封装培养,利特异性荧光底物对菌株的次级代谢产物、一些特异性酶类的产量进行荧光筛选和定量,通过筛选平台对荧光强度符合筛选阈值的液滴进行分选收集,最终获得大量备选液滴。该筛选方法的通量高、特异性强,但其使用难点在于特异性荧光底物的合成。
发明内容
本发明的目的在于克服上述现有技术存在的问题,提供一种聚氨酯降解酶特异性荧光探针、其制备方法及应用。
为实现上述技术目的,本发明采用如下技术方案:
一种聚氨酯降解酶特异性荧光探针,其具有如下所示的结构式:
本发明的另一目的在于提供上述聚氨酯降解酶特异性荧光探针的制备方法,包括:
利用4,4’-亚甲基双(异氰酸苯酯)与1,4’-丁二醇合成聚氨酯骨架结构;
将苯并噻吨二羧酸酐与3-氨基丙酸反应使苯并噻吨二羧酸酐引入羧基;
引入羧基的苯并噻吨二羧酸酐与所述聚氨酯骨架结构上的羟基发生酯化反应,得到所述聚氨酯降解酶特异性荧光探针。
本发明的又一目的在于提供上述聚氨酯降解酶特异性荧光探针在聚氨酯降解酶液滴微流控筛选中的应用。
具体的,将所述聚氨酯降解酶特异性荧光探针和待筛选酶共同封装在微液滴中,以荧光信号强度表征酶对荧光底物的水解效果,筛选荧光强度超出预设阈值的微液滴,其封装的酶即为聚氨酯降解酶。
作为一种优选的实施方式,所述聚氨酯降解酶特异性荧光探针使用时激发波长为537nm,吸收波长为440nm。
作为一种优选的实施方式,所述聚氨酯降解酶特异性荧光探针使用时浓度为1-50μM。
作为一种优选的实施方式,所述聚氨酯降解酶特异性荧光探针使用时浓度为25-50μM。
本发明设计的聚氨酯降解酶特异性荧光探针可用于液滴微流控筛选途径的聚氨酯降解酶/菌株高效、特异性筛选,该探针以4,4’-MDI和1,4-丁二醇为骨架单元,可最大限度模拟聚氨酯塑料的结构,在该聚氨酯骨架结构的基础上利用3-氨基丙酸结合酯化反应引入荧光基团,可以最大限度模拟真实的聚氨酯塑料结果,进而有利于筛选出对真实聚氨酯有降解效果的酶。该荧光探针具有荧光增强效应,原因在于该荧光探针的溶解性较低,初始状态下荧光较弱,当被聚氨酯降解酶水解后,荧光基团后探针的可溶性增强,使得荧光信号增强。利用该原理,可将探针应用于聚氨酯降解酶的筛选。
本发明选用大肠杆菌K12,铜绿假单胞菌PAO1和酿酒酵母EBY100用于检测聚氨酯降解酶特异性荧光探针毒性,并检测不同类型酶对聚氨酯降解酶特异性荧光探针的水解效果,结果显示,本发明设计的聚氨酯降解酶特异性荧光探针生物毒性非常低,可用于聚氨酯降解微生物的筛选,此外,该聚氨酯降解酶特异性荧光探针对聚氨酯水解酶具有很强的特异性。
本发明基于设计的聚氨酯降解酶特异性荧光探针,构建了一套荧光强度激活的液滴筛选系统(Fluorescence-Activated Droplet Sorting,FADS),可用于高效特异性筛选聚氨酯降解微生物。应用本发明的聚氨酯降解酶特异性荧光探针结合FADS进行聚氨酯降解微生物的筛选,精度高、通量大、特异性强,克服了目前DLN平板筛选技术筛选效率低,且无法排除蛋白酶和脂肪酶干扰的问题。
附图说明
图1是聚氨酯降解酶特异性荧光探针(Fluorescent Polyurethane AnalogueProbe,PFAP)的合成路线。
图2是PFAP的最优激发和吸收波长以及最优使用浓度。
图3是不同浓度PFAP对常见微生物生长的影响情况。
图4是已报道的聚氨酯降解酶对PFAP特异性的验证。
图5是利用液滴微流控系统验证探针的筛选效率。
具体实施方式
本发明通过借助液滴微流控筛选平台,通过设计聚氨酯塑料结构类似的荧光探针,利用FADS技术的原理,通过验证探针的生物毒性以及对常见聚氨酯降解酶的水解反应,验证探针用于聚氨酯降解菌株筛选的特异性。随后利用液滴封装技术,使用水-油相互剪切形成油包水的微液滴(droplet),并将已报道的聚氨酯降解酶LCC与底物共同封装在微液滴中。经过一段时间的培养,LCC酶会对PFAP进行降解释放荧光基团,从而导致液滴中的荧光发生变化。随后使用筛选平台,设置一定的筛选阈值,超过这一阈值的液滴可被收集起来用于后续进一步鉴定。本发明的整理操作流程如图5中A所示。
下面结合具体实施例,进一步陈述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
下述实施例中铜绿假单胞菌PAO1、大肠杆菌K12、酿酒酵母EBY100均为商业菌株。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例用于说明聚氨酯荧光探针PFAP的合成路线。
基于对聚氨酯塑料结构的认识,本实施例选择利用4,4’-亚甲基双(异氰酸苯酯)(4,4’-Methylene diphenyl diisocyanate, 4,4’-MDI)与1,4’-丁二醇(1,4-butanediol)为原料合成聚氨酯的骨架结构。随后利用荧光分子与3-氨基丙酸反应使得荧光分子带上羧基,随后与聚氨酯骨架上的羟基发生酯化反应,使聚氨酯的骨架结构的两侧都带上荧光基团(图1),荧光分子选用苯并噻吨二羧酸酐。该荧光探针具有与聚氨酯塑料结构高度相似,因此可以最大限度的可能筛选出可以真实降解聚氨酯塑料的微生物及其产生的酶。
具体合成方式为在氮气保护下,将50 g的4,4’-亚甲基双(异氰酸苯酯)溶于20 mL的无水DMF中,然后将混合溶液缓慢滴加到200 mL的1,4-丁二醇中,并加热回流8小时后冷却至室温;进一步,在减压条件下除去未反应的1,4-丁二醇,并加入200 mL去离子水,通过真空抽滤,获得粘性白色固体;所得白色固体在无水乙醇中进行多次重结晶,并再次使用真空抽滤,获得高纯度白色固体,利用鼓风干燥获得32 g白色固体。最终,将荧光分子和所合成白色固体按照2:1的摩尔比例溶解在三乙胺和二氯甲烷中反应生成荧光探针。
需要指出的是聚氨酯骨架结构上的两个基团之间由于空间具体相互靠近,会因此产生聚集荧光淬灭效应(aggregation-caused quenching,ACQ),导致PFAP自身结构完整时处于一种低荧光的状态,一旦PFAP被水解酶水解后,荧光基团被释放,从而表现出显著的荧光增强效应。同时PFAP结构中的酯键和氨基甲酸酯键都是降解酶的水解位点,无论哪个位点被水解酶攻击都会释放荧光基团。PFAP的合成路线如图1所示,探针合成后,经过NMR检测发现所有设计的特征基团都被NMR成功检测到,表明成功合成了PFAP探针。
实施例2
本实施例用于说明PFAP的最优激发/吸收波长以及最优使用浓度。
在成功获得了PFAP荧光探针后,本实施例检测了探针的特征参数,便于后续实施例的开展。本实施例选择了全波长扫描仪对探针的最优激发和吸收波长进行了测定,通过全波长扫描发现PFAP在440 nm处具有最高吸收峰,在537 nm出具有最高激发峰。在明确PFAP的最优激发/吸收波长后,本实施例进一步探究的不同浓度的PFAP在最优激发光下的荧光强度的差异。结果如图2所示,在1-50 μM低浓度的范围内,PFAP探针的荧光强度与浓度呈正相关关系。然而,一旦探针浓度超过50 μM后,荧光强度会发生明显的降低。这也PFAP探针荧光基团之间出现的聚集荧光淬灭效应(Aggregation-Caused Quenching, ACQ)相一致,PFAP浓度过高后会发生分子间相互聚集,导致荧光强度的降低。因此本实施例明确了后续PFAP使用过程中的最优浓度为50 μM。
实施例3
本实施例用于说明FPAP具有较低的生物毒性,可用于微生物的筛选。
探针的生物毒性是其应用于微生物筛选的关键,探针的毒性太高会抑制微生物的生长从而导致探针无法用于聚氨酯降解微生物的筛选。因此,本实施例选用3种具有代表性的常见微生物:铜绿假单胞菌PAO1、大肠杆菌K12、酿酒酵母EBY100,用于检测不同浓度的PFAP对其生长的影响。通过在培养体系中添加不同浓度的PFAP并经过15 h培养,在培养过程中不间断检测菌株的OD600值用于测定其生物量。结果如图4所示,在15 h的培养过程中发现不同浓度的PFAP对菌株的生物量均无明显差异,表明PFAP具有非常低的生物毒性,可用于聚氨酯降解微生物的筛选。
实施例4
本实施例用于检测不同类型酶对PFAP探针的水解效果,用于说明探针对聚氨酯水解酶的特异性。
传统基于DLN平板水解圈的筛选手段的一个重要缺陷在于无法分辨是酯酶还是蛋白酶或是脂肪酶对DLN底物的水解,因为一些脂肪酶和蛋白酶对DLN底物也有很好的水解活性。
为了测试本发明的合成的荧光探针PFAP的特异性,本实施例选用了自灰色链霉菌的蛋白酶和猪胰腺来源的脂肪酶作为本发明中用于检测酯酶对探针特异性的负对照。同时选用已报道的具有聚氨酯降解活性的LCC酶作为正对照,用于检测探针的特异性。
当LCC酶与PFAP共同孵育反应后对反应体系的荧光强度进行测定后发现,与不加LCC酶的对照相比,经过LCC酶的水解,反应体系的荧光强度出现了显著增强(图4),表明LCC酶可以作用于PFAP探针,释放荧光基团。然而,当把反应体系中的LCC酶替换成蛋白酶或脂肪酶后,反应体系中的荧光强度与对照组相比没有明显的差异,表明实施例中的蛋白酶和脂肪酶对PFAP探针没有水解效果(图4)。以上结果表明,PFAP探针对聚氨酯水解酶具有很强的特异性。
实施例5
本实施例用于说明PFAP探针在液滴微流控筛选系统中的可行性和工作效率。
为了测试荧光探针在液滴微流控筛选系统中的可行性和工作效率,本实施例基于实施例4中的特异性聚氨酯降解酶LCC构建了两类液滴,用于后续微流控筛选:一是含有LCC酶和PFAP探针共孵育的正对照微球;二是只含PFAP荧光探针的负对照微球。将这两类微球分别以液滴进行包裹培养后,利用分选平台进行信号收集和分选,同时对产生的荧光信号进行统计。
结果如图5所示,正对照微球产生的GFP荧光信号的范围为2.0 a.u.到2.5 a.u.,其信号主要集中在2.25 a.u.附近。然而负对照微球产生的GFP信号则要显著低于正对照微球,其范围区间为0.5 a.u.到0.75 a.u.。此外,当将液滴微流控系统的筛选阈值设定在1.75 a.u.后,对超过筛选阈值的微球进行了收集和统计。结果表明,收集到的微球中具有明显荧光增强效应的微球比例达到95%。这些结果表明PFAP探针在液滴微流控系统中具有非常强的特异性,可以识别不同液滴的信号强度,因而可以应用于液滴微流控平台筛选。
Claims (7)
2.权利要求1所述聚氨酯降解酶特异性荧光探针的制备方法,其特征在于,包括:
利用4,4’-亚甲基双(异氰酸苯酯)与乙二醇合成聚氨酯骨架结构;
将苯并噻吨二羧酸酐与3-氨基丙酸反应使苯并噻吨二羧酸酐引入羧基;
引入羧基的苯并噻吨二羧酸酐与所述聚氨酯骨架结构上的羟基发生酯化反应,得到所述聚氨酯降解酶特异性荧光探针。
3.权利要求1所述聚氨酯降解酶特异性荧光探针在聚氨酯降解酶液滴微流控筛选中的应用。
4.根据权利要求3所述的应用,其特征在于,将所述聚氨酯降解酶特异性荧光探针和待筛选酶共同封装在微液滴中,以荧光信号强度表征酶对荧光底物的水解效果,筛选荧光强度超出预设阈值的微液滴,其封装的酶即为聚氨酯降解酶。
5.根据权利要求3所述的应用,其特征在于,所述聚氨酯降解酶特异性荧光探针使用时激发波长为537nm,吸收波长为440nm。
6.根据权利要求3或5所述的应用,其特征在于,所述聚氨酯降解酶特异性荧光探针使用时浓度为1-50μM。
7.根据权利要求6所述的应用,其特征在于,所述聚氨酯降解酶特异性荧光探针使用时浓度为25-50μM。
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