CN113045607A - 一种用于检测乙烯的荧光探针及其制备方法与应用 - Google Patents
一种用于检测乙烯的荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种乙烯荧光探针化合物的合成及其作为分子探针在植物和环境样品中的应用,属于荧光探针技术领域。
背景技术
乙烯是一种重要的气态植物激素,它参与控制种子萌发、细胞伸长、果实成熟、种子传播等多种生长发育过程。此外,乙烯还在抵御病原体和应对外界胁迫因子的免疫反应中起着重要作用。在果蔬储存领域,根据储存类型的不同,乙烯浓度需要控制在不同的范围内。通常在成熟室中,乙烯浓度要求维持在10至200ppm之间;在储存室中,乙烯浓度要求低于1ppm。因此,乙烯浓度的检测和监测不仅可以提供有关收获管理的信息,而且还可以优化储存和运输条件。当前,检测乙烯的方法主要包括气相色谱法,激光光谱法和电化学方法等。这些方法灵敏度较高,但这些方法所用仪器存在体积笨重,操作不便,价格昂贵,无法提供实时监测等缺点。
基于功能金属配合物的分子荧光探针,广泛应用于复杂生物分子的修饰与检测,是解决上述常用方法不足之处的可行性策略之一。一价银离子和一价铜离子作为两种重要的金属离子,它们可以可逆地与乙烯配体结合,利用这一特性,研究者成功开发出了多种金属配合物探针,用于外源乙烯的检测。此外,依据烯烃复分解反应,制备得到的含钌金属配合物探针,可用于内源乙烯的检测。虽然基于金属配合物的乙烯荧光探针已经取得了一些进展,但是这些探针仍然存在灵敏度较低、生物相容性较差等缺点,不适用于复杂的生物系统中。因此,克服时空限制,提供一种灵敏度高、特异性好、生物相容性好的乙烯探针具有很大的应用价值。
发明内容
针对现有乙烯探针灵敏度较低、生物相容性较差等问题,本发明提供了一种灵敏度高、低检测限、特异性好的可用于检测植物和环境样品中乙烯的荧光探针。
本发明采用如下技术方案:
一种检测乙烯的荧光探针,简称CEP,其结构式如式(I)所示:
式中,R1、R2、R3和R4各自独立选自氢、卤素、羟基、硝基、二氟甲基、三氟甲基、C1-6的烷基、C1-6的烷氧基,以及由1-3个卤素取代的C1-6的烷基和由1-3个卤素取代的C1-6的烷氧基。
优选的,所述乙烯荧光探针的R1、R2、R3和R4基均为H。
本发明提供一种上述乙烯荧光探针合成方法,该方法产率高,操作简单,方法如下:
(1)将N-香豆素氧基酰胺和二氯(五甲基环戊二烯基)合铑(III)二聚体溶于乙腈溶剂中,加入碳酸钾,在惰性气体的保护下,搅拌反应得到目标化合物。
(2)制备步骤中,所述反应N-香豆素氧基酰胺和二氯(五甲基环戊二烯基)合铑(III)二聚体优选的摩尔比为1∶0.55
(3)制备步骤中,所述的碳酸钾反应量与N-香豆素氧基酰胺优选的摩尔比为1∶1。
(4)制备步骤中,优选的反应温度为25℃,反应时间为8-12h。
(5)制备步骤中,所述惰性气体为氮气和氩气的一种。
(6)制备步骤中还包括对产物分离提纯的步骤:反应结束后过滤出粗产物,再用乙醚冲洗,干燥后得到产物。
目标荧光探针化合物的合成路线如下:
本发明所述的荧光探针可用于检测植物和环境样品中乙烯,其优良效果包括:
(1)本发明的荧光探针具有很好的水溶性,以醇类(甲醇,乙醇,正丙醇,正丁醇,乙二醇)作为溶剂,可溶于PBS buffer溶液,具有很好的生物相容性。
(2)本发明的荧光探针可检测ppb级别的乙烯,因此灵敏度高。
(3)本发明的荧光探针具有很好的选择性,特异性识别乙烯,其他烯烃类物质对其没有干扰效果。
(4)本发明的荧光探针可检测植物和环境样品中的乙烯,可用于生物成像。
(5)本发明的荧光探针合成方法简便,原料易得,产率高。
附图说明
图1为荧光探针对不同体积的乙烯的荧光强度响应图。探针的浓度为100μM,乙烯的体积为0-250μL,检测体系为乙二醇和PBS buffer的混合溶液(体积比为1∶9),激发波长为365nm。
图2为荧光探针对不同烯烃的荧光强度响应图。探针的浓度为100μM,法尼醇、香茅醇、双戊烯、月桂烯、α-蒎烯和萜品油烯的浓度均为6mM。
图3和图4为荧光探针用于检测拟南芥叶片上乙烯的荧光成像。
具体实施方式:
以下通过实施例说明本发明的具体步骤,但不受实施例限制。
实施例1荧光探针CEP-1的合成
将N-香豆素氧基酰胺(21.9mg,0.1mmol)、二氯(五甲基环戊二烯基)合铑(III)二聚体(34.0mg,0.055mmol)和碳酸钾(13.8mg,0.1mmol)加入到2mL乙腈溶剂中。混合物在氮气保护下,25℃室温条件下,搅拌反应8h后过滤,用乙醚洗涤,得到目标探针化合物CEP-1,产率95%。1H NMR(500MHz,methanol-d4)δ7.83(d,J=9.3Hz,1H),7.20(d,J=8.2Hz,1H),6.90-6.69(m,1H),6.15(d,J=9.4Hz,1H),2.22(s,3H),1.66(s,15H);13C NMR(125MHz,methanol-d4)δ170.5,165.2,164.2,159.2,145.7,125.8,113.8,110.0,105.1,96.7,21.42,8.3;ESI-HRMS m/z:456.0696([M+H]+).
实施例2不同体积乙烯下荧光探针CEP-1的荧光强度
通过气体注射器量取10、20、50、100、150、200和250μL体积的乙烯分别加入到CEP-1探针溶液(100μM)中,反应1min后进行荧光检测(λex=365nm,λem=460nm),检测各体系中荧光强度,以相对荧光强度-乙烯的体积作曲线,如图1所示,最终得到该探针对乙烯的最低检测限为52ppb。
实施例3乙烯探针的选择性
植物或环境中的烯烃类物质对乙烯的检测可能存在干扰,因而要确定合成的乙烯荧光探针在识别乙烯时能否具备良好的选择性。
取9个样品瓶,均加入100μM CEP-1荧光探针的溶液,1个样品瓶作为空白对照,其他8个分别加入1mL的乙烯(浓度约为2mM),6mM法尼醇、6mM香茅醇、6mM双戊烯、6mM月桂烯、6mMα-蒎烯和6mM萜品油烯,反应1min后,记录不同反应体系的荧光强度,结果如图2所示。由图2可知,该探针溶液仅对乙烯有响应,且荧光强度增加接近50倍,而其他反应体系的荧光强度没有明显变化,这说明该荧光探针对乙烯有高度的选择性。
实施例4荧光探针CEP-1应用于植物中乙烯的检测
将生长4周的拟南芥叶片用镊子进行去皮处理,放入透明的96孔板中,加入100μL超纯水,室温条件下孵育12h去除损伤压力。加入不同的试剂对不同的拟南芥叶片进行处理,分别包括1mM的1-氨基环丙烷羧酸(ACC,乙烯前体物质)、5μM flg22(flg22为含有22个氨基酸的序列,可以激活植物的免疫机制,导致植物乙烯量增加)和1μM nlp20(nlp20为含有20个氨基酸的序列,可以通过细胞毒性和非细胞毒性引起各种十字花科植物的免疫激活,导致植物乙烯量增加),进一步孵育6h,移除上述溶剂,然后将叶片分别加入到100μMCEP-1探针溶液(50μL)孵育半小时,用水冲洗三遍后,进行荧光显微成像。结果如图3和图4所示。由图可知,植物叶片未受1-氨基环丙烷羧酸(ACC)、flg22和nlp20刺激时,植物叶片荧光很微弱,当植物叶片受到1-氨基环丙烷羧酸(ACC)、flg22和nlp20刺激后,植物叶片荧光明显增强。这表明该探针可用于植物中乙烯的检测。
Claims (8)
2.根据权利要求1所述荧光探针的制备方法,其特征在于包括:将取代的N-香豆素氧基酰胺和二氯(五甲基环戊二烯基)合铑(III)二聚体溶于乙腈溶剂中,加入碳酸钾,在惰性气体的保护下,搅拌反应得到目标探针化合物。
3.根据权利要求2所述荧光探针的制备方法,其特征在于,所述的反应生成目标探针时,其反应条件为25℃下搅拌反应8-12h。
4.根据权利要求2所述荧光探针的制备方法,其特征在于,所述的反应生成目标探针时,对产物分离提纯的步骤包括:反应结束后过滤出粗产物,再用乙醚冲洗,干燥后得到产物。
5.根据权利要求2所述荧光探针的制备方法,其特征在于,所述反应物N-香豆素氧基酰胺与二氯(五甲基环戊二烯基)合铑(III)二聚体摩尔比为1∶0.5。
6.根据权利要求2所述荧光探针的制备方法,其特征在于,所述的碳酸钾反应量与N-香豆素氧基酰胺摩尔比为1∶1。
7.根据权利要求2所述荧光探针的制备方法,其特征在于,所述惰性气体为氮气和氩气的一种。
8.根据权利要求1所述的荧光探针在检测乙烯中的应用,其特征在于,所述的荧光探针用于检测植物和环境样品中的乙烯。
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