CN110981841A - 一种基于香豆素的检测革兰氏阳性菌的免洗荧光探针及应用 - Google Patents
一种基于香豆素的检测革兰氏阳性菌的免洗荧光探针及应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及表面活性剂及检测革兰氏阳性菌的荧光探针制备领域,具体涉及一种基于香豆素的检测革兰氏阳性菌的免洗荧光探针及应用。
背景技术
在过去的几十年中,细菌感染性疾病对人类健康提出了严峻挑战,并日益引起医学界和公众的关注。全世界每年都会有数百万人的感染。此外,占医院感染25%的手术部位感染(SSI) 主要是由革兰氏阳性细菌引起的。同时,肿瘤中的某些革兰氏阳性细菌可以明显增强化学耐药性并降低化疗药物的疗效,并促进肿瘤的生长和转移。考虑到革兰氏阳性细菌的巨大威胁,迅速地区分革兰氏阳性细菌成为了首要任务。
革兰氏染色法是区分革兰氏阳性菌和革兰氏阴性菌的一种常用的传统方法,但是这种策略需要繁琐的工作并且缺乏实现对活菌原位检测的能力。其他常规细菌鉴定方法,包括聚合酶链反应在内的文献报道(PCR),DNA芯片,免疫技术,质量光谱法,表面增强拉曼光谱 (SERS)和生物传感技术通常耗时、成本和能源消耗大,并且需要复杂的仪器,这些缺点极大地限制了它们在临床的应用。
目前常规检测采用传统培养方法,每种病菌需进行单独的培养,单独进行检测,工作量大;而使用荧光定量单个检测方法逐个操作,配置反应体系数目多,要消耗大量的试剂、人力、物力和时间;免疫学方法检测需要寻找高灵敏度和特异性的抗原,且试剂昂贵,操作复杂。虽然现在有探针可以区分革兰氏阳性菌和革兰氏阴性菌,但哺乳动物细胞也容易被染色。
因此,开发易合成、选择性好,且不对细胞产生干扰,实现迅速地区分革兰氏阳性细菌且不对哺乳动物细胞产生干扰的探针尤为重要。
发明内容
本发明提出了一种基于香豆素的检测革兰氏阳性菌的免洗荧光探针及应用,以香豆素为母体构建荧光探针。探针是以香豆素为母体,主要官能团为羧基,由于羧基的转动在强极性条件下荧光很弱,该探针对阳离子表面活性剂有荧光增强型响应,进而做了细菌筛选,发现探针能特异性检测革兰氏阳性菌。
实现本发明的技术方案是:
一种基于香豆素的检测革兰氏阳性菌的免洗荧光探针,结构式如下所示:
所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针,制备步骤如下:
(1)将草酰乙二酸二乙酯和3-(二甲基氨基)苯酚或3-(二乙基氨基)苯酚的混合物在 120℃下搅拌3小时,冷却后,将反应混合物用CH2Cl2稀释,将其通过硅胶柱色谱法纯化,得橙色固体;
(2)将步骤(1)得到的橙色固体溶于乙醇中,之后加入氢氧化钠溶液,将得到的混合物回流后冷却至室温,向混合物加入水,调节pH,得到沉淀,将沉淀过滤、洗涤得到荧光探针。
所述步骤(1)中草酰乙二酸二乙酯和3-(二甲基氨基)苯酚或3-(二乙基氨基)苯酚的摩尔比为1:1。
所述步骤(2)加入氢氧化钠溶液调节pH至2-3。
所述步骤(2)中回流时间为3h,pH调节至2-3。
所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针在检测革兰氏阳性菌中的应用。
将革兰氏阴性菌和革兰氏阳性菌分别用含有荧光探针的Stock溶液孵育0.5分钟,在405nm 激发下,荧光发射波长收集范围为490-700nm。
所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针在监测表面活性剂中的应用。
分别测试探针储存液加入1,4-二氧六环、二氯甲烷、乙酸乙酯、四氢呋喃、二甲基亚砜、 N,N-二甲基甲酰胺、乙二醇、乙醇、乙腈、甲醇和水溶液中的荧光光谱(激发波长为390nm )。观察紫外可见吸收光谱和荧光光谱的变化。
分别测试探针加入含有阴离子表面活性剂、阳离子离子表面活性剂、中性表面活性剂、两性表面活性剂的PBS缓冲溶液中前后的紫外可见吸收光谱和荧光发射光谱的变化(激发波长为390nm),观察紫外及荧光图谱变化及其加入表面活性剂前后荧光的变化。逐渐加入不同浓度的阳离子表面活性剂后,探针在550nm处的荧光强度逐渐增强,并在达到临界胶束浓度 CMC值后探针的荧光达到最大值,且探针的荧光随表面活性剂浓度增加保持不变。加入阴离子表面活性剂、中性表面活性剂、两性表面活性剂后,荧光强度未发生明显变化。表面活性剂在日常生活中扮演着重要的角色,由于其特殊的物理化学性质,如润湿、增溶、洗涤、防腐等,被广泛应用于各个领域。对于大多数表面活性剂而言,当其浓度达到0.1mg/L时就会对周围的环境造成污染,严重者会影响人类和生物的生存质量。本探针可以对阴离子表面活性剂特异性检测。
细菌免洗成像:将革兰氏阴性菌、革兰氏阳性菌分别用含有探针的Stock溶液孵育0.5分钟,在405nm激发下,荧光发射波长收集范围为490-700nm,革兰氏阳性菌显示非常强的荧光,革兰氏阴性菌没有荧光,说明探针只对革兰氏阳性菌特异性响应。
本发明的有益效果是:(1)以香豆素为基本结构作为荧光基团,具有光稳定性好、量子产率高等优点,本发明专利涉及4-羧基香豆素的衍生物具有大的斯托克斯位移、光学性质易调控等优点;(2)在毫摩尔级的表面活性剂的存在下,探针与阳离子表面活性剂能很好的反应,能快速检测水溶液中的表面活性剂;(3)本发明中的探针合成简单,产率高,能快速检测革兰氏阳性菌,革兰氏阴性菌、真菌和哺乳动物细胞未有明显干扰,并且细胞毒性小。探针还具有选择性好、灵敏度高、细胞毒性小等优点。为检测革兰氏阳性菌及解决细菌污染问题提供了一种新的思路和方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是在不同极性溶剂体系中,4μM探针1与不同极性溶剂作用的荧光光谱图。
图2是在PBS缓冲(10mM,pH=7.4)体系中,4μM探针1与不同浓度CTAB(0-10mM) 作用的荧光发射光谱图。
图3是在PBS缓冲(10mM,pH=7.4)体系中。4μM探针1与浓度范围分别在(0-10mM)的阳离子表面活性剂【CTAB(十六烷基三甲基溴化铵)、DTAB(月桂基三甲基溴化铵)、CTAC(十六烷基三甲基氯化铵)】、阴离子表面活性剂【SDS(十二烷基硫酸钠)】、中性表面活性剂【BS-12(十二烷基二甲基胺乙内酯)】、两性表面活性剂【OP(十二烷基磷酸酯)】作用时,分别在534nm、560nm、548nm处的荧光强度随着表面活性剂浓度的变化趋势总图。
图4是探针1分别与革兰氏阳性菌、革兰氏阴性菌、和细胞的成像图。(a1)是在荧光场中,S.aureus中加入探针1(4.0μM)的成像图;(a2)是在叠加场中,S.aureus中加入探针1(4.0μM)的成像图;b1)是在荧光场中,E.coli中加入探针1(4.0μM)的成像图;(b2)是在叠加场中,E.coli中加入探针1(4.0μM)的成像图;c1)是在荧光场中,C.albicans中加入探针1(4.0μM)的成像图;(c2)是在叠加场中C.albicans中加入探针1(4.0μM)的成像图;(d1)是在荧光场中,HepG-2细胞中加入探针1(4.0μM)的成像图;(d2)是在叠加场中,HepG-2细胞中加入探针1(4.0μM)的成像图;比例尺:25μm。
图5是探针1与革兰氏阳性菌和革兰氏阴性菌的混合成像图和普通细胞的成像图。(a1) 是在明场中,S.aureus和E.coli混合细菌的成像图;(a2)是在荧光场中,S.aureus和E.coli混合细菌的成像图;(a3)是在叠加场中,S.aureus和E.coli混合细菌的成像图;(b1)是在明场中,S.aureus和E.coli混合细菌加入探针1(4.0μM)的成像图;(b2)是在荧光场中,S.aureus和E.coli混合细菌加入探针1(4.0μM)的成像图;(b3)是在叠加场中,S.aureus和E.coli混合细菌加入探针1(4.0μM)的成像图;比例尺:25μm。
图6是用探针1不同浓度(0、2、4、6、8、10、15、20μM)处理HepG-2后细胞的存活率。
具体实施方式
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
探针1结构式如下:
探针1的制备步骤如下:将草酰乙二酸二乙酯(1.818g,10mM)和3-(二甲基氨基)苯酚(1.3718g,10mM)的混合物在120℃下搅拌3小时。冷却后,将反应混合物用CH2Cl2稀释,将其通过硅胶柱色谱法纯化(CH2Cl2),得橙色固体(1.07g,45%收率)。
用2N氢氧化钠(0.75ml)处理橙色固体(0.37g,mmol)的乙醇(7.5ml)溶液。将混合物回流3小时,冷却至室温后,向混合物中加入20ml水,然后用盐酸将所得溶液的pH调节至2-3,直至沉淀出7-乙基氨基香豆素-4-羧酸。在大量的棕色沉淀物中过滤沉淀物,用CH2Cl2洗涤,得到所需产物,为棕色固体(0.24g,73%收率)。
实施例2
探针2的结构式如下:
探针2的制备步骤如下:将草乙二酸二乙酯(1.818g,10mM)和3-(二乙基氨基)苯酚(1.3718g,10mM)的混合物在120℃下搅拌3小时。冷却后,将反应混合物用CH2Cl2稀释,将其通过硅胶柱色谱法纯化(CH2Cl2),得a橙色固体(1.07g,45%收率)。
用2N氢氧化钠(0.75ml)处理a(0.37g,mmol)的乙醇(7.5ml)溶液。将混合物回流3小时,冷却至室温后,向混合物中加入20ml水,然后用盐酸将所得溶液的pH调节至 2-3,直至沉淀出7-乙基氨基香豆素-4-羧酸。在大量的棕色沉淀物中过滤沉淀物,用CH2Cl2洗涤,得到所需产物,为棕色固体(0.24g,73%收率)。
实施例1制备的探针的应用
1.探针与不同极性溶剂作用的荧光强度变化。
配制pH=7.4的PBS(10mM)缓冲溶液;称取探针,用DMSO溶解,准确配制2mM的探针储存液;分别向比色皿中加入2mL的1,4-二氧六环、二氯甲烷、乙酸乙酯、四氢呋喃、二甲基亚砜、N,N-二甲基甲酰胺、乙二醇、乙醇、乙腈、甲醇和PBS缓冲溶液后,加入4μL浓度为2mM的探针储存液,进行荧光光谱测试。如图1所示,A为探针在390nm激发下在不同极性的溶液中荧光发射光谱图,B为探针在525nm波长处的荧光发射强度图,A、B说明该探针发射波长的荧光强度有一定差异,说明极性对该探针有一定的影响。
2.探针与表面活性剂CTAB作用的荧光强度随表面活性剂浓度的变化
向2mL的PBS缓冲(10mM,pH=7.4)体系中,加入4μL浓度为2mM的探针储存液,再加入不同浓度CTAB(0-20mM),以390nm光激发,进行荧光光谱测定。随着表面活性剂浓度的增加,荧光强度逐渐增加。如图2所示,可以应用于表面活性剂的检测。
3.探针与不同表面活性剂作用
探针与不同表面活性剂(阳离子表面活性剂:CTAB、DTAB、CTAC;阴离子表面活性剂:SDS、LAS;中性表面活性剂BS-12;两性表面活性剂:OP)作用时,分别在534nm、560 nm、548nm处的荧光强度随着表面活性剂浓度的变化趋势总图(如图3)。
4.探针分别与革兰氏阳性菌、革兰氏阴性菌、和细胞作用的成像图
如图4所示,(a1)是在荧光场中,S.aureus中加入探针1(4.0μM)的成像图;(a2)是在叠加场中,S.aureus中加入探针1(4.0μM)的成像图;b1)是在荧光场中,E.coli中加入探针1(4.0μM) 的成像图;(b2)是在叠加场中E.coli中加入探针1(4.0μM)的成像图;c1)是在荧光场中, C.albicans中加入探针1(4.0μM)的成像图;(c2)是在叠加场中C.albicans中加入探针1(4.0μM)的成像图;(d1)是在荧光场中,HepG-2细胞中加入探针1(4.0μM)的成像图;(d2)是在叠加场中,HepG-2细胞中加入探针1(4.0μM)的成像图(如图4);选用405nm激发波长,收集450-600nm 的波长;比例尺:25μm。
5.探针1与革兰氏阳性菌和革兰氏阴性菌的混合成像图和普通细胞的成像图
(a1)是在明场中,S.aureus和E.coli混合细菌的成像图;(a2)是在荧光场中,S.aureus和E. coli混合细菌的成像图;(a3)是在叠加场中,S.aureus和E.coli混合细菌的成像图;(b1)是在明场中,S.aureus和E.coli混合细菌加入探针1(4.0μM)的成像图;(b2)是在荧光场中,S.aureus 和E.coli混合细菌加入探针1(4.0μM)的成像图;(b3)是在叠加场中,S.aureus和E.coli混合细菌加入探针1(4.0μM)的成像图;选用405nm作为激发波长,收集450-600nm的波长。比例尺:25μm。
6.探针的细胞毒性实验
用探针不同浓度(0、2、4、6、8、10、15、20μM)处理HepG-2后细胞的存活率(如图 6)。说明此探针细胞毒性较小。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
2.根据权利要求1所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针,其特征在于制备步骤如下:
(1)将草酰乙二酸二乙酯和3-(二甲基氨基)苯酚或3-(二乙基氨基)苯酚的混合物在120℃下搅拌3小时,冷却后,将反应混合物用CH2Cl2稀释,将其通过硅胶柱色谱法纯化,得橙色固体;
(2)将步骤(1)得到的橙色固体溶于乙醇中,之后加入氢氧化钠溶液,将得到的混合物回流后冷却至室温,向混合物加入水,调节pH,得到沉淀,将沉淀过滤、洗涤得到荧光探针。
3.根据权利要求1所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针,其特征在于:所述步骤(1)中草酰乙二酸二乙酯和3-(二甲基氨基)苯酚或3-(二乙基氨基)苯酚的摩尔比为1:1。
4.根据权利要求1所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针,其特征在于:所述步骤(2)加入氢氧化钠溶液调节pH至2-3。
5.根据权利要求1所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针,其特征在于:所述步骤(2)中回流时间为3h,pH调节至2-3。
6.权利要求1-5任一项所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针在检测革兰氏阳性菌中的应用。
7.根据权利要求6所述的应用,其特征在于步骤如下:将革兰氏阴性菌和革兰氏阳性菌分别用含有荧光探针的Stock溶液孵育0.5分钟,在405nm激发下,荧光发射波长收集范围为490-700nm。
8.权利要求1-5任一项所述的基于香豆素的检测革兰氏阳性菌的免洗荧光探针在监测表面活性剂中的应用。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103476761A (zh) * | 2011-03-30 | 2013-12-25 | 3M创新有限公司 | 荧光原性化合物或荧光性化合物及其用途 |
WO2018210334A1 (en) * | 2017-05-19 | 2018-11-22 | The Hong Kong University Of Science And Technology | Aiegens for cancer cells and gram-positive bacteria discrimination and killing |
WO2019044736A1 (ja) * | 2017-08-28 | 2019-03-07 | 静岡県公立大学法人 | コリバクチンおよびコリバクチン産生菌の検出方法および検出プローブ |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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WO2018210334A1 (en) * | 2017-05-19 | 2018-11-22 | The Hong Kong University Of Science And Technology | Aiegens for cancer cells and gram-positive bacteria discrimination and killing |
WO2019044736A1 (ja) * | 2017-08-28 | 2019-03-07 | 静岡県公立大学法人 | コリバクチンおよびコリバクチン産生菌の検出方法および検出プローブ |
Non-Patent Citations (2)
Title |
---|
ROBERT J. VON TREBRA ET AL.: "Photochemistry of coumarin laser dyes: the role of singlet oxygen in the photo-oxidation of coumarin 311", 《JOURNAL OF PHOTOCHEMISTRY》 * |
SHUAI WANG ET AL.: "A colorimetric and ratiometric fluorescent sensor for sequentially detecting Cu2+ and arginine based on a coumarin–rhodamine B derivative and its application for bioimaging", 《RSC ADV.》 * |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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CN114031593B (zh) * | 2021-08-30 | 2023-10-13 | 中国科学院长春应用化学研究所 | 一种纳米荧光材料、纳米荧光探针及其制备方法和应用 |
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