CN114195775A - 一种荧光染料及其制备方法和在细菌染色中的应用 - Google Patents
一种荧光染料及其制备方法和在细菌染色中的应用 Download PDFInfo
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Abstract
本发明公开了一种荧光染料及其制备方法和在细菌染色中的应用，本发明的荧光染料易于合成，并且显示出良好的红色荧光强度，对金黄色葡萄球菌染色发红色荧光、对大肠杆菌不染色不发荧光，可作为荧光染料用于革兰氏阳性菌和革兰氏阴性菌的鉴别，及用于革兰氏阳性菌荧光成像，在染色检验技术领域具有相当重要的意义与价值。本发明合成的荧光染料除了具有使用方便，结果判定明了的优点外，还具有便于贮存、运输等优点，它适合各检测单位实验室里使用。
Description
技术领域
本发明属于染色检验技术领域，涉及一种荧光染料及其制备方法和在细菌染色中的应用。
背景技术
在微生物检验工作中，细菌染色是不可缺少的重要步骤。因为菌体微小，无色透明，不易观察。通过染色可观察到细菌构造，这对细菌鉴定起着重要作用。
常用的染料有4',6-二脒基-2-苯基吲哚二盐酸盐（DAPI）、碘化丙啶（PI）、草酸铵结晶紫-碘液-番红等。但现有技术还存在一些不足，如DAPI只染活菌，PI只染死菌，草酸铵结晶紫-碘液-番红染色虽可区分革兰氏阳性菌和革兰氏阴性菌，但其在染色过程中有脱色和复染步骤，控制不好就不容易区分结果是蓝紫色还是红色，影响鉴定。
发明内容
本发明的第一个目的是提供一种荧光染料，解决现有技术中的革兰氏阳性菌和革兰氏阴性菌区分鉴别的不易，以及革兰氏阳性菌荧光成像的问题。
本发明解决技术问题所采用的技术方案是：一种荧光染料，所述荧光染料的结构式如式（I）所示：
式（I）
本发明的第二个目的是提供上述的荧光染料的制备方法，该制备方法的工艺如下：以4-硼酸三苯胺和4,7-二溴-2,1,3-苯并噻二唑为原料，在碳酸钾和氮气氛条件下，四(三苯基膦)钯催化发生偶联反应得到前体化合物；前体化合物与5-醛基-2-噻吩硼酸在四(三苯基膦)钯催化发生偶联反应得到中间化合物；中间化合物与1-乙基-4-甲基喹啉碘化物缩合后，再与六氟磷酸钾进行离子交换得到权利要求1所述的荧光染料，
其中，前体化合物为
;
其中，中间化合物为
;
具体反应式如下：
具体制备步骤为：
步骤A：将4-硼酸三苯胺、4,7-二溴-2,1,3-苯并噻二唑、碳酸钾、四(三苯基膦)钯置于四氢呋喃/水(体积比10/1)溶剂中，氮气氛下70-80℃（优选75℃）加热回流20-28小时（优选24小时），反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱，得到前体化合物；
步骤B：将所述前体化合物、5-醛基-2-噻吩硼酸、碳酸钾、四(三苯基膦)钯置于四氢呋喃/水(体积比10/1)溶剂中，氮气氛下70-80℃（优选75℃）加热回流20-28小时（优选24小时），反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱，得到中间化合物；
步骤C：将所述中间化合物、1-乙基-4-甲基喹啉碘化物、哌啶置于无水乙醇中，氮气氛下加热回流20-28小时（优选24小时）后，加入3倍体积(乙醇的3倍体积)的乙醚静置0.5-1.5小时（优选1小时），抽滤，滤渣溶于甲醇，加入饱和的六氟磷酸钾水溶液进行离子交换反应，搅拌0.5-1.5小时（优选1小时），得到结构式（I）所示的最终产品的荧光染料。
优选，步骤A 中4-硼酸三苯胺、4,7-二溴-2,1,3-苯并噻二唑、碳酸钾、四(三苯基膦)钯的物质的量的比为1:1.2:10:0.05。
优选，步骤B 中所述的前体化合物、5-醛基-2-噻吩硼酸、碳酸钾、四(三苯基膦)钯的物质的量的比为1:1.2:10:0.02。
优选，步骤C 中所述的中间化合物、1-乙基-4-甲基喹啉碘化物、哌啶的物质的量的比为1:1.2:1.5。
优选，步骤A和步骤B中，过硅胶柱时正己烷：二氯甲烷为5:1到2:1梯度洗脱。
本发明的第三个目的是提供上述的结构式如式（I）所示的荧光染料在细菌染色中的应用。
优选，用于革兰氏阳性菌和革兰氏阴性菌的区分鉴别，革兰氏阳性菌被染色发红色荧光，革兰氏阴性菌不染色。更为优选，能鉴别革兰氏阳性菌和革兰氏阴性菌的荧光染料为金黄色葡萄球菌红色荧光染色剂。
另一优选，用于革兰氏阳性菌荧光成像。
本发明的第四个目的是提供一种用于细菌染色的荧光染料，该荧光染料含有结构式如式（I）所示的物质作为活性成分。
与现有技术相比，本发明具有如下有益效果：
本发明的荧光染料易于合成，并且显示出良好的红色荧光强度，对金黄色葡萄球菌染色发红色荧光、对大肠杆菌不染色不发荧光，可作为荧光染料用于革兰氏阳性菌和革兰氏阴性菌的鉴别，及用于革兰氏阳性菌荧光成像，在染色检验技术领域具有相当重要的意义与价值。本发明合成的荧光染料除了具有使用方便，结果判定明了的优点外，还具有便于贮存、运输等优点，它适合各检测单位实验室里使用。
附图说明
图1是荧光染料I的紫外-可见光吸收光谱（浓度：10μM）；
图2是荧光染料I的荧光光谱（浓度：10μM，激发波长：540nm）；
图3是荧光染料I对金黄色葡萄球菌和大肠杆菌的荧光成像图（菌浓度：109 CFU/mL，荧光染料I浓度：10μM，染色时间：10min）。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例
一种荧光染料，六氟磷酸化(E)-4-(2-(5-(7-(4-(二苯胺)苯基)苯并[c][1,2,5]噻二唑-4-基)噻吩-2-基)乙烯基)-1-乙基喹啉（I）的制备步骤：
步骤A：将4.0 mmol 4-硼酸三苯胺、4.8 mmol 4,7-二溴-2,1,3-苯并噻二唑、40mmol碳酸钾、0.2 mmol四(三苯基膦)钯置于100 mL四氢呋喃/水(10/1)溶剂中，氮气氛下75℃加热回流24小时，反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱（正己烷：二氯甲烷，5:1到2:1梯度洗脱），得到前体化合物；
步骤B：将所述2.0 mmol前体化合物、2.4 mmol 5-醛基-2-噻吩硼酸、20 mmol碳酸钾、0.04 mmol四(三苯基膦)钯置于50 mL四氢呋喃/水(10/1)溶剂中，氮气氛下75℃加热回流24小时，反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱（正己烷：二氯甲烷，5:1到2:1梯度洗脱），得到中间化合物；
步骤C：将所述1.0 mmol中间化合物、1.2 mmol 1-乙基-4-甲基喹啉碘化物、1.5mmol哌啶置于20 mL无水乙醇中，氮气氛下75℃加热回流24小时后，加入3倍体积的乙醚静置1小时，抽滤，滤渣溶于40 mL甲醇，加入20 mL饱和的六氟磷酸钾水溶液进行离子交换反应，搅拌1小时，过滤，水洗，干燥，得到橙色的目标产物（I），收率80%。
核磁表征数据（Bruker, AVANCE IIITM HD）：1H NMR (600 MHz, DMSO) δ 9.01(d, J = 6.4 Hz, 1H, pyridine), 8.60 (d, J = 8.5 Hz, 1H, benzene), 8.42 (d, J= 8.0 Hz, 1H, benzene), 8.23 (t, J = 7.7 Hz, 1H, benzene), 8.10 (m, 3H,benzene), 7.94 (d, J = 6.0 Hz, 1H, pyridine), 7.92 (d, J = 8.0 Hz, 2H,benzene), 7.84 (d, J = 4.0 Hz, 1H, thiophene), 7.53 (d, J = 3.8 Hz, 1H,thiophene), 7.46 (d, J = 4.1 Hz, 2H, ethylene), 7.36 (t, J = 5.0 Hz, 4H,benzene), 7.11 (m, 8H, benzene), 4.80 (q, J = 8.0 Hz, 2H, ethyl), 1.57 (t, J= 8.0 Hz, 3H, methyl)。电喷雾高分辨质谱（Bruker, maXis impact）：ESI-HRMS m/z:643.1986 [M]+。
实验数据：对实验中所用的仪器设备的型号和相关使用参数最好再细化一下，标黄的那3个仪器。
目标产物（I）的光学特性研究：
目标产物（I）的紫外-可见光吸收光谱（Thermo Scientific, NanoDrop One）如图1所示。最大吸收波长在550nm，在波长350nm-650nm范围内有良好的吸收，可用于可见光激发荧光研究。
为了评估目标产物（I）在可见光光照下荧光发射的能力，利用540nm进行激发检测荧光信号。如图2所示，目标产物（I）有很强的荧光发射，最大发射峰在640nm，为红光。这意味着，在染色检验技术方面，目标产物（I）可作为荧光染料，将具有很好的荧光成像性能。
目标产物（I）的荧光成像测试：
采用荧光显微镜（Nikon, Eclipse Ni）测定目标产物（I）的荧光成像性能，测定实施例制备的目标产物（I））对金黄色葡萄球菌（Staphylococcus aureus ATCC 6538P，革兰氏阳性菌）、大肠杆菌（Escherichia coli ATCC 8739，革兰氏阴性菌）的荧光成像性能。
微生物培养：
细菌用水解酪蛋白胨肉汤（MH）培养基培养，取1 mL浓度为109 cfu/mL的菌液，8000rpm离心1分钟收集菌体，用磷酸盐缓冲溶液（PBS）重悬。
每990μL的109 cfu/mL的PBS菌液中加入10μL的1mmol/L的目标化合物（终浓度10μmol/L），染色10分钟。处理结束后，分别取10μL菌液滴在载玻片上，盖上盖玻片压平，放于荧光显微镜下观察，拍照，发红色荧光为染色成功，无光为不染色。
结果显示：
如图3所示，染色10分钟时，10μM浓度的目标产物（I）几乎可以染色所有的金黄色葡萄球菌。同样，在染色10分钟时，10μM浓度的目标产物（I）几乎不染大肠杆菌。在金黄色葡萄球菌和大肠杆菌的混合菌液中，也只染金黄色葡萄球菌。这些结果表明目标产物（I）对革兰氏阳性菌和革兰氏阴性菌具有很强的鉴别效果，并能够对革兰氏阳性菌荧光成像。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种荧光染料，其特征在于，所述的荧光染料的结构式如式（I）所示：
2.如权利要求1所述的一种荧光染料的制备方法，其特征在于，包括以下步骤：以4-硼酸三苯胺和4,7-二溴-2,1,3-苯并噻二唑为原料，在碳酸钾和氮气氛条件下，四(三苯基膦)钯催化发生偶联反应得到前体化合物；前体化合物与5-醛基-2-噻吩硼酸在四(三苯基膦)钯催化发生偶联反应得到中间化合物；中间化合物与1-乙基-4-甲基喹啉碘化物缩合后，再与六氟磷酸钾进行离子交换得到权利要求1所述的荧光染料。
3.根据权利要求2所述的一种荧光染料的制备方法，其特征在于，包括以下步骤：
步骤A：将4-硼酸三苯胺、4,7-二溴-2,1,3-苯并噻二唑、碳酸钾、四(三苯基膦)钯置于四氢呋喃和水的溶剂中，氮气氛下70-80℃加热回流20-28小时，反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱，得到前体化合物；
步骤B：将前体化合物、5-醛基-2-噻吩硼酸、碳酸钾、四(三苯基膦)钯置于四氢呋喃和水的溶剂中，氮气氛下70-80℃加热回流20-28小时，反应结束后，冷却，减压旋蒸，二氯甲烷萃取，水洗，有机相用无水硫酸镁干燥后，过滤，减压旋蒸，过硅胶柱，得到中间化合物；
步骤C：将中间化合物、1-乙基-4-甲基喹啉碘化物、哌啶置于无水乙醇中，氮气氛下70-80℃加热回流20-28小时，加入3倍体积的乙醚静置0.5-1.5小时，抽滤，滤渣溶于甲醇，加入饱和的六氟磷酸钾水溶液进行离子交换反应，搅拌0.5-1.5小时，得到结构式（I）所示的荧光染料。
4.根据权利要求2所述的一种荧光染料的制备方法，其特征在于，步骤A 中4-硼酸三苯胺、4,7-二溴-2,1,3-苯并噻二唑、碳酸钾、四(三苯基膦)钯的物质的量的比为1:1.2:10:0.05。
5.根据权利要求2所述的一种荧光染料的制备方法，其特征在于，步骤B中前体化合物、5-醛基-2-噻吩硼酸、碳酸钾、四(三苯基膦)钯的物质的量的比为1:1.2:10:0.02。
6.根据权利要求2所述的一种荧光染料的制备方法，其特征在于，其制备步骤为：步骤C中间化合物、1-乙基-4-甲基喹啉碘化物、哌啶的物质的量的比为1:1.2:1.5。
7.如权利要求1所述的一种荧光染料在细菌染色中的应用。
8.根据权利要求7所述的一种荧光染料在细菌染色中的应用，其特征在于，用于革兰氏阳性菌和革兰氏阴性菌的区分鉴别，革兰氏阳性菌被染色发红色荧光，革兰氏阴性菌不染色。
9.根据权利要求7所述的一种荧光染料在细菌染色中的应用，其特征在于，用于革兰氏阳性菌荧光成像。
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