CN110330518B - 一种荧光核酸探针的制备方法 - Google Patents
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Abstract
本发明涉及核酸检测材料制备技术领域，具体涉及一种高效低毒荧光核酸探针的制备方法，包括以下步骤：配置一定浓度的溴化‑3,8‑二氨基‑5‑乙基‑6‑苯基菲啶鎓二氯甲烷溶液A；配置一定浓度的四苯硼盐或四苯硼衍生物盐水溶液B；将溶液A和溶液B按照一定体积比混合后遮光反应至反应完全；反应完后加水后分液，用二氯甲烷溶液萃取二氯甲烷相并收集；收集的二氯甲烷相除去水分后进行柱色谱分离，收集第一个红色荧光色带，干燥，得到所述的高效低毒荧光核酸探针。本发明的制备方法制备的高效低毒荧光核酸探针，弥补溴化乙锭在光稳定性和细胞毒性方面的不足，满足科研对于低毒性、高灵敏度、高稳定性且价格低廉的DNA荧光探针的需求。
Description
技术领域
本发明涉及核酸检测材料制备技术领域，具体涉及一种荧光核酸探针的制备方法。
背景技术
荧光传感器由于具有响应快，灵敏度高，操作简单等特点，近些年来受到了极大的关注。荧光传感器的特点使其得以弥补仪器分析的不足之处，对于及时性检测和现场应急监测有着明显的优势，尤其是滤纸条方面的应用，更是有着诱人的前景。但传统荧光传感器因为存在刚性平面结构，分子内形成π-π相互作用导致聚集诱导荧光淬灭(ACQ)现象，在高浓度或者固态的时候，荧光强度急剧降低甚至消失。科学家们采用了各种物理或化学的方法来减少或者防止荧光分子的聚集，如引入螺环骨架或大位阻基团防止有机荧光分子聚集、增强分子内电荷转移跃迁、交叉堆积、J-聚集态、偶极交叉堆积等多种方法来消除聚集荧光猝灭效应的影响。但这些修饰都是在分子骨架上进行修改，造成了合成过程繁琐，极大的增加了合成成本。
溴化乙锭(EB)是在核酸检测领域应用最广泛的荧光探针。溴化乙锭的菲啶环端可以插入双链DNA的疏水腔体中，其复合物荧光强度较之游离态溴化乙锭要高出数倍，且伴随着最大发射波长的红移，从而实现对DNA的检测。低浓度溴化乙锭染色法作为检测凝胶电泳实验中微量DNA的方法，已经使用了数十年。在推荐的溴化乙锭工作浓度下(0.5mg/L),检出限低至10ng。溴化乙锭由于其价格便宜，灵敏度高，操作简单而被广泛应用。但溴化乙锭的细胞毒性，且是强诱变剂，近些年来受到越来越多的重视[1]。同时，溴化乙锭具有易光漂白的特点，无法满足长程追踪成像的需求。因此，越来越多的商品化溴化乙锭替代物被开发出来，例如SYBR系列染料分子，Goldview，Gelred等。但这些商业化分子仍有许多无法克服的缺点，无法完全满足核酸检测的需求[2-4]。例如SYBR系列仍有细胞毒性，光稳定性较差，对于低于50bp的DNA片段不具备染色能力，低于100bp的DNA片段检测灵敏度低；Goldview使用的吖啶橙(AO)分子具有高细胞毒性和诱变性，且具有较高的染色背景和较低的灵敏度；Gelred系列价格昂贵，极大提高检测成本；双联EB(EthD)虽能极大的提高EB的检出限，但也提高了EB的分子毒性，且存在交联配合，影响DNA片段检测的准确性等。由于这些常用的商品化EB替代品明显的缺点，开发低毒性，高灵敏度，高稳定性，价格低廉的DNA荧光探针仍迫在眉睫。
发明内容
本发明的目的在于提供一种荧光核酸探针的制备方法，制备方法简单，成本低廉，合成产物具有低毒性、高灵敏度、高稳定性。
本发明解决上述技术问题所采用的方案是：一种高效低毒荧光核酸探针的制备方法，包括以下步骤：
(1)配置一定浓度的溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓二氯甲烷溶液A；
(2)配置一定浓度的四苯硼盐或四苯硼衍生物盐水溶液B；
(3)将溶液A和溶液B按照一定体积比混合后遮光反应至反应完全；
(4)反应完后加水后分液，用二氯甲烷溶液萃取二氯甲烷相并收集；
(5)收集的二氯甲烷相除去水分后进行柱色谱分离，收集第一个红色荧光色带，干燥，得到所述的高效低毒荧光核酸探针。
优选地，所述步骤(1)中，溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓的浓度为(2.0-3.0)×10-4mol/mL。
优选地，所述步骤(2)中，四苯硼盐和四苯硼衍生物盐的浓度均为(1.0-1.5)×10- 4mol/mL。
优选地，所述步骤(2)中，四苯硼盐为四苯硼钠，四苯硼衍生物盐为四(3,5-二(三氟甲基)苯基)硼酸钠。
优选地，当所述四苯硼盐为四苯硼钠时，所得高效低毒荧光核酸探针的最佳激发波长为530nm，荧光最大发射波长在638nm，当所述四苯硼衍生物盐为四(3,5-二(三氟甲基)苯基)硼酸钠时，最佳激发波长为514nm，荧光最大发射波长在606nm。
优选地，所述步骤(3)中，二氯甲烷溶液A与水溶液B的体积比为0.9-1.1:2。
优选地，所述步骤(5)中，柱色谱分离的淋洗剂为甲醇：二氯甲烷＝1：20。
优选地，所述高效低毒荧光核酸探针的粒径为60-95nm。
优选地，还包括步骤(6)，将所述高效低毒荧光核酸探针制备成纳米颗粒。
优选地，所述步骤(6)的具体方法为：将所述步骤(5)制备的高效低毒荧光核酸探针溶于DMSO中，配置成1g/L的探针母液，取一定量母液，摇晃下加入所取母液30倍体积的水，摇晃10秒后再加入相同体积的水，重复同样操作3次后，定容后再次摇晃10秒后得到所述纳米颗粒溶液。
本发明的制备方法利用原料溴化乙锭一步反应得到本发明的高效低毒荧光核酸探针，弥补溴化乙锭在光稳定性和细胞毒性方面的不足，满足科研对于低毒性、高灵敏度、高稳定性且价格低廉的DNA荧光探针的需求。利用商品化且价格低廉的溴化乙锭，通过一步法将其对离子溴改变成了具有扭曲结构的有机阴离子，合成出了本发明的高效低毒荧光核酸探针，从而实现了其在聚集状态下不发生荧光淬灭。
经DLS粒径分析，本发明的高效低毒荧光核酸探针的粒径分布在60-95nm。研究表明，人体皮肤角质层无法吸收粒径大于40nm的纳米颗粒，而本发明的高效低毒荧光核酸探针的最小粒径均远大于40nm，故无法通过皮肤屏障。同时，本发明的高效低毒荧光核酸探针具有极低的细胞毒性。本发明的高效低毒荧光核酸探针的纳米颗粒溶液在无法通过皮肤屏障的同时，又具有低毒性，给实验人员的安全提供了双重保障，弥补了溴化乙锭及相应商品化替代品在毒性方面的缺点。
附图说明
图1为本发明实施例3中探针1和探针2形成的纳米颗粒粒径测试图；
图2为本发明实施例4中探针1加入DNA前后荧光强度变化图；
图3为本发明实施例4中探针2加入DNA前后荧光强度变化图；
图4为本发明实施例5中探针1、探针2与溴化乙锭凝胶电泳成像的条带对比图；
图5为本发明实施例6中探针1、探针2以及商业化溴化乙锭的细胞毒性对比图；
图6为本发明实施例7中探针1、探针2以及商业化溴化乙锭的光稳定性测试图。
具体实施方式
为更好的理解本发明，下面的实施例是对本发明的进一步说明，但本发明的内容不仅仅局限于下面的实施例。
实施例1
合成路线如下：
将200mg溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓溶于2mL二氯甲烷中置于25mL圆底烧瓶，将180mg四苯硼钠溶于2mL去离子水后，搅拌下加入到二氯甲烷溶液中，遮光反应12个小时后加入10mL去离子水，用200mL二氯甲烷分四次萃取水相，分液后收集。母液用无水硫酸钠干燥半小时后过滤去除硫酸钠，柱色谱分离，淋洗剂为甲醇/二氯甲烷＝1/20，收集第一个红色荧光色带，旋干干燥，得到190mg红色固体，为探针1，产率为62％。
实施例2
合成路线如下：
将200mg溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓溶于2mL二氯甲烷中置于25mL圆底烧瓶，将466mg四(3,5-二(三氟甲基)苯基)硼酸钠溶于4mL去离子水后，搅拌下加入到二氯甲烷溶液中，遮光反应12个小时后加入10mL去离子水，用200mL二氯甲烷分四次萃取水相，分液后收集。母液用无水硫酸钠干燥半小时后过滤去除硫酸钠，柱色谱分离，淋洗剂为甲醇/二氯甲烷＝1/20，收集第一个红色荧光色带，旋干干燥，得到325mg红色固体，为探针2，产率为60％。
本发明实施例中收集有机相后，是用无水Na2SO4干燥，也可以采用其他干燥剂，只要能除去有机相中的水分且不与有机相反应即可。
实施例3
实施例1-2中探针1和探针2纳米颗粒溶液的制备
将1mg的探针(1或2)溶于1mL DMSO中，配置1g/L的母液保存。取0.1mL母液，摇晃下加入3mL去离子水(蒸馏水也可)，摇晃10秒后再加入3mL去离子水，重复同样操作。加入三次3mL去离子水后，定容至10mL，再次摇晃10秒后得到操作用纳米颗粒溶液，浓度为0.1g/L。图1为探针1和探针2形成的纳米颗粒粒径测试；测试仪器为动态光散射仪(DLS)，型号为WyattTechnology Dynapro Titan TC；其中探针1的粒径为85nm-95nm，探针2的粒径为60nm-80nm。
实施例4
探针1和探针2的纳米颗粒的荧光强度的测定
将实施例3中制备的纳米颗粒溶液摇晃5秒后，取100mL待测溶液(分为两组含DNA片段和不含DNA片段)直接加入3mL直接纳米颗粒溶液中，混合均匀后即可立刻测荧光强度改变。由图2可知，探针1最佳激发波长为530nm，荧光最大发射波长在638nm；由图3可知，探针2最佳激发波长为514nm，荧光最大发射波长在606nm。加入DNA后，两种种荧光探针的荧光强度都增加了约5倍，并伴随着轻微的蓝移，这是由于溴化乙锭母体嵌入DNA的疏水腔中导致的，加入DNA后明显的荧光增强可以帮助我们高灵敏的检测DNA片段。
实施例5
实施例里1-2中制备的探针1和探针2及溴化乙锭的凝胶电泳分析
将1mg的探针(1或2)溶于1mL DMSO中，配置1g/L的母液保存。取30μL母液，摇晃下加入冷却至50℃以下的琼脂糖凝胶溶液中，晃动溶液半分钟使其分布均匀后制胶，加入DNA样品，用7V/cm的电场进行凝胶电泳后，使用伯乐凝胶成像系统进行成像(任意品牌凝胶电泳成像仪均可，具体操作参考各个品牌的仪器说明书)。
本实施例检测了凝胶电泳下探针1、探针2以及溴化乙锭的凝胶电泳成像应用效果。从图4中可以看出，在相同曝光时间和感光度的条件下，探针1和探针2的条带亮度大于溴化乙锭的条带亮度。尤其是低分子量的条带，探针1和探针2成像清晰度和亮度要优于溴化乙锭，说明探针1和2的灵敏度要远高于商业化溴化乙锭，且对于低分子量DNA有着更好的响应性。
实施例6
实施例1-2中制备的探针1和探针2的细胞毒性研究
将10mg/L的探针1和探针2的纳米颗粒溶液和Hela细胞置于商业化的DMEM培养基中，其中含有10％体积比(相较于培养基)的小牛胚胎血清和1％的青霉素-链霉素(相较于DMEM的质量比)，于37℃下置于96孔板中培养24小时后，后用移液枪移去培养液，并用无菌PBS缓冲液清洗后，使用商业化的MTT法检测细胞存活率(使用探针购于KeyGEN BioTECH公司)。由图5可知探针1和探针2的细胞存活率分别在97％和86％，均远大于同条件下商品化溴化乙锭(购于TCI公司)59％的存活率。而测试浓度10mg/L远大于使用浓度0.5mg/L，说明探针1和探针2具有极低的细胞毒性。
实施例7
实施例1-2中制备的探针1，2以及溴化乙锭的光稳定性
将探针1、探针2以及溴化乙锭按实施例3方法配置成溶液后，用35W绿光光源持续照射三种溶液，并每600秒取样测试荧光强度，共照射3600秒，等间隔取样6次。如图6所示，在用绿光光源持续照射1小时后，溴化乙锭的荧光强度从100％下降到了73％，而探针1和探针2则分别为初始强度的98.4％和83.3％。证明探针1和探针2，尤其是探针1的光稳定性要远强于溴化乙锭。
以上所述是本发明的优选实施方式而已，当然不能以此来限定本发明之权利范围，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和变动，这些改进和变动也视为本发明的保护范围。
Claims (8)
1.一种荧光核酸探针的制备方法，其特征在于，包括以下步骤：
(1)配置一定浓度的溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓二氯甲烷溶液A；
(2)配置一定浓度的四苯硼盐或四(3,5-二(三氟甲基)苯基)硼酸钠水溶液B；
(3)将溶液A和溶液B按照一定体积比混合后遮光反应至反应完全；
(4)反应完后加水后分液，用二氯甲烷溶液萃取二氯甲烷相并收集；
(5)收集的二氯甲烷相除去水分后进行柱色谱分离，收集第一个红色荧光色带，干燥，得到所述的荧光核酸探针化合物；
(6)将所述步骤(5)制备的荧光核酸探针化合物溶于DMSO中，配置成1g/L的探针母液，取一定量母液，摇晃下加入所取母液30倍体积的水，摇晃10秒后再加入相同体积的水，重复同样操作3次后，定容后再次摇晃10秒后得到纳米颗粒溶液。
2.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述步骤(1)中，溴化-3,8-二氨基-5-乙基-6-苯基菲啶鎓的浓度为(2.0-3.0)×10-4mol/mL。
3.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述步骤(2)中，四苯硼盐和四(3,5-二(三氟甲基)苯基)硼酸钠的浓度均为(1.0-1.5)×10-4mol/mL。
4.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述步骤(2)中，四苯硼盐为四苯硼钠。
5.根据权利要求4所述的荧光核酸探针的制备方法，其特征在于：当所述四苯硼盐为四苯硼钠时，所得荧光核酸探针的最佳激发波长为530nm，荧光最大发射波长在638nm，当为四(3,5-二(三氟甲基)苯基)硼酸钠时，最佳激发波长为514nm，荧光最大发射波长在606nm。
6.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述步骤(3)中，二氯甲烷溶液A与水溶液B的体积比为0.9-1.1:2。
7.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述步骤(5)中，柱色谱分离的淋洗剂为甲醇：二氯甲烷＝1：20。
8.根据权利要求1所述的荧光核酸探针的制备方法，其特征在于：所述荧光核酸探针的粒径为60-95nm。
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