CN112209877A - 一种二价铁荧光探针及其应用 - Google Patents
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Abstract
本发明公开了一种二价铁荧光探针及其应用。本发明的二价铁荧光探针的结构式如式I所示；首次将二价铁荧光探针技术应用于功能微生物选育中，利用三价铁在细菌细胞内被还原成二价铁的显色过程结合流式细胞仪进行特异筛选功能活性细菌。本发明建立了一套全新的基于化学荧光标记技术的微生物选育研究模式，可更高效、直观、特异地对单个细胞进行快速、高灵敏分析分选，能为功能微生物的高效选育提供新的工具，也能为功能微生物单细胞研究和未培养微生物的深入挖掘提供技术支撑。
Description
技术领域
本发明属于功能微生物选育技术领域，具体涉及一种二价铁荧光探针及其应用。
背景技术
铁矿物广泛地存在于缺氧的地下环境中，可作为许多微生物的电子受体。虽然微生物的铁还原过程是地球上最古老的呼吸形式之一，但是铁还原微生物所具有的独特的电子转移方式(如细菌的纳米导线、矿物介导的种间电子转移和直接的种间/细胞间电子转移等)近年来才被发现报道。在自然环境中，具有铁还原能力的微生物引发的电子转移是硫化物、有机物氧化和许多其他生物地球化学过程的重要驱动力。例如，厌氧条件下铁的异化还原过程会强烈影响各种有机无机污染物的环境行为。在铁还原微生物的异化作用中，Fe(III)被用作电子受体，还原产生的Fe(II)，偶联多种有机无机物的氧化。异化铁还原微生物具有强大的代谢能力，铁还原菌可以影响到除Fe(III)之外的很多重金属以及放射性核素，如Mn(IV)、Cr(VI)、 Ag(I)、Au(III)、Hg(II)、V(V)、Sr(II)、Co(II)等生物的地球化学循环，在厌氧地层的生物修复中起到重要作用。这些很多重金属以及放射性核素在以高价态存在时往往会形成对环境有害的污染物，一旦被还原为低价态后或形成沉淀，或与Fe(II)形成共沉淀，使得其毒性降低。因此异化Fe(III)还原在污染环境的生物修复中表现出了很大的优势。此外，铁还原能力的微生物在废水处理、生物电化学系统(BESs)等工程过程或生物反应器中起着关键作用。因此，开展铁还原微生物的鉴定和分离及其特性研究将有助于深入了解微生物铁还原过程，不仅可以丰富污染修复的生物材料，而且为污染修复应用提供重要的理论依据。
鉴定具有铁还原能力的微生物是分离筛选铁还原微生物的前提，目前最常用的鉴定方法是Fe2+的分光光度法：让Fe3+的水溶液和微生物共培养，然后具有铁还原能力的微生物把Fe3+还原成Fe2+，然后用Fe2+与邻菲罗啉在pH 3.5～7下反应形成橙红色的络合物，在分光光度计下测510nm的吸光度。然而，当暴露在空气中时，橙红色的络合物的颜色并不稳定。此外，该方法不能用于从复杂的环境样本或在单个细胞水平上识别、定位或量化铁还原菌。目前，还没有方法能从原位或者混合环境中可视化鉴定铁还原微生物。荧光探针标记技术的出现为可视化原位鉴定铁还原微生物提供了技术支持。荧光探针标记技术不仅可以快速可视化鉴定混合环境中的铁还原微生物，还可以相对定量地鉴别铁还原微生物的还原能力，为现实环境样品中筛选铁还原微生物提供了有效的依据。
分离选育具有铁还原能力的微生物，是开展环境治理和修复，加速重金属污染物去除的核心工作。传统平板分离筛选方法所获得的单个细胞的性状和功能需要在单个克隆增殖后以群体的形式体现从而导致其功能表征的滞后性和低灵敏性。而以群体形式存在的微生物细胞之间的异质性又导致其功能表征的不确定性，从而降低了筛选的准确性。而且，传统筛选方法只能对在特定培养基中生长较快的功能微生物进行筛选，对于难培养或生长较慢的微生物难以实现分析筛选。因此，在单个细胞水平实现对微生物功能活性的快速、准确地分析与追踪筛选是功能微生物高效选育的关键。
相较于传统的细胞功能代谢活性分析检测手段，如酶活的检测、代谢产物分析，荧光标记技术是一种快速、方便的生物分析手段。荧光激活细胞分选(流式细胞仪)可以对单个细胞进行快速、高灵敏度分析分选。但过去的菌株筛选研究中使用的荧光探针多是通用型的核酸探针，难以对微生物的功能活性进行追踪。利用纳米荧光探针追踪微生物，其定位性强，荧光物质不易泄露，这可以解决微生物功能细胞非特异追踪问题。利用荧光能量转移技术构建目标污染物纳米荧光探针，实现对单个功能细胞快速、准确地分析与追踪筛选，并且结合流式分选，建立高效直观、特异性追踪功能活性菌株的高通量筛选方法，将显著提高功能微生物分离选育的效率，不仅为功能微生物单细胞水平研究和为培养功能微生物资源的深入挖掘提供重要的技术支撑，也将为环境污染的微生物治理和修复提供宝贵的菌种资源。
发明内容
本发明的目的是克服现有技术中存在的上述不足，提供一种二价铁荧光探针及其在鉴定和选育具有铁还原能力微生物中的应用。
本发明的二价铁荧光探针，其结构式如式I所示：
本发明还提供所述的二价铁荧光探针在鉴别和筛选具有铁还原活性的微生物中的应用。
优选，所述的应用，包括以下步骤：
培养并收集待鉴定微生物菌体，用PBS缓冲液洗涤后接种到含有3mM柠檬酸铁的无机盐培养基中厌氧培养7h以上，加入终浓度为50μM的所述的二价铁荧光探针培养15min，然后用激光共聚焦扫描显微镜观察荧光进行鉴定。
所述的激光共聚焦扫描显微镜使用激发波长为488nm，发射波长为530nm；观察到的荧光强度越强表示微生物的铁还原能力越强。
优选，所述的应用，包括以下步骤：
培养并收集待鉴定微生物菌体，用PBS缓冲液洗涤后接种到含有3mM柠檬酸铁的无机盐培养基中厌氧培养8h以上，加入终浓度为50μM的所述的二价铁荧光探针培养15min，然后利用流式细胞仪进行筛选。
所述的利用流式细胞仪进行筛选，是使用Sysmex Partec CyFlow Cube 6流式细胞仪的 FL1检测器：激发波长为488nm，发射波长为536/40nm；在488nm激发波长下激发，流量是50μL/min，运行2min，信号收集通道选用DsRed通道。
相对于现有技术，本发明具有如下优点和有益效果：
本发明具有铁还原活性微生物的鉴别方法是以二价铁为目标化合物，以铁还原菌株为实验客体，首次将二价铁荧光探针技术应用于功能微生物选育中，利用三价铁在细菌细胞内被还原成二价铁的显色过程结合流式细胞仪进行特异筛选功能活性细菌。本发明建立了一套全新的基于化学荧光标记技术的微生物选育研究模式，更高效、直观、特异地对单个细胞进行快速、高灵敏度分析分选，能为功能微生物的高效选育提供新的工具，也能为功能微生物单细胞研究和深入挖掘培养微生物提供技术支撑，对于胞外电子传递机制解释以及污染物防治有重大的实践意义和应用潜力。
附图说明
图1是二价铁荧光探针的GC-MS图：上图是气象色谱保留时间图，下图是保留时间为 43min的峰的质谱图。
图2是二价铁荧光探针的光谱分析；A为二价铁荧光探针和不同浓度Fe2+溶液作用的荧光光谱图；B为不同浓度Fe2+溶液和二价铁荧光探针作用后在发射波长为530nm处的荧光关系图；C为Fe2+溶液浓度的对数和在发射波长为530nm处荧光的线性关系图。
图3是二价铁荧光探针用激光共聚焦扫描显微镜鉴别铁还原菌的激光共聚焦图片(激发波长488nm)；A为根瘤菌F217；B为鞘脂菌C1；C为芽孢杆菌Iβ8；D为变型赖氨酸芽孢杆菌GY32；E为类芽孢杆菌Iβ12；F为脱色希瓦氏菌S12；G为脱色希瓦氏菌S12的突变株 S22(敲除了细胞色素c的相关基因，不能进行铁还原)；H为用传统分光光度计法测的以上不同菌的铁还原能力图；图3A-G的标尺：5μm。
图4是铁还原功能微生物的流式细胞仪分析图；A为铁还原菌脱色希瓦氏菌S12和非铁还原菌鞘脂菌C1混合菌液在没有加二价铁荧光探针前的流式细胞散点图；B为铁还原菌脱色希瓦氏菌S12和非铁还原菌鞘脂菌C1混合菌液在没有加二价铁荧光探针前的FL1通道(激发波长为488nm，发射波长为536/40nm)荧光强度分析图；C为铁还原菌脱色希瓦氏菌S12 和非铁还原菌鞘脂菌C1混合菌液在加入二价铁荧光探针后的流式细胞散点图；D为铁还原菌脱色希瓦氏菌S12和非铁还原菌鞘脂菌C1混合菌液在加入二价铁荧光探针后的FL1通道的荧光强度分析图。
图5是二价铁荧光探针合成步骤及荧光显色原理示意图。
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。
本发明的二价铁荧光探针合成步骤及荧光显色原理如图5所示。
实施例1
二价铁荧光探针的合成：
二价铁荧光探针合成的第一步：在250mL的单口烧瓶中加入4.2g 4-溴,1-8-萘酐和180 mL无水乙醇，超声8min后，将6mL正丁胺溶于10mL无水乙醇中，边快速搅拌边慢慢滴加正丁胺溶液到单口烧瓶中，在80℃下加热回流搅拌6h，冷却结晶后抽滤并用无水乙醇洗涤晶体三次，晶体溶解在乙酸乙酯中，通过硅胶柱分离(洗脱液：乙酸乙酯/石油醚＝1：50)，得到中间产物4-溴-N-丁基-1-8-萘亚胺4.8g，产率80.1％。
二价铁荧光探针合成的第二步：在150ml三口圆底烧瓶中加入0.3g联苯二碲和20mL 无水乙醇，超声5min后，放入冰浴中一边磁力搅拌一边通氮气20min。称0.08g硼氢化钠溶于4mL无水乙醇中，在氮气氛围下慢慢滴加入三口圆底烧瓶中，直到三口烧瓶中的溶液由红色变成无色透明，然后马上将三口圆底烧瓶放到83℃的水浴锅中，期间三口圆底烧瓶里一直通着氮气。事先分别称好0.18g第一步产物4-溴-N-丁基-1-8-萘亚胺和0.14g CuI于两个离心管中，再分别加上4mL无水乙醇溶解。用注射器先快速加入第一步产物的乙醇溶液到三口圆底烧瓶里，5min后再用注射器快速加入CuI乙醇溶液，在氮气的氛围下继续加热搅拌回流半小时后抽滤，滤液经过减压蒸馏后得到橙黄色固体，通过硅胶柱分离(洗脱液：乙酸乙酯 /石油醚＝1：125,)，得到产物二价铁荧光探针0.72g，产率88％。
经气相色谱质谱联用仪(图1)鉴定，该二价铁荧光探针的结构式如式I所示：
实施例2
二价铁荧光探针的光谱：
将二价铁荧光探针溶于乙腈中，测试其对二价铁的响应荧光光谱。二价铁荧光探针本身不会发出强荧光，但是当它遇到二价铁后，连在萘亚胺上的Te-C键就会断裂，该荧光探针就会发出较强的荧光。为了探究该荧光探针对二价铁的荧光响应灵敏度，本实施例用2mM二价铁荧光探针和不同浓度下的二价铁溶液混合然后测其荧光强度。荧光光谱测试使用荧光分光光度计(Perkin Elmer/LS-45)，激发波长530nm，发射波长范围480-600nm。
图2是二价铁荧光探针对二价铁响应灵敏度的荧光光谱分析，从图中可以看出，随着二价铁浓度的增加，该二价铁荧光探针的荧光强度越强，而且荧光强度和二价铁浓度的对数成正比关系。
实施例3
二价铁荧光探针示踪纯菌：
为了验证该二价铁荧光探针对铁还原菌的鉴别能力，本实验选用了脱色希瓦氏菌S12(脱色希瓦氏菌S12，该菌株是现有技术中的菌株，公开于文献：Meiying Xu,Jun Guo,Guoqu Zeng, Xiaoyan Zhong,Guoping Sun，Decolorization of anthraquinone dyeShewanella decolorationis S12， Appl Microbiol Biotechnol,2006,71:246～251)作为阳性对照，脱色希瓦氏菌S12的突变株S22 (敲除了细胞色素c的相关基因，不能进行铁还原)作为阴性对照，然后再选取了鞘脂菌C1，根瘤菌F217，芽孢杆菌Iβ8，类芽孢杆菌Iβ12，变型赖氨酸芽孢杆菌GY32来鉴别它们的铁还原能力。将以上各菌种分别接种至40mL Luria-Bertani培养基(LB培养基：胰蛋白胨 (Tryptone)10g/L，酵母提取物(Yeast extract)5g/L，氯化钠(NaCl)10g/L，pH＝7.4)的锥形瓶，摇床隔夜培养确保生长完全，再分别将10μL各预培养菌液转接到4mL新鲜LB培养基的锥形瓶中摇床下继续培养8h，分别取出离心(7500rmp，10min,25℃)收获菌体用PBS(phosphate buffer saline：137mM NaCl，2.7mMKCl，10mM Na2HPO4，2mM KH2PO4，pH＝7.4)缓冲液洗涤三次后接种到含有3mM柠檬酸铁的厌氧乳酸盐培养基(LM培养基：2.0g/L乳酸钠， 0.2g/L酵母提取物，12.8g/L Na2HPO4·7H2O，3g/L KH2PO4，0.5g/L NaCl和1.0g/L NH4Cl) 中，厌氧培养7h后，取出5mL菌液，然后分别加入50μM的二价铁荧光探针培养15min 后用激光共聚焦扫描显微镜观察荧光。
图3为以上各种纯菌在柠檬酸铁的LM培养基中厌氧培养7h后加入二价铁荧光探针的激光共聚焦照片，从图中可以看出，阳性对照菌S12(图3F)可以进行铁还原所以显示出比较强的荧光，而阴性对照菌S22(图3G)没有铁还原功能则几乎没有显示出荧光。另外五个未知铁还原能力的纯菌中，只有类芽孢杆菌Iβ12显示出绿色荧光，但其荧光强度比阳性对照菌S12稍微弱一点，这证明类芽孢杆菌Iβ12具有一定的铁还原能力，但是其铁还原能力比S12稍微弱一点，而其他四个纯菌均没有铁还原能力。此结果与传统的铁还原菌鉴别方法的结果一致(图3H)。
实施例4
铁还原功能微生物的流式分析：
将二价铁荧光探针应用到复配菌群中去，利用流式细胞仪进行功能活性特异性分析分选。将已知具有铁还原功能的脱色希瓦氏菌S12，以及无铁还原功能的鞘脂菌C1分别培养至对数期，然后加入含有3mM柠檬酸铁的LM培养液中，再厌氧培养8h，再将这两种菌液等量混合然后加入50μM二价铁荧光探针培育15min，再利用流式细胞仪进行分析。流式分析使用 Sysmex Partec CyFlow Cube 6流式细胞仪的FL1检测器(激发波长为488nm，发射波长为 536/40nm)，在488nm激发波长下激发，流量是50μL/min，运行2min。图4是铁还原菌脱色希瓦氏菌S12和非铁还原菌鞘脂菌C1的流式细胞仪结果，从图4A和B可以看出，在没加入二价铁荧光探针前，脱色希瓦氏菌S12和鞘脂菌C1在FL1荧光通道都几乎没荧光，而加入了二价铁荧光探针后再经流式细胞仪分析后，从图4C和D可以看出，非铁还原菌鞘脂菌C1在FL1的荧光通道中还是几乎没荧光，而铁还原菌脱色希瓦氏菌S12则显示出较强的FL1荧光。这就证明该二价铁荧光探针可以在混合样品中分选出铁还原菌。
Claims (6)
1.一种二价铁荧光探针，其特征在于，其结构式如式I所示：
2.权利要求1所述的二价铁荧光探针在鉴别和筛选具有铁还原活性的微生物中的应用。
3.根据权利要求2所述的应用，其特征在于，包括以下步骤：
培养并收集待鉴定微生物菌体，用PBS缓冲液洗涤后接种到含有3mM柠檬酸铁的无机盐培养基中厌氧培养7h以上，加入终浓度为50μM的所述的二价铁荧光探针培养15min，然后用激光共聚焦扫描显微镜观察荧光进行鉴定。
4.根据权利要求3所述的应用，其特征在于，所述的激光共聚焦扫描显微镜使用激发波长为488nm，发射波长为530nm；观察到的荧光强度越强表示微生物的铁还原能力越强。
5.根据权利要求2所述的应用，其特征在于，包括以下步骤：
培养并收集待鉴定微生物菌体，用PBS缓冲液洗涤后接种到含有3mM柠檬酸铁的无机盐培养基中厌氧培养8h以上，加入终浓度为50μM的所述的二价铁荧光探针培养15min，然后利用流式细胞仪进行筛选。
6.根据权利要求5所述的应用，其特征在于，所述的利用流式细胞仪进行筛选，是使用Sysmex Partec CyFlow Cube 6流式细胞仪的FL1检测器：激发波长为488nm，发射波长为536/40nm；在488nm激发波长下激发，流量是50μL/min，运行2min，信号收集通道选用DsRed通道。
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| CN108003862A (zh) * | 2017-11-28 | 2018-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | 一种核壳型纳米二氧化硅荧光探针及其合成方法和应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108003862A (zh) * | 2017-11-28 | 2018-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | 一种核壳型纳米二氧化硅荧光探针及其合成方法和应用 |
Non-Patent Citations (2)
| Title |
|---|
| ZONGJIN QU ET AL.: "A turn-on fluorescent chemodosimeter based on detelluration for detecting ferrous iron (Fe2+) in living cells", 《J. MATER. CHEM. B》 * |
| 林海龙 等: "《厌氧环境微生物学》", 31 October 2014, 哈尔滨工业大学出版社 * |
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