CN110031436B - 一种检测脂滴的有机硅荧光探针 - Google Patents
一种检测脂滴的有机硅荧光探针 Download PDFInfo
- Publication number
- CN110031436B CN110031436B CN201910293090.0A CN201910293090A CN110031436B CN 110031436 B CN110031436 B CN 110031436B CN 201910293090 A CN201910293090 A CN 201910293090A CN 110031436 B CN110031436 B CN 110031436B
- Authority
- CN
- China
- Prior art keywords
- reaction
- fluorescent probe
- probe
- compound
- organic silicon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/14—Macromolecular compounds
- C09K2211/1441—Heterocyclic
- C09K2211/1466—Heterocyclic containing nitrogen as the only heteroatom
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
技术领域
本发明属于分析化学技术领域,具体涉及一种检测脂滴的荧光探针及其应用。
背景技术
脂滴由于其特殊的结构和生理功能,最近得到广大研究者的关注。脂滴作为富含脂质的亚细胞器,在真核细胞中广泛存在,由中性脂质的核心组成,所述中性脂质由安装相关蛋白质的单层磷脂包围。除了作为能量储存器外,脂滴还参与各种生理过程,包括细胞活化,迁移,增殖,凋亡等。越来越多的证据表明脂滴的异常与癌症发展有关。据报道,癌细胞中脂滴的数量高于正常细胞,因为脂滴提供的大量能量是癌细胞更快的增殖所必需的。此外,由于癌细胞中脂质代谢的特定改变,癌细胞中脂滴的极性低于正常细胞。因此,结合两个指标(脂滴的数量和极性)能够定位癌变细胞。然而,目前通过监测脂滴的数量和极性变化来定位癌变细胞的研究尚未实现。
传统检测癌细胞的方法有许多的不方便与限制性,荧光探针因其快速方便、灵敏度高、即时检测和响应迅速等众多优势,近年来再快速发展。这些优点使得其在化学、生物医学等学科领域都有着较为广泛的应用,尤其是在生物医学领域,荧光探针不仅可以用于体外分析还可以用于活体的影像学研究。近年来,检测癌细胞的小分子荧光探针被大量报道。然而,其中许多探针水溶性差、灵敏度低、pH的变化对检测的效果影响较大,另有一些探针生物毒性大、细胞膜渗透能力差,这些缺陷很大程度上影响了小分子探针的应用。因此发展稳定灵敏的检测癌细胞的探针具有十分重要的意义。
发明内容
针对目前探针水溶性差、灵敏度低、pH的变化对检测的效果影响较大,探针生物毒性大、细胞膜渗透能力差的问题,本发明提供一种检测脂滴的有机硅荧光探针,响应速度快、抗干扰能力强。
本发明的另一目的是提供一种上述荧光探针在检测细胞内脂滴的应用。
为实现上述目的,本发明采用如下技术方案。
一种检测脂滴的有机硅荧光探针,简称TR,其化学结构式如式(I)所示:
式(I)。
上述有机硅荧光探针的制备方法,包括以下步骤:
(1)氨丙基硅油和4-溴-1,8-萘酐在乙醇中加热回流反应,反应结束后过滤,滤渣以体积比为1:20的甲醇:二氯甲烷为淋洗液过硅胶柱,然后干燥得化合物1:
(2)保护气氛下,化合物1和4-硼酸三苯胺在甲苯中在K2CO3存在下以四(三苯基磷)钯催化加热回流反应,反应结束后以体积比为1:10的甲醇:二氯甲烷为淋洗液过硅胶柱,然后干燥得有机硅荧光探针:
所述氨丙基硅油和4-溴-1,8-萘酐的质量比为5:1。
所述化合物1和4-硼酸三苯胺的质量比为1:0.8-1。
步骤(1)和步骤(2)中,反应温度为80℃。
一种上述有机硅荧光探针在制备检测细胞脂滴数量和极性试剂中的应用。
本发明的机理如下:
本发明的探针TR具有AIE效应,且在脂滴中溶解度差。当细胞中脂滴含量增加时会使细胞极性降低,从而使细胞质中的探针TR含量相对提高,引起荧光强度增大,且发射波长发生蓝移。通过判断荧光的强弱可以区分不同的细胞。
本发明具有以下优点:
本发明提供的荧光探针,通过极性变化分辨癌细胞与正常细胞,该结果及其现象为生物学成像应用奠定了理论基础,预示其在激光激发荧光生物标记领域具有潜在的应用价值。相应的,利用本发明的探针通过荧光成像技术可于评价和研究癌细胞的生理功能,对研究获得癌细胞的生理功能有潜在的应用价值。
附图说明
图1是探针的核磁谱图;
图2是探针在不同极性的溶剂中的荧光光谱。其中激发波长为405 nm;探针的浓度:10 µM;
图3是探针在不同比例的石油醚与四氢呋喃的混合溶液中的荧光光谱。其中激发波长为405 nm;探针的浓度10 µM;
图4是探针孵育24h后对细胞毒性;
图5是探针在小鼠成纤维细胞(3T3)与小鼠乳腺癌细胞(4T-1)的细胞成像测试。探针浓度为10 μM,激发波长为405 nm。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制,化合物编号对应发明内容中反应式。
实施例1荧光探针的合成
(1)先将2 g氨丙基硅油溶于20 mL的乙醇中,再将0.4 g 4-溴-1,8-萘酐溶于40mL的乙醇中,共同加入到100 mL的茄型反应瓶中,升温至80℃,回流反应8h。反应结束后,过滤得到固体,通过柱层析法提纯(甲醇:二氯=1:20),干燥后得到化合物1;
(2)将0.37 g的化合物A溶于20 mL的甲苯中,再将0.346 g的4-硼酸三苯胺溶于20mL的甲苯中,共同加入100 mL的茄型反应瓶中。再向其中加入0.058 g的四(三苯基磷)钯,2mL的K2CO3的水溶液(2 mol/L)。氮气保护,80℃反应搅拌10 h。反应结束后,通过柱层析法提纯(甲醇:二氯=1:10),干燥后得到化合物TR。其1H NMR图谱如图1。
实施例2荧光探针在不同极性的溶剂中的荧光光谱
配制10 mL浓度为1 mM的本发明所述检测极性的高分子荧光探针TR母液作为备用。加入20 μL探针母液于2 mL不同极性的溶剂中(探针浓度为10 μM)。溶剂按照极性从小到大排列依次为:甲苯,二氯,四氢呋喃,1,4-二氧六环,乙腈,DMF,DMSO。激发波长为405nm。结果如图2所示:探针TR的最大发射峰随着溶剂极性的增大,逐渐发生红移。
实施例3荧光探针在不同比例石油醚与四氢呋喃中的荧光光谱
配制10 mL浓度为1 mM的本发明所述检测极性的高分子荧光探针TR母液作为备用。分别配置2 mL不同比例的石油醚与四氢呋喃混合溶剂,石油醚:四氢呋喃=0,10%,20%,30%,40%,50%,60%,70%,80%,90%,95%。将10 μL的母液加入2 mL不同极性溶剂中测试荧光光谱,激发波长为405 nm,结果如图3所示:随着石油醚体积在容积中含量的增加,荧光强度逐渐增大。这是由于探针在石油醚中溶解度很差,增加石油醚在溶剂中的百分比,相当于使探针TR进行聚集,强度增大,说明探针TR具有AIE效应,且发射波长发生蓝移。
实施例4荧光探针对细胞的毒性
将细胞密度为8000个/mL的HeLa细胞接种到96孔板的部分孔内,剩余孔则用无细胞的培养基填充,并在不同的条件下在CO2培养箱中孵育细胞。实验组为用含1-30 μM的TR的培养基孵育24小时后的细胞样品,对照组为不加染料的含细胞样品,空白组为无细胞的培养基样品。待孵育完成后,用新鲜的培养基换掉细胞培养液,并在每个培养孔中加入10μL的MTT,再孵育细胞4小时。孵育完成后,移除培养基,每孔加入200μL的DMSO,并用摇床晃动其10min以溶解甲瓒。使用酶标仪测试每个孔在440nm处的吸光度,细胞存活率可通过下述公式计算得到:
其中,Asample为实验组吸光度,Ac为对照组吸光度,Ab为空白组的吸光度。如图4所示,染色24小时后,TR探针的浓度为10μM以下时,细胞存活率达90%以上,说明探针的毒性很低。
实施例5荧光探针在不同细胞中的成像应用
配制探针TR母液,其浓度为1 mM,将10 μM的探针母液分别加入3T3和4T-1细胞中孵育20 min。待培养结束后,用荧光显微镜在单光子模式下分别拍摄3T3和4T-1细胞荧光照片,结果如图5所示:探针TR在小鼠乳腺癌细胞(4T-1)中进行聚集,发射出绿色的荧光,而在小鼠成纤维细胞(3T3)中,探针TR没有发出荧光。由于4T-1细胞中脂滴的数量高于正常细胞,并且脂滴的极性远低于3T3细胞中的脂滴,所以探针TR在4T-1细胞中聚集因AIE效应荧光强度增大。
Claims (4)
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)和步骤(2)中,反应温度为80℃。
4.一种如权利要求1所述的有机硅荧光探针在制备检测细胞脂滴数量和极性试剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910293090.0A CN110031436B (zh) | 2019-04-12 | 2019-04-12 | 一种检测脂滴的有机硅荧光探针 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910293090.0A CN110031436B (zh) | 2019-04-12 | 2019-04-12 | 一种检测脂滴的有机硅荧光探针 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110031436A CN110031436A (zh) | 2019-07-19 |
CN110031436B true CN110031436B (zh) | 2021-04-20 |
Family
ID=67238110
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910293090.0A Expired - Fee Related CN110031436B (zh) | 2019-04-12 | 2019-04-12 | 一种检测脂滴的有机硅荧光探针 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110031436B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110372738A (zh) * | 2019-07-30 | 2019-10-25 | 济南大学 | 一种定位脂滴检测硫化氢的荧光探针及其制备方法和应用 |
CN110643355A (zh) * | 2019-09-19 | 2020-01-03 | 济南大学 | 一种检测内质网极性的荧光探针及其制备方法和应用 |
CN111393441B (zh) * | 2020-04-28 | 2021-05-04 | 四川大学 | 基于嘌呤骨架的聚集诱导型脂滴靶向染色试剂及其制备方法和应用 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013180181A1 (ja) * | 2012-05-30 | 2013-12-05 | 国立大学法人 東京大学 | 高感度膵液検出用蛍光プローブ、及び膵液検出方法 |
CN108069908B (zh) * | 2016-11-15 | 2021-07-27 | 香港科技大学 | 荧光探针及其应用 |
CN107325814B (zh) * | 2017-06-22 | 2018-11-09 | 东南大学 | 一种荧光硅纳米点及其制备方法与应用 |
CN108822081B (zh) * | 2018-08-09 | 2020-10-09 | 济南大学 | 一种同时检测线粒体和dna的荧光探针 |
CN108822019B (zh) * | 2018-08-21 | 2019-08-13 | 济南大学 | 一种检测脂滴极性的荧光探针及其制备方法和应用 |
CN108997289B (zh) * | 2018-09-25 | 2022-03-22 | 山东大学 | 一种靶向脂滴的次氯酸比率荧光探针及其应用 |
CN109336915A (zh) * | 2018-10-11 | 2019-02-15 | 贺州学院 | 一种半胱氨酸荧光探针及其制备方法和应用 |
-
2019
- 2019-04-12 CN CN201910293090.0A patent/CN110031436B/zh not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN110031436A (zh) | 2019-07-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110031436B (zh) | 一种检测脂滴的有机硅荧光探针 | |
CN109053549B (zh) | 一种定位线粒体检测粘度的双光子荧光探针及其合成方法和应用 | |
CN108484590B (zh) | 一种基于咔唑的双光子粘度荧光探针及其制备方法和用途 | |
Ji et al. | A rhodamine-based “turn-on” fluorescent probe for Fe 3+ in aqueous solution | |
Ma et al. | A water-soluble tetraphenylethene based probe for luminescent carbon dioxide detection and its biological application | |
CN105801479B (zh) | 一种双光子黏度荧光探针及其制备方法和用途 | |
CN110003173B (zh) | 一种基于咔唑的双光子极性荧光探针及其制备方法和用途 | |
CN104710816A (zh) | 一种大斯托克斯位移和近红外荧光发射的新型罗丹明类荧光染料及其合成方法 | |
CN106432348A (zh) | 一种基于铕配合物的可见光可激发的比率荧光温敏探针及其制备方法和应用 | |
CN109722059A (zh) | 基于嘌呤骨架的免洗类聚集诱导型细胞膜靶向染色试剂及其制备方法和用途 | |
CN114163463A (zh) | 一类针对肿瘤过程中过氧化氢实时变化的近红外荧光双光子荧光探针设计及其合成方法 | |
CN106892870B (zh) | 一种溶酶体靶向的双光子黏度荧光探针及其制备方法和用途 | |
CN103773060A (zh) | 有机荧光染料分子及其合成方法和应用 | |
CN102660254B (zh) | 一种用于汞离子检测的含铱配合物磷光材料及其制备方法 | |
CN110357896B (zh) | 一类化合物及制备与其在检测二价铜离子和强酸pH中的应用 | |
CN114276356B (zh) | 一种线粒体靶向的荧光探针及其合成方法和应用 | |
CN113072534B (zh) | 一种rna荧光探针及其制备方法和应用 | |
CN113493465B (zh) | 一种基于苯并噁嗪可视有机分子比率型荧光探针及其制备方法和细胞成像应用 | |
WO2022147872A1 (zh) | 酰胺衍生物中性线粒体荧光标记物及其制备方法和应用 | |
CN111961072B (zh) | 一种溶酶体靶向的红外二窗发射荧光染料及其制备方法和应用 | |
CN103333676A (zh) | 一种硒离子荧光探针及其制备方法和用途 | |
CN106146342B (zh) | 芴基水杨醛联肼类衍生物及其制备方法和应用 | |
Arroyo‐Pieck et al. | Bichromophoric sensors for ratiometric measurements of molecular microenvironments through the interplay of charge transfer and energy transfer channels | |
CN111334068B (zh) | 一种基于SNAP-tag技术的自闪烁超分辨荧光染料及其合成和应用 | |
Potaniec et al. | Controlled Synthesis of Luminescent Xanthene Dyes and Use of Ionic Liquid in Thermochromic Reaction |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210420 |
|
CF01 | Termination of patent right due to non-payment of annual fee |