CN116925025B - 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 - Google Patents
一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 Download PDFInfo
- Publication number
- CN116925025B CN116925025B CN202310997910.0A CN202310997910A CN116925025B CN 116925025 B CN116925025 B CN 116925025B CN 202310997910 A CN202310997910 A CN 202310997910A CN 116925025 B CN116925025 B CN 116925025B
- Authority
- CN
- China
- Prior art keywords
- probe
- nitroreductase
- near infrared
- rapidly detecting
- infrared fluorescent
- 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.)
- Active
Links
- 102000004459 Nitroreductase Human genes 0.000 title claims abstract description 38
- 108020001162 nitroreductase Proteins 0.000 title claims abstract description 38
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000000523 sample Substances 0.000 claims abstract description 38
- 241000252212 Danio rerio Species 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 6
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000003208 petroleum Substances 0.000 claims description 4
- MHSGOABISYIYKP-UHFFFAOYSA-N (4-nitrophenyl)methyl carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(COC(Cl)=O)C=C1 MHSGOABISYIYKP-UHFFFAOYSA-N 0.000 claims description 3
- GPRYKVSEZCQIHD-UHFFFAOYSA-N 1-(4-aminophenyl)ethanone Chemical compound CC(=O)C1=CC=C(N)C=C1 GPRYKVSEZCQIHD-UHFFFAOYSA-N 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 15
- 206010021143 Hypoxia Diseases 0.000 abstract description 11
- 230000007954 hypoxia Effects 0.000 abstract description 8
- 238000003384 imaging method Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 230000008859 change Effects 0.000 abstract description 3
- 238000013399 early diagnosis Methods 0.000 abstract description 3
- 206010027476 Metastases Diseases 0.000 abstract description 2
- 230000009401 metastasis Effects 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000011534 incubation Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000001146 hypoxic effect Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- -1 HSO 3 - Chemical compound 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006184 cosolvent Substances 0.000 description 2
- DOBMPNYZJYQDGZ-UHFFFAOYSA-N dicoumarol Chemical compound C1=CC=CC2=C1OC(=O)C(CC=1C(OC3=CC=CC=C3C=1O)=O)=C2O DOBMPNYZJYQDGZ-UHFFFAOYSA-N 0.000 description 2
- 229960001912 dicoumarol Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108020000284 NAD(P)H dehydrogenase (quinone) Proteins 0.000 description 1
- 102000004960 NAD(P)H dehydrogenase (quinone) Human genes 0.000 description 1
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010066657 azoreductase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- 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
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种快速检测硝基还原酶（Nitroreductase,NTR）的近红外荧光探针及其制备和应用。探针式1具有制备简便、稳定性好、灵敏度高、抗干扰能力强的特点；且能够达到快速检测NTR的目的（在体外光谱测试中与NTR的反应在5分钟内可结束）；同时在斑马鱼乏氧活体成像、Hela细胞成像中，该探针可以检测常氧、乏氧状况下NTR的含量变化。该探针在肿瘤的早期诊断、治疗肿瘤转移以及肿瘤缺氧分析等方面具有良好的应用前景。
Description
技术领域
本发明涉及一种快速检测硝基还原酶的近红外荧光探针及其制备和应用，属于有机小分子荧光探针领域。
背景技术
据统计，癌症已经成为导致人类死亡的重要原因之一，目前癌症的检测手段主要依靠组织切片活检，其检测手段复杂，检测时间长。因此发展用于癌症的早期诊断与识别的方法具有重要意义。乏氧是肿瘤组织的一个特征，一些实体瘤的中位氧（O2）浓度为4%，甚至有些局部组织可能降至0%。正常组织恶化成为肿瘤后，肿瘤细胞快速增殖，血管结构和功能异常，导致氧气和营养物质供应不足，产生缺氧区域，即乏氧。肿瘤乏氧会导致还原酶的高表达，主要包括硝基还原酶、醌还原酶和偶氮还原酶等。因此，通过检测硝基还原酶的含量就可以间接性评价肿瘤区域情况。
荧光探针因其高的特异性和优良生物相容性被广泛应用于硝基还原酶的检测。目前已经报道的检测硝基还原酶的荧光探针，波长大多数在600 nm左右，其波长较短穿透性弱，且对生物伤害性较大；水溶性较差需要用较多的助溶剂助溶，而较多的助溶剂对生物体伤害大，而水溶性好的探针不需要大量化学试剂溶解，可以通过水运输作用进入体内，对生物伤害小；其次，大多数探针与硝基还原酶的反应时间较长，不够灵敏，不能达到快速检测的效果。因此，设计出一种快速检测硝基还原酶的近红外荧光探针有极大的意义。
发明内容
针对现有检测硝基还原酶探针大多水溶性不佳、发射波长较短的问题，本发明目的在于提供一种快速检测硝基还原酶的近红外荧光探针，具有特异性高、灵敏度高、稳定性好、穿透性强，对生物体伤害小的特点。
本发明的另一目的在于提供一种快速检测硝基还原酶的近红外荧光探针的制备方法，合成步骤简单，产率高。
本发明的再一目的在于提供一种快速检测硝基还原酶的近红外荧光探针可于肿瘤的早期诊断、肿瘤转移的治疗以及肿瘤缺氧分析的用途。
一种快速检测硝基还原酶的近红外荧光探针式1，其结构式如下图所示：
一种快速检测硝基还原酶的近红外荧光探针的制备方法，包括以下步骤：
步骤1：将4-二乙氨基酮酸和4-氨基苯乙酮，溶于浓硫酸加热回流，反应结束滴加在冰水混合物中再加入高氯酸静置沉淀析出，抽滤得到所需化合物TM；
步骤2：将TM、氯甲酸对硝基苄酯、N,N-二异丙基乙胺溶于二氯甲烷中，室温下反应，反应结束后柱层析分离得到所需探针式1，洗脱剂为石油醚、乙酸乙酯和甲醇混合溶剂。
本路线包含的合成步骤如下：
一种快速检测硝基还原酶的近红外荧光探针可用于Hela细胞以及斑马鱼中硝基还原酶的检测，具体检测方法如下。
斑马鱼成像：取10 μL式1（1 mM）加入到1 mL含有PBS（pH=7.4）水溶液中，将斑马鱼置于上述溶液中孵育20 min，在荧光倒置显微镜下观察其荧光强度。观察发现鱼逐渐死亡的过程形成乏氧环境，探针的荧光强度逐渐增强。这是由于在斑马鱼形成乏氧过程中硝基还原酶含量会逐渐增多，因此探针的荧光强度也会逐渐增强。
Hela细胞成像：将密度为2×104个/mL的Hela细胞接种到35 mm培养皿中，置于培养箱（温度为37 °C，5% CO2）中培养，待细胞贴壁后，分成三组培养：一组氧气含量21%，一组氧气含量10%，另一组氧气含量1%。培养12小时后，分别向两种培养条件下的细胞加入探针式1（10 μM），继续培养30分钟，将细胞培养液弃掉，PBS洗3次，用共聚焦显微镜分别拍摄三种培养条件下的细胞。
本发明有益效果为：
本发明提供的一种快速检测硝基还原酶的近红外荧光探针，其制备方法简单，具有良好的水溶性，波长可达到近红外波段；灵敏度高，抗干扰能力强且与硝基还原酶反应速度快（可在5分钟内完成检测）；可实现在斑马鱼体内、Hela细胞中乏氧环境中硝基还原酶的检测；在生物活体成像分析、肿瘤检测领域具有广阔的应用前景。
附图说明
图1是探针式1的反应机理图。
图2是探针式1的核磁共振氢谱图。
图3是探针式1的质谱图。
图4是探针式1、探针式1+NADH、探针式1+NADH+硝基还原酶的紫外及荧光光谱图。
图5是探针式1内与硝基还原酶在0-5 min反应的紫外及荧光光谱图。
图6是探针式1的抗干扰能力测试图。探针式1浓度10 μM，金属离子（Na+，K+，Mg2+）、氧化还原物质（抗坏血酸、葡萄糖、HSO3 -、SO3 2-、HS-、H2O2、GSH）氨基酸（Cys、Trp、Asp、Pro）浓度均为1 mM。
图7是不同氧含量条件下（21%、10%、1%）的HeLa细胞与探针式1 (10 μM)共培养30分钟后的共聚焦图像。
图8是探针式1检测斑马鱼体内硝基还原酶含量的成像图。其中第一组为对照组；第二组为加入探针式1（10 μM）；第三组为加入探针式1放置30 min后斑马鱼由常氧变为乏氧；第四组为加入探针式1（10 μM）及硝基还原酶抑制剂50 μM双香豆素（dicoumarol）；其中斑马鱼孵化时间均为30 min。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述。所描述的实施例仅仅是本发明一部分实施例，并非全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做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：合成式1
将4-二乙氨基酮酸（318.3 mg，1 mM）和4-氨基苯乙酮（135.4 mg，1 mM）置于25 mL圆底烧瓶中加入5 mL浓硫酸，90 °C反应2 h，待冷却到室温后滴加在20 g冰水混合物中再加入1 mL70%高氯酸静置沉淀析出，抽滤得所需化合物TM，为紫色固体。
将TM（0.0831 g，0.2 mmol），N, N-二异丙基乙胺（0.0939 g，0.4 mmol）和氯甲酸对硝基苄酯（0.0498 g，0.23 mmol）置于50 mL圆底烧瓶中，量取25 mL无水二氯甲烷作为溶剂，0 °C下搅拌反应20 min，后室温下反应。薄层色谱法实时监测，直至反应完全结束。减压旋蒸得粗产物。柱色谱法分离纯化（洗脱剂为石油醚:乙酸乙酯:甲醇=5:5:1），得紫色固体。
实施例2：式1的光谱测试
探针用二甲基亚砜配置成1 mM的储备液，硝基还原酶用pH=7.4的PBS配置成500 μg/mL的储备液，NADH配置成1 mM的储备液。
为了探究探针式1对硝基还原酶的响应，取10 μM探针式1、500 μM NADH、10 μg/mL硝基还原酶、10 mM磷酸盐缓冲液（pH=7.4）用高纯水稀释至500 μL，测定紫外-可见吸收光谱和荧光发射光谱。
实施例3：式1的选择性测试
10 μM的探针式1分别与1 mM的金属离子（Na+，K+，Mg2+）、氧化还原物质（抗坏血酸、葡萄糖、HSO3-、SO3 2-、HS-、H2O2、GSH）、氨基酸（Cys、Trp、Asp、Pro）37 °C下反应5分钟，然后测定荧光发射光谱。
实施例4：探针式1的细胞成像应用
将密度为2×104 个/mL的Hela细胞接种到35 mm培养皿中，置于培养箱（温度为37°C，5% CO2）中培养，待细胞贴壁后，分成三组培养：氧气含量分别为21%、10%、1%，培养12小时后，分别向三种培养条件下的细胞加入10 μM探针式1，继续培养30分钟，将细胞培养液弃掉，用PBS清洗3次后，用共聚焦显微镜分别拍摄三种培养条件下的细胞，如图6所示。从图中发现在含氧量为10%条件下培养的细胞和在1%条件下的细胞与探针作用后荧光强度明显更强。证明在乏氧状况下，细胞硝基还原酶的含量发生了变化，该探针能够有效检测Hela细中硝基还原酶的变化。
实施例5：探针式1的斑马鱼成像
在浓度为10 μM的探针式1以及PBS溶液中孵化20 min，进行荧光显微成像。鱼在脱离水体观察的30 min过程中，其鱼从常氧状态变为乏氧状态，体内硝基还原酶的含量发生变化，荧光强度逐渐增强（如图7所示探针式1在斑马鱼体内特定部位有很强的荧光）；而在另一组加入硝基还原酶抑制剂50 μM双香豆素（dicoumarol）其体内硝基还原酶被清除后没有强的荧光。说明该探针在生物体内具有较高选择性和灵敏度。
Claims (3)
1.一种快速检测硝基还原酶的近红外荧光探针，其结构特征在于，其结构式如式1所示，
。
2.权利要求1中所述的一种快速检测硝基还原酶的近红外荧光探针的制备方法，其特征在于包含以下步骤：
（1）将4-二乙氨基酮酸和4-氨基苯乙酮，溶于浓硫酸中加热回流，反应结束后滴加在冰水混合物中再加入高氯酸静置沉淀析出，抽滤得到所需化合物TM，
，
（2）将化合物TM、氯甲酸对硝基苄酯、N, N-二异丙基乙胺溶于二氯甲烷中，室温下反应，反应结束后柱层析分离得到所需探针式1，洗脱剂为石油醚、乙酸乙酯和甲醇的混合溶剂，
。
3.权利要求1中所述的一种快速检测硝基还原酶的近红外荧光探针在制备用于检测Hela细胞及斑马鱼中硝基还原酶的试剂中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310997910.0A CN116925025B (zh) | 2023-08-09 | 2023-08-09 | 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310997910.0A CN116925025B (zh) | 2023-08-09 | 2023-08-09 | 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116925025A CN116925025A (zh) | 2023-10-24 |
| CN116925025B true CN116925025B (zh) | 2024-05-14 |
Family
ID=88392435
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310997910.0A Active CN116925025B (zh) | 2023-08-09 | 2023-08-09 | 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116925025B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749153A (zh) * | 2016-12-19 | 2017-05-31 | 华东理工大学 | 硝基还原酶的特异性荧光探针及其制备和用于肿瘤靶向荧光成像和监测肿瘤缺氧程度的应用 |
| CN111303102A (zh) * | 2019-11-29 | 2020-06-19 | 福建医科大学孟超肝胆医院(福州市传染病医院) | 一种硝基还原酶响应的乏氧探针化合物及其制备与应用 |
| CN114149448A (zh) * | 2021-11-12 | 2022-03-08 | 山东第一医科大学(山东省医学科学院) | 一种用于检测硝基还原酶的近红外荧光探针及应用 |
-
2023
- 2023-08-09 CN CN202310997910.0A patent/CN116925025B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749153A (zh) * | 2016-12-19 | 2017-05-31 | 华东理工大学 | 硝基还原酶的特异性荧光探针及其制备和用于肿瘤靶向荧光成像和监测肿瘤缺氧程度的应用 |
| CN111303102A (zh) * | 2019-11-29 | 2020-06-19 | 福建医科大学孟超肝胆医院(福州市传染病医院) | 一种硝基还原酶响应的乏氧探针化合物及其制备与应用 |
| CN114149448A (zh) * | 2021-11-12 | 2022-03-08 | 山东第一医科大学(山东省医学科学院) | 一种用于检测硝基还原酶的近红外荧光探针及应用 |
Non-Patent Citations (1)
| Title |
|---|
| Designing a Brightness-Restored Rhodamine Derivative bythe Ortho-CompensationEffect for AssessingDrug-InducedAcute KidneyInjury;Haowei Guo et al.;Analytical Chemistry;第95卷;6863−6870 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116925025A (zh) | 2023-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111303102B (zh) | 一种硝基还原酶响应的乏氧探针化合物及其制备与应用 | |
| CN106946902B (zh) | 一种二氧化硫近红外-双光子比率荧光探针及其制备方法 | |
| CN112745303B (zh) | 一种乏氧荧光探针及其应用 | |
| CN110982513B (zh) | 一种荧光碳点的制备方法及其在细胞成像中的应用 | |
| CN113999219B (zh) | 一种双位点荧光探针及其合成方法和应用 | |
| CN112961177B (zh) | 一种检测还原型烟酰胺腺嘌呤二核苷酸nadh的近红外荧光探针及其制备方法和应用 | |
| CN107056618B (zh) | 一种检测硝基还原酶的荧光探针 | |
| CN113234014B (zh) | 一种检测氨肽酶n的聚集诱导发射荧光探针及其制备 | |
| CN113461609B (zh) | 一种硫酸酯酶响应的aie纳米探针及其制备方法与应用 | |
| CN112500386A (zh) | 基于吡啰红肟的近红外HClO荧光探针、制备及其应用 | |
| CN116925025B (zh) | 一种快速检测硝基还原酶的近红外荧光探针及其制备和应用 | |
| CN113004258B (zh) | 一种基于esipt效应的硫化氢比率型荧光分子探针的制备方法及其应用 | |
| CN111518546A (zh) | 一种乏氧微环境响应的荧光探针及其制备方法与应用 | |
| CN111635385B (zh) | 一种线粒体靶向的双光子激发近红外发射的硫化氢荧光探针及其制备方法和应用 | |
| CN114835658B (zh) | 一种用于检测硫化氢的荧光探针及其制备方法和应用 | |
| CN113773265B (zh) | 一种用于检测cyp450的荧光探针及其制法和应用 | |
| CN112457360B (zh) | 一种肝靶向的过氧亚硝酸根荧光探针及制备方法和应用 | |
| CN110229203B (zh) | 一种氨基己糖酶荧光探针及其制备方法和应用 | |
| CN115043893B (zh) | 一种肝细胞靶向的次氯酸近红外荧光探针及其制备方法和应用 | |
| CN112694469A (zh) | 基于吡啰红肼的HOCl荧光探针、制备方法及应用 | |
| CN117603203B (zh) | 一种比率荧光探针及其制备方法和应用 | |
| CN114539268B (zh) | 基于Cardipy染料的溶酶体靶向荧光探针及其制备方法和应用 | |
| CN113135904B (zh) | 羟自由基近红外荧光分子探针及其制备方法与应用 | |
| Nakahara et al. | Fluorophotometric determination of hydrogen peroxide and other reactive oxygen species with fluorescein hydrazide (FH) and its crystal structure | |
| CN110156630B (zh) | 一种双光子超低背景荧光探针及其制备方法和应用 |
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 |