阅读说明：本技术 一种聚联乙炔脂质体在水环境中Pb2+的可视化检测方法 (A kind of poly- diacetylene liposome Pb in water environment2+Visible detection method ) 是由 陈淑伟 陈曦鹏 李杨 杨宁 姬广昊 陈文帅 胡飞虎 于 2019-08-20 设计创作，主要内容包括：本发明公开了一种聚联乙炔脂质体在水环境中Pb~(2+)的可视化检测方法,包括以下步骤：首先准备好实验所需的化学试剂原料和干燥的玻璃仪器,接着依次进行PCDA-NS的合成、PCDA-EDEA的合成和PCDA-TAA的合成,并且将合成得到的化合物放在相应的样品瓶中,然后对聚联乙炔脂质体进行制备,制备好后通过透射电子显微镜对不同比例制备的PDA脂质体的形态进行观察,而且在加入Pb~(2+)前后都进行拍照,通过其形态和样貌的特征以及颜色变化得出最优的PDA脂质体传感器。该聚联乙炔脂质体在水环境中Pb~(2+)的可视化检测方法,无需大型仪器设备,也不需繁琐的样品预处理,且分析周期短,可以实现快速实时分析,为工作人员的操作带来便利,也增加了该检测实验的实用性。(The invention discloses a kind of poly- diacetylene liposome Pb in water environment 2+ Visible detection method, the following steps are included: being ready to test required chemical reagent raw material and dry glass apparatus first, then synthesis, the synthesis of PCDA-EDEA and the synthesis of PCDA-TAA of PCDA-NS are successively carried out, and the compound that synthesis obtains is placed in corresponding sample bottle, then it prepared by poly- diacetylene liposome, it is observed after preparing by form of the transmission electron microscope to PDA liposome prepared by different proportion, and Pb is being added 2+ Front and back is all taken pictures, and optimal PDA lipid body sensor is obtained by the feature and color change of its form and complexion.Poly- diacetylene liposome Pb in water environment 2+ Visible detection method, be not necessarily to large-scale instrument and equipment, be also not required to cumbersome sample pretreatment, and analytical cycle is short, analysis in real time may be implemented quickly, offer convenience for the operation of staff, also increase the practicability of the test experience.)

1. a kind of poly- diacetylene liposome Pb in water environment²⁺Visible detection method, comprising:

Step 1: getting out the round-bottomed flask and other glass apparatus of chemical reagent raw material, multiple dryings

Round-bottomed flask is fixed on corresponding position, and to flask carry out sequence label, such as 1,2,3,4,5,6,7,8；

Step 2: accurately weighing the raw materials such as NHS, EDCHCl

Weighed raw material is stored in No. 1 round-bottomed flask；

Step 3: preparing anhydrous DCM solvent

Ready anhydrous DCM solvent is added in No. 1 round-bottomed flask, is dissolved NHS and EDCHCl with anhydrous DCM；

Step 4: addition 10,12- pentacosane diacetylenic acid

10,12- pentacosane diacetylenic acid is added in No. 1 round-bottomed flask, mixes it with dissolved substance, and It is stirred at room temperature；

Step 5: vacuum concentration

Stirring mixture is concentrated in vacuo with Rotary Evaporators, and the mixture after concentration is moved into separatory funnel and is carried out Extraction；

Step 6: washing is dried

The organic layer obtained after extraction is merged, is washed with saturated salt solution, is dried after washed, concentration, Obtain PCDA-NS white solid；

Step 7: preparing anhydrous DCM solvent and appropriate PCDA-NS

It accurately weighs 229mg PCDA-NS to be dissolved in the anhydrous DCM solvent of 10mL, and is put into spare in No. 2 flasks；

Step 8: mixed processing

The flask that the material by injection device injection of dissolution is added in the anhydrous DCM of 8mL of 710mg EDEA, and is reacted is No. 3, so Afterwards after completion of dropwise addition, stir 3 hours at room temperature；

Step 9: vacuum concentration, washing, dry and purification process

It is concentrated in vacuo using Rotary Evaporators, and the mixture after concentration is moved in separatory funnel and is extracted, closed And organic phase, NaCl solution is then added and washs 3 times, anhydrous MgSO is added after washed₄It is dried, by crude product after concentration With column chromatography separating purification, white solid PCDA-EDEA is obtained；

The synthesis of step 10:PCDA-TAA

Weighed 30mg NHS and 54mg EDCHCl are added in No. 4 flasks, DMF solvent is then added and is mixed, warp Vacuum concentration, extraction, drying are crossed, obtains white solid PCDA-TAA, and be placed in sample bottle.

Step 11: the preparation of poly- diacetylene liposome

PCDA-TAA the and PCDA mixed liquor of different proportion is dissolved in the chloroform solvent of 1mL, and is reacted in sample bottle, Navy blue poly- diacetylene liposome solutions are obtained after the reaction such as heating, ultrasound, cooling.

Step 12: the observation of poly- diacetylene liposome

Observed using form of the transmission electron microscope to the poly- diacetylene liposome of preparation, and to its color change into Row is taken pictures.

2. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: NHS the and EDCHCl weight in the step 2 is respectively 76mg and 137.5mg.

3. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: the anhydrous DCM volume in the step 3 is 20mL.

4. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: the quality of 10,12- pentacosane diacetylenic acid is 215mg in the step 4, and it is small for 2 that the time is stirred at room temperature When.

5. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: the extraction mode in the step 5 are as follows: 20mLEA is added in separatory funnel and extracts 3-4 times, merges organic phase.

6. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: washing, drying mode in the step 6 are as follows: organic layer is with saturated common salt water washing 3 times, then with anhydrous MgSO₄ It is dried, removes solvent, it is final to obtain 229mg PCDA-NS white solid.

7. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: vacuum concentration, washing, drying and purification process mode in the step 9 are as follows: mixture is put into Rotary Evaporators In be concentrated in vacuo, and extracted 3 times in separatory funnel with 20mL DCM, merge organic phase, then washed with saturation NaCl solution It washs 3 times, then with anhydrous MgSO₄It is dried, by crude product column chromatography separating purification, eluant, eluent DCM:MeOH after concentration (15:1, v/v), it is final to obtain 192mg white solid, yield 78.4%.

8. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: the synthesis mode of PCDA-TAA in the step 10 are as follows: weighs 30mg NHS and 54mg EDCHCl and is added to No. 4 In flask, 20mL DMF solution is then added, then weigh 41mg thymidine -1- acetic acid and be added in above-mentioned solution, then exists It stirs at room temperature 3 hours, then screws out solvent and be placed in separatory funnel, residue 20mL EA is extracted 3-4 times, is associated with Machine phase, then washed with saturation NaCl solution, wherein organic layer Na₂SO₄It dries, filters, removes solvent and obtain residue, later 25mL DMF solution, stirring are added into above-mentioned residue, then accurately weighs 113.4mg PCDA-EDEA and is added to above-mentioned solution In, it is stirred overnight at room temperature, to after reaction, removes solvent, extracted 3 times with 30mL DCM, merge organic phase, and with satisfying It is washed 3 times with NaCl solution, anhydrous Na₂SO₄It is dry, be concentrated to get crude product, crude product through column Chromatographic purification, eluant, eluent (DCM: MeOH, 10:1, v/v) purifying, obtain 113mg white solid PCDA-TAA, yield 73%.

9. a kind of poly- diacetylene liposome Pb in water environment according to claim 1²⁺Visible detection method, it is special Sign is: the preparation method of poly- diacetylene liposome in the step 11 are as follows: weigh different mol ratio example (0:10,1:9,2:8, 3:7,4:6) PCDA-TAA and PCDA mixed liquor is dissolved in the chloroform solvent of 1mL, marks respectively, then, removed with nitrogen stream Organic solvent is removed, suitable ultra-pure water is added, obtained solution ultrasound 20 minutes under 80 DEG C of bath temperatures, it is clear to obtain Or translucent solution, concentration are the liposome of 1mM, are formed by monomer dispersion liquid and are placed in 4 DEG C of refrigerator that stand 6 small When, placement is then taken out to room temperature, is carried out photoinduction under the ultraviolet light irradiation of 254nm later and is polymerize 15 minutes, obtains navy blue Poly- diacetylene liposome solutions.

Technical field

The present invention relates to poly- diacetylene liposome-based detection technical field, specially a kind of poly- diacetylene liposome is in water environment Middle Pb²⁺Visible detection method.

Background technique

Poly- diacetylene (PDA) is a kind of conjugated polymer with special optical performance, is widely used in sensor Field, the monomer of PDA is easy to be dispersed and be can be carried out to be self-assembled into liposome structure in aqueous solution, in 254nm ultraviolet light Under irradiation, the inside of liposome can accelerate polymerization to form polymer, to obtain from the colorless and transparent PDA polymerization for becoming blue Object, after adding outside stimulus in system, PDA can be undergone by blue to red ratio colour transition and from non-fluorescence to fluorescence Variation therefore ultraviolet-visible absorption spectroscopy and fluorescence spectrum not only can be used and easily detect its optical change, can also be real Now naked eyes identify.

These characteristics make PDA become design and construct the ideal material of various sensors, in recent years, it has been reported that many The biosensor and chemical sensor of optic response based on poly- diacetylene, these sensors can realize the identification of analyte And analysis, including temperature, metal ion, anion, surfactant, amine, water, gas, sugar, hydrocarbon, virus, liver Element, enzyme, bacterium and cancer need to be identified and be detected as the scientific and technological development maked rapid progress is constantly progressive with social Object gradually becomes many kinds of and distinct, thus, for expand the identification object of poly- diacetylene and improve its detection and point Performance is analysed, needs further to develop the novel excellent sensor based on poly- diacetylene, constructs high sensitivity, discrimination is more preferable Poly- diacetylene identify system.

And traditional detection Pb²⁺Main method include atomic absorption spectrography (AAS), inductively coupled plasma body atomic emissions Spectroscopic methodology and inductively coupled plasma mass spectrometry etc., such method can sensitively detect Pb²⁺, but need large-scale instrument Equipment, complicated sample pretreatment, interminable analytical cycle etc. all limit Pb²⁺Quick real-time analysis detection, although being used for Detect Pb²⁺Small-molecule fluorescent probe design principle it is simple, detection sensitivity is high, but its synthesis process usually needs multistep anti- It answers, it is often difficult with function dough, it is difficult to device, meanwhile, water-soluble poor, the fluorescence parent nucleus of most small-molecule fluorescent probes Luminosity be highly prone to the influence of water environment, these all seriously constrain small-molecule fluorescent probe answering in water environment With so we have proposed a kind of poly- diacetylene liposome Pb in water environment²⁺Visible detection method, on solving The problem of stating middle proposition.

Summary of the invention

The purpose of the present invention is to provide a kind of poly- diacetylene liposome Pb in water environment²⁺Visible detection method, Low, the not high phenomenon of discrimination that there are sensitivity to solve to propose existing poly- diacetylene liposome in above-mentioned background technique, and And to poly- diacetylene liposome Pb in water environment²⁺Visual retrieval be easy by water environmental impact, cause seriously to constrain The problem of application of the small-molecule fluorescent probe in water environment.

To achieve the above object, the invention provides the following technical scheme: a kind of poly- diacetylene liposome Pb in water environment²⁺ Visible detection method, including

Step 1: preparing the round-bottomed flask of multiple dryings

Multiple flasks are fixed on corresponding position, and to flask carry out sequence label, such as 1,2,3,4,5,6,7,8；

Step 2: accurately weighing the raw materials such as NHS, EDCHCl

Weighed raw material is stored in No. 1 round-bottomed flask；

Step 3: preparing anhydrous DCM solvent

Ready anhydrous DCM solvent is added in No. 1 round-bottomed flask, is dissolved NHS and EDCHCl with anhydrous DCM；

Step 4: addition 10,12- pentacosane diacetylenic acid

10,12- pentacosane diacetylenic acid is added in No. 1 round-bottomed flask, mixes it with dissolved substance, And it is stirred at room temperature；

Step 5: vacuum concentration

Stirring mixture is concentrated in vacuo with Rotary Evaporators, and the mixture after concentration is moved into separatory funnel It is extracted；

Step 6: washing is dried

The organic layer obtained after extraction is merged, is washed with saturated salt solution, is dried after washed, at concentration Reason, obtains PCDA-NS white solid；

Step 7: preparing anhydrous DCM solvent and appropriate PCDA-NS

It accurately weighs 229mg PCDA-NS to be dissolved in the anhydrous DCM solvent of 10mL, and is put into spare in No. 2 flasks；

Step 8: mixed processing

The flask that the material by injection device injection of dissolution is added in the anhydrous DCM of 8mL of 710mg EDEA, and is reacted is 3 Number, then after completion of dropwise addition, stir 3 hours at room temperature；

Step 9: vacuum concentration, washing, dry and purification process

It is concentrated in vacuo using Rotary Evaporators, and the mixture after concentration is moved in separatory funnel and is extracted It takes, merges organic phase, NaCl solution is then added and washs 3 times, anhydrous MgSO is added after washed₄It is dried, it will after concentration Crude product column chromatography separating purification obtains white solid PCDA-EDEA；

The synthesis of step 10:PCDA-TAA

Weighed 30mg NHS and 54mg EDCHCl are added in No. 4 flasks, DMF solvent mixing is then added and stirs It mixes, by vacuum concentration, extraction, drying, obtains white solid PCDA-TAA, and be placed in sample bottle.

Step 11: the preparation of poly- diacetylene liposome

PCDA-TAA the and PCDA mixed liquor of different proportion is dissolved in the chloroform solvent of 1mL, and is carried out in sample bottle Reaction obtains navy blue poly- diacetylene liposome solutions after the reaction such as heating, ultrasound, cooling.

Step 12: the observation of poly- diacetylene liposome

It is observed using form of the transmission electron microscope to the poly- diacetylene liposome of preparation, and its color is become Change is taken pictures.Preferably, NHS the and EDCHCl mass in the step 2 is respectively 76mg and 137.5mg.

Preferably, the anhydrous DCM volume in the step 3 is 20mL.

Preferably, the quality of 10,12- pentacosane diacetylenic acid is 215mg in the step 4, and when being stirred at room temperature Between be 2 hours.

Preferably, the extraction mode in the step 5 are as follows: 20mL EA is added in separatory funnel and extracts 3-4 times, merges Organic phase.

Preferably, the washing in the step 6, drying mode are as follows: then organic layer is used with saturated common salt water washing 3 times Anhydrous MgSO₄It is dried, removes solvent, it is final to obtain 229mg PCDA-NHS white solid.

Preferably, the vacuum concentration in the step 9, washing, drying and purification process mode are as follows: rotate mixture Evaporimeter is concentrated in vacuo, and is extracted 3 times in separatory funnel with 20mL DCM, and organic phase is merged, then molten with saturation NaCl Liquid washs 3 times, then with anhydrous MgSO₄It is dried, then by crude product column chromatography separating purification, eluant, eluent DCM: MeOH (15:1, v/v), it is final to obtain 192mg white solid, yield 78.4%.

Preferably, in the step 10 PCDA-TAA synthesis mode are as follows: weigh 30mg NHS and 54mg EDCHCl and add Enter into No. 4 flasks, 20mL DMF solvent be then added, then weigh 41mg thymidine -1- acetic acid and be added in above-mentioned solution, Then it is stirred at room temperature 3 hours, then screws out solvent, residue 20mL EA is extracted 3-4 times, merges organic phase, then use It is saturated NaCl solution washing, wherein organic layer Na₂SO₄It dries, filters, removes solvent and obtain residue, later to above-mentioned residual 25mL DMF solvent, stirring are added in object, then accurately weighs 113.4mg PCDA-EDEA and is added in above-mentioned solution, at room temperature It is stirred overnight, to after reaction, remove solvent, is extracted 3 times with 30mL DCM, merge organic phase, and molten with saturation NaCl Liquid washs 3 times, anhydrous Na₂SO₄It is dry, it is concentrated to get crude product, crude product is through column Chromatographic purification, eluant, eluent (DCM:MeOH, 10:1, v/ V) it purifies, obtains 113mg white solid PCDA-TAA, yield 73%.

Preferably, in the step 11 poly- diacetylene liposome preparation method are as follows: weigh different mol ratio example (0:10, 1:9,2:8,3:7,4:6) PCDA-TAA and PCDA mixed liquor is dissolved in the chloroformic solution of 1mL, marks respectively, then, uses Nitrogen stream removes organic solvent, adds suitable ultra-pure water, obtained solution ultrasound 20 minutes under 80 DEG C of bath temperatures, Obtain clear or translucent solution, concentration is the liposome of 1mM, is formed by monomer dispersion liquid and is placed in 4 DEG C of refrigerator 6 hours are stood, placement is then taken out to room temperature, carries out photoinduction under the ultraviolet light irradiation of 254nm later and polymerize 15 minutes, obtain Navy blue poly- diacetylene liposome solutions.

Compared with prior art, the beneficial effects of the present invention are: poly- diacetylene liposome Pb in water environment²⁺It is visual Change detection method；

(1) due to Pb²⁺Generally it is prevalent in aqueous phase system, and general organic matter sensor can not achieve in pure water Detection in phase, but poly- diacetylene liposome hydrophily is preferable, can realize and detect to analyte in pure water phase, thus sharp The poly- of thymidine -1- acetic acid modification is prepared for by the design and synthesis to the poly- diacetylene monomer of modification with this advantage Acetylene liposome, and explored, optimized and improved by the condition to detection architecture, utilize the poly- of this novel functionalization Diacetylene sensor realizes in water phase to Pb²⁺Colorimetric and fluorescent dual detection and analysis bring for the practice of later period people It is convenient；

(2) design has synthesized diacetylene the monomer PCDA-TAA, Pb of thymidine -1- molecular acid modification²⁺With oxygen atom With very strong coordination, therefore, thymidine -1- aceticoceptor is introduced in the water-wet side of connection polyacetylene monomer, it can With Pb²⁺Form T-Pb²⁺T-type complex, to reach to Pb²⁺Recognition detection, pass through PCDA-TAA and 10,12- pentadiene two Sour (PCDA) first disperses to be self-assembly of a kind of new PDA liposome chemical sensor again in water phase, is used for Pb²⁺Colorimetric and Fluorimetric analysis, after different metal ions is added in liposome solutions, only Pb²⁺It can cause from blue to the bright of red Aobvious color change, and can be along with the enhancing of fluorescence, and then construct one kind and identify Pb in water phase²⁺It is sensitiveer and single-minded Sensor, for the identification high sensitivity of poly- diacetylene system, discrimination is more preferable；

(3) compared to traditional detection Pb²⁺Method, be not necessarily to large-scale instrument and equipment, do not need cumbersome specimen preprocessing yet Reason, and analytical cycle is short, and analysis in real time may be implemented quickly, thus make Pb²⁺Detection it is more convenient quickly, not be only work The operation of personnel offers convenience, and also increases the practicability of the test experience.

Specific embodiment

The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.