阅读说明：本技术 一种对膜电荷响应的比率荧光探针及其在细菌检测的应用 (The ratio fluorescent probe that a kind of pair of membrane charge responds and its application in Bacteria Detection ) 是由 徐兆超 苗露 龙双双 于 2018-05-22 设计创作，主要内容包括：本发明提供一种对膜电荷响应的比率荧光探针及其在细菌检测的应用,其结构为二联咪唑盐两端连接有两个芘荧光团,能够发射芘单体或芘激基复合物荧光,分别在375nm和482nm,其结构如(1)所示。该化合物带有2个正电荷,在水溶液中由于疏水作用形成纳米聚集体而导致荧光淬灭。细菌表面带有大量的电荷,与探针纳米聚集体作用将其解聚引起荧光大幅度增强。由于细菌表面电荷分布不同,导致探针形成不同程度的单体和二聚体,从而产生芘单体和激基复合物荧光的比率变化(I<Sub>482</Sub>/I<Sub>375</Sub>)。以探针聚合物与细菌结合后产生的荧光增强,以及单体与激基复合物荧光比率变化作为荧光信号,能够识别革兰氏阴性菌或阳性菌,并且能够区分不同种类的细菌。<Image he="309" wi="700" file="DDA0001668136050000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention provides the ratio fluorescent probe of a kind of pair of membrane charge response and its application in Bacteria Detection, its structure is that there are two pyrene fluorogens for the connection of bigeminy imidazole salts both ends, pyrene monomer or pyrene exciplex fluorescence can be emitted, respectively in 375nm and 482nm, structure is such as shown in (1).The compound has 2 positive charges, leads to fluorescent quenching since hydrophobic effect forms Micelle-like Nano-structure of Two in aqueous solution.Bacterium surface has a large amount of charge, acts on probe nano aggregation and causes fluorescence to be greatly enhanced its depolymerization.Since bacterium surface distribution of charges is different, probe is caused to form different degrees of monomer and dimer, to generate the rate of change (I of pyrene monomer and exciplex fluorescence 482 /I 375 ).Using probe polymer in conjunction with bacterium after the fluorescence enhancement that generates and monomer and exciplex ratio fluorescent change as fluorescence signal, can identify Gram-negative bacteria or positive bacteria, and different types of bacterium can be distinguished.)

1. the ratio fluorescent probe of a kind of pair of membrane charge response, it is characterised in that the probe molecule structural formula is as follows:

2. according to claim 1 to the synthetic method of the ratio fluorescent probe of membrane charge response, it is characterised in that specific step It is rapid as follows:

(1) under nitrogen protection, pyrene and bromoacetyl bromide are dissolved in methylene chloride, maintain the temperature at -5-0 DEG C, into reaction solution It is slowly added to aluminum chloride powder, three's the mass ratio of the material is pyrene: bromoacetyl bromide: aluminium chloride=1:0.5-3:0.5-3, stirs 1-3 Dark solution is obtained after hour；After temperature is raised to room temperature and stirs 6-10 hours, extra aluminium chloride is quenched with ice water, then Isolated crude product is extracted with dichloromethane, passes through the isolated 2- bromo -1- pyrenyl ethyl ketone of silica gel column chromatography；

(2) under nitrogen protection, Lithium Aluminium Hydride and aluminium chloride are dissolved in ether, will be then dissolved in the 2- bromo -1- of methylene chloride Pyrenyl ethyl ketone is slowly added in reaction solution, obtains white precipitate after being stirred at room temperature 0.5-3 hours；It is extra to be quenched with ice water Aluminium chloride and Lithium Aluminium Hydride adjust reaction solution to acidity with hydrochloric acid, isolated crude product are then extracted with dichloromethane, passes through The isolated 1- of silica gel column chromatography (2- bromoethyl)-pyrene；

(3) under nitrogen protection, two bisglyoxalines and 1- (2- bromoethyl)-pyrene are mixed in second by the mass ratio of the material example 1:0.5-3 In nitrile, it is warming up to 75-90 DEG C of reflux, generates precipitating after stirring 12-48 hours, precipitating is washed with ether；Then precipitating is dissolved in In methanol, slowly Potassium Hexafluorophosphate is instilled in solution and obtains white precipitate product, i.e., the ratio fluorescent of membrane charge response visited Needle di-PYIM.

3. the synthetic method of the ratio fluorescent probe according to claim 2 to membrane charge response, it is characterised in that: press object The amount of matter is than Lithium Aluminium Hydride: aluminium chloride: 2- bromo -1- pyrenyl ethyl ketone=1:0.5-4:0.1-1.

4. the application of the ratio fluorescent probe according to claim 1 to membrane charge response, it is characterised in that the fluorescence is visited For detecting different type surfactant, specific detection method is needle: surveying after surfactant is mixed with fluorescence probe Fluorescence spectrum；Change (I with fluorescence enhancement and ratio fluorescent₄₈₂/I₃₇₅) it is used as probe response signal, it is different types of for distinguishing Surfactant.

5. the application of the ratio fluorescent probe according to claim 1 to membrane charge response, it is characterised in that the fluorescence is visited Needle is for distinguishing different types of bacterium；The specific detection method is as follows:

(1) Bacteria Culture；

(2) bacterium is collected: being centrifuged 5-10 minutes under the conditions of 10000-14000rpm, supernatant is abandoned, with 20mM, pH=7.4 HEPES buffer solution is cleaned bacterium 2-3 times, and bacterium finally is resuspended with HEPES；

(3) it is detected after probe being added in bacterium with Fluorescence Spectrometer.

6. the application of the ratio fluorescent probe according to claim 5 to membrane charge response, it is characterised in that: step (1) Middle Bacteria Culture is to OD₆₀₀=0.1-2.0.

7. the application of the ratio fluorescent probe according to claim 5 to membrane charge response, it is characterised in that: step (2) It is middle to be resuspended with HEPES to the OD of bacterium₆₀₀=0.05-2.0, final concentration of 5-20 μM of probe.

8. the application of the ratio fluorescent probe according to claim 5 to membrane charge response, it is characterised in that: step (3) In, fluorescence is detected under 345nm excitation wavelength and obtains fluorescence spectra, and (I is changed with fluorescence enhancement and ratio fluorescent₄₈₂/I₃₇₅) As probe response signal, for distinguishing variety classes bacterium.

9. the application for the ratio fluorescent probe that membrane charge is responded according to claim any in claim 5~8, It is characterized in that bacterium is gram-positive bacteria or Gram-negative bacteria to the fluorescence probe for identification.

Technical field

The invention belongs to bioanalysis detection fields, and in particular to the ratio fluorescent probe of a kind of pair of membrane charge response and its In the application of Bacteria Detection.

Background technique

Bacterium, either pathogenic bacteria or non-pathogenic bacteria, are all prevalent in living nature.The life for being grown on us of bacterium Work is closely bound up, probably has the different types of bacterial number of 500-1000 kind to account for all work of human body in everyone body The 90% of cell, the repeats itself of these bacterium human bodies exercise respective function.But it is meanwhile food-borne caused by germ contamination Disease is also most distinct issues in food safety.The etesian food posioning event in China accounts for food poisoning The 30%-90% of sum, number account for the 60%-90% of food poisoning total number of persons.Therefore, the identification of bacterium is examined with detection in medical treatment Many fields such as disconnected, biology and food safety are all extremely important.In addition, Bacteria Identification is in the body outburst that finds the cause of disease, monitoring sense It also plays an important role in terms of the appearance of dye trend and identification new threat.Currently used Bacteria Detection method includes: polymerase chain The biological methods such as formula reaction, the sequencing of unicellular and genomic DNA and targeting specific immunoassays.But these traditional inspections There are the disadvantages such as time-consuming, cumbersome, and expensive or poor repeatability, sensitivity are low in survey method.Therefore, it is used for bacterium The exploitation of the new method of detection still suffers from huge challenge.

Fluorescence detection because have many advantages, such as it is quick, simple, sensitive, can real-time monitoring due to by development and application in bacterium Detection.The Fluorometric assay bacterium registered is divided into specific detection and non-specific two kinds of detection: specific recognition is exactly pair The single-minded fluorescence detection of the peculiar target molecules of bacterium, such as the specific protease of bacterial secretory, the mannose receptor of bacterial flagellum Or N-acetyl-glucosamine of bacterium surface etc., however, this probe can only a certain bacterium of single detection, do not have pervasive Property.The carried charge difference because of caused by the difference of the surface texture of different bacterium and composition is utilized in non-specificity detection, passes through The fluorescence signal that the electrostatic interaction of probe and bacterium generates carrys out discriminating bacteria.In view of non-specific approach generality and Simplicity is of great significance to the selectivity identification of different bacterium.

In recent years, the fluorescence probe based on electrostatic interaction identification bacterium has been reported, effectively such as Bunz study group Devise it is a kind of based on polymer/peptide complexes sensor array, for quickly identifying 14 kinds of bacteriums in mankind's urine；One The supermolecule nano component of kind layer assembly is exploited for response bacterium and eliminates and effective Bacteria Detection；In addition, using pair Different bacterium generates multiple aggregation-induced emission probes of different response signals, can identify eight kinds of bacteriums by Array Method.So And the fluorescence probe reported at present is all based on array approach detection bacterium, use can while needing multiple compounds Realize differentiation to different genera fine difference, this method is there are cumbersome, poor repeatability, the small problem of the scope of application, And it is merely able to distinguish variety classes bacterium, the ability without identification bacterium such as identifies Gram-negative or the positive Bacterium.Therefore, have and identify and distinguish between the single fluorescence probe of variety classes bacterium ability, and have Bacteria Detection it is simple, quickly and The novel fluorescence probe of sensitivity characteristics still has to be developed.

Ratio fluorescent probe usually contains two launch wavelengths, and when detection can be qualitative by the rate of change of launch wavelength Or quantitative detection target molecule.Pyrene is a traditional fluorogen, the monomer fluorescence of transmitting 400nm or so；And two pyrenes Fluorogen exciplex easy to form and the long wavelength's fluorescence for emitting 480nm or so.Pass through the distance between control pyrene molecule, energy The change in fluorescence of pyrene monomer and exciplex is enough generated, therefore pyrene is usually used in designed ratios fluorescence probe.The present invention is exactly benefit Different types of bacterium is detected by rate of change with this property of pyrene.

Summary of the invention

The object of the present invention is to provide the ratio fluorescent probe of a kind of pair of membrane charge response and its in the application of Bacteria Detection. This probe is used as signal detection bacterium with different charge by fluorescence enhancement and ratio fluorescent variation, can identify gram Negative and positive bacteria, and variety classes bacterium can be distinguished, have preparation method simple, detects sensitive quick feature, performance Better than existing Bacteria Detection probe.

The ratio fluorescent probe of a kind of pair of membrane charge response of the present invention, the probe are the connection of bigeminy imidazole salts both ends There are two pyrene fluorogens, can emit pyrene monomer or pyrene exciplex fluorescence, respectively in 375nm and 482nm；The compound band There are 2 positive charges, leads to fluorescent quenching since hydrophobic effect forms Micelle-like Nano-structure of Two in aqueous solution, the following institute of structural formula Show:

A kind of synthetic method of the ratio fluorescent probe of pair of membrane charge response, the specific steps are as follows:

(1) under nitrogen protection, pyrene and bromoacetyl bromide are dissolved in methylene chloride, maintain the temperature at -5-0 DEG C, Xiang Fanying Aluminum chloride powder is slowly added in liquid, three's the mass ratio of the material is pyrene: bromoacetyl bromide: aluminium chloride=1:0.5-3:0.5-3, stirring Dark solution is obtained after 1-3 hours；After temperature is raised to room temperature and stirs 6-10 hours, extra aluminium chloride is quenched with ice water, Isolated crude product is then extracted with dichloromethane, passes through the isolated 2- bromo -1- pyrenyl ethyl ketone of silica gel column chromatography；

(2) under nitrogen protection, Lithium Aluminium Hydride and aluminium chloride are dissolved in ether, will be then dissolved in the 2- bromine of methylene chloride Generation -1- pyrenyl ethyl ketone is slowly added in reaction solution, obtains white precipitate after being stirred at room temperature 0.5-3 hours；By the amount of substance Than Lithium Aluminium Hydride: aluminium chloride: 2- bromo -1- pyrenyl ethyl ketone=1:0.5-4:0.1-1；

Extra aluminium chloride and Lithium Aluminium Hydride are quenched with ice water, adjusts reaction solution to acidity with hydrochloric acid, then uses dichloromethane Alkane extraction and separation obtain crude product, pass through the isolated 1- of silica gel column chromatography (2- bromoethyl)-pyrene；

(3) under nitrogen protection, two bisglyoxalines and 1- (2- bromoethyl)-pyrene are mixed by the mass ratio of the material example 1:0.5-3 In acetonitrile, it is warming up to 75-90 DEG C of reflux, generates precipitating after stirring 12-48 hours, precipitating is washed with ether；It then will precipitating It is dissolved in methanol, slowly Potassium Hexafluorophosphate is instilled in solution and obtains white precipitate product, i.e., to the ratio of bacterial membrane charge response Rate fluorescence probe di-PYIM.

Its route synthesized are as follows:

The application of the ratio fluorescent probe of a kind of pair of membrane charge response, the fluorescence probe are living for detecting different type surface Property agent, specific detection method is to survey fluorescence spectrum after mixing surfactant with fluorescence probe；With fluorescence enhancement and fluorescence Rate of change (I₄₈₂/I₃₇₅) it is used as probe response signal, for distinguishing different types of surfactant.

The application of the ratio fluorescent probe of a kind of pair of membrane charge response, for identification and distinguishes difference by its rate of change The bacterium of type；The fluorescence probe is for distinguishing different types of bacterium；The specific detection method is as follows:

(1) Bacteria Culture；

(2) bacterium is collected: being centrifuged 5-10 minutes under the conditions of 10000-14000rpm, supernatant is abandoned, with 20mM, pH=7.4 HEPES buffer solution clean bacterium 2-3 time, finally with HEPES resuspension bacterium；

(3) it is detected after probe being added in bacterium with Fluorescence Spectrometer.

Bacteria Culture is to OD in step (1)₆₀₀=0.1-2.0.

It is resuspended with HEPES to the OD of bacterium in step (2)₆₀₀=0.05-2.0, final concentration of 5-20 μM of probe.

In step (3), fluorescence is detected under 345nm excitation wavelength and obtains fluorescence spectra, with fluorescence enhancement and fluorescence ratio Rate changes (I₄₈₂/I₃₇₅) it is used as probe response signal, for distinguishing variety classes bacterium.

The application of the ratio fluorescent probe of a kind of pair of membrane charge response, bacterium is gram sun to the fluorescence probe for identification Property bacterium or Gram-negative bacteria.

The advantages and benefits of the present invention are:

Bacterium surface has a large amount of charge, acts on probe nano aggregation and causes fluorescence significantly to increase its depolymerization By force.Since bacterium surface distribution of charges is different, probe is caused to form different degrees of monomer and dimer, to generate pyrene monomer With the rate of change (I of exciplex fluorescence₄₈₂/I₃₇₅).With probe polymer in conjunction with bacterium after the fluorescence enhancement that generates, with And monomer and the variation of exciplex ratio fluorescent are used as fluorescence signal, can identify Gram-negative bacteria or positive bacteria, and Different types of bacterium can be distinguished.

Detailed description of the invention

Fluorescence probe nuclear magnetic spectrogram hydrogen spectrum prepared by Fig. 1 embodiment 1；

Fluorescence probe nuclear magnetic spectrogram carbon spectrum prepared by Fig. 2 embodiment 1；

It is molten that Fig. 3 is that the fluorescence probe (concentration be 10 μM) prepared described in embodiment 2 by embodiment 1 is buffered in HEPES The fluorescence spectras and two dimension acted on respectively with different surfaces activating agent and (400 μM) of various ions in liquid (20mM, pH=7.4) Ratio chart.

It is molten that Fig. 4 is that the fluorescence probe (concentration be 10 μM) prepared described in embodiment 3 by embodiment 1 is buffered in HEPES It is dynamic with surfactant B S-12, Triton X-100, DTAB or SDS effect front and back respectively in liquid (20mM, pH=7.4) The transmission electron microscope picture of state light scattering diagram spectrum and fluorescence probe and SDS effect front and back.

Fig. 5 is the fluorescence probe described in embodiment 4 prepared by embodiment 1 in HEPES buffer solution (20mM, pH= 7.4) in different bacterium (OD₆₀₀=0.1) fluorescence spectra of effect front and back and two-dimentional ratio chart.

Specific embodiment

The following examples will be further described the present invention, but not thereby limiting the invention.