CN105503768A - Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples - Google Patents

Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples Download PDF

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CN105503768A
CN105503768A CN201610050322.6A CN201610050322A CN105503768A CN 105503768 A CN105503768 A CN 105503768A CN 201610050322 A CN201610050322 A CN 201610050322A CN 105503768 A CN105503768 A CN 105503768A
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陈�光
付强
王亦琳
李秀
刘玉霞
王桦
张书圣
尤进茂
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Qufu Normal University
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    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract

The invention relates to a preparation method of an alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of the alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples. The preparation method includes the steps that NBD-Cl is dissolved in chloroform, the dissolution concentration is 0002-0.012 g/ml, then a hydrazine hydrate-methanol solution with the volume concentration being 0.2-1.2% is added in and uniformly mixed, brown precipitate is obtained through stirring at room temperature and filtered, a filter cake is washed through ethyl acetate and dried, and the brown product alpha-oxoglutarate fluorescent/ultraviolet molecular probe is obtained. The alpha-oxoglutarate fluorescent/ultraviolet molecular probe is applicable to qualitatively and quantitatively analyzing alpha-oxoglutarate in the biological samples, and detection is sensitive, accurate and fast. The biological samples mainly comprise serum, living cells, muscle tissue and the like. The alpha-oxoglutarate fluorescent/ultraviolet molecular probe can be applied to analytical chemistry, life organic analysis, disease pre-diagnosis, medical clinical inspection and other related fields. The alpha-oxoglutarate fluorescent/ultraviolet molecular probe has the advantages of being good in response performance and high in data accuracy, repeatability and precision, equipment is convenient, fast and easy to operate, operability is high, and the alpha-oxoglutarate fluorescent/ultraviolet molecular probe is particularly suitable for large-batch sample combined screening and other large-data research.

Description

Fluorescence/the preparation method of ultraviolet molecular probe of alpha-KG and the application in biological specimen thereof
Technical field
The invention belongs to analytical chemistry field, relate to a kind of fluorescence/preparation method of ultraviolet molecular probe of alpha-KG and the application in biological specimen thereof.
Background technology
At present, the analytical chemistry method for alpha-KG mainly comprises gas phase (GC), high performance liquid chromatography (HPLC) and mass spectrum (MS), UV-detector (UV-detector) etc.And fluorescent molecular probe detection method quantity is very few, comprises and utilize nitro diazosulfide hydrazine (nitrobenzothiadiazolehydrazineDT) and naphthalimide hydrazine substituent (hydrazino-substitutednaphthalimide) fluorescent molecular probe to monitor alpha-KG.
Chromatographic detection method is applied in a lot of field, but is not best selection for the rapid batch of the biological sample of clinical medicine detects.Its reason is: first, and technological step is complicated, and sample handling procedure is various, workload is large, the operating time is long, inefficiency etc.This is completely not in the cards for medical biotechnology sample, particularly viable cell, In vivo detection.Secondly, sample requirements is large, and reagent consumption is large.Therefore and be not suitable for trace sample analysis the feature of biological specimen is that sample size is small, and the method that chromatogram associating large-scale instrument detects then often needs the sample to be tested of pre-treatment larger amt to reach the object of target compound purifying, enrichment, separation.Finally, the cost of large-scale instrument own is high, and it is careful to operate often step, is not easy to grasp and promote, and in the allegro modern society of high-level efficiency, the application of these class methods seems to reach the bottleneck phase of development.
Fluorescent molecular probe detects highly to be selected to detect to determinand mainly through design fluorescent molecular probe, thus produces the change of fluorescence response, and utilizes the difference character of optical signalling to carry out highly sensitive detection to determinand.Step is summarised as substantially: molecular designing, sample detection etc.Based on fluorescent molecular probe detection with application be technology more novel in analytical technology, its principle is by molecular designing thought, is connected by luminophore obtains molecular probe with reactive group.During detection, probe identifies target compound by the specific reaction of reactive group, and the change of molecular structure will impact luminophore, and fluorescent signal is changed, thus the detection realized target compound or bio-imaging.Be that selectivity is good from its Cleaning Principle this class methods advantage visible, do not need determinand to be separated, easy and simple to handle, be suitable for the batch quantity analysis of sample.Institute enjoys high praise in life analysis and research field in this way.But for alpha-KG, these class methods are considerably less sees, illustrate that the current research degree of academia is also nowhere near.From current two kinds of reported methods, there is following scarce limit:
First, fluorescence response is poor, and sensitivity is lower, and principle of luminosity is unfavorable for detection of biological samples.From existing report, after existing probe and determinand alpha-KG react, Fluorescence Increasing is about 7-9 doubly, is difficult to realize highly sensitive detection.Although realize Fluorescence Increasing by the method for adding tensio-active agent, destruction coenocorrelation can be brought larger error by this method simultaneously, is thus not suitable for the actual detection of biological specimen.Therefore, the Molecular Structure Design of probe itself haves much room for improvement.
Secondly, detection accuracy, the accurately index such as close are to be improved.As everyone knows, the signal stabilization higher relative to large-scale instrument, bio-sensing often brings comparatively big error.Although the signal self-correcting function of fluorescent probe can effectively improve this shortcoming.But existing report is not implemented to improve to this shortcoming.And existing probe molecule principle of luminosity is unfavorable for the strong increase of molecular fluorescence.So functional design and the luminescence mechanism of probe all have much room for improvement.
Again, the convenient degree of detection still has much room for improvement.At present, in life organic analysis detection field, emerge a lot of detection means fast and easily, comprise colorimetry, naked eyes monitoring etc.These methods widely use in the field such as cell analysis, monitored in vivo, but the colorimetric of alpha-KG/naked-eye observation technology is still in the blank of research.Therefore, detection method improved further, the convenient degree of experimental implementation being continued to improve is the actual demand that alpha-KG detection method is developed.
Finally, the research to alpha-KG imaging technique is lacked.Cell/tissue imaging technique is that the uniqueness of fluorescent probe advantage embodies.This technology can realize visualizing monitor and the treatment of disease, and the discovery for major diseases such as cancers has outstanding meaning with diagnosis.But do not relate to this application in the method that forefathers report.Thus, exploitation is suitable for the molecular probe of cell imaging, and setting up visualizing monitor platform will inherently change the research mode of alpha-KG, is expected to realize the visual research to Cancerous disease caused by alpha-KG.Therefore, imaging technique and application are urgently carried out.
Summary of the invention
The present invention is directed to the above-mentioned problems in the prior art, a kind of preparation method of fluorescence/ultraviolet molecular probe of alpha-KG is provided, the molecular probe prepared can be applicable to detection of biological samples, have that responsiveness is good, the accuracy of data, circulation ratio, precision advantages of higher, equipment is convenient easy to operate, exploitativeness is strong, is particularly suitable for the large data researches such as sample combined sorting in enormous quantities.
Technical solution of the present invention is as follows:
A kind of preparation method of fluorescence/ultraviolet molecular probe of alpha-KG, step is: by NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) be dissolved in chloroform, concentration of ordinary dissolution 0.002 ~ 0.012g/mL, then hydrazine hydrate-methanol solution that volumetric concentration is 0.2 ~ 1.2% is added, mix, stirred at ambient temperature obtains brown precipitate, filter, filter cake washs through ethyl acetate, dry, obtain the fluorescence/ultraviolet molecular probe of brown product alpha-KG.
In above-mentioned preparation method, described NBD-Cl is dissolved in the preferred 0.006g/mL of concentration of ordinary dissolution in chloroform; Hydrazine hydrate-methanol solution volumetric concentration preferably 0.6%.
Fluorescence/ultraviolet the molecular probe of described alpha-KG, Detection results index is as follows:
Detection sensitivity: detectability 0.9 μm of oL/L; Fluorescence Increasing multiple is 20 ~ 60 times;
Absorbing wavelength changes: absorb during detection and move 100nm to ruddiness;
Colour-change: color becomes redness from light green under fluorescent light; Color under ultraviolet lamp becomes green from colourless;
Dual quantitative correction: have fluorescent quantitation function and absorption peak ratio quantitative function concurrently;
Optical Mechanism index: have PET fluorescent switch function concurrently and absorb red shift function.
The application of the fluorescence/ultraviolet molecular probe of above-mentioned alpha-KG: the qualitative and quantitative analysis being applicable to alpha-KG in biological specimen, detects sensitive, accurate, quick; Wherein biological specimen mainly comprises serum, viable cell, muscle tissue etc., can be applicable to analytical chemistry, life organic analysis, disease examine and the association area such as clinical medicine detection in advance.
In the fluorescence/ultraviolet molecular probe quantitative analysis biological specimen of alpha-KG of the present invention during alpha-KG, be applicable to detect alpha-KG content in serum.
Fluorescence of the present invention/ultraviolet molecular probe detects the method for alpha-KG content in serum, and step comprises:
1) obtain solution
Probe HNBD storing solution is prepared: the fluorescence/ultraviolet molecular probe accurately taking alpha-KG is dissolved in anhydrous acetonitrile, and compound concentration is 2 × 10 -4the probe HNBD storing solution of M;
Target compound alpha-KG to be measured (α-KA) storing solution is prepared: accurately take target compound alpha-KG to be measured and be dissolved in anhydrous acetonitrile, compound concentration is 1 × 10 -3the alpha-KG storing solution of M;
Serum storing solution is prepared: by the 5:1 mixing by volume of serum and anhydrous acetonitrile, the centrifugal 20min of rotating speed 5000rpm, gets supernatant liquor and crosses 0.2 μm of film process and obtain serum storing solution;
2) linear equation of serum/alpha-KG standard substance is set up
The target compound alpha-KG storing solution dilution to be measured of getting step 1) preparation obtains the alpha-KG standard solution that gradient concentration is 2 ~ 900 μMs, then after after getting 200 μ L dilutions respectively, 2 ~ 900 μMs of alpha-KG standard solutions mix with 100 μ L probe HNBD storing solutions and 650 μ L serum storing solutions, adding 50 μ L degree is 10mM, the PBS damping fluid of pH7.4, jolting mixes, 60min is placed at 37 DEG C, then detect through spectrophotofluorometer and ultraviolet spectrometer, set up the linear equation of serum/α-KA concentration and fluorescence signal intensity or the linear equation of serum/α-KA concentration and uv-absorbing strength of signal,
Fluorescence or UV process detect alpha-KG content in serum testing sample
A) fluoroscopic examination testing sample: after 1000 μ L testing samples are injected quartz colorimetric utensil, Scanning Detction is carried out in fluorescence detector, the intensity data collecting fluorescent emission position substitutes into the linear equation of serum/α-KA concentration and fluorescence signal intensity, calculates alpha-KG content;
B) ultraviolet detection testing sample: after 1000 μ L testing samples are injected quartz colorimetric utensil, insert ultraviolet spectrophotometer, collect the intensity of maximum absorption wavelength position, obtain maximum absorption band volume efficiency before and after reaction and substitute into serum/α-KA concentration and uv-absorbing strength of signal, calculating alpha-KG content.
Above-mentioned fluorescence/ultraviolet molecular probe detects the application of alpha-KG content in serum, and optimal detection scope is fluorescence 3 ~ 900 μm of ol/L, ultraviolet 80 ~ 200 μm of ol/L.With the method for fluoroscopic examination, ultraviolet detection, parallel repeated detection (n=7) is carried out to determinand respectively, and calibrate with standard substance, obtain the optimal detection scope of fluorescence/ultraviolet detection, thus the concentration range of determinand selects optimized detection means to carry out quantitatively contained by different sample.
In the fluorescence/ultraviolet molecular probe qualitative analysis biological specimen of alpha-KG of the present invention during alpha-KG, be applicable to the naked eyes monitoring of alpha-KG in serum sample, the monitoring of alpha-KG in the monitoring of alpha-KG and muscle tissue in viable cell.
1, the naked eyes monitoring of alpha-KG in serum sample, method is: by test serum sample and anhydrous acetonitrile 5:1 mixing by volume, the centrifugal 20min of rotating speed 5000rpm, get supernatant liquor and cross 0.2 μm of film process, get gained supernatant liquor 200 μ L and add 100 μ L probe HNBD storing solutions and 200 μ Lalpha-ketoisocaproic storing solutions successively, then 1000 μ L are settled to the PBS damping fluid of pH7.4, gained solution preserves 50 ~ 70min at 37 DEG C, imaging is observed under being placed in fluorescent microscope, whether judge in serum sample containing alpha-KG according to luminous situation,
Color determination methods: if incarnadine under showing green or fluorescent lamp under ultraviolet lamp, then containing alpha-KG.
Described probe HNBD storing solution is identical with the method for alpha-KG content in above-mentioned detection serum with alpha-KG storing solution compound method.
2, the monitoring of alpha-KG in viable cell, method is: inserted by viable cell to be measured in substratum and cultivate 18 ~ 26h, viable cell to be measured reaches 2 × 10 in the medium 7~ 9 × 10 7individual/mL time, the probe HNBD-acetonitrile solution adding 3 ~ 5 times of volumes 20 μMs cultivates 10 ~ 12h altogether in 37 DEG C, with PBS buffer solution three times, under being placed in fluorescent microscope, observe imaging, whether judge in viable cell to be measured containing alpha-KG according to luminous situation;
Determination methods: aobvious green under ultraviolet lamp, then containing alpha-KG.
Described viable cell preferred yeast mycetocyte; Described substratum optimization protein peptone agar glucose.
3, the monitoring of alpha-KG in muscle tissue, method is: lamella muscle tissue to be measured being cut into thick 1 ~ 1.5 ㎜, with the probe HNBD-acetonitrile solution of 3 ~ 5 times of volumes 20 μMs, in substratum, 1 ~ 2h is cultivated altogether at 37 DEG C, then PBS buffer solution is used, observe imaging under being placed in fluorescent microscope, judge whether to treat in muscle tissue containing alpha-KG according to luminous situation;
Determination methods: aobvious green under ultraviolet lamp, then containing alpha-KG.
Described substratum is cultivated 20min by the aqueous solution of 1wt% fat extract, 2wt% peptone and 2wt% glucose at 121 DEG C and is obtained.
Fluorescence/ultraviolet the molecular probe of alpha-KG prepared by the present invention detects mechanism:
The design of the fluorescence/ultraviolet molecular probe of alpha-KG, principle is: by outstanding fluorescence female ring NBD, utilizes photo induced electron transfer mechanism (PET) to make its fluorescent quenching; This quenching group possesses good response characteristic to determinand alpha-KG simultaneously, requires that the molecule advanced linear orbital energy of product has change by a relatively large margin to cause PET mechanism and is prevented from, thus discharge hyperfluorescenceZeng Yongminggaoyingguang signal and improve sensitivity after reaction; Advanced linear orbital energy difference should reduce to some extent in addition, and to make absorbing wavelength red shift, thus to demonstrate visible light colors be that naked-eye observation is given security.
Fluorescence female ring NBD molecule has the advantage such as longwave absorption and transmitting, high quantum production rate, excellent Cell permeable, hypotoxicity and good availability.In alpha-KG molecular structure there is a carbonyl in carboxylic acid ortho position, this group can with electron-donating group (as-amino) react, change the electronics of group for imitating.Therefore, parent Luminous Ring is made with NBD, the elemental chlorine of NBD-Cl molecule No. 7 positions is replaced and becomes diazanyl, obtain the fluorescence/ultraviolet molecular probe (i.e. probe HNBD) of alpha-KG of the present invention, then be applied to detection of biological samples, mechanism is (wherein in HNBD-alpha-KA fraction, luminophore color is green) as shown in Figure 1.
As can be seen from Fig. 1 to the energy balane of this probe when excited state, in HNBD molecule, amino as the highest occupied molecular orbital energy (-11.34eV) of its excited energy (-8.92eV) of electron-donating group (Donor1) far above determinand alpha-KG, thus can cause the generation of PET electronic transfer process easily, thus make probe itself be in fluorescent quenching state.And once after being combined with determinand, electron-donating group becomes the Donor2 shown in figure, its energy is significantly reduced under Acceptor energy, causes PET process suppressed, thus fluorescence is released.After this process will make probe and target compound react, significantly promoting appears in fluorescence intensity, significantly improves the sensitivity of detection signal.It should be noted that, after probe HNBD and target compound alpha-KG are combined into product HNBD-alpha-KA, the difference of its advanced linear orbital energy will diminish in addition.Therefore, as can be seen from theoretic, the absorption spectrum of product likely moves to long-wavelength region, and this point has great significance about realizing naked-eye observation technical problem for solution is above-mentioned.Therefore, this organic molecule of HNBD is built practical as the polyfunctional molecule probe of alpha-KG.
Technical solution of the present invention beneficial effect is:
1) sensitivity is higher:
Current reported in literature congenic method detects and is limited to 6 μMs, and this detection limit reached after add the additional materials such as tensio-active agent in system, and in the inventive method, detection can be realized be limited to 0.9 μM under the condition not adding any tensio-active agent, sensitivity improves greatly, avoid the interpolation of additional materials simultaneously, decrease source of error.
2) multi-channel detection, accuracy is higher:
The probe that the inventive method is developed has fluorescent switch function and wavelength shift function concurrently simultaneously, this function be in the past report and detect in the method for determinand α-KA not available with fluorescent probe.The inventive method can respond enhancing caused by open mode give quantitatively by closing to by fluorescence, volume efficiency caused by absorbing wavelength displacement also can be utilized to give quantitatively simultaneously.Wherein, ratio quantivative approach effectively can reduce background interference, and dosing accuracy is improved, and uv-absorbing quantitatively can correct with fluorescent quantitation mutually simultaneously, gets final product self-correcting function, thus substantially increases the accuracy of detected result.
3) luminescence mechanism is novel unique, is more suitable for optical detection:
The luminescence mechanism of probe of the present invention is PET photo induced electron transfer mechanism and the combination absorbing red shift, and principle is very novel.Wherein PET process makes the probe fluorescence after chance determinand being originally in cancellation state significantly strengthen, and can bring highly sensitive response; Further, the absorption red shift that probe has concurrently is with the wave number of UV, visible light 380nm for starting point shifts to red light district, thus the color of product is significantly changed, and thus the probe of mechanism more single than other is more suitable for the detection of optical signal sensing assays.And fluorescence, thus can not interference ratio chromatographic analysis without transmitting displacement in process.
4) multiple biological sample imaging:
The successful implementation imaging of determinand in viable cell, muscle tissue, this is equally also to fail in forefathers' method to realize.The realization of imaging plays very large pushing effect for the further investigation of this biomarker of α-KA.
5) naked eyes monitoring, convenient and swift:
Probe of the present invention and detection method have filled up the blank of α-KA detection method, and making the naked eyes of determinand monitor (without the need to instrument, directly observing) becomes possibility, and testing process is more quick than average probe.
Accompanying drawing explanation
Fig. 1 is that the fluorescence/ultraviolet molecular probe of alpha-KG of the present invention detects and principle of luminosity schematic diagram;
Fig. 2 is the high performance liquid chromatography-uv-spectrogram of embodiment 1 probe HNBD and HNBD-α-KA: (a) probe molecule HNBD, (b) HNBD-α-KA, (c) probe HNBD, HNBD-α-KA;
Fig. 3 is high phase liquid chromatography-one-level (MS) and secondary (MS/MS) mass spectrum of embodiment 1 probe HNBD and HNBD-α-KA: probe HNBD (a:MS; B:MS/MS); HNBD-α-KA (c:MS; D:MS/MS);
Fig. 4 is the linear equation of embodiment 2 serum/α-KA concentration and fluorescence signal intensity;
Fig. 5 is the linear equation of embodiment 2 serum/α-KA concentration and uv-absorbing strength of signal;
Fig. 6 is different determinand α-KA concentration (1:0 μM, 2:5 μM, 3:100 μM, 4:300 μM in embodiment 2; 5:400 μM, 6:600 μM, 7:700 μM; 8:900 μM, 9:1000 μM) ultraviolet (on) and fluorescent lamp (under) irradiate under photo (HNBD:20 μM; PBSbuffer (pH7.4);
Fig. 7 is the illuminated diagram of serum sample under ultraviolet lamp and fluorescent lamp, 1: blank serum, 2: blank+HNBD, 3: blank+α-KA, 4: blank+HNBD+ α-KA, wherein HNBD:20 μM; α-KA:200 μM, PBSbuffer (pH7.4);
Fig. 8 is the linear relationship of determinand α-KA and fluorescent signal in serum; 1 be PBS solution data, 2 is data in serum;
Fig. 9 is yeast cell imaging experiment: a (blank), b (blank+200 μMs of α-KA), c (blank+20 μMs of HNBD) and d (blank+20 μMs of HNBD+200 μM of α-KA);
Figure 10 is the experiment of fresh pork imaging of tissue: c (blank), d (blank+200 μMs of α-KA), g (blank+20 μMs of HNBD) and h (blank+20 μMs of HNBD+200 μM of α-KA);
Figure 11 is different concns probe HNBD response intensity figure;
Figure 12 is that probe HNBD is responding with/without the fluorescent signal under determinand 200 μMs of α-KA existence conditions: up/down two Trendline are respectively probe molecule and are responding with/without the fluorescent signal under determinand 200 μMs of α-KA existence conditions;
Figure 13 is probe HNBD (20 μMs) is the fluorescence response in 5.4 ~ 7.4 with α-KA (200 μMs) in pH scope;
Figure 14 temperature affects the fluorescent signal that probe HNBD (20 μMs) and α-KA (200 μMs) react;
Figure 15 ionic strength is on the impact of probe HNBD (20 μMs) with α-KA (100 μMs) reaction system;
Figure 16 probe HNBD is to the fluorescence response figure (α-KA concentration: 0 ~ 900 μM) of the target compound α-KA to be measured of different concns;
Figure 17 probe HNBD is to different concns pH value fluorescence response figure;
Figure 18 probe HNBD is to determinand α-KA and other matter selective control experiment results.
Embodiment
By describing the present invention in conjunction with specific embodiments, without departing from the idea case in the present invention described above, the various replacement made according to ordinary skill knowledge and customary means or change, include within the scope of the invention.
Utilize Agilent1290 to connect Agilent6460 triple quadrupole bar mass spectrometer system (Agilent by high performance liquid phase-mass spectroscopy in the embodiment of the present invention, USA), and being equipped with AgilentJetStream electrospray system. it is by SBC18 post (2.1mm × 50mm that high-level efficiency liquid chromatography is separated, 1.8 μm of i.d., Agilent, USA) come.Gao Xiao Ye Xiang – ultra-violet analysis experiment is the online vacuum suction device of employing Agilent1260, automatically carries out sample device, fluorimetric detector associated working.Be separated gradient and be set to 0 minute: 70%A+30%B; 10 minutes: 0%A+100%B; 15 minutes: 0%A+100%B; Wherein A and B is respectively 0.5% formic acid+5% acetonitrile and 100% acetonitrile solution.Fluoroscopic examination utilizes Hitachi HitachiF-7000 fluorescence spectrophotometer to carry out, and excitation wavelength is 475nm, and emission wavelength is 540nm, excites and launch slit width to be 10.0nm, voltage 400V, sweep velocity 2400 nm/min.Uv-vis spectra is undertaken by Cary300Bio ultraviolet-visual spectrometer, and sweep limit is 350-700 nanometer.Fluorescence imaging observation is undertaken by Olympus, IX73-DP80 (Japan) inverted fluorescence microscope.The separation and purification of compound adopts thin-layer chromatography silicagel column to realize (filler 300-400 order).
Embodiment 1: the fluorescence/ultraviolet molecular probe preparing alpha-KG
0.3gNBD-Cl is dissolved in 50mL chloroform, then hydrazine hydrate-the methanol solution of 50mL volumetric concentration 0.6% is added, mix, stirred at ambient temperature 30min, obtains brown precipitate, filter, filter cake washs through ethyl acetate, dries, obtains 0.29g brown product, be the fluorescence/ultraviolet molecular probe (probe HNBD) of alpha-KG, yield is 97%.
Fluorescence/ultraviolet molecular probe effect the judge index of the alpha-KG prepared is as follows:
Detection sensitivity: detectability 0.9 μm of ol/L; Fluorescence Increasing multiple is 20 ~ 60 times;
Absorbing wavelength changes: absorb during detection and move 100nm to ruddiness;
Two channels colour-change: color becomes redness from light green under fluorescent light; Color under ultraviolet lamp becomes green from colourless;
Dual quantitative correction: have fluorescent quantitation function and absorption peak ratio quantitative function concurrently;
Optical Mechanism index: have PET fluorescent switch function concurrently and absorb red shift function.
Probe HNBD prepared by embodiment 1 and alpha-KG react feasibility and verify: get 0.1 gram of probe HNBD and be dissolved in 80mL acetonitrile, add 2 times of equivalent alpha-KGs wherein and at room temperature stir 1h, obtain product HNBD-α-KA, products therefrom is separated (chromatographic condition: Wates600controller through half preparative chromatography; Detector: waters2489; Elution requirement: condition: 0-5 minute 100%A, 5-7 minute 5%A+95%B; 7-15 minute: 0%A+100%B; 15-18 minute: 0%A+100%B).
Characterized by high-level efficiency liquid chromatography-ultraviolet, High Performance Liquid Chromatography/Mass Spectrometry, visible product HNBD-α-KA is comprehensively formed by the diazanyl of probe HNBD7 position and the carbonyl of alpha-KG just.High-level efficiency liquid chromatography-ultraviolet, the High Performance Liquid Chromatography/Mass Spectrometry of probe HNBD and product HNBD-α-KA are shown in Fig. 2 and Fig. 3 respectively.
Embodiment 2: detect alpha-KG content in serum (fluorescence/ultraviolet molecular probe of the alpha-KG adopting embodiment 1 to prepare)
Get after cancer patients's serum sample product (esophageal carcinoma, right side breast cancer, right side colon, the rectum cancer) add alpha-KG standard substance and make alpha-KG serum model (100 μMs) as detected object:
Obtain solution
Probe HNBD storing solution is prepared: the fluorescence/ultraviolet molecular probe accurately taking alpha-KG is dissolved in anhydrous acetonitrile, and compound concentration is 2 × 10 -4the probe HNBD storing solution of M;
Target compound alpha-KG to be measured (α-KA) storing solution is prepared: accurately take target compound alpha-KG to be measured and be dissolved in anhydrous acetonitrile, compound concentration is 1 × 10 -3the alpha-KG storing solution of M;
Serum storing solution is prepared: by the 5:1 mixing by volume of serum and anhydrous acetonitrile, the centrifugal 20min of rotating speed 5000rpm, gets supernatant liquor and crosses 0.2 μm of film process and obtain serum storing solution;
Set up the linear equation of serum/alpha-KG standard substance
The target compound alpha-KG storing solution dilution to be measured of getting step 1) preparation obtains gradient concentration and is respectively 0 μM, , 5 μMs, 100 μMs, 300 μMs, 400 μMs, 600 μMs, 700 μMs, the alpha-KG standard solution of 900 μMs and 1000 μMs, then after after getting 200 μ L dilutions respectively, above-mentioned alpha-KG standard solution mixes with 100 μ L probe HNBD storing solutions and 650 μ L serum storing solutions, adding 50 μ L degree is 10mM, the PBS damping fluid of pH7.4, jolting mixes, 60min is placed at 37 DEG C, then detect through spectrophotofluorometer and ultraviolet spectrometer, set up the linear equation (Fig. 4) of serum/α-KA concentration and fluorescence signal intensity or the linear equation (Fig. 5) of serum/α-KA concentration and uv-absorbing strength of signal,
Fluorescence or UV process detect alpha-KG content in serum testing sample
A) fluoroscopic examination testing sample: after 1000 μ L test serum samples are injected quartz colorimetric utensil, Scanning Detction is carried out in fluorescence detector, the intensity data collecting fluorescent emission position substitutes into the linear equation of serum/α-KA concentration and fluorescence signal intensity, and calculating alpha-KG content is 99.07 ~ 99.34 μMs;
B) ultraviolet detection testing sample: after 1000 μ L test serum samples are injected quartz colorimetric utensil, insert ultraviolet spectrophotometer, collect the intensity of maximum absorption wavelength position, obtain maximum absorption band volume efficiency before and after reaction and substitute into serum/α-KA concentration and uv-absorbing strength of signal, calculating alpha-KG content is 99.45 ~ 99.79 μMs.
The present embodiment: four kinds of serum model sample fluorescence (FLD) and ultraviolet (UV) detection level are respectively: the esophageal carcinoma: FLD:99.07, UV:99.45; Right side breast cancer: FLD:99.20, UV:99.58; Right side colon: FLD:99.34, UV:99.48; The rectum cancer: FLD:99.31, UV:99.79; Limit of error: FLD:-0.93% ~-0.56%; UV:-0.55% ~-0.21%.
Result shows, alpha-KG (α-KA) in the concentration range of 2 ~ 900 μMs (than 10 of bibliographical information -3~ 10 -5the wider Ind.Eng.Chem.Res.2015 of M, 54,2886 2893) response signal caused probe HNBD of the present invention presents linear relationship (Fig. 4), detect and be limited to 0.9 μm of ol/L(and be better than 6 μMs of Ind.Eng.Chem.Res.2015 that document reports, 54,, and in wider concentration range, show good relation conefficient (r 2886 2893) 2=0.9921.).
Meanwhile, the volume efficiency before and after uv-absorbing wavelength moves presents linear dependence (Fig. 5) to testing concentration, and this makes quantitative accuracy greatly improve.And consider the concentration rank of the determinand α-KA that may exist in biological sample, uv-absorbing ratio gained linearity range (80 ~ 190 μMs) is just adapted in biological sample quantitative, thus improves the biological suitability of method.In addition, significantly absorbing red shift makes the color of system become red by green transition, this acute variation ensure that naked eyes monitoring (Fig. 6) of trace determinand, this characteristic detects in the field of α-KA at fluorescent probe and occurs first, the detection method that forefathers report is all without this characteristic (Chem.Sci., 2014,5,4012)
1 is prepared respectively: blank serum, 2: blank+HNBD, 3: blank+α-KA, 4: blank+HNBD+ α-KA, four kinds of solution to be measured, wherein HNBD:20 μM according to embodiment 2 solution manufacturing method; α-KA:200 μM, PBSbuffer (pH7.4), preserve 60min under 37 DEG C of conditions.Visible by contrasting, the sample of blank+HNBD does not present absorption red shift, and under ultraviolet lamp, fluorescence is in cancellation state all the time simultaneously, and this shows that serum matrix can not cause interference to this probe.And after adding determinand α-KA, occur obviously green under ultraviolet lamp, show that fluorescence is opened (PET is prohibited) and occurred orange red color (Fig. 6) under fluorescent light simultaneously, show that the target compound α-KA that can realize in serum sample carries out color comparison of naked eye monitoring (Fig. 7).
Embodiment 3: naked eyes monitoring (sample is with embodiment 2) of alpha-KG in serum sample
Method is: by test serum sample and anhydrous acetonitrile 5:1 mixing by volume, the centrifugal 20min of rotating speed 5000rpm, get supernatant liquor and cross 0.2 μm of film process, get gained supernatant liquor 200 μ L and add 100 μ L probe HNBD storing solutions and 200 μ Lalpha-ketoisocaproic storing solutions successively, then 1000 μ L are settled to the PBS damping fluid of pH7.4, gained solution preserves 60min at 37 DEG C, imaging is observed under being placed in fluorescent microscope, color is: for green under ultraviolet lamp, be light red under fluorescent lamp, prove containing alpha-KG.
Probe HNBD storing solution is identical with embodiment 2 with alpha-KG storing solution compound method.
By the sample of Fig. 7 blank+HNBD, can find out that not absorbing red shift occurs, under ultraviolet lamp, fluorescence is in cancellation state all the time simultaneously, and this shows that serum matrix can not cause interference to this probe.And after adding determinand α-KA, occur obviously green under ultraviolet lamp, show that fluorescence is opened (PET is prohibited) and occurred orange red color under fluorescent light simultaneously, this shows that the target compound α-KA that can realize in serum sample carries out color comparison of naked eye observation now.Simultaneously, as can be seen from the linear relationship (Fig. 8) of determinand α-KA in serum with fluorescent signal, in human serum solution, within the scope of the determinand of 0 ~ 900 μM, fluorescence wire size presents linear dependence, and the relation simultaneously set up with uv-absorbing also meets linear variability law.
Embodiment 4: the monitoring (for yeast) of alpha-KG in viable cell
Method is: inserted by yeast cell in Yeast protein peptone agar glucose and cultivate 24h, treat that yeast cell reaches 2 × 10 in the medium 7~ 9 × 10 7individual/mL time, then the probe HNBD-acetonitrile solution adding 3 times of volumes 20 μMs cultivates 12h altogether in 37 DEG C, with PBS buffer solution three times, imaging is observed under inserting fluorescent microscope, in viable cell to be measured, alpha-KG color is: green, and other control samples (a, b in Fig. 9, c) all without color, then under this condition, green presents provable containing alpha-KG.
Comparison test analysis: under ultraviolet wavelength excites, yeast cell (as shown in Figure 9) respectively with blank, HNBD (20 μMs) and α-KA (200 μMs) in 37 DEG C, fluorescent emission is not presented after cultivating 24h altogether, wherein blank solution is by cell at fat extract (1%), cultivates 20min and make in peptone (2%) and glucose (2%) mixture.This shows that probe HNBD is optics immunity for the various materials in cell, and namely cell matrix does not cause optical interference.And when adding the determinand α-KA of 10 times amount, under ultraviolet lamp, there is obvious launching effect, the imaging of green fluorescence is observed smoothly.This illustrates that probe HNBD has excellent membrane penetrating, and probe HNBD can be applied to cell imaging aspect simultaneously.
Embodiment 5: the monitoring (being organized as example with fresh pork) of alpha-KG in muscle tissue
Method is: lamella fresh pork tissue being cut into thick 1 ㎜, at 37 DEG C, 1h is cultivated with the probe HNBD-acetonitrile solution of 3 times of volumes 20 μMs, (aqueous solution of 1wt% fat extract, 2wt% peptone and 2wt% glucose is cultivated 20min at 121 DEG C and is obtained), then PBS buffer solution is used, imaging is observed under inserting fluorescence microscopy device, in fresh pork tissue, alpha-KG color is: green (under ultraviolet lamp), proves containing alpha-KG.
Simultaneous test is verified: experimental result (as shown in Figure 10) (37 DEG C, cultivate 24h altogether, c (blank), d (blank+200 μMs of α-KA), g (blank+20 μMs of HNBD) and h (blank+20 μMs of HNBD+200 μM of α-KA), wherein blank solution is that muscle tissue obtains at 121 DEG C of cultivation 20min at the aqueous solution of 1wt% fat extract, 2wt% peptone and 2wt% glucose.Show that, when not adding determinand, blank sample and probe almost do not present fluorescent emission after cultivating altogether.After cultivating 60min altogether with 10 times of equivalent determinand α-KA, can be observed fluorescent emission and significantly strengthen, present histofluorescence image clearly.These phenomenons confirm that this probe is organized for sturdy grain of meat and still had good penetrance, are thus suitable for muscle tissue imaging.
The experimental verification of the fluorescence/ultraviolet molecular probe all technical of alpha-KG of the present invention, specific as follows:
Probe dosage optimization
Probe consumption is related to the important indicator such as sensitivity and reagent consumption of detection, and the primary factor often as inspection optimization is investigated.According to the luminous characteristics of this probe, its sensitivity should belong to higher, and therefore when reality detects, required concentration and probe concentration should be lower.As higher in given concentration, by occurring that self-quenching makes information strength reduce.Therefore, the concentration range that have selected 10 ~ 50 μMs is optimized.Result shows, when testing concentration is 200 μMs, concentration is that the probe of 20 μMs has and responds the most by force.This concentration will be brought into follow-up optimization experiment process (Figure 11).
Reaction times is optimized
Reaction times will affect reaction efficiency and the level of response of probe molecule HNBD and determinand α-KA, also will determine the stabilized soil pavement of final signal simultaneously.Therefore, after determining concentration and probe concentration, be optimized the time of reaction, temperature: 37 DEG C, buffered soln PBS, pH are 7.4.As can be seen from Figure 12, before the reaction in 60 minutes, to plateau after fluorescent signal is in rising trend, thus can determine that the reaction times is 60 minutes.By contrast, then there is not the change of fluorescence intensity in the probe solution not containing determinand, and this illustrates that signal response is caused by determinand.
The optimization of pH value of reaction system
PH value often has certain influence to the optical property of organic molecular probe, is thus controlled often through buffered soln in the reaction thus meets experiment needs.For application of the present invention (serum, cell, tissue) own characteristic, have studied the pH value (5.4 ~ 7.4) that may reach under animal body physiological environment.Visible from Figure 13 (37 DEG C, 60min), in physiological range pH value minor fluctuations for the fluorescence intensity that the mixed system of target compound and probe presents respond affect very little.Therefore, in common living things system, when namely pH value is 7.4, organism acidity environment can not impact the application of probe.
The optimization of temperature of reaction
The impact of temperature on chemical reaction is most important, and the biological specimen studied for the present invention is as all the more so in viable cell, organizational framework.The temperature of usual animal body is generally about 37 DEG C, and can probe have good response to be related to the success or failure of whole experiment to target compound to be measured at this temperature.As shown in Figure 14 (buffered soln: PBSbuffer (pH7.4), temperature: 37 DEG C, time: 60min), we investigate the fluorescence response that temperature is brought in the reaction of 10 ~ 40 DEG C of scopes.Therefrom be not difficult to find, this probe 37 DEG C time and determinand react and provide good fluorescence response, be thus particularly suitable for the study and practice of biological specimen.
Ionic strength is on the impact of fluorescent signal
Ionic strength, also likely causes certain influence to fluorescent signal as factor distinctive in solution.In living things system, ionogen is based on sodium-chlor, and therefore the present invention determines the electrolyte environment of simulating actual sample system with the sodium chloride concentration (100mM) under physiological condition, thus proves the feasibility of present method in practical systems further.Figure 15 (NaCl:100mM, buffered soln: PBSbuffer (pH7.4), temperature: 37 DEG C, time: 60min) in visible, the existence of sodium-chlor does not almost impact for fluorescent signal, and this illustrates that this probe is adapted in life system ionic strength atmosphere completely to determinand examinations.
Optical property and Mechanism Validation
The present invention is intended to invent a kind of polyfunctional molecule probe possessing novel luminescent mechanism.The intensity of emission spectra of probe molecule HNBD is very faint (as shown in figure 16), shows to have occurred in this molecule to allow the electronic behavior of the effective cancellation of fluorescence.
For confirming that this behavior belongs to PET mechanism, the present invention has carried out simulating (density general sea theory, the horizontal B3LYP6-31g (d, p) of base group to probe excited state with Quantum chemical calculation 1).From the results of view, in probe molecule, amino energy is enough to cause effective PET mechanism to occur, thus quenching fluorescence.And after HNBD is in conjunction with determinand, its PET process is drawn up, then fluorescence release.In probe and the reacted mixed solution of determinand, fluorescence intensity has obvious lifting, maximumly strengthens 60 times.This illustrates rational PET Fluorescence Mechanism design fluorescent probe the is possessed handoff functionality of close/open.Moreover, for verifying this mechanism further, the present invention demonstrates different pH value to be affected the fluorescence of probe HNBD.Consider HNBD alkalescence with diazanyl, devise with in hydrogen ion and the experiment of amino electronics.From Figure 17, when pH value is in neutral Acidity Range, affect by PET, the fluorescence activity of probe HNBD is lower; When pH reduces, the hydrogen ion increased gradually causes strong effect to probe, prevents the electronics on amino to move, thus fluorescence is strengthened greatly.This phenomenon has reconfirmed the existence of PET mechanism, also illustrate that the reasonableness of this probe design simultaneously.Otherwise if not PET mechanism, then likely there is blue shift in the probe maximum absorption spectrum of HNBD, but this hypothesis obviously and experimental observation to phenomenon be not inconsistent.
In fact, HNBD and target compound α-KA reacted product HNBD-α-KA demonstrates and absorbs red shift greatly, this may be due to after being converted to product, the difference of the advanced linear orbital energy of HNBD-α-KA reduces to some extent than the difference of the advanced linear orbital energy of probe HNBD molecule, thus result in endergonic reduction, namely there is the red shift of absorption spectrum.Meanwhile, do not occur the Red Shift Phenomena of emmission spectrum, this shows fluorescent switch+absorption red shift mechanism that this probe light emitting machine ought to belong to very novel.Therefore, by means of these optical characteristics, the present invention can realize sensitive detection response, naked eyes monitoring, or even contribution ratios correct detection.This also shows that the design of this probe is very successful simultaneously.
Probe molecule detects the selectivity analysis of α-KA
Following substances storing solution compound method: respectively with redistilled water Dissolved Amino Acids (Ala, Cys, His, Met, Arg, Gln, Ile, Phe, Asn, Leu, Pro, Asp, Gly, Lys, Sar, Ser, Thr, Trp, Val, Glu), glucose (Glucose), seminose (Galactose), oxalic dialdehyde (Glyoxal), hydrogen peroxide (H- 2o 2), Sodium.alpha.-ketopropionate (sodiumpyruvate (PAS)), phenylglyoxal (phenylglyoxal) and pyruvic aldehyde (methylglyoxal), phenyl-pyruvic acid (phenylpyruvicacid), obtain the storing solution of above-mentioned substance, for subsequent analysis, in subsequent experimental, required low concentration solution all dilutes obtained on this storing solution basis.
Based on the fluorescence response that this probe is stronger, the present invention analyzes the selectivity of probe HNBD to α-KA, and its comparison other comprises glucose (Glucose), seminose (Galactose), oxalic dialdehyde (Glyoxal), hydrogen peroxide (H -2o 2), prepared by Sodium.alpha.-ketopropionate (sodiumpyruvate (PAS)), phenylglyoxal (phenylglyoxal), pyruvic aldehyde (methylglyoxal), phenyl-pyruvic acid (phenylpyruvicacid).To determinand α-KA and other matter selective control experiment results as shown in figure 18, in figure, other materials comprise probe HNBD: in a figure, 100 micro-ly rub/liter amino acid: 1:Ala2:Cys3:His4:Met5:Arg6:Gln7:Ile8:Phe9:Asn10: α-KA11:Leu12:Pro13:Asp14:Gly15:Lys16:Sar17:Ser18:Thr19:Trp 20:Val21:Glu; In b figure 5 millis rub/liter α-KA, 200 micro-ly to rub/liter semi-lactosi (Galactose (Gal)), oxalic dialdehyde Glyoxal (GO), hydrogen peroxide (H -2o 2), Sodium.alpha.-ketopropionate sodiumpyruvate (PAS), phenylglyoxal phenylglyoxal (PGO), pyruvic aldehyde (methylglyoxal) and phenyl-pyruvic acid (phenylpyruvicacid).
First, relative to determinand α-KA, HNBD goes out the response of extreme difference to amino acids exhibit, this should be due to amino acid and α-KA structurally have obvious different caused by.When add excessive aldose, ketose, oxalic dialdehyde, pyruvate salt and phenylglyoxal time, HNBD still maintains the selectivity outstanding to determinand α-KA.Although the pyruvic aldehyde of 200 μMs causes the small response of HNBD, this can't bring the interference of detection, and the concentration that pyruvic aldehyde may exist in vivo is after all generally less than 2 μMs.Therefore, the application that outstanding selectivity makes HNBD be adapted in biological specimen completely., found by pH titration experiments, be the interval range of 5.4 ~ 7.4 in pH value, the intensity of fluorescence does not appear at noticeable change meanwhile, and this shows that this probe is suitable for completely when coenocorrelation and pH are 7.4.In addition, temperature experiment also demonstrate that this probe is highly suitable for biological sample.

Claims (10)

1. the preparation method of the fluorescence/ultraviolet molecular probe of an alpha-KG, it is characterized in that, step is: be dissolved in by NBD-Cl in chloroform, concentration of ordinary dissolution 0.002 ~ 0.012g/mL, then adds hydrazine hydrate-methanol solution that volumetric concentration is 0.2 ~ 1.2%, mix, stirred at ambient temperature obtains brown precipitate, and filter, filter cake washs through ethyl acetate, dry, obtain the fluorescence/ultraviolet molecular probe of brown product alpha-KG.
2. preparation method according to claim 1, is characterized in that: described NBD-Cl is dissolved in the preferred 0.006g/mL of concentration of ordinary dissolution in chloroform; Hydrazine hydrate-methanol solution volumetric concentration preferably 0.6%.
3. preparation method according to claim 1, is characterized in that: the fluorescence/ultraviolet molecular probe of described alpha-KG, and Detection results index is as follows:
Detection sensitivity: detectability 0.9 μm of oL/L; Fluorescence Increasing multiple is 20 ~ 60 times;
Absorbing wavelength changes: absorb during detection and move 100nm to ruddiness;
Colour-change: color becomes redness from light green under fluorescent light; Color under ultraviolet lamp becomes green from colourless;
Dual quantitative correction: have fluorescent quantitation function and absorption peak ratio quantitative function concurrently;
Optical Mechanism index: have PET fluorescent switch function concurrently and absorb red shift function.
The application of the fluorescence/ultraviolet molecular probe of 4.alpha-ketoisocaproic, is characterized in that: the qualitative and quantitative analysis being applicable to alpha-KG in biological specimen; Wherein biological specimen comprises serum, viable cell, muscle tissue, can be applicable to analytical chemistry, life organic analysis, disease is examined in advance and clinical medicine detects.
5. application according to claim 4, is characterized in that: in quantitative analysis biological specimen during alpha-KG, is applicable to detect alpha-KG content in serum; In qualitative analysis biological specimen during alpha-KG, be applicable to the naked eyes monitoring of alpha-KG in serum sample, the monitoring of alpha-KG in the monitoring of alpha-KG and muscle tissue in viable cell.
6. application according to claim 5, is characterized in that, detect the method for alpha-KG content in serum, step comprises:
1) obtain solution
Probe HNBD storing solution is prepared: the fluorescence/ultraviolet molecular probe accurately taking alpha-KG is dissolved in anhydrous acetonitrile, and compound concentration is 2 × 10 -4the probe HNBD storing solution of M;
Target compound alpha-KG to be measured (α-KA) storing solution is prepared: accurately take target compound alpha-KG to be measured and be dissolved in anhydrous acetonitrile, compound concentration is 1 × 10 -3the alpha-KG storing solution of M;
Serum storing solution is prepared: by the 5:1 mixing by volume of serum and anhydrous acetonitrile, the centrifugal 20min of rotating speed 5000rpm, gets supernatant liquor and crosses 0.2 μm of film process and obtain serum storing solution;
2) linear equation of serum/alpha-KG standard substance is set up
The target compound alpha-KG storing solution dilution to be measured of getting step 1) preparation obtains the alpha-KG standard solution that gradient concentration is 2 ~ 900 μMs, then after after getting 200 μ L dilutions respectively, 2 ~ 900 μMs of alpha-KG standard solutions mix with 100 μ L probe HNBD storing solutions and 650 μ L serum storing solutions, adding 50 μ L degree is 10mM, the PBS damping fluid of pH7.4, jolting mixes, 60min is placed at 37 DEG C, then detect through spectrophotofluorometer and ultraviolet spectrometer, set up the linear equation of serum/α-KA concentration and fluorescence signal intensity or the linear equation of serum/α-KA concentration and uv-absorbing strength of signal,
3) fluorescence or UV process detect alpha-KG content in serum testing sample
A) fluoroscopic examination testing sample: after 1000 μ L testing samples are injected quartz colorimetric utensil, Scanning Detction is carried out in fluorescence detector, the intensity data collecting fluorescent emission position substitutes into the linear equation of serum/α-KA concentration and fluorescence signal intensity, calculates alpha-KG content;
B) ultraviolet detection testing sample: after 1000 μ L testing samples are injected quartz colorimetric utensil, insert ultraviolet spectrophotometer, collect the intensity of maximum absorption wavelength position, obtain maximum absorption band volume efficiency before and after reaction and substitute into serum/α-KA concentration and uv-absorbing strength of signal, calculating alpha-KG content.
7. application according to claim 6, is characterized in that:: optimal detection scope is fluorescence 3 ~ 900 μm of ol/L, ultraviolet 80 ~ 200 μm of ol/L.
8. application according to claim 5, it is characterized in that, the naked eyes monitoring of alpha-KG in serum sample, method is: by test serum sample and anhydrous acetonitrile 5:1 mixing by volume, the centrifugal 20min of rotating speed 5000rpm, get supernatant liquor and cross 0.2 μm of film process, get gained supernatant liquor 200 μ L and add 100 μ L probe HNBD storing solutions and 200 μ Lalpha-ketoisocaproic storing solutions successively, then 1000 μ L are settled to the PBS damping fluid of pH7.4, gained solution preserves 50 ~ 70min at 37 DEG C, imaging is observed under being placed in fluorescent microscope, whether judge in serum sample containing alpha-KG according to luminous situation,
Color determination methods: if incarnadine under showing green or fluorescent lamp under ultraviolet lamp, then containing alpha-KG.
9. application according to claim 5, is characterized in that, the monitoring of alpha-KG in viable cell, and method is: inserted by viable cell to be measured in substratum and cultivate 18 ~ 26h, viable cell to be measured reaches 2 × 10 in the medium 7~ 9 × 10 7individual/mL time, add 3 ~ 5 times of volumes, 20 μMs of probe HNBD-acetonitrile solutions and cultivate 10 ~ 12h altogether in 37 DEG C, with PBS buffer solution three times, under being placed in fluorescent microscope, observe imaging, whether judge in viable cell to be measured containing alpha-KG according to luminous situation;
Determination methods: aobvious green under ultraviolet lamp, then containing alpha-KG;
Described viable cell preferred yeast mycetocyte; Described substratum optimization protein peptone agar glucose.
10. application according to claim 5, it is characterized in that, the monitoring of alpha-KG in muscle tissue, method is: lamella muscle tissue to be measured being cut into thick 1 ~ 1.5 ㎜, with the probe HNBD-acetonitrile solution of 3 ~ 5 times of volumes 20 μMs, at 37 DEG C, in substratum, cultivate 1 ~ 2h altogether, then use PBS buffer solution, observe imaging under being placed in fluorescent microscope, judge whether to treat in muscle tissue containing alpha-KG according to luminous situation;
Determination methods: aobvious green under ultraviolet lamp, then containing alpha-KG;
Described substratum is cultivated 20min by the aqueous solution of 1wt% fat extract, 2wt% peptone and 2wt% glucose in 121 DEG C and is obtained.
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CN107382900B (en) * 2017-06-30 2020-05-19 曲阜师范大学 Preparation method and application of pH fluorescent probe
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CN109748970A (en) * 2019-01-24 2019-05-14 华东理工大学 α-ketoglutaric acid optical probe and its preparation method and application
CN109748970B (en) * 2019-01-24 2022-07-05 华东理工大学 Alpha-ketoglutaric acid optical probe and preparation method and application thereof
CN116148231A (en) * 2023-02-17 2023-05-23 广东兴昶未来食品科技有限公司 On-line monitoring system and method for freezing and preserving of prefabricated vegetables
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