CN106009760B - Half cyanine fluorochrome - Google Patents
Half cyanine fluorochrome Download PDFInfo
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- CN106009760B CN106009760B CN201610343736.8A CN201610343736A CN106009760B CN 106009760 B CN106009760 B CN 106009760B CN 201610343736 A CN201610343736 A CN 201610343736A CN 106009760 B CN106009760 B CN 106009760B
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- 0 CC(*c1c(CCc2ccccc2)cccc1)=C Chemical compound CC(*c1c(CCc2ccccc2)cccc1)=C 0.000 description 1
- CNKHPZHLGMCESM-TUXUZCGSSA-N CC(C)C1C=CC(C[C@](C)(Cc2ccccc2)c(cccc2)c2S)=CC1 Chemical compound CC(C)C1C=CC(C[C@](C)(Cc2ccccc2)c(cccc2)c2S)=CC1 CNKHPZHLGMCESM-TUXUZCGSSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/14—Styryl dyes
- C09B23/145—Styryl dyes the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
- C09B23/146—(Benzo)thiazolstyrylamino dyes
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
Abstract
The present invention discloses a kind of half cyanine fluorochrome, has the structure of general formula I.In general formula I, R1Selected from H, C1‑6Alkyl, phenyl, C1‑6Phenyl that alkyl arbitrarily replaces, SO3R5And COOR5；The R5Selected from H and C1‑6Alkyl；R2Selected from H, C1‑6Alkyl, SO3R6And COOR6；The R6Selected from H and C1‑6Alkyl；R3And R4It is each independently selected from H and C1‑6Alkyl；Y‑For Cl‑、Br‑Or I‑.Half cyanine fluorochrome of the present invention has human serum albumins the launch wavelength of good response speed, reversible thermal stability, excellent selectivity and near-infrared, and can reach the minimum detection limit of 1.73mg/L.The fluorescent dye of the present invention may be used on sero-abluminous detection and the imaging of living cells confocal fluorescent in human urine.
Description
Technical field
The invention belongs to field of fine chemical, are related to a kind of fluorescent dye that seralbumin detects suitable for biological sample
And its application in cell imaging.
Background technology
Human serum albumins (Human Serum Albumin, abbreviation HSA) is protein important in human plasma, is accounted for about
The 60% of Total plasma protein.Human serum albumins, which not only plays, in biosystem, in body fluid maintains blood normal osmotic pressure to make
With, but also carry the transportation functions such as steroid hormone, BILE PIGMENTS, drug molecule and metabolite.In addition, people's blood in blood plasma
The exception of pure protein content and some diseases are closely related, such as hepatitis, hepatic sclerosis and kidney trouble etc..
Medically human serum albumins can be used for treatment burn and shock, for supplement because operation, contingency or
Blood loss caused by massive haemorrhage, also can be as the bulking agent of blood plasma.For healthy population, seralbumin containing in serum
It measures as 35~50g/L, the content in urine is less than 30mg/L.Clinically, serum albumin levels be considered as one about
The reliable index of biological function and relevant disease of liver and kidney.For example, by detecting in vitro urine specimen, urine is measured
There is excessive seralbumin in sample, available for the auxiliary diagnosis of renal impairment, play the role of early warning.
Fluorescence probe has many advantages, such as that good high sensitivity, selectivity, real-time in-situ, detection limit low and Visual retrieval, and
And the shortcomings of complicated sample pretreatment process present in conventional method, instrument price costliness can also be overcome, can not be analyzed in real time,
Therefore cause extensive concern in field of biological detection.However up to the present, in spite of some for human serum albumins
Fluorescence probe has been reported, but due to its detection limit it is low (>30mg/L), stability difference and transmitted wave length (<600nm), greatly
Most probes are all difficult to detect micro human serum albumins in complicated biological sample.Therefore, it develops to human seralbumin egg
The small-molecule fluorescent probe of white function admirable has great importance.
Invention content
Present invention firstly provides a kind of half cyanine fluorochromes, have the structure of general formula I：
In general formula I,
R1Selected from H, C1-6Alkyl, phenyl, C1-6Phenyl that alkyl arbitrarily replaces, SO3R5And COOR5；The R5Selected from H and
C1-6Alkyl；
R2Selected from H, C1-6Alkyl, SO3R6And COOR6；The R6Selected from H and C1-6Alkyl；
R3And R4It is each independently selected from H and C1-6Alkyl；
Y-For Cl-、Br-Or I-。
On the other hand, the present invention provides the preparation method of above-mentioned half cyanine fluorochrome, and described method includes following steps：
(1) compound 1 and compound 2 are according to molar ratio 1:1.2~1.5 reaction prepare compounds 3；
(2) compound 3 and compound 4 are according to molar ratio 1:1.0~1.2 reactions prepare compounds of formula I,
Above-mentioned half cyanine fluorochrome of the present invention is that there are multiple contortion sites using in structure by the present inventor
Hemicyanine dye for development platform, based on distortion Intramolecular electron transfer (Twisted Intramolecular Charge
Transfer, abbreviation TICT) mechanismic design synthesis one kind can be combined with human serum albumins hydrophobic cavity it is enhanced glimmering
Light probe.Such fluorescence probe has good response speed (5s), reversible thermal stability (5~60 to human serum albumins
DEG C), the launch wavelength of excellent selectivity and near-infrared (>680nm), and the minimum detection limit of 1.73mg/L can be reached.And
And the imaging of living cells confocal fluorescent can also be applied to.
Based on this, the present invention provides half cyanine fluorochrome of the invention described above answering in albumin identifies and detects
With.
Specifically, half cyanine fluorochrome of present invention offer the invention described above is preparing albumin identification and detection reagent
In application.Further, the purpose of the present invention, which is lain also in, provides a kind of albumin detection reagent, half cyanines containing the invention described above
Fluorochrome.Half cyanine fluorochrome of the present invention containing effective dose in the albumin detection reagent.It can
For the detection of human serum albumins in laboratory or clinical biospecimens.The biological sample can illustrate but be not limited in vitro
Urine sample.
Description of the drawings
25 width of attached drawing of the present invention, it is as follows：
Fig. 1 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, add in 100 μM
HSA excitation spectrums and emission spectrum.Abscissa is wavelength (nm), and ordinate is normalized intensity.The a length of 580nm of excitation light wave.
Fig. 2 is 10 μM of probe NIR-HSA of the present invention in the fluorescent emission spectrogram (Fig. 2 a) of different solvents and accordingly not
With the absorbance (Fig. 2 b) of solvent.Abscissa be wavelength (nm), a length of 580nm of excitation light wave.
Fig. 3 be the present invention 10 μM of probe NIR-HSA-2 in PBS (10mM pH 7.4) buffer solution, add in 20 μM
After HSA stirrings, the ultraviolet-visible absorption spectroscopy (Fig. 3) after scanning combination.Abscissa is wavelength (nm), and ordinate is absorbance.
Fig. 4 is that 10 μM of probe NIR-HSA of the invention fluorescence in different glycerine/PBS (v/v) mixed solution systems is sent out
Penetrate collection of illustrative plates (Fig. 4).The a length of 580nm of excitation light wave.
Fig. 5 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, add in 0.1,0.2,
0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、2.0、3.0、4.0、5.0、6.0、8.0、10.0、15.0、20.0、30.0、
50.0th, the variation of 70.0,90.0,95.0,100.0,105.0,110.0 μM of HSA fluorescence intensities.Abscissa is wavelength (nm),
The a length of 580nm of excitation light wave.
Fig. 6 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, add in 0.1,0.2,
0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、2.0、3.0、4.0、5.0、6.0、8.0、10.0、15.0、20.0、30.0、
50.0th, the variation of 70.0,90.0,95.0,100.0,105.0,110.0 μM of HSA UV absorptions.Abscissa is wavelength (nm).
Fig. 7 be the present invention 10 μM of probe NIR-HSA-2 in PBS (10mM pH 7.4) buffer solution, add in 0.2,
0.4th, 0.6,0.8,1.0,1.2,1.4,1.6,1.8,4.0,6.0,8.0,10.0,12.0,14.0,16.0,18.0,20.0 μM
The variation of HSA fluorescence intensities.Abscissa be wavelength (nm), a length of 580nm of excitation light wave.
Fig. 8 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, extract fluorescence intensity
In F680Nm is with the variation diagram of different HSA additions.Abscissa be different HSA additions (μM), a length of 580nm of excitation light wave.
Fig. 9 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, add in 0.1,0.2,
0.3rd, the variation of 0.4,0.5,0.6,0.7,0.8,0.9,1.0 μM of HSA fluorescence intensities.The a length of 580nm of excitation light wave, ordinate
For fluorescence intensity.
10 μM of probe NIR-HSA-2 of Figure 10 present invention in PBS (10mM pH 7.4) buffer solution, add in 0.2,
0.4th, the variation of 0.6,0.8,1.0,1.2,1.4,1.6,1.8 μM of HSA fluorescence intensities.The a length of 580nm of excitation light wave, ordinate
For fluorescence intensity.
Figure 11 is 10 μM of probe NIR-HSA of the invention time stability in PBS (10mM pH 7.4) buffer solution,
Every 5min run-down fluorescence intensities.Abscissa be the different time (min), a length of 580nm of excitation light wave.
Figure 12 be the present invention probe NIR-HSA Job, s Plot curves.[dyestuff+HSA]=10 μM, abscissa [dye
Material]/([dyestuff+HSA]) respectively from 0.1 to 1.0 map, a length of 580nm of excitation light wave.
Figure 13 be 5 μM of probe NIR-HSA of the present invention in PBS (10mM pH 7.4) buffer solution, add in 50 μM
Every 1s surface sweepings once fluorescence intensity in 0~400s after the HSA response times.The a length of 580nm of excitation light wave.
Figure 14 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, be separately added into 1mM
Sodium ion (2), 1mM magnesium ions (3), 1mM calcium ions (4), 1mM zinc ions (5), 1mM potassium ions (6), 1mM lead ions (7),
F when 1mM manganese ions (8), 1mM ammonium ions (9), 1mM nickel ions (10), 100 μM of HSA (11)680Fluorescence intensity at nm
Variation.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 15 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, be separately added into 1mM
Fluorine ion (2), 1mM chlorions (3), 1mM bromide ions (4), 1mM iodide ions (5), 1mM nitrate ions (6), 1mM nitrite anions
Ion (7), 1mM perchlorates (8), 1mM sulfate radicals (9), 1mM thiocyanate ions (10), 1mM acetates (11), 1mM
F when phosphoric acid hydrogen radical ion (12), 1mM carbanions (13), 1mM bicarbonate radicals (14), 100 μM of HSA (15)680It is glimmering at nm
The variation of luminous intensity.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 16 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, be separately added into 50 μ
M histidines (2), 50 μM of glycine (3), 50 μM of tryptophans (4), 50 μM of phenylalanines (5), 50 μM of serines (6), 50 μM of asparagus ferns
When amide (7), 50 μM of glutamine (8), 50 μM of asparatates (9), 50 μM of cysteines (10), 100 μM of HSA (11)
F680The variation of fluorescence intensity at nm.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 17 be the present invention 10 μM of probe NIR-HSA-2 in PBS (10mM pH 7.4) buffer solution, be separately added into
1mM sodium ions (2), 1mM magnesium ions (3), 1mM calcium ions (4), 1mM zinc ions (5), 1mM potassium ions (6), 1mM lead ions
(7), F when 1mM manganese ions (8), 1mM ammonium ions (9), 1mM nickel ions (10), 100 μM of HSA (11)680Fluorescence at nm is strong
The variation of degree.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 18 be the present invention 10 μM of probe NIR-HSA-2 in PBS (10mM pH 7.4) buffer solution, be separately added into
1mM fluorine ions (2), 1mM chlorions (3), 1mM bromide ions (4), 1mM iodide ions (5), 1mM nitrate ions (6), 1mM nitrous
Acid ion (7), 1mM perchlorates (8), 1mM sulfate radicals (9), 1mM thiocyanate ions (10), 1mM acetates
(11), when 1mM phosphoric acid hydrogen radical ions (12), 1mM carbanions (13), 1mM bicarbonate radicals (14), 100 μM of HSA (15)
F680The variation of fluorescence intensity at nm.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 19 be the present invention 10 μM of probe NIR-HSA-2 in PBS (10mM pH 7.4) buffer solution, be separately added into
50 μM of histidines (2), 50 μM of glycine (3), 50 μM of tryptophans (4), 50 μM of phenylalanines (5), 50 μM of serines (6), 50 μM
When asparagine (7), 50 μM of glutamine (8), 50 μM of asparatates (9), 50 μM of cysteines (10), 100 μM of HSA (11)
F680The variation of fluorescence intensity at nm.Wherein 1 be blank control, a length of 580nm of excitation light wave.
Figure 20 be the present invention 5 μM of probe NIR-HSA and 5 μM of probe NIR-HSA add in 15 μM of HSA pH value be 4,
5th, 6,7,7.4,8,9 when F680Nm fluorescence intensities.The a length of 580nm of excitation light wave.
Figure 21 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution, in different temperatures
Under fluorescence intensity.Figure 21 a are temperature from 5 DEG C~60 DEG C of fluorescence pattern；Figure 21 b are 60 DEG C~5 DEG C of fluorescence patterns.Excitation
The a length of 580nm of light wave.
Figure 22 is 10 μM of probe NIR-HSA of the invention thermal stability in PBS (10mM pH 7.4) buffer solution.Figure
22a be temperature in the environment of 60 DEG C, scan fluorescence spectrum every 5min；Figure 22 b are extraction F697Fluorescence at nm is strong
Degree, abscissa be the time (min), a length of 580nm of excitation light wave.
Figure 23 be the present invention 10 μM of probe NIR-HSA in PBS (10mM pH 7.4) buffer solution 5 DEG C fluorescence it is strong
Degree.1 fluorescence intensity for being heated to take 5 DEG C during 60 DEG C for 5 DEG C；2 are heated to take 5 DEG C of fluorescence strong during 5 DEG C for 60 DEG C
Degree；3 is cool to 5 DEG C of fluorescence intensity, a length of 580nm of excitation light wave after continuing 2h under 60 DEG C of environment.
Figure 24 is the cell imaging experiment of probe NIR-HSA of the present invention.With 10 μM of probe NIR-HSA and 100 μM of HSA mixing
After serum free medium is incubated mono- hour of normal liver cell HL-7702, cell imaging (d-f) is then carried out.A-c is serum-free
The cell imaging of medium culture.A, two figures of b are HL-7702 cell imagings, and a, d are fluorogram, and b, e are light field figure.C, f is phase
The stacking chart's cell imaging answered.
Figure 25 is that probe of the present invention seralbumin in human urine sample is detected is tested.
Specific embodiment
Half cyanine fluorochrome of the present invention has the structure of general formula I：
In general formula I,
The R1Selected from H, C1-6Alkyl, phenyl, C1-6Phenyl that alkyl arbitrarily replaces, SO3R5And COOR5；The R5Choosing
From H and C1-6Alkyl；It is preferred that the R1Selected from H, C1-6Alkyl, phenyl and C1-6The phenyl that alkyl arbitrarily replaces；It is more highly preferred to
Ground, the R1Selected from H, phenyl and C1-4Alkyl；Most preferably, the R1It is methyl or phenyl.
The R2Selected from H, C1-6Alkyl, SO3R6And COOR6；The R6Selected from H and C1-6Alkyl；It is preferred that the R2Choosing
From H and C1-6Alkyl.
The R3Selected from H and C1-6Alkyl, preferably C1-4Alkyl；
The R4Selected from H and C1-6Alkyl, preferably C1-4Alkyl；
Y-For Cl-、Br-Or I-, preferably Br-Or I-。
More specifically in embodiment, half cyanine fluorochrome is selected from following compound：
On the other hand, the present invention provides the preparation method of half cyanine fluorochrome, includes the following steps：
(1) compound 1 and compound 2 are according to molar ratio 1:1.2~1.5 reaction prepare compounds 3；
(2) compound 3 and compound 4 are according to molar ratio 1:1.0~1.2 reactions prepare compounds of formula I：
In specific embodiment, the preparation method of half cyanine fluorochrome includes the following steps：
(1) using acetonitrile as reaction dissolvent, compound 1 and compound 2 are according to molar ratio 1:1.2~1.5 back flow reactions, thin plate
Thin layer chromatography (TCL) monitoring reaction carry out degree, system is cooled to room temperature after reaction completely；After solid completely precipitation, vacuum
It filters, the solid of gained washs with ether, and obtained solid product finally is placed on vacuum drying chamber is dried overnight to get to change
Close object 3；
For volatile compound 2, preferably in N in the reaction process2It is carried out under gas shield；
(2) using acetic anhydride as solvent, compound 3 and compound 4 are according to molar ratio 1:1.0~1.2, in N2Protection is lower to flow back
Reaction, thin plate chromatography chromatography (TCL) monitoring reaction carry out degree, system is cooled to room temperature after reaction completely, adds in ice water and is quenched
Reaction；Ethyl acetate extracts three times, and then with brine organic phase three times, anhydrous sodium sulfate is dried overnight, and decompression filters, rotation
It steams；It purifies to obtain compounds of formula I through post separation technology.
The following examples can make those of ordinary skill in the art that present disclosure be more fully appreciated, but not
Add and limit the invention in any way.
Embodiment 1
The synthesis of compound N IR-HSA
The synthesis step of compound N IR-HSA is as follows：
(1) synthesis of compound 3
2- methylbenzothiazoles (0.50g, 3.93mM), iodoethane (0.62g, 4mM) are dissolved in 15ml acetonitriles, in N2It is lazy
Property gas shield under, flow back 8h.After reaction system is cooled to room temperature, the ether of 20ml is added in, reaction system has a large amount of solid
Body Precipitation.Then, decompression filters, and after being washed three times with the ether of 5ml, is placed on vacuum drying chamber and is dried overnight.It obtains
The compound 3 of 0.84g, yield 70%.
(2) synthesis of compound N IR-HSA
Compound 3 (0.31g, 1mM), 4- dimethylaminocinnamaldehydes (0.18,1mM) are dissolved in 10ml acetic anhydrides, heated
It flows back after 30min, treats that reaction system is cooled to room temperature, add in 15ml distilled water and reaction is quenched.Then, with the ethyl acetate of 20ml
Extraction three times, then adds in anhydrous sodium sulfate and is dried overnight.Decompression filters, and silicagel column separating-purifying is used after Rotary Evaporators evaporation
Obtain blue-green solid 0.30g, yield 65%.The polarity of eluant, eluent：Methylene chloride/methanol is from 100/1-50/1 gradient elutions.
The structure of compound is characterized by nuclear-magnetism and high resolution mass spectrum.
1H NMR(400MHz,DMSO):δ 8.34 (d, J=8.1,1H), 8.19 (d, J=8.4,1H), 8.02 (dd, J=
), 14.4,11.0,1H 7.81 (t, J=7.7,1H), 7.71 (t, J=7.7,1H), 7.55 (d, J=8.8,2H), 7.47 (d, J
=15.0,1H), 7.37 (d, J=14.5,1H), 7.22 (dd, J=14.9,11.0,1H), 6.80 (d, J=8.8,2H),
4.74 (q, J=7.2,2H), 3.05 (s, 6H), 1.44 (t, J=7.1,3H)
13C NMR(101MHz,MeOD):δ170.13,152.28,151.73,148.37,140.87,130.61,
129.26,127.74,127.63,124.23,122.94,122.29,115.92,112.25,112.01,111.62,43.77,
13.76.
HRMS-ESI:M/z theoretical values：C21H23N2S+,335.1576；Measured value：335.1582.
Embodiment 2
The synthesis of compound N IR-HAS-2
The synthesis step of compound N IR-HAS-2 is as follows：
(1) synthesis of compound 6
2- methylbenzothiazoles (0.50g, 3.93mM), cylite (0.68g, 4mM) are dissolved in 15ml toluene, in N2It is lazy
Property gas shield under, flow back 8h.After reaction system is cooled to room temperature, the ether of 20ml is added in, reaction system has a large amount of solid
Body Precipitation.Then, decompression filters, and after being washed three times with the ether of 5ml, is placed on vacuum drying chamber and is dried overnight.It obtains
The compound 6 of 0.81g, yield 65%.
Compound 6 (0.32g, 1mM), 4- dimethylaminocinnamaldehydes (0.18,1mM) are dissolved in 10ml acetic anhydrides, heated
It flows back after 30min, treats that reaction system is cooled to room temperature, add in 15ml distilled water and reaction is quenched.Then, with the ethyl acetate of 20ml
Extraction three times, then adds in anhydrous sodium sulfate and is dried overnight.Decompression filters, and silicagel column separating-purifying is used after Rotary Evaporators evaporation
Obtain blue-green solid 0.30g, yield 65%.The polarity of eluant, eluent：Methylene chloride/methanol is from 100/1~50/1 gradient elution.
The structure of compound is characterized by nuclear-magnetism and high resolution mass spectrum.
1H NMR(400MHz,MeOD):δ 8.20-8.12 (m, 1H), 8.05 (dd, J=14.2,11.1Hz, 1H), 7.95
(d, J=8.1Hz, 1H), 7.78-7.69 (m, 1H), 7.67 (dd, J=11.2,4.1Hz, 1H), 7.55 (d, J=9.0Hz,
2H), 7.40 (dd, J=9.3,3.6Hz, 3H), 7.28 (d, J=6.8Hz, 2H), 7.25-7.16 (m, 2H), 7.16-7.08 (m,
1H), 6.75 (d, J=9.0Hz, 2H), 5.96 (s, 3H), 5.96 (s, 2H), 3.08 (s, 6H)
13C NMR(126MHz,MeOD):δ173.10,154.84,154.52,152.11,142.90,134.63,
132.48,130.61,130.51,129.91,129.14,128.85,127.67,124.77,124.73,123.18,117.02,
113.22,111.31,52.22,40.21.
HRMS-ESI:M/z theoretical values：C21H23N2S+,397.1733；Measured value：397.1745.
Embodiment 3
Compound N IR-HSA excitation spectrums after PBS (10mM pH 7.4) is with reference to HSA are tested with emission spectrum normalization：
The probe NIR-HSA synthesized using example 1 is first made into mother liquor with dimethyl sulfoxide solvent in volumetric flask.Then micro-sampling is used
Device samples, and with PBS (10mMpH 7.4) by the concentration dilution of fluorescence probe NIR-HSA mother liquors to 10 μM, and ensures dimethyl Asia
The addition volume of sulfone is less than the one thousandth of total volume.It then adds in 50 μM of HSA to be uniformly mixed, changes different excitation waves
It is long, from 520~600nm at interval of 10nm, run-down fluorescence spectrum.Then maximum fluorescence emission wavelengths, counter-scanning are selected
The excitation wavelength of dyestuff, dyestuff excitation spectrum and the emission spectrum of normalized are shown in Fig. 1.Left curve be excitation spectrum, right song
Line is emission spectrum.
Instrument is Agilent sepectrophotofluorometer (model：G9800A, number MY15210003).
Embodiment 4
Probe NIR-HSA is tested in the fluorescent emission of different solvents with ultraviolet-visible absorption spectroscopy：
Sampled from probe NIR-HSA mother liquors with microsyringe, be added separately to Isosorbide-5-Nitrae-dioxane, dichloromethane,
Ethyl acetate, acetonitrile, methanol, ethyl alcohol, n,N-Dimethylformamide and PBS (10mMpH 7.4), are finally diluted to 10 μM of NIR-
HSA solution, by the use of 580nm as excitation wavelength, test result is as shown in Figure 2 a.The probe molecule is found from the interpretation of result of test
More sensitive to the polarity of solution, fluorescence is most strong in methylene chloride, most weak in PBS (10mM pH 7.4) on the contrary.Pass through test
The compound different solvents ultraviolet-visible absorption spectra figure, as a result, it has been found that 10 μM of NIR-HSA probes occur in methylene chloride
Red shift, and absorption intensity highest；There is blue shift, and absorption intensity is minimum in PBS (10mM pH 7.4).In addition to this, exist
Ultravioletvisible absorption collection of illustrative plates is similar in other solvents.
Instrument is Agilent ultraviolet-uisible spectrophotometer (model：G6860A, number MY1523004) and Agilent
Sepectrophotofluorometer (model：G9800A, number MY15210003).
Embodiment 5
Probe NIR-HSA-2 is combined rear ultra-violet absorption spectrum experiment with HSA：
The probe NIR-HSA-2 synthesized using example 2 is first made into mother liquor with dimethyl sulfoxide solvent in volumetric flask.Then
It is sampled with microsyringe, with PBS (10mM pH 7.4) by the concentration dilution of fluorescence probe NIR-HSA mother liquors to 10 μM, and really
The addition volume for protecting dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.20 μM of HSA is then added in be uniformly mixed, with it is ultraviolet can
See that spectrophotometer test probe NIR-HSA-2 is combined rear ultra-violet absorption spectrum with HSA cavitys.Absorption maximum is found by test
Peak about near 575nm (Fig. 3).
Embodiment 6
Probe NIR-HSA viscosity experiments：
Measure the volume of glycerine and PBS (10mM, pH 7.4) respectively with syringe, the glycerine prepared respectively/PBS (10mM
PH 7.4) mixed solution of (v/v) volume ratio 0/10,1/9,2/8,3/7,4/6,5/5,6/4,7/3,8/2,9/1,10/0 treats
With.Then probe NIR-HSA is taken to add in above-mentioned prepared mixed liquor, the probe finally prepared from mother liquor with microsyringe
A concentration of 10 μM.Ultrasound 10 minutes, stands after removing bubble therein, it is measured on sepectrophotofluorometer and emits light
Spectrum.As Fig. 4 be test fluorescence spectrum as a result, it is seen that with glycerine proportion increase, the fluorescence intensity of probe NIR-HSA
Increase.May be because the viscosity with solution system increases, the existing torsion of probe molecule is inhibited.The energy of excitation state
It is discharged to reverse to dissipate to be converted in the form of heat radiation with fluorescent form.
Embodiment 7
Probe compound NIR-HSA is to the concentration titrations fluorescence spectrum experiments of HSA：
It is sampled from mother liquor with microsyringe, is diluted to 10 μM PBS (10mM pH 7.4) solution, and ensure diformazan
The addition of base sulfoxide is less than the one thousandth of total volume.Sequentially add 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,
0.9、1.0、2.0、3.0、4.0、5.0、6.0、8.0、10.0、15.0、20.0、30.0、50.0、70.0、90.0、95.0、
100.0th, 105.0,110.0 μM of HSA solution.Often HSA of addition needs to wait for more than 10s and detects its change in fluorescence situation again.
As shown in figure 5, the addition with HSA is more, fluorescence intensity constantly increases test result, while intensity increase,
Launch wavelength slightly blue-shifted phenomenon, blue shift 20nm or so.
Embodiment 8
Probe compound NIR-HSA tests the concentration titrations uv-vis spectra of HSA：
It is sampled from mother liquor with microsyringe, is diluted to 10 μM PBS (10mM pH 7.4) solution, and ensure diformazan
The addition of base sulfoxide is less than the one thousandth of total volume.Sequentially add 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,
0.9、1.0、2.0、3.0、4.0、5.0、6.0、8.0、10.0、15.0、20.0、30.0、50.0、70.0、90.0、95.0、
100.0th, 105.0,110.0 μM of HSA solution.Often HSA of addition needs to wait for more than 10s and detects uv-vis spectra variation again
Situation.Test result is as shown in fig. 6, the addition with HSA is more, under the uv-vis spectra absorption intensity at 512nm
Drop, while declining, slightly Red Shift Phenomena, red shift 13nm or so.
Embodiment 9
Probe compound NIR-HSA-2 is to the concentration titrations fluorescence spectrum experiments of HSA：
It is sampled from probe NIR-HSA-2 mother liquors with microsyringe, it is molten to be diluted to 10 μM of PBS (10mM pH 7.4)
Liquid, and ensure that the addition of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Sequentially add 0.2,0.4,0.6,0.8,1.0,
1.2nd, 1.4,1.6,1.8,4.0,6.0,8.0,10.0,12.0,14.0,16.0,18.0,20.0 μM of HSA solution.Often add in one
Secondary HSA needs to wait for more than 10s and detects its change in fluorescence situation again.Test result as shown in fig. 7, the addition with HSA is more,
Its fluorescence intensity constantly increases.
Embodiment 10
Probe compound NIR-HSA tests the concentration titrations saturability of HSA：
Specific experiment is operated on the basis of example 6, extracts the fluorescence intensity at 680nm as HSA additions are mapped,
As shown in figure 8, the addition with HSA is bigger, F680Nm fluorescence intensities are bigger, glimmering about when its addition reaches 100 μM
Luminous intensity reaches maximum value.
Embodiment 11
Probe compound NIR-HSA limits experiment to the detection of HSA：
Microsyringe is sampled from probe NIR-HSA mother liquors, is diluted to 10 μM PBS (10mM pH 7.4) solution, and
Ensure that the addition of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Sequentially add 0.1,0.2,0.3,0.4,0.5,0.6,
0.7th, 0.8,0.9,1.0 μM of HSA solution.Often HSA of addition needs to wait for more than 10s and detects fluorescence spectrum situation of change again.
Then the fluorescence intensity at 700nm is taken as shown in figure 9, as HSA additions are bigger, and fluorescence intensity incrementss are in a linear relationship.
Fluorescence intensity and HSA concentration can be done into straight line, linear coefficient R2=0.9978.Five blank controls are tested, pass through detection
Calculation formula the DL=3 σ/k, wherein σ of limit are the standard variance of five blank controls, and k is the slope of fitting a straight line.And then it can count
Calculation obtains probe NIR-HSA and is limited to 1.73mg/L to the detection of HSA.
Embodiment 12
Probe compound NIR-HSA-2 limits experiment to the detection of HSA：
Microsyringe is sampled from probe NIR-HSA-2 mother liquors, is diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Sequentially add 0.2,0.4,0.6,0.8,1.0,1.2,
1.4th, 1.6,1.8 μM of HSA solution.Often HSA of addition needs to wait for more than 10s and detects fluorescence spectrum situation of change again.Then
Take the fluorescence intensity at 700nm as shown in Figure 10, as HSA additions are bigger, fluorescence intensity incrementss are in a linear relationship.It will be glimmering
Luminous intensity can be straight line, linear coefficient R with HSA concentration2=0.9993.Five blank controls are tested, by detecting limit
Calculation formula DL=3 σ/k, wherein σ are the standard variance of five blank controls, and k is the slope of fitting a straight line.And then it can calculate
0.930mg/L is limited to the detection of HSA to probe NIR-HSA.
Embodiment 13
Probe compound NIR-HSA stabilities of solution are tested：
The mother liquor of probe molecule NIR-HSA is diluted to 10 μM PBS (10mM pH 7.4) solution, and ensures dimethyl
The addition volume of sulfoxide is less than the one thousandth of total volume.With sepectrophotofluorometer every 5min run-down fluorescence spectrums,
70min is scanned altogether, its fluorescence intensity at 700nm is extracted, draws the scatter plot with time change, such as row's point institute under Figure 11
Show.Then it adds in 100 μM of HSA to be uniformly mixed, equally be carried with sepectrophotofluorometer every 5min run-down fluorescence spectrums
Its fluorescence intensity at 680nm is taken to draw with the scatter plot of time change, as shown in row's point on Figure 11.
Embodiment 14
Probe compound NIR-HSA is tested with HSA coordination ratios：
By the mother liquor of probe molecule NIR-HSA and HSA mother liquors in PBS (10mM pH 7.4) solution according to [dyestuff]/([dye
Material]+[HSA])=0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0 ratio relation is prepared, wherein [dye
Material]+[HSA]=10 μM, it then scans the fluorescence spectrum of above-mentioned different mixed systems respectively with sepectrophotofluorometer, extracts
Fluorescence intensity at 680nm is mapped with [dyestuff]/([dyestuff]+[HSA]) ratios, and [dyestuff] as shown in figure 12/([dyestuff]+
[HSA])=0.5 when two fitting a straight lines cross, it is 1 that can measure NIR-HSA and HSA coordination ratios：1.
Embodiment 15
Probe compound NIR-HSA is tested with the HSA response times：
The mother liquor of probe molecule NIR-HSA is diluted to 5 μM PBS (10mM pH 7.4) solution, and ensures dimethyl Asia
The addition volume of sulfone is less than the one thousandth of total volume.Then time sweep fluorescence intensity is carried out with sepectrophotofluorometer, often
Every 1s run-downs, 400s is scanned altogether.50 μM of HSA is added in 35s, continues to carry out the time with sepectrophotofluorometer to sweep
It retouches, finally obtains the fluorescence intensity at 680nm and do a curve with the time, obtain Figure 13.It can be seen from the figure that after adding in HSA
Fluorescence has the tendency that enhancing at once, and fluorescence intensity can reach maximum after 5s.Thus illustrate probe NIR-HSA to external HSA
Respond rapidly to.
Embodiment 16
Probe compound NIR-HSA cation selectives are tested：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Be then respectively adding 100 μM of HSA, 1mM sodium from
Son, magnesium ion, calcium ion, zinc ion, potassium ion, lead ion, manganese ion, ammonium ion, nickel ion.It is scanned after standing 1 minute
Fluorescence spectrum extracts the fluorescence intensity at its 680nm and does block diagram.Test result such as Figure 14, from the results, it was seen that the probe
Other cationic ions are not responded to substantially, and it is stronger to the response of HSA.
Embodiment 17
Probe compound NIR-HSA anion selectivities are tested：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Be then respectively adding 100 μM of HSA, 1mM fluorine from
Son, chlorion, bromide ion, iodide ion, nitrate ion, nitrite ion, perchlorate, sulfate radical, thiocyanate radical
Ion, acetate, phosphoric acid hydrogen radical ion, carbanion, bicarbonate radical.Fluorescence spectrum is scanned after standing 1 minute, extracts it
Fluorescence intensity at 680nm does block diagram.Test result such as Figure 15, from the results, it was seen that the probe to other anion from
Subbase does not originally respond to, and stronger to the response of HSA.
Embodiment 18
Probe compound NIR-HSA amino acid is selectively tested：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.It is then respectively adding 100 μM of HSA, 50 μM of group ammonia
Acid, glycine, tryptophan, phenylalanine, serine, asparagine, glutamine, asparatate, cysteine, acetic acid
Root, phosphoric acid hydrogen radical ion, carbanion, bicarbonate radical.Fluorescence spectrum is scanned after standing 1 minute, is extracted glimmering at its 680nm
Luminous intensity does block diagram.Test result such as Figure 16, from the results, it was seen that the probe does not ring other anionic ions substantially
Should, and it is stronger to the response of HSA, illustrate that probe NIR-HSA has HSA ions selectivity well.
Embodiment 19
Probe compound NIR-HSA-2 cation selectives are tested：
The NIR-HSA-2 mother liquors that preparation is taken with microsyringe are a little, and it is molten to be diluted to 10 μM of PBS (10mM pH 7.4)
Liquid, and ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.It is then respectively adding 20 μM of HSA, 1mM sodium
Ion, magnesium ion, calcium ion, zinc ion, potassium ion, lead ion, manganese ion, ammonium ion, nickel ion.It is swept after standing 1 minute
Fluorescence spectrum is retouched, the fluorescence intensity at its 680nm is extracted and does block diagram.Test result such as Figure 17, from the results, it was seen that the spy
Needle NIR-HSA-2 does not respond to other cationic ions substantially, and stronger to the response of HSA.
Embodiment 20
Probe compound NIR-HSA-2 anion selectivities are tested：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Be then respectively adding 20 μM of HSA, 1mM fluorine from
Son, chlorion, bromide ion, iodide ion, nitrate ion, nitrite ion, perchlorate, sulfate radical, thiocyanate radical
Ion, acetate, phosphoric acid hydrogen radical ion, carbanion, bicarbonate radical.Fluorescence spectrum is scanned after standing 1 minute, extracts it
Fluorescence intensity at 680nm does block diagram.Test result such as Figure 18, from the results, it was seen that probe NIR-HSA-2 is to other
Anionic ion does not respond to substantially, and stronger to the response of HSA.
Embodiment 21
Probe compound NIR-HSA-2 amino acid is selectively tested：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM PBS (10mM pH 7.4) solution,
And ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.It is then respectively adding 20 μM of HSA, 50 μM of group ammonia
Acid, glycine, tryptophan, phenylalanine, serine, asparagine, glutamine, asparatate, cysteine, acetic acid
Root, phosphoric acid hydrogen radical ion, carbanion, bicarbonate radical.Fluorescence spectrum is scanned after standing 1 minute, is extracted glimmering at its 680nm
Luminous intensity does block diagram.Test result such as Figure 19, from the results, it was seen that the probe does not ring other anionic ions substantially
Should, and it is stronger to the response of HSA, illustrate that probe NIR-HSA-2 has HSA ions selectivity well.
Embodiment 22
Probe compound NIR-HSA influences to test in different pH value:
PH value is adjusted with hydrochloric acid and sodium hydroxide, is configured to the buffer solution of pH=4,5,6,7,7.4,8,9.Then with micro-
Amount injector takes the NIR-HSA mother liquors of preparation a little, is diluted to 5 μM of solution respectively, and ensure the addition body of dimethyl sulfoxide (DMSO)
Product is less than the one thousandth of total volume.Fluorescence spectrum is scanned after standing 1 minute, the fluorescence intensity at its 680nm is extracted and does column
Figure.Such as Figure 20 (left one side of something), show that fluorescence intensity is not influenced substantially by pH value.Then add in 15 μM of HAS, mixing with it is quiet
Fluorescence spectrum is scanned after putting 1 minute, the fluorescence intensity at its 680nm is extracted and does block diagram.Such as Figure 20 (right one side of something), in physiological pH
During value, ideal fluorescence intensity is obtained.
Embodiment 23
Probe compound NIR-HSA is in alternating temperature process experiment:
The probe NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM of PBS (10mM pH 7.4)
Solution, and ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Adjust the constant temperature of sepectrophotofluorometer
Control device sets 5,10,15,20,25,30,35,40,50,60 DEG C of temperature gradients respectively.At the corresponding temperature, 5min is stood
To reach stable system.Then fluorescence spectrum is scanned with sepectrophotofluorometer, such as Figure 21 a, shows the raising with temperature, it is right
The fluorescence intensity answered reduces.In turn, come probe into heating after invertibity, taken from above-mentioned 60 DEG C gradient cooling mode come
Probe into influence of the temperature to probe.Specific temperature setting is 60,50,40,35,30,25,20,15,10,5 DEG C, stands 5min
To reach stable system.Then fluorescence spectrum is scanned with sepectrophotofluorometer, such as Figure 21 b, shows the reduction with temperature, it is right
The fluorescence intensity increase answered.
Embodiment 24
Probe compound NIR-HSA stability experiments under high temperature environment
The probe NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM of PBS (10mM pH 7.4)
Solution, and ensure that the addition volume of dimethyl sulfoxide (DMSO) is less than the one thousandth of total volume.Adjust the constant temperature of sepectrophotofluorometer
Control device is arranged to 60 DEG C, is put into sample cell, stands 5min to reach system temperature stabilization, then uses fluorescence spectrophotometry
Meter scans 120min, test result such as Figure 22 a altogether every 5min run-down fluorescence spectrums.Extract the fluorescence intensity at 700nm
It maps with the time, such as Figure 22 b, shows that probe NIR-HSA has good thermal stability at high temperature.After 2h, regulating thermostatic
Control device, set temperature are 5 DEG C, treat that temperature continues to stand 5min to reach system temperature stabilization after reaching, then with glimmering
Light spectrophotometer scans fluorescence spectrum, takes the fluorescence intensity at 700nm with 17 two process middle probe NIR-HSA of example 5
DEG C the mapping of 700nm fluorescence intensity levels, such as Figure 23 shows that structure is not destroyed probe NIR-HSA after 2h at high temperature, is still had
There is the invertibity of temperature.
Embodiment 25
The living cells experiment of probe NIR-HSA：
The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, and 10 μM of NIR-HSA are diluted to serum free medium
With 100 μM of HSA mixed solution cultures, at 37 DEG C, 5%CO2It lower incubation mono- hour of normal liver cell HL-7702, then uses
Laser co-focusing carries out cell imaging experiment.Representative area is chosen, is observed with oil mirror (60 ×), in triplicate.Excitation wavelength：
559nm, receiving wave range are：600 arrive 700nm.Imaging results two figure of such as Figure 24, a, d is the fluorogram of HL-7702 cell imagings,
B, e is light field figure, and c, f are the stacking chart of two channels.Wherein a-c is that the liver cell HL-7702 of serum free medium culture is empty
White control.Fluorogram shows that probe NIR-HSA combination HSA compounds easily enter cell, and glimmering available for living cells
Light is imaged.
Embodiment 26
Experiments of the probe NIR-HSA in human urine sample：
The freshly voided urine of male volunteers is collected, the NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10
μM urine solution, and ensure dimethyl sulfoxide (DMSO) addition volume be less than total volume one thousandth.Urine wherein in urine is micro-
The content of amount albumin is tested in the attached Second Academy of Dalian Medical Univ, and test result is 0.41 μM (27mg/L).Respectively again
Toward be diluted to 10 μM urine solution add in 1,2,3,4,5,6,7,8,9,10,11,13,15,17,19,21,26,31,36,41,
51st, 61,71,81,91,101 μM of HSA are uniformly mixed, and stand 2min to reach system temperature stabilization, then with fluorescence spectrophotometer light
Degree meter scans its fluorescence spectrum.As shown in Figure 25 test results, with the increase of human serum albumins, fluorescence intensity is with increasing
By force.The fluorescence intensity at 700nm is taken to be fitted straight line, linear good relationship R with concentration2=0.9994, show to lead to
The human serum albumins in this method detection human urine is crossed, and then speculates that its renal function is likely to occur damage, plays early warning
With the effect of identification.
In order to investigate accuracys of the probe NIR-HSA in urine detection human serum albumins, following experiment is also carried out and has surveyed
Examination.The NIR-HSA mother liquors that preparation is taken with microsyringe are a little, are diluted to 10 μM of above-mentioned urine solution, and ensure dimethyl
The addition volume of sulfoxide is less than the one thousandth of total volume.20 μM, 30 μM of human serum albumins are separately added into, are uniformly mixed, it is quiet
2min is put to reach system temperature stabilization, then scans its fluorescence spectrum with sepectrophotofluorometer, the sample of each concentration is surveyed
Examination is three times.The Fluorescence Fluorescence intensity at 700nm is taken, with the linear relation equation y=2.13x+36.89 of fitting, calculates it
In concentration be respectively 22.00 ± 0.22 μM, 32.61 ± 0.03 μM, recovery of standard addition is respectively 108.79%, 107.23%,
Corresponding relative standard deviation is respectively 1.00%, 0.09%.The serum that probe NIR-HSA can be accurately detected in human urine is white
The content of albumen has practical application value.
Claims (4)
1. application of half cyanine fluorochrome in albumin identification and detection reagent is prepared, half cyanine fluorochrome,
Structure with general formula I：
In general formula I,
R1Selected from phenyl or C1-6The phenyl that alkyl arbitrarily replaces；
R2Selected from H, C1-6Alkyl, SO3R6And COOR6；The R6Selected from H and C1-6Alkyl；
R3And R4It is each independently selected from H and C1-6Alkyl；
Y-For Cl-、Br-Or I-。
2. application according to claim 1, which is characterized in that the R2Selected from H and C1-6Alkyl.
3. application according to claim 1, which is characterized in that the R3And R4It is each independently selected from C1-4Alkyl.
4. application according to claim 1, half cyanine fluorochrome is selected from following compound：
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