CN105838355B - Small molecule fluorescence probe and its application - Google Patents

Small molecule fluorescence probe and its application Download PDF

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CN105838355B
CN105838355B CN201610285502.2A CN201610285502A CN105838355B CN 105838355 B CN105838355 B CN 105838355B CN 201610285502 A CN201610285502 A CN 201610285502A CN 105838355 B CN105838355 B CN 105838355B
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hsa
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CN105838355A (en
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杜健军
朱涛
彭孝军
樊江莉
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Dalian University of Technology
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Abstract

Small molecule fluorescence probe and its application, the fluorescence probe are based on TICT mechanism, the structure with general formula I.Small-molecule fluorescent probe of the present invention background fluorescence in PBS buffer solution is weaker, and after the hydrophobic cavity of human serum albumin HSA is entered, fluorescence intensity shows significantly to enhance.Its selectivity is preferable, is not interfered by other common amino acids and protein molecule.In addition, the response time is shorter after it is acted on HSA, and can be combined with the FA1 locus specificities of HSA.In PBS buffer solution, the concentration of fluorescence intensity and HSA show good linear relationship, and can receive orders to human serum albumins HAS detection.Under the test environment of true urine, fluorescence intensity has good linear relationship with albumin concentration agriculture poplar in urine, therefore with good biologic applications prospect.

Description

Small molecule fluorescence probe and its application
Technical field
The present invention relates to field of fine chemical fluorescent probe, preparation method and use more particularly to one kind to be based on The small-molecule fluorescent probe of TICT mechanism, preparation method and its application in albumin label and quantitative context of detection.
Background technology
Detection technique of fluorescence has many advantages, such as that high sensitivity, selectivity are good, method is simple, in situ and detection in real time, has become For essential detection means in modern biotechnology and life science.In protein labeling technology, fluorescent marker Method has apparent advantage relative to other traditional labeling methods, increasingly causes the concern of researcher.
Human serum albumin HSA is the protein that content is most in serum, and the content in serum is on a 30-50g/L left sides It is right.Due to the Dialysis of kidney, the content of albumin is in below 30mg/L in urine.The physiologic function of albumin includes dimension Hold the osmotic pressure, balanced nutritious, transport drug and metabolin of blood vessel.Meanwhile the content of microalbumin and kidney disease in urine Disease is related with diabetes, can be as kidney trouble early treatment and the key index of intensification therapy of diabetes mellitus patients.Therefore it is being urinated Accurate detection in liquid has very important biology and medical significance.
Imaging-PAM provides the low trace analysis of a kind of good selectivity, high sensitivity, detection limit for people, It has been widely used in the analysis detection of large biological molecule such as protein, DNA and RNA.The side's of having been reported acids in document Dyestuff, BODIPY classes dyestuff and polar sensitive probe are used for the detection of albumin in water phase.But it was found that with good fluorescence property (such as high quantum production rate, long wavelength and property stablize), fluorescence probe convieniently synthesized, that albumin can be detected at low concentrations according to So face the challenge.We have synthesized a kind of HSA fluorescence probes based on TICT mechanism, can be realized in PBS buffer solution The fluorescence response of OFF-ON.Such probe is convieniently synthesized, only needs one-step or two-step reaction i.e. available, therefore with good warp Ji effect.In addition, such probe has TICT structures, while to HSA inner apolar environment sensitives, the freedom of intramolecular Rotation can be inhibited by protein cavity, have relatively low detection limit and good sensitivity.In addition to this, by introducing Luo Dan Peaceful electron withdrawing group realizes specific binding of the probe to HSA FA1 sites, is carried for research dyestuff with protein combination New approach is supplied.
Invention content
The present invention is intended to provide a kind of small-molecule fluorescent probe based on TICT mechanism, available for human seralbumin egg in water phase The quantitative detection of white HSA.
Present invention firstly provides small molecule fluorescence probes, have the following structure general formula I：
In general formula I：
R1-R7It is each independently selected from H, C1-8Alkyl, substitution or unsubstituted phenyl；
The substituted-phenyl is arbitrarily replaced by following group：CN、COOH、NH2、NO2、OH、SH、C1-6Alkoxy, C1-6Alkyl Amino, C1-6Amide groups, halogen or C1-6Halogenated alkyl；
X is oxygen or sulphur；
N is selected from the integer of 1-8.
On the other hand, the present invention provides the preparation method of above-mentioned small-molecule fluorescent probe, is in toluene, ammonium acetate and ice vinegar In the mixed system of acid, compounds of formula II reacts gained with compounds of formula III：
Fluorescence probe of the present invention background fluorescence in PBS buffer solution is weaker, after the hydrophobic cavity of HSA is entered, With apparent Fluorescence Increasing, and its fluorescence intensity has good linear relationship, while to other big point with protein concentration Sub- protein and each amino acid have good selectivity, and are not interfered by internal common zwitterion.In addition, its with The response time is shorter after HSA effects, and can be combined with the FA1 locus specificities of HSA.In PBS buffer solution, fluorescence intensity Good linear relationship is shown with the concentration of HSA, can receive orders to human serum albumins HAS detection.In the test of true urine Under environment, fluorescence intensity has good linear relationship with albumin concentration agriculture poplar in urine, therefore with good biology Application prospect.In consideration of it, the small-molecule fluorescent probe that is designed to provide of further aspect of the present invention is preparing albumen inspection Survey the application in composition.And based on this, a kind of composition for albumin detection is provided, the composition includes above-mentioned hair Bright small-molecule fluorescent probe.The composition is particularly suitable for micro white in human serum albumins (HSA) and urine sample The detection of albumen.
TICT mechanism small-molecule fluorescent probe of the present invention has following significant feature：
(1) the almost unstressed configuration in PBS buffer solutions, into protein cavity after have apparent Fluorescence Increasing；
(2) good linear relationship is presented with protein concentration in fluorescence intensity of the probe in buffer solution, available for white egg White quantitative detection；
(3) selectivity is good, to other protein and amino acid almost without response；
(4) it can be combined with the FA1 locus specificities of albumin, the mode of action for research dyestuff and protein provides Powerful method；
(5) convieniently synthesized, product is easy to get.
Description of the drawings
8 width of attached drawing of the present invention,
Fig. 1 be performance measurement experiment 1 in fluorescent probe compounds ES1, ES2, ES3 to human serum albumins (HSA) fluorescence Titration experiments test chart.A concentration of 5 μM of probe molecule, test system are probe in PBS buffer solutions (pH=7.4,10mM) The excitation wavelength of molecule ES1 is 474nm, and the excitation wavelength of ES2 is 507nm, and the excitation wavelength of ES3 is 430nm.
Fig. 2 be performance measurement experiment 2 in fluorescent probe compounds ES1 to human serum albumins (HSA) and different aminoacids Selectivity experiment block diagram.A concentration of 5 μM of a concentration of 5 μM of probe molecule ES1, HSA, a concentration of 50 μM of other amino acid. Excitation wavelength is 474nm, acquires the fluorescence intensity at 547nm.
Fig. 3 be performance measurement experiment 3 in fluorescent probe compounds ES1 to human serum albumins (HSA) and different proteins Selectivity experiment block diagram.A concentration of 5 μM of probe molecule ES1, HSA and a concentration of of other protein are 5 μM.Excitation wave A length of 474nm records the fluorescence intensity at 547nm.
Fig. 4 is fluorescent probe compounds ES1 and human serum albumin HSA combination Ratio Experiments figure in performance measurement experiment 4. The total concentration of fixed probe molecule ES1 and HSA are 10 μM, constantly the concentration ratio of change probe and HSA, respectively 1:9、2:8、3: 7、4:6、5:5、6:4、7:3、8:2、9:1.Excitation wavelength is 474nm, records the fluorescence intensity at 547nm.
Fig. 5 is the positioning experiment figure of fluorescent probe compounds ES1 and human serum albumin HSA in performance measurement experiment 5.It visits Needle molecular concentration is 10 μM, and adding in 2 μM of HSA makes itself and probe mixing 1h.The concentration for replacing drug is respectively 5,10,15,20, 30,40,60,80μM。
Fig. 6 be in performance measurement experiment 6 fluorescent probe compounds ES1 and human serum albumin HSA compound system to temperature Response diagram.The concentration of probe molecule and HSA are 5 μM, and temperature chooses 20,25,30,35,40,45,50 degrees Celsius respectively.
Fig. 7 is that fluorescent probe compounds ES1 schemes the time response of human serum albumin HSA in performance measurement experiment 7.It visits A concentration of 5 μM of needle molecule, a concentration of 5 μM of the HSA of addition.Abscissa is the time (min), and ordinate is fluorescence intensity.
Fig. 8 is that fluorescent probe compounds ES1 is real to the detection sensitivity of human serum albumin HSA in performance measurement experiment 8 Test figure.A concentration of 5 μM of probe molecule.Scheme the fluorescence intensity that a is (pH=7.4,10mM) middle probe molecule in PBS buffer solutions With the linear relationship between HSA concentration；It is linear between the fluorescence intensity of true urine middle probe molecule and HSA concentration to scheme b Relationship.Excitation wavelength is 474nm, records the fluorescence intensity at 547nm.
Specific embodiment
Unless otherwise stated, term used herein has following meanings.
Unless otherwise stated, term used herein has following meanings.
Term " alkyl " used herein is including straight chained alkyl and branched alkyl.As mentioned by single alkyl such as " propyl ", Straight chained alkyl is then only refered in particular to, as mentioned by single branched alkyl such as " isopropyl ", then only refers in particular to branched alkyl.For example, " C1-4Alkyl " Including methyl, ethyl, n-propyl, isopropyl, normal-butyl and tertiary butyl etc..Similar rule is also applied for using in this specification Other groups.
Small-molecule fluorescent probe of the present invention has the following structure general formula I：
In general formula I：R1-R7It is each independently selected from H, C1-8Alkyl, substitution or unsubstituted phenyl；
The substituted-phenyl is arbitrarily replaced by following group：CN、COOH、NH2、NO2、OH、SH、C1-6Alkoxy, C1-6Alkyl Amino, C1-6Amide groups, halogen or C1-6Halogenated alkyl；
In specific embodiment, the R1-R4It is each independently selected from H or C1-4Alkyl, preferably H or methyl are optimal Select H.
In specific embodiment, the R5And R6It is each independently selected from C1-4Alkyl, preferably methyl or ethyl are optimal Select methyl.
In specific embodiment, the R7Selected from H or C1-4Alkyl, preferably H or methyl, most preferably H.
In general formula I, the X is oxygen or sulphur, preferably S.
In general formula I, the n is selected from the integer of 1-8, the preferably integer of 1-3, particularly preferred 1 or 2, most preferably 1.
Each preferred feature described above can be combined with each other, and gained technical solution should be included in this hair completely Bright addressed range.
One of the example of the combination of preferred feature, be for specific embodiment of the present invention, it is of the present invention small Fluorescence probe, selected from following compound：
On the other hand, the present invention provides the preparation method of the small-molecule fluorescent probe, is in toluene, ammonium acetate and ice In the mixed system of acetic acid, compounds of formula II reacts gained with compounds of formula III：
In specific embodiment, the molar ratio of the compound II and compound III is 0.1-1000:1, preferably For 0.5-100:1, more preferable 0.5-10:1, further preferably 1-5:1, most preferably 1.5:1, condensation reaction, generating structure occur for the two General formula is the target compound of I.
Above reaction can carry out in aqueous organic solvent or anhydrous organic solvent.It is preferably anhydrous organic molten Agent.Organic solvent includes but are not limited to toluene, benzene, dichloromethane, tetrahydrofuran or acetonitrile etc., and preferably reaction dissolvent is first Benzene.Reaction process judges the terminal of reaction by thin-layer chromatography (TLC).
Above-mentioned reaction temperature is -10 DEG C -150 DEG C, preferably 111 DEG C -130 DEG C；Reacting optional acid or basic catalyst is Triethylamine, piperidines, ammonium acetate, sodium acetate and glacial acetic acid, preferred catalyst are ammonium acetate and glacial acetic acid mixed system.
The method of purification of the small molecule fluorescent dyestuff of the present invention is not particularly limited using conventional method.In general, reaction knot Shu Hou utilizes chromatographic column separating-purifying product after directly filtering or boiling off solvent.
Gained fluorescent dye can be recycled by separation well known in the art and purification technique, to reach the purity of needs.
The various raw materials used in the present invention are commercially available or can be by the way that well known to a person skilled in the art methods Or disclosed method is simply prepared by raw material well known in the art in the prior art.
It should be understood that the various ring substituents in the compounds of this invention have some before above-mentioned steps progress or just complete Cheng Hou is introduced or is generated by conventional modified with functional group by the aromatics substitution reaction of standard, this is included in the present invention Method and step in terms of.This reaction and modification include such as substituent group by the introducing of aromatics substitution reaction, substituent group also The former, alkylation of substituent group and the oxidation of substituent group.It is well known to chemical field for the reagent and reaction condition of these processes. The specific example of aromatics substitution reaction includes introducing nitro with concentrated nitric acid, is existed with such as carboxylic acid halides and lewis acid (such as alchlor) Acyl group is introduced under the conditions of Friedel Crafts, with alkyl halide and lewis acid (such as alchlor) in Friedel Crafts items Alkyl is introduced under part and introduces halogen group.The specific example of modification is included for example, by carrying out catalytic hydrogenation with Raney nickel Or heated in presence of hydrochloric acid with iron, nitro is reduced into amino；Alkylthio group is oxidized to alkyl sulphinyl Or alkyl sulphonyl.
Small-molecule fluorescent probe as described herein based on TICT mechanism has apparent fluorescence after albumin cavity is entered Restore, the quantitative detection available for microalbumin in urine.
Following non-limiting examples can make those of ordinary skill in the art be more fully understood the present invention, but not with Any mode limits the present invention.
Embodiment 1
The synthesis of fluorescent probe compounds ES1：
Dimethylamino benzaldehyde (1.49g, 10mmol) and rhodanine (1.33g, 10mmol) will be added in equipped with 10mL first In the round-bottomed flask of benzene, 200mg ammonium acetates and 1mL glacial acetic acids are added as catalyst, is warming up to 115 DEG C of back flow reactions.Instead Should solvent, the isolated red solid ES1 (78.25%) of chromatographic column be removed under reduced pressure for 24 hours.1H NMR (500MHz, DMSO-D6), δ:3.04 (s, 6H), 4.27 (s, 1H), 6.82 (t, J=10Hz, 2H), 7.42 (t, J=10Hz, 2H), 7.53 (s, 1H)；13C NMR(500MHz,DMSO-D6),δ:112.172,117.313,119.757,132.872,133.253,151.759, 169.371,176.645,194.973,105.280ppm；TOF MS:m/zcalcd for[M+H]+:265.0469,found: 265.0458.
Embodiment 2
The synthesis of fluorescent probe compounds ES2：
4- di methyl amino cinnamaldehydes (1.75g, 10mmol) and rhodanine (1.33g, 10mmol) are added in equipped with 12mL In the round-bottomed flask of toluene, 200mg ammonium acetates and 1mL glacial acetic acids are added as catalyst, is warming up to 115 DEG C of back flow reactions. Reaction for 24 hours, removes solvent, the isolated dark brown solid ES2 (65.46%) of chromatographic column under reduced pressure.1H NMR(500MHz,DMSO- D6), δ:2.99 (s, 6H), 3.10 (q, J=25Hz, 1H), 6.72 (d, J=5Hz, 3H), 7.21 (q, J=45Hz, 2H), 7.51 (d, J=10Hz, 2H)；13C NMR(500MHz,DMSO-D6),δ:45.75,111.89,112.10,118.48,123.31, 129.41,129.73,130.97,145.41,151.45ppm；TOFMS:m/z calcd for[M+H]+:291.0626, found:291.0618.
Embodiment 3
The synthesis of fluorescent probe compounds ES3：
Dimethylamino benzaldehyde (1.49g, 10mmol) and 2,4- thiazolidinediones (1.17g, 10mmol) will be added in and filled In the round-bottomed flask for having 10mL toluene, 200mg ammonium acetates and 1mL glacial acetic acids are added as catalyst, is warming up to 115 DEG C of reflux Reaction.Reaction for 24 hours, removes solvent, the isolated yellow solid ES3 (85.60%) of chromatographic column under reduced pressure.1H NMR(500MHz, DMSO-D6), δ:3.01 (s, 6H), 6.83 (d, J=10Hz,
2H), 7.42 (d, J=5Hz, 2H), 7.67 (s, 1H), 12.31 (s, 1H)；13C NMR(500MHz,DMSO-D6), δ:110.79,110.91,111.95,120.02,115.63,119.79,132.10,132.91,133.82,139.29, 151.44,
167.48,168.11ppm；TOF MS:m/z calcd for[M-H]-:247.0541,found:247.0545.
Embodiment 4
Fluorescent probe compounds ES1, ES2 and ES3 test human serum albumins (HSA) fluorescence titration
5 μM of small-molecule fluorescent probes ES1, ES2 and ES3 are added in PBS buffer solutions (pH=7.4,10mM), gradually The HSA of various concentration is added in, as shown in Figure 1,1a, 1b and 1c are respectively dyestuff ES1, ES2 and ES3 to the glimmering of haemocyanin HSA Photoresponse figure.The excitation wavelength of three dyestuffs is respectively 474nm, 507nm and 430nm.With the increase of HSA concentration, probe point Fluorescence intensity at sub- maximum emission peak gradually enhances.
Embodiment 5
Fluorescent probe compounds ES1 tests the selectivity of human serum albumins (HSA) and different aminoacids
The selectivity to different aminoacids is evaluated using the compound ES1 of above-mentioned synthesis：By 5 μM of probe compound ES1 and 5 μM HSA and 50 μM of variety classes amino acid (glutamic acid, glycine, histidine, tryptophan, arginine, tyrosine, asparagine Acid, asparagine, cysteine and lysine) it is added in PBS buffer solution (pH=7.4,10mM), the excitation wavelength of probe is 474nm, selection launch wavelength are 547nm, record corresponding fluorescence intensity change.Test result is as shown in Fig. 2, adding in 5 μM After human serum albumin HSA, the fluorescence intensity of probe increases 72 times, and the addition of 50 μM of variety classes amino acid can't influence The fluorescence intensity of probe.
Embodiment 6
Fluorescent probe compounds ES1 tests the selectivity of human serum albumins (HSA) and different proteins
The selectivity to different proteins is evaluated using the compound ES1 of above-mentioned synthesis：By 5 μM of probe compound ES1 and 5 μM HSA and variety classes protein (bovine serum albumin(BSA) BSA, chymotrypsin, protease, lysozyme, chymotrypsinogen A, hemoglobin and histone) it is added in PBS buffer solution (pH=7.4,10mM), the excitation wavelength of probe is 474nm, is chosen Launch wavelength is 547nm, records corresponding fluorescence intensity change.Test result as shown in figure 3, due to have similar structure with Biological property, probe show the high selectivity to HSA and BSA, i.e., just there is probe compound when only adding in HSA and BSA Apparent Fluorescence Increasing.In addition, by comparing fluorescence responses different with BSA to HSA probe compound ES1, it has been found that add After entering 5 μM of HSA, the fluorescence intensity of probe increases 60 times, and 5 μM of BSA are only capable of making the fluorescence intensity of probe to increase 6 times, explanation Probe compound ES1 has better choice to HSA.
Embodiment 7
Fluorescent probe compounds ES1 and human serum albumin HSA combination Ratio Experiments
The combination ratio of probe molecule and human serum albumins is verified using the compound ES1 of above-mentioned synthesis：Job’s Plot experiments are a kind of methods for determining two kinds of substance stoichiometric ratios.PBS buffer solution (pH=7.4,10
MM in test system), the total concentration of fixed probe molecule ES1 and HSA are 10 μM, constantly change probe and HSA Concentration ratio, respectively 1:9、2:8、3:7、4:6、5:5、6:4、7:3、8:2、9:1, test the change of its corresponding fluorescence spectrum Change.As shown in figure 4, when the concentration ratio of probe molecule and HSA are 5:When 5, fluorescence intensity is most strong, this illustrate probe molecule ES1 with HSA is with 1:What 1 ratio combined.
Embodiment 8
The positioning experiment of fluorescent probe compounds ES1 and human serum albumin HSA
The binding site of probe molecule and human serum albumins is verified using the compound ES1 of above-mentioned synthesis：Four kinds of selection Different pharmaceutical, i.e. warfarin, brufen, Propofol and hemin replace experimental drug as drug, they respectively can be with people's blood FA7, FA3/4 and FA6 of pure albumen HSA, FA3/4 and FA5 and FA1 sites combine.PBS buffer solution (pH=7.4, In test system 10mM), probe molecule concentration is 10 μM, and adding in 2 μM of HSA makes itself and probe mixing 1h, ensures the complete of the two Full effect.Then to four kinds of medicines that various concentration (5,10,15,20,30,40,60,80 μM) is added in above-mentioned supramolecular system Object, the variation of the fluorescence intensity of recording responses.As shown in figure 5, add in various concentration into the mixed system of probe molecule and HSA Warfarin, brufen and Propofol the fluorescence intensity of mixed system is had little effect, and body can be significantly quenched in hemin The fluorescence of system illustrates that probe compound ES1 is combined with human serum albumin HSA in FA1 sites, for research dye molecule with The interaction of protein provides new method.
Embodiment 9
Temperature-responsive is tested after fluorescent probe compounds ES1 and human serum albumin HSA effect
To temperature-responsive reality after being acted on using the compound ES1 evaluation probe molecules of above-mentioned synthesis with human serum albumins It tests.According to the literature, the fluorescence intensity of the molecule with TICT properties often shows the sensibility to temperature.Delay in PBS In the test system of fliud flushing (pH=7.4,10mM), the concentration of probe molecule and human serum albumins is 5 μM, respectively from height to Temperature that is low and adjusting test system from low to high, the temperature of selection is respectively 20,25,30,35,40,45 and 50 degrees Celsius.By It is found that when temperature gradually rises, probe molecule and the fluorescence intensity of protein complex system continuously decrease Fig. 6 a, by Fig. 6 b, When temperature continuously decreases, probe molecule and the fluorescence intensity of protein complex system gradually rise.
Embodiment 10
Fluorescent probe compounds ES1 tests the response time of human serum albumin HSA
Use the compound ES1 evaluation probe molecules of above-mentioned synthesis and the response time of HSA effects：In PBS buffer solution (pH =7.4,10mM) test system in, probe molecule concentration be 5 μM, excitation wavelength 474nm, record 547nm at fluorescence intensity Variation.As shown in Figure 7, after 5 μM of protein are added in, probe molecule is quite rapid to the response of human serum albumin HSA, 10s or so fluorescence intensities can reach balance, and remained unchanged in subsequent at least 60min.
Embodiment 11
Fluorescent probe compounds ES1 is to the detection sensitivity of human serum albumin HSA
Detection sensitivity of the probe molecule to HSA is evaluated using the compound ES1 of above-mentioned synthesis：First in PBS buffer solution In the test system of (pH=7.4,10mM), a concentration of 10 μM of dyestuff, a concentration of 0.002 is separately added into above-mentioned system, 0.004,0.006,0.008,0.013,0.018,0.023,0.028 and 0.033mg/mL, excitation wavelength 474nm, record Fluorescence intensity change at 547nm.As shown in Figure 8 a, the fluorescence intensity of probe gradually enhances with the increase of HSA concentration, and its Fluorescence intensity is with human serum albumin HSA in 0-0.033
There is good linear relationship (R in the range of mg/mL2=0.997).It calculates to detect by 3 σ/k simultaneously and be limited to 0.132mg/L, therefore probe molecule ES1 can be used for the quantitative detection of low concentration HSA.In addition, we have also investigated compound ES1 In true urine test system to the response condition of HSA.We select the urine specimen that healthy male volunteers provide, using facing The content that bed process measures microalbumin in the urine specimen is 0.0035
Mg/mL, and as benchmark, 10 μM of probe molecule ES1 are added in into above-mentioned system, then add in various concentration The HSA of (0.005,0.01,0.015,0.025,0.035,0.045 and 0.055mg/mL), excitation wavelength 474nm, record Fluorescence intensity change at 547nm.As shown in Figure 8 b, side, the fluorescence of probe molecule ES1 are strong in the test system of true urine Degree is similary with the concentration of human serum albumin HSA to have good linear relationship (R2=0.999), illustrate probe compound ES1 With good biologic applications.

Claims (6)

1. application of the small molecule fluorescence probe in Protein Detection composition is prepared, small-molecule fluorescent probe therein, tool Just like lower structure general formula I：
In general formula I：
R1-R7It is each independently selected from H, C1-8Alkyl, substitution or unsubstituted phenyl；
The substituted-phenyl is arbitrarily replaced by following group：CN、COOH、NH2、NO2、OH、SH、C1-6Alkoxy, C1-6Alkyl ammonia Base, C1-6Amide groups, halogen or C1-6Halogenated alkyl；
X is oxygen or sulphur；
N is selected from the integer of 1-8.
2. application according to claim 1, which is characterized in that the R1-R7It is each independently selected from H or C1-4Alkane Base.
3. application according to claim 2, which is characterized in that the R1-R4、R7It is each independently selected from H or methyl.
4. application according to claim 2, which is characterized in that the R5And R6It is each independently selected from C1-4Alkyl.
5. application according to claim 1, which is characterized in that the n is selected from the integer of 1-3.
6. application according to claim 1, the small-molecule fluorescent probe is selected from following compound：
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