CN106699734B - Fluorescent molecular probe and nano probe as well as preparation method and application thereof - Google Patents
Fluorescent molecular probe and nano probe as well as preparation method and application thereof Download PDFInfo
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- CN106699734B CN106699734B CN201611177239.1A CN201611177239A CN106699734B CN 106699734 B CN106699734 B CN 106699734B CN 201611177239 A CN201611177239 A CN 201611177239A CN 106699734 B CN106699734 B CN 106699734B
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a fluorescent molecular probe and a nano probe as well as a preparation method and application thereof, wherein the fluorescent probe molecule contains a multi-phenyl-substituted imidazole ring and a quaternary ammonium salt structure, the fluorescent nano material is easy to self-assemble into a non-fluorescent nano material in aqueous solution, can be subjected to de-assembly after being combined with albumin, probe molecules enter a hydrophobic cavity of the albumin, hydrogen bonds and hydrophobic interaction exist between the probe and partial amino acid residues in the cavity, so that the rotation of the probe molecule is limited to generate fluorescence, the content of albumin can be reflected by detecting the intensity of the emitted light of the fluorescence, the quantitative detection of the albumin is realized, the method has the advantages of high sensitivity, strong anti-interference and the like, is particularly suitable for the quantitative detection of the albumin in a chemical solution system, a patient blood sample and the like, has important practical value for the detection of trace albumin, the content detection of albumin in blood of patients and clinical medication guidance thereof.
Description
Technical Field
The invention relates to a fluorescent molecular probe and a nano probe, in particular to a water-soluble fluorescent molecular probe and a fluorescent nano probe with aggregation-induced emission effect, and preparation methods thereof, and particularly relates to application of the fluorescent molecular probe in albumin detection, belonging to the technical field of chemical analysis and biological analysis detection.
Background
Human Serum Albumin (HSA)) Is a main protein in human blood, and plays a key role in maintaining plasma colloid osmotic pressure and transporting endogenous and exogenous molecules including a plurality of medicines. Studies have shown that the concentration of HSA in body fluids is closely related to the progression of a number of diseases including cancer, rheumatoid arthritis, myocardial ischemia and liver failure (g.fanali, a.dimani, v.trezza, m.marino, m.fasano, p.ascenzi, Mol accessories med.2012,33,209; v.aroyo, r.garcia-Martinez, x.salvatella, j.hepatol.2014,61,396; c.e.ha, n.v.bhagavan, BBA-gen.subjects 2013,1830,5486; j.rozga, t.piatek, p.malkski, ann.transpl.2013,18,205.). The level of HSA in humans is a reliable indicator of health and is normally about 35-55 g L-1(527-828μmolL-1). Low levels of albumin in plasma are closely associated with cirrhosis, chronic hepatitis and hepatic failure. In contrast, the presence of excess HSA in urine is recognized as an indicator for the diagnosis of diabetes, hypertension, cardiovascular disease and renal disease. Therefore, the method has great significance in clinical diagnosis for detecting the HSA content in human body fluid, especially the serum content, with high selectivity, high sensitivity and accuracy.
So far, dye-bound fluorescence assays, antibody-based (immunohistochemical) methods, and mass spectrometry-based proteomics techniques have been included for the quantitative detection of albumin in blood samples. Among the many methods, fluorescence-based assays have been widely used for the detection of HAS content in different samples due to their excellent characteristics of being rapid, sensitive, non-destructive, and suitable for high-throughput detection. However, the fluorescent probe that has been reported has some limitations, for example, some probes have poor solubility in aqueous solution, making it difficult to apply in biological systems (x.fan, q.he, s.sun, h.li, y.pei, y.xu, chem.commun.2016,52,1178.). In addition, some of the reported probes in the literature detect albumin by binding to a thiol or amino group of the protein via a covalent bond (P.Anees, S.Sreejith, A.Ajayaghosh, J.Am.chem.Soc.2014,136, 13233; M.Cieplak, K.Szwabinska, M.Sosnoroska, B.K.C.Chandra, P.Borowicz, K.Noworta, F.D' Souza, W.Kutner, biosens.Bioeleectron.2015, 74,960.). However, this irreversible binding may lead to denaturation of the protein. In addition, most of the fluorescent probes for detecting albumin utilize conventional organic dye molecules as a fluorescence emitting group, and such molecules are easy to aggregate in physiological buffer, however, fluorescence quenching is caused by non-radiative relaxation of the excited state of the aggregate after aggregation. To overcome this drawback, researchers have to use extremely dilute probe solutions and highly soluble probes for protein detection. However, the probe in a dilute solution also suffers from weak fluorescence emission, poor sensitivity, and the like. Furthermore, the effect of quenching due to aggregation even in dilute solutions cannot be completely avoided, because fluorescent probe molecules may accumulate on the surface of hydrophobic cavities of biological macromolecules and proteins in bioassays, resulting in fluorescence quenching. Therefore, it is very critical to design fluorescent probes with complete water solubility and to be able to overcome the drawback of aggregation-induced quenching for the detection of human serum albumin.
Disclosure of Invention
Aiming at the defects of the performance of the existing fluorescent probe for detecting serum protein, the first purpose of the invention is to provide a fluorescent molecular probe which has good water solubility and aggregation induction effect.
The second purpose of the invention is to provide a fluorescent nano probe formed by self-assembling a fluorescent molecular probe with good water solubility and aggregation induction effect in aqueous solution.
The third purpose of the invention is to provide a method for preparing the fluorescent molecular probe, which is simple to operate and has easily available raw materials.
The fourth purpose of the invention is to provide a method for preparing the fluorescent nanoprobe, which is simple to operate and has mild conditions.
The fifth purpose of the invention is to provide the application of the fluorescent nanoprobe in detecting albumin in blood; the fluorescent molecular probe is self-assembled in an aqueous solution to form loose nanoparticles, is disassembled after being acted with albumin, enters a hydrophobic cavity of protein to limit the rotation of the probe molecule and emit fluorescence to realize the detection of the protein, and has the characteristics of high sensitivity and good selectivity.
In order to achieve the technical purpose, the invention provides a fluorescent molecular probe with water solubility and aggregation-induced emission effect, which has a structure shown in formula 1:
wherein,
R1、R2and R3Independently selected from hydrogen, halogen, alkyl, hydroxyl, alkoxy, nitro, carboxyl or amino;
R4is selected from C1～C13Fatty chains or C8～C13An alkyl chain containing an aryl group.
Preferred embodiment, R2And R3Are all selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino; more preferred embodiment, R2And R3Are all selected from hydrogen.
Preferred embodiment, R4Is selected from C2～C8An alkyl chain or a 1, 4-dimethylenephenyl group.
Preferred embodiment, R1Selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino, and the preferable scheme is that R is1Selected from hydrogen.
The invention also provides a fluorescent nano probe which is formed by self-assembly of the fluorescent molecular probe. The fluorescent molecular probe with the structure of formula 1 forms aggregation-state nanoparticles through self-assembly in an aqueous solution, and the aggregation-state nanoparticles mainly comprise fluorescent molecular probe monomers, oligomers and polymers.
The invention also provides a method for preparing the fluorescent probe molecule with water solubility and aggregation-induced emission effect, which comprises the following steps:
1) 4-formyl pyridine, aniline compounds in a formula 2 and benzil compounds in a formula 3 are subjected to cyclization reaction in an acetic acid/ammonium acetate system to obtain an intermediate in a formula 4;
2) carrying out nucleophilic substitution reaction on the intermediate of the formula 4 and a dibromo compound of the formula 5 to obtain an intermediate of a formula 6;
3) carrying out nucleophilic substitution reaction on the intermediate of the formula 6 and pyridine to obtain the intermediate;
wherein,
R1、R2and R3Independently selected from hydrogen, halogen, alkyl, hydroxyl, alkoxy, nitro, carboxyl or amino;
R4is selected from C1～C13Fatty chains or C8～C13An alkyl chain containing an aryl group.
In a preferred embodiment, the reaction process in 1) is as follows: stirring the 4-formylpyridine and the aniline compound in a glacial acetic acid solvent for 0.5-1.5 h, adding the benzil compound and ammonium acetate, and reacting at the temperature of 110-130 ℃ for 8-16 h.
In a preferred embodiment, the reaction conditions in 2) are: acetonitrile is used as a solvent, and the reaction is carried out for 6-10 hours at the temperature of 80-95 ℃.
In a preferred embodiment, the reaction conditions in 3) are: pyridine is used as a solvent, and the reaction is carried out for 6-10 hours at the temperature of 80-95 ℃.
The invention also provides a method for preparing the fluorescent nano probe, which comprises the steps of dissolving the fluorescent molecular probe in an organic solvent, adding the solution into an aqueous solution, and carrying out ultrasonic treatment to obtain the fluorescent nano probe.
In a preferred embodiment, the organic solvent is at least one selected from methanol, ethanol, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, and acetonitrile.
Preferably, the aqueous solution is selected from pure water, physiological saline, phosphoric acid buffer solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution or tris (hydroxymethyl) aminomethane hydrochloride buffer solution.
The invention also provides an application of the fluorescent nano probe, and the fluorescent nano probe is applied to the detection of serum albumin.
In a preferred embodiment, the fluorescent nanoprobe is applied to a chemical solution system, and the fluorescent quantitative analysis and detection of albumin in blood or biological tissue of a patient.
The fluorescent probe molecule is a typical bola type molecule, has a multi-phenyl substituted imidazole ring and a quaternary ammonium salt structure, and is connected with the middle part through a hydrophobic alkyl chain. The probe molecules have good water solubility, form organic nanoparticles by self-assembly in water, and have no fluorescence. After recognition with albumin, disassembly occurs, the probe molecules enter into the hydrophobic cavities of the protein, and hydrogen bonding and hydrophobic interaction occur with the amino acid residues of the protein, finally resulting in the restriction of the rotation of the probe molecules to generate fluorescence. The detection principle is shown in fig. 5.
The preparation route of the fluorescent molecular probe with water solubility and aggregation induction effect is as follows (R is used as1、R2And R3Is hydrogen, R4＝C2、C4、C6、C8、C10、C12Alkyl chains are specifically illustrated as examples):
the specific preparation method of the fluorescent molecular probe with water solubility and aggregation induction effect is realized by the following steps:
a. dissolving 4-formyl pyridine and aniline in glacial acetic acid, stirring at normal temperature for 1 hour, then sequentially adding benzil and ammonium acetate, uniformly mixing, continuously heating at 120 ℃, carrying out reflux reaction overnight, adjusting the pH value to be nearly neutral by using a sodium hydroxide solution after the TLC detection reaction is completed, generating a precipitate, carrying out reduced pressure filtration to obtain a filter cake, drying, and carrying out column chromatography separation and purification to obtain a compound 1;
b. dissolving the compound 1 in acetonitrile, adding a dibromo compound (1, 2-dibromoethane, 1, 4-dibromobutane, 1, 6-dibromohexane, 1, 4-di (bromomethyl) benzene, 1, 8-dibromooctane, 1, 10-dibromodecane or 1, 12-dibromododecane); after the system is uniformly mixed, heating and refluxing at 90 ℃, after the reaction is finished, distilling under reduced pressure to remove the solvent, and separating and purifying by column chromatography to obtain a compound 2, a compound 4, a compound 6, a compound 8, a compound 10, a compound 12 or a compound 14;
c. dissolving the compound 2, the compound 4, the compound 6, the compound 8, the compound 10, the compound 12 or the compound 14 in pyridine, heating the system to 90 ℃, carrying out reflux reaction, distilling under reduced pressure to remove the solvent after the reaction is finished, and carrying out column chromatography separation and purification to obtain the target product.
The preparation method of the fluorescent nano probe is realized by the following steps:
fully dissolving a target product in an organic solvent to prepare a mother solution, then sucking the mother solution by using a liquid-transferring gun, adding the mother solution into a certain amount of aqueous solution under an ultrasonic condition, and stirring the system at normal temperature for 30min to obtain the organic nanoparticles for detecting the serum albumin. The particle size and morphology of the formed nanoparticles were observed by Dynamic Light Scattering (DLS) and projection electron microscopy (TEM).
According to the technical scheme, the method for detecting the albumin based on the fluorescent nano probe fully utilizes the self-assembly of the fluorescent molecular probe in the aqueous solution to form the nanoparticles, the formed nanoparticle solution has no fluorescence, the nanoparticles are disassembled in the presence of the albumin, the probe molecules enter a hydrophobic cavity of the protein and perform hydrogen bond action and hydrophobic action with amino acid residues of the protein, and finally the rotation of the probe molecules is limited to generate the fluorescence. The method for detecting albumin is established based on the method, has strong anti-interference and high sensitivity, and can be widely popularized and applied.
The fluorescent nano probe can be used for detecting albumin in a chemical simulation biological system; the method can also be used for detecting albumin in serum in clinical medicine.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
1) the fluorescent molecular probe has the characteristics of good water solubility and capability of forming non-fluorescent nanoparticles by self-assembly in water, has an aggregation-induced emission effect, is subjected to de-assembly when being acted with albumin to generate 480nm fluorescence, and is particularly suitable for fluorescence detection.
2) The fluorescent probe molecule and the fluorescent nano probe have simple preparation method and low cost, and are beneficial to large-scale production.
3) The fluorescent nano probe is used for detecting albumin in serum and the like, has good selectivity on albumin, does not interfere with the detection of albumin by trypsin, papain, pepsin, immunoglobulin, glucose oxidase, histidine, cysteine, homocysteine, lysine, glutathione, ATP, glucose and the like, has good linear relation between the fluorescence intensity of a nano probe solution and the concentration of albumin within a certain concentration range (the concentration range of albumin is 0-15 mu M), shows quantitative detection characteristics, and can completely meet the requirement of clinical detection on the content of albumin in blood of a patient.
4) The fluorescent nanoprobe has short response time (15s) to albumin, high measurement sensitivity, strong anti-interference capability, simple operation and low cost, so that the fluorescent nanoprobe is suitable for popularization and application.
Drawings
FIG. 1 is a dynamic light scattering diagram of the fluorescent nanoprobe prepared in example 1 of the present invention.
Fig. 2 is a projection electron microscope image of the fluorescent nanoprobe prepared in example 1 of the present invention.
FIG. 3 is a linear relationship between the fluorescence intensity of the fluorescent nanoprobe and the albumin concentration in example 1 of the present invention, wherein the abscissa represents the albumin concentration and the ordinate represents the fluorescence intensity.
FIG. 4 shows the selectivity of the fluorescent nanoprobe for albumin in example 1 of the present invention.
FIG. 5 is a schematic diagram of the fluorescent nanoprobe for detecting albumin.
Detailed Description
The following embodiments are intended to further illustrate the present invention and are not intended to limit the present invention.
Example 1
Target 3 (R in the examples) of the present invention with enhanced aggregation-induced fluorescence emission1、R2、R3＝H、R4＝C2) The synthetic route is as follows:
synthesis of Compound 1: 4-formylpyridine (1.06g,10mmol) and aniline (0.91mL, 10mmol) were each weighed out and dissolved in 100mL glacial acetic acid and stirred at room temperature for one hour. Benzil (2.1g, 10mmol) and ammonium acetate (3.55g, 46mmol) are sequentially added into a reaction system, the compound reacts at 120 ℃ overnight, after the reaction is finished, the reaction system is poured into 300mL of ice water, the pH value of the system is adjusted to be neutral by 0.1mmol/L sodium hydroxide solution, the mixture is filtered and washed by water for three times, and after vacuum drying, the product is separated and purified by silica gel column chromatography to obtain the product. The yield was 42%. The results of the nmr analysis were:1H NMR(400MHz,CDCl3,δ):8.48–8.47(d,J＝2H),7.58-7.60(d,J＝2H),7.31-7.35(m,4H),7.22-7.29(m,7H),7.13-7.14(d,2H),7.09-7.10(d,2H)；13c NMR (100MHz, CDCl3, delta.) 149.71,143.84,139.20,137.78,136.65,133.96,132.46,131.05,130.00,129.48,129.01,128.47,128.34,128.30,128.27,127.32,126.97,122.32 HRMS (ESI) m/z calculated C26H19N3,373.1579[M](ii) a It was found that 374.1651[ M + H]+.
Synthesis of Compound 2: compound 1(0.19g,0.50mmol) and compound 1, 2-dibromoethane (93mg,0.50mmol) were weighed out and dissolved in 15mL acetonitrile, stirred well at room temperature, the reaction system was refluxed well for 10 hours at 90 ℃, after completion of the reaction was monitored by TLC plate, the solvent was distilled off under reduced pressure, and the product 2 was isolated by silica gel column chromatography. The yield was 45%. The results of the nmr analysis were:1H NMR(400MHz,DMSO-d6,δ):9.09–9.11(d,2H),7.81-7.82(d,2H),7.51-7.55(m,7H),7.25-7.42(m,8H),3.48-3.50(t,2H),3.40-3.43(t,2H)；13C NMR(100MHz,DMSO-d6,δ):145.39,144.84,141.81,140.85,140.41,137.23,137.04,136.75,135.92,133.49,133.41,131.35,130.79,129.16,128.96,127.06,123.72,123.48,60.74,32.08.
synthesis of Compound 3: weighing Compound 2(110mg, 0.20mmol) and dissolving in 10mL pyridine solutionAnd fully stirring at room temperature, fully refluxing the reaction system at 90 ℃ for 8 hours, monitoring the reaction by a TLC plate, removing the solvent by reduced pressure distillation, and separating by silica gel column chromatography to obtain a product 3. The yield was 53%. The results of the nmr analysis were:1H NMR(400MHz,CDCl3,δ):9.14-9.15(d,2H),8.94-8.96(d,2H),8.68-8.72(t,1H),8.19-8.23(t,2H)7.77-7.79(d,2H),7.50-7.55(m,7H),7.27-7.37(m,8H),5.29-5.32(t,2H),5.20-5.23(t,2H),1.88-1.92(m,4H),1.23-1.32(m,4H)；13C NMR(100MHz,DMSO-d6,δ):147.08,145.80,145.48,144.77,140.57,140.43,136.80,135.84,133.45,131.37,130.66,130.47,130.11,129.78,129.15,128.93,128.87,128.58,128.06,127.01,124.12,59.58,59.05.
example 2
Target 5 (R in the examples) of the present invention with enhanced aggregation-induced fluorescence emission1、R2、R3＝H、R4＝C4) The synthetic route is as follows:
synthesis of Compound 1: 4-formylpyridine (1.06g,10mmol) and aniline (0.91mL, 10mmol) were each weighed out and dissolved in 100mL glacial acetic acid and stirred at room temperature for one hour. Benzil (2.1g, 10mmol) and ammonium acetate (3.55g, 46mmol) are sequentially added into a reaction system, the compound reacts at 120 ℃ overnight, after the reaction is finished, the reaction system is poured into 300mL of ice water, the pH value of the system is adjusted to be neutral by 0.1mmol/L sodium hydroxide solution, the mixture is filtered and washed by water for three times, and after vacuum drying, the product is separated and purified by silica gel column chromatography to obtain the product. The yield was 42%. The results of the nmr analysis were:1H NMR(400MHz,CDCl3,δ):8.48–8.47(d,J＝2H),7.58-7.60(d,J＝2H),7.31-7.35(m,4H),7.22-7.29(m,7H),7.13-7.14(d,2H),7.09-7.10(d,2H)；13c NMR (100MHz, CDCl3, delta.) 149.71,143.84,139.20,137.78,136.65,133.96,132.46,131.05,130.00,129.48,129.01,128.47,128.34,128.30,128.27,127.32,126.97,122.32 HRMS (ESI) m/z calculated C26H19N3,373.1579[M](ii) a It was found that 374.1651[ M + H]+.
Synthesis of Compound 4: compound 1(0.19g,0.50mmol) and compound 1, 4-dibromobutane (0.106g,0.50mmol) were weighed and dissolved in 15mL acetonitrile, stirred well at room temperature, the reaction system was refluxed well for 10 hours at 90 ℃, after completion of the reaction was monitored by TLC plate, the solvent was distilled off under reduced pressure, and product 4 was isolated by silica gel column chromatography. The yield was 51%. The results of the nmr analysis were:1H NMR(400MHz,DMSO-d6,δ):8.90-8.92(d,2H),7.79-7.81(d,2H),7.48-7.53(m,7H),7.26-7.36(m,8H),4.50-4.54(t,2H),3.54-3.57(t,2H),1.98-2.01(m,2H),1.79-1.82(m,2H)；13C NMR(100MHz,DMSO-d6,δ):144.99,144.17,140.55,140.33,136.51,135.96,133.54,131.38,130.63,129.71,129.30,129.12,128.98,128.91,127.99,127.01,124.12,59.60,34.36,29.61,29.16.
synthesis of Compound 5: weighing compound 4(120mg, 0.20mmol), dissolving in 10mL pyridine solution, stirring thoroughly at room temperature, refluxing the reaction system at 90 deg.C for 8 hr, monitoring the reaction by TLC plate, distilling under reduced pressure to remove solvent, and separating by silica gel column chromatography to obtain product 5. The yield was 53%. The results of the nmr analysis were:1H NMR(400MHz,DMSO-d6,δ):9.29-9.30(d,2H),9.10-9.12(d,2H),8.61-8.65(t,1H),8.16-8.20(t,2H)7.77-7.79(d,2H),7.49-7.53(m,7H),7.26-7.37(m,8H),4.76-4.80(t,2H),4.65-4.69(t,2H),1.91-1.98(m,4H)；13C NMR(100MHz,DMSO-d6,δ):144.67,144.05,143.82,142.73,139.19,138.93,135.12,134.58,132.18,130.01,129.29,129.07,128.35,127.94,127.76,127.54,127.21,126.61,125.63,122.61,58.62,57.91,26.25,25.89.
example 3
Target 7 (R in the example) of the present invention with enhanced aggregation-induced fluorescence emission1、R2、R3＝H、R4＝C6) The synthetic route is as follows:
synthesis of Compound 1: separately weighed 4-formyl pyridine (1.06g,10mmol) and aniline (0.91ml, 10mmol) to dissolveThe solution was stirred at room temperature for one hour in 100mL of glacial acetic acid. Benzil (2.1g, 10mmol) and ammonium acetate (3.55g, 46mmol) are sequentially added into a reaction system, the compound reacts at 120 ℃ overnight, after the reaction is finished, the reaction system is poured into 300mL of ice water, the pH value of the system is adjusted to be neutral by 0.1mmol/L sodium hydroxide solution, the mixture is filtered and washed by water for three times, and after vacuum drying, the product is separated and purified by silica gel column chromatography to obtain the product. The yield was 42%. The results of the nmr analysis were:1H NMR(400MHz,CDCl3,δ):8.48–8.47(d,J＝2H),7.58-7.60(d,J＝2H),7.31-7.35(m,4H),7.22-7.29(m,7H),7.13-7.14(d,2H),7.09-7.10(d,2H)；13c NMR (100MHz, CDCl3, delta.) 149.71,143.84,139.20,137.78,136.65,133.96,132.46,131.05,130.00,129.48,129.01,128.47,128.34,128.30,128.27,127.32,126.97,122.32 HRMS (ESI) m/z calculated C26H19N3,373.1579[M](ii) a It was found that 374.1651[ M + H]+.
Synthesis of Compound 6: compound 1(0.19g,0.50mmol) and compound 1, 6-dibromohexane (0.12g,0.50mmol) were weighed out and dissolved in 15mL of acetonitrile, stirred well at room temperature, the reaction system was refluxed well for 10 hours at 90 ℃, after completion of the reaction was monitored by TLC plate, the solvent was distilled off under reduced pressure, and the product 6 was isolated by silica gel column chromatography. The yield was 54%. The results of the nmr analysis were:1H NMR(500MHz,DMSO-d6,δ):8.91-8.93(d,2H),7.79-7.80(d,2H),7.49-7.54(m,7H),7.28-7.37(m,8H),4.46-4.50(t,2H),3.51-3.54(t,2H),1.78-1.91(m,2H),1.78-1.81(m,2H),1.24-1.43(m,4H)；13C NMR(125MHz,DMSO-d6,δ):143.36,142.41,138.93,138.61,134.80,134.32,131.90,129.74,129.00,128.78,128.08,127.67,127.51,127.35,127.29,126.35,125.36,122.40,58.74,33.83,30.66,30.14,25.66,23.30.
synthesis of compound 7: weighing compound 6(110mg, 0.21mmol), dissolving in 10mL pyridine solution, stirring thoroughly at room temperature, refluxing the reaction system at 90 deg.C for 8 hr, monitoring the reaction completion by TLC plate, distilling off solvent under reduced pressure, and separating by silica gel column chromatography to obtain product 3. The yield was 53%. The results of the nmr analysis were:1H NMR(400MHz,DMSO-d6,δ):9.29-9.30(d,2H),9.10-9.12(d,2H),8.61-8.65(t,1H),8.16-8.20(t,2H)7.77-7.79(d,2H),7.49-7.53(m,7H),7.26-7.37(m,8H),4.76-4.80(t,2H),4.65-4.69(t,2H),1.91-1.98(m,4H)；13C NMR(100MHz,DMSO-d6,δ):145.13,144.56,144.34,143.21,139.78,139.43,135.62,135.15,132.74,130.58,129.85,129.62,128.91,128.52,128.33,128.19,128.11,127.71,127.17,126.18,123.15,59.97,59.24,29.97,29.65,24.24,24.20.
example 4
Preparing a fluorescent organic nano probe:
mu.l of compound 7(1mM) in dimethylsulfoxide was taken using a pipette and added to 2mL of phosphate buffer (10mM, pH 7.4) under ultrasonic conditions to give a final concentration of compound 3 of 10. mu.m. When the system is stirred for 30min at room temperature, opalescence is generated, and in order to verify the nano-aggregation behavior, the average particle size is 101nm as determined by a dynamic light scattering experiment, as shown in figure 1.
Example 5
Projection electron microscope (TEM) testing of the prepared fluorescent nanoprobes:
one drop of the solution prepared in example 4 was taken, dropped on a copper mesh, filtered to remove water, air dried naturally, and observed in a transmission electron microscope. The projection electron microscope picture is shown in fig. 2.
Example 6
Linear relationship of fluorescence intensity of fluorescent nanoprobe to albumin concentration:
to the system prepared in example 4, 10. mu.L of each albumin solution was added to achieve a final concentration of albumin of 1.5. mu.M, 3.0. mu.M, 4.5. mu.M, 6.0. mu.M, 7.5. mu.M, 9.0. mu.M, 10.5. mu.M, 12.0. mu.M, 13.5. mu.M, 15.0. mu.M, 18.0. mu.M, 21. mu.M, respectively. After all test solutions were formulated, they were mixed well using a vortex apparatus. After incubation at room temperature for 1min, the fluorescence emission intensity at 480nm was measured. The obtained results are shown in FIG. 3, and the fluorescence intensity at 480nm in the system has a good linear relationship with the concentration of albumin between 0 and 15. mu.M.
Example 7
Selectivity of fluorescent nanoprobes for albumin detection:
the solution prepared in example 4 was used as a fluorescent nanoprobe having an excitation wavelength of 380nm, and the selectivity of the probe for albumin was evaluated. The concentration of compound 3 in the system is 10 mu M, albumin is added into the system respectively to enable the concentration to be 15 mu M, 5 times of molar equivalent of trypsin, papain, pepsin, immunoglobulin, glucose oxidase, histidine, cysteine, homocysteine, lysine, glutathione, ATP and glucose are mixed fully, after incubation for 1min at room temperature, the fluorescence emission spectrum is measured, and the intensity of fluorescence emission at 480nm is recorded. As shown in FIG. 5, only the system to which albumin was added produced strong blue-green fluorescence, while little blue-green fluorescence was observed in the solution to which the other species was added. The results show that the fluorescent organic nanoparticles have good selectivity and practical applicability in albumin detection.
Finally, it is specifically intended that the foregoing examples be construed as merely illustrative of certain embodiments of the present invention. It is obvious that the invention is not limited to the examples described above, and all variations which can be derived or suggested from the disclosure of the invention by a person skilled in the art are to be considered within the scope of protection of the invention.
Claims (8)
1. A fluorescent molecular probe with water solubility and aggregation-induced emission effect is characterized in that: has the structure of formula 1:
wherein,
R2and R3Are all selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino; r4Is selected from C2～C8An alkyl chain;
R1selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino.
2. A fluorescent nanoprobe, characterized in that: formed by self-assembly of the fluorescent molecular probe of claim 1.
3. A method for preparing the fluorescent molecular probe with water solubility and aggregation-induced emission effect as claimed in claim 1, wherein: the method comprises the following steps:
1) 4-formyl pyridine, aniline compounds in a formula 2 and benzil compounds in a formula 3 are subjected to cyclization reaction in an acetic acid/ammonium acetate system to obtain an intermediate in a formula 4;
2) carrying out nucleophilic substitution reaction on the intermediate of the formula 4 and the dibromo compound of the formula 5 to obtain an intermediate of a formula 6;
3) carrying out nucleophilic substitution reaction on the intermediate of the formula 6 and pyridine to obtain the intermediate;
BrR4Br
formula 5
Wherein,
R2and R3Are all selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino; r4Is selected from C2～C8An alkyl chain;
R1selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino.
4. The method for preparing a fluorescent molecular probe with water solubility and aggregation-induced emission effect as claimed in claim 3, wherein:
1) the reaction process in (1) is as follows: stirring 4-formyl pyridine and aniline compounds in a glacial acetic acid solvent for 0.5-1.5 h, adding a benzil compound and ammonium acetate, and reacting at the temperature of 110-130 ℃ for 8-16 h;
2) the reaction conditions in (1) are: acetonitrile is used as a solvent, and the reaction is carried out for 6-10 hours at the temperature of 80-95 ℃;
3) the reaction conditions in (1) are: pyridine is used as a solvent, and the reaction is carried out for 6-10 hours at the temperature of 80-95 ℃.
5. A method of preparing the fluorescent nanoprobe of claim 2, characterized in that: dissolving the fluorescent probe molecule of claim 1 in an organic solvent, adding the solution into an aqueous solution, and performing ultrasonic treatment to obtain the fluorescent nano probe.
6. The method of preparing a fluorescent nanoprobe according to claim 5, characterized in that: the organic solvent is selected from at least one of methanol, ethanol, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and acetonitrile;
the aqueous solution is selected from pure water, normal saline, phosphoric acid buffer solution, 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution or tris (hydroxymethyl) aminomethane hydrochloride buffer solution.
7. Use of the fluorescent molecular probe of claim 1 in the preparation of a fluorescent nanoprobe for albumin detection.
8. Use of the fluorescent molecular probe according to claim 7 for the preparation of fluorescent nanoprobes for albumin detection, characterized in that: the method is applied to the preparation of fluorescent nano-probes for the fluorescent quantitative analysis and detection of albumin in a chemical solution system and blood or biological tissues of a patient.
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