CN106632305B - A kind of fluorescence probe and nanoparticle and preparation method and application with water-soluble and aggregation inducing transmitting effect - Google Patents

A kind of fluorescence probe and nanoparticle and preparation method and application with water-soluble and aggregation inducing transmitting effect Download PDF

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CN106632305B
CN106632305B CN201611130837.3A CN201611130837A CN106632305B CN 106632305 B CN106632305 B CN 106632305B CN 201611130837 A CN201611130837 A CN 201611130837A CN 106632305 B CN106632305 B CN 106632305B
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曾文彬
高瑭
杨舒琪
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Abstract

The invention discloses a kind of fluorescence probe with water-soluble and aggregation inducing transmitting effect and nanoparticle and preparation method and application, the imidazole ring and quaternary ammonium salt structure feature that the fluorescence probe replaces containing more phenyl, there is dispersibility well in water, and have the characteristics that aggregation inducing transmitting effect, launch wavelength are long, it is adapted to Fluorometric assay；Under the induction of heparin, fluorescence probe meeting self assembly, to assemble generation red fluorescence, reflect the content of heparin by the intensity of detection transmitting light, realize the quantitative detection of heparin, have many advantages, such as high sensitivity, strong interference immunity, the detection especially suitable for the heparin in the environment such as chemical solution system, biological sample.This method has important practical value for the detection of trace heparin and the real-time monitoring based on heparin in blood samples of patients and its clinical application guidance.

Description

Fluorescent probe and nanoparticle with water solubility and aggregation-induced emission effect, and preparation method and application thereof
Technical Field
The invention relates to a fluorescent probe and a nanoparticle, in particular to a fluorescent probe or nanoparticle with water solubility and aggregation-induced emission effect, and preparation methods thereof, and particularly relates to application of the fluorescent probe nanoparticle in detection of heparin, belonging to the technical field of chemical analysis and analysis detection.
Background
Fluorescent Organic Nanoparticles (FONs) are generally formed by self-assembly of oligomers in a monodisperse state or of fluorophores in a conjugated structure. Compared with inorganic nanoparticles, the nano-particle has the advantages of flexible synthesis method, biodegradability, low toxicity, stability in aqueous solution and the like, and is widely applied to the fields of chemical sensing, optical materials, coatings, cell imaging, biological monitoring, in-vivo imaging, photodynamic therapy and the like (X.Zhang, X.Y.Zhang, L.Tao, Z.G.Chi, J.R.Xu, and Y.Wei.J.Mater.Chem.B.,2014,2, 4398. supplement 4414). However, the conventional fluorophore will be quenched when it is aggregated by self-assembly in aqueous solution, and this will limit its application in organic light emitting materials and biological imaging. Fluorophores having aggregation-induced emission or enhanced aggregation-induced emission compared to conventional fluorophores exhibit strong fluorescence in both the aggregate and solid state conditions. The unique behavior of aggregation-induced emission molecules has attracted great attention of scientists, and has become a hotspot in the field of research of multifunctional optical materials. The aggregation-induced emission type nanoparticles have great application value in the fields of biosensing and biological imaging due to good biocompatibility and high fluorescence intensity (R.T.K.Kwok, C.W.T.Leung, J.W.Y.Lam, and B.Z.Tang, chem.Soc.Rev.,2014,13, 4228-one-well 4238).
Heparin is a linear, unbranched, sulfated glycosaminoglycan, consisting primarily of trisulfated disaccharide repeating units. Heparin plays a key role in regulating various physiological processes in the organism, such as cell growth, cell differentiation, inflammation, immune defense, lipid transport, and the like. Heparin is used as a high-efficiency anticoagulant, is widely applied to anticoagulant treatment of pregnancy patients clinically and surgical prevention of thrombus formation, and can also prevent venous embolism of patients of acute myocardial infarction. Another important clinical application of heparin is to maintain the patient's blood circulation in vitro during cardiac surgery, renal dialysis, etc. In addition, heparin may also be usedCan be used for treating Disseminated Intravascular Coagulation (DIC), rheumatoid arthritis, glomerulonephritis, and nephrotic syndrome. The anticoagulant effect of heparin is mainly achieved by its interaction with the thrombin protein inhibitor (antithrombin III). Clinically required doses of heparin for cardiovascular surgery and post-operative long-term care were 2-8U mL, respectively-1(17-67. mu.M) and 0.2-1.2U mL-1(1.7-10. mu.M), so the dosage of heparin used in clinic is strictly controlled. Excessive heparin dosage can cause spontaneous bleeding, and clinical manifestations include various mucosal bleeding, joint cavity hematocele, wound bleeding, etc. Heparin-induced thrombocytopenia is drug-induced thrombocytopenia and is a serious complication in heparin therapy. In view of the serious side effects that excessive heparin may cause, the development of a highly sensitive and selective method for detecting heparin content is urgently required.
There are many methods for detecting heparin. Activated Partial thromboplastin Time (APPT) is commonly used clinically. The method comprises adding calcium ion, cephalin and other activating substances such as kaolin into plasma, which activate intrinsic coagulation cascade system to promote coagulation of plasma, and Activating Partial Thromboplastin Time (APTT) is the time required for coagulation of plasma. The dosage of heparin can be monitored according to the length of APTT time, so that the optimal dosage range can be found. Although the APTT method is simple and cheap to operate, the APTT method is an indirect detection method and cannot directly detect the content of heparin in blood plasma of patients in situ, and in addition, the method for Activating Partial Thromboplastin Time (APTT) can only be used for detecting common heparin and cannot be used for detecting low molecular weight heparin. Therefore, it is highly desirable to develop a new method for monitoring heparin with high accuracy and reliability. In recent years, fluorescent detection and analysis methods are attracting great attention due to their characteristics of simplicity, rapidness, high sensitivity, no need of expensive instruments, and the like. Many fluorescent probes for detecting heparin based on small organic molecules, polymers and biological macromolecules are reported. Since heparin itself has abundant negative charges, probes themselves are designed to have positive charges to detect heparin through the action of electrostatic attraction. This strategy is often used to design fluorescent probes for detection of heparin. Cong Yu et al reported that a polycation-induced fluorescent probe for benzo [ g, h, i ] pyrene detects heparin by aggregating fluorophores via electrostatic forces between the negatively charged fluorophores and the polycations, and upon addition of highly negatively charged heparin, competitively binds to the polycations with the fluorophores, and ratiometrically detects heparin via changes in the aggregation state of the fluorophores (M.D.Yang, J.Chen, H.P.ZHou, W.Y.Li, Y.Wang, J.M.Li, C.Y.ZHang, C.B.ZHou and C.Yu, Biosens bioelectrtron, 2016,75,404, 410.). Although this probe can sensitively detect heparin, it requires the addition of Polycation (PDA) to detect heparin through a two-step reaction, and the negatively charged biomacromolecule in serum can also competitively bind to PDA, which causes great interference to heparin detection. In addition, heparin fluorescent probes have been reported to have a short emission wavelength (less than 600nm), and biological macromolecules with fluorescence in blood can also affect the fluorescence quantification.
Disclosure of Invention
Aiming at the defects of the performance of the existing fluorescent probe for detecting heparin, the first purpose of the invention is to provide a fluorescent probe which has good water solubility and aggregation induction effect.
The second purpose of the invention is to provide a fluorescent probe nanoparticle with aggregation induction effect.
The third purpose of the invention is to provide a method for preparing the fluorescent probe, which is simple to operate and has easily obtained raw materials.
The fourth purpose of the invention is to provide a method for preparing the fluorescent probe, which is simple to operate and mild in conditions.
The fifth purpose of the invention is to provide an application of the fluorescent probe nanoparticle in detection of heparin, and the fluorescent probe is aggregated to form an aggregation state to emit fluorescence to realize detection of heparin by utilizing the water solubility and aggregation induction effect of the fluorescent probe nanoparticle and the electrostatic interaction force of heparin, so that the fluorescent probe nanoparticle has the characteristics of sensitivity and high selectivity.
In order to achieve the technical purpose, the invention provides a fluorescent probe with water solubility and aggregation-induced emission effect, which has a structure shown in formula 1:
wherein,
X-is selected from I-、ClO4 -、Br-Or PF6 -；
R1、R2And R3Independently selected from hydrogen, halogen, alkyl, hydroxyl, alkoxy, nitro, carboxyl or amino.
Preferred embodiment, R in formula 12And R3Are all selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino.
Preferred embodiment, R in formula 11Selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, hydroxyl, methoxyl, nitryl, carboxyl or dimethylamino.
The invention also provides a fluorescent probe nanoparticle which is formed by gathering the fluorescent probe.
The invention also provides a method for preparing the fluorescent probe with water solubility and aggregation-induced emission effect, which comprises the following steps:
1) carrying out N substitution reaction on 2-methylbenzothiazole and methyl iodide to obtain an intermediate shown in a formula 2;
2) performing cyclization reaction on terephthalaldehyde, aniline compounds in a formula 3 and benzil compounds in a formula 4 in an acetic acid/ammonium acetate system to obtain an intermediate in a formula 5;
3) carrying out condensation reaction on the intermediate of the formula 2 and the intermediate of the formula 5 under the action of piperidine to obtain a target product of a formula 6, or carrying out ion exchange on the target product of the formula 6 and perchlorate, bromide or hexafluorophosphate to obtain a target product containing perchlorate ions, bromide ions or hexafluorophosphate ions;
wherein,
R1、R2and R3Independently selected from hydrogen, halogen, alkyl, hydroxyl, alkoxy, nitro, carboxyl or amino.
In a preferred embodiment, the reaction conditions in 1) are: and (3) reacting for 4-8 h at 80-95 ℃ by adopting absolute ethyl alcohol as a solvent.
In a preferred embodiment, the reaction process in 2) is as follows: stirring terephthalaldehyde 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 3) are: the method comprises the steps of reacting for 6-10 hours at 80-95 ℃ by using absolute ethyl alcohol as a solvent and piperidine as a catalyst.
The invention also provides a method for preparing the fluorescent probe nanoparticle, which comprises the steps of dissolving the fluorescent probe in an organic solvent, adding the organic solvent into an aqueous solution, and carrying out ultrasonic treatment to obtain the fluorescent probe nanoparticle.
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 application of the fluorescent probe nanoparticle to detection of heparin.
The preferable scheme applies the fluorescent probe nano-particle to the fluorescent quantitative analysis and detection of heparin in a chemical solution system, biological body fluid or biological tissue.
The fluorescent probe has an imidazole ring and a quaternary ammonium salt structure, the quaternary ammonium salt has positive charges and is in a dispersed state in water, the whole molecule has no fluorescence, and after the fluorescent probe is identified with heparin, the fluorescent probe is aggregated under the induction of the electrostatic acting force between molecules due to the characteristic of high negative charges of the heparin, so that strong red fluorescence is emitted. The detection principle is shown in figure 1.
The preparation route of the fluorescent probe with water solubility and aggregation induction effect is as follows (using R)1、R2And R3Specifically illustrated for hydrogen):
the specific preparation method of the fluorescent probe with water solubility and aggregation induction effect is realized by the following steps:
a. dissolving 2-methylbenzothiazole in absolute ethyl alcohol, adding methyl iodide, heating and refluxing at 90 ℃, distilling the system under reduced pressure to remove the solvent, and recrystallizing diethyl ether to obtain a compound 1;
b. dissolving terephthalaldehyde and aniline in glacial acetic acid, stirring at normal temperature for 1 hour, sequentially adding benzil and ammonium acetate, uniformly mixing, continuously heating at 120 ℃ for reflux reaction overnight, adjusting the pH value to nearly neutral by using a sodium hydroxide solution after TLC detection reaction is complete, generating precipitate, filtering under reduced pressure to obtain a filter cake, drying, and performing column chromatography separation and purification to obtain a compound 2.
c. Dissolving the compound 2 in absolute ethyl alcohol, sequentially adding the compound 1 and a piperidine system, uniformly mixing, heating and refluxing at 90 ℃, removing the solvent by reduced pressure distillation after the reaction is finished, and separating and purifying by column chromatography to obtain a compound 3;
d. dissolving the compound 3 in acetone, stirring at normal temperature, adding saturated aqueous solution of sodium perchlorate or potassium hexafluorophosphate, stirring the system for 30min, filtering to obtain a filter cake, washing with distilled water, and drying to obtain the corresponding anion exchange product compound 4 or 5.
The preparation method of the fluorescent probe nanoparticle is realized by the following steps:
fully dissolving the compound 3,4 or 5 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 the ultrasonic condition, and stirring the system at normal temperature for 30min to obtain the organic nanoparticles for detecting heparin. The particle size and morphology of the nanoparticles formed were observed by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM).
The method for detecting heparin fully utilizes the dispersibility of the fluorescent probe provided by the invention in aqueous solution, the dispersed state has no fluorescence, and when heparin exists, the fluorescent probe is aggregated to form an aggregation state to emit fluorescence through the electrostatic interaction force of the heparin, and the emission wavelength is more than 600 nm.
The fluorescent probe nanoparticle can be used for detecting heparin in a chemical simulation biological system; can also be used for clinically detecting the concentration of heparin in blood and detecting the heparin in blood, serum or tissues in clinical medicine.
Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:
1) the fluorescent probe and the nanoparticle have the characteristics of good water solubility and dispersibility in water, have an aggregation-induced emission effect, emit fluorescence with a wavelength of more than 600nm in an aggregation state, and are particularly suitable for fluorescence detection.
2) The preparation method of the fluorescent probe and the nanoparticle is simple, low in cost and beneficial to large-scale production.
3) The fluorescent probe nanoparticle is used for detecting heparin in blood, the probe has good selectivity on heparin, human serum albumin, hyaluronic acid, chondroitin sulfate, ATP, amino acid and the like do not interfere detection, and the fluorescence intensity of the nano probe solution and the concentration of the heparin are within a certain concentration range (the concentration range of the heparin is 0-1.2 UmL)-1) Has good linear relation, shows quantitative detection characteristic and can completely meet the clinical requirement on the detection of the heparin concentration in postoperative long-term care.
4) The fluorescent nano probe provided by the invention has the advantages of short response time to heparin, high detection sensitivity and small interference of biomacromolecules in blood with emission wavelength in a red light region, so that the fluorescent nano probe is suitable for popularization and application.
Drawings
FIG. 1 is a dynamic light scattering diagram of the fluorescent probe nanoparticles prepared in example 1 of the present invention;
FIG. 2 is a scanning electron microscope image of the fluorescent probe nanoparticles prepared in example 1 of the present invention;
fig. 3 shows a linear relationship between the fluorescence intensity of the fluorescent probe nanoparticle and the heparin concentration in example 1 of the present invention, where the abscissa represents the heparin concentration and the ordinate represents the fluorescence intensity;
FIG. 4 shows the selectivity of the fluorescent probe nanoparticles to heparin in example 1 of the present invention;
FIG. 5 is a schematic diagram of fluorescent probe nanoparticles used for heparin detection.
Detailed Description
The following examples are intended to further illustrate the present disclosure, but not to limit the scope of the claims.
Example 1
Compound 3 (R in the examples) of the present invention which induces enhancement of fluorescence emission by aggregation1、R2And R3H, X ═ I), the synthetic route is as follows:
synthesis of Compound 1: weighing 2-methylbenzothiazole (298mg, 2mmol), dissolving in 10mL acetonitrile, adding iodomethane (572mg, 4mmol) into the system, refluxing the reaction mixed system at 90 ℃ for 6 hours, monitoring the reaction by a TLC plate, distilling under reduced pressure to remove the solvent, recrystallizing with diethyl ether, filtering, and adding 30mL ethyl ether
Washing with ether for three times, drying to obtain compound 1, and directly carrying out the next reaction without further purification.
Synthesis of Compound 2: terephthalaldehyde (1.34g,10mmol) and aniline (930mg, 10mmol) were dissolved in 100mL glacial acetic acid and stirred at room temperature for one hour. Benzil (2.1g, 10mmol) and ammonium acetate (5.4g, 70mmol) 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 40%. The results of the nmr analysis were:1H NMR(500MHz，CDCl3，ppm)δ＝9.98(s,1H)，7.76-7.78(d,2H)，7.62-7.65(m,4H)，7.36–7.23(m，9H)，7.16(m，2H)，7.09–7.11(d，2H).13C NMR(125MHz，CDCl3，ppm)δ＝191.70,145.34,135.47,130.23,129.39,128.78,128.37,128.26,126.91.
synthesis of Compound 3: compound 2(200mg, 0.5mmol) and compound (220.5mg, 0.5mmol) were weighed and dissolved in 10mL of anhydrous ethanol, 1 drop of piperidine was added, the reaction was stirred well at room temperature, the reaction was refluxed well at 90 ℃ for 8 hours, the completion of the reaction was monitored by TLC plate, the solvent was distilled off under reduced pressure, and the product 3 was isolated by silica gel column chromatography. The yield was 51%. The results of the nmr analysis were:1HNMR(500MHz,DMSO-D6):δ＝8.44-8.46(d,1H，J＝7.7Hz)，8.28-8.26(d,1H,J＝8.7Hz)，8.20-8.16(d,1H,J＝15.7Hz)，8.06-8.02(d,1H,J＝15.7Hz)，8.00-7.98(d,2H,J＝8.4Hz)，7.92-7.88(t,1H)，7.83-7.79(t，1H)，7.55-7.52(m，4H),7.41-7.27(d，13H)，4.35(s，3H).13C NMR(125MHz,DMSO-D6) δ 172.13,147.73,145.27,142.54,130.01,128.91,124.79,117.43,115.01,55.39,40.38,36.93,31.17,29.84,27.01,22.68 HRMS (m/z): calculated value C37H28IN3S, 673.1049; discovery of [ M-I]+,546.2007.
Example 2
Preparing water-soluble fluorescent organic nanoparticles:
mu.l of compound 3(1mM) in acetonitrile was taken with a pipette and added to 2mL of phosphate buffer solution (10mM, pH 7.4) under ultrasonic conditions to give a final concentration of compound 3 of 10 μ 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 3
Scanning Electron Microscope (SEM) testing of the prepared fluorescent organic nanoparticles:
one drop of the solution prepared in example 2 was taken, dropped on a copper mesh, filtered to remove water, air dried naturally, and observed in a scanning electron microscope. The scanning electron microscope picture is shown in figure 2.
Example 4
The linear relationship between the fluorescence intensity of the fluorescent nanoprobe and the heparin concentration is as follows:
10 μ L of heparin sodium injection was added to the systems prepared in example 2 to achieve final heparin concentrations of 0.05UmL-1、0.1UmL-1、0.15UmL-1、0UmL-1.2UmL-1、0.3UmL-1、0.4UmL-1、0.5UmL-1、0.6UmL-1、0.7UmL-1、0.8UmL-1、0.9UmL-1、1.0UmL-1、1.2UmL-1、1.4UmL-1、1.6UmL-1、1.8UmL-1、2.0UmL-1. After all test solutions were formulated, they were mixed well using a vortex apparatus. After incubation at room temperature for 10min, the fluorescence emission intensity at 650nm was measured. The obtained result is shown in figure 3, and the fluorescence intensity of 650nm and the concentration of heparin in the system are 0-1.2 UmL-1There is a good linear relationship between them.
Example 5
Selectivity of fluorescent organic nano-meter to heparin detection:
the solution prepared in example 2 was used as a fluorescent nanoprobe having an excitation wavelength of 380nm, and the selectivity of the probe to heparin was evaluated. The concentration of compound 3 in this system was 10. mu.M, and heparin was added to this system so that its concentration was 2.0UmL-1And 5 times of single amount of phosphate radical, adenosine triphosphate, sulfate radical acetate radical, hyaluronic acid, glutamic acid and cysteine, fully mixing, and incubating at room temperature 1After 0min, the fluorescence emission spectrum was measured and the intensity of the fluorescence emission at 650nm was recorded. As shown in FIG. 5, only the system to which heparin was added produced strong red fluorescence, while almost no red fluorescence was observed in the solution to which other species were added. The results show that the fluorescent organic nanoparticles have good selectivity and practical applicability for detecting heparin.
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 probe with water solubility and aggregation-induced emission effect is characterized in that: has the structure of formula 1:
wherein,
X-is selected from I-；
R1、R2And R3Selected from hydrogen.
2. A fluorescent probe nanoparticle is characterized in that: a nanoparticle comprising the fluorescent probe of claim 1 aggregated.
3. A method for preparing a fluorescent probe with water solubility and aggregation-induced emission effect according to claim 1, characterized in that: the method comprises the following steps:
1) carrying out N substitution reaction on 2-methylbenzothiazole and methyl iodide to obtain an intermediate shown in a formula 2;
2) performing cyclization reaction on terephthalaldehyde, aniline compounds in a formula 3 and benzil compounds in a formula 4 in an acetic acid/ammonium acetate system to obtain an intermediate in a formula 5;
3) carrying out condensation reaction on the intermediate of the formula 2 and the intermediate of the formula 5 under the action of piperidine to obtain a target product of a formula 6;
wherein,
R1、R2and R3Independently selected from hydrogen.
4. The method for preparing a fluorescent probe with water solubility and aggregation-induced emission effect as claimed in claim 3, wherein:
1) the reaction conditions in (1) are: reacting for 4-8 h at 80-95 ℃ by adopting absolute ethyl alcohol as a solvent;
2) the reaction process in (1) is as follows: stirring terephthalaldehyde 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;
3) the reaction conditions in (1) are: the method comprises the steps of reacting for 6-10 hours at 80-95 ℃ by using absolute ethyl alcohol as a solvent and piperidine as a catalyst.
5. A method for preparing the fluorescent probe nanoparticle of claim 2, wherein the method comprises the following steps: dissolving the fluorescent probe of claim 1 in an organic solvent, adding the solution into an aqueous solution, and performing ultrasonic treatment to obtain the fluorescent probe nanoparticle.
6. The method for preparing fluorescent probe nanoparticles according to claim 5, wherein: 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. The use of the fluorescent probe nanoparticle of claim 2, wherein: the method is used for preparing the fluorescent probe applied to heparin detection.
8. The use of the fluorescent probe nanoparticle of claim 2, wherein: the fluorescent probe is used for preparing the fluorescent probe applied to the fluorescent quantitative analysis and detection of heparin in a chemical solution system, biological body fluid or biological tissues.
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