A cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene and its application

Abstract

The present invention discloses a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene and its application, wherein the fluorescent supramolecular probe is named J3-Ad, and its structural formula is shown in the following formula (I). After the fluorescent supramolecular probe J3-Ad is configured into a molecular probe stock solution with a concentration of 5mmol / L, the host-guest interaction mechanism of the cyanine dye fluorescent supramolecular probe J3-Ad and H2-cyclodextrin is used to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene. The fluorescent supramolecular probe has the advantages of less background interference, strong tissue penetration and less tissue damage. In addition, the fluorescent supramolecular probe also has good solubility, and its absorption wavelength is also in the near-infrared region, which can detect pyrene and 1-hydroxypyrene in real time with high efficiency, rapidity, high selectivity and high sensitivity, and has strong anti-interference ability, and can realize visual identification of pyrene and 1-hydroxypyrene.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent molecular probes and applications, and in particular to a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene and an application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) refer to compounds containing two or more benzene rings connected in a fused ring form. PAHs can be produced naturally or artificially, and humans can ingest PAHs through different routes, such as inhalation, skin contact, and ingestion. Some PAHs are widely known as carcinogens, mutagens, and teratogens, such as pyrene, 1-hydroxypyrene, benzo(a)pyrene, and benzo(a)anthracene. These PAHs pose a major threat to human health when ingested by people through various routes. Therefore, it is necessary to develop an efficient and convenient method for detecting PAHs.

[0003] Existing methods for detecting PAHs include gas chromatography-mass spectrometry, high performance liquid chromatography, capillary electrophoresis analysis, thin layer scanning and fluorescence spectrophotometry. Although these methods have the advantages of high sensitivity and specificity, they are time-consuming, poorly portable, and require expensive instruments and professional operating methods. Unlike these methods, fluorescent probes have some special advantages, including excellent selectivity and sensitivity, real-time detection, simple operation and low cost. Compared with single probes, cyanine dye supramolecular has attracted extensive attention and favor from researchers because of its high molar absorption coefficient, wide spectral range and high fluorescence quantum yield. Therefore, it is very valuable to develop a cyanine dye supramolecular probe that can detect PAHs. Summary of the invention

[0004] The object of the present invention is to provide a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene and an application thereof. The fluorescent supramolecular probe has the advantages of small background interference, strong tissue penetration and small tissue damage. In addition, the fluorescent supramolecular probe also has good solubility, and its absorption wavelength is also in the near-infrared region. It can detect pyrene and 1-hydroxypyrene in real time with high efficiency, rapidity, high selectivity and high sensitivity, and has strong anti-interference ability, so as to realize visual identification of pyrene and 1-hydroxypyrene.

[0005] In order to achieve the above object, the present invention provides a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene. The fluorescent supramolecular probe is named J3-Ad, and its structural formula is shown in the following formula (I):

[0006]

[0007] Wherein, X is one of O, N, S, Se, Te; R1 and R3 are hydrogen atoms or methyl groups; R2 is (CH2) n SO3 or (CH2) n COOH, n is an integer from 1 to 4.

[0008] The present invention also provides an application of a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene for qualitative or quantitative detection of pyrene and 1-hydroxypyrene, and utilizes the host-guest interaction mechanism between the cyanine dye fluorescent supramolecular probe J3-Ad and H2-cyclodextrin to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene, wherein the H2-cyclodextrin is a dimer modified by two cyclodextrins, and its structural formula is shown in the following formula (II):

[0009]

[0010] Furthermore, the specific steps of using the fluorescent supramolecular probe to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene include:

[0011] (1) The fluorescent supramolecular probe J3-Ad was dissolved in DMSO to prepare a molecular probe solution stock solution with a concentration of 5 mmol / L, and diluted to 500 μmol / L with ultrapure water before use;

[0012] Dissolve H2-cyclodextrin in ultrapure water to prepare a mother solution with a concentration of 10 mmol / L, and dilute it with ultrapure water to 1 mmol / L when using;

[0013] (2) adding different concentrations of pyrene or 1-hydroxypyrene standard solution to a PBS buffer solution, then adding a certain volume of 1 mmol / L H2-cyclodextrin, and finally adding 500 μmol / L of the fluorescent supramolecular probe J3-Ad stock solution to make its working concentration 5-20 μmol / L, incubating for 10-15 minutes to obtain a pyrene or 1-hydroxypyrene standard sample reaction solution, testing the fluorescence spectrum or ultraviolet spectrum signal of the pyrene or 1-hydroxypyrene standard sample reaction solution, and constructing the fluorescence or ultraviolet relationship between different concentrations of pyrene or 1-hydroxypyrene standard sample reaction solutions and the fluorescent supramolecular probe J3-Ad;

[0014] (3) Pre-treating the sample to be tested according to the method of step (2), and then testing the fluorescence spectrum or ultraviolet spectrum signal in the pre-treated sample to be tested. According to the fluorescence intensity or ultraviolet intensity of the sample to be tested, combined with the linear relationship between the reaction solution of the standard sample of pyrene or 1-hydroxypyrene of different concentrations constructed in step (2) and the fluorescent supramolecular probe J3-Ad, the concentration of pyrene or 1-hydroxypyrene in the sample to be tested can be detected.

[0015] Preferably, in step (2), the pyrene standard solution is first prepared into a mother solution with a concentration of 10 mmol / L by tetrahydrofuran, and then diluted step by step according to a concentration gradient to a concentration of 1 to 50 μmol / L of the pyrene standard solution.

[0016] Preferably, in step (2), the 1-hydroxypyrene standard solution is first prepared into a mother solution with a concentration of 10 mmol / L by methanol, and then diluted step by step according to a concentration gradient to a concentration of 0.3 to 90 μmol / L of the 1-hydroxypyrene standard solution.

[0017] The detection mechanism of the fluorescent supramolecular probe J3-Ad based on host-guest interaction of the present invention for detecting pyrene and 1-hydroxypyrene is as follows: in a PBS solution, when H2-cyclodextrin is present, it can act with the cyanine dye fluorescent supramolecular probe J3-Ad, prompting the cyanine dye fluorescent probe molecules to undergo aggregate changes, that is, to be converted from H-aggregates to J-aggregates. At the same time, the fluorescence will also be enhanced. When pyrene or hydroxypyrene is added again, it will compete with J3-Ad, and host-guest interaction will occur with H2-cyclodextrin, so that the J-aggregate is converted into H-aggregates again, and the fluorescence will also be weakened. The use of H2-cyclodextrin will enhance the fluorescence signal of J3-Ad, while the addition of pyrene or 1-hydroxypyrene will weaken its fluorescence signal, and an "off-on-off" sensing platform is constructed. Different concentrations of pyrene and 1-hydroxypyrene can cause the square acid cyanine dye J3-Ad to produce different fluorescence response intensities, and by constructing a linear relationship, specific and highly sensitive detection of pyrene and 1-hydroxypyrene is achieved.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The present invention utilizes the host-guest interaction mechanism of a squaraine dye fluorescent supramolecular probe J3-Ad and H2-cyclodextrin to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene. The fluorescent supramolecular probe has the advantages of less background interference, strong tissue penetration and less tissue damage. In addition, the fluorescent supramolecular probe also has good solubility, and its absorption wavelength is also in the near-infrared region. It can efficiently, rapidly, highly selectively and highly sensitively detect pyrene and 1-hydroxypyrene in real time, and has strong anti-interference ability, and can realize visual identification of pyrene and 1-hydroxypyrene, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The fluorescence spectra of JH aggregates regulated by different concentrations of pyrene were measured using a fluorescence spectrometer;

[0021] Figure 2 is a linear curve of the fluorescence intensity at 676 nm varying with the pyrene concentration and a linear relationship diagram of the fluorescence intensity at 676 nm and the pyrene concentration;

[0022] Figure 3This is the specific recognition diagram of pyrene by the fluorescent supramolecular probe J3-Ad;

[0023] Figure 4 The fluorescence spectra of JH aggregates regulated by 1-hydroxypyrene at different concentrations were measured using a fluorescence spectrometer;

[0024] Figure 5 is a linear curve of the fluorescence intensity at 676 nm varying with the concentration of 1-hydroxypyrene and a linear relationship diagram of the fluorescence intensity at 676 nm and the concentration of 1-hydroxypyrene;

[0025] Figure 6 This is the specific recognition diagram of the fluorescent supramolecular probe J3-Ad to 1-hydroxypyrene;

[0026] Figure 7 It is a visualization of the color changes of the fluorescent supramolecular probe J3-Ad, JH aggregates, and JH aggregates + 1-hydroxypyrene in 1× PBS;

[0027] Figure 8 This is the absorption spectrum of the fluorescent supramolecular probe J3-Ad in different substances;

[0028] Fig. 9 This is the fluorescence intensity spectrum of the fluorescent supramolecular probe J3-Ad in different substances at 676nm. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0030] In the present invention, the substance obtained by the combination of the fluorescent supramolecular probe J3-Ad and cyclodextrin H2 is named "JH aggregate".

[0031] When X is S, R1 and R3 are hydrogen atoms, and R2 is (CH2)3SO3, the structural formula of the squarylium cyanine dye fluorescent supramolecular probe used in this embodiment is shown in the following formula (III):

[0032]

[0033] Its synthetic route is as follows:

[0034]

[0035] Reagents and conditions used in the synthesis route of the compound: (i) 1-ethyl-(3-dimethylamino)carbodiimide (EDC), N-hydroxysuccinimide (NHS), triethylamine, room temperature, 24h; (ii) 1,3-propane sultone, 120°C, 4h; (iii) squaric acid, n-butanol, pyridine, 115°C, 6h.

[0036] The specific preparation process is:

[0037] Preparation process of compound 3: 1-NH2 adamantane (1.02 g, 6.21 mmol) and 2-methylbenzothiazole-5-carboxylic acid (0.8 g, 4.41 mmol) were dissolved in 50 ml of chloroform, and 3 ml of triethylamine was added; 1.6 g of EDC and 0.95 g of NHS were added, and the reaction was allowed to proceed overnight. The column ratio was EA / PE = 1 / 8 to EA / PE = 1 / 2, and 1.58 g of product was obtained, with a yield of 77.94%. 1HNMR (400MHz, CDCl3) δ = 8.27 (s, 1H), 7.89 (d, J = 8.3, 1H), 7.83 (d, J = 8.2, 1H), 7.29 (s, 2H) ,5.93(s,1H),2.91(s,3H),2.64(s,2H),2.17(s,9H),1.83–1.67(m,6H),1.38–1.20(m,2H). HRMS(ESI):[C 19 H 22 N2OS + The calculated m / z value was 326.46 and the measured value was 327.1521.

[0038] Preparation process of compound 4: Weigh compound 3 (0.7 g, 2.144 mmol) and 1,3-propane sultone (0.725 g, 6.099 mol) in a flask, set the temperature to 90-120°C, and react in a silicone oil bath for 4-8 hours; after the reaction, add an appropriate amount of methanol to dissolve the product, then drop it into ether, and a precipitate is obtained. After filtration, 0.5045 g of solid is obtained, and the yield is 54.15%. HRMS (ESI): [C 22 H 28 N2O4S2]+ m / z calculated value is 448.60, measured value is 471.1381.

[0039] Preparation process of compound J3-Ad: Compound 4 (0.1000 g, 0.230 mmol) and squaric acid (0.0312 g, 0.115 mmol) were weighed into a flask and reacted in an argon atmosphere; then 20 ml of n-butanol and 3-6 ml of pyridine were added and refluxed at 115°C for 6-10 h. After the reaction, solids were generated, and the precipitate was washed with ether several times, and finally filtered to obtain 0.02 g of a blue-purple solid. 1H NMR (400MHz, DMSO) δ = 9.10 (d, J = 5.7, 2H), 8.59 (d, J = 8.0, 1H), 8.32 (s, 1H), 8.17 (d, J = 7.0, 2H), 7.89 (d, J = 8.2, 1H), 7.70 (d, J = 1 1.6,2H),5.88(s,1H),4.75(t,J=7.0,2H),4.51(s,2H),2.45–2.33(m,2H),2.14(s,6H),2.08(s,6H),1.68(s,6H).HRMS(ESI):[C 48 H 52 N4O 10 S4 2- ] + , m / z calculated value is 973.20, and the measured value is 972.2573.

[0040] The J3-Ad probe is used for detecting pyrene and 1-hydroxypyrene based on host-guest interaction.

[0041] The preparation process of H2-cyclodextrin used in the following examples is as follows: H2-cyclodextrin is dissolved in ultrapure water to prepare a 10 mmol / L stock solution, and then diluted to a 1 mmol / L application solution; 1 mmol / L H2-cyclodextrin application solution is added to 1×PBS buffer to make the concentration of H2-cyclodextrin in the buffer system 30 μmol / L.

[0042] The preparation process of the pyrene standard sample solution used in the following examples is as follows: the pyrene standard sample is dissolved in tetrahydrofuran to prepare a 10 mmol / L mother solution.

[0043] The preparation process of the 1-hydroxypyrene standard sample solution used in the following examples is as follows: the 1-hydroxypyrene standard sample is dissolved in methanol to prepare a 10 mmol / L mother solution.

[0044] The specific steps of using the fluorescent supramolecular probe J3-Ad to detect pyrene include:

[0045] (1) The fluorescent supramolecular probe J3-Ad was dissolved in DMSO to prepare a mother solution with a concentration of 5 mmol / L. When used, it was diluted to 500 μmol / L with ultrapure water, and 5-15 μL was added to the reaction system to make its working concentration 10 μmol / L;

[0046] (2) Take 14 containers of the same size, make the working concentration of J3-Ad in the reaction system to be 10μmol / L, the working concentration of H2-cyclodextrin to be 30μmol / L, prepare the test solution with a pyrene concentration gradient of 0μmol / L, 1μmol / L, 3μmol / L, 5μmol / L, 6μmol / L, 9μmol / L, 12μmol / L, 14μmol / L, 16μmol / L, 18μmol / L, 20μmol / L, 30μmol / L, 40μmol / L, and 50μmol / L in a certain volume of 1×PBS solvent system, and the constant volume solvent is PBS. J3-Ad, H2-cyclodextrin and pyrene solution are added at the same time, mixed and centrifuged, and reacted for 10 minutes; then test the fluorescence spectra of the 14 samples, and use a fluorescence spectrometer to measure the fluorescence spectra of JH aggregates regulated by different concentrations of pyrene (see Figure 1 ) and draw a linear curve of the fluorescence intensity at 676 nm versus pyrene concentration (see Figure 2 ), thereby constructing the fluorescence linear relationship between different concentrations of pyrene samples and the host-guest polymer JH aggregates.

[0047] Depend on Figure 1 It can be seen that the fluorescence intensity spectrum of JH aggregates is affected by the pyrene concentration. The fluorescence intensity of JH aggregates in PBS gradually decreases with the increase of pyrene concentration. Figure 2 As shown in the figure, when the concentration of pyrene is 0.0-6.0 μmol / L, the fluorescence intensity of pyrene at 676 nm is linear (y = -325.37265x + 4261.54981, where y is the fluorescence intensity, x is the pyrene concentration, and the correlation coefficient R 2 =0.9995), which shows that the fluorescence intensity has a good linear relationship with the pyrene concentration. The detection limit of pyrene is 1.76nmol / L calculated by the formula 3σ / slope.

[0048] (3) According to the method described in step (2), the pyrene in the sample to be tested is measured by fluorescence spectroscopy. H2-cyclodextrin is added to the sample to make the H2-cyclodextrin concentration in the sample to be tested 30 μmol / L. Then, J3-Ad stock solution is added to the sample to make its working concentration 10 μmol / L. The pyrene in the sample to be tested will compete with the J3-Ad probe to preferentially form a complex with H2-cyclodextrin. After incubation for 20 minutes, the fluorescence spectrum of the sample system to be tested is measured using a fluorescence spectrometer. According to the fluorescence intensity of the sample to be tested, combined with the linear relationship between the fluorescence intensity of pyrene and JH aggregates in step (2), the content of pyrene in the sample to be tested can be detected.

[0049] Specific recognition and verification process for pyrene: Prepare 17 1.5 mL microcentrifuge tubes, add a certain volume of pyrene or other ion stock solution to the PBS buffer solution, then add J3-Ad and H2-cyclodextrin stock solutions respectively, and finally make the concentration of pyrene and metal ions 30 μmol / L, the concentration of J3-Ad 10 μmol / L, and the concentration of H2-cyclodextrin 30 μmol / L. After incubation for 10 minutes at room temperature under yellow light, measure the fluorescence spectrum.

[0050] In order to verify the specificity of JH aggregates in response to pyrene, fluorescence spectrometer was used to measure the fluorescence of different metal ions (0: JH aggregates, 1: Ca 2+ , 2: Cu 2+ , 3: Hg 2+ , 4: K + , 5: Mg 2+ , 6: Co 2+ 7: Mn 2+ , 8: Cd 2+ , 9: Cr 3+ , 10: Ni 2+ , 11: Pd 2+ , 12: Sn 2+ , 13: Fe 3+ , 14: Ba 2+ , 15: Zn 2+ , 16: Na + , 17: pyrene) on the fluorescence intensity of JH aggregates, such as Figure 3 The figure shows the fluorescence intensity bar graph at 676 nm. The effect of other metal ions on the fluorescence intensity of JH aggregates is almost negligible. Only pyrene can significantly reduce the fluorescence intensity of JH aggregates, which indicates that the J3-Ad probe can specifically detect pyrene.

[0051] The specific steps of using the fluorescent supramolecular probe J3-Ad probe to detect 1-hydroxypyrene include:

[0052] (1) The fluorescent supramolecular probe J3-Ad was dissolved in DMSO to prepare a mother solution with a concentration of 5 mmol / L. When used, it was diluted to 500 μmol / L with ultrapure water, and 5-15 μL was added to the reaction system to make its working concentration 10 μmol / L;

[0053] (2) Take 19 containers of the same size, make the working concentration of J3-Ad in the reaction system to be 10 μmol / L, the working concentration of H2-cyclodextrin to be 30 μmol / L, prepare the test solution with a concentration gradient of 1-hydroxypyrene of 0 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.8 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 9 μmol / L, 14 μmol / L, 16 μmol / L, 18 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, 80 μmol / L, and 90 μmol / L in a certain volume of 1×PBS solvent system, and the constant volume solvent is PBS. J3-Ad, H2-cyclodextrin and 1-hydroxypyrene solution were added simultaneously, mixed and centrifuged, and reacted for 10 minutes; then the fluorescence spectra of 19 samples were tested, and the fluorescence spectra of JH aggregates regulated by 1-hydroxypyrene at different concentrations were measured using a fluorescence spectrometer (see Figure 4 ) and draw a linear curve of the fluorescence intensity at 676nm versus the concentration of 1-hydroxypyrene (see Figure 5 ), thereby constructing the fluorescence linear relationship between 1-hydroxypyrene samples of different concentrations and the host-guest polymer JH aggregates.

[0054] Depend on Figure 4 It can be seen that the fluorescence intensity spectrum of JH aggregates is affected by the concentration of 1-hydroxypyrene. The fluorescence intensity of JH aggregates in PBS gradually decreases with the increase of 1-hydroxypyrene concentration. Figure 5 As shown in Figure 2, when the concentration of 1-hydroxypyrene is 2.0-20.0 μmol / L, the fluorescence intensity of 1-hydroxypyrene at 676 nm is linear (y=3851.58323-94.49853x, where y is the fluorescence intensity, x is the concentration of 1-hydroxypyrene, and the correlation coefficient R 2 =0.99280), which shows that the fluorescence intensity has a good linear relationship with the concentration of 1-hydroxypyrene. The detection limit of pyrene is calculated by the formula 3σ / slope to be 67.02nmol / L.

[0055] (3) According to the method described in step (2), 1-hydroxypyrene in the sample to be tested is measured by fluorescence spectroscopy. H2-cyclodextrin is added to the sample to make the H2-cyclodextrin concentration in the sample to be tested 30 μmol / L. Then, J3-Ad stock solution is added to the sample to make its working concentration 10 μmol / L. 1-hydroxypyrene in the sample to be tested will compete with the J3-Ad probe to preferentially form a complex with H2-cyclodextrin. After incubation for 20 minutes, the fluorescence spectrum of the sample system to be tested is measured using a fluorescence spectrometer. According to the fluorescence intensity of the sample to be tested, combined with the linear relationship between the fluorescence intensity of 1-hydroxypyrene and JH aggregates in step (2), the content of 1-hydroxypyrene in the sample to be tested can be detected.

[0056] Specific recognition and verification process of 1-hydroxypyrene: prepare 15 1.5 mL microcentrifuge tubes, add a certain volume of 1-hydroxypyrene or other ion stock solution to PBS buffer solution, then add J3-Ad and H2-cyclodextrin stock solution respectively, and finally make the concentration of 1-hydroxypyrene and metal ions 30 μmol / L, the concentration of J3-Ad 10 μmol / L, and the concentration of H2-cyclodextrin 30 μmol / L. After incubation for 10 minutes under yellow light environment at room temperature, measure the fluorescence spectrum.

[0057] In order to verify the specificity of JH aggregates in response to 1-hydroxypyrene, fluorescence spectrometer was used to measure the fluorescence of different metal ions (0: JH aggregates, 1: HPO4 2- , 2: H2PO4 - 3: SO4 2- 4: S2O4 2- 5: CO3 2- 6: HCO3 - , 7: Cl - 8: ClO 2- 9: CH3COO - 10: NO2 - , 11: Br - , 12: SCN - , 13, SO3 2- , 14: I - , 15:1-hydroxypyrene) on the fluorescence intensity of JH aggregates, such as Figure 6 The figure shows the fluorescence intensity bar graph at 676 nm. The effect of other metal anions on the fluorescence intensity of JH aggregates is almost negligible. Only 1-hydroxypyrene can significantly reduce the fluorescence intensity of JH aggregates, which indicates that the J3-Ad probe can specifically detect 1-hydroxypyrene.

[0058] The specific steps of using the fluorescent supramolecular probe J3-Ad probe to detect 1-hydroxypyrene in human urine include:

[0059] (1) Collect 5-10 mL of urine samples from two healthy adult volunteers, take the urine samples out of the refrigerator, and place them at room temperature; add 500 μL of urine sample to the reaction container, then add 5 μL of β-glucuronidase / arylsulfatase, vortex to mix, and place in a 37°C constant temperature shaker for enzymatic hydrolysis for 8-10 hours; after the enzymatic hydrolysis is completed, add 500 μL of acetonitrile to the reaction system to denature and precipitate the protein, vortex to mix, and centrifuge at 12,000 rpm at 4°C for 10 minutes. After the centrifugation is completed, take the supernatant, which is the sample solution.

[0060] (2) The method was used to detect that the urine samples of two volunteers did not contain 1-hydroxypyrene. Then, different concentrations of 1-hydroxypyrene (5 μmol / L, 10 μmol / L, 15 μmol / L) were added to the urine samples of each volunteer, and then J3-Ad fluorescent probe and H2-cyclodextrin were added. After mixing, the fluorescence spectrum was tested and the concentration of 1-hydroxypyrene in the sample was calculated. Each group of urine samples was repeated three times. The results are shown in Table 1. The recoveries of these 1-hydroxypyrene spiked samples were 95.32% to 115.40%, and the relative standard deviation (RSD) was 0.86% to 4.34%, indicating that the method has good practicality for detecting 1-hydroxypyrene in urine.

[0061] Table 1 Detection results of 1-hydroxypyrene in urine samples (n=3)

[0062]

[0063] The specific steps of using the fluorescent supramolecular probe J3-Ad probe to detect 1-hydroxypyrene in human blood include:

[0064] (1) Collect 5-10 mL of blood samples from two healthy adult volunteers, take the blood samples out of the refrigerator and place them at room temperature; take 1 mL of the blood sample and add it to a centrifuge tube, add a mixture of n-hexane and acetone in a volume ratio of 1:1, mix well, perform ultrasonic extraction, and finally centrifuge at 10,000 r / min for 8 min to obtain the supernatant; then pass through methanol and water-activated C18 solid phase extraction columns in turn, purify the obtained supernatant through a C18 solid phase extraction column, slowly elute with a mixture of n-hexane and dichloromethane into a centrifuge tube, and then concentrate to 1 mL in a vacuum drying oven.

[0065] (2) Different concentrations of 1-hydroxypyrene (4 μmol / L, 8 μmol / L, 12 μmol / L) were added to the blood samples of each volunteer, and then the J3-Ad fluorescent probe and H2-cyclodextrin were added. After mixing, the fluorescence spectrum was tested and the concentration of 1-hydroxypyrene in the sample was calculated. Each group of blood samples was repeated three times. The results are shown in Table 2. The recoveries of these 1-hydroxypyrene spiked samples were 96.25% to 105.40%, and the relative standard deviation (RSD) was 1.76% to 5.36%, indicating that this method has good practicality for detecting 1-hydroxypyrene in blood.

[0066] Table 2 Detection results of 1-hydroxypyrene in blood samples (n=3)

[0067]

[0068] The specific steps of using the fluorescent molecular probe to visually identify 1-hydroxypyrene include:

[0069] (1) Take three liquid storage bottles of the same volume, add 40 μL of 500 μmol / L fluorescent supramolecular probe J3-Ad and 960 μL of 1×PBS solution to the first tube; add 40 μL of 500 μmol / L fluorescent supramolecular probe J3-Ad, 50 μL of 1 mmol / L H2-cyclodextrin, and 910 μL of 1×PBS solution to the second tube; add 40 μL of 500 μmol / L fluorescent supramolecular probe J3-Ad, 50 μL of 1 mmol / L H2-cyclodextrin, 60 μL of 10 mmol / L 1-hydroxypyrene solution, and 850 μL of 1×PBS solution to the third tube; shake thoroughly to mix. Figure 7 As shown, it can be observed with the naked eye that the colors of the three tubes of solution change from pink to blue and then to pink, thereby realizing the detection of 1-hydroxypyrene.

[0070] (2) Detect the absorption and fluorescence spectra of the above three samples respectively. Figure 8 As shown in the figure, the J3-Ad probe has a main absorption peak at 535nm. After adding H2-cyclodextrin, it red-shifts to an absorption peak at 680nm, and exists in the form of a monomer. When 1-hydroxypyrene is added, it blue-shifts to an absorption peak at 540nm. Fig. 9 As shown, the excitation wavelength is 590nm, the fluorescence intensity of the J3-Ad probe is zero, and after adding H2-cyclodextrin, the fluorescence intensity is enhanced by 4000 times. After adding 1-hydroxypyrene again, the fluorescence intensity is significantly reduced. According to the changes in the absorption peak and fluorescence intensity, the detection of 1-hydroxypyrene can be achieved.

Claims

1. A cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene, characterized in that: The fluorescent supramolecular probe is named J3-Ad, and its structural formula is shown in the following formula (III): 。 2. The use of a cyanine dye fluorescent supramolecular probe for detecting pyrene and 1-hydroxypyrene according to claim 1 for qualitative or quantitative detection of pyrene and 1-hydroxypyrene, wherein the host-guest interaction mechanism between the cyanine dye fluorescent supramolecular probe J3-Ad and H2-cyclodextrin is used to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene, wherein the H2-cyclodextrin is a dimer modified by two cyclodextrins, and its structural formula is shown in the following formula (II): 。 3. The use according to claim 2, characterized in that: The specific steps of using the fluorescent supramolecular probe to qualitatively or quantitatively detect pyrene and 1-hydroxypyrene include: (1) Dissolve the fluorescent supramolecular probe J3-Ad in DMSO to prepare a molecular probe solution stock solution with a concentration of 5 mmol / L, and dilute it with ultrapure water to 500 μmol / L when used; Dissolve H2-cyclodextrin in ultrapure water to prepare a mother solution with a concentration of 10 mmol / L, and dilute it with ultrapure water to 1 mmol / L when using; (2) Add different concentrations of pyrene or 1-hydroxypyrene standard solution to PBS buffer solution, then add a certain volume of 1 mmol / L H2-cyclodextrin, and finally add 500 μmol / L fluorescent supramolecular probe J3-Ad stock solution to make its working concentration 5-20 μmol / L, incubate for 10-15 minutes to obtain pyrene or 1-hydroxypyrene standard sample reaction solution, test the fluorescence spectrum or ultraviolet spectrum signal of the pyrene or 1-hydroxypyrene standard sample reaction solution, and establish the fluorescence or ultraviolet relationship between different concentrations of pyrene or 1-hydroxypyrene standard sample reaction solution and the fluorescent supramolecular probe J3-Ad; (3) Pre-treating the sample to be tested according to the method of step (2), and then testing the fluorescence spectrum or ultraviolet spectrum signal in the pre-treated sample to be tested. According to the fluorescence intensity or ultraviolet intensity of the sample to be tested, combined with the linear relationship between the reaction solution of the standard sample of different concentrations of pyrene or 1-hydroxypyrene constructed in step (2) and the fluorescent supramolecular probe J3-Ad, the concentration of pyrene or 1-hydroxypyrene in the sample to be tested can be detected.

4. The use according to claim 3, characterized in that: In step (2), the pyrene standard solution is first prepared into a mother solution with a concentration of 10 mmol / L by tetrahydrofuran, and then diluted step by step according to a concentration gradient until the concentration of the pyrene standard solution is 1 to 50 μmol / L.

5. The use according to claim 3, characterized in that: In step (2), the 1-hydroxypyrene standard solution is first prepared into a mother solution with a concentration of 10 mmol / L by methanol, and then diluted step by step according to a concentration gradient to a concentration of 0.3 to 90 μmol / L of the 1-hydroxypyrene standard solution.

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