Near-infrared luminescent biomass quantum dot and NIR ratio fluorescence probe and preparation method and application thereof
阅读说明：本技术 近红外发光的生物质量子点和nir比率荧光探针及其制备方法和应用 (Near-infrared luminescent biomass quantum dot and NIR ratio fluorescence probe and preparation method and application thereof ) 是由 赵书林 林鹏翔 张亮亮 赵晶瑾 黄勇 于 2019-09-18 设计创作，主要内容包括：本发明提供了近红外(NIR)发光的生物质量子点(BQDs)和对过氧亚硝酸根(ONOO<Sup>-</Sup>)特异性响应的NIR比率荧光探针的制备方法和应用。NIR比率荧光探针及其制备方法,包括先从冬青树叶(Holly leaves)中制备叶绿素粗提取液,再制备生物质量子点(HL-BQDs),最后制备NIR比率荧光探针：HL-BQDs-Cy7。本发明提出的NIR比率荧光探针,对ONOO<Sup>-</Sup>具有特异性响应,检定细胞线粒体内ONOO<Sup>-</Sup>含量表现出高的灵敏性和好的选择性,实现了小鼠体内内源性ONOO<Sup>-</Sup>的比率荧光成像。(The present invention provides Near Infrared (NIR) luminescent Biomass Quantum Dots (BQDs) and peroxynitrite (ONOO) ‑ ) A preparation method and application of a NIR ratiometric fluorescent probe with specific response. An NIR ratiometric fluorescence probe and its preparation method comprises preparing chlorophyll crude extract from folium Ilicis Purpureae (Holly leaves), and preparing biomassQuantum dots (HL-BQDs), finally NIR ratiometric fluorescent probes were prepared: HL-BQDs-Cy 7. The NIR ratiometric fluorescent probe provided by the invention is used for the ONOO ‑ With specific response, assaying the intracellular ONOO ‑ The content shows high sensitivity and good selectivity, and realizes endogenous ONOO in mice ‑ Fluorescence imaging of the ratio (c).)
1. A preparation method of near-infrared luminous biomass quantum dots is characterized by comprising the following process steps of:
s11, preparing a chlorophyll crude extraction solution, crushing 18-22g of cleaned and air-dried holly leaves and 18-22mL of absolute ethanol in a crushing device, pouring the crushed holly leaf solution into a beaker, adding 8-11mL of acetone, stirring uniformly, standing, and filtering to obtain a chlorophyll crude extraction solution;
s12 adding 18-21mL of oleic acid and 0.04-0.06g of NH into a round-bottom three-neck flask2-PEG-NH2Argon is filled, the temperature is raised to 245-255 ℃ under stirring, the heating is stopped after the solution becomes orange red, 4-6mL of chlorophyll crude extraction liquid is added after the solution is cooled to room temperature, the temperature is continuously raised to 175-185 ℃ after the solution is uniformly stirred, the solution reacts for 160-200min under stirring, 12mol/L of HCl is added after the solution is cooled to the room temperature until the solution is strongly acidic, and the solution is stirred and reacts for 11-13h at the temperature of 27-33 ℃;
s13: transferring the mixed solution to a separating funnel, adding 0.8-1.2mL of ultrapure water, shaking up, standing for layering, transferring the lower layer solution to a beaker, adjusting the pH of the solution to be neutral by using a saturated NaOH solution, and filtering by using a water system filter membrane to remove large-particle substances in the solution;
s14: transferring the filtrate into a dialysis bag, and dialyzing in ultrapure water for 22-25h to obtain a biomass quantum dot (HL-BQDs) solution derived from folium Ilicis Purpureae.
2. The method for preparing the near-infrared luminescent biomass quantum dot according to claim 1, characterized in that:
in step S13, filtering with 0.2-0.4 μm water system filter membrane;
in step S14, the model of the dialysis bag is MWCO:800-2000 Da.
3. The near-infrared luminescent biomass quantum dot is characterized by being prepared according to the preparation method of the near-infrared luminescent biomass quantum dot as claimed in any one of claims 1-2.
A method for preparing an NIR ratiometric fluorescent probe, characterized in that it comprises the steps of:
s21, adding 32-38 mu L of a 10mL HL-BQDs solution with the concentration of 3.7-3.9mmol/L of Cy7NHS ester, stirring for reaction for 55-65min, transferring the solution into a dialysis bag of MWCO 1800-2500Da for dialysis for 23-25h to remove redundant Cy7 NHSester;
s22: adding 0.08-0.11g of 1-ethyl- (3-dimethylaminopropyl) carbon into the dialyzed solutionDiimine hydrochloride (EDC) and 0.008-0.011g N-hydroxysuccinimide (NHS), stirring for 28-35min, adding 32-36 μ L Cy7-NH with concentration of 2.2-2.3mmol/L3 +Stirring for 55-65min, transferring the solution into a dialysis bag with MWCO of 1800 and 2500Da for dialysis for 22-25h, and removing excessive Cy7-NH3 +Thus obtaining HL-BQDs-Cy7 solution.
An NIR ratiometric fluorescent probe, characterized in that it is prepared according to the preparation method of claim 4.
Use of NIR ratiometric fluorescent probes for the determination of ONOO in vivo-The content of (a).
7. Use of an NIR ratiometric fluorescent probe according to claim 6, characterized in that the in vivo ONOO is determined by a ratiometric imaging analysis-The content of (a).
Technical Field
The invention relates to the technical field of chemistry and biomedicine, in particular to a material for detecting biological information in organisms and cells, and specifically relates to near-infrared luminescent biomass quantum dots and NIR ratio fluorescent probes, and a preparation method and application thereof.
Background
Peroxynitrite (ONOO)-) Is an important active oxygen in the organism, and consists of nitric oxide free radical (NO) and superoxide anion free radical (O) in the organism2 -Etc.) and its production site is mainly in mitochondria in cells. Due to ONOO-Has strong oxidizing property and nucleophilicity, and can react with various proteins, liposomes, nucleic acids and the like to cause cell damage.
Accurate determination of ONOO-Can provide precise biological information for the molecular level of the degradation of ONOO-The relevant mechanisms of injury are of great significance. In addition, long-term accumulation of cellular damage is closely related to cardiovascular diseases, neurodegenerative diseases, metabolic diseases, inflammation and even cancer. Therefore, for ONOO-The imaging and quantitative analysis of (A) facilitates ONOO-Early diagnosis of the associated disease. Currently detected ONOO-The methods include electron paramagnetic resonance spectroscopy (EPR), ultraviolet-visible absorption spectroscopy, chromatography, etc., and the existing detection methods are expensive in experimental equipment, low in sensitivity and selectivity due to the detection substances, and not suitable for use in cells and in vivo ONOO-Imaging detection, etc.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a near-infrared luminescent biomass quantum dot and an NIR ratiometric fluorescent probe, which can realize low-cost detection of ONOO in cell mitochondria and in vivo-And (4) content. The invention relates to a preparation method and application of a near-infrared luminescent biomass quantum dot and an NIR ratio fluorescent probe. Assaying cellular mitochondria in vivo and in vivo ONOO-The content can show high sensitivity and good selectivity.
The invention provides a preparation method of a near-infrared luminescent biomass quantum dot. The preparation method comprises the following steps of:
s11, preparing a chlorophyll crude extraction solution, crushing 18-22g of cleaned and air-dried holly leaves and 18-22mL of absolute ethanol in a crushing device, pouring the crushed holly leaf solution into a beaker, adding 8-11mL of acetone, stirring uniformly, standing, and filtering to obtain a chlorophyll crude extraction solution;
s12 adding 18-21mL of oleic acid and 0.04-0.06g of NH into a round-bottom three-neck flask2-PEG-NH2Argon is filled, the temperature is raised to 245-255 ℃ under stirring, the heating is stopped after the solution becomes orange red, 4-6mL of chlorophyll crude extraction liquid is added after the solution is cooled to room temperature, the temperature is continuously raised to 175-185 ℃ after the solution is uniformly stirred, the solution reacts for 160-200min under stirring, 12mol/L of HCl is added after the solution is cooled to the room temperature until the solution is strongly acidic, and the solution is stirred and reacts for 11-13h at the temperature of 27-33 ℃;
s13: transferring the mixed solution to a separating funnel, adding 0.8-1.2mL of ultrapure water, shaking up, standing for layering, transferring the lower layer solution to a beaker, adjusting the pH of the solution to be neutral by using a saturated NaOH solution, and filtering by using a water system filter membrane to remove large-particle substances in the solution;
s14: transferring the filtrate into a dialysis bag, and dialyzing in ultrapure water for 22-25h to obtain HL-BQDs solution.
Specifically, in step S13, the mixture is filtered through a 0.2 to 0.4 μm aqueous membrane; in step S14, the model of the dialysis bag is MWCO:800-2000 Da.
In a second aspect, the present invention provides a Near Infrared (NIR) luminescent biomass quantum dot prepared according to the method for preparing the same.
The third aspect of the invention provides a preparation method of an NIR ratiometric fluorescence probe, which comprises the following steps, wherein the material parts are matched according to the parts:
s21, adding 32-38 mu L of a 10mL HL-BQDs solution with the concentration of 3.7-3.9mmol/L of Cy7NHS ester, stirring for reaction for 55-65min, transferring the solution into a dialysis bag of MWCO 1800-2500Da for dialysis for 23-25h to remove redundant Cy7NHS ester;
s22: adding 0.08-0.11g EDC and 0.008-0.011g NHS into the dialyzed solution, stirring for 28-35min, adding 32-36 μ L Cy7-NH with concentration of 2.2-2.3mmol/L3 +Stirring for 55-65min, transferring the solution into a dialysis bag with MWCO of 1800 and 2500Da for dialysis for 22-25h, and removing excessive Cy7-NH3 +Thus obtaining HL-BQDs-Cy7 solution.
In a fourth aspect, the present invention provides an NIR ratiometric fluorescent probe prepared according to the above-described NIR ratiometric fluorescent probe preparation method.
The fifth aspect of the invention also provides the application of the NIR ratiometric fluorescence probe for determining the ONOO in the mitochondria of the organism cells-The content of (a).
Further, in vivo ONOO determination by ratio imaging analysis-The content of (a).
In one aspect, due to the presence of a mitochondrial targeting group on Cy7, HL-BQDs-Cy7 can target to mitochondria in cells, while Cy7 in HL-BQDs-Cy7 can specifically recognize ONOO produced in mitochondria in cells-(ii) a On the other hand, since the fluorescence emission spectra of HL-BQDs overlap with the absorption of Cy7 molecules, they are a pair of donor and acceptor for Fluorescence Resonance Energy Transfer (FRET). After the HL-BQDs donor is covalently coupled with the Cy7 acceptor, the fluorescence intensity of the HL-BQDs is reduced, and the fluorescence intensity of the Cy7 is increased based on FRET, so that the constructed novel HL-BQDs-Cy7 fluorescent probe has double-emission characteristics. When there is ONOO in the system-When the probe molecule exists, the carbon-carbon double bond of the Cy7 specific site is broken, so that the fluorescence intensity of Cy7 is reduced, and the fluorescence intensity of HL-BQDs is increased. Therefore, the probe can be used as a ratiometric fluorescent probe for detecting ONOO in mitochondria of cells-And exhibits high sensitivity and good selectivity. The HL-BHQs-Cy7 probe can be used because the double-emission fluorescent wavelength is in the near infrared regionIn situ ratiometric fluorescence imaging to track viable single cells and endogenous ONOO in vivo-Is generated. In addition, the leaves of the holly are easy to obtain, and the cost is low.
The invention will be illustrated by way of example and with reference to the accompanying drawings.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the design and application of HL-BQDs-Cy7 nanoprobes in the invention to ONOO-Schematic diagram of detection and in vivo generation process tracking;
FIG. 2 is a transmission electron microscope and high resolution transmission electron microscope photographs of HL-BQDs in the present invention;
FIG. 3 is an X-ray diffraction (XRD) pattern of HL-BQDs and HL-BQDs-Cy7 in accordance with the present invention;
FIG. 4 is an X-ray photoelectron spectroscopy (XPS) chart of HL-BQDs in the present invention;
FIG. 5 is a Fourier Infrared Spectroscopy (FIRT) chart of HL-BQDs in the present invention;
FIG. 6 is a UV-VISIBLE ABSORPTION SPECTRUM of HL-BQDs and HL-BQDs-Cy7 in accordance with the present invention;
FIG. 7 is a diagram showing the fluorescence excitation spectrum, fluorescence emission spectrum and Cy7 absorption spectrum of HL-BQDs in the present invention;
FIG. 8 is a fluorescence emission spectrum of the HL-BQDs covalently coupled with Cy7 in the present invention;
FIG. 9 shows HL-BQDs-Cy7 at 0-15. mu. mol/L ONOO in the present invention-A spectrum of fluorescence in the presence;
FIG. 10 shows fluorescence intensity ratio (I) in the present invention670/I780) Logarithmic value of and ONOO-A linear plot of concentration;
FIG. 11 is a graph showing the selectivity of HL-BQDs-Cy7 probes in the present invention for other bioactive molecules;
FIG. 12 is a comparison of cytotoxicity of HL-BQDs and HL-BQDs-Cy7 nanoprobes in accordance with the present invention;
FIG. 13 is a co-localization imaging graph of subcellular organelles after co-incubation of HL-BQDs-Cy7 nanoprobes and localization reagents with RAW264.7 cells in the invention;
FIG. 14 is a drawing of exogenous ONOO in cells of the present invention-The two-channel fluorescence imaging graph of (1);
FIG. 15 shows the different concentrations of exogenous ONOO in the present invention-A two-channel fluoroscopic image of the living cells present;
FIG. 16 is a graph showing the mean fluorescence intensity of cells in the presence of different concentrations of SIN-1 in two channels of the present invention;
FIG. 17 is a graph of the log of the mean fluorescence intensity ratio of two channels in the presence of different concentrations of SIN-1 in accordance with the present invention plotted linearly with the concentration of SIN-1;
FIG. 18 is a graph showing the endogenous ONOO in living cells of the present invention-The two-channel fluorescence imaging;
FIG. 19 is a graph showing the endogenous ONOO in a single living cell in accordance with the present invention-Generating a real-time imaging map;
FIG. 20 is a graph of the fluorescence intensity of single cells in two channels of the present invention as a function of time;
FIG. 21 is a graph of the log of the ratio of fluorescence intensity of two-channel cells in the present invention versus time;
FIG. 22 is a graph showing the in vivo fluorescence stability of HL-BQDs-Cy7 nanoprobes of the present invention;
FIG. 23 shows the ONOO in the abdominal cavity of a mouse under the stimulation of paracetamol (APAP) drugs with HL-BQDs-Cy7 nanoprobes of the invention-Generating a real-time in-situ ratio fluorescence imaging map;
FIG. 24 is a graph showing fluorescence intensities of HL-BQDs-Cy7 nanoprobes at 700nm channel and 790nm channel at different times in accordance with the present invention;
FIG. 25 is a graph showing the increase of the ratio of fluorescence intensity of HL-BQDs-Cy7 nanoprobes in two channels (I700 nm and I790 nm) with time according to the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and thus the scope of the present invention is not limited by the specific embodiments disclosed below.
In a specific embodiment provided in a first aspect of the present invention, a method for preparing a near-infrared (NIR) luminescent biomass quantum dot is provided, which is characterized by comprising the following steps, wherein the material parts are matched in parts:
s11, preparing a chlorophyll crude extraction solution, crushing 18-22g of cleaned and air-dried holly leaves and 18-22mL of absolute ethanol in a crushing device, pouring the crushed holly leaf solution into a beaker, adding 8-11mL of acetone, uniformly stirring, standing for 20-40min, and filtering to obtain a chlorophyll crude extraction solution;
s12 adding 18-21mL of oleic acid and 0.04-0.06g of NH into a round-bottom three-neck flask2-PEG-NH2Argon is filled, the temperature is raised to 245-255 ℃ under stirring, the heating is stopped after the solution becomes orange red, 4-6mL of chlorophyll crude extraction liquid is added after the solution is cooled to room temperature, the temperature is continuously raised to 175-185 ℃ after the solution is uniformly stirred, the solution reacts for 160-200min under stirring, 12mol/L of HCl is added after the solution is cooled to the room temperature until the solution is strongly acidic, and the solution is stirred and reacts for 11-13h at the temperature of 27-33 ℃;
s13: transferring the mixed solution to a separating funnel, adding 0.8-1.2mL of ultrapure water, shaking up, standing for layering, transferring the lower layer solution to a beaker, adjusting the pH value of the solution to be neutral by using a saturated NaOH solution, and filtering by using a 0.2-0.4 mu m water system filter membrane to remove large-particle substances in the solution;
s14: transferring the filtrate into a dialysis bag of MWCO: 800-.
The HL-BQDs solution prepared by the preparation method is the biomass carbon quantum dot with Near Infrared (NIR) luminescence.
In a second aspect of the invention, a specific embodiment is provided for the ONOO-A method for preparing a specific corresponding NIR ratiometric fluorescent probe, characterized in that the method comprises the following steps,wherein the materials are matched according to the parts:
s21, adding 32-38 mu L of a 10mL HL-BQDs solution with the concentration of 3.7-3.9mmol/L of Cy7NHS ester, stirring for reaction for 55-65min, transferring the solution into a dialysis bag of MWCO 1800-2500Da for dialysis for 23-25h to remove redundant Cy7NHS ester;
s22: adding 0.08-0.11g EDC and 0.008-0.011g NHS into the dialyzed solution, stirring for 28-35min, adding 32-36 μ L Cy7-NH with concentration of 2.2-2.3mmol/L3 +Stirring for 55-65min, transferring the solution into a dialysis bag with MWCO of 1800 and 2500Da for dialysis for 22-25h, and removing excessive Cy7-NH3 +Thus obtaining HL-BQDs-Cy7 solution.
The HL-BQDs-Cy7 solution prepared according to the steps S21 and S22 is the pair ONOO-Specific corresponding NIR ratiometric fluorescent probes.
Experimental methods were used to verify the preparation of prepared biomass quantum dots emitting Near Infrared (NIR) light and on ONOO-Specific corresponding NIR ratiometric fluorescent probes.
The experimental reagents prepared were: oleic acid, polyoxyethylene diamine (NH)2-PEG-NH2MW 2000), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), potassium superoxide (KO)2) 3-morphinanimide Hydrochloride (3-Morpholino sydnimine Hydrochloride, SIN-1) and 4 '-hydroxy-3' -methoxyacetophenone (apocynin) were purchased from Shanghai Allantin Biotechnology Ltd; 2- (N, N-diethylamino) -diazene-2-oxydiethanammonium salt (NONOATE), t-butyl hydroperoxide (tBuOOH), Diethylenetriamine/nitric oxide adduct (Diethylenamine/nitric oxide adduct, NOC-18), minocycline hydrochloride (minocycline), Menadione Sodium Bisulfite (MSB) and 1400W dihydrochloride (1400W) are available from Sigma-Aldrich. Gamma-Interferon (Interferon-gamma, INF-gamma), dialysis bag (MWCO:1000 Da; MWCO:2000Da) purchased from Shanghai Biotechnology engineering, Inc.; MitoLiteTMRED FX600，LysoBriteTMRED，Cyanine 7amine(Cy7-NH3 +)，Cyanine7monosuccinimidyl ester (Cy 7NHS ester) are available from AAT Bioquest, Inc. NucViewTM is available from GeneCoporia. Cell lines used in the experiments: RAW264.7 cells (mouse monocyte macrophage leukemia cells) were purchased from cell banks of the culture Collection type of the Chinese academy of sciences/cell resource center of Shanghai Life sciences institute of Chinese academy of sciences; all nude mice were purchased from slagochada laboratory animals limited, Hunan; the animal experiments were approved with the approval of the animal ethics Committee of the university of Guangxi Master (No. 20150325-XC). Other chemical reagents of the experiment are all domestic analytical pure, and the water for the experiment is 18.2M omega cm.
The prepared experimental instrument comprises: a Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA) was used for the determination of fluorescence spectra, a Cary-60 UV-Vis spectrophotometer (Agilent Technologies, USA) was used for the determination of UV-Vis absorption spectra; fourier transform Infrared Spectroscopy (Perkin-Elmer Instruments, USA) was used for the characterization of BQDs surface groups; x-ray powder diffractometer XRD (Rigaku, Japan); edinburgh FLS980 time resolved fluorescence spectrometer (Edinburgh, England) for fluorescence lifetime determination; a transmission electron microscope (TEM, Philips, Netherlands) is used for the determination and characterization of the particle size range of the BQDs; x-ray photoelectron spectroscopy (XPS, USA) for BQDs elemental analysis; EL × 800 type microplate reader (Bio Tekinstruments, USA) for cytotoxicity determination; the Zeiss LSM710 laser scanning confocal microscope system (CLSM, Zeiss, Germany) was used for the determination of live cell imaging; an SZCL-3B digital display intelligent temperature control magnetic stirrer (Steud City Zeihua Instrument, Inc.); PHS-3C type pH meter (Shanghai apparatus, electrosciences Instrument, Inc.); kodak in vivo FX Pro imaging system (Bruk).
The specific preparation process steps of one part of HL-BQDs-Cy7 solution are as follows, wherein the mass and the volume or the volume can be changed according to the same proportion as required:
s11, taking 20g of cleaned and air-dried holly leaves, putting the leaves into a crusher, adding 20mL of absolute ethanol, crushing for 3min, pouring the crushed holly leaf solution into a beaker, adding 10mL of acetone, stirring uniformly, standing for 30min, and filtering to obtain a chlorophyll crude extraction solution;
s12: a round bottom three-neck flask was charged with 20mL of oleic acid and 0.05g of NH2-PEG-NH2Argon is filled, the temperature is raised to 250 ℃ under stirring, heating is stopped after the solution becomes orange red, 5mL of chlorophyll crude extraction liquid is added after the solution is cooled to room temperature, the temperature is continuously raised to 180 ℃ after the solution is uniformly stirred, the reaction is carried out for 3h under stirring, 12mol/L HCl is added after the solution is cooled to room temperature until the solution is strongly acidic, and the reaction is carried out for 12h under stirring at 30 ℃;
s13: transferring the mixed solution to a separating funnel, adding 1mL of ultrapure water, shaking uniformly, standing for layering, transferring the lower layer solution to a beaker, adjusting the pH of the solution to be neutral by using a saturated NaOH solution, and filtering by using a 0.22-micron water system filter membrane to remove large-particle substances in the solution;
14: transferring the filtrate into a dialysis bag with MWCO of 1000Da, dialyzing in ultrapure water for 24h to obtain HL-BQDs solution;
s21: taking 10mL of HL-BQDs solution, adding 35 mu L of 3.794mmol/L of Cy7NHS ester, stirring for reacting for 1h, transferring the solution into a dialysis bag of MWCO:2000Da for dialysis for 24h to remove the redundant Cy7NHS ester;
s22: adding 0.1g EDC and 0.01g NHS into the dialyzed solution, stirring for 30min, adding 35 μ L2.27 mmol/L Cy7-NH3 +After stirring for 1h, the solution was transferred to a dialysis bag of MWCO:2000Da for dialysis for 24h, removing excess Cy7-NH3 +Thus obtaining HL-BQDs-Cy7 solution.
As shown in FIG. 1, a biomass quantum dot emitting light in the near infrared, i.e., HL-BQDs solution, prepared by the method in the above example was coupled with a near infrared dye Cy7 to obtain an HL-BQDs-Cy7 solution, i.e., on ONOO-Specific corresponding NIR ratiometric fluorescent probes. Because the fluorescence emission spectrum of HL-BQDs overlaps with the absorption of Cy7 molecules, after the HL-BQDs is covalently coupled with Cy7, the fluorescence intensity of the HL-BQDs is reduced based on a FRET mechanism, and the fluorescence intensity of Cy7 is increased, so that the constructed novel HL-BQDs-Cy7 fluorescent nano-probe has double emission characteristics. When there is ONOO in the system-When the probe molecule exists, the carbon-carbon double bond of the Cy7 specific site in the probe molecule is broken, so that the conjugated system of Cy7 is destroyed, the fluorescence intensity is reduced, and the fluorescence intensity of HL-BQDs is increased. The other partyFurthermore, because Cy7 has a mitochondrion targeting group, the HL-BQDs-Cy7 probe can target to mitochondrion in cells, therefore, the probe can be used as a ratiometric fluorescent probe for detecting ONOO in the mitochondrion of cells-And (4) content. Because the double-emission fluorescent wavelength is in the near infrared region, HL-BHQs-Cy7 can be used for in-situ ONOO-Ratiometric fluorescence imaging of (1) and tracking of viable single cells to neutralize endogenous ONOO in vivo-Is generated.
HL-BQDs and HL-BQDs-Cy7 were characterized by experiments as follows.
Transmission Electron Microscopy (TEM) is adopted to respectively represent the morphology, the dispersion degree and the particle size of the HL-BQDs. As can be seen from FIG. 2, HL-BQDs have good dispersibility, and the diameter is about 2 nm. FIG. 2 (b) is a High Resolution Transmission Electron Micrograph (HRTEM) of HL-BQDs, which has a lattice constant of 0.22 μm and corresponds to the crystal plane of graphene (100), and shows that the graphitization and the crystallinity of HL-BQDs are relatively high.
The crystal forms of HL-BQDs and HL-BQDs-Cy7 are inspected by XRD, in the figure 3, (a) and (b) are XRD patterns of HL-BQDs and HL-BQDs-Cy7 respectively, and from the figure, an obvious wide diffraction peak is found at the position of 23.43
XPS is adopted to represent the surface element composition of HL-BQDs, as shown in figure 4, the HL-BQDs mainly consists of C, N, O three elements, the proportion of which is 57.31 percent, 4.33 percent and 31.67 percent respectively; a strong O1s characteristic peak appears at the position of 532.7eV, a second strong C1s characteristic peak appears at the position of 285.1eV, and a weak N1s characteristic peak appears at the position of 399.1 eV.
The composition of the functional groups on the surfaces of HL-BQDs is characterized by a Fourier transform infrared spectrometer, and as shown in FIG. 5, curves a, b and c are respectively the infrared spectrums of Cy7, HL-BQDs and HL-BQDs-Cy 7. 3410cm in the figure-1The peak corresponds to a-O-H/N-H stretching vibration peak, and HL-BQDs-Cy7 is obviously enhanced compared with HL-BQDs, which indicates that Cy7 is covalently coupled with the HL-BQDs through amido bond, and the experimental principle is consistent; 2848cm-1Corresponding to the telescopic vibration of C-H, 1634cm-11360 and 1020cm corresponding to C and C bending vibration-1The absorption of corresponding C-O and C-N, HL-BQDs-Cy7 is obviously enhanced compared with that of HL-BQDs, and 1190cm-1The absorption of the broad peak near the position is increased, and the broad peak is a characteristic absorption peak of sulfonic acid group, which indicates that Cy7 is successfully modified on HL-BQDs.
The optical properties of HL-BQDs-Cy7 were examined by experimental data below.
In order to examine the optical properties of HL-BQDs and HL-BQDs-Cy7, the ultraviolet-visible absorption spectra of HL-BQDs and HL-BQDs-Cy7 were first examined, and as shown in FIG. 6, an obvious absorption peak is formed at 279nm in the ultraviolet-visible absorption spectra, which is the pi-pi electron transition formation of C ═ O in HL-BQDs. HL-BQDs-Cy7 of Cy7 modified by covalent coupling has an obvious absorption peak at 750nm, which is a characteristic absorption peak of Cy7, and further illustrates that Cy7 is successfully coupled on the surface of the HL-BQDs.
Then, the feasibility of the system for generating FRET is researched, and as shown in FIG. 7, in the fluorescence spectrum of HL-BQDs, curves 1 and 2 in the figure are respectively the fluorescence excitation spectrum and the emission spectrum of HL-BQDs, and a
After the HL-BQDs are covalently coupled with the Cy7, a fluorescence emission spectrum is shown in FIG. 8, wherein an emission peak of the HL-BQDs is shown at 670nm, an emission peak of the Cy7 is shown at 780nm, the fluorescence intensity of the HL-BQDs is gradually reduced along with the increase of the adding amount of the Cy7, and the fluorescence intensity of the Cy7 is gradually increased. Indicating that HL-BQDs are successfully coupled with Cy7, and a dual-emission near-infrared ratiometric fluorescent probe is formed through FRET.
The following discussion of the fluorescence detection of ONOO by using HL-BQDs-Cy7 nanoprobe ratio through experimental data results-：
Mixing a certain amount of HL-BQDs-Cy7 solution with different concentrations of ONOO-The solutions were mixed and the probe pair ONOO was investigated-The results are shown in fig. 9 and 10. In FIG. 9 with ONOO-Increase in concentration, ProbeThe fluorescence intensity at 780nm decreased, while the fluorescence intensity at 670nm increased. As shown in FIG. 10, the ratio of the logarithm of the fluorescence intensity at two wavelengths to the ratio of the fluorescence intensity at two wavelengths is shown-The concentration of (B) shows a good linear relationship in the range of 0.02 to 15. mu. mol/L. The linear regression equation is:
log(F670/F780)＝0.05788CONOO-+0.4474，R2＝0.995。
the detection limit was 8.5nmol/L (S/N ═ 3).
The specificity of HL-BQDs-Cy7 nanoprobes was examined experimentally as follows.
The specificity of the HL-BQDs-Cy7 nanoprobe to ONOO-is experimentally examined, the result is shown in FIG. 11, and it can be seen from the figure that different bioactive molecules with the concentration of 15 mu mol/L are respectively added into the HL-BQDs-Cy7 solution relative to the blank control group, and only ONOO-The fluorescence ratio of the group is changed, other active oxygen small molecules are hardly changed, and the result shows that HL-BQDs-Cy7 nano-probe is used for detecting ONOO-Has high specificity.
The cytotoxicity of HL-BQDs-Cy7 nanoprobes was examined by the following experimental results:
in order to examine the application possibility of the HL-BQDs and the HL-BQDs-Cy7 nanoprobes in organisms, the cytotoxicity of the HL-BQDs and the HL-BQDs-Cy7 nanoprobes was investigated by MTT method analysis using RAW264.7 cells, and the results are shown in FIG. 12. As can be seen from the figure, the cell survival rate is about 99% when the concentration of HL-BQDs and HL-BQDs-Cy7 is 40 mu g/mL, and the cell survival rate is reduced from 99% to 83% when the concentration is increased from 40 mu g/mL to 200 mu g/mL, so that the high-molecular-density magnetic nano-particles have good biocompatibility and can be used for imaging living cells and in vivo biomolecules.
The use of nanoprobes for cell imaging is illustrated by experimental data below.
1. Subcellular organelle co-localization imaging
Since endogenous peroxynitrite is produced in mitochondria of living cells, it is necessary to confirm that nanoprobes can precisely enter the mitochondria of cells through cell co-localization experiments. After RAW264.7 cells are incubated for 20h, HL-BQDs-Cy7 nanoprobes and lysosome, cell nucleus and mitochondrion positioning reagents are respectively added, and incubation is continued for 8 h; the HL-BQDs-Cy7 nanoprobe collects fluorescence (probe channel) in the range of 650-650 nm under the excitation of 405nm laser, the excitation wavelength of lysosome and mitochondrial positioning agent is 533nm, and the fluorescence (positioning agent channel) in the range of 560-650nm is collected. The excitation wavelength of the nuclear localization agent is 488nm, and the fluorescence (localization agent channel) in the range of 500-550nm is collected. As can be seen from FIG. 13, the HL-BQDs-Cy7 nanoprobes have good mitochondrial targeting (co-localization coefficient of 0.89), while the lysosome and nucleus co-localization coefficients are only 0.56 and 0.58.
2. Exogenous ONOO in living cells-Ratio imaging of
Experiment researches exogenous ONOO in cells-In the imaging situation, RAW264.7 cells with appropriate density are inoculated in a 35mm confocal imaging dish, and after 20h of incubation, HL-BQDs-Cy7 nanoprobe is added, and the incubation is continued for 6 h. SIN-1 as ONOO-Donor, MSB as O2 -Donor, NOC-18 as NO donor, 50. mu.L PBS in blank control group A; adding SIN-1 with final concentration of 100 μmol/L into group B; adding SIN-1 with final concentration of 1mmol/L into group C; adding SIN-1 and minocycline (minocycline) inhibitor at final concentration of 1mmol/L and 100 μmol/L; adding group E into LMSB with final concentration of 100 μmol/LMSB; group F was added to NOC-18 at a final concentration of 500. mu. mol/L. After further incubation for 2h, the cells were imaged on two channels and the results are shown in FIG. 14. Compared with the blank control group A, after 100 mu mol/L SIN-1 is added into the group B, the fluorescence of a channel with 780nm is reduced, and the fluorescence of a channel with 670nm is increased; after 1mmol/L SIN-1 is added into the group C, the fluorescence of a channel with the wavelength of 780nm almost disappears, and the fluorescence of a channel with the wavelength of 670nm is obviously enhanced; the fluorescence intensity of two channels of the D group is almost the same as that of the A group when 1mmol/L SIN-1 is added and the inhibitor minocycline is added, which indicates that the minocycline completely inhibits the ONOO-And (4) generating. In groups E and F, due to the addition of O separately2 -Failure to form ONOO with NO donors-Therefore, the fluorescence intensity of both channels was also the same as that of group A. The results of these experiments demonstrate that HL-BQDs-Cy7 nanoprobes can specifically recognize ONOO in living cells-And for ONOO-And (6) imaging.
To explore the detection of ONOO in living cells by HL-BQDs-Cy7 nanoprobes-The kinetic range of (1) is that RAW264.7 cells with proper density are respectively inoculated in 5 35mm confocal imaging dishes, HL-BQDs-Cy7 nanoprobes are respectively added after 20h of incubation, and then 50mL of PBS, 100 mu mol/L, 300 mu mol/L, 600 mu mol/L and 1mmol/L of SIN-1 are respectively added in 5 dishes. After the incubation is continued for 2h, the two-channel imaging is carried out on each group of cells, the result is shown in FIG. 15, and it can be seen from the figure that the fluorescence of the channel of 780nm is gradually weakened and the fluorescence of the channel of 670nm is gradually strengthened along with the increase of the concentration of SIN-1, as shown in FIG. 16; the log value of the ratio of the fluorescence intensities of the two channels and the SIN-1 concentration showed good linear relationship in the range of 0-1000. mu. mol/L (FIG. 14C). The linear regression equation is:
log(F670/F780)＝4.453×10-4CSIN-1+0.1765，R2＝0.9464。
3. endogenous ONOO in living cells-Ratio imaging of
Investigation of endogenous ONOO in Living cells-Imaging conditions, experiment using Lipopolysaccharide (LPS), gamma-interferon (INF-gamma) and PMA to stimulate cells and induce the production of ONOO-. RAW264.7 cells of appropriate density were seeded in 4 35mm confocal imaging dishes A, B, C, D, etc., respectively, and after 20h of incubation, HL-BQDs-Cy7 nanoprobes were added, 50mLPBS was added to group A, 50ng/mL INF- γ and 1 μ g/mL LPS were added to the other three groups, and incubation was carried out for 4 h. Then 4 μ L PBS was added to blank group a; adding 25nmol/L PMA and 2 mu L PBS into group B; adding 25nmol/L PMA and 100 μmol/L1400W into group C; group D was supplemented with 25nmol/L PMA and 100. mu. mol/L apocynin. After further incubation for 0.5h, the cells of each group were imaged on two channels, and the results are shown in FIG. 18. Compared with the blank control group A, after the PMA is added into the group B, the fluorescence of a 780nm channel is reduced, and the fluorescence of a 670nm channel is increased; while in PMA stimulated macrophages ONOO-Can be regulated by nitric oxide synthase (iNOS) and NADPH Oxidase (NOX). When 1400W (inhibition of iNOS production) and apocynin (inhibition of NOx production) which are inhibitors of these were added to C, D groups, respectively, the fluorescence intensities of both channels were almost the same as those of group A.
4. Endogenous ONOO in a single living cell-Real-time ratio imaging
Experiment Using the probe of the invention to image and track endogenous ONOO in single living cell in real time-Is generated. RAW264.7 cells were seeded in 35mm confocal imaging dishes, and after incubation to the appropriate density HL-BQDs-Cy7 nanoprobes were added and incubated for 6 h. 50ng/mL INF-. gamma.and 1. mu.g/mL LPS were then added and incubation continued for 4 h. After the subsequent addition of 25nmol/L PMA, single cells were imaged on a confocal microscopy imaging system at 0, 5, 10, 20, 30, 40min of incubation, respectively, and the results are shown in FIG. 19, where it can be seen that the fluorescence intensity of the cells in the 780nm channel gradually decreased and the fluorescence intensity of the cells in the 670nm channel gradually increased with the increase of the incubation time, as shown in FIG. 20. The logarithmic value of the ratio of the fluorescence intensities of the two-channel cells and the incubation time showed a good linear relationship within 0-40min, as shown in FIG. 21. The linear regression equation is:
log(F670/F780)＝0.01383T(min)+0.08668，R2＝0.9995。
the results prove that the HL-BQDs-Cy7 nanoprobe can accurately track the endogenous ONOO in single cells-And endogenous ONOO is found within 0-40min-Is increased linearly.
The use of nanoprobes for in vivo ratio imaging is illustrated by experimental data below.
Fluorescence stability of nanoprobe in living body:
to realize the application of the nanoprobe to the ONOO in vivo-The fluorescence stability of the nanoprobe in vivo was first examined. Two male nude mice (10 weeks, the weight is about 20 g/mouse), one is injected with normal saline (150 muL) intraperitoneally, the other is injected with HL-BQDs-Cy7 nanoprobe (3mg/mL, 150 muL intraperitoneally), the nanoprobe is anesthetized with isoflurane, then on a small animal imaging system, the fluorescence of 700 +/-30 nm and 790 +/-30 nm emission channels is collected respectively at the excitation wavelength of 650nm at 0.5, 1.5 and 4h after the injection of HL-BQDs-Cy7, the result is shown in figure 22, the fluorescence intensity in the two channels can be seen in the figure and is kept stable within 4h, which indicates that the nanoprobe is suitable for livingIn vivo ONOO-The ratio (c) is long.
In situ real-time ratio imaging tracking of ONOO in vivo-Generation of (a):
APAP was used as an induction ONOO in the experiment-The generated reagent researches HL-BQDs-Cy7 nanoprobes for in-situ real-time ratio imaging to track ONOO in living body-Feasibility of production. Injecting HL-BQDs-Cy7 nanoprobe (3mg/mL, 150 μ L) into abdominal diaphragm of male nude mouse, and injecting APAP (administration amount is 500mg/kg) after 30 min. The fluorescence of 700 + -30 nm and 790 + -30 nm emission channels were collected respectively at 650nm as excitation wavelength at 10, 30, 50 and 70min after APAP injection on a small animal imaging system, and the results are shown in FIG. 23, where it can be seen that the fluorescence intensity of 700 + -30 nm channel gradually increases and the fluorescence intensity of 790 + -30 nm channel gradually decreases as the time after APAP injection into the body increases, as shown in FIG. 24. Ratio of fluorescence intensities of two channels (I)700/I790) Gradually increased with time, but the rate of increase slowed down after 50min, as shown in fig. 25. The results prove that the HL-BQDs-Cy7 nano probe can be used for in-situ ratio imaging to monitor in-vivo ONOO in real time-And possibly for tracking and ONOO-The in vivo development of the associated disease.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the invention shall be included in the protection scope of the invention.