CN110894201B - Single-molecule fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid and preparation and application thereof - Google Patents
Single-molecule fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a monomolecular fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid, and preparation and application thereof. The fluorescent probes of the invention can selectively enter cell mitochondria and interact with hydrogen peroxide, protein hydrophobic regions and nucleic acids to produce distinguishable fluorescence spectra for three-channel imaging. The three fluorescence emission wavelengths are longer, and the method is suitable for analyzing the spatial distribution of hydrogen peroxide, protein and nucleic acid in mitochondria and the interaction among molecules by super-resolution imaging. The single-molecule three-channel fluorescence imaging fluorescent probe overcomes the problems of serious cytotoxicity brought by simultaneously using multiple fluorescence probes for imaging, complication of biological distribution and spectrum differentiation of the fluorescence probes in cells and the like.
Description
Technical Field
The invention relates to a monomolecular fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid, and preparation and application thereof, and belongs to the field of analysis and detection.
Background
Mitochondria play an important role in various biological functions of cells and themselves as one of the most complex organelles. Firstly, mitochondria can be used as an energy supply station of cells, not only can supply energy to the cells by oxidizing glucose, fat and other biomolecules, but also can generate Reactive Oxygen Species (ROS) in the process, and the generation of the ROS can further activate other signal paths to regulate the functions of the mitochondria; secondly, proteins or enzymes are the most important life substances in mitochondria, and almost participate in all mitochondrial biological behaviors; third, mitochondria contain their own nucleic acids, which play a crucial role in controlling mitochondrial replication, senescence, and protein expression. Like the genetic material in the nucleus, nucleic acids in the mitochondria can also often form nucleic acid-protein complexes with proteins to perform many important biological functions in the mitochondria in concert.
On the other hand, these important biological components in mitochondria are often surrounded by double-layer mitochondrial membranes, and thus a precise and advanced network structure is formed, which will greatly limit our understanding of the interaction between these bioactive components. However, the key problem to solve is the lack of an effective method to monitor these important biomolecules simultaneously in real time in living cells. Conventionally, the ultra-high resolution technique of transmission electron microscopy has been used for the fine structure study of organelles, but this technique has limitations in that it is neither possible to label multiple biomolecules simultaneously nor to study the interaction relationship between these biomolecules in living cells. Super-resolution fluorescence nanoscopy by combining fluorescent markers with various biomarker technologies has become a powerful tool in molecular cell biology research today. However, the simultaneous tracking of different biomolecules in a particular organelle remains a serious challenge for fluorescent probes and labeling techniques, mainly due to the severe cytotoxicity, complexity of biodistribution and spectral discrimination problems caused by the simultaneous use of multiple fluorescent probes. Therefore, the development of an effective single-molecule fluorescent probe capable of simultaneously carrying out spatial localization observation on ROS, protein and nucleic acid in mitochondria and knowing the influence of ROS on a nucleic acid-protein complex is important in understanding the physiological function of mitochondria.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a monomolecular fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, protein and nucleic acid, and preparation and application thereof. The fluorescent probes of the invention can selectively enter cell mitochondria and interact with hydrogen peroxide, protein hydrophobic regions and nucleic acids to produce distinguishable fluorescence spectra for three-channel imaging. The three fluorescence emission wavelengths are longer, and the method is suitable for analyzing the spatial distribution of hydrogen peroxide, protein and nucleic acid in mitochondria and the interaction among molecules by super-resolution imaging. The single-molecule three-channel fluorescence imaging fluorescent probe solves the problems of serious cytotoxicity brought by simultaneously using a plurality of fluorescence probes for imaging, complication of biological distribution and spectrum differentiation of the fluorescence probes in cells and the like.
The invention relates to a monomolecular fluorescent probe, which is a cationic quinoline-vinyl-N, N-dimethylaniline borate derivative and has the following structural formula:
the preparation method of the monomolecular fluorescent probe comprises the following steps:
step 1: preparation of intermediate products
Dissolving 4-methylquinoline and 4-bromomethyl phenylboronic acid pinacol ester in an acetonitrile solution, wherein the feeding molar ratio is 1;
step 2: preparation of fluorescent probes
Dissolving the intermediate product and p-dimethylaminobenzaldehyde in absolute ethanol at a feeding molar ratio of 1.
According to the monomolecular fluorescent probe, the phenylboronate is connected to quinoline-vinyl-N, N-dimethylaniline through a single bond, and the fluorescent emission of the probe can be inhibited due to the dissipation excitation energy caused by the free rotation of the single bond in the molecule.
According to the invention, phenylboronate in the monomolecular fluorescent probe is used as a reaction site of hydrogen peroxide, and can be cracked with quinoline-vinyl-N, N-dimethylaniline to eliminate depletion of excitation energy caused by free rotation of a single bond, so that fluorescence emission is caused.
The quinoline-vinyl-N, N-dimethylaniline in the monomolecular fluorescent probe has higher hydrophobic property.
The hydrophobic characteristic of quinoline-vinyl-N, N-dimethylaniline in the monomolecular fluorescent probe can interact with amino acid residues in a hydrophobic cavity of protein, so that the single bond space rotation of a borate part is limited to cause fluorescence emission.
The monomolecular fluorescent probe has positive charges and large pi conjugation characteristics, can have various action modes (such as hydrogen bonds, electrostatic action and pi-pi accumulation) with nucleic acid, and is aggregated on nucleic acid molecules to form J-like aggregates, so that the fluorescence emission red shift is caused.
The single-molecule fluorescent probe is used as a detection reagent in the detection process of hydrogen peroxide, protein and nucleic acid in mitochondria to perform super-resolution fluorescence imaging analysis, shows three different long-wavelength emissions with distinguishable spectrums in response to the hydrogen peroxide, the protein and the nucleic acid, and can avoid the interference of autofluorescence of biomolecules.
The single-molecule fluorescent probe provided by the invention has high specificity on detection of hydrogen peroxide, protein and nucleic acid, and has basically no toxicity to cells.
The single-molecule fluorescent probe has mitochondria targeting specificity.
The fluorescent probe provided by the invention shows that the protein with the hydrophobic cavity is located on the cristae of mitochondria through super-resolution fluorescence imaging, the nucleic acid is uniformly distributed in the matrix of the mitochondria, and in addition, part of the protein and the nucleic acid are fused together to form a nucleic acid-protein complex.
The fluorescent probe disclosed by the invention shows that hydrogen peroxide serving as an upstream signal molecule can regulate the dissociation of a nucleic acid-protein complex in mitochondria through super-resolution fluorescence imaging.
The invention has the beneficial effects that:
1. the phenylboronate in the monomolecular fluorescent probe is connected to quinoline-vinyl-N, N-dimethylaniline through a single bond, the excitation energy can be exhausted through the rotation of the single bond in a molecule, so that the fluorescent emission is inhibited, and the Turn-on type fluorescent emission is shown when the monomolecular fluorescent probe reacts with hydrogen peroxide, protein and nucleic acid, so that the sensitivity of the probe is improved, and a false positive signal is avoided.
2. The responsiveness of the single-molecule fluorescent probe to hydrogen peroxide, protein and nucleic acid depends on three different response mechanisms, and three different fluorescence emission spectra with distinguishable spectra are shown, so that the simultaneous fluorescence imaging of the probe to the hydrogen peroxide, the protein and the nucleic acid is facilitated.
3. The monomolecular fluorescent probe shows higher selectivity on hydrogen peroxide, protein and nucleic acid, is easy to penetrate cell membrane targeting mitochondria and basically has no cytotoxicity, and increases the applicability of the probe in cells to simultaneous fluorescence imaging of the hydrogen peroxide, the protein and the nucleic acid in the mitochondria.
4. The monomolecular fluorescent probe shows long-wavelength emission to the response spectrum of hydrogen peroxide, protein and nucleic acid, is suitable for super-resolution fluorescence imaging analysis, and increases the applicability of the probe in the super-resolution space distribution fluorescence imaging analysis of the hydrogen peroxide, the protein and the nucleic acid in mitochondria.
5. The fluorescent probe provided by the invention is a monomolecular probe which can be used for simultaneously detecting hydrogen peroxide, protein and nucleic acid, and has the advantage of avoiding the problems of serious cytotoxicity, complex distribution of biological distribution, spectral region and the like caused by the simultaneous use of a plurality of probes.
Drawings
FIG. 1 shows (a) the structure of a mitochondrial-targeted fluorescent probe molecule and the mechanism for detecting hydrogen peroxide, proteins and nucleic acids, and (b) the simultaneous detection of hydrogen peroxide, proteins and nucleic acids in mitochondria by the fluorescent probe molecule.
FIG. 2 shows a fluorescent probe 1 H NMR analysis.
FIG. 3 shows a fluorescent probe 13 C NMR analysis.
FIG. 4 is HR-MS analysis of fluorescent probes.
FIG. 5 shows a fluorescent probe directed to hydrogen peroxide (H) 2 O 2 ) The ultraviolet absorption spectrum of the responsiveness of protein (BSA) and nucleic acid (RNA).
FIG. 6 shows a schematic view of a graph showing a difference (a) between H and H 2 O 2 (b) BSA, (c) change in fluorescence intensity of the fluorescent probe at RNA and (d) DNA concentrations.
FIG. 7 shows a fluorescent probe and H 2 O 2 After reaction product 1 H NMR analysis.
FIG. 8 shows a fluorescent probe and H 2 O 2 After reaction product 13 C NMR analysis.
FIG. 9 shows a fluorescent probe and H 2 O 2 And (4) performing HR-MS analysis on a product after the reaction.
FIG. 10 is the change in fluorescence intensity after competitive displacement of fluorescent probes in BSA by warfarin.
FIG. 11 is a graph showing that the fluorescence spectrum of the fluorescent probe is significantly red-shifted as the concentration of the fluorescent probe increases.
FIG. 12 is a selective assay of fluorescent probes for various assay substrates contained within cells.
FIG. 13 is a fluorescent probe pair H 2 O 2 Protein and nucleic acid responsive spectral overlay analysis, (a) H 2 O 2 A channel: exciting at 405nm, and collecting at 500-560nm; (b) protein channels: exciting at 530nm, and collecting at 550-590nm; (c) a nucleic acid channel: excitation is carried out at 610nm, and collection is carried out at 710-770nm.
FIG. 14 is a fluorescent probe cytotoxicity assay.
FIG. 15 is a fluorescent probe pair H 2 O 2 And protein and nucleic acid are detected and analyzed in the coexistence of the three.
FIG. 16 is a negative control of fluorescent probe cell imaging assays (a) and (b) using Dithiothreitol (DTT) and Nuclease (Nuclease) in cells.
FIG. 17 shows (a) exogenous H in cells 2 O 2 And (b) endogenous H 2 O 2 Control experiment for imaging.
FIG. 18 is a co-localized imaging analysis of fluorescent probes in cells.
FIG. 19 is H in mitochondria by fluorescent probe 2 O 2 Protein and nucleic acid super-resolution fluorescence imaging. (a) Standard confocal fluorescence imaging, (b, c) H in mitochondria 2 O 2 Ultra-high resolution images of proteins and nucleic acids.
FIG. 20 is a super-resolution fluorescence imaging analysis H 2 O 2 Effects on nucleic acid-protein complexes in mitochondria. (a) Exogenous different doses of H 2 O 2 Graph of the effect on nucleic acid-protein complexes in mitochondria. (b) a the overlap factor of protein and nucleic acid in graph and H 2 O 2 Dose correlation, (c) PMA induced endogenous different doses of H 2 O 2 Map of the effect on nucleic acid-protein complexes in mitochondria, (d) map of protein-to-nucleic acid overlap factor and H 2 O 2 Dose dependence, (e) H 2 O 2 Cleared super-resolution fluorescence imaging, (f) H 2 O 2 Fluorescence intensity of the channel and overlap factor of protein and nucleic acid under different treatments.
Detailed Description
The technical content of the present invention is further illustrated by the following examples, but the essence of the present invention is not limited to the following examples, and those skilled in the art can and should understand that any simple transformation or replacement based on the spirit of the invention should fall into the protection scope of the present invention.
Example 1: preparation of intermediate products
4-methylquinoline (0.51g, 3.60mmol) and 4-bromomethylbenzeneboronic acid pinacol ester (1.60g, 5.40mmol) were dissolved in 100mL of acetonitrile solution, and the mixed solution was refluxed at 90 ℃ for 12 hours, cooled to room temperature, and a white precipitate was collected as an intermediate product. 1 H NMR(DMSO-d 6 ,400MHz,δ):9.63(d,1H,quinoline H),8.56(d,1H,quinoline H),8.38(d,1H,quinoline H),8.21-8.14(m,2H,quinoline H),8.01(t,1H,quinoline H),7.68-7.66(d,2H,ArH),7.35-7.33(d,2H,ArH),6.36(s,2H,-CH 2 ),3.06(s,3H,-CH 3 ),1.27(s,12H,-CH 3 ). 13 C NMR(DMSO-d 6 ,400MHz,δ):19.84,24.59,59.56,83.81,119.62,122.98,126.45,127.28,129.17,129.66,135.01,135.19,137.28,149.28,159.73.HR-MS(m/z,ESI):Cal.for[C 23 H 27 BNO 2 ] + ,m/z＝360.2129；[M] + found,m/z＝360.2163.
Example 2: preparation of fluorescent probes
The intermediate (0.60g, 1.36mmol) and p-dimethylaminobenzaldehyde (0.21g, 1.40mmol) were dissolved in 100mL of anhydrous ethanol, and two drops of piperidine were added dropwise to the solution. And refluxing the mixed solution at 70 ℃ for 6h, and separating the crude product by silica gel column chromatography to obtain the fluorescent probe. 1 H NMR(DMSO-d 6 ,400MHz,δ):9.35(d,1H,quinoline H),9.04(d,1H,quinoline H),8.46(d,1H,quinoline H),8.26(d,1H,quinoline H),8.19(d,1H,quinoline H),8.07(d,1H,quinoline H),8.02(d,1H,ArH),7.91-7.85(m,3H,ArH),7.65(d,2H,ArH),7.31(d,2H,-CH＝CH-),6.82(d,2H,ArH),6.19(s,2H,-CH 2 ),3.07(s,6H,-CH 3 ),1.25(s,12H,-CH 3 ). 13 C NMR(DMSO-d 6 ,400MHz,δ):24.58,40.12,58.44,83.78,111.94,113.05,114.11,123.08,126.22,128.44,131.69,134.75,135.02,137.95,145.85,146.78,152.55.HR-MS(m/z,ESI):Calcd for[C 32 H 36 BN 2 O 2 ] + ,m/z＝491.2864；[M-Br] + found,m/z＝491.2865.
Example 3: measurement of ultraviolet absorption Spectroscopy
To 3ml of PBS buffer solution (0.01M, pH 7.4) was added fluorescent probe molecules to a concentration of 10. Mu.M, followed by dropwise addition of hydrogen peroxide (H) 2 O 2 100 μ M), or bovine serum albumin (BSA, 1 mg/mL), or ribonucleic acid/deoxyribonucleic acid (RNA/DNA, 20 μ M), the change in the UV absorption spectrum was recorded, and as can be seen from FIG. 5, the fluorescent probe molecule had a strong absorption peak at 530nm, and H was added 2 O 2 Then, the absorption peak at 530nm disappears, and a new absorption peak appears at 380nm, which indicates that the probe molecule can react with H 2 O 2 The reaction is carried out. When BSA was added, the peak at 530nm was enhanced, indicating that the probe interacted with BSA. When RNA is added, the absorption peak at 530nm has obvious red shift, which indicates that the probe has interaction with the RNA.
Example 4: fluorescent probes at different H 2 O 2 Change in fluorescence intensity at BSA or RNA concentration
To test fluorescent probe molecules at different H 2 O 2 Strong fluorescence under BSA or RNA conditionsExamination of degree Change, fluorescent probe molecules were added to 1ml of PBS buffer (0.01M, pH 7.4) to give a concentration of 10. Mu.M, and then H was added thereto 2 O 2 (0-100. Mu.M), BSA (0-3 mg/mL) or RNA (0-100. Mu.M), and the change in fluorescence intensity was examined, as shown in FIG. 6, with H 2 O 2 The fluorescence intensity at 580nm gradually increased with increasing concentration. When adding H 2 O 2 The final concentration of the fluorescent probe is 100 mu M, and the increase multiple of the fluorescent probe molecule can reach 44 times. The fluorescence intensity at 632nm gradually increased with increasing BSA concentration. When BSA is added to the solution at a final concentration of 3mg/mL, the increase of the fluorescent probe molecule can reach 582 times. The fluorescence intensity at 688nm gradually increased with increasing RNA concentration. When the final concentration of the added RNA is 100 mu M, the increase multiple of the fluorescent probe molecule can reach 172 times.
Example 5: fluorescent probe pair H 2 O 2 Investigating the mechanism of BSA or RNA luminescence
To explore the fluorescent Probe pair H 2 O 2 By the fluorescent light-emitting mechanism of (1), we refer to H 2 O 2 The products after reaction with the fluorescent probe were separated and subjected to nuclear magnetic and mass spectrometry, as shown in FIGS. 7,8, and 9: h 2 O 2 Causing cleavage of the borate ester and further decomposition of the quinoline-vinyl-N, N-dimethylaniline, eliminating depletion of excitation energy by single bond rotation, resulting in fluorescence emission.
To explore the "turn-on" mechanism of BSA for fluorescent probes, a competition experiment was performed with the classical compound warfarin (bound to the hydrophobic cavity of BSA), as shown in FIG. 10, where warfarin was added to the pre-mixed solution of probe and BSA, the fluorescence of the fluorescent probe gradually decreased at 632nm, indicating that the probe was bound to the hydrophobic region of BSA. This result indicates that the fluorescent response of the probe to BSA is due to the spatial rotation of the fluorescent molecule limited by the interaction of the probe with amino acid residues in the hydrophobic cavity of the protein.
Fluorescent probes contain positive charges and pi-conjugation features that provide multiple interactions (e.g., hydrogen bonding, electrostatic interactions, pi-pi stacking, etc.) with negatively charged nucleic acid strands. However, unlike the hydrophobic regions of proteins, DNA or RNA strands can bind multiple probe molecules through these interactions. Thus, the nucleic acid strand acts like a compressor, causing the fluorescent probe to accumulate on the nucleic acid strand, resulting in a red-shift in the fluorescent emission. To help understand this effect, we measured the fluorescence spectrum of a high concentration fluorescent probe solution, and as can be seen in fig. 11, as the probe concentration increases, the fluorescence spectrum undergoes a significant red shift phenomenon, similar to the formation of J-aggregated crystals of organic dye molecules.
Example 6: selective exploration of fluorescent probes
To test fluorescent probe molecules against other ROS and biomolecules contained within the cell such as: the selectivity of inorganic salts, amino acids, polypeptides and other proteins or enzymes was measured by adding fluorescent probes (10. Mu.M) to PBS buffer (0.01M, pH 7.4), and then adding biomolecules, such as hydrogen peroxide, hydroxyl radicals, superoxide anions, nitrous oxide ions, nitric oxide, hypochlorous acid, singlet oxygen, calcium ions, magnesium ions, cysteine, serine, lysine, glutathione, bovine Serum Albumin (BSA), human Serum Albumin (HSA), lysozyme, hemoglobin, trypsin, immunoglobulin G, pepsin, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), which are commonly found in cells, respectively, after reacting with the probes. As can be seen from FIG. 12, only H is added 2 O 2 BSA, HSA, RNA and DNA, the fluorescence intensity of the fluorescent probe molecule changes significantly at 580nm,632nm or 688 nm. The probe has good selectivity.
Example 7: spectral overlay analysis
To explore the fluorescent Probe pair H 2 O 2 And performing spectral overlap analysis on BSA and RNA responses, and selecting 405nm,530nm and 610nm respectively to excite the response of the fluorescent probe and the three substances. As shown in fig. 13, spectral overlap was suppressed below 5% in the selected fluorescence collection range.
Example 8: cytotoxicity assays
Human hepatoma cells (Hep G2) at 1 × 10 per well 4 The individual cells were inoculated into a 96-well plate, and the 96-well plate was placed in a cell incubatorThe culture conditions are as follows: 37 deg.C, 5% CO 2 And culturing for 24 hours under saturated humidity to ensure that the cells are completely attached to the wall. Then, replacing the fresh culture solution, adding 20 mu L of fluorescent probe dispersion liquid with different concentrations, after culturing for 12h, adding MTT solution (5 mg/mL) into each hole, continuing to incubate for 4h, then adding 150 mu L of DMSO into each hole, placing the 96-hole plate on a horizontal oscillation table, oscillating for 10min, setting the wavelength to be 570nm on a microplate reader, measuring the absorbance (OD value) of the solution in each hole of the 96-hole plate, and calculating the cell survival rate according to the following formula: cell viability = (OD) Group to be tested －OD Blank group )/(OD Cell group －OD Blank group ) X100%. As seen in FIG. 14, the fluorescent probe was almost non-cytotoxic in the range of 2-20. Mu.M.
Example 9: detection of three substances by fluorescent probe
We investigated the ability of the probe to spectrally discriminate three analytes in a hybrid system before cellular imaging was performed. As shown in fig. 15, when H is added 2 O 2 When BSA and RNA were added together to the probe solution, fluorescence emissions at 580, 632 and 688nm were detected, respectively, indicating that H could be detected separately with the fluorescent probe in a co-existing system 2 O 2 Proteins and nucleic acids.
Example 10: live cell imaging of fluorescent probes
Hep G2 cells were seeded into glass-bottom culture dishes and cultured at 37 ℃ for 24h. Subsequently, a probe (10. Mu.M) was added to the cells and incubated at 37 ℃ for 30min, followed by 3 washes with PBS. Finally, exogenous H is added 2 O 2 After incubation for 30min (100. Mu.M) or 1h of phorbol 12-myristate 13-acetate (PMA, 2. Mu.g/mL), cells were washed 3 times with PBS and imaged with confocal laser microscopy, with the results shown in FIGS. 16, 17: three channels of fluorescence signals come from the probe and H in the cell 2 O 2 Protein and nucleic acid responses.
Example 11: intracellular co-localization imaging
In co-localization experiments, hep G2 cells were first incubated with fluorescent probes (10. Mu.M) for 30min, followed by sequential incubation with 2. Mu.g/mL PMA for 1h, 2. Mu.M mitochondrial quotientThe staining reagent was incubated for 20min. Finally, cells were washed 3 times with PBS solution (pH 7.4) and imaged with laser confocal microscopy. As can be seen from FIG. 18, the fluorescent probes co-localized in mitochondria and simultaneously addressed H in the mitochondria of cells 2 O 2 And protein and nucleic acid are subjected to fluorescence imaging.
Example 12: super-resolution imaging of fluorescent probes
The fluorescence probe was subjected to super-resolution nanomicroscopy under confocal microscopy, excited under super-resolution laser, and the emission signal was collected using a HyD reflectance detector. The preparation method of the super-resolution imaging living cell is consistent with the imaging of a common confocal microscope. The super-resolution micrographs were further processed using Huygens specialty software (version: 16.05) under authorized permission. As can be seen from fig. 19, the protein having the hydrophobic cavity is located on the cristae of mitochondria, the nucleic acid is distributed relatively uniformly in the matrix of mitochondria, and a part of the protein is fused with the nucleic acid to form a nucleic acid-protein complex.
Example 13: h 2 O 2 Effect on nucleic acid-protein complexes in mitochondria
To explore H 2 O 2 Triggering the dissociation of mitochondrial nucleic acid-protein complexes in living cells, first, varying concentrations of H 2 O 2 The cells were treated for 2h, then incubated with fluorescent probes for 30min, and then, after washing 3 times with PBS buffer, the cells were subjected to super-resolution fluorescence imaging in a laser confocal microscope. Each kind of H 2 O 2 Six pictures were taken at the dose, and then two 20 μm pictures were randomly selected on each super-resolution picture 2 And calculating the overlap coefficient of the protein and nucleic acid channels according to the region, thus obtaining twelve overlap coefficient data under each dose, and finally carrying out statistical analysis according to the twelve data. In addition, the light from H is collected during the photographing process 2 O 2 Fluorescence intensity of the channel. PMA-induced endogenous H was studied by a similar method 2 O 2 Production, effect on mitochondrial nucleic acid-protein complexes. As can be seen from FIG. 20, hydrogen peroxide as an upstream signaling molecule can trigger nucleic acid-protein complexes in mitochondriaDissociation of (2).
Claims (6)
1. A monomolecular fluorescent probe for simultaneous super-resolution imaging of mitochondrial hydrogen peroxide, proteins and nucleic acids is characterized by the following structural formula:
2. the method for preparing the monomolecular fluorescent probe according to claim 1, which is characterized by comprising the following steps:
step 1: preparation of intermediate products
Dissolving 4-methylquinoline and 4-bromomethyl phenylboronic acid pinacol ester in acetonitrile solution, carrying out reflux reaction for 12 hours at 90 ℃, cooling to room temperature, and collecting white precipitate to obtain an intermediate product;
step 2: preparation of fluorescent probes
And dissolving the intermediate product and p-dimethylamine benzaldehyde in absolute ethyl alcohol, then dropwise adding two drops of piperidine, carrying out reflux reaction at 70 ℃ for 6 hours, and carrying out chromatographic separation on the crude product by using a silica gel column to obtain the target product.
3. The method of claim 2, wherein:
in the step 1, the feeding molar ratio of the 4-methylquinoline to the 4-bromomethylbenzeneboronic acid pinacol ester is 1.5.
4. The production method according to claim 2, characterized in that:
in step 2, the feeding molar ratio of the intermediate product to p-dimethylamine benzaldehyde is 1.
5. Use of the monomolecular fluorescent probe according to claim 1 for preparing a detection reagent, characterized in that:
the detection reagent is used for detecting hydrogen peroxide, protein and nucleic acid in mitochondria.
6. Use according to claim 5, characterized in that:
through super-resolution fluorescence imaging analysis, the single-molecule fluorescent probe shows three different long-wavelength emissions with spectrally distinguishable responses to hydrogen peroxide, protein and nucleic acid, and can avoid interference of autofluorescence of biomolecules.
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