Preparation and multi-element detection application of nano probe based on metal organic framework material
Patent Information
- Application Number
- CN202411402344.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-02-28
Smart Images
Figure QLYQS_1 
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biochemical analysis, and more specifically, relates to the preparation and multivariate detection application of a nanoprobe based on metal organic framework materials. Background Art
[0002] Adenosine triphosphate (ATP), as the "currency molecular unit" of cellular energy transfer, is the energy source of cellular metabolism and biochemical processes, participates in a variety of cellular activities, and is considered to be an important chemical signal agent for cell vitality and damage. In the process of mitochondria producing ATP, there is always the appearance of byproduct reactive oxygen species (ROS). Excessive ROS will attack mitochondria or other organelles or other substances in the cell, causing inflammation or direct cell apoptosis. In order to prevent the fatal effects of excessive ROS, cells will produce responsive reducing substances to cope with oxidative stress. Glutathione (GSH) is one of the most important antioxidants in the cellular antioxidant system. It can protect cells from free radicals or reactive oxygen species and plays an important role in maintaining the redox homeostasis of the human body. Changes in the levels of both ATP and GSH have been found in tumor cells. Given the correlation between ATP and GSH, it is of great significance to simultaneously detect these two active molecules to better understand their changes at the cellular level during disease progression.
[0003] Common fluorescent nanoprobes include organic small molecule fluorophores, upconversion nanoparticles, carbon dots, quantum dots, aggregation-induced emission (AIE) fluorescent molecules, metal organic frameworks (MOFs), etc. They have many advantages such as high sensitivity, strong anti-interference ability, fast response, simple synthesis, low cost, and non-destructiveness. However, most of the fluorescent nanoprobes reported so far are only for single GSH or single ATP detection, and there are very few fluorescent nanoprobes that can quantify these two substances at the same time. Summary of the invention
[0004] The purpose of the present invention is to provide a preparation of a composite fluorescent nanoprobe for simultaneous detection of GSH and ATP and for fluorescent imaging of intracellular GSH and ATP.
[0005] On the one hand, the present invention provides a method for preparing a composite fluorescent nanoprobe, comprising: mixing zirconium tetrachloride (ZrCl4), an organic ligand having a maleimide group (H2L1) and copper nitrate (Cu(NO3)2) to prepare Cu-Mi-UiO-66, and then mixing Cu-Mi-UiO-66 with an ATP aptamer labeled with a fluorescent dye to synthesize a composite fluorescent nanoprobe.
[0006] The fluorescent dye labeled by the ATP aptamer satisfies that its fluorescence emission can be quenched by Cu-Mi-UiO-66, and the fluorescence emission peak does not overlap with Cu-Mi-UiO-66, such as Cy5, Cy3, and ROX.
[0007] In the above preparation method, Cu-Mi-UiO-66 has an octahedral structure, which is similar to the classic UiO-66 structure, but it has two improvements: 1) maleimide group modification; 2) incorporation of the metal element Cu.
[0008] The incorporation of the Cu element is specifically as follows: Cu combines with the -OH / -OH2 ligands in UiO-66 and MOF, covering the defect sites on the Zr oxide clusters to form Cu-Mi-UiO-66.
[0009] The incorporation of Cu during the synthesis of the composite fluorescent probe of the present invention reduces the bulk fluorescence of Mi-UiO-66, achieves a greater fluorescence turn-on effect, and improves the detection sensitivity. The composite fluorescent nanoprobe designed by the present invention has the characteristics of dual fluorescence emission, and can detect GSH and ATP without interfering with each other. In addition, intracellular fluorescence imaging of GSH and ATP can also be achieved.
[0010] Further, the synthesis of H2L1 is as follows:
[0011]
[0012] Further, the preparation of Cu-Mi-UiO-66 is as follows:
[0013] Zirconium tetrachloride (ZrCl4), an organic ligand with a maleimide group (H2L1) and copper nitrate are added to DMF. The final concentrations of ZrCl4, H2L1 and copper nitrate in the obtained mixed solution are all 10-50 mmol / L. After complete dissolution, an appropriate amount of acidic substance 0.5-5 ml is added. The acidic substance includes but is not limited to acetic acid, hydrochloric acid, etc., and then reacted in an environment of 120±20℃ for 4 hours or more. Finally, a light green product, namely Cu-Mi-UiO-66, is obtained after centrifugal washing and vacuum drying;
[0014] Furthermore, when the fluorescent dye labeled by the ATP aptamer is Cy5, the specific preparation steps of the composite fluorescent nanoprobe are as follows:
[0015] The sequence of the ATP aptamer labeled with Cy5 fluorescent dye is:
[0016] 5'-ACCTGGGGGAGTATTGCGGAGGAAGGT-3', both ends can be labeled with Cy5;
[0017] 100-240 μg / ml of Cu-Mi-UiO-6 was mixed with 10-200 nM of ATP aptamer sequence labeled with Cy5 fluorescent dye, and oscillated at low speed at 37°C for not less than 3 min. The supernatant was then removed by centrifugation and the precipitate was collected to form a composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5.
[0018] The aptamer-Cy5 in the composite fluorescent nanoprobe is combined with the MOF material through π-π stacking and electrostatic adsorption.
[0019] The composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 has dual fluorescence emission characteristics, namely blue fluorescence (400-600nm) excited by UiO-66 at around 320-390nm and red fluorescence (620-720nm) excited by Cy5 at around 540-610nm.
[0020] The composite fluorescent nanoprobe synthesized by the present invention can respond to GSH and ATP at the same time. The composite fluorescent nanoprobe synthesized by the present invention can realize intracellular imaging of GSH and ATP without interfering with each other.
[0021] The composite fluorescent nanoprobe synthesized by the invention has good cell compatibility and has little effect on cell survival.
[0022] On the other hand, the present invention also discloses the application of the composite fluorescent nanoprobe in the synchronous detection of GSH and ATP and intracellular imaging. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 It is a schematic diagram of the synthesis of Cu-Mi-UiO-66 / aptamer-Cy5 and its dual fluorescence response principle to GSH and ATP;
[0024] Figure 2 is the fluorescence response of Mi-UiO-66 to GSH before and after Cu doping;
[0025] Figure 3 The fluorescence responses of Cu-Mi-UiO-66 and Cu-Mi-UiO-66 / aptamer-Cy5 to GSH (A) and ATP (B) were verified respectively. DETAILED DESCRIPTION
[0026] The technical scheme of the present invention is further described in detail below in conjunction with specific embodiments, but does not constitute any limitation to the present invention. Unless otherwise specified, the reagents, equipment and methods used in the present invention are conventional commercially available reagents, equipment and conventional methods in the art.
[0027] Example 1 Synthesis and characterization of composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5
[0028] The preparation steps are as follows:
[0029] Step 1. Synthesis of organic ligand with maleimide group (H2L1)
[0030]
[0031] The reaction was divided into two steps: (1) N-substitution reaction: Weigh 2-aminobenzene dimethyl carboxylate (20 mmol) and maleic anhydride (20 mmol) in a round-bottom flask and add 15-20 ml of ionic liquid (Bmin) (PF6). The mixture was placed on a magnetic heater and stirred at a constant temperature of 140°C for 2 hours and cooled to room temperature. The product was then extracted with anhydrous ether for several times, and the ether phase was evaporated under reduced pressure using a rotary evaporator to obtain a crude product. The product was then purified by column chromatography (silica gel, CH2Cl2) to obtain a yellow solid intermediate ester powder; (2) Ester hydrolysis reaction: Weigh the intermediate ester (3.36 mmol) and dissolve it in 40 ml of a tetrahydrofuran / methanol (THF / MeOH=1 / 1) mixed organic solution, and then slowly add 10 ml of NaOH or KOH solution (50-254 mmol) dropwise. The mixture was stirred at room temperature in the dark for 1 hour and then rotary evaporated to remove the reaction solvents THF and MeOH. Then, 10 ml of water was added and the remaining solution was heated until it was completely dissolved. The pH of the solution was adjusted with dilute HCl. When the pH was less than 2, a large amount of precipitate was precipitated. The precipitate was collected with a vacuum filter and washed with deionized water several times. Finally, it was dried in a vacuum drying oven to obtain the final product, a light yellow powder ligand H2L1.
[0032] Step 2. Zirconium tetrachloride (0.4 mmol), the ligand H2L1 (0.4 mmol) synthesized in step 1 and copper nitrate (0.4 mmol) were added to 16 ml of DMF, and 1.2 ml of acetic acid was added after complete dissolution, and then reacted at 120°C for 24 hours. Finally, a light green product, namely Cu-Mi-UiO-66, was obtained after centrifugation, washing and vacuum drying.
[0033] Step 3. The sequence of the ATP aptamer labeled with Cy5 fluorescent dye is
[0034] 5'-Cy5-ACCTGGGGGAGTATTGCGGAGGAAGGT-3';
[0035] Step 4. Mix the product Cu-Mi-UiO-6 (100-240 μg / ml) synthesized in step 2 with the ATP aptamer sequence (100 nM) described in step 3 (total volume: 1 ml), and oscillate at low speed at 37°C for not less than 3 minutes, then centrifuge to remove the supernatant and collect the precipitate to form a composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5, which is stored in a 4°C environment.
[0036] H NMR ( 1 HNMR) and high-resolution mass spectrometry were used to characterize the successful synthesis of the intermediate ester and ligand H2L1.
[0037] SEM, TEM, EDS mapping, DLS particle size test, N2 adsorption-desorption, PXRD, XPS and FTIR were used to characterize UiO-66, Mi-UiO-66 and Cu-Mi-UiO-66. The results showed that the obtained Cu-Mi-UiO-66 particles had uniform particle size distribution (50-100nm) and clear faces and edges. The introduction of maleimide groups and Cu made the Brunauer-Emmett-Teller surface areas of Mi-UiO-66 and Cu-Mi-UiO-66 smaller than that of classical UiO-66, but it did not affect the structural motif of UiO-type MOF.
[0038] TEM, EDS mapping, DLS particle size test and zeta potential test were used to characterize Cu-Mi-UiO-66 / aptamer-Cy5. The appearance of P element in EDS mapping and the change of zeta potential compared with Cu-Mi-UiO-66 indicate the successful preparation of Cu-Mi-UiO-66 / aptamer-Cy5.
[0039] In step 4, 120 μg / mL of Cu-Mi-UiO-66 is preferably used to obtain the best aptamer-Cy5 loading efficiency.
[0040] Step 4 preferably uses an incubation time of 10 min.
[0041] The synthesized composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 has good stability in pure water, HEPES buffer and HepG2 cell lysate.
[0042] Example 2 Fluorescence detection of GSH and ATP by Cu-Mi-UiO-66 / aptamer-Cy5
[0043] Step 1. Prepare a 1 mg / ml Cu-Mi-UiO-66 solution, mix it evenly with GSH solutions (800 μM) of different concentration gradients, and perform fluorescence spectrum test after equilibration in the dark for no less than 80 minutes;
[0044] Step 2. Take the probe prepared in Example 1 and mix it evenly with GSH solution (800 μM) of different concentrations, keep it away from light for equilibration for not less than 80 minutes, and then perform fluorescence spectrum test;
[0045] Step 3. Take the composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 prepared in Example 1 and mix it evenly with ATP solution (800 μM) of different concentrations. After incubation in a shaker at 37°C in the dark for not less than 80 minutes, centrifuge and take the supernatant for fluorescence spectrum testing.
[0046] The GSH test conditions were set as follows: excitation wavelength 360 nm, emission spectrum scanning range 380 nm to 600 nm.
[0047] The ATP test conditions were set as follows: excitation wavelength 570 nm, emission spectrum scanning range 600 nm to 720 nm.
[0048] All fluorescence tests were performed in triplicate.
[0049] The fluorescence intensity enhancement rate was calculated as Where F is the actual measured fluorescence intensity and F0 is the background fluorescence intensity.
[0050] The fluorescence intensity enhancement rates at 455 nm and 670 nm were linearly fitted with the concentrations of GSH and ATP to prepare standard curves.
[0051] The present invention has screened out that the most suitable buffer for GSH and ATP detection is HEPES buffer (pH=7.4).
[0052] The composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 can respond to GSH and ATP simultaneously. The detection principle is as follows Figure 1 As shown:
[0053] Due to the PET effect between the maleimide group and the benzene ring in the MOF ligand and the 4+ Due to the charge transfer between the metal centers, the blue fluorescence of UiO-66 and the red fluorescence of Cy5 in Cu-Mi-UiO-66 / aptamer-Cy5 were quenched.
[0054] When GSH and ATP are present, GSH can undergo Michael addition reaction with the maleimide group on the MOF skeleton, blocking the PET process and turning on the blue fluorescence of the probe. ATP binds to aptamer-Cy5 and causes it to detach from MOF, turning on the red fluorescence of Cy5. The content of GSH and ATP can be determined by the strength of blue and red fluorescence.
[0055] The PET effect in the MOF ligand is that the HOMO in H2L1 is basically present in the whole molecule delocalization, but the LUMO is mainly located in the maleimide group. This difference allows intramolecular charge transfer from the aromatic group to the maleimide part in H2L1, resulting in fluorescence quenching. When GSH is present, it undergoes a Michael addition reaction with the maleimide group, causing the maleimide group to partially become HOMO, blocking the PET process and restoring the blue fluorescence of Cu-Mi-UiO-66.
[0056] The quenching of Cy5 fluorescence is specifically the quenching of the fluorescence intensity of Cy5 by the transfer of the fluorescence from Cy5 to the MOF metal center Zr 4+ When aptamer-Cy5 binds to ATP and falls off the MOF surface, the distance effect causes Cy5 to bind to Zr 4+ The PET blocking during the experiment restored its red fluorescence.
[0057] The Cu-Mi-UiO-66 / aptamer-Cy5 composite fluorescent nanoprobe can realize intracellular imaging of GSH and ATP without interfering with each other. The Cu-Mi-UiO-66 / aptamer-Cy5 composite fluorescent nanoprobe has good cell compatibility and has little effect on cell survival.
[0058] The fluorescence sensing performance of MOF in response to GSH changes after Cu doping. Figure 2 As shown in the figure, Cu-Mi-UiO-66 has a lower initial fluorescence than Mi-UiO-66 at the same concentration. After adding GSH, the fluorescence enhancement of Cu-Mi-UiO-66 is greater than that of Mi-UiO-66. Cu-doped MOF exhibits a better fluorescence turn-on effect.
[0059] The cross-response verification of Cu-Mi-UiO-66 / aptamer-Cy5 fluorescence sensing of GSH and ATP was performed. Figure 3 When ATP was added alone, the fluorescence emission of the probe at 455nm did not change significantly; when GSH was added alone, the change in the fluorescence emission of the probe at 670nm was also negligible; only when a mixed solution of GSH and ATP was added, obvious fluorescence enhancement emission was observed at 455nm and 670nm.
[0060] The fluorescence emission of the target probe at 455 nm increased with the increase of the added GSH concentration (5-800 μM), the linear response range was: 5-450 μM, and the LOD value was calculated to be 2.17 μM.
[0061] The fluorescence signal of the target probe at 670 nm increased with the increase of ATP concentration (1-800 μM), and showed a good linear relationship in the range of 1-50 μM and 50-800 μM. The LOD value was calculated to be 0.635 μM.
[0062] The detection selectivity of the target probe for GSH and ATP was evaluated. Only GSH can significantly cause an enhanced change in the fluorescence intensity of the probe. Since the concentration of GSH (mM level) in the biological system is much higher than that of Cys (μM level), the weak fluorescence enhancement reaction induced by Cys can be ignored. Similarly, thanks to the high specific affinity of the aptamer for ATP,
[0063] Cu-Mi-UiO-66 / aptamer-Cy5 exhibited highly selective fluorescence on-response among ATP and its analogs (including GTP, CTP, UTP, AMP, ADP, etc.).
[0064] In order to demonstrate the actual sensing ability of the composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 in complex biological systems, the present invention implemented a standard addition experiment on samples in cell lysate and performed recovery analysis. However, the actual sample is not limited to cell lysate only, such as artificial serum.
[0065] The present invention implements a standard addition experiment on samples in cell lysate and performs recovery analysis, compares the detection results of the composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 designed by the present invention with those of commercial GSH and ATP ELISA detection kits, and analyzes the results by paired T test, and finds that the p value is greater than the significance level (p=0.05), indicating that the quantitative results of the constructed composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5 fluorescent sensor and the standard kit are basically consistent, and there is no systematic difference.
[0066] Example 3: Verification of intracellular GSH and ATP fluorescence imaging by Cu-Mi-UiO-66 / aptamer-Cy5
[0067] The present invention selects human liver cancer cells (HepG2) as the research object, but is not limited to HepG2 cells. Corresponding cells can be selected according to actual detection needs, and the present invention does not impose too many restrictions on this, such as liver cancer cells.
[0068] Cytotoxicity test. The MTT colorimetric method was used to evaluate the effects of Mi-UiO-66, Cu-Mi-UiO-66, and the target probe Cu-Mi-UiO-66 / aptamer-Cy5 on the survival rate of HepG2.
[0069] Cell laser confocal imaging. In addition to the addition of Cu-Mi-UiO-66 / aptamer-Cy5 during cell culture, HepG2 cell nuclei were stained with green cyanine SYTO nuclear dye.
[0070] The parameters of the laser confocal microscope were set as follows: green channel (excitation wavelength: 500 nm; detection wavelength: 530 nm); red channel (excitation wavelength: 650 nm; detection wavelength: 670 nm); blue channel (excitation wavelength: 360 nm; detection wavelength: 460 nm).
[0071] After Cu-Mi-UiO-66 / aptamer-Cy5 was co-incubated with cells for different time periods, laser confocal cell imaging was used to image the cells. The ability of cells to take up Cu-Mi-UiO-66 / aptamer-Cy5 was evaluated by comparing the blue and red fluorescence intensities at different incubation times.
[0072] After treating the same cells with GSH inhibitor N-ethylmaleimide (NEM), promoter α-lipoic acid (ALA), ATP inhibitor oligomycin and ATP inducer etoposide, Cu-Mi-UiO-66 / aptamer-Cy5 was added for co-incubation to verify the imaging ability of the target fluorescent probe for different levels of GSH and ATP in the cells.
[0073] Different types of cells can be co-cultured with Cu-Mi-UiO-66 / aptamer-Cy5 to further verify the imaging capability of the target fluorescent probe designed by the present invention on the levels of GSH and ATP in different cells.
[0074] To ensure the accuracy of the imaging results, the cells can be lysed, and the GSH and ATP in the cell lysate can be detected using commercial GSH and ATP ELISA kits, and the detection results can be compared with the cell imaging results.
[0075] Example 4 Synthesis of Cu-Mi-UiO-66 / aptamer-Cy3 and simultaneous detection of intracellular GSH and ATP and fluorescence imaging verification
[0076] The Cy5 label of the ATP aptamer sequence in Example 1 was replaced with a Cy3 label.
[0077] The ATP test conditions in Example 2 were set as follows: an excitation wavelength of 510 nm and an emission spectrum scanning range of 530 nm to 630 nm.
[0078] The remaining steps are then the same as in Example 1 and Example 2.
Claims
1. A method for preparing a composite fluorescent nanoprobe, characterized in that: include: Zirconium tetrachloride, an organic ligand having a maleimide group and copper nitrate are mixed to prepare Cu-Mi-UiO-66, and then the Cu-Mi-UiO-66 is mixed with an ATP aptamer labeled with a fluorescent dye to synthesize a composite fluorescent nanoprobe; The fluorescent dye labeled by the ATP aptamer satisfies that its fluorescence emission can be quenched by the Cu-Mi-UiO-66, and the fluorescence emission peak does not overlap with the Cu-Mi-UiO-66.
2. The preparation method according to claim 1, characterized in that: The fluorescent dye labeled with the ATP aptamer is Cy5 or Cy3 or ROX.
3. The preparation method according to claim 1, characterized in that: The synthesis of the organic ligand having a maleimide group is as follows:
4. The preparation method according to claim 1 or 3, characterized in that: The preparation of the Cu-Mi-UiO-66 is as follows: Zirconium tetrachloride, an organic ligand having a maleimide group and copper nitrate are added to DMF, and the final concentrations of zirconium tetrachloride, the organic ligand having a maleimide group and copper nitrate in the obtained mixed solution are all 10-50 mmol / L. After complete dissolution, 0.5-5 ml of an acidic substance is added, followed by reaction in an environment of 120±20°C, and finally centrifugation, washing and vacuum drying are performed to obtain Cu-Mi-UiO-66.
5. The preparation method according to claim 4, characterized in that: The acidic substance is acetic acid or hydrochloric acid.
6. The preparation method according to claim 1, characterized in that: The fluorescent dye labeled by the ATP aptamer is Cy5, and the sequence of the ATP aptamer labeled with the Cy5 fluorescent dye is: 5'-ACCTGGGGGAGTATTGCGGAGGAAGGT-3', both ends can be labeled with Cy5; 100-240 μg / ml of Cu-Mi-UiO-6 was mixed with 10-200 nM of ATP aptamer sequence labeled with Cy5 fluorescent dye and shaken at low speed at 37°C. The supernatant was removed by centrifugation and the precipitate was collected to form a composite fluorescent nanoprobe Cu-Mi-UiO-66 / aptamer-Cy5.
7. A composite fluorescent nanoprobe for simultaneous detection of GSH and ATP synthesized by the preparation method according to any one of claims 1 to 6.
8. Use of the composite fluorescent nanoprobe according to claim 7 in the simultaneous detection of GSH and ATP.
9. Use of the composite fluorescent nanoprobe according to claim 8 in intracellular imaging.