Super-resolution imaging probe of GQDs-double fluorescent dye based on FRET (Fluorescence Resonance Energy Transfer) effect

Abstract

The invention relates to the technical field of super-resolution optical imaging, in particular to a super-resolution imaging probe of GQDs-double fluorescent dye based on an FRET effect. The probe takes graphene quantum dots as donors and double fluorescent dyes as receptors, the surfaces of the graphene quantum dots are modified with polyethylene glycol, and the probe is used for super-resolution optical imaging of specific organelles through antibody specific targeting. According to the super-resolution imaging probe of the GQDs-double fluorescent dye based on the FRET effect, provided by the invention, single-laser excited double-channel super-resolution optical imaging is realized, and the super-resolution imaging probe can be used for super-resolution optical imaging based on a single-molecule positioning method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of super-resolution optical imaging technology, and particularly relates to a GQDs-double fluorescent dye super-resolution imaging probe based on FRET effect. BACKGROUND

[0002] In the past few decades, a series of imaging technologies that can break through the 200 nm resolution limit of optical microscopy have emerged, which is called super-resolution imaging microscopy technology. Super-resolution imaging technology is mainly based on two methods, which are: (1) Super-resolution imaging technology based on light modulation, such as stimulated emission depletion microscopy (STED) and structured illumination microscopy (SIM); (2) Super-resolution imaging technology based on single molecule localization (SMLM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). The principles of PALM and STORM are very similar, and the imaging principle is to let only a small number of probes (fluorescent protein or fluorescent dye molecules) emit light and collect fluorescent signals at the same time, and the super-resolution image is reconstructed by using single molecule localization technology through cyclic repetition.

[0003] Fluorescence resonance energy transfer (FRET) is a non-destructive optical detection method with wide application prospects, which refers to the phenomenon that the donor fluorescence energy of two different fluorescent groups (donor and acceptor) is transferred to the acceptor. The conditions for FRET to occur include: the emission spectrum of the donor and the absorption spectrum of the acceptor partially overlap, and the donor and the acceptor are at a suitable distance (usually less than 100 Å). Nanoprobes based on FRET effect have been widely used in DNA folding and catalysis and other fields.

[0004] Quantum dots (QDs) are nanometer particles with extremely small size and radius less than or close to its Bohr radius. Quantum dots are generally spherical and have very small particle size, about 1-10 nm. Because the electrons and holes are quantum confined, they become energy level structures with molecular characteristics, and can be excited to produce fluorescence under laser irradiation.

[0005] Graphene quantum dots (GQDs) are zero-dimensional graphene / oxidized graphene nanomaterials with edge effect or defect state (surface state) related luminescent properties. Surface state makes excitation-dependent photoluminescence possible, and the functional groups on the surface of GQDs can make them have good water solubility. Compared with other quantum dots, the main advantages of GQDs include excellent fluorescence properties, biocompatibility, water solubility and low toxicity, and have been widely used in optics, biomedical imaging and biosensing and other aspects.

[0006] To this end, the application provides a GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect, which provides another technical solution to the above technical problems. SUMMARY

[0007] Therefore, it is necessary to provide a GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect to solve the technical problems in the background art.

[0008] To solve the above technical problems, the application adopts the following technical solutions: The GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect is characterized in that graphene quantum dots are used as donors, bifluorescent dyes are used as receptors, the graphene quantum dots are surface-modified with polyethylene glycol, and the probe is specifically targeted by antibodies.

[0009] As a preferred embodiment of the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect provided by the application, the graphene quantum dots are surface-modified with PEG with amino groups at both ends, specifically .

[0010] As a preferred embodiment of the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect provided by the application, the graphene quantum dots are connected with bifluorescent dyes through EDC / NHS cross-linking reaction.

[0011] As a preferred embodiment of the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect provided by the application, the bifluorescent dyes are Alexa Fluor NHS ester.

[0012] The preparation method of the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect includes the following steps: A. preparing graphene oxide; B. preparing graphene quantum dots; C. surface-modifying PEG on the graphene quantum dots through EDC / NHS cross-linking reaction; D. connecting bifluorescent dyes on the graphene quantum dots; E. labeling antibodies on the graphene quantum dots.

[0013] As a preferred embodiment of the preparation method of the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect provided by the application, FRET effect occurs between the graphene quantum dots and the bifluorescent dyes, and both channels have fluorescence flickering characteristics.

[0014] The use method of the super-resolution imaging probe of GQDs-double fluorescent dye based on FRET effect, which is labeled by EDC / NHS cross-linking reaction.

[0015] The application of the super-resolution imaging probe of GQDs-double fluorescent dye based on FRET effect, which is targeted to the super-resolution optical imaging of specific organelles by antibodies.

[0016] It can be seen without doubt that the above technical solutions of the present application can solve the technical problems to be solved by the present application.

[0017] Meanwhile, the above technical solutions have at least the following beneficial effects: The super-resolution imaging probe of GQDs-double fluorescent dye based on FRET effect provided by the present application realizes single laser excitation double-channel super-resolution optical imaging, can be used for super-resolution optical imaging based on single molecule localization method, and is applied to PALM and STORM imaging. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 The schematic diagram of the graphene quantum dot-double fluorescent dye super-resolution imaging probe based on FRET effect of the present application; Figure 2 The preparation process diagram of the graphene quantum dot-double fluorescent dye super-resolution imaging probe based on FRET effect proposed by the present application; Figure 3 The fluorescence spectrum of the probe in the embodiment of the present application; Figure 4 The FTIR spectrum of the probe in the embodiment of the present application. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0021] In order to make those skilled in the art better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings.

[0022] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0023] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0024] Example 1 Reference Figures 1-4 , A super-resolution imaging probe based on GQDs-dual fluorescent dyes using the FRET effect.

[0025] Figure 1 This invention provides a graphene quantum dot-dye dual-dye super-resolution fluorescent probe based on the FRET effect, wherein the structures are labeled as 1, graphene quantum dot; 2, and 3. 3. Fluorescent dye 1; 4. Fluorescent dye 2; 5. Antibody.

[0026] The PBS buffer (Phosphate Buffer Saline) involved in this embodiment has a concentration of 10 mM (unit is millimoles per liter, which can also be written as mmol / L) and pH = 7.4.

[0027] Using Alexa Fluor 488 (AF 488) and Alexa Fluor 568 (AF 568) as fluorescent dye 1 and fluorescent dye 2, respectively, and thus having different emission wavelengths, a graphene quantum dot-dual fluorescent dye super-resolution imaging probe based on the FRET effect is developed. Figure 2 As shown, it includes the following steps: Step 1: Preparation of graphene oxide Add 1 g of graphite powder to 23 mL of concentrated sulfuric acid (98% by mass) and stir overnight. Transfer the mixture to an ice bath, then carefully add 4 g of potassium permanganate and continue stirring for 1 hour. Heat the mixture to 40 °C and stir for 30 minutes. Continue heating the mixture to 100 °C and dilute it to 100 mL, stirring for 30 minutes. Add 10 mL of hydrogen peroxide (30% by mass), and the mixture immediately turns bright yellow. Continue stirring and cooling to room temperature. Remove the reaction solution, centrifuge to remove the supernatant, and continue centrifuging and washing at least three times with deionized water and 5% dilute hydrochloric acid solution until the solution pH=7. Sonicate the obtained solution for 30 minutes using an ultrasonic probe, then centrifuge and wash again to obtain the graphene oxide solution (GO).

[0028] Step 2: Preparation of graphene quantum dots The graphene oxide solution prepared in step one was dispersed in dimethylformamide (DMF) at a concentration of 300 mg / mL. The mixture was sonicated for 40 minutes using an ultrasonic probe (120 W). After sonication, the mixture was placed in a high-pressure reactor (lined with polytetrafluoroethylene) and reacted at 200 °C for 5 hours. After cooling to room temperature, the mixture was removed and filtered through a microporous membrane to remove the precipitate, resulting in a brown solution. Residual DMF was removed using a rotary evaporator. The resulting GQDs solution was dialyzed using a dialysis membrane (molecular weight cutoff of 500 D). The dialysis process required multiple changes of deionized water. Dialysis further removed residual DMF, and the GQDs solution was obtained after dialysis.

[0029] Step 3: PEG (polyethylene glycol) modification of GQDs Add 4 mL of GQDs solution to 12 mg EDC and 18 mg NHS, stir for 30 minutes, then add 1 mL of GQDs solution. A 50 mg / mL solution was prepared and stirred for another 2 hours. The mixture was dialyzed through a dialysis membrane (3500 D), and the resulting solution was concentrated using an ultrafiltration tube (Amicon Ultra-15, 3KD, Millipore) and finally dispersed to obtain 5 mL of GQDs-PEG solution.

[0030] Step 4: Connect the dual fluorescent dyes 1 μL Alexa Fluor NHS Ester ( M) was added to the GQD-PEG solution in step 3, and the mixture was stirred at room temperature for 4 hours. Excess Alexa Fluor dye was removed by dialysis through a dialysis membrane (molecular weight cutoff 3500 D), and the resulting solution was concentrated and redispersed in PBS solution through an ultrafiltration tube (Amicon Ultra-15, 3KD, Millipore).

[0031] Step 5: Label with specific antibodies 100 μL of Goat anti-mouse IgG was added to 2 mL of EDC and NHS pretreated GQDs-Alexa Fluor dyes solution, and the mixture was stirred at room temperature for 4 hours. The reaction was continued overnight. The resulting solution was concentrated through an ultrafiltration tube (Amicon Ultra-15, 3KD, Millipore) and redispersed in PBS solution.

[0032] The fluorescence spectrum of the probe prepared in step four of this embodiment is as follows: Figure 3As shown, under 405 nm laser irradiation, two emission peaks at 525 nm and 600 nm appeared, corresponding to the fluorescence emission peaks of Alexa Fluor 488 and Alexa Fluor 568, respectively. Figure 4 is the FTIR spectrum of the probe prepared in step four, after the surface amino-modification of GQDs, two new characteristic peaks at 1551 and 2957 appeared, corresponding to the asymmetric stretching vibration of N-H bond and the asymmetric stretching vibration of C-H, and the stretching vibration peak (3430 ) and the bending vibration peak (1365 ) of O-H became more obvious In this embodiment, the probe uses graphene quantum dots (GQDs) as the donor, and double-fluorescent dyes as the acceptor, the surface of GQDs is modified with polyethylene glycol (PEG), the probe targets specifically through antibodies, and the surface ligand of the probe is also an antibody, so that the probe can be used for immunofluorescence super-resolution imaging.

[0033] In this embodiment, the double-fluorescent dyes are Alexa Fluor NHS ester (containing carboxyl groups).

[0034] Embodiment Two The probe prepared in Embodiment One is labeled with antibodies through EDC / NHS cross-linking reaction.

[0035] Embodiment Three The probe prepared in Embodiment One enables FRET effect between GQDs and double-fluorescent dyes, and both channels have fluorescence blinking characteristics.

[0036] Embodiment Four The probe prepared in Embodiment One is targeted by antibodies for super-resolution optical imaging of specific organelles.

[0037] The preferred embodiments of the application disclosed above are only used to help explain the application. The preferred embodiments do not describe all the details, nor limit the application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the present application. The present application selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application. The present application is limited only by the claims and their full scope and equivalents.

Claims

1. A GQDs-bifluorescent dye based super-resolution imaging probe based on FRET effect, characterized in that, The probe takes graphene quantum dots as a donor, double fluorescent dyes as an acceptor, graphene quantum dots are modified by polyethylene glycol, and the probe is specifically targeted by antibodies.

2. The GQDs-bifluorescent dye based super-resolution imaging probe based on FRET effect according to claim 1, characterized in that, The graphene quantum dots are modified by PEG with amino groups at both ends.

3. The GQDs-bifluorescent dye based FRET effect based super-resolution imaging probe according to claim 1, characterized in that, The graphene quantum dots are connected with double fluorescent dyes through EDC / NHS cross-linking reaction.

4. The GQDs-bifluorescent dye based FRET effect based super-resolution imaging probe according to claim 1, characterized in that, The double fluorescent dyes are Alexa Fluor NHS ester.

5. A method for preparing a GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect, characterized in that, The steps are as follows: A. Preparation of graphene oxide; B. Preparation of graphene quantum dots; C. Modification of PEG on the surface of graphene quantum dots through EDC / NHS cross-linking reaction; D. Connection of double fluorescent dyes on the surface of graphene quantum dots; E. Labeling of antibodies on the surface of graphene quantum dots.

6. The method for preparing the GQDs-bifluorescent dye based super-resolution imaging probe based on FRET effect according to claim 5, characterized in that, FRET effect occurs between the graphene quantum dots and the double fluorescent dyes, and both channels have fluorescence flickering characteristics.

7. A method for using the GQDs-bifluorescent dye super-resolution imaging probe based on FRET effect, characterized in that, The probe is labeled by antibodies through EDC / NHS cross-linking reaction.

8. Use of GQDs-bifluorescent dye super-resolution imaging probes based on FRET effect, characterized in that, The probe is targeted by antibodies for super-resolution optical imaging of specific organelles.

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