Membrane tension response type composite molecular sensor as well as preparation method and application thereof

Abstract

The invention discloses a membrane tension response type composite molecular sensor and a preparation method and application thereof, the membrane tension response type composite molecular sensor comprises a DNA tetrahedral framework formed by a plurality of single-stranded DNAs through complementary base pairing, and the single-stranded DNAs comprise single-stranded DNAs modified with vibration luminescent molecules, single-stranded DNAs modified with cholesterol molecules and unmodified single-stranded DNAs; the vibration luminescent molecule is loaded on the end point of one single-stranded DNA through covalent modification, and the cholesterol molecule is loaded on the end point of the other single-stranded DNA through covalent modification; the vibration light-emitting molecules are excited by single laser, switching between bending conformation and torsional conformation occurs, and then cell membrane tension is responded through emission wavelength intensity change. The membrane tension response type composite molecular sensor has the advantages of being controllable in size, high in brightness, high in cell membrane targeting performance and the like, fluorescence proportion imaging of cell membrane tension is achieved, and the huge potential of the membrane tension response type composite molecular sensor in research and application of the life process of stem cells is revealed.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological nanotechnology, and particularly relates to a membrane tension response type composite molecular sensor and a preparation method and application thereof. BACKGROUND

[0002] Cell membrane tension is of great significance for stem cell stemness maintenance, regeneration and development, and can indicate embryonic development status and respond to external environment and self-structure changes. Cell membrane tension mainly includes cytoskeleton tension, membrane-cortex attachment force and lateral membrane tension. Among them, lateral membrane tension is doubly regulated by membrane-cortex attachment force and membrane skeleton reconstruction, and as the weakest mechanical parameter, lateral membrane tension detection is more difficult. At the same time, cell membrane tension can regulate stem cell differentiation, and the cell membrane surface tension decreases during osteogenic differentiation. Traditional detection methods (such as atomic force microscope, microsyringe, optical tweezers and magnetic tweezers) need to extract local membrane structure invasively, and it is difficult to distinguish membrane-cortex attachment force and lateral tension, and the measured is mostly composite tension, which limits the accurate research of lateral tension in living cell membrane. The latest optical detection method of membrane tension is Flipper-TR, which relies on fluorescence lifetime for detection, but requires high equipment.

[0003] Therefore, it is urgent to develop a new tool for lateral membrane tension monitoring with wide application range, high cell membrane targeting, mechanical sensitivity and non-invasiveness for studying stem cell membrane tension in full field of view. SUMMARY

[0004] The application aims to provide a membrane tension response type composite molecular sensor and a preparation method and application thereof, so as to solve the problems of traditional invasive membrane tension detection method in the prior art, such as invasiveness, inability to distinguish lateral membrane tension and membrane-cortex attachment force, high requirement of the latest Flipper-TR optical detection probe equipment, and poor water solubility, poor cell membrane targeting and easy endocytosis and exocytosis of the existing membrane tension probe.

[0005] In order to solve the above technical problems, the application adopts the following technical solutions:

[0006] According to a first aspect of the present invention, a membrane tension-responsive composite molecular sensor is provided, comprising a DNA tetrahedral framework formed by base complementary pairing of multiple single-stranded DNAs, wherein the single-stranded DNA includes: a single-stranded DNA modified with a vibrational luminescent molecule, a single-stranded DNA modified with a cholesterol molecule, and unmodified single-stranded DNA; the vibrational luminescent molecule is covalently modified and loaded at the endpoint of the single-stranded DNA, and the cholesterol molecule is covalently modified and loaded at the endpoint of another single-stranded DNA; the cholesterol molecule anchors the composite molecular sensor to the outer side of the cell membrane of a living cell through its lipophilicity; the vibrational luminescent molecule can be excited by a single laser to switch between bending and twisting conformations, thereby responding to cell membrane tension by changes in emission wavelength intensity.

[0007] In a preferred embodiment, the site of the DNA single-strand chemical modification vibrational luminescent molecule can be either the 3' end or the 5' end.

[0008] In a preferred embodiment, the site at which the DNA single-strand chemically modifies the cholesterol molecule can be either the 3' end or the 5' end.

[0009] In a preferred embodiment, the total number of all the DNA single-strand chemically modified vibrational luminescent molecules can be 1, 2, or 3.

[0010] Further preferably, the number of all DNA single-strand chemically modified vibrational luminescent molecules is 2, and the number of cholesterol molecules is 1.

[0011] According to a preferred embodiment of the present invention, a membrane tension-responsive composite molecular sensor, TDF-VIE2-Chol, was prepared. S1 was unmodified, S2 was modified with azide at the 3' end, S3 with cholesterol at the 5' end, and S4 with azide at the 3' end. The azide was then modified with vibrational luminescent molecules via a click reaction. In practical biological applications, this molecular sensor has been shown to have good imaging performance.

[0012] According to a second aspect of the present invention, a method for preparing the membrane tension-responsive composite molecular sensor is provided, comprising the following steps:

[0013] 1) Preparation of single-stranded DNA modified with vibrational luminescent molecules: The vibrational luminescent molecules modified with alkyne groups are mixed with single-stranded DNA modified with azide groups through a click reaction to obtain single-stranded DNA modified with vibrational luminescent molecules.

[0014] 2) Constructing a DNA tetrahedral framework: The single-stranded DNA modified with vibrational luminescent molecules, the single-stranded DNA modified with cholesterol molecules, and the unmodified single-stranded DNA obtained in step 1) were mixed in phosphate buffer and subjected to denaturation annealing to obtain the membrane tension-responsive composite molecular sensor.

[0015] In a preferred embodiment, the denaturing annealing process is as follows: maintaining at 95°C for 5 minutes, followed by cooling to room temperature by decreasing the temperature by 0.1°C every 3 seconds.

[0016] In a preferred embodiment, in step 1), the reaction system of the click reaction further includes sodium ascorbate, triphenyltriazole methylamine-monovalent copper complex and dimethyl sulfoxide; the click reaction conditions are 40°C in the dark, oscillating at 300 rpm for 4 hours.

[0017] In a preferred embodiment, step 1) further includes purifying the single-stranded DNA modified with vibrational luminescent molecules by high-performance liquid chromatography after the click reaction.

[0018] According to an embodiment of the present invention, a method for verifying the successful synthesis of a composite molecular sensor is provided, the method comprising the following steps:

[0019] 1) Provide the aforementioned composite molecular sensor;

[0020] 2) The composite molecular sensor was subjected to electrophoresis using a PAGE gel to confirm its synthesis;

[0021] 3) The composite molecular sensor was characterized using atomic force microscopy to confirm its synthesis.

[0022] According to an embodiment of the present invention, a method for verifying the membrane tension response of a composite molecular sensor in living cells is provided, the method comprising the following steps:

[0023] 1) LUVs vesicles were prepared using DOPC solution, and the composite molecular sensor was co-incubated with the prepared vesicles;

[0024] 2) Place the vesicles with the attached composite molecular sensor in a quartz cuvette with a 1 cm optical path and test their fluorescence emission spectrum under 405 nm excitation light. The photophysical properties of the composite molecular sensor are obtained by analyzing the measurement results.

[0025] 3) The composite molecular sensor was co-incubated with live cells. By adjusting different osmotic pressures, the intensity changes of the emitted light from the composite molecular sensor under different tension conditions at the same excitation wavelength were observed. The change in fluorescence intensity ratio was used to indicate the change in tension.

[0026] According to a third aspect of the present invention, an application of the membrane tension-responsive composite molecular sensor in fluorescence imaging under different tension conditions in living cells is provided, comprising the following steps:

[0027] 1) Culture mesenchymal stem cells, mix the composite molecular sensor with the mesenchymal stem cells, and incubate at 37°C for 8-12 min. The composite molecular sensor will anchor to the cell membrane through the lipophilicity of cholesterol molecules, and at the same time, the vibrational luminescent molecules it carries will leave a fluorescent signal on the cell surface. Wash 3 times with phosphate buffer.

[0028] 2) Use membrane-targeting dye to blend with the mesenchymal stem cells obtained in step 1), incubate at 37°C for 1-3 min, and wash 3 times with phosphate buffer;

[0029] 3) Use a laser confocal microscope to perform fluorescence imaging on cells, and use the fluorescence imaging results to determine the targeting of the composite molecular sensor to live cells. The laser wavelength of the fluorescence confocal microscope used is 405 nm.

[0030] According to a fourth aspect of the present invention, an application of the membrane tension-responsive composite molecular sensor in measuring changes in cell membrane tension of mesenchymal stem cells at different differentiation stages is provided, comprising the following steps:

[0031] 1) Culture mesenchymal stem cells and use osteogenic induction differentiation culture medium to induce directed differentiation of mesenchymal stem cells to obtain cells at different differentiation stages at 0 days, 7 days, 14 days, and 21 days.

[0032] 2) The composite molecular sensor was incubated with mesenchymal stem cells at different differentiation stages at 37°C for 10 min, and then washed three times with phosphate buffer.

[0033] 3) Excited with a 405nm laser, the fluorescence intensity ratio of the 450-550nm and 550-700nm channels was detected by laser confocal microscopy, and the cell membrane tension of mesenchymal stem cells at different differentiation stages was determined based on the change in fluorescence intensity ratio.

[0034] According to a fifth aspect of the present invention, an application is provided for measuring changes in cell membrane tension of mesenchymal stem cells induced by osteogenic differentiation under the action of metal ions, comprising the following steps:

[0035] 1) Culture mesenchymal stem cells, use induction differentiation culture medium and lithium chloride to induce directed differentiation of cells, and obtain mesenchymal stem cells at different differentiation stages;

[0036] 2) The above-mentioned composite molecular sensor was mixed with pretreated mesenchymal stem cells and incubated at 37°C for 8-12 min, followed by washing three times with phosphate buffer.

[0037] 3) Excited with a 405nm laser, the fluorescence intensity ratio of the 450-550nm and 550-700nm channels was detected by laser confocal microscopy, and the cell membrane tension induced by osteogenic differentiation under the action of metal ions was determined based on the change in fluorescence intensity ratio.

[0038] According to a sixth aspect of the present invention, the application of the membrane tension-responsive composite molecular sensor described above in the preparation of reagents for measuring cell membrane tension is provided.

[0039] The key inventive point of this invention lies in the first-ever fabrication of a membrane tension-responsive composite molecular sensor, composed of single-stranded DNA linked to vibrational luminescent molecules and cholesterol molecules, with the structure shown below. Figure 1 As shown. Single-stranded DNA possesses editability and addressability, allowing for the connection of multifunctional groups. Furthermore, vibrational luminescent molecules exhibit single-excitation and double-emission characteristics; that is, VIE molecules (such as N,N'-disubstituted-dihydrodibenzo[a,c]phenazine) exhibit a saddle-shaped configuration in the ground state, and their conjugated backbone has a certain degree of flexibility, undergoing a configurational change in the excited state, transforming into a twist configuration. Simultaneously, cholesterol molecules utilize their superior lipophilic properties, enabling the anchoring of nanomachines to the cell membrane of living cells through their flexible lipophilic groups. By employing the base complementarity pairing principle, single-stranded DNA modified with different functional groups are denatured and annealed to construct a composite molecular sensor with a DNA tetrahedral structure. This composite molecular sensor can respond to changes in the surface tension of the cell membrane by varying the emission wavelength intensity of the vibrational luminescent molecules, and thus by the ratio of emitted laser wavelengths. It should be understood that the hydrophilic DNA nucleic acid framework of this invention is limited to the DNA tetrahedral structure. It should also be understood that unmodified single-stranded DNA is indispensable in the construction of this composite molecular sensor.

[0040] As is well known, existing DNA framework nucleic acid tetrahedral probes often rely on single-stranded DNA modified with a single functional group due to the difficulty of strand modification and the interactions between strands, followed by the combination of DNA containing that single functional group. The composite molecular sensor of this invention adds a biomimetic cholesterol molecule to the existing single-stranded sensor, providing both a functional group and improved biocompatibility. However, no prior art has ever disclosed such a "vibrational luminescent molecule-cholesterol-DNA" ternary structure, nor has it provided any technical inspiration for migrating the functional end to the 5'-end to utilize membrane steric hindrance to improve stability. In terms of preparation, the cholesterol-modified DNA single strand does not require the introduction of an additional protecting group, making the synthesis process simpler and significantly more efficient.

[0041] Another key inventive point of this invention lies in achieving excellent imaging capabilities by introducing the synergistic effect of cholesterol and vibrational luminescent molecules at specific sites. Considering the morphology and steric hindrance after DNA framework nucleic acid simulation, the vibrational luminescent molecules and cholesterol-modified strands of this invention are located at both the 5' and 3' ends, ultimately resulting in a more stable tetrahedral framework structure. For example, in the TDF-VIE2-Chol prepared in the embodiments of this invention, the azide modification of S2 is at the 3' end, the cholesterol modification of S3 is at the 5' end, and the azide modification of S4 is at the 3' end. Combined with experimental data, it is shown that, compared with fluorescence spectroscopy analysis, in practical biological applications, the composite molecular sensor structure containing one cholesterol unit and two vibrational luminescent molecules exhibits superior imaging performance.

[0042] Compared with the prior art, the main advantages of the present invention include:

[0043] 1) The preparation and application of the membrane tension-responsive composite molecular sensor provided by the present invention solves the problems of poor water solubility, poor cell membrane targeting, and small molecular structure that is easily endocytosed and exocytotic in existing membrane tension probes.

[0044] 2) The membrane tension-responsive composite molecular sensor prepared according to the present invention has the advantages of controllable size, high brightness, high efficiency in cell membrane targeting, and good biocompatibility. It has significant advantages over existing nano fluorescent probes and provides a new tool for bioimaging.

[0045] 3) According to the membrane tension-responsive composite molecular sensor provided by the present invention, fluorescence ratio imaging of cell membrane tension is realized, revealing that this type of composite functional molecular sensor has great potential in the study of life processes of living cells, especially stem cells. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the structure of a composite molecular sensor provided by the present invention;

[0047] Figure 2 These are the agarose gel electrophoresis characterization results of the composite molecular sensor; among them, a is the Page gel image of the TDF-VIE2-Chol synthesis, from left to right: Marker, S1 DNA single strand, S1 DNA single strand mixed with S2 DNA single strand (modified with VIE molecules), S1 DNA single strand mixed with S2 DNA single strand (modified with VIE molecules) mixed with S3 DNA single strand (modified with cholesterol molecules), and tetrahedrons formed by mixing four single strands of S1, S2 (containing VIE), S3 (containing cholesterol molecules), and S4 (containing VIE) and denaturing and annealing; b is the Page gel image of DNA tetrahedrons formed by unmodified DNA single strands and three forms of TDF-VIEx-Cholx, from left to right: Marker, DNA tetrahedrons without modified functional groups, TDF-VIE-Chol2, TDF-VIE2-Chol, and TDF-VIE3;

[0048] Figure 3 These are the atomic force microscopy characterization results of the composite molecular sensor;

[0049] Figure 4 This is the fluorescence emission spectrum after co-incubation of the composite molecular sensor with DOPC vesicles;

[0050] Figure 5 This is the result of biocompatibility testing of the composite molecular sensor;

[0051] Figure 6 This is a colocalization map of cell membrane components of the composite molecular sensor in mesenchymal stem cells;

[0052] Figure 7 This is a fluorescence imaging image of mesenchymal stem cells under different osmotic pressure environments using a composite molecular sensor;

[0053] Figure 8 This is a fluorescence imaging image of mesenchymal stem cells at different osteogenic differentiation stages obtained by a composite molecular sensor;

[0054] Figure 9 The results are fluorescence imaging and analysis of subcellular regions of mesenchymal stem cells at different osteogenic differentiation stages using a composite molecular sensor.

[0055] Figure 10 The results are fluorescence imaging and analysis of mesenchymal stem cells at different osteogenic differentiation stages using a composite molecular sensor. Detailed Implementation

[0056] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0057] Through extensive and in-depth research and screening, the inventors have, for the first time, developed a design and method for a composite molecular sensor with a mechanical response containing multiple modifying groups. This invention constructs an atomically precise amphiphilic composite molecular sensor. By confining the size of the composite molecular sensor using DNA tetrahedra, the flexible luminescent groups can deeply probe the coupling interaction mechanisms of various cell membrane tension components in living cells, thus completing this invention.

[0058] Example 1

[0059] According to this embodiment, a method for preparing a size-controllable tetrahedral composite molecular sensor based on DNA nanotechnology is provided, thereby synthesizing a membrane tension-responsive composite molecular sensor.

[0060] Step 1: All DNA single strands were dissolved in ultrapure water and quantified to 100 μM using a UV-Vis spectrophotometer. 20 mg of sodium ascorbate was dissolved in 1 mL of ultrapure water to prepare a 20 mg / mL working solution. Then, 7OD Biotech's custom-designed azide-modified single-stranded DNA powder was dissolved in 100 μL of ultrapure water, and 40 μL of DMSO was added. Next, 140 μL of a 5.68 mg / mL vibrational luminescent molecule VIE (N,N'-disubstituted-dihydrodibenzo[a,c]phenazine, provided by Professor Tian He's research group at East China University of Science and Technology) solution dissolved in DMSO, 20 μL of a triphenyltriazole methylamine-monovalent copper complex (TBTA / Cu(I)) solution, and 100 μL of the aforementioned sodium ascorbate working solution were added sequentially to prevent oxidation of the monovalent copper. Finally, after thorough mixing, the mixture was reacted at 300 rpm for 4 h in a constant temperature mixer at 40 ℃ in the dark to obtain a crude suspension of functionalized single-stranded DNA, namely single-stranded DNA-VIE conjugate.

[0061] Step 2: The crude ssDNA-VIE conjugate obtained from the aforementioned click reaction was directly loaded into an Agilent 1260 Infinity II high-performance liquid chromatography system. Elution was performed on a C18 reversed-phase column using a triethylamine acetate buffer-acetonitrile gradient. The triethylamine acetate buffer was prepared by diluting 5.6 mL of glacial acetic acid and 13.86 mL of triethylamine to 800 mL of ultrapure water and adjusting the pH to 7.4 with alkali. The target peak fraction was collected to obtain the high-purity ssDNA-VIE conjugate. The collected product was then freeze-dried using a freeze dryer.

[0062] Step 3: Dissolve all DNA single strands in ultrapure water and quantify to 100 μM using a UV-Vis spectrophotometer. First, dissolve the DNA single strands in PBS buffer (10 mM PBS, 1 mM MgSO4). 2+ Dilute the DNA tetrahedron (pH 7.4) to a working concentration of 1 μM. Then, mix the four strands at an equimolar concentration of 1:1:1:1. Place the prepared mixture into a PCR instrument and obtain DNA tetrahedra by high-temperature annealing. Specifically, heat to 95°C, hold for 10 minutes, then cool to 25°C at a rate of 0.1°C every 3 seconds. Store the obtained DNA tetrahedra at room temperature. Figure 1 DNA tetrahedral nucleic acid complex molecular probes were analyzed by agarose gel electrophoresis.

[0063] Results: Agarose gel electrophoresis ( Figure 2 The results showed that TDF-VIE-Chol2, TDF-VIE2-Chol, and TDF-VIE3 were all successfully synthesized.

[0064] In this embodiment, the DNA sequences used are shown in Table 1 below:

[0065] Table 1

[0066]

[0067] Example 2

[0068] According to this embodiment, a detection method based on a DNA tetrahedral composite molecular sensor is provided.

[0069] 30 μL of 10 mM NiCl2 solution was dropped onto the freshly peeled mica surface and allowed to stand for 30 seconds. It was then thoroughly rinsed with deionized water and dried with nitrogen gas. Subsequently, the surface was treated with NiCl2... 2+ 10 μL of 5 nM sample solution was dropped onto the modified mica. After adsorption at room temperature for 5 minutes, 40 μL of TM buffer was added. Immediately, scanning imaging was completed in the liquid phase environment using PeakForce tapping mode atomic force microscopy to achieve high-resolution characterization of the sample.

[0070] Results: Atomic force microscopy images show ( Figure 3 The TDF-VIE2-Chol tetrahedron was synthesized, and its edge length is about 5nm, which is consistent with the theoretical value.

[0071] Example 3

[0072] According to this embodiment, a method for detecting cell membrane anchoring based on a DNA tetrahedral composite molecular sensor is provided.

[0073] First, LUVs vesicles were prepared using DOPC. A chloroform solution containing 30 mM DOPC was rotary evaporated under reduced pressure at room temperature for 4 h to form a uniform lipid membrane. Then, 1 mL of 250 mM sucrose solution was added, and the membrane was stirred at 55 ℃ and 800 rpm for 30 min to fully hydrate it. The resulting crude emulsion was flash-frozen in liquid nitrogen for 2 min and then completely thawed in a 55 ℃ water bath. This freeze-thaw cycle was repeated 5 times. Subsequently, the sample was continuously extruded through a 400 nm pore size polycarbonate membrane to obtain a large monolayer liposome stock solution with uniform particle size.

[0074] LUVs vesicles were stained using three of the aforementioned DNA tetrahedral composite molecular sensors at a working concentration of 1 μM, and then scanned using a microplate reader.

[0075] Results: The fluorescence emission spectrum showed that ( Figure 4 The DNA tetrahedral composite molecular sensor at various scales produced similar emission peaks, consistent with the expected increase. The fluorescence emission curves showed two distinct emission peaks at 520 nm and 610 nm, corresponding to the Bent and Twist configurations of the VIE molecule, respectively.

[0076] Example 4

[0077] According to this embodiment, a biocompatibility detection method based on a DNA tetrahedral composite molecular sensor is provided.

[0078] The survival rate of human mesenchymal stem cells after probe labeling was evaluated using the Calcein-AM / PI live / dead staining kit: MSCs seeded in confocal culture dishes were washed twice with 1×PBS, then incubated in serum-free medium at 37 ℃ with the specified amount of Calcein-AM and propidium iodide in the dark for 30 min. After washing again with PBS and immersion in Hank's balanced salt solution, images were immediately acquired using a confocal microscope. The proliferation and survival status of cells were detected by counting green live cells and red dead cells.

[0079] Results: Live and dead staining fluorescence imaging showed that ( Figure 5 The number of green live cells accounted for more than 93% of all cells in all three groups, and there was no significant difference among the three groups. This indicates that all three DNA tetrahedral composite molecular sensors have good biocompatibility.

[0080] Example 5

[0081] According to this embodiment, a cell membrane colocalization detection method based on a DNA tetrahedral composite molecular sensor is provided.

[0082] First, mesenchymal stem cells (purchased from Cybio (Shanghai) Biotechnology Co., Ltd.) were cultured, and the following experiments were performed using fifth-generation cells. 3000 cells were seeded in 35mm diameter laser confocal dishes and incubated at 37°C in a CO2 incubator for 12 hours. After incubation with TDF-VIE2-Chol for ten minutes, the cells were washed twice with PBS, followed by incubation with DIL membrane dye for 1-3 minutes, and then washed three times with PBS. Imaging was performed at 405nm excitation and 450-550nm reception to show the localization of TDF-VIE2-Chol on the cells; at 561nm excitation and 570-610nm reception to show the localization of DIL on the cells (e.g., ...). Figure 6 As shown in Figure a). The line drawn indicates the fluorescence intensity along the line, and a line graph is created (e.g., ...). Figure 6 (As shown in b).

[0083] Example 6

[0084] According to this embodiment, a method for detecting cell membrane tension based on a DNA tetrahedral composite molecular sensor is provided.

[0085] First, mesenchymal stem cells were cultured, and fifth-generation cells were used for the following experiments. Then, sucrose-regulated osmotic pressure was prepared. Different proportions of sucrose buffer were mixed with the basal culture medium to create three osmotic pressures: low osmotic pressure, isotonic pressure, and high osmotic pressure. Next, cells were stained with a DNA tetrahedral complex molecular sensor for 10 minutes and washed three times with 1×PBS. Subsequently, the cell culture medium was replaced with the three osmotic pressure regulators mentioned above, and the cells were imaged using a laser confocal microscope. Changes in membrane tension were determined by comparing the intensity ratios at different wavelengths (channel 1: 450-550 nm; channel 2: 550-700 nm).

[0086] Results: Fluorescence imaging and calculated fluorescence intensity ratio plots ( Figure 7 The changes in the fluorescence intensity ratio under different osmotic pressures were observed. Under high osmotic pressure, the fluorescence intensity ratio (channel 1:channel 2) was greater, and the pseudo-color image of the fluorescence intensity ratio approached yellow-red, indicating low cell surface membrane tension. Under isotonic pressure, the fluorescence intensity ratio was smaller, and the pseudo-color image of the fluorescence intensity ratio approached blue-green, indicating high cell surface membrane tension. These results demonstrate that the DNA tetrahedral composite molecular sensor is suitable for detecting membrane tension at different levels in living cells.

[0087] Example 7

[0088] According to this embodiment, a method for detecting mesenchymal stem cell membrane tension at different osteogenic differentiation stages is provided based on a DNA tetrahedral composite molecular sensor.

[0089] First, mesenchymal stem cells were cultured, and fourth-generation cells were used for the following experiments. Then, osteogenic differentiation was induced in the cells using osteogenic induction medium, and cells at days 0, 7, 14, and 21 were selected for further tension assays. Next, cells were stained with a DNA tetrahedral composite molecular sensor for 10 minutes and washed three times with 1×PBS. Subsequently, the cells were imaged using a laser confocal microscope. Changes in membrane tension were determined by comparing the intensity ratios at different wavelengths (channel 1: 450-550 nm; channel 2: 550-700 nm).

[0090] Results: Fluorescence imaging and calculated fluorescence intensity ratio plots ( Figure 8 This allows observation of changes in fluorescence intensity and ratio on the cell membrane surface at different osteogenic induction differentiation stages. Furthermore, by subdividing and cutting the fluorescence map (…),… Figure 9 The ratio of fluorescence intensity in different regions of the cell also showed differences in pseudocolor images. Its heterogeneity initially increased and then decreased with increasing induction.

[0091] Example 8

[0092] According to this embodiment, a method for detecting cell membrane tension of mesenchymal stem cells at different osteogenic differentiation stages under the participation of lithium metal ions is provided based on a DNA tetrahedral composite molecular sensor.

[0093] First, mesenchymal stem cells were cultured, and fourth-generation cells were used for the following experiments. Then, osteogenic differentiation was induced in the cells using either lithium chloride-containing or lithium chloride-free osteogenic differentiation-inducing medium. Cells at 0, 7, 14, and 21 days were selected for further tension assays. Next, cells were stained with a DNA tetrahedral complex molecular sensor for 10 minutes and washed three times with 1×PBS. Subsequently, the cells were imaged using a laser confocal microscope. Changes in membrane tension were determined by comparing the intensity ratios at different wavelengths (channel 1: 450-550 nm; channel 2: 550-700 nm).

[0094] Results: Fluorescence imaging and calculated fluorescence intensity ratio plots ( Figure 10 The fluorescence intensity and ratio of cell membranes at different osteogenic induction differentiation stages were observed. Significant differences were observed between the two groups on day 7 in the osteogenic induction differentiation medium containing lithium chloride. These results suggest that lithium chloride promotes osteogenic differentiation and that this process is closely related to membrane tension.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A membrane tension-responsive composite molecular sensor, characterized in that, The device comprises a DNA tetrahedral framework formed by base pairing of multiple single-stranded DNA molecules. The single-stranded DNA includes: single-stranded DNA modified with vibrational luminescent molecules, single-stranded DNA modified with cholesterol molecules, and unmodified single-stranded DNA. The vibrational luminescent molecules are covalently loaded onto the ends of the single-stranded DNA molecules, and the cholesterol molecules are covalently loaded onto the ends of another single-stranded DNA molecule. The cholesterol molecules anchor the composite molecular sensor to the outer surface of the cell membrane of a living cell through their lipophilicity. The vibrational luminescent molecules can be excited by a single laser, switching between bending and twisting conformations, and thus responding to cell membrane tension through changes in emission wavelength intensity.

2. The membrane tension-responsive composite molecular sensor according to claim 1, characterized in that, The site of the single-stranded DNA chemically modified vibrational luminescent molecule is at the 3' end or the 5' end; the site of the single-stranded DNA chemically modified cholesterol molecule is at the 3' end or the 5' end.

3. The membrane tension-responsive composite molecular sensor according to claim 1, characterized in that, The total number of vibrational luminescent molecules chemically modified on all single-stranded DNA is 1, 2, or 3.

4. A method for preparing a membrane tension-responsive composite molecular sensor according to any one of claims 1-3, characterized in that, Includes the following steps: 1) Preparation of single-stranded DNA modified with vibrational luminescent molecules: The vibrational luminescent molecules modified with alkyne groups are mixed with single-stranded DNA modified with azide groups through a click reaction to obtain single-stranded DNA modified with vibrational luminescent molecules. 2) Constructing a DNA tetrahedral framework: The single-stranded DNA modified with vibrational luminescent molecules, the single-stranded DNA modified with cholesterol molecules, and the unmodified single-stranded DNA obtained in step 1) were mixed in phosphate buffer and subjected to denaturation annealing to obtain the membrane tension-responsive composite molecular sensor.

5. The preparation method according to claim 4, characterized in that, In step 1), the reaction system of the click reaction also includes sodium ascorbate, triphenyltriazole methylamine-monovalent copper complex and dimethyl sulfoxide; the click reaction conditions are 40°C in the dark, oscillating at 300 rpm for 4 hours.

6. The preparation method according to claim 4, characterized in that, Step 1) also includes purifying the single-stranded DNA modified with vibrational luminescent molecules by high-performance liquid chromatography after the click reaction.

7. An application of the membrane tension-responsive composite molecular sensor according to any one of claims 1-3 in fluorescence imaging of living cells under different tension environments, characterized in that, Includes the following steps: 1) Culture mesenchymal stem cells, mix the composite molecular sensor with the mesenchymal stem cells, and incubate at 37°C for 8-12 min. The composite molecular sensor will anchor to the cell membrane through the lipophilicity of cholesterol molecules, and at the same time, the vibrational luminescent molecules it carries will leave a fluorescent signal on the cell surface. Wash 3 times with phosphate buffer. 2) Use membrane-targeting dye to blend with the mesenchymal stem cells obtained in step 1), incubate at 37°C for 1-3 min, and wash 3 times with phosphate buffer; 3) Use a laser confocal microscope to perform fluorescence imaging on cells, and use the fluorescence imaging results to determine the targeting of the composite molecular sensor to live cells. The laser wavelength of the fluorescence confocal microscope used is 405 nm.

8. An application of the membrane tension-responsive composite molecular sensor according to any one of claims 1-3 in measuring changes in cell membrane tension of mesenchymal stem cells at different differentiation stages, characterized in that, Includes the following steps: 1) Culture mesenchymal stem cells and use osteogenic induction differentiation culture medium to induce directed differentiation of mesenchymal stem cells to obtain cells at different differentiation stages at 0 days, 7 days, 14 days, and 21 days. 2) The composite molecular sensor was incubated with mesenchymal stem cells at different differentiation stages at 37°C for 8-12 min, and then washed 3 times with phosphate buffer. 3) Excited with a 405nm laser, the fluorescence intensity ratio of the 450-550nm and 550-700nm channels was detected by laser confocal microscopy, and the cell membrane tension of mesenchymal stem cells at different differentiation stages was determined based on the change in fluorescence intensity ratio.

9. An application of the membrane tension-responsive composite molecular sensor according to any one of claims 1-3 in measuring changes in cell membrane tension of mesenchymal stem cells induced by osteogenic differentiation under the action of metal ions, characterized in that, Includes the following steps: 1) Culture mesenchymal stem cells, use induction differentiation culture medium and lithium chloride to induce directed differentiation of cells, and obtain mesenchymal stem cells at different differentiation stages; 2) The above-mentioned composite molecular sensor was mixed with pretreated mesenchymal stem cells and incubated at 37°C for 8-12 min, followed by washing three times with phosphate buffer. 3) Excited with a 405nm laser, the fluorescence intensity ratio of the 450-550nm and 550-700nm channels was detected by laser confocal microscopy, and the cell membrane tension induced by osteogenic differentiation under the action of metal ions was determined based on the change in fluorescence intensity ratio.

10. The application of a membrane tension-responsive composite molecular sensor according to any one of claims 1-3 in the preparation of reagents for measuring cell membrane tension.

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