Nucleic acid multicolor fluorescent probe and preparation and application thereof in real-time in-situ observation of sequential activation of p53-mediated apoptosis pathway

Abstract

The application discloses a nucleic acid multicolor fluorescent probe and application thereof in preparation and in-situ real-time observation of sequential activation of a P53-mediated cell apoptosis pathway. A P53 fixed chain, a Bax fixed chain and a Cyt c fixed chain are modified on the surface of S-AuNSs@SiO2 through an amide bond, and a complementary chain modified with different fluorescent groups, an aptamer chain are connected through a complementary pairing rule to construct a multicolor fluorescent probe. In the presence of a target, DNA double strands are forced to be opened, and a fluorescent group is released from the system to restore quenched fluorescence. Due to 20 symmetrical 'hot spots', a stronger local electric field can be generated, and the probe based on S-AuNSs shows better optical performance. The S-AuNSs@SiO2-P of the application has high integration, is easy to obtain, and realizes in-situ real-time observation of sequential activation of a P53-mediated apoptosis pathway in living cells, and has application prospects in mechanism research, drug discovery, dynamic monitoring of an apoptosis process and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanomaterials and life science, and particularly relates to a nucleic acid multicolor fluorescent probe and preparation and application thereof in real-time in-situ observation of sequential activation of P53-mediated cell apoptosis pathway. BACKGROUND

[0002] There are two different but ultimately converging apoptosis pathways in mammalian cells: the Bcl-2-regulated (also known as intrinsic, mitochondrial or stress) pathway and the death receptor (also known as extrinsic) pathway. In the Bcl-2-regulated pathway, P53, Bax and cytochrome C (Cyt c) are in close upstream and downstream relationship and are determined as attractive targets for monitoring cell apoptosis. P53 protein is a tumor suppressor and is a key factor in DNA damage response. Activation of P53 for cell apoptosis involves the intrinsic apoptosis pathway to eliminate stressed cells containing DNA damage. Bax is a member of the Bcl-2 family and is a core regulator of the intrinsic pathway of apoptosis. After apoptosis stimulation, they are activated and oligomerized at the outer membrane of mitochondria to mediate its permeability, thereby causing the translocation of Cyt c from the intermembrane space to the cytoplasm, followed by the activation of the caspase cascade. Real-time detection of the sequential activation of P53, Bax and Cyt c and elucidation of the upstream and downstream relationships of these caspases are crucial for understanding the process of cell apoptosis and evaluating the therapeutic effect of cancer treatment.

[0003] The currently widely used method for analyzing genes and proteins in signaling pathways is based on traditional techniques such as Northern blot hybridization, real-time reverse transcription-PCR (RT-PCR), microarray hybridization, Western blotting and immunohistochemistry. However, these methods require a large amount of time, tedious steps and a large amount of cell sample. In addition, the cell lysis or fixation process used in these methods makes it impossible to study the dynamic changes and natural state of these molecules. Therefore, it is very important to develop a non-invasive method to detect multiple molecules in the signaling pathway. While the advantages of fluorescence imaging analysis in rapid, high-resolution and direct visualization in intracellular monitoring make it have great potential in simultaneously monitoring genes and proteins involved in the signaling pathway of living cells and providing useful information about the cell signaling pathway.

[0004] However, in the probe based on gold nanomaterials, Au-S bond is easily cleaved by biological thiols including glutathione (GSH) and cysteine, which exist in relatively high concentration, and inevitably leads to false positive results. Amide bond (-CO-NH-) as the backbone of peptide, widely exists in the whole physiological system, and plays a crucial role in human life. Due to inert chemical properties, amide bond is considered as one of the most stable chemical groups. Importantly, amide bond can be easily formed by direct condensation of amino group (-NH2) and carboxyl group (-COOH). Therefore, amide bond can be a good substitute for Au-S bond. So developing a kind of nano probe with simple operation, high stability and in-situ real-time intracellular related signal pathway will have good application prospect. SUMMARY

[0005] To solve the above technical problems, the present application provides a nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P and a preparation method thereof and application thereof in P53 mRNA, Bax mRNA and Cyt c detection. The method of the present application combines the dual advantages of nucleic acid and gold nanomaterials, and can realize real-time in-situ monitoring of P53-mediated apoptosis pathway in cells. The material is simple to prepare, stable in nature, high in sensitivity and high in biocompatibility.

[0006] The first object of the present application is to provide a nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P, comprising a silica-coated symmetrical gold nanostar, a target fixation chain and a target recognition chain.

[0007] The diameter of the silica-coated symmetrical gold nanostar (S-AuNSs@SiO2) is 260-300 nm; the target fixation chain is selected from one or more of P53 fixation chain (P53-cDNA), Bax fixation chain (Bax-cDNA) and Cyt c fixation chain (Cyt c-cDNA); the target recognition chain is selected from one or more of P53 recognition chain (P53-rDNA), Bax recognition chain (Bax-rDNA) and Cyt c aptamer chain (Cyt c-apt); the target fixation chain and the target recognition chain are connected by base complementary pairing.

[0008] The P53 fixation chain, Bax fixation chain or Cyt c fixation chain is labeled with a carboxyl group, and the P53 recognition chain, Bax recognition chain or Cyt c aptamer chain is labeled with different fluorescent groups.

[0009] In an embodiment of the present application, the different fluorescent groups are selected from one or more of FAM, TAMRA and Cy5.

[0010] In one embodiment of the present application, the P53 fixed strand sequence is: 5'-COOH-AAAAAGCTTTGAGGTGC-3';

[0011] Bax fixed strand: 5'-COOH-AAAAAAGAGGCGGGGG-3';

[0012] Cyt c fixed strand: 5'-COOH-AAAAAGCAACAACGTAC-3';

[0013] P53 recognition strand: 5'-GCACAAACACGCACCTCAAAGC-fluorescent group-3';

[0014] Bax recognition strand: 5'-CTCAGAGCTGGTGGGCCCCCCGCCTCT-fluorescent group-3';

[0015] Cyt c aptamer strand: 5'-CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-fluorescent group-3'.

[0016] In one embodiment of the present application, the silica-coated symmetrical gold nanostar is prepared by the following method:

[0017] S1: heat the PVP solution to reflux, add the DEG solution of HAuCl4·3H2O and react, after the reaction is completed, wash and solid-liquid separation to obtain a solid phase, and disperse the solid phase in an organic solvent to obtain an I-AuNPs solution;

[0018] S2: add the DMA solution and hydrochloric acid solution to the PVP solution and stir to mix uniformly, add the I-AuNPs solution obtained in S1, then add the HAuCl4 solution, and heat to react to obtain a symmetrical gold nanostar;

[0019] S3: add CTAC to the aqueous solution of the symmetrical gold nanostar obtained in S2 and stir, add the NaOH solution, and add the TEOS at least three times, and react to obtain the silica-coated symmetrical gold nanostar.

[0020] In one embodiment of the present application, in step S1, the mass ratio of PVP to HAuCl4·3H2O is 5-20:1-3.

[0021] In one embodiment of the present application, in step S2, the mass-volume ratio of PVP to DMA is 0.5-1.5g:60-140μL.

[0022] The second object of the present application is to provide a preparation method of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P, comprising the following steps:

[0023] (1) Ammonification of silica-coated symmetric gold nanostars (S-AuNSs@SiO2) to obtain the amino-functionalized silica-coated symmetric gold nanostars S-AuNSs@SiO2;

[0024] (2) Taking the target fixation chain, adding EDC and NHS and incubating, adding the amino-functionalized silica-coated symmetric gold nanostars to react, and obtaining the cDNA modified S-AuNSs@SiO2;

[0025] (3) Mixing and incubating the target recognition chain with the cDNA modified S-AuNSs@SiO2 obtained in step (2) to obtain the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P.

[0026] In an embodiment of the present application, in step (2), the molar ratio of EDC, NHS and S-AuNSs@SiO2 is 40000-60000:40000-60000:1-4; and the molar ratio of the target fixation chain and the amino-functionalized silica-coated symmetric gold nanostars S-AuNSs@SiO2 is 600-1000:1-4.

[0027] In an embodiment of the present application, in step (3), the molar ratio of the target recognition chain and the cDNA modified S-AuNSs@SiO2 is 300-700:1-4.

[0028] A third object of the present application is to provide the application of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P in the quantitative or qualitative detection of P53 mRNA, Bax mRNA or Cyt c.

[0029] A fourth object of the present application is to provide the application of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P in the real-time in-situ monitoring of the P53-mediated apoptosis pathway in living cells.

[0030] In an embodiment of the present application, the detection method is as follows: the culture medium containing S-AuNSs@SiO2-P is used to incubate cells, then the culture medium containing toxins is added for incubation, and fluorescence imaging is used to collect data. The use of toxins induces apoptosis in cells, so that the content of P53 mRNA, Bax mRNA or Cyt c in the cells changes (increases).

[0031] In an embodiment of the present application, the concentration of nano S-AuNSs@SiO2-P in the culture medium is 20-80 pM.

[0032] In an embodiment of the present application, the S-AuNSs@SiO2-P is incubated with the cells for 4-8 hours.

[0033] In an embodiment of the present application, the cells include one or more of human cervical cancer cells Hela, human colorectal adenocarcinoma cells Caco-2, human hepatoma cells HepG2 and human breast cancer cells.

[0034] In an embodiment of the present application, the toxin is selected from one or more of T-2 toxin, aflatoxin B1, vomitoxin, zearalenone and ochratoxin.

[0035] The nucleic acid multicolor fluorescence probe based on DNA strand unwinding is based on the difference in binding affinity. Due to the 20 symmetrical "hot spots", S-AuNSs can generate a stronger local electric field. By wrapping a stable silicon shell on its surface and coating a large number of amino (-NH2) groups. Amino modification enables S-AuNSs@SiO2 to couple with carboxyl-labeled DNA (P53-cDNA, Bax-cDNA, Cyt c-cDNA) sequences through simple but strong amide bonds. Then, dye (FAM, TAMRA and Cy5) labeled recognition DNA strands (P53-rDNA, Bax-rDNA, Cyt c-apt) hybridize with the attached cDNA to form a covalent multicolor fluorescence probe S-AuNSs@SiO2-P, resulting in fluorescence quenching. In the presence of a target, rDNA and apt tend to form more stable duplexes or complexes with the target, and the dye molecules are released from the surface of S-AuNSs@SiO2, resulting in fluorescence enhancement.

[0036] The above technical solutions of the present application have the following advantages compared with the prior art:

[0037] (1) Compared with traditional detection methods, the S-AuNSs@SiO2-P proposed in the present application has the characteristics of fast response, simple preparation, stable properties, high biocompatibility and low cost. In addition, different DNA single strands and gold nanomaterials can be designed individually, and the method can be improved and optimized.

[0038] (2) Due to the strong amide bond and highly stable silicon shell, the S-AuNSs@SiO2-P nano system almost does not show non-specific signals in the presence of high concentrations of biological environment (such as nucleases, biological thiols), showing good stability and anti-interference performance.

[0039] (3) S-AuNSs@SiO2-P can be practically applied to biological samples such as cells, and in-situ imaging and detection of P53 mRNA, Bax mRNA and Cyt c sequential activation in the P53-mediated apoptosis pathway during apoptosis are realized, which is conducive to clarifying the upstream and downstream relationships of these molecules, understanding the cell apoptosis process and evaluating the treatment effect of cancer.

[0040] (4) S-AuNSs@SiO2-P is a non-destructive method, which has the advantages of rapidity, high resolution and direct visualization in monitoring cells.

[0041] (5) The nano S-AuNSs@SiO2-P has universality in tracing and sensing P53 mRNA, Bax mRNA and Cyt c in different cell lines. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0043] Figure 1 TEM electron micrograph and ultraviolet-visible absorption spectrum of the symmetric gold prepared for Example 1 of the present application;

[0044] Figure 2 TEM electron micrograph and ultraviolet-visible absorption spectrum of S-AuNSs prepared for Example 1 of the present application;

[0045] Figure 3 TEM electron micrograph of S-AuNSs@SiO2 prepared for Example 1 of the present application;

[0046] Figure 4 Statistical diagram of the particle size of S-AuNSs and S-AuNSs@SiO2 prepared in Example 1 by NanoMeasurer software of the present application;

[0047] Figure 5 HPLC spectrum of the supernatant after coupling of carboxyl DNA with S-AuNSs@SiO2 in Example 2 of the present application, Zeta potential and infrared spectrum of S-AuNSs@SiO2 and its modification;

[0048] Figure 6 Fluorescence spectrum and fluorescence lifetime and Raman spectrum of double-stranded DNA before and after hybridization on the surface of S-AuNSs@SiO2 in Example 2 of the present application;

[0049] Figure 7 Optimization of the concentration of fluorescent strands in Example 2 of the present application;

[0050] Figure 8The salt stability test results of S-AuNSs@SiO2-P in Example 3 of the present application;

[0051] Figure 9 The stability and reproducibility test results of S-AuNSs@SiO2-P in Example 3 of the present application;

[0052] Figure 10 The spectral detection results of the responsiveness of S-AuNSs@SiO2-P to the target in Example 4 of the present application;

[0053] Figure 11 The linear fitting results of the responsiveness of S-AuNSs@SiO2-P to the target in Example 4 of the present application;

[0054] Figure 12 The specificity detection results of S-AuNSs@SiO2-P in Example 4 of the present application;

[0055] Figure 13 The results of Hela cells infected by T-2 toxin with different concentrations in Example 5 of the present application;

[0056] Figure 14 The cytotoxicity of S-AuNSs@SiO2-P with different concentrations and Hela cells co-cultured for different times in Example 6 of the present application;

[0057] Figure 15 The incubation time of S-AuNSs@SiO2-P and Hela cells is optimized by ICP-MS in Example 8 of the present application;

[0058] Figure 16 The contents of P53 mRNA, Bax mRNA and Cyt c when the apoptosis of living cells is determined by a kit after Hela cells are infected by T-2 toxin for different times in Example 9 of the present application;

[0059] Figure 17 The laser confocal imaging of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P in living cells in Example 10 of the present application;

[0060] Figure 18 The relationship between the confocal fluorescence intensity and the incubation time of T-2 toxin in Example 10 of the present application;

[0061] Figure 19 The fluorescence imaging of S-AuNSs@SiO2-P in different cell lines in Example 11 of the present application;

[0062] Figure 20 The detection principle diagram of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2 designed in the present application. DETAILED DESCRIPTION

[0063] The present application is further described in connection with the following drawings and embodiments so that it can be better understood and carried out by those skilled in the art. The embodiments are not intended to limit the present application.

[0064] Example 1 Synthesis and characterization of silica-coated symmetrical gold nanostars (S-AuNSs@SiO2)

[0065] First, icosahedral gold seeds (I-AuNPs) were synthesized. 0.05-0.2 g PVP was dissolved in 25 mL DEG solution, which was heated to boiling reflux in a flask. After 5 min, 2 mL of DEG solution containing 10-30 mg HAuCl4·3H2O was quickly injected. After 10 min, the reaction was stopped and cooled to room temperature. The product was centrifuged and washed twice with DMF. Finally, I-AuNPs were dispersed in 27 mL DMF solution. I-AuNPs were characterized by TEM micrograph and UV-Vis absorption spectrum, the results were as follows Figure 1 .

[0066] Symmetrical gold nanostars (S-AuNSs) were synthesized in the presence of DMA. 0.5-1.5 g PVP was dissolved in 15 mL DMF. Then 60-140 μL of 40% DMA and 120-200 μL of 2.5 M HCl solution were added. Subsequently, 1 mL of I-AuNPs solution was added, followed by the addition of 10-30 μL of 0.5 M HAuCl4solution. Finally, the reaction solution was gently stirred at 80 °C in an oil bath for 4 h. The product was centrifuged and washed twice with ethanol, and finally dispersed in 2 mL of water. S-AuNSs were characterized by TEM micrograph and UV-Vis absorption spectrum, the results were as follows Figure 2 .

[0067] Silica shell coating was performed on S-AuNSs. 2.0 mM of CTAC (10-40 μL) was added to 2.5 mL of S-AuNFs solution and stirred overnight, followed by the addition of 15-20 μL of NaOH (0.1 M). Subsequently, 5-10 μL of 20% TEOS prepared with methanol was added three times at 30 min intervals. After 24 h, the product was centrifuged at 8000 rpm for 5 min and washed three times with ethanol. Finally, the obtained S-AuNSs@SiO2 was re-dispersed in 2.5 mL of ethanol. S-AuNSs@SiO2 were characterized by TEM micrograph and UV-Vis absorption spectrum, and the particle size was measured by NanoMeasurer 1.2 software, the results were as follows Figure 2 、 3 、4.

[0068] From Figure 1It can be seen that the icosahedral gold seeds (I-AuNPs) are well dispersed and uniform in appearance, showing a plasmonic resonance peak at about 570 nm, and the average particle size is 97 nm.

[0069] From Figure 2 It can be seen that all the S-AuNSs synthesized are highly symmetrical, with ten identical arm projection profiles and significant monodispersity. At the same time, S-AuNSs have a wide plasmonic resonance scattering peak, which matches well with the absorption peak of most fluorescent groups, so that the process of fluorescence resonance energy transfer can occur, resulting in resonance quenching of the fluorescent group.

[0070] Figure 3 It can be seen that the silica is successfully coated on the S-AuNSs (S-AuNSs@SiO2).

[0071] Figure 4 It can be seen that the average particle size of S-AuNSs is 204 nm, and the average particle size of S-AuNSs@SiO2 is 280 nm.

[0072] Example 2 Preparation of S-AuNSs@SiO2-based DNA probe (S-AuNSs@SiO2-P)

[0073] First, 10-15 μL of ammonia (25%) and 2.5 mL of S-AuNSs@SiO2 were mixed and stirred for 30 min, then 4-8 μL of APTES was added, and stirred at 35°C for 3 h, then stirred at 65°C for 1 h. Subsequently, the product was centrifuged at 8000 rpm for 5 min and washed 3 times. The obtained amino-functionalized S-AuNSs@SiO2 was re-dispersed in 2.5 mL of PBS (pH = 7.4, 10 mM). An appropriate amount of carboxyl-labeled DNA (P53-cDNA, Bax-cDNA, Cyt c-cDNA) was taken respectively and mixed, 50 μL of 0.2M EDC / NHS was added and incubated at room temperature for 30 min. Subsequently, 920 μL of amino-functionalized S-AuNSs@SiO2 was added and shaken overnight. Characterized by high performance liquid chromatography, Zeta potential and infrared spectroscopy, and the results are shown in Figure 5

[0074] ​Finally, the product was centrifuged at 8000 rpm for 5 min, washed 3 times, and then dispersed in 10 mL of PBS. Then, the fluorescent group-labeled DNA (P53-rDNA, Bax-rDNA, Cyt c-apt) was added to the above solution and incubated at 37 °C for 3 h. The excess DNA was removed by repeated centrifugation, and the precipitate was resuspended in PBS buffer to obtain the nano-multicolor S-AuNSs@SiO2-P (S-AuNSs@SiO2-P). The results of the characterization by fluorescence spectra and lifetime, Raman spectra are shown in Figure 6 .

[0075] To obtain the maximum loading, the concentration of the fluorescent chain solution was changed for optimization. The difference in fluorescence intensity before and after the hybridization of the fluorescent chain on the surface of S-AuNSs@SiO2 proved the complementary hybridization of the two chains. The results are shown in Figure 7 .

[0076] As can be seen in Figure 5 , the characteristic peak of COOH-DNA (260 nm) in the supernatant was significantly reduced Figure 5 (A) before and after incubation with S-AuNSs@SiO2. As shown in Figure 5 B, S-AuNSs exhibited a positive charge due to the adsorption of CTAC. After silica coating, the surface charge became negative due to the silicon hydroxyl group. After amino functionalization with the -NH2 group, the surface charge became positive. When COOH-DNA was covalently bonded to the surface of S-AuNSs@SiO2, the surface charge became negative again. At the same time, the characteristic peaks at 1554 and 1634 cm -1 significantly increased, corresponding to the N-H deformation and C=O stretching vibration, also confirming the successful covalent bonding between COOH-DNA and S-AuNSs@SiO2.

[0077] As can be seen in Figure 6 , after the hybridization of the fluorescent (FAM, TAMRA, Cy5) labeled DNA and S-AuNSs@SiO2-DNA, the fluorescence of the fluorescent group was quenched and the Raman signal was enhanced. At the same time, the fluorescence lifetime of the fluorescent group was significantly reduced after hybridization, which also indicated that energy transfer occurred between S-AuNSs@SiO2 and the fluorescent group.

[0078] The concentration of the fluorescent chain was optimized using the fluorescence signal (as shown in Figure 7 ). The decrease in fluorescence intensity in the system before and after hybridization indirectly reflects the number of successfully hybridized fluorescent chains. With the increase of the concentration of the three fluorescent chains, the value first increases and then remains unchanged. It is speculated that the hybridization of the three chains has been saturated, so 1 μM is selected as the optimal concentration, respectively.

[0079] Example 3 Stability experiment of nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P

[0080] Finally, the Raman spectra of S-AuNSs@SiO2-P at 20 different points were collected to evaluate the signal reproducibility thereof.

[0081] (1) UV-Vis spectra were used to characterize the salt stability of S-AuNSs@SiO2-P:

[0082] The prepared S-AuNSs@SiO2-P was centrifuged and resuspended in PBS buffer containing different concentrations of NaCl (100 mM, 200 mM, 300 mM, 400 mM, 500 mM), vortexed, and allowed to stand for 10 min, and then the UV-Vis absorption spectra thereof were observed. The results are shown in Figure 8

[0083] (2) Fluorescence experiments were used to evaluate the anti-enzyme cutting and anti-displacement stability of S-AuNSs@SiO2-P:

[0084] Three groups of nano S-AuNSs@SiO2-P were placed in a 96-well fluorescence microplate, and DNase I (1 U / mL), Rnase A (1 mg / mL), and GSH (5 mM) were added, respectively. After monitoring the fluorescence intensity for a period of time, the target was added, and the fluorescence signal was continuously monitored to evaluate the anti-enzyme cutting and anti-displacement stability. The results are shown in Figure 9 A-C.

[0085] (3) pH stability of S-AuNSs@SiO2-P:

[0086] S-AuNSs@SiO2-P was centrifuged and resuspended in buffers with different pH values (5, 6, 7, 8, 9), and the fluorescence intensity of the samples was continuously monitored. After a period of time, the target was added to each group. The fluorescence intensity of the samples was continuously monitored. The results are shown in Figure 9 D-F.

[0087] (4) Raman experiments were used to evaluate the medium stability of S-AuNSs@SiO2-P:

[0088] S-AuNSs@SiO2-P was centrifuged and resuspended in buffer, medium, and serum, respectively, and incubated for 0, 3, 6, 9, 12, and 15 h, and then the changes in the three fluorescence signals were detected. The results are shown in Figure 9 G-I.

[0089] (5) Raman spectra were used to characterize the signal reproducibility of bimodal nano S-AuNSs@SiO2-P:

[0090] ​Raman spectra of S-AuNSs@SiO2-P were collected at 20 different points to evaluate the signal reproducibility. The results are shown in Figure 9 J-L.

[0091] As shown in Figure 8 , even if the concentration of NaCl is as high as 400 mM (much higher than the ionic strength in cells), the position and intensity of the absorption peak of S-AuNSs@SiO2-P remain essentially unchanged, because the modification of a large number of biological DNA molecules on the surface of the probe prevents the aggregation of S-AuNSs@SiO2-P.

[0092] There is a certain amount of nuclease in cells, so high nuclease resistance is essential for intracellular sensing systems. DNase I and Rnase A were used as model enzymes to study the stability of the probe. Figure 9 A-C found that the fluorescence of S-AuNSs@SiO2-P fluctuated little. Due to the high local salt ion concentration and surface negative charge, the proposed nanosystem protects the DNA probe from nuclease degradation, thereby reducing non-specific signal fluctuations. In addition to the interference of enzymes, abundant biological thiols, such as glutathione, are also a big challenge to traditional DNA nanoprobes, which can significantly replace the attached DNA probes, leading to unreliable sensing results. The anti-GSH interference ability of S-AuNSs@SiO2-P was also studied. Figure 9 A-C shows that the fluorescence intensity of S-AuNSs@SiO2-P changes little over a long period of time under high concentration GSH (5 mM) treatment, which is attributed to the strong amide bond between DNA and S-AuNSs@SiO2.

[0093] Apoptosis can cause intracellular acidification, and the effect of pH in the range of 5-9 on S-AuNSs@SiO2-P was studied. Figure 9 D-F shows that the effect of pH on the detection effect can be ignored.

[0094] As shown in Figure 9 G-I, as the solvent, the culture medium, fetal bovine serum has little effect on the fluorescence signal of S-AuNSs@SiO2-P over time.

[0095] As shown in Figure 9 J-L, the relative standard deviation (RSD) of the SERS peak intensity corresponding to different fluorescent groups is about 5%, which shows that the proposed nano S-AuNSs@SiO2-P has good signal reproducibility.

[0096] Example 4 In vitro response of S-AuNSs@SiO2-P

[0097] S-AuNSs@SiO2-P (0.5 nM) and target RNAs (P53 mRNA and Bax mRNA, final concentration of 150 nM) and Cyt c (final concentration of 300 nM) were mixed at 37 °C, and the reaction time was first optimized. The fluorescence intensity was measured at different times (0, 20, 40, 60, 80, 100, 120 min). S-AuNSs@SiO2-P with a concentration of 1.5 nM was incubated with different concentrations of target RNAs (0, 20, 40, 60, 80, 100, 120, 140, 60, 180 nM) and different concentrations of Cyt c (0, 100, 150, 200, 250, 300, 350, 400, 450 nM) at 37 °C for 1 h. The fluorescence spectrum collection range was 500-650, 560-700 and 655-750 nm at the excitation wavelengths of 490, 553 and 638 nm, respectively. All fluorescence measurements were repeated at least 3 times. The spectral results are shown in Figure 10 , respectively. The fluorescence intensity at 520 nm, 580 nm and 670 nm was linearly fitted with the concentration of P53 mRNA, Bax mRNA and Cyt c, and the results are shown in Figure 11 .

[0098] In order to determine whether S-AuNSs@SiO2-P can specifically recognize two kinds of mRNA and Cyt c, their analogues and other important biological components (1 mM) were selected for comparison. The analogues of the two kinds of mRNA include T P53 mismatched , T Bax mismatched , T VEGF , T HCV , T miR-21 , T P21 , T Bcl-2 , T PTEN , bovine serum albumin (BSA), ascorbic acid (AA), L-cysteine (L-cys) and magnesium chloride (MgCl). The analogues of Cyt c include glucose (Glu), BSA, immunoglobulin G (IgG), bovine serum albumin (BSA), caspase 3 (Casp-8), alpha-lactalbumin (a-LA), beta-lactoglobulin (b-LG), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), cytosolic linker protein (Keap1), adenosine triphosphate (ATP), AA, L-cys, MgCl. The operation method is the same as above, and the results are shown in Figure 12 .

[0099] As shown in Figure 10 , 11 , the fluorescence intensity gradually increased with the increase of the concentration of the target Figure 10). There was a good linear relationship between the fluorescence emission of FAM and the concentration of P53 mRNA Figure 11 A). The regression equation was y = 2286.15 · X - 463.12, the linear coefficient R 2 = 0.98456. The limit of detection (LOD) was determined to be 0.47 nM. Similarly, the fluorescence intensity of TAMRA showed a good linear relationship with the concentration of Bax mRNA between 0.5-4 nM Figure 11 B). The regression equation was y = 1238.13 · X + 8.54572, R 2 = 0.99621, and the LOD was 0.60 nM. Figure 11 C showed a good proportional relationship between the concentration of Cyt c and the fluorescence signal of Cy5, with a linear response range of 300-4000 nM, the regression equation was y = 1.22 · X - 95.33, R 2 = 0.99697, and the LOD was 259.60 nM. The proposed S-AuNSs@SiO2-P showed acceptable performance in mRNA and Cytc detection, while covering the minimum values of RNA and Cytc in the cytoplasm (1 nM and 1 μM).

[0100] As Figure 12 shown, under the same conditions, S-AuNSs@SiO2-P was treated with nucleic acid mismatched chains, biological proteins and other bioactive substances to evaluate the selectivity of the nano probe. Among the analytes, S-AuNSs@SiO2-P only produced the highest fluorescence response to the three targets. In contrast, other interferents did not cause significant fluorescence recovery.

[0101] Example 5 Determination of the working concentration of T-2 toxin:

[0102] The CCK-8 method was used to determine the optimal concentration of T-2 toxin co-cultured with cells. The T-2 toxin stock solution was diluted with cell culture medium to different concentrations. Hela cells were seeded at a density of 5000 cells per well in 100 μL cell suspension in a 96-well plate. After incubating the culture plate in an incubator for 24 h (37 °C, 5% CO2), 10 μL of T-2 toxin at different concentrations was added to the culture plate, so that the final concentration of T-2 toxin in the culture medium was 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. After incubation in the incubator for 2.5 h, 10 μL of CCK-8 solution was added to each well and incubated for another 4 h, and then the absorbance at 450 nm was measured. The relative survival rate of cells was calculated using the following formula. (Wherein, the toxin can stimulate cells to undergo apoptosis, up-regulate the expression of P53 mRNA, Bax mRNA and Cyt c in cells, and also maintain the activity of cells.)

[0103] Relative cell viability = (Experimental group absorbance - Blank group absorbance) / (Control group experimental absorbance - Blank group absorbance) × 100%. The IC50 of T-2 toxin on HeLa cells was fitted using Origin software. 50 The result is as follows Figure 13 As shown.

[0104] like Figure 13 As shown, HeLa cell viability decreased sequentially with increasing T-2 toxin concentration. The IC50 of T-2 toxin was obtained by fitting the data using Origin software. 50 The value was approximately 5 μM. To ensure that T-2 toxin exhibited a certain degree of toxicity to HeLa cells while maintaining a certain cell viability, 5 μM T-2 toxin was used as the working concentration for subsequent experiments.

[0105] Example 6: Evaluation of the cytotoxicity of S-AuNSs@SiO2-P using the CCK-F method:

[0106] HeLa cells were seeded at a density of 5000 cells per well in 96-well fluorescent plates. After cell adhesion at 37°C and 5% CO2, the culture medium was discarded, and the cells were washed three times with PBS. Then, DMEM basal medium containing different concentrations of S-AuNSs@SiO2-P (100 pM, 50 pM, 25 pM, 12.50 pM) was added to each well, and the cells were cultured for different times (6 h, 12 h, 24 h, 36 h). The culture medium was then discarded, and the cells were washed with PBS buffer. After discarding the PBS buffer, 100 μL of CCK-F reagent was added to each well, and the cells were cultured for another 30 min. The fluorescence intensity of each well was then measured using a microplate reader. Untreated cells served as a control, with 100% viability. Cell viability was calculated at different probe concentrations and treatment times. Calcein AM and PI were used in combination for double fluorescence staining of cells incubated with the probes. The results are as follows: Figure 14 As shown.

[0107] Depend on Figure 14 It was found that even stimulation of HeLa cells with 100 pnM S-AuNSs@SiO2-P for 36 hours could maintain HeLa cell viability above 90%. Dual fluorescence staining revealed that, compared to the control, only green fluorescence was observed in the field of view, with no red fluorescence. These data confirm that S-AuNSs@SiO2-P has low toxicity and is suitable for cellular environments.

[0108] Example 7: Optimization of incubation time of S-AuNSs@SiO2-P with cells using ICP-MS:

[0109] To further verify that S-AuNSs@SiO2-P had indeed been ingested by the cells, the gold content in the cells after incubation with the probe was quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The specific procedure was as follows: HeLa cells were incubated with 50 pM S-AuNSs@SiO2-P at 37°C for different times. Then, the cells were separated from the culture dish, sonicated, and dissolved in aqua regia overnight. The solution was diluted to 10 mL with pure water for ICP-MS detection. Simultaneously, different concentrations of S-AuNSs@SiO2-P were digested with aqua regia, appropriately diluted, and then analyzed by ICP-MS. The peak mass response (CPS) was obtained, and a standard curve was fitted. Therefore, the amount of probe in each cell can be determined using the standard curve measured by ICP-MS. The results are as follows: Figure 15 As shown.

[0110] Depend on Figure 15 It can be seen that the uptake of S-AuNSs@SiO2-P by HeLa cells reaches saturation at approximately 6 hours. At this point, at a concentration of 1×10⁻⁶... 4 The concentration of S-AuNSs@SiO2-P in cells was approximately 16 pM.

[0111] Example 8: Kit for determining the levels of p53 mRNA, Bax mRNA, and Cyt c during apoptosis in live cells:

[0112] According to the specified protocol, total cellular RNA was extracted from T-2 toxin-treated cells using a total RNA extraction kit, and cDNA samples were then prepared using reverse transcription (RT) using a HiScript kit. cDNA qPCR analysis was performed using a qPCR kit. A 20 μL reaction solution contained 2 μL cDNA sample, 10 μL SYBR qPCR Master Mix, 2 μL primer mixture, and 6 μL enzyme-free water. PCR conditions were as follows: initial 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. GAPDH was used as an internal control, and 2... -ΔΔCt Methods: P53 mRNA and Bax mRNA were evaluated. Cyt c levels in toxin-treated HeLa cells were simultaneously determined using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit. Standards and lysed cell supernatant were added to wells pre-coated with anti-Cyt c antibody. After incubation, biotin-labeled anti-Cyt c antibody was added. This antibody then bound to HRP-labeled streptavidin. After incubation and washing to remove unbound enzyme, the chromogenic substrate TMB was added, producing a blue color, which was converted to a final yellow color under acidic conditions. Finally, the absorbance of the reaction wells was measured at 450 nm. The Cyt c concentration in the sample was directly proportional to the absorbance, and the Cyt c concentration in the sample was calculated by plotting a standard curve. Results are as follows:Figure 16 As shown.

[0113] Depend on Figure 16 It was found that in HeLa cells, the levels of p53 mRNA, Bax mRNA, and Cyt c increased in a toxin incubation time-dependent manner.

[0114] Example 9: Nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P used for fluorescence imaging during live cell apoptosis:

[0115] First, HeLa cells were pre-cultured in confocal culture dishes for 24 hours until 80% coverage was achieved. S-AuNSs@SiO2-P was then added to the culture dishes. After incubation at 37°C for 6 hours, the cells were washed three times with PBS buffer, then T-2 toxin (5 μM) was added, and the culture dishes were immediately placed in the cell workstation of a confocal laser scanning microscope (CLSM). The microscope was focused on a group of cells, and multi-channel image acquisition was performed. The results are as follows: Figure 17 As shown in the figure. Simultaneously, the fluorescence intensity of each cell was analyzed using ImageJ software. The results are as follows. Figure 18 As shown.

[0116] like Figure 17 , 18 As shown, no background fluorescence was observed in the cells before stimulation with T-2 toxin. After 30 min of incubation, FAM (green, P53 mRNA) fluorescence first appeared in HeLa cells and gradually increased with the progression of apoptosis. TAMRA fluorescence (yellow, Bax mRNA) was observed after 60 min of incubation. The time-dependent fluorescence signal associated with Cy5 and Cyt c activation was not detected until 90 min of T-2 toxin treatment, and then gradually increased with the progression of apoptosis.

[0117] Example 10: Verification of the universality of S-AuNSs@SiO2-P in other cell lines:

[0118] The universality of S-AuNSs@SiO2-P in relevant human cell lines was determined by selecting HepG2 liver cancer cells, MCF-7 breast cancer cells, and Caco-2 colorectal adenocarcinoma cells. After incubation with HepG2, MCF-7, and Caco-2 cells, S-AuNSs@SiO2-P was used to stimulate the cells with T-2 toxin. Following staining and fixation, laser confocal imaging was performed immediately. Results are shown below. Figure 19 As shown.

[0119] Depend on Figure 19As shown, the intracellular fluorescence signal was weak before incubation with T-2 toxin. After 2.5 hours of incubation, the fluorescence signals of the three intracellular channels were enhanced. It can be concluded that the proposed nucleic acid multicolor fluorescent probe has excellent versatility and can be used to monitor apoptosis pathways in various human cell lines.

[0120] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P, characterized in that, The nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P comprises a symmetric gold nanostar coated by silicon dioxide, a target fixing chain and a target recognizing chain. The diameter of the symmetric gold nanostar coated by silicon dioxide is 260-300 nm; the target fixing chain is a P53 fixing chain, a Bax fixing chain and a Cyt c fixing chain; the target recognizing chain is a P53 recognizing chain, a Bax recognizing chain and a Cyt c aptamer chain; the target fixing chain and the target recognizing chain are connected through base complementary pairing; The P53 fixing chain, the Bax fixing chain or the Cyt c fixing chain is labeled with a carboxyl group, the P53 recognizing chain, the Bax recognizing chain or the Cyt c aptamer chain is labeled with different fluorescent groups, the sequence of the P53 fixing chain is 5'-COOH-AAAAAGCTTTGAGGTGC-3', the sequence of the Bax fixing chain is 5'-COOH-AAAAAAGAGGCGGGGG-3', the sequence of the Cyt c fixing chain is 5'-COOH-AAAAAGCAACAACGTAC-3', the sequence of the P53 recognizing chain is 5'-GCACAAACACGCACCTCAAAGC-fluorescent group-3', the sequence of the Bax recognizing chain is 5'-CTCAGAGCTGGTGGGCCCCCCGCCTCT-fluorescent group-3', and the sequence of the Cyt c aptamer chain is 5'-CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-fluorescent group-3'; The preparation method of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P comprises the following steps: (1) The symmetric gold nanostar coated by silicon dioxide is aminated to obtain the amino-functionalized symmetric gold nanostar coated by silicon dioxide S-AuNSs@SiO2; (2) The target fixing chain is taken, EDC and NHS are added and incubated, the amino-functionalized symmetric gold nanostar coated by silicon dioxide is added for reaction to obtain the cDNA-modified S-AuNSs@SiO2; (3) The target recognizing chain is mixed with the cDNA-modified S-AuNSs@SiO2 obtained in step (2) for incubation to obtain the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P; The molar ratio of the target fixing chain to the amino-functionalized symmetric gold nanostar coated by silicon dioxide S-AuNSs@SiO2 is 600-1000:1-4, and the molar ratio of the target recognizing chain to the cDNA-modified S-AuNSs@SiO2 is 300-700:1-4; The symmetric gold nanostar coated by silicon dioxide is prepared by the following method: S1: The PVP solution is heated to reflux, the DEG solution of HAuCl4·3H2O is added and reacted, the reaction is stopped, the solid phase is obtained after washing and solid-liquid separation, and the solid phase is dispersed in an organic solvent to obtain an I-AuNPs solution; S2: The DMA solution and the hydrochloric acid solution are added to the PVP solution and stirred to mix uniformly, the I-AuNPs solution obtained in S1 is added, then the HAuCl4 solution is added, and the symmetric gold nanostar is obtained after heating and reaction. S3: adding CTAC into the aqueous solution of the symmetrical gold nanostar obtained in S2 and stirring, adding NaOH solution, and adding TEOS at least in three times, and the silica-coated symmetrical gold nanostar is obtained by reaction. 2.The nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P of claim 1, wherein, The different fluorescent groups are selected from FAM, TAMRA and Cy5.

3. Use of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P according to claim 1 or 2 in the preparation of a kit for quantitative or qualitative detection of P53 mRNA, Bax mRNA and Cyt c.

4. Use of the nucleic acid multicolor fluorescent probe S-AuNSs@SiO2-P according to claim 1 or 2 in the preparation of a kit for real-time in situ monitoring of P53-mediated apoptosis pathway in living cells.

5. Use according to claim 4, characterized in that, The cells include one or more of human cervical cancer cells Hela, human colorectal adenocarcinoma cells Caco-2, human hepatoma cells HepG2 and human breast cancer cells.

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