Nanoprobe for simultaneously detecting and inhibiting VCAM-1mRNA expression as well as preparation method and application of nanoprobe

Abstract

The invention relates to the technical field of medical detection, in particular to a nanoprobe for simultaneously detecting and inhibiting VCAM-1mRNA expression as well as a preparation method and application of the nanoprobe. The nano particle takes a nano Au particle as a core, and p-mercaptocyanophenyl (4-MBN) serving as a Raman signal internal standard molecule is modified on the surface of the Au core; the nano particle takes a gold layer as a shell, and a VCAM-1mRNA targeting recognition sequence segment is fixed on the Au shell; and the tail end of the VCAM-1 mRNA targeted recognition sequence segment is modified with a Raman reporter molecule Cy5. The recognition sequence segment on the nanoprobe can be specifically combined with VCAM-1mRNA to form a rigid double strand, so that Cy5 is far away from the surface of the gold core-shell nanoparticle, the SERS signal strength of Cy5 is weakened, the position of 4-MBN located on a spacing layer is unchanged, and the SERS signal of the 4-MBN is also unchanged. The expression of the VCAM-1 protein can be inhibited by the accompanying mRNA consumption in the detection process, so that the synchronous monitoring and treatment intervention of the atherosclerosis disease closely associated with the expression of the VCAM-1 are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, and particularly relates to a nano probe for simultaneously detecting and inhibiting VCAM-1 mRNA expression, and a preparation method and application thereof. BACKGROUND

[0002] Atherosclerosis (AS) is a chronic inflammatory disease mainly involving large and medium-sized arteries, which can lead to serious complications such as cardiovascular disease, cerebrovascular disease and organ dysfunction. At present, the clinical diagnosis mainly relies on imaging examinations such as ultrasound, X-ray computed tomography (CT) and magnetic resonance imaging (MRI), but these methods can usually only detect formed plaques and are difficult to achieve early diagnosis, which cannot meet the clinical needs of precise risk assessment. For example, although traditional carotid ultrasound is often used to detect plaques, the plaques often cause blood flow restriction during clinical diagnosis. Therefore, it is urgent to develop a method that can realize early diagnosis and timely intervention of atherosclerosis.

[0003] VCAM-1 is a 100-110 kDa type I transmembrane protein with seven C2-type immunoglobulin domains, and is an important cell adhesion molecule. In atherosclerotic plaques, the expression of VCAM-1 in endothelial cells, macrophages and smooth muscle cells is significantly increased, promoting the recruitment of leukocytes to the vascular wall. This up-regulation phenomenon exists in all stages of pathological tissues, making VCAM-1 an important imaging biomarker for tracking atherosclerotic inflammation. In addition, studies have shown that VCAM-1 is a key anti-inflammatory therapeutic target, and inhibiting its expression can effectively delay the progression of atherosclerosis. It has been reported that VCAM-1 antibodies can alleviate the atherosclerotic condition of ApoE(- / -) mice by improving plaque stability, reducing inflammatory response and inhibiting inflammatory cell adhesion. Therefore, it is of great significance to establish a method that can simultaneously detect and inhibit the expression of VCAM-1 mRNA for AS diagnosis and treatment. However, since the expression level of VCAM-1 in macrophages is extremely low, even after AS occurs, its content is still negligible, so it is a great challenge to accurately detect VCAM-1 in macrophages.

[0004] Surface-enhanced Raman scattering (SERS) is an in-situ, non-destructive and highly sensitive fingerprint spectroscopy technique, which is particularly suitable for the detection of intracellular biomarkers such as proteins and nucleic acids, with a sensitivity of single molecule level. For example, Wang et al. developed a gold-silver alloy nanocage SERS probe, which realized in-situ and non-destructive detection of hydrogen sulfide (H2S) in living cells, with a detection limit as low as 0.36 nM; and the digital colloidal enhanced Raman spectroscopy (dCERS) technology proposed by Ye's group realized the ultra-sensitive detection of various biomolecules through single molecule counting strategy, with a quantitative detection limit of less than 1 fM.

[0005] In view of the problems in synchronous detection and inhibition of VCAM-1 mRNA expression, the application constructs a gold core-shell structure isometric SERS nanoprobes, which can realize accurate detection and efficient inhibition of VCAM-1 expression in macrophages. SUMMARY

[0006] The application aims to solve the problems in the prior art and provides a nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression as well as a preparation method and application thereof. The probe is modified with a VCAM-1 target recognition sequence (labeled Raman reporter Cy5) on the surface of a gold shell and 4-MBN is fixed in the gap layer between the core and the shell. After recognizing the target, the rigid double-stranded body is formed to make Cy5 away from the metal surface, resulting in a decrease in the SERS signal of Cy5, while the space-limited 4-MBN maintains stable signal intensity. The isometric SERS signal of Cy5 / 4-MBN can be monitored to realize accurate quantitative detection of VCAM-1 mRNA. More importantly, the consumption of mRNA in the detection process can inhibit the expression of VCAM-1 protein, thereby realizing early monitoring and therapeutic intervention of atherosclerosis.

[0007] In order to achieve the above object, the application adopts the following technical scheme:

[0008] A nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression, wherein the nanoprobe is a gold nanoparticle with a core-shell structure, the nanoparticle has a nanometer Au particle as a core, and 4-mercaptobenzoic nitrile (4-MBN) is modified on the surface of the Au core as a Raman signal internal standard molecule.

[0009] The nanoparticle has a gold layer as a shell, and a VCAM-1 mRNA target recognition sequence is fixed on the Au shell.

[0010] The VCAM-1 mRNA target recognition sequence segment is modified with a Raman reporter Cy5 at the end.

[0011] Preferably, the gold nanoparticle with a core-shell structure has a particle size of 60 nm.

[0012] Preferably, the volume and concentration of the VCAM-1 mRNA target recognition sequence segment modified on the Au shell are 50 μL and 1 μM, respectively.

[0013] A preparation method of a nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression, comprising the following steps:

[0014] S1, synthesis of gold core-shell nanoparticles: first, gold nanoparticles (AuNPs) were synthesized by the classic sodium citrate reduction method, in order to graft the Raman reporter molecule 4-MBN to the surface of AuNPs, 4.35 μL of PEG-SH solution with a concentration of 10 mg / mL was mixed with 10 mL of gold nanoparticles, and the Au@PEG particles were obtained after 6 h of vigorous stirring, followed by centrifugation (12,000 g, 30 min) and resuspension of the precipitate in 2 mL of DMF; then 2 mL of the Au@PEG solution was mixed with 300 μL of 1.5 mM 4-MBN solution in 10 mL of DMF, and after 2 h of stirring at room temperature, the Au@MBN particles were collected by centrifugation (12,000 g, 15 min) and stored in 2 mL of deionized water;

[0015] The Au@MBN solution was mixed with a solution containing 50 mL of 1% PVP and 5 mL of 100 mM CTAB at room temperature, and 1.5 mL of 10 mM AgNO3 solution and 1.25 mL of 100 mM ascorbic acid (AA) were added dropwise, followed by the addition of 2.5 mL of 100 mM sodium hydroxide, and within 15 min the solution changed from colorless to orange yellow, indicating the formation of Au@MBN@Ag nanoparticles, which were stored in 10 mL of 1% PVP solution after centrifugation (7,000 g, 20 min); then the solution was combined with a mixture of 100 mL of 1% PVP and 10 mL of 100 mM CTAB, and 5 mL of 0.4 mg / mL chloroauric acid solution was added dropwise, and the solution turned dark blue after 15 min of stirring; 5 mL of 100 mM AA was added and the stirring was continued for 15 min, and the product was collected by centrifugation (12,000 g, 12 min) to obtain gold core-shell nanoparticles;

[0016] S2, preparation of the nanoprobe: the VCAM-1 mRNA recognition sequence was heated in a water bath at 90℃ for 5 min, and then naturally cooled to room temperature (1 h), 50 μL of the above sequence (1 μM) was added to 1 mL of the gold core-shell nanoparticles solution prepared as described above, and the mixed solution was subjected to oscillation reaction at 37℃; the solution after reaction was centrifuged (4000 rpm, 15 min) to remove the excess DNA chains that were not modified on the gold core-shell nanoparticles, and the nanoprobe was obtained.

[0017] Preferably, in S2, the reaction time of the gold core-shell nanoparticles with the VCAM-1 mRNA targeting recognition sequence segment is 12 h.

[0018] The application also provides a nanoprobe for simultaneously detecting and inhibiting the expression of VCAM-1 mRNA, which is obtained by the preparation method.

[0019] By adopting the technical scheme, the recognition sequence segment on the nano probe can specifically bind with VCAM-1 mRNA to form a rigid double strand, so that the Cy5 is far away from the surface of the gold core-shell nanoparticle, and the SERS signal intensity of the Cy5 is weakened, while the 4-MBN located in the spacer layer is not changed, and the SERS signal of the 4-MBN is not changed. According to the equal-ratio signal change of the Cy5 / 4-MBN, the trace VCAM-1 mRNA in the macrophage can be accurately detected. The mRNA consumption in the detection process can inhibit the expression of the VCAM-1 protein, so that the synchronous monitoring and treatment intervention of the atherosclerosis disease closely related to the VCAM-1 expression can be realized.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1, The present application utilizes the plasmonic coupling effect generated by the connection of the nanoparticles in the gold nano assembly to form a strong electromagnetic field, so that the Raman internal standard molecule signal located in this area is significantly stronger than the signal on a single nano structure, highlighting the key role of the nano assembly in signal amplification.

[0022] 2, The present application measures the signal intensity ratio of two different Raman displacements under a single excitation wavelength, and the ratio type SERS probe can effectively overcome the background interference such as non-specific binding, illumination fluctuation and uneven delivery / washing.

[0023] 3, The nano probe prepared by the present application can realize the detection and expression inhibition of the VCAM-1 mRNA synchronously, and the synchronous diagnosis and treatment of atherosclerosis can be completed without additional therapeutic drugs, so that the serious side effects caused by drugs are avoided. DETAILED DESCRIPTION

[0024] Figure 1 The structure and working principle diagram of the nano probe prepared by the present application;

[0025] Figure 2 (a) is a transmission electron microscope diagram of AuNPs synthesized by the present application, (b) is a transmission electron microscope diagram of gold core-shell nanoparticles synthesized, (c) is a transmission electron microscope diagram of the nano probe synthesized;

[0026] Figure 3 It is the ultraviolet absorption spectrum diagram of DNA, AuNPs, Au@MBN@Au and nano probe;

[0027] Figure 4 It is the Zeta potential diagram of AuNPs, Au@MBN@Au and nano probe;

[0028] Figure 5 It is the nano probe and the concentration 1.0*10 -12SERS spectra of the nanoprobes before and after reacting with VCAM-1 mRNA;

[0029] Figure 6 (a) is the SERS spectra of the nanoprobes after reacting with VCAM-1 mRNA of different concentrations in Example 4 of the present application, (b) is the linear relationship diagram of VCAM-1 mRNA concentration and (I0-I 1594cm-1 ) / I 2226cm-1 ;

[0030] Figure 7 is the selectivity diagram of the nanoprobes prepared in the present application for detecting different kinds of cytokines;

[0031] Figure 8 is the diagram of the peak intensity ratio of the nanoprobes at I 1594cm-1 / I 2226cm-1 versus the storage time;

[0032] Figure 9 (a) is the SERS spectra of the nanoprobes after reacting with VCAM-1 mRNA in RAW264.7 cells stimulated by different concentrations of LPS, (b) is the histogram of (I0-I 1594cm-1 ) / I 2226cm-1 versus the LPS concentration;

[0033] Figure 10 (a) is the SERS spectra of the nanoprobes after reacting with VCAM-1 mRNA in RAW264.7 cells stimulated by LPS for different time, (b) is the histogram of (I0-I 1594cm-1 ) / I 2226cm-1 versus the LPS used time;

[0034] Figure 11 is the WB diagram of the expression of VCAM-1 protein in RAW264.7 cells after being treated with PBS, Au@MBN@Au and the nanoprobes respectively;

[0035] Figure 12 is the RT-PCR diagram of the expression of VCAM-1 mRNA in RAW264.7 cells after being treated with PBS, Au@MBN@Au and the nanoprobes respectively;

[0036] Figure 13(a) is a schematic diagram of VCAM-1 mediated atherosclerosis formation and plaque progression mechanism, (b) is a fluorescence imaging diagram of GSDME in RAW264.7 cells after the PBS, Au@MBN@Au and nanoprobes respectively act on the cells, (c)-(f) are the expression contents of (c) caspase-1, (d) IL-1β, (e) IL-18 and (f) IL-6 in RAW264.7 cells respectively co-incubated with PBS, Au@MBN@Au, 0.01 nM nanoprobes and 0.03 nM nanoprobes. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings, so that the persons skilled in the art can better understand the advantages and features of the present application, and the protection scope of the present application can be defined more clearly. The described embodiments of the present application are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by the persons skilled in the art without creative labor fall within the protection scope of the present application.

[0038] Embodiment 1: A preparation method of a nanoprobes for simultaneously detecting and inhibiting VCAM-1 mRNA expression, comprising the following steps:

[0039] S1, synthesis of gold core-shell nanoparticles: first, gold nanoparticles (AuNPs) were synthesized by the classic sodium citrate reduction method. To graft the Raman reporter molecule 4-MBN to the surface of AuNPs, 4.35 μL of PEG-SH solution with a concentration of 10 mg / mL was mixed with 10 mL of gold nanoparticles, and the Au@PEG particles were obtained after 6 h of vigorous stirring. Then, the particles were centrifuged (12,000 g, 30 min) and resuspended in 2 mL of DMF. Subsequently, 2 mL of the Au@PEG solution was mixed with 300 μL of 1.5 mM 4-MBN solution in 10 mL of DMF, and the resulting Au@MBN particles were collected by centrifugation (12,000 g, 15 min) after 2 h of stirring at room temperature and stored in 2 mL of deionized water.

[0040] The Au@MBN solution was mixed with a solution containing 50 mL of 1% PVP and 5 mL of 100 mM CTAB at room temperature. Then, 1.5 mL of 10 mM MgNO3 solution and 1.25 mL of 100 mM ascorbic acid (AA) were added dropwise, followed by 2.5 mL of 100 mM sodium hydroxide. Within 15 min, the solution changed from colorless to orange-yellow, indicating the formation of Au@MBN@Ag nanoparticles. After centrifugation (7,000 g, 20 min), the solution was stored in 10 mL of 1% PVP solution. This solution was then combined with a mixture of 100 mL of 1% PVP and 10 mL of 100 mM CTAB, and 5 mL of 0.4 mg / mL chloroauric acid solution was added dropwise. After stirring for 15 min, the solution turned deep blue. 5 mL of 100 mM CTAB was added, and stirring continued for 15 min. The product was collected by centrifugation (12,000 g, 12 min), yielding gold core-shell nanoparticles.

[0041] S2. Preparation of Nanoprobes: The VCAM-1 mRNA recognition sequence was heated in a 90℃ water bath for 5 min, then naturally cooled to room temperature (1 h). 50 μL of the above sequence (1 μM) was added to 1 mL of the previously prepared gold core-shell nanoparticle solution, and the mixture was shaken at 37℃ for 12 h. The reacted solution was centrifuged (4000 rpm, 15 min) to remove unmodified excess DNA strands from the gold core-shell nanoparticles, yielding the nanoprobes.

[0042] Example 2: This example characterizes and analyzes the nanoprobes prepared in Example 1. The analysis is as follows:

[0043] 1) Morphology analysis: The morphology of AuNPs, gold core-shell nanoparticles, and nanoprobes was observed using transmission electron microscopy, such as... Figure 2 As shown in Figure a, the synthesized gold cores are spherical and uniformly distributed, with an average particle size of approximately 18 nm. The gold core-shell nanoparticles prepared by seed-mediated growth have a particle size of approximately 49 nm, and a distinct gold shell structure surrounding the gold core can be clearly observed. After further modification with the targeted recognition sequence segment, the resulting nanoprobes still maintain excellent monodispersity. Figure 2 c) indicates that it has good stability in aqueous solution.

[0044] 2) Ultraviolet absorption spectroscopy analysis: Ultraviolet-visible absorption spectroscopy showed that the gold nanocore exhibited a maximum absorption peak at 520 nm. After the formation of gold core-shell nanoparticles, this absorption peak red-shifted to 550 nm and broadened. Figure 3 The final prepared nanoprobe exhibited both the characteristic absorption peak of gold core-shell nanoparticles (548 nm) and the characteristic absorption peak of DNA capture (265 nm), indicating the successful preparation of the nanoprobe.

[0045] 3) Zeta potential analysis: The Zeta potentials of AuNPs, Au@MBN@Au, and nanoprobes were measured, and the results are as follows: Figure 4 As shown, compared with the pure gold nanocore (-1.04 mV), the surface potential of the gold core-shell nanoparticles becomes more negative (-3.16 mV), while the potential of the nanoprobe turns positive (+1.13 mV). This is attributed to the fact that positively charged recognition sequence segments are modified on the surface of the gold core-shell nanoparticles to form nanoprobes.

[0046] Example 3: This example verifies the feasibility of detecting VCAM-1 mRNA using the nanoprobe constructed in Example 1. The verification method and results are as follows:

[0047] The feasibility of detecting VCAM-1 mRNA using nanoprobes was verified using SERS spectroscopy. Figure 5 As shown, with VCAM-1 mRNA (1.0 × 10⁻⁶) -12 After the M) reaction, the SERS signal intensity of the characteristic peak corresponding to Cy5 generally decreased, while that of 4-MBN at 2226 cm⁻¹ decreased. -1 The peak intensity at 2226 cm⁻¹ remained essentially unchanged. This phenomenon arises from the formation of a rigid double-stranded structure after the recognition sequence on the nanoprobe surface specifically binds to the target VCAM-1 mRNA. This structural change increases the distance between the Cy5 molecule and the gold shell surface, leading to a decrease in its SERS signal. Meanwhile, the 4-MBN molecule located within the nano-intervals maintained its position before and after the reaction, thus its peak intensity at 2226 cm⁻¹ remained essentially unchanged. -1 The intensity of the characteristic peak at this location changed very little. The results indicate that the relative variation between the Cy5 and 4-MBN characteristic peaks can be used to detect VCAM-1 mRNA.

[0048] Example 4: This example examines the detection performance of the nanoprobe from Example 1. The analysis method is as follows:

[0049] 1) Sensitivity test of nanoprobes: Nanoprobes were used to detect different concentrations of VCAM-1 mRNA in aqueous solution. Results are as follows: Figure 6 As shown in figure a, with the increase of VCAM-1 mRNA concentration, 1594 cm -1 The SERS signal strength gradually decreased at 2226 cm⁻¹, while at 2226 cm⁻¹... -1 The signal strength at that location remained unchanged. Further analysis revealed that at 1594cm... -1 Signal strength change at 2226cm -1 The ratio of signal intensity at each location showed a good linear relationship with the concentration of VCAM-1 mRNA. Figure 6 (b) The linear regression equation is y = 16.3 + 1.2x, with a correlation coefficient of 0.956. When the signal-to-noise ratio is 3, the detection limit for VCAM-1 mRNA using this method is 1.0 × 10⁻⁶. -13M, indicating that the SERS nanoprobe has high detection sensitivity.

[0050] 2) Selectivity test of the nanoprobe: To verify the selectivity of the nanoprobe, VCAM-1 mRNA and a series of possible interfering substances were detected in this embodiment. The results are shown in Figure 7 Compared with the blank control group, other interfering substances only caused a slight change in the value of (I0-I 1594cm-1 ) / I 2226cm-1 , while the target molecule VCAM-1 mRNA caused a significant change in the value, indicating that the nanoprobe has excellent selectivity for VCAM-1 mRNA detection.

[0051] 3) Stability test of the nanoprobe: The prepared nanoprobe was stored at room temperature for different time and then subjected to SERS detection. The results are shown in Figure 8 The peak intensity ratio of 1596 cm -1 and 2226 cm -1 changed little over time, demonstrating that the nanoprobe has good stability.

[0052] Example 5: This embodiment verifies whether the nanoprobe of Example 1 can monitor the VCAM-1 mRNA in RAW264.7 cells dynamically, and the experimental method is as follows:

[0053] After RAW264.7 cells were incubated in 10% fetal bovine serum medium for 24 h, the cells were treated with LPS at different concentrations (0, 1, 10, 50 and 100 μg / mL) for 12 h. Then the LPS-treated cells were incubated with 0.03 nM nanoprobe for 4 h. After washing with PBS for three times, the cells in the glass dish were subjected to SERS spectrum collection. The experimental results are shown in Figure 9 a, with the increase of LPS concentration on RAW264.7 cells, the SERS signal of Cy5 on the nanoprobe gradually weakened. In addition, the value of (I0-I 1594cm-1 ) / I 2226cm-1 was closely related to the LPS concentration Figure 9 b). These results indicate that LPS induces the expression of VCAM-1 mRNA in macrophages in a concentration-dependent manner. Similarly, this embodiment monitored the change of VCAM-1 mRNA level in RAW264.7 cells after LPS treatment for different time, and the results are shown in Figure 10 a, the longer the treatment time, the more VCAM-1 mRNA expressed in the cells. The above results show that the nanoprobe prepared in the present application can successfully monitor the dynamic change of VCAM-1 mRNA level in cells.

[0054] Example 6: This example verifies whether the nanoprobe of Example 1 can inhibit the expression of VCAM-1 mRNA and VCAM-1 protein in RAW264.7 cells, and the experimental method is as follows:

[0055] Western blot (WB) and reverse transcription polymerase chain reaction (RT-PCR) were used to analyze the changes in VCAM-1 expression in RAW264.7 cells treated with different nanomaterials. After treatment, the VCAM-1 protein and mRNA in the cells were separated for analysis. The results of WB analysis are shown in Figure 11 Compared with the control group treated with PBS, the total protein level of VCAM-1 in the cells treated with the nanoprobe was significantly reduced. Simultaneous RT-PCR was performed to analyze the expression of VCAM-1 mRNA, and the results are shown in Figure 12 Compared with the control group, the use of Au@Au alone reduced the mRNA level by 22%, while co-incubation with the nanoprobe reduced the mRNA level by 43%. Analysis of the above experimental results shows that the nanoprobe can inhibit the expression of VCAM-1 mRNA and protein in the cells.

[0056] Example 7: This example tests the effect of the nanoprobe on slowing down the development of atherosclerosis, and the experimental method is as follows:

[0057] To verify whether the nanoprobe can delay the progression of atherosclerosis (AS), this example establishes a model of macrophage inflammation induced by LPS to study its regulatory effect on key upstream and downstream biomarkers (proteins and cytokines) related to AS. First, enzyme-linked immunosorbent (ELISA) kits were used to detect the changes in caspase-1 levels in macrophages treated with different nanomaterials. Compared with the control group, the core-shell nanoparticles caused a slight decrease in caspase-1 content Figure 13 c), while the high-concentration nanoprobe group showed a significant decrease, confirming that the nanoprobe can effectively inhibit the expression of caspase-1. Since activated caspase-1 (an upstream regulator of GSDME-mediated pyroptosis) can cleave gasdermin E (GSDME) to produce its N-terminal fragment (GSDME-N), this example evaluates the cleavage of GSDME in RAW264.7 cells exposed to different nanomaterials by immunofluorescence staining. Figure 13b shows that the fluorescence intensity of GSDME in the cells treated with the nanoprobes is enhanced, indicating that the proteolysis process of GSDME to GSDME-N is inhibited. Since pyroptosis is usually accompanied by the secretion of inflammatory cytokines interleukin-18 (IL-18) and interleukin-1β (IL-1β), this embodiment analyzes the RAW264.7 cells treated with the nanomaterials, and the contents of the two cytokines in the culture supernatant. Compared with the control group, the nanoprobes significantly reduce the secretion levels of IL-18 and IL-1β Figure 13 d&e), proving that the nanoprobes can inhibit the pyroptosis pathway closely related to the progression of AS. This embodiment further monitors the expression level of the inflammatory cytokine IL-6 which plays a key role in the development of AS. Compared with other groups, high-concentration nanoprobes significantly reduce the expression of IL-6 in cells, indicating that they can effectively alleviate the inflammatory response of macrophages. All the above research results show that the nanoprobes can block the pyroptosis pathway and alleviate the inflammatory response by inhibiting the expression of VCAM-1 mRNA, thereby slowing down the development process of atherosclerosis.

[0058] In summary, the probe prepared in the present application is modified with a VCAM-1 targeted recognition sequence (labeled with Raman reporter Cy5) on the surface of the gold shell, and 4-MBN is fixed in the gap layer between the core and shell. When the target is recognized, the formation of the rigid duplex makes Cy5 away from the metal surface, resulting in a decrease in its SERS signal, while the space-limited 4-MBN maintains a stable signal intensity. By monitoring the Cy5 / 4-MBN ratio SERS signal, VCAM-1 mRNA can be accurately quantitatively detected. More importantly, the consumption of mRNA during the detection process will inhibit the expression of VCAM-1 protein, thereby achieving early monitoring and therapeutic intervention of atherosclerosis.

[0059] The description and practice disclosed in the present application are easy to think and understand for ordinary skilled persons in the technical field, and several improvements and refinements can be made without departing from the principles of the present application. Therefore, the modifications or improvements made without departing from the spirit of the present application should also be considered within the protection scope of the present application.

Claims

1. A nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression, characterized in that, The nanoprobe is a core-shell structured gold nanoparticle. The nanoparticle has a core of Au nanoparticles and is modified with p-mercaptobenzonitrile 4-MBN as an internal standard molecule for Raman signal on the surface of the Au core. The nanoparticles have a gold shell on which the VCAM-1 mRNA targeting recognition sequence is immobilized. The VCAM-1 mRNA target recognition sequence segment is modified with Raman reporter molecule Cy5 at its end.

2. The nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression according to claim 1, characterized in that, The core-shell structured gold nanoparticles have a particle size of 60 nm.

3. The nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression according to claim 1, characterized in that, The volume and concentration of the VCAM-1 mRNA target recognition sequence modified on the Au shell were 50 μL and 1 μM, respectively.

4. The method for preparing a nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression according to claim 1, characterized in that, Includes the following steps: S1. Synthesis of gold core-shell nanoparticles: First, gold nanoparticles (AuNPs) were synthesized using the classic sodium citrate reduction method. To graft the Raman reporter molecule 4-MBN onto the surface of AuNPs, 4.35 μL of a 10 mg / mL PEG-SH solution was mixed with 10 mL of gold nanoparticles and stirred vigorously for 6 h to obtain dispersed Au@PEG particles. The precipitate was then centrifuged and resuspended in 2 mL of DMF. Subsequently, 2 mL of this Au@PEG solution was mixed with 300 μL of a 1.5 mM 4-MBN solution in 10 mL of DMF. After stirring at room temperature for 2 h, the resulting Au@MBN particles were collected by centrifugation and stored in 2 mL of deionized water. Au@MBN solution was mixed with a solution containing 50 mL of 1% PVP and 5 mL of 100 mM CTAB at room temperature. Then, 1.5 mL of 10 mM MgNO3 solution and 1.25 mL of 100 mM ascorbic acid (AA) were added dropwise, followed by 2.5 mL of 100 mM sodium hydroxide. Within 15 min, the solution changed from colorless to orange-yellow, indicating the formation of Au@MBN@Ag nanoparticles. After centrifugation, the nanoparticles were stored in 10 mL of 1% PVP solution. This solution was then combined with a mixture of 100 mL of 1% PVP and 10 mL of 100 mM CTAB, and 5 mL of 0.4 mg / mL chloroauric acid solution was added dropwise. After stirring for 15 min, the solution turned deep blue. Finally, 5 mL of 100 mM CTAB was added, and stirring continued for 15 min. The product was collected by centrifugation, yielding gold core-shell nanoparticles. S2. Preparation of nanoprobes: The VCAM-1 mRNA recognition sequence was heated in a 90℃ water bath for 5 min, and then naturally cooled to room temperature. 50 μL of the above sequence (1 μM) was added to 1 mL of the gold core-shell nanoparticle solution prepared above. The mixed solution was shaken at 37℃. After the reaction, the solution was centrifuged to remove the unmodified excess DNA strands on the gold core-shell nanoparticles to obtain nanoprobes.

5. The method for preparing a nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression according to claim 4, characterized in that, In S2, the reaction time between the gold core-shell nanoparticles and the VCAM-1 mRNA target recognition sequence is 12 hours.

6. The application of a nanoprobe for simultaneously detecting and inhibiting VCAM-1 mRNA expression, obtained by the preparation method according to any one of claims 4-5, in the early diagnosis and treatment of atherosclerosis.

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