Molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 (vascular endothelial growth factor receptor 2) and application

Abstract

The invention discloses a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 (vascular endothelial growth factor receptor 2) and application of the molecular probe. The invention discloses a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 (vascular endothelial growth factor receptor 2). The nucleotide sequence of the molecular probe is as shown in SEQ ID NO. 1. The invention successfully proves that a novel electrochemical method is used for detecting site-specific phosphorylation of VEGFR2 tyrosine residues, which is crucial for evaluating the risk of cerebral hemorrhage induced by preeclampsia of pregnant women. By means of VEGFR2 targeting peptide molecular probe and copper ion mediated double-tyrosine crosslinking, detection of the characteristics and sensitivity of the phosphorylation state of VEGFR2 in a clinical sample is achieved.

Description

Technical Field

[0001] The invention belongs to the field of probe application, and particularly relates to a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 and an application thereof. Background Art

[0002] In pregnant women with preeclampsia, the major fatal risk is due to cerebral hemorrhage. Hypertension induced by preeclampsia can cause cerebral hemorrhage, and recent biomedical studies have shown that preeclampsia can also increase the permeability of the blood-brain barrier, which may underlie the risk of clinically observed cerebral hemorrhage. At the molecular level, this increase in blood-brain barrier permeability is closely related to the biological function of vascular endothelial growth factor receptor 2 (VEGFR2), especially its phosphorylation, which further regulates the proliferation of endothelial cells. Studies have shown that phosphorylation of tyrosine 951 on the VEGFR2 molecule can inhibit the normal function of VEGFR2, thereby inhibiting the proliferation of endothelial cells and increasing the permeability of the blood-brain barrier. Conversely, phosphorylation of tyrosine 1175 can enhance the function of VEGFR2, thereby controlling the permeability of the blood-brain barrier. Detecting these specific structural biological changes at the molecular level can provide a valuable clinical intervention window for early assessment of the risk of death in patients with clamp syndrome. However, for a long time, simple bioassays using molecular probes can only quantitatively measure the concentration and enzyme activity of protein biomarkers. Detailed chemical information about amino acid residues at specific sites of protein molecules, such as chemical modifications of side chains at specific sites, often requires more complex experimental equipment, such as mass spectrometry-liquid chromatography, leading to inevitable time and cost issues. To a certain extent, electrochemical technology can enable simple biological detection methods, even handheld portable instruments, to glimpse the complex chemical information of protein disease biomarkers.

[0003] Compared with detection techniques such as fluorescence and colorimetry, the unique advantage of electrochemistry is that it can chemically modify certain redox-active functional groups on macromolecules such as proteins and nucleic acids in a reliable and controllable manner. Therefore, electrochemistry has been used for sample preparation for protein mass spectrometry-liquid chromatography since the end of the last century. In recent years, this feature has gradually regained attention. The use of electrochemical methods to introduce artificial modification groups at specific sites of peptides and proteins has become a hot topic in related research fields such as protein prodrug design, ordered peptide nanoassembly, and peptide-protein asymmetric catalysis. Taking advantage of this unique advantage of electrochemistry, it is convenient to achieve peptide and protein modification, and even main chain reconstruction, under relatively mild non-denaturing experimental conditions in polar, aqueous solutions similar to in vivo conditions, without the need for toxic organic reagents and harsh reaction temperatures in the past liquid-phase synthesis, coupling and side chain modification. This technical feature creates new opportunities for electrochemical biosensing, making it possible to obtain specific chemical modification information of target protein molecules directly using this simple and rapid detection method, even without relying on complex biological agents such as antibodies and enzymes, but simply by electrochemically controlling valence and redox potential. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular probe and application for detecting phosphorylation of two specific tyrosine residues of VEGFR2 in view of the defects of the prior art.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2, wherein the nucleotide sequence of the molecular probe is shown in SEQ ID NO.1.

[0006] Use of a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 in the preparation of a diagnostic / predictive reagent for diagnosing the risk of cerebral hemorrhage induced by preeclampsia in pregnant women, wherein the nucleotide sequence of the molecular probe is shown in SEQ ID NO.1.

[0007] Furthermore, the diagnostic / predictive reagent detects the electrochemical oxidation signal of VEGFR2 protein in the peripheral blood of the subjects. The electrochemical oxidation signal of VEGFR2 protein is significantly downregulated compared with the healthy control group, thereby judging whether the subjects are at risk of cerebral hemorrhage.

[0008] A diagnostic / prediction kit for diagnosing the risk of cerebral hemorrhage induced by preeclampsia in pregnant women, comprising the above-mentioned molecular probe.

[0009] A method for determining the phosphorylation of two specific tyrosine residues of VEGFR2, comprising the following steps:

[0010] (1) A conductive glass slide coated with indium tin oxide is used as a substrate, and the surface is modified with the molecular probe described in claim 1;

[0011] (2) The conductive glass slide modified with the molecular probe was incubated with the VEGFR2 protein isolated from the subjects and the healthy control group at room temperature, and then the electrochemical potential was scanned;

[0012] (3) The electrochemical anodic dissolution method was used to compare the electrochemical oxidation signals of the subjects and the healthy control group. The lower the electrochemical oxidation signal value of the subjects was than that of the healthy control group, the risk of cerebral hemorrhage can be predicted.

[0013] Furthermore, VEGFR2 protein was obtained by separating the cell components through differential centrifugation to separate the cell membrane, nuclear and cytoplasmic fractions.

[0014] Based on previous studies on electrochemical cleavage of phosphorylated tyrosine, this study involves electrochemical cleavage of VEGFR2 protein, phosphorylated at two different tyrosine sites into fragments of different lengths. In addition, by electrochemically adjusting the valence and coordination affinity of copper ions, we use copper ion-mediated tyrosine oxidative cross-linking to quantitatively analyze the length of protein fragments, achieving the purpose of distinguishing two site-specific tyrosine phosphorylations. The basic principle is similar to mass spectrometry measurement of protein fragment mass.

[0015] The present invention eliminates the reliance on complex preparations such as antibodies, enzymes and nanomaterials. It only requires simple short-chain peptides and metal ions. By utilizing electrochemically controlled redox reactions, site-specific chemical modifications of proteins can be achieved. This detection of complex chemical information is more complex than previous protein quantification and biological activity detection, enabling bedside detection using simple instruments to provide rich chemical information similar to that from large professional instruments, adapting to the increasingly in-depth and complex trend of molecular pathology.

[0016] This paper successfully demonstrated a novel electrochemical method for detecting site-specific phosphorylation of tyrosine residues of VEGFR2, which is critical for assessing the risk of preeclampsia-induced intracerebral hemorrhage in pregnant women. By utilizing a VEGFR2-targeted peptide molecular probe and copper ion-mediated dual tyrosine cross-linking, we achieved characteristic and sensitive detection of VEGFR2 phosphorylation status in clinical samples. The validity of the tyrosine method was verified by the distinct electrochemical signals generated by phosphorylation of residues 951 and 1175, which correlated with the biological functions and pathological significance of VEGFR2 in regulating endothelial cell proliferation and blood-brain barrier permeability. This method not only simplifies the detection process by eliminating the need for complex biologics, but also provides rapid and reliable insights into molecular modifications, thus providing a potential tool for early clinical intervention. The ability to quantitatively assess phosphorylation status in peripheral blood samples emphasizes its applicability in bedside diagnosis, consistent with the growing number of advanced molecular pathology technologies that can provide comprehensive chemical information using simple, portable instruments. This innovation represents a major advance in the field of electrochemical biosensing, providing a powerful platform for early detection and management of preeclampsia and other potential related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The isothermal titration calorimetry results in the examples are shown (a is a record of titrating 0.01 mM target protein (VEGFR2) with 0.1 mM peptide probe, b is a record of titrating 0.1 mM copper (II) ions to 0.01 mM peptide probe, and the integrated thermal peak value as a function of the molar ratio is shown below each figure).

[0018] Figure 2 are the electrochemical characteristics of the target protein detected in the embodiment (a is the result of dityrosine oxidation, b is the result of the change in copper ion valence during dityrosine cross-linking, and c is the oxidation of phosphotyrosine).

[0019] Figure 3 is the electrochemical evaluation of the potential for tyrosine phosphorylation of denatured target proteins in the examples (a is a recombinant protein sample diluted with standard serum, phosphorylated and unphosphorylated denatured tandem electrochemical double tyrosine cross-linking, b is the same set of samples after electrochemical cleavage treatment of peptide bonds at tyrosine residues, c and d are from clinical samples).

[0020] Figure 4 : are the performance analysis diagrams in the embodiments (a and b are the quantification of VEGFR2, wherein a is the IE line curve, b is the working curve of peak current and protein concentration, the inset in b represents the standard deviation (n=3), c and d are the completely denatured target proteins used as controls to determine the location of phosphorylation).

[0021] Figure 5is a statistical distribution diagram of the signal output detected in the pregnant woman's cell sample according to the embodiment. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0023] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or apparatuses.

[0024] Based on previous studies on electrochemical cleavage of phosphorylated tyrosine, this study involves electrochemical cleavage of VEGFR2 protein, phosphorylated at two different tyrosine sites into fragments of different lengths. In addition, by electrochemically adjusting the valence and coordination affinity of copper ions, we use copper ion-mediated tyrosine oxidative cross-linking to quantitatively analyze the length of protein fragments, achieving the purpose of distinguishing two site-specific tyrosine phosphorylations. The basic principle is similar to mass spectrometry measurement of protein fragment mass.

[0025] Specifically, the first step is to capture VEGFR2 from the peripheral blood of pregnant women onto the sensing interface using a specific molecular probe (as shown in SEQ ID NO. 1), facilitating the subsequent impurity elution, cleavage and peptide length measurement steps. The recognition sequence of the VEGFR2 molecular probe is only 12 amino acids long and contains a tyrosine. By fixing the molecular probe on the sensing interface, they can temporarily capture VEGFR2 from the peripheral blood sample non-covalently. Another functional module on the molecular probe is a DAHK sequence consisting of only 4 amino acids. Under the action of electrochemical potential scanning, the valence change of the copper ions complexed by this sequence mediates the covalent cross-linking of the proximal 12-peptide sequence with the VEGFR2 protein. VEGFR2 is more than 1000 amino acids long and contains a large number of tyrosines, a few of which are exposed on the protein surface, becoming the binding site for VEGF and the 12-peptide molecular probe from VEGF. The phosphorylation of these exposed tyrosines can interfere with the hydrophobic interaction between VEGF and VEGFR, achieving the above-mentioned result biological activity regulation. The double tyrosine cross-linking of the target protein and the molecular probe can generate specific fluorescence and electrochemical oxidation signals, which can be used as output signals for protein quantification.

[0026] Next, we can roughly assess the degree of phosphorylation of the target protein. According to previous studies, phosphorylated tyrosine can produce specific irreversible oxidation signals. Excess recombinant target protein is treated with different concentrations of tyrosine kinase, and the phosphorylated tyrosine signal obtained can be combined with the above-mentioned fluorescence and electrochemical oxidation signals to quantitatively detect the phosphorylation degree of the target protein.

[0027] To determine the location of tyrosine, in another set of parallel samples, after the target protein VEGFR2 and the 12-peptide molecular probe were covalently cross-linked, the sensing interface was thoroughly denatured and eluted to remove interfering proteins and denature the captured VEGFR2 protein to extend to the long-chain peptides fixed on the sensing interface. Subsequently, these extended long-chain peptides were again subjected to the above-mentioned double tyrosine electrochemical cross-linking. Since the hydrophobic core of the target protein exposes a large number of new tyrosines, an amplified double tyrosine fluorescence signal and an electrochemical oxidation signal can be generated. This signal difference ratio between the cross-linking signal generated by the target protein-molecular probe mentioned above can reflect the signal intensity of the fully cross-linked complete long-chain peptide. Within the electrochemical oxidation potential scanning range of irreversible oxidation of phosphotyrosine, the long chain of the target protein will be cut on the peptide bonds around the phosphorylated tyrosine to form fragments. The unmodified tyrosine peptide bond is cleaved at the carboxyl end, but the double tyrosine cross-linking can keep the short peptide fragments connected by double tyrosine. Therefore, the target protein phosphorylated at two different sites will produce fragments of different lengths after the above-mentioned electrochemical oxidation cleavage operation. The number of fragments that have been cross-linked with dityrosine will be different, generating different fluorescent / electrochemical signals, thereby distinguishing pregnant women with differential VEGFR2 phosphorylation sites in peripheral blood and further assessing the risk of increased blood-brain barrier permeability.

[0028] The advantage of this design is that it eliminates the reliance on complex preparations such as antibodies, enzymes and nanomaterials. It only requires simple short-chain peptides and metal ions. By utilizing electrochemically controlled redox reactions, site-specific chemical modifications of proteins can be achieved. This detection of complex chemical information is more complex than previous protein quantification and bioactivity detection, enabling bedside detection using simple instruments to provide rich chemical information similar to that from large professional instruments, adapting to the increasingly in-depth and complex trend of molecular pathology.

[0029] 2. Experimental

[0030] 2.1 Chemicals and reagents

[0031] The designed peptide molecular probes and all reagents used in this study were synthesized and verified to have a purity of more than 95%. This included VEGFR2 targeting peptides and copper ion chelating peptide sequences. All reagents were prepared in 100 mM phosphate buffered saline (PBS) with a pH of 7.4. The water used in the preparation of the reagents was obtained through a Milli-Q water purification system after double distillation to ensure a resistance of 18 MΩcm. Clinical samples were collected after approval by the local ethics committee and followed the standard guidelines for clinical laboratories.

[0032] 2.2 Cell and tissue sample processing

[0033] Human endothelial cells were obtained from placental biopsies and cultured briefly in vitro. The cells were placed in culture dishes in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (penicillin / streptomycin) and incubated at 37°C in a 5% carbon dioxide atmosphere for 24 hours.

[0034] Peripheral blood samples were collected from patients with preeclampsia and healthy pregnant women. Platelets were separated by centrifugation and briefly cultured in platelet-rich plasma (PRP) at 37°C in a 5% carbon dioxide atmosphere for 24 hours. To induce specific signaling pathways, cultured endothelial cells and platelets were treated with vascular endothelial growth factor A (VEGF-A) at a concentration of 100 ng / mL for 30 minutes to simulate physiological response conditions.

[0035] After VEGF-A treatment, cells and tissue samples were processed using a cell separation kit. The samples were lysed and the cellular components were separated by differential centrifugation to isolate the cell membrane, nuclear, and cytoplasmic fractions. The separated proteins were then prepared for subsequent analysis.

[0036] 2.3 Fabrication of the sensing surface

[0037] Conductive glass slides coated with indium tin oxide serve as substrates. Each slide is thoroughly cleaned using ultrasonic treatment in water and alcohol and then dried under high-purity nitrogen. The slide surface is modified with molecular probes. VEGFR2 targeting peptides are initially immobilized to ensure that the probes specifically target VEGFR2. Next, a layer of tetrapeptide sequences is added, designed to chelate divalent copper ions. This layer enables the subsequent binding and detection of copper ions. A drying and curing process is performed after each application to ensure that the biosensing molecules are stably attached to the glass surface.

[0038] 2.4 Sample preparation and application

[0039] VEGFR2 samples of endothelial cells were prepared using a cell isolation kit. VEGFR2 was isolated from samples of preeclampsia and healthy pregnant women and then incubated with the prepared biosensor slide at room temperature. The incubation process allowed the VEGFR2 targeting peptide to direct the probe to its target. After sufficient incubation, a gentle electrochemical potential scan was performed to ensure the interaction of the molecular probe with copper ions.

[0040] 2.5 Detection process

[0041] After the binding phase, the slides were subjected to another gentle electrochemical potential scan to activate the copper-containing peptides and promote covalent cross-linking of the captured proteins. The slides were then washed thoroughly with a denaturing detergent to remove non-specifically bound proteins, ensuring that only the specifically captured VEGFR2 protein remained on the surface.

[0042] 2.6 Electrochemical analysis

[0043] Final detection of bound proteins was performed using electrochemical anodic dissolution. This involves applying a controlled oxidative potential to the slide, resulting in oxidation of surface bound proteins and the generation of a measurable current. This current is recorded and analyzed using an electrochemical workstation equipped with a three-electrode setup. These data provide a detailed profile of the presence and activity of VEGFR2 in the sample, represented by the peaks of the electrochemical signal. This approach allows us to gain real-time insight into the effectiveness of detecting VEGFR2 and assessing its phosphorylation status under various physiological and pathological conditions.

[0044] 3. Results and Discussion

[0045] Figure 1 The isothermal titration calorimetry results show that the protein and metal ion binding modules in the designed probe interact with the target, respectively. (a) records the titration of 0.01 mM target protein (VEGFR2) with 0.1 mM peptide probe, while (b) shows the result of titrating 0.1 mM copper (II) ions into 0.01 mM peptide probe. In both panels, the bottom row shows the integrated heat peak plotted as a function of the molar ratio.

[0046] 3.1 Binding efficiency of peptide modules

[0047] We first investigated the ability of the two functional modules of our designed peptide molecular probes - two short peptide sequences - to effectively bind their target substances in solution. Isothermal titration calorimetry was used to record the heat released during the binding interaction between the molecular probe and its target protein or ion. When the peptide targeting VEGFR2 and the peptide targeting copper ions each interacted with each other, the two interactions produced typical saturation kinetic curves with strong affinity.

[0048] 3.2 Combination of functional sequences

[0049] These two functional sequences were then combined to form the target molecular probe used in this study. Figure 1 As shown in Figs. a and 1b, both molecular recognition sequences form specific, high-affinity complexes with the target substance without interference, even in a peptide-rich environment. Therefore, in subsequent experiments, the molecular probe was pre-labeled with divalent copper ions to promote its-mediated electrochemical oxidative coupling reaction of tyrosine.

[0050] 3.3 Probe immobilization and target detection

[0051] Figure 2The electrochemical characteristics of the detected target proteins (as shown in the figure) are: (a) is the result of dityrosine oxidation, (b) is the result of the change in copper ion valence during dityrosine cross-linking, and (c) is the oxidation of phosphotyrosine.

[0052] The copper-labeled peptide molecular probe was fixed on a conductive glass substrate to construct a sensing interface for detecting VEGFR2 protein in the sample. Figure 2 As shown in a, a specific double tyrosine cross-linking signal induced by the interaction between the target protein and the protein targeting sequence in the peptide molecular probe was detected. This confirms that the peptide sequence within the molecular probe specifically targets VEGFR2, effectively bringing the protein close enough to covalently couple with the probe. This covalent coupling is achieved through the electrochemical redox activity of copper ions, such as Figure 2 b. Covalent coupling not only generates a detectable signal output but also stably immobilizes the target protein on the electrode surface, allowing thorough cleaning of the sensing interface to reduce the impact of interfering proteins in clinical samples.

[0053] 3.4 Tyrosine phosphorylation detection

[0054] Next, we performed preliminary electrochemical oxidation detection of potential tyrosine phosphorylation on the target protein. Figure 2 As shown in c, the detected electrochemical oxidation peak signal of phosphotyrosine is roughly correlated with the concentration of the target protein in the gradient dilution, indicating that the phosphorylated target protein was successfully detected.

[0055] 3.5 Site-specific tyrosine phosphorylation detection

[0056] Figure 3 The potential for tyrosine phosphorylation of a denatured target protein is evaluated electrochemically to determine the location of phosphorylation. (a) is a sample of recombinant protein diluted in standard serum, phosphorylated and unphosphorylated denatured in series electrochemical double tyrosine cross-linking, and (b) is the same set of samples after electrochemical cleavage of peptide bonds at tyrosine residues. (c) and (d) are from clinical samples (see text for details) showing the difference between the signals after the same denaturation, cross-coupling and cleavage steps as described above.

[0057] Given that the biological function of VEGFR2 involves different degrees of autophosphorylation in response to different VEGFs, and that the overall phosphorylation level is not directly related to biological activity, we attempted a preliminary electrochemical discrimination of tyrosine phosphorylation of VEGFR2. According to the detection principle described previously, we first needed to establish a background control by denaturing and electrochemically inducing dual-tyrosine coupling on the non-phosphorylated target protein. Different concentrations of human recombinant target protein (non-phosphorylated) were incubated with the sensing interface, and their dual-tyrosine signals were detected. This step also covalently immobilized the target protein on the sensing interface. The sensing interface was then thoroughly washed with detergents and denaturants to remove interfering proteins. After this treatment, the captured target protein unfolded into an extended polypeptide chain, exposing the previously hidden tyrosines to the aqueous phase. The subsequent dual-tyrosine coupling produced a significantly stronger electrochemical signal compared to the folded state, as shown in Figure 2. Figure 3 As shown in a.

[0058] Using this background signal, we then electrochemically cleave the peptide bond on the carboxyl side of tyrosine and compare the remaining peptide fragment signals to identify the tyrosine phosphorylation site. Figure 3 As shown in b, after cleavage, the control protein (lacking phosphorylated tyrosine) maintained a consistent electrochemical signal level, indicating a stable dityrosine-linked peptide chain despite cleavage. This non-phosphorylated standard protein can be used as a baseline for subsequent site-specific tyrosine phosphorylation detection.

[0059] 3.6 Validation using clinical samples

[0060] Since VEGF stimulation does not induce different tyrosine phosphorylation at different sites, we used cell-based experiments to validate our approach. Endothelial cells from pregnant placentas were obtained by biopsy and briefly cultured in vitro. The experimental groups were stimulated with serum from women with preeclampsia. The cell samples were then subjected to the electrochemical detection process described previously. Figure 3 As shown in c and 3d, the final electrochemical signal of the experimental group (stimulated by preeclampsia serum) was lower than that of the control group, indicating that the peptide fragments were shorter due to site-specific tyrosine phosphorylation.

[0061] 3.7 Quantitative detection and optimization

[0062] Figure 4 The performance of the assay: (a) and (b) are quantification of VEGFR2, where (a) represents the IE curve, (b) represents the peak current vs. protein concentration working curve, and the inset represents the standard deviation (n=3). Similarly, (c) and (d) use fully denatured target proteins as controls to determine the location of phosphorylation.

[0063] Under the optimized reaction conditions, the quantitative detection curve of the target protein was first established, such as Figure 4 a and Figure 4As shown in b. Figure 4 a shows the relationship between the peak value of the electrochemical reaction and the concentration of the target protein in the gradient dilution. Figure 4 b plots the logarithm of the concentration versus the peak current, showing a low detection limit, a wide dynamic detection range, and good reproducibility and stability. Figure 4 c and Figure 4 d constructed a phosphorylation baseline control for clinical test samples. Figure 3 resemblance, Figure 4 c Electrochemical signals of cross-linked amino acids of maximally unfolded and denatured target proteins were recorded at different concentrations. Figure 4 d The logarithms of these signals and concentrations are plotted for easy reference in clinical sample testing.

[0064] Figure 5 is the statistical distribution of the signal output detected in the maternal cell sample, Figure 5 Detection of site-specific tyrosine phosphorylation of VEGFR2 in clinical samples from pregnant women with preeclampsia is shown. These women presented with signs of miscarriage and underwent routine placental biopsies to provide placental endothelial cell samples. The control group consisted of relatively healthy pregnant women with signs of miscarriage but no symptoms of preeclampsia, who also underwent placental biopsies to obtain endothelial cell samples. Comparison of the signals obtained from these two groups using the described detection process revealed a significantly lower electrochemical signal in the preeclampsia samples. This raises the question of whether site-specific tyrosine phosphorylation exists in pregnant women with preeclampsia that inhibits the normal function of VEGFR2. Lower levels of VEGFR2 were found in women with preeclampsia, which is consistent with previous reports. By calculating the signal from the two groups according to the described detection process, the electrochemical signal was significantly lower in the preeclampsia samples. Figure 5 By normalizing the protein concentration by neutralizing the signal, it can be clearly seen that the preeclampsia samples contain shorter protein fragments, indicating the presence of site-specific tyrosine phosphorylation, which inhibits the normal function of VEGFR.

[0065] 4. Conclusion

[0066] This study successfully demonstrated a novel electrochemical method for detecting site-specific phosphorylation of tyrosine residues of VEGFR2, which is critical for assessing the risk of preeclampsia-induced intracerebral hemorrhage in pregnant women. By utilizing a VEGFR2-targeted peptide molecular probe and copper-mediated dual-tyrosine crosslinking, we achieved characteristic and sensitive detection of VEGFR2 phosphorylation status in clinical samples. The effectiveness of the tyrosine method was verified by the distinct electrochemical signals generated by phosphorylation of residues 951 and 1175, which correlated with the biological functions and pathological significance of VEGFR2 in regulating endothelial cell proliferation and blood-brain barrier permeability. This method not only simplifies the detection process by eliminating the need for complex biologics, but also provides rapid and reliable insights into molecular modifications, thus providing a potential tool for early clinical intervention. The ability to quantitatively assess phosphorylation status in peripheral blood samples emphasizes its applicability in bedside diagnosis, in contrast to molecular pathology techniques, which can provide comprehensive chemical information using simple, portable instrumentation. This innovation represents a major advance in the field of electrochemical biosensing, providing a powerful platform for early detection and management of preeclampsia and other potential related diseases.

[0067] It should be understood that the present invention is described by way of example only and can be modified within the scope and spirit of the present invention. The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary skills in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2, characterized in that: The nucleotide sequence of the molecular probe is shown in SEQ ID NO.

1.

2. Use of a molecular probe for detecting phosphorylation of two specific tyrosine residues of VEGFR2 in the preparation of a diagnostic / predictive reagent for diagnosing the risk of cerebral hemorrhage induced by preeclampsia in pregnant women, characterized in that: The nucleotide sequence of the molecular probe is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The diagnostic / predictive reagent detects the electrochemical oxidation signal of VEGFR2 protein in the peripheral blood of the subject, and the electrochemical oxidation signal of VEGFR2 protein is significantly downregulated compared with the healthy control group, thereby judging that the subject has the risk of cerebral hemorrhage.

4. A diagnostic / prediction kit for diagnosing the risk of cerebral hemorrhage induced by preeclampsia in pregnant women, characterized in that: Comprising the molecular probe according to claim 1.

5. A method for determining the phosphorylation of two specific tyrosine residues of VEGFR2, characterized in that The following steps are involved: (1) A conductive glass slide coated with indium tin oxide is used as a substrate, and the surface is modified with the molecular probe described in claim 1; (2) The conductive glass slide modified with the molecular probe was incubated with the VEGFR2 protein isolated from the subjects and the healthy control group at room temperature, and then the electrochemical potential was scanned; (3) The electrochemical anodic dissolution method was used to compare the electrochemical oxidation signals of the subjects and the healthy control group. The lower the electrochemical oxidation signal value of the subjects was than that of the healthy control group, the risk of cerebral hemorrhage can be predicted.

6. The method for determining the phosphorylation of two specific tyrosine residues of VEGFR2 according to claim 5, characterized in that: The VEGFR2 protein is obtained by separating cell components through differential centrifugation, and separating cell membrane, cell nucleus and cytoplasm.

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