A drug evaluation and companion diagnostic biosensing system based on dual-target linkage monitoring probe

Abstract

The application provides a biosensing probe for double-target linkage monitoring, which is based on an NH2-UiO-66@AuNPs nanocomposite material, double-monitoring marker recognition aptamers are connected to the base material, the double-monitoring marker aptamers are used to connect monitoring markers, the monitoring markers are IL-6 and CRP; AuNPs are in-situ synthesized on the surface of NH2-UiO-66 to form an NH2-UiO-66@AuNPs nanocomposite material as a base material; IL-6 aptamers and CRP aptamers are connected to the NH2-UiO-66@AuNPs through Au-S bonds; the IL-6 aptamers are complementary to IL-6 cDNA to form a double-stranded structure, and a first fluorescent dye is connected to the IL-6 cDNA; the CRP aptamers are complementary to CRP cDNA to form a double-stranded structure, and a second fluorescent dye is connected to the CRP cDNA. The double-target linkage monitoring biosensing probe provided by the application provides a promising platform for the synchronous tracking of double indicators, which can not only be used for mechanism verification in drug discovery, but also be used for companion diagnosis to tailor treatment programs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a new product and method for medical biosensor detection. Background Technology

[0002] Cardiovascular disease is a heart and blood vessel disorder characterized by high morbidity and mortality. Among the many types of cardiovascular disease, hypertension is the most common, affecting approximately one-third of adults worldwide. Hypertension can also induce a range of metabolic syndromes; due to dysfunction in different metabolic pathways, it increases the risk of cerebral ischemia, atherosclerosis, cardiovascular disease, and inflammatory diseases, thus often being called a "silent killer." Hypertension generally lacks obvious symptoms and is traditionally diagnosed by measuring systolic and diastolic blood pressure. However, in most cases, this form of diagnosis is too late, as the disease has already caused organ damage. Therefore, early diagnosis of hypertension by monitoring subtle changes in specific biochemical markers in body fluids can minimize the risk of organ damage and improve patients' quality of life.

[0003] Early detection by monitoring subtle changes in specific biochemical markers in bodily fluids can significantly reduce the risk of organ damage and improve quality of life. Studies have identified specific inflammatory markers, such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which are elevated in patients with hypertension. These markers provide valuable insights into disease-related inflammatory pathways and can serve as potential biomarkers for early detection. Standard analytical methods have been used to quantify inflammatory factors, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and surface-enhanced Raman scattering (SERS). However, monitoring them alone only provides an incomplete picture of inflammation in the progression of hypertension. High IL-6 levels indicate persistent inflammation, but it is difficult to assess the severity of inflammation without correlating it with other markers. Similarly, measuring CRP alone may miss the role of cytokines that initiate the inflammatory cascade. It is difficult to measure the full impact of treatment on the inflammatory mechanisms leading to hypertension by monitoring only IL-6 or CRP. Furthermore, monitoring only one biomarker fails to capture the dynamic relationship between cytokines and acute-phase proteins, resulting in an incomplete assessment of the inflammatory state and its potential role in the progression of hypertension. Therefore, developing a method for simultaneously detecting inflammatory biomarkers is crucial for advancing the clinical management of hypertension and developing more effective targeted treatments. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a biosensing probe based on NH2-UiO-66@AuNPs for the coordinated monitoring of IL-6 and CRP; and to establish a multifunctional platform to support drug discovery and companion diagnostics. This invention pre-designs a dual-target coordinated monitoring probe (DTLM probe), which is the dual-target coordinated monitoring biosensing probe described in this invention. IL-6 and CRP are key inflammatory markers. This invention uses DTLM probes prepared with IL-6 and CRP aptamers to construct a drug evaluation platform and / or companion diagnostic platform for related diseases such as diabetes, depression, osteoarthritis, coronary heart disease, and hypertension. By observing changes in the expression levels of IL-6 and CRP inflammatory markers during medication or companion diagnostics, the effectiveness of the drug or companion diagnostic method can be determined. In a preferred embodiment of this invention, the efficacy of Qishen Yiqi Dripping Pills in relieving hypertensive cardiac damage is used as an example to verify the accuracy and effectiveness of the drug evaluation and / or companion diagnostic platform provided by this invention. The dual-target linkage monitoring biosensor probe, drug evaluation and / or companion diagnostic monitoring system, and drug evaluation and / or companion diagnostic platform provided by this invention are not limited to hypertension, nor are they limited to drug evaluation and companion diagnostic evaluation for diseases such as diabetes, depression, osteoarthritis, coronary heart disease, and hypertension. In fact, any inflammation-related disease in which the expression levels of IL-6 and CRP change during medication or companion diagnostics falls within the scope of this invention.

[0005] First, NH2-UiO-66 was synthesized in a one-pot process using a targeted immobilization strategy, providing it with abundant NH2 groups. Subsequently, AuNPs were synthesized in situ on NH2-UiO-66, forming NH2-UiO-66@AuNPs nanocomposites via Au-N bonds. Next, thiol-modified IL-6 and CRP aptamers were immobilized onto NH2-UiO-66@AuNPs via Au-S bonds for specific recognition of various targets. The DTLM probe was constructed by combining a FAM-modified IL-6 signaling chain and a TAMRA-modified CRP signaling chain. The DTLM probe was also optimized to achieve the best performance for the coordinated monitoring of IL-6 and CRP. In the presence of IL-6 and CRP, the target analyte binds to the corresponding aptamer, replacing the corresponding signaling chain, thereby enhancing the fluorescence signal in solution. The dual signal outputs do not interfere with each other, enabling the simultaneous detection of IL-6 and CRP. Furthermore, depending on specific needs, IL-6 or CRP can also be detected separately, enabling multiplex detection in a single sample injection. For individual detection, the detection range for IL-6 is 1 pg / mL to 1 μg / mL, with a limit of detection (LOD) of 0.350 pg / mL, while the detection range for CRP is 1 pg / mL to 10 μg / mL, with an LOD of 0.315 pg / mL. For simultaneous detection, the detection ranges for IL-6 and CRP remain unchanged, while the LODs are 0.355 pg / mL and 0.367 pg / mL, respectively. Under optimized parameters, subtle differences in serum IL-6 and CRP levels in healthy and hypertensive rats under different treatments can be rapidly identified. The dual-target linked monitoring biosensor probe described in this invention successfully explored the anti-inflammatory effects of the traditional Chinese medicine "Qi Shen Yi Qi" and evaluated its effectiveness in alleviating hypertensive cardiac damage. Moreover, the dual-target linked monitoring biosensor probe accurately distinguished serum IL-6 and CRP levels in healthy donors and hypertensive patients. More importantly, the biosensor probes for dual-target linked monitoring capture subtle changes in serum levels after treatment, ensuring that anti-inflammatory therapy can be accurately applied to patients who need it most. These biosensor probes provide a promising platform for the simultaneous tracking of two indicators, not only for mechanism validation in drug discovery but also for companion diagnostics to tailor treatment regimens.

[0006] To achieve the above objectives, this invention provides a biosensing probe for dual-target linked monitoring, using NH2-UiO-66@AuNPs nanocomposite material as the substrate material. A dual-target monitoring marker recognition aptamer is attached to the substrate material, and the dual-target monitoring marker aptamer is used to connect monitoring markers, namely IL-6 and CRP. AuNPs are synthesized in situ on the surface of NH2-UiO-66 to form NH2-UiO-66@AuNPs nanocomposite material as the substrate material. The IL-6 aptamer and CRP aptamer are linked to NH2-UiO-66@AuNPs via Au-S bonds. The IL-6 aptamer is complementary to IL-6 cDNA to form a double-stranded structure, and a first fluorescent dye is attached to the IL-6 cDNA. The CRP aptamer is complementary to CRP cDNA to form a double-stranded structure, and a second fluorescent dye is attached to the CRP cDNA.

[0007] Preferably, the 5' end of the IL-6 aptamer or CRP aptamer contains a thiol group modification.

[0008] Preferably, the IL-6 aptamer is a nucleotide sequence as shown in SEQ ID NO: 1, wherein the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 contains a thiol group modification, and the structural formula of the modification group is as follows:

[0009]

[0010] Preferably, the IL-6cDNA contains the nucleotide sequence shown in SEQ ID NO: 3.

[0011] Preferably, the CRP aptamer is a nucleotide sequence as shown in SEQ ID NO: 2, wherein the 5' end of the nucleotide sequence shown in SEQ ID NO: 2 contains a thiol group modification, and the structural formula of the modification group is:

[0012]

[0013] Preferably, the CRP cDNA contains a nucleotide sequence as shown in SEQ ID NO: 4.

[0014] Preferably, the excitation spectra of the first fluorescent dye and the second fluorescent dye should not completely overlap; the emission spectra of the first fluorescent dye and the second fluorescent dye are separated to avoid signal interference; and the maximum emission wavelength of one of the first fluorescent dyes and the second fluorescent dye is different from the maximum excitation wavelength of the other dye.

[0015] Preferably, the first fluorescent dye and the second fluorescent dye are a combination of TAMRA and FAM, or a combination of Cy3 and Cy5.

[0016] In a preferred embodiment of the present invention, the first fluorescent dye attached to the IL-6 cDNA is preferably 6-FAM; and the second fluorescent dye attached to the CRP cDNA is preferably TAMRA.

[0017] The present invention also provides a method for preparing the biosensing probe described in any one of the above claims, comprising the following steps:

[0018] Step 1: Synthesize NH2-UiO-66;

[0019] Step 2: AuNPs were synthesized in situ on the surface of NH2-UiO-66 to obtain NH2-UiO-66@AuNPs;

[0020] Step 3: Link the IL-6 aptamer and CRP aptamer to NH2-UiO-66@AuNPs to obtain...

[0021] NH2-UiO-66@AuNPs@Apt IL-6&CRP ;

[0022] Step 4: Ligate IL-6 cDNA and CRP cDNA linked with fluorescent dye to NH2-UiO-66@AuNPs@Apt. IL-6&CRP We obtained NH2-UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP .

[0023] Preferably, in step 1, the method for synthesizing NH2-UiO-66 is as follows:

[0024] Zirconium chloride, benzoic acid, and aminoterephthalic acid were completely dissolved in DMF using ultrasound. The mixture was then transferred to a hydrothermal reactor lined with polytetrafluoroethylene and reacted at 120°C for 24 h. The product was then collected by centrifugation and washed with DMF and methanol. Finally, the precipitate was dried to remove residual organic solvents, yielding NH2-UiO-66.

[0025] In a preferred embodiment of the present invention, the method for synthesizing NH2-UiO-66 is as follows:

[0026] 120 mg zirconium chloride, 1.9 g benzoic acid, and 110 mg aminoterephthalic acid were completely dissolved in 10 mL of DMF using ultrasound. The mixture was then transferred to a hydrothermal reactor lined with polytetrafluoroethylene and reacted at 120 °C for 24 h. The product was then collected by centrifugation at 12,000 rpm for 15 min and washed three times with DMF and methanol. Finally, the precipitate was vacuum dried overnight at 65 °C to remove residual organic solvents, yielding NH2-UiO-66, which was collected for later use.

[0027] Preferably, in step 2, the method for in-situ synthesis of AuNPs on the surface of NH2-UiO-66 to obtain NH2-UiO-66@AuNPs is as follows:

[0028] The NH2-UiO-66 obtained in step 1 was dispersed in deionized water, and then HAuCl4·3H2O was slowly added dropwise while stirring; then NaBH4 was added and stirred, and the NH2-UiO-66@AuNPs product was collected.

[0029] In a preferred embodiment of the present invention, the method for synthesizing NH2-UiO-66@AuNPs is as follows:

[0030] 10 mg of the obtained NH2-UiO-66 was dispersed in 50 mL of deionized water. Then, 5 mL of 6 mM HAuCl4·3H2O was slowly added dropwise under stirring. After stirring for 4 h, 1 mL of 0.5 M NaBH4 was added and stirred for 15 min. The NH2-UiO-66@AuNPs product was collected, washed three times with deionized water by centrifugation, and then resuspended in 4 mL of deionized water for later use.

[0031] Preferably, in step 3, NH2-UiO-66@AuNPs@Apt IL-6&CRP The preparation method is as follows:

[0032] The IL-6 aptamer and the CRP aptamer were added together to the NH2-UiO-66@AuNPs suspension and allowed to react at room temperature. Then, 10mM PBS buffer containing 2M NaCl was added dropwise to the solution until the final NaCl concentration was 0.2M. The reaction was allowed to continue at room temperature, and then the reaction product was centrifuged to obtain NH2-UiO-66@AuNPs@Apt. IL-6&CRP product.

[0033] In any of the above-mentioned preferred embodiments, the concentration of the IL-6 aptamer or the CRP aptamer is 1 to 30 μM, preferably 1, 5, 10, 15, 20, 25, 30 μM and the range thereof, and most preferably 20 μM.

[0034] Preferably, the incubation time of NH2-UiO-66@AuNPs and the aptamer is 10 to 18 hours, more preferably 10, 12, 14, 16, 18 hours and the range thereof; and more preferably 14 hours is the optimal incubation time for NH2-UiO-66@AuNPs and the aptamer.

[0035] Preferably, in step 4, NH2-UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The preparation method is as follows:

[0036] IL-6 cDNA and CRP cDNA were inoculated into NH2-UiO-66@AuNPs@Apt. IL-6&CRP The reaction takes place in a suspension. The result is NH2-UiO-66@AuNPs@Apt. IL-6&CRP @cDNA IL-6&CRP product.

[0037] In a preferred embodiment of the present invention, the biosensing probe for dual-target linkage monitoring is an IL-6 and CRP dual-target biosensing probe, and the construction method is as follows:

[0038] 20 μL of 20 μM IL-6 aptamer and 20 μL of 20 μM CRP aptamer were added to 100 μL of NH2-UiO-66@AuNPs suspension and allowed to stand at room temperature for 14 h. Then, 10 mM PBS buffer containing 2 M NaCl was added dropwise to the solution until the final NaCl concentration was 0.2 M. After standing at room temperature for 24 h, the reaction product was centrifuged and washed three times with ultrapure water. The resulting NH2-UiO-66@AuNPs@Apt... IL-6&CRP The product was resuspended in 140 μL of ultrapure water.

[0039] Subsequently, 70 μL of 10 μM IL-6 cDNA and 70 μL of 10 μM CRP cDNA were added dropwise to the above suspension, and the reaction was carried out at 37 °C for 1.5 h. Finally, the obtained NH2-UiO-66@AuNPs@Apt was... IL-6&CRP @cDNA IL-6&CRP The product was repeatedly washed and finally resuspended in 280 μL of ultrapure water and stored at 4°C for later use.

[0040] This invention also provides a biosensor monitoring system constructed from the biosensor probes described in any of the preceding claims. The monitoring system is a drug evaluation and companion diagnostic biosensor system based on dual-target linked monitoring probes. By monitoring changes in the expression levels of IL-6 and CRP inflammatory markers during medication or companion diagnostics, it determines whether the drug or treatment method produces the corresponding effect.

[0041] Preferably, when the monitoring system detects IL-6 alone, the limit of detection (LOD) for IL-6 is 0.350 pg / mL.

[0042] Preferably, when the monitoring system detects CRP alone, the limit of detection (LOD) for CRP is 0.315 pg / mL.

[0043] Preferably, when the monitoring system detects IL-6 and CRP simultaneously, the LOD of IL-6 is 0.355 pg / mL and the LOD of CRP is 0.367 pg / mL.

[0044] In any of the above-mentioned preferred embodiments, when the monitoring system detects IL-6 alone, the linear regression equation for F-F0 and the logarithm of IL-6 concentration is y = 188.975 + 409.075 × lg C. IL-6 R 2 =0.995, y is the detected fluorescence intensity, C IL-6 To detect the concentration of IL-6 in the sample. Preferably, the IL-6 concentration range is 1 pg / mL to 1 μg / mL.

[0045] Preferably, when the monitoring system detects CRP alone, the linear regression equation between F-F0 and the logarithm of CRP concentration is y = 17.189 + 26.078 × lg C CRP R 2 =0.992, y is the detected fluorescence intensity, C CRP To detect the concentration of CRP in the sample, preferably, the CRP concentration range is 1 pg / mL to 10 μg / mL.

[0046] Preferably, when the monitoring system simultaneously detects IL-6 and CRP, the linear regression equation for F-F0 and the logarithm of IL-6 concentration is y = 188.919 + 408.883 × lg C. IL-6 R 2 =0.993, y is the detected fluorescence intensity, C IL-6 To detect the concentration of IL-6 in the sample, the linear regression equation for F-F0 and the logarithm of CRP concentration is y = 17.164 + 26.065 × lg C. CRP R 2 =0.993, y is the detected fluorescence intensity, C CRP To detect the concentration of CRP in the sample, preferably, the IL-6 concentration range is 1 pg / mL to 1 μg / mL, and the CRP concentration range is 1 pg / mL to 10 μg / mL.

[0047] F and F0 represent the fluorescence intensity in the presence and absence of IL-6 and CRP.

[0048] The present invention also provides the application of the biosensing monitoring system described in any of the above claims in the construction of a drug evaluation and / or companion diagnostic platform.

[0049] In a preferred embodiment of the present invention, the application effect of the monitoring system in constructing a drug evaluation and companion diagnostic platform was verified. Using the biosensor probe with dual-target linkage monitoring described in the present invention, the therapeutic efficacy of Qishen Yiqi Dripping Pills for clinical hypertension patients was verified through the system or platform described in the present invention, demonstrating the application prospects of the present invention in drug evaluation and companion diagnostic platforms for inflammation-related diseases. Attached Figure Description

[0050] Figure 1 The morphology of NH2-UiO-66 and NH2-UiO-66@AuNPs observed by TEM and SEM in preferred embodiment 1 of the present invention is shown.

[0051] Figure 2 This is a feasibility verification result for the biosensor probe for hypertension dual-target linkage monitoring described in the preferred embodiment 2 of the present invention.

[0052] Figure 3 This is the result of optimizing the aptamer incubation conditions in the preferred embodiment 3 of the present invention.

[0053] Figure 4 The fluorescence spectrum and calibration curve of the biosensor probe for hypertension dual-target linkage monitoring described in the preferred embodiment 4 of the present invention.

[0054] Figure 5 The results of the analysis of rat serum samples in preferred embodiment 5 of the present invention are shown.

[0055] Figure 6 The biosensor probe for hypertension dual-target linkage monitoring described in the preferred embodiment 5 of the present invention is consistent with the ELISA detection of (A) IL-6 and (B) CRP in rat serum.

[0056] Figure 7 The results are analyzed in clinical serum samples using the biosensor probe for hypertension dual-target linkage monitoring as described in the preferred embodiment 6 of the present invention.

[0057] Figure 8 The biosensor probe for hypertension dual-target linkage monitoring described in the preferred embodiment 6 of the present invention is consistent with the ELISA detection of (A) IL-6 and (B) CRP in clinical serum. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0059] Example 1

[0060] To establish a stable sensing platform, AuNPs were synthesized in situ on the surface of NH2-UiO-66, forming an NH2-UiO-66@AuNPs nanocomposite material as the substrate. The morphology of the synthesized NH2-UiO-66 and NH2-UiO-66@AuNPs was observed using field emission transmission electron microscopy (TEM) and field emission scanning electron microscopy (SEM).

[0061] The synthesis method of NH2-UiO-66 is as follows:

[0062] 120 mg zirconium chloride, 1.9 g benzoic acid, and 110 mg aminoterephthalic acid were completely dissolved in 10 mL of DMF using ultrasound. The mixture was then transferred to a hydrothermal reactor lined with polytetrafluoroethylene and reacted at 120 °C for 24 h. The product was then collected by centrifugation at 12,000 rpm for 15 min and washed three times with DMF and methanol. Finally, the precipitate was vacuum dried overnight at 65 °C to remove residual organic solvents, yielding NH2-UiO-66, which was collected for later use.

[0063] The synthesis method of NH2-UiO-66@AuNPs is as follows:

[0064] 10 mg of the obtained NH2-UiO-66 was dispersed in 50 mL of deionized water. Then, 5 mL of 6 mM HAuCl4·3H2O was slowly added dropwise under stirring. After stirring for 4 h, 1 mL of 0.5 M NaBH4 was added and stirred for 15 min. The NH2-UiO-66@AuNPs product was collected, washed three times with deionized water by centrifugation, and then resuspended in 4 mL of deionized water for later use.

[0065] Figure 1 The images show the morphology of NH2-UiO-66 and NH2-UiO-66@AuNPs observed by TEM and SEM, respectively. In the images, A is a TEM image of NH2-UiO-66, B is a SEM image of NH2-UiO-66, C is a TEM image of NH2-UiO-66@AuNPs, and D is a SEM image of NH2-UiO-66@AuNPs. Figure 1The NH2-UiO-66 samples exhibited a uniform morphology with a typical octahedral structure and a diameter of approximately 200 nm. Compared to NH2-UiO-66, the TEM and SEM images of NH2-UiO-66@AuNPs clearly showed a large number of uniformly distributed AuNPs on the surface of NH2-UiO-66. These AuNPs were observed to be uniformly shaped and spherical in size, with a diameter of approximately 10 nm. The abundant in-situ synthesized AuNPs provided rich binding sites for subsequent DNA ligation. The TEM and SEM characterization results of NH2-UiO-66 and NH2-UiO-66@AuNPs indicate that the preparation of NH2-UiO-66@AuNPs was successful.

[0066] Example 2

[0067] Example 2 verified the feasibility of the sensing strategy of the present invention using fluorescence detection. To design a target-response biosensor probe, aptamers for IL-6 and CRP were linked to NH2-UiO-66@AuNPs via Au-S bonds. Then, 6-FAM-labeled IL-6 cDNA and TAMRA-labeled CRP cDNA were added. The partial complementarity between the aptamers and cDNA resulted in a double-stranded structure. In the presence of IL-6 and CRP, due to the strong interaction between the target and the aptamers, the target competes with the cDNA, displacing the fluorescently labeled cDNA and generating a fluorescence signal.

[0068] The method for constructing a dual-target biosensor probe for IL-6 and CRP is as follows:

[0069] 20 μL of 20 μM IL-6 aptamer and 20 μL of 20 μM CRP aptamer were added to 100 μL of NH2-UiO-66@AuNPs suspension and allowed to stand at room temperature for 14 h. Then, 10 mM PBS buffer containing 2 M NaCl was added dropwise to the solution until the final NaCl concentration was 0.2 M. After standing at room temperature for 24 h, the reaction product was centrifuged and washed three times with ultrapure water. The resulting NH2-UiO-66@AuNPs@Apt... IL-6&CRP The product was resuspended in 140 μL of ultrapure water.

[0070] Subsequently, 70 μL of 10 μM IL-6 cDNA and 70 μL of 10 μM CRP cDNA were added dropwise to the above suspension, and the reaction was carried out at 37 °C for 1.5 h. Finally, the obtained NH2-UiO-66@AuNPs@Apt was... IL-6&CRP @cDNA IL-6&CRP The product was repeatedly washed and finally resuspended in 280 μL of ultrapure water and stored at 4°C for later use.

[0071] In Example 2, the IL-6 aptamer is a nucleotide sequence as shown in SEQ ID NO: 1, with a thiol group modified at the 5' end of the nucleotide sequence shown in SEQ ID NO: 1. The structural formula of the modified group is as follows:

[0072]

[0073] In Example 2, the IL-6 cDNA has the nucleotide sequence shown in SEQ ID NO: 3.

[0074] In Example 2, the CRP aptamer is a nucleotide sequence as shown in SEQ ID NO: 2, with a thiol group modified at the 5' end of the nucleotide sequence shown in SEQ ID NO: 2. The structural formula of the modified group is as follows:

[0075]

[0076] In Example 2, the CRP cDNA is a nucleotide sequence as shown in SEQ ID NO: 4.

[0077] The sequence described in Example 2 is shown in the table below:

[0078]

[0079] The thiol modification method of IL-6 aptamer or CRP aptamer is a prior art method, referenced from: https: / / store.sangon.com / page / center / cart / class_TCEP_reduction.html, however, the present invention is not limited thereto.

[0080] In Example 2, the measurement method includes the following steps:

[0081] IL-6 measurement: 30 μL of IL-6 at different concentrations was mixed with 30 μL of NH2-UiO-66@AuNPs@Apt. IL-6&CRP @cDNA IL-6&CRP The suspensions were mixed and incubated at 37°C for 1.5 h. Finally, the mixture was centrifuged, and the fluorescence signal of 6-FAM in the supernatant was detected using a microplate reader under 494 nm excitation.

[0082] CRP measurement: 30 μL of CRP at different concentrations was mixed with 30 μL of NH2-UiO-66@AuNPs@Apt. IL-6&CRP @cDNA IL-6&CRP Mix the suspensions. Incubate at 37°C for 1.5 h. Finally, centrifuge the mixture and detect the fluorescence signal of TAMRA in the supernatant under 552 nm excitation using a microplate reader.

[0083] Measurement of IL-6 and CRP: 30 μL of a mixture of IL-6 and CRP at different concentrations was added to 30 μL of NH2-UiO-66@AuNPs@Apt. IL-6&CRP @cDNA IL-6&CRP Mix the suspensions. Incubate at 37°C for 1.5 h, centrifuge the mixture, and detect the fluorescence signals of 6-FAM and TAMRA in the supernatant under excitation at 494 nm and 552 nm, respectively, using a microplate reader.

[0084] Figure 2 The results of the feasibility verification of the biosensor probe for hypertension dual-target linkage monitoring described in the preferred embodiment 2 of the present invention are as follows: A is the FAM fluorescence measurement when IL-6 is present (red line) and absent (blue line); B is the TAMRA fluorescence measurement when CRP is present (red line) and absent (blue line); C is the FAM and TAMRA fluorescence measurement when IL-6 and CRP are both present (red line) and absent (blue line).

[0085] Figure 2 The results demonstrate the feasibility of the biosensor probe for hypertension dual-target linkage monitoring obtained in Example 2 for simultaneously detecting 1 ng / mL IL-6 and CRP. As shown in A, fluorescence detection shows that when IL-6 is present, the characteristic absorption peak of 6-FAM at 520 nm is much stronger than when IL-6 is absent, proving the feasibility of the biosensor probe for detecting IL-6. As shown in B, when CRP is present, TAMRA can detect a strong characteristic absorption peak at 580 nm, while when CRP is absent, only a weak background absorption peak is detected, proving the feasibility of the biosensor probe for detecting CRP. In addition, Example 2 also studied the fluorescence response of the biosensor probe in the presence or absence of IL-6 and CRP. As shown in C, it can be seen that when IL-6 and CRP are present, the fluorescence signals at 520 nm and 580 nm are much higher than when IL-6 and CRP are absent, indicating that the biosensor probe can effectively detect IL-6 and CRP simultaneously.

[0086] Example 3

[0087] To achieve higher sensing performance, Example 3 optimized the reaction conditions for IL-6 and CRP detection. Considering that the concentrations of IL-6 and CRP aptamers are the main factors controlling reaction efficiency, Example 3 first optimized these conditions. Figure 3 The figure shows the optimization results of aptamer incubation conditions, where A represents the optimized aptamer concentration and B represents the concentration for incubation time.

[0088] like Figure 3As shown in -A, when detecting 1 ng / mL IL-6 and CRP, Example 3 observed a gradual increase in the F-F0 fluorescence signal (F and F0 represent the fluorescence intensity in the presence and absence of IL-6 and CRP) as the aptamer concentration increased from 1 μM to 20 μM. However, the fluorescence signal decreased with further increases in aptamer concentration. This may be because excessively high aptamer concentrations lead to nonspecific adsorption and increased steric hindrance, reducing the recognition between the target and the aptamer, resulting in decreased fluorescence intensity. Therefore, 20 μM is preferably the optimal aptamer concentration in this invention.

[0089] Subsequently, the incubation time of NH2-UiO-66@AuNPs and the aptamer was optimized by comparing the fluorescence signal intensities generated at different times. Figure 3 As shown in Figure -B, when detecting 1 ng / mL IL-6 and CRP, increasing the incubation time significantly improved the fluorescence signal intensity, with the highest signal intensity obtained at 14 h. However, after 14 h, further incubation may cause some aptamers to detach from NH2-UiO-66@AuNPs, resulting in a decrease in fluorescence signal. Therefore, 14 h was selected as the optimal incubation time for NH2-UiO-66@AuNPs and the aptamers.

[0090] Example 4

[0091] To evaluate the analytical performance of the biosensor probe for hypertension dual-target linkage monitoring provided by this invention under optimal experimental conditions, a series of concentrations of IL-6 and CRP were detected separately and simultaneously using the prepared biosensor probe. Figure 4 The following are examples: (A) fluorescence spectra and (B) calibration curves for a series of IL-6 concentrations (1 pg / mL-1 μg / mL); (C) fluorescence spectra and (D) calibration curves for a series of CRP concentrations (1 pg / mL-10 μg / mL); (E) fluorescence spectra and (F) calibration curves for different concentrations of IL-6 (1 pg / mL-1 μg / mL) and CRP (1 pg / mL-10 μg / mL); error bars are SD (n=3).

[0092] First, the analytical performance of the biosensing probes for detecting IL-6 and CRP separately was investigated. For IL-6 detection and analysis, such as... Figure 4 As shown in Figure A, with increasing IL-6 concentration, the binding of IL-6 to the aptamer also increases, leading to the displacement of more 6-FAM-labeled cDNA and a gradual increase in fluorescence intensity at 520 nm. Example 4 also observed a significant linear relationship between F-F0 and the logarithm of IL-6 concentration (1 pg / mL - 1 μg / mL). Figure 4 The linear regression equation for F-F0 in -B is y = 188.975 + 409.075 × lg CIL-6 (R 2 =0.995). For CRP detection analysis, such as Figure 4 -C and Figure 4 As shown in Figure -D, the fluorescence intensity of TAMRA at 580 nm gradually increases with increasing CRP concentration. The linear regression equation of F-F0 and the logarithm of CRP concentration in the range of 1 pg / mL-10 μg / mL is y = 17.189 + 26.078 × lg C CRP (R 2 =0.992). Using 3S D The limits of detection (LODs) for IL-6 and CRP calculated using the standard were as low as 0.350 pg / mL and 0.315 pg / mL, respectively. D (where m represents the standard deviation of 20 blank measurements and m is the slope of the calibration plot). The analytical performance of the biosensing probe for simultaneous detection of IL-6 and CRP was then investigated, and the results are as follows: Figure 4 -E and Figure 4 As shown in Figure -F, compared with individual detection, the detection ranges of IL-6 and CRP remained consistent when detected simultaneously, and the LOD did not change significantly. The linear regression equation for IL-6 in the range of 1 pg / mL-1 μg / mL is y = 188.919 + 408.883 × lgC IL-6 (R 2 =0.993), LOD was 0.355 pg / mL, and the linear regression equation for CRP in the range of 1 pg / mL-10 μg / mL was y = 17.164 + 26.065 × lg C CRP (R 2 =0.993), and LOD was 0.367 pg / mL, indicating that there was no significant cross-interference when using different aptamers and cDNA labeled with different fluorescence during the detection and analysis process. This also shows that the constructed biosensor probe has the advantages of high sensitivity, high specificity, good cost-effectiveness, and simultaneous analysis.

[0093] Example 5

[0094] To explore the applicability of the obtained biosensing probe and the efficacy and mechanism of action of Qishen Yiqi Dripping Pills in relieving hypertension and cardiac damage, Example 5 investigated rat serum samples.

[0095] Figure 5 The following are the analysis results of rat serum samples: thermograms of fluorescence signals of (A) IL-6 and (D) CRP in each group measured by biosensor probe method; thermograms of OD values ​​of (B) IL-6 and (E) CRP in each group measured by ELISA method; expression levels of (C) IL-6 and (F) CRP in each group measured by biosensor probe method and ELISA method; each sample was tested three times.

[0096] The expression levels of IL-6 and CRP in the serum samples of rats in each group were detected by two methods: the obtained biosensor probes and enzyme-linked immunosorbent assay (ELISA). Figure 5 -A and Figure 5 -B represent the signal response results of IL-6 measured by the two methods. It can be seen that the fluorescence response intensity and OD value response magnitude of IL-6 are as follows: Mod group > QSYQ-L group (low-dose group) > QSYQ-H group (high-dose group) > Con group. This indicates that the biosensing method and ELISA method show significant differences in signal when detecting IL-6 in different groups of rat serum, and both can be used to distinguish different levels of the inflammatory factor IL-6. Furthermore, from... Figure 5 As shown in Figure -C, compared with the Con group, the serum IL-6 expression level in the Mod group was significantly increased. Compared with the Mod group, Qishen Yiqi Dripping Pills significantly reduced IL-6 concentration, and the high-dose group of Qishen Yiqi Dripping Pills showed a better effect on reducing IL-6 than the low-dose group. Similarly, in the CRP detection results, as... Figure 5 -D to Figure 5 As shown in Example 5, the fluorescence response, OD value, and concentration of CRP in the serum of the Mod group were all higher than those of the Con group. Qishen Yiqi Dripping Pills also significantly reduced the fluorescence response, OD value, and concentration of CRP in the serum, with the QSYQ-H group showing better results than the QSYQ-L group. These results indicate that the biosensor probe constructed in this invention has good applicability, and that Qishen Yiqi Dripping Pills can alleviate hypertensive cardiac damage by regulating the inflammatory response, while also demonstrating dosage compliance.

[0097] Example 5 also investigated the consistency between the biosensing probe constructed in this invention and ELISA detection. Figure 6 The biosensing probe is consistent with the ELISA detection of (A) IL-6 and (B) CRP in rat serum.

[0098] like Figure 6 -A and Figure 6 As shown in Figure -B, the two methods exhibit high consistency in the detection of IL-6 and CRP, further demonstrating the feasibility and reliability of using NH2-UiO-66@AuNPs-based biosensor probes for sensitive detection of IL-6 and CRP in complex samples. Furthermore, compared to traditional ELISA methods, the biosensor probe method significantly reduces detection time and lowers detection costs.

[0099] Example 6

[0100] To verify the therapeutic efficacy of Qishen Yiqi Dripping Pills in clinical hypertension patients, Example 6 selected 60 clinical patients: 20 healthy donors (Healthy), 20 patients receiving routine hypertension treatment (Con), and 20 patients receiving routine treatment combined with Qishen Yiqi Dripping Pills (QSYQ). The levels of IL-6 and CRP in serum samples from healthy donors (Healthy), patients receiving routine hypertension treatment (Con), and patients receiving routine treatment combined with Qishen Yiqi Dripping Pills (QSYQ) were measured before (B-Con) and after (A-Con) treatment using the biosensor probes and ELISA method.

[0101] Figure 7 For clinical serum sample analysis: thermograms of serum (A) IL-6 and (D) CRP fluorescence signals measured by biosensor probe method; thermograms of serum (B) IL-6 and (E) CRP OD values ​​measured by ELISA method; expression levels of serum (C) IL-6 and (F) CRP measured by biosensor probe method and ELISA method; each sample was tested three times.

[0102] Figure 7 -A and Figure 7 -B shows the fluorescence response and OD value of IL-6 in serum. The two signals differed significantly across the five clinical sample groups, indicating that both biosensing and ELISA methods are sensitive to IL-6 and can be used for the detection of IL-6 in clinical samples. Figure 7 As shown in Figure C, the Healthy group had the lowest serum IL-6 levels, while the B-Con and B-QSYQ groups had the highest. After conventional treatment and conventional combined treatment with Qishen Yiqi Dripping Pills, serum IL-6 levels decreased, and the decrease in IL-6 levels was greater in the A-QSYQ group than in the A-Con group, indicating that Qishen Yiqi Dripping Pills can reduce IL-6 levels in hypertensive patients. Similarly, as... Figure 7 -D to Figure 7 As shown in Figure -F, the fluorescence response and OD values ​​of CRP in serum varied among different groups. The B-Con and B-QSYQ groups exhibited the highest CRP concentrations, while the Healthy group showed the lowest. The A-QSYQ group showed a greater decrease in CRP levels than the A-Con group, which is similar to the IL-6 detection results, indicating that Qishen Yiqi Dripping Pills have a regulatory effect on the inflammatory factor CRP. These results demonstrate that the biosensor probe constructed in this invention has good applicability in clinical samples and further validates that Qishen Yiqi Dripping Pills can reduce inflammatory responses.

[0103] Furthermore, Example 6 also investigated the consistency between the biosensing probe constructed in this invention and the ELISA method. Figure 8The biosensing probe demonstrated consistency with ELISA detection of (A) IL-6 and (B) CRP in clinical serum. Figure 8 -A and Figure 8 As shown in Figure -B, the two methods are highly consistent, further demonstrating the feasibility and reliability of the obtained biosensor probe in sensitively detecting IL-6 and CRP in clinical serum samples. The sensor probe and monitoring system described in this invention can be used for hypertension drug evaluation and / or companion diagnostic platforms. The drugs are not limited to Qishen Yiqi Dripping Pills; conventional Western medicine treatments for hypertension, such as CCBs, ACEIs, and ARBs, are also applicable to this invention.

[0104] The accuracy and effectiveness of the dual-target linked monitoring biosensor probe and monitoring system provided by this invention in drug evaluation and companion diagnostics were verified through its application in the evaluation of hypertension drugs and / or companion diagnostics. This invention is applicable to drug evaluation and companion diagnostics for inflammatory diseases, including but not limited to diabetes, depression, osteoarthritis, coronary heart disease, and hypertension. Any inflammatory disease in which changes in the expression levels of IL-6 and CRP occur during medication or companion diagnostics fall within the scope of this invention.

[0105] The above embodiments are merely illustrative examples 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. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A biosensor probe for dual-target linkage monitoring, taking NH2-UiO-66@AuNPs nanocomposites as a base material, wherein a monitoring marker is connected to the base material, and characterized in that, The substrate material is connected with a double monitoring marker adapter, which is used to connect the monitoring marker; the double monitoring marker adapter is an IL-6 adapter and a CRP adapter; the monitoring marker is IL-6 and CRP; AuNPs are synthesized in situ on the surface of NH2-UiO-66 to form NH2-UiO-66@AuNPs nanocomposites as the substrate material; the IL-6 adapter and the CRP adapter are connected to NH2-UiO-66@AuNPs through Au-S bonds; the IL-6 adapter is complementary to IL-6 cDNA to form a double-stranded structure, and the IL-6 cDNA is connected with a first fluorescent dye; the CRP adapter is complementary to CRP cDNA to form a double-stranded structure, and the CRP cDNA is connected with a second fluorescent dye.

2. The biosensing probe of claim 1, wherein, The IL-6 adapter is a nucleotide sequence as shown in SEQ ID NO: 1, and a thiol group is modified at the 5' end of the nucleotide sequence as shown in SEQ ID NO: 1; the structure of the modification group is:

3. The biosensing probe of claim 2, wherein, The IL-6 cDNA comprises a nucleotide sequence as shown in SEQ ID NO:

3.

4. The biosensing probe of claim 1, wherein, The CRP adapter is a nucleotide sequence as shown in SEQ ID NO: 2, and a thiol group is modified at the 5' end of the nucleotide sequence as shown in SEQ ID NO: 2; the structure of the modification group is:

5. The biosensing probe of claim 1, wherein, The CRP cDNA comprises a nucleotide sequence as shown in SEQ ID NO:

4.

6. The preparation method of the biosensor probe according to any one of claims 1-5, comprising the following steps: Step 1: Synthesis of NH2-UiO-66; Step 2: Synthesis of AuNPs in situ on the surface of NH2-UiO-66 to obtain NH2-UiO-66@AuNPs; Step 3: Connecting the IL-6 adapter and the CRP adapter to NH2-UiO-66@AuNPs to obtain NH2-UiO-66@AuNPs@Apt IL-6&CRP ; Step 4: IL-6 cDNA conjugated with fluorescent dye and CRP cDNA conjugated with fluorescent dye were connected to NH2-UiO-66@AuNPs@Apt IL-6&CRP , to obtain NH2-UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP , namely the biosensor probe for dual-target linkage monitoring.

7. A biosensor monitoring system constructed using the biosensor probe according to any one of claims 1-6.

8. The monitoring system according to claim 7, wherein ①When the monitoring system detects IL-6 alone, the detection limit LOD of IL-6 is 0.350 pg / mL; ②When the monitoring system detects CRP alone, the detection limit LOD of CRP is 0.315 pg / mL; ③When the monitoring system detects IL-6 and CRP simultaneously, the LOD of IL-6 is 0.355 pg / mL, and the LOD of CRP is 0.367 pg / mL.

9. The monitoring system according to claim 7, wherein When the monitoring system detects IL-6 alone, the linear regression equation of F-F0 versus the logarithm of IL-6 concentration is y = 188.975 + 409.075 x lg C IL-6 , R 2 = 0.995, where y is the detected fluorescence intensity and C IL-6 is the concentration of IL-6 in the detection sample. The linear regression equation of F-F0 and the logarithm of CRP concentration is y = 17.189 + 26.078 x lg C when the monitoring system detects CRP alone CRP , R 2 = 0.992, y is the detected fluorescence intensity, and C CRP is the concentration of CRP in the detected sample. The linear regression equation of F-F0 versus the logarithm of IL-6 concentration is y = 188.919 + 408.883 x lg C IL-6 , R 2 = 0.993, where y is the detected fluorescence intensity and C IL-6 is the concentration of IL-6 in the detected sample; and the linear regression equation of F-F0 versus the logarithm of CRP concentration is y = 17.164 + 26.065 x lg C CRP , R 2 = 0.993, where y is the detected fluorescence intensity and C CRP is the concentration of CRP in the detected sample.

10. Use of the biosensor monitoring system according to any one of claims 7-9 in constructing a drug evaluation and / or companion diagnosis platform.

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