Drug evaluation and adjoint diagnosis biosensing system based on double-target linkage monitoring probe

Abstract

The invention provides a biosensing probe for double-target linkage monitoring, which is characterized in that an NH2-UiO-66 (at) AuNPs nano composite material is used as a substrate material, the substrate material is connected with a double monitoring marker recognition aptamer, the double monitoring marker recognition aptamer is used for connecting monitoring markers, and the monitoring markers are IL-6 and CRP; the preparation method comprises the following steps: synthesizing AuNPs on the surface of NH2-UiO-66 in situ to form a NH2-UiO-66 (at) AuNPs nano composite material as a substrate material; the preparation method comprises the following steps: connecting an IL-6 (Interleukin-6) aptamer and a CRP (C-Reactive Protein) aptamer to NH2-UiO-66 (at) AuNPs through an Au-S bond; the IL-6 aptamer and the IL-6cDNA are complementary to form a double-chain structure, and the IL-6cDNA is connected with a first fluorescent dye; the CRP aptamer and the CRP cDNA are complementary to form a double-chain structure, and the CRP cDNA is connected with a second fluorescent dye. The double-target linkage monitoring biosensing probe provided by the invention provides a promising platform for synchronous tracking of double indexes, is not only used for mechanism verification in drug discovery, but also can be used for accompanying diagnosis so as to customize a treatment scheme.

Description

Technical Field

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

[0002] Cardiovascular disease is a disease of the heart and blood vessels that is characterized by high morbidity and mortality. Among the many types of cardiovascular disease, hypertension is the most common one, affecting about one-third of adults worldwide. At the same time, hypertension can induce a series of metabolic syndromes. Since dysfunction of different metabolic pathways can lead to an increased risk of cerebral ischemia, atherosclerosis, cardiovascular and inflammatory diseases, hypertension is often called the "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 and 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 the quality of life of patients.

[0003] Early detection by monitoring subtle changes in specific biochemical markers in body fluids can significantly reduce the risk of organ damage and improve quality of life. Studies have found 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 the inflammatory pathways involved in the disease 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, surface plasmon resonance (SPR), and surface-enhanced Raman scattering (SERS). However, monitoring them alone can only provide an incomplete picture of inflammation in the progression of hypertension. High IL-6 levels indicate that inflammation persists, but it is difficult to assess the severity of inflammation without correlating it with other markers. Similarly, measuring CRP alone may miss the effects of cytokines that initiate the inflammatory cascade. By monitoring only IL-6 or CRP, it is difficult to measure the full impact of treatment on the inflammatory mechanisms that lead to hypertension. In addition, monitoring only one of the biomarkers fails to capture the dynamic relationship between cytokines and acute phase proteins, resulting in an incomplete assessment of the inflammatory status and its potential role in the progression of hypertension. Therefore, the development of a method to simultaneously detect inflammatory markers is essential to facilitate the clinical management of hypertension and develop more effective targeted treatments. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a 2-UiO-66@AuNPs biosensor probe for linkage monitoring of IL-6 and CRP; establish a multifunctional platform to support drug discovery and companion diagnosis. The present invention pre-designs a dual-target linkage monitoring probe (DTLM probe), that is, a biosensor probe for dual-target linkage monitoring according to the present invention. IL-6 and CRP are key inflammatory markers. The DTLM probe prepared by the present invention using IL-6 and CRP aptamers is used to construct a drug evaluation platform and / or companion diagnosis platform for related diseases such as diabetes, depression, osteoarthritis, coronary heart disease, and hypertension. By changing the expression levels of IL-6 and CRP inflammatory markers during medication or companion diagnosis, it is determined whether the drug or companion diagnosis method produces the corresponding effect. In a preferred embodiment of the present invention, taking the efficacy of Qishen Yiqi Drop Pills in alleviating hypertensive heart damage as an example, the accuracy and effectiveness of the drug evaluation and / or companion diagnosis platform provided by the present invention are verified. 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 the present 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, all inflammation-related diseases and diseases in which the expression levels of IL-6 and CRP change during medication or companion diagnostics fall within the scope of the present invention.

[0005] First, for the directed immobilization strategy, NH 2 -UiO-66, which provides rich NH 2 Subsequently, in NH 2 AuNPs were in situ synthesized on UiO-66 to form NH 2 Next, the thiol-modified IL-6 aptamer and CRP aptamer were immobilized on the NH 2-UiO-66@AuNPs for specific identification of each target. The construction of the DTLM probe was completed by combining the FAM-modified IL-6 signal chain and the TAMRA-modified CRP signal chain. The optimization of the DTLM probe was also studied to achieve the best performance for the linkage monitoring of IL-6 and CRP. In the presence of IL-6 and CRP, the target binds to the corresponding aptamer and replaces the corresponding signal chain, thereby enhancing the fluorescence signal in the solution. The dual signal outputs do not interfere with each other and can detect IL-6 and CRP simultaneously. In addition, according to specific needs, IL-6 or CRP can also be detected separately, so that multiple detections can be completed in a single sample injection. For individual detection, the detection range of IL-6 is 1pg / mL to 1μg / mL, with a limit of detection (LOD) of 0.350pg / mL, and the detection range of CRP is 1pg / mL to 10μg / mL, with an LOD of 0.315pg / mL. For simultaneous detection, the detection ranges of IL-6 and CRP remained unchanged, while the detection limits were 0.355pg / mL and 0.367pg / mL, respectively. Under the optimized parameters, subtle differences in serum IL-6 and CRP levels between healthy rats and hypertensive rats under different treatments were quickly identified. The dual-target linkage monitoring biosensor probe of the present invention successfully explored the anti-inflammatory effect of the traditional Chinese medicine "Qishen Yiqi" and evaluated its effectiveness in reducing hypertensive heart damage. In addition, the dual-target linkage monitoring biosensor probe accurately distinguished the serum IL-6 and CRP levels of healthy donors and hypertensive patients. More importantly, the dual-target linkage monitoring biosensor probe captures slight changes in serum levels after treatment, ensuring that anti-inflammatory treatment can be accurately applied to patients with the greatest need. The dual-target linkage monitoring biosensor probe provides a promising platform for the simultaneous tracking of dual indicators, which is not only used for mechanism verification in drug discovery, but also for companion diagnosis to tailor treatment plans.

[0006] In order to achieve the above-mentioned object, the present invention provides a biosensor probe for dual-target linkage monitoring, with NH 2 -UiO-66@AuNPs nanocomposite material is a base material, and a dual monitoring marker recognition aptamer is connected to the base material. The dual monitoring marker aptamer is used to connect the monitoring marker, and the monitoring marker is IL-6 and CRP; in NH 2 -AuNPs were synthesized in situ on the surface of UiO-66 to form NH 2 -UiO-66@AuNPs nanocomposite was used as the substrate; IL-6 aptamer and CRP aptamer were connected to NH 2-UiO-66@AuNPs; the IL-6 aptamer and IL-6 cDNA complement each other to form a double-stranded structure, and the IL-6 cDNA is connected to a first fluorescent dye; the CRP aptamer and CRP cDNA complement each other to form a double-stranded structure, and the CRP cDNA is connected to a second fluorescent dye.

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

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

[0009]

[0010] Preferably, in any of the above items, the IL-6 cDNA comprises the nucleotide sequence shown in SEQ ID NO:3.

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

[0012]

[0013] Preferably, in any of the above items, the CRP cDNA comprises a nucleotide sequence as shown in SEQ ID NO:4.

[0014] Any of the above items is preferably that 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 not produce signal interference; the maximum emission wavelength of one of the first fluorescent dye and the second fluorescent dye is different from the maximum excitation wavelength of the other dye.

[0015] Preferably, in any of the above items, the first fluorescent dye and the second fluorescent dye are preferably selected from 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 connected to the IL-6 cDNA is preferably 6-FAM; the second fluorescent dye connected to the CRP cDNA is preferably TAMRA.

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

[0018] Step 1: Synthesis of NH 2 -UiO-66;

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

[0020] Step 3: Connect IL-6 aptamer and CRP aptamer to NH 2 -UiO-66@AuNPs, obtained

[0021] NH 2 -UiO-66@AuNPs@Apt IL-6&CRP ;

[0022] Step 4: Connect the IL-6 cDNA and CRP cDNA linked to the fluorescent dye to NH 2 -UiO-66@AuNPs@Apt IL-6&CRP , get NH 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP .

[0023] Preferably, in step 1, NH 2 -The method of UiO-66 is:

[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 hours. The product was then collected by centrifugation and washed with DMF and methanol. Finally, the precipitate was dried to remove the residual organic solvent, and the resulting precipitate was NH 2 -UiO-66.

[0025] In a preferred embodiment of the present invention, NH 2 -The synthesis method of UiO-66 is:

[0026] 120 mg of zirconium chloride, 1.9 g of benzoic acid and 110 mg of aminoterephthalic acid were completely dissolved in 10 mL of DMF using ultrasound. The mixture was then transferred to a polytetrafluoroethylene-lined hydrothermal reactor and reacted at 120 ° C for 24 h. The product was then collected by centrifugation at 12,000 rpm for 15 min, and the product was washed three times with DMF and methanol. Finally, the precipitate was vacuum dried at 65 ° C overnight to remove the residual organic solvent, and the precipitate obtained was NH 2 -UiO-66, and collect the precipitate for later use.

[0027] Preferably, in step 2, in NH 2 -AuNPs were synthesized in situ on the surface of UiO-66 to obtain NH 2 -UiO-66@AuNPs method is:

[0028] The NH obtained in step 1 2 -UiO-66 was dispersed in deionized water, and then HAuCl was slowly added dropwise 4 ·3H 2 O, stir; then add NaBH 4 , stirring, collecting NH 2 -UiO-66@AuNPs product.

[0029] In a preferred embodiment of the present invention, NH 2 -The synthesis method of UiO-66@AuNPs is:

[0030] 10 mg of the obtained NH 2 -UiO-66 was dispersed in 50 mL of deionized water, and then 5 mL of 6 mM HAuCl was slowly added under stirring. 4 ·3H 2 O, continue stirring for 4 h, and then add 1 mL 0.5 M NaBH 4 After stirring for 15 min, NH 2 -UiO-66@AuNPs product was 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, NH 2 -UiO-66@AuNPs@Apt IL-6&CRP The preparation method is:

[0032] The IL-6 aptamer and the CRP aptamer were added to NH 2 -UiO-66@AuNPs suspension, let it stand at room temperature for reaction, then add 10mM PBS buffer containing 2M NaCl to the solution until the final NaCl concentration is 0.2M. Let it stand at room temperature for reaction, then centrifuge the reaction product to obtain NH 2 -UiO-66@AuNPs@Apt IL-6&CRP product.

[0033] Preferably, in any of the above items, 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 therebetween, and most preferably 20 μM.

[0034] Any of the above is preferably, NH 2 The incubation time of -UiO-66@AuNPs and aptamers is 10 to 18 h, more preferably 10, 12, 14, 16, 18 h and the range therebetween; 14 h is more preferably NH 2 -Optimal incubation time of UiO-66@AuNPs and aptamers.

[0035] Preferably, in step 4, NH 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The preparation method is:

[0036] IL-6 cDNA and CRP cDNA were dripped into NH 2 -UiO-66@AuNPs@Apt IL-6&CRP The reaction was carried out in a suspension. 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP product.

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

[0038] 20 μL of 20 μM IL-6 aptamer and 20 μL of 20 μM CRP aptamer were added to 100 μL NH 2 -UiO-66@AuNPs suspension was allowed to react at room temperature for 14 h, and 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. 2 -UiO-66@AuNPs@Apt IL-6&CRP The product was resuspended in 140 μL ultrapure water.

[0039] Subsequently, 70 μL of 10 μM IL-6 cDNA and 70 μL of 10 μM CRP cDNA were added to the above suspension and reacted at 37°C for 1.5 h. 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The product was washed repeatedly, and finally resuspended in 280 μL ultrapure water and stored at 4°C for later use.

[0040] The present invention also provides a biosensor monitoring system constructed by any of the above-mentioned biosensor probes. The monitoring system is a drug evaluation and companion diagnostic biosensor system based on dual-target linkage monitoring probes, which monitors the changes in the expression levels of IL-6 and CRP inflammatory markers during medication or companion diagnostics, thereby determining whether the drug or treatment method has produced the corresponding effect.

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

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

[0043] Preferably, in any of the above items, 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] Any of the above is preferably that when the monitoring system detects IL-6 alone, regarding FF 0 The linear regression equation with 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 a sample, preferably, the concentration of IL-6 is in the range of 1 pg / mL to 1 μg / mL.

[0045] Any of the above is preferably that when the monitoring system detects CRP alone, about FF 0 The linear regression equation with 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 a sample, preferably, the concentration of CRP ranges from 1 pg / mL to 10 μg / mL.

[0046] Any of the above is preferably that when the monitoring system detects IL-6 and CRP simultaneously, 0 The linear regression equation of 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; about FF 0The linear regression equation with 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 concentration range of IL-6 is 1 pg / mL to 1 μg / mL, and the concentration range of CRP is 1 pg / mL to 10 μg / mL.

[0047] F and F 0 The fluorescence intensity in the presence and absence of IL-6 and CRP is shown.

[0048] The present invention also provides the use of any of the above-mentioned biosensor monitoring systems in constructing 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. By utilizing the biosensor probe for dual-target linkage monitoring described in the present invention, the system or platform described in the present invention was used to verify the therapeutic effectiveness of Qishen Yiqi Drops on clinical hypertension patients, demonstrating the application prospects of the present invention in drug evaluation and companion diagnostic platforms for inflammatory-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 NH observed by TEM and SEM in the preferred embodiment 1 of the present invention 2 -UiO-66 and NH 2 -UiO-66@AuNPs morphology.

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

[0052] Figure 3 This is the optimization result of 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 dual-target linkage monitoring of hypertension described in the preferred embodiment 4 of the present invention.

[0054] Figure 5 This is the analysis result of the rat serum sample in the preferred embodiment 5 of the present invention.

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

[0056] Figure 7 This is the analysis result of the biosensor probe for dual-target linkage monitoring of hypertension in clinical serum samples as described in the preferred embodiment 6 of the present invention.

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

[0058] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0059] Example 1

[0060] In order to establish a stable sensing platform, the NH 2 -AuNPs were synthesized in situ on the surface of UiO-66 to form NH 2 -UiO-66@AuNPs nanocomposite was used as the substrate material. The synthesized NH 2 -UiO-66 and NH 2 -UiO-66@AuNPs were morphologically observed.

[0061] NH 2 -The synthesis method of UiO-66 is:

[0062] 120 mg of zirconium chloride, 1.9 g of benzoic acid and 110 mg of aminoterephthalic acid were completely dissolved in 10 mL of DMF using ultrasound. The mixture was then transferred to a polytetrafluoroethylene-lined hydrothermal reactor and reacted at 120 ° C for 24 h. The product was then collected by centrifugation at 12,000 rpm for 15 min, and the product was washed three times with DMF and methanol. Finally, the precipitate was vacuum dried at 65 ° C overnight to remove the residual organic solvent, and the precipitate obtained was NH 2 -UiO-66, and collect the precipitate for later use.

[0063] NH 2 -The synthesis method of UiO-66@AuNPs is:

[0064] 10 mg of the obtained NH 2 -UiO-66 was dispersed in 50 mL of deionized water, and then 5 mL of 6 mM HAuCl was slowly added under stirring. 4 ·3H 2 O, continue stirring for 4 h, and then add 1 mL 0.5 M NaBH 4 After stirring for 15 min, NH2 -UiO-66@AuNPs product was washed three times with deionized water by centrifugation and then resuspended in 4 mL of deionized water for later use.

[0065] Figure 1 Shown are TEM and SEM observations of NH 2 -UiO-66 and NH 2 -UiO-66@AuNPs morphology. Where A is NH 2 -TEM image of UiO-66, B is NH 2 -SEM image of UiO-66, C is NH 2 -TEM image of UiO-66@AuNPs, D is NH 2 -SEM image of UiO-66@AuNPs. Figure 1 NH 2 -UiO-66 has a uniform morphology and a typical octahedral structure with a diameter of about 200nm. 2 -UiO-66 compared to NH 2 NH-UiO-66@AuNPs can be clearly identified in the TEM and SEM images. 2 A large number of AuNPs are evenly distributed on the surface of -UiO-66. It can be observed that the AuNPs are spherical with uniform shape and size, with a diameter of about 10nm. A large number of in-situ synthesized AuNPs provide abundant binding sites for subsequent DNA connection. 2 -UiO-66 and NH 2 -UiO-66@AuNPs TEM and SEM characterization results showed that NH 2 -UiO-66@AuNPs preparation was successful.

[0066] Example 2

[0067] Example 2 verified the feasibility of the sensing strategy of the present invention by fluorescence detection. In order to design a target-responsive biosensor probe, the aptamers of IL-6 and CRP were connected to NH 2 -UiO-66@AuNPs, and then add 6-FAM-labeled IL-6 cDNA and TAMRA-labeled CRP cDNA. The partial complementarity between the aptamer and the cDNA will form a double-stranded structure. When IL-6 and CRP are present, due to the strong interaction between the target and the aptamer, the target will compete with the cDNA and displace the fluorescently labeled cDNA, thereby generating a fluorescent signal.

[0068] The construction method of IL-6 and CRP dual-target biosensor probe 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 NH 2 -UiO-66@AuNPs suspension was allowed to react at room temperature for 14 h, and 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. 2 -UiO-66@AuNPs@Apt IL-6&CRP The product was resuspended in 140 μL ultrapure water.

[0070] Subsequently, 70 μL of 10 μM IL-6 cDNA and 70 μL of 10 μM CRP cDNA were added to the above suspension and reacted at 37°C for 1.5 h. 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The product was washed repeatedly, and finally resuspended in 280 μL 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, and the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 contains a thiol modification, and the structural formula of the modification group is:

[0072]

[0073] In Example 2, the IL-6 cDNA is 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, and the 5' end of the nucleotide sequence shown in SEQ ID NO: 2 contains a thiol modification, and the structural formula of the modification group is:

[0075]

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

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

[0078]

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

[0080] In Example 2, the measuring method comprises the following steps:

[0081] IL-6 measurement: 30 μL of IL-6 of different concentrations were mixed with 30 μL of NH 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The suspension was mixed and incubated for 1.5 h at 37° C. 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 of different concentrations was mixed with 30 μL NH 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The suspension was mixed and incubated at 37°C for 1.5 h. Finally, the mixture was centrifuged and the fluorescence signal of TAMRA in the supernatant was detected by an ELISA reader under 552 nm excitation.

[0083] Measurement of IL-6 and CRP: 30 μL of IL-6 and CRP mixtures of different concentrations were mixed with 30 μL NH 2 -UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP The suspension was mixed and incubated at 37°C for 1.5 h, the mixture was centrifuged, and the fluorescence signals of 6-FAM and TAMRA in the supernatant were detected using an ELISA reader under excitation at 494 nm and 552 nm, respectively.

[0084] Figure 2 These are the feasibility verification results of the biosensor probe for dual-target linkage monitoring of hypertension in the preferred embodiment 2 of the present invention, wherein A is the FAM fluorescence measurement in the presence (red line) and absence (blue line) of IL-6; B is the TAMRA fluorescence measurement in the presence (red line) and absence (blue line) of CRP; C is the FAM and TAMRA fluorescence measurement in the presence (red line) and absence (blue line) of IL-6 and CRP.

[0085] Figure 2The results shown prove the feasibility of the biosensor probe for dual-target linkage monitoring of hypertension obtained in Example 2 to detect 1ng / mL IL-6 and CRP separately and simultaneously. As shown in A, fluorescence detection shows that when IL-6 is present, the characteristic absorption peak of 6-FAM at 520nm is much stronger than when IL-6 is not present, proving the feasibility of the biosensor probe to detect IL-6; as shown in B, when CRP is present, TAMRA can detect a strong characteristic absorption peak at 580nm, while when CRP is not present, only a weaker background absorption peak is detected, proving the feasibility of the biosensor probe to detect CRP. In addition, Example 2 also studies the fluorescence response of the biosensor probe when IL-6 and CRP are present or not at the same time. As shown in C, it can be seen that when IL-6 and CRP are present at the same time, the fluorescence signals at 520nm and 580nm are much higher than when IL-6 and CRP are not present, indicating that the biosensor probe can effectively detect IL-6 and CRP at the same time.

[0086] Example 3

[0087] In order to obtain higher sensing performance, the reaction conditions for IL-6 and CRP detection were optimized in Example 3. Considering that the concentrations of IL-6 and CRP aptamers are the main factors controlling the reaction efficiency, Example 3 first optimized them. Figure 3 The results of optimization of aptamer incubation conditions are shown, where A is the optimization of aptamer concentration; B is the concentration of incubation time.

[0088] like Figure 3 -A, when detecting 1 ng / mL IL-6 and CRP, when the aptamer concentrations of IL-6 and CRP increased from 1 μM to 20 μM, FF was observed in Example 3 0 (F and F 0 The fluorescence signal gradually increases, but as the concentration of the aptamer increases, the fluorescence signal decreases. This may be because an excessively high aptamer concentration will lead to increased nonspecific adsorption and steric hindrance, reducing the recognition between the target and the aptamer, resulting in a decrease in fluorescence intensity. Therefore, the present invention preferably uses 20 μM as the optimal concentration of the aptamer.

[0089] Subsequently, the NH 2 -Incubation time of UiO-66@AuNPs and aptamers. Figure 3 -B, when detecting 1ng / mL IL-6 and CRP, increasing the incubation time can significantly increase the fluorescence signal intensity, and the highest signal intensity is obtained at 14h. However, after 14h, the extension of the incubation time may cause some aptamers to detach from the NH 2-UiO-66@AuNPs, resulting in a decrease in fluorescence signal. Therefore, 14h was selected as the NH 2 -Optimal incubation time of UiO-66@AuNPs and aptamers.

[0090] Example 4

[0091] In order to evaluate the analytical performance of the biosensor probe for dual-target linkage monitoring of hypertension provided by the present 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 Shown are (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 biosensor probe when detecting IL-6 and CRP separately was studied. For IL-6 detection analysis, Figure 4 As shown in Figure 1, as the concentration of IL-6 increases, the binding of IL-6 to the aptamer also increases, so that more 6-FAM-labeled cDNA is displaced, resulting in a gradual increase in the fluorescence intensity at 520 nm. 0 There is an obvious linear relationship between the logarithm of IL-6 concentration (1pg / mL-1μg / mL). Figure 4 - About FF in B 0 The linear regression equation is y = 188.975 + 409.075 × lg C IL-6 (R 2 =0.995). For CRP detection analysis, Figure 4 -C and Figure 4 -D, the fluorescence intensity of TAMRA at 580 nm gradually increased with the increase of CRP concentration, FF 0 The linear regression equation with the logarithm of CRP concentration in the range of 1pg / mL-10μg / mL is y=17.189+26.078×lg C CRP (R 2 =0.992). Using 3S D The limits of detection (LOD) of IL-6 and CRP calculated by the standard were as low as 0.350 pg / mL and 0.315 pg / mL (S DThe analytical performance of the biosensor probe for simultaneous detection of IL-6 and CRP was then investigated. Figure 4 -E and Figure 4 -F shows that compared with the single detection, the detection range of IL-6 and CRP in the simultaneous detection is consistent with that in the single detection, and the LOD does not change much. The linear regression equation of IL-6 in the range of 1pg / mL-1μg / 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 of CRP in the range of 1 pg / mL-10 μg / mL became y=17.164+26.065×lg C CRP (R 2 =0.993), and the LOD was 0.367 pg / mL, indicating that there was no obvious cross-interference when using different aptamers and cDNA labeled with different fluorescence during the detection and analysis process. It also showed that the constructed biosensor probe has the advantages of high sensitivity, high specificity, good cost-effectiveness, and simultaneous analysis.

[0093] Example 5

[0094] In order to explore the applicability of the obtained biosensor probe and the efficacy and mechanism of Qishen Yiqi Dropping Pills in alleviating hypertensive heart damage, rat serum samples were studied in Example 5.

[0095] Figure 5 Figure 2 shows the analysis of rat serum samples: heat map of fluorescence signals of (A) IL-6 and (D) CRP in serum of each group measured by biosensor probe method; heat map of OD values ​​of (B) IL-6 and (E) CRP in serum of each group measured by ELISA method; expression levels of (C) IL-6 and (F) CRP in serum of each group measured by biosensor probe method and ELISA method; each sample was tested three times.

[0096] The obtained biosensor probe and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression levels of IL-6 and CRP in the serum samples of each group of rats. Figure 5 -A and Figure 5 -B are the signal response results of IL-6 measured by two methods. It can be seen that the fluorescence response intensity and OD value response size of IL-6 are Mod group>QSYQ-L group (low dose group)>QSYQ-H group (high dose group)>Con group, which shows that the biosensor method and ELISA method have significant differences in the signal when detecting IL-6 in the serum of different groups of rats, and both can be used to distinguish different contents of inflammatory factor IL-6. In addition, from Figure 5-C, compared with the Con group, the Mod group had a significantly higher serum IL-6 expression level. Compared with the Mod group, the Qishen Yiqi Dropping Pills could significantly reduce the IL-6 concentration, and the high-dose Qishen Yiqi Dropping Pills group had a better effect on reducing IL-6 than the low-dose group. Similarly, in the CRP test results, Figure 5 -D to Figure 5 -F, Example 5 also shows that the fluorescence response, OD value and concentration of CRP in serum of Mod group are higher than those of Con group, and Qishen Yiqi Drop Pills can also significantly reduce the fluorescence response, OD value and concentration of CRP in serum, and the effect of QSYQ-H group is better than that of QSYQ-L group. The above results show that the biosensor probe constructed by the present invention has good applicability, and Qishen Yiqi Drop Pills can alleviate hypertensive heart damage by regulating inflammatory response, while showing dosage compliance.

[0097] Example 5 also explored the consistency between the biosensor probe constructed by the present invention and the ELISA test. Figure 6 The consistency between the biosensor probe and ELISA in detecting (A) IL-6 and (B) CRP in rat serum.

[0098] like Figure 6 -A and Figure 6 As shown in Figure 2B, the two methods were highly consistent in the detection of IL-6 and CRP, further demonstrating that the NH 2 -UiO-66@AuNPs biosensor probes are feasible and reliable for sensitive detection of IL-6 and CRP in complex samples. In addition, compared with the traditional ELISA method, the biosensor probe method greatly shortens the detection time and has lower detection costs.

[0099] Example 6

[0100] In order to verify the therapeutic effectiveness of Qishen Yiqi Drop Pills for clinical hypertension patients, Example 6 selected 20 healthy donors (Healthy), 20 conventional hypertension patients (Con) and 20 conventional combined Qishen Yiqi Drop Pills for hypertension patients (QSYQ), a total of 60 clinical patients to verify the clinical effectiveness of Qishen Yiqi Drop Pills. The biosensor probe and ELISA method were used to detect the levels of IL-6 and CRP in serum samples of healthy donors (Healthy), conventional hypertension patients before treatment (B-Con) and after treatment (A-Con), and conventional combined Qishen Yiqi Drop Pills for hypertension patients before treatment (B-QSYQ) and after treatment (A-QSYQ).

[0101] Figure 7Analysis of clinical serum samples: heat map of the fluorescence signals of (A) IL-6 and (D) CRP in serum of each group measured by the biosensor probe method; heat map of the OD values ​​of (B) IL-6 and (E) CRP in serum of each group measured by the ELISA method; expression levels of (C) IL-6 and (F) CRP in serum of each group measured by the 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 are significantly different in the five groups of clinical samples, indicating that the biosensor method and ELISA method are sensitive to IL-6 and can be used for the detection of IL-6 in clinical samples. Figure 7 -C shows that the IL-6 level in the serum samples of the Healthy group was the lowest, and the highest in the B-Con and B-QSYQ groups. After conventional treatment and conventional combined treatment with Qishen Yiqi Dropping Pills, the IL-6 level in the serum decreased, and the IL-6 level in the A-QSYQ group decreased more than that in the A-Con group, indicating that Qishen Yiqi Dropping Pills can reduce the IL-6 level in patients with hypertension. Similarly, Figure 7 -D to Figure 7 -F, the fluorescence response and OD value of CRP in serum of different groups are also different. The CRP concentration of B-Con and B-QSYQ groups is the highest level, the Healthy group is the lowest level, and the CRP level of A-QSYQ group is more decreased than that of A-Con group, which is similar to the IL-6 detection result, indicating that Qishen Yiqi Drop Pills have the effect of regulating the inflammatory factor CRP. The above results show that the biosensor probe constructed by the present invention also has good applicability in clinical samples, and once again verifies that Qishen Yiqi Drop Pills can reduce inflammatory response.

[0103] In addition, Example 6 also explored the consistency between the biosensor probe constructed by the present invention and the ELISA method. Figure 8 The consistency of the biosensor probe and ELISA in detecting (A) IL-6 and (B) CRP in clinical serum is shown. Figure 8 -A and Figure 8 -B shows that the two methods are highly consistent, further proving the feasibility and reliability of the obtained biosensor probe for sensitive detection of IL-6 and CRP in clinical serum samples. The sensor probe and monitoring system of the present invention can be used for the evaluation of hypertension drugs and / or the application of companion diagnostic platforms. The drugs are not limited to Qishen Yiqi Drop Pills, and conventional Western medicine hypertension treatments, such as CCBs, ACEI, and ARB drugs, are all applicable to the present invention.

[0104] Through the application of hypertension drug evaluation and / or companion diagnostic platform, the accuracy and effectiveness of the dual-target linkage monitoring biosensor probe and monitoring system provided by the present invention in drug evaluation and companion diagnostic applications are verified. The present invention is applicable to drug evaluation and companion diagnostic platform applications for inflammation-related diseases including, but not limited to, diabetes, depression, osteoarthritis, coronary heart disease, and hypertension. All inflammation-related diseases, diseases in which the expression levels of IL-6 and CRP change during medication or companion diagnostics, fall within the scope of the present invention.

[0105] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

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

2. The biosensor probe according to claim 1, characterized in that The IL-6 aptamer is a nucleotide sequence as shown in SEQ ID NO: 1, and the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 contains a thiol modification, and the structural formula of the modification group is:

3. The biosensor probe according to claim 2, characterized in that The IL-6 cDNA comprises the nucleotide sequence shown in SEQ ID NO:

3.

4. The biosensor probe according to claim 1, characterized in that The CRP aptamer is a nucleotide sequence as shown in SEQ ID NO: 2, and the 5' end of the nucleotide sequence shown in SEQ ID NO: 2 contains a thiol modification, and the structural formula of the modification group is:

5. The biosensor probe according to claim 1, characterized in that The CRP cDNA comprises the nucleotide sequence shown in SEQ ID NO:

4.

6. The method for preparing the biosensor probe according to any one of claims 1 to 5, comprising the following steps: Step 1: Synthesis of NH2-UiO-66; Step 2: In situ synthesis of AuNPs on the surface of NH2-UiO-66 to obtain NH2-UiO-66@AuNPs; Step 3: Connect the IL-6 aptamer and CRP aptamer to NH2-UiO-66@AuNPs to obtain NH2-UiO-66@AuNPs@Apt IL-6&CRP ; Step 4: Connect IL-6 cDNA and CRP cDNA linked to fluorescent dye to NH2-UiO-66@AuNPs@Apt IL-6&CRP , and obtain NH2-UiO-66@AuNPs@Apt IL-6&CRP @cDNA IL-6&CRP , that is, a biosensor probe for dual-target linkage monitoring.

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

8. The monitoring system according to claim 7, characterized in that: ① When the monitoring system detects IL-6 alone, the detection limit LOD of IL-6 is 0.350 pg / mL; ② When the monitoring system detects CRP alone, the detection limit LOD of CRP is 0.315 pg / mL; ③ 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.

9. The monitoring system according to claim 7, characterized in that: ① 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; ② When the monitoring system detects CRP alone, the linear regression equation for 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; ③ When the monitoring system detects IL-6 and CRP simultaneously, 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 the logarithm of F-F0 and 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.

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

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