Tacrolimus colorimetric sensor based on aptamer nano-enzyme and application of tacrolimus colorimetric sensor

Abstract

The invention provides a tacrolimus colorimetric sensor based on aptamer nano enzyme and application thereof, the tacrolimus colorimetric sensor is used for rapid, accurate, visual and portable detection of tacrolimus drug concentration, and the sensor shows excellent magnetic enrichment amplification effect and Fe3O4 (at) CeO2 bimetallic nano POD enzyme catalytic efficiency. The aptamer nano-enzyme colorimetric sensor disclosed by the invention has the advantages of simplicity and convenience in operation, high response speed and high cost effectiveness. No expensive reading equipment is needed, and macroscopic qualitative detection can be realized through color change. The method has good accuracy and anti-interference performance in TAC detection of saliva and blood samples.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensing, and relates to a colorimetric detection method for immunosuppressants, specifically to a colorimetric sensor for detecting the concentration of tacrolimus (TAC) drug, especially a colorimetric sensor based on aptamer nanozyme, which is applicable to the monitoring of anti-rejection drugs after organ transplantation. Background Art

[0002] The success rate of organ transplantation surgery highly depends on postoperative management, especially the precise use of immunosuppressive drugs. Tacrolimus (TAC), as a first-line immunosuppressant, plays a crucial role in the applications after heart, liver, and kidney transplantation. However, there is currently a lack of a portable bedside monitoring method for the concentration of tacrolimus drug. The existing immunosuppressant detection methods mainly include two schemes: immunoassay and biochemical technology. Among them, immunoassay has problems such as high cost, complex operation, and cross-immune reaction, while biochemical technology methods such as liquid mass spectrometry can effectively reflect the blood drug concentration of immunosuppressants, but rely on expensive foreign instruments and the operation of professional technical personnel, and are difficult to be applicable to medical institutions below the secondary level and patients for home use. At the present stage, there is an urgent need to develop new portable immunosuppressant monitoring technologies.

[0003] Nanozyme sensors have received extensive attention due to their simplicity, cost-effectiveness, visualization, and potential for bedside detection applications, especially in the field of small molecule monitoring. Nanozymes, as a kind of nanomaterials with mimetic enzyme activity, stand out for their low cost, easy preparation, and high stability. In particular, nanozymes with peroxidase mimetic activity can catalyze the oxidation of chromogenic substrates, such as 3,3',5,5'-tetramethylbenzidine (TMB), in the presence of hydrogen peroxide (H2O2), and this property makes them show great potential in the development of colorimetric biosensors. Therefore, developing nanozymes with high catalytic activity and high substrate selectivity is still the main challenge currently faced, which limits their wide application in the field of medical monitoring.

[0004] Summary of the Invention: The purpose of the present invention is to provide a tacrolimus colorimetric sensor based on aptamer nanozyme for rapid, accurate, visual, and portable detection of tacrolimus drug concentration. This sensor exhibits excellent magnetic enrichment amplification effect and Fe3O4@CeO2 bimetallic nano POD enzyme catalytic efficiency.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A tacrolimus colorimetric sensor based on aptamer nanozyme, having an Apt-CS-Fe3O4@CeO2@PCPyYSNs structure, which includes an Apt-CS shell with excellent magnetic enrichment amplification effect, a yolk structure YSN with a more suitable catalytic microenvironment, and a Fe3O4@CeO2 bimetallic nano-core.

[0007] Another object of the present invention is to provide a preparation method of a tacrolimus colorimetric sensor based on aptamer nanozyme, the method comprising the following steps:

[0008] (1) Synthesis of Fe3O4@CeO2: First, a Fe3O4@CeO2 core-shell structure is prepared by a ligand exchange method, and the reaction solution is placed in a water bath at 50-90 °C for condensation reflux for 2-10 h, which involves mixing Fe3O4 nanoparticles and CeO2 nanoparticles under this condition to form a Fe3O4@CeO2 bimetallic nanostructure.

[0009] (2) Modification step of Fe3O4@CeO2 bimetallic nanozyme: Polyethyleneimine-polyethylene glycol (PEI-PEG) is introduced onto the surface of Fe3O4@CeO2 to form a Fe3O4@CeO2@PEI-PEG complex. Then, a polydopamine (PDA) layer is grown on the surface of Fe3O4@CeO2@PEI-PEG by chemical polymerization to obtain Fe3O4@CeO2@PDA nanoparticles, and this step is carried out at a temperature of 25-60 °C. Further, a polypyrrole (PCPy) layer is grown on the surface of Fe3O4@CeO2@PDA by chemical oxidative polymerization to form Fe3O4@CeO2@PDA@PCPy nanoparticles, and the polymerization reaction is carried out at 50-90 °C.

[0010] (3) Loading of aptamer: By carboxyl activation and aptamer conjugation technology, the aptamer and complementary strand (CS) are fixed on the surface of Fe3O4@CeO2@PCPy nanoparticles to form an Apt-CS-Fe3O4@CeO2@PCPyYSNs sensor, and this step is carried out at 40-70 °C.

[0011] The third object of the present invention is to provide a method for detecting the concentration of tacrolimus (TAC) drug using the above colorimetric sensor, the method comprising the following steps:

[0012] 1. The obtained aptamer-functionalized Fe3O4@CeO2 bimetallic nanozyme was added to multiple buffer samples containing different concentrations of tacrolimus for incubation, and then H2O2 and the chromogenic substrate 3,3',5,5'-tetramethylbenzidine were added for reaction. Next, the aptamer-functionalized Fe3O4@CeO2 bimetallic nanozyme was mixed with the processed samples in different concentration ranges and reacted under appropriate conditions. The nanozyme catalyzed the oxidation of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), and in the presence of H2O2, a color change occurred. Subsequently, a microplate reader was used to detect the optical absorption value at 652 nm of the reaction system. Based on the change in the optical absorption value, the drug concentration of tacrolimus could be quantitatively analyzed. By establishing a standard curve, the optical absorption value of the sample was converted into the concentration value of tacrolimus.

[0013] 2. According to the detected linear relationship between the tacrolimus concentration and the absorbance value A obtained above and the absorbance value of the sample to be tested containing tacrolimus, the concentration of tacrolimus in the sample to be tested was obtained.

[0014] For the qualitative detection of tacrolimus drug concentration based on the above blue change, preferably, the aptamer-functionalized Fe3O4@CeO2 bimetallic nanozyme was first mixed with the sample to ensure that the nanozyme concentration was 10 μg / mL. Then, at 37 °C, the reaction system was mixed with 0.1 mol / L H2O2 and 0.5 mol / L TMB chromogenic substrate and reacted for 30 minutes. The color change of the reaction system was observed at a wavelength of 650 nm. If blue appeared within 30 minutes, the presence of tacrolimus was qualitatively judged; if there was no color change, it indicated that the tacrolimus concentration was below the detection limit.

[0015] For the above colorimetric detection method of antibiotics based on the regulation of the catalytic activity of nanozymes by nucleic acid aptamers, preferably, the immunosuppressant is tacrolimus, the nucleic acid aptamer has the nucleotide sequence shown in SEQ 1, and the detected linear regression equation of the ratio of tacrolimus concentration to absorbance (A - A0) / A0 is:

[0016] y = 0.2322x - 0.0652 (1)

[0017] R 2 = 0.9961

[0018] In formula (1), A represents the absorbance value, A0 represents the absorbance value of the blank group, X is the tacrolimus concentration, and the unit corresponding to the concentration value is pM. The correlation coefficient R of formula (1) 2 = 0.995, the detected linear range of tacrolimus is 0.01 nM to 100 nM, and the detection limit is 2.3 pM.

[0019] Preferably, the sequence of SEQ1 is GCUGUGUGAC UCCUGCAAAC UACUGGUGUU CCUUGCCCUG U.

[0020] Preferably, the concentration of H2O2 during the incubation process is 0.1 - 2.5 mol / L, and the concentration of 3,3',5,5'-tetramethylbenzidine (TMB) is 0.5 - 2.5 mol / L.

[0021] Preferably, for the qualitative detection of tacrolimus drug concentration based on the blue change, the concentration of the nanozyme is 10 μg / mL, the color reaction time is 30 min - 3 h, and the temperature is 15 - 25 °C.

[0022] The present invention has the following beneficial effects. The aptamer nanozyme colorimetric sensor invented has the following advantages:

[0023] Benefiting from the high POD enzyme catalytic activity of the Fe3O4 / CeO2 double-metal core and the more active sites provided by the yolk structure, the detection of TAC in the examples has the advantages of simple operation and fast response speed; at the same time, the high stability and easy availability of the nanozyme compared with the bioenzyme make it have the advantages of high cost-effectiveness, etc.

[0024] The high reactivity provided by the above-mentioned nanostructure with high catalytic performance enables the examples to achieve naked-eye visible qualitative detection through the color change of oxTMB without expensive readout equipment.

[0025] The specific binding of the Apt / CS aptamer to TAC enables the examples to have good accuracy and anti-interference performance in the detection of TAC in saliva and blood samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the characterization results of the nanomaterials.

[0027] Figure 2 Shows the standard curve for monitoring the concentration of tacrolimus by the aptamer Fe3O4@CeO2 double-metal nanozyme colorimetric sensor.

[0028] Figure 3 Shows the day-to-day stability of the particle size of Fe3O4@CeO2@PPyYSNs.

[0029] Figure 4 Shows the Deta potential stability of Fe3O4@CeO2@PPyYSNs.

[0030] Figure 5 Shows the mechanism of Apt1-CS inhibiting the catalytic activity of Fe3O4@CeO2@PPyYSNs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The following examples are intended to further illustrate the implementation modes of the present invention, but should not be construed as limiting the present invention.

[0032] Example 1: Preparation of the aptamer Fe3O4@CeO2 bimetallic sensor.

[0033] (1) Synthesis of Fe3O4@CeO2

[0034] Fe3O4@OA was obtained through commercial channels. Under the hydrothermal conditions of a mixed solvent, the OA molecules on the surface of Fe3O4@OA were replaced by the CA molecules on the surface of ultrasmall CeO2@CA NPs, and Fe3O4@CeO2 was obtained after being modified with PEI-PEG. The CeO2@CA colloidal solution was dissolved in acetone, and Fe3O4@OA was dissolved in n-hexane. Then the two were mixed and mechanically stirred thoroughly. The reaction solution was placed in a water bath at 62 °C for condensation reflux for 5 h, and stirred continuously during this process. Fe3O4@CA@CeO2 NPs were obtained by centrifugation and magnetic separation, and then resuspended in pure water and thoroughly dialyzed. The Fe3O4@CA@CeO2 NPs solution was filtered through a Millipore 220 nm filter membrane and stored at room temperature for later use.

[0035] 288 mg of polyethyleneimine-polyethylene glycol (PEI-PEG) was dissolved in 200 ml of deionized water and ultrasonically dispersed, and then this mixture was added to a 500 mL three-necked flask. The Fe3O4@CeO2@CA aqueous solution (50 mg Fe) was diluted to 150 mL with deionized water respectively. After the three-necked flask was fixed, mechanical stirring (1200 rpm / min) was maintained, and ultrasonic treatment was carried out at 80 Hz at the same time. The diluted aqueous solution of nanoparticles was slowly dropped into the PEI-PEG solution. After the dropping was completed, ultrasonic treatment was continued. After stirring for 20 min, the ultrasonic was turned off, and after 1.5 h, the stirring was turned off. The obtained Fe3O4@CA@CeO2@PEI-PEG (Fe3O4@CeO2) was concentrated to 15 mL through a Millipore (MWCO: 50 kDa) ultrafiltration membrane, and after measuring the concentration, it was placed in a refrigerator at 4 °C for later use.

[0036] (2) Synthesis of Fe3O4@CeO2@PDA

[0037] To prepare Fe3O4@CeO2@PDA nanoparticles. 100 mg of Fe3O4@CeO2 was ultrasonically dispersed in 100 ml of Tris-HCl buffer (20 mmol, pH 8.5), and then 100 mg of ascorbic acid was added. 100 mg of ascorbic acid and 150 mg of DA·HCl were added, and then the mixed solution was shaken for 12 hours. Then the mixed solution was shaken for 12 hours. After washing several times with ultrapure water, the Fe3O4@CeO2@PDA nanoparticles were dried under vacuum at 45 °C overnight. Dried under vacuum at 45 °C overnight.

[0038] (3) Synthesis of Fe3O4@CeO2@PDA@PCPy nanoparticles and Fe3O4@CeO2@PCPyYSN

[0039] Fe3O4@CeO2@PDA@PCPy nanoparticles were synthesized by chemical oxidative polymerization of pyrrole-2-carboxylic acid. 100 mg of Fe3O4@CeO2@PDA was dispersed in 50 ml of aqueous solution together with 0.3 g of pyrrole-2-carboxylic acid. Then, 12 mg of sodium dodecyl sulfate, 150 mg of ammonium persulfate, and 200 mg of FeCl3·6H2O were successively added to the suspension. The temperature was raised to 50 °C and polymerization was carried out for 8 h. After repeated washing, finally Fe3O4@CeO2@PDA@PCPy was obtained. Then the synthesized Fe3O4@CeO2@PDA@PCPy was dispersed in 100 mL of H2O, and 50 mg of NaOH was added. After stirring at 55 °C for 4 hours, the mixture was separated and washed with water until the pH reached neutral. Finally, after 12 hours of freeze-drying, Fe3O4@CeO2@PCPyYSN was obtained.

[0040] (4) Preparation of Apt-CS-Fe3O4@CeO2@PCPyYSNs

[0041] Normally, first, 0.5 mg of the synthesized Fe3O4@CeO2@PCPyYSNs was ultrasonically dispersed in 0.3 ml of PBS solution (5 mmol, pH = 7.4), then 50 μL of EDC (200 mg / ml) and NHS (100 mg / ml) solutions were added to the above mixture, and shaken for 30 min to activate the carboxyl group. After shaking at 37 °C for 4 hours, Apt-NH2 and CS-NH2 solutions were added. The nanozyme was magnetically separated to remove the unconjugated Apt and its CS, and resuspended in STE buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA), and incubated for 2 hours to completely load CS with Apt. Finally, the synthesized Apt / CS-Fe3O4@CeO2@PCPyYSNs were magnetically separated, washed twice, and after being characterized by techniques such as TEM without error (Figure 1 ) and redispersed in PBS buffer for subsequent experiments.

[0042] Example 2: Detection performance test of the aptasensor

[0043] Prepare tacrolimus standard solutions with a concentration range from 0 pM to 70 pM, place the standard solutions in a cuvette, and then measure the absorbance (A) of each standard solution and the unknown sample using a UV-visible spectrophotometer at a specific wavelength. Select a reference wavelength of 652 nm to ensure that the absorbance of the blank control (PBS solution) is close to zero at this wavelength; for each standard solution sample, calculate the ratio of the change in absorbance to the initial absorbance, i.e., (A - A0) / A0, where A0 is the absorbance of the blank control; then plot a graph of (A - A0) / A0 versus the concentration C (pM) of the standard solution; through linear regression analysis, determine the equation of the standard curve, i.e., y = 0.2322x - 0.0652, where y represents (A - A0) / A0 and x represents the concentration C (pM); use the standard curve equation ( Figure 2 ) to quantitatively analyze the concentration of the unknown sample.

[0044] Example 3: Stability of the aptasensor

[0045] (1) Test the change in Zeta potential of the Apt / CS-Fe3O4@CeO2@PCPy composite material at different time points (0, 1, 3, 5, 7 days) to evaluate the dispersion stability of the material in the aqueous phase. By measuring the Zeta potential at different time points, the change in the surface charge characteristics of the material over time can be observed ( Figure 3 ), thereby inferring its stability and possible aggregation tendency. In the experiment, the Zeta potential of the material was between -15 mV and -20 mV, indicating that the composite material maintained good stability in the aqueous phase within the tested time range.

[0046] (2) Test the change in hydrodynamic size of the Apt / CS-Fe3O4@CeO2@PCPy composite material at different time points (0, 1, 3, 5, 7 days) to evaluate the size stability of the material in the aqueous phase. By measuring the hydrodynamic size at different time points, the change in the size of the material over time can be observed, thereby inferring its stability and possible aggregation or dissolution behavior in the solution ( Figure 4 ). The experimental results showed that within the tested time range, the hydrodynamic size of the composite material remained between 70 - 80 nanometers, indicating that its size was relatively stable.

[0047] Example 4: Monitoring mechanism of the aptasensor

[0048] Prepare four different modified Fe3O4@CeO2@PCPy nanoparticle samples: unmodified Fe3O4@CeO2@PCPy, CS-modified CS-Fe3O4@CeO2@PCPy, Apt-modified Apt-Fe3O4@CeO2@PCPy, and Apt / CS dual-modified Apt / CS-Fe3O4@CeO2@PCPy; secondly, dissolve each sample in an appropriate solvent to ensure consistent concentration for easy comparison; next, use a UV-visible spectrophotometer to measure the absorbance of each sample in the wavelength range of 500 - 750 nm; record the absorbance data of each sample and plot a graph of absorbance versus wavelength ( Figure 5 ). The experimental results show that the modification of Apt / CS has a significant reducing effect on the color development ability of Fe / Ce nanoparticles (NPs), which may be related to the influence of the Apt / CS modification layer on the absorption or scattering characteristics of light. By comparing the absorbance curves of different materials, the influence of different modifications on the optical properties of the materials can be analyzed.

Claims

1. A tacrolimus colorimetric sensor based on aptamer nanozyme, characterized in that: It consists of an Apt-CS shell, an egg-yolk structured YSN, and a Fe3O4@CeO2 bimetallic nanocore.

2. The method for preparing the tacrolimus colorimetric sensor according to claim 1, characterized in that: The following steps are involved: Preparation of Fe3O4@CeO2 bimetallic nanozymes; The Fe3O4@CeO2 surface was modified by polyethyleneimine-polyethylene glycol (PEI-PEG) to form a Fe3O4@CeO2@PEI-PEG complex; A polydopamine (PDA) layer was grown on the surface of Fe3O4@CeO2@PEI-PEG to obtain Fe3O4@CeO2@PDA nanoparticles; A polypyrrole (PCPy) layer was grown on the surface of Fe3O4@CeO2@PDA to form Fe3O4@CeO2@PDA@PCPy nanoparticles; Through carboxyl activation and aptamer conjugation technology, the aptamer (Apt) and complement (CS) were immobilized on the surface of Fe3O4@CeO2@PCPy nanoparticles to form Apt-CS-Fe3O4@CeO2@PCPyYSNs sensor.

3. The preparation method according to claim 2, characterized in that: The preparation method of the Fe3O4@CeO2 bimetallic nanozyme comprises: The Fe3O4@CeO2 core-shell structure was prepared by ligand exchange method, and the reaction solution was placed in a 50-90°C water bath for condensation and reflux for 2-10 hours; Fe3O4 nanoparticles were mixed with CeO2 nanoparticles to form Fe3O4@CeO2 bimetallic nanostructures.

4. The preparation method according to claim 2, characterized in that: A polypyrrole (PCPy) layer was grown on the surface of Fe3O4@CeO2@PDA by chemical oxidation polymerization to form Fe3O4@CeO2@PDA@PCPy nanoparticles, and the polymerization reaction was carried out at 50-90°C.

5. The preparation method according to claim 2, characterized in that: The aptamer has the nucleotide sequence shown in SEQ 1.

6. A method for detecting the concentration of tacrolimus (TAC) drug using the colorimetric sensor according to claim 1, characterized in that: The following steps are involved: The aptamer-functionalized Fe3O4@CeO2 bimetallic nanozymes were mixed with buffer samples containing different concentrations of tacrolimus; H2O2 and the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) were added to react; The light absorption value of the reaction system was detected by an enzyme-labeled instrument at a wavelength of 652 nm to quantitatively analyze the drug concentration of tacrolimus.

7. The method according to claim 6, characterized in that The concentration of H2O2 is 0.1-2.5 mol / L, and the concentration of TMB is 0.5-2.5 mol / L.

8. The method according to claim 6, characterized in that The detection linear range of the colorimetric sensor is 0.01 nM to 100 nM, and the detection limit is 2.3 pM.

9. The method according to claim 6, characterized in that The aptamer-functionalized Fe3O4@CeO2 bimetallic nanozyme was mixed with a buffer sample containing tacrolimus, and the nanozyme concentration in the resulting mixed solution was 10 μg / mL.

10. The method according to claim 6, characterized in that The color development reaction time is 30min-3h and the temperature is 15-25℃.

Images