An electrochemiluminescence sensor for detecting tacrolimus and a preparation method and application thereof

Abstract

This invention provides an electrochemiluminescence sensor for detecting tacrolimus, its preparation method, and its application, belonging to the field of electrochemiluminescence sensing technology. The invention modifies gold nanocages to have numerous amino groups on their surface, making them positively charged. Gold and silver nanoclusters are adsorbed into the gold nanocages using electrostatic adsorption. Then, amide bonds are used to connect aptamers to the surface of the gold nanocages, encapsulating them and preventing redox reactions between the gold and silver nanoclusters and potassium persulfate, thus suppressing the electrochemiluminescence signal. The electrochemiluminescence sensor based on gold nanocages and gold-silver nanocluster aptamers provided by this invention exhibits high sensitivity, selectivity, and accuracy, enabling sensitive and rapid detection of tacrolimus.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence sensing and detection technology, specifically to an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, its preparation method, and its application in the detection of tacrolimus. Background Technology

[0002] Tacrolimus is a first-line drug for immunosuppressive therapy after organ transplantation, and in my country, it is mainly used clinically to treat rejection in liver, kidney, heart, lung, and bone marrow transplants. The efficacy and side effects of tacrolimus vary from person to person. Even with the same drug and dosage, blood drug concentrations may differ between patients due to variations in metabolic rate, drug tolerance, and other factors. Because patients lack convenient and timely means of monitoring blood drug concentrations, during long-term treatment, sometimes low blood drug concentrations lead to poor efficacy, while sometimes high blood drug concentrations cause toxic reactions. For example, excessively high tacrolimus blood concentrations can easily lead to liver and kidney toxicity and central nervous system toxicity.

[0003] Currently, methods for monitoring tacrolimus blood concentrations mainly include microparticle enzyme immunoassay, fluorescence polarization immunoassay, and high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). However, methods such as microparticle enzyme immunoassay and fluorescence polarization immunoassay have drawbacks such as high cost, requirements for reagents and instruments, and low sensitivity. While HPLC and LC-MS offer high accuracy, their slow detection speed hinders widespread clinical application. Therefore, developing a highly efficient, sensitive, simple-to-operate, and clinically applicable biosensor for drug concentration monitoring is of great significance for the use of immunosuppressive drugs. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an electrochemiluminescence sensor for detecting tacrolimus, its preparation method, and its application. The electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters provided by this invention has high sensitivity, selectivity, and accuracy, enabling sensitive and rapid detection of tacrolimus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers, comprising an electrode, a gold nanocage loaded on the surface of the electrode, gold-silver nanoclusters located within the gold nanocage, and a tacrolimus aptamer chemically connected to the surface of the gold nanocage via amide bonds.

[0006] Preferably, the electrode comprises a screen-printed electrode.

[0007] Preferably, the gold nanocage has a particle size of 60-80 nm; the molar ratio of gold to silver in the gold-silver nanoclusters is 1-2:1, and the particle size of the gold-silver nanoclusters is 2-3 nm.

[0008] This invention provides a method for preparing the above-mentioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, comprising the following steps: Gold nanocages were mixed with lipoic acid solution and incubated for the first time to obtain lipoic acid modified gold nanocages. The thioctic acid-modified gold nanocages, carboxyl activator, and buffer solution were mixed to carry out a carboxyl activation reaction. Then, a polylysine solution was added to carry out an amidation reaction to obtain polylysine-modified gold nanocages. The polylysine-modified gold nanocage was mixed with a gold and silver nanocluster solution and subjected to a second incubation to obtain a gold and silver nanocluster-gold nanocage composite. The gold-silver nanocluster-gold nanocage complex was mixed with a tacrolimus aptamer solution and subjected to a third incubation to obtain the gold-silver nanocluster-gold nanocage-aptamer complex; the 5' end of the tacrolimus aptamer was modified with a carboxyl group. An electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers was obtained by drop-coating a solution of gold-silver nanocluster-gold nanocage-aptamer composite onto the electrode surface.

[0009] Preferably, the method for preparing the gold nanocage includes the following steps: Sodium sulfide, HCl, PVP and ethylene glycol were mixed and silver trifluoroacetate solution was added to carry out a precipitation reaction to obtain silver nanocubes. The silver nanocubes were heated and mixed with PVP solution, and HAuCl4 solution was added to carry out a template sacrificial reaction to obtain gold nanocages. The preparation method of the gold and silver nanoclusters includes the following steps: HAuCl4, AgNO3 and water were mixed, and glutathione, inorganic base and ascorbic acid were added in sequence to carry out a reduction reaction to obtain gold and silver nanoclusters.

[0010] Preferably, the second incubation temperature is 0~4℃ and the time is 8~12h; After mixing the gold-silver nanocluster-gold nanocage composite with a tacrolimus aptamer solution, the final concentration of the tacrolimus aptamer solution was 30-200 nM; the third incubation temperature was 25-32℃ and the time was 3-8 h. The concentration of the gold-silver nanocluster-gold nanocage-aptamer complex solution is 500~4500 ng / mL, and the drop volume is 5~15 μL.

[0011] This invention provides the application of the above-mentioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters in the detection of tacrolimus for non-diagnostic purposes.

[0012] The present invention provides a kit for detecting tacrolimus, comprising the above-mentioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers, and potassium persulfate solution.

[0013] This invention provides a method for detecting tacrolimus for non-diagnostic purposes, comprising the following steps: The liquid to be tested was applied to the surface of an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, and incubated to obtain the incubated electrochemiluminescence sensor. Potassium persulfate solution was added to the surface of the incubated electrochemiluminescence sensor, and cyclic voltammetry was performed to test the ECL signal of the first peak of electrochemiluminescence of the electrochemiluminescence sensor. The content of tacrolimus in the test liquid was obtained based on the ECL signal and a predetermined standard curve. The standard curve is a linear relationship curve between the ECL signal and tacrolimus concentration.

[0014] Preferably, the incubation time is 5 to 60 minutes; The concentration of the potassium persulfate solution is 10~200 mM; after adding the potassium persulfate solution, the pH value of the resulting detection system is 3.27~8.13; The scanning voltage of the cyclic voltammetric scan is -2~2V, and the scanning speed is 100~300mV / s.

[0015] This invention provides an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers (referred to as the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor), comprising an electrode, a gold nanocage loaded on the electrode surface, gold-silver nanoclusters located within the gold nanocage, and a tacrolimus aptamer chemically linked to the surface of the gold nanocage via amide bonds. This invention modifies the gold nanocage to have a large number of amino groups on its surface, making it positively charged overall. Gold-silver nanoclusters are adsorbed into the gold nanocage using electrostatic adsorption, and then the aptamer is linked to the surface of the gold nanocage using amide bonds. This encapsulates the gold nanocage, preventing the gold-silver nanoclusters from undergoing a redox reaction with potassium persulfate, thus suppressing the electrochemiluminescence signal. Based on this, it is drop-coated onto the electrode surface, completing the construction of the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor.

[0016] This invention utilizes aptamers as gating mechanisms to construct a "Signal-Off-On" detection strategy. The sensing principle is as follows: Gold and silver nanoclusters possess excellent electrochemiluminescence properties. When gold and silver nanoclusters enter a gold nanocage, the electrochemiluminescence and oxidation peak are enhanced, indicating that the gold nanocage can amplify the electrochemiluminescence signal of gold and silver nanoclusters by approximately 1.6 times, promote a positive shift in the oxidation peak potential, and increase the current value by approximately 1.6 times. The gold nanocage promotes the reduction of gold and silver nanoclusters. Due to the severe obstruction of electron transfer by the aptamer, the signal drops sharply from 6500 to around 600 after the aptamer is introduced, the oxidation current in the CV curve decreases, and the potential remains almost unchanged. After the addition of tacrolimus, it specifically binds to the aptamer, the aptamer conformation changes, exposing the gap to form a channel, the blocking effect weakens, and the gold and silver nanoclusters undergo a redox reaction with the co-reactants. The electrochemical signal (CV) recovers from 400, and the oxidation peak potential shifts positively. Gold and silver nanoclusters, acting as reducing agents, react with potassium persulfate to enhance the conductivity of screen printing. The gold-silver nanocluster-gold nanocage further improves conductivity. The aptamer forms an electron transport barrier; upon binding with tacrolimus, the aptamer conformation changes, weakening the barrier effect, and both the oxidation current and ECL signal recover synchronously. Throughout the detection process, the first ECL peak is used as the quantitative signal peak, fully demonstrating the construction logic of a "Signal-Off-On" type electrochemiluminescence biosensor.

[0017] The results of the examples show that the present invention obtained two standard curves in tacrolimus standard solutions with concentrations ranging from 1 to 100 ng / mL. The first curve is for a range of 1 to 25 ng / mL, with ECL = 22.02 C + 840.16; R 2 = 0.9950; second range 25~100 ng / mL, ECL=9.808C+1157.8; R 2=0.9988; the detection limit of this invention is as low as 6.38×10 -3 The sensor, with a concentration of ng / mL (7.94 f mol / L), exhibits ultra-high sensitivity, accuracy, selectivity, stability, repeatability, and reproducibility. Furthermore, this electrochemiluminescence sensor, based on gold nanocages and gold-silver nanoclusters, enables rapid and convenient detection of tacrolimus, possessing significant market potential. Attached Figure Description

[0018] Figure 1 Microstructure diagrams of different materials; Figure 2 The zeta potentials of different materials; Figure 3 XPS images of gold-silver nanoclusters-gold nanocages composites and gold-silver nanoclusters-gold nanocages-aptamer composites. Figure 4 The UV absorption spectra of gold and silver nanoclusters, gold nanocages, and silver nanocubes are shown. Figure 5 The fluorescence excitation and emission peak spectra of gold-silver nanoclusters and gold nanocages are shown. Figure 6 Electrochemical verification results for Au-AgNCs@Aucages-Apt122; Figure 7 CV plots and oxidation peaks at each stage of the assembly process of Au-AgNCs@Aucages-Apt122; Figure 8 EIS images of various nanomaterials; Figure 9 The results show the optimization conditions for key parameters that affect the ECL signal; Figure 10 The results are for the performance testing of Au-AgNCs@Aucages-Apt122. Detailed Implementation

[0019] The present invention provides an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, comprising an electrode, a gold nanocage loaded on the surface of the electrode, gold-silver nanoclusters located within the gold nanocage, and a tacrolimus aptamer chemically connected to the surface of the gold nanocage via amide bonds.

[0020] In this invention, the electrode preferably comprises a screen-printed electrode.

[0021] In this invention, the gold nanocage is preferably a polylysine-modified gold nanocage; the particle size of the gold nanocage is preferably 60-80 nm, more preferably 70 nm; the molar ratio of gold to silver in the gold-silver nanoclusters is preferably 1-2:1, more preferably 1.5:1, and the particle size of the gold-silver nanoclusters is preferably 2-3 nm. In this invention, the gold-silver nanoclusters are fixed in the pores inside the gold nanocage by electrostatic adsorption.

[0022] In this invention, the tacrolimus aptamer is preferably a tacrolimus aptamer Apt122 with a carboxyl group modified at the 5' end, and the sequence is preferably as shown in SEQ ID NO.1: SEQ ID NO.1: 5'-COOH-GCT GTG TGA CTC CTG CAAACT ACT GGT GTT CCT TGC CCT GTT TCA ATA AAT CGT GTG TGC CGC AGC TGT ATC TTGTCT CC-3'.

[0023] This invention provides a method for preparing the above-mentioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, comprising the following steps: Gold nanocages were mixed with lipoic acid solution and incubated for the first time to obtain lipoic acid-modified gold nanocages. The thioctic acid-modified gold nanocages, carboxyl activator, and buffer solution were mixed to carry out a carboxyl activation reaction. Then, a polylysine solution was added to carry out an amidation reaction to obtain polylysine-modified gold nanocages. The polylysine-modified gold nanocage was mixed with a gold and silver nanocluster solution and subjected to a second incubation to obtain a gold and silver nanocluster-gold nanocage composite. The gold-silver nanocluster-gold nanocage complex was mixed with a tacrolimus aptamer solution and subjected to a third incubation to obtain the gold-silver nanocluster-gold nanocage-aptamer complex; the 5' end of the tacrolimus aptamer was modified with a carboxyl group. An electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers was obtained by drop-coating a solution of gold-silver nanocluster-gold nanocage-aptamer composite onto the electrode surface.

[0024] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0025] In this invention, the method for preparing the gold nanocage preferably includes the following steps: Sodium sulfide, HCl, PVP and ethylene glycol were mixed and silver trifluoroacetate solution was added to carry out a precipitation reaction to obtain silver nanocubes. The silver nanocubes were heated and mixed with PVP solution, and HAuCl4 solution was added to carry out a template sacrificial reaction to obtain gold nanocages.

[0026] In this invention, the sodium sulfide is preferably provided in the form of an ethylene glycol solution, and the concentration of the sodium sulfide solution is preferably 2-5 mM, more preferably 3-4 mM; the HCl is preferably provided in the form of an aqueous solution, and the concentration of the HCl is preferably 20-50 mM, more preferably 30-40 mM; the molecular weight of the PVP is preferably 5800-10000, more preferably 6500-8500, and the PVP is preferably provided in the form of an ethylene glycol solution, and the concentration of the PVP solution is preferably 15-30 mg / mL, more preferably 20-25 mg / mL. In this invention, the preferred volume ratio of sodium sulfide solution, HCl solution, PVP solution, and ethylene glycol is 0.1:0.1:(3~6):(6~18). The preferred mixing method is magnetic stirring, with a stirring temperature of 120~180℃, more preferably 140~160℃, and a stirring speed of 300~600 rpm. In this invention, the preferred solvent for the silver trifluoroacetate solution is ethanol, the preferred concentration of the silver trifluoroacetate solution is 0.28M, and the preferred volume ratio of the silver trifluoroacetate solution to the sodium sulfide solution is 2~6:1, more preferably 4:1. In this invention, the preferred temperature for the precipitation reaction is 150℃, and the preferred time is 3 hours. After the precipitation reaction, the preferred method is to place the precipitation reaction system in an ice-water bath, add acetone to terminate the reaction, and then centrifuge and wash the terminated precipitation reaction system. In this invention, the preferred particle size of the silver nanocubes is 60~80 nm, more preferably 70 nm.

[0027] The silver nanocubes are heated and mixed with a PVP solution, and then HAuCl4 solution is added to carry out a template sacrifice reaction to obtain gold nanocages. In this invention, the concentration of the PVP solution is preferably 0.5~2 mg / mL, more preferably 1 mg / mL; the silver nanocubes are preferably provided in the form of an aqueous dispersion, and the volume ratio of the silver nanocube dispersion to the PVP solution is preferably 0.1:1~3, more preferably 0.1:1~2. In this invention, the heating and mixing is preferably carried out by boiling, and the time is preferably 8~30 min, more preferably 10~20 min. In this invention, the concentration of the HAuCl4 solution is preferably 0.1~5 mM, more preferably 0.5~2 mM; the HAuCl4 solution is added dropwise, and the amount added is based on obtaining a stable blue solution. After obtaining a stable blue solution, the template sacrifice reaction is considered complete. After the template sacrifice reaction, the resulting reaction solution is preferably centrifuged and washed, and the washing is preferably performed sequentially by washing with saturated NaCl solution and water.

[0028] In this invention, the method for preparing the gold and silver nanoclusters preferably includes the following steps: HAuCl4, AgNO3 and water were mixed, and glutathione, inorganic base and ascorbic acid were added in sequence to carry out a reduction reaction to obtain gold and silver nanoclusters.

[0029] In this invention, the HAuCl4 and AgNO3 are preferably provided in the form of aqueous solutions. The concentration of the HAuCl4 solution is preferably 5-20 mM, more preferably 10 mM; the concentration of the AgNO3 solution is preferably 5-20 mM, more preferably 10 mM; the volume ratio of the HAuCl4 solution, AgNO3 solution and water is preferably (0.5-1):(0.5-1):(7-30), more preferably (0.5-0.8):(0.5-0.8):(10-20); in this invention, the mixing is preferably carried out under magnetic stirring conditions. In this invention, the glutathione is preferably added in solution form, and the concentration of the glutathione solution is preferably 80 mM; the inorganic base is preferably NaOH, and the concentration is preferably 0.5~2 M, more preferably 1 M; the ascorbic acid is preferably provided in solution form, and the concentration of the ascorbic acid solution is preferably 0.2~2.5 M, more preferably 0.5~2 M; the volume ratio of the HAuCl4 solution, the glutathione solution, the NaOH solution, and the ascorbic acid solution is preferably 0.5:(1~2):(0.4~1.6):(1~2); the temperature of the reduction reaction is preferably 50~120 ℃, more preferably 80~100 ℃, and the time is preferably 5~8 h, more preferably 6~7 h. After the reduction reaction, the resulting reduction reaction solution is preferably dialyzed, the molecular weight cutoff for dialyzed is preferably 3.5 kDa, the dialyzed temperature is preferably room temperature, and the time is preferably ≥24 h.

[0030] This invention involves mixing gold nanocages with a lipoic acid solution and performing a first incubation to obtain lipoic acid-modified gold nanocages. In this invention, the concentration of the lipoic acid solution is preferably 1 mM, and the first incubation is preferably carried out under light-protected conditions, preferably at room temperature, and preferably for 4 hours. After the first incubation, the resulting product is preferably centrifuged and redispersed in PBS solution.

[0031] This invention involves mixing lipoic acid-modified gold nanocages, a carboxyl activator, and a buffer solution to perform a carboxyl activation reaction. Following this, a polylysine solution is added to initiate an amidation reaction, yielding polylysine-modified gold nanocages. In this invention, the carboxyl activator is preferably EDC and NHS, with a preferred mass ratio of EDC to NHS of 4:6; the buffer solution is preferably MES buffer. In this invention, the carboxyl activation reaction is preferably performed at room temperature for 15–60 min, more preferably 30–40 min. After the carboxylation reaction, this invention preferably removes excess EDC / NHS by centrifugation and redisperses the precipitate in PBS solution. In this invention, the molecular weight of the polylysine is preferably 15,000-30,000 Da, more preferably 20,000-25,000 Da. As a specific embodiment of this invention, the polylysine is manufactured by Maclean, with batch number C17498140. The concentration of the polylysine solution is preferably 1-5 mg / mL, more preferably 2-4 mg / mL. The amidation reaction temperature is preferably room temperature, and the time is preferably 0.5-3 h, more preferably 1-2 h. After the amidation reaction, the product is preferably centrifuged to completely remove unreacted polylysine, and the precipitate is redispersed in PBS solution.

[0032] In this invention, polylysine-modified gold nanocages are mixed with a gold-silver nanocluster solution and subjected to a second incubation to obtain a gold-silver nanocluster-gold nanocage composite. In this invention, the mass ratio of the polylysine-modified gold nanocages to the gold-silver nanoclusters is preferably 1:1. In this invention, the temperature of the second incubation is preferably 4°C, and the time is preferably 8-12 h, more preferably 10-11 h. After the second incubation, this invention preferably involves centrifugation to disperse the resulting solid in PBS solution for later use. In this invention, the centrifugation rate is preferably 8000 rpm, and the time is preferably 10 min.

[0033] In this invention, the gold-silver nanoclusters-gold nanocages complex is mixed with a tacrolimus aptamer solution and subjected to a third incubation to obtain the gold-silver nanoclusters-gold nanocages-aptamer complex; the tacrolimus aptamer is modified with a carboxyl group at its 5' end. In this invention, after mixing the gold-silver nanoclusters-gold nanocages complex with the tacrolimus aptamer solution, the final concentration of the tacrolimus aptamer solution is preferably 30-200 nM, more preferably 50-150 nM, further preferably 60-80 nM, and most preferably 75 nM. In this invention, the temperature of the third incubation is preferably 4°C, and the time is 2-5 h, more preferably 3 h. After the third incubation, the obtained gold-silver nanoclusters-gold nanocages-aptamer complex solution is preferably centrifuged at a speed of 8500 rpm for 10 min, and the precipitate is retained after centrifugation to remove unbound aptamers.

[0034] This invention involves drop-coating a gold-silver nanocluster-gold nanocage-aptamer composite solution onto an electrode surface to obtain an electrochemiluminescence sensor based on gold nanocages and gold-silver nanocluster aptamers. In this invention, the concentration of the gold-silver nanocluster-gold nanocage-aptamer composite solution is preferably 500~4500 ng / mL, more preferably 1000~3000 ng / mL, and the drop-coating volume is preferably 5~15 μL, more preferably 10 μL. After drop-coating, the solution is preferably allowed to air dry naturally.

[0035] This invention provides the application of the above-mentioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters in the detection of tacrolimus for non-diagnostic purposes.

[0036] This invention provides a kit for detecting tacrolimus, comprising the aforementioned electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers, and a potassium persulfate solution. In this invention, the concentration of the potassium persulfate solution is preferably 10-200 mM, more preferably 50-180 mM, and even more preferably 100-180 mM. In this invention, the kit also preferably includes a buffer solution, which preferably includes one or more of PBS buffer, MES buffer, and TE buffer.

[0037] This invention provides a method for detecting tacrolimus for non-diagnostic purposes, comprising the following steps: The liquid to be tested was applied to the surface of the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters and incubated to obtain the incubated electrochemiluminescence sensor. The incubated electrochemiluminescence sensor was placed in a potassium persulfate solution, and cyclic voltammetry was performed to test the ECL signal of the first peak of electrochemiluminescence of the electrochemiluminescence sensor. The content of tacrolimus in the test liquid was obtained based on the ECL signal and a predetermined standard curve. The standard curve is a linear relationship curve between the ECL signal and tacrolimus concentration.

[0038] This invention involves applying a test liquid to the surface of an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters, followed by incubation to obtain an incubated electrochemiluminescence sensor. In this invention, the test liquid is preferably blood. The test liquid is preferably applied dropwise, with a preferred volume of 50-200 μL, more preferably 100-150 μL. The incubation temperature is preferably 25-32°C (room temperature), and the incubation time is preferably 5-60 min, more preferably 20-50 min, and even more preferably 30-40 min. After incubation, the electrochemiluminescence sensor is preferably rinsed and dried.

[0039] In this invention, potassium persulfate solution is added dropwise to the surface of the incubated electrochemiluminescence sensor, and cyclic voltammetry is performed to measure the ECL signal of the first peak of electrochemiluminescence from the sensor. The content of tacrolimus in the test liquid is obtained based on the ECL signal and a predetermined standard curve. In this invention, the concentration of the potassium persulfate solution is preferably 10-200 mM, more preferably 50-180 mM, and even more preferably 100-180 mM. After adding the potassium persulfate solution, the pH value of the resulting detection system is preferably 3.27-8.13, more preferably 6-7.

[0040] In this invention, the scanning voltage of the cyclic voltammetric scan is preferably -2 to 2V, and the scanning speed is preferably 100 to 300 mV / s, more preferably 150 to 200 mV / s, and even more preferably 150 to 180 mV / s.

[0041] In this invention, the method for obtaining the standard curve preferably includes the following steps: Provides tacrolimus standard solutions with gradient concentrations; Using the gradient concentrations of tacrolimus standard solutions as test samples, the samples were applied to the surface of the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters and incubated. Then, potassium persulfate solution was added, and cyclic voltammetry was performed to obtain the ECL signals corresponding to different concentrations of tacrolimus standard solutions. A standard curve was plotted with the tacrolimus solution concentration as the abscissa and the ECL signal value as the ordinate.

[0042] The following detailed description, in conjunction with embodiments, illustrates the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters provided by the present invention, its preparation method, and its application in the detection of tacrolimus. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1 (1) Preparation of gold nanocages: Add 6 mL of ethylene glycol to a 30 mL sample vial and place it in an oil bath at 150 °C with magnetic stirring (40 min, 300 rpm). Immediately add 0.1 mL of freshly prepared sodium sulfide (3 mM, ethylene glycol solution) and react for 4 min. Then add 0.1 mL of HCl and 3 mL of PVP (20 mg / mL, ethylene glycol solution) and react for 2 min. Next, add 0.4 mL of silver trifluoroacetate (0.28 M, ethanol solution) to the mixture and react for 3 h, yielding a yellow-greenish-brown turbid liquid. At the end of the reaction, stop the reaction by adding 3 mL of acetone in an ice-water bath and centrifuging at 3000 rpm for 30 min. After the first centrifugation, discard the supernatant, add 2 mL of ultrapure water, wash three times, and finally disperse in 3 mL of ultrapure water. Add 5 mL of PVP (1 mg / mL) aqueous solution and 500 μL of the prepared silver nanocubes to a 30 mL sample vial. Heat to boiling for 10 min with magnetic stirring (300 rpm). Slowly add 0.5 mM HAuCl4 dropwise while observing the color change until a stable blue solution is obtained. Cool the prepared solution to room temperature and separate the product by centrifugation at 14000 rpm for 10 min. Dissolve the reaction mixture in an equal volume of saturated NaCl solution to remove impurities, then wash twice with ultrapure water. Redisperse the obtained gold nanocages in 10 mL of ultrapure water and store at 4 °C.

[0044] (2) Preparation of gold-silver nanoclusters (Au-AgNCs) Gold and silver nanoclusters were prepared using a green dual-reduction method. The specific procedure was as follows: 7 mL of ultrapure water, 0.5 mL of HAuCl4 (10 mM), and 0.5 mL of AgNO3 (10 mM) were added to a 30 mL sample vial. The mixture was magnetically stirred (300 rpm) at room temperature for 3 min. Then, 1 mL of glutathione (GSH) (80 mM) was rapidly added, and the reaction proceeded for 5 min. Next, 0.4 mL of NaOH (1 M) was rapidly added, and the reaction proceeded for 2 min. Finally, 1 mL of icy ascorbic acid (0.5 M) was rapidly added. The solution was heated to 80 °C and reacted for 6 hours, yielding a clear, pale yellow liquid. The solution was then naturally cooled to room temperature. The prepared Au-AgNCs were dialyzed in ultrapure water using a 3.5 kDa dialysis bag for at least 24 hours, with the water changed every 6 hours to remove all small impurities. Larger precipitates formed during dialysis were removed using a 0.22 μm filter. The purified Au-AgNCs solution was stored at 4 °C.

[0045] (3) Preparation of polylysine-modified gold nanocages (AuNCages-LA-PLL) Take 2 mL of AuNCages dispersion, centrifuge at 10000 rpm for 15 minutes, and discard the supernatant. Redisperse the precipitate in 2 mL of 1 mM lipoic acid solution. Incubate the mixture at room temperature in the dark with shaking for 4 hours. After incubation, centrifuge at 10000 rpm for 15 minutes and redisperse in 2 mL of PBS. Take the above solution, centrifuge, and disperse in 2 mL of 0.1 M MES buffer. Add 4 mg EDC and 6 mg NHS, and gently shake at room temperature for 30 minutes to activate the carboxyl groups. Centrifuge to remove excess EDC / NHS, and redisperse the precipitate in 2 mL of PBS. Immediately add 1 mL of 2 mg / mL polylysine solution. Shake and react at room temperature for 2 hours. After the reaction is complete, centrifuge and wash three times to completely remove unreacted PLL. Finally, disperse in 2 mL of PBS to obtain AuNCages-LA-PLL.

[0046] (4) Preparation of gold-silver nanoclusters-gold nanocages composites (Au-AgNCs@Aucages) Take 1 mL of AuNCages-LA-PLL solution, centrifuge, discard the supernatant, add 1 mL of gold and silver nanoclusters solution, and incubate overnight at 4°C. Centrifuge the incubated product, and disperse the precipitate in 1 mL of PBS solution.

[0047] (5) Preparation of gold and silver nanoclusters-gold nanocages-aptamer complexes Tacrolimus aptamer (Apt122) was mixed with TE buffer to prepare an aptamer solution with a concentration of 2500 nM. 300 μL of Au-AgNCs@Aucages PBS dispersion was added to the aptamer solution to bring the final concentration of the aptamer solution to 75 nM. The solution was incubated at room temperature for 6 h. After centrifugation, the supernatant was discarded, and 300 μL of PBS was added to obtain the gold-silver nanoclusters-gold nanocages-aptamer complex.

[0048] (6) Fabrication of an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters (Au-AgNCs@Aucages-Apt122) Take a screen-printed electrode and drop-coat the Au-AgNCs@Aucages-Apt122 solution prepared in the previous step onto the electrode surface. The drop-coating volume is 10 μL. After air drying, an electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters is obtained.

[0049] Structural characterization (I) The morphology, composition, and structure of gold and silver nanoclusters and gold nanocages during preparation and assembly of electrochemiluminescence sensors were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectrophotometry (UV-Vis), and fluorescence spectrophotometry (FL), as detailed below: Microstructure diagrams of different materials as follows Figure 1 As shown, Figure 1 In the image, (A) is a SEM image of gold nanocages, (B) is a TEM image of gold and silver nanoclusters, (C) is a TEM image of gold and silver nanoclusters-gold nanocages composites, and (D) is a TEM image of gold and silver nanoclusters-gold nanocages-aptamer composites.

[0050] Depend on Figure 1 As can be seen in (A), gold nanocages with a particle size of approximately 60–80 nm were accurately obtained using the silver nanocube template sacrificial method; Figure 1 As can be seen from (B), the particle size of the gold and silver nanoclusters is approximately 3 nm. The particle size of the gold and silver nanoclusters is significantly smaller than that of the gold nanocages, thus satisfying the rigid condition for fitting gold and silver nanoclusters into gold nanocages. From Figure 1 As can be seen from (C), the gold and silver nanoclusters are adsorbed into the gold nanocage, and as can be seen from (D), the aptamer is successfully attached to the gold nanocage.

[0051] The zeta potential of different materials, such as Figure 2 As shown, zeta potential detection revealed that both the gold nanocage and the gold-silver nanoclusters had negative potentials. To enable the gold-silver nanoclusters to autonomously enter the gold nanocage using electrostatic adsorption, the charge of the gold nanocage needed to be altered. Lipoic acid was attached to the gold nanocage via a gold-sulfur bond (Au-S), and polylysine containing multiple amino groups was attached to the gold nanocage via an amide bond (-CO-NH-), thus giving the gold nanocage a positive charge and allowing the gold-silver nanoclusters to successfully enter it.

[0052] XPS images of gold-silver nanocluster-gold nanocage composites and gold-silver nanocluster-gold nanocage-aptamer composites are shown below. Figure 3 As shown, when Apt122 is attached, the phosphorus content increases from 0.96% to 8.48%, proving that the aptamer has successfully attached to the gold nanocage.

[0053] UV absorption spectra of gold and silver nanoclusters, gold nanocages, and silver nanocubes are shown below. Figure 4 As shown, Figure 4In the diagram, red represents gold nanocages; green represents gold-silver nanoclusters; and blue represents silver nanocubes. It can be seen that the ultraviolet absorption peak of silver nanoclusters is around 275 nm, which does not overlap with the ultraviolet absorption of gold nanocages, suggesting it may not be a metal plasmon resonance peak. It shows almost no characteristic absorption in the visible light region; the quantum confinement effect of the ultra-small nanoclusters may cause its ultraviolet absorption to appear below 300 nm. The ultraviolet absorption peak of gold nanocages is between 550 and 600 nm, exhibiting a broadened surface plasmon resonance (SPR) that shifts towards longer wavelengths, consistent with the characteristics of hollow multi-cell structures, and may show tailing absorption in the near-infrared region (>600 nm). The silver nanocubes show a relatively narrow peak width between 420 and 450 nm. The spectral characteristics of Au-AgNCs in the visible light region are significantly different from those of individual Au cages and Ag nanocubes, indicating the successful formation of gold-silver composite nanomaterials.

[0054] The fluorescence excitation and emission peaks of gold and silver nanoclusters and gold nanocages are as follows: Figure 5 As shown, Figure 5 In the image, the left image shows the excitation peak, and the right image shows the emission peak. (From...) Figure 5 It can be seen that the fluorescence excitation peak of the gold-silver nanoclusters is at 400 nm, with a full width at half maximum (FWHM) of approximately 80–100 nm, consistent with the typical fluorescence characteristics of metal nanoclusters. Its emission peak is at a longer wavelength of 690 nm, further confirming that Au-AgNCs are ultrasmall nanoclusters (nucleus number <25) rather than plasmonic nanoparticles. The fluorescence of Au-AgNCs may originate from the quantum confinement luminescence of the gold-silver nanoclusters, the luminescence of new surface states generated at the Au-Ag interface, and the intermetallic interaction of electron transfer from ligands to the gold-silver nuclei. Fluorescence and electrochemiluminescence usually share the same excited state. If Au-AgNCs produce luminescence near 690 nm under electrochemical excitation, the ECL wavelength is likely also around this wavelength, with a peak position of 680–700 nm indicating an excited state energy level of 1.77–1.82 eV.

[0055] (II) To further verify the successful construction of the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor and to confirm that gold nanocages can indeed amplify the electrochemiluminescence of gold and silver nanoclusters, electrochemical verifications were performed. The sensor assembly steps were characterized using electrochemiluminescence (ECL) and cyclic voltammetry (CV). For ECL measurements, the test parameters were set as follows: high voltage 1000 V, measurement rate 66.67 mV. For CV measurements, the voltage range was -2 to 2 V, and the test parameters were set as follows: scan rate 0.15 V / s, sampling interval 0.01 V, forward scan direction, current range 1.e-0.3 A. The results are shown below. Figure 6 As shown. Figure 6In the diagram, (A) shows nine consecutive ECL peaks detected by Au-AgNCs@Aucages-Apt122. It can be seen that the RSD of the nine consecutive ECL signals is 3.97%, indicating that the signal after Au-AgNCs@Aucages-Apt122 assembly is relatively stable. (B) shows the ECL at each stage during the assembly process of Au-AgNCs@Aucages-Apt122, where a represents Au-AgNCs, b represents Au-AgNCs@GNCs, c represents Au-AgNCs@GNCs - Apt122, and d represents Au-AgNCs@GNCs-Apt122-TAC. It can be seen that three consecutive ECL signal peaks appear in the detection segment at each stage of the sensor assembly process. The maximum RES of the three consecutive peaks is 3.73%. However, for quantification, this invention uses the ECL value of the first peak. It is evident that loading gold and silver nanoclusters into gold nanocages significantly amplifies the ECL signal of the gold and silver nanoclusters. The possible reason for this change is the synergistic effect of localized surface plasmon resonance (LSPR) and electromagnetic field enhancement caused by the combination of gold and silver nanoclusters and gold nanocages. Gold nanocages have tunable hollow structures and plasmon resonance absorption characteristics. When gold and silver nanoclusters enter gold nanocages, a strong plasmon resonance energy transfer (PRET) or electromagnetic field coupling effect is generated. The LSPR effect on the surface of the gold nanocage generates a highly localized enhanced electromagnetic field around it, accelerating the electron-hole recombination rate, promoting the radiative transition of excited-state electrons in the gold and silver nanoclusters to the ground state, increasing the radiative transition probability, enhancing photon emission efficiency, suppressing non-radiative relaxation channels, promoting electron transfer dynamics, and accelerating the electron transfer process between the electrode surface and co-reactants. Compared to solid nanoparticles, the hollow porous structure of gold nanocages offers unique advantages: a larger specific surface area provides more active sites for interaction with gold and silver nanoclusters; plasmon coupling exists between the inner and outer surfaces, allowing the LSPR fields on both the inner and outer walls to couple simultaneously with the gold and silver nanoclusters, thus improving light scattering / trapping efficiency; and the hollow structure enhances the ability to capture the luminescence signal of the excitation light. However, the addition of aptamers reduces the ECL signal, and steric hindrance hinders electron transfer. The aptamers form a thicker organic layer on the surface of the gold nanocage / gold-silver nanoclusters, impeding the absorption of S2O8. 2- Diffusion to the electrode surface inhibits the direct transfer of electrons between the electrode and the nanoclusters, leading to a decrease in ECL excitation efficiency and an increase in interfacial impedance. The negatively charged phosphate backbone of the aptamer forms an electrostatic repulsion layer on the electrode surface, increasing the interfacial charge transfer resistance (Rct) and reducing the electrochemical reaction rate. Upon addition of tacrolimus, it specifically binds to the aptamer Apt122, causing a conformational change in the aptamer that exposes interstitial spaces, forming channels. This weakens the blocking effect, allowing the gold and silver nanoclusters to undergo redox reactions with the co-reactants, thus restoring the electrochemiluminescence signal.

[0056] (C) shows the FTIR analysis of Au-AgNCs@Aucages-Apt122, which indicates that at 3380 cm⁻¹... -1 (N–H stretching), 1646 cm -1 (C=O stretching vibration), 1180-1039 cm -1 (Stretching vibrations of CO and PO bonds) and the low wavenumber region 636-865 cm⁻¹ -1 Characteristic peaks were observed at locations such as the Au-S bond. This indicates that the Au-AgNCs@Aucages-Apt122 solution was indeed drop-coated onto the electrode, preserving the basic structure of Au-AgNCs@Aucages-Apt122. (D) shows the voltage-electrochemiluminescence diagram during the assembly of Au-AgNCs@Aucages-Apt122. Chemiluminescence occurred at -2V, and the electrochemiluminescence change is consistent with... Figure 6 (B) in the middle.

[0057] Figure 7 The CV curves and oxidation peaks at various stages of the Au-AgNCs@Aucages-Apt122 assembly process are shown. The results indicate that the oxidation peak is enhanced after the gold and silver nanoclusters enter the gold nanocages, showing that the gold nanocages can effectively amplify the electrochemiluminescence signal of the gold and silver nanoclusters by about 1.6 times, promote the positive shift of the oxidation peak potential, and increase the current value by about 1.6 times. The gold nanocages promote the reduction of gold and silver nanoclusters. When the aptamer is introduced, the oxidation current in the CV curve decreases, while the potential remains almost unchanged, possibly because the aptamer severely hinders electron transfer. After the addition of tacrolimus, it specifically binds to the aptamer Apt122, the aptamer conformation changes, the gap is exposed to form a channel, the blocking effect is weakened, and redox reactions occur with the co-reactants, resulting in an increase in the oxidation peak. Figure 8 The image shows the EIS spectra of various nanomaterials. This invention performs resistance measurements at each stage, and the resistance at each stage corresponds to the changes in ECL and oxidation peak heights. Figure 8 The results showed that, compared with the bare electrode, the conductivity was significantly improved after surface modification with metal nanomaterials. The resistance of the entire system increased after the introduction of the aptamer. Although simply coating gold-silver nanoclusters or gold nanocages on the electrode surface reduced the electrode resistance to some extent, neither was as significant as the reduction in resistance after the gold-silver nanoclusters were incorporated into the gold nanocages. This further verified that the incorporation of gold-silver nanoclusters into the gold nanocages promotes electron transfer. To verify that the drop-coating method could firmly connect the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor to the electrode surface and form a pathway, the electrochemiluminescence sensing system was successfully constructed.

[0058] Example 2 The optimal preparation conditions for electrochemiluminescence detection of gold nanocages and gold nanocages were obtained by systematically optimizing the pH, volume of the gold-silver nanocage-gold nanocage composite solution, concentration of potassium persulfate, and scan rate throughout the detection environment. The optimization conditions for key parameters affecting the ECL signal are as follows: Figure 9 As shown. Figure 9 In the table, (A) is the concentration of potassium persulfate; (B) is the volume of the gold-silver nanocluster-gold nanocage-aptamer complex solution; (C) is the pH of the detection environment; (D) is the scan rate; (E) is the aptamer concentration; and (F) is the binding time between tacrolimus and the aptamer.

[0059] (1) Optimization of conditions for potassium persulfate K2S2O8, as a co-reactant, is reduced on the electrode surface to generate highly oxidizing sulfate radicals (SO4). - ·), can undergo electron transfer reactions with gold and silver nanoclusters, exciting the luminescent material to generate ECL signals. Figure 9 In (A), within the concentration range of 0–180 mM, the ECL intensity of the system showed a significant increasing trend with increasing K₂S₂O₈ concentration. This is because increasing the concentration of co-reactants can effectively enhance the activity of free radicals (such as SO₄²⁻) in the system. - The generation efficiency of K2S2O8 is increased, thereby enhancing the luminescence efficiency of the ECL reaction. When the concentration of K2S2O8 reaches 180 mM, the concentration is close to saturation. When the concentration continues to increase to 200 mM, the ECL intensity decreases significantly. This may be due to the quenching effect of the co-reactants themselves at high concentrations, the aggravation of side reactions, or the inhibitory effect of excessively high ionic strength on the electron transfer process on the electrode surface.

[0060] (2) Condition optimization of gold-silver nanoclusters-gold nanocages-aptamer complex To obtain the optimal ECL luminescence efficiency, the effect of the solution volume (5–15 μL) of the gold-silver nanocluster-gold nanocage-aptamer complex on the luminescence intensity was systematically investigated. The solution volume of the complex affects the coverage density of active sites on the electrode surface, electron transfer efficiency, and spatial accessibility of co-reactants. Figure 9 As shown in (B), the ECL intensity exhibits a parabolic trend of first increasing and then decreasing with the increase of the volume of the gold-silver nanocluster-gold nanocage-aptamer complex. During the dropwise addition of 5~15 μL of the gold-silver nanocluster-gold nanocage-aptamer complex solution, an effective catalytic layer is formed on the electrode surface, the number of active centers increases, and the co-reactants (such as S2O8) are promoted. 2-The reduction of gold nanoclusters reached its peak at a volume of 10 μL. Further increasing the volume to 15 μL resulted in an excessively thick film, hindering electron conduction and increasing impedance. At high concentrations, energy transfer between cage cluster molecules led to non-radiative decay, and mass transfer from the co-reactants to the electrode surface was impeded, causing a sharp signal attenuation. Based on the experimental results, 10 μL was determined to be the optimal feed volume for the gold-silver nanoclusters-gold nanocages-aptamer composite. Under this condition, the ECL intensity reached its maximum, and the error bars showed good reproducibility.

[0061] (3) pH condition optimization pH affects both the stability of gold and silver nanoclusters and the electron transfer rate during the reaction. Acidic conditions increase the risk of structural disintegration, protonation of gold and silver nanocluster ligands, inhibition of photoinduced electron transfer (PET), changes in the surface charge of gold nanocages, and failure of electrostatic stability. Excessive alkalinity leads to structural damage, destruction of the gold and silver core structure, ligand dissociation, and luminescence quenching. This invention investigated the electrochemiluminescence behavior at pH 3.27–8.13. Figure 9 The results (C) show that a neutral to slightly acidic environment is best for the electrochemiluminescence signal of gold nanocages of gold and silver nanoclusters. Therefore, this invention selects a pH 7.01 environment as the optimal condition, and the ECL signal reaches more than 7500.

[0062] (4) Conditional optimization of scan rate Changes in the scan rate directly modulate the electric double layer structure and mass transfer efficiency of the electrode surface. Under low-speed conditions, the gold and silver nanoclusters and the co-reactant potassium persulfate have sufficient diffusion time to fully contact the electrode surface, and the redox reaction is more likely to reach a steady state, thus generating a high-intensity ECL signal. However, under high-speed scanning, the diffusion rate of the substance lags behind the potential scan rate, resulting in a decrease in surface concentration and a corresponding decrease in ECL intensity. Figure 9Figure (D) shows that within the scan rate range of 100–150 mV / s, the ECL intensity exhibits a significant decreasing trend. When the scan rate increases from 100 mV / s to 150 mV / s, the ECL signal intensity drops sharply from approximately 12000 au to around 8000 au. This is mainly due to limitations imposed by electrochemical kinetics and mass transfer processes. Higher scan rates accelerate the pace of potential changes, shorten the residence time of electroactive substances on the electrode surface, leading to a decrease in the effective enrichment of luminescent material and co-reactants on the electrode surface. Furthermore, the electron transfer process struggles to reach a sufficient equilibrium, ultimately weakening the luminescence efficiency of the ECL reaction. When the scan rate continues to increase and exceeds 150 mV / s (to the 180–200 mV / s range), the ECL intensity shows a slight rebound, stabilizing at around 7500–7800 au. This indicates that the inhibitory effect of the scan rate on the mass transfer process gradually stabilizes at this point, and the system enters a relatively stable response phase. Considering the ECL signal strength, experimental detection sensitivity, and the kinetic compatibility of the system, this invention selects 150 mV / s as the optimal scan rate.

[0063] (5) Optimization of aptamer concentration conditions This invention uses an aptamer as an encapsulation barrier layer. Figure 9 As shown in (E), in the low concentration range of 30–50 nM, the aptamer modification density on the electrode surface is low, and electron transfer and co-reactant diffusion are not significantly hindered, resulting in a strong luminescent signal. When the aptamer concentration increases to the 60–80 nM range, the ECL intensity decreases significantly, reaching its lowest point at 75 nM. At this point, the aptamer is over-modified on the electrode surface, forming a dense steric hindrance layer that hinders electron transfer and co-reactant diffusion to the electrode surface, thus significantly suppressing the ECL reaction efficiency. When the concentration continues to increase to 100 nM, the ECL intensity recovers and tends to stabilize. The aptamer “oversaturation” leads to conformational changes / spatial rearrangements, and some active sites are re-exposed. At the same time, the aptamer Apt122 itself is negatively charged, exhibiting electrostatic repulsion, which makes the nanomaterials more uniformly dispersed and reduces ECL quenching caused by particle aggregation. As the aptamer concentration continues to increase, the aptamer modification density tends to saturate, the surface adsorption reaches a dynamic equilibrium, the signal no longer changes sensitively, and the system enters a relatively stable response stage. Taking into account signal strength, steric hindrance, and the number of effective binding sites for the target analyte, this invention selects 75 nM as the optimal working concentration for the aptamer Apt122. This condition ensures a high initial ECL signal while reserving sufficient reaction sites for the specific binding of the target analyte and the aptamer, laying a crucial foundation for constructing a highly sensitive "Signal-Off-On" tacrolimus ECL sensing platform.

[0064] (6) Binding time of aptamer Apt122 to tacrolimus Aptamer Apt122 can specifically bind to tacrolimus. The binding time between tacrolimus and aptamer is a key factor affecting the clinical application of subsequent methods. The binding of the drug and aptamer was examined over a period of 5–60 min. Figure 9 In the incubation period (F) of 0–40 min, the ECL intensity of the system showed a significant increasing trend with increasing incubation time. This phenomenon originates from the dynamic process of the specific binding reaction between the aptamer and tacrolimus. As the incubation time increases, more and more tacrolimus molecules specifically recognize and bind to the aptamer on the electrode surface, forming an aptamer-target complex, which in turn leads to a gradual enhancement of the ECL signal. When the incubation time exceeds 40 min (extended to 60 min), the ECL intensity basically remains stable and no longer changes significantly with time, indicating that the specific binding reaction between the aptamer and tacrolimus has basically reached dynamic equilibrium, and the binding sites on the electrode surface have been fully occupied by the target. Further extending the incubation time cannot further improve the ECL response intensity. Therefore, this invention selects 40 min as the optimal incubation time for the aptamer and tacrolimus. Under these conditions, the target and aptamer can be fully bound together to obtain a stable and significant ECL response signal, while avoiding the reduction in detection efficiency caused by excessively long incubation time. This lays a key experimental foundation for building a rapid and sensitive tacrolimus ECL sensing detection method.

[0065] Example 3 Tacrolimus standard solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 35 ng / mL, 50 ng / mL, 75 ng / mL, and 100 ng / mL were prepared using the standard addition method. The gold-silver nanoclusters-gold nanocages-aptamer composites prepared under optimal conditions were drop-coated onto a screen-printed electrode (10 μL). After drying, different concentrations of tacrolimus standard solutions were added, and the electrode was incubated for 40 min. Excess tacrolimus solution was aspirated, and the electrode was slowly rinsed twice. After drying, 180 mM potassium persulfate solution was added, and the ECL signal value was detected. The linear regression equation was calculated. Subsequently, the standard deviation of the response values ​​in 10 blank solutions was continuously measured, and the limits of quantitation and detection were calculated using the formulas LOD = 3.3σ / S and LOQ = 10σ / S. The performance test results of the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor are as follows: Figure 10 As shown. Figure 10In the table, (A) represents the response of ECL to solutions with different tacrolimus concentrations; (B) represents the standard curve of Au-AgNCs@Aucages-Apt122; (C) represents the repeatability of Au-AgNCs@Aucages-Apt122; (D) represents the reproducibility of Au-AgNCs@Aucages-Apt122; (E) represents the stability of Au-AgNCs@Aucages-Apt122; and (F) represents the selectivity of Au-AgNCs@Aucages-Apt122.

[0066] (1) Standard curve and detection limit like Figure 10 As shown in (A), after tacrolimus specifically binds to the aptamer, it alters the aptamer conformation, exposing channels. This allows the gold-silver nanoclusters to bind with potassium persulfate, increasing the ECL signal. With increased specific binding of tacrolimus to the aptamer, more channels are exposed, and the more potassium persulfate binds to the gold-silver nanoclusters, the stronger the ECL signal. Finally, two standard curves of tacrolimus concentration versus electrochemiluminescence signal were obtained using the gold-silver nanocluster-gold nanocage aptamer electrochemiluminescence detection system, as shown in Figure (A). Figure 10 As shown in (B), it can be seen that in the tacrolimus concentration range of 1~25 ng / mL, ECL = 22.02 C + 840.16; R 2 = 0.9950, concentration in the range of 25~100 ng / mL, ECL=9.808C+1157.8; R 2 =0.9988. Using the formulas LOD=3.3 σ / S and LOQ=10 σ / S (σ is the standard deviation of the response values ​​in 10 blank solutions, and S is the slope), the LOD was calculated to be 6.38 × 10. -3 ng / mL (7.94 f mol / L), LOQ is 19.33×10 -3 The result was ng / mL (24.0 fmol / L), which demonstrates the excellent sensitivity of the gold-silver nanocluster-gold nanocage aptamer electrochemiluminescence detection system.

[0067] (2) Reproducibility, repeatability and stability Under optimal conditions, six electrodes from the same batch were used to repeatedly detect 100 ng / mL tacrolimus. The results were as follows: Figure 10 As shown in (C), the RSE of the six detections was 3.04%, indicating that the electrochemiluminescence detection system of gold-silver nanoclusters-gold nanocage aptamers has good repeatability.

[0068] Two batches of electrodes manufactured on different dates (June 14, 2024 and February 13, 2025) were used to detect tacrolimus at concentrations of 1, 5, and 50 ng / mL, respectively. The results for the three groups are as follows: Figure 10 As shown in (D), the RSD of the group with the largest concentration difference between the two different batches of electrodes was 2.18%, indicating that the gold-silver nanocluster-gold nanocage aptamer electrochemiluminescence detection system has good reproducibility. The screen-printed electrode with the gold-silver nanocluster-gold nanocage-aptamer composite was placed in a petri dish and stored at 4°C without any other special treatment. The ECL value of 100 ng / mL tacrolimus stock solution was measured at different storage days. The ECL value measured on the seventh day was 98% of that on the first day. Figure 10 The (E) in the figure suggests the good stability of the gold-silver nanocluster-gold nanocage aptamer electrochemiluminescence detection system.

[0069] (3) Selectivity To meet the needs of detecting tacrolimus concentrations in blood under various scenarios, especially in blood, the anti-interference capability of the gold-silver nanoclusters-gold nanocage aptamers electrochemiluminescence detection system of this invention is crucial. This invention investigated common interfering substances in the blood matrix, such as dopamine, glucose, ascorbic acid, and two common immunosuppressive drugs, mycophenolic acid and rapamycin. A 1 μM anti-interference solution was added to the screen-printed electrode surface of the pre-coated gold-silver nanoclusters-gold nanocage aptamers electrochemiluminescence detection system solution. After waiting 40 min, excess solution was aspirated, and the system was slowly rinsed twice. Changes in ECL were then detected. Figure 10 As shown in (F), the above interfering substances have a minimal impact on ECL. It is worth noting that 1 μM rapamycin causes a slight increase in ECL signal, but this increase is less than that caused by 1 ng / mL (1.24 nM) tacrolimus. Furthermore, clinically, rapamycin and tacrolimus are usually not used simultaneously due to their antagonistic effects. Therefore, the specificity of the gold-silver nanocluster-gold nanocage aptamer electrochemiluminescence detection system can meet the clinical needs for tacrolimus concentration detection.

[0070] Example 4 This invention utilizes a standard addition method to add different amounts of tacrolimus to human whole blood and calculates the recovery rate. The accuracy of the Au-AgNCs@Aucages-Apt122 electrochemiluminescence sensor method is verified through three parallel assays. The recovery rates of Au-AgNCs@Aucages-Apt122 are shown in Table 1. As can be seen from Table 1, the tacrolimus concentration measured in the biological samples is in good agreement with the added concentration.

[0071] Table 1 Recovery rates of Au-AgNCs@Aucages-Apt122

[0072] In addition, the obtained discarded human samples were tested and compared with the results of chemiluminescence immunoassay (CMIA) at the hospital. The results are shown in Table 2. Table 2. Detection of real samples by Au-AgNCs@Aucages-Apt122

[0073] The results above show that the electrochemiluminescence sensor provided by this invention has a high recovery rate (97.2~102.8%) and a low relative standard deviation (RSD<5.0%, n=3), verifying that the Au-AgNCs@Aucages aptamer electrochemiluminescence detection method can reliably quantify tacrolimus.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers, characterized in that, It includes an electrode, a gold nanocage loaded on the surface of the electrode, gold and silver nanoclusters located within the gold nanocage, and a tacrolimus aptamer chemically linked to the surface of the gold nanocage via amide bonds.

2. The electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters according to claim 1, characterized in that, The electrodes include screen-printed electrodes.

3. The electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters according to claim 1 or 2, characterized in that, The gold nanocage has a particle size of 60-80 nm; the molar ratio of gold to silver in the gold-silver nanoclusters is 1-2:1, and the particle size of the gold-silver nanoclusters is 2-3 nm.

4. The method for preparing the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Gold nanocages were mixed with lipoic acid solution and incubated for the first time to obtain lipoic acid-modified gold nanocages. The thioctic acid-modified gold nanocages, carboxyl activator, and buffer solution were mixed to carry out a carboxyl activation reaction. Then, a polylysine solution was added to carry out an amidation reaction to obtain polylysine-modified gold nanocages. The polylysine-modified gold nanocage was mixed with a gold and silver nanocluster solution and subjected to a second incubation to obtain a gold and silver nanocluster-gold nanocage composite. The gold-silver nanocluster-gold nanocage complex was mixed with a tacrolimus aptamer solution and subjected to a third incubation to obtain the gold-silver nanocluster-gold nanocage-aptamer complex; the 5' end of the tacrolimus aptamer was modified with a carboxyl group. An electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters aptamers was obtained by drop-coating a solution of gold-silver nanocluster-gold nanocage-aptamer composite onto the electrode surface.

5. The preparation method according to claim 4, characterized in that, The preparation method of the gold nanocage includes the following steps: Sodium sulfide, HCl, PVP and ethylene glycol were mixed and silver trifluoroacetate solution was added to carry out a precipitation reaction to obtain silver nanocubes. The silver nanocubes were heated and mixed with PVP solution, and HAuCl4 solution was added to carry out a template sacrificial reaction to obtain gold nanocages. The preparation method of the gold and silver nanoclusters includes the following steps: HAuCl4, AgNO3 and water were mixed, and glutathione, inorganic base and ascorbic acid were added in sequence to carry out a reduction reaction to obtain gold and silver nanoclusters.

6. The preparation method according to claim 4, characterized in that, The second incubation temperature is 0~4℃, and the time is 8~12h; After mixing the gold-silver nanocluster-gold nanocage composite with a tacrolimus aptamer solution, the final concentration of the tacrolimus aptamer solution is 30~200 nM; the third incubation temperature is 25~32℃ and the time is 3~8h. The concentration of the gold-silver nanocluster-gold nanocage-aptamer complex solution is 500~4500 ng / mL, and the drop volume is 5~15 μL.

7. The application of the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters according to any one of claims 1 to 3, or the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters prepared by the preparation method according to any one of claims 4 to 6, in the detection of tacrolimus for non-diagnostic purposes.

8. A kit for detecting tacrolimus, characterized in that, The invention includes the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters as described in any one of claims 1 to 3, or the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters prepared by the preparation method described in any one of claims 4 to 6, and potassium persulfate solution.

9. A method for detecting tacrolimus for non-diagnostic purposes, characterized in that, Includes the following steps: The liquid to be tested is applied to the surface of the electrochemiluminescence sensor based on gold nanocages and gold-silver nanoclusters as described in any one of claims 1 to 3, and incubated to obtain the incubated electrochemiluminescence sensor. Potassium persulfate solution was added to the surface of the incubated electrochemiluminescence sensor, and cyclic voltammetry was performed to test the ECL signal of the first peak of electrochemiluminescence of the electrochemiluminescence sensor. The content of tacrolimus in the test liquid was obtained based on the ECL signal and a predetermined standard curve. The standard curve is a linear relationship curve between the ECL signal and tacrolimus concentration.

10. The detection method according to claim 9, characterized in that, The incubation time is 5-60 minutes; The concentration of the potassium persulfate solution is 10~200mM; after adding the potassium persulfate solution, the pH value of the resulting detection system is 3.27~8.13; The scanning voltage of the cyclic voltammetric scan is -2~2V, and the scanning speed is 100~300mV / s.

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