Carbon-based optical-addressable potentiometric sensor for detection of low-density lipoprotein based on opd@ngqds

Abstract

A carbon-based light-addressable potentiometric sensor for detecting low-density lipoprotein based on OPD@NGQDs, which utilizes the good photoelectric conversion ability and high specific surface area of OPD@NGQDs and rGO composite material, LDL aptamer can specifically recognize and bind LDL protein, leading to the change of the surface potential of the sensitive unit of the carbon-based light-addressable potentiometric sensor, and then affecting the size of photocurrent, the current-voltage (I-V) curve produces corresponding offset. According to the size change of I-V offset, the linear relationship between offset and LDL concentration is established, realizing the quantitative detection of LDL with high sensitivity and good selectivity. The method is simple, low cost and has a low detection limit of 6.43 ng / mL.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a method for detecting low-density lipoprotein based on a carbon-based light-addressable potentiometric sensor. BACKGROUND

[0002] Low-density lipoprotein (LDL) is a common marker of cardiovascular and cerebrovascular diseases. Common detection methods mainly include direct method-surfactant removal method, chemical precipitation method, Friedewald indirect calculation method, ultracentrifugation method, electrophoresis method, immunoseparation method, and electrochemical sensor. However, the above-mentioned detection methods all need special experimental equipment, are time-consuming, and are complicated to operate, and are not suitable for routine clinical detection. The patent for invention with publication number CN111269962B develops a low-density lipoprotein kit, and the method involves too many reagents, is time-consuming, and is complicated to operate. The patent for invention with publication number CN105784801B relates to a method for detecting LDL by silver deposition through the catalysis of two enzymes, in which a gold nano-particle and an apolipoprotein B-100 antibody are modified on an electrode, and hydrogen peroxide is generated under the catalysis of cholesteryl esterase and cholesterol oxidase, and then silver ions are deposited, so that the dissolution voltammetric current of silver is detected to realize rapid detection of LDL. However, the antibody LDL used is expensive and has harsh storage conditions. The light-addressable potentiometric sensor (LAPS) is an electrochemical sensor with semiconductor photoelectric properties based on a field effect structure. Due to its simple preparation process, good stability, high sensitivity, and short response time, the LAPS has attracted extensive research and application, and is expected to become a new method for detecting low-density lipoprotein. SUMMARY

[0003] The application provides a method for detecting low-density lipoprotein based on a carbon-based light-addressable potentiometric sensor (C-LAPS) with an o-phenylenediamine@nitrogen-doped graphene quantum dot (OPD@NGQDs) as a photosensitive layer and reduced graphene oxide (rGO) as a light-addressable potentiometric sensor (C-LAPS) with an indium tin oxide conductive glass (ITO) as a carrier substrate. First, an o-phenylenediamine@nitrogen-doped graphene quantum dot (OPD@NGQDs) with high quantum fluorescence efficiency is designed and synthesized; the OPD@NGQDs, rGO, and low-density lipoprotein aptamer (LDL Apt ) are modified on the ITO glass to form a biological sensitive unit, and a light-addressable potentiometric sensor capable of specifically recognizing low-density lipoprotein in serum is designed. The method has high stability, high sensitivity, short detection time, and requires a small amount of sample. The method shows a good linear relationship when the LDL concentration is 0.01-0.15 μg / mL, and the detection limit can reach 6.43 ng / mL.

[0004] In order to solve the problem: the C-LAPS test substrate, light source drive circuit, signal amplification circuit and LabVIEW platform constitute the LAPS real-time test system. OPD@NGQDs, rGO and LDL Apt Layer by layer modification on the ITO glass substrate forms a biological sensitive unit, and the LDL solution is added on the biological sensitive unit to form a C-LAPS test substrate. The C-LAPS test substrate is placed in the LAPS test system, and under the action of the excitation light source and the bias voltage, the OPD@NGQDs generates a detectable photocurrent, and the LDL on the C-LAPS test substrate is specifically combined with LDL Apt , resulting in a change in the surface potential of the sensitive unit, thereby affecting the size of the photocurrent, and thus the I-V curve produces a corresponding offset. According to the size change of the I-V offset, a linear relationship between the offset and the LDL concentration is established, and the quantitative detection of LDL with high sensitivity and good selectivity is realized.

[0005] The present application is carried out according to the following steps:

[0006] Step 1: Preparation of OPD@NGQDs

[0007] (1) Preparation of nitrogen-doped graphene quantum dots (NGQDs): A certain amount of citric acid (CA) and urea (Uric) are dissolved in water in a certain proportion, and stirred until completely dissolved. Then the mixed solution is transferred to a reaction kettle and heated for a period of time, and after the reaction is completed, it is cooled to room temperature. Centrifugal washing with a large amount of ethanol and drying to obtain NGQDs powder.

[0008] (2) Preparation of OPD@NGQDs: A certain amount of NGQDs powder and o-phenylenediamine (OPD) are dissolved in N,N-dimethylformamide (DMF) solvent in a certain proportion, and stirred until OPD is completely dissolved. Then the mixed solution is transferred to a reaction kettle and heated for a period of time, and after the reaction is completed, it is cooled to room temperature. A large amount of pure water is added to the solution, and transferred to a rotary evaporator to remove DMF from the solution. The solution after removing DMF is dialyzed with a dialysis bag to remove impurities, and then freeze-dried to obtain OPD@NGQDs powder.

[0009] Step 2: Construction of C-LAPS test substrate sensitive unit

[0010] (1) Preparation of reduced graphene oxide (rGO): A certain amount of graphene oxide (GO) powder is added to pure water and broken using an ultrasonic cell crusher to form a suspension, and then a certain amount of ascorbic acid (AA) is added and magnetically stirred to obtain an rGO solution.

[0011] (2) OPD@NGQDs / rGO / ITO electrode assembly: First, the ITO glass was immersed in acetone, ethanol and pure water respectively and cleaned by ultrasonic, and finally washed with pure water. Then, the ITO glass was immersed in a mixed solution of hydrogen peroxide, ammonia and pure water for a period of time. The activated ITO electrode was immersed in 3-aminopropyl triethoxysilane (APTES) to make the surface aminosilane, and then cleaned. Finally, the ITO electrode was activated by carbodiimide / N-hydroxysuccinimide (EDC / NHS); rGO solution, OPD@NGQDs solution were added to the surface of ITO glass in turn, and incubated in a constant temperature incubator for a period of time, then washed with water to increase the photocurrent size and increase the loading of LDL Apt of OPD@NGQDs / rGO / ITO electrode.

[0012] (3) Construction of C-LAPS test substrate sensitive unit: LDL Apt was added to the OPD@NGQDs / rGO / ITO electrode, and incubated in a constant temperature incubator for a period of time. Non-specific sites were blocked with bovine serum albumin (BSA), and then washed with phosphate buffered saline (PBS) to obtain LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit, which was dried for standby use.

[0013] Step 3: Preparation of LDL standard curve

[0014] (1) Different concentrations of LDL standard solution were added to the surface of LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit, and incubated in a constant temperature incubator for a period of time to form a C-LAPS test substrate.

[0015] (2) The C-LAPS test substrate was placed in a test pool containing phosphate buffered saline (PBS), and then introduced into the LAPS real-time test system. Under the action of excitation light source and bias voltage, the current-voltage (I-V) curve produced a corresponding shift, and the voltage shift was recorded.

[0016] (3) According to the relationship between the voltage shift value of the LAPS system and the concentration of LDL, a standard curve was drawn, and the minimum detection limit of the method was calculated.

[0017] Step 4: Detection of LDL in actual serum samples

[0018] (1) Actual serum sample solution was added to the surface of LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit, and incubated in a constant temperature incubator for a period of time to form a C-LAPS test substrate.

[0019] (2) Place the C-LAPS test substrate into a test cell containing phosphate-buffered saline (PBS) and import it into the LAPS real-time testing system. Under the action of the excitation light source and bias voltage, the IV curve will produce a corresponding shift, and the voltage shift will be recorded.

[0020] (3) Calculate the concentration of LDL in the serum sample based on the LDL standard curve obtained in step 3.

[0021] Furthermore, in step 1, the molar ratio of citric acid to urea is 1:3.

[0022] Furthermore, in step 1, the temperature in the reactor is 160°C and the time is 4 hours.

[0023] Furthermore, in step 2, the volume ratio of hydrogen peroxide, ammonia, and pure water is 1:1:5.

[0024] Furthermore, in step 2, the concentration of the rGO solution is 1.0 mg / mL; the concentration of the NaOH solution is 1.0 mol / L; and the concentration of EDC / NHS is 0.1 mol / L.

[0025] Furthermore, in step 2, LDL Apt The base sequence is 5′-C6-ACCTCGATTTTATATTATTTCGCTTACCAACAACTGCAGA-3′, LDL Apt The concentration was 0.5 μM, the incubation temperature was 25 °C, and the time was 30 min.

[0026] Furthermore, in step 2, the concentration of BSA is 1%, the incubation temperature is 25°C, and the incubation time is 30 minutes.

[0027] Furthermore, in steps 3 and 4, the light source wavelength is 405nm, and the bias voltage scanning range is -2.0V to 2.0V.

[0028] Preferably, the incubation temperature of LDL (or serum) in steps 3 and 4 is 25°C, the incubation time is 80 min, the concentration of PBS is 0.1 mol / L, and the pH value is 6.5.

[0029] In step 1, a green fluorescent OPD@NGQDs with high quantum fluorescence yield was prepared. OPD@NGQDs exhibit high photocurrent conversion capability, improving the sensitivity of the testing system. In step 2, the high specific surface area of ​​rGO enhances the specific capacitance of the sensor and allows for loading more LDL. Apt Using LDL Apt Using LDL as a sensitive unit in the C-LAPS assay substrate constructed for recognizing molecules can improve the selectivity of the system. Step 3 utilizes LDL and LDL AptThe specific binding of LDL causes the change of the space structure of the sensitive unit of the C-LAPS test substrate, resulting in the change of the surface potential of the sensitive unit, and then affecting the size of the photocurrent, so that the I-V curve produces a corresponding offset, and the quantitative detection of LDL is realized through the voltage offset. The LDL calibration curve of step 3 provides a calculation basis for the determination of the concentration of LDL in the actual serum sample of step 4. It can be seen that steps 1-4 support each other and work together to establish a C-LAPS for high-selectivity detection of LDL by using the photoelectric conversion ability of OPD@NGQDs / rGO.

[0030] The present application has the following advantages over the prior art:

[0031] 1. The method uses the high fluorescence performance of OPD@NGQDs, the large specific surface area and loading capacity of rGO to develop a simple and efficient carbon-based photosensitive layer, and combines high-affinity LDL Apt , and successfully prepares a C-LAPS based on OPD@NGQDs, providing a new method for the detection of LDL in serum.

[0032] 2. The sensor uses LDL aptamer as a recognition probe to detect LDL, has the characteristics of small background interference, and can effectively improve the detection accuracy. The sensor has good specificity, stability and reproducibility, and can specifically detect the LDL level in serum, with a minimum detection limit of 6.43 ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Principle diagram of C-LAPS based on OPD@NGQDs for detecting LDL;

[0034] Figure 2 (A) NGQDs transmission electron microscope (TEM) image, (B) OPD@NGQDs TEM image;

[0035] Figure 3 Fluorescence spectra of NGQDs and OPD@NGQDs;

[0036] Figure 4 SEM characterization diagram of C-LAPS based on OPD@NGQDs at different stages, (A) ITO, (B) APTES / ITO, (C) rGO / APTES / ITO, (D) OPD@NGQDs / rGO / APTES / ITO, (E) LDL Apt / OPD@NGQDs / rGO / APTES / ITO, (F) LDL / LDL Apt / OPD@NGQDs / rGO / APTES / ITO;

[0037] Figure 5I-V curves of C-LAPS under different LDL concentrations. DETAILED DESCRIPTION

[0038] The application will be described in detail below with reference to the drawings and specific embodiments.

[0039] A method for detecting LDL based on OPD@NGQDs C-LAPS, the detection schematic diagram is shown in Figure 1. First, on the surface of the activated ITO, amino silanization is performed using APTES to make the surface contain a large number of amino groups. Then rGO, OPD@NGQDs, LDL Apt are assembled layer by layer on the amino-silanized ITO surface to construct LDL Apt / OPD@NGQDs / rGO / APTES / ITO sensitive unit. Among them, OPD@NGQDs / rGO has the advantages of high photoelectric conversion efficiency, large specific surface area, high specific capacitance and high conductivity. Drop LDL solution on the biological sensitive unit to form a C-LAPS test substrate. Put the C-LAPS test substrate into the LAPS test system. Under the action of excitation light source and bias voltage, OPD@NGQDs generates a detectable photocurrent. LDL on the C-LAPS test substrate specifically binds with LDL Apt , resulting in a change in the surface potential of the sensitive unit, which in turn affects the size of the photocurrent, so the current-voltage (I-V) curve produces a corresponding shift. According to the size change of the I-V shift, a linear relationship between the shift and the LDL concentration is established to realize the quantitative detection of LDL with high sensitivity and good selectivity. The implementation steps are as follows:

[0040] 1. Preparation of OPD@NGQDs

[0041] (1) Dissolve 2.1 g (1 moL) of citric acid and 1.8 g (3 moL) of urea in a ratio of 1:3 in 10 mL of pure water, and place it in a water bath at a temperature of 60°C for heating and stirring until it becomes a transparent solution.

[0042] (2) Then transfer the transparent solution to a 20 mL high-pressure reaction kettle, heat to 160°C and keep for 8 h, then cool to room temperature. Then add anhydrous ethanol to the solution, then put it into a high-speed refrigerated centrifuge, centrifuge at a speed of 6000 r / min for 5 min, then extract the supernatant to separate it from the precipitate, continue for several times until no precipitate is produced, freeze-dry the obtained solution to obtain NGQDs. Figure 2 A is the transmission electron microscope image of NGQDs, and NGQDs show good dispersibility with a particle size below 10 nm.

[0043] (3) 10 mg NGQDs powder was dissolved in 50 mg o-phenylenediamine in 20 mL N,N-dimethylformamide by stirring until completely dissolved, and then transferred to a 20 mL high-pressure reactor, heated to 200 °C and kept for 12 h, and then cooled to room temperature. The reaction product was transferred to a rotary evaporator, and a large amount of pure water was added, heated to 70 °C to remove the solvent to obtain an OPD@NGQDs solution. The OPD@NGQDs solution was placed in a high-speed refrigerated centrifuge, centrifuged at 9000 r / min for 5 min, and the precipitate was removed, repeated several times until no precipitate was produced, and the resulting clear solution was freeze-dried to obtain OPD@NGQDs powder. The OPD@NGQDs powder was dissolved in pure water to prepare a 0.5 μg / mL OPD@NGQDs solution.

[0044] Figure 2 B is a transmission electron micrograph of OPD@NGQDs, the particle size of OPD@NGQDs is about 5 nm, and good dispersibility is exhibited.

[0045] 2. Construction of a sensing unit of a C-LAPS test substrate

[0046] (1) 10 mg GO was ultrasonically dispersed in 10 mL pure water. 100 mg ascorbic acid was added to the GO dispersion liquid, and placed on a magnetic stirrer for stirring for 12 h, and finally the mixture was transferred to a high-speed centrifuge and centrifuged at 6000 r / min for 5 min, the supernatant was removed, and the obtained precipitate was dried in a drying oven at 60 °C for 2 h to obtain rGO black powder. The rGO powder was dispersed in pure water to prepare a 1.0 mg / mL rGO dispersion liquid.

[0047] (2) The ITO electrode was ultrasonically cleaned by immersing it in acetone, ethanol and pure water, respectively, and finally washed with pure water. The ITO electrode was immersed in a mixed solution of hydrogen peroxide, ammonia water and pure water in a volume ratio of 1:1:5 for a period of time for activation. The activated ITO was immersed in APTES (1%, 20 mL) to silanize the surface with amino groups.

[0048] (3) Under the action of EDC / NHS (0.1 M, 10 μL) coupling agent, 10 μL of rGO dispersion liquid was added to the surface of the aminated ITO electrode, dried to form an rGO / ITO electrode. 10 μL of OPD@NGQDs solution was added to the surface of the rGO / ITO electrode to form an OPD@NGQDs / rGO / ITO electrode.

[0049] (4) 5 μL LDL AptThe solution was added to the OPD@NGQDs / rGO / ITO electrode and incubated at 25°C for 30 min. After the end, non-specific sites were blocked using BSA (1%, 5 μL), washed, and dried to obtain the C-LAPS test substrate sensitive unit.

[0050] The electrode construction process was characterized by a scanning electron microscope (SEM) SU8020 produced by Hitachi, Japan, as shown in Figure 4 . Figure 4 A is untreated ITO, and the electrode surface is smooth and flat. Figure 4 B is APTES / ITO, and small particles uniformly distributed on the surface appear. Figure 4 C is rGO / APTES / ITO, and obvious wrinkle-like structures can be seen. Figure 4 D is OPD@NGQDs / rGO / ITO, and the electrode surface shows the presence of particles of about 100 nm, which may be formed after the agglomeration of OPD@NGQDs. Figure 4 E is LDL Apt / OPD@NGQDs / rGO / ITO, and the electrode surface has wrinkles, particles, and strips, indicating that LDL Apt has successfully adhered to the electrode surface to form new structures, which indicates that the C-LAPS test substrate sensitive unit has been successfully prepared.

[0051] 3. Preparation of the LDL working curve

[0052] (1) On the C-LAPS test substrate sensitive unit prepared in step 2, 5 μL of LDL protein solution of different concentrations (0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.15 μg / mL, 0.2 μg / mL) was added, and incubated at 25°C for 80 min. to form a C-LAPS test substrate. Figure 4 F is the SEM image of the C-LAPS test substrate. Compared with Figure 4 E, the test substrate surface has a cluster structure, which is due to the reaction of LDL and LDL Apt to form a unique structure.

[0053] (2) The C-LAPS test substrate was placed in a ring-shaped test pool containing 300 μL of phosphate buffer (PBS, 0.1 mol / L, pH 6.5), and then the lower end of the Ag / AgCl reference electrode and the platinum wire counter electrode were immersed in the buffer. Then the electrode and test pool were introduced into the LAPS real-time test system, and the C-LAPS test substrate was connected to the working electrode of the C-LAPS system,

[0054] (3) Start the irradiation light source, and apply a bias voltage between the working electrode and the reference electrode (-0.6V-2.0V), record the I-V curve and the voltage offset. The I-V curve of the C-LAPS for the detection of different LDL concentrations is shown in Figure 2. Figure 5 As shown in Figure 2, with the increase of the LDL concentration, the I-V curve is shifted to the left, and the voltage offset value gradually increases, showing a positive correlation.

[0055] (4) According to the relationship between the voltage offset value of the LAPS system and the LDL concentration, the working curve is drawn. When the LDL protein concentration is in the range of 0.01 μg / mL-0.15 μg / mL, the relationship between the offset of the carbon-based LDL aptamer light-addressable potentiometric sensor and the LDL concentration is linear, and the working curve is ΔV=1819.42C-11.33 (where ΔV is the voltage offset of the I-V curve, and C is the LDL concentration), and the correlation coefficient R 2 =0.9902, and the lowest detection limit of the sensor is 6.43 ng / mL.

[0056] 4. Detection of LDL in actual serum samples

[0057] (1) Collect serum samples with known LDL concentrations. During the collection of the samples, the requirements of the Ethics Committee of the 924 th Hospital of the Chinese People's Liberation Army in Guangxi Zhuang Autonomous Region were strictly followed to ensure the quality and safety of the samples.

[0058] (2) Dilute the serum samples 10000 times with ultrapure water, and select nine serum samples with concentrations of 0.1275 μg / mL, 0.1082 μg / mL, 0.1237 μg / mL, 0.1542 μg / mL, 0.1469 μg / mL, 0.1550 μg / mL, 0.0817 μg / mL, 0.0806 μg / mL, and 0.0815 μg / mL. Detect the concentration of LDL in the serum according to the method of detecting the LDL standard solution in step 3, and record the voltage offset in the I-V curve. Each serum sample is tested three times.

[0059] (3) According to the LDL standard curve ΔV=1819.42C-11.33 obtained in step 3, the corresponding LDL concentration in the actual serum sample can be calculated, and the detection results are shown in Table 1. The relative error between the LDL concentration measured by the constructed C-LAPS and the LDL concentration measured clinically in the serum is between 0.91%-7.89%, and the relative standard deviation is 1.16%-8.92%, which reflects good detection performance. The results show that the sensor can be used for the detection of clinical serum samples.

[0060] Table 1 Results of C-LAPS detection of LDL in actual serum samples

[0061]

[0062] (Note: The LDL concentration in serum samples was determined by the direct method-surfactant clearing method by the 924th Hospital of the Chinese People's Liberation Army Joint Logistics Support Force.)

Claims

1. A method for detecting low-density lipoprotein for non-diagnostic and / or therapeutic purposes based on o-phenylenediamine@nitrogen-doped graphene quantum dots (OPD@NGQDs), characterized by: The following steps are taken: Step 1: Preparation of OPD@NGQDs (1) Preparation of nitrogen-doped graphene quantum dots NGQDs: A certain amount of citric acid and urea are dissolved in water in a certain proportion and stirred to dissolve; then the mixed solution is transferred to a reaction kettle and heated for a period of time, and cooled to room temperature after the reaction is completed; centrifugal washing with a large amount of ethanol and drying to obtain NGQDs powder; (2) Preparation of o-phenylenediamine@nitrogen-doped graphene quantum dots OPD@NGQDs: Dissolve NGQDs powder and o-phenylenediamine OPD in N,N-dimethylformamide DMF solvent in a certain proportion, stir until OPD is completely dissolved; then transfer the mixed solution to a reaction kettle and heat for a period of time, and cool to room temperature after the reaction is completed; add a large amount of pure water to the solution, and transfer to a rotary evaporator to remove DMF from the solution; dialysis bag dialysis is used to remove impurities from the solution after removing DMF, and freeze-drying is used to obtain OPD@NGQDs powder; Step 2: Construction of C-LAPS test substrate sensitive unit (1) Preparation of reduced graphene oxide rGO: Take a certain amount of graphene oxide GO powder, add pure water and use an ultrasonic cell crusher to break it into a suspension, then add a certain amount of ascorbic acid and stir magnetically to obtain a rGO solution; (2) Assembly of OPD@NGQDs / rGO / ITO electrode: First, immerse the ITO glass in acetone, ethanol and pure water and ultrasonically clean it, and finally rinse it with pure water; then, immerse the ITO glass in a mixed solution of hydrogen peroxide, ammonia and pure water in a certain proportion and activate it for a period of time; immerse the activated ITO electrode in 3-aminopropyltriethoxysilane (APTES) to silanize the surface amino group, and clean it; finally, activate the ITO electrode with carbodiimide / N-hydroxysuccinimide EDC / NHS; add the rGO solution and OPD@NGQDs solution to the surface of the ITO glass in turn, incubate, and wash with water to obtain the OPD@NGQDs / rGO / ITO electrode; (3) Construction of sensitive unit of C-LAPS test substrate: drop LDL on OPD@NGQDs / rGO / ITO electrode Apt Incubate, block non-specific sites with bovine serum albumin BSA, and wash with phosphate buffer PBS to obtain LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit, and air dry for standby use; Step 3: Drawing of LDL standard curve (1) in LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit surface drop of different concentrations of LDL standard solution, incubation, C-LAPS test substrate formation; (2) Place the C-LAPS test substrate in a PBS test pool and introduce it into the LAPS real-time test system; under the action of excitation light source and bias voltage, the current-voltage I-V curve produces a corresponding shift, and the voltage shift is recorded; (3) According to the relationship between the voltage shift value of the LAPS system and the concentration of LDL, a standard curve is drawn, and the minimum detection limit of the method is calculated; Step 4: Detection of LDL in actual serum samples (1) in LDL Apt / OPD@NGQDs / rGO / ITO sensitive unit surface drop of actual serum samples, incubation, C-LAPS test substrate formation (2) Place the C-LAPS test substrate in a PBS test pool and introduce it into the LAPS real-time test system; under the action of excitation light source and bias voltage, the I-V curve produces a corresponding shift, and the voltage shift is recorded; (3) According to the LDL standard curve obtained in step 3, calculate the concentration of LDL in the serum sample.

2. The method of claim 1, wherein: The mass ratio of citric acid to urea in step 1 is 2.1:1.8, the reaction temperature is 160℃, and the reaction time is 8h; the mass ratio of NGQDs to OPD is 1:5, the reaction temperature is 200℃, and the reaction time is 12h.

3. The method of claim 1, wherein: LDL in step 2 Apt The base sequence of LDL is 5'-C6-ACCTCGATTTTATATTATTTCGCTTACCAACAACTGCAGA-3', and the concentration of LDL is 0.5 μM. Apt The base sequence of LDL is 5'-C6-ACCTCGATTTTATATTATTTCGCTTACCAACAACTGCAGA-3', and the concentration of LDL is 0.5 μM.

4. The method of claim 1, wherein: The incubation temperature in step 2 is 25℃, the incubation time is 30 minutes, and the pH of PBS is 6.

5.

5. The method of claim 1, wherein: The incubation temperature in step 3 is 25℃, the incubation time is 80 minutes, and the pH of PBS is 6.5.

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