CN115753716A - Fluorescence biosensor for detecting Golgi protein 73 - Google Patents

Abstract

A fluorescent biosensor for GP73 detection based on fluorescence resonance energy transfer, using GP73 aptamers as recognition probes, GP73 aptamers can specifically recognize and bind GP73 proteins, based on nitrogen and sulfur doped graphene quantum dots Based on the principle of fluorescence resonance energy transfer between N,S‑GQDs‑GP73 aptamer and molybdenum disulfide@reduced graphene oxide MoS ₂ @RGO, a fluorescent biosensor for detecting GP73 was established to detect GP73 in human serum content. The method simplifies the entire detection process based on the one-step reaction principle, and has short detection cycle time, low cost and strong experimentability.

Description

A Fluorescent Biosensor for Detecting Golgi Protein 73

technical field

The invention belongs to the technical field of optical sensing, in particular to a fluorescent biosensor for GP73 detection based on fluorescence resonance energy transfer.

Background technique

Golgi protein 73 (GP73), also known as Golgi membrane protein Ⅰ or Golgi phosphorylated protein 2, is a transmembrane protein expressed in the Golgi apparatus of epithelial cells discovered in recent years. Invention patent CN 114113604A discloses a method for detecting the marker GP73 based on flow fluorescence technology, using magnetic fluorescent coded microspheres as a carrier, coupling the fluorescent marker with the target antibody to form a fluorescent immune complex, and detecting the complex However, the preparation of magnetic fluorescent-encoded microspheres used in this method is cumbersome and inconvenient to operate. Invention patent CN 111273033A discloses a assay kit for Golgi protein 73, which combines magnetic particle technology with acridinium ester labeling technology to achieve chemiluminescent assay of the target. In this technology, the GP73 antibody is coupled with the target Linking requires a lower temperature environment and a longer incubation time. Fluorescence resonance energy transfer (FRET) refers to the phenomenon of energy transfer between the two when the emission wavelength of a fluorescent molecule overlaps with the excitation wavelength of another fluorescent substance. It is necessary to construct novel fluorescent biosensors with high detection accuracy relying on the FRET phenomenon.

Contents of the invention

The technical problem to be solved _by the present invention is to provide a kind of fluorescence resonance energy transfer ( FRET) fluorescent biosensor for GP73 detection.

In order to solve this technical problem, N,S-GQDs were used as fluorescent substances, and N,S-GQDs were combined with aminated GP73 aptamer (GP73 _Apt ) through amide bonds to form fluorescently labeled N,S-GQDs-GP73 _Apt complex. When MoS ₂ @RGO is added to the N,S-GQDs-GP73 _Apt complex, the N,S-GQDs-GP73 _Apt complex is combined with MoS ₂ @RGO through van der Waals force and π-π conjugation, and the fluorescence resonance energy occurs When FRET is transferred, the fluorescence intensity of the whole system will become lower, forming N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor; after adding GP73 protein, due to the specificity of GP73 _Apt to GP73, GP73 preferentially interacts with N ,S-GQDs-GP73 _Apt combined to form N,S-GQDs-GP73 _Apt -GP73 complex, which was detached from the bottom surface of MoS ₂ @RGO, interrupting the fluorescence resonance energy transfer, so that the fluorescence of N,S-GQDs-GP73 _Apt was obtained recover. According to the change of the recovery degree of the fluorescence intensity in the system, the linear relationship between the concentration of GP73 and the change of the fluorescence intensity of N,S-GQDs-GP73 _Apt was established, and the rapid, sensitive and selective quantitative detection of GP73 was realized.

The present invention carries out according to the following steps:

Step 1: Preparation of fluorescence resonance donor N,S-GQDs-GP73 _Apt

(1) Preparation of N,S-GQDs: Weigh citric acid and thiourea, add pure water to constant volume, stir evenly, heat at high temperature for a certain period of time, add ethanol after cooling, mix and stir, after stirring completely, dialyze for a period of time, and The dialyzed solution was freeze-dried to obtain N,S-GQDs solid;

(2) Preparation of N,S-GQDs-GP73 _Apt : Measure N,S-GQDs and GP73 aptamer GP73 _Apt , use EDAC/NHS cross-linking agent to activate cross-linking, at room temperature and under the condition of avoiding light, Stir and incubate for a certain period of time to obtain a N,S-GQDs-GP73 _Apt solution.

Step 2: Construction of fluorescent biosensor based on fluorescence resonance energy transfer

(1) Weigh the MoS ₂ powder, add N,N-dimethylformamide solution (DMF) to constant volume, put it into an ultrasonic cell crusher, and crush it until the MoS ₂ powder is completely dispersed in the DMF to obtain the MoS ₂ dispersion liquid;

(2) Weigh graphene oxide (GO) powder, add pure water to constant volume, put it into an ultrasonic cell crusher, and crush it until the GO powder is completely dispersed in pure water to obtain a GO dispersion; add Ascorbic acid (AA), after stirring and mixing for a certain period of time, a redox graphene (RGO) dispersion is obtained;

(3) The MoS ₂ dispersion and the RGO dispersion were mixed and stirred for a certain period of time to obtain a MoS ₂ @RGO solution. The obtained solution was centrifuged, and the separated solid was washed and dried to obtain MoS ₂ @RGO. Weigh MoS ₂ @RGO, add pure water and stir to get MoS ₂ @RGO dispersion;

(4) Mix the MoS ₂ @RGO solution and the N,S-GQDs-GP73 _Apt solution, mix and incubate for a period of time to quench the fluorescence of N,S-GQDs and form N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor. Scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368nm, measure the fluorescence intensity at 450nm, and record it as F ₀ .

Step 3: Drawing of GP73 working curve

(1) Add different concentrations of GP73 solutions to the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor, incubate at a certain temperature for a period of time, and detect the peak change at 450nm; use a fluorescence spectrophotometer Carry out scanning, fix the excitation wavelength at 368nm, measure the fluorescence intensity at 450nm, record it as F ₁ ;

(2) Using (F ₁ -F ₀ )/F ₀ as the ordinate and the concentration of GP73 as the abscissa, draw a working curve to calculate the minimum detection limit of the fluorescent biosensor.

Step 4: Detection of GP73 in real samples

(1) Add the sample to be tested to the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor in step 2, incubate at a certain temperature for a period of time, scan with a fluorescence spectrophotometer, and fix the excitation The wavelength is 368nm, record the fluorescence intensity at 450nm;

(2) According to the working curve of GP73 obtained in step 3, calculate the concentration of GP73 in the sample to be tested.

Further, the amount of citric acid in the step 1 is 2.1g, and the amount of thiourea is 2.3g;

Further, after adding pure water in the step 1, the volume was fixed at 10mL;

Further, heating at 200°C for 160min in the step 1;

Further, the volume of ethanol added in the step 1 is 50mL;

Further, in the step 1, a dialysis bag with a molecular weight of 300 was used for dialysis for 6 hours;

Further, the concentration of N,S-GQDs in the step 1 is 1.0mg/mL;

Further, the DNA sequence of GP73 _Apt in the step 1 is 5′-NH ₂ -C ₆ -GCAGTTGATCCTTTGGATACCCTGG-3′, and the concentration is 1.0 μM;

Further, the crosslinking agent solution in step 1 contains carbodiimide (EDAC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDAC is 0.7668 mg/mL, and the concentration of NHS is 2.1713 mg/mL;

Further, the volume ratio of N,S-GQDs solution, GP73 _Apt solution and crosslinking agent solution in step 1 is 10:10:1;

Further, in the step 1, the incubation temperature is 25°C, and the incubation time is 1h;

Further, in the step 2, the concentration of _MoS2 is 1.0mg/mL, and the concentration of GO is 1.0mg/mL;

Further, the mass ratio of AA added in step 2 to GO is 10:1;

Further, the stirring time after adding AA in the step 2 is 12h;

Further, in the step ₂ , the MoS solution and the RGO solution volume ratio are 1:1

Further, the stirring time after mixing _the MoS solution and the RGO solution in the step 2 is 24h;

Further, in the step 2, the centrifuge is centrifuged at a speed of 5000r/min for 5min;

Further, the concentration of the MoS ₂ @RGO solution in step 2 is 100.0 μg/mL;

Further, the volume ratio of the MoS ₂ @RGO solution and the N,S-GQDs-GP73 _Apt solution in step 2 is 1:1;

Further, the incubation temperature in step 2 is 25°C, and the incubation time is 60 minutes;

Further, the excitation wavelength of the fluorescence spectrophotometer in the step 2, step 3 and step 4 is 368nm, and the emission wavelength is 450nm;

Further, the incubation temperature in step 3 and step 4 is 25° C., and the incubation time is 60 min.

Wherein, step 1 provides a blue fluorescent N,S-GQDs nanomaterial and N,S-GQDs-GP73 _Apt probe, which provide the fluorescent donor of the fluorescence resonance energy transfer reaction system for step 2. Step 2 provided MoS ₂ @RGO nanomaterials as fluorescence energy transfer acceptors; due to the van der Waals force and π-π conjugation between N,S-GQDs-GP73 _Apt and MoS ₂ @RGO, the N,S-GQDs -GP73 _Apt and MoS ₂ @RGO are close to each other, triggering the FRET process, resulting in the fluorescence quenching of the system. Step 3 is a further extension of step 2. When GP73 protein is added to the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent sensor, its original conformation is changed due to the preferential binding of GP73 _Apt to GP73, and N,S-GQDs -The interaction force between GP73 _Apt and MoS ₂ @RGO is greatly weakened. Therefore, N,S-GQDs-GP73 _Apt was separated from MoS ₂ @RGO, and the FRET process was interrupted, thereby restoring the fluorescence of N,S-GQDs-GP73 _Apt . The working curve of GP73 in step 3 provides a calculation basis for the determination of the concentration of GP73 in the actual sample in step 4. It can be seen that steps 1-4 support each other and work together to use the fluorescence resonance energy transfer phenomenon between N,S-GQDs-GP73 _Apt and MoS ₂ @RGO to establish N,S-GQDs-GP73 _Apt /MoS ₂ that can detect GP73 @RGO FRET fluorescent biosensor.

Compared with the prior art, the present invention has the following advantages:

1. Utilize the strong fluorescence quenching ability of MoS ₂ @RGO and the strong fluorescence stability of N,S-GQDs to reduce the error of fluorescent biosensors and improve the accuracy of fluorescent biosensors; MoS ₂ @RGO itself has a high specific surface area and Abundant groups with strong affinity can effectively adsorb more aptamer chains, strengthen the quenching ability of fluorescent donor N,S-GQDs, and improve the detection range of fluorescent biosensors (0.1ng/mL ~10.0 μg/mL). The novel fluorescent biosensor constructed on the principle of fluorescence resonance energy transfer has simple detection process, short detection cycle, wide detection range and low cost.

2. The system uses a fluorescent biosensor constructed with GP73 aptamer to detect GP73. This biodetection sensor has the characteristics of strong anti-interference and strong adaptability to ensure the accuracy of detection.

Description of drawings

Fig. 1 Schematic diagram of the N,S-GQDs/MoS ₂ @RGO FRET fluorescent biosensor based on N,S-GQDs and MoS ₂ @RGO to detect GP73;

Fig. 2 Fluorescence spectra of N, S-GQDs and N, S-GQDs-GP73 _Apt ;

Figure 3A is the TEM image of N,S-GQDs; B is the SEM image of MoS ₂ @RGO;

Figure 4. Fluorescence recovery intensity diagram of N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor under different GP73 concentrations.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

A fluorescent biosensor based on N,S-GQDs/MoS ₂ @RGO fluorescence resonance energy transfer combined with a suitable ligand to detect GP73, the detection principle is shown in Figure 1. The first is to use N,S-GQDs as a fluorescent group, and combine the activated N,S-GQDs with GP73 _Apt with amino groups through CO-NH bonds to form fluorescently labeled N,S-GQDs-GP73 _Apt probes ; Adding MoS ₂ @RGO, due to the strong conjugation between MoS ₂ @RGO and N,S-GQDs, MoS ₂ @RGO and N,S-GQDs-GP73 _Apt are close together, presenting the N ,The fluorescence quenching of S-GQDs-GP73 _Apt ; after adding the target GP73 protein, GP73 _Apt specifically binds to GP73 protein, thus keeping N,S-GQDs-GP73 _Apt away from MoS ₂ @RGO,N,S-GQDs- The fluorescence of GP73 _Apt was restored, and a fluorescent biosensor for detecting GP73 protein was established. Quantitative analysis of GP73 protein can be effectively realized by measuring the change of fluorescence intensity by a fluorescence spectrophotometer. The implementation steps are as follows:

1. Preparation of N,S-GQDs-GP73 _Apt

(1) 2.1g of citric acid and 2.3g of thiourea are fixed to 10mL with pure water, stirred until completely dissolved, put into a high-temperature reaction kettle lined with Teflon, and sent into a blast drying oven Heat at 200°C for 160min.

(2) After cooling the fully reacted solution to room temperature, add 50 mL of ethanol, absorb the solution after fully shaking and stirring evenly, and use a dialysis bag with a molecular weight of 300 for dialysis for 6 hours, and place the solution obtained after dialysis in a freeze dryer for 12 hours to obtain N,S-GQDs solid.

(3) Weigh 10 mg of N,S-GQDs, add pure water to 10 mL to obtain a 1 mg/mL N,S-GQDs solution. Weigh 7.668 mg of carbodiimide (EDAC) and 2.1713 mg of N-hydroxysuccinimide (NHS), mix them, add pure water to 10 mL, and stir evenly to obtain a crosslinking agent solution. Measure 200 μL of N,S-GQDs, 200 μL of 1 μM GP73 _Apt and 20 uL of cross-linking agent solution, mix them evenly, put them in a light-proof environment with a room temperature of 25 °C, shake evenly, and incubate for 1 hour to obtain the fluorescently labeled complex N, S-GQDs-GP73 _Apt solution. Figure 2 shows the fluorescence spectra of N,S-GQDs and N,S-GQDs-GP73 _Apt . The fluorescence spectra of the two are basically the same, and the fluorescence intensity of N,S-GQDs-GP73 _Apt is relatively small, indicating that N,S-GQDs Connected successfully with GP73 _Apt . Figure 3A is a transmission electron microscope image (TEM) of N,S-GQDs, which shows that the prepared N,S-GQDs have uniform size, good dispersion, and a particle size of about 7nm.

2. Construction of fluorescent biosensors based on fluorescence resonance energy transfer

(1) Weigh 30 mg of MoS ₂ solid with a precision electronic balance and put it into a beaker, put it into N,N-dimethylformamide solution (DMF) and set the volume to 30 mL, put it into an ultrasonic cell disruptor and ultrasonically break it for 1 hour , after the crushing is completed, a MoS ₂ dispersion with a concentration of 1 mg/mL is obtained.

(2) Use a precision electronic balance to weigh 30 mg of single-layer graphene oxide (GO) solids into a beaker, add pure water to make the volume 30 mL, then stir the solution completely, put it into an ultrasonic cell disruptor for ultrasonic crushing for 3 hours, and crush After completion, 300 mg of ascorbic acid (AA) was added, placed on a magnetic stirrer and continuously stirred for 12 hours to obtain a RGO dispersion with a concentration of 1 mg/mL.

(3) Put 20mL of RGO dispersion into a 50mL beaker, add 20mL of ultrasonically crushed MoS ₂ dispersion, put it on a magnetic stirrer and continue stirring for 24 hours, so that MoS ₂ is fully adsorbed on RGO; after the stirring is complete, take out the mixed liquid Put it in a centrifuge and centrifuge at a speed of 5000r/min for 5min. After centrifugation, wash the precipitate repeatedly with pure water, and dry it in a vacuum low-temperature drying oven to obtain a black MoS ₂ @RGO solid. Weigh 1mg of MoS ₂ @RGO, add pure water to 10mL to obtain a 100ug/mL MoS ₂ @RGO solution. Figure 3B is the scanning electron microscope image (SEM) of MoS ₂ @RGO. It can be seen from the figure that RGO is well extended in layers with MoS ₂ as the substrate, which increases the surface specific area of the overall material, which is beneficial to N,S- Adsorption of GQDs-GP73 _Apt .

(4) Take 100 μL of 100 μg/mL MoS ₂ @RGO and 100 μL of 1 μM N,S-GQDs-GP73 _Apt and mix, shake evenly and incubate at 25°C for 60 min to obtain N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET Fluorescent biosensors. Scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368nm, measure the fluorescence intensity at 450nm, and record it as F ₀ .

3. Drawing of GP73 working curve

The N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor after the fluorescence intensity was measured in step 2 was evenly divided into 6 groups, and then 200 μL of GP73 protein solution (0.1 ng/mL, 1.0 ng/mL, mL, 10.0ng/mL, 100.0ng/mL, 1.0μg/mL, 10.0μg/mL), shake and mix evenly, react at 25°C for 60min, scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368nm, measure its The fluorescence intensity at 450nm is recorded as F ₁ . The fluorescence spectra of N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensors at different GP73 concentrations are shown in Fig. 4. It can be seen that the fluorescence recovery intensity of the fluorescent biosensors ((F ₁ -F ₀ )/F ₀ ) was positively correlated with the concentration of GP73. When the GP73 protein concentration ranged from 0.1 ng/mL to 10 μg/mL, the relationship between the fluorescence recovery value of the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor and the GP73 concentration was log-linear , the working curve is Y=0.03027lgX+0.012572 (Y represents the fluorescence recovery intensity, X represents the concentration of GP73 protein), and the correlation coefficient is R ² =0.99206.

4. Detection of GP73 in actual serum samples

Prepare three kinds of serum samples, which are the serum of normal people, the serum of liver cirrhosis patients and the serum of liver cancer patients, and the concentration of GP73 obtained by clinical detection GP73 method (enzyme-linked immunosorbent assay ELISA) is 58.85ng/mL, respectively, 106.74ng/mL, 306.32ng/mL. 200 μL of serum was added to the prepared N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor, mixed evenly and incubated for 1 h, then the fluorescence intensity was measured, and each serum sample was measured three times. According to the working curve Y=0.03027lgX+0.012572 obtained in step 3, the concentration of GP73 in the corresponding actual serum sample can be calculated and obtained, and the detection results are shown in Table 1. It can be seen from Table 1 that, compared with the ELISA method, the relative error of the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor for the detection of GP73 in serum is between 0.75% and 7.13%, and the relative standard deviation Between 4.57% and 6.38%, it shows that the fluorescent biosensor can be applied to the detection of actual serum samples.

Table 1 The detection results of GP73 in actual serum samples

(Note: Serum samples came from the Guangxi Key Laboratory of Metabolic Disease Research, the 924th Hospital of the Chinese People's Liberation Army (Guilin, China), and followed the requirements of the Ethics Committee of the Guangxi Key Laboratory of Metabolic Disease Research, the 924th Hospital of the Chinese People's Liberation Army. Sample 1: normal serum, AFP =2.45ng/mL, GP73=58.85ng/mL; sample 2: liver cirrhosis serum, AFP=9.30ng/mL, GP73=106.74ng/mL; sample 3: liver cancer serum, AFP=173.99ng/mL, GP73=306.32 ng/mL).

Claims (7)

1. A fluorescent biosensor based on fluorescence resonance energy transfer for GP73 detection is constructed according to the following steps: Step 1: Preparation of fluorescence resonance donor nitrogen, sulfur doped graphene quantum dots-GP73 aptamer N,S-GQDs-GP73 _Apt (1) Preparation of nitrogen and sulfur doped graphene quantum dots N,S-GQDs: Weigh citric acid and thiourea, add pure water to constant volume, stir evenly, heat, cool; add ethanol to stir, and dialyze with a dialysis bag; The dialyzed solution was freeze-dried to obtain N,S-GQDs solid; (2) Preparation of N,S-GQDs-GP73 _Apt : Measure N,S-GQDs and GP73 aptamer GP73 _Apt , use a cross-linking agent to activate and cross-link, and incubate to obtain N,S-GQDs-GP73 _Apt solution ; Step 2: Construction of fluorescent biosensor based on fluorescence resonance energy transfer (1) Weigh the MoS ₂ powder, add N,N-dimethylformamide DMF solution to constant volume, and disperse the MoS ₂ powder in DMF to obtain the MoS ₂ dispersion; (2) Weigh the graphene oxide GO powder, add pure water to constant volume, and disperse the GO powder in pure water to obtain a GO dispersion; add ascorbic acid AA to the GO dispersion, stir and mix to obtain reduced graphene oxide RGO Dispersions; (3) Mix the MoS ₂ dispersion and the RGO dispersion, and stir for a certain period of time to obtain a molybdenum disulfide@reduced graphene oxide MoS ₂ @RGO solution, centrifuge the obtained solution, wash and dry the separated solid to obtain MoS ₂ @RGO; Weigh the MoS ₂ @RGO solid, add pure water and stir to get the MoS ₂ @RGO dispersion; (4) Mix the MoS ₂ @RGO solution and the N,S-GQDs-GP73 _Apt solution, mix and incubate for a period of time to quench the fluorescence of N,S-GQDs and form N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor; scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368nm, measure the fluorescence intensity at 450nm, and record it as F ₀ ; Step 3: Drawing of GP73 working curve (1) Add different concentrations of GP73 solutions to the N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET fluorescent biosensor, incubate, and detect the peak change at 450nm; scan with a fluorescence spectrophotometer, and fix the excitation wavelength at 368nm, measure the fluorescence intensity at 450nm, denoted as F ₁ ; (3) Use (F ₁ -F ₀ )/F ₀ as the ordinate, and the concentration of GP73 as the abscissa, draw a working curve, and calculate the minimum detection limit of the fluorescent biosensor; Step 4: Detection of GP73 in real samples (1) Add the actual sample to the fluorescent biosensor of N,S-GQDs-GP73 _Apt /MoS ₂ @RGO FRET in step 2, incubate, scan with a fluorescence spectrophotometer, fix the excitation wavelength at 368nm, and record at 450nm the fluorescence intensity; (2) According to the working curve of GP73 obtained in step 3, calculate the concentration of GP73 in the sample to be tested. 2. The fluorescent biosensor according to claim 1, characterized in that: in step 1, the molecular weight of the dialysis bag is 300, and the dialysis time is 6 hours. 3. The fluorescent biosensor according to claim 1, characterized in that: in step 1, the concentration of N, S-GQDs is 1.0 mg/mL; the concentration of GP73 _Apt is 1.0 μM; the cross-linking agent solution contains carbodiimide EDAC With N-hydroxysuccinimide NHS, the concentration of EDAC is 0.7668mg/mL, and the concentration of NHS is 2.1713mg/mL. 4. The fluorescent biosensor according to claim 1, characterized in that: in step 1, the volume ratio of N,S-GQDs solution, GP73 _Apt solution and cross-linking agent solution is 10:10:1, and the incubation time is 1 h. 5. The fluorescent biosensor according to claim 1, characterized in that: the concentration of _MoS2 in step 2 is 1.0 mg/mL; the concentration of GO is 1.0 mg/mL; the mass ratio of AA and GO added is 10:1 ; Stirring time is 12h. 6. The fluorescent biosensor according to claim 1, wherein the volume ratio of the MoS ₂ @RGO solution and the N,S-GQDs-GP73 _Apt solution in step 2 is 1:1. 7. The fluorescent biosensor according to claim 1, characterized in that: the incubation temperature in step 2, step 3 and step 4 is 25°C, and the incubation time is 60min.

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