CN115753716A - Fluorescence biosensor for detecting Golgi protein 73 - Google Patents
Fluorescence biosensor for detecting Golgi protein 73 Download PDFInfo
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Abstract
A fluorescence biosensor for GP73 detection is constructed based on fluorescence resonance energy transfer, a GP73 aptamer is used as an identification probe, the GP73 aptamer can specifically identify and combine GP73 protein, and the fluorescence biosensor is based on a nitrogen and sulfur doped graphene quantum dot N, S-GQDs-GP73 aptamer and molybdenum disulfide @ reductive graphene oxide MoS 2 The principle of fluorescence resonance energy transfer between @ RGO is used for establishing a fluorescence biosensor for detecting GP73, and the fluorescence biosensor is used for detecting the content of GP73 in human serum. The method simplifies the whole detection process according to a one-step reaction principle, and has the advantages of short detection period, low cost and strong experimentability.
Description
Technical Field
The invention belongs to the technical field of optical sensing, and particularly relates to a fluorescence biosensor for GP73 detection based on fluorescence resonance energy transfer.
Background
Golgi protein 73 (GP 73), also known as Golgi membrane protein I or Golgi phosphorylated protein 2, is a transmembrane protein expressed in the Golgi apparatus of epithelial cells which has been discovered in recent years. The invention patent CN 114113604A discloses a method for detecting a marker GP73 based on a flow fluorescence technology, which takes magnetic fluorescence coding microspheres as a carrier, couples a fluorescence marker with a target antibody to form a fluorescence immune complex, and calculates the concentration of the target by detecting the fluorescence intensity of the complex. The invention patent CN 111273033A discloses a determination kit of Golgi protein 73, which combines a magnetic particle technology with an acridinium ester labeling technology to realize chemiluminescence determination of a target substance, wherein a low-temperature environment and a long incubation time are required for coupling a GP73 antibody with the target substance. Fluorescence Resonance Energy Transfer (FRET) refers to a phenomenon of energy transfer between a fluorescent molecule and another fluorescent substance when the emission wavelength of the fluorescent molecule overlaps with the excitation wavelength of the fluorescent substance.
Disclosure of Invention
The invention aims to solve the technical problem of providing a nitrogen-sulfur-doped graphene quantum dot (N, S-GQDs) and molybdenum disulfide @ reductive graphene oxide (MoS) 2 @ RGO) for GP73 detection.
In order to solve the technical problem, N, S-GQDs are used as fluorescent substances, and the N, S-GQDs are combined with an aminated GP73 aptamer (GP 73) Apt ) The fluorescent-labeled N, S-GQDs-GP73 is formed by combining amide bonds Apt And (3) a compound. In N, S-GQDs-GP73 Apt Adding MoS into the compound 2 @RGO，N,S-GQDs-GP73 Apt Composite and MoS 2 The @ RGO is bound to pi-pi conjugation by van der Waals force to cause fluorescence resonance energy transfer FRET, and the fluorescence intensity of the whole system becomes low to form N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescent biosensor; after the addition of GP73 protein, due to GP73 Apt Specificity for GP73, GP73 preferentially binds to N, S-GQDs-GP73 Apt Combine to form N, S-GQDs-GP73 Apt GP73 complex from MoS 2 The @ RGO bottom surface is separated, and the fluorescence resonance energy transfer is interrupted, so that N, S-GQDs-GP73 Apt The fluorescence of (2) is recovered. Establishing GP73 concentration and N, S-GQDs-GP73 according to the change of the recovery degree of the fluorescence intensity in the system Apt The linear relation of the change of the fluorescence intensity realizes the rapid, sensitive and highly selective quantitative detection of GP 73.
The invention is carried out according to the following steps:
step 1: fluorescence resonance donor N, S-GQDs-GP73 Apt Preparation of
(1) Preparation of N, S-GQDs: weighing citric acid and thiourea, adding pure water to a constant volume, uniformly stirring, heating at a high temperature for a certain time, cooling, adding ethanol, mixing and stirring, completely stirring, dialyzing for a period of time, and freeze-drying the dialyzed solution to obtain a solid N, S-GQDs;
(2)N,S-GQDs-GP73 Apt the preparation of (1): measuring N, S-GQDs and GP73 aptamer GP73 Apt Performing activation crosslinking by using an EDAC/NHS crosslinking agent, and stirring and incubating for a certain time under the conditions of room temperature and dark place to obtain N, S-GQDs-GP73 Apt And (3) solution.
Step 2: construction of fluorescence biosensor based on fluorescence resonance energy transfer
(1) Weighing MoS 2 Adding N, N-Dimethylformamide (DMF) solution to constant volume, and crushing in ultrasonic cell crusher to MoS 2 The powder is completely dispersed in DMF to obtain MoS 2 A dispersion liquid;
(2) Weighing Graphene Oxide (GO) powder, adding pure water to a constant volume, and crushing in an ultrasonic cell crusher until the GO powder is completely dispersed in the pure water to obtain a GO dispersion liquid; adding Ascorbic Acid (AA) into the GO dispersion liquid, and stirring and mixing for a certain time to obtain a Redox Graphene (RGO) dispersion liquid;
(3) Mixing MoS 2 Mixing the dispersion and the RGO dispersion, and stirring for a certain time to obtain MoS 2 @ RGO solution, centrifuging the obtained solution, washing the separated solid and drying to obtain MoS 2 @ RGO. Weighing MoS 2 @ RGO, adding pure water, stirring and mixing to obtain MoS 2 @ RGO dispersion;
(4) Mixing MoS 2 @ RGO solution and N, S-GQDs-GP73 Apt Mixing the solutions, standing and incubating for a period of time after mixing to quench the fluorescence of N, S-GQDs to form N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescent biosensor. Scanning with a fluorescence spectrophotometer at a fixed excitation wavelength of 368nm, measuring the fluorescence intensity at 450nm, and recording as F 0 。
And step 3: drawing of GP73 working curve
(1) GP73 solutions of different concentrations were added to N, S-GQDs-GP73 Apt /MoS 2 The @ RGO FRET fluorescence biosensor is incubated and reacted for a period of time at a certain temperature, and the peak value change at 450nm is detected; scanning with a fluorescence spectrophotometer at a fixed excitation wavelength of 368nm, measuring the fluorescence intensity at 450nm, and recording as F 1 ；
(2) With (F) 1 -F 0 )/F 0 The ordinate is the GP73 concentration, and the abscissa is the GP73 concentration, and the minimum detection limit of the fluorescence biosensor is calculated by plotting a working curve.
And 4, step 4: detection of GP73 in real samples
(1) Adding the sample to be tested into the N, S-GQDs-GP73 in the step 2 Apt /MoS 2 The @ RGO FRET fluorescence biosensor is incubated and reacted for a period of time at a certain temperature, a fluorescence spectrophotometer is adopted for scanning, the fixed excitation wavelength is 368nm, and the fluorescence intensity at 450nm is recorded;
(2) And (4) calculating the concentration of GP73 in the sample to be detected according to the working curve of GP73 obtained in the step (3).
Further, the amount of citric acid in the step 1 is 2.1g, and the amount of thiourea is 2.3g;
further, adding pure water in the step 1, and then fixing the volume to 10mL;
further, heating at 200 ℃ for 160min in the step 1;
further, the volume of the ethanol added in the step 1 is 50mL;
further, in the step 1, dialyzing for 6 hours by using a dialysis bag with the molecular weight of 300;
further, the concentration of N, S-GQDs in the step 1 is 1.0mg/mL;
further, the GP73 in the step 1 Apt The DNA sequence of (A) is 5' -NH 2 -C 6 -GCAGTTGA tcctttggatacctgg-3' at a concentration of 1.0 μ Μ;
further, the crosslinking agent solution in the step 1 contains carbodiimide (EDAC) and N-hydroxysuccinimide (NHS), wherein the concentration of the EDAC is 0.7668mg/mL, and the concentration of the NHS is 2.1713mg/mL;
further, the solution of N, S-GQDs, GP73 in the step 1 Apt The volume ratio of the solution to the cross-linking agent solution is 10;
further, the incubation temperature in the step 1 is 25 ℃, and the incubation time is 1h;
further, moS in the step 2 2 The concentration is 1.0mg/mL, and the GO concentration is 1.0mg/mL;
further, the mass ratio of AA to GO added in the step 2 is 10:1;
further, stirring for 12 hours after adding AA in the step 2;
further, moS in the step 2 2 Volume ratio of solution to RGO solution 1
Further, mixing MoS in the step 2 2 Stirring the solution and the RGO solution for 24 hours;
further, the centrifuge in the step 2 centrifuges for 5min at the rotating speed of 5000 r/min;
further, moS in the step 2 2 @ RGO solution concentration is 100.0. Mu.g/mL;
further, moS in the step 2 2 @ RGO solution and N, S-GQDs-GP73 Apt The volume ratio of the liquid is 1;
further, the incubation temperature in the step 2 is 25 ℃, and the incubation time is 60min;
further, the excitation wavelength of the fluorescence spectrophotometer in the step 2, the step 3 and the step 4 is 368nm, and the emission wavelength is 450nm;
further, the incubation temperature in the step 3 and the step 4 is 25 ℃, and the incubation time is 60min.
Wherein step 1 providesA nanometer material of N, S-GQDs emitting blue fluorescence and N, S-GQDs-GP73 Apt And (3) a probe which is used for providing a fluorescence donor of the fluorescence resonance energy transfer reaction system for the step 2. Step 2 provides MoS 2 The @ RGO nano material is used as a fluorescence energy transfer receptor; due to N, S-GQDs-GP73 Apt And MoS 2 Van der Waals' force and pi-pi conjugation exist between @ RGO to make N, S-GQDs-GP73 Apt And MoS 2 @ RGO approaches each other, initiating the FRET process, resulting in quenching of the fluorescence of the system. Step 3 is a further extension of step 2 when GP73 protein is added to N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescent sensor due to GP73 Apt Preferentially bind GP73 and alter its native conformation, N, S-GQDs-GP73 Apt And MoS 2 The interaction between @ RGO is greatly attenuated. Thus, N, S-GQDs-GP73 Apt And MoS 2 @ RGO separation, disruption of FRET process, and thus N, S-GQDs-GP73 Apt The fluorescence of (2) is recovered. The working curve of GP73 in step 3 provides a basis for calculating the concentration of GP73 in the actual sample in step 4. It can be seen that the steps 1-4 are mutually supported and act together to utilize N, S-GQDs-GP73 Apt And MoS 2 Fluorescence resonance energy transfer phenomenon between @ RGO, establishing N, S-GQDs-GP73 capable of detecting GP73 Apt /MoS 2 @ RGO FRET fluorescent biosensor.
Compared with the prior art, the invention has the following advantages:
1. using MoS 2 The fluorescence of @ RGO strong fluorescence quenching ability and N, S-GQDs strong stability reduces the error of the fluorescence biosensor and improves the accuracy of the fluorescence biosensor; moS 2 The @ RGO has high specific surface area and rich groups, has strong affinity, can effectively adsorb more aptamer chains, enhances the quenching capability of fluorescence donors N and S-GQDs, and improves the detection range (0.1 ng/mL-10.0 mu g/mL) of the fluorescence biosensor. The novel fluorescence biosensor constructed by the fluorescence resonance energy transfer principle has the advantages of simple detection process, short detection period, wide detection range and low cost.
2. The system adopts a fluorescence biosensor constructed by a GP73 aptamer to detect GP73, and the biosensor has the characteristics of strong anti-interference and strong adaptability and ensures the detection accuracy.
Drawings
FIG. 1 is based on N, S-GQDs and MoS 2 N, S-GQDs/MoS constructed by @ RGO 2 Schematic diagram of @ RGO FRET fluorescence biosensor for detecting GP 73;
FIG. 2N, S-GQDs and N, S-GQDs-GP73 Apt A fluorescence spectrum of (a);
FIG. 3A is a transmission electron micrograph of N, S-GQDs; b is MoS 2 Scanning electron micrographs of @ RGO;
FIG. 4N, S-GQDs-GP73 at different GP73 concentrations Apt /MoS 2 Graph of fluorescence recovery intensity for @ RGO FRET fluorescent biosensor.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Based on N, S-GQDs/MoS 2 The fluorescence resonance energy transfer of @ RGO is combined with a fluorescence biosensor for detecting GP73 by using a suitable ligand, and the detection principle is shown in figure 1. Firstly, N, S-GQDs are taken as fluorescent groups, and activated N, S-GQDs and GP73 with amino are taken as fluorescent groups Apt The fluorescent marked N, S-GQDs-GP73 is formed by the combination of CO-NH bonds Apt A probe; adding MoS 2 @ RGO due to MoS 2 The strong conjugation exists between @ RGO and N, S-GQDs, so that MoS 2 @ RGO and N, S-GQDs-GP73 Apt In close proximity, exhibit N, S-GQDs-GP73 Apt Quenching the fluorescence of (a); after the target GP73 protein is added, GP73 Apt Binds with GP73 protein specifically, thereby leading N, S-GQDs-GP73 to react Apt Distance from MoS 2 @RGO，N,S-GQDs-GP73 Apt The fluorescence is recovered, and a fluorescence biosensor for detecting GP73 protein is established. The change of fluorescence intensity is measured by a fluorescence spectrophotometer, so that quantitative analysis on GP73 protein can be effectively realized. The implementation steps are as follows:
1、N,S-GQDs-GP73 Apt preparation of
(1) 2.1g of citric acid and 2.3g of thiourea were dissolved in 10mL of pure water, stirred until completely dissolved, and then placed in a high temperature reaction vessel lined with Teflon, and the vessel was heated in an air-blown drying oven at 200 ℃ for 160min.
(2) And cooling the completely reacted solution to room temperature, adding 50mL of ethanol, fully shaking and stirring uniformly, sucking the solution, dialyzing for 6 hours by using a dialysis bag with the molecular weight of 300, and drying the dialyzed solution in a freeze dryer for 12 hours to obtain the N, S-GQDs solid.
(3) Weighing 10mg of N, S-GQDs, adding pure water to fix the volume to 10mL, and obtaining 1mg/mL of N, S-GQDs solution. 7.668mg of carbodiimide (EDAC) and 2.1713mg of N-hydroxysuccinimide (NHS) were weighed, mixed, and added with pure water to a constant volume of 10mL, and stirred uniformly to obtain a crosslinking agent solution. 200 μ L of N, S-GQDs and 200 μ L of 1 μ M GP73 were measured Apt And 20uL of cross-linking agent solution, uniformly mixing, placing into a dark environment with the room temperature of 25 ℃, uniformly oscillating, and incubating for 1h to obtain a fluorescence-labeled complex N, S-GQDs-GP73 Apt And (3) solution. FIG. 2 shows N, S-GQDs and N, S-GQDs-GP73 Apt The fluorescence spectra of the two are basically consistent, N, S-GQDs-GP73 Apt The fluorescence intensity of (A) is smaller, indicating that N, S-GQDs and GP73 Apt The connection has been successful. FIG. 3A is a Transmission Electron Micrograph (TEM) of N, S-GQDs, which shows that the prepared N, S-GQDs have uniform size, good dispersibility and a particle size of about 7 nm.
2. Construction of fluorescence biosensor based on fluorescence resonance energy transfer
(1) 30mg MoS was weighed using a precision electronic balance 2 Putting the solid into a beaker, adding N, N-Dimethylformamide (DMF) solution to a constant volume of 30mL, putting the solid into an ultrasonic cell disruption instrument for ultrasonic disruption for 1h, and obtaining MoS with the concentration of 1mg/mL after the disruption is finished 2 And (3) dispersing the mixture.
(2) Weighing 30mg of single-layer Graphene Oxide (GO) solid by using a precision electronic balance, putting the single-layer Graphene Oxide (GO) solid into a beaker, adding pure water to a constant volume of 30mL, then completely stirring the solution, putting the solution into an ultrasonic cell crusher for ultrasonic crushing for 3 hours, adding 300mg of Ascorbic Acid (AA) after the crushing is finished, putting the crushed solution on a magnetic stirrer, and continuously stirring for 12 hours to obtain an RGO dispersion liquid with the concentration of 1 mg/mL.
(3) Taking 20mL of the LRGO dispersion liquid, putting the dispersion liquid into a 50mL beaker, and adding 20mL of MoS subjected to ultrasonication 2 Dispersion liquid ofStirring for 24h on a magnetic stirrer to ensure that MoS 2 Sufficiently adsorbing RGO thereon; stirring completely, taking out the mixed liquid, centrifuging at 5000r/min for 5min, repeatedly washing the obtained precipitate with pure water, and drying at low temperature in a vacuum low-temperature drying oven to obtain black MoS 2 @ RGO solid, 1mg of MoS was weighed 2 @ RGO, adding pure water to constant volume of 10mL to obtain 100ug/mL MoS 2 @ RGO solution. FIG. 3B is MoS 2 Scanning Electron Microscopy (SEM) of @ RGO, it can be seen that RGO is well organized as MoS 2 The substrate is extended in a layered way, so that the specific surface area of the whole material is increased, and N, S-GQDs-GP73 is facilitated Apt Adsorption of (2).
(4) 100 μ L of 100 μ g/mL MoS was taken 2 @ RGO and 100. Mu.L 1. Mu.M N, S-GQDs-GP73 Apt Mixing, shaking, and incubating at 25 deg.C for 60min to obtain N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescent biosensor. Scanning with a fluorescence spectrophotometer at a fixed excitation wavelength of 368nm, measuring the fluorescence intensity at 450nm and recording as F 0 。
3. Drawing of GP73 working curve
Measuring the fluorescence intensity of the N, S-GQDs-GP73 obtained in the step 2 Apt /MoS 2 @ @ RGO FRET fluorescence biosensor is divided into 6 groups evenly, then 200 μ L GP73 protein solution (0.1 ng/mL,1.0ng/mL,10.0ng/mL,100.0ng/mL,1.0 μ g/mL,10.0 μ g/mL) is added according to concentration gradient, after shaking and mixing evenly, reaction is carried out for 60min at 25 ℃, scanning is carried out by a fluorescence spectrophotometer, the excitation wavelength is fixed to be 368nm, the fluorescence intensity of 450nm is measured, and is recorded as F 1 . N, S-GQDs-GP73 at different GP73 concentrations Apt /MoS 2 The fluorescence spectrum of the @ RGO FRET fluorescence biosensor is shown in FIG. 4, and the fluorescence recovery intensity of the fluorescence biosensor can be seen ((F) 1 -F 0 )/F 0 ) Is in positive correlation with the concentration of GP 73. When the concentration range of GP73 protein is 0.1 ng/mL-10 mu g/mL, N, S-GQDs-GP73 Apt /MoS 2 The relationship between the fluorescence recovery value of the @ RGO FRET fluorescence biosensor and the concentration of GP73 is logarithmically linear, and the working curve is Y =0.03027lgX +0.012572 (Y represents the fluorescence recoveryIntensity, X represents the concentration of GP73 protein), correlation coefficient is R 2 ＝0.99206。
4. Detection of GP73 in actual serum samples
Three serum samples, namely, the serum of a normal person, the serum of a cirrhosis patient and the serum of a liver cancer patient are prepared, and the GP73 concentration is respectively 58.85ng/mL,106.74ng/mL and 306.32ng/mL by the method for clinically detecting GP73 (enzyme-linked immunosorbent assay ELISA). Adding 200 μ L serum into the prepared N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescence biosensor, mixed well and incubated for 1h, its fluorescence intensity was measured, each serum sample was assayed 3 times. According to the working curve Y =0.03027lgX +0.012572 obtained in step 3, the corresponding GP73 concentration in the actual serum sample can be obtained by calculation, and the detection result is shown in Table 1. As can be seen from Table 1, N, S-GQDs-GP73 in comparison with the ELISA method Apt /MoS 2 The relative error of the @ RGO FRET fluorescence biosensor for detecting GP73 in serum is between 0.75% and 7.13%, and the relative standard deviation is between 4.57% and 6.38%, which indicates that the fluorescence biosensor can be applied to the detection of actual serum samples.
TABLE 1 detection of GP73 in real serum samples
(Note: the serum sample is from the key laboratory of Guangxi Metabolic disease research at hospital 924 of the people's liberation force (Guilin, china) and complies with the ethical Committee for the key laboratory of Guangxi Metabolic disease research at hospital 924 of the people's liberation force.1: normal serum, AFP =2.45ng/mL, GP73=58.85ng/mL, sample 2: serum for liver cirrhosis, AFP =9.30ng/mL, GP73=106.74ng/mL, sample 3: serum for liver cancer, AFP =173.99ng/mL, GP73=306.32 ng/mL).
Claims (7)
1. A fluorescence biosensor for GP73 detection is constructed based on fluorescence resonance energy transfer, and the method comprises the following steps:
step 1: fluorescence resonance donor nitrogenSulfur-doped graphene quantum dot-GP 73 aptamer N, S-GQDs-GP73 Apt Preparation of (2)
(1) Preparing nitrogen and sulfur doped graphene quantum dots N, S-GQDs: weighing citric acid and thiourea, adding pure water to a constant volume, uniformly stirring, heating and cooling; adding ethanol, stirring, and dialyzing with dialysis bag; freeze-drying the dialyzed solution to obtain N, S-GQDs solid;
(2)N,S-GQDs-GP73 Apt the preparation of (1): measuring N, S-GQDs and GP73 aptamer GP73 Apt Activated crosslinking is carried out by using a crosslinking agent, and incubation is carried out to obtain N, S-GQDs-GP73 Apt A solution;
step 2: construction of fluorescence biosensor based on fluorescence resonance energy transfer
(1) Weighing MoS 2 Adding N, N-dimethylformamide DMF solution to constant volume to obtain MoS 2 Dispersing the powder in DMF to obtain MoS 2 A dispersion liquid;
(2) Weighing graphene oxide GO powder, adding pure water to a constant volume, and dispersing the GO powder in the pure water to obtain a GO dispersion liquid; adding ascorbic acid AA into the GO dispersion liquid, and stirring and mixing to obtain a Reduced Graphene Oxide (RGO) dispersion liquid;
(3) Mixing MoS 2 Mixing the dispersion liquid and the RGO dispersion liquid, and stirring for a certain time to obtain molybdenum disulfide @ reducing graphene oxide MoS 2 @ RGO solution, centrifuging the obtained solution, washing the separated solid and drying to obtain MoS 2 @ RGO; weighing MoS 2 @ RGO solid, adding pure water, stirring and mixing to obtain MoS 2 @ RGO dispersion;
(4) Mixing MoS 2 @ RGO solution and N, S-GQDs-GP73 Apt Mixing the solutions, standing and incubating for a period of time after mixing to quench the fluorescence of N, S-GQDs to form N, S-GQDs-GP73 Apt /MoS 2 @ RGO FRET fluorescent biosensor; scanning with a fluorescence spectrophotometer at a fixed excitation wavelength of 368nm, measuring the fluorescence intensity at 450nm, and recording as F 0 ；
And 3, step 3: drawing of GP73 working curve
(1) GP73 solutions of different concentrations were added to N, S-GQDs-GP73 Apt /MoS 2 @RGO FRAn ET fluorescence biosensor is incubated, and the peak value change at 450nm is detected; scanning with a fluorescence spectrophotometer at a fixed excitation wavelength of 368nm, measuring the fluorescence intensity at 450nm, and recording as F 1 ；
(3) With (F) 1 -F 0 )/F 0 As a vertical coordinate, the GP73 concentration is taken as a horizontal coordinate, a working curve is drawn, and the lowest detection limit of the fluorescence biosensor is calculated;
and 4, step 4: detection of GP73 in real samples
(1) Adding the actual sample to the N, S-GQDs-GP73 in step 2 Apt /MoS 2 The @ RGO FRET fluorescence biosensor is incubated, a fluorescence spectrophotometer is adopted for scanning, the fixed excitation wavelength is 368nm, and the fluorescence intensity at 450nm is recorded;
(2) And (4) calculating the concentration of GP73 in the sample to be detected according to the working curve of GP73 obtained in the step (3).
2. The fluorescent biosensor of claim 1, wherein: in the step 1, the molecular weight of the dialysis bag is 300, and the dialysis time is 6h.
3. The fluorescent biosensor of claim 1, wherein: in the step 1, the concentration of N, S-GQDs is 1.0mg/mL; GP73 Apt The concentration is 1.0 mu M; the cross-linking agent solution contains carbodiimide EDAC and N-hydroxysuccinimide NHS, wherein the concentration of the EDAC is 0.7668mg/mL, and the concentration of the NHS is 2.1713mg/mL.
4. The fluorescent biosensor of claim 1, wherein: n, S-GQDs solution, GP73 in step 1 Apt The volume ratio of the solution to the cross-linking agent solution is 10:10:1, and the incubation time is 1h.
5. The fluorescent biosensor of claim 1, wherein: moS in step 2 2 The concentration is 1.0mg/mL; the GO concentration is 1.0mg/mL; the mass ratio of the added AA to the added GO is 10:1; the stirring time was 12h.
6. The fluorescent biosensor of claim 1, wherein: moS in step 2 2 @ RGO solution and N, S-GQDs-GP73 Apt The volume ratio of the liquid is 1:1.
7. the fluorescent biosensor of claim 1, wherein: the incubation temperature in step 2, step 3 and step 4 is 25 ℃, and the incubation time is 60min.
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