CN114751400B - Nitrogen-zinc co-doped graphene quantum dot, ratio immunosensor and preparation method and application thereof - Google Patents
Nitrogen-zinc co-doped graphene quantum dot, ratio immunosensor and preparation method and application thereof Download PDFInfo
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- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/184—Preparation
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The application discloses a nitrogen-zinc co-doped graphene quantum dot, a ratio immunosensor and a preparation method and application thereof, wherein the method comprises the following steps: uniformly mixing GO powder and L-cysteine in water by ultrasonic, adjusting the pH value to 8-9, then performing high-temperature heating reaction to obtain supernatant, removing water, and performing freeze drying to obtain N-doped graphene quantum dot powder; and heating the N-doped graphene quantum dot powder, dropwise adding a zinc ion-containing solution under stirring to react in a dark place, centrifuging to obtain precipitate, washing, preparing the solution by using deionized water after freeze drying to obtain the nitrogen-zinc co-doped graphene quantum dot, so that the fluorescence intensity of the quantum dot is improved, and the quantum yield of the quantum dot is also improved. Based on graphene oxide and nitrogen-zinc co-doped graphene quantum dot composite materials, a photoelectric dual-response ratio immunosensor is constructed, and the photoelectric dual-response ratio immunosensor is used for detecting disease markers, and has good results and low detection limit.
Description
Technical Field
The application relates to the technical field of novel nanocomposite and biosensing detection, in particular to a nitrogen-zinc co-doped graphene quantum dot, a ratio immunosensor and a preparation method and application thereof.
Background
In recent years, the prevalence rate of chronic diseases of middle-aged and elderly people is continuously rising, the health and life quality of the chronic diseases are seriously affected, and the economic burden of society is increased. The chronic diseases mainly comprise the following steps: hypertension, diabetes, cardiovascular and cerebrovascular diseases, etc. Therefore, early detection and diagnosis of the above chronic diseases are one of the current research hotspots.
Graphene Oxide (GO) is an ideal material of an electrochemical biosensor, and the biosensor prepared from the graphene oxide has good application prospects in medical detection of proteins, glucose and the like; GO also has excellent light transmission properties, photon transmittance is 97.4% higher, more importantly, graphene Oxide (GO) has a higher specific surface area, and its two-dimensional structure also allows other substances to exist on its surface. Because of these unique properties, graphene oxide has become a good carrier in biological applications. And the graphene quantum dot prepared by taking GO as a raw material has excellent physical and chemical characteristics, so that the graphene quantum dot can have great application potential in the aspect of biological detection. The fluorescence intensity and quantum yield of the existing graphene quantum dots are not high;
therefore, it is necessary to develop a graphene quantum dot with fluorescence intensity and quantum yield.
Disclosure of Invention
The application aims to provide a nitrogen-zinc co-doped graphene quantum dot, a ratio immunosensor and a preparation method and application thereof. And a photoelectric dual-response ratio immunosensor is constructed based on graphene oxide and nitrogen-zinc co-doped graphene quantum dot composite material, and is used for detecting disease markers, good results are obtained, and the detection limit is low. Has higher selectivity and strong stability compared with the electrochemical sensor and the photochemical sensor.
In order to achieve the above purpose, the present application adopts the following technical scheme:
in a first aspect of the application, a method for preparing nitrogen-zinc co-doped graphene quantum dots is provided, the method comprising:
uniformly mixing GO powder and L-cysteine in water by ultrasonic, adjusting the pH value to 8-9, then performing high-temperature heating reaction to obtain supernatant, removing water to obtain yellowish solid, and performing freeze drying to obtain N-doped graphene quantum dot powder (N-GQDs);
heating the N-doped graphene quantum dot powder, dropwise adding a zinc ion-containing solution under stirring to react in a dark place, centrifuging to obtain precipitate, washing, freeze-drying, and preparing the solution with deionized water to obtain the N-zinc co-doped graphene quantum dot, namely the N, zn-GQDs nanocomposite.
Further, the mass ratio of the GO powder to the L-cysteine is 1: (1-6).
Further, the temperature of the high-temperature heating reaction is 190-210 ℃, and the time of the high-temperature heating reaction is 3-5 h.
Further, heating the N-doped graphene quantum dot powder to 35-45 ℃; the temperature of the light-shielding reaction is 25-80 ℃, and the time of the light-shielding reaction is 1-5 h.
Further, the zinc ion-containing solution comprises one of zinc acetate, zinc sulfate, zinc chloride, zinc nitrate, zinc fluosilicate, zinc fluoborate and zinc gluconate; the volume ratio of the N-GQDs to the zinc ion-containing solution is 1: (1-10).
In a second aspect of the application, a nitrogen-zinc co-doped graphene quantum dot obtained by the method is provided.
In a third aspect of the present application, there is provided a method of preparing a ratiometric immunosensor, the method comprising:
and (3) dropwise adding a GO suspension to the surface of the activated electrode, forming a film, dropwise adding the nitrogen-zinc co-doped graphene quantum dots to the surface of the GO, and sequentially combining an antibody, a sealing agent and an antigen after drying to obtain the ratio immunosensor.
Further, the GO suspension is prepared by dispersing powder in deionized water; the concentration of the suspension of GO is 0.1-10 mg/mL, and the concentration of N, zn-GQDs is 0.1-20 mg/mL; and the concentration ratio of the GO suspension to the N, zn-GQDs is 1: (1-10).
Further, the antibody comprises one of cardiac troponin, abeta protein, myoglobin, procalcitonin, albumin, total protein, urinary beta 2 microglobulin, clusterin and antibodies corresponding to prostate specific antigen; the blocking agent comprises one of Bovine Serum Albumin (BSA), serum, casein, skim milk powder, and a protein-free compound; the antigen is one of cardiac troponin, abeta protein, myoglobin, procalcitonin, albumin, total protein, urinary beta 2 microglobulin, clusterin and prostate specific antigen.
In a fourth aspect of the application, there is provided a ratiometric immunosensor prepared by the method.
In a fourth aspect of the application, there is provided the use of the ratiometric immunosensor in the detection of a biomarker in a human serum sample.
One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:
1. the application provides a nitrogen-zinc co-doped graphene quantum dot and a preparation method thereof, wherein graphene oxide is used as a raw material, nitrogen-doped graphene quantum dots (N-GQDs) are synthesized through a high-temperature hydrothermal method reaction, and then the N-GQDs are reacted with zinc acetate to synthesize the nitrogen-zinc co-doped graphene quantum dot, so that the fluorescence intensity of the quantum dot is improved, and the quantum yield of the quantum dot is improved to 31.5%.
2. The application provides a ratio immunosensor and a preparation method thereof, and the ratio immunosensor is constructed based on graphene oxide and nitrogen-zinc co-doped graphene quantum dot composite material, is used for detecting disease markers, obtains good results and has lower detection limit. Compared with an electrochemical sensor and a photochemical sensor, the fluorescent dye has higher selectivity and strong stability, and can be further applied to detection of various disease biomarkers.
3. The application provides an application of a ratio immunosensor in detection of a biomarker in a human serum sample, and adopts a ratio analysis method of photoelectric dual-signal response. By analyzing the ratio of photochemical signal to current signal rather than just detecting the content of a disease biomarker based on one of them, this method has been gradually applied in the fields of protein detection, virus detection and detection of other bioactive substances. Compared with other analysis technologies, the analysis method has the advantages of simplicity, rapidness, high sensitivity, strong practicability and the like, and provides wider application prospects for development and research of biological sensors.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a scanning electron microscope and transmission electron microscope image of GO; wherein FIG. 1A is a scanning electron microscope and FIG. 1B is a transmission electron microscope.
FIG. 2 is a scanning electron microscope image and a transmission electron microscope image of N, zn-GQDs; wherein fig. 2A is a scanning electron microscope, and fig. 2B is a transmission electron microscope.
FIG. 3 is an XPS plot of N, zn-GQDs nanocomposite.
FIG. 4 is a fluorescence spectrum and an ultraviolet spectrum of N-GQDs and N, zn-GQDs; wherein FIG. 4A is a fluorescence spectrum; fig. 4B is a uv spectrum.
Fig. 5 shows the results of the concentration of standard solutions of cardiac troponin I at various concentrations of the ratiometric immunosensor, wherein fig. 5A is a graph of ECL signal, fig. 5B is a graph of DPV signal, and fig. 5C is a graph of the ratio of ECL signal to DPV signal versus concentration of standard solution of cardiac troponin I.
Fig. 6 is a graph showing the results of the concentration of standard aβ protein solution at different concentrations for a ratio immunosensor, wherein fig. 6A is a graph of ECL signal, fig. 6B is a graph of DPV signal, and fig. 6C is a graph of the ratio of ECL signal to DPV signal versus concentration of standard aβ protein solution.
Detailed Description
The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present application are commercially available or may be obtained by existing methods. The steps S1, S2, S3 and … … of the present application do not represent a strict sequence relationship, and the sequence may be appropriately adjusted as required.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
according to an exemplary embodiment of the present application, there is provided a method for preparing a nitrogen-zinc co-doped graphene quantum dot, the method including:
step S101, uniformly mixing GO powder and L-cysteine in water by ultrasonic, adjusting the pH value to 8-9, then performing high-temperature heating reaction to obtain supernatant, removing water to obtain yellowish solid, and performing freeze drying to obtain N-doped graphene quantum dot powder (N-GQDs);
in the step S101 of the above-mentioned process,
the mass ratio of the GO powder to the L-cysteine is 1: (1-6). If the mass ratio is too small, the reaction may not be completely and the like; if the mass ratio is too large, the stability of the prepared sample is adversely affected;
the GO powder is graphene oxide powder, and can be directly purchased or prepared in the following manner: accurately measuring 30mL of concentrated sulfuric acid, adding 1g of graphite and 0.8g of sodium nitrate into 30mL of concentrated sulfuric acid, placing the mixture in an ice bath, continuously stirring for 4h, slowly adding 4g of potassium permanganate into the mixed solution to prevent rapid oxidation-reduction reaction from causing bumping, stirring for 1h at room temperature, adding 100mL of deionized water into the solution after the solution turns green, heating for 1h at 37 ℃, transferring to an oil bath pot, heating to 95 ℃ and refluxing for 2h, and stopping further reaction by 25mL of 30w/v% hydrogen peroxide after observing that the reaction solution turns yellow. And finally, washing the prepared GO with 5% hydrochloric acid and deionized water for multiple times, and vacuum drying the washed GO for 24 hours.
The temperature of the high-temperature heating reaction is 190-210 ℃, and the time of the high-temperature heating reaction is 3-5 h. If the temperature of the high-temperature heating reaction is too small, the reaction is not easy to be completely carried out, and if the temperature is too large, the fluorescence yield of the quantum dots is not easy to be improved, and the temperature is preferably 200 ℃.
And S102, heating the N-doped graphene quantum dot powder, dropwise adding a zinc ion-containing solution under stirring to react in a dark place, centrifuging to obtain precipitate, washing, and preparing a solution by using deionized water after freeze drying to obtain the N-zinc co-doped graphene quantum dot, namely the N, zn-GQDs nanocomposite.
In the step S102 of the above-mentioned process,
heating the N-doped graphene quantum dot powder to 35-45 ℃; heating to 35-45 ℃ and then dropwise adding a zinc ion-containing solution under strong stirring to facilitate the doping of zinc ions; preferably 40 ℃.
The temperature of the light-shielding reaction is 25-80 ℃, and the time of the light-shielding reaction is 1-5 h. If the temperature of the light-shielding reaction is too low, the formation of a nitrogen-zinc covalent bond is not facilitated, and if the temperature of the light-shielding reaction is too high, the fluorescence stability of the quantum dots is not facilitated;
the zinc ion-containing solution comprises one of zinc acetate, zinc sulfate, zinc chloride, zinc nitrate, zinc fluosilicate, zinc fluoborate and zinc gluconate; the volume ratio of the N-GQDs to the zinc ion-containing solution is 1: (1-10). If the volume ratio is too low, the successful doping of zinc ions is not facilitated, and if the volume ratio is too high, the fluorescence stability of the quantum dots is not facilitated;
according to another exemplary embodiment of the present application, there is provided a nitrogen-zinc co-doped graphene quantum dot obtained by the method. According to the application, graphene oxide is used as a raw material, nitrogen-doped graphene quantum dots (N-GQDs) are synthesized through a high-temperature hydrothermal method reaction, and then N-GQDs are reacted with zinc acetate to synthesize the nitrogen-zinc co-doped graphene quantum dots, so that not only is the fluorescence intensity of the quantum dots improved, but also the quantum yield of the quantum dots is improved.
According to another exemplary embodiment of the present application, there is provided a method of manufacturing a ratio-type immunosensor, the method including:
and (3) dropwise adding a GO suspension to the surface of the activated electrode, forming a film, dropwise adding the nitrogen-zinc co-doped graphene quantum dots to the surface of the GO, and sequentially combining an antibody, a sealing agent and an antigen after drying to obtain the ratio immunosensor.
The GO suspension is prepared by dispersing powder in deionized water; the concentration of the GO suspension is 0.1-10 mg/mL, and the suspension is favorable for loading N, zn-GQDs under the concentration range.
The concentration of the N, zn-GQDs is 0.1-20 mg/mL; this concentration range is advantageous for capturing N, zn-GQDs with respect to protein antibodies.
And the concentration ratio of the GO suspension to the N, zn-GQDs is 1: (1-10). This concentration ratio facilitates the reaction to completion; if the concentration ratio is too small, the utilization of GO surface space is not facilitated, and if the concentration ratio is too large, the capture of N, zn-GQDs on protein antibodies is not facilitated.
The antibody comprises one of antibodies corresponding to cardiac troponin, abeta protein, myoglobin, procalcitonin, albumin, total protein, urinary beta 2 microglobulin, clusterin and prostate specific antigen; the blocking agent comprises one of Bovine Serum Albumin (BSA), serum, casein, skim milk powder, and a protein-free compound; the antigen is one of cardiac troponin, abeta protein, myoglobin, procalcitonin, albumin, total protein, urinary beta 2 microglobulin, clusterin and prostate specific antigen.
The application constructs the photoelectric dual-response ratio immunosensor based on graphene oxide and nitrogen-zinc co-doped graphene quantum dot composite material, which is used for detecting disease markers and has good results and lower detection limit. Compared with an electrochemical sensor and a photochemical sensor, the fluorescent dye has higher selectivity and strong stability, and can be further applied to detection of various disease biomarkers.
The following describes a ratio immunosensor of the present application, and a method for making and using the same, in detail, with reference to examples, comparative examples, and experimental data.
Example 1
The embodiment of the application provides a preparation method of a nitrogen-zinc co-doped graphene quantum dot, which comprises the following steps:
1. preparation of Graphene Oxide (GO): accurately measuring 30mL of concentrated sulfuric acid, adding 1g of graphite and 0.8g of sodium nitrate into 30mL of concentrated sulfuric acid, placing the mixture in an ice bath, continuously stirring for 4h, slowly adding 4g of potassium permanganate into the mixed solution to prevent rapid oxidation-reduction reaction from causing bumping, stirring for 1h at room temperature, adding 100mL of deionized water into the solution after the solution turns green, heating for 1h at 37 ℃, transferring to an oil bath pot, heating to 95 ℃ and refluxing for 2h, and stopping further reaction by 25mL of 30w/v% hydrogen peroxide after observing that the reaction solution turns yellow. And finally, washing the prepared GO with 5% hydrochloric acid and deionized water for multiple times, and vacuum drying the washed GO for 24 hours. And (3) grinding the prepared graphene oxide sheets to be in powder form for later use by using a mortar, wherein a scanning electron microscope image and a transmission electron microscope image of the obtained GO result are shown in the attached figure 1.
2. Accurately weighing 15mg of GO powder and 45mg of L-cysteine, mixing, adding 15mL of deionized water, performing ultrasonic treatment for 30min to uniformly disperse the powder, and adding 100 mu L of ammonia water to adjust the pH value of the mixed solution to be between 8 and 9. Then, after ultrasonic dispersion for 10min in an ultrasonic cleaner, transferring the solution to an autoclave lined with polytetrafluoroethylene, placing the autoclave in a muffle furnace for heating reaction for 4h at 200 ℃, cooling the autoclave to room temperature, transferring the autoclave to a glass bottle for light-proof ultrasonic dispersion for 30min, and in order to disperse quantum dots attached between unreacted and complete graphene oxide sheets into an aqueous solution, filtering black precipitates and collecting supernatant, wherein due to fluorescence characteristics of the quantum dots, light-proof conditions are required to be maintained in the whole process to prevent fluorescence quenching. Transferring the supernatant to a round-bottom flask, removing water at 60 ℃ by using a rotary evaporator to obtain a yellowish solid, and freeze-drying the obtained yellowish solid to obtain yellowish N-doped graphene quantum dot powder (N-GQDs).
3. Accurately weighing 1mg of the N-GQDs prepared in the step (1) in a glass bottle, heating to 40 ℃, then slowly adding 2mL of 0.1g/mL zinc acetate solution dropwise under high-speed magnetic stirring, and reacting for 3h in a dark place. After the completion of the reaction, the mixture was placed in a 4mL centrifuge tube, followed by centrifugation at 10000rpm in the centrifuge for 14min, the supernatant was discarded, and the precipitate was washed three to four times with deionized water to obtain N, zn-GQDs.
4. The scanning electron microscope image and the transmission electron microscope image of the N, zn-GQDs composite material are shown in figure 2, the precipitate is prepared into a solution of 1mg/mL by deionized water after freeze drying, the XPS spectrum of the obtained composite material N, zn-GQDs is shown in figure 3, the fluorescence spectrum and the ultraviolet spectrum of the obtained composite material N, zn-GQDs are shown in figure 4, and the obtained aqueous solution is stored in a dark place for later use. Indicating that the composite material of N, zn-GQDs is successfully prepared.
5. Quantum yield was calculated. The quantum yield of the application was improved to 31.5% by calculation. The calculation formula is that Wherein (1)>For quantum yield, I and I S Fluorescence peak areas of N, zn-GQDs and reference substance solutions, A and A S Is the absorbance, eta and eta of the N, zn-GQDs and reference substance solution s Refractive index of N, zn-GQDs and reference solution (1.33 in aqueous solution); in this embodiment, i=0.6; i S ＝0.3；A s ＝40302.5；A＝1387206.5；η＝1.33；η s ＝1.33。
Example 2
The embodiment of the application provides a preparation method and application of a myocardial troponin I ratio immunosensor, which comprises the following steps:
1. 4.5mg of N, zn-GQDs prepared in example 1 above was weighed out and dispersed in a PBS solution (pH=7.4) of 1mg/mL EDC with stirring at high speed, and stirred for 2 hours to activate carboxyl groups on the surface of N, zn-GQDs.
2. Continuously polishing the glassy carbon electrode by using 0.3 mu m and 0.05 mu m aluminum oxide powder, sequentially immersing the electrode into water and ethanol, ultrasonically cleaning, and drying. The cleaned electrode is activated by cyclic voltammetry in 0.1mol/L PBS solution and 50mmol/L potassium ferricyanide solution respectively until the cyclic voltammetry current is stable. And cleaning the activated electrode in ultrapure water and ethanol again, and drying for later use.
3. And (3) uniformly dripping 5uL of 1mg/mL GO suspension on the surface of the electrode pretreated in the step (2), drying at normal temperature, uniformly dripping 5 mu L N of GO after the GO forms a film on the surface of the electrode, and drying Zn-GQDs on the surface of the GO. After the above materials were completely dried, 5. Mu.L (100. Mu.g/mL) of an antibody against cardiac troponin I (anti-cTnI) was added dropwise to the surface thereof, followed by incubation at 37℃for 3 hours, so that the amino group on anti-cTnI and the carboxyl group on N, zn-GQDs could form an amide bond. Next, 5. Mu.L of BSA (1%) was incubated at 4℃for 2 hours to block non-specific binding sites, unbound BSA was washed off, dried, and 5. Mu.L (100. Mu.g/mL) of a cardiac troponin I (cTnI) solution was added dropwise to incubate at 4℃for 12 hours to be assayed.
4. Determination of the Rate biosensor for the cardiac troponin I (cTnI) content
Drawing a standard curve: the concentration of the prepared cTnI antigen is sequentially increased from top to bottom (respectively 0.001, 0.02, 0.03, 0.05, 0.08 and 0.10 ng/mL), a standard curve is drawn through the ratio of the ECL optical signal to the DPV current signal and the concentration of the cTnI, and the concentration of the cTnI is measured according to the standard curve, and the specific method comprises the following steps:
a series of cTnI antigen solutions (5 mu L) with different concentrations are dripped on the surface of the antibody modified glassy carbon electrode to react for 120min, and DPV signals and ECL signals are detected by using a differential pulse voltammetry method under the optimized experimental conditions. DPV detection conditions are that the scanning range is-0.6-0.8V; the potential increment was 4m V; pulse width 0.1s; the pulse amplitude was 50m V. ECL detection conditions: the application voltage is-2-0V (Ag/AgC 1), the scanning speed is 100mV/s, the photomultiplier is set to 800V, and the amplification level is 3.
As shown in FIG. 5, the prepared ratio immunosensor can realize sensitive detection of cTnI, and the detection limit is as low as 4.59pg/L.
Example 3
The embodiment of the application provides a preparation method and application of an Abeta ratio immunosensor, which are characterized by comprising the following steps:
1. the working electrode was pretreated in the same manner as in example 2, 5ul of a suspension of 1mg/mL GO was uniformly dropped on the pretreated electrode surface, dried at normal temperature, and after GO was formed into a film on the electrode surface, 5 μ L N, zn-GQDs were uniformly dropped on the GO surface, and dried. After the above materials were completely dried, 5. Mu.L (100. Mu.g/mL) of Abeta amyloid antibody (anti-Abeta) was dropped to the surface thereof, followed by incubation at 25℃for 3 hours, so that the amino group on the anti-Abeta and the carboxyl group on the N, zn-GQDs could form an amide bond. Next, 5. Mu.L of BSA (1%) was incubated at 4℃for 2 hours to block non-specific binding sites, unbound BSA was washed off, dried, and 5. Mu.L (100. Mu.g/mL) of Abeta antigen solution was added dropwise thereto to incubate at 37℃for 3 hours to be measured.
2. Determination of amyloid beta content (aβ) by a ratio biosensor.
Drawing a standard curve: the concentration of the prepared antigen is sequentially increased (1, 2, 30, 50, 80 and 100 ng/mL) from top to bottom, a standard curve is drawn, and the concentration of Abeta is measured according to the standard curve.
A series of Abeta antigen solutions (5 mu L) with different concentrations are dripped on the surface of the antibody modified electrode to react for 120min, and Differential Pulse Voltammetry (DPV) signals and ECL signal intensities are detected under optimized experimental conditions. DPV detection conditions are that the scanning range is-0.6-0.8V; the potential increment was 4m V; pulse width 0.1s; the pulse amplitude was 50m V. ECL detection conditions: the application voltage is-2-0V (Ag/AgC 1), the scanning speed is 100mV/s, the photomultiplier is set to 800V, and the amplification level is 3.
As shown in FIG. 6, it was found that the ratio immunosensor prepared as described above was capable of detecting Abeta protein in human serum with a detection limit as low as 52pg/L.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. The preparation method of the nitrogen-zinc co-doped graphene quantum dot is characterized by comprising the following steps of:
uniformly mixing GO powder and L-cysteine in water by ultrasonic, adjusting the pH value to 8-9, then performing high-temperature heating reaction to obtain supernatant, removing water to obtain yellowish solid, and performing freeze drying to obtain N-doped graphene quantum dot powder (N-GQDs); wherein, the mass ratio of the GO powder to the L-cysteine is 1: (1-6); the temperature of the high-temperature heating reaction is 190-210 ℃, and the time of the high-temperature heating reaction is 3-5 h;
heating the N-doped graphene quantum dot powder, dropwise adding a zinc ion-containing solution under stirring to react in a dark place, centrifuging to obtain precipitate, washing, preparing the solution with deionized water after freeze drying to obtain a nitrogen-zinc co-doped graphene quantum dot, namely an N, zn-GQDs nanocomposite; wherein the N-doped graphene quantum dot powder is heated to 35-45 ℃; the temperature of the light-shielding reaction is 25-80 ℃, and the time of the light-shielding reaction is 1 h-5 h; the zinc ion-containing solution comprises one of zinc acetate, zinc sulfate, zinc chloride, zinc nitrate, zinc fluosilicate, zinc fluoborate and zinc gluconate; the volume ratio of the N-GQDs to the zinc ion-containing solution is 1: (1-10).
2. A nitrogen-zinc co-doped graphene quantum dot prepared by the method of claim 1.
3. A method of making a ratiometric immunosensor, the method comprising:
and (3) dropwise adding a GO suspension to the surface of the activated electrode, forming a film, dropwise adding the nitrogen-zinc co-doped graphene quantum dot according to claim 2 to the surface of the GO, drying, and sequentially combining an antibody, a blocking agent and an antigen to obtain the ratio immunosensor.
4. The method of claim 3, wherein the GO suspension is prepared by dispersing powder in deionized water; the concentration of the suspension of GO is 0.1-10 mg/mL, and the concentration of N, zn-GQDs is 0.1-20 mg/mL; and the concentration ratio of the GO suspension to the N, zn-GQDs is 1: (1-10).
5. A ratiometric immunosensor made by the method of any one of claims 3-4.
6. Use of a ratiometric immunosensor as claimed in claim 5 for detection of a biomarker in a human serum sample.
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Application publication date: 20220715 Assignee: Wuhan geditai Health Technology Co.,Ltd. Assignor: HUBEI University OF TECHNOLOGY Contract record no.: X2023980047145 Denomination of invention: A nitrogen zinc co doped graphene quantum dot, ratio type immunosensor and its preparation method and application Granted publication date: 20230825 License type: Common License Record date: 20231120 |