Fluorescent immunoassay based on carbon quantum dots for detection of ovalbumin concentration

Abstract

The application belongs to the field of nanometer material in environmental monitoring application, and discloses a fluorescence immunoassay method for detecting ovalbumin concentration based on carbon quantum dots, which comprises the following steps: using L-glutamic acid as a precursor, one-step preparation of carbon quantum dots with amino groups by ultrasonic hydrothermal method; using a reduced graphene oxide method to prepare graphene sheets, and optimizing the graphene by modifying polydiallyldimethylammonium chloride to obtain graphene sheets which can have good fluorescence resonance energy transfer with the carbon quantum dots with amino groups; and coupling the prepared carbon quantum dots with amino groups with a monoclonal antibody. The application directly prepares carbon quantum dots with amino groups by one-step hydrothermal method, and the quantum dots can be directly coupled with a monoclonal antibody. The carbon quantum dots involved in the application avoid the aminoization step, and the linear range can reach 0.1 ng / mL-3000 ng / mL.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nanomaterials in environmental monitoring applications, and particularly relates to a fluorescent immunoassay method for detecting vitellogenin (Vtg) concentration based on carbon quantum dots. BACKGROUND

[0002] With the rapid development of modern industry and agriculture, environmental estrogens such as pesticides, plastics, polychlorinated biphenyls, bisphenols, etc. have been widely used. These chemicals enter rivers, lakes, seas and soil, causing environmental pollution. Environmental estrogens can exhibit cumulative effects in the body, interfere with the normal function of the endocrine, immune, nervous and other systems, cause endocrine disorders, metabolic disorders and reproductive system damage and other diseases, and have immeasurable harm to animals and humans. Animals in water bodies are particularly affected by environmental estrogens, and the toxicological effects exposed thereby have become a global environmental problem. The Organization for Economic Cooperation and Development has recommended vitellogenin (Vtg) of aquatic vertebrates or invertebrates as one of the biomarkers of estrogen interference. Sensing detection methods with high sensitivity, high specificity, wide detection range, low detection limit and fast testing speed are of great significance for the detection of environmental estrogens. Detecting vitellogenin concentration can reflect the quality, health and safety of water bodies.

[0003] Due to the high specificity of the immune reaction between antigens and antibodies, immunosensors have inherent specificity. For immunosensors based on monoclonal antibodies, certain antibodies can recognize specific antigens among many substances, so that immunosensor technology based on monoclonal antibodies has become the development direction of complex environmental monitoring.

[0004] Traditional immunological techniques include enzyme-linked immunoassay, radioimmunoassay, Western blots, etc. In recent years, electrochemical immunosensors, optical sensing methods, immunocolloidal gold technology, etc. have been developed. Traditional immunological techniques show high specificity and sensitivity, but have some shortcomings, such as enzyme-linked immunoassay which must use enzymes, and the preparation and preservation of enzymes require higher environmental conditions; radioimmunoassay requires the use of radioactive elements, which may cause harm to the environment or people; and immunological Western blots method requires the use of secondary antibodies. These methods generally take a long time and are not suitable for large-scale detection in a short time. In recent years, electrochemical and optical sensors have been used for detection of ovalbumin concentration. Electrochemical immunosensors based on impedance spectroscopy show a wide measurement range (1000-8000 ng / mL), but have low sensitivity (420 ng / mL); electrochemical immunosensors based on current show low detection limit (0.09 ng / mL) but small linear range (0.25-7.8 ng / mL); optical waveguide spectroscopy sensors show a wide measurement range (100-10000 ng / mL) and low detection limit (0.1 ng / mL); surface-enhanced Raman scattering technology has very low detection limit (5 pg / mL) but narrow linear range (about 0.2 ng / mL). Immunocolloidal gold technology requires the use of nanogold, and gold is a precious metal with high cost.

[0005] The fluorescence resonance energy transfer immunofluorescence sensor for detecting ovalbumin (Vtg) based on graphene quantum dots (GQDs) and reduced graphene oxide (RGO) first needs to amino-functionalize the graphene quantum dots, and then effectively couple the graphene quantum dots with antibodies.

[0006] To solve the above technical problems, the present application proposes a fluorescence immunological method for detecting ovalbumin concentration, which mainly includes the following steps: amino-functionalization of graphene quantum dots; coupling of graphene quantum dots with monoclonal antibodies; obtaining water-soluble graphene by reducing graphene oxide; and detection of ovalbumin concentration, etc. The method has high sensitivity, good repeatability and strong anti-interference ability.

[0007] Through the above analysis, the problems and defects of the prior art are:

[0008] (1) The graphene quantum dots involved in the prior art cannot avoid the amino-functionalization step, which consumes time and energy, increases the cost of material preparation, and the prior art cannot further save time. In the prior art, 50 mL of 1 mg / mL GQDs aqueous solution is mixed with 50 mL of 25% ammonia solution, and the mixture is stirred uniformly at room temperature. The solution is transferred to a polytetrafluoroethylene container and heated at 200°C for 10 hours, and then cooled at room temperature. The solution is further heated to 95°C for one hour, the excess ammonia is evaporated, and then purified by dialysis in pure water for 24 hours. Therefore, the prior art has high preparation cost and long process time.

[0009] (2) The linear detection range of the prior art is generally 0.001-1500 ng / mL, and the applicability is limited.

[0010] (3) The fluorescence recovery rates of 1.5 μg / ml chicken egg white and bovine serum albumin in the prior art are 23.70% and 20.51%. SUMMARY

[0011] To overcome the problems in the related art, the present application provides a fluorescence immunoassay method for detecting the concentration of egg yolk protamine based on carbon quantum dots.

[0012] The technical solution is as follows: a fluorescence immunoassay method for detecting the concentration of egg yolk protamine based on carbon quantum dots includes:

[0013] Using L-glutamic acid as a precursor, carbon quantum dots with amino groups are prepared by a one-step ultrasonic hydrothermal method;

[0014] Graphene sheets are prepared by a method of reducing graphene oxide, and graphene is optimized by modifying polydiallyldimethylammonium chloride (PDDA) to obtain graphene sheets (RGO_PDDA) that can cause fluorescence resonance energy transfer with carbon quantum dots with amino groups;

[0015] The prepared carbon quantum dots with amino groups are coupled with monoclonal antibodies to obtain purified antibody-carbon quantum dot conjugates as fluorescence nanoprobes, which are then used to determine the optimal dosage of graphene sheets, establish a fluorescence recovery linear curve, and measure repeatability, anti-interference, and precision.

[0016] In one embodiment, the use of L-glutamic acid as a precursor, carbon quantum dots with amino groups are prepared by a one-step ultrasonic hydrothermal method, which includes:

[0017] 60 mL of 15% L-glutamic acid aqueous solution is subjected to ultrasonic hydrothermal reaction at 250℃ for 4-5 hours, cooled to room temperature, filtered, then dialyzed, and then the sample is freeze-dried to obtain a powder sample.

[0018] In one embodiment, the use of a method of reducing graphene oxide to prepare graphene sheets, and the use of polydiallyldimethylammonium chloride (PDDA) to optimize graphene to obtain graphene (RGO_PDDA) that can cause good fluorescence resonance energy transfer with carbon quantum dots includes the following steps:

[0019] Step 1) configure a graphene oxide aqueous solution, ultrasonic dispersion; under stirring, add ammonia water, adjust the pH value to 9, then add polyvinylpyrrolidone, stir to make the polyvinylpyrrolidone uniformly dispersed in the solution, drop ascorbic acid solution, under magnetic stirring, water bath heating reduction;

[0020] Step 2) centrifuge the prepared solution, wash twice with water to remove residual polyvinylpyrrolidone, and finally use ethanol to centrifuge and clean, dry the reduced graphene oxide in a constant temperature oven to obtain a solid graphene powder (RGO), which can be uniformly dispersed in water;

[0021] Step 3) prepare RGO and polydiallyldimethylammonium chloride (PDDA) into a suspension according to the mass ratio of 1:1, fully stir, wash with water and centrifuge three times to remove excess PDDA; finally dry in a constant temperature drying oven into a graphene solid powder that can occur fluorescence resonance energy transfer with amino-containing carbon quantum dots, denoted as RGO_PDDA.

[0022] In one embodiment, the amino-containing carbon quantum dots coupled with the monoclonal antibody include the following steps:

[0023] (1) prepare 2-morpholinoethanesulfonic acid (MES) solution, then add 1-ethyl-(3-dimethylpropyl) carbonyl diimide (EDC) reagent and N-hydroxysuccinimide (NHS) to the MES solution respectively, mix and stir to form a crosslinking agent (EDC-NHS) solution for later use;

[0024] (2) take the antibody, add the EDC-NHS solution to it, stir to activate the antibody; mix the activated antibody and the phosphate buffered saline solution (PBS) of the carbon quantum dots in equal volume, incubate to form an antibody-carbon quantum dot conjugate;

[0025] (3) obtain the purified antibody-carbon quantum dot conjugate as a fluorescent nanoprobes.

[0026] In one embodiment, before step (3) obtains the purified antibody-carbon quantum dot conjugate, the antibody-carbon quantum dot conjugate mixed solution obtained in step (2) is washed with PBS buffer and ultracentrifuged three times.

[0027] In one embodiment, the optimal dosage of the graphene sheet comprises the following steps: taking multiple activated antibody-carbon quantum dot conjugates, mixing with modified PDDA graphene (RGO_PDDA) phosphate buffered saline (PBS) solutions with concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 μg / ml respectively, and adding phosphate buffered saline (PBS) to a total volume of 1 mL, shaking and then standing, and the antibody-carbon quantum dot conjugate is adsorbed on the surface of RGO_PDDA by electrostatic attraction and π-π stacking; measuring the fluorescence spectrum under different RGO_PDDA concentrations, analyzing fluorescence resonance energy transfer; and when the fluorescence quenching degree reaches saturation, the corresponding concentration of the graphene sheet is the optimal dosage of the graphene.

[0028] In one embodiment, the antibody-carbon quantum dot / RGO_PDDA is used as an immune sensor for detecting a target.

[0029] In one embodiment, the establishment of the fluorescence recovery linear curve comprises the following steps: adding different concentrations of antigens to multiple antibody-carbon quantum dot / RGO_PDDA solutions, and adding phosphate buffered saline (PBS) to constant volume, and the concentration of the antigen is in the range of 1-15000 ng / mL; and the linear range is 0.1-3000 ng / mL.

[0030] After incubation at room temperature, the fluorescence spectrum of the mixture is measured, and the measurement is repeated three times; and a standard curve of the logarithmic concentration of the antibody versus the fluorescence intensity is drawn.

[0031] In one embodiment, the detection of the concentration of the egg yolk protein in the solution specifically comprises: taking an egg yolk protein solution, adding an antibody-carbon quantum dot / RGO_PDDA aqueous solution, and then adding phosphate buffered saline, incubating at room temperature, measuring the fluorescence spectrum of the mixture, reading the fluorescence peak intensity, and obtaining the concentration of the egg yolk protein from the established standard curve of the logarithmic concentration of the antigen versus the fluorescence intensity.

[0032] In one embodiment, in the repeatability and anti-interference measurement and detection accuracy, six groups of antibody-carbon quantum dot / RGO_PDDA parallel samples are used to detect the same concentration of egg yolk protein, and the repeatability of the measurement is analyzed.

[0033] In one embodiment, chicken egg white (OVA) and bovine serum albumin (BSA) are used as interferents, and the fluorescence recovery experiment is repeated, and the anti-interference of the fluorescence immune sensor is analyzed, and the experiment is repeated three times.

[0034] In combination with all the above technical solutions, the application has the following advantages and positive effects:

[0035] First, in view of the technical problems existing in the prior art and the difficulty in solving the problems, the technical solution of the present application and the results and data obtained during the research and development are combined closely, and the technical problems solved by the technical solution of the present application and the technical effects brought about after the problems are solved are analyzed in detail and profoundly. The specific description is as follows:

[0036] The present application is improved on the basis of the previous research. The carbon quantum dots carrying amino groups are directly prepared by one-step hydrothermal method in the present application. The quantum dots can be directly coupled with monoclonal antibodies. The fluorescence resonance energy transfer immunofluorescence sensor based on graphene quantum dots and reduced graphene oxide for detecting ovalbumin first needs to aminoate the graphene quantum dots, and then the graphene quantum dots can be effectively coupled with the antibodies. The carbon quantum dots involved in the present application avoid the aminoation step, saving time and cost. In order to make the fluorescence probe (the coupling product of carbon quantum dots and monoclonal antibodies) and the reduced graphene oxide have high-efficiency fluorescence resonance energy transfer, and expand the detection range, the reduced graphene oxide is further modified (modified by polydiallyldimethylammonium chloride (PDDA)) in the present application. The linear detection range (0.1-3000ng / mL) of the present application is wider than that (0.001-1500ng / mL) of the previous research, and the applicability is wide.

[0037] Second, from the perspective of the product as a whole, the technical effects and advantages of the technical solution of the present application are described as follows:

[0038] The purpose of the present application is to make up for the shortcomings of the prior art. The carbon quantum dots coupled with monoclonal antibodies are used as fluorescence probes. When the distance between the carbon quantum dots-antibody and RGO_PDDA is less than 10nm, fluorescence resonance energy transfer phenomenon occurs between the carbon quantum dots-antibody and RGO_PDDA. The fluorescence emitted by the carbon quantum dots-antibody is absorbed by RGO_PDDA and quenched. When the antigen (ovalbumin) is added, due to the strong immune reaction between the antigen and the antibody, the carbon quantum dots-antibody conjugate is far away from RGO_PDDA, so that the quenched fluorescence can be recovered. Since the fluorescence recovery intensity is positively correlated with the concentration of the added antigen, the concentration of the antigen can be quantitatively detected. Ovalbumin is one of the biomarkers of environmental estrogens. The detection of the concentration of ovalbumin can reflect the quality of the water body.

[0039] Third, as the auxiliary evidence for the creativity of the claims of the present application, it is also embodied in the following important aspects:

[0040] (1) The expected income and commercial value of the technical scheme of the present application after transformation are: carbon quantum dots emit fluorescence, are resistant to light bleaching, have good fluorescence stability at room temperature, are green, environmentally friendly, non-toxic, non-polluting, have good water solubility and good biocompatibility, and can be prepared by a hydrothermal method at low cost and can be used for large-flux detection;

[0041] (2) The technical scheme of the present application fills the domestic and foreign technical blank: the present scheme first uses carbon quantum dots for detection of ovalbumin concentration in the world. BRIEF DESCRIPTION OF DRAWINGS

[0042] The accompanying drawings, which are incorporated into and form part of the specification, illustrate embodiments consistent with the present disclosure and, together with the specification, serve to explain the principles of the present disclosure.

[0043] Figure 1 is a flow chart of a fluorescence immunoassay method for detecting ovalbumin concentration based on carbon quantum dots provided by the embodiments of the present application;

[0044] Figure 2 is a graph for judging whether carbon quantum dots carry amino groups or not by Fourier transform infrared spectroscopy thereof provided by the embodiments of the present application;

[0045] Figure 3A is a total spectrum graph for judging whether carbon quantum dots carry amino groups or not by X-ray photoelectron spectroscopy thereof provided by the embodiments of the present application;

[0046] Figure 3B is an N1s high-resolution spectrum graph for judging whether carbon quantum dots carry amino groups or not by X-ray photoelectron spectroscopy thereof provided by the embodiments of the present application;

[0047] Figure 4A is a fluorescence quenching graph between graphene sheets and carbon quantum dots before modification of PDDA provided by the embodiments of the present application;

[0048] Figure 4B is a fluorescence quenching graph between graphene sheets and carbon quantum dots after modification of PDDA provided by the embodiments of the present application;

[0049] Figure 4C is a graph of fluorescence resonance energy transfer efficiency between graphene sheets and carbon quantum dots before and after modification of PDDA provided by the embodiments of the present application;

[0050] Figure 5 is a graph for judging that the surface of graphene sheets before and after modification of PDDA changes in electric charge property by Zeta potential analysis provided by the embodiments of the present application;

[0051] Figure 6A is a graph for judging successful coupling of carbon quantum dots and monoclonal antibodies by ultraviolet-visible absorption spectroscopy (the conjugate is a fluorescent probe) provided by the embodiments of the present application;

[0052] Figure 6B is a graph provided by an embodiment of the present application, which shows that the successful coupling of carbon quantum dots and monoclonal antibodies is determined by the change of fluorescence spectrum;

[0053] Figure 6C is a graph provided by an embodiment of the present application, which shows that the fluorescence intensity of the fluorescent probe changes with the excitation wavelength;

[0054] Figure 7A is a graph provided by an embodiment of the present application, which shows that when the concentration of graphene sheets (RGO / PDDA) reaches 8 μg / ml, the fluorescence of the antibody-carbon quantum dot (fluorescent probe) is completely quenched;

[0055] Figure 7B is a graph provided by an embodiment of the present application, which shows that 8 μg / ml of graphene sheets (RGO / PDDA) is the optimal dosage;

[0056] Figure 8A is a graph provided by an embodiment of the present application, which shows that the quenching fluorescence is restored and becomes stronger with the increase of the concentration of ovalbumin;

[0057] Figure 8B is a graph provided by an embodiment of the present application, which shows the relationship between the fluorescence recovery intensity (I) and the logarithmic concentration (log c) of ovalbumin;

[0058] Figure 9A is a graph provided by an embodiment of the present application, which shows that the same concentration (10 ng / mL) of ovalbumin is detected by 6 groups of antibody-carbon quantum dot / graphene sheet (RGO_PDDA) parallel samples, and the fluorescence spectrum of the mixture is determined after standing at room temperature for 15 minutes;

[0059] Figure 9B is a graph provided by an embodiment of the present application, which shows the results of repeated fluorescence recovery experiments with chicken egg white (OVA) and bovine serum albumin (BSA) as interferents, which are 100 times the concentration of ovalbumin. DETAILED DESCRIPTION

[0060] In order to make the above-mentioned objects, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application. However, the present application can be implemented in many different ways other than those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0061] I. Explanation of Embodiments

[0062] This invention provides a fluorescence immunoassay method based on carbon quantum dots for detecting vitellogenin concentration, comprising: using L-glutamic acid as a precursor, preparing amino-containing carbon quantum dots in one step by ultrasonic hydrothermal method;

[0063] Graphene sheets were prepared using the reduced graphene oxide method, and graphene was optimized by modifying polydiallyl dimethylammonium chloride (PDDA) to obtain graphene (RGO_PDDA) that can undergo good fluorescence resonance energy transfer with carbon quantum dots.

[0064] The prepared amino-containing carbon quantum dots were conjugated with monoclonal antibodies to obtain purified antibody-carbon quantum dot conjugates, which were then used as fluorescent nanoprobes to determine the optimal dosage of RGO_PDDA, establish fluorescence recovery linear curves, and measure repeatability, anti-interference, and accuracy.

[0065] The technical solution of the present invention will be further described below with reference to specific examples.

[0066] Example 1

[0067] like Figure 1 As shown, the fluorescence immunoassay method for detecting vitellogenin concentration based on carbon quantum dots provided in this embodiment of the invention includes:

[0068] S101, Preparation of amino-containing carbon quantum dots: Using L-glutamic acid as a precursor, amino-containing carbon quantum dots were prepared in one step by ultrasonic hydrothermal method.

[0069] 60 mL of 15% L-glutamic acid aqueous solution was subjected to ultrasonic hydrothermal reaction at 250 °C for 4-5 hours, cooled to room temperature, filtered, dialyzed, and then freeze-dried to obtain a powder sample.

[0070] S102, Preparation of graphene capable of fluorescence resonance energy transfer with amino-containing carbon quantum dots: Graphene sheets were prepared using the reduced graphene oxide method, and graphene was optimized by modifying polydiallyldimethylammonium chloride (PDDA) to obtain graphene (RGO_PDDA) capable of fluorescence resonance energy transfer with amino-containing carbon quantum dots.

[0071] The steps include: preparing a 1 mg / 8 mL aqueous solution of graphene oxide, ultrasonic dispersion for 20 min. Under stirring, add 25% ammonia water by mass fraction, adjust the pH value to 9, then add polyvinylpyrrolidone (PVP), so that the mass fraction of PVP in the solution is 1%, stir to uniformly disperse PVP in the solution, drop 0.02 mol / L ascorbic acid solution, and reduce under magnetic stirring and water bath heating to 80°C. In this process, the color of the solution gradually deepens from brown to dark black, indicating that the graphene oxide is reduced to graphene (RGO). Centrifuge the prepared solution, wash with water twice to remove residual PVP, and finally use ethanol to centrifuge and clean, and dry the RGO in a 45°C constant temperature oven to obtain a solid RGO powder. The prepared RGO and PDDA are mixed in a mass ratio of 1:1 to prepare a suspension, stirred for 30 min, washed with water and centrifuged three times to remove excess PDDA. Finally, dry into a solid powder in a constant temperature drying oven at 45°C, and record as RGO_PDDA.

[0072] S103, carbon quantum dots with amino groups are coupled with monoclonal antibodies (fluorescent probes): prepare a 0.1 mol / L MES solution, then take 0.4 mg of 1-ethyl-(3-dimethylpropyl) carbonyl diimide (EDC) reagent and 1.1 mg of N-hydroxysuccinimide (NHS) into 0.1 mL of the prepared MES solution, mix and stir to form a crosslinking agent (EDC-NHS) solution for later use. Take 0.99 mL of antibody, add 0.01 mL of EDC-NHS solution, and stir at 27°C for 15 min to activate the antibody. Mix equal volumes of the activated antibody and the PBS solution of carbon quantum dots, incubate at 37°C for 1 hour to form antibody-carbon quantum dot conjugates. To remove unconjugated molecules, the obtained mixed solution is washed with PBS buffer and ultracentrifuged three times to obtain purified antibody-carbon quantum dot conjugates as fluorescent nanoprobes.

[0073] S104, the optimal dosage of graphene sheet: take multiple 0.5 ml of activated antibody-carbon quantum dots conjugate, and 0.1 mL of RGO_PDDA phosphate buffered saline (PBS) with concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μg / mL, respectively, are mixed, and PBS is added to a total volume of 1 mL. After appropriate shaking, stand for a period of time (about 30 min), wait for the antibody-carbon quantum dots conjugate to be adsorbed on the surface of RGO_PDDA by electrostatic attraction and π-π stacking. Measure the fluorescence spectrum under different RGO_PDDA concentrations, and observe the fluorescence resonance energy transfer phenomenon. Under the irradiation of excitation light, due to the dipole-dipole interaction, the energy (photons) emitted by the fluorescent probe is non-radiatively transferred to RGO_PDDA, resulting in fluorescence quenching. When the degree of fluorescence quenching reaches saturation, the corresponding RGO_PDDA concentration is the optimal dosage of graphene. Antibody-carbon quantum dots / RGO_PDDA can be used as an immunosensor for detecting targets.

[0074] S105, establishment of fluorescence recovery linear curve: add 0.1 ml of different concentrations of antigen to multiple 0.6 ml of antibody-carbon quantum dots / RGO_PDDA solution, and dilute to 1 mL with PBS buffer. The concentration of the antigen is between 0 and 15000 ng / mL. Incubate at room temperature for 15 min, measure the fluorescence spectrum of the mixture, and observe the fluorescence recovery. The fluorescence measurement conditions are the same as above. The above experiment is repeated three times. Draw the standard curve of antibody logarithmic concentration-fluorescence intensity. Read the linear range, measurement error, and the lowest detection limit from the standard curve.

[0075] S106, repeatability and anti-interference measurement and detection accuracy: use 6 groups of antibody-carbon quantum dots / RGO_PDDA parallel samples to detect the same concentration of ovalbumin, and observe the repeatability of the measurement.

[0076] Take chicken egg white (OVA) and bovine serum albumin (BSA) with a concentration 100 times that of ovalbumin as interferents, repeat the fluorescence recovery experiment, and prove the anti-interference of the fluorescence immunosensor. The experiment is repeated three times.

[0077] S107, ovalbumin concentration detection: take the ovalbumin solution, add antibody-carbon quantum dots / RGO_PDDA, and then add phosphate buffered saline. Incubate at room temperature, measure the fluorescence spectrum, repeat three times, read the average intensity of the fluorescence peak, and obtain the ovalbumin concentration from the established standard curve.

[0078] Through the above embodiments, the graphene is modified and improved, the optimal dosage of graphene, the coupling of carbon quantum dots and monoclonal antibodies, the establishment of a fluorescence recovery linear curve and other technical schemes. The effects obtained include: the linear detection range is wide (0.1-3000 ng / mL), the detection limit is low (LOD, 0.038 ng / mL), the sensitivity is high (28833.63 CPS / (ng / mL), the repeatability is good (the relative mean square deviation of 6 measurements is 1.63%), the measurement speed is fast (<15 min), and the anti-interference performance is strong.

[0079] Example 2

[0080] Based on the fluorescence immunoassay method for detecting the concentration of ovalbumin provided in Example 1 of the present application, further, the present application can provide an improved graphene quantum dot and reduced graphene oxide fluorescence resonance energy transfer immunofluorescence sensor for detecting ovalbumin.

[0081] In the above embodiments, the description of each embodiment has its own focus, and the parts not described or recorded in a certain embodiment can be referred to the related description of other embodiments.

[0082] II. Application Examples

[0083] Application Example 1

[0084] Based on the improved graphene quantum dot and reduced graphene oxide fluorescence resonance energy transfer immunofluorescence sensor obtained by the fluorescence immunoassay method for detecting the concentration of ovalbumin provided in the present application, the sensor is used for detecting pesticides.

[0085] Application Example 2

[0086] Based on the improved graphene quantum dot and reduced graphene oxide fluorescence resonance energy transfer immunofluorescence sensor obtained by the fluorescence immunoassay method for detecting the concentration of ovalbumin provided in the present application, the sensor is used for detecting polychlorinated biphenyl.

[0087] Application Example 3

[0088] Based on the improved graphene quantum dot and reduced graphene oxide fluorescence resonance energy transfer immunofluorescence sensor obtained by the fluorescence immunoassay method for detecting the concentration of ovalbumin provided in the present application, the sensor is used for detecting bisphenol.

[0089] III. Evidence of Effects Related to Examples

[0090] Experiment

[0091] The application provides a fluorescent immunoassay method for detecting yolk protein concentration based on carbon quantum dots, which comprises the following steps:

[0092] Step 1, preparation of carbon quantum dots with amino groups;

[0093] The carbon quantum dots with amino groups are prepared by using L-glutamic acid as a precursor through an ultrasonic hydrothermal method.

[0094] 60mL of 15% L-glutamic acid aqueous solution is subjected to ultrasonic hydrothermal reaction at 250 DEG C for 4-5 hours, cooled to room temperature, filtered, then subjected to dialysis, and finally freeze-dried to obtain a powder sample.

[0095] Whether the carbon quantum dots carry amino groups is determined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, as shown in Figure 2 and Fig. 3, Figure 2 which is Fourier transform infrared spectroscopy, and peaks at about 3400cm -1 and 1530cm -1 represent the stretching vibration of amino N-H, indicating that the carbon quantum dots have amino groups. Fig. 3 is X-ray photoelectron spectroscopy, Figure 3A which is a full spectrum analysis, indicating that the carbon quantum dots have a high nitrogen content, Figure 3B and N1s spectrum, which can indicate the existence of N-H bond through peak separation.

[0096] Step 2, preparation of graphene capable of good fluorescence resonance energy transfer with the carbon quantum dots;

[0097] The preparation process comprises the following steps: configuring 1mg / 8mL of graphene oxide aqueous solution, ultrasonic dispersion for 20min, adding 25% ammonia water under stirring to adjust the pH value to 9, then adding polyvinylpyrrolidone (PVP) so that the mass fraction of PVP in the solution is 1%, stirring to uniformly disperse the PVP in the solution, and dropwise adding 0.02mol / L ascorbic acid solution, and reducing under magnetic stirring and water bath heating to 80 DEG C. In this process, the color of the solution gradually deepens from brown to dark black, indicating that the graphene oxide is reduced to graphene. The prepared solution is centrifuged, washed with water twice to remove residual PVP, and finally washed with ethanol by centrifugation. The reduced graphene oxide is placed in a 45 DEG C constant temperature box for drying to obtain solid graphene (RGO) powder, which can be uniformly dispersed in water. The prepared RGO and PDDA are configured into a suspension liquid in a mass ratio of 1:1, fully stirred for 30min, and washed with water and centrifuged three times to remove excess PDDA. Finally, the solid powder is dried in a constant temperature drying box at 45 DEG C, and is recorded as RGO_PDDA.

[0098] The fluorescence resonance energy transfer efficiency between the graphene sheet and the carbon quantum dots is determined by photoluminescence spectroscopy. As shown in Fig. 4, Figure 4A and Figure 4BThe fluorescence quenching graphs between the graphene sheets and carbon quantum dots before and after modification of PDDA, respectively, corresponding to the quenching efficiency as shown in Figure 4C It can be seen that the graphene sheets modified by PDDA can have good fluorescence resonance energy transfer with carbon quantum dots. The charge properties of the graphene sheets and carbon quantum dots before and after modification of PDDA are determined by Zeta potential analysis, as shown in Figure 5 It can be seen that the potential of the graphene sheets modified by PDDA changes, and it is easier for them to approach the carbon quantum dots through electrostatic attraction, so that the fluorescence resonance energy transfer between them is more likely to occur.

[0099] Step 3, coupling of carbon quantum dots and monoclonal antibody (fluorescent probe);

[0100] Prepare 0.1 mol / L of MES solution. Measure the pH value of the MES solution using a pH meter, and adjust the pH to about 5 by adding hydrochloric acid. Then, take 0.4 mg of 1-ethyl-(3-dimethylpropyl) carbonyl diimide (EDC) and 1.1 mg of N-hydroxysuccinimide (NHS) into 0.1 mL of the MES solution, mix and stir to form an EDC-NHS solution for later use. In order to activate the antibody, take 0.99 mL of the antibody, and add 0.01 mL of the EDC-NHS solution to it, and stir at 27°C for 15 min to activate the antibody. Mix the activated antibody with an equal volume of the PBS solution of carbon quantum dots, and incubate at 37°C for 1 h to form an antibody-carbon quantum dot conjugate. In order to remove the unconjugated molecules, wash the obtained mixed solution with PBS buffer and ultracentrifuge three times to obtain the purified antibody-carbon quantum dot conjugate as a fluorescent nanoprobe.

[0101] The successful coupling of carbon quantum dots and monoclonal antibody (fluorescent probe) is determined by changes in ultraviolet-visible absorption spectrum Figure 6A ) and fluorescence spectrum Figure 6B ), the fluorescence intensity of the fluorescent probe changes with the excitation wavelength Figure 6C , and the strongest fluorescence corresponds to the excitation wavelength selected as the excitation wavelength (350 nm) for subsequent experiments.

[0102] Step 4, optimal dosage of graphene sheets;

[0103] Take multiple 0.5 ml of antibody-carbon quantum dots conjugate, with 0.1 mL of concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μg / ml RGO_PDDA phosphate buffered saline solution mixed, and add PBS to a total volume of 1 mL, appropriate shaking after standing for a period of time (about 30 min), to wait for the antibody-carbon quantum dots conjugate by electrostatic attraction and π-π stacking effect adsorbed on the surface of RGO_PDDA. Measure the fluorescence spectrum under different RGO_PDDA concentration, observe fluorescence resonance energy transfer phenomenon. Under the irradiation of excitation light, due to the dipole-dipole interaction, the energy (photons) emitted by the fluorescent probe is non-radiatively transferred to RGO_PDDA, resulting in fluorescence quenching phenomenon. When the degree of fluorescence quenching reaches saturation, the corresponding RGO_PDDA concentration is the optimal dosage of graphene. Antibody-carbon quantum dots / RGO_PDDA can be used as an immunosensor for detecting targets.

[0104] As shown in Figure 7, when the concentration of RGO_PDDA reaches 8 μg / ml, the fluorescence of antibody-carbon quantum dots (fluorescent probe) is completely quenched, as shown in Figure 7A ; 8 μg / ml RGO_PDDA as the optimal dosage. Figure 7B The corresponding concentration of quenching efficiency is shown.

[0105] Step 5, establishment of fluorescence recovery linear curve;

[0106] To multiple 0.6 ml of antibody-carbon quantum dots / RGO_PDDA solution, add 0.1 ml of different concentrations of antigen, and add PBS buffer to 1 mL. The concentration of the antigen is between 0-15000 ng / mL. Incubate at room temperature for 15 min, measure the fluorescence spectrum of the mixture, and observe the fluorescence recovery. The fluorescence measurement conditions are the same as above. The above experiment is repeated three times. Draw the standard curve of antibody logarithmic concentration-fluorescence intensity. Read the linear range, measurement error and the lowest detection limit from the standard curve.

[0107] As Figure 8A , the fluorescence increases with the increase of the concentration of vitellogenin (Vtg), and the relationship curve between the fluorescence recovery intensity (I) and the logarithmic concentration of Vtg (log c) is shown in Figure 8B . The mathematical expression can be written as I = 54324.76 + 28833.63 log c Vtg, and the regression coefficient (R 2 ) is 0.99968. The lowest detection limit determined by the signal-to-noise ratio of 4 (S / N = 4) is 0.038 ng / mL, the linear detection range is 0.1-3000 ng / mL, and the sensitivity is 28833.63 CPS / (ng / mL).

[0108] Step 6, repeatability and anti-interference measurement and detection accuracy;

[0109] The same concentration (10 ng / mL) of ovalbumin was detected by 6 groups of antibody-carbon quantum dots / RGO_PDDA parallel samples, and the fluorescence spectrum of the mixture was determined after standing at room temperature for 15 min (Fig. 4b), and the relative standard error was 1.63%, indicating that the developed immunosensor had good repeatability. Figure 9A

[0110] Chicken egg white (OVA) and bovine serum albumin (BSA) with 100 times the concentration of ovalbumin were used as interferents, and the fluorescence recovery experiment was repeated, and the results are shown in Fig. 4c. Figure 9B The fluorescence recovery rate of 10 ng / mL ovalbumin was 41.75%, while the fluorescence recovery rates of 1 μg / ml chicken egg white and bovine serum albumin were only 4.23% and 2.86%, respectively, proving that the fluorescence immunosensor has good anti-interference performance.

[0111] Figure 9B Fig. 4c is a fluorescence recovery experiment result graph of chicken egg white and bovine serum albumin with 100 times the concentration of ovalbumin (Vtg) as interferents, respectively, according to the embodiment of the present application. Wherein curve 1: fluorescence spectrum of antibody-carbon quantum dot conjugate / ovalbumin; 2: fluorescence spectrum of antibody-carbon quantum dot conjugate; 3: fluorescence recovery spectrum of 10 ng / mL ovalbumin; 4: fluorescence recovery spectrum of 1.0 μg / mL bovine serum albumin; 5: fluorescence recovery spectrum of 1.0 μg / mL chicken egg white; 6: fluorescence spectrum of antibody-carbon quantum dot / RGO_PDDA.

[0112] The above is only a more preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principle of the present application, should be covered within the protection scope of the present application.​

Claims

1. A fluorescent immunoassay method for detecting the concentration of ovalbumin based on carbon quantum dots, characterized by, The fluorescence immunoassay method for detecting the concentration of ovalbumin based on carbon quantum dots comprises the following steps: The amino-carbon quantum dots are prepared by using L-glutamic acid as a precursor through ultrasonic hydrothermal method; 60 mL of 15% L-glutamic acid aqueous solution is subjected to ultrasonic hydrothermal reaction at 250 DEG C for 4-5 hours, cooled to room temperature, filtered, then dialyzed, and finally freeze-dried to obtain a powder sample; The graphene sheets are prepared by using a reduced graphene oxide method, and the graphene is optimized by modifying polydiallyldimethylammonium chloride to obtain graphene sheets capable of fluorescence resonance energy transfer with the amino-carbon quantum dots; The prepared amino-carbon quantum dots are coupled with a monoclonal antibody to obtain purified antibody-carbon quantum dot conjugates as fluorescent nanoprobes, which are used for determining the graphene sheet dosage, establishing a fluorescence recovery linear curve, and measuring the repeatability, anti-interference and precision; the fluorescence spectra under different graphene concentrations are measured, and the fluorescence resonance energy transfer is analyzed; when the fluorescence quenching degree reaches saturation, the corresponding graphene sheet concentration is the optimal dosage of graphene. 2.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 1, characterized in that, The graphene sheets capable of fluorescence resonance energy transfer with the amino-carbon quantum dots are prepared by using a reduced graphene oxide method, and the graphene is optimized by modifying polydiallyldimethylammonium chloride, comprising the following steps: Step 1) prepare an aqueous solution of graphene oxide and ultrasonically disperse; under stirring, add ammonia water to adjust the pH value to 9, then add polyvinylpyrrolidone, stir to uniformly disperse the polyvinylpyrrolidone in the solution, and drop ascorbic acid solution under magnetic stirring and water bath heating reduction; Step 2) centrifuge the prepared solution, wash twice with water to remove residual polyvinylpyrrolidone, and finally centrifuge with ethanol to clean the reduced graphene oxide, and dry the reduced graphene oxide in a constant temperature oven to obtain a solid graphene powder, which can be uniformly dispersed in water; Step 3) prepare the graphene sheets and polydiallyldimethylammonium chloride into a suspension liquid with a mass ratio of 1:1, fully stir, centrifuge three times with water to remove excess polydiallyldimethylammonium chloride; finally, dry in a constant temperature drying oven to obtain a solid graphene powder capable of fluorescence resonance energy transfer with the carbon quantum dots. 3.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 1, characterized in that, The coupling of the amino-carbon quantum dots with the monoclonal antibody comprises the following steps: (1) prepare a 2-morpholinoethanesulfonic acid solution, then add 1-ethyl-(3-dimethylpropyl) carbonyl diimide reagent and N-hydroxysuccinimide to the prepared 2-morpholinoethanesulfonic acid solution respectively, mix and stir to form a crosslinking agent solution for later use; (2) take the antibody, add the crosslinking agent solution, stir to activate the antibody; mix the activated antibody and the carbon quantum dot phosphate buffer solution in equal volume, incubate to form an antibody-carbon quantum dot conjugate; (3) obtain the purified antibody-carbon quantum dot conjugate as a fluorescent nanoprobe. 4.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 3, characterized in that, Before obtaining the purified antibody-carbon quantum dot conjugate in step (3), the antibody-carbon quantum dot conjugate mixed solution obtained in step (2) is washed with a phosphate buffer solution and ultracentrifuged three times. 5.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 1, characterized in that, In the graphene sheets capable of fluorescence resonance energy transfer with the amino-carbon quantum dots, the graphene sheet dosage comprises: Take multiple activated antibody-carbon quantum dots conjugates, respectively, with the concentration of 1.0 μg / ml, 2.0 μg / ml, 3.0 μg / ml, 4.0 μg / ml, 5.0 μg / ml, 6.0 μg / ml, 7.0 μg / ml, 8.0 μg / ml of modified polydiallydimethylammonium chloride after the graphene phosphate buffered saline solution is mixed, and the phosphate buffered saline solution is added to the total volume of 1 mL, after shaking, the antibody-carbon quantum dots conjugate is adsorbed on the surface of graphene by electrostatic attraction and π-π stacking effect. 6.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 5, characterized in that, The activated antibody-carbon quantum dots conjugate uses antibody-carbon quantum dots / graphene as the detection target of the immunosensor. 7.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 1, wherein, The establishment of the fluorescence recovery linear curve includes the following steps: Add different concentrations of antigens to multiple antibody-carbon quantum dots / graphene solutions, and dilute with phosphate buffered saline. Incubate at room temperature, measure the fluorescence spectrum of the mixture three times, and draw the standard curve of the logarithmic concentration of the antigen versus the fluorescence intensity. The concentration of the antigen is in the range of 1 ng / mL-15000 ng / mL, and the linear range is 0.1 ng / mL-3000 ng / mL. 8.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 7, characterized in that, In the antibody-carbon quantum dots / graphene solution, the concentration of the egg yolk protein is detected by adding the egg yolk protein solution to the antibody-carbon quantum dots / graphene aqueous solution, then adding phosphate buffered saline to dilute, incubating at room temperature, measuring the fluorescence spectrum of the mixture, reading the fluorescence peak intensity, and obtaining the concentration of the egg yolk protein from the established standard curve of the logarithmic concentration of the antigen versus the fluorescence intensity. 9.The carbon quantum dots-based fluorescent immunoassay method for detecting the concentration of vitellin according to claim 1, wherein, In the repeatability and anti-interference measurement and detection accuracy, six groups of antibody-carbon quantum dots / graphene parallel samples are used to detect the same concentration of egg yolk protein, chicken egg white and bovine serum albumin are used as interferents at 100 times the concentration of the egg yolk protein, the fluorescence recovery experiment is repeated, and the anti-interference of the fluorescence immunosensor is analyzed. The experiment is repeated three times.

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