Fluorescence immunoassay and immunosensor for detecting vitellogenin concentration

Abstract

The present invention belongs to the technical field of application of nanomaterials in environmental monitoring, and discloses a fluorescent immunoassay method and an immunosensor for detecting the concentration of vitellogenin. The fluorescent immunoassay method for detecting the concentration of vitellogenin includes: coupling aminated graphene quantum dots with monoclonal antibodies, reducing graphene oxide, and transferring the energy emitted by fluorescent nanoprobes to graphene sheets without radiation for fluorescence quenching. When the degree of fluorescence quenching reaches saturation, the corresponding graphene sheet concentration is the optimal amount of graphene; adding different concentrations of antigens to multiple portions of antibody-aminated graphene quantum dots / graphene aqueous solutions obtained based on the optimal amount of graphene to obtain an antigen logarithmic concentration-fluorescence intensity standard curve. The present invention has a wide linear detection range, a low detection limit, high sensitivity, good repeatability, fast measurement speed, and strong anti-interference.

Description

Technical Field

[0001] The invention belongs to the technical field of application of nano materials in environmental monitoring, and in particular relates to a fluorescent immunoassay method and an immunosensor for detecting vitellogenin concentration. Background Art

[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. Through fruits, vegetables, meat and water, these estrogens accumulate in humans or animals, and ultimately threaten human or animal health, such as interfering with the endocrine system, leading to abnormal testicular development, gender ratio deviation and other adverse phenomena, so estrogen detection has attracted widespread attention. The Organization for Economic Cooperation and Development (OECD) and the US EPA have established screening test guidelines with fish as test organisms and vitellogenin (Vtg) as the main indicator, and recommend vitellogenin from 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 test speed are of great significance for the detection of environmental estrogens.

[0003] The accurate determination of vitellogenin is an important prerequisite for the correct evaluation of the estrogenic activity of compounds. Since the immune reaction between antigens and antibodies is highly specific, immunosensors have inherent specificity. For immunosensors based on monoclonal antibodies, certain antibodies can recognize specific antigens among many substances, so immunosensor technology based on monoclonal antibodies has become the development direction of complex environmental monitoring.

[0004] Traditional immunoassay techniques include enzyme-linked immunosorbent assay, radioimmunoassay, Western blots, etc. In recent years, electrochemical immunosensors, optical sensing methods, immunocolloidal gold techniques, etc. have been developed. However, fish vitellogenin is prone to degradation, which will cause errors in quantitative results and is not suitable for use as a standard for quantitative detection experiments. Compared with vitellogenin, the main degradation product of vitellogenin, lipovitellin (Lv), did not degrade under conditions such as high temperature treatment, long-term storage at -80°C, and repeated freezing and thawing, showing good stability; immunological experiments showed that Lv and Vtg had the same immunogenicity, and Lv and Vtg had the same binding rate to anti-Lv antibodies (literature on the same binding rate of brown flounder Vtg and Lv with anti-Lv antibodies: Zhenzhong Zhang, Jun Wang, Ming Gao, Xuefu Li, Yuqi Cheng, Xiaona Zhang, Hua Tian, ​​Wei Wang, Shaoguo Ru. New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin, Ecotoxicology and Environmental Safety 180 (2019) 624–631.), so Lv is more suitable as a standard than Vtg, and the immunosensor established based on this can be used to detect Vtg. Monoclonal antibodies are more effective than polyclonal antibodies.

[0005] Through the above analysis, the problems and defects of the existing technology are as follows: traditional immunoassays show high specificity and sensitivity, but there are some disadvantages, such as the enzyme-linked immunosorbent assay must use enzymes, and the preparation and preservation of enzymes have high requirements on environmental conditions; the radioimmunoassay method requires the use of radioactive elements, which may cause harm to the environment or humans; the immunological Western blots method requires the use of secondary antibodies. These methods are generally time-consuming and not suitable for large-scale detection in a short period of time. In recent years, electrochemical and optical sensors have been used for vitellogenin concentration detection. The electrochemical immunosensor based on impedance spectroscopy shows a wide measurement range (1000-8000 ng / mL), but has a low sensitivity (420 ng / mL); the electrochemical immunosensor based on current has a low detection limit (0.09 ng / mL) but a small linear range (0.25-7.8 ng / mL); the optical waveguide spectroscopy sensor shows a wide measurement range (100-10000 ng / mL) and a low detection limit (0.1 ng / mL), but cannot detect Vtg below 100 ng / mL; the surface enhanced Raman scattering technology has a very low detection limit (5 pg / mL), but a very narrow linear range (~0.2 ng / mL). Immunocolloidal gold technology requires the use of nanogold, which is a precious metal and is expensive. Summary of the invention

[0006] In order to overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a fluorescent immunoassay method and an immunosensor for detecting the concentration of vitellogenin.

[0007] The technical scheme is as follows: A fluorescent immunoassay for detecting vitellogenin concentration comprises:

[0008] The aminated graphene quantum dots are coupled with monoclonal antibodies to obtain a purified antibody-aminated graphene quantum dot conjugate as a fluorescent nanoprobe; graphene oxide is reduced to obtain water-soluble graphene; the energy emitted by the fluorescent nanoprobe is transferred to the graphene sheet without radiation to perform fluorescence quenching, and the corresponding graphene sheet concentration when the fluorescence quenching degree reaches saturation is the optimal amount of graphene; different concentrations of antigens are added to multiple antibody-aminated graphene quantum dots / graphene aqueous solutions obtained based on the optimal amount of graphene, incubated, the antigen and the antibody specifically react to restore the quenched fluorescence, the restored fluorescence intensity is positively correlated with the amount of antigen added, the fluorescence spectrum of the mixture is measured, and a standard curve of antigen logarithmic concentration-fluorescence intensity is obtained.

[0009] In one embodiment, the fluorescent immunoassay method for detecting vitellogenin concentration specifically comprises the following steps:

[0010] Step 1, amination of graphene quantum dots;

[0011] Step 2, coupling the amino graphene quantum dots with monoclonal antibodies;

[0012] Step 3, reducing graphene oxide to obtain water-soluble graphene;

[0013] Step 4, determining the optimal amount of graphene;

[0014] Step 5, establishment of fluorescence recovery linear curve;

[0015] Step 6: Repeatability and anti-interference measurement;

[0016] Step seven, detection of vitellogenin concentration.

[0017] In one embodiment, the amination of graphene quantum dots in step 1 specifically includes: mixing an aqueous solution of graphene quantum dots (GQDs) with an aqueous ammonia solution, and stirring at room temperature to evenly mix; transferring the solution to a polytetrafluoroethylene container and heating it to evaporate excess ammonia, and then dialyzing and purifying it in pure water, and freeze-drying the dialyzed amination graphene quantum dots to obtain a powder sample.

[0018] In one embodiment, in step 2, the coupling of the aminated graphene quantum dots with the monoclonal antibody specifically includes: taking 1-ethyl-(3-dimethylpropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) respectively, adding them to a 4-morpholineethanesulfonic acid solution, mixing and stirring to form an EDC-NHS solution for standby use; taking an antibody, adding an EDC-NHS reagent, and stirring to activate the antibody; mixing the activated antibody with an aminated graphene quantum dot (afGQDs) solution, incubating, and forming an antibody-aminated graphene quantum dot conjugate; centrifuging to remove free uncoupled antibodies, and during the centrifugation process, washing the conjugate with a PBS buffer; obtaining a purified antibody-aminated graphene quantum dot conjugate as a fluorescent nanoprobe.

[0019] In one embodiment, the specific steps of reducing graphene oxide to obtain water-soluble graphene in step three include: configuring 1mg / 10mL of graphene oxide aqueous solution, ultrasonic dispersion for 20 minutes. Add 25% ammonia water under stirring, adjust the pH value to 9, then add polyvinyl pyrrolidone (PVP), make the mass fraction of PVP and solution to be 1%, stir to make PVP uniformly dispersed in the solution, drip 0.02mol / L ascorbic acid solution, heat to 80°C in a water bath under magnetic agitation, and reduce for 80min. In this process, the color of the solution gradually deepens from brown to dark black, indicating that graphene oxide is reduced to graphene. The prepared solution is centrifuged, washed twice with water to remove residual PVP, finally cleaned by ethanol centrifugation, and the reduced graphene oxide is placed in a 40°C thermostat and dried to obtain solid graphene powder, and the powder sample can be evenly dispersed in water.

[0020] In one embodiment, the optimal amount of graphene in step four specifically includes: taking multiple portions of antibody-amino-graphene quantum dot solution of the same volume, adding graphene solutions of different concentrations respectively, stirring and dispersing, and then adding PBS aqueous solution, and incubating at room temperature; then measuring the fluorescence spectrum of the above solution and observing fluorescence quenching; when the fluorescence quenching degree reaches saturation, the corresponding graphene concentration is the optimal amount of graphene.

[0021] In one embodiment, the optimal amount of graphene is 4 μg / mL.

[0022] In one embodiment, the establishment of the fluorescence recovery linear curve in step five specifically includes: adding different concentrations of antigen to multiple antibody-amino graphene quantum dots / graphene aqueous solutions, and then adding PBS buffer, the concentration of the antigen is 0-1500.0 ng / mL; incubating at room temperature, measuring the fluorescence spectrum of the mixture, and repeating three times; drawing a standard curve of antigen logarithmic concentration-fluorescence intensity; reading the linear range, measurement error and minimum detection limit from the standard curve.

[0023] In one embodiment, the mathematical expression of the antigen logarithmic concentration-fluorescence intensity standard curve is I=26407.85log c Lv+85145.31, and the regression coefficient (R 2 ) is 0.9994.

[0024] In one embodiment, the repeatability and anti-interference measurement in step six specifically includes: using egg white and bovine serum albumin as interference substances, repeating the fluorescence recovery experiment;

[0025] In step seven, the detection of vitellogenin concentration specifically includes: taking vitellogenin solution, adding antibody-amino-graphene quantum dots / graphene aqueous solution, then adding PBS buffer, incubating at room temperature, measuring the fluorescence spectrum, reading the fluorescence peak intensity, and obtaining the vitellogenin concentration from the established standard curve.

[0026] Another object of the present invention is to provide an immunosensor for detecting target antigens based on antibody-amino-graphene quantum dots / graphene, wherein the immunosensor implements the fluorescent immunoassay for detecting vitellogenin concentration.

[0027] Combining all the above technical solutions, the advantages and positive effects of the present invention are as follows:

[0028] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, the technical solutions to be protected by the present invention and the results and data in the research and development process are closely combined to analyze in detail and deeply how the technical solutions of the present invention solve the technical problems, and some creative technical effects brought about after solving the problems. The specific description is as follows: The problem of the poor accuracy of the detection method and the possible false positive results caused by the easy degradation of vitellogenin as a standard in the existing method is successfully solved. The problem that graphene sheets are easy to agglomerate and not easily soluble in water in the process of obtaining graphene by reducing graphene oxide is successfully solved. This method has high measurement accuracy and fast measurement speed.

[0029] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are described in detail as follows:

[0030] The present invention makes up for the deficiencies of the prior art, utilizes the strong fluorescence property of graphene quantum dots (graphene quantum dots are graphene sheets with a size less than 10nm, and their edges often have oxygen-containing groups, and graphene quantum dots can be obtained or prepared by a variety of methods), and uses the conjugate after the amino graphene quantum dots are coupled with the monoclonal antibody as a fluorescent probe. When the distance between the fluorescent probe (i.e., the amino graphene quantum dot-antibody conjugate) and the graphene sheet is less than 10nm, the fluorescence of the fluorescent probe (i.e., the amino graphene quantum dot-antibody conjugate) will be absorbed by the graphene sheet and quenched (fluorescence resonance energy transfer); when the antigen is added, due to the strong immune reaction between the antigen and the antibody, the fluorescent probe (i.e., the amino graphene quantum dot-antibody conjugate) is urged to stay away from the graphene sheet, so that the quenched fluorescence can be restored. Since the fluorescence recovery intensity is positively correlated with the concentration of the added antigen, it can be used to detect the concentration of the antigen. Existing results show that the main degradation product of vitellogenin, namely yolk lipophosphoprotein, is more stable than vitellogenin and similar to vitellogenin, has the same binding efficiency with monoclonal antibody (Anti-Lv-mAb). Therefore, yolk lipophosphoprotein can be used as a standard, and the antigen logarithmic concentration-fluorescence intensity standard curve established by this can be used to measure the concentration of vitellogenin with higher accuracy. In the present invention, the fluorescent probe refers to the conjugate of amino graphene quantum dots and antibodies, which also fluoresces and can be used as a fluorescent probe.

[0031] The innovative points of the present invention are: the conjugate of amino graphene quantum dots and monoclonal antibodies also fluoresces and can be used as a fluorescent probe; the optimal amount of graphene for fluorescence quenching; and the establishment of a linear curve for fluorescence recovery. Through the above innovative technical solutions, the present invention has a wide linear detection range (0.001-1500ng / mL), a low detection limit (LOD, 0.9pg / mL), a high sensitivity (26407.8CPS / (ng / mL), good repeatability (relative mean square deviation of five measurements is 1.2%), a fast measurement speed (<12min), and strong anti-interference.

[0032] Third, the inventiveness of the claims of the present invention is also reflected in that the present invention has a fast measurement speed, a simple measurement method, and can perform high-throughput detection (measuring multiple samples in a short time). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0034] Figure 1 is a flow chart of a fluorescent immunoassay method for detecting vitellogenin concentration provided by an embodiment of the present invention;

[0035] Figure 2 It is a UV-visible absorption spectrum of the anti-Lv-monoclonal antibody, graphene quantum dots, aminated graphene quantum dots and aminated graphene quantum dots / anti-Lv-monoclonal antibody conjugate (fluorescent probe) provided in the embodiments of the present invention;

[0036] Figure 3 It is a fluorescence spectrum diagram of graphene quantum dots, aminated graphene quantum dots and aminated graphene quantum dots / anti-Lv-monoclonal antibody conjugates provided by the embodiments of the present invention; CPS in the figure is the abbreviation of counts per second, which is the number of photons measured by the detector per second;

[0037] Figure 4 The fluorescence of the amino graphene quantum dots / anti-Lv-monoclonal antibody conjugate provided by the embodiment of the present invention varies with the excitation wavelength, wherein the fluorescence is strongest when excited at 360nm; CPS in the figure is the abbreviation of counts per second, which is the number of photons measured by the detector per second;

[0038] Figure 5A This is a fluorescence spectrum diagram of the fluorescence of the fluorescent probe provided by the embodiment of the present invention being gradually quenched by the graphene sheet; in the figure, CPS is the abbreviation of counts per second, which is the number of photons measured by the detector per second;

[0039] Figure 5BIt is a schematic diagram of an embodiment of the present invention that when the concentration of graphene reaches 4 μg / mL, the fluorescence intensity of the fluorescent probe is completely quenched, and 4 μg / mL graphene is used as the optimal dosage;

[0040] Fig. 6A It is a schematic diagram of the fluorescence intensity increasing with the increase of the concentration of yolk lipophosphoprotein provided by the embodiment of the present invention; in the figure, CPS is the abbreviation of counts per second, which is the number of photons measured by the detector per second;

[0041] Figure 6B 1 is a graph showing the relationship between the fluorescence recovery intensity (I) and the logarithmic concentration (logc) of yolk lipophosphoprotein (Lv) provided in an embodiment of the present invention; CPS in the graph is the abbreviation of counts per second, which is the number of photons measured by the detector per second;

[0042] Figure 7 The embodiment of the present invention provides a graph of the repeatability of the fluorescence spectra of five 1 ng / mL antigen (Lv) / antibody-amino-graphene quantum dots / graphene aqueous solutions respectively measured and observed. In the graph, CPS is the abbreviation of counts per second, which is the number of photons measured by the detector per second.

[0043] Figure 8 The present invention provides an embodiment in which egg white and bovine serum albumin (1.5 μg / mL) at a concentration 150 times that of egg yolk lipophosphoprotein (Lv) are used as interferences to repeat the fluorescence recovery experiment result graph. In the graph, CPS is the abbreviation of counts per second, which is the number of photons measured by the detector per second. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0045] 1. Explanation of the Embodiments

[0046] like Figure 1 As shown, the fluorescent immunoassay method for detecting the concentration of vitellogenin provided by the embodiment of the present invention includes:

[0047] S101, amination of graphene quantum dots; 50 mL of 1 mg / mL GQDs aqueous solution was mixed with 50 mL of 25% ammonia aqueous solution, and stirred at room temperature to mix evenly. The solution was transferred to a polytetrafluoroethylene container, heated at 200°C for 10 hours, and cooled at room temperature. The solution was further heated to 95°C for one hour to evaporate excess ammonia, and then dialyzed in pure water for 24 hours, with a molecular weight cutoff of 1000 in the dialysis bag. The amination graphene quantum dots after dialysis were freeze-dried to obtain a powder sample.

[0048] S102, graphene quantum dots coupled with monoclonal antibodies (fluorescent probe); 8 mg of 1-ethyl-(3-dimethylpropyl) carbodiimide (EDC) and 22 mg of N-hydroxysuccinimide were added to 2 mL of 0.1 mol / L 4-morpholineethanesulfonic acid (MES) solution, mixed and stirred to form an EDC-NHS solution for standby use. In order to activate the antibody, 19.8 mL of antibody with a concentration of 4 μg / mL was taken, 0.2 mL of EDC-NHS reagent was added thereto, and stirred for 15 min at 27°C to form an active antibody. The activated antibody was mixed with a 10 μg / mL amino graphene quantum dot solution in a volume ratio of 1:1, incubated at 37°C for 30 minutes, and the antibody and the amino graphene quantum dots were amidated to form an antibody-amino graphene quantum dot conjugate. On this basis, the free uncoupled antibody was removed by centrifugation, and the conjugate was washed 3 times with PBS buffer during the centrifugation process. Finally, the purified antibody-amino graphene quantum dot conjugate was used as a fluorescent nanoprobe and the volume was fixed to 20 mL with PBS buffer.

[0049] S103, reducing graphene oxide to obtain water-soluble graphene; in one embodiment, the specific steps of reducing graphene oxide to obtain water-soluble graphene in step three include: configuring 1mg / 10mL of graphene oxide aqueous solution, and ultrasonically dispersing for 20 minutes. Add 25% ammonia water under stirring, adjust the pH value to 9, add polyvinyl pyrrolidone (PVP), make the mass fraction of PVP and solution to 1%, stir to make PVP evenly dispersed in the solution, add 0.02mol / L ascorbic acid solution, heat to 80°C in a water bath under magnetic stirring, and reduce for 80min. In this process, the color of the solution gradually deepens from brown to dark black, indicating that graphene oxide is reduced to graphene. The obtained solution is centrifuged, washed twice with water to remove residual PVP, and finally centrifuged and cleaned with ethanol, and the reduced graphene oxide is placed in a 40°C thermostat to dry to obtain solid graphene powder, which can be evenly dispersed in water.

[0050] S104, the optimal amount of graphene sheets; take more than 0.5mL of antibody-amino graphene quantum dot solution, add 0.1mL of different concentrations of graphene solution (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0μg / mL) respectively, stir and disperse, add 0.4mL PBS aqueous solution, and incubate at room temperature for 30 minutes to promote the antibody-amino graphene quantum dots to be adsorbed on the surface of the graphene sheet through electrostatic attraction and π-π stacking. Then measure the fluorescence spectrum of the above solution and observe the fluorescence quenching phenomenon. Under the irradiation of excitation light, due to the dipole-dipole interaction, the energy (photons) emitted by the fluorescent probe is transferred to the graphene sheet without radiation, resulting in fluorescence quenching. The corresponding graphene sheet concentration when the fluorescence quenching degree reaches saturation is the optimal amount of graphene. The antibody-amino graphene quantum dot / graphene sheet complex can be used as an immunosensor for detecting targets.

[0051] S105, establishment of fluorescence recovery linear curve; add 0.2 mL of antigen of different concentrations to multiple 0.6 mL antibody-amino graphene quantum dots / graphene aqueous solutions, and then add 0.2 mL of PBS buffer to make the total volume 1 mL, and the concentration of the antigen is between 0-1500.0 ng / mL. After 12 minutes of incubation at room temperature, measure the fluorescence spectrum of the mixture, observe the fluorescence recovery, and the fluorescence measurement conditions are the same as S104. The above experiment is repeated three times. Draw a standard curve of antibody logarithmic concentration-fluorescence intensity. Read the linear range, measurement error and minimum detection limit from the standard curve.

[0052] S106, repeatability and anti-interference measurement; using 1.5 μg / mL chicken egg white and bovine serum albumin as interferences, the above fluorescence recovery experiment was repeated to prove the anti-interference property of the fluorescent immunosensor. The experiment was repeated three times.

[0053] Fluorescence spectra of five 1 ng / mL antigen / antibody-amino-modified graphene quantum dots / graphene aqueous solutions were measured respectively to observe the repeatability of the measurements.

[0054] S107, detection of vitellogenin concentration; because yolk lipophosphoprotein is one of the main components of vitellogenin and is more stable than vitellogenin, it is more suitable as a standard substance than vitellogenin; and because yolk lipophosphoprotein and vitellogenin have the same immunogenicity and both have the same binding rate with anti-Lv-monoclonal antibody, the standard curve established with yolk lipophosphoprotein as the standard substance is also applicable to the detection of vitellogenin.

[0055] Take 0.2 mL of unknown concentration of vitellogenin solution and add it to the above 0.6 mL of antibody-amino-graphene quantum dots / graphene aqueous solution, then add 0.2 mL of PBS buffer, incubate at room temperature for 12 minutes, measure the fluorescence spectrum, and read the fluorescence peak intensity. Repeat the above experiment three times, take the average of the three fluorescence peak intensities, and read the vitellogenin concentration from the established standard curve.

[0056] 2. Application Examples

[0057] For tap water containing 1 ng / mL vitellogenin, the fluorescence recovery rates measured three times were 95.53%, 99.18% and 101.23% respectively, that is, the measured concentrations were 0.9553 ng / mL, 0.9918 ng / mL, and 1.0123 ng / mL, and the relative measurement error was within 4.5%.

[0058] 3. The effects of the present invention are further described below in conjunction with evidence of the effects of the embodiments.

[0059] experiment

[0060] Normally, Vtg is only present in female fish during the yolk production period, but male fish and juvenile fish can also synthesize Vtg under the induction of environmental estrogens because they contain the Vtg gene. Therefore, the estrogenic effect of chemical substances can be evaluated by detecting the Vtg content in male fish or juvenile fish.

[0061] The fish species in the experiment was brown flounder, and the preparation and separation of Vtg and Lv and the preparation of anti-Lv monoclonal antibodies were public technologies (for the separation and purification of brown flounder Vtg, Lv, and the preparation technology of anti-Lv monoclonal antibodies, see: Zhenzhong Zhang, Jun Wang, Zongbao Pan, Yabin Zhang, Xiaona Zhang, Hua Tian, ​​Wei Wang, Shaoguo Ru. Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens, Aquatic Toxicology 216 (2019) 105321.).

[0062] Literature on the same binding rate between Vtg and Lv of brown flounder and anti-Lv antibodies: Zhenzhong Zhang, Jun Wang, Ming Gao, Xuefu Li, Yuqi Cheng, Xiaona Zhang, Hua Tian, ​​Wei Wang, Shaoguo Ru. New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin, Ecotoxicology and Environmental Safety 180 (2019) 624–631.

[0063] 1. Amination of graphene quantum dots:

[0064] Mix 50 mL of 1 mg / mL graphene quantum dot aqueous solution with 50 mL of 25% ammonia solution, stir and mix evenly at room temperature. Transfer the solution to a polytetrafluoroethylene container, heat at 200°C for 10 hours, and cool at room temperature. The solution is further heated to 95°C for one hour to evaporate excess ammonia, and then dialyzed in pure water for 24 hours, with a molecular weight cutoff of 1000 in the dialysis bag. The dialyzed amino graphene quantum dots are freeze-dried to obtain a powder sample.

[0065] 2. Graphene quantum dots coupled with monoclonal antibodies (fluorescent probes):

[0066] 8 mg of 1-ethyl-(3-dimethylpropyl) carbodiimide (EDC) and 22 mg of N-hydroxysuccinimide (NHS) were added to 2 mL of 0.1 mol / L 4-morpholineethanesulfonic acid (MES) solution, mixed and stirred to form an EDC-NHS solution for standby use. In order to activate the antibody, 19.8 mL of 4 μg / mL brown flounder anti-Lv-monoclonal antibody was added with 0.2 mL of EDC-NHS reagent, and stirred for 15 min at 27 ° C to form an active antibody. The activated antibody was mixed with a 10 μg / mL amino graphene quantum dot solution in a volume ratio of 1:1, incubated at 37 ° C for 30 minutes, and the antibody and the amino graphene quantum dots were amidated to form an antibody-amino graphene quantum dot conjugate. On this basis, centrifugation was performed to remove free unconjugated antibodies, and the conjugate was washed 3 times with PBS buffer during the centrifugation process. Finally, the purified antibody-amino graphene quantum dot conjugate was used as a fluorescent nanoprobe and the volume was fixed to 20 mL with PBS buffer.

[0067] The successful conjugation of graphene quantum dots with monoclonal antibodies (fluorescent probes) was confirmed by UV-visible absorption spectroscopy ( Figure 2 ) and fluorescence spectrum changes to judge ( Figure 3 ), the fluorescence intensity of the fluorescent probe changes with the excitation wavelength ( Figure 4 ), and the excitation wavelength corresponding to the strongest fluorescence was selected as the excitation wavelength for subsequent experiments (360 nm).

[0068] 3. Reduce graphene oxide to obtain water-soluble graphene: In one embodiment, the specific steps of reducing graphene oxide to obtain water-soluble graphene in step three include: configuring 1mg / 10mL of graphene oxide aqueous solution, and ultrasonically dispersing for 20 minutes. Add 25% ammonia water under stirring, adjust the pH value to 9, add polyvinyl pyrrolidone (PVP), make the mass fraction of PVP and solution to be 1%, stir to make PVP evenly dispersed in the solution, drop 0.02mol / L ascorbic acid solution, heat to 80°C in a water bath under magnetic stirring, and reduce for 80min. In this process, the color of the solution gradually deepens from brown to dark black, indicating that graphene oxide is reduced to graphene. The obtained solution is centrifuged, washed twice with water to remove residual PVP, and finally centrifuged and cleaned with ethanol, and the reduced graphene oxide is placed in a 40°C thermostat to dry to obtain solid graphene powder, which can be evenly dispersed in water.

[0069] 4. Optimal dosage of graphene sheets:

[0070] Take more than 0.5mL of antibody-amino graphene quantum dot solution, add 0.1mL of different concentrations of graphene solution (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0μg / mL) respectively, stir and disperse, add 0.4mL PBS aqueous solution, incubate at room temperature for 30 minutes to promote antibody-amino graphene quantum dots to be adsorbed on the surface of graphene sheets by electrostatic attraction and π-π stacking. Then measure the fluorescence spectrum of the above solution and observe the fluorescence quenching phenomenon. Under the irradiation of excitation light, due to the dipole-dipole interaction, the energy (photon) emitted by the fluorescent probe is transferred to the graphene sheet without radiation, resulting in the fluorescence quenching phenomenon. The corresponding graphene sheet concentration when the fluorescence quenching degree reaches saturation is the optimal amount of graphene. Antibody-amino graphene quantum dot / graphene sheet complex can be used as an immunosensor for detecting targets. Figure 5A is a fluorescence spectrum diagram showing that the fluorescence of the fluorescent probe provided by an embodiment of the present invention is gradually quenched by a graphene sheet; Figure 5B It is a schematic diagram of an embodiment of the present invention that when the concentration of graphene sheets reaches 4 μg / mL, the fluorescence intensity of the fluorescent probe is completely quenched, and 4 μg / mL graphene sheets is used as the optimal dosage.

[0071] 4. Establishment of fluorescence recovery linear curve:

[0072] Add 0.2 mL of different concentrations of brown flounder egg yolk phosphoprotein to multiple 0.6 mL portions of antibody-amino-graphene quantum dots / graphene aqueous solution, and then add 0.2 mL of PBS buffer to make the total volume 1 mL, and the concentration of egg yolk phosphoprotein is between 0-1500.0 ng / mL. After 12 minutes of incubation at room temperature, measure the fluorescence spectrum of the mixture, observe the fluorescence recovery, and the fluorescence measurement conditions are the same as above. Repeat the above experiment three times. Draw a standard curve of antigen logarithmic concentration-fluorescence intensity. Read the linear range, measurement error, and minimum detection limit from the standard curve.

[0073] like Fig. 6A The fluorescence becomes stronger with the increase of yolk lipophosphoprotein concentration. The relationship curve between the fluorescence recovery intensity (I) and the logarithmic concentration (log c) of yolk lipophosphoprotein (Lv) is shown in Figure 6B , the mathematical expression is written as I = 26407.85log c Lv + 85145.31, the regression coefficient (R 2 ) was 0.9994. The lowest detection limit determined by the signal-to-noise ratio of 4 (S / N=4) was 0.9 pg / mL, the linear detection range was 1.0 pg / mL–1500 ng / mL, and the sensitivity was 26407.8 CPS / (ng / mL).

[0074] 5. Repeatability and anti-interference measurement and detection accuracy:

[0075] The fluorescence spectra of five 1 ng / mL antigen / antibody-amino-graphene quantum dots / graphene aqueous solutions were measured to observe the repeatability of the measurements. The measurement results are shown in Figure 7 , relative error is 1.2%, indicating that the sensor has good repeatability.

[0076] The fluorescence recovery experiment was repeated using egg white and bovine serum albumin (1.5 μg / mL) at concentrations 150 times that of Lv as interferences. The results are shown in Figure 8 The fluorescence recovery rate of 10 ng / mL Lv was 62.13%, and the fluorescence recovery rates of 1.5 μg / mL chicken egg white and bovine serum albumin were 23.70% and 20.51%, respectively, which proved that the fluorescent immunosensor has good anti-interference ability.

[0077] Figure 8The results of the fluorescence recovery experiment are shown in the embodiment of the present invention, with egg white and bovine serum albumin (1.5 μg / mL) at a concentration 150 times that of egg yolk lipophosphoprotein (Lv) as interferences. Curve 1: Fluorescence spectrum of amino graphene quantum dots / anti-Lv-monoclonal antibody conjugate; 2: Fluorescence recovery spectrum of 10 ng / mL egg yolk lipophosphoprotein; 3: Fluorescence recovery spectrum of 1.5 μg / mL bovine serum albumin; 4: Fluorescence recovery spectrum of 1.5 μg / mL egg white; 5: Fluorescence spectrum of amino graphene quantum dots / anti-Lv-monoclonal antibody / graphene.

[0078] 6. Vitellogenin concentration detection:

[0079] Take 0.2 mL of unknown concentration of brown flounder vitellogenin solution, add the above 0.6 mL of antibody-amino-graphene quantum dots / graphene aqueous solution, then add 0.2 mL of PBS buffer, incubate at room temperature for 12 minutes, measure the fluorescence spectrum, and read the fluorescence peak intensity. Repeat the above experiment three times, take the average of the three fluorescence peak intensities, read the logarithmic concentration logc of vitellogenin from the established standard curve, and further calculate the vitellogenin concentration c.

[0080] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the protection scope of the present invention.

Claims

1. A fluorescent immunoassay for detecting vitellogenin concentration, characterized in that: The fluorescent immunoassay for detecting vitellogenin concentration comprises: Step 1, amination of graphene quantum dots; Step 2: coupling graphene quantum dots with monoclonal antibodies, coupling the aminated graphene quantum dots with the monoclonal antibodies to obtain purified antibody-aminated graphene quantum dot conjugates as fluorescent nanoprobes; Step 3, reducing graphene oxide to obtain water-soluble graphene, wherein the water-soluble graphene is a graphene sheet; Step 4, determining the optimal amount of graphene sheets, using the energy emitted by the fluorescent nanoprobe to transfer to the graphene sheets without radiation to perform fluorescence quenching, and the corresponding graphene sheet concentration when the fluorescence quenching degree reaches saturation is the optimal amount of graphene; Step 5, establishing a standard curve of antigen logarithmic concentration-fluorescence intensity, adding different concentrations of antigen to multiple antibody-amino-graphene quantum dots / graphene aqueous solutions obtained based on the optimal amount of graphene, incubating, the antigen and antibody specifically react to restore the fluorescence quenching, the restored fluorescence intensity is positively correlated with the amount of antigen added, measuring the fluorescence spectrum of the mixture, and obtaining a standard curve of antigen logarithmic concentration-fluorescence intensity; Step six, using the antigen logarithmic concentration-fluorescence intensity standard curve to detect the vitellogenin concentration.

2. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step one, the amination of graphene quantum dots specifically includes: mixing the graphene quantum dot aqueous solution with the ammonia aqueous solution, and stirring at room temperature to evenly mix; transferring the solution to a polytetrafluoroethylene container and heating it to evaporate excess ammonia, and then dialyzing and purifying it in pure water, and freeze-drying the dialyzed amination graphene quantum dots to obtain a powder sample.

3. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step 2, the coupling of graphene quantum dots and monoclonal antibodies specifically includes: taking 1-ethyl-(3-dimethylpropyl)carbodiimide and N-hydroxysuccinimide respectively, adding them to a 4-morpholineethanesulfonic acid solution, mixing and stirring, forming an EDC-NHS solution for standby use; taking an antibody, adding an EDC-NHS reagent, and stirring to form an active antibody; mixing the activated antibody with an amino graphene quantum dot solution, incubating, and forming an antibody-amino graphene quantum dot conjugate; centrifuging to remove free uncoupled antibodies, and during the centrifugation process, washing the conjugate with a PBS buffer solution; obtaining a purified antibody-amino graphene quantum dot conjugate as a fluorescent nanoprobe.

4. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step three, during the process of reducing graphene oxide to obtain water-soluble graphene, a surfactant is added to the graphene oxide aqueous solution to prevent graphene sheets from agglomerating; the surfactant is polyvinyl pyrrolidone, which accounts for 1% of the total mass of the graphene oxide aqueous solution and polyvinyl pyrrolidone.

5. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step 4, the optimal amount of graphene sheets specifically includes: taking multiple portions of antibody-amino graphene quantum dot solution of the same volume, adding graphene solutions of different concentrations respectively, stirring and dispersing, and then adding PBS aqueous solution, and incubating at room temperature; then measuring the fluorescence spectrum of the above solution, the fluorescence of the antibody-amino graphene quantum dots is quenched by graphene, and the corresponding graphene sheet concentration when the fluorescence quenching degree reaches saturation is the optimal amount of graphene.

6. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 5, characterized in that: The optimal dosage of graphene sheets is 4 μg / mL.

7. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step 5, the establishment of the antigen logarithmic concentration-fluorescence intensity standard curve specifically includes: adding different concentrations of antigen to multiple antibody-amino-graphene quantum dots / graphene aqueous solutions, and then adding PBS buffer, the concentration of the antigen is 0-1500.0ng / mL; incubating at room temperature, measuring the fluorescence spectrum of the mixture, and repeating three times; drawing the antigen logarithmic concentration-fluorescence intensity standard curve; reading the linear range, measurement error and minimum detection limit from the standard curve; the mathematical expression of the antigen logarithmic concentration-fluorescence intensity standard curve is I=26407.85log c Lv+85145.31, and the regression coefficient R 2 is 0.9994.

8. The fluorescent immunoassay for detecting vitellogenin concentration according to claim 1, characterized in that: In step six, the detection of vitellogenin concentration specifically includes: taking the vitellogenin solution, adding the antibody-amino-graphene quantum dots / graphene aqueous solution, then adding PBS buffer, incubating at room temperature, measuring the fluorescence spectrum, reading the fluorescence peak intensity, and obtaining the vitellogenin concentration from the established standard curve.

9. An immunosensor for detecting target antigens based on antibody-amino-graphene quantum dots / graphene sheets complex, characterized in that: The immunosensor implements the fluorescent immunoassay for detecting the concentration of vitellogenin according to any one of claims 1 to 8.

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