A method for quantitative detection of myoantigen based on L-Met@AuNCs near-infrared chemiluminescence

Abstract

The present application relates to a kind of methods for detecting MYO antigen based on L-Met@AuNCs near-infrared chemiluminescence, belong to the field of chemiluminescence immunoassay technology.The method of the present application uses water-soluble L-Met coated gold nanoclusters as chemiluminescence reagent, uses oxidant trigger method, uses deionized aqueous solution of (NH4)2S2O8 with a specific concentration as excitation agent, constructs flash type L-Met gold nanocluster / (NH4)2S2O8 chemiluminescence system, carries out immunodetection to target, reaches the detection level of better than picogram / milliliter, uses step-by-step method, effectively reduces experimental error, and simultaneously has low detection limit, high sensitivity, wide detection range.

Description

Technical Field

[0001] This invention relates to a method for quantitative detection of MYO antigen based on L-Met@AuNCs near-infrared chemiluminescence, belonging to the field of chemiluminescence immunoassay technology. Background Technology

[0002] Chemiluminescence immunoassay (CLIA) originated in 1977 (Halmann, M. et al. Appl. Environ. Microbiol. 1977, 34, 473; Arakawa, H. et al. Bunseki Kagaku 1977, 26, 322) and has become one of the mainstream detection technologies in the field of in vitro diagnostics (IVD). Since 2004, the research on nanomaterials as chemiluminescence reagents has attracted widespread attention from scholars (Nano Lett. 2004, 4, 693), but to date, the luminescent reagents used in commercial CLIA products are still molecular substances such as acridinium esters, adamantane, and luminol (Angew. Chem. Int. Ed. 2022, 61,e202210057). For molecular substances, the limit of detection (LOD) is close to the theoretical limit due to the energy release from a single bond breaking, and ultra-high sensitivity detection has not yet been achieved.

[0003] With continuous exploration and regulation of nanomaterials, the use of nanoparticles as luminescent labels has enabled sensitive detection of target proteins at LOD levels of picograms / mL (Anal. Chem. 2022, 94, 6902). Currently, research on nanoparticles as CLIA markers is still primarily in the basic research stage and cannot yet meet the requirements of automated commercial applications. Furthermore, the tendency of nanomaterial labeling to precipitate and its poor storage stability make it difficult to meet the reagent performance requirements of the IVD field, thus limiting the application of nanomaterials in commercial immunoassay. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, especially the tendency of nanomaterial-based luminescent reagents to generate precipitation and the limitations of existing automated immunoassay methods in achieving high-sensitivity detection of target analytes, this invention provides a method for quantitative detection of MYO antigen based on L-Met@AuNCs near-infrared chemiluminescence.

[0005] The method of this invention uses water-soluble L-Met-coated gold nanoclusters as chemiluminescent reagents and employs an oxidant-triggered method. A deionized aqueous solution of (NH4)2S2O8 at a specific concentration is used as an exciter to construct a flash-type L-Met gold nanocluster / (NH4)2S2O8 chemiluminescent system for immunoassay of target analytes. The method achieves a detection level better than picograms per milliliter. The stepwise method effectively reduces experimental errors and features low detection limits, high sensitivity, and a wide detection range.

[0006] Terminology Explanation:

[0007] DDW: Deionized water, also known as Double-distilled water.

[0008] L-Met: L-methionine, also known as L-methionine, is abbreviated as L-Met.

[0009] HAuCl4‧3H2O: Gold acid chloride trihydrate, abbreviated as HAuCl4.

[0010] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, abbreviated as EDC.

[0011] NHS: N-hydroxysuccinimide, English name 1-Hydroxypyrrolidine-2, 5-dione, abbreviated as NHS.

[0012] Gly: Glycine, abbreviated as Gly.

[0013] (NH4)2S2O8: Ammonium persulfate, abbreviated as (NH4)2S2O8.

[0014] MYO: Myoglobin, abbreviated as MYO.

[0015] Magnetic bead-labeled primary antibody (SA-Ab1): The magnetic bead-labeled primary antibody (SA-Ab1) of this invention refers to magnetic beads that label the corresponding antibody of myoglobin antigen (MYO). This invention has better effect on monoclonal antibodies corresponding to the above antigen.

[0016] Secondary antibody (MYO-Ab2): The secondary antibody in this invention refers to the corresponding secondary antibody to the MYO antigen and the primary antibody.

[0017] PBS: Phosphate buffered saline, abbreviated as PBS, mainly consists of K2HPO4, KH2PO4, and KCl.

[0018] This invention is achieved through the following technical solution:

[0019] A method for quantitative detection of MYO antigen for non-disease diagnosis and treatment purposes based on L-Met@AuNCs near-infrared chemiluminescence includes the following steps:

[0020] (1) Preparation of L-Met coated gold nanoclusters

[0021] L-methionine (L-Met) and NaOH solution were added to deionized water and sonicated until the precipitate dissolved. HAuCl4 solution was added under stirring. The reaction was first carried out at room temperature, and then at 40-60 °C. After the reaction was completed, the mixture was purified by centrifugation with isopropanol and dispersed in deionized water to obtain a solution of gold nanoclusters coated with L-Met as a stabilizer, i.e., the purified water-soluble L-Met@AuNCs solution.

[0022] (2) Preparation of immune complexes

[0023] 1) Add PBS solution containing EDC and PBS solution containing NHS sequentially to the purified water-soluble L-Met@AuNCs solution for activation. After activation, add MYO secondary antibody solution, mix well, incubate, and then add blocking solution for blocking. Purify to remove unlinked gold nanoclusters and MYO secondary antibody to obtain L-Met@AuNCs-labeled MYO secondary antibody. Disperse the MYO secondary antibody in PBS solution to obtain L-Met@AuNCs-labeled MYO secondary antibody solution.

[0024] 2) Mix the magnetic bead-labeled primary antibody (SA-Ab1) solution with a standard MYO antigen solution of known concentration, shake to react, wash and then reconstitute in PBS buffer solution to obtain a magnetic bead solution conjugated with MYO antigen and primary antibody.

[0025] 3) Mix the L-Met@AuNCs-labeled MYO secondary antibody solution and the magnetic bead solution conjugated with MYO antigen and primary antibody, and shake the mixture to obtain an immune complex solution;

[0026] (3) Place the reaction cups of the known MYO antigen immune complexes at different concentrations in the test position of the semi-automatic instrument, use the aqueous solution of (NH4)2S2O8 as the excitation solution, use the CLiC-180-I semi-automatic chemiluminescence instrument to collect the chemiluminescence signal, plot the total photon value curve of chemiluminescence corresponding to different antigen concentrations, and obtain the working curve.

[0027] (4) Perform chemiluminescence total photon value test on each sample solution according to the method in step (3), and detect the MYO antigen concentration in the sample solution based on the obtained chemiluminescence total photon value and working curve.

[0028] According to a preferred embodiment of the present invention, in step (1), the mass-volume ratio of L-methionine (L-Met) to deionized water is (55-65):(3-5), with units of mg / mL.

[0029] According to a preferred embodiment of the present invention, in step (1), the concentration of the NaOH solution is 0.5-1.5 M, and the volume ratio of the NaOH solution to the deionized water is (0.2-0.4):(3-5).

[0030] According to a preferred embodiment of the present invention, in step (1), the concentration of the HAuCl4 solution is 80-120 mmol / L, and the volume ratio of the HAuCl4 solution to deionized water is (0.1-0.3):(3-5).

[0031] According to a preferred embodiment of the present invention, in step (1), the reaction time at room temperature is 4-10 min, and the reaction is carried out at 40-60 °C for 10-14 h.

[0032] According to a preferred embodiment of the present invention, in step (1), the concentration of the purified water-soluble L-Met@AuNCs solution is 1.5-2.5 mg / mL.

[0033] According to a preferred embodiment of the present invention, in step 1), the preparation method of the PBS buffer solution is as follows:

[0034] Weigh 0.1867 g K2HPO4, 0.0259 g KH2PO4 and 0.0749 g KCl and dissolve them in 100 mL of deionized water to prepare a 100 mmol / L PBS buffer solution.

[0035] According to a preferred embodiment of the present invention, in step 1), the EDC concentration of the PBS solution containing EDC is 50-150 mg / mL, the solvent is a 100 mmol / L PBS buffer solution with pH=6.0, and the volume ratio of the PBS solution containing EDC to the purified water-soluble L-Met@AuNCs solution is (15-25):500.

[0036] According to a preferred embodiment of the present invention, in step 1), the NHS concentration of the PBS solution containing NHS is 50-150 mg / mL, the solvent is a 100 mmol / L PBS buffer solution with pH=6.0, and the volume ratio of the PBS solution containing NHS to the purified water-soluble L-Met@AuNCs solution is (15-25):500.

[0037] According to a preferred embodiment of the present invention, in step 1), activation is performed by activating the carboxyl group at 37 °C for 30 minutes.

[0038] According to the present invention, in step 1), the concentration of the MYO secondary antibody solution is 80-120 μg / mL, and most preferably, the concentration of the MYO secondary antibody solution is 100 μg / mL; the solvent is 100 mmol / L PBS buffer solution with pH=7.4.

[0039] According to a preferred embodiment of the present invention, in step 1), the volume ratio of the added MYO secondary antibody solution to the purified water-soluble L-Met@AuNCs solution is (60-100):500, and the incubation is carried out at 37 °C for 3 hours, utilizing the coupling of the amino group on the MYO secondary antibody with the activated carboxyl group on the L-Met@AuNCs.

[0040] Most preferably, in step 1), the volume ratio of the added MYO secondary antibody solution to the purified water-soluble L-Met@AuNCs solution is 80:500.

[0041] According to a preferred embodiment of the present invention, in step 1), the sealing liquid is added and reacted at 37 °C for 30 min.

[0042] According to a preferred embodiment of the present invention, in step 1), the concentration of the L-Met@AuNCs-labeled MYO secondary antibody solution is 1-10 mg / mL.

[0043] According to a preferred embodiment of the present invention, in step 2), the concentration of the magnetic bead-labeled primary antibody (SA-Ab1) solution is 0.6-0.8 mg / mL, and most preferably, the concentration of the magnetic bead-labeled primary antibody (SA-Ab1) solution is 0.75 mg / mL; the solvent is 100 mmol / L PBS buffer solution with pH=7.4;

[0044] According to a preferred embodiment of the present invention, in step 2), the solvent for the standard known concentration MYO antigen solution is a 100 mmol / L PBS buffer solution with pH=7.4; the volume ratio of the magnetic bead-labeled primary antibody (SA-Ab1) solution to the standard known concentration MYO antigen solution is (1-3):(4-6).

[0045] According to a preferred embodiment of the present invention, in step 2), the concentration of the magnetic bead solution for linking the MYO antigen and the primary antibody is 1-10 mg / mL.

[0046] According to a preferred embodiment of the present invention, in step 2), the magnetic bead solution solvent for linking the MYO antigen and the primary antibody is a 100 mmol / L PBS buffer solution with pH=7.4.

[0047] According to a preferred embodiment of the present invention, in step 3), the volume ratio of the magnetic bead solution conjugated with MYO antigen and primary antibody to the MYO secondary antibody solution labeled with L-Met@AuNCs is (9-11):(6-8).

[0048] According to a preferred embodiment of the present invention, in step 3), specifically, 25-40 μL of L-Met@AuNCs-labeled MYO secondary antibody solution is added to the magnetic bead solution conjugated with MYO antigen and primary antibody, the mixture is shaken at 37°C for 30 min, placed on a magnetic rack, and washed 5 times with PBS buffer solution to obtain the immune complex solution.

[0049] According to the preferred embodiment of the present invention, the magnetic bead-labeled primary antibody, MYO secondary antibody, and MYO antigen are conventional commercially available products, which are sold by Shanghai Modis Biotechnology Co., Ltd.

[0050] According to a preferred embodiment of the present invention, in step (3), the concentration of (NH4)2S2O8 in the (NH4)2S2O8 aqueous solution is 5.0-10.0 mmol / L, and more preferably, the concentration of (NH4)2S2O8 in the (NH4)2S2O8 aqueous solution is 7.5 mmol / L.

[0051] According to a preferred embodiment of the present invention, in step (3), the volume ratio of the (NH4)2S2O8 aqueous solution to the known MYO antigen immune complexes of different concentrations is (100-300): (50-500).

[0052] Technical features and advantages of the present invention:

[0053] 1. The detection method of the present invention uses L-Met as a stabilizer coating strategy to synthesize water-soluble L-Met coated gold nanoclusters. The water-soluble L-Met coated gold nanoclusters can be stably stored for more than 3 months under storage conditions of 4 °C.

[0054] 2. In the detection method of the present invention, water-soluble L-Met-coated gold nanoclusters are used as chemiluminescent reagents, and an oxidant triggering strategy is adopted to achieve flash-type chemiluminescence. The total number of photons can reach 140 M in a 10 s sampling time.

[0055] 3. In the detection method of the present invention, water-soluble L-Met-coated gold nanoclusters are used as chemiluminescent reagents, and a chemiluminescent system is constructed with (NH4)2S2O8 to achieve near-infrared chemiluminescence with a wavelength of 834 nm.

[0056] 4. The immune complex constructed in the detection method of the present invention has a wide detection range and sensitive signal response, achieving highly sensitive detection of the target analyte and excellent picogram / mL detection level. The detection method uses a semi-automatic chemiluminescence analyzer, sets specific parameters, and adopts a stepwise method. The reaction time of the immune complex of the present invention is within 10 seconds.

[0057] 5. The immune complex constructed in the detection method of the present invention exhibits a signal response in the concentration range of 50 fg / mL-1000 pg / mL, with a detection limit of 10 fg / mL, breaking through the traditional molecular-level picogram / mL detection level, and realizing a wide range and high sensitivity detection of semi-automated chemiluminescent immunoassay.

[0058] 6. The detection method of the present invention uses the commercially available CLiC-180-I semi-automatic chemiluminescence analyzer from Nanjing Renmai Biotechnology Co., Ltd. for testing. Compared with purely scientific research testing devices, it is more in line with the testing standards in the field of in vitro diagnostics. Attached Figure Description

[0059] Figure 1 The image shows the chemiluminescence intensity curves of the L-Met@AuNCs / (NH4)2S2O8 chemiluminescence system composed of aqueous solutions of (NH4)2S2O8 at different concentrations and L-Met@AuNCs solutions in Experimental Example 1; the horizontal axis represents time and the vertical axis represents the chemiluminescence intensity value.

[0060] Figure 2 The image shows the chemiluminescence intensity curves of the chemiluminescence system composed of (NH4)2S2O8 and L-Met@AuNCs solutions prepared with different buffer solutions in Experimental Example 2; the horizontal axis represents the chemiluminescence intensity curve, and the vertical axis represents the chemiluminescence intensity.

[0061] Figure 3 The graph shows the chemiluminescence intensity curve of the chemiluminescence system in Experiment Example 3; the horizontal axis represents time, and the vertical axis represents chemiluminescence intensity.

[0062] Figure 4 The image shows the chemiluminescence spectrum of the chemiluminescence system in Experiment Example 3; the horizontal axis represents wavelength, and the vertical axis represents chemiluminescence intensity.

[0063] Figure 5 The image shows the chemiluminescence intensity curve of the L-Met@AuNCs-labeled MYO secondary antibody in Experiment Example 4; the horizontal axis represents time, and the vertical axis represents chemiluminescence intensity.

[0064] Figure 6 The graph shows the effect of different concentrations of MYO antigen on the luminescence intensity detected in Experiment Example 5; the horizontal axis represents the MYO antigen concentration, and the vertical axis represents the total number of photons in the chemiluminescence light.

[0065] Figure 7 The graph shows the effect of different types of antigens on the luminescence intensity in Experiment Example 6; the horizontal axis represents the names of different antigens, and the vertical axis represents the total number of photons in the chemiluminescence light. Detailed Implementation

[0066] The present invention is further illustrated below by way of examples, but is not limited thereto.

[0067] In the examples, the chemiluminescence intensity curves and total chemiluminescence photon values ​​were obtained by a CLiC-180-I semi-automatic chemiluminescence analyzer manufactured by Nanjing Renmai Biotechnology Co., Ltd.

[0068] In the examples, the magnetic beads labeled with MYO primary antibody, MYO secondary antibody, and MYO antigen are conventional commercially available products, sold by Shanghai Modis Biotechnology Co., Ltd.

[0069] Preparation of PBS buffer solution in the example: Weigh 0.1867 g K2HPO4, 0.250 g KH2PO4 and 0.0749 g KCl and dissolve them in 100 mL deionized water to prepare a 100 mmol / L PBS buffer solution.

[0070] Example 1

[0071] A method for quantitative detection of MYO antigen based on L-Met@AuNCs near-infrared chemiluminescence comprises the following steps:

[0072] (1) Preparation of L-Met coated gold nanoclusters

[0073] Add 59.7 mg L-Met and 300 μL 1mol / L sodium hydroxide to 4.0 mL of deionized water, sonicate until the precipitate dissolves, then add 230 μL 96 mmol / L HAuCl4 solution under stirring. React at room temperature first, then at 50 ℃ for 12 h. After the reaction is complete, purify by centrifugation with isopropanol and disperse in deionized water to obtain a 2 mg / mL solution of gold nanoclusters coated with L-Met as a stabilizer, i.e., the purified water-soluble L-Met@AuNCs solution.

[0074] (2) Preparation of immune complexes

[0075] 1) Add 20 μL of PBS solution containing EDC (100 mg / mL) and 20 μL of PBS solution containing NHS (100 mg / mL) to 500 μL of purified water-soluble L-Met@AuNCs solution. Activate the carboxyl groups at 37 °C for 30 min. After activation, add 80 μL of 100 μg / mL MYO secondary antibody solution, mix well, and incubate at 37 °C for 3 h. Utilize the amino groups on the MYO secondary antibody to couple with the activated carboxyl groups on L-Met@AuNCs. After incubation, add blocking buffer and react at 37 °C for 30 min. Purify to remove unlinked gold nanoclusters and MYO secondary antibody to obtain L-Met@AuNCs-labeled MYO secondary antibody. Disperse the MYO secondary antibody in PBS solution to obtain a 1 mg / mL L-Met@AuNCs-labeled MYO secondary antibody solution.

[0076] 2) Mix 20 μL of 0.75 mg / mL magnetic bead-labeled primary antibody (SA-Ab1) solution with 50 μL of standard MYO antigen solution of known concentration, shake to react, wash and then reconstitute in PBS buffer solution to obtain a magnetic bead solution with 2 mg / mL MYO antigen and primary antibody conjugated.

[0077] 3) Mix 90 μL of the L-Met@AuNCs-labeled MYO secondary antibody solution from step 1) and 70 μL of the magnetic bead solution from step 2) which is used to link the MYO antigen and primary antibody, and shake the mixture to obtain the immune complex solution.

[0078] (3) Place 50 μL of reaction cups containing known concentrations of MYO antigen immune complexes in the test position of a semi-automatic instrument, use 200 μL of 7.5 mmol / L (NH4)2S2O8 aqueous solution as excitation solution, collect chemiluminescence signals using a CLiC-180-I semi-automatic chemiluminescence analyzer, plot the total photon count curves of chemiluminescence corresponding to different antigen concentrations, and obtain the working curve.

[0079] (4) Perform chemiluminescence total photon value test on each sample solution according to the method in step (3), and detect the MYO antigen concentration in the sample solution based on the obtained chemiluminescence total photon value and working curve.

[0080] Example 2

[0081] The method described in the same way as in Example 1 differs in that:

[0082] In step (3), the concentration of (NH4)2S2O8 in the aqueous solution of (NH4)2S2O8 is 6.5 mmol / L, and the rest is carried out as in Example 1.

[0083] Example 3

[0084] The method described in the same way as in Example 1 differs in that:

[0085] In step 1), the concentration of the L-Met@AuNCs-labeled MYO secondary antibody solution was 1 mg / mL, and the rest was carried out as in Example 1.

[0086] Example 4

[0087] The method described in the same way as in Example 1 differs in that:

[0088] In step 3), 100 μL of the L-Met@AuNCs-labeled MYO secondary antibody solution from step 1) and 70 μL of the magnetic bead solution linking the MYO antigen and primary antibody from step 2) are mixed and shaken to obtain an immune complex solution; the rest is carried out as in Example 1.

[0089] Experimental Example 1

[0090] Different concentrations of (NH4)2S2O8 aqueous solutions of varying concentrations (5 mmol / L, 7.5 mmol / L, 10 mmol / L, 12.5 mmol / L, 15 mmol / L, 17.5 mmol / L, and 20 mmol / L) were used as activators.

[0091] The purified water-soluble L-Met@AuNCs solution at a concentration of 2 mg / mL prepared in Example 1 was used as the luminescent reagent;

[0092] Take 200 μL of aqueous solutions of (NH4)2S2O8 at different concentrations and mix them with 100 μL of L-Met@AuNCs solution with a concentration of 2 mg / mL to obtain the L-Met@AuNCs / (NH4)2S2O8 chemiluminescent system.

[0093] Chemiluminescence signals were acquired using a CLiC-180-I semi-automatic chemiluminescence analyzer. The luminescence intensities of the L-Met@AuNCs / (NH4)2S2O8 chemiluminescence system, composed of (NH4)2S2O8 aqueous solutions of different concentrations and L-Met@AuNCs solutions, are shown below. Figure 1 As shown.

[0094] Depend on Figure 1 It can be seen that L-Met@AuNCs can produce flash-type chemiluminescence when using aqueous solutions of (NH4)2S2O8 at different concentrations as exciters. The chemiluminescence intensity values ​​are different. With the increase of (NH4)2S2O8 concentration, the maximum chemiluminescence intensity value produced by L-Met@AuNCs shows a trend of first increasing and then decreasing. The maximum chemiluminescence intensity is reached when the (NH4)2S2O8 concentration is 7.5 mmol / L.

[0095] Experiment Example 2

[0096] The solvents of the water-soluble L-Met@AuNCs solution in Example 1 were replaced with 0.1 mol / L pH 7.4 PBS, 0.1 mol / L pH 9.2 CB, 0.1 mol / L pH 7.4 BB, and 0.1 mol / L pH 6.5 C, respectively. i B, 0.1 mol / L pH 6.0 AA, and 0.1 mol / L pH 7.4 HEPE were used to prepare 7.5 mmol / L (NH4)2S2O8 solutions, which were used as activators.

[0097] The purified water-soluble L-Met@AuNCs solution at a concentration of 2 mg / mL prepared in Example 1 was used as the luminescent reagent;

[0098] Take 200 μL of each of the different buffer solutions containing 7.5 mmol / L (NH4)2S2O8 as the exciter, and mix them with 100 μL of L-Met@AuNCs solution with a concentration of 2 mg / mL to obtain the L-Met@AuNCs / (NH4)2S2O8 chemiluminescent system.

[0099] Chemiluminescence signals were acquired over 1800 s using a CLiC-180-I semi-automatic chemiluminescence analyzer. The luminescence intensities of the L-Met@AuNCs / (NH4)2S2O8 chemiluminescence system, composed of an exciter prepared with different buffer solutions of 7.5 mmol / L (NH4)2S2O8 and an L-Met@AuNCs solution, are shown below. Figure 2 As shown.

[0100] Depend on Figure 2 It can be seen that the maximum chemiluminescence intensity of L-Met@AuNCs varies when the solutions prepared with different buffers of 7.5 mmol / L (NH4)2S2O8 are used as exciters. Under different buffer solution conditions, L-Met@AuNCs can produce the previous flash chemiluminescence, but the maximum chemiluminescence intensity value decreases. The maximum chemiluminescence intensity is reached when DDW is used as the exciter.

[0101] Experimental Example 3

[0102] A 7.5 mmol / L aqueous solution of (NH4)2S2O8 prepared with deionized water was used as the activator;

[0103] The purified water-soluble L-Met@AuNCs solution at a concentration of 2 mg / mL prepared in Example 1 was used as the luminescent reagent;

[0104] Take 200 μL of an aqueous solution with a concentration of 7.5 mmol / L (NH4)2S2O8 and mix it with 100 μL of an L-Met@AuNCs solution with a concentration of 2 mg / mL. Collect the chemiluminescence signal of the labeled L-Met@AuNCs using a CLiC-180-I semi-automatic chemiluminescence analyzer. Figure 3 As shown.

[0105] Take 200 μL of an aqueous solution containing 7.5 mmol / L (NH4)2S2O8 and mix it with 100 μL of L-Met@AuNCs solution with a concentration of 2 mg / mL. Collect the chemiluminescence spectrum of the labeled L-Met@AuNCs as shown below. Figure 4 As shown.

[0106] Depend on Figure 3 It is known that L-Met@AuNCs can trigger flash-type chemiluminescence, with a maximum intensity of 6 million within 10 seconds. (From...) Figure 4 It is known that the chemiluminescence radiation band generated by the L-Met@AuNCs / (NH4)2S2O8 system of the present invention is located at around 834 nm, which belongs to near-infrared chemiluminescence.

[0107] Experiment Example 4

[0108] Take 100 μL of the 1 mg / mL L-Met@AuNCs-labeled MYO secondary antibody solution from step 1) of Example 1, place it in a chemiluminescence reaction cuvette, and use a CLiC-180-I semi-automatic chemiluminescence analyzer to collect the chemiluminescence intensity curve within 10 s after injection of the excitation solution, as shown in the figure. Figure 5 As shown.

[0109] Depend on Figure 5 It can be seen that the chemiluminescence intensity curve of the L-Met@AuNCs-labeled MYO secondary antibody also shows a flash-type pattern, indicating that labeling with MYO antibody does not affect the chemiluminescence properties of L-Met@AuNCs.

[0110] Experimental Example 5

[0111] In step (3) of Example 1, MYO antigen solutions of different concentrations were prepared, namely 0 fg / mL, 10 fg / mL, 50 fg / mL, 100 fg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 100 pg / mL, and 1000 pg / mL; an aqueous solution containing 7.5 mmol / L (NH4)2S2O8 was used as the activation solution, and the stepwise method was adopted, with an incubation time of 30 min to form immune complexes.

[0112] In step (3) of Example 1, a CLIC-180-I semi-automatic chemiluminescence analyzer was used to collect chemiluminescence spectrum curves and chemiluminescence signals within a 10-second sampling time. The signal values ​​obtained for different MYO antigen concentrations were plotted as working curves as shown below. Figure 6 As shown.

[0113] Depend on Figure 6 It is understood that the semi-automated chemiluminescence immunoassay method for detecting MYO of the present invention can achieve detection in the range of 50 fg / mL to 1000 pg / mL, with a detection limit of 10 fg / mL.

[0114] Experimental Example 6

[0115] The detection method is the same as that described in Example 1, except that:

[0116] Replace the MYO antigen with blank, carbohydrate antigen (CA125), carcinoembryonic antigen (CEA), procalcitonin antigen (PCT), prostate-specific antigen (PSA), thyroid-stimulating hormone (TSH), MYO antigen, carbohydrate antigen (CA125) and a mixture of carcinoembryonic antigen (CEA), procalcitonin antigen (PCT), prostate-specific antigen (PSA), thyroid-stimulating hormone (TSH) and MYO antigen.

[0117] Chemiluminescent responses of different antigens, such as Figure 7 As shown. By Figure 7 As shown, the detection method of the present invention has good selectivity for MYO antigen, and other antigen proteins do not interfere with MYO antigen, indicating that the detection method of the present invention has high specificity for MYO antigen detection.

Claims

1. A method for quantitative detection of MYO antigen for non-disease diagnosis and treatment purposes based on L-Met@AuNCs near-infrared chemiluminescence immunoassay, comprising the following steps: (1) Preparation of L-Met coated gold nanoclusters L-methionine (L-Met) and NaOH solution were added to deionized water and sonicated until the precipitate dissolved. HAuCl4 solution was added under stirring. The reaction was first carried out at room temperature, and then at 40-60 °C. After the reaction was completed, the mixture was purified by centrifugation with isopropanol and dispersed in deionized water to obtain a solution of gold nanoclusters coated with L-Met as a stabilizer, i.e., the purified water-soluble L-Met@AuNCs solution. (2) Preparation of immune complexes 1) Add PBS solution containing EDC and PBS solution containing NHS sequentially to the purified water-soluble L-Met@AuNCs solution for activation. After activation, add MYO secondary antibody solution, mix well, incubate, and then add blocking solution for blocking. Purify to remove unlinked gold nanoclusters and MYO secondary antibody to obtain L-Met@AuNCs-labeled MYO secondary antibody. Disperse the MYO secondary antibody in PBS solution to obtain L-Met@AuNCs-labeled MYO secondary antibody solution. 2) Mix the magnetic bead-labeled primary antibody (SA-Ab1) solution with a standard MYO antigen solution of known concentration, shake to react, wash and then reconstitute in PBS buffer solution to obtain a magnetic bead solution conjugated with MYO antigen and primary antibody. 3) Mix the L-Met@AuNCs-labeled MYO secondary antibody solution and the magnetic bead solution conjugated with MYO antigen and primary antibody, and shake the mixture to obtain an immune complex solution; (3) Place the reaction cups of the known MYO antigen immune complexes at different concentrations in the test position of the semi-automatic instrument, use the aqueous solution of (NH4)2S2O8 as the excitation solution, use the CLiC-180-I semi-automatic chemiluminescence instrument to collect the chemiluminescence signal, plot the total photon value curve of chemiluminescence corresponding to different antigen concentrations, and obtain the working curve. (4) Perform chemiluminescence total photon value test on each sample solution according to the method in step (3), and detect the MYO antigen concentration in the sample solution based on the obtained chemiluminescence total photon value and working curve.

2. The method according to claim 1, characterized in that, In step (1), the mass-volume ratio of L-methionine (L-Met) to deionized water is (55-65):(3-5), unit mg / mL, the concentration of NaOH solution is 0.5-1.5 M, and the volume ratio of NaOH solution to deionized water is (0.2-0.4):(3-5).

3. The method according to claim 1, characterized in that, In step (1), the concentration of HAuCl4 solution is 80-120 mmol / L, the volume ratio of HAuCl4 solution to deionized water is (0.1-0.3):(3-5), the reaction time is 4-10 min at room temperature, the reaction is carried out at 40-60 ℃ for 10-14 h, and the concentration of purified water-soluble L-Met@AuNCs solution is 1.5-2.5 mg / mL.

4. The method according to claim 1, characterized in that, In step 1), the EDC-containing PBS solution has an EDC concentration of 50-150 mg / mL and is in the solvent of 100 mmol / L PBS buffer solution with pH=6.

0. The volume ratio of the EDC-containing PBS solution to the purified water-soluble L-Met@AuNCs solution is (15-25):

500. The NHS-containing PBS solution has an NHS concentration of 50-150 mg / mL and is in the solvent of 100 mmol / L PBS buffer solution with pH=6.

0. The volume ratio of the NHS-containing PBS solution to the purified water-soluble L-Met@AuNCs solution is (15-25):

500.

5. The method according to claim 1, characterized in that, In step 1), activation is performed by activating the carboxyl group at 37 °C for 30 minutes. The concentration of the MYO secondary antibody solution is 80-120 μg / mL, and most preferably, the concentration of the MYO secondary antibody solution is 100 μg / mL. The solvent is 100 mmol / L PBS buffer solution with pH=7.

4.

6. The method according to claim 1, characterized in that, In step 1), the volume ratio of the added MYO secondary antibody solution to the purified water-soluble L-Met@AuNCs solution is (60-100):

500. The incubation is carried out at 37 °C for 3 hours. The amino group on the MYO secondary antibody is coupled with the activated carboxyl group on L-Met@AuNCs. In step 1), the concentration of the L-Met@AuNCs-labeled MYO secondary antibody solution is 1-10 mg / mL.

7. The method according to claim 1, characterized in that, In step 2), the concentration of the magnetic bead-labeled primary antibody (SA-Ab1) solution is 0.6-0.8 mg / mL, and the solvent is 100 mmol / L PBS buffer solution with pH=7.4; the solvent for the standard MYO antigen solution of known concentration is 100 mmol / L PBS buffer solution with pH=7.

4. The volume ratio of the magnetic bead-labeled primary antibody (SA-Ab1) solution to the standard MYO antigen solution of known concentration is (1-3):(4-6), and the concentration of the magnetic bead solution connecting the MYO antigen and the primary antibody is 1-10 mg / mL.

8. The method according to claim 1, characterized in that, In step 3), the volume ratio of the magnetic bead solution conjugated with MYO antigen and primary antibody to the MYO secondary antibody solution labeled with L-Met@AuNCs is (9-11):(6-8).

9. The method according to claim 1, characterized in that, In step (3), the concentration of (NH4)2S2O8 in the (NH4)2S2O8 aqueous solution is 5.0-10.0 mmol / L.

10. The method according to claim 1, characterized in that, In step (3), the volume ratio of (NH4)2S2O8 aqueous solution to known MYO antigen immune complexes of different concentrations is (100-300): (50-500).

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