Luminescent competitive ratio-based double-antibody sandwich immunosensor based on the same co-reactants

Abstract

The present invention belongs to the technical field of core electronic industries and relates to a luminescent body competition ratio type double antibody sandwich immunosensor for the same co-reactant. It includes a sensing electrode, a second antibody bioconjugate, and a luminescent body; a first antibody and a first catalytic component are attached to the surface of the sensing electrode, and the second antibody bioconjugate is formed by covalently connecting a second catalytic component and a second antibody; both the first antibody and the second antibody can specifically bind to the antigen to be detected, so that the second antibody bioconjugate can be connected to the sensing electrode through the specific binding of the first antibody and the second antibody to the antigen to be detected; both the first catalytic component and the second catalytic component are composites of gold nanoparticles and reduced graphene oxide, the average particle size of the gold nanoparticles in the first catalytic component is 12-14 nm, and the average particle size of the gold nanoparticles in the second catalytic component is 3.3-3.5 nm; the luminescent body is Ru(bpy)3 2+ .

Description

Technical Field

[0001] The present invention belongs to the technical field of core electronic industry, and relates to a luminophore competition ratio type double antibody sandwich immunosensor of the same co-reactant. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] According to the inventors' research, the method of electrochemiluminescence (ECL) combined with resonance energy transfer (RET) involves energy transfer between a pair of ECL photophores or between a photophore and an intermediate reagent. However, it is difficult for some ECL-RET systems to achieve sufficient spectral overlap between donors and acceptors, and ideal energy transfer cannot be achieved. At the same time, the lack of energy-tunable materials limits its development and application. Based on the co-reactant competition strategy, the reverse signal of the dual potential resolution ECL emitter is triggered for detection, usually using H 2 O 2 and O 2 As a co-reactant. However, due to the instability of the two in the solution, this method is also difficult to achieve more convenient application. The internal reference system that can achieve good accuracy, reliability and sensitivity obtains quantitative determination of the target concentration through a distinguishable ECL signal and an internal reference signal. However, it is often difficult to find an internal reference substance with excellent performance, and the system construction is more difficult. In the simpler single-luminophore system, since there is only one co-reactant, the cathode and anode signals will rise simultaneously after antigen incubation, and there is a lack of intuitive signal intensity changes, which makes the detection results difficult to be accurate, hindering its application. Therefore, it is necessary to develop a detection system that can obtain intuitive luminescence changes through a single luminophore and a co-reactant that is easy to obtain and prepare. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a luminophore competitive ratio-type double-antibody sandwich immunosensor with the same co-reactant, so as to construct a simpler and more accurate single luminescence ratio immunosensor system.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] On the one hand, a luminophore competitive ratio-type double antibody sandwich immunosensor for the same co-reactant comprises a sensing electrode, a second antibody bioconjugate and a luminophore; a first antibody and a first catalytic component are attached to the surface of the sensing electrode, and the second antibody bioconjugate is formed by connecting the second catalytic component and the second antibody through a covalent bond; the first antibody and the second antibody can both specifically bind to the antigen to be detected, so that the second antibody bioconjugate can be connected to the sensing electrode through the specific binding of the first antibody and the second antibody to the antigen to be detected; the first catalytic component and the second catalytic component are both composite materials of gold nanoparticles and reduced graphene oxide, the average particle size of the gold nanoparticles in the first catalytic component is 12-14 nm, and the average particle size of the gold nanoparticles in the second catalytic component is 3.3-3.5 nm; the luminophore is Ru(bpy) 3 2+ .

[0007] The present invention has found that a composite material of gold nanoparticles with an average particle size of 3.3 to 3.5 nm and reduced graphene oxide has a moderate ORR catalytic ability, and at the same time has excellent cathode co-reactant performance, increases cathode ECL luminescence, and has the effect of reducing the anode signal; a composite material of gold nanoparticles with an average particle size of 12 to 14 nm and reduced graphene oxide has a moderate-weak OER catalytic ability, and at the same time has excellent anode co-reactant performance, increases anode ECL luminescence.

[0008] Studies have shown that when the sensor constructed in the present invention detects the antigen to be tested, as the concentration of the antigen to be tested increases, the anode ECL intensity decreases, while the cathode ECL intensity increases, and there is a strong linear relationship between the anode-cathode ECL intensity ratio (Ia / Ic) and the logarithmic concentration of CEA, thereby achieving the above-mentioned purpose.

[0009] On the other hand, a method for preparing the above-mentioned luminophore competitive ratio type double antibody sandwich immunosensor of the same co-reactant comprises:

[0010] Preparation of the sensing electrode: incubate the first antibody on the bare electrode, and then dropwise add the dispersion of the second catalytic component to obtain;

[0011] Preparation of the second antibody bioconjugate: Add the first catalytic component to the EDC / NHS solution for activation, and then add the second antibody for incubation.

[0012] In a third aspect, a luminophore competitive ratio-type double antibody sandwich immunosensor of the above-mentioned same co-reactant is used in detecting CEA or preparing a system for detecting CEA.

[0013] In a fourth aspect, a detection kit is provided, comprising the above-mentioned luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant and a buffer solution.

[0014] The beneficial effects of the present invention are:

[0015] The present invention uses an electrochemiluminescence immunoassay method to propose a simple and accurate single luminescence ratio immunosensor system. It improves the defects of current immunoassays such as the difficulty in finding co-reactants and unstable performance, and designs co-reactants with simple preparation process, stable performance and excellent cathode and anode luminescence. At the same time, the design system of the present invention simplifies and improves the common complex design of immunosensors, uses only one luminophore, and achieves a direct and clear detection luminescence effect, realizing accurate, reliable, intuitive and highly sensitive detection, greatly improving the detection efficiency of ECL. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0017] Figure 1 is a schematic diagram of an immunosensor in an embodiment of the present invention;

[0018] Figure 2 is an optimization curve of the luminophore concentration of the immunosensor in the embodiment of the present invention;

[0019] Figure 3 is an optimization curve of the co-reactant concentration of the immunosensor in the embodiment of the present invention;

[0020] Figure 4 is an optimization curve of the pH of the detection solution of the immunosensor in the embodiment of the present invention;

[0021] Figure 5 : is the ECL signal response curve to different concentrations of CEA in the embodiment of the present invention;

[0022] Figure 6 Graph showing a linear relationship between the logarithmic value of the anode to cathode ECL intensity ratio (Ia / Ic) and the logarithmic concentration of CEA in an embodiment of the present invention;

[0023] Figure 7 is a bar graph of the selective detection of the immunosensor in an embodiment of the present invention;

[0024] Figure 8 This is a stability characterization diagram of the immunosensor in the embodiment of the present invention. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0027] In view of the defects of existing electrochemiluminescence immunoassay such as difficulty in selecting co-reactants and unstable performance, the present invention proposes a luminophore competition ratio-type double antibody sandwich immunosensor with the same co-reactants.

[0028] A typical embodiment of the present invention provides a luminophore competitive ratio-type double antibody sandwich immunosensor for co-reactants, comprising a sensing electrode, a second antibody bioconjugate and a luminophore; a first antibody and a first catalytic component are attached to the surface of the sensing electrode, and the second antibody bioconjugate is formed by connecting the second catalytic component and the second antibody through a covalent bond; the first antibody and the second antibody can both specifically bind to the antigen to be detected, so that the second antibody bioconjugate can be connected to the sensing electrode through the specific binding of the first antibody and the second antibody to the antigen to be detected; the first catalytic component and the second catalytic component are both composite materials of gold nanoparticles and reduced graphene oxide, the average particle size of the gold nanoparticles in the first catalytic component is 12 to 14 nm, and the average particle size of the gold nanoparticles in the second catalytic component is 3.3 to 3.5 nm; the luminophore is Ru(bpy) 3 2+ .

[0029] In some embodiments, bovine serum albumin (BSA) is also attached to the surface of the sensing electrode, which can block non-specific adsorption and improve detection accuracy and sensitivity.

[0030] Another embodiment of the present invention provides a method for preparing the above-mentioned luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant, comprising:

[0031] Preparation of the sensing electrode: incubate the first antibody on the bare electrode, and then dropwise add the dispersion of the first catalytic component to obtain;

[0032] Preparation of the second antibody bioconjugate: Add the second catalytic component to the EDC / NHS solution for activation, and then add the second antibody for incubation.

[0033] In some embodiments, the first antibody is incubated on the bare electrode at a temperature of 35-40° C. and a time of 0.5-1.5 h.

[0034] In some embodiments, the first antibody is incubated on the bare electrode and then immersed in a BSA solution, and then the dispersion of the first catalytic component is added dropwise. The concentration of the BSA solution is preferably 0.5-1.5 wt%.

[0035] In some embodiments, the temperature for incubation with the addition of the second antibody is 3 to 5° C., and the incubation time is 8 to 12 hours.

[0036] In some embodiments, after adding the secondary antibody for incubation, a BSA solution is added to block non-specific sites.

[0037] In some embodiments, after the addition of the secondary antibody and incubation, the secondary antibody is added to an EDC / NHS solution for activation.

[0038] In some embodiments, the sensing electrode is added to the antigen solution to be detected, incubated, washed, and then a second antibody bioconjugate is added for incubation.

[0039] In one or more embodiments, the sensing electrode is added to the antigen solution to be tested and incubated at a temperature of 35 to 40° C. for a time of 0.5 to 1.5 h.

[0040] In one or more embodiments, the temperature for incubation after adding the second antibody bioconjugate is 35-40° C., and the incubation time is 0.5-1.5 h.

[0041] A third embodiment of the present invention provides an application of the above-mentioned luminophore competition ratio type double antibody sandwich immunosensor of the same co-reactant in detecting CEA or preparing a system for detecting CEA.

[0042] The application of the present invention may be for the purpose of diagnosis and treatment of diseases, or for the purpose of targeted treatment of non-diseases such as scientific research.

[0043] The detection process is as follows: the sensing electrode is added to the CEA solution to be tested for incubation, the second antibody bioconjugate is added for incubation after washing, and the incubated electrode is added to the luminescent material Ru(bpy) 3 2+ Electrochemiluminescence detection was performed in solution.

[0044] A fourth embodiment of the present invention provides a detection kit, comprising the above-mentioned luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant and a buffer solution.

[0045] In some embodiments, ultrapure water is also included for cleaning the sensing electrode after incubation with the antigen solution to be tested.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0047] Example

[0048] Materials preparation:

[0049] 10mL 2mg / mL graphene oxide (GO) and 10mL hydrazine hydrate were placed on a stirrer, covered with a large glass cup, and stirred at room temperature for 6h. Hydrazine hydrate reduced GO to graphene oxide (rGO). Two groups of 2mL 2mg / mL rGO were added with 2.5μL and 7.5μL 1% HAuCl respectively under stirring. 4 Solution. Sodium borohydride and sodium citrate were then added as reducing agents. Finally, two Au / reduced graphene oxides with AuNPs of different particle sizes were synthesized, namely Au-rGO-1 (AuNPs average particle size is 13nm) and Au-rGO-2 (AuNPs average particle size is 3.4nm). The study found that Au / rGO-2 with moderate ORR catalytic ability has superior cathode co-reactant performance, increasing cathode ECL luminescence and having the effect of reducing the anode signal, while Au / rGO-1 with moderate-weak OER catalytic ability has superior anode co-reactant performance and increases anode ECL luminescence.

[0050] Construction of a luminophore competitive ratio-type double antibody sandwich immunosensor for the same co-reactants:

[0051] 1.120μL Au-rGO-2 was added to 120μL EDC (0.01mol / L) / NHS (0.002mol / L)) and 120μL antibody 2 (Ab2, 10μg / mL, 9μl Ab2 (detection antibody) + 111μL 0.1% sodium azide) was added to the mixture and centrifuged at 4℃ for 10h. After centrifugation, 120μL 1% BSA solution was added to block non-specific sites, and then the mixture was shaken at 4℃ for 2h, and then centrifuged for 10 minutes to obtain Au-rGO-2-Ab2 (Ab2 bioconjugate).

[0052] 2. The Au-rGO-2-Ab2 obtained in step 1 was treated with EDC (0.01 mol / L) / NHS (0.002 mol / L) solution at 37°C for 30 min to activate the carboxyl groups of Au-rGO-2. The Au-rGO-2-Ab2 was washed with ultrapure water and then stored.

[0053] 3. Antibody 1 (Ab1, 10 μg / mL, named capture antibody) was incubated on the polished glassy carbon electrode for 1 hour (37°C). Then the excess reagent was removed and the electrode was soaked with 1.0 wt% BSA to block the nonspecific adsorption sites. Then Au-rGO-1 was dropped on the electrode surface.

[0054] 4. Add the CEA solution (5 μL) to be detected to the electrode obtained in step 3 and incubate at 37°C for 1 hour. After washing with ultrapure water, add 5 μL of Ab2 bioconjugate prepared in step 2 and incubate for 1 hour (37°C). Due to the specific binding effect of antigen and antibody, antibody 2 (Ab2) and antibody 1 (Ab1) are connected through CEA as an antigen, and Au-rGO-2-Ab2 is connected to the anode modified with Ab1 and Au-rGO-1, competing for the luminescent material Ru (bpy) common to the cathode and anode in the solution. 3 2+ As the concentration of the detected substance CEA increases, the anode ECL intensity decreases, while the cathode ECL intensity increases. There is a strong linear relationship between the anode-cathode ECL intensity ratio (Ia / Ic) and the logarithmic concentration of CEA. From this point on, the ratio ECL immunosensor was constructed. Figure 1 shown.

[0055] The detection process is: put the electrode prepared in step 4 into the luminescent solution in the dark box of the ECL detector to obtain the ECL detection effect image.

[0056] Parameter optimization:

[0057] In order to achieve excellent performance in the ECL detection of CEA, several experimental parameters such as the pH value of the detection solution (CEA solution to be tested), the concentration of co-reactants and luminophores were optimized. Figures 2 to 4 shown. Figure 2-3 The optimal concentrations of the luminophore and cathode co-reactant (Au-rGO-2) were determined to be 1 mM and 0.25 mg mL -1 , which improves the sensitivity of the ratio immunosensor. Figure 4 Determine the Au-rGO-1 and Au-rGO-2 in Ru(bpy) 3 2+ The optimum pH for achieving both the best performance and the maximum anodic or cathodic emission is pH = 6.

[0058] Linear detection:

[0059] Under optimal conditions, different concentrations of CEA were incubated on the immunosensor to study Ru(bpy) 3 2+The cathodic and anodic ECL signals of AuNPs-rGO-2 and AuNPs-rGO-1. Figure 5 As shown in Figure 2, as the CEA concentration increases, the anodic ECL intensity decreases, while the cathodic ECL intensity increases. In the range of 1fg / ml to 1ng / ml, Figure 6 It shows that there is a strong linear relationship between the anode to cathode ECL intensity ratio (Ia / Ic) and the logarithmic concentration of CEA. The regression equation is Ig(Ia / Ic)=-0.26lgC-1.43(ng / mL), and the correlation coefficient is R 2 =0.9995. Therefore, the CEA immunosensor has excellent analytical performance, with a detection limit (LOD) of 0.33 fg / mL (S / N=3).

[0060] Select, stable, and real samples:

[0061] To evaluate the selectivity of the composite immunosensor, other TMs (EGFR2, ERa and MHP22, with a concentration of 10 - 2 ng / mL) were used as interfering substances. Figure 7 As shown, the ECL intensities of EGFR2, ERa, and MHP22 were very weak compared to the obvious ECL intensity of CEA, which reflects the superior selectivity.

[0062] The current immunosensor has good specificity for CEA detection. Under the condition of 1pg / mL HE4, this example also conducted a 10-cycle stability evaluation of the ECL ratio strategy, such as Figure 8 The RSDs of the cathode emission and the anode emission were 0.18% and 0.48%, respectively, indicating that the immunosensor has good stability.

[0063] In order to evaluate the applicability and reliability of the constructed immunosensor in real samples, human serum samples were tested using the standard addition method. As shown in Table 1, the recovery rate was tested using human serum samples containing standard concentrations of 0.1fg / mL and 10fg / mL, and the recovery rates were 100.7% to 106.7%, with an RSD value of less than 5.9%, indicating the feasibility of the immunosensor in practical applications.

[0064] Table 1 Recovery and RSD of authentic samples

[0065]

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A luminophore competitive ratio-type double antibody sandwich immunosensor for the same co-reactant, Its characteristics are: The invention comprises a sensing electrode, a second antibody bioconjugate and a luminophore; a first antibody and a first catalytic component are attached to the surface of the sensing electrode, and the second antibody bioconjugate is formed by connecting the second catalytic component and the second antibody through a covalent bond; the first antibody and the second antibody can both specifically bind to the antigen to be detected, so that the second antibody bioconjugate can be connected to the sensing electrode through the specific binding of the first antibody and the second antibody to the antigen to be detected; the first catalytic component and the second catalytic component are both composite materials of gold nanoparticles and reduced graphene oxide, the average particle size of the gold nanoparticles in the first catalytic component is 12-14 nm, and the average particle size of the gold nanoparticles in the second catalytic component is 3.3-3.5 nm; the luminophore is Ru(bpy) 3 2+ .

2. The luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant as claimed in claim 1, Its characteristics are: BSA is also attached to the surface of the sensing electrode.

3. A method for preparing the luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant as claimed in claim 1, Its characteristics are: include: Preparation of the sensing electrode: incubate the first antibody on the bare electrode, and then dropwise add the dispersion of the second catalytic component to obtain; Preparation of the second antibody bioconjugate: Add the first catalytic component to the EDC / NHS solution for activation, and then add the second antibody for incubation.

4. The preparation method according to claim 3, Its characteristics are: The first antibody is incubated on the bare electrode at a temperature of 35 to 40° C. and a time of 0.5 to 1.5 h.

5. The preparation method according to claim 3, Its characteristics are: After the first antibody is incubated on the bare electrode, the electrode is immersed in a BSA solution, and then the dispersion of the second catalytic component is added dropwise.

6. The preparation method according to claim 3, Its characteristics are: The temperature for incubation after adding the second antibody is 3 to 5°C, and the incubation time is 8 to 12 hours.

7. The preparation method according to claim 3, Its characteristics are: After incubation with the secondary antibody, BSA solution was added to block nonspecific sites.

8. The preparation method according to claim 3, Its characteristics are: The sensing electrode is added to the antigen solution to be tested, incubated, washed, and then the second antibody bioconjugate is added for incubation.

9. Use of the luminophore competitive ratio-type double antibody sandwich immunosensor of the same co-reactant as claimed in claim 1 or 2 in detecting CEA or preparing a system for detecting CEA.

10. A detection kit, Its characteristics are: The invention comprises the luminophore competition ratio type double antibody sandwich immunosensor of the same co-reactant as claimed in claim 1 or 2, and a buffer solution.

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