A method for modifying gold-plated immunomagnetic microspheres and its application in immunoassay.

Abstract

This invention discloses a method for modifying gold-plated immunomagnetic microspheres and its application in immunoassay. The method proposes a gold-plated immunomagnetic microsphere modification method that can be used in electrochemiluminescence immunoassay. Compared to non-conductive magnetic microspheres, this method overcomes the limitations of the low oxidation potential path in electrochemiluminescence, increasing the electrochemiluminescence intensity by nearly 20 times and significantly improving electrochemiluminescence efficiency. Combined with electrochemiluminescence imaging technology, it realizes a particle size-resolved multiplex immunoassay method, which improves detection throughput compared to Roche Diagnostics.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic technology, specifically relating to a method for modifying gold-plated immunomagnetic microspheres and its application in immunoassay. Background Technology

[0002] Electrochemiluminescence is a luminescent process caused by electrochemical reactions, and therefore is unaffected by photobleaching and autofluorescence. Due to its characteristics such as low background, high sensitivity, fast response speed, and spatiotemporal controllability, electrochemiluminescence has become a versatile and powerful analytical technique in many fields, from basic research to commercial clinical and biological applications.

[0003] Currently, commercially available electrochemiluminescence methods include those from Roche Diagnostics and Meso Scale Discovery (MSD). Roche Diagnostics uses an immunoassay to label luminescent molecules onto the surface of magnetic microspheres, enabling the detection of disease biomarkers. However, this method suffers from several drawbacks due to its low oxidation potential pathway: (1) the magnetic microspheres used are non-conductive, resulting in low electrochemiluminescence efficiency; (2) the equipment used is an electron multiplier tube, which collects the ECL of all the magnetic beads, limiting the detection to only one biomarker at a time and resulting in low throughput; and (3) the magnetic microspheres used are mostly ~2.8 μm in size, which is unsuitable for immunoassay using electrochemiluminescence imaging.

[0004] Therefore, it is of great significance to overcome the limitations of electrochemiluminescence principles in low oxidation potential pathways and develop a high-efficiency electrochemiluminescence immunoassay method. Summary of the Invention

[0005] To address the aforementioned issues, this invention discloses a method for modifying gold-plated immunomagnetic microspheres and its application in immunoassay, overcoming the limitations of electrochemiluminescence principles based on low oxidation potential pathways. Compared to non-conductive magnetic microspheres, the electrochemiluminescence intensity of conductive gold-plated immunomagnetic microspheres is increased by nearly 20 times, significantly improving electrochemiluminescence efficiency. Combined with electrochemiluminescence imaging technology, a particle size-resolved multiplex immunoassay method is realized. Compared to Roche Diagnostics, this method increases detection throughput.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for modifying conductive gold-plated immunomagnetic microspheres, the specific steps of which are as follows:

[0008] (1) The gold-plated magnetic microspheres were cleaned with plasma, then immersed in an ethanol solution of 3-mercaptopropionic acid, and allowed to stand at 4°C for 18 h. Finally, after ultrasonic dispersion, they were collected and washed to obtain carboxyl-functionalized gold-plated magnetic microspheres.

[0009] (2) The carboxyl-functionalized gold-plated magnetic microspheres obtained in step (1) were washed with deionized water and morpholine ethanesulfonic acid buffer, and then incubated with carboxyl activator at room temperature for 30 min. After magnetic separation and washing, activated carboxyl-functionalized gold-plated magnetic microspheres were obtained.

[0010] (3) The activated carboxyl-functionalized gold-plated magnetic microspheres obtained in step (2) were incubated with the capture antibody at 37 °C for 3 h. After magnetic separation and washing, they were incubated with 6-mercapto-1-hexanol at 37 °C for 1 h. Finally, after magnetic separation and washing, the gold-plated magnetic microspheres modified with the capture antibody were obtained.

[0011] (4) The gold-plated magnetic microspheres modified with the capture antibody obtained in step (3) were incubated with the antigen and biotin-labeled detection antibody at 37 °C for 30 min. After magnetic separation and washing, they were reacted with the luminescent molecule Ru(bpy)3. 2+ Labeled streptavidin was incubated at 37 °C for 10 min, and finally obtained by magnetic separation and washing to yield Ru(bpy)3. 2+ Marking gold-plated magnetic microspheres.

[0012] As an improvement of the present invention, the plasma cleaning conditions in step (1) are air atmosphere and the cleaning time is 3 min.

[0013] As an improvement of the present invention, the concentration of the ethanol solution of 3-mercaptopropionic acid in step (1) is 1 mM.

[0014] As an improvement of the present invention, the carboxyl activator in step (2) is a morpholine ethanesulfonic acid buffer containing 10 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL of N-hydroxythiosuccinimide sodium salt.

[0015] As an improvement of the present invention, the concentration of 6-mercapto-1-hexanol in step (3) is 1 mM.

[0016] The present invention also provides a conductive gold-plated immunomagnetic microsphere prepared by the above method.

[0017] Another objective of this invention is to provide the application of conductive gold-plated immunomagnetic microspheres prepared by the above method in electrochemiluminescence immunoassay.

[0018] As an improvement of the present invention, the conductive gold-plated immunomagnetic microspheres are used as carriers for electrochemiluminescence immunoassay to improve electrochemiluminescence efficiency.

[0019] The beneficial effects of this invention are as follows:

[0020] 1. The conductive gold-plated immunomagnetic microspheres of the present invention overcome the limitations of the electrochemiluminescence principle of the low oxidation potential path. Compared with the magnetic beads used in Roche Diagnostics, the electrochemiluminescence intensity of the gold-plated magnetic microspheres is increased by nearly 20 times, which significantly improves the electrochemiluminescence efficiency.

[0021] 2. By overcoming the limitations of the electrochemiluminescence principle based on the low oxidation potential pathway, a particle size-resolved electrochemiluminescence multiplex immunoassay method was established, which improved the detection throughput. Attached Figure Description

[0022] Figure 1 This is an electrochemiluminescence imaging image of the immunoassay based on gold-plated magnetic microspheres and non-conductive magnetic beads according to the present invention.

[0023] Figure 2 This is a statistical graph of electrochemiluminescence intensity based on immunoassay of gold-plated magnetic microspheres and non-conductive magnetic beads according to the present invention.

[0024] Figure 3 This is an electrochemiluminescence imaging image of particle size-resolved multiplex immunoassay based on gold-plated magnetic microspheres according to the present invention. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0026] Example 1: Preparation of conductive gold-plated immunomagnetic microspheres

[0027] (1) The gold-plated magnetic microspheres were cleaned with plasma, then immersed in an ethanol solution of 3-mercaptopropionic acid with a concentration of 1 mM, and allowed to stand at 4 °C for 18 h. Finally, after ultrasonic dispersion, the gold-plated magnetic microspheres were collected and washed to obtain carboxyl-functionalized gold-plated magnetic microspheres. Preferably, the plasma cleaning conditions were air atmosphere and the cleaning time was 3 min.

[0028] (2) The carboxyl-functionalized gold-plated magnetic microspheres obtained in step (1) are washed with deionized water and morpholine ethanesulfonic acid buffer, and then incubated with a carboxyl activator at room temperature for 30 min. After magnetic separation and washing, activated carboxyl-functionalized gold-plated magnetic microspheres are obtained. Preferably, the carboxyl activator is a morpholine ethanesulfonic acid buffer containing 10 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL of N-hydroxythiosuccinimide sodium salt.

[0029] (3) The activated carboxyl-functionalized gold-plated magnetic microspheres obtained in step (2) were incubated with the capture antibody at 37 °C for 3 h. After magnetic separation and washing, they were incubated with 1 mM 6-mercapto-1-hexanol at 37 °C for 1 h. Finally, after magnetic separation and washing, the gold-plated magnetic microspheres modified with the capture antibody were obtained.

[0030] (4) The gold-plated magnetic microspheres modified with the capture antibody obtained in step (3) were incubated with the antigen and biotin-labeled detection antibody at 37 °C for 30 min. After magnetic separation and washing, they were reacted with the luminescent molecule Ru(bpy)3. 2+ Labeled streptavidin was incubated at 37 °C for 10 min, and finally obtained by magnetic separation and washing to yield Ru(bpy)3. 2+ Marking gold-plated magnetic microspheres.

[0031] Example 2: Ru(bpy)3 2+ Electrochemiluminescence imaging test of labeled gold-plated magnetic microspheres:

[0032] The luminescent molecule Ru(bpy)3 is delivered via an immune sandwich reaction. 2+ Labels were applied to the surfaces of gold-plated magnetic microspheres (Ru@GMB) and non-conductive beads (Ru@MMB), respectively, and dispersed in phosphate-buffered saline (PBS, pH 7.4). 5 μL of the dispersion was dropped onto the surface of a glassy carbon electrode. In a homemade electrolytic cell, 0.1 M PBS (pH 7.4) containing 100 mM tri-n-propylamine (TPrA) was added, and a potential of 1.3 V vs Ag / AgCl was applied. Electrochemiluminescence images were acquired using an upright microscope (Nikon, ECLIPSE Ni-u) equipped with an electron multiplication charge-coupled device (EMCCD, Andor iXon Ultra 897). Figure 1 As shown, significant electrochemiluminescence was observed on Ru@GMB. Figure 2 As shown, statistical analysis of light intensity indicates that the light intensity of the Ru@GMB group is approximately 12 times that of the Ru@MMB group.

[0033] Ru(bpy)3 on the surface of a single microsphere was tested using ICP-OES (brand: Agilent, model: ICP-OES 730). 2+ The content was analyzed, as shown in Table 1. The results indicate that Ru(bpy)3 on the Ru@MMB surface... 2+ The content is about twice that of Ru@GMB surface, so the gold-plated magnetic microspheres increase the light intensity of electrochemiluminescence by nearly 20 times, significantly improving the efficiency of electrochemiluminescence.

[0034] Table 1. Surface loading of luminescent molecules Ru(bpy)3 onto the surfaces of individual gold-plated magnetic microspheres and non-conductive magnetic beads.2+ The content of.

[0035] Beads <![CDATA[ N beads ]]> [Ru] / (μg / L) <![CDATA[ N Ru ]]> GMB-3 7.79E+06 94 7.10E+07 MMB-3 7.78E+06 188 1.43E+08

[0036] in: N beads [Ru] represents the number of microspheres in the sample, and [Ru] represents the Ru(bpy)3 loading on the surface of the microspheres. 2+ The total content, N Ru Ru(bpy)3 loaded on the surface of a single microsphere 2+ The quantity.

[0037] Example 3: Size-resolved multiplex immunoassay electrochemiluminescence imaging of conductive gold-plated immunomagnetic microspheres:

[0038] Four myocardial infarction markers—cardiac troponin I (cTnI), fatty acid-binding protein (FABP), C-reactive protein (CRP), and myoglobin (Myo)—were detected using gold-plated magnetic microspheres of 3, 5, 8, and 11 μm, respectively. The captured antibody-modified gold-plated magnetic microspheres were incubated for 30 min in a mixed solution containing cTnI (2.0 ng / mL), FABP (6 ng / mL), CRP (10 ng / mL), and Myo (50 ng / mL) and their corresponding biotin-labeled detection antibodies. The microspheres were washed three times with PBS and then incubated with Ru(bpy)3. 2+ The labeled streptavidin was incubated for 10 min. The product was washed three times with PBS and dispersed in PBS. Images were acquired using the electrochemiluminescence imaging method described in Example 2. Figure 3 As shown, this imaging method can capture four different sizes of gold-plated magnetic microspheres at once, thus enabling qualitative analysis of the target type; at the same time, the luminescence images of the four different sizes of gold-plated magnetic microspheres can also be clearly seen from the electrochemiluminescence image.

[0039] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, several improvements and modifications can be made on the basis of the above embodiments without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for modifying conductive gold-plated immunomagnetic microspheres, characterized in that, The specific steps are as follows: (1) The gold-plated magnetic microspheres were cleaned with plasma, then immersed in an ethanol solution of 3-mercaptopropionic acid, and allowed to stand at 4 °C for 18 h. Finally, after ultrasonic dispersion, they were collected and washed to obtain carboxyl-functionalized gold-plated magnetic microspheres. The concentration of the ethanol solution of 3-mercaptopropionic acid was 1 mM. (2) The carboxyl-functionalized gold-plated magnetic microspheres obtained in step (1) are washed with deionized water and morpholine ethanesulfonic acid buffer, and then incubated with a carboxyl activator at room temperature for 30 min. After magnetic separation and washing, activated carboxyl-functionalized gold-plated magnetic microspheres are obtained. The carboxyl activator is a morpholine ethanesulfonic acid buffer containing 10 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL of N-hydroxythiosuccinimide sodium salt. (3) The activated carboxyl-functionalized gold-plated magnetic microspheres obtained in step (2) were incubated with the capture antibody at 37 °C for 3 h. After magnetic separation and washing, they were incubated with 6-mercapto-1-hexanol at 37 °C for 1 h. Finally, after magnetic separation and washing, the gold-plated magnetic microspheres modified with the capture antibody were obtained. The concentration of the 6-mercapto-1-hexanol was 1 mM. (4) The gold-plated magnetic microspheres modified with the capture antibody obtained in step (3) were incubated with the antigen and biotin-labeled detection antibody at 37 °C for 30 min. After magnetic separation and washing, they were reacted with the luminescent molecule Ru(bpy)3. 2+ Labeled streptavidin was incubated at 37 °C for 10 min, and finally obtained by magnetic separation and washing to yield Ru(bpy)3. 2+ Marking gold-plated magnetic microspheres.

2. The method for modifying conductive gold-plated immunomagnetic microspheres according to claim 1, characterized in that: The plasma cleaning conditions in step (1) are air atmosphere and the cleaning time is 3 min.

3. A conductive gold-plated immunomagnetic microsphere, characterized in that: The conductive gold-plated immunomagnetic microspheres were prepared according to the method described in any one of claims 1-2.

4. The application of conductive gold-plated immunomagnetic microspheres prepared according to any one of claims 1-2 in electrochemiluminescence immunoassay.

5. The application of conductive gold-plated magnetic microspheres in electrochemiluminescence immunoassay according to claim 4, characterized in that, The conductive gold-plated immunomagnetic microspheres are used as carriers for electrochemiluminescence immunoassay to improve the efficiency of electrochemiluminescence materials.

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