Biological immunodetection method based on LEDs light-emitting chip

Abstract

The invention provides a biological immunodetection method based on an LEDs light-emitting chip, and solves the technical problems of high instrument cost, relatively tedious operation steps and low sensitivity in the existing immunodetection technology. The method comprises the following steps: preparing an LED light-emitting chip substrate modified by a characteristic antibody and gold nanoparticles modified by the characteristic antibody, and dropwise adding a biomarker to be detected and the gold nanoparticles on the LED light-emitting chip substrate to form a double-antibody sandwich structure. The gold particles adsorbed on the substrate of the LEDs light-emitting chip are used as resonant nano antennas, surface plasmon transmitted on the light-emitting chip is converted into a local surface plasmon mode, the local surface plasmon mode is effectively and outwards coupled and radiated to a far field, and a bright point is presented at the adsorbed gold nano particles, so that the light-emitting effect of the LED light-emitting chip is greatly improved, and the service life of the LED light-emitting chip is prolonged. And calculating the number of the gold nanoparticles under a microscope to obtain a detection result. The method can be used for quantitative detection of biomarkers, and is convenient for detection, low in cost, good in visibility and high in detection sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of immune detection, and relates to a biological immune detection method based on LEDs light-emitting chip and application thereof. Background Art

[0002] Bioimmunoassay technology is a detection method based on immunological principles, which is used to detect antigens, antibodies or other biological molecules in biological samples. However, traditional detection technologies usually require the use of expensive detection equipment, which is costly and limits its application in certain specific scenarios. At the same time, the sample processing and inspection operation procedures of traditional detection technologies are relatively complicated, and operators need to undergo professional training or have certain professional knowledge and skills. As a low-cost special light source, LEDs light-emitting chips can evenly emit the light emitted by LEDs through a reflective film and a diffusion film, and further add an extinction layer on the surface of the diffusion film to prevent the emission of light. The gold nanoparticles adsorbed on the surface of the extinction layer are used as resonant antennas to convert the surface plasmons propagating on the LEDs light-emitting chip into localized surface plasmon modes, effectively coupling the light outward and radiating it to the far field. Therefore, detection technology based on LEDs light-emitting chips is a new method that does not rely on the light source of the detection system and can improve the detection sensitivity.

[0003] Chinese patent application CN202410188582.4 proposes an immunoassay method based on fluorescent microspheres, which includes preparing a substrate, modifying fluorescent microspheres, preparing a sandwich structure, detecting fluorescent signals, and analyzing and processing the collected information. However, during the detection process, this method still relies on the light source module of the microscope as the excitation light to make the fluorescent microspheres emit light, which is relatively expensive and not conducive to the rapid detection of biological molecules in special environments. Summary of the invention

[0004] The purpose of the present invention is to provide a biological immune detection method based on LEDs light-emitting chip. The method has the advantages of low cost, simple operation, high sensitivity, high visualization, etc.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A biological immune detection method based on LEDs light-emitting chip, the specific steps are as follows:

[0007] S1 prepares LEDs light-emitting chip substrate;

[0008] S2 modified gold nanoparticles;

[0009] S3 prepares a sandwich structure, which includes an LED light-emitting chip substrate, a biomarker to be detected, and gold nanoparticles from bottom to top;

[0010] S4 detects the transmission signal;

[0011] The prepared sandwich structure was placed on a common microscope stage, and the signal was collected through a 50x objective lens and a CCD camera without turning on the microscope light source;

[0012] S5 analyzes and processes the collected information.

[0013] Furthermore, step S1 is specifically as follows:

[0014] S1.1 Place the cleaned LED backlight panel in an ultra-high vacuum thermal evaporation film deposition device at a pressure less than 10 -5 Pa, 2nm chromium film and 50nm gold film were evaporated in sequence. The evaporation rate of the chromium film was 0.02nm / s, the evaporation rate of the first 3nm of the gold film was 0.02nm / s, and the evaporation rate of the gold film after the thickness reached 3nm was 0.05nm / s.

[0015] S1.2 Soak the substrate in a mixed solution of 3-mercaptopropionic acid solution and deionized water, and shake it overnight on a shaker at room temperature;

[0016] S1.3 rinse the substrate with deionized water and blow dry, add 50uL of a 50mM mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10mM N-hydroxysuccinimide (NHS) (the solvent is phosphate buffer solution PBS), leave it at room temperature for 1 hour, and then rinse with deionized water and blow dry;

[0017] S1.4 Add 50 μL of 1 mg / mL immunoglobulin antibody solution to the substrate, let it react at room temperature for 2 hours, and then rinse with deionized water;

[0018] S1.5 add 50 μL of phosphate buffer solution (PBS), wherein the concentration of bovine serum albumin (BSA) in the phosphate buffer solution (PBS) is 2%, and after 1 hour at room temperature, rinse with deionized water for later use.

[0019] Furthermore, step S2 is specifically as follows:

[0020] S2.1 Take 500 μL of gold nanoparticle (Au NPs) solution (particle size 150 nm or other diameter) and add it to a low adsorption centrifuge tube;

[0021] S2.2 Take 30 μL of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) solution and add it to 500 μL of gold nanoparticle (Au NPs) solution, mix well with a pipette, and then shake on a shaker at room temperature for 1 hour;

[0022] S2.3 Take 60 μL of SH-PEG solution and add it to the above solution, mix well with a pipette, and then shake on a shaker at room temperature for 1 hour;

[0023] S2.4 After centrifuging the above solution, remove the supernatant with a pipette, and then resuspend in 500 μL of a mixed solution of 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (EDC) and 10 mM N-hydroxysuccinimide solution (NHS), wherein the solvent is a phosphate buffer solution, mix well, and react at room temperature for 1 hour;

[0024] S2.5 After centrifuging the above solution, use a pipette to remove the supernatant, then resuspend in 500 μL of phosphate buffer solution (PBS), mix evenly with a pipette, take 10 μL of 1 ug / mL immunoglobulin antibody solution (solvent is phosphate buffer solution) and add it to the gold nanoparticle (Au NPs) solution, mix well and place at room temperature to react for 2 hours.

[0025] S2.6 After centrifuging the above solution, use a pipette to remove the supernatant, then resuspend it in 500 μL of phosphate buffer solution (mass fraction of 2%) dissolved with bovine serum albumin, mix it evenly with a pipette to obtain a modified gold nanoparticle solution, and store it at 4°C for use.

[0026] Furthermore, step S3 is specifically as follows:

[0027] S3.1 Add immunoglobulin phosphate buffer with concentrations ranging from 1600 pg / mL to 100 pg / mL to the substrate prepared in S1 in sequence, with an addition amount of 50 μL per square centimeter, leave it at room temperature for 2 hours, rinse with deionized water, and blow dry with nitrogen gas.

[0028] S3.2 Add the modified gold nanoparticle solution in S2 onto the substrate of S3.1 at a drop size of 50 μl per square centimeter, place at room temperature for 2 hours, rinse with deionized water, and blow dry with nitrogen gas.

[0029] Furthermore, step S5 is specifically as follows:

[0030] The number of polymer microspheres imaged in the images collected by S4 was statistically analyzed using ImageJ software. The number of immunoglobulins and gold nanoparticles imaged at concentrations ranging from 1600 pg / mL to 100 pg / mL showed a positive correlation, and the detection limit of immunoglobulins was 10 -7 mg per ml, and has a good linear relationship, R 2 It is 0.98.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The LEDs light-emitting chip technology route adopted by the present invention is not only mature, stable and reliable, but also can be prepared on a large scale with relatively low cost, and has high practicality and market potential.

[0033] 2. The technology of the present invention can directly detect immunoglobulins under CCD, without the need to use other complex precision detection equipment such as microscope light source, etc. It is simple to operate and more suitable for popularization and promotion.

[0034] 3. The gold particle self-luminescence detection technology based on plasmon resonance is not easily interfered by other signals during the detection process, so it has less immune interference and stronger specificity. It can be observed through CCD and ordinary microscope, without the need for special microscope, and the results can be observed directly, with high visualization and low cost.

[0035] 4. Compared with the prior art, the detection technology based on LEDs light-emitting chips proposed in this application does not rely on the external light source of the detection system, which can further reduce the system complexity and reduce costs; the plasmon resonance based on gold particles can effectively couple the light outward to the far field, which can further reduce external environmental interference and improve detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the specific implementation modes of the present invention, the drawings required for use in the specific implementation modes will be briefly introduced below. Obviously, the drawings described below are only some specific implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 This is a dark field microscopic image of gold particles based on LEDs light-emitting chip;

[0038] Figure 2 This is a self-luminous microscopic image of gold particles based on LEDs light-emitting chip;

[0039] Figure 3 This is a graph showing the relationship between human immunoglobulin concentration and the number of gold particle signals using a microscopic imaging detection method based on LEDs light-emitting chips. DETAILED DESCRIPTION

[0040] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0041] A biological immune detection method based on LEDs light-emitting chip, the specific steps are as follows:

[0042] S1 prepares LEDs light-emitting chip substrate.

[0043] S1.1 Place the cleaned LED backlight panel in an ultra-high vacuum thermal evaporation film deposition device at a pressure less than 10 -5 Pa, 2nm chromium film and 50nm gold film were evaporated in sequence. The evaporation rate of the chromium film was 0.02nm / s, the evaporation rate of the first 3nm of the gold film was 0.02nm / s, and the evaporation rate of the gold film after the thickness reached 3nm was 0.05nm / s.

[0044] S1.2 Soak the substrate in a mixed solution of 3-mercaptopropionic acid solution and deionized water, and shake it overnight on a shaker at room temperature;

[0045] S1.3 rinse the substrate with deionized water and blow dry, add 50uL of a 50mM mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10mM N-hydroxysuccinimide (NHS) (the solvent is phosphate buffer solution PBS), leave it at room temperature for 1 hour, and then rinse with deionized water and blow dry;

[0046] S1.4 Add 50 μL of 1 mg / mL immunoglobulin antibody solution to the substrate, let it react at room temperature for 2 hours, and then rinse with deionized water;

[0047] S1.5 add 50 μL of phosphate buffer solution (PBS), wherein the concentration of bovine serum albumin (BSA) in the phosphate buffer solution (PBS) is 2%, and after 1 hour at room temperature, rinse with deionized water for later use.

[0048] S2 modified gold nanoparticles.

[0049] S2.1 Take 500 μL of gold nanoparticle (Au NPs) solution (particle size 150 nm or other diameter) and add it to a low adsorption centrifuge tube;

[0050] S2.2 Take 30 μL of thiol-polyethylene glycol-carboxyl (SH-PEG-COOH) solution and add it to 500 μL of gold nanoparticle (Au NPs) solution, mix well with a pipette, and then shake on a shaker at room temperature for 1 hour;

[0051] S2.3 Take 60 μL of SH-PEG solution and add it to the above solution, mix well with a pipette, and then shake on a shaker at room temperature for 1 hour;

[0052] S2.4 After centrifuging the above solution, remove the supernatant with a pipette, and then resuspend in 500 μL of a mixed solution of 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (EDC) and 10 mM N-hydroxysuccinimide solution (NHS), wherein the solvent is a phosphate buffer solution, mix well, and react at room temperature for 1 hour;

[0053] S2.5 After centrifuging the above solution, use a pipette to remove the supernatant, then resuspend in 500 μL of phosphate buffer solution (PBS), mix evenly with a pipette, take 10 μL of 1 ug / mL immunoglobulin antibody solution (solvent is phosphate buffer solution) and add it to the gold nanoparticle (Au NPs) solution, mix well and place at room temperature to react for 2 hours.

[0054] S2.6 After centrifuging the above solution, use a pipette to remove the supernatant, then resuspend it in 500 μL of phosphate buffer solution (mass fraction of 2%) dissolved with bovine serum albumin, mix it evenly with a pipette to obtain a modified gold nanoparticle solution, and store it at 4°C for use.

[0055] S3 prepares a sandwich structure, which includes an LED light-emitting chip substrate, a biomarker to be tested, and gold nanoparticles from bottom to top.

[0056] S3.1 Add immunoglobulin phosphate buffer with concentrations ranging from 1600 pg / mL to 100 pg / mL to the substrate prepared in S1 in sequence, with an addition amount of 50 μL per square centimeter, leave it at room temperature for 2 hours, rinse with deionized water, and blow dry with nitrogen gas.

[0057] S3.2 Add the modified gold nanoparticle solution in S2 onto the substrate of S3.1 at a drop size of 50 μl per square centimeter, place at room temperature for 2 hours, rinse with deionized water, and blow dry with nitrogen gas.

[0058] S4 detects the transmission signal.

[0059] The prepared sandwich structure was placed on an ordinary microscope stage, and the signal was collected through a 50x objective lens and a CCD camera without turning on the light source.

[0060] S5 analyzes and processes the collected information.

[0061] The dark field microscopic image of LEDs-light-emitting chip-gold particles captured by CCD camera is shown in the figure. Figure 1 As shown in the self-luminous microscopy image Figure 2As shown. It can be seen that the photon outcoupling using the plasmon nanoantenna can effectively radiate light outward. The particles of the self-luminous detection technology are less disturbed by impurities and have a high degree of visualization. The detection effect is consistent with that using a microscope light source. The number of images of polymer microspheres in the images collected by S4 was statistically analyzed using ImageJ software. The number of immunoglobulins and gold nanoparticles with concentrations of 1600pg / mL to 100pg / mL showed a positive correlation. The detection limit of immunoglobulins is 10 -7 mg per ml, and has a good linear relationship, R 2 It is 0.98.

[0062] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0063] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A biological immune detection method based on LEDs light-emitting chip, characterized in that: The specific steps are as follows: S1 prepares LEDs light-emitting chip substrate; S2 modified gold nanoparticles; S3 prepares a sandwich structure, which includes an LED light-emitting chip substrate, a biomarker to be detected, and gold nanoparticles from bottom to top; S4 detects the transmission signal; The prepared sandwich structure was placed on a common microscope stage, and signals were collected through a 50x objective lens and a CCD camera without turning on the microscope light source; S5 analyzes and processes the collected information.

Images