CN103575896B - Highly sensitive disposable multicomponent chemical luminescence imaging immunosensor - Google Patents

Abstract

The invention relates to a high-sensitivity disposable multi-component chemiluminescent imaging immunosensor. Using screen printing technology, a 4×12 array was constructed on a silanized glass slide, and a disposable multi-component immunosensing array was constructed by coating different capture antibodies on the array spots. Preparation of multilayer DNase functionalization by simultaneously immobilizing biotinylated capture DNA and signal DNA with multiple G-quadruplex sequence repeats on the surface of gold nanoparticles, using G-quadruplex signal DNA combined with heme to form DNase Gold nanoparticle probes. Based on the sandwich immunoassay, sandwich immune complexes are formed on the sensor array, and multilayer DNase-functionalized gold nanoparticle probes are labeled to different immune complexes through a biotin-avidin reaction. Utilizing the peroxidase properties of DNase to catalyze the reaction between the chemiluminescent substrate H ₂ O ₂ -luminol to obtain sensitive chemiluminescent signals and realize highly sensitive image immunoassays of various proteins. The immunosensor has the advantages of simple design, low cost, high sensitivity, high throughput, and good reproducibility, and has certain clinical application value.

Description

Highly sensitive disposable multicomponent chemiluminescent imaging immunosensor

1. Technical field

The invention is a high-sensitivity disposable multi-component chemiluminescent imaging immunosensor, which involves the simultaneous immunoanalysis of multiple components to be measured in multiple samples. By constructing a disposable multi-component immunosensing array, a new type of immunosensor is designed. Multilayer DNase-functionalized gold nanoparticle probes for chemiluminescence signal amplification for highly sensitive image immunoassays of various proteins.

2. Background technology

As an analytical method with high selectivity and high sensitivity, immunoassay has been widely used in environmental monitoring, clinical diagnosis, food safety and other fields. According to the different immunoassay steps, it can be divided into heterogeneous immunoassay and homogeneous immunoassay, the latter is widely used because of its higher sensitivity. Chemiluminescence immunoassay combines chemiluminescence assay technology with immunoassay, and has the advantages of simple equipment, high sensitivity, fast analysis speed, wide linear range, and no interference in the measurement of emitted light intensity. In practical applications, it is often necessary to determine the content of multiple components in complex samples, and there is an increasing need for highly sensitive analytical methods to improve the accurate detection of low-abundance components. In most immunoassay methods, natural enzymes such as horseradish peroxidase and alkaline phosphatase are loaded onto nanoparticles to achieve signal amplification, but natural enzymes often affect their catalytic activity due to slight conformational changes, resulting in deterioration, and the preparation and purification of natural enzymes is time-consuming and expensive.

To determine the content of multiple components in complex systems, parallel single-component analysis is traditionally used. However, this method requires a long time, consumes a lot of reagents, and requires a lot of work. It is not an ideal analysis mode. Simultaneous or quasi-simultaneous detection of multiple components in a single analysis process has outstanding advantages such as high analysis throughput, short time required, less sample consumption, and low analysis cost. The chemiluminescence immunosensing mode based on the spatial resolution mode has received extensive attention in the field of multi-component immunoassay in recent years. The immune reaction of the components occurs simultaneously in different spatial positions of the reactor, and then the array detector is used for signal collection to realize the simultaneous detection of multiple components. In recent years, the research on protein detection arrays has attracted great attention. However, protein arrays are mostly prepared by photolithography or lattice technology, and the price is relatively expensive. In addition, due to the insensitivity of chemiluminescence detection signals, protein arrays often need to be equipped with Sensitive array detector.

3. Contents of the invention

The purpose of the present invention is to combine screen printing and chemical coating technology to construct a disposable multi-component immunosensing array, and simultaneously design a multi-layer DNA enzyme functionalized gold nanoparticle probe for chemiluminescent signal amplification. A highly sensitive disposable multi-component chemiluminescent imaging immunosensor and a corresponding immunoassay method.

The disposable multi-component chemiluminescent imaging immunosensor proposed by the present invention is shown in Figure 1. It consists of a disposable multi-component immunosensing array (1), a multilayer DNase functionalized gold nanoparticle probe (2) and signal generating system (3). Prepared by the following technical scheme:

1) The disposable multi-component immunosensing array is based on the spatial resolution mode. A 4×12 silanized array is printed on the glass slide, and a certain amount of capture antibody is added dropwise to each row. The epoxy group on the silanized glass slide is covalently bonded. After the reaction is completed, rinse with the washing solution, and then add the blocking solution dropwise. After the sealing is completed, rinse with the washing solution and dry to obtain an array that can capture a total of 4 target antigens. (figure 2);

2) The multilayer DNase-functionalized gold nanoparticle probe is made by simultaneously immobilizing biotinylated capture DNA and signal DNA with multiple G-quadruplex sequence repeats on the surface of gold nanoparticles, and the latter binds heme to form DNA Enzymes, as shown in Figure 3;

3) The signal generation system utilizes the peroxidase property of DNase to catalyze the reaction between the chemiluminescence substrate H ₂ O ₂ -luminol to generate a sensitive chemiluminescence signal.

How the sensor works:

1) Drop the sample to be detected on the surface of each sensing point of the disposable 4×12 multi-component immunosensing array, and incubate; after washing with the washing solution, drop the corresponding biological agent on the surface of each sensing point. Kittenylated antibody for sandwich immune reaction;

2) After washing with washing solution, add streptavidin dropwise on the surface of each sensing array for incubation, then wash with washing solution, and add multilayer DNase functionalized gold nanoparticle probes dropwise, and the reaction ends Rinse with flushing solution afterwards;

3) Dropping a chemiluminescence substrate on the surface of the sensor array, and performing chemiluminescence image detection by CCD;

4) The chemiluminescent signal of each sensor is positively correlated with the concentration of the corresponding detection component, so the standard solution can be used to obtain the working curve, and the concentration of different antigens in the sample can be obtained, so as to realize the simultaneous high detection of multiple antigens in various samples. Sensitive Image Immunoassay.

In the above analysis method, the blocking solution is 0.01M phosphate buffer containing 5% bovine serum albumin (BSA), pH 7.4; the washing solution is 0.01M phosphate buffer containing 0.05% Tween-20, pH 7.4.

Compared with the prior art, the present invention has the following characteristics:

Combining multi-component chemiluminescent image immunodetection means, the present invention designs a multi-layer DNase-functionalized gold nanoparticle probe to amplify chemiluminescent signals, and proposes a highly sensitive A multicomponent chemiluminescent image immunosensing approach. Compared with the existing detection system, it has the following characteristics:

(1) The sensor is a disposable multi-component immunosensing array based on a silanized glass slide, which is simple to prepare and low in cost. At the same time, since the array has 4×12 array sensing points, 48 The immunoassay of samples has the advantages of high throughput, good stability and reproducibility.

(2) Design novel nanoprobes loaded with a high proportion of DNase on the surface, combined with the catalysis of DNase on the H ₂ O ₂ -luminol reaction, to amplify the signal and improve the detection sensitivity, making it more suitable for low abundance Antigen detection.

(3) The surface of the designed nanoprobe is rich in biotin, and based on the biotin-avidin reaction, it can be used as a common beacon probe for the sandwich immunoreaction of four antigens.

(4) The detection system detects through chemiluminescent signals, does not need an external light source, and is very simple to prepare and operate.

4. Description of drawings

Figure 1. Schematic diagram of highly sensitive multi-component chemiluminescent image immunoassay

Figure 2. Schematic diagram of preparation of immunosensor and sandwich immunoreaction, beacon binding reaction and chemiluminescent signal generation and acquisition

Figure 3. Schematic diagram of the preparation of multilayer DNase-functionalized gold nanoparticle probes

5. Specific implementation

Embodiment 1: In conjunction with accompanying drawing 3, the preparation of the gold nanoparticle probe of multilayer DNase functionalization

Synthesize gold nanoparticles with a particle size of 13nm, and two kinds of DNA single strands modified with sulfhydryl groups at the 5' end (including: signal DNA single strands containing multiple G-quadruplex sequence repeats, and capture DNA single strands modified with biotin at the 3' end Chain) according to a certain ratio, through the gold-sulfhydryl bond loaded on the gold nanoparticles. The specific synthesis details are as follows: add 40 μL 1 μM biotinylated capture DNA single strand and 24 μL 100 μM signal DNA single strand to 1 mL of 13 nm particle size gold nanoparticle solution, the ratio of capture DNA and signal DNA is 1:60, stir at room temperature for 16 Hour. Then, 0.01M PBS solution containing 2M NaCl was added dropwise to the above reaction solution to make the final concentration of NaCl in the reaction solution reach 0.1M. Subsequently, 0.1 mL of 0.01 M PBS solution containing 0.1 M KCl was added to the above reaction solution, and reacted for 2 hours. Unbound DNA single strands were removed by centrifugation, and the prepared DNA functionalized gold nanoparticles were distributed in 0.01M PBS containing 0.1M KCl. Finally, react the DNA-functionalized gold nanoparticles prepared above with excess heme at 4°C for 1.5 hours, centrifuge to remove unbound heme, and prepare multilayer DNase-functionalized gold nanoparticle probes, which are dispersed in 1 mL 0.01M PBS and stored at 4°C until use.

Example 2: In combination with accompanying drawing 2, preparation of immunosensor, sandwich immunoreaction, probe binding reaction and chemiluminescent signal generation and collection process

Screen-print 4×12 silanized arrays on glass slides, drop 1.5 μL of a certain concentration of capture antibody on the surface of each array point in each row, and let it absorb overnight at 4°C and 100% humidity; The amino group is covalently bonded to the epoxy group on the silanized glass slide, rinsed after the reaction, and after drying, drip blocking solution on the surface of each sensor, rinse with rinse solution after blocking for 1 hour, and dry to obtain a capture A total of 4 arrays of target antigens.

Drop the sample on the surface of the immunosensing array for incubation, bind the antigen to be tested, then wash the sample with the washing solution and add the biotinylated antibody dropwise for incubation to form a sandwich immune complex and wash away the unbound biotinylated Antibody, then add avidin dropwise for incubation, wash off unbound avidin after biotin and avidin are combined, then add the multilayer DNase functionalized gold nanoparticle probe prepared in Example 1, continue Incubation. After the nanoprobes are bound to the sandwich immune complex, the unbound nanoprobes are washed away, and finally the chemiluminescent substrate H ₂ O ₂ -luminol is dropped on the surface of the immunosensor, and the DNase catalyzes the oxidation of luminol by hydrogen peroxide. Minol produces chemiluminescence.

Embodiment 3: in conjunction with accompanying drawing 2, take AFP, β-HCG, CA 125, CEA as example, illustrate the application of this highly sensitive multi-component chemiluminescent image immunosensor

(1) Drop 1.5 μL of 10 μg/mL AFP, β-HCG, CA 125 and CEA capture antibodies on the silanized surface of the array, the amino groups of the antibodies are covalently bonded to the epoxy groups of the slide, and rinse after the reaction. Add blocking solution dropwise on the surface of each sensing point, rinse and dry after sealing to make a disposable multi-component immune sensing array (Figure 2);

(2) Add 1.5 μL of different concentrations of standard antigen or serum to be tested on the surface of each sensing point of the prepared immunosensing array, incubate at room temperature for 15 minutes, and wash with washing solution; Add 1.5 μL 2 μg/mL biotinylated AFP, β-HCG, CA 125 and CEA antibodies to the surface of the sensor respectively, incubate at room temperature for 20 minutes and wash with washing solution;

(3) Add 1.5 μL 2 μg/mL avidin dropwise to each sensing point for biotin-avidin reaction, incubate for 15 minutes, wash with washing solution, and then add dropwise 1.5 μL prepared in Example 1 The multi-layer DNase functionalized gold nanoparticle probe was washed with washing solution after reacting for 30 minutes;

(4) Add a chemiluminescent substrate on the surface of the sensor, use CCD to detect multi-component chemiluminescent images, set the shooting mode to integral shooting, accumulate 3 times, each exposure time is one minute, according to the recorded chemiluminescence value, detect Concentrations of AFP, β-HCG, CA 125 and CEA in the sample to be tested.

Claims (7)

1. A highly sensitive disposable multicomponent chemiluminescence imaging immunosensor is characterized in that the sensor consists of a disposable multicomponent immune sensing array (1), a multilayer DNA enzyme functionalized gold nanoparticle probe ( 2) and signal generation system (3): the disposable multi-component immunosensing array is printed on a glass slide with a silanized array of 4×12, and a capture antibody is chemically coated in each row to produce DEK arrays capture a total of 4 target antigens; the multilayer DNase-functionalized gold nanoparticle probe is simultaneously immobilized on the surface of gold nanoparticles with biotinylated capture DNA and multiple G-quadruplex repeats that bind heme signal DNA; the signal generation system uses the peroxidase properties of DNase to catalyze the reaction between the chemiluminescent substrate H ₂ O ₂ -luminol to generate sensitive chemiluminescent signals, which are obtained by the data acquisition system (4) Immunosensing image signals. 2. The sensor according to claim 1, characterized in that the disposable multi-component immunosensing array is respectively coated and captured on the glass slide through the printed 4-row silanized array according to the spatial resolution mode. Antibody 1, Capture Antibody 2, Capture Antibody 3, and Capture Antibody 4 were prepared. 3. The sensor according to claim 1, characterized in that the multilayer DNase functionalized gold nanoparticle probe is repeated by multiple G-quadruplex sequences immobilized on the gold nanoparticle surface at a ratio of 60: 1. Signal DNA and biotinylated capture DNA are made by combining G-quadruplex DNA with heme to form DNase. 4. The sensor according to claim 1, characterized in that the multilayer DNase-functionalized gold nanoparticle probe has peroxidase properties and can catalyze the reaction between the chemiluminescence substrate H ₂ O ₂ -luminol reaction to obtain a sensitive chemiluminescent signal. 5. The sensor according to claim 1, characterized in that the multilayer DNA enzyme functionalized gold nanoparticle probe contains biotinylated capture DNA, and the biotin-avidin reaction can be used to bind the probe to The sandwich immune complex formed on the sensor array is used to perform highly sensitive multi-component chemiluminescence image immunoassay. The specific analysis steps are as follows: (1) Dropping the sample to be detected on the surface of each sensing point of the disposable multi-component immunosensing array, and incubating; washing with the washing solution, adding the corresponding biotinylated antibody dropwise, and performing a sandwich immune reaction; (2) After rinsing with rinsing solution, add streptavidin dropwise on the surface of each sensing array for incubation, then rinse with rinsing solution, and add multilayer DNase functionalized gold nanoparticle probes dropwise, and react Rinse with flushing solution after finishing; (3) Dropping a chemiluminescence substrate on the surface of the sensing array, and performing chemiluminescence image detection by a CCD; (4) Use the standard solution to obtain the working curve, and calculate the concentration of different antigens in the sample. 6. The sensor according to claim 5, characterized in that 1-12 columns of the sensor array can drop 1-12 different samples respectively, and detect the concentration of each component in 1-12 different samples at the same time. 7. The sensor according to claim 5, characterized in that the flushing solution is 0.01M phosphate buffer containing 0.05% Tween-20, pH 7.4.