阅读说明：本技术 一种aie免疫层析试纸 (AIE immunochromatography test paper ) 是由 张兵波 徐琰 杨维涛 曾维薇 刘凯 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种AIE免疫层析试纸,该试纸包括结合物垫、测试线及质控线,结合物垫上设有已标记检测抗体或检测抗原的AIE纳米粒子,测试线包被有用于捕获待检测样品中目标物的捕获抗体或捕获抗原,质控线包被有用于识别结合物垫上已标记在AIE纳米粒子上的检测抗体或检测抗原的抗体。与现有技术相比,本发明利用AIE材料独特聚集诱导发光性能,有效增强免疫层析试纸的发光效率,间接降低背景干扰信号,极大提升了对待检测物的检测灵敏度和定量准确性。(The invention relates to an AIE immunochromatographic test paper which comprises a conjugate pad, a test line and a quality control line, wherein AIE nano particles marked with detection antibodies or detection antigens are arranged on the conjugate pad, the test line is coated with capture antibodies or capture antigens used for capturing target objects in a sample to be detected, and the quality control line is coated with antibodies used for identifying the detection antibodies or the detection antigens marked on the AIE nano particles on the conjugate pad. Compared with the prior art, the invention utilizes the unique aggregation-induced emission performance of the AIE material, effectively enhances the luminous efficiency of the immunochromatographic test paper, indirectly reduces background interference signals, and greatly improves the detection sensitivity and the quantitative accuracy of the object to be detected.)

1. An AIE immunochromatographic test strip comprising a conjugate pad (3), characterized in that AIE nanoparticles labeled with a detection antibody or a detection antigen are provided on the conjugate pad (3).

2. The AIE immunochromatographic test strip according to claim 1, further comprising a test line (6) and a quality control line (7), wherein the test line (6) is coated with a capture antibody or a capture antigen for capturing a target in a sample to be detected, and the quality control line (7) is coated with an antibody for recognizing a detection antibody or a detection antigen labeled on AIE nanoparticles on the conjugate pad (3).

3. The AIE immunochromatographic test strip according to claim 2, further comprising a base plate (1), a sample pad (2), a nitrocellulose membrane (4) and a water absorption pad (5), wherein the base plate (1) is sequentially lapped with the sample pad (2), the conjugate pad (3), the nitrocellulose membrane (4) and the water absorption pad (5) along the length direction of the base plate (1), and the test line (6) and the quality control line (7) are sequentially arranged on the nitrocellulose membrane (4) along the sample chromatography direction.

4. An AIE immunochromatographic test strip according to claim 3, wherein the sample pad (5) is a glass fiber membrane or a polyester fiber membrane, the conjugate pad (3) is a glass fiber membrane, and the base plate (1) is a polyvinyl chloride rubber plate.

5. The AIE immunochromatographic test strip according to claim 3, wherein a card shell is provided outside the test strip, a sample application zone for dropping a sample to be tested and a color development zone for observing or reading a signal are provided on the card shell, and the sample to be tested is one or more of serum, whole blood, urine or interstitial fluid.

6. The AIE immunochromatographic test strip according to claim 1, wherein the AIE nanoparticles have a particle size of 10 to 300 nm.

7. The AIE immunochromatographic strip of claim 1, wherein the AIE nanoparticles are prepared from one or more of the following compounds: tetrastyrene AIE derivatives, silole AIE derivatives, 1, 4-stilbenes AIE derivatives, five-membered heterocyclic AIE derivatives and organic boron AIE derivatives.

8. The AIE immunochromatographic strip according to claim 1, wherein the labeling process of the AIE nanoparticles comprises the following steps:

1) preparing active ester NHS modified AIE nano particles or aminated AIE nano particles;

2) and labeling a detection antibody or a detection antigen on the AIE nano particle modified by the NHS of the active ester or the aminated AIE nano particle.

9. The AIE immunochromatographic strip of claim 1 in which the AIE nanoparticles emit an optical signal under the irradiation of an excitation light having a wavelength of 254-530nm and an irradiation power of 10-300mW/cm²。

Technical Field

The invention belongs to the technical field of immunochromatographic test paper, and relates to AIE immunochromatographic test paper and a detection method thereof.

Background

In Vitro Diagnosis (IVD) refers to the determination of disease or function of a body by obtaining diagnostic information through the detection of biological samples such as blood, body fluids, and tissues. Rapid, sensitive and quantitative detection of biological samples is the direction of current in vitro diagnostic development. The biomedical detection based on the immunochromatographic test strip has the advantages of rapidness, simplicity, convenience, intuition, low cost and the like, and has great social and economic benefits in the aspects of early diagnosis, prevention and the like of diseases. The Immunochromatographic test paper technology (ICA) is based on the principle of antigen-antibody specific immunological reaction and chromatographic reaction, and an indicator marked with an antibody can specifically recognize and combine a specific substance to be detected in a biological sample such as a urine sample, a blood sample or saliva, and the substance is retained and developed in a test paper detection area and can be combined with a reading instrument for quantitative detection, so that the purpose of quickly and accurately detecting the substance to be detected is achieved.

In recent years, due to continuous innovation and progress of medical industry, the quantitative detection requirement of the immunochromatographic test strip is increased. The immunochromatographic test paper based on colloidal gold as an indicator is applied to the field of early pregnancy detection for the first time, and is currently expanded to a plurality of fields such as biomedicine, food safety, environment, pesticide residue and the like, thereby achieving great success. However, it has not been able to detect with accuracy and ultrasensitiveness, and at best it can only be defined as a semi-quantitative conventional detection. Therefore, based on the rapid development of society, one or more immunochromatographic test strip methods capable of rapid, sensitive and quantitative detection are urgently needed.

Although the traditional immunochromatographic test paper taking the fluorescent nanoparticles based on the organic dye molecules as the indicator is improved in quantitative and sensitive detection, the traditional immunochromatographic test paper has higher requirements for detection items with higher detection sensitivity and accuracy, such as infectious diseases, myocardial injuries, infection and other diseases, and generally the traditional immunochromatographic test paper is difficult to meet the detection requirements. The reason for this is that the organic dye has good fluorescence in a dilute solution state, but these compounds have a defect of Aggregation-induced Quenching (ACQ) in a thin film state or a high concentration state. The application of the organic dye in the aspect of immunochromatographic test paper is severely limited by the ACQ, quenching can occur when the concentration is too high in the using process, but the problems of weak fluorescence signal, serious background signal interference and the like can be caused when the concentration is too low, so that a great amount of time has to be spent on finding a balance value.

With the advent of Aggregation-Induced Emission (AIE) materials, the drawback of ACQ in practical applications of conventional fluorescent materials is fundamentally solved, and people's knowledge of organic luminescent materials is turned to a new height. Compared with the traditional organic fluorescent dye, the AIE material has some remarkable advantages in the aspects of fluorescence detection, biological imaging and the like: the fluorescence intensity of the fluorescent material is gradually enhanced along with the increase of the concentration due to the high luminous efficiency in the aggregation state, so that the interference of background signals is indirectly reduced, and the high-contrast fluorescence detection can be realized; the light stability is good, and the photobleaching can be effectively solved; the fluorescent indicator has strong modifiability and can be used for designing a fluorescent indicator with response capability.

Therefore, the design and preparation of the immunochromatographic test paper and the quantitative detection method which take the AIE nano particles as the luminescent indicator and have high sensitivity, accuracy and good light stability have important significance.

Disclosure of Invention

The aim of the invention is to overcome the defects of the prior art and provide the AIE immunochromatographic test paper which can effectively eliminate interfering luminescent signals and improve the sensitivity and accuracy of quantitative detection.

The purpose of the invention can be realized by the following technical scheme:

an AIE immunochromatographic test strip comprises a conjugate pad, wherein AIE nano-particles marked with a detection antibody or a detection antigen are arranged on the conjugate pad.

Furthermore, the test strip also comprises a test line and a quality control line, wherein the test line is coated with a capture antibody or a capture antigen for capturing a target object in a sample to be detected, and the quality control line is coated with an antibody for identifying a detection antibody or a detection antigen which is marked on the AIE nano particles on the conjugate pad.

Furthermore, the test paper still include bottom plate, sample pad, nitrocellulose membrane and water absorption pad, the bottom plate on along bottom plate length direction overlap joint in proper order have sample pad, conjugate pad, nitrocellulose membrane and water absorption pad, test line and quality control line set gradually on nitrocellulose membrane along sample chromatography direction.

Furthermore, the sample pad is a glass fiber film or a polyester fiber film, the conjugate pad is a glass fiber film, and the bottom plate is a polyvinyl chloride rubber plate.

Furthermore, a clamping shell is arranged outside the test paper, a sample adding area for dropwise adding a sample to be detected and a color developing area for observing or reading signals are arranged on the clamping shell, and the sample to be detected is one or more of serum, whole blood, urine or tissue fluid.

Furthermore, the particle size of the AIE nano particles is 10-300 nm.

Further, the AIE nano-particles are prepared from one or more of the following compounds: tetrastyrene AIE derivatives, silole AIE derivatives, 1, 4-stilbenes AIE derivatives, five-membered heterocyclic AIE derivatives and organic boron AIE derivatives.

The tetrastyrene AIE derivative is preferably one of the following structural formulae:

silole-type AIE derivatives are preferably as follows:

in the above formula, R₁、R₂One of the following combinations of groups is preferred:

the 1, 4-stilbenes AIE derivatives are preferably one of the following structural formulas:

the five-membered heterocyclic AIE derivative is preferably one of the following structural formulas:

the organoboron AIE derivative is preferably one of the following structural formulae:

other hydrocarbon AIE derivatives may also be used in the compounds for preparing AIE nanoparticles, such as:

further, the labeling process of the AIE nanoparticles comprises the following steps:

1) preparing active ester NHS modified AIE nano particles or aminated AIE nano particles;

2) and labeling a detection antibody or a detection antigen on the AIE nano particle modified by the NHS of the active ester or the aminated AIE nano particle.

Furthermore, the AIE nano-particles emit optical signals under the irradiation of exciting light, the wavelength of the exciting light is 254-530nm, and the irradiation power is 10-300mW/cm²。

The fluorescence intensity of the AIE nano particles on the chromatographic test paper is far stronger than that of the dilute solution.

The invention provides the AIE immunochromatographic test paper with high detection sensitivity and high detection accuracy by using AIE nano particles as an indicator. The specific scheme is that an antibody or an antigen of a detection target object is connected to AIE nano particles through a chemical bond, and then the AIE nano particles marked with the antibody or the antigen are arranged on a conjugate pad and used for detecting the target object in a sample to be detected; the test line set on the nitrocellulose membrane is coated with a capture antibody or antigen aiming at a target object in advance; the quality control line set on the nitrocellulose membrane is coated with an antibody which is marked on the AIE nano particles and used for detecting the antibody or the antigen in advance. The conjugate pad, the nitrocellulose membrane coated with the test line and the quality control line, and other sample pads, water absorption pads, card shells and base plates are assembled according to a traditional test paper manufacturing method to form the immunochromatographic test paper.

The detection method using the immunochromatographic test paper of the invention comprises the following steps:

(1) dripping a sample to be detected on the sample pad;

(2) irradiating the test line and the quality control line with exciting light;

(3) and reading the luminous signals of the test line and the quality control line by using a luminous reading instrument.

In the above scheme, the wavelength of the excitation light is 254-530nm, and the irradiation power is 10-300mW/cm²。

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

according to the invention, the AIE nano particles are adopted to replace the traditional fluorescent nano particles for marking the antibody or the antigen, and as the AIE molecules have the characteristics of more aggregation and more luminescence and excellent light stability, after the conjugate pad coated with the AIE nano particles absorbs the energy irradiated by the excitation light, the emission light intensity is obviously enhanced compared with that of the traditional fluorescent nano particles, and is far stronger than the fluorescence of the nitrocellulose membrane on the test strip and the biological sample, so that the background interference is reduced; in addition, the material has strong photobleaching resistance, so that great convenience is brought to the transfer and storage of the test sample. Therefore, the invention effectively enhances the luminous efficiency of the detection material, indirectly reduces the interference signals generated by the stimulation of the nitrocellulose membrane and the biological sample, greatly improves the detection sensitivity and the quantitative accuracy of the object to be detected, and simultaneously provides a new way for the convenient transfer and storage of the immunochromatographic test paper.

Drawings

FIG. 1 is a schematic structural diagram of the AIE immunochromatographic test strip of the present invention;

the notation in the figure is:

1-bottom plate, 2-sample pad, 3-conjugate pad, 4-nitrocellulose membrane, 5-absorbent pad, 6-test line, 7-quality control line.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.