CN116819088A - Fluorescent probe, multichannel detection kit and method - Google Patents
Fluorescent probe, multichannel detection kit and method Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 57
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- 238000006243 chemical reaction Methods 0.000 claims description 36
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- WGTODYJZXSJIAG-UHFFFAOYSA-N tetramethylrhodamine chloride Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C(O)=O WGTODYJZXSJIAG-UHFFFAOYSA-N 0.000 claims description 3
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/58—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing copper, silver or gold
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
Abstract
The invention discloses a fluorescent probe, a multichannel detection kit and a multichannel detection method, and belongs to the technical field of biological detection. The probe is of a sphere structure and comprises: the plasmon nanoparticle comprises plasmon nanoparticles, a silicon dioxide layer wrapped on the plasmon nanoparticles, and antibodies distributed on the outer wall of the silicon dioxide layer according to preset requirements; fluorescent substances are embedded at predetermined positions of the silicon dioxide layer. The invention uses the difference value of fluorescence as the correlation coefficient of the concentration of the biological marker, solves the dependence on absolute fluorescence intensity, and reduces the batch difference of products; the portable homogeneous immunofluorescence detection is realized, and the defects that the chromatography method is easy to be interfered by the environment and the like are avoided; and plasmon enhanced fluorescence is adopted, the fluorescence intensity is improved by 100-10000 times compared with the initial fluorescence, and the detection precision is high.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a fluorescent probe, a multichannel detection kit and a method.
Background
Immunofluorescence currently the dominant techniques can be divided into two main categories: heterogeneous immunofluorescence and homogeneous immunofluorescence. Heterogeneous immunofluorescence is represented by a fluorescent immunochromatography technique, and a fluorescent immunochromatography detection technique is a membrane detection technique based on an antigen-antibody specific immune reaction. The technology uses strip fiber chromatographic materials fixed with detection lines (coated antibodies or coated antigens) and quality control lines (anti-antibodies) as stationary phases, test liquid as mobile phases, fluorescent labeled antibodies or antigens are fixed on a connecting pad, and analytes are enabled to move on chromatographic strips through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with multiple antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely an object to be detected is firstly combined with a fluorescent labeled antibody under the action of a mobile phase, and then is combined with a coated antibody to form a sandwich-type double-antibody sandwich when reaching a detection line. Homogeneous fluoroimmunoassay is an immunofluorescent analytical technique developed based on the Homogeneous Enzyme Immunoassay (HEI) established by Rubenstein et al in 1972. By "homogeneous" is meant that the antigen and antibody are bound in the same liquid medium environment, which greatly increases the capture efficiency of the antigen-antibody. Homogeneous fluoroimmunoassay is a variety of test methods designed and established based on the fact that some special physicochemical properties of fluorescence (e.g., excitation, absorption, quenching, etc.) are utilized, and that changes occur after the labeled antigen is bound to a specific antibody.
Among these two types of immunofluorescence techniques, heterogeneous immunofluorescence mainly uses chromatography techniques, and the method is suitable for portable field detection, can be applied to basic-level inspection scenes, and has relatively low equipment cost. However, the sensitivity of the detection device and fluorescence itself is limited and the efficiency of solid-liquid capture is not high due to the large environmental impact of chromatography, so the detection accuracy of the fluorescence immunochromatography technology is relatively low. In addition, after the small molecule antigen is combined with the fluorescent labeled antibody, the small molecule antigen is difficult to be combined with the coated antibody on the detection line due to steric hindrance.
However, homogeneous immunofluorescence is generally used together with an automatic instrument, the capture efficiency of antigen and antibody is very high in the method, the instrument can accurately sample, clean, warm bath, detect and the like, the detection result is relatively accurate, and the sensitivity is relatively high. But is limited by an automatic instrument, the use place of the product is limited, the instrument cost is higher, and the failure rate is not guaranteed. How to develop a portable high-sensitivity immunofluorescence detection technology under a homogeneous system is a current academic and industrial problem.
Disclosure of Invention
The invention aims to: in order to solve the problems, the invention provides a fluorescent probe, a multi-channel detection kit and a method.
The technical scheme is as follows: a fluorescent probe, said probe being of a spherical structure comprising: the plasmon nanoparticle comprises plasmon nanoparticles, a silicon dioxide layer wrapped on the plasmon nanoparticles, and antibodies distributed on the outer wall of the silicon dioxide layer according to preset requirements; fluorescent substances are embedded at predetermined positions of the silicon dioxide layer.
In a further embodiment, the antibodies on the fluorescent probes of two adjacent sets are coupled by an antigen.
In another technical scheme, a preparation method of the fluorescent probe is provided, and the preparation method comprises the following steps:
step one, preparing a kernel: taking metal nano particles with the particle size of 40-100 nm, and carrying out oxidation-reduction reaction on the metal nano particles to obtain plasmon nano particle cores;
step two, wrapping a silicon dioxide layer on the outer surface of the inner core of the plasmon nanoparticle to obtain the plasmon nanoparticle@SiO 2 : is prepared by hydrolyzing triaminopropyl triethoxy silane for 10-30 minThe concentration of the aminopropyl triethoxysilane is 0.5-2%mol/L;
step three, modifying fluorescent substances on the outer surface of the silicon dioxide layer in the step two: the surface of the silicon dioxide layer is adsorbed by adopting an electrostatic adsorption mode, and the plasmon nano particles @ SiO 2 Dropwise adding 1-5% of fluorescent substances in mass fraction under a vigorous stirring state, and carrying out plasmon nano particle@SiO 2 The volume ratio of the fluorescent material to the fluorescent material is (10-50): 1, obtaining plasmon nano particles @ SiO 2 A @ fluorescent substance;
step four, wrapping a silicon dioxide layer on the outer layer of the fluorescent substance, wherein the thickness of the silicon dioxide layer is 3-10 nm, and obtaining the plasmon nanoparticle@SiO 2 Fluorescent material @ SiO 2 ；
Step five, modifying the corresponding antibody on the silicon dioxide layer in the step four: adding an antibody with carboxyl into a cross-linking agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution, uniformly mixing, adding an N-hydroxysuccinimide solution, uniformly mixing, and activating for 15-30 minutes; then adding the plasmon nano particle @ SiO with amino 2 Fluorescent material @ SiO 2 And (5) coupling in a dark place to obtain the fluorescent probe.
In a further embodiment, the metal nanoparticles in the first step are gold nanoparticles or silver nanoparticles.
In a further embodiment, the thickness of the silicon dioxide layer in the second step is 3-10 nm.
In a further embodiment, the fluorescent material in the third step is: fluorescein isothiocyanate FITC, tetraethylrhodamine RIB200, tetramethylrhodamine TRITC, lanthanide chelate, or quantum dot.
In a further embodiment, the chemical reactions of the first to fifth steps are all completed under a magnetic stirrer, and the coated or modified materials of the second to fifth steps are all cleaned by low-speed multiple centrifugation to remove the uncoated or modified materials.
In another aspect, there is provided a multi-channel assay kit comprising:
the device comprises a body, a reaction chamber and a reaction chamber, wherein at least one sample injection chamber and N reaction chambers are arranged in the body;
at least two groups of cover plates are correspondingly arranged on two sides of the body; one of the cover plates is provided with a sample inlet which is communicated with the liquid inlet cavity;
the reaction cavity is used for containing a detection reagent and a sample liquid; the detection reagent is a fluorescent probe prepared by the preparation method.
In another technical scheme, a detection method based on the multichannel detection kit is provided, and the method comprises the following steps:
the fluorescent probe is filled into the reaction cavity in a freeze-dried form; adding sample liquid into the sample injection cavity from the sample injection port, and correspondingly flowing into the N reaction cavities;
inserting the multichannel detection kit into a constant temperature detector, and carrying out warm bath on the multichannel detection kit;
the fluorescent probe in the reaction cavity is redissolved with the sample liquid; the constant temperature detector detects the light transmittance change of the complex solution in the reaction cavity in real time through illumination and outputs the light transmittance change in the form of analog signals; and drawing a fluorescence spectrum based on the analog signal.
The beneficial effects are that:
(1) The difference value of fluorescence is used as a correlation coefficient of biomarker concentration, so that dependence on absolute fluorescence intensity is solved, and the batch difference of products is reduced;
(2) The portable homogeneous immunofluorescence detection is realized, and the defects that the chromatography method is easy to be interfered by the environment and the like are avoided;
(3) The plasmon is adopted to enhance fluorescence, the fluorescence intensity is improved by 100-10000 times compared with the initial fluorescence, and the detection precision is high;
(4) The detection reagent is packaged in a multi-channel detection kit in a freeze-dried form, so that the on-site detection of the biomarker can be realized;
(5) The 6 reaction chambers of the kit determine that 1-6 biomarkers can be detected by one sample;
(6) Because the enhanced fluorescence based on the self fluorescence is detected, the background fluorescence interference in the sample is relatively small, and the product is beneficial to being applied to a real whole blood sample.
Drawings
FIG. 1 is a schematic diagram of a multi-channel detection kit;
FIG. 2 is a block diagram of a fluorescent probe;
FIG. 3 is a state diagram of fluorescent probes without specific biomarkers;
FIG. 4 is a state diagram of fluorescent probe and marker coupling;
FIG. 5 is a graph showing fluorescence spectra of troponin I at various concentrations.
Each labeled in fig. 1-5 is: the device comprises a body 10, a sample injection cavity 11, a reaction cavity 12, a cover plate 20, a sample injection port 21, a fluorescent probe 30, plasmon nano particles 31, a silicon dioxide layer 32, fluorescent substances 33 and antibodies 34.
Detailed Description
Example 1
This embodiment provides a fluorescent probe 30, which has a sphere structure, comprising: plasmonic nanoparticles 31, a silica layer 32 wrapped around the plasmonic nanoparticles 31, and antibodies 34 distributed on the outer wall of the silica layer 32 according to predetermined requirements; the silica layer 32 is embedded with a fluorescent substance 33 at a predetermined position.
When antigen is present in the sample, the antibodies 34 on the fluorescent probes 30 of adjacent two sets are coupled by the antigen to form a "sandwich-like" structure.
Example 2
This example provides a method for preparing the fluorescent probe 30 according to example 1, wherein the method for preparing the fluorescent probe 30 by chemical synthesis comprises the following steps:
in the first step, the inner core of the fluorescent probe 30 is plasmon nanoparticle 31, typically gold or silver nanoparticle. The nano particles are prepared by a relatively conventional oxidation-reduction method, the particle size is 40-100 nanometers, if the gold nano particles adopt sodium citrate to reduce chloroauric acid, and the silver nano particles adopt sodium citrate to reduce silver nitrate;
step two, wrapping a silicon dioxide layer 32 outside the inner core, wherein the thickness of the silicon dioxide layer 32 is 3-10 nm, and the silicon dioxide layer is prepared by hydrolyzing triaminopropyl triethoxysilane (APTEOS), the thickness of the thin layer is regulated and controlled mainly by controlling the hydrolysis time and the concentration of the APTEOS, and the concentration of the APTEOS is generally 0.5-2% when the hydrolysis time is controlled for 10-30 minutes;
step three, modifying fluorescent molecules or fluorescent substances 33 such as fluorescein isothiocyanate FITC, tetraethylrhodamine RIB200, tetramethylrhodamine TRITC, lanthanide chelate, quantum dots and the like on the surface of the silicon dioxide, wherein the fluorescent molecules or fluorescent substances 33 are generally adsorbed on the surface of the silicon dioxide in an electrostatic adsorption mode, and the plasmon nano particles 31@SiO 2 Dropwise adding 1% -5% of fluorescent substance 33 in mass fraction under intense stirring, and finally obtaining plasmon nanoparticle 31@SiO 2 The volume ratio of the fluorescent substance 33 is between 10:1 and 50:1;
step four, continuously wrapping the silicon dioxide layer 32 on the outer layer of the fluorescent molecule, wherein the thickness of the silicon dioxide layer 32 is 3-10 nm, and the wrapping method and the inner core wrap SiO 2 The modes are consistent;
step five, the corresponding antibody 34 is modified on the outermost layer for identifying a specific biomarker, and the antibody 34 is modified on the surface of silicon dioxide by adopting a conventional method: the carboxyl group of antibody 34 forms an active ester bond with N-hydroxysuccinimide (NHS) in the presence of the crosslinker 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), which in turn forms an amide bond with the amino group on the silica surface. The core step is that 5ml of antibody 34 with carboxyl and 1% mass fraction is added with 0.2-0.5 ml of 10mg/ml EDC solution, evenly mixed, then added with 2-5 ml of 10mg/ml NHS solution, evenly mixed and activated for 15-30 minutes. Then 5ml of 10% mass fraction of plasmon nano particles 31@SiO2@fluorescent substance 33@SiO with amino groups are added 2 Coupling for 2 hours at 37 ℃ in a dark place;
the whole chemical synthesis can be completed under a magnetic stirrer, and the coated materials in each step are required to be cleaned by adopting low-speed repeated centrifugation to remove the uncoated materials.
Example 3
The present embodiment provides a multi-channel detection kit (hereinafter referred to as kit), and the overall structure of the kit is shown in fig. 1, and the kit includes a body 10 and two sets of cover plates 20. The kit is divided into 3 pieces and is formed by laser bonding.
The body 10 is internally provided with a sample injection cavity 11 and N reaction cavities 12 communicated with the sample injection cavity 11. Two sets of cover plates 20 are correspondingly arranged on two sides of the body 10; one of the cover plates 20 is provided with a sample inlet 21, and the sample inlet 21 is communicated with the liquid inlet cavity.
The reaction chamber 12 is used for containing detection reagent and sample liquid; the detection reagent is the fluorescent probe prepared in the example 2.
The corresponding position of the reaction cavity 12 is made of transparent materials. The multichannel detection kit is inserted into a constant temperature detector during detection.
The detection reagent is disposed in each reaction chamber 12. The fluorescent probe 30 is in a lyophilized form.
In a further embodiment, the detection reagent comprises, in addition to the fluorescent probe 30: protecting agents and excipients for lyophilization, such as PEG, bovine serum albumin.
1-6 detection reagent balls or reagent powder can be arranged in the reaction cavity 12 of the kit, and 1-6 biomarkers can be detected correspondingly. The sample or diluted sample is introduced from the liquid inlet and flows into the 6 reaction chambers 12. The filter column of the sample inlet 21 can filter part of red blood cells and the like in whole blood to prevent the channel from being blocked. The sample flows into the reaction chamber 12, and the freeze-dried reagent (ball or powder) in the reaction chamber 12 is reconstituted, so that subsequent immunocapture and fluorescence detection can be performed.
The detection reagent (ball or powder) in the reaction chamber 12 has a main component of a fluorescent probe 30 as shown in FIG. 2.
Example 4
The embodiment provides a detection method based on the multi-channel detection kit described in embodiment 3, comprising the following steps: the fluorescent probe is filled in the reaction chamber 12 in a freeze-dried form; adding sample liquid from a sample inlet 21, flowing into the sample injection cavity 11, and correspondingly flowing into the N reaction cavities 12;
inserting the multichannel detection kit into a constant temperature detector, and carrying out warm bath on the multichannel detection kit;
the fluorescent probe in the reaction cavity 12 is redissolved with the sample liquid; the constant temperature detector is used for carrying out light transmittance change of the complex solution in the reaction cavity 12 in real time through illumination and outputting the complex solution in the form of analog signals; and drawing a fluorescence spectrum based on the analog signal.
The concrete explanation is as follows:
6 different fluorescent probes 30 can be placed in the 6 reaction chambers 12 of the kit, and the main difference is that different antibodies 34 are modified, so that different detection markers can be aimed at.
When there is no specific biomarker in the sample, as shown in fig. 3, the fluorescent probe 30 will not form coupling, and when excited by the excitation light, the fluorescence emitted by the fluorescent molecule itself is detected.
When a specific biomarker is present in the sample, as shown in fig. 4, the antigen-antibody 34 specifically binds, forming a "sandwich" structure. When excited by excitation light, the plasmonic nanoparticle 31 develops an electromagnetic field of plasmon coupling because the distance is less than 10 nanometers after the nanoparticle is pulled by the antigen; the stable multiple nano particles are close to each other in distance, and the coupling effect occurs after the mutual influence of the respective electromagnetic fields; the coupling effect is better within 10 nanometers, the closer the coupling effect is, but the contact cannot occur; only the respective electromagnetic fields interact to form a locally amplified electromagnetic field.
Excitation of fluorescent molecules is positively correlated with coupling efficiency, as follows: enhancement factor e=c Excitation *C Emission of The method comprises the steps of carrying out a first treatment on the surface of the The excited fluorescence is further emitted in the coupling light field, which is also positively correlated with the coupling efficiency. The resulting synergistically enhanced fluorescence is received by the detector.
As fluorescence intensity increases with time from the onset of reconstitution, specific biomarkers are present and the rate and endpoint intensity of fluorescence intensity increase is positively correlated with biomarker concentration; if the fluorescence intensity does not increase with time, a specific biomarker is not present.
The target organism troponin I (cTnI) to be detected is further described below.
The fluorescent material in the fluorescent probe 30 is carbon quantum dots, troponin I antibody 34 (cTnI-Ab) is modified on the surface of the probe, and the fluorescent probe 30 is freeze-dried and then packaged in the reaction cavity 12 of the microfluidic kit. The gradient diluted sample containing the biomarker cTnI is added from a sample adding port, the reagent balls are dissolved again in the sample flow channel reaction cavity 12, the kit is put into a warm bath device for warm bath at 30 ℃, and the endpoint fluorescence spectrum is collected, as shown in figure 5. The concentration of the biomarker cTnI in the samples is 0 respectively; 0.05;0.1;0.5;1, a step of; 5, a step of; 10;50; the fluorescence intensity is gradually increased along with the concentration of the sample, so that the detection range of the method for cTnI is 0.1-50 ng/ml. Therefore, the detection method has high sensitivity, and the existing fluorescence detection technology is difficult to reach the detection range of 0.1 ng/ml.
Claims (10)
1. The fluorescent probe is characterized in that the probe has a sphere structure and comprises: the plasmon nanoparticle comprises plasmon nanoparticles, a silicon dioxide layer wrapped on the plasmon nanoparticles, and antibodies distributed on the outer wall of the silicon dioxide layer according to preset requirements; fluorescent substances are embedded at predetermined positions of the silicon dioxide layer.
2. The fluorescent probe of claim 1, wherein antibodies on two adjacent sets of fluorescent probes are coupled by an antigen.
3. A method of preparing a fluorescent probe according to any one of claims 1 to 2, comprising the steps of:
step one, taking metal nano particles with the particle size of 40-100 nm, and carrying out oxidation-reduction reaction on the metal nano particles to obtain plasmon nano particle cores;
step two, wrapping a silicon dioxide layer on the outer surface of the inner core of the plasmon nanoparticle to obtain the plasmon nanoparticle@SiO 2 The thickness of the silicon dioxide layer is 3-10 nm;
step three, modifying fluorescent substances on the outer surface of the silicon dioxide layer in the step two: plasmon nanoparticle @ SiO 2 Dropwise adding 1 under stirringFluorescent substances accounting for percent to 5 percent of the mass percent are stirred to obtain plasmon nano particles@SiO 2 A @ fluorescent substance;
step four, wrapping a silicon dioxide layer on the outer layer of the fluorescent substance, wherein the thickness of the silicon dioxide layer is 3-10 nm, and obtaining the plasmon nanoparticle@SiO 2 Fluorescent material @ SiO 2 ；
Step five, modifying the corresponding antibody on the silicon dioxide layer in the step four: adding the antibody into a cross-linking agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution, uniformly mixing, then adding an N-hydroxysuccinimide solution, uniformly mixing, and activating for 15-30 minutes; then adding plasmon nano particles @ SiO 2 Fluorescent material @ SiO 2 And (5) coupling in a dark place to obtain the fluorescent probe.
4. The portable multi-channel assay kit of claim 1, wherein the metal nanoparticles in step one are gold nanoparticles or silver nanoparticles.
5. The portable multichannel detection kit of claim 1, wherein the step three plasmon nanoparticle @ SiO 2 The volume ratio of the fluorescent material to the fluorescent material is (10-50): 1.
6. the portable multi-channel assay kit of claim 1, wherein the fluorescent material in step three is: fluorescein isothiocyanate FITC, tetraethylrhodamine RIB200, tetramethylrhodamine TRITC, lanthanide chelate, or quantum dot.
7. A multi-channel assay kit comprising: the device comprises a body, a reaction chamber and a reaction chamber, wherein at least one sample injection chamber and N reaction chambers are arranged in the body;
at least two groups of cover plates are correspondingly arranged on two sides of the body; one of the cover plates is provided with a sample inlet which is communicated with the liquid inlet cavity;
the reaction cavity is used for containing a detection reagent and a sample liquid; the detection reagent is the fluorescent probe prepared by the method of claim 3.
8. The multi-channel assay kit of claim 7, wherein the reaction chamber is formed of a transparent material.
9. A multi-channel assay kit according to claim 7, wherein said multi-channel assay kit is inserted into a isothermal detector during testing.
10. A detection method based on a multi-channel detection kit according to any one of claims 7 to 9, characterized in that the detection method comprises the steps of:
the fluorescent probe is filled into the reaction cavity in a freeze-dried form; adding sample liquid into the sample injection cavity from the sample injection port, and correspondingly flowing into the N reaction cavities;
inserting the multichannel detection kit into a constant temperature detector, and carrying out warm bath on the multichannel detection kit;
the fluorescent probe in the reaction cavity is redissolved with the sample liquid; the constant temperature detector detects the light transmittance change of the complex solution in the reaction cavity in real time through illumination and outputs the light transmittance change in the form of analog signals; and drawing a fluorescence spectrum based on the analog signal.
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