CN115746827A - Membranous multilayer quantum dot fluorescent material, preparation method and immunochromatography application thereof - Google Patents
Membranous multilayer quantum dot fluorescent material, preparation method and immunochromatography application thereof Download PDFInfo
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Abstract
The invention provides a membranous multilayer quantum dot fluorescent material, which comprises a membranous carrier and a fluorescent layer arranged on the membranous carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially superposed, and the cationic polymer self-assembly layer in the fluorescent layer is contacted with the membrane-shaped carrier. Compared with the prior art, the film-shaped multilayer quantum dot fluorescent material provided by the invention is a three-dimensional fluorescent nano film, and has larger surface area, higher fluorescent signal, better dispersibility and stability compared with the traditional spherical fluorescent marker, thereby greatly improving the detection sensitivity of the test strip, enabling the constructed fluorescent LFA biosensor to simultaneously and sensitively quantify SARS-CoV-2, influenza A virus and human adenovirus, and having low detection limit, short detection time, good reproducibility and high accuracy.
Description
Technical Field
The invention belongs to the technical field of fluorescence immunochromatography, and particularly relates to a membranous multilayer quantum dot fluorescent material, a preparation method and an immunochromatography application thereof.
Background
The lateral flow immunoassay (LFA) has the characteristics of rapidness, easiness in use, portability, low cost, multi-target detection capability and the like, is considered as the most promising point-to-point detection (POCT) technology, and is widely applied to the fields of clinical examination, personal health self-detection, food safety monitoring and the like.
The immunochromatography technology is characterized in that: (1) The device does not need professional operators, and can be widely applied to different places such as hospitals, community medical institutions, airports, stations, schools, families and the like. (2) The detection result can be directly and rapidly provided (generally <20 min) without complex instruments. (3) The LFA technique can analyze multiple targets in one test by arranging multiple test lines on one strip, thereby effectively simplifying the detection process. However, due to the complex composition of traditional spherical nano-labels and respiratory tract samples (saliva, throat swab, sputum, etc.), poor performance in detecting respiratory viruses (e.g., instability, low signal intensity, poor dispersion or large size), currently developed LFA methods still exhibit limited quantification capability, low sensitivity (typically >0.1 ng/mL) and low flux (< 2) in detecting respiratory viruses. To date, although many colloidal gold-based LFA kits and more sensitive LFA techniques have been proposed, including fluorescence-based, chemiluminescence-based, and raman signal-based strategies for the detection of new coronavirus (SARS-CoV-2) and influenza a virus, these methods still fail to simultaneously sensitively detect multiple respiratory viruses. In order to meet the requirement of multiplex detection of respiratory viruses, a novel LFA system with signal amplification capability, immunological binding efficiency and stability needs to be developed.
In recent years, two-dimensional (2D) thin film type nanomaterials, such as graphene and its derivatives, molybdenum disulfide, black phosphorus, etc., have shown great potential in the field of rapid diagnosis due to their unique structural characteristics (such as large surface area, ultra-thin structure, abundant surface active groups) and extraordinary electronic and optical properties. Therefore, the invention provides a membranous multilayer fluorescent nano label and introduces the membranous multilayer fluorescent nano label into an immunochromatography system for detecting respiratory viruses, so that rapid, ultrasensitive and quantitative analysis of various common respiratory viruses is realized on one test strip.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a membranous multilayer quantum dot fluorescent material, a preparation method thereof and an immunochromatography application thereof, the membranous multilayer quantum dot fluorescent material can be used for ultrasensitive simultaneous detection of three target respiratory viruses (SARS-CoV-2, influenza a virus and human adenovirus) in a complex biological specimen, and the problem of insufficient performance of the existing nano-label material for immunochromatography detection is solved, especially the problem of insufficient sensitivity when applied to simultaneous detection of multiple respiratory viruses.
The invention provides a membranous multilayer quantum dot fluorescent material, which comprises a membranous carrier and a fluorescent layer arranged on the membranous carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially stacked, and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the membrane-shaped carrier.
Preferably, the number of the cationic polymer self-assembly layers and the number of the electronegative quantum dot layers in the fluorescent layer are both greater than or equal to 2, and the cationic polymer self-assembly layers and the electronegative quantum dot layers are arranged alternately.
Preferably, the membrane-shaped carrier is a single layer of graphene oxide; the cationic polymer self-assembly layer is a polyethyleneimine self-assembly layer; the electronegative quantum dot layer is a carboxylated cadmium selenide/zinc sulfide core-shell quantum dot self-assembly layer or a 3-mercaptopropionic acid coated cadmium selenide/zinc sulfide quantum dot self-assembly layer.
The invention also provides a preparation method of the membranous multilayer quantum dot fluorescent material, which comprises the following steps:
s1) mixing the membrane-shaped carrier with a cationic polymer solution and performing ultrasonic treatment to obtain a solution containing the membrane-shaped carrier loaded with a cationic polymer self-assembled layer;
s2) mixing the electronegative quantum dot solution with a solution of a film-shaped carrier which loads a cationic polymer self-assembly layer, and performing ultrasonic treatment to obtain the film-shaped multilayer quantum dot fluorescent material.
Preferably, the film-shaped carrier is prepared by the following method:
carrying out ultrasonic treatment on the monolayer graphene oxide dispersion liquid, centrifuging, collecting precipitates, and suspending the precipitates in water to obtain a film-shaped carrier; the thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion liquid is 1-2 nm; the sheet diameter is larger than 200nm; the power of the ultrasonic treatment is 500-1000W; the rotating speed of the centrifugation is 10000-20000 g.
Preferably, the concentration of the cationic polymer in the cationic polymer solution is 0.1-5 mg/mL; the molecular weight of the cationic polymer in the cationic polymer solution is 3000-100000;
the concentration of the electronegative quantum dot solution is 1-50 ng/mL; the volume ratio of the cationic polymer solution to the electronegative quantum dot solution is (300-800): 1.
preferably, the power of the mixed ultrasound in the step S1) and the step S2) is 500-1000W; the time of mixing and ultrasonic treatment is 10-60 min.
Preferably, after the ultrasonic mixing in the step S2), the sediment is collected by centrifugation, and the step S1) and the step S2) are repeated by replacing the membranous carrier; the number of repetitions is 1 to 5.
The invention also provides application of the film-shaped multilayer quantum dot fluorescent material in immunochromatography.
The invention also provides a membranous multilayer quantum dot nano label which comprises the membranous multilayer quantum dot fluorescent material and a detection antibody modified on the surface of the membranous multilayer quantum dot fluorescent material.
The invention provides a membranous multilayer quantum dot fluorescent material, which comprises a membranous carrier and a fluorescent layer arranged on the membranous carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially superposed, and the cationic polymer self-assembly layer in the fluorescent layer is contacted with the membrane-shaped carrier. Compared with the prior art, the film-shaped multilayer quantum dot fluorescent material provided by the invention is a three-dimensional fluorescent nano film, has larger surface area, higher fluorescent signal and better dispersity and stability compared with the traditional spherical fluorescent marker, thereby greatly improving the detection sensitivity of the test paper strip, enabling the constructed fluorescent LFA biosensor to simultaneously and sensitively quantify SARS-CoV-2, influenza A virus and human adenovirus, and having low detection limit, short detection time, good reproducibility and high accuracy.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a film-shaped multi-layer quantum dot fluorescent material and a method for modifying an antibody according to the present invention;
FIG. 2 is a transmission electron microscope image of various structural characterization data in the preparation process of the film-like multilayer quantum dot fluorescent material in embodiment 1 of the present invention, wherein a, b, c, d are GO, GO @ QD, GO @ DQD and GO @ TQD nanosheets, respectively; e. f and g are respectively amplified TEM images of the local forms of GO @ QD, GO @ DQD and GO @ TQD nanosheets; h. i and j are respectively scanning electron microscope images of GO @ QD, GO @ DQD and GO @ TQD; k is the element surface scanning analysis result of GO @ TQD; m, n and o are fluorescence property contrast of QD, GO @ QD, GO @ DQD and GO @ TQD nanosheets; l is a particle size distribution result graph of GO, GO @ QD, GO @ DQD and GO @ TQD nanosheets;
fig. 3 is a stability test result diagram of the film-like multilayer quantum dot fluorescent material obtained in example 1 of the present invention, wherein a is a high salt stability test result diagram; b is a graph of the acid-base stability test result; c is a time stability test result chart;
fig. 4 is a comparison of performances of three film-like multilayer quantum dot materials (go @ QD, go @ dqd, and go @ tqd) with different numbers of QD shell layers on an immunochromatography system in example 2 of the present invention, in which a is a test strip visualization fluorescence result diagram for detecting new coronavirus NP protein with different concentrations based on immunochromatography of the three film-like materials; b is a test strip fluorescence analysis result and a linear range diagram for detecting the new coronavirus NP protein with different concentrations by immunochromatography based on three membranous materials;
fig. 5 is an experimental flowchart of the detection of new coronavirus, influenza a virus and influenza b virus by using the film-like multilayer quantum dot fluorescent material of embodiment 3 as a high-performance film-like fluorescent label in combination with an immunochromatographic system;
FIG. 6 is a graph showing the results of detecting new coronavirus, influenza A virus and influenza B virus by the immunochromatographic system based on the membranous multilayer quantum dot labels in example 3 of the present invention, in which a is the result of a fluorescent photograph of simultaneously detecting three viruses; b, the graph is the fluorescence values of 3 detection lines recorded by a fluorescence spectrometer; and c, a graph shows the results of a fluorescence numerical value-virus concentration fitting curve for detecting three viruses.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a membranous multilayer quantum dot fluorescent material, which comprises a membranous carrier and a fluorescent layer arranged on the membranous carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially stacked, and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the film-shaped carrier.
In the present invention, the thickness of the film-like support is preferably 1 to 2nm; the sheet diameter of the membranous carrier is preferably 500-800 nm; the membrane-like support is preferably a single layer of Graphene Oxide (GO). The two-dimensional single-layer graphene oxide is large in area and has good flexibility when being used as a carrier.
A fluorescent layer is arranged on the film-shaped carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially overlapped; the cationic polymer self-assembly layer is preferably a polyethyleneimine self-assembly layer; the electronegative quantum dot layer is preferably a carboxylated cadmium selenide/zinc sulfide core-shell quantum dot (CdSe @ ZnS-COOH QD) self-assembly layer or a 3-mercaptopropionic acid coated cadmium selenide/zinc sulfide quantum dot (CdSe @ ZnS-MPA QD) self-assembly layer.
In the invention, the number of the cationic polymer self-assembly layer and the number of the electronegative quantum dot layer in the fluorescent layer are both preferably greater than or equal to 2, more preferably 2 to 5, still more preferably 2 to 4, and most preferably 3; and the cationic polymer self-assembly layer and the electronegative quantum dot layer are arranged alternately. The multi-layer cationic polymer self-assembly layer is used as an interlayer, so that the dispersity of the nanosheets can be greatly improved, and a large number of electronegative quantum dots can be effectively adsorbed; a multi-layered set of electronegative quantum dots layer contains thousands of electronegative quantum dots that produce fluorescence signals thousands of times greater than that produced by a single quantum dot and provide a large number of antibody coupling surface sites. Compared with the commonly used spherical fluorescent material, the film-shaped multilayer quantum dot fluorescent material provided by the invention has larger surface area, more excellent fluorescence property, better dispersibility and stability, thereby greatly improving the detection sensitivity of the test strip.
The invention also provides a preparation method of the film-shaped multilayer quantum dot fluorescent material, which comprises the following steps: s1) mixing the membrane-shaped carrier with a cationic polymer solution and performing ultrasonic treatment to obtain a solution containing the membrane-shaped carrier loaded with a cationic polymer self-assembled layer; s2) mixing the electronegative quantum dot solution with a solution of a film-shaped carrier which loads a cationic polymer self-assembly layer, and performing ultrasonic treatment to obtain the film-shaped multilayer quantum dot fluorescent material.
In the present invention, the sources of all raw materials are not particularly limited and are commercially available.
In the present invention, the film-shaped support is preferably prepared according to the following method: carrying out ultrasonic treatment on the single-layer graphene oxide dispersion liquid, centrifuging, collecting precipitates, and suspending the precipitates in water to obtain a membranous carrier; the thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion liquid is preferably 1-2 nm; the sheet diameter is preferably more than 200nm; the concentration of the single-layer graphene oxide dispersion liquid is preferably 0.5-3 mg/mL, and more preferably 1-2 mg/mL; the power of the ultrasonic treatment is preferably 500-1000W, more preferably 600-900W, and still more preferably 700-800W; the time of the ultrasonic treatment is preferably 5 to 30min, more preferably 10 to 20min, and still more preferably 15min; the graphene oxide can be completely dispersed through ultrasonic treatment; the rotating speed of the centrifugation is preferably 10000-20000 g, more preferably 11000-16000 g, still more preferably 11000-14000 g, and most preferably 12000g; the time of centrifugation is preferably 3-10 min, more preferably 5-8 min, and further preferably 6min; graphene oxide with undersize sheet diameter in supernatant can be removed by centrifugation (< 400 nm), and graphene oxide nanosheets with uniform sheet diameter are separated; collecting the precipitate and re-suspending the precipitate in water to obtain graphene oxide nanosheets with a flake diameter ranging from 500 nm to 800nm; the ratio of the volume of water used for resuspension to the volume of the monolayer graphene oxide dispersion is preferably 1: (0.5 to 2), more preferably 1: (0.8 to 1.5), more preferably 1:1.
mixing the membranous carrier with a cationic polymer solution and carrying out ultrasonic treatment; the membrane-shaped carrier is mixed with the cationic polymer solution in a form of being suspended in water; the ratio of the volume of water in which the film-like carrier is suspended to the volume of the cationic polymer solution is preferably 1: (0.5 to 2), more preferably 1: (0.8 to 1.5), more preferably 1:1; the concentration of the cationic polymer in the cationic polymer solution is preferably 0.1-5 mg/mL, more preferably 0.5-3 mg/mL, and still more preferably 1-2 mg/mL; the molecular weight of the cationic polymer in the cationic polymer solution is preferably 3000 to 100000; the cationic polymer is preferably polyethyleneimine, more preferably polyamino branched polyethyleneimine; the power of the ultrasonic mixing is preferably 500-1000W, more preferably 600-900W, and still more preferably 700-800W; the ultrasonic mixing time is preferably 10-60 min, more preferably 20-40 min, and still more preferably 30min; and (3) rapidly self-assembling the cationic polymer on the surface of the monolayer graphene oxide by intense ultrasound to obtain a solution containing the membrane-shaped carrier loaded with the cationic polymer self-assembled layer.
Mixing the electronegative quantum dot solution with a solution of a membranous carrier containing a loaded cationic polymer self-assembled layer and performing ultrasonic treatment; the concentration of the electronegative quantum dot solution is preferably 1-50 ng/mL, more preferably 5-50 ng/mL, still more preferably 10-40 ng/mL, and most preferably 20-30 ng/mL; the volume ratio of the cationic polymer solution to the electronegative quantum dot solution is preferably (300-800): 1, more preferably (400 to 700): 1, more preferably (500 to 600): 1, most preferably 500:1; the power of the ultrasonic mixing is preferably 500-1000W, more preferably 600-900W, and still more preferably 700-800W; the ultrasonic mixing time is preferably 10-60 min, more preferably 20-40 min, and still more preferably 30min; in the process, the electronegative quantum dots are adsorbed to the surface of the polyethyleneimine self-assembly layer in a large amount through electrostatic adsorption.
After ultrasonic mixing, preferably centrifuging to obtain a film-shaped multi-layer quantum dot fluorescent material of which the fluorescent layer is provided with a single-layer cationic polymer self-assembly layer and a single-layer electronegative quantum dot layer; the rotating speed of the centrifugation is preferably 4000-8000 g, more preferably 5000-7000 g, and even more preferably 6000g; the time for the centrifugation is preferably 3 to 10min, more preferably 5 to 8min, and still more preferably 6min.
In the present invention, after the precipitate is collected by centrifugation, the steps S1) and S2) are repeated with it in place of the membrane-shaped carrier; the number of repetition is preferably 1 to 5 times, more preferably 1 to 4 times, still more preferably 1 to 3 times, and most preferably 2 times. By repeating the steps S1) and S2) for multiple times, more layers of electronegative quantum dot layers can be coated continuously by using layer-by-layer self-assembly mediated by cationic polymers according to needs.
The invention adopts an ultrasonic-mediated electrostatic adsorption method to prepare a membranous multilayer quantum dot fluorescent material, and sequentially coats three layers of compact electronegative quantum dots on the surface of a two-dimensional single-layer graphene oxide sheet through layer-by-layer self-assembly mediated by electrostatic adsorption. The method converts the two-dimensional nano structure into the three-dimensional fluorescent nano film, and has the advantages of larger surface area, higher fluorescent signal, better dispersibility and stability compared with the traditional spherical fluorescent marker. The constructed fluorescence LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus and human adenovirus, and has the advantages of low detection limit, short detection time, good reproducibility and high accuracy.
The invention also provides application of the film-shaped multilayer quantum dot fluorescent material in immunochromatography, preferably application of the film-shaped multilayer quantum dot fluorescent material serving as a nano label of fluorescence immunochromatography.
The invention also provides a membranous multilayer quantum dot nano label, which comprises the membranous multilayer quantum dot fluorescent material and a detection antibody modified on the surface of the membranous multilayer quantum dot fluorescent material; the detection antibody is preferably a monoclonal antibody; the detection antibody is preferably an antibody of one or more of new coronavirus, influenza A virus and influenza B virus; the detection antibody can be directly coupled and modified by activating a membranous multilayer quantum dot fluorescent material through a carboxyl activating reagent; the carboxyl activating reagent is not particularly limited as long as it is known to those skilled in the art, but EDC/NHS is preferred in the present invention.
The invention introduces the membrane-shaped multilayer quantum dot fluorescent material modified by the detection antibody into an immunochromatography system, can provide fluorescence performance, stability, dispersibility and a reaction interface superior to those of spherical quantum dot microspheres, and can realize high-sensitivity, rapid and quantitative detection of a target object in the immunochromatography system.
The film-shaped multilayer quantum dot nano label provided by the invention can be prepared according to the following method: respectively modifying antibodies of target objects to be detected (new coronaviruses, influenza A viruses and influenza B viruses) on the surface of the film-shaped multilayer quantum dot fluorescent material, then dripping the antibodies on a glass fiber film, and freeze-drying the glass fiber film to be used as a fluorescent nano label for immunochromatography. Three detection lines (T1, T2 and T3) are constructed by respectively spraying the capture antibodies of the three target viruses on a nitrocellulose membrane (NC membrane) so as to capture corresponding virus-membrane-shaped label immune complexes. Simultaneously spraying goat anti-mouse IgG on a quality control line of the NC membrane to fix redundant membrane-shaped labels; assembling the NC membrane, the sample pad, the water absorption pad and the bottom plate into an immunochromatographic test strip; and uniformly mixing a sample to be detected with the running buffer solution, dropwise adding the mixture onto a sample pad of the immunochromatographic test strip, and reading fluorescence signals at 3T lines of the immunochromatographic test strip after 15-20 min, so as to realize high-sensitivity detection of the new coronavirus, the influenza A virus and the influenza B virus at the same time.
Compared with the prior art, the invention has the advantages that:
(1) From the aspect of performance, the membranous multilayer quantum dot fluorescent material provided by the invention integrates excellent dispersibility and flexible structure of a single-layer GO nano sheet and strong fluorescence emission capability of a multilayer quantum dot shell, the overall light stability, fluorescence intensity, dispersibility and stability of the membranous multilayer quantum dot fluorescent material are far superior to those of other fluorescent microsphere materials, and the sensitivity of immunochromatography detection can be obviously improved;
(2) In terms of structure, the membrane-shaped multilayer quantum dot fluorescent material provided by the invention is a typical membrane-shaped multilayer nano structure, has larger relative surface area, larger reaction interface and lighter weight compared with spherical nano particles, can easily flow on a chromatography test strip, and is very suitable for the construction of a multi-channel immunochromatography system;
(3) The invention provides a method for realizing layer-by-layer self-assembly of a QD shell on the surface of a GO nanosheet by adopting an ultrasonic-mediated electrostatic adsorption method, and the method is simple, high in reliability and capable of realizing large-scale stable production;
(4) The electrostatic adsorption mediated layer-by-layer self-assembly method for preparing the membranous multilayer quantum dot material is a preparation method of a membranous multilayer structure, continuous coating of a QD (quantum dot) shell on the surface of GO can be realized by controlling the reaction times, the number of layers of the QD shell can be continuously superposed, and further the quantum dot carrying capacity of a single structure is continuously increased;
(5) The surface carboxyl of the film-shaped multilayer quantum dot fluorescent material can be used for directly modifying biological recognition molecules such as antibodies, aptamers, antibiotics, polypeptides and the like, so that biological functionalization is conveniently realized;
(6) The film-shaped multilayer quantum dot fluorescent material provided by the invention has wide application prospect, and can be applied in the field of on-site rapid detection, biosensing, in-vivo imaging, clinical examination, food analysis and other fields;
(7) The film-shaped multilayer quantum dot fluorescent material provided by the invention is used as a high-performance film-shaped fluorescent label for immunochromatography detection, can provide stronger and stable fluorescent signals, better stability and dispersibility and more surface active sites, and can effectively improve the detection performance and detection flux of the fluorescence immunochromatography technology.
In conclusion, the electrostatic adsorption mediated layer-by-layer self-assembly method for preparing the membranous multilayer quantum dot label has the characteristics of novel structure, good performance, simple method and high efficiency. Compared with a spherical fluorescent material, the prepared film-shaped multilayer quantum dot fluorescent material has higher quantum dot loading capacity, larger surface area, more excellent fluorescence performance, better stability and dispersibility, and has wide application prospect in the field of on-site rapid detection, particularly in the aspect of high-sensitivity fluorescence immunochromatography detection.
In order to further illustrate the present invention, the following detailed description is made with reference to the examples for a film-like multi-layer quantum dot fluorescent material, its preparation method and immunochromatography application.
The reagents used in the following examples are all commercially available; NP protein was purchased from Yiqiao Shenzhou, catalog #40588-V08B; influenza a virus antibodies and influenza b virus antibodies were purchased from china and american and modern biotechnology limited, wherein influenza a: catalog # FluA-001; fluA-002; b, flow: catalog # FluB-001; fluB-002.
Example 1
The membranous multilayer quantum dot fluorescent material prepared by the invention is mainly prepared through three steps, firstly, the GO nano sheets are centrifugally separated, secondly, GO @ QD, GO @ DQD and GO @ TQD are prepared in a mode of one PEI layer and one QD layer through the layer-by-layer self-assembly effect of electrostatic adsorption, and finally, the antibody is coupled on the surface of GO @ TQD through an amido bond by utilizing the carboxyl on the surface of the membranous multilayer quantum dot material.
A method for preparing the film-like multi-layer quantum dot material and the antibody coupling method of the embodiment are shown in fig. 1, and include the following steps:
(1) Preparing GO nano sheets:
carrying out ultrasonic treatment on 10mL of GO nano-sheet aqueous solution (with the concentration of 1 mg/mL) for 15 minutes, centrifuging for 6 minutes at 12000g, collecting precipitate, and suspending in 10mL of deionized water to obtain the GO nano-sheet aqueous solution with the particle size of 500-800 nm for later use.
(2) Preparation of go @ qd nanoplates:
under ultrasonic treatment, after 10mLGO nanosheets are mixed with 10mL of PEI solution with the concentration of 1mg/mL, 800W is subjected to violent ultrasonic reaction for half an hour to enable PEI to be rapidly self-assembled on the GO surface. 20 μ L of carboxylated QD (20 ng/mL CdSe @ ZnS-COOH QD solution) was then added and vigorous sonication continued for half an hour. In the process, a large amount of carboxylated QDs are adsorbed to the surface of GO @ PEI through electrostatic adsorption to form GO @ QD nanosheets; go @ qd was collected by 6000g centrifugation for 6 minutes, and dispersed into 10mL deionized water for use.
(3) Preparing GO @ DQD nanosheets:
after the GO @ QD nanosheet is mixed with 10mL of PEI solution with the concentration of 1mg/mL, 800W violent ultrasonic reaction is carried out for half an hour to enable PEI to be rapidly self-assembled on the surface of GO @ QD. Subsequently 20. Mu.L of carboxylated QD (20 ng/mL CdSe @ ZnS-COOH QD solution) was added and vigorous sonication was continued for half an hour. In the process, a large amount of carboxylated QDs are adsorbed to the surfaces of GO @ QDs @ PEI through electrostatic adsorption to form GO @ DQD nanosheets; GO @ DQD was collected by centrifugation at 6000g for 6 minutes and dispersed into 10mL deionized water for use.
(4) Preparing GO @ TQD nanosheets:
after mixing the prepared GO @ DQD nanosheet with 10mL of PEI solution with the concentration of 1mg/mL, carrying out 800W violent ultrasonic reaction for half an hour to enable PEI to be rapidly self-assembled on the surface of GO @ DQD. 20 μ L of carboxylated QD (20 ng/mL CdSe @ ZnS-COOH QD solution) was then added and vigorous sonication continued for half an hour. In the process, a large amount of carboxylated QDs are adsorbed to the surface of GO @ DQD @ PEI through electrostatic adsorption to form GO @ TQD nanosheets; GO @ TQD was collected by centrifugation at 6000g for 6 minutes, dispersed into 10mL deionized water for use.
(5) Preparation of antibody-modified go @ tqd fluorescent labels:
first, 1mL of GO @ TQD was centrifuged and resuspended in 500. Mu.L of 1mL of 2- (N-morpholine) ethanesulfonic acid buffer (0.1M, pH 5.5). Followed by addition of 10. Mu.L of N-hydroxysuccinimide solution and 5. Mu.L of carbodiimide solution and incubation for activation for 15 minutes. Activated GO @ TQD was collected from the solution by centrifugation and resuspended in 200. Mu.L of PBS (0.01M, pH 7.4). 10 μ g of the new coronavirus NP antibody, influenza A virus antibody and influenza B virus antibody were added, respectively, and incubation and shaking were continued for 2 hours. Followed by addition of 100. Mu.L of 10% BSA (w/v) for 1 hour of blocking. Finally, three kinds of antibody modified GO @ TQD are resuspended in preservation solution through centrifugation, and are dripped to a glass fiber membrane for freeze-drying and assembly of subsequent immunochromatographic test strips.
FIG. 2 shows high resolution transmission electron microscopy images (HRTEM) and scanning electron microscopy results of the GO @ QD nanosheets prepared in step (1), the GO @ QD nanosheets prepared in step (2), the GO @ DQD nanosheets prepared in step (3), and the GO @ TQD nanosheets prepared in step (4) of this embodiment; scanning and analyzing the element surface of the GO @ TQD nanosheet obtained through preparation; the prepared membranous multilayer quantum dot and the common quantum dot have the fluorescence property comparison result and the particle size distribution result.
The stability characterization result of the film-shaped multi-layer quantum dot fluorescent material prepared in the embodiment is shown in fig. 3. The film-shaped multilayer quantum dot fluorescent material provided by the invention has excellent salt stability, acid-base stability and long-term stability. As shown in FIG. 3a, the fluorescence signal of the prepared membrane-like GO @ TQD remained stable in high salt solution (0-1000 mM NaCl) (24 h at room temperature). As shown in FIG. 3b, the fluorescence signal of membrane-like GO @ TQD618 nm wavelength remains stable in the environment of pH 4-14 (the concentration of the added solution in water is 1mg/mL, aqueous solutions with different pH values are provided by HCl or NaOH, and the solution is placed at room temperature for 24 hours); as shown in FIG. 3c, the fluorescence signal at room temperature at the wavelength of membrane-like GO @ TQD618 nm remained stable for 60 days (storage under dark conditions).
Example 2
The surface of the film-shaped multilayer quantum dot fluorescent material provided by the invention is modified with an antibody, and then the film-shaped multilayer quantum dot fluorescent material can be used for immunochromatography detection. The QD with more layers can be loaded to effectively improve the detection performance of the membranous multilayer quantum dot on immunochromatography. The performance of loading 1 ~ 3 layers of membranous multilayer quantum dot label (GO @ QD, GO @ DQD and GO @ TQD) for immunochromatography is shown in FIG. 4. The visualized fluorescence signals of ordinary QDs (namely CdSe @ ZnS-COOH) and GO @ QDs, GO @ DQD and GO @ TQD as fluorescence labels for immunochromatography detection of new coronavirus NP protein are 0.5, 0.05 and 0.01ng/mL in sequence. The fluorescence intensity of all test strips was quantified using a commercial fluorescence analyzer and these values were then used to plot a calibration curve for 4 detection platforms (fig. 4 b). Calculate the test paper strip detection limit based on ordinary QD and GO @ QD, GO @ DQD, GO @ TQD and be 111, 52, 16 and 8pg/mL in proper order. This result demonstrates that the use of go @ tqd with multiple QD layers as the fluorescent label can effectively improve the detection sensitivity of LFAs.
Example 3
The film-shaped multilayer quantum dot fluorescent material provided by the invention can be used as a high-performance film-shaped fluorescent label for a multi-channel immunochromatographic system after a target respiratory virus antibody is modified on the surface. The present example employs antibodies to the novel coronavirus NP protein, influenza a virus and influenza b virus.
The modified membrane-shaped GO @ TQD label is used as an immunochromatography system label to detect a mixed sample containing three target viruses with different concentrations. FIG. 5 is a flow chart of the experiment for rapidly detecting three target respiratory viruses by using the membrane-shaped GO @ TQD tag and the immunochromatographic system in this embodiment. FIG. 6 is a test analysis result of three target respiratory viruses detected by a three-channel immunochromatographic system based on a membranous multilayer quantum dot fluorescent material. According to the fluorescence signal of the test strip and the calculation of the corresponding fitting curve result, the detection limits of simultaneously detecting the new coronavirus NP protein, the influenza A virus and the influenza B virus by immunochromatography based on the membranous GO @ TQD label can respectively reach 8pg/mL, 471copies/mL and 488copies/mL.
The above description is only for the purpose of illustrating the technical idea and features of the present invention, and it is intended for the skilled person to understand the content of the present invention and implement the same, and any modifications, equivalent substitutions, improvements, etc. should be included in the protection scope of the present invention.
Claims (10)
1. The membranous multilayer quantum dot fluorescent material is characterized by comprising a membranous carrier and a fluorescent layer arranged on the membranous carrier; the fluorescent layer comprises a cationic polymer self-assembly layer and an electronegative quantum dot layer which are sequentially stacked, and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the membrane-shaped carrier.
2. The film-like multilayer quantum dot fluorescent material according to claim 1, wherein the number of the cationic polymer self-assembled layers and the number of the electronegative quantum dot layers in the fluorescent layer are both 2 or more, and the cationic polymer self-assembled layers and the electronegative quantum dot layers are alternately arranged.
3. The film-like multilayer quantum dot fluorescent material according to claim 1, wherein the film-like support is a single layer of graphene oxide; the cationic polymer self-assembly layer is a polyethyleneimine self-assembly layer; the electronegative quantum dot layer is a carboxylated cadmium selenide/zinc sulfide core-shell quantum dot self-assembled layer or a 3-mercaptopropionic acid-coated cadmium selenide/zinc sulfide quantum dot self-assembled layer.
4. The preparation method of the membranous multilayer quantum dot fluorescent material is characterized by comprising the following steps:
s1) mixing the membrane-shaped carrier with a cationic polymer solution and performing ultrasonic treatment to obtain a solution containing the membrane-shaped carrier loaded with a cationic polymer self-assembled layer;
s2) mixing the electronegative quantum dot solution with a solution of a film-shaped carrier which loads a cationic polymer self-assembly layer, and performing ultrasonic treatment to obtain the film-shaped multilayer quantum dot fluorescent material.
5. The production method according to claim 4, wherein the film-shaped support is produced by:
carrying out ultrasonic treatment on the monolayer graphene oxide dispersion liquid, centrifuging, collecting precipitates, and suspending the precipitates in water to obtain a film-shaped carrier; the thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion liquid is 1-2 nm; the sheet diameter is more than 200nm; the power of the ultrasonic treatment is 500-1000W; the rotating speed of the centrifugation is 10000-20000 g.
6. The method according to claim 4, wherein the concentration of the cationic polymer in the cationic polymer solution is 0.1 to 5mg/mL; the molecular weight of the cationic polymer in the cationic polymer solution is 3000-100000;
the concentration of the electronegative quantum dot solution is 1-50 ng/mL; the volume ratio of the cationic polymer solution to the electronegative quantum dot solution is (300-800): 1.
7. the preparation method according to claim 1, wherein the power of the mixed ultrasound in the steps S1) and S2) is 500-1000W; the time of mixing and ultrasonic treatment is 10-60 min.
8. The method according to claim 1, wherein the steps S1) and S2) are repeated by using the mixture of the ultrasonic waves in step S2) instead of the membrane-shaped carrier after the precipitate is collected by centrifugation; the number of repetitions is 1 to 5.
9. The use of the film-like multilayer quantum dot fluorescent material according to any one of claims 1 to 3 or the film-like multilayer quantum dot fluorescent material prepared by the preparation method according to any one of claims 4 to 8 in immunochromatography.
10. A membranous multilayer quantum dot nano-label is characterized by comprising the membranous multilayer quantum dot fluorescent material of any one of claims 1 to 3 or the membranous multilayer quantum dot fluorescent material prepared by the preparation method of any one of claims 4 to 8 and a detection antibody modified on the surface of the membranous multilayer quantum dot fluorescent material.
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