CN115746827A - Membranous multilayer quantum dot fluorescent material, preparation method and immunochromatography application thereof - Google Patents

Abstract

The invention provides a film-shaped multilayer quantum dot fluorescent material, which includes a film-shaped carrier and a fluorescent layer arranged on the film-shaped carrier; the fluorescent layer includes a cationic polymer self-assembly layer and an electronegative quantum dot layer stacked in sequence , and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the film carrier. Compared with the prior art, the film-like multilayer quantum dot fluorescent material provided by the present invention is a three-dimensional fluorescent nanofilm, which has larger surface area, higher fluorescent signal, and better dispersion compared with traditional spherical fluorescent labels and stability, thereby greatly improving the detection sensitivity of the test strip, so that the constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus and human adenovirus, with low detection limit and detection Short time, good reproducibility and high accuracy.

Description

A film-like multilayer quantum dot fluorescent material, its preparation method and application in immunochromatography

technical field

The invention belongs to the technical field of fluorescence immunochromatography, and in particular relates to a film-shaped multilayer quantum dot fluorescent material, a preparation method thereof and an application of immunochromatography.

Background technique

Lateral flow immunoassay (LFA) is fast, easy-to-use, portable, low-cost and multi-target detection capability, and is considered to be the most promising point-to-point detection (POCT) technology, widely used in clinical examination, personal health self-check and food safety monitoring.

The characteristics of immunochromatography technology are: (1) It does not require professional operators, and can be widely used in different places such as hospitals, community medical institutions, airports, stations, schools, and families. (2) Test results can be provided directly and quickly (generally <20 minutes) without complicated instruments. (3) LFA technology can analyze multiple targets in one test by setting multiple test lines on one strip, which effectively simplifies the detection process. However, due to the poor performance (such as instability, low signal intensity, poor dispersibility or large size) of traditional spherical nanolabels and respiratory samples (saliva, throat swab, sputum, etc.) in the detection of respiratory viruses, the currently developed The LFA method still exhibits limited quantitative capability, low sensitivity (typically >0.1 ng/mL), and low throughput (≤2) in the detection of respiratory viruses. So far, although many colloidal gold-based LFA kits and more sensitive LFA technologies, including fluorescence-based, chemiluminescence-based, and Raman signal-based strategies, have been proposed for SARS-CoV-2 ) and influenza A virus detection, but these methods still cannot detect multiple respiratory viruses sensitively at the same time. In order to meet the needs of multiplex detection of respiratory viruses, it is necessary to develop a novel LFA system with signal amplification ability, immune binding efficiency and stability.

In recent years, two-dimensional (2D) film-type nanomaterials, such as graphene and its derivatives, molybdenum disulfide, black phosphorus, etc., have been widely used due to their unique structural features (such as large surface area, ultrathin structure, abundant surface active groups, etc.) ) and extraordinary electronic and optical properties, showing great potential in the field of rapid diagnostics. Therefore, the present invention considers to provide a film-like multilayer fluorescent nano-label and introduce it into the immunochromatography system for the detection of respiratory viruses, so as to realize rapid, ultrasensitive and rapid detection of various common respiratory viruses on a test strip. quantitative analysis.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a film-like multilayer quantum dot fluorescent material, its preparation method and application in immunochromatography. The film-like multilayer quantum dot fluorescent material can be used for ultra- Sensitive and simultaneous detection of three target respiratory viruses (SARS-CoV-2, influenza A virus, and human adenovirus), which solves the problem of insufficient performance of existing nanolabel materials for immunochromatographic detection, especially for a variety of Insufficient sensitivity for simultaneous detection of respiratory viruses.

The invention provides a film-shaped multilayer quantum dot fluorescent material, which comprises a film-shaped carrier and a fluorescent layer arranged on the film-shaped carrier; the fluorescent layer includes a cationic polymer self-assembly layer and negatively charged quantum dots which are sequentially stacked. layer, and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the film carrier.

Preferably, the number of cationic polymer self-assembly layers and negatively charged quantum dot layers in the fluorescent layer is greater than or equal to 2, and the cationic polymer self-assembled layers and negatively charged quantum dot layers are arranged alternately.

Preferably, the film-like carrier is a single-layer graphene oxide; the cationic polymer self-assembly layer is a polyethyleneimine self-assembly layer; the negatively charged quantum dot layer is carboxylated cadmium selenide/zinc sulfide core-shell Quantum dot self-assembly layer or 3-mercaptopropionic acid coated cadmium selenide/zinc sulfide quantum dot self-assembly layer.

The present invention also provides a method for preparing a film-like multilayer quantum dot fluorescent material, comprising the following steps:

S1) mixing the film-like carrier and the cationic polymer solution with ultrasound to obtain a solution containing a film-like carrier loaded with a cationic polymer self-assembly layer;

S2) Mix and sonicate the negatively charged quantum dot solution and the solution containing the film-like carrier loaded with cationic polymer self-assembly layer to obtain a film-like multilayer quantum dot fluorescent material.

Preferably, the film carrier is prepared according to the following method:

The single-layer graphene oxide dispersion is ultrasonically treated, centrifuged, and the precipitate is collected and resuspended in water to obtain a film carrier; the thickness of the single-layer graphene oxide in the single-layer graphene oxide dispersion is 1-2 nm; the sheet diameter is larger than 200nm; the power of the ultrasonic treatment is 500-1000W; the rotational speed of the centrifuge is 10000-20000g.

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 500-1000 W; the time of the mixed ultrasound is 10-60 minutes.

Preferably, after mixing the ultrasound in step S2), the precipitate is collected by centrifugation, and then repeats step S1) and step S2) by replacing the membrane carrier; the number of repetitions is 1 to 5 times.

The present invention also provides the application of the film-like multilayer quantum dot fluorescent material in immunochromatography.

The present invention also provides a film-like multi-layer quantum dot nano-label, comprising the film-like multi-layer quantum dot fluorescent material and a detection antibody modified on the surface of the film-like multi-layer quantum dot fluorescent material.

The invention provides a film-shaped multilayer quantum dot fluorescent material, which comprises a film-shaped carrier and a fluorescent layer arranged on the film-shaped carrier; the fluorescent layer includes a cationic polymer self-assembly layer and an electronegative quantum dot layer stacked in sequence , and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the film carrier. Compared with the prior art, the film-like multilayer quantum dot fluorescent material provided by the present invention is a three-dimensional fluorescent nanofilm, which has larger surface area, higher fluorescent signal, and better dispersion compared with traditional spherical fluorescent labels and stability, thereby greatly improving the detection sensitivity of the test strip, so that the constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus and human adenovirus, with low detection limit and high detection efficiency. Short time, good reproducibility and high accuracy.

Description of drawings

Fig. 1 is the schematic diagram of the preparation method and the antibody modification method of the film-like multilayer quantum dot fluorescent material provided by the present invention;

Figure 2 is the various structural characterization data in the preparation process of the film-like multilayer quantum dot fluorescent material in Example 1 of the present invention, where a, b, c, and d are GO, GO@QD, GO@DQD and GO@TQD nano TEM image of the sheet; e, f, g are enlarged TEM images of the local morphology of GO@QD, GO@DQD and GO@TQD nanosheets; h, i, j are GO@QD, GO@DQD and GO Scanning electron microscope image of @TQD; k is the elemental surface scanning analysis result of GO@TQD; m, n, o are the comparison of fluorescence properties of QD, GO, GO@QD, GO@DQD and GO@TQD nanosheets; l is The particle size distribution results of GO, GO@QD, GO@DQD and GO@TQD nanosheets;

Fig. 3 is the stability test result figure of the film-shaped multilayer quantum dot fluorescent material obtained in the embodiment of the present invention 1, wherein a figure is a high salt stability test result figure; b figure is an acid-base stability test result figure; c The picture shows the time stability test results;

Figure 4 is a performance comparison of three film-like multilayer quantum dot materials (GO@QD, GO@DQD and GO@TQD) with different QD shell layers in Example 2 of the present invention on the immunochromatographic system, where a It is a visual fluorescence result diagram of the test strip based on the immunochromatography of three membrane materials to detect different concentrations of the new coronavirus NP protein; Figure b is the detection of different concentrations of the new coronavirus NP protein based on the three membrane materials of immunochromatography Test strip fluorescence analysis results and its linear range diagram;

Fig. 5 is a flow chart of an experiment in which the film-like multilayer quantum dot fluorescent material of Example 3 of the present invention is used as a high-performance film-like fluorescent label in combination with an immunochromatography system to detect novel coronavirus, influenza A virus and influenza B virus;

Figure 6 is the results of the detection of new coronavirus, influenza A virus and influenza B virus by the immunochromatographic system based on the film-like multi-layer quantum dot label in Example 3 of the present invention, wherein a is the fluorescence of the simultaneous detection of the three viruses Photo results; Figure b shows the fluorescence values of the three detection lines recorded by the fluorescence spectrometer; Figure c shows the results of the fluorescence value-virus concentration fitting curves for the detection of three viruses.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The invention provides a film-shaped multilayer quantum dot fluorescent material, which comprises a film-shaped carrier and a fluorescent layer arranged on the film-shaped carrier; the fluorescent layer includes a cationic polymer self-assembly layer and negatively charged quantum dots which are sequentially stacked. layer, and the cationic polymer self-assembly layer in the fluorescent layer is in contact with the film carrier.

In the present invention, the thickness of the film-like carrier is preferably 1-2 nm; the sheet diameter of the film-like carrier is preferably 500-800 nm; the film-like carrier is preferably a single-layer graphene oxide (GO). Two-dimensional single-layer graphene oxide has a large area and good flexibility as a carrier.

The film carrier is provided with a fluorescent layer; the fluorescent layer includes a cationic polymer self-assembly layer and an electronegative quantum dot layer that are stacked in sequence; 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 3-mercaptopropionic acid coated cadmium selenide/zinc sulfide quantum dot ( CdSe@ZnS-MPA QD) self-assembled layer.

In the present invention, the number of layers of the cationic polymer self-assembly layer and the electronegative quantum dot layer in the fluorescent layer is preferably greater than or equal to 2, more preferably 2-5, more preferably 2-4, most preferably 3; And the cationic polymer self-assembly layer and the negatively charged quantum dot layer are arranged alternately. Multi-layer cationic polymer self-assembly layer as an interlayer can greatly improve the dispersion of nanosheets, and effectively adsorb a large number of negatively charged quantum dots; the multi-layered negatively charged quantum dot layer contains thousands of negatively charged quantum dots, these quantum dots generate The fluorescent signal is thousands of times larger than that generated by a single quantum dot and provides a large number of surface sites for antibody conjugation. Compared with commonly used spherical fluorescent materials, the film-shaped multilayer quantum dot fluorescent materials provided by the invention have larger surface area, better fluorescent performance, better dispersion and stability, thereby greatly improving the durability of test strips. Detection sensitivity.

The present invention also provides a method for preparing the above-mentioned film-like multilayer quantum dot fluorescent material, which includes the following steps: S1) mixing the film-like carrier and the cationic polymer solution with ultrasound to obtain a film-like carrier containing a self-assembled layer of the cationic polymer Carrier solution; S2) Mix and sonicate the negatively charged quantum dot solution and the film carrier solution containing the cationic polymer self-assembly layer to obtain a film multilayer quantum dot fluorescent material.

Among them, the present invention has no special limitation on the sources of all raw materials, which can be commercially available.

In the present invention, the film-like carrier is preferably prepared according to the following method: ultrasonically treat the single-layer graphene oxide dispersion, centrifuge, collect and resuspend the precipitate in water to obtain a film-like carrier; the single-layer graphene oxide dispersion The thickness of single-layer graphene oxide in the medium is preferably 1～2nm; The sheet diameter is preferably greater than 200nm; The concentration of the single-layer graphene oxide dispersion is preferably 0.5～3mg/mL, more preferably 1～2mg/mL; The power of the ultrasonic treatment is preferably 500-1000W, more preferably 600-900W, and more preferably 700-800W; the time of the ultrasonic treatment is preferably 5-30min, more preferably 10-20min, and more preferably 15min; The treatment can completely disperse the graphene oxide; the rotational speed of the centrifugation is preferably 10000-20000g, more preferably 11000-16000g, more preferably 11000-14000g, most preferably 12000g; the centrifugation time is preferably 3-10min, More preferably 5～8min, more preferably 6min; Can remove the graphene oxide (<400nm) that sheet diameter is too small in the supernatant by centrifugation, sort out the graphene oxide nanosheet of sheet diameter uniform; Collect precipitation and resuspend in In water, graphene oxide nanosheets with sheet diameters in the range of 500 to 800 nm can be obtained; the volume ratio of the volume of water used for resuspension to the volume ratio of the monolayer graphene oxide dispersion is preferably 1: (0.5 to 2), more preferably 1:(0.8-1.5), more preferably 1:1.

Mix the film carrier with the cationic polymer solution and ultrasonically; the film carrier is mixed with the cationic polymer solution in the form of being suspended in water; the volume ratio of the water suspended with the film carrier to the cationic polymer solution is preferably 1 : (0.5～2), more preferably 1:(0.8～1.5), more preferably 1:1; the concentration of the cationic polymer in the cationic polymer solution is preferably 0.1～5mg/mL, more preferably 0.5～ 3mg/mL, more preferably 1-2mg/mL; the molecular weight of the cationic polymer in the cationic polymer solution is preferably 3000-100000; the cationic polymer is preferably polyethyleneimine, more preferably polyamino branched chain polyethyleneimine; the power of the ultrasonic mixing is preferably 500-1000W, more preferably 600-900W, and more preferably 700-800W; the time of the ultrasonic mixing is preferably 10-60min, more preferably 20-40min , and preferably 30 min; rapid self-assembly of the cationic polymer on the surface of the single-layer graphene oxide by vigorous ultrasound to obtain a solution containing a film-like carrier loaded with a self-assembled layer of the cationic polymer.

The electronegative quantum dot solution is mixed with the solution containing the film-like carrier of the cationic polymer self-assembly layer and ultrasonically mixed; the concentration of the negatively charged quantum dot solution is preferably 1 to 50 ng/mL, more preferably 5 to 50 ng/mL, More preferably 10～40ng/mL, most preferably 20～30ng/mL; The volume ratio of described cationic polymer solution and electronegative quantum dot solution is preferably (300～800):1, more preferably (400～700 ): 1, preferably (500-600): 1, most preferably 500: 1; the power of the ultrasonic mixing is preferably 500-1000W, more preferably 600-900W, and more preferably 700-800W; The time for ultrasonic mixing is preferably 10-60 minutes, more preferably 20-40 minutes, and more preferably 30 minutes; during this process, a large amount of negatively charged quantum dots are adsorbed to the surface of the polyethyleneimine self-assembly layer through electrostatic adsorption.

After ultrasonic mixing, preferably centrifuged, the film-like multilayer quantum dot fluorescent material with a single-layer cationic polymer self-assembly layer and a single-layer negatively charged quantum dot layer can be obtained; the rotational speed of the centrifuge is preferably 4000 ~ 8000g, More preferably, it is 5000-7000g, and even more preferably 6000g; the centrifugation time is preferably 3-10min, more preferably 5-8min, and more preferably 6min.

In the present invention, after the precipitate is collected by centrifugation, step S1) and step S2) are repeated by replacing the film carrier; the number of repetitions is preferably 1 to 5 times, more preferably 1 to 4 times, and more preferably 1 to 3 times , most preferably 2 times. By repeating step S1) and step S2) multiple times, cationic polymer-mediated layer-by-layer self-assembly can be used to continuously coat more layers of negatively charged quantum dot layers.

The invention adopts the ultrasonic-mediated electrostatic adsorption method to prepare film-like multilayer quantum dot fluorescent materials, and through the layer-by-layer self-assembly mediated by electrostatic adsorption, three layers of dense negatively charged quantum dots are sequentially coated on the two-dimensional single-layer graphite oxide. vinyl flake surface. This method converts two-dimensional nanostructures into three-dimensional fluorescent nanofilms, which have larger surface area, higher fluorescent signal, better dispersion and stability compared with traditional spherical fluorescent labels. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limit, short detection time, good reproducibility, and high accuracy.

The present invention also provides an application of the film-like multilayer quantum dot fluorescent material in immunochromatography, preferably as a nano-label of fluorescent immunochromatography.

The present invention also provides a film-like multi-layer quantum dot nano-label, comprising the above-mentioned film-like multi-layer quantum dot fluorescent material and a detection antibody modified on the surface of the film-like multi-layer quantum dot fluorescent material; the detection antibody is preferably a monoclonal Antibody; the detection antibody is preferably one or more antibodies in the new coronavirus, influenza A virus and influenza B virus; Co-modification; the carboxyl activating reagent is a carboxyl activating reagent well known to those skilled in the art, and there is no special limitation. In the present invention, it is preferably EDC/NHS.

The present invention introduces the membrane-like multi-layer quantum dot fluorescent material modified by the detection antibody into the immune chromatography system, which can provide fluorescent performance, stability, dispersion and reaction interface superior to spherical quantum dot microspheres, and can realize the immune chromatography system Highly sensitive, rapid and quantitative detection of medium and medium targets.

The film-shaped multi-layer quantum dot nano-label provided by the present invention can be prepared according to the following method: the surface of the film-shaped multi-layer quantum dot fluorescent material is respectively modified on the target object (new coronavirus, influenza A virus and influenza B virus) The antibody was added dropwise on the glass fiber membrane, freeze-dried, and used as a fluorescent nano-label for immunochromatography. The capture antibodies of the three target viruses were sprayed on the nitrocellulose membrane (NC membrane) to construct three detection lines (T1, T2, T3) to capture the corresponding virus-membrane tag immune complexes. Goat anti-mouse IgG is sprayed onto the quality control line of the NC membrane at the same time to fix the excess-membrane label; the NC membrane, sample pad, absorbent pad, and bottom plate are assembled into an immunochromatographic test strip; the sample to be tested is Mix with the running buffer, add dropwise to the sample pad of the immunochromatography test strip, and read the fluorescent signals at the 3 T lines of the immunochromatography test strip after 15 to 20 minutes to realize the detection of new coronavirus, A Simultaneous, highly sensitive detection of influenza type 2 and influenza B viruses.

Compared with the prior art, the present invention has the advantages of:

(1) In terms of performance, the film-like multilayer quantum dot fluorescent material proposed by the present invention integrates the excellent dispersion of single-layer GO nanosheets, the flexible structure and the strong fluorescence emission ability of the multilayer quantum dot shell, and the overall photostability The property, fluorescence intensity, dispersibility and stability are far superior to other fluorescent microsphere materials, which can significantly improve the sensitivity of immunochromatographic detection;

(2) From a structural point of view, the film-like multilayer quantum dot fluorescent material proposed by the present invention is a typical film-like multilayer nanostructure, which has a larger relative surface area, a larger reaction interface and lighter The weight can easily flow on the chromatography test strip, which is very suitable for the construction of multi-channel immunochromatography system;

(3) The present invention proposes the use of ultrasonic-mediated electrostatic adsorption method to realize the layer-by-layer self-assembly of QD shells on the surface of GO nanosheets. The method is simple, high in reliability, and can realize large-scale stable production;

(4) The layer-by-layer self-assembly method mediated by electrostatic adsorption proposed by the present invention to prepare film-like multi-layer quantum dot materials is a preparation method of a film-like multi-layer structure. By controlling the number of reactions, the continuous QD shell on the GO surface can be realized. Coating, the number of layers of the QD shell can be continuously superimposed, and then continue to increase the quantum dot loading of a single structure;

(5) The carboxyl groups on the surface of the film-like multilayer quantum dot fluorescent material can be used to directly modify biorecognition molecules such as antibodies, aptamers, antibiotics, and polypeptides, so as to facilitate the realization of biological functionalization;

(6) The film-like multilayer quantum dot fluorescent material proposed by the present invention has broad application prospects, including applications in many fields such as on-site rapid detection, biosensing, in vivo imaging, clinical testing, and food analysis;

(7) The film-shaped multilayer quantum dot fluorescent material proposed by the present invention is used as a high-performance film-shaped fluorescent label for immunochromatographic detection, which can provide stronger and stable fluorescent signals, better stability and dispersion, and more Therefore, it can effectively improve the detection performance and detection throughput of fluorescence immunochromatography.

In summary, the electrostatic adsorption-mediated layer-by-layer self-assembly method of the present application to prepare film-like multilayer quantum dot labels has the characteristics of novel structure, good performance, and simple and efficient method. Compared with spherical fluorescent materials, the prepared film-like multilayer quantum dot fluorescent materials have higher quantum dot loading, larger surface area, better fluorescent performance, better stability and dispersibility, especially in the field of on-site rapid detection. It has broad application prospects in the detection of high-sensitivity fluorescence immunochromatography.

In order to further illustrate the present invention, a film-like multilayer quantum dot fluorescent material provided by the present invention, its preparation method and immunochromatographic application are described in detail below in conjunction with the examples.

The reagents used in the following examples are all commercially available; the NP protein in the examples was purchased from Yiqiao Shenzhou, Catalog #40588-V08B; the influenza A virus antibody and the influenza B virus antibody were purchased from Zhongmeixin New Biotechnology Co., Ltd. Science and Technology Co., Ltd., wherein, stream A: Catalog#FluA-001; FluA-002; stream B: Catalog#FluB-001; FluB-002.

Example 1

The film-like multi-layer quantum dot fluorescent material prepared by the present invention is mainly prepared through three steps, firstly, the centrifugal separation of GO nanosheets, and secondly, the layer-by-layer self-assembly of electrostatic adsorption is prepared in the form of one layer of PEI and one layer of QD. GO@QD, GO@DQD and GO@TQD, and finally use the carboxyl groups on the surface of the film-like multilayer quantum dot material to couple the antibody on the surface of GO@TQD through amide bonds.

A preparation method and antibody coupling method of the above-mentioned film-like multilayer quantum dot material of the present embodiment, as shown in Figure 1, comprises the following steps:

(1) Preparation of GO nanosheets:

10 mL of an aqueous solution of GO nanosheets (concentration 1 mg/mL) was sonicated for 15 minutes, and the precipitate was collected by centrifugation at 12,000 g for 6 minutes and resuspended in 10 mL of deionized water to obtain an aqueous solution of GO nanosheets with a particle size in the range of 500-800 nm for use.

(2) Preparation of GO@QD nanosheets:

Under ultrasonic treatment, after mixing 10 mL of GO nanosheets with 10 mL of PEI solution with a concentration of 1 mg/mL, the 800 W vigorous ultrasonic reaction for half an hour allowed PEI to rapidly self-assemble on the GO surface. Then 20 μL of carboxylated QDs (20 ng/mL of CdSe@ZnS-COOH QD solution) was added, and vigorous sonication was continued for half an hour. During this process, the carboxylated QDs were largely adsorbed to the surface of GO@PEI by electrostatic adsorption to form GO@QD nanosheets; GO@QDs were collected by centrifugation at 6000g for 6 minutes and dispersed into 10 mL deionized water for use.

(3) Preparation of GO@DQD nanosheets:

After GO@QD nanosheets were mixed with 10 mL of PEI solution with a concentration of 1 mg/mL, the PEI was rapidly self-assembled on the surface of GO@QD by 800 W vigorous ultrasonic reaction for half an hour. Then 20 μL of carboxylated QDs (20 ng/mL of CdSe@ZnS-COOH QD solution) was added, and vigorous sonication was continued for half an hour. During this process, the carboxylated QDs were massively adsorbed to the surface of GO@QD@PEI through electrostatic adsorption to form GO@DQD nanosheets; GO@DQDs were collected by centrifugation at 6000 g for 6 min and dispersed into 10 mL of deionized water for later use.

(4) Preparation of GO@TQD nanosheets:

After mixing the prepared GO@DQD nanosheets with 10 mL of PEI solution with a concentration of 1 mg/mL, 800 W vigorous ultrasonic reaction for half an hour made PEI self-assemble rapidly on the surface of GO@DQD. Then 20 μL of carboxylated QDs (20 ng/mL of CdSe@ZnS-COOH QD solution) was added, and vigorous sonication was continued for half an hour. During this process, the carboxylated QDs were largely adsorbed to the surface of GO@DQD@PEI through electrostatic adsorption to form GO@TQD nanosheets; GO@TQD was collected by centrifugation at 6000 g for 6 min, and dispersed into 10 mL deionized water for later use.

(5) Preparation of antibody-modified GO@TQD fluorescent labels:

First, 1 mL of GO@TQD was centrifuged and resuspended in 500 μl of 1 mL 2-(N-morpholine)ethanesulfonic acid buffer (0.1 M, pH 5.5). Then add 10 μL N-hydroxysuccinimide solution and 5 μL carbodiimide solution, and incubate for 15 minutes for activation. The activated GO@TQDs were collected from the solution by centrifugation and resuspended in 200 μL of PBS (0.01 M, pH 7.4). Add 10 μg of the new coronavirus NP antibody, influenza A virus antibody and influenza B virus antibody respectively, and continue to incubate and shake for 2 hours. Subsequently, 100 μL of 10% BSA (w/v) was added to block for 1 hour. Finally, the three kinds of antibody-modified GO@TQD were resuspended in the preservation solution by centrifugation, and dropped onto the glass fiber membrane to freeze-dry for subsequent assembly of immunochromatographic test strips.

Figure 2 shows the GO nanosheets made in step (1) of this example, the GO@QD nanosheets made in step (2), the GO@DQD nanosheets made in step (3), and the GO@DQD nanosheets made in step (4). High-resolution transmission electron microscopy (HRTEM) and scanning electron microscope results of GO@TQD nanosheets; element surface scanning analysis results of prepared GO@TQD nanosheets; comparison of fluorescence properties between prepared film-like multilayer quantum dots and ordinary quantum dots Results and particle size distribution results.

The stability characterization results of the film-like multilayer quantum dot fluorescent material prepared in this example are shown in FIG. 3 . The film-like multilayer quantum dot fluorescent material proposed by the present invention exhibits excellent salt stability, acid-base stability and long-term stability. As shown in Figure 3a, the fluorescence signal of the prepared film-like GO@TQD remained stable in high-salt solution (0–1000 mM NaCl) (24 h at room temperature). As shown in Figure 3b, the fluorescence signal of film-like GO@TQD at 618 nm wavelength remains stable in the environment of pH 4-14 (the concentration in water is 1 mg/mL, the aqueous solutions with different pH values are provided by HCl or NaOH, and left at room temperature for 24 hours ); as shown in Figure 3c, the fluorescence signal of film-like GO@TQD at 618 nm wavelength remained stable within 60 days (stored in dark conditions) at room temperature.

Example 2

After the surface of the membrane-like multilayer quantum dot fluorescent material proposed by the invention is modified with antibodies, it can be used for immunochromatographic detection. QDs loaded with more layers can effectively improve the detection performance of film-like multilayer quantum dots on immunochromatography. The performance of the membrane-like multilayer quantum dot labels (GO@QD, GO@DQD and GO@TQD) loaded with 1 to 3 layers for immunochromatography is shown in Figure 4. Ordinary QD (i.e. CdSe@ZnS-COOH) and GO@QD, GO@DQD, GO@TQD are used as fluorescent labels for immunochromatographic detection of the NP protein of the new coronavirus. The visualized fluorescent signals are 0.5, 0.5, 0.05, 0.01ng/ mL. The fluorescence intensities of all tested bands were quantified using a commercial fluorescence analyzer, and these values were then used to draw calibration curves for the 4 detection platforms (Fig. 4b). The calculated detection limits of test strips based on ordinary QD and GO@QD, GO@DQD, and GO@TQD were 111, 52, 16, and 8 pg/mL, respectively. This result confirmed that the use of GO@TQD with multiple QD layers as fluorescent labels can effectively improve the detection sensitivity of LFA.

Example 3

The film-shaped multilayer quantum dot fluorescent material proposed by the present invention can be used as a high-performance film-shaped fluorescent label for a multi-channel immune chromatography system after the surface of the target respiratory virus antibody is modified. In this embodiment, antibodies to the NP protein of the new coronavirus, antibodies to influenza A virus and antibodies to influenza B virus were used.

The modified membrane-like GO@TQD label was used as the label of the immunochromatography system to detect the mixed samples containing different concentrations of the three target viruses. Fig. 5 is an experimental flow chart for the rapid detection of three target respiratory viruses by using the film-like GO@TQD label shown in this example and the immunochromatography system. Fig. 6 is the test and analysis results of three target respiratory viruses detected by the three-channel immunochromatography system based on the film-like multilayer quantum dot fluorescent material. According to the calculation of the fluorescent signal of the test strip and the corresponding fitting curve results, the detection limits of the novel coronavirus NP protein, influenza A virus and influenza B virus can reach 8pg by immunochromatography based on the membrane-like GO@TQD label. /mL, 471copies/mL and 488copies/mL.

The above description is only to illustrate the technical concept and characteristics of the present invention, and its purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. Any modifications, equivalent replacements, improvements, etc., should be included in the within 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.

Images