Multilayer MXene aptamer sensor, paper-based analysis device as well as preparation method and application of paper-based analysis device

Abstract

The invention belongs to the technical field of fluorescent aptamer sensors and simple low-cost detection devices thereof, and relates to a multi-layer MXene aptamer sensor, a paper-based analysis device and a preparation method and application of the paper-based analysis device. The multi-layer MXene aptamer sensor comprises a multi-layer Nb2C MXene sensor and a fluorescent aptamer probe, and the multi-layer Nb2C MXene sensor comprises a multi-layer Nb2C MXene sensor and a fluorescent aptamer probe; the fluorescent aptamer probe is a carbon quantum dot coupled with a target protein nucleic acid aptamer. The invention provides a convenient and low-cost instant detection means, and meets the rapid detection requirements of novel coronavirus and influenza virus antigens. The technology belongs to a novel fluorescence sensing technology combining carbon quantum dots, a multilayer MXene nano material, aptamer sensing and paper-based analysis, and is expected to provide a powerful universal tool for the fields of disease prevention and control, environment monitoring and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent aptamer sensors and simple and low-cost detection devices thereof, and specifically relates to a multilayer MXene aptamer sensor, a paper-based analysis device loaded with the multilayer MXene aptamer sensor, and their preparation methods and applications. Background Art

[0002] On-site instant detection of biomolecules has problems such as high cost, complex operation, and insufficient sensitivity. Signal-enhanced fluorescence sensors based on nanoquenchers provide great opportunities for efficient and convenient on-site rapid detection. These nanoquenchers, such as metal-organic frameworks, graphene oxide, cobalt oxide (CoOOH), etc., can adsorb fluorescently modified aptamer probes and quench their fluorescence, while the presence of analytes causes the probes to be released, resulting in fluorescence recovery. This sensing mode does not require other signal amplification steps, such as nucleic acid amplification, but mainly relies on the interaction between the nanoquencher and the nucleic acid component of the aptamer to work. When this method is applied to the solid-phase detection mode, the fixed amount of nanoquencher is limited, and the amount of released fluorescent probe is low, which is easily insufficient to be read by traditional detectors. Therefore, the choice of nanoquencher is crucial. MXenes are emerging transition metal carbides, nitrides or carbonitrides with two-dimensional nanolayered structures. They have good conductivity, which is conducive to photoinduced electron transfer, as well as the advantages of large surface area and numerous mass transfer channels, making them good candidates for the manufacture of fluorescent biosensors. However, the research on fluorescent aptamer sensors based on MXene nanomaterials is still in its infancy, solid-phase fluorescence detection systems are rarely reported, and only traditional organic dye-labeled aptamer probes are used.

[0003] Considering the attractive idea of ​​loading multi-layer two-dimensional MXene onto a paper-based analytical device, the present invention introduces nano-modification engineering to obtain highly stable fluorescent aptamer probes. Traditional organic fluorescent dyes are often limited in biosensing and paper-based detection applications due to their small Stokes shift and easy photobleaching. Carbon quantum dots, as a new type of zero-dimensional nanomaterial, have attracted widespread attention due to their excellent photoluminescence performance, excellent photostability, simple synthesis, good water solubility and high biocompatibility, and have become a promising candidate material in the field of fluorescent labeling and biosensing. In particular, for the nano-modification engineering of aptamers, their particle size, which is usually less than 10nm, and their zero-dimensional characteristics enable carbon quantum dot-labeled aptamer probes to better maintain their original specific recognition function and free dispersion state compared to other larger fluorescent nanoparticles. Compared with traditional quantum dots containing heavy metal elements such as lead and cadmium, carbon quantum dots are mainly composed of carbon, which is safer and more environmentally friendly. Therefore, it is expected to combine MXene materials with recognition probes labeled with carbon quantum dots to construct a robust and reliable fluorescent sensor. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a technical method for introducing multilayer MXene nanoquenchers and stable luminescent carbon quantum dots into the construction of a paper-based fluorescent aptamer sensor, which can be used for on-site rapid detection of biological large novel coronavirus S protein and influenza virus HA protein.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a multilayer MXene aptamer sensor, which comprises a multilayer Nb2C MXene sensor and a fluorescent aptamer probe; the fluorescent aptamer probe is a carbon quantum dot coupled with a target protein nucleic acid aptamer.

[0006] According to the present invention, the multilayer Nb2C MXene sensor has an accordion-like microstructure formed by loosely stacked nanosheets under a scanning electron microscope, has a high specific surface area, and has high ultraviolet light absorption in the wavelength range of 200-800nm.

[0007] According to a preferred embodiment of the present invention, the multilayer Nb2C MXene sensor is a niobium carbide MXene two-dimensional material (Nb2CMXene) obtained by etching niobium aluminum carbide (Nb2AlC) powder with a hydrofluoric acid solution to remove the aluminum layer in the niobium aluminum carbide.

[0008] The concentration of the hydrofluoric acid solution used is preferably 35-45%; the etching conditions are preferably sealed and stirred in a water bath at 50-70° C. for 18-32 hours; and the ultrasonic treatment time is preferably 10-15 hours.

[0009] According to a specific embodiment of the present invention, niobium aluminum carbide powder (Nb2AlC) powder (1g) is slowly added to 40mL HF (40%) solution in batches within 10min, and then centrifuged (10000rpm, 10min) after stirring for 24h in a 60°C water bath and sealed conditions to collect the precipitate. After that, the precipitate is mixed with 10mL HF (40%), and after ultrasonic treatment for 13h, it is centrifuged again (10000rpm, 10min), the precipitate is collected, and washed with ultrapure water until neutral. Finally, the precipitate is frozen at -80°C for 12h and then freeze-dried to obtain accordion-type multilayer Nb2C MXene, which can be stored for a long time in a nitrogen environment at 4°C for standby use.

[0010] The present invention uses carbon quantum dots as the luminescent component of the fluorescent aptamer probe, and the carbon quantum dots are preferably green light quantum dots G-CDs and / or blue light carbon quantum dots B-CDs.

[0011] The above carbon quantum dots are commercially available and can also be prepared.

[0012] According to one embodiment of the present invention, a narrow emission peak width, high luminescence blue light carbon quantum dot B-CDs is prepared by direct hydrothermal synthesis, and the specific method is as follows: 0.11g L-glutamic acid and 0.16g o-phenylenediamine are weighed respectively, and mixed with 25mL formamide, and the mixed suspension is transferred to a polytetrafluoroethylene-lined high-pressure reactor, and reacted in an electric blast drying oven for 12h (200°C). After the reaction is completed, when cooled to room temperature, the solution in the reactor is transferred to a centrifuge tube for centrifugation (15000rpm, 10min), the supernatant is filtered with a 0.22μm filter membrane, and dialyzed with a dialysis bag of MW=1000Da for 24h and then freeze-dried, and the obtained carbon quantum dot powder is stored in the dark at 4°C. The obtained carbon quantum dots have uniform particle size and well-resolved lattice fringes, and the preferred particle size distribution is 1.8-2.8nm.

[0013] The target protein nucleic acid aptamer is determined according to the target protein to be tested. In one embodiment of the present invention, the target protein is the new coronavirus S protein (S-RBD), and the target protein nucleic acid aptamer is a specific nucleic acid aptamer of the new coronavirus S protein. In another embodiment of the present invention, the target protein is the influenza virus HA protein, and the target protein nucleic acid aptamer is a specific nucleic acid aptamer of the influenza virus HA protein. The above-mentioned specific nucleic acid aptamer can be an aptamer of a reported known sequence, or a newly designed aptamer, as long as the aptamer has specific binding ability with the target protein.

[0014] The carbon quantum dots and the target protein nucleic acid aptamer can be connected by covalent bonds. In a preferred embodiment of the present invention, the carboxyl carbon quantum dots are group activated by EDC / NHS, and the activated carboxyl carbon quantum dots react with the amino-modified nucleic acid aptamer to obtain a fluorescent aptamer probe.

[0015] In the embodiments of the present invention, the novel coronavirus S protein aptamer and the influenza virus HA protein aptamer are used to obtain the fluorescent aptamer probes G-CDs@anti-SRBD Apt and B-CDs@anti-HA Apt.

[0016] According to a specific embodiment of the present invention, the preparation of the new coronavirus S protein fluorescent probe includes the following steps: First, 5 mg EDC and 5 mg NHS are dispersed in 1 mL 10×PBS buffer (pH 7.4). Then, 100 μL of G-CDs (0.5-4 mg / mL) solution is mixed with 100 μL of EDC / NHS solution and ultrasonicated at room temperature for 30 min. Take 20 μL of the ultrasonic solution and 80 μL of the new coronavirus S protein (S-RBD) aptamer (5 μM), incubate at 37 ° C for 12 h, and filter the incubation solution with an ultrafiltration tube (10 kDa) (10000 rpm, 5 min) to obtain the fluorescent probe G-CDs@anti-SRBD Apt.

[0017] According to a specific embodiment of the present invention, the preparation method of the influenza virus HA protein fluorescent probe comprises the following steps: First, 5 mg EDC and 5 mg NHS are dispersed in 1 mL 10×PBS buffer (pH 7.4). Then, 100 μL of G-CDs (0.5-4 mg / mL) solution is mixed with 100 μL of EDC / NHS solution, and ultrasonic treatment is performed at room temperature for 30 min. 20 μL of the ultrasonic solution is mixed with 80 μL of influenza virus hemagglutinin protein (HA) aptamer (5 μM), incubated at 37°C for 12 h, and the incubation solution is filtered (10000 rpm, 5 min) by ultrafiltration tube (10 kDa) to obtain the fluorescent probe G-CDs@anti-HA Apt.

[0018] A second aspect of the present invention provides a method for preparing the above-mentioned multilayer MXene aptamer sensor, comprising the following steps:

[0019] Step 1: Niobium aluminum carbide powder is mixed with hydrofluoric acid solution, etched, and then ultrasonically treated. The precipitate is then collected, washed, and freeze-dried to obtain multilayer Nb2C MXene nanomaterials.

[0020] Step 2: Activate the carboxyl group of the carboxyl carbon quantum dots by EDC / NHS and couple them with the amino-modified aptamer to prepare the fluorescent aptamer probe.

[0021] The concentration of hydrofluoric acid, etching conditions and ultrasonic treatment conditions are as described above and will not be repeated here.

[0022] The working concentrations of EDC and NHS in the coupling reaction can be 2-3 mg / mL, and the reaction conditions are 37° C. for 10-16 h. The obtained fluorescent aptamer probe can be purified by an ultrafiltration tube (10 kDa).

[0023] The present invention also includes mixing Nb2C MXene and carbon quantum dot fluorescence detection probes, and pre-incubating to form a signal-enhanced MXene / CDs@Apt sensor.

[0024] A third aspect of the present invention provides a paper-based analytical device based on multilayer MXene fluorescent aptamer sensing, on which the above-mentioned multilayer MXene aptamer sensor is loaded.

[0025] According to the present invention, the loading amount of the multilayer MXene aptamer sensor on the paper substrate needs to be controlled to obtain good detection results. Preferably, the loading amount of the multilayer MXene aptamer sensor on the paper substrate is controlled to obtain good detection results. 2 The paper substrate is loaded with 50-70 ng of multilayer Nb2CMXene sensors and 0.6-1.0 pmol of fluorescent aptamer probes.

[0026] A fourth aspect of the present invention provides a method for preparing the paper-based analytical device, comprising the following steps:

[0027] Step 1: Mix niobium aluminum carbide powder with hydrofluoric acid solution, etch, and then ultrasonicate, then collect the precipitate, wash, and freeze-dry to obtain multilayer Nb2C MXene nanomaterials;

[0028] Step 2: Activate the carboxyl group of carboxyl carbon quantum dots by EDC / NHS and couple them with amino-modified aptamers to prepare fluorescent aptamer probes;

[0029] Step 3: Mix and incubate the multilayer Nb2C MXene nanomaterial, the fluorescent aptamer probe, and the buffer to obtain an incubation solution;

[0030] Step 4: Add the incubation solution dropwise onto the paper substrate and dry it in the dark to obtain a paper-based multilayer MXene aptamer sensor.

[0031] The conditions of step 1 and step 2 are as described above and will not be repeated here.

[0032] In step 3, the buffer used is preferably 1×PBS buffer (pH=7.4), and the Nb2C multilayer MXene nanomaterial and the carbon quantum dot aptamer probe are mixed in 1×PBS buffer (pH=7.4), and the mixture is mixed by blowing and pre-incubated for later use.

[0033] The paper substrate used in the present invention can be any conventional paper substrate for detection in the art, such as a mixed fiber filter (MCE).

[0034] According to a specific embodiment of the present invention, a 4 mm biological puncher is used to punch a mixed fiber filter (MCE) to obtain a small round paper with a diameter of 4 mm as a paper base. The incubation solution is dripped onto the paper base, and the amount of incubation solution dripped onto a single small round paper is 5 μL. After drying in the dark for at least 5 minutes, a paper-based aptamer sensor is obtained.

[0035] A fifth aspect of the present invention provides a method for detecting viral antigens, using the above-mentioned multilayer MXene aptamer sensor for detection, comprising the following steps:

[0036] S1, mixing and incubating the multilayer Nb2C MXene nanomaterial, the fluorescent aptamer probe, and the buffer solution to obtain a detection solution;

[0037] S2, mixing the test solution with the test solution, incubating the mixture, and then detecting the mixture using a fluorescence spectrophotometer;

[0038] S3, substituting the fluorescence intensity value detected in step S2 into the standard working curve to calculate the target substance content in the solution to be tested;

[0039] The standard working curve is a fitting curve based on target standard solutions with different concentration gradients and their corresponding fluorescence intensity values.

[0040] The present invention optimizes various conditions. Preferably, in step S2, the concentration of the multilayer Nb2C MXene nanomaterial in the detection solution is 1-2 mg / mL, the concentration of the fluorescent aptamer probe is 4.5-5.5 μM, the pH of the buffer solution is 7-8, and the mixing and incubation time is more than 30 minutes. Preferably, the mixing and incubation temperature in step S3 is 37°C and the time is 20-30 minutes.

[0041] The test solution and the multilayer MXene aptamer sensor were mixed and incubated in an EP tube and then detected using a fluorescence spectrophotometer.

[0042] According to a specific embodiment of the present invention, the target protein is the new coronavirus S protein, and the detection method includes the following steps: add 50 μL ultrapure water, 10 μL 10×PBS buffer (pH=7.4), 10 μL G-CDs@anti-SRBD Apt solution (20 μmol / L), and 10 μL multilayer Nb2C MXene solution (1.5 mg / mL) to the EP tube, blow and mix, and place on a horizontal shaker to incubate in the dark (37°C) for 25 minutes, then add 20 μL of the test solution containing the new coronavirus S protein, continue to incubate in the dark (37°C) for 25 minutes, and then measure the fluorescence value. (Fluorescence spectrophotometer parameter settings: excitation 470nm, bandwidth 10nm, gain 900V). The fluorescence intensity obtained by detection is substituted into the standard working curve to calculate the content of the new coronavirus S protein in the test solution; the standard working curve is a fitting curve based on the new coronavirus S protein standard solution with different concentration gradients and its corresponding fluorescence intensity.

[0043] According to another specific embodiment of the present invention, the target protein is influenza virus HA protein, and the detection method includes the following steps: add 50 μL ultrapure water, 10 μL 10×PBS buffer (pH=7.4), 10 μL B-CDs@anti-HAApt solution (20 μmol / L), and 10 μL multilayer Nb2CMXene solution (1.5 mg / mL) to the EP tube, blow and mix, and place on a horizontal shaker to incubate in the dark (37°C) for 25 minutes, then add 20 μL of the test solution containing influenza virus HA protein, continue to incubate in the dark (37°C) for 25 minutes, and then measure the fluorescence value. (Fluorescence spectrophotometer parameter settings: excitation 470nm, bandwidth 10nm, gain 900V). The fluorescence intensity obtained by detection is substituted into the standard working curve to calculate the content of influenza virus HA protein in the test solution; the standard working curve is a fitting curve based on influenza virus HA protein standard solutions with different concentration gradients and their corresponding fluorescence intensities.

[0044] The sixth aspect of the present invention provides a method for detecting viral antigens, which uses the above-mentioned paper-based analysis device based on multilayer MXene fluorescent aptamer sensing for detection, comprising the following steps:

[0045] S1. Add the solution to be tested dropwise onto the paper-based analysis device, incubate for a period of time, place in a dark box, take photos and record the fluorescence image, and obtain the RGB value; preferably, the incubation time is 20 to 30 minutes;

[0046] S2, substituting the RGB value into the standard working curve to calculate the target content in the test solution;

[0047] The standard working curve is a fitting curve based on target standard solutions with different concentration gradients and their corresponding RGB values. The method for making the standard working curve is the method described in step S1.

[0048] According to a preferred embodiment of the present invention, in step S1, 5 μL of the solution to be tested is added dropwise to the paper-based analysis device, incubated for 25 min, and then placed in a dark box with a 365 nm excitation UV lamp for observation, and the fluorescence image is photographed and recorded, and the RGB value is obtained using Image J software.

[0049] In one embodiment of the present invention, B-CDs and G-CDs are used to prepare a dual-color fluorescent paper-based detection device to achieve qualitative and quantitative detection of dual targets. The detection method may include the following steps: take 1 EP tube (1.5mL), add 50μL ultrapure water, 10μL 10×PBS buffer (pH=7.4), 10μL B-CDs@Apt solution (20μmol / L) or G-CDs@Apt solution (60μmol / L), 10μL multilayer Nb2C MXene suspension (1.5mg / mL) to the tube, and blow and mix well and incubate for 25min for standby use. Use a 4mm biological puncher to punch the mixed fiber filter membrane (MCE) to obtain a small round paper with a diameter of 4mm. Take 5μL of the incubation solution in the tube and drop it on the MCE small round paper. After drying in the dark for 5min, the test paper to be used is obtained. After adding the target, incubate at room temperature for 25 minutes and then place in a 365nm excitation UV dark box, take a photo to record the fluorescence image of the MCE small circle paper, and use Image J software to quantify the RGB value. Substitute the RGB value obtained by the test into the standard working curve to calculate the target content in the test solution; the standard working curve is a fitting curve based on the target standard solution with different concentration gradients and its corresponding RGB value.

[0050] The multilayer MXene aptamer sensor or the paper-based analytical device of the present invention can be used to prepare a viral antigen detection reagent.

[0051] The present invention provides a sensor based on multilayer MXene nanoquenchers and carbon quantum dot functionalized aptamers and a paper-based fluorescence analysis device, wherein the paper-based analysis device is loaded with multilayer MXene nanoquenchers at low density, and due to its multilayer nanosheet stacking structure, it can carry abundant fluorescent aptamers, providing ample opportunities for analyte-aptamer contact; the numerous channels between the sheet structures are conducive to the passage of the aptamer / analyte complex; the low-density distribution of MXene makes it easy for the released fluorescent probe to fall into the non-quenching area without MXene. The present invention provides a convenient and low-cost instant detection method, which meets the needs of on-site rapid detection of novel coronavirus and influenza virus antigens. For the novel coronavirus, the detection time is less than 30 minutes, and the detection limit is 8pg / test. This technology belongs to a new type of fluorescence sensing technology that combines carbon quantum dots, multilayer MXene nanomaterials, aptamer sensing and paper-based analysis, and is expected to provide a powerful general tool for disease prevention and control, environmental monitoring and other fields.

[0052] In addition, the universal paper-based detection method proposed in the present invention can detect different antigens by replacing the aptamer. It is simple to operate, highly sensitive, and has the potential for parallel detection, which is of great significance in the rapid detection of pathogens.

[0053] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0055] Figure 1 1 is a SEM image of an aluminum niobium carbide precursor (A) and niobium carbide (B) in one embodiment of the present invention.

[0056] Figure 2 The SEM image of a multilayer Nb2C MXene sensor on a paper substrate in one embodiment of the present invention and a comparative experimental diagram with a single-layer Nb2C MXene-loaded test paper.

[0057] Figure 3 This is a schematic diagram of the working principle of the fluorescent paper-based aptamer sensor of the present invention.

[0058] Figure 4 This is the result of condition optimization of the paper-based analysis method for the novel coronavirus S protein in one embodiment of the present invention.

[0059] Figure 5The fluorescence images of the novel coronavirus S protein aptamer modified by different concentrations of G-CDs (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg / mL) before ultrafiltration under ultraviolet light (365 nm) in one embodiment of the present invention (A), the fluorescence images of the novel coronavirus S protein aptamer modified by different concentrations of G-CDs (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg / mL) after ultrafiltration under ultraviolet light (365 nm) (B) and the fluorescence images of the MCE filter membrane loaded with anti-SRBD Apt (5 μM) modified by different concentrations of G-CDs (0.5-4 mg / mL) under ultraviolet light (365 nm) (C).

[0060] Figure 6 This is the test result of detecting the new coronavirus S protein using an in-tube fluorescence detection method in one embodiment of the present invention.

[0061] Figure 7 This is the test result of detecting the new coronavirus S protein using a paper-based analysis method in one embodiment of the present invention.

[0062] Figure 8 This is the test result of detecting influenza virus HA protein using a paper-based analytical method in one embodiment of the present invention. DETAILED DESCRIPTION

[0063] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0064] Example 1

[0065] This example is used to illustrate the preparation and characterization of the fluorescence quenching material and the fluorescence aptamer probe of the present invention. The preparation method is as follows:

[0066] (1) Preparation of B-CDs, synthesized by direct hydrothermal method. The specific method is as follows: weigh 0.11g L-glutamic acid and 0.16g o-phenylenediamine respectively, and mix with 25mL formamide, transfer the mixed solution to a polytetrafluoroethylene-lined high-pressure reactor, and react in an electric blast drying oven for 12h (200℃). After the reaction is completed, when cooled to room temperature, transfer the solution in the reactor to a centrifuge tube for centrifugation (15000rpm, 10min), take the supernatant and filter it with a 0.22μm filter membrane, and dialyze it with a dialysis bag with MW=1000Da for 24h and then freeze-dry it. The obtained carbon quantum dot powder is stored in the dark at 4℃.

[0067] (2) Preparation of niobium carbide MXene two-dimensional material (Nb2C MXene), Nb2C MXene is obtained by removing the aluminum layer of niobium aluminum carbide by hydrofluoric acid etching. The specific method is as follows: Niobium aluminum carbide (Nb2AlC) powder (1g) is slowly added to 40mL HF (40%) solution in batches within 10min, and then stirred in a 60℃ water bath and sealed conditions for 24h and centrifuged (10000rpm, 10min) to collect the precipitate. After that, the precipitate is mixed with 10mL HF (40%) and ultrasonically treated for 13h, and then centrifuged again (10000rpm, 10min), the precipitate is collected, and washed with ultrapure water until neutral. Finally, the precipitate is frozen at -80℃ for 12h and then freeze-dried to obtain accordion-type Nb2C MXene, which can be stored for a long time in a nitrogen environment at 4℃ for use.

[0068] (3) Preparation of the novel coronavirus S protein fluorescent probe. The specific method is as follows: First, 5 mg EDC and 5 mg NHS were dispersed in 1 mL 10× PBS buffer (pH 7.4). Then, 100 μL of G-CDs (Suzhou Xingshuo Nanotechnology Co., Ltd., product number CQDs-572-Water) (0.5-4 mg / mL) solution was mixed with 100 μL of EDC / NHS solution and ultrasonicated at room temperature for 30 minutes. Take 20 μL of the ultrasonic solution and 80 μL of the novel coronavirus S protein aptamer (nucleic acid sequence: 5'-NH2-C6-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGC GTTAATGGACA-3', SEQ ID NO: 1) (5 μM), mix, incubate at 37°C for 12 hours, and filter the incubation solution with an ultrafiltration tube (10 kDa) (10000 rpm, 5 min) to obtain the fluorescent probe G-CDs@anti-SRBD Apt.

[0069] (4) Preparation of influenza virus HA protein fluorescent probe. The specific method is as follows: First, 5 mg EDC and 5 mg NHS were dispersed in 1 mL 10×PBS buffer (pH = 7.4). Then, 100 μL B-CDs (1 mg / mL) solution was mixed with 100 μL EDC / NHS solution and ultrasonicated at room temperature for 30 h. Take 20 μL of the ultrasonicated solution and 80 μL influenza virus HA protein aptamer (nucleic acid sequence: 5'-NH2-C6-TTGGGGTTATTTGGGAGGGCGGGGGTT-3', SEQ ID NO: 2) and mix them. Incubate at 37°C for 12 hours, filter the incubation solution with an ultrafiltration tube (30 kDa) (10000 rpm, 5 min) to obtain the fluorescent probe B-CDs@anti-HA Apt.

[0070] (5) Characterization results

[0071] TEM was used to characterize the morphology, structure and size of B-CDs. The particle size of B-CDs is uniform, spherical and well dispersed. High-resolution transmission electron microscopy (HRTEM) images show that B-CDs have well-resolved lattice fringes with a spacing of 0.21nm, which is consistent with the (100) lattice distance of graphitic carbon. The particle size of B-CDs ranges from 1.8 to 2.8nm and is normally distributed with an average particle size of (2.22±0.24)nm. The chemical composition of B-CDs was studied by Fourier transform infrared spectrometer (FT-IR) and X-ray photoelectron spectroscopy (XPS). Infrared spectra show that B-CDs have abundant polar functional groups, such as COC (1066cm -1 )、CN(1250cm -1 )、C=O(1394cm -1 )、CH(2988cm -1 ) and OH / NH(3675cm -1 ) stretching vibration and NH(892cm -1 ) bending vibration. XPS results show that the three typical peaks of B-CDs at 285eV, 400eV and 531eV correspond to C1s, N1s and O1s, respectively, indicating that it is composed of the above elements.

[0072] The morphology and structure of Nb2AlC and Nb2CMXene were characterized by cold field emission high-resolution scanning electron microscopy (SEM). Figure 1 ). From the SEM image of Nb2AlC, it can be seen that Nb2AlC is an irregular block composed of many dense layers stacked together, and there are also some tiny particles distributed on the surface of these dense layers ( Figure 1 A). From the SEM image of Nb2C MXene, it can be seen that after Nb2AlC is etched with hydrofluoric acid, the F ions in HF cut the weaker Nb-Al bonds in Nb2AlC, resulting in the removal of its Al layer, causing the dense MAX phase to evolve into an accordion-like structure composed of loosely stacked nano-thick sheets, which significantly increases the specific surface area of ​​the material ( Figure 1 In addition, UV-visible spectrophotometer characterization revealed that Nb2C MXene has high absorbance in the 200-800nm ​​wavelength range and is an excellent fluorescence quenching material.

[0073] Example 2

[0074] This example is used to illustrate the optimization of the use conditions of the aptamer sensor.

[0075] In order to achieve the best performance of the constructed aptamer sensor, the B-CDs@anti-HAApt solution prepared in Example 1 and multilayer Nb2C MXene were used to evaluate the relevant experimental parameters, including aptamer concentration, quenching material concentration, fluorescence quenching time, and incubation time, incubation temperature, and buffer pH of the detection system.

[0076] (1) Aptamer concentration

[0077] Based on the optimal concentration of B-CDs fluorescence, different concentrations of aptamers were used for modification to observe the modification effect. When the aptamer concentration reached 5 μM, further increasing the aptamer concentration did not significantly increase the probe fluorescence intensity, so the aptamer concentration in subsequent experiments was 5 μM.

[0078] (2) Concentration of quenching material

[0079] Since the quenching platform plays a vital role in the constructed sensing strategy, the optimal concentration of the fluorescence quenching material Nb2CMXene was experimented. With the increase of Nb2C MXene concentration, the fluorescence intensity value gradually decreased and stabilized at 1.5 mg / mL. This is because as the concentration of Nb2CMXene increases, the aptamer in the solution is gradually adsorbed onto Nb2CMXene through π-π interactions, resulting in gradual quenching of fluorescence. Therefore, 1.5 mg / mL was selected as the optimal concentration for aptamer fluorescence signal quenching and used for further experiments.

[0080] (3) pH of the test system buffer

[0081] The changes in the fluorescence intensity of B-CDs and B-CDs@Apt in different pH buffers (pH = 4-10) were optimized. The fluorescence of B-CDs and B-CDs@Apt was strongest under the condition of pH = 7-8. At the same time, the effect of different pH buffers on the quenching of B-CDs@Apt fluorescence by Nb2C MXene was also investigated. The results showed that the quenching effect of Nb2C MXene was best under the condition of pH = 7-8.

[0082] (4) Fluorescence quenching time

[0083] It can be observed that the degree of fluorescence quenching changes with the extension of time, and the fluorescence intensity does not decrease significantly with the increase of time after 30 minutes. The fluorescent probe in the sensing system has been fully quenched by Nb2C MXene when incubated for up to 30 minutes. Therefore, 30 minutes is selected as the optimal fluorescence quenching time.

[0084] (5) Incubation time and temperature of the detection system

[0085] The effects of incubation temperature and time on the fluorescence recovery of B-CDs@Apt after the addition of the target were studied, and the optimal incubation temperature was determined to be 37°C and the optimal incubation time was 25 min.

[0086] Example 3

[0087] This example is used to illustrate the detection mechanism of the multilayer MXene aptamer sensor and paper-based analytical device of the present invention.

[0088] The B-CDs@anti-HA Apt solution prepared in Example 1 and multilayer Nb2CMXene were mixed according to the preferred conditions of Example 2 to obtain a MXene / B-CDs@Apt sensor. The mixed fiber filter membrane (MCE) was punched using a 4 mm bio-puncher to obtain a small MCE circular paper with a diameter of 4 mm.

[0089] The MXene / B-CDs@Apt sensor was dropped onto the MCE small disc, so that the loading of Nb2C and B-CDs@Apt on a single small disc was 750ng and 10pmol, respectively. The prepared paper-based small disc loaded with MXene / B-CDs@Apt sensor was subjected to SEM test. SEM results ( Figure 2 A) shows that MXene nanoquenchers are distributed at low density on the paper substrate. Due to its multi-layer nanosheet stacking nanostructure, these low-density distributed MXenes can carry a large number of fluorescent aptamers, providing more opportunities for analyte-aptamer contact. The numerous channels between the nanosheets vary in size, with the largest channel spacing exceeding 100nm ( Figure 2 The release of the fluorescent aptamer / analyte complex may be related to the electrostatic repulsion between it and MXene, while the low density distribution of MXene makes the released fluorescent complex easily fall into the MXene-free zone ( Figure 2 D). In contrast, when the same mass concentration of a single layer of Nb2C-MXene nanoquencher (purchased from Xianfeng Nano) (750 ng) and a fluorescent probe B-CDs@anti-HAApt (10 pmol) were loaded on the MCE film, the fluorescence signal was weak regardless of whether the target was added ( Figure 2 After reducing the amount of Nb2C added to the MCE paper (75 ng), many B-CDs@anti-HAApt probes were not effectively adsorbed by the monolayer Nb2C nanoquencher and produced high fluorescence regardless of the presence of the analyte ( Figure 2 F).

[0090] The working principle of the detection using fluorescent paper-based analytical device is as follows Figure 3As shown. The fluorescent aptamer is distributed in the MXene nanoquencher, and the fluorescence is quenched. When the analyte is added, the analyte binds to the fluorescent aptamer and is released from the MXene nanoquencher, and the fluorescence is restored. By measuring the fluorescence intensity, the qualitative or quantitative detection of the analyte can be achieved.

[0091] Example 4

[0092] This example is used to illustrate the preparation and condition optimization of the fluorescent paper-based analysis device of the present invention.

[0093] Preparation of fluorescent paper-based analytical device: Take 1 EP tube (1.5 mL), add ultrapure water, PBS buffer (pH = 7.4), B-CDs@anti-HA Apt solution or G-CDs@anti-SRBD Apt solution, and multilayer Nb2C MXene suspension into the tube, mix well by pipetting, and incubate for later use.

[0094] G-CDs@anti-SRBD Apt was used to optimize the conditions of the paper-based analysis method. The optimization results are as follows: Figure 4 As shown: the G-CDs connection concentration is preferably 2-2.5 mg / mL (A), the Nb2C MXene loading concentration is preferably 1.5 mg / mL (B), the pre-incubation time after the G-CDs@anti-SRBD Apt / Nb2C material is added is preferably 25-30 min (C), and the reaction time after the test sample is added is preferably 25 min (D).

[0095] The fluorescent paper-based analytical device was prepared according to the above preferred conditions: 1 EP tube (1.5 mL) was taken, and 50 μL of ultrapure water, 10 μL of 10× PBS buffer (pH=7.4), and 10 μL of B-CDs@anti-HAApt solution (20 μmol L -1 ) or G-CDs@anti-SRBD Apt solution (60 μmol L -1 ), 10 μL of multilayer Nb2C MXene suspension (1.5 mg / mL), and mix well by blowing and incubate for 25 min. Use a 4 mm biological puncher to punch the mixed fiber filter (MCE) to obtain a small round paper with a diameter of 4 mm. Take 5 μL of the incubation solution in the tube and drop it on the MCE small round paper. After drying in the dark for 5 minutes, the test paper for use is obtained.

[0096] According to the above method, MCE small round paper discs targeting the new coronavirus S protein and MCE small round paper discs targeting the influenza virus HA protein were prepared respectively.

[0097] Example 5

[0098] This example uses the novel coronavirus S protein as an example to illustrate the detection method of the present invention.

[0099] The novel coronavirus S protein solutions were prepared according to various concentration gradients; the fluorescence spectrophotometer method and the MCE paper-based method were used for detection respectively. Figure 5 The fluorescence images of the novel coronavirus S protein aptamer modified by different concentrations of G-CDs (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mg / mL) before ultrafiltration under ultraviolet light (365 nm) (A), the fluorescence images of the novel coronavirus S protein aptamer modified by different concentrations of G-CDs (0.5-4.0 mg / mL) after ultrafiltration under ultraviolet light (365 nm) (B), and the fluorescence images of the MCE filter membrane loaded with anti-SRBD Apt (5 μM) modified by different concentrations of G-CDs (0.5-4 mg / mL) under ultraviolet light (365 nm) (C). It can be seen that the fluorescence intensity on the MCE filter membrane is consistent with the fluorescence intensity change trend of the solution after ultrafiltration, which proves the feasibility of paper-based fluorescence visualization analysis.

[0100] Fluorescence spectrophotometer method: Add the concentration gradient of the new coronavirus S protein standard solution to the fluorescence detection system, and obtain the fluorescence signal result at the corresponding concentration after 25 minutes (fluorescence spectrophotometer parameter settings: excitation 470nm, bandwidth 10nm, gain 900V, Figure 6 A); use the concentration of the new coronavirus S protein standard solution as the horizontal axis and the fluorescence recovery value (F-F0) as the vertical axis to draw a standard curve ( Figure 6 B). The concentration of the novel coronavirus S protein standard is 1-100ng mL -1 When the fluorescence intensity is within the range of 2.5, there is a good linear relationship between the fluorescence intensity and the S protein concentration. The standard curve equation is y = 232.10919 + 23.66935x, R 2 =0.953.

[0101] MCE paper-based method: Add the concentration gradient of the new coronavirus S protein standard solution onto the MCE small round paper, incubate for 25 minutes, put it into a 365nm excitation UV dark box, take pictures to record the fluorescence image of the MCE small round paper, and use Image J software to quantify the RGB value ( Figure 7 A, B), with the concentration of the new coronavirus S protein standard solution as the horizontal axis and the RGB value (G value) as the vertical axis, draw a standard curve ( Figure 7 C). The concentration of the novel coronavirus S protein standard is 10-80ng mL -1When the G value is within the range of 0.04477 / cm3, there is a good linear relationship between the G value and the concentration of the new coronavirus S protein. The standard curve equation is y=126.41211+0.59465x, R 2 =0.973, detection limit is 1.6ng mL -1 , that is 8pg / test.

[0102] Example 6

[0103] This example uses influenza virus HA protein as an example to illustrate the detection method of the present invention.

[0104] Influenza virus HA protein solutions were prepared according to various concentration gradients and tested by MCE paper-based method.

[0105] The concentration gradient of influenza virus HA protein standard solution was added dropwise onto the MCE small round paper disc, and the fluorescence images at the corresponding concentrations were obtained after incubation for 25 min. Figure 8 A); with the concentration of influenza virus HA protein standard solution as the horizontal axis and the RGB value (G value) as the vertical axis, a standard curve was drawn ( Figure 8 B). The concentration of influenza virus HA protein standard is 1-80ng mL -1 When the total fluorescence value is within the range of 1ng / mL, there is a positive correlation between the total fluorescence value and the concentration of the new coronavirus S protein, which is visible to the naked eye under ultraviolet light. -1 The fluorescence intensity of samples above the concentration (5 pg / test) was significantly stronger than that of the negative control.

[0106] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A multilayer MXene aptamer sensor, characterized in that: The multilayer MXene aptamer sensor comprises a multilayer Nb2C MXene sensor and a fluorescent aptamer probe; the fluorescent aptamer probe is a carbon quantum dot coupled with a target protein nucleic acid aptamer.

2. The multilayer MXene aptamer sensor according to claim 1, characterized in that: The multilayer Nb2C MXene sensor has an accordion-like microstructure formed by loosely stacked nanosheets and exhibits ultraviolet light absorption in the wavelength range of 200-800nm.

3. The multilayer MXene aptamer sensor according to claim 2, characterized in that: The multilayer Nb2C MXene sensor is prepared by etching niobium aluminum carbide powder with a hydrofluoric acid solution to remove the aluminum layer in the niobium aluminum carbide.

4. The multilayer MXene aptamer sensor according to claim 1, characterized in that: The carbon quantum dots are green light quantum dots G-CDs and / or blue light carbon quantum dots B-CDs; The target protein nucleic acid aptamer is a specific nucleic acid aptamer for the new coronavirus S protein or the influenza virus HA protein.

5. The method for preparing the multilayer MXene aptamer sensor according to any one of claims 1 to 4, comprising the following steps: Step 1: Mix niobium aluminum carbide powder with hydrofluoric acid solution, etch, and then ultrasonicate, then collect the precipitate, wash, and freeze-dry to obtain multilayer Nb2C MXene nanomaterials; Step 2: Activate the carboxyl group of carboxyl carbon quantum dots by EDC / NHS and couple them with amino-modified aptamers to prepare fluorescent aptamer probes; Preferably, in step 1, the concentration of the hydrofluoric acid solution is 35-45%; the etching conditions are sealed and stirred in a water bath at 50-70°C for 18-32 hours; the ultrasonic treatment time is 10-15 hours; Preferably, in step 2, the working concentrations of EDC and NHS are independently 2-3 mg / mL, and the coupling conditions are reaction at 37° C. for 10-16 hours.

6. A paper-based analytical device based on multilayer MXene fluorescent aptamer sensing, characterized in that: The paper-based analysis device is loaded with a multilayer MXene aptamer sensor according to any one of claims 1 to 4; preferably, each mm 2 The paper substrate is loaded with 50-70 ng of multilayer Nb2C MXene sensors and 0.6-1.0 pmol of fluorescent aptamer probes.

7. The method for preparing the paper-based analytical device according to claim 6, comprising the following steps: Step 1: Mix niobium aluminum carbide powder with hydrofluoric acid solution, etch, and then ultrasonicate, then collect the precipitate, wash, and freeze-dry to obtain multilayer Nb2C MXene nanomaterials; Step 2: Activate the carboxyl group of carboxyl carbon quantum dots by EDC / NHS and couple them with amino-modified aptamers to prepare fluorescent aptamer probes; Step 3: Mix and incubate the multilayer Nb2C MXene nanomaterial, the fluorescent aptamer probe, and the buffer to obtain an incubation solution; Step 4: Add the incubation solution dropwise onto the paper substrate and dry it in the dark to obtain a paper-based multilayer MXene aptamer sensor.

8. A method for detecting viral antigens using the multilayer MXene aptamer sensor according to any one of claims 1 to 5, comprising the following steps: S1. Mix and incubate the multilayer Nb2C MXene nanomaterial, the fluorescent aptamer probe, and the buffer solution to obtain a detection solution; preferably, the concentration of the multilayer Nb2C MXene nanomaterial in the detection solution is 1-2 mg / mL, the concentration of the fluorescent aptamer probe is 4.5-5.5 μM, and the pH of the buffer solution is 7-8; the mixed incubation time is more than 30 min; S2, mixing and incubating the test solution with the detection solution, and then detecting with a fluorescence spectrophotometer; preferably, the temperature of the mixed incubation is 37° C., and the time is 20 to 30 minutes; S3, substituting the fluorescence intensity value detected in step S2 into the standard working curve to calculate the target substance content in the solution to be tested; The standard working curve is a fitting curve based on target standard solutions with different concentration gradients and their corresponding fluorescence intensity values.

9. A method for detecting viral antigens, using the paper-based analytical device based on multilayer MXene fluorescent aptamer sensing as described in claim 6 for detection, comprising the following steps: S1. Add the solution to be tested dropwise onto the paper-based analysis device, incubate for a period of time, place in a dark box, take photos and record the fluorescence image, and obtain the RGB value; preferably, the incubation time is 20 to 30 minutes; S2, substituting the RGB value into the standard working curve to calculate the target content in the test solution; The standard working curve is a fitting curve based on target standard solutions with different concentration gradients and their corresponding RGB values.

10. Use of the multilayer MXene aptamer sensor according to any one of claims 1 to 5 or the paper-based analytical device according to claim 7 in the preparation of a viral antigen detection reagent.

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