Immunochromatography test strip based on nano-robot as well as preparation method and application of immunochromatography test strip

Abstract

The invention discloses an immunochromatography test strip based on a nano-robot as well as a preparation method and application thereof. The immunochromatography test strip comprises a bottom plate, and a sample pad, filter paper, a nitrocellulose membrane and absorbent paper which are sequentially overlapped and adhered to the bottom plate, when in use, an FPSN (at) Ab buffer solution is dropwise added on the surface of the sample pad; the FPSN (at) Ab buffer solution is prepared from Fe3O4 (at) mPDAamp; the preparation method comprises the following steps: preparing an mSiO2 (at) Nanozyme nano composite material and an antibody to be marked; the Fe3O4 (at) is mPDAamp; the mSiO2 (at) Nanozyme nano composite material is nano particles with an asymmetric structure, one end of the asymmetric structure is a core-shell structure, the core is Fe3O4, the shell is mesoporous polydopamine particles, the other end is rod-like mesoporous SiO2, and noble metal nano particles are loaded on the surfaces of Fe3O4 and mesoporous SiO2; according to the invention, the nano-robot is used as the probe to construct the immunochromatography test strip, so that the detection sensitivity and the detection speed of the test strip are improved, the binding efficiency between the probe and the to-be-detected object is optimized, and the problem that in the prior art, accurate detection of disease markers is difficult to realize under the conditions of small sample size and low concentration is solved.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an immunochromatographic test strip based on nanorobots, its preparation method, and its application. Background Technology

[0002] Immunochromatographic test strips, as a rapid and convenient diagnostic tool, have been widely used in clinical medicine, environmental monitoring, and food safety. Their basic principle is to utilize the specific binding between antigens and antibodies, achieving qualitative or quantitative detection of target substances through visualization or signal amplification systems. Traditional immunochromatographic test strips have become a widely adopted detection method due to their ease of operation, lack of specialized equipment, and low cost. However, traditional immunochromatographic test strips often exhibit low sensitivity and slow reaction speed when dealing with low-concentration samples, making them unsuitable for certain high-precision, high-sensitivity detection tasks.

[0003] With the rapid development of nanotechnology, especially the advancements in nanoparticle and nanorobot technologies, new methods and materials have emerged, providing new insights for improving the performance of immunochromatographic test strips. Nanoparticles, due to their large specific surface area and unique physicochemical properties, can significantly enhance signals and improve detection sensitivity. Meanwhile, nanorobots, as nanotechnology products with autonomous movement capabilities, can precisely control their motion in response to external stimuli (such as temperature, light, pH, etc.), further optimizing the capture efficiency and binding kinetics of target substances. Summary of the Invention

[0004] To overcome the limitations of traditional immunochromatographic test strips in detecting low concentrations of target substances, this invention aims to provide an immunochromatographic test strip based on nanorobots, its preparation method, and its application. By using nanorobots as probes to construct the immunochromatographic test strip, not only can the detection sensitivity of the immunochromatographic test strip be improved, but the reaction process can also be accelerated, and the binding efficiency between the probe and the analyte can be optimized. This solves the problem that existing technologies struggle to achieve accurate detection of disease biomarkers when sample volume is small and concentration is low.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] An immunochromatographic test strip based on nanorobots includes a base plate and a sample pad, filter paper, nitrocellulose membrane, and absorbent paper sequentially overlapped and pasted onto the base plate. In use, FPSN@Ab buffer is dropped onto the surface of the sample pad. The FPSN@Ab buffer is prepared from Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material and the antibody to be labeled. The Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material consists of asymmetric nanoparticles. One end of the asymmetric structure is a core-shell structure, with the core being Fe3O4 and the shell being mesoporous polydopamine particles. The other end is rod-shaped mesoporous SiO2, and noble metal nanoparticles are loaded onto the surfaces of the Fe3O4 and mesoporous SiO2.

[0007] The FPSN@Ab buffer is prepared by mixing 1-20 mg of Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material with 5 μg of antibody to be labeled, incubating for 20-120 min, adding blocking agent, continuing incubation at room temperature for 20-120 min, centrifuging to collect the precipitate, and reconstituted.

[0008] The blocking agent is bovine serum albumin with a mass concentration of 3-20%.

[0009] The antibody to be labeled is one of monoclonal antibodies, polyclonal antibodies, nanobodies, or phage expression antibodies.

[0010] The Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material was prepared by adding 10-100 mg of Fe3O4@mPDA&mSiO2Janus nanoparticles to 200-500 μL of a 10 mg / mL noble metal nanoparticle solution, reacting under nitrogen protection for 4-8 h, then washing with ethanol at least 3 times, and drying.

[0011] The noble metal nanoparticles are one of Pt, Pd, and Rh.

[0012] The nitrocellulose membrane has a detection line and a control line arranged sequentially along the chromatography direction. Tumor marker detection antibodies of adjusted concentration are sprayed onto the surface of the nitrocellulose membrane as detection lines; anti-mouse antibodies or anti-rabbit antibodies of adjusted concentration are sprayed onto the surface of the nitrocellulose membrane as control lines; the detection lines and control lines are spaced a certain distance apart, and the spraying volume for both is 0.05–0.85 μL / cm; the nitrocellulose membrane with the detection lines and control lines is dried and then stored at room temperature in a dry environment for later use.

[0013] The detection process of the immunochromatographic test strip is as follows:

[0014] After processing, the test sample is added to the prepared immunochromatographic test strip at a volume of 50-200 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 1-20 min. Quantitative detection is achieved by reading the signal data of the test line and control line on the test strip and calculating the concentration of the test sample using the built-in standard curve. Qualitative judgment is achieved by directly observing whether the test line and control line are dark brown.

[0015] A method for preparing an immunochromatographic test strip based on nanorobots involves sequentially overlapping and pasting a sample pad with FPSN@Ab buffer solution on its surface, filter paper, a nitrocellulose membrane with a detection antibody sprayed with a tumor marker as a detection line and an anti-mouse antibody or anti-rabbit antibody sprayed as a quality control line, and absorbent paper onto a base plate, thus completing the assembly.

[0016] The present invention also provides the application of the above-mentioned nanorobot-based immunochromatographic test strip in immunochromatographic detection.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The immunochromatographic test strip provided by this invention can detect the breast cancer tumor marker CA153 within 10 minutes, which has a relatively fast detection speed.

[0019] 2. The immunochromatographic test strip provided by this invention, based on the driving performance of nanorobots, solves the problems of disordered probe movement, low efficiency of binding with analytes and low sensitivity in rapid detection. The detection limit for CA153 is as low as 0.0125 U / mL, and it has extremely high detection sensitivity.

[0020] 3. The immunochromatographic test strip provided by the present invention has three signal output modes: catalytic, colorimetric, and photothermal. It can perform qualitative detection of CA153 based on colorimetry, and can also determine the concentration of CA153 in blood by detecting the temperature signal on the T line of the test strip through near-infrared light irradiation and temperature sensor detection, thereby realizing quantitative detection of CA153 in serum.

[0021] 4. The immunochromatographic test strips provided by this invention require readily available and inexpensive raw materials, and do not require large and expensive testing instruments; the preparation process is simple, requiring only basic experimental steps such as sonication and centrifugation, without complex experimental operations, and is easy to operate. It has potential applications in food safety, medical testing, and environmental pollutant monitoring.

[0022] In summary, this invention, by constructing immunochromatographic test strips using nanorobots as probes, not only improves the detection sensitivity of the immunochromatographic test strips but also accelerates the reaction process and optimizes the binding efficiency between the probe and the analyte, thus solving the problem of the difficulty in achieving accurate detection of disease biomarkers in the case of small sample volumes and low concentrations in existing technologies. Attached Figure Description

[0023] Figure 1 This is a TEM image of Fe3O4 prepared in this invention.

[0024] Figure 2 This is a TEM image of Fe3O4&mSiO2Janus prepared in this invention.

[0025] Figure 3 This is a TEM image of Fe3O4@mPDA&mSiO2Janus prepared in this invention.

[0026] Figure 4 This is a TEM image of Fe3O4@mPDA&mSiO2Janus@Pt prepared in this invention.

[0027] Figure 5 This is the ultraviolet spectrum of Fe3O4@mPDA&mSiO2Janus@Pt prepared in this invention.

[0028] Figure 6 This is a schematic diagram of the detection method for the immunochromatographic test strips prepared according to the present invention.

[0029] Figure 7 This describes the catalytic activity of Fe3O4@mPDA&mSiO2Janus@Pt prepared in this invention.

[0030] Figure 8 The photothermal properties of Fe3O4@mPDA&mSiO2Janus@Pt prepared by this invention are shown.

[0031] Figure 9 This is the motion trajectory of Fe3O4@mPDA&mSiO2Janus@Pt prepared in this invention.

[0032] Figure 10 This is a graph showing the change in the intensity of the detection line of the immunochromatographic test strip prepared by this invention at different times.

[0033] Figure 11 These are physical images of the immunochromatographic test strips prepared according to the present invention under three detection modes at different CA153 concentrations.

[0034] Figure 12It is the linear equation of the standard curve of the immunochromatographic test strip prepared by this invention.

[0035] Figure 13 This is a specificity test of the immunochromatographic test strip prepared by the present invention.

[0036] Figure 14 This is the detection of real samples using the immunochromatographic test strips prepared according to the present invention. Detailed Implementation

[0037] The technical solution of the present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0038] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0039] An immunochromatographic test strip constructed based on nanorobots includes a base plate and a sample pad, filter paper, nitrocellulose membrane, and absorbent paper sequentially overlapped and adhered to the base plate. In use, FPSN@Ab buffer is dropped onto the surface of the sample pad. The FPSN@Ab buffer is prepared from FPSN asymmetric intelligent micro / nanorobots and the antibody to be labeled. The FPSN asymmetric intelligent micro / nanorobots are Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite materials, abbreviated as "FPSN nanocomposite materials." The FPSN nanocomposite materials are asymmetric nanoparticles with a core-shell structure at one end, the core being Fe3O4 and the shell being mesoporous polydopamine particles (mPDA), and the other end being rod-shaped mesoporous SiO2. Noble metal nanoparticles are loaded on the surfaces of the Fe3O4 and mesoporous SiO2. The antibody to be labeled is one of monoclonal antibody, polyclonal antibody, nanobody, or phage expression antibody. The noble metal nanoparticles are one of platinum (Pt), palladium (Pd), or rhodium (Rh).

[0040] The preparation process of the FPSN@Ab buffer is as follows:

[0041] Mix 1-20 mg of FPSN nanocomposite material with 5 μg of the antibody to be labeled, incubate for 20-120 min, add 3-20% blocking agent, continue incubation at room temperature for 20-120 min, centrifuge and collect the precipitate. The precipitate is reconstituted with 0.01 mol of phosphate-buffered saline (PBS) at pH 7.0 to 1 / 10 of the initial volume of FPSN nanocomposite material to prepare FPSN@Ab buffer.

[0042] In this embodiment, CA153 monoclonal antibody (CA153-mAb) was used as the antibody to be labeled. The FPSN@Ab buffer was prepared by mixing 5 mg of FPSN nanocomposite material with 5 μg of antibody to be labeled, incubating for 1 h, adding 10% bovine serum albumin (BSA), and continuing to incubate at room temperature for 1 h. The precipitate was collected by centrifugation, and the precipitate was reconstituted with 0.01 mol of phosphate-buffered saline (PBS) at pH 7.0 to 1 / 10 of the initial volume of the FPSN nanocomposite material.

[0043] The preparation process of the FPSN nanocomposite material is as follows:

[0044] (1) Synthesis of Fe3O4 nanoparticles;

[0045] 3.2g FeCl3·H2O, 1.3g trisodium citrate and 6.0g sodium acetate trihydrate were dissolved in 100mL ethylene glycol and stirred for 2h. The mixture was then transferred to a 200mL Teflon-lined stainless steel autoclave and reacted at 200℃ for 10h. After cooling to room temperature, the mixture was washed with distilled water and ethanol.

[0046] Depend on Figure 1 It can be seen that the synthesized Fe3O4 nanoparticles have a spherical structure and uniform particle size.

[0047] (2) Synthesize Fe3O4&mSiO2Janus nanoparticles, denoted as FS;

[0048] 75 mg of Fe3O4 nanoparticles and 190 mg of hexadecyltrimethylammonium bromide (CTAB) as a template were dissolved in 40 mL of distilled water. The mixture was sonicated for 30 min and then magnetically stirred at 40 °C for 10 min. 2.0 mL of ammonia and 360 μL of tetraethoxysilane (TEOS) were added, and the reaction was allowed to proceed for 36 h. The reaction product was washed by centrifugation with water and ethanol, and then refluxed at 60 °C for 6 h to remove the CTAB template. The extraction was repeated three times, and the final product was washed with ethanol.

[0049] Depend on Figure 2 It can be seen that the synthesized Fe3O4&mSiO2Janus nanoparticles exhibit an asymmetrical head-tail structure, with the head being spherical Fe3O4 and the tail being rod-shaped mesoporous SiO2.

[0050] (3) Synthesize Fe3O4@mPDA&mSiO2 Janus nanoparticles, denoted as FPS;

[0051] 30.0 mg of Fe3O4 & mSiO2 Janus nanoparticles were dissolved in 250 mL of distilled water and 200 mL of ethanol. After sonication for 10 min, 2.0 g of Pluronic F-127 and 500 mg of dopamine hydrochloride were added and stirred for 30 min. 4 mL of 3,3',5,5'-tetramethylbenzidine (LTMB) was added and stirring was continued for 30 min. 30 mg of tris(hydroxymethyl)aminomethane (Tris) was added and the reaction was carried out for 4 h. Finally, the product was washed with water and ethanol.

[0052] Depend on Figure 3 It can be seen that a layer of polydopamine particles (mPDA) with a mesoporous structure was selectively grown on the surface of Fe3O4 in the synthesized Fe3O4&mSiO2 Janus nanoparticles, thus synthesizing Fe3O4@mPDA&mSiO2 Janus nanoparticles.

[0053] (4) Synthesize Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material, denoted as FPSN;

[0054] 10-100 mg of Fe3O4@mPDA&mSiO2Janus nanoparticles were added to 200-500 μL of a 10 mg / mL noble metal nanoparticle solution and reacted under nitrogen protection for 4-8 h. Then, the mixture was washed with ethanol at least 3 times and finally dried at 60 °C for 3 h to obtain the FPSN nanocomposite material. The noble metal nanoparticles were one of Pt, Pd, and Rh.

[0055] In this embodiment, 50 mg of Fe3O4@mPDA&mSiO2 Janus nanoparticles were added to 400 μL of a 10 mg / mL Pt nanoparticle solution and reacted for 6 h under nitrogen protection. The mixture was then washed three times with ethanol and finally dried at 60 °C for 3 h to obtain the Fe3O4@mPDA&mSiO2@Pt nanocomposite material, denoted as FPSP. Figure 4 It can be seen that Pt particles with a particle size of approximately 6.5 nm are loaded on the surface of the synthesized Fe3O4@mPDA&mSiO2Janus nanoparticles.

[0056] This invention compares the absorption spectra of Fe3O4 nanoparticles, FS, FPS, and FPSP at the same particle concentration, such as... Figure 5As shown, the results indicate that with the gradual modification of mPDA and Pt nanoparticles, the color of the solution gradually deepens and the absorbance gradually increases, indicating that the absorbance of the FPSP prepared in this invention is significantly enhanced under the collective absorption effect of Fe3O4 nanoparticles, mPDA and Pt nanoparticles.

[0057] The nitrocellulose membrane has a detection line and a control line sequentially arranged along the chromatography direction. The detection line is coated with a detection antibody for the tumor marker; the control line is coated with an anti-mouse antibody or an anti-rabbit antibody. The tumor marker detection antibody, at an adjusted concentration, is sprayed onto the surface of the nitrocellulose membrane as the detection line (T line); the anti-mouse antibody, at an adjusted concentration, is sprayed onto the surface of the nitrocellulose membrane as the control line (C line). The detection line and the control line are spaced a certain distance apart, and the spraying volume for both is 0.05–0.85 μL / cm. The nitrocellulose membrane coated with the detection line and control line is dried overnight at 37°C and then stored in a dry environment at room temperature for later use.

[0058] In this embodiment, the tumor marker used is the breast cancer marker CA153. The concentration of CA153-mAb was diluted to 1 mg / mL with 0.01M pH7.5 PBS, and the resulting solution was sprayed onto a nitrocellulose membrane to form a detection line. The concentration of the anti-mouse antibody was diluted to 0.7 mg / mL, and the resulting solution was sprayed onto a nitrocellulose membrane to form a control line. The spray volume of both lines was 0.74 μL / cm. The detection line was spaced 10 mm from the top edge of the nitrocellulose membrane, and the two lines were spaced 5 mm apart. The membrane was dried at 37°C for 12 h and then stored in a desiccator for later use.

[0059] The assembly process of the immunochromatographic test strip based on nanorobots is as follows:

[0060] (1) The sample pad for adding FPSN@Ab buffer should be 1×30cm;

[0061] (2) The filter paper has a size of 0.8×30cm;

[0062] (3) The specifications of the nitrocellulose membrane with the detection line and quality control line sprayed on are 2.5×30cm;

[0063] (4) The absorbent paper has a size of 1.5×30cm;

[0064] (5) The PVC base plate has a size of 5.5×30cm.

[0065] Paste the above materials in sequence according to the positions of each component in the test strip structure diagram. After assembly, cut the strips into 4×55mm test strips to obtain the assembled immunochromatographic test strip plate using Fe3O4@mPDA&mSiO2@Nanozyme as immunomarkers. Place the strips into a plastic card, press them tightly, put them into an aluminum foil bag, add desiccant, seal and store. The shelf life is 12 months at room temperature.

[0066] The detection process of the immunochromatographic test strip is as follows:

[0067] After processing, the test sample is added to the prepared immunochromatographic test strip at a volume of 50-200 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 1-20 min. Quantitative detection is achieved by reading the signal data of the test line and control line on the test strip and calculating the concentration of the test sample using the built-in standard curve. Qualitative judgment is achieved by directly observing whether the test line and control line are dark brown.

[0068] In the presence of CA153, the FPSN@mAb tag captures the antigen under 808 nm near-infrared (NIR) irradiation and is magnetically enriched, then redispersed in PBS for test strip testing. The immune complex then flows capillarily through which the anti-CA153 polyclonal antibody (pAb) is retained at the T line, resulting in color development. In the absence of CA153, the FPSN@mAb tag binds directly to the antibody at the C line, resulting in color development at the C line. After the experiment, the test strip is immersed in H2O2 and TMB solution for FPSN-based enzymatic deposition to amplify the colorimetric signal. After 10 minutes of color development, the appearance of only the C line indicates a negative result, while the simultaneous appearance of the T and C lines indicates a positive result. The local enzymatic deposition of an insoluble brown-blue product at the T line, catalyzed by the FPSN nanomotor, significantly enhances the intensity of the colorimetric signal. Colorimetric, photothermal, and catalytic outputs are read in their respective detection modes.

[0069] In this embodiment, the immunochromatographic test strip prepared above is used to detect CA153 in a real blood sample, such as... Figure 6 As shown, specifically:

[0070] 1. First, mix the prepared FPSP@Ab buffer with the blood sample, then irradiate it under near-infrared light at 808 nm for 5 min. Then, enrich the obtained FPSP@mAb-CA153 complex magnetically for 30 s, and then add it to 100 μL of phosphate buffer for recombination. Drop the 100 μL phosphate buffer-treated test sample mixture onto the sample pad and react for 15 min.

[0071] 2. Insert the immunochromatographic test strip into the detection window of the reader. The strength of the test line and control line signals will be displayed on the screen as numerical values. Based on the standard curve already entered in the instrument, the content of CA153 in the test sample can be calculated, thus realizing the quantitative detection of CA153 in the test sample.

[0072] The standard curves were entered as follows: spiked in the negative matrix, the CA153 concentration in the standard curve was 0-25.6 U / mL, and the linear regression equations were calculated as follows: y = 7681x + 9943 (colorimetric + FPSP nanorobot test strip), y = 8919x + 19706 (catalytic + FPSP nanorobot test strip), y = 19.3x + 31.8 (photothermal + FPSP nanorobot test strip).

[0073] See Figure 7 The signal amplification efficiency of catalytic colorimetric nanoparticles is typically assessed by their peroxidase-like activity; Figure 7 It can be seen that FPSP exhibits excellent peroxidase-like activity, and its absorbance at 652 nm is higher than that of Fe3O4, FS and FPS.

[0074] See Figure 8 The FPSP asymmetric intelligent micro-nano robot prepared by this invention has excellent photothermal conversion efficiency of 38.12%, providing a power source for subsequent photothermal drive.

[0075] See Figure 9 The motion speed of the FPSP asymmetric intelligent micro / nanorobot prepared in this invention can be controlled by adjusting the intensity of the near-infrared laser, and its motion trajectory was recorded at different laser power densities. As the laser power increases, the length of the motion trajectory also increases. Analysis suggests that the enhanced motion capability is due to the temperature increase caused by near-infrared irradiation. This indicates that increasing the near-infrared laser power density significantly improves the motion range of the FPSP asymmetric intelligent micro / nanorobot, enhancing detection performance and facilitating efficient antigen-antibody binding and autonomous capture in complex biological samples.

[0076] See Figure 10 The immunochromatographic test strip constructed based on FPSP asymmetric intelligent micro-nano robots of this invention shows that as the reaction time increases, the signal intensity of the detection line (T line) gradually increases and tends to stabilize at 10 min, indicating that the immunochromatographic test strip can complete the detection within 10 min and has a fast detection speed.

[0077] See Figure 11An immunochromatographic test strip constructed based on FPSP asymmetric intelligent micro / nanorobots showed a strong linear correlation between different CA153 concentrations and measured signal intensities. In the colorimetric-standard curve image, the visually observable limit of detection was 0.1 U / mL. -1 In the catalysis-standard curve chromatogram, the visually observable limit of detection is 0.00313 U / mL. -1 In the photothermal-standard curve image, the detection limit observed by the naked eye is 0.05 U / mL. -1 It can achieve quantitative detection of CA153 in serum under all three signal output modes, and the catalysis-standard curve has the highest sensitivity.

[0078] See Figure 12 In the figure: the blue line represents the catalytic-linear curve, with a maximum linear range of 0.0125 U / mL. -1 -12.8U mL -1 The orange line represents the colorimetric-linear curve, with a linear range of 0.05 μmL. -1 -6.4U mL -1 The red line represents the photothermal-linear curve, with a linear range of 0.1 U / mL. -1 -12.8 UmL -1 The immunochromatographic test strips prepared in this invention exhibit a wide linear range and excellent detection performance in all three signal output modes; especially in the catalytic signal output mode, they not only have high detection sensitivity but also a wide linear range.

[0079] See Figure 13 Specificity is crucial for developing a reliable and robust test strip. Analysis of interfering proteins such as CA199, CA125, AFP, and CEA revealed that the FPSP immunochromatographic test strip accurately identifies only CA-153, highlighting the superior specificity of the immunochromatographic test strip prepared in this invention for detecting CA-153.

[0080] See Figure 14 The diagnostic efficacy of the immunochromatographic test strip prepared in this invention for the breast cancer biomarker CA-153 was evaluated using real serum samples from 27 breast cancer patients and 28 healthy individuals. In the detection of CA153, the results of the immunochromatographic test strip prepared in this invention were consistent with those of the existing chemiluminescent immunoassay (CLIA) analysis used in clinical testing, indicating that the immunochromatographic test strip prepared in this invention has high detection sensitivity and good clinical application prospects.

Claims

1. An immunochromatographic test strip based on nanorobots, comprising a base plate and a sample pad, filter paper, nitrocellulose membrane, and absorbent paper sequentially overlapped and adhered to the base plate, characterized in that: In use, FPSN@Ab buffer is dropped onto the surface of the sample pad; the FPSN@Ab buffer is prepared from Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material and the antibody to be labeled; the Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material is asymmetric nanoparticles, one end of which is a core-shell structure, with Fe3O4 as the core and mesoporous polydopamine particles as the shell, and rod-shaped mesoporous SiO2 as the other end, with noble metal nanoparticles loaded on the surface of Fe3O4 and mesoporous SiO2.

2. The immunochromatographic test strip based on nanorobots according to claim 1, characterized in that: The FPSN@Ab buffer is prepared by mixing 1-20 mg of Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material with 5 μg of antibody to be labeled, incubating for 20-120 min, adding blocking agent, continuing incubation at room temperature for 20-120 min, centrifuging to collect the precipitate, and reconstituted.

3. An immunochromatographic test strip based on nanorobots according to claim 2, characterized in that: The blocking agent is bovine serum albumin with a mass concentration of 3-20%.

4. An immunochromatographic test strip based on nanorobots according to claim 2, characterized in that: The antibody to be labeled is one of monoclonal antibodies, polyclonal antibodies, nanobodies, or phage expression antibodies.

5. An immunochromatographic test strip based on nanorobots according to claim 2, characterized in that, The Fe3O4@mPDA&mSiO2@Nanozyme nanocomposite material was prepared by adding 10-100 mg of Fe3O4@mPDA&mSiO2Janus nanoparticles to 200-500 μL of a 10 mg / mL noble metal nanoparticle solution, reacting under nitrogen protection for 4-8 h, then washing with ethanol at least 3 times, and drying.

6. An immunochromatographic test strip based on nanorobots according to claim 5, characterized in that: The noble metal nanoparticles are one of Pt, Pd, and Rh.

7. An immunochromatographic test strip based on nanorobots according to claim 1, characterized in that, The nitrocellulose membrane has a detection line and a control line arranged sequentially along the chromatography direction. Tumor marker detection antibodies of adjusted concentration are sprayed onto the surface of the nitrocellulose membrane as detection lines; anti-mouse antibodies or anti-rabbit antibodies of adjusted concentration are sprayed onto the surface of the nitrocellulose membrane as control lines; the detection lines and control lines are spaced a certain distance apart, and the spraying volume for both is 0.05–0.85 μL / cm; the nitrocellulose membrane with the detection lines and control lines is dried and then stored at room temperature in a dry environment for later use.

8. The immunochromatographic test strip based on nanorobots according to claim 1, characterized in that, The detection process of the immunochromatographic test strip is as follows: After processing, the test sample is added to the prepared immunochromatographic test strip at a volume of 50-200 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 1-20 min. Quantitative detection is achieved by reading the signal data of the test line and control line on the test strip and calculating the concentration of the test sample using the built-in standard curve. Qualitative judgment is achieved by directly observing whether the test line and control line are dark brown.

9. A method for preparing an immunochromatographic test strip based on nanorobots, characterized in that: The sample pad with FPSN@Ab buffer solution, filter paper, nitrocellulose membrane with tumor marker detection antibody as the detection line and anti-mouse antibody or anti-rabbit antibody as the quality control line, and absorbent paper are sequentially overlapped and pasted onto the base plate to complete the assembly.

10. The application of a nanorobot-based immunochromatographic test strip according to any one of claims 1-7 in immunochromatographic detection.

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