Immunochromatography test strip based on asymmetric near-infrared driven nano-motor as well as preparation method and application of immunochromatography test strip

Abstract

The invention discloses an immunochromatography test strip based on an asymmetric near-infrared driving nano motor and 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 pasted to the bottom plate, and when the immunochromatography test strip is used, a PZSA-Ab buffer solution is dropwise added to the surface of the sample pad; the PZSA (at) Ab buffer solution is prepared from a PS-SiO2 (at) Au nano composite material and an antibody to be marked; the PS-SiO2 (at) Au nano composite material is nanoparticles of an asymmetric snowman-shaped structure, polystyrene nanospheres are arranged at one end of the asymmetric snowman-shaped structure, hemispherical SiO2 nanoparticles are arranged at the other end of the asymmetric snowman-shaped structure, and Au nanoparticles are loaded on the surface of the asymmetric snowman-shaped structure. According to the present invention, the PS-SiO2 (at) Au nano-composite material is adopted as the nano-motor to be combined with the antibody to be labeled, and the near-infrared light is adopted to drive the snowman-shaped nano-motor to directionally move so as to achieve the active capture and enrichment of the tumor marker, such that the detection sensitivity and the detection speed of the immunochromatography test strip can be significantly improved.

Description

Technical Field

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

[0002] With the development of rapid diagnostic technologies, immunochromatographic test strips have been widely used in disease detection, food safety, and biomarker analysis due to their advantages such as ease of operation, rapid response, and no need for specialized instruments. However, traditional immunochromatographic test strips typically rely on capillary action to drive the flow of liquid on the test strip, lacking an external active driving mechanism. This makes them susceptible to the influence of sample viscosity, ambient temperature, and carrier materials, resulting in problems such as low sensitivity, long response time, and poor accuracy in quantitative analysis.

[0003] In recent years, nanomotors, as an emerging intelligent actuation material, have gradually attracted attention in the fields of biosensing and nanomedicine due to their ability to achieve autonomous or externally controlled motion at the micro- and nano-scale. Among them, near-infrared driven nanomotors, with their excellent penetration, biocompatibility, and non-contact actuation characteristics, have shown great potential as active fluid actuators in in vitro diagnostic systems. However, most current nanomotors have symmetrical structures, resulting in low dynamic efficiency and difficulty in precisely controlling their motion behavior in complex biological systems. Near-infrared driven nanomotors, designed based on asymmetric structures, can achieve directional motion, improve fluid propulsion efficiency, and have significant application value on immunochromatographic platforms.

[0004] Chinese invention patent application No. 202511004707.4 discloses an immunochromatographic test strip based on nanorobots, its preparation method, and its application. The strip 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. Pt, Pd, or Rh nanoparticles are loaded on the surfaces of the Fe3O4 and mesoporous SiO2. However, the asymmetric structure of this FPSN exhibits certain fluid resistance, especially limiting its movement efficiency in complex biological fluids, resulting in low detection sensitivity. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides an immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor, its preparation method, and its application. By optimizing the amount of MPS, a snowman-shaped PS-SiO2@Au nanocomposite material is prepared. The PS-SiO2@Au nanocomposite material is used as a nanomotor to bind with the antibody to be labeled. Near-infrared light is used to drive the snowman-shaped nanomotor to perform directional movement, thereby achieving active capture and enrichment of tumor markers. This significantly improves the sensitivity and detection speed of the immunochromatographic test strip, solving the technical problems of difficulty in detecting low-abundance tumor markers and high detection limits in existing immunochromatographic detection methods.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor 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, PZSA@Ab buffer is added to the surface of the sample pad. The PZSA@Ab buffer is prepared from PS-SiO2@Au nanocomposite material and the antibody to be labeled. The PS-SiO2@Au nanocomposite material consists of asymmetric snowman-shaped nanoparticles, with one end of the asymmetric snowman-shaped structure being a polystyrene nanosphere and the other end being a hemispherical SiO2 nanoparticle. Au nanoparticles are loaded onto the surface of the asymmetric snowman-shaped structure.

[0008] The polystyrene nanospheres have a diameter of 400nm-450nm, and the hemispherical SiO2 nanoparticles have a chord length of 220nm-280nm.

[0009] The PZSA@Ab buffer is prepared by mixing 1-20 mg of PS-SiO2@Au nanocomposite material with 5 μg of antibody to be labeled, incubating for 30-150 min, adding blocking agent, continuing incubation at room temperature for 20-120 min, centrifuging to collect the precipitate, and then reconstituted.

[0010] The blocking agent is casein with a mass concentration of 3-20%.

[0011] The antibody to be labeled is one of monoclonal antibodies, polyclonal antibodies, nanobodies, or phage expression antibodies.

[0012] The preparation process of the PS-SiO2@Au nanocomposite material is as follows:

[0013] (1) Synthesis of PS-SiO2 nanoparticles;

[0014] 1.0 g of polystyrene (PS) nanospheres were uniformly dispersed in 20 ml of ultrapure water, and then 8.0 μL of concentrated ammonia was added and stirred for 1 hour. The mixture was then heated to 70 °C and stirred for 1 hour to obtain concentrated ammonia containing PS nanospheres. 20.0 mg of KPS was dissolved in 12 ml of ultrapure water, and then 0.02 g of SDS and 1.0 g of MPS were added sequentially. After ultrasonic treatment, an emulsion was formed. The emulsion was slowly dripped into the concentrated ammonia containing PS nanospheres over 20 minutes at 70 °C and 300 rpm, and then polymerized at 70 °C for 12 hours. Finally, the mixture was separated by centrifugation and washed with ethanol to obtain snowman-shaped PS-SiO2 nanoparticles.

[0015] (2) Synthesis of PS-SiO2@Au nanocomposite materials;

[0016] 1 g of PS-SiO2 nanoparticles were dispersed in 20 mL of ethanol, followed by the addition of 0.01 g of APTES and 1 mL of 28 wt% ammonia solution. The mixture was reacted at 75 °C for 12 hours. After washing, aminated Janus particles were obtained. 1 mg of aminated Janus particles were dissolved in 5 mL of ultrapure water, followed by the addition of 5-20 mg of Au nanoparticles. The mixture was stirred at room temperature for 6-12 hours to obtain the final product.

[0017] 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.

[0018] The detection process of the immunochromatographic test strip is as follows:

[0019] After processing, the test sample is added to the prepared immunochromatographic test strip at a volume of 50-150 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 15 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 brownish-red.

[0020] An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor is prepared by sequentially overlapping and pasting a sample pad with PZSA@Ab buffer solution on its surface, filter paper, a nitrocellulose membrane with a detection antibody sprayed with a tumor marker as the detection line and an anti-mouse antibody or anti-rabbit antibody sprayed as the quality control line, and absorbent paper onto a base plate, thus completing the assembly.

[0021] The present invention also provides the application of the above-mentioned immunochromatographic test strip based on asymmetric near-infrared driven nanomotor in immunochromatographic detection.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The immunochromatographic test strip provided by this invention can detect CEA in the serum of patients with malignant tumors within 7 minutes. It is often used as a tumor marker for clinical detection and disease monitoring, and has a relatively fast detection speed.

[0024] 2. The PS-SiO2@Au nanocomposite material prepared in this invention has an asymmetric snowman-like structure. On the one hand, the asymmetric snowman-like structure has better hydrodynamic performance. Its streamlined shape helps to reduce eddy current resistance generated during movement, significantly reducing the movement resistance in the serum of malignant tumor patients, and helping to improve the propulsion efficiency of nanomotors. On the other hand, the asymmetric snowman-like structure has a more defined direction of movement, which is conducive to achieving rapid movement in complex media, thereby increasing the number of immune complexes and improving detection sensitivity.

[0025] 3. The immunochromatographic test strip provided by the present invention can perform qualitative detection of CEA based on colorimetry and realize quantitative detection of CEA in serum. The detection limit for CEA is as low as 0.00815 ng / mL, and it has extremely high detection sensitivity.

[0026] 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.

[0027] In summary, this invention, by optimizing the amount of MPS, prepares a snowman-shaped PS-SiO2@Au nanocomposite material. This PS-SiO2@Au nanocomposite material is used as a nanomotor to bind with the antibody to be labeled. Near-infrared light is used to drive the snowman-shaped nanomotor to perform directional movement, achieving active capture and enrichment of tumor markers. This significantly improves the sensitivity and detection speed of immunochromatographic test strips, solving the technical problems of difficulty in detecting low-abundance tumor markers and high detection limits in existing immunochromatographic detection methods. Attached Figure Description

[0028] Figure 1 This is a TEM image of the PS nanospheres prepared in Example 1.

[0029] Figure 2 The image shows a TEM image of the PS-SiO2 prepared in Example 1.

[0030] Figure 3 The image shows a TEM image of PS-SiO2@Au prepared in Example 1.

[0031] Figure 4 The image shows a SEM image of PS-SiO2@Au prepared in Example 1.

[0032] Figure 5 The photothermal performance of PS-SiO2@Au prepared in Example 1 is compared with that of PS-SiO2 nanoparticles and PS nanospheres.

[0033] Figure 6 This is a TEM image of the PXSA prepared in Comparative Example 1.

[0034] Figure 7 This is a TEM image of the PDSA prepared in Comparative Example 2.

[0035] Figure 8 The images show the trajectory diagrams of PZSA prepared in Example 1 and the comparative example under near-infrared light irradiation.

[0036] Figure 9 The mean square displacements of the PZSA prepared in Example 1 and the comparative example under the same power of near-infrared light irradiation are shown.

[0037] Figure 10 This is a schematic diagram of the detection method of the immunochromatographic test strip of the present invention.

[0038] Figure 11 The mean square displacement of PZSA prepared in Example 1 under different near-infrared light powers is shown.

[0039] Figure 12 This is a photograph of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1, showing detection at different CEA concentrations.

[0040] Figure 13 It is the linear equation of the standard curve of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1.

[0041] Figure 14 This is a graph showing the change in detection line intensity of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1 at different times.

[0042] Figure 15 This is a specificity test of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1.

[0043] Figure 16 These are the actual sample detection results of the immunochromatographic test strip based on the PZSA nanomotor prepared in Example 1.

[0044] Figure 17 This is a regression analysis of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1 and the detection of CEA by chemiluminescent immunoassay. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings. 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.

[0046] 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.

[0047] An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor 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, PZSA@Ab buffer is added to the surface of the sample pad. The PZSA@Ab buffer is prepared from PS-SiO2@Au nanocomposite material and the antibody to be labeled. The PS-SiO2@Au nanocomposite material consists of asymmetric snowman-shaped nanoparticles, with one end of the asymmetric snowman-shaped structure being a polystyrene nanosphere and the other end being a hemispherical SiO2 nanoparticle. Au nanoparticles are loaded onto the surface of the asymmetric snowman-shaped structure. The antibody to be labeled is one of monoclonal antibody, polyclonal antibody, nanobody, or phage expression antibody. The PZSA nanocomposite material proposed in this invention is a PZSA asymmetric snowman-shaped nanomotor.

[0048] The preparation process of the PZSA@Ab buffer is as follows:

[0049] Mix 1-20 mg of PS-SiO2@Au nanocomposite material with 5 μg of antibody to be labeled, incubate for 30-150 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 PZSA nanocomposite material to prepare PZSA@Ab buffer.

[0050] In Example 1, a carcinoembryonic antigen monoclonal antibody (CEA-mAb) was used as the antibody to be labeled. The PZSA@Ab buffer was prepared by mixing 5 mg of PS-SiO2@Au nanocomposite material with 5 μg of the antibody to be labeled, incubating for 1 h, adding 5% casein, and continuing incubation at room temperature for another 1 h. The precipitate was collected by centrifugation, and the precipitate was reconstituted with 0.01 mol of PBS at pH 7.0 to 1 / 10 of the initial volume of the PZSA nanocomposite material.

[0051] Example 1

[0052] The preparation process of the PZSA nanocomposite material is as follows:

[0053] (1) Synthesis of PS nanospheres;

[0054] 80 mL of styrene solution (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was thoroughly washed with 20 mL of 10% NaOH solution for 12 hours; then washed five times with deionized water to remove the stabilizer hydroquinone; next, 35 mL of the washed styrene was added to a 500 mL round-bottom flask containing 250 mL of deionized water and 0.3 g of PVP. After purging with nitrogen for 30 minutes, the mixture was magnetically stirred and refluxed at 75 °C for 30 minutes. Then, potassium persulfate (KPS) solution was quickly added to the flask, and the mixture was reacted at 75 °C for 24 hours. After cooling to room temperature, the mixture was filtered to obtain monodisperse colloidal polystyrene (PS) nanospheres; the potassium persulfate solution was prepared by dissolving 0.1 g of potassium persulfate (KPS) in 30 mL of deionized water.

[0055] Depend on Figure 1 It can be seen that the synthesized PS nanospheres have a spherical structure and uniform particle size.

[0056] (2) Synthesis of PS-SiO2 nanoparticles;

[0057] 1.0 g of PS nanospheres were dispersed in 20 ml of ultrapure water and magnetically stirred for 2 hours. Then, 8.0 μL of concentrated ammonia was added to the PS solution and magnetically stirred for 1 hour. Subsequently, the mixed solution was heated to 70 °C and magnetically stirred for 1 hour to obtain concentrated ammonia containing PS nanospheres.

[0058] Add 20.0 mg KPS to 12 ml of ultrapure water and treat under sonication for 2 minutes. Then add 0.02 g sodium dodecyl sulfate (SDS) and 1.0 g methacrylatetrimethoxysilane (MPS) in sequence and treat under sonication for 1 minute to form an emulsion.

[0059] The emulsion was slowly dripped into concentrated ammonia water containing PS nanospheres over 20 minutes at 70°C and magnetic stirring speed of 300 rpm, followed by polymerization at 70°C for 12 hours; finally, PS-SiO2 nanoparticles were obtained by centrifugation and washing with ethanol.

[0060] Depend on Figure 2 As can be seen, the PS-SiO2 nanoparticles synthesized in Example 1 exhibit an asymmetric snowman-like structure, with spherical PS nanoparticles on one side and hemispherical SiO2 nanoparticles on the other side. The diameter of the PS nanospheres is larger than the chord length of the SiO2 hemispheres. The diameter of the PS nanospheres is 400nm-450nm, and the chord length of the SiO2 hemispheres is between 220nm-280nm. This size is defined as the median size and denoted as PZS.

[0061] (3) Synthesize PS-SiO2@Au nanocomposite material, denoted as PZSA;

[0062] First, 1 g of PS-SiO2 nanoparticles were dispersed in 20 mL of ethanol; then 0.01 g of triethoxypropylamine (APTES) and 1 mL of ammonia were added, and the reaction was carried out at 75 °C for 12 hours. The reaction product was washed with ethanol and deionized water to obtain amination-modified Janus particles; then 1 mg of amination-modified Janus particles were dissolved in 5 mL of ultrapure water, followed by the addition of 15 mg of Au nanoparticles, and the mixture was stirred at room temperature for 8 hours; the mass concentration of the ammonia was 28 wt%.

[0063] Depend on Figure 3 and Figure 4 It can be seen that Au nanoparticles were loaded on the entire surface of PS-SiO2 nanoparticles, and PS-SiO2@Au nanocomposite materials were successfully synthesized.

[0064] like Figure 5As shown, the prepared PZS nanoparticles rapidly increased in temperature to 48.6℃ within 600s after loading Au nanoparticles, exhibiting significantly better photothermal performance than simple PS nanospheres and PS-SiO2 nanoparticles. This is because Au nanoparticles instantly generate heat upon near-infrared irradiation, causing a rapid increase in the local temperature around them. This creates a temperature gradient in the snowman-shaped region, driving the snowman-shaped nanomotors to move directionally from the PS nanospheres to the SiO2 nanoparticles. This superior photothermal performance provides the power source for subsequent photothermal actuation, driving the labeled antibody bound to the snowman-shaped nanomotors to move over a wider area.

[0065] Comparative Example 1

[0066] The preparation process and raw materials of Comparative Example 1 are the same as those of Example 1, except that in step (2), the amount of MPS used is 0.5g; Figure 6 As shown, in the PS-SiO2@Au nanocomposite material prepared in Comparative Example 1, the diameter of the PS nanospheres is 400nm-450nm, and the chord length of the SiO2 hemispheres is between 50nm-150nm. The size is relatively small, and it is denoted as PXSA.

[0067] Comparative Example 2

[0068] The preparation process and raw materials of Comparative Example 2 are the same as those of Example 1, except that in step (2), the amount of MPS used is 1.5g; Figure 7 As shown, in the PS-SiO2@Au nanocomposite material prepared in Comparative Example 2, the diameter of the PS nanospheres is 400nm-450nm, and the chord length of the SiO2 hemispheres is between 350nm-500nm. The size is relatively large, and it is denoted as PDSA.

[0069] When the amounts of KPS and concentrated ammonia are fixed, i.e. the kinetic rates of the two polymerization reactions are fixed, increasing the amount of MPS is equivalent to increasing the concentration of reactants, promoting the formation of more silica, which in turn significantly increases the size (diameter and width) of SiO2, forming a significant and balanced asymmetric snowman-like structure. The driving force of the photothermal self-propelled motor often comes from the temperature gradient caused by the asymmetric structure and the autothermal force generated therefrom. Therefore, by optimizing the amount of MPS, the best motor motion performance can be obtained.

[0070] like Figure 8 and 9 As shown, at 1W / cm 2Under near-infrared light irradiation with the same power and time, the motion trajectories of four nanomotors—PS@Au, PXSA, PZSA, and PDSA—were recorded, and their mean square displacements were analyzed. Among them, the PZSA nanomotor had a larger motion range, a straighter motion trajectory, and a significantly increased mean square displacement. This indicates that the SiO2 size in the PZSA nanomotor prepared in Example 1 is moderate, forming a significant and balanced asymmetry, which has a significant advantage in motor motion and can better convert random Brownian motion into directional motion.

[0071] 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.

[0072] In Example 1, the tumor marker used was CEA-mAb. The concentration of CEA-mAb was diluted to 1 mg / mL with 0.01 M, pH 7.5 PBS buffer, and the resulting solution was sprayed onto a nitrocellulose membrane to form a detection line. The concentration of anti-mouse antibody was diluted to 0.9 mg / mL with 0.01 M, pH 7.5 PBS buffer, and the resulting solution was sprayed onto a nitrocellulose membrane to form a control line. The spray volume for both lines was 0.75 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 stored in a desiccator for later use.

[0073] The assembly process of the immunochromatographic test strip based on the asymmetric near-infrared driven nanomotor is as follows:

[0074] (1) The size of the sample pad for adding PZSA@Ab buffer is 1×30cm;

[0075] (2) The filter paper has a size of 0.8×30cm;

[0076] (3) The specifications of the nitrocellulose membrane with the detection line and quality control line sprayed on are 2.5×30cm;

[0077] (4) The absorbent paper has a size of 1.5×30cm;

[0078] (5) The PVC base plate has a size of 5.5×30cm.

[0079] Paste the above materials in the order of the components in the test strip structure diagram. After assembly, cut the strips into 4×55mm test strips to obtain the assembled immunochromatographic test strip plate. Place the strips into a plastic card, press them tightly, put them into an aluminum foil bag, add a desiccant, seal and store. The shelf life is 12 months at room temperature.

[0080] The detection process of the immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor is as follows:

[0081] After processing, the test sample is added to the prepared immunochromatographic test strip at a volume of 50-150 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 15 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 brownish-red.

[0082] In the presence of CEA, the PZSA@mAb tag captures the antigen under 808 nm near-infrared (NIR) irradiation and is then used for test strip analysis. The immune complex subsequently flows through a capillary-driven flow past the anti-CEA polyclonal antibody (pAb), which is retained at the T line, resulting in color development. In the absence of CEA, the PZSA@mAb tag binds directly to the antibody at the C line, resulting in color development at the C line. After a 10-minute colorimetric reaction, the appearance of only the C line indicates a negative result, while the simultaneous appearance of both the T and C lines indicates a positive result. Finally, ImageJ software is used for data processing of the test strips.

[0083] In Example 1, the immunochromatographic test strips prepared above were used to detect CEA in blood samples from real malignant tumor patients. Figure 10 As shown, specifically:

[0084] 1. First, mix the prepared PZSA@Ab buffer with the blood sample, then irradiate it under 808nm near-infrared light for 3 min to carry out the active capture reaction. Add the resulting PZSA@mAb-CEA complex droplet onto the sample pad at a volume of 100μL / strip and react for 15 min.

[0085] 2. Place the immunochromatographic test strip in the instrument. The test line and control line will be captured and recorded. Based on the ImageJ data signal processing and the previously established standard curve, the CEA content in the test sample can be calculated, thus realizing the quantitative detection of CEA in the test sample.

[0086] The standard curve was entered: the negative matrix was spiked, and the CEA concentration in the standard curve was 0-10.0 ng / mL. The linear regression equation was calculated as: y = 5105lg(x) + 10964.

[0087] See Figure 11 The PZSA nanomotor prepared in Example 1 possesses tunable photothermal driving capability, and its movement speed can be precisely controlled by adjusting the power of the near-infrared laser. With increasing laser intensity, the mean square displacement of the nanomotor significantly increases, and its trajectory gradually lengthens. Analysis indicates that this enhanced movement capability is mainly attributed to the local temperature increase induced by laser irradiation. This phenomenon demonstrates that enhanced laser power density can effectively expand the movement range of the nanomotor, thereby improving its detection efficiency in complex biological samples and promoting rapid and efficient binding between antigens and antibodies.

[0088] See Figure 12 The immunochromatographic test strip based on PZSA nanomotor prepared in Example 1 showed a strong linear correlation between different CEA concentrations and the measured signal intensity. The detection limit observed visually in the standard curve graph was 0.01 ng / mL.

[0089] See Figure 13 The colorimetric signal output mode of the immunochromatographic test strip based on PZSA nanomotor prepared in Example 1 has a wide linear range and excellent detection performance. The red dots represent the standard curve of the test strip, while the inserted part represents the linear equation of the method. According to the linear equation, the test strip based on PZSA nanomotor prepared in Example 1 has excellent detection performance.

[0090] See Figure 14 The immunochromatographic test strip constructed based on PZSA nanomotors showed that the intensity of the T-line signal gradually increased with the extension of reaction time and tended to stabilize at 7 minutes, indicating that the immunochromatographic test strip of the present invention can complete the detection within 7 minutes.

[0091] See Figure 15 Specificity is a key factor in ensuring the stability and accuracy of the test strip detection system. Analysis of common interfering proteins such as CA153, CA199, CA125, AFP, and PSA showed that the immunochromatographic test strip specifically recognizes CEA protein without being affected by other interfering molecules. This fully demonstrates that the immunochromatographic test strip constructed from the PZSA nanomotor prepared in Example 1 has excellent recognition ability and high specificity in CEA detection.

[0092] See Figure 16To evaluate the diagnostic capability of the PZSA nanomotor-based immunochromatographic test strip prepared in Example 1 for CEA detection, real serum samples from 27 breast cancer patients and 28 healthy individuals were selected for testing. The results showed that the test strip's CEA detection results were highly consistent with those of clinically commonly used chemiluminescent immunoassay (CLIA), demonstrating good detection sensitivity and accuracy, further validating its potential and feasibility in practical clinical applications.

[0093] See Figure 17 The immunochromatographic test strip based on PZSA nanomotors prepared in Example 1 showed a similar slope (0.94) to the commonly used chemiluminescent immunoassay in CEA detection in regression analysis, with a 95% confidence interval ranging from 0.943 to 0.991. This indicates a high degree of consistency between the compared measurements, further demonstrating that the immunochromatographic test strip based on PZSA nanomotors prepared in Example 1 has high accuracy in real samples.

Claims

1. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor, 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, PZSA@Ab buffer is dropped onto the surface of the sample pad; the PZSA@Ab buffer is prepared from PS-SiO2@Au nanocomposite material and antibody to be labeled; the PS-SiO2@Au nanocomposite material is asymmetric snowman-shaped nanoparticles, one end of the asymmetric snowman-shaped structure is a polystyrene nanosphere, and the other end is a hemispherical SiO2 nanoparticle, and Au nanoparticles are loaded on the surface of the asymmetric snowman-shaped structure.

2. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to claim 1, characterized in that: The polystyrene nanospheres have a diameter of 400nm-450nm, and the hemispherical SiO2 nanoparticles have a chord length of 220nm-280nm.

3. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to claim 1, characterized in that: The PZSA@Ab buffer is prepared by mixing 1-20 mg of PS-SiO2@Au nanocomposite material with 5 μg of antibody to be labeled, incubating for 30-150 min, adding blocking agent, continuing incubation at room temperature for 20-120 min, centrifuging to collect the precipitate, and then reconstituted.

4. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to claim 3, characterized in that: The blocking agent is casein with a mass concentration of 3-20%.

5. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to claim 1, characterized in that: The antibody to be labeled is one of monoclonal antibodies, polyclonal antibodies, nanobodies, or phage expression antibodies.

6. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to claim 1, characterized in that, The preparation process of the PS-SiO2@Au nanocomposite material is as follows: (1) Synthesis of PS-SiO2 nanoparticles; 1.0 g of polystyrene (PS) nanospheres were uniformly dispersed in 20 ml of ultrapure water, and then 8.0 μL of concentrated ammonia was added and stirred for 1 hour. The mixture was then heated to 70 °C and stirred for 1 hour to obtain concentrated ammonia containing PS nanospheres. 20.0 mg of KPS was dissolved in 12 ml of ultrapure water, and then 0.02 g of SDS and 1.0 g of MPS were added sequentially. After ultrasonic treatment, an emulsion was formed. The emulsion was slowly dripped into the concentrated ammonia containing PS nanospheres over 20 minutes at 70 °C and 300 rpm, and then polymerized at 70 °C for 12 hours. Finally, the mixture was separated by centrifugation and washed with ethanol to obtain snowman-shaped PS-SiO2 nanoparticles. (2) Synthesis of PS-SiO2@Au nanocomposite materials; 1 g of PS-SiO2 nanoparticles were dispersed in 20 mL of ethanol, followed by the addition of 0.01 g of APTES and 1 mL of 28 wt% ammonia solution. The mixture was reacted at 75 °C for 12 hours. After washing, aminated Janus particles were obtained. 1 mg of aminated Janus particles were dissolved in 5 mL of ultrapure water, followed by the addition of 5-20 mg of Au nanoparticles. The mixture was stirred at room temperature for 6-12 hours to obtain the final product.

7. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor 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. An immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor 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 50-150 μL / strip. The strip is then irradiated with near-infrared light for 1-10 min, and the reaction time is 15 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 brownish-red.

9. A method for preparing an immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor, characterized in that, The sample pad with PZSA@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 an immunochromatographic test strip based on an asymmetric near-infrared driven nanomotor according to any one of claims 1-7 in immunochromatographic detection.

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