A multi-mode portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflower

Abstract

The application relates to a multi-mode portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflower, the application uses a MnO2NFs-labeled detection antibody as a detection probe, and carcinoembryonic antigen and the probe are sequentially added to a paper base which is modified with a capture antibody; due to a specific antigen-antibody reaction, the three form a sandwich complex. At this time, the concentration of the carcinoembryonic antigen is positively correlated with the amount of the MnO2NFs finally fixed to the paper base. The multi-mode portable carcinoembryonic antigen detection sensor can realize detection of carcinoembryonic antigen through three visual signals of color, temperature and distance. The three signals are complementary to each other and are mutually verified, thus providing a new method and thought for development of the sensor. The construction method is simple, the sensitivity is high, the specificity is good, the detection limit is low, and the linear range is wide, and the carcinoembryonic antigen CEA can be sensitively detected, the linear range is 2.5-100 ng / mL, and the detection limits of the three signals are 1.4 ng / mL, 1.5 ng / mL and 2.5 ng / mL respectively.

Description

Technical Field

[0001] This invention relates to a multimode portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflowers, belonging to the field of detection and analysis technology. Background Technology

[0002] Cancer is a chronic disease that threatens public health. Over the past few decades, its incidence has been steadily increasing, becoming the second leading cause of death worldwide. Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker that can indicate the presence of various tumors. While CEA levels are low in the serum of healthy adults, they are significantly elevated in patients with breast cancer, colorectal cancer, and gastric cancer. Therefore, CEA detection is crucial for cancer diagnosis and treatment. Currently, analytical methods for detecting CEA mainly include fluorescence methods, colorimetric methods, and electrochemical methods. However, these methods often require complex and sophisticated equipment and highly skilled technicians, and are typically only suitable for large hospitals and laboratories. Furthermore, the poor stability and high cost of natural enzymes greatly limit their application in practical detection. Therefore, there is a need to develop simple, portable, and user-friendly new detection methods for daily home care.

[0003] Point-of-care testing (POCT) technology is an inexpensive, easy-to-operate, and user-friendly analytical method with broad application prospects in biological detection. Currently, POCT's simple signal output modes mainly include color, temperature, pressure, and distance, which can be measured using portable tools or instruments such as smartphones, thermometers, pressure gauges, and rulers. However, single-mode signal output often leads to false positive or false negative results due to factors such as operating conditions. Therefore, portable sensors with multi-mode signal output have attracted widespread attention.

[0004] In recent years, nanozymes have made groundbreaking progress in cancer treatment, anti-inflammatory and antibacterial applications, and biodetection due to their excellent stability and high catalytic activity. To date, reported nanozymes include carbon nanomaterials, noble metals, and metal oxides. Among them, manganese dioxide (MnO2) is a non-toxic, stable, and low-cost nanomaterial. MnO2 nanoflowers (NFs) are three-dimensional nanomaterials. Compared with nanowires, nanosheets, and nanorods, MnO2NFs have a larger specific surface area and atomic utilization, making them one of the best nanostructures to date. MnO2 can catalyze the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine (TMB) without the need for H2O2, laying the foundation for its application in immunoassay. Furthermore, TMB is oxidized to form oxTMB, which not only involves a color change but also allows for the detection of target analytes through the photothermal effect of oxTMB and other visual signals such as temperature changes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a multimodal portable carcinoembryonic antigen (CEA) detection sensor based on manganese dioxide nanoflowers. This ECA detection sensor enables real-time, multimodal detection of the tumor marker CEA without the need for complex instruments and operating procedures.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a multimodal portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflowers includes the following steps:

[0008] (1) Dissolve KMnO4 in hydrochloric acid solution to obtain KMnO4 solution.

[0009] (2) Add citric acid solution dropwise to the KMnO4 solution prepared in step (1), stir at room temperature, the solution changes from purple to brown and brown particles are produced, centrifuge, wash the brown particles with deionized water until the supernatant becomes colorless and transparent, and vacuum dry the particles to obtain MnO2 NFs.

[0010] (3) Disperse MnO2 NFs in deionized water and sonicate to obtain MnO2 NFs dispersion;

[0011] (4) Add EDC and NHS in MES buffer solution to MnO2 NFs dispersion, use EDC and NHS to activate the carboxyl groups on MnO2 NFs, then wash, redisperse, and obtain activated MnO2 NFs.

[0012] (5) Mix the detection antibody solution corresponding to carcinoembryonic antigen with the activated MnO2 NFs and incubate. Then wash with PBS buffer. After washing, add BSA solution and incubate together to block non-specific adsorption. Disperse in PBS buffer solution to obtain antibody-modified MnO2 NFs probe solution.

[0013] (6) Add a mixed solution of NaIO4 and LiCl to the cellulose chromatography paper every 30 min, repeat the addition step, wash with deionized water to remove unbound NaIO4 and LiCl, then add the capture antibody solution evenly, incubate, remove unreacted capture antibody, add BSA solution to block unbound reaction sites, remove excess BSA, and obtain the paper substrate for immunoassay.

[0014] (7) Add the carcinoembryonic antigen solution to the paper substrate for immunoassay, incubate at room temperature, wash, add the antibody-modified MnO2 NFs probe solution prepared in step (5), incubate at room temperature, and obtain a multimode portable carcinoembryonic antigen detection sensor.

[0015] According to a preferred embodiment of the present invention, in step (1), the concentration of the hydrochloric acid solution is 0.1-0.3M.

[0016] According to a preferred embodiment of the present invention, in step (1), the concentration of the KMnO4 solution is 1-5 mg / mL.

[0017] According to a preferred embodiment of the present invention, in step (2), the concentration of the citric acid solution is 0.05-0.15M.

[0018] According to a preferred embodiment of the present invention, in step (2), the volume ratio of KMnO4 solution to citric acid solution is 35-45:1.

[0019] According to a preferred embodiment of the present invention, in step (2), the stirring time at room temperature is 40-60 min, the centrifugation is performed at a speed of 8000-12000 rpm for 5-20 min, and the vacuum drying temperature is 50-70℃.

[0020] According to a preferred embodiment of the present invention, in step (3), the concentration of the MnO2 NFs dispersion is 0.4-0.6 mg / mL.

[0021] According to a preferred embodiment of the present invention, in step (4), the concentration of EDC in the MES buffer solution of EDC and NHS is 1-5 mg / mL, the concentration of NHS is 3-8 mg / mL, and the pH is 4-6.5.

[0022] According to a preferred embodiment of the present invention, in step (5), the concentration of the detection antibody solution is 90-110 μg / mL.

[0023] According to a preferred embodiment of the present invention, in step (5), the volume ratio of the detection antibody solution to the activated MnO2 NFs is (1-2):(1-2).

[0024] According to a preferred embodiment of the present invention, in step (5), the incubation is performed at 37°C for 1 hour.

[0025] According to a preferred embodiment of the present invention, in step (5), the mass concentration of the BSA solution is 0.4-0.6%, and the volume ratio of activated MnO2NFs to BSA solution is (1-2):(1-2).

[0026] According to a preferred embodiment of the present invention, in step (5), the concentration of the antibody-modified MnO2 NFs probe in the PBS buffer solution is 0.4-0.6 mg / mL.

[0027] According to a preferred embodiment of the present invention, in step (6), the diameter of the cellulose chromatography paper is 5-7 mm.

[0028] According to a preferred embodiment of the present invention, in step (6), the addition of the mixed solution of NaIO4 and LiCl is carried out at a temperature of 55-65°C.

[0029] According to a preferred embodiment of the present invention, in step (6), the concentration of NaIO4 in the mixed solution of NaIO4 and LiCl is 40-60 mM and the concentration of LiCl is 650-750 mM, and the amount added is 10-30 μL.

[0030] According to a preferred embodiment of the present invention, in step (6), the concentration of the capture antibody solution is 30-50 μg / mL.

[0031] According to a preferred embodiment of the present invention, in step (6), the incubation is carried out at 37°C for 0.5-2 hours.

[0032] According to a preferred embodiment of the present invention, in step (6), in order to make the binding more complete, the amount of the capture antibody solution added is 4-10 μL.

[0033] According to a preferred embodiment of the present invention, in step (6), removing unreacted capture antibodies involves washing three times with PBS buffer, and removing excess BSA involves washing three times with PBS buffer.

[0034] According to a preferred embodiment of the present invention, in step (7), the amount of carcinoembryonic antigen solution added is 4-10 μL.

[0035] The detection antibody solution, capture antibody solution, and carcinoembryonic antigen solution of the present invention are all prepared using PBS buffer solution, which is prepared according to existing technology.

[0036] A multimode portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflowers was prepared using the method described above.

[0037] The method for detecting carcinoembryonic antigen using the aforementioned multimodal portable carcinoembryonic antigen detection sensor includes the following steps:

[0038] 1) Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multimode portable CEA detection sensor according to the preparation method of a multimode portable CEA detection sensor. Add 6 μL TMB solution to the sensor and react for 8-12 min. Take a picture in a light box to record the color signal of the paper substrate. As the CEA concentration increases, the color of the paper substrate gradually turns blue. Plot the working curve based on the relationship between the ΔR value of the image and the concentration of the standard antigen solution.

[0039] 2) Construct a multimode portable carcinoembryonic antigen (CEA) detection sensor according to the preparation method of the multimode portable CEA detection sensor, and perform detection according to the method in step 1). Detect the concentration of antigen in the CEA sample based on the ΔR value of the image and the working curve.

[0040] The method for detecting carcinoembryonic antigen using the aforementioned multimodal portable carcinoembryonic antigen detection sensor includes the following steps:

[0041] I. Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multimode portable CEA detection sensor according to the preparation method of the multimode portable CEA detection sensor. Add 6 μL of TMB solution to the sensor and react for 8-12 min. Under 808 nm laser irradiation, use a portable thermal imager to capture a thermal image of the paper substrate and record the temperature signal. Based on the relationship between the temperature signal and the concentration of the standard antigen solution, plot the working curve.

[0042] II. Construct a multimode portable carcinoembryonic antigen (CEA) detection sensor according to the preparation method of the multimode portable CEA detection sensor, and perform detection according to the method in step I. Detect the concentration of antigen in the CEA sample based on the temperature signal and the working curve.

[0043] This invention can also combine temperature-responsive hydrogels with a multimodal portable carcinoembryonic antigen (CEA) detection sensor to achieve distance-based detection of CEA, as detailed below:

[0044] a. Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multimode portable CEA detection sensor according to the preparation method of the multimode portable CEA detection sensor. Add 6 μL of TMB solution to the sensor and react for 8-12 min. Place the reacted paper substrate and gelatin hydrogel block in the circular sample loading area of ​​the paper substrate distance sensor. Immediately after irradiation with an 808 nm laser, measure the distance signal generated by it using vernier calipers. Plot the working curve based on the relationship between the water flow distance signal and the concentration of the standard antigen solution.

[0045] b. Construct a multi-mode portable carcinoembryonic antigen (CEA) detection sensor according to the preparation method of the multi-mode portable CEA detection sensor, and perform detection according to the method in step a. Detect the concentration of antigen in the sample based on the signal of the water flow distance and the working curve.

[0046] According to a preferred embodiment of the present invention, the paper-based distance sensor includes a PMMA substrate with grooves and a pH test paper. The pH test paper is disposed in the grooves of the PMMA substrate and its shape matches the shape of the grooves of the PMMA substrate. The pH test paper includes a circular sample-carrying area and a rectangular straight channel connected to the sample-carrying area.

[0047] According to a preferred embodiment of the present invention, the irradiation time by the 808nm laser is 60-120s.

[0048] According to a preferred embodiment of the present invention, the hydrogel block is prepared by the following method:

[0049] Disperse the gelatin in water, heat and stir until dissolved, and quickly fill it into a plastic syringe while hot. Refrigerate at 4°C for later use. Cut it into uniformly thick hydrogel cylinders and let it stand at room temperature for 20 minutes before use.

[0050] According to a preferred embodiment of the present invention, the concentration of gelatin is 4-6 wt%, and the heating temperature is 30-50°C.

[0051] The detection principle of this invention is as follows:

[0052] This invention uses a detection antibody labeled with MnO2 NFs as a detection probe. Carcinoembryonic antigen (CEA) and the probe are sequentially added to a paper substrate modified with a capture antibody. Due to a specific antigen-antibody reaction, the three form a sandwich complex. The concentration of CEA is positively correlated with the amount of MnO2 NFs ultimately immobilized on the paper substrate. Since MnO2 NFs possess oxidase activity, they can catalyze the generation of oxTMB from TMB. The color and temperature signals generated by oxTMB can be acquired using a smartphone and a thermal imager, respectively. Furthermore, the paper substrate generating oxTMB is placed on a paper-based distance sensor, and a temperature-responsive gelatin hydrogel is placed in the sample area. When irradiated with near-infrared light, the photothermal effect of oxTMB and the gel-sol transition and viscosity change of the temperature-responsive hydrogel cause it to flow along the rectangular channel of the paper-based distance sensor, thereby detecting CEA through the distance signal. The sensor of this invention not only provides a simple, portable, and easy-to-operate analytical method in resource-limited environments but also helps to broaden its application in the detection of various biomarkers.

[0053] Beneficial effects

[0054] 1. The multi-mode portable carcinoembryonic antigen detection sensor of the present invention is simple to construct, has high sensitivity, good specificity, low detection limit, and wide linear range, and can sensitively detect carcinoembryonic antigen CEA with a linear range of 2.5 to 100 ng / mL.

[0055] 2. The multimode portable carcinoembryonic antigen detection sensor of the present invention is constructed based on manganese dioxide nanoflowers. It has the advantages of simple operation, low cost, and user-friendliness, which effectively solves the problems of complexity and high cost of existing detection methods and provides a promising prospect for clinical diagnosis and application.

[0056] 3. The multi-mode portable carcinoembryonic antigen detection sensor of the present invention can detect carcinoembryonic antigen through three visual signals: color, temperature and distance. The detection limits of the three signals are 1.4 ng / mL, 1.5 ng / mL and 2.5 ng / mL, respectively. The three signals complement and corroborate each other, providing new methods and ideas for sensor development. Attached Figure Description

[0057] Figure 1 This is a schematic diagram illustrating the principle of the multi-mode portable carcinoembryonic antigen detection sensor of the present invention for detecting carcinoembryonic antigen.

[0058] Figure 2 The images show the morphology of MnO2 NFs in Example 1: (a) TEM image and (b) SEM image.

[0059] Figure 3 The UV-vis spectra of the TMB, MnO2, and MnO2+TMB solutions in Example 1 are shown.

[0060] Figure 4 This is a TEM image of the probe in Example 2.

[0061] Figure 5 The FT-IR spectra of (i) MnO2 NFs and (ii) probe in Example 2 are shown.

[0062] Figure 6 This is a graph showing the linear relationship between the ΔR value and the logarithm of the carcinoembryonic antigen concentration in Example 3. The inset is an optical photograph of paper substrates treated with different concentrations of carcinoembryonic antigen.

[0063] Figure 7 The following are examples from Example 4: (a) thermal images of paper substrates obtained under different concentrations of carcinoembryonic antigen (CEA); and (b) calibration curves between temperature change and the logarithm of CEA concentration.

[0064] Figure 8 This is a schematic diagram of the paper-based distance sensor device in Example 4.

[0065] Figure 9 The images shown in Example 5 are: (a) photographs of paper-based distance sensors obtained under different concentrations of carcinoembryonic antigen (CEA); and (b) the linear relationship between the flow distance and the logarithm of the CEA concentration.

[0066] Figure 10 To illustrate the results of detecting carcinoembryonic antigen (CEA) in serum in the application experiment, the following images are presented: (a) optical photographs, ΔR values, and (b) thermal images and temperature changes of the paper substrate in response to different concentrations of CEA in the actual sample; (c) photographs and (d) flow distance of the paper-based distance sensor in response to different concentrations of CEA in the actual sample. Detailed Implementation

[0067] The technical solution of the present invention will be further described below with reference to the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0068] Potassium permanganate (KMnO4) and hydrochloric acid (HCl) used in the examples are available from Sinopharm Chemical Reagent Co., Ltd. Citric acid, phosphate-buffered saline (PBS) buffer, and 4-morpholinoethanesulfonic acid (MES) are available from Sigma-Aldrich. Carcinoembryonic antigen (CEA), CEA detection antibodies, and CEA capture antibodies are available from Shanghai Leading Biotechnology Co., Ltd., China. Whatman Grade 1 chromatography paper is available from Cytiva.

[0069] Unless otherwise specified, the drugs and reagents involved in this embodiment are all commercially available products.

[0070] Example 1: Preparation of MnO2 NFs

[0071] 40 mg of KMnO4 was dissolved in 20 mL of 0.1 M hydrochloric acid solution to obtain a uniform purple solution. Then, 0.5 mL of 0.1 M citric acid solution was added dropwise, and the mixture was stirred at room temperature for 30 min. At this point, the solution changed from purple to brown, and brown particles were produced. The product was centrifuged at 10,000 rpm for 10 min and washed several times with deionized water until the supernatant became colorless and transparent. Finally, the brown particles obtained by centrifugation were vacuum dried at 60 °C to obtain MnO2 NFs.

[0072] The prepared MnO2 NFs were characterized using TEM and SEM, such as... Figure 2 As shown, MnO2 NFs exhibit a morphology similar to a "large pom-pom" flower, with a large specific surface area, which can effectively increase the antibody loading.

[0073] Because MnO2 NFs possess oxidase-like activity, they can oxidize TMB to blue oxTMB in the absence of H2O2 (see...). Figure 3 ).

[0074] Example 2: Fabrication of a multimodal portable carcinoembryonic antigen detection sensor based on manganese dioxide nanoflowers

[0075] (1) 0.5 mg MnO2 NFs were dispersed in 1.0 mL of deionized water and sonicated for 1 h to obtain MnO2 NFs dispersion. Then, the carboxyl groups on MnO2 NFs were activated using a MES buffer solution (10 mM, pH=6) of EDC (2 mg / mL) and NHS (4 mg / mL). After reacting at room temperature for 1 h, the mixture was washed 3 times and redispersed to obtain activated MnO2 NFs.

[0076] (2) Mix 1 mL of detection antibody solution (100 μg / mL) with activated MnO2 NFs at a volume ratio of 1:1 and incubate at 37 °C for 1 h. Wash the product three times to remove unbound detection antibody and incubate at 37 °C with 1 mL of BSA solution (0.5%) for 1 h to block non-specific adsorption. Finally, wash the MnO2 NFs-detection antibody complex three times and disperse it in 1 mL of PBS buffer solution to obtain antibody-modified MnO2 NFs probe solution, which is stored at 4 °C for later use.

[0077] The morphology of the antibody-modified MnO2 NFs probes was characterized using TEM. The results are as follows: Figure 4 As shown, the surface of the antibody-modified MnO2 NFs is coated with a layer of material. Figure 5 As shown in the infrared spectrum of MnO2 NFs, at 550 cm⁻¹-1 The peak at 1637 cm⁻¹ can be attributed to the stretching vibration of Mn-O; -1 and 3400cm -1 The two characteristic peaks originate from the stretching vibrations of C=O and OH, indicating the presence of carboxyl functional groups in the MnO2 NFs material, which is helpful for subsequent modification of the detection antibody. In the probe's infrared spectrum, the peak at 1640 cm⁻¹... -1 and 1540cm -1 The absorption peaks corresponding to amide bands I and II, respectively, indicate that the activated carboxyl group of MnO2 NFs has combined with the amino group on the antibody to form an amide bond, and also indicate that the detection antibody has been successfully modified onto MnO2 NFs.

[0078] (3) At 60℃, a mixed solution of NaIO4 and LiCl was added dropwise to cellulose chromatography paper with a diameter of 6 mm every 30 min. The concentration of NaIO4 in the mixed solution was 50 mM and the concentration of LiCl was 700 mM. The dropwise volume was 20 μL. The dropwise addition step was repeated. The paper was washed with deionized water to remove unbound NaIO4 and LiCl. Then, 6 μL of capture antibody solution was added evenly. The paper was incubated at 37℃ for 1 h to remove unreacted capture antibody. BSA solution was added to block unbound reaction sites. Excess BSA was removed to obtain the paper substrate for immunoassay.

[0079] (4) Add 6 μL of carcinoembryonic antigen solution to the paper substrate for immunoassay, incubate at room temperature for 30 min, wash, add 6 μL of antibody-modified MnO2 NFs probe solution prepared in step (2), incubate at room temperature for 30 min, and obtain a multimode portable carcinoembryonic antigen detection sensor.

[0080] Example 3: Color-based detection of carcinoembryonic antigen

[0081] 1) Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multi-mode portable CEA detection sensor according to the method in Example 2. Add 6 μL TMB solution to the sensor and react for 10 min. Use a smartphone to take a picture in a light box to record the color signal. As the CEA concentration increases, the paper substrate gradually turns blue. Plot the working curve based on the relationship between the ΔR value of the image and the concentration of the standard antigen solution.

[0082] 2) Construct a multi-mode portable carcinoembryonic antigen detection sensor for the sample to be tested according to the method in Example 2, and perform detection according to the method in step 1). Detect the concentration of antigen in the sample to be tested based on the ΔR value of the image and the working curve.

[0083] Based on the oxidase-like activity of MnO2 NFs and the color signal of the product oxTMB, from Figure 6As can be seen in the illustration, the paper substrate gradually turns blue as the carcinoembryonic antigen (CEA) concentration increases. This is because a higher concentration of the target analyte results in a greater amount of MnO2 NFs immobilized on the paper substrate, leading to a higher content of TMB oxidized to the blue product oxTMB. Simultaneously, the ΔR value of the image also increases with increasing CEA concentration. Figure 6 As shown, when the concentration of carcinoembryonic antigen (CEA) is in the range of 2.5–100 ng / mL, there is a good linear relationship between the value of ΔR and the logarithm of the CEA concentration (R0). 2 =0.998), and the detection limit is 1.4 ng / mL (3σ / k).

[0084] Example 4: Temperature-based detection of carcinoembryonic antigen

[0085] I. Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multi-mode portable CEA detection sensor according to the method in Example 2. Add 6 μL of TMB solution to the sensor and react for 10 min. Under 808 nm laser irradiation, use a portable thermal imager to capture a thermal image of the paper substrate and record the temperature signal. Based on the relationship between the temperature signal and the concentration of the standard antigen solution, plot the working curve.

[0086] II. Construct a multi-mode portable carcinoembryonic antigen detection sensor for the sample to be tested according to the method in Example 2, and perform detection according to the method in Step I. Detect the concentration of antigen in the sample to be tested based on the temperature signal and the working curve.

[0087] Based on the photothermal effect of oxTMB, a handheld thermal imager was used to record the temperature signal. Under irradiation with an 808nm laser, the temperature of the reaction system gradually increased with increasing carcinoembryonic antigen concentration. Figure 7 (a)). For example Figure 7 As shown in (b), when the concentration of carcinoembryonic antigen (CEA) is in the range of 2.5–100 ng / mL, the temperature change of the reaction system shows a good linear relationship with the logarithm of the CEA concentration (R0). 2 =0.998), and the detection limit is 1.5 ng / mL (3σ / k).

[0088] Example 5: Distance-based detection of carcinoembryonic antigen

[0089] To broaden the application of biosensors in the POCT field, a temperature-responsive hydrogel was combined with a portable sensor mediated by MnO2 nanoflowers to achieve distance-based detection of carcinoembryonic antigen, thus providing a simpler and more intuitive quantitative method for the detection of biomolecules.

[0090] a. Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multi-mode portable CEA detection sensor according to the method in Example 2. Add 6 μL of TMB solution to the sensor and react for 10 min. Place the reacted paper substrate and gelatin hydrogel block in the circular sample loading area of ​​the paper substrate distance sensor. Immediately after irradiation with an 808 nm laser, measure the distance signal generated by the sensor with vernier calipers. Plot the working curve based on the relationship between the water flow distance signal and the concentration of the standard antigen solution.

[0091] b. Construct a multi-mode portable carcinoembryonic antigen detection sensor for the sample to be tested according to the method in Example 2, and perform detection according to the method in step a. Detect the concentration of antigen in the sample to be tested based on the signal of the water flow distance and the working curve.

[0092] The device of paper-based distance sensor, such as Figure 8 As shown, it includes a PMMA substrate iv with grooves and a pH test strip iii. The pH test strip is disposed in the groove of the PMMA substrate and its shape matches the shape of the groove of the PMMA substrate. The pH test strip includes a circular sample loading area and a rectangular straight channel connected to the sample loading area.

[0093] pH test strips are cut by an embossing tool into a shape consisting of a circular sample area (6 mm in diameter) and a rectangular straight channel (50 mm × 2 mm); grooves are engraved on the PMMA base layer (72 mm × 16 mm × 5 mm) using a laser cutter, with the groove shape being a circular sample area (7 mm in diameter) and a rectangular straight channel (60 mm × 2.3 mm).

[0094] During detection, the reacted paper substrate ii and gelatin hydrogel block i were placed within the circular sample area of ​​the paper-based distance sensor, and the circular sample area was irradiated with an 808nm laser. At this time, as the carcinoembryonic antigen concentration increased, the temperature of the sample area gradually rose, causing the hydrogel to undergo varying degrees of gel-sol phase transition and viscosity changes, thus generating water flow distances of different lengths along the long channel of the pH test paper. Figure 9 (a)). For example Figure 9 As shown in (b), when the concentration of carcinoembryonic antigen (CEA) is 2.5–100 ng / mL, there is a good linear relationship between the flow distance and the logarithm of the CEA concentration (R0). 2 =0.997), and the detection limit is 2.5 ng / mL.

[0095] The principle of the detection method of this invention is as follows: Figure 1As shown, this sensor immobilizes the capture antibody on a paper substrate and then sequentially binds it to carcinoembryonic antigen (CEA) and a detection antibody labeled with MnO2 NFs to form a sandwich complex. The oxTMB generated by MnO2 NFs-catalyzed TMB not only produces a color signal but also exhibits a photothermal effect, enabling the detection of CEA through color and temperature signals. Furthermore, combining gelatin hydrogel with this system allows for the detection of CEA by creating a water flow distance on pH test paper due to the temperature responsiveness of gelatin and its gel-sol phase transition. The biosensor constructed using this strategy is not only simple and inexpensive to prepare, but also provides complementary signals and allows for the modification of different detection antibodies with MnO2 NFs, offering a universal method for the detection of target analytes.

[0096] Application Example 1: Detection of Carcinoembryonic Antigen in Serum

[0097] Normal human serum was diluted 10-fold with PBS buffer, and then carcinoembryonic antigen solution was added to obtain serum standards with concentrations of 0 ng / mL, 3 ng / mL, 30 ng / mL, and 60 ng / mL. These standards were then tested according to the detection methods described in Examples 3, 4, and 5. The results are as follows: Figure 10 As shown.

[0098] Depend on Figure 10 It is known that different color, temperature, and distance signals are output as the concentration of carcinoembryonic antigen (CEA) increases. Therefore, the multi-mode portable sensor based on manganese dioxide nanoflowers provided by this invention can detect CEA in serum through three visual signals: color, temperature, and distance.

[0099] The above description is merely a specific embodiment of the present invention. The scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for detecting carcinoembryonic antigen (CEA) for non-diagnostic purposes using a multimode portable CEA detection sensor. This method combines a temperature-responsive hydrogel with a multimode portable CEA detection sensor to achieve distance-based CEA detection. The specific method is as follows: a. Prepare carcinoembryonic antigen (CEA) solutions of different standard concentrations. Using CEA solutions of different standard concentrations, construct a multimode portable CEA detection sensor according to the preparation method of the multimode portable CEA detection sensor. Add 6 μL of TMB solution to the sensor and react for 8-12 min. Place the reacted paper substrate and gelatin hydrogel block in the circular sample loading area of ​​the paper substrate distance sensor. Immediately after irradiation with an 808 nm laser, measure the distance signal generated by it using vernier calipers. Plot the working curve based on the relationship between the water flow distance signal and the concentration of the standard antigen solution. b. Construct a multi-mode portable carcinoembryonic antigen (CEA) detection sensor according to the preparation method of the multi-mode portable CEA detection sensor, and perform detection according to the method in step a. Detect the concentration of antigen in the CEA sample based on the signal of the water flow distance and the working curve. The paper-based distance sensor includes a grooved PMMA substrate and a pH test paper. The pH test paper is placed in the groove of the PMMA substrate and its shape matches the shape of the groove of the PMMA substrate. The pH test paper includes a circular sample carrying area and a rectangular straight channel connected to the sample carrying area. The irradiation time by an 808nm laser is 60-120s. The hydrogel block was prepared by the following method: Disperse the gelatin in water, heat and stir until dissolved, and quickly fill it into a plastic syringe while hot. Refrigerate at 4°C for later use. Cut it into uniformly thick hydrogel cylinders and let it stand at room temperature for 20 minutes before use. The concentration of the gelatin is 4-6 wt%, and the heating temperature is 30-50°C. The multimodal portable carcinoembryonic antigen detection sensor is prepared according to the following method: (1) Dissolve KMnO4 in hydrochloric acid solution to obtain KMnO4 solution. (2) Add citric acid solution dropwise to the KMnO4 solution prepared in step (1), stir at room temperature, the solution changes from purple to brown and brown particles are produced, centrifuge, wash the brown particles with deionized water until the supernatant becomes colorless and transparent, and vacuum dry the particles to obtain MnO2 NFs. (3) Disperse MnO2 NFs in deionized water and sonicate to obtain MnO2 NFs dispersion; (4) Add a MES buffer solution of EDC and NHS to the MnO2 NFs dispersion, use EDC and NHS to activate the carboxyl groups on the MnO2 NFs, then wash and redisperse to obtain activated MnO2 NFs. (5) Mix the detection antibody solution corresponding to carcinoembryonic antigen with the activated MnO2 NFs and incubate. Then wash with PBS buffer. After washing, add BSA solution and incubate together to block non-specific adsorption. Disperse in PBS buffer solution to obtain antibody-modified MnO2 NFs probe solution. (6) Add a mixed solution of NaIO4 and LiCl to the cellulose chromatography paper every 30 min, repeat the addition step, wash with deionized water to remove unbound NaIO4 and LiCl, then add the capture antibody solution evenly, incubate, remove unreacted capture antibody, add BSA solution to block unbound reaction sites, remove excess BSA, and obtain the paper substrate for immunoassay. (7) Add the carcinoembryonic antigen solution to the paper substrate for immunoassay, incubate at room temperature, wash, add the antibody-modified MnO2 NFs probe solution prepared in step (5), incubate at room temperature, and obtain a multimode portable carcinoembryonic antigen detection sensor.

2. The method according to claim 1, characterized in that, In step (1), the concentration of hydrochloric acid solution is 0.1M-0.3M, and the concentration of KMnO4 solution is 1-5mg / mL.

3. The method according to claim 1, characterized in that, In step (2), the concentration of citric acid solution is 0.05-0.15M, the volume ratio of KMnO4 solution to citric acid solution is 35-45:1, the stirring time at room temperature is 40-60min, the centrifugation is performed at 8000-12000rpm for 5-20min, and the vacuum drying temperature is 50-70℃.

4. The method according to claim 1, characterized in that, In step (3), the concentration of the MnO2 NFs dispersion is 0.4-0.6 mg / mL.

5. The method according to claim 1, characterized in that, In step (4), the concentration of EDC in the MES buffer solution of EDC and NHS is 1-5 mg / mL, the concentration of NHS is 3-8 mg / mL, and the pH is 4-6.

5.

6. The method according to claim 1, characterized in that, In step (5), the concentration of the detection antibody solution is 90-110 μg / mL, the volume ratio of the detection antibody to the activated MnO2 NFs is (1-2):(1-2), the incubation is carried out at 37℃ for 1 h, the mass concentration of the BSA solution is 0.4-0.6%, the volume ratio of the activated MnO2 NFs to the BSA solution is (1-2):(1-2), and the concentration of the antibody-modified MnO2 NFs probe in the PBS buffer solution is 0.4-0.6 mg / mL.

7. The method according to claim 1, characterized in that, In step (6), the diameter of the cellulose chromatography paper is 5-7 mm. The mixed solution of NaIO4 and LiCl is added at a temperature of 55-65℃. The concentration of NaIO4 in the mixed solution is 40-60 mM and the concentration of LiCl is 650-750 mM. The amount added is 10-30 μL. The concentration of the capture antibody solution is 30-50 μg / mL. The incubation is carried out at 37℃ for 0.5-2 h. The amount added of the capture antibody solution is 4-10 μL. Unreacted capture antibody is removed by washing three times with PBS buffer. Excess BSA is removed by washing three times with PBS buffer. In step (7), the amount added of carcinoembryonic antigen solution is 4-10 μL.

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