A rapid detection carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals and its preparation method

Abstract

The present invention discloses a perovskite nanocrystal-based immunochromatographic test strip for rapid detection of carcinoembryonic antigen (CEA) and a preparation method thereof. The immunochromatographic test strip comprises a base plate, a sample pad, a conjugate pad, and a nitrocellulose membrane. The preparation method comprises: pretreating the sample pad and conjugate pad; preparing gold nanoparticles by a trisodium citrate reduction method; then coupling the gold nanoparticles with CEA antibodies by electrostatic adsorption to prepare a gold nanoparticle immunoprobe, which is then fixed to the conjugate pad; synthesizing perovskite nanocrystals by a ligand-assisted reprecipitation method; spraying the perovskite nanocrystals onto the nitrocellulose membrane to form a test line and a control line; then spraying BSA-blocked CEA onto the test line; and finally, assembling the test strip. The test strip of the present invention has the advantages of rapid detection, low cost, high sensitivity, low detection limit, high selectivity, and no need for large instruments or professional operators, and has good application prospects in point-of-care diagnosis.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test strip and a preparation method thereof, and in particular to an immunochromatographic test strip for rapid detection of carcinoembryonic antigen based on perovskite nanocrystals and a preparation method thereof. Background Art

[0002] Early screening and diagnosis of cancer are crucial. Carcinoembryonic antigen (CEA) in patient plasma is a common cancer marker used in the clinical diagnosis of pancreatic, colorectal, and lung cancers. Abnormally elevated levels of CEA in serum may increase the risk of cancer.

[0003] Previous reports have typically used methods such as fluorescence, chemiluminescence, electrochemistry, and surface-enhanced Raman scattering to detect CEA levels in patient serum. These methods rely on laboratory infrastructure and are cumbersome to perform on-site. Therefore, to enable rapid and convenient monitoring of CEA levels in serum, point-of-care (POCT) diagnostics have emerged. POCT can rapidly, sensitively, and quantitatively detect CEA, which is of great significance for diagnosis in resource-poor areas. Currently, there is an urgent need to find a test method that can rapidly, sensitively, and quantitatively detect CEA. Summary of the Invention

[0004] Purpose of the invention: The present invention aims to provide a method for preparing a dual-readout signal immunochromatographic test strip based on perovskite nanocrystals that can achieve rapid and sensitive detection of carcinoembryonic antigen.

[0005] Technical solution: The perovskite nanocrystal-based rapid detection of carcinoembryonic antigen immunochromatographic test strip described in the present invention includes a base plate, a sample pad, a conjugate pad and a nitrocellulose membrane. The conjugate pad is fixed with a gold nanoparticle immune probe, and the nitrocellulose membrane is coated with a test line and a control line. The test line is obtained by spraying a perovskite nanocrystal solution and carcinoembryonic antigen blocked with bovine serum albumin, and the control line is obtained by spraying a perovskite nanocrystal solution.

[0006] Preferably, the spraying concentration of the perovskite nanocrystal solution is 1-3 mg / mL, and the spraying concentration of the bovine serum albumin-blocked carcinoembryonic antigen is 2-20 μg / mL.

[0007] Preferably, the distance between the test line and the control line is 1-2 cm, and the width of the overlapping parts of the components on the immunochromatographic test strip is 2-4 mm.

[0008] The method for preparing the rapid detection carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals comprises the following steps:

[0009] (1) Soak the sample pad in a buffer solution, then take it out and dry it in a vacuum oven for later use;

[0010] (2) Soaking the conjugate pad in a buffer solution, then taking it out and drying it in a vacuum oven for later use; then fixing the gold nanoparticle immunoprobe on the pretreated conjugate pad, and then drying it in a vacuum oven for later use;

[0011] (3) spraying a perovskite nanocrystal solution and carcinoembryonic antigen blocked with bovine serum albumin (BSA) onto a nitrocellulose membrane to form a test line; spraying an equal volume of the perovskite nanocrystal solution onto a nitrocellulose membrane to form a control line; then, drying the nitrocellulose membrane in a vacuum oven for later use;

[0012] (4) The pretreated components and absorbent paper are assembled in sequence onto a PVC base plate that has been cut into a suitable width to obtain a perovskite nanocrystal-based rapid detection of carcinoembryonic antigen dual-readout signal immunochromatographic test strip.

[0013] Preferably, the method for preparing the gold nanoparticle immune probe in step (2) comprises the following steps: adjusting the pH of the gold nanoparticle solution with potassium carbonate, then adding an antibody against carcinoembryonic antigen to the solution and incubating the mixture, then adding bovine serum albumin and incubating the mixture to block excess binding sites, and finally washing the solution with a phosphate buffer solution and redispersing the mixture to obtain a gold nanoparticle immune probe solution.

[0014] Preferably, the method for preparing the gold nanoparticle solution comprises the following steps: heating a chloroauric acid solution and deionized water, then rapidly adding a trisodium citrate solution to the solution, and immediately stopping heating after the reaction is complete; washing the solution after cooling it to room temperature, and finally redispersing it in distilled water to obtain a gold nanoparticle solution.

[0015] Preferably, the preparation method of the perovskite nanocrystals in step (3) comprises the following steps: dissolving lead bromide and cesium bromide in a solvent and stirring, adding octylamine grafted polyacrylic acid amphiphilic polymer and oleylamine, and stirring until the solution becomes clear from turbidity, i.e., forming a precursor solution; then rapidly injecting the above-mentioned precursor solution into toluene, washing after the reaction is complete, and drying to obtain perovskite nanocrystals; dispersing the perovskite nanocrystals in distilled water, adding glutaraldehyde for activation, then adding bovine serum albumin for incubation, washing, and then re-dispersing to obtain a perovskite nanocrystal solution.

[0016] Preferably, the buffer solution in step (1) is 10-20 mM PBS, pH = 7.2-7.6, 1-2% BSA, 0.2-0.5% Tween-20 and 4-5% sucrose, and the buffer solution in step (2) is 10-20 mM PBS, pH = 7.2-7.6, 1-2% BSA and 0.1-0.5% Tween-20.

[0017] Preferably, the soaking time in the buffer solution in step (1) and step (2) is 2-5 hours.

[0018] Preferably, the drying temperature in step (1), step (2) and step (3) is 35-37°C.

[0019] Principle of the invention: First, perovskite nanocrystals are synthesized using an improved ligand-assisted reprecipitation method with octylamine-grafted polyacrylic acid amphiphilic polymer and oleylamine as ligands. They are then coupled with BSA via glutaraldehyde coupling to form BSA-blocked perovskite nanocrystals. They are then immobilized on the test line and control line of a nitrocellulose membrane, and the BSA-blocked carcinoembryonic antigen is then immobilized on the test line. An immunoprobe coupled with gold nanoparticles and antibodies to carcinoembryonic antigen is immobilized on the binding pad of an immunochromatographic test strip. When the analyte does not contain carcinoembryonic antigen, the gold nanoparticle immunoprobe is captured by the corresponding antigen on the test line, forming a red gold nanoparticle line. The green fluorescence of the perovskite nanocrystal fixed on the test line is quenched by the fluorescent inner filter effect (IFE), while the fluorescence on the control line remains bright and is used for comparison with the fluorescence of the test line. When carcinoembryonic antigen is present in the analyte, the carcinoembryonic antigen in the sample competes with the carcinoembryonic antigen on the test line for binding to the gold nanoparticle immunoprobe, causing the red band on the test line to gradually "close" while the green fluorescence signal slowly "opens." Due to the localized surface plasmon resonance effect of gold nanoparticles, the color bands were observed with the naked eye. The changes in the perovskite fluorescence signal were observed in a dark box using a 365nm UV lamp, achieving dual-mode quantitative detection.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] (1) The prepared perovskite nanocrystal-based dual-readout immunochromatographic test strip for rapid detection of carcinoembryonic antigen can, on the one hand, perform qualitative analysis through the colorimetric signal of gold nanoparticles, and on the other hand, perform quantitative analysis through the fluorescent signal generated by the fluorescent inner filter effect between the perovskite nanocrystals and gold nanoparticles. This colorimetric-fluorescence dual-readout immunochromatographic test strip has the advantages of rapidity, low cost, high sensitivity, low detection limit, and high selectivity.

[0022] (2) The perovskite nanocrystals sprayed on the nitrocellulose membrane in the prepared perovskite nanocrystal-based rapid carcinoembryonic antigen dual-readout immunochromatographic test strip exhibited excellent stability. After 40 days of storage in air, the fluorescence intensity at the test line maintained 96.69% of the initial intensity, and the fluorescence intensity at the control line maintained 95.99% of the initial intensity.

[0023] (3) The colorimetric and fluorescence signals generated by the prepared perovskite nanocrystal-based rapid detection of carcinoembryonic antigen dual-readout immunochromatographic test strip can be qualitatively evaluated by the naked eye under both natural light and ultraviolet light. It can also be recorded by taking a photo with a mobile phone, and the photo capture test results can be analyzed by colorimetry and fluorescence intensity using Fjij software, thereby achieving quantitative detection of carcinoembryonic antigen. It does not require laboratory infrastructure or professional operators, and has good application prospects in POCT diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a physical picture of a dual-readout signal immunochromatographic test strip;

[0025] Figure 2 This is a diagram showing the effect of different antibody conjugation amounts on the detection effect of dual-readout signal immunochromatographic test strips;

[0026] Figure 3 This is a graph showing the fluorescence intensity changes of perovskite nanocrystals sprayed on nitrocellulose membrane for 40 days;

[0027] Figure 4 This is a diagram showing the test results of carcinoembryonic antigen using a dual-readout signal immunochromatographic test strip for rapid detection of carcinoembryonic antigen based on perovskite nanocrystals. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0029] Example 1

[0030] 1. Preparation of gold nanoparticles

[0031] Add 0.5 mL of 1 wt% chloroauric acid solution and 50 mL of deionized water to a three-necked flask and heat. When the temperature reaches 110°C, quickly add 2.5 mL of 1 mg / mL trisodium citrate solution to the solution. After 10 minutes of reaction, stop heating and allow the solution to cool to room temperature. Finally, rinse with deionized water three times, redisperse in 20 mL of deionized water, and store at 4°C until ready to use.

[0032] 2. Preparation of gold nanoparticle immunoprobes

[0033] Potassium carbonate (0.1 mol / L) was added dropwise to 1 mL of the gold nanoparticle solution. Antibodies against carcinoembryonic antigen (containing 0.5% BSA) were mixed with the solution and incubated at 37°C for 1 hour. 100 μL of 10% BSA was then added and incubated for an additional 30 minutes to block unreacted sites. The incubated solution was centrifuged at 10,000 rpm for 10 minutes, the supernatant discarded, and the suspension resuspended in 200 μL of buffer (10 mM PBS, pH 7.4, 1% BSA, 5% sucrose, 0.5% Tween-20) and stored at 4°C until use.

[0034] 3. Preparation of perovskite nanocrystals

[0035] 0.4 mmol of lead bromide and 0.4 mmol of cesium bromide were added to a single-necked flask containing 10 mL of N,N-dimethylformamide and stirred. After the solids in the flask were completely dissolved, 2 mg of an amphiphilic polymer grafted with octylamine (OPA) and 0.5 mL of oleylamine were added. After stirring for 10 minutes, the solution turned clear from turbidity, forming a precursor solution. 0.5 mL of this precursor solution was quickly injected into 10 mL of toluene, causing the solution to turn green. After 10 minutes of reaction, the solution was centrifuged at 8000 rpm for 5 minutes and washed three times with toluene. The solution was then dried in a vacuum oven at 60°C for 2 hours to obtain perovskite nanocrystals. The perovskite nanocrystals were dispersed in aqueous solution and activated with 10 μL of 5% glutaraldehyde for 30 minutes. Then, 100 μL of BSA (100 mg / mL) was added and incubated for 2 hours. The incubated solution was centrifuged at 8000 rpm for 5 minutes and rinsed three times with ultrapure water. Finally, the BSA-sealed perovskite nanocrystals were dissolved in 500 μL of ultrapure water and stored at 4°C for later use.

[0036] 4. Preparation of a dual-readout immunochromatographic test strip for rapid detection of carcinoembryonic antigen based on perovskite nanocrystals

[0037] (1) Soak the sample pad in a buffer solution (10 mM PBS, pH = 7.4, 1% BSA, 0.2% Tween-20, and 4% sucrose) for 2 h, then take it out and dry it in a vacuum oven at 37°C for use;

[0038] (2) Soak the conjugate pad in a buffer solution (10 mM PBS, pH = 7.4, 1% BSA, 0.1% Tween-20) for 2 h, then take it out and dry it in a vacuum oven at 37°C for later use; then immobilize the gold nanoparticle immunoprobe on the pretreated conjugate pad, and then dry it in a vacuum oven at 37°C for later use;

[0039] (3) Spraying the perovskite nanocrystal solution and BSA-blocked carcinoembryonic antigen onto a nitrocellulose membrane to form a test line; spraying an equal volume of the perovskite nanocrystal solution onto a nitrocellulose membrane to form a control line; then, drying the nitrocellulose membrane in a vacuum oven at 37°C for later use;

[0040] (4) The pre-treated components and absorbent paper were assembled onto a PVC base plate cut into 5 mm width to obtain a dual-readout immunochromatographic test strip for rapid detection of carcinoembryonic antigen based on perovskite nanocrystals. Figure 1 .

[0041] 5. Carcinoembryonic antigen detection based on perovskite nanocrystal dual-readout signal immunochromatographic test strips

[0042] The sample pad on the prepared test strip was inserted into 80 μL of a standard solution containing CEA at a range of concentrations and allowed to react for 10 minutes. The results were qualitatively evaluated visually under both natural and ultraviolet light and recorded using a mobile phone camera. The photographic results were then analyzed using Fjij software for colorimetric and fluorescence intensity analysis, enabling quantitative CEA detection.

[0043] Example 2

[0044] The preparation method is similar to that of Example 1, except that the amount of carcinoembryonic antigen antibody coupled to the gold nanoparticles is adjusted during the preparation of the gold nanoparticle immune probe.

[0045] The specific operation is as follows: 20 μL, 40 μL, 60 μL, 80 μL, and 100 μL of carcinoembryonic antigen antibody (100 μg / mL) were mixed with an equal amount of gold nanoparticle solution, and incubated at 37°C for 1 hour.

[0046] The dual-readout signal immunochromatographic test strips based on perovskite nanocrystals prepared in the above embodiments were used to detect carcinoembryonic antigen. Figure 4 Shown are the test results of carcinoembryonic antigen at different concentrations.

[0047] Depend on Figure 4 As can be seen, on the one hand, as the concentration of carcinoembryonic antigen increases, the red band of gold nanoparticles on the detection line gradually fades and disappears, and the visual limit of detection (VLOD) of carcinoembryonic antigen in colorimetric visual mode is 25ng / mL. On the other hand, under ultraviolet light, as the concentration of carcinoembryonic antigen increases, the green fluorescence signal of the perovskite nanocrystals on the detection line "turns on", and the fluorescence intensity gradually increases. The VLOD of carcinoembryonic antigen in fluorescence visual mode is 10ng / mL.

[0048] Depend on Figure 2 It can be seen that 80 μL of antibody (100 μg / mL) is the optimal dose, at which point the gold nanoparticle immunoprobe is captured the most.

[0049] Depend on Figure 3 This shows that the fluorescence of perovskite nanocrystals sprayed onto the nitrocellulose membrane remains highly stable. After 40 days of storage in air, the fluorescence intensity at the control line and the test line remained at 96.69% and 95.99% of their initial intensity, respectively.

[0050] It can be seen that the dual-readout immunochromatographic test strip based on perovskite nanocrystals prepared by the present invention has high stability and low detection limit, and is expected to be applied to the point-of-care (POCT) diagnosis of carcinoembryonic antigen levels in human serum.

Claims

1. A rapid detection of carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals, characterized in that: The invention comprises a base plate, a sample pad, a conjugation pad and a nitrocellulose membrane, wherein a gold nanoparticle immune probe is fixed on the conjugation pad, and a detection line and a control line are coated on the nitrocellulose membrane, wherein the detection line is obtained by spraying a perovskite nanocrystal solution and a carcinoembryonic antigen blocked by bovine serum albumin, and the control line is obtained by spraying a perovskite nanocrystal solution. The preparation method of the perovskite nanocrystal comprises the following steps: dissolving lead bromide and cesium bromide in a solvent and stirring, adding an octylamine-grafted polyacrylic acid amphiphilic polymer and oleylamine, and stirring until the solution becomes clear from turbidity, thus forming a precursor solution; then rapidly injecting the precursor solution into toluene, washing and drying after the reaction is complete, thereby obtaining perovskite nanocrystals; and dispersing the perovskite nanocrystals in distilled water, adding glutaraldehyde for activation, then adding bovine serum albumin for incubation, washing, and then redispersing to obtain a perovskite nanocrystal solution.

2. The rapid detection of carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 1, characterized in that: The spraying concentration of the perovskite nanocrystal solution is 1-3 mg / mL, and the spraying concentration of the bovine serum albumin-blocked carcinoembryonic antigen is 2-20 μg / mL.

3. The rapid detection of carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 1, characterized in that: The distance between the test line and the control line is 1-2 cm, and the width of the overlapping parts of the components on the immunochromatographic test strip is 2-4 mm.

4. A method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Soak the sample pad in a buffer solution and then dry it for later use; (2) Soaking the conjugate pad in a buffer solution and then drying it for later use; then fixing the gold nanoparticle immunoprobe on the pretreated conjugate pad and drying it for later use; (3) Spraying the perovskite nanocrystal solution and carcinoembryonic antigen blocked with bovine serum albumin onto a nitrocellulose membrane to form a test line; spraying the perovskite nanocrystal solution onto the nitrocellulose membrane to form a control line, and then drying the membrane in a vacuum oven for later use; (4) Assemble the pretreated components and absorbent paper on the bottom plate in sequence to obtain the immunochromatographic test strip.

5. The method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 4, characterized in that: The preparation method of the gold nanoparticle immune probe in step (2) comprises the following steps: adjusting the pH of the gold nanoparticle solution with potassium carbonate, then adding an antibody against carcinoembryonic antigen to the solution and incubating the mixture, then adding bovine serum albumin and incubating the mixture to block excess binding sites, and finally washing the solution with a phosphate buffer solution and redispersing the solution to obtain a gold nanoparticle immune probe solution.

6. The method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 5, characterized in that: The preparation method of the gold nanoparticle solution comprises the following steps: heating a chloroauric acid solution and deionized water, then rapidly adding a trisodium citrate solution to the solution, and immediately stopping heating after the reaction is complete; washing the solution after cooling it to room temperature, and finally redispersing it in distilled water to obtain the gold nanoparticle solution.

7. The method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 4, characterized in that: The buffer solution described in step (1) is 10-20 mM PBS, pH = 7.2-7.6, 1-2% BSA, 0.2-0.5% Tween-20 and 4-5% sucrose, and the buffer solution described in step (2) is 10-20 mM PBS, pH = 7.2-7.6, 1-2% BSA and 0.1-0.5% Tween-20.

8. The method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 4, characterized in that: In step (1) and step (2), the immersion time in the buffer solution is 2-5 h.

9. The method for preparing a rapid carcinoembryonic antigen immunochromatographic test strip based on perovskite nanocrystals according to claim 4, characterized in that: In step (1), step (2) and step (3), the drying temperature is 35-37 °C.

Images