Chromatographic test strip and method for detecting carbohydrate antigen 15-3

Abstract

The invention discloses a chromatographic test strip and method for detecting carbohydrate antigen 15-3, the chromatographic test strip is provided with a sample pad, a combination pad and a detection pad which are sequentially arranged along the chromatographic direction, and gold-silver composite nanoparticles with beacon molecules are stored in the combination pad. The surfaces of the gold-silver composite nanoparticles are coupled with a first antibody which can be specifically combined with a carbohydrate antigen 15-3, and a second antibody which is specifically combined with the carbohydrate antigen 15-3 is stored in the detection pad. According to the application, the concentration of the carbohydrate antigen 15-3 can be responded at the same time based on double signals of Raman and temperature, the content of the carbohydrate antigen 15-3 in a to-be-detected sample can be rapidly, sensitively, accurately and quantitatively detected in practical application, and the operation is simple and convenient.

Description

Technical Field

[0001] The present application relates to the field of biochemical analysis technology, and in particular to a chromatographic test strip and method for detecting carbohydrate antigen 15-3. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. Currently, the main methods for screening breast cancer include clinical breast examination, mammography, breast ultrasound, and breast magnetic resonance imaging (MRI). These methods can achieve the purpose of early screening, but they often involve privacy and radiation, and the examination time is long and the cost is high.

[0003] In recent years, the detection of tumor markers has gradually become one of the important methods for screening breast cancer because of its simplicity, rapidity, good specificity and diagnostic accuracy. Carbohydrate antigen 15-3 (CA15-3) is currently the most recognized, important and most commonly used macromolecular breast cancer specific tumor marker.

[0004] In the prior art, the method for detecting CA15-3 is divided into two categories according to the number of output signals: single-mode and dual-mode methods. Among them, the single-mode method is a single-signal detection method based on enzyme-linked immunosorbent assay (ELISA), electrochemical method, electrochemiluminescence method, fluorescence method, and SERS method. Since there is only one detection signal, it is easy to cause false negatives and false positives. The dual-mode method mainly includes electrochemical-fluorescence method and colorimetric-electrochemical method. However, fluorescence and electrochemical techniques often require the use of corresponding large-scale equipment for detection, which makes it difficult to achieve direct on-site observation; colorimetric technology can achieve direct on-site observation without the help of other instruments, and the displayed color is relatively single, which makes it difficult to perform accurate quantitative identification. Summary of the invention

[0005] In view of the problems of the prior art, the present application provides a chromatographic test strip and method for detecting carbohydrate antigen 15-3, so as to quickly, sensitively and accurately quantitatively detect the content of carbohydrate antigen 15-3 in a sample to be tested.

[0006] The chromatographic test strip for detecting carbohydrate antigen 15-3 in the present application comprises a sample pad, a conjugation pad and a detection pad arranged in sequence along the chromatographic direction, the conjugation pad stores gold-silver composite nanoparticles with beacon molecules, the surface of the gold-silver composite nanoparticles is coupled with a first antibody that can specifically bind to the carbohydrate antigen 15-3, and the detection pad stores a second antibody that specifically binds to the carbohydrate antigen 15-3.

[0007] Optionally, the gold-silver composite nanoparticles are a core-shell structure in which a silver layer wraps the surface of a gold spherical nanoparticle.

[0008] Optionally, the diameter of the gold spherical nanoparticles is 30-40 nm; the thickness of the silver layer is 1-3 nm, preferably 1.5-2.5 nm.

[0009] Optionally, the detection pad includes a base plate and a cellulose acetate membrane fixed on the surface of the base plate and used for chromatographic filtration.

[0010] Optionally, the detection pad stores a third antibody that specifically binds to the first antibody.

[0011] Optionally, the third antibody is goat anti-mouse IgG.

[0012] Optionally, the beacon molecule is at least one of 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid, 4-mercaptobenzonitrile, and 4-mercaptobenzenethiol, preferably 4-ATP.

[0013] Optionally, the preparation method of gold-silver composite nanoparticles with beacon molecules includes:

[0014] Providing a gold nanoparticle solution, adding a beacon molecule thereto for reaction, centrifuging after the reaction is completed, removing the supernatant to obtain a precipitate, and then adding water for redissolution to obtain a redissolution;

[0015] The complex solution is heated, and sodium citrate solution and silver nitrate solution are added thereto for reaction to obtain the gold-silver composite nanoparticles with beacon molecules.

[0016] Optionally, the gold-silver composite nanoparticles with beacon molecules are mixed with the first antibody for reaction under strong alkaline conditions, and a site blocking agent is added after the reaction is completed; the supernatant is removed by centrifugation to separate the first antibody-gold-silver composite nanoparticles-beacon molecule conjugate (Au / 4-NTP@Ag NPs).

[0017] Optionally, the site blocking agent is bovine serum albumin (BSA).

[0018] The present application also provides a method for detecting carbohydrate antigen 15-3, comprising the following steps:

[0019] Providing the chromatography test strip;

[0020] The sample to be tested is added to the sample pad, and after the reaction is completed, the detection pad is irradiated with laser, the Raman signal of the detection pad and / or the temperature difference signal before and after irradiation are collected, and the content of carbohydrate antigen 15-3 in the sample to be tested is calculated.

[0021] Optionally, a standard curve of Raman signal and carbohydrate antigen 15-3 concentration and / or a standard curve of temperature difference signal and carbohydrate antigen 15-3 concentration is constructed, and the content of carbohydrate antigen 15-3 in the sample to be tested is calculated according to the standard curve.

[0022] Compared with the existing technology, the detection method provided in the present application can simultaneously respond to the concentration of carbohydrate antigen 15-3 based on the dual signals of Raman and temperature. In practical applications, it can quickly, sensitively, accurately and quantitatively detect the content of carbohydrate antigen 15-3 in the sample to be tested, and the operation is simple and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The structure and detection principle diagram of the chromatography test strip in this application;

[0024] Figure 2A The UV-visible spectra of the nanoparticles in Preparation Example 1, wherein a is gold nanoparticles (AuNPs), and b is gold-silver composite nanoparticles with 4-ATP (Au / 4-ATP@Ag NPs);

[0025] Figure 2B The Raman spectra of the Au / 4-ATP@Ag NPs test samples, where a is a sample containing 100 U / mL CA15-3 antigen, and b is a blank sample;

[0026] Figure 2C Transmission electron microscopy image of Au / 4-ATP@Ag NPs;

[0027] Figure 2D Characterization diagram of silver element in Au / 4-ATP@Ag NPs;

[0028] Figure 2E Characterization diagram of gold element in Au / 4-ATP@Ag NPs;

[0029] Figure 2F Characterization diagram of mixed elements of gold and silver in Au / 4-ATP@Ag NPs;

[0030] Figure 3A It is the result figure of the specificity test in Example 1;

[0031] Figure 3B The result diagram of the stability test in Example 2;

[0032] Figure 4A is the Raman intensity of CA15-3 at different concentrations at the T line (1077 cm -1 ) and the temperature difference of the corresponding points measured;

[0033] Figure 4B A standard linear curve of CA15-3 was constructed for the data measured by the ELISA kit method;

[0034] Figure 5A The figure is the chromatography result of the human serum dilution in Example 4;

[0035] Figure 5B The Raman signal diagram of human serum dilution at five points on the chromatography test strip;

[0036] Figure 5C It is a temperature diagram of human serum dilution at five points on the chromatography test strip;

[0037] Figure 5D The RSD graph of Raman signal and temperature change of human serum dilution compared with negative control at five points on the chromatography test strip;

[0038] Figure 5E The Raman signal diagram of three sets of parallel experiments to detect human serum dilutions;

[0039] Fig. 5F Temperature diagram of three sets of parallel tests of human serum dilutions;

[0040] Figure 5G The RSD graphs of Raman signals and temperatures of three sets of human serum dilutions detected by the Pingxin test. DETAILED DESCRIPTION

[0041] The technical solution described in this application is further described below in conjunction with specific implementation methods, but the application is not limited thereto.

[0042] Preparation Example 1

[0043] 1.1 Synthesis of colloidal gold solution

[0044] Using the Tris-base solution-assisted method, the synthesis steps are as follows:

[0045] (1) When 140 mL of ultrapure water is heated to 100° C. in an oil bath, 10 mL of a 1 wt % sodium citrate solution is added. After 40 min, 1.0 mL of a 1 wt % tetrachloroauric acid solution is added.

[0046] (2) After 1 minute, 5.0 mL of 0.1 M Tris-base solution was added for reaction. After half an hour of reaction, the temperature was lowered to 100° C., and 1.0 mL of 1 wt % tetrachloroauric acid was added, and the reaction was continued for 30 minutes;

[0047] (3) Repeat step (2);

[0048] (4) After the reaction is completed, stop heating and allow the mixture to cool naturally to room temperature while stirring.

[0049] 1.2 Synthesis of Au / 4-ATP@Ag NPs

[0050] (1) Add 10 μL of 10 mM 4-aminothiophenol (4-ATP) to 10 mL of colloidal gold solution, react for 1 h, centrifuge at 9000 rpm for 10 min, discard the supernatant, and redissolve the precipitate with 20 mL of deionized water to obtain a reconstituted solution;

[0051] (2) The reconstituted solution was placed in an oil bath at 125°C and heated to a boil. A 10 wt% sodium citrate solution was added under vigorous stirring. After 3 minutes, 20 μL of 10 mM silver nitrate was added every 30 seconds, for a total of 320 μL. The solution was heated and boiled for another 15 minutes.

[0052] (3) After stopping heating, remove from oil bath, cool naturally to room temperature while stirring, and store away from light.

[0053] The optical properties of nanoparticles were characterized by UV-visible spectroscopy, such as Figure 2A As shown, when the outer layer of the gold ball is wrapped with a silver layer, the UV-visible absorption peak of the gold ball moves from the original 520nm to 500nm, and the UV-visible absorption peak of silver appears at 384nm, indicating that the silver layer is successfully wrapped.

[0054] In order to further verify the uniformity of the silver layer wrapping, the material was characterized by transmission electron microscopy (TEM). Figure 2C As shown, Au NPs are spherical and are characterized by mapping elements ( Figure 2D to Figure 2F ) proves that the core element of the particle is gold, the shell is silver, and it is a silver-coated gold core-shell structure. The particle size of the gold sphere nanoparticles is 30-40nm, and the thickness of the silver layer is 1.5-2.5nm.

[0055] In addition, by detecting 100U / mL CA15-3 antigen, we found an obvious 4-ATP characteristic peak in the Raman spectrum at the T line ( Figure 2B ), which also proves the successful connection of 4-ATP.

[0056] In summary, it was demonstrated that Au / 4-ATP@Ag NPs were successfully synthesized.

[0057] Preparation Example 2

[0058] See also Figure 1 The chromatography test strip is provided with a sample pad, a binding pad, a detection pad and a water absorption pad in sequence along the chromatography direction, wherein a cellulose acetate membrane is fixed on the detection pad, and a T line and a C line are provided on the cellulose acetate membrane. The specific preparation steps are as follows:

[0059] (1) Using a contact automatic film stripper, 0.1 mg / mL CA15-3 capture antibody was coated at the T line position, and 0.25 mg / mL goat anti-mouse IgG was coated at the C line position, and after drying at 37°C, they were assembled on the top surface of the PVC base plate;

[0060] (2) Add 0.1M K into 1.0mL Au / 4-ATP@Ag NPs solution 2 CO 3 25 μL of the solution was mixed and allowed to stand for 5 min, then 10.0 μL of 0.1 mg / mL CA15-3 labeled antibody was added, and the mixture was reacted at room temperature for 30 min. Then, 10 wt% bovine serum albumin (BSA) was added and reacted for 1 h. The mixture was centrifuged at 4°C and 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was redissolved in 100 μL of a resolubilization solution (containing 1.0% BSA, 1.0% sucrose, 0.5% Tween-20 and 10 mM Tris-HCl buffer) to prepare a SERS probe solution.

[0061] (3) evenly dripping the SERS probe solution onto the treated conjugate pad, and drying it at 37°C after soaking;

[0062] (4) The conjugate pad is attached to the end of the cellulose acetate membrane close to the T line, with the two overlapping by about 1 mm. The sample pad and the conjugate pad are overlapped by about 2 mm and glued to the PVC base plate. The absorbent pad is attached to the end close to the C line.

[0063] Example 1

[0064] 60 μL of blank sample, 0.060 μg / mL carcinoembryonic antigen (CEA), 39 μg / mL total prostate-specific antigen (t-PSA), 1.0 μg / mL carbohydrate antigen 125 (CA125), and 30 U / mL carbohydrate antigen 15-3 (CA15-3) solution were respectively dropped on the paper strip sample pad for specificity test. After 15 minutes of reaction, a portable Raman spectrometer and an infrared thermometer were used to detect the SERS signal on the T line and the temperature signal before and after laser (785 nm) irradiation, and the Raman signal (1340 cm -1 (4-ATP characteristic peak, -NO 2 stretching vibration) and temperature measurements, calculate the △I of each antigen 1340cm -1 and △T, the calculation formulas are:

[0065] △I 1340cm -1 =I 1340cm -1 / I 1340cm -1 (空白) ;

[0066] ΔT=T 2 -T 1 , T 1 is the temperature of the point on the T line before laser irradiation, T 2 It is the temperature of the point corresponding to the T line during laser irradiation.

[0067] See also Figure 3A , measured CA15-3 obtained △ I 1340cm -1 The signal ratios of ΔT and ΔT are much higher than those of CEA, t-PSA and CA125 at higher concentrations, which shows that the chromatographic test strip provided by the present application has good specificity.

[0068] Example 2

[0069] The 0.30U / mL CA15-3 standard solution was tested using chromatographic test strips after being stored for 1, 2, 3, 70, 95, and 120 days.

[0070] The results are as follows Figure 3B As shown, the results are Figure 3B As shown, within 120 days, △I 1340cm -1 The changes in ΔT and ΔT are small. The test results show that the chromatographic test strip of the present application has good stability and can be stably used in the detection of CA15-3.

[0071] Example 3

[0072] 60 μL of CA15-3 solution with different concentrations (0.030 U / mL, 0.30 U / mL, 3.0 U / mL, 30 U / mL, 100 U / mL) was added dropwise to the sample pad. Under the action of chromatography, the sample combined with the SERS probe on the binding pad and flowed through the T line and C line in sequence. After 15 minutes of reaction, a portable Raman spectrometer and an infrared thermometer were used to detect the SERS signal and temperature signal on the T line, respectively, and the Raman signal (1340 cm -1 (4-ATP characteristic peak, -NO 2 stretching vibration) and temperature measurements.

[0073] See also Figure 4A , construct the standard curve of different concentrations of CA15-3 and Raman signal, △I 1340cm -1 There is a good linear relationship between the logarithm of CA15-3 concentration and the linear range is 0.03~100U / mL. The linear equation is: SERS =3.48LgC CA15-3 +1.21(R 2 =0.95).

[0074] The standard curves of different concentrations and temperatures of CA15-3 were constructed. Compared with the blank sample, there was a good linear relationship between △T and the logarithm of CA15-3 concentration. The linear range was 0.03-100 U / mL, and the linear equation was: Y T =2.73LgC CA15-3 +1.18(R 2 =0.89).

[0075] The test results show that the chromatographic test strip provided in this application has good detection performance, and the minimum detection limit of CA15-3 is 0.03U / mL, which is lower than the positive judgment standard (>30U / mL).

[0076] Example 4

[0077] like Figure 5A As shown, the chromatography detection device includes two test strips, each of which is composed of a PVC base plate, a sample pad, a binding pad, a detection pad and a water absorbent pad, wherein the detection pad is fixed with a cellulose acetate membrane, and the cellulose acetate membrane is provided with a T line and a C line.

[0078] The content of CA15-3 in human serum samples was detected using a chromatographic detection device, and the specific steps were as follows:

[0079] Before use, dilute the human serum sample 3 times with PBS (10mM, pH=7.4), take 120μL of the dilution and add it to the chromatography detection device. After 15 minutes of reaction, use a portable Raman spectrometer and infrared thermometer for detection. Use the blank sample as the negative control group, measure five points on each chromatography test strip, and perform three parallel experiments.

[0080] like Figure 5B to Figure 5D As shown in Figure 2, the RSD of Raman signal and temperature change are both less than 5%. Figures 5E to 5F As shown, the RSD of the Raman signal and temperature change of each group in the parallel experiments was less than 6%, indicating that the detection method of the present application has good uniformity and reproducibility when detecting reagent samples.

[0081] Table 1 is a comparison table of the detection method of the present application and the ELISA method in determining the concentration of CA15-3 in serum. From the results in Table 1, it can be seen that the results obtained by the detection method of the present application are equivalent to those of the ELISA method, but the detection time and RSD are greatly improved, which can prove that the temperature-SERS immunochromatographic test strip has a better application prospect in actual detection.

[0082] Table 1 Test results

[0083]

[0084] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A chromatographic test strip for detecting carbohydrate antigen 15-3, comprising a sample pad, a conjugate pad and a detection pad arranged in sequence along the chromatographic direction, characterized in that: The conjugate pad stores gold-silver composite nanoparticles with beacon molecules, the surface of the gold-silver composite nanoparticles is coupled with a first antibody that can specifically bind to the carbohydrate antigen 15-3, and the detection pad stores a second antibody that specifically binds to the carbohydrate antigen 15-3.

2. The chromatography test strip according to claim 1, characterized in that The gold-silver composite nanoparticles are core-shell structures in which the surface of the gold sphere nanoparticles is wrapped by a silver layer.

3. The chromatography test strip according to claim 2, characterized in that The particle size of the gold spherical nanoparticles is 30-40 nm, and the thickness of the silver layer is 1-3 nm.

4. The chromatography test strip according to claim 1, characterized in that: The detection pad comprises a bottom plate and a cellulose acetate membrane fixed on the surface of the bottom plate and used for chromatographic filtration.

5. The chromatography test strip according to claim 1, characterized in that: The detection pad stores a third antibody that specifically binds to the first antibody.

6. The chromatography test strip according to claim 1, characterized in that: The preparation method of the gold-silver composite nanoparticle coupled to the first antibody comprises: The gold-silver composite nanoparticles with beacon molecules are mixed with the first antibody for reaction under strong alkaline conditions, and after the reaction is completed, a site blocking agent is added; The supernatant was removed by centrifugation to separate the conjugate of the first antibody-gold-silver composite nanoparticles-beacon molecule.

7. A method for detecting carbohydrate antigen 15-3, characterized in that: The steps include: Providing a chromatography test strip as claimed in any one of claims 1 to 6; The sample to be tested is added to the sample pad, and after the reaction is completed, the detection pad is irradiated with laser, the Raman signal of the detection pad and / or the temperature difference signal before and after irradiation are collected, and the content of carbohydrate antigen 15-3 in the sample to be tested is calculated.

8. The method according to claim 7, characterized in that It includes constructing a standard curve of Raman signal and carbohydrate antigen 15-3 concentration, and / or a standard curve of temperature difference signal and carbohydrate antigen 15-3 concentration, and calculating the content of carbohydrate antigen 15-3 in the sample to be tested according to the standard curve.

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