Chromatographic test strip and method for detecting microcystic toxins

Abstract

The invention discloses a chromatographic test strip and a method for detecting microcystic toxins, the chromatographic test strip is provided with a sample pad, a combination pad and a detection pad which are sequentially arranged along a chromatographic direction, and silver-gold composite nanoparticles with beacon molecules are stored in the combination pad. The surfaces of the silver-gold composite nanoparticles are coupled with a first antibody which can be specifically bound with the microcystic toxins, and an aptamer which is specifically bound with the microcystic toxins is stored in the detection pad; a sample to be detected is added to the sample pad, after the reaction is completed, the detection pad is irradiated with laser, a Raman signal of the detection pad is collected, and the content of microcystic toxins in the sample to be detected is calculated according to the Raman signal. The detection method provided by the invention can rapidly realize quantitative detection of microcystic toxins in water, and has the advantages of high detection sensitivity, strong specificity and good accuracy.

Description

Technical Field

[0001] The present application relates to the field of biochemical analysis technology, and in particular to a chromatography test strip and method for detecting microcystin. Background Art

[0002] Microcystins (MCs) are a class of cyclic heptapeptide cyanobacterial toxins produced by cyanobacteria and widely found in rivers and lakes. It is reported that it has a significant inhibitory effect on protein phosphatases, so MCs can cause liver failure and even death in humans. Due to the different compositions of the two variable amino acids in the polypeptide, MCs have multiple isomers, and more than 90 isomers of MCs have been found. Among them, microcystin LR (MC-LR) is the most widely distributed, most toxic and most harmful toxin in the MCs family. It contains 7 amino acids, with leucine (L) and arginine (R) at the two variable positions. Humans may be exposed to MC-LR through drinking water or ingestion of contaminated aquatic products. For this reason, my country has stipulated in the Chinese Drinking Water Hygiene Standard (GB 5749-2006) that the content of microcystins in drinking water is limited to 1.0μg / L. Therefore, on-site rapid detection of MC-LR in freshwater sources is essential to ensure compliance with safety standards.

[0003] At present, the main detection methods for MC-LR include high performance liquid chromatography / mass spectrometry (HPLC), liquid chromatography-mass spectrometry (HPLC-MS), enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay (PPIA), etc. Although these detection methods have high detection sensitivity, they are difficult to meet the needs of rapid detection of MC-LR due to disadvantages such as high analysis cost, expensive instruments, and long detection time. Summary of the invention

[0004] In view of the problems of the prior art, the present application provides a chromatographic test strip and method for detecting microcystins, which can realize on-site rapid detection of microcystins in water samples.

[0005] The chromatographic test strip for detecting microcystin 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 silver-gold composite nanoparticles with beacon molecules, the surface of the silver-gold composite nanoparticles is coupled with a first antibody that can specifically bind to the microcystin, and the detection pad stores an aptamer that specifically binds to the microcystin.

[0006] Optionally, the silver-gold composite nanoparticles are a core-shell structure in which a gold layer wraps the surface of a silver sphere nanoparticle.

[0007] Optionally, the particle size of the silver sphere nanoparticles is 30-40 nm, and the thickness of the gold layer is 1-2 nm.

[0008] Optionally, the microcystin is microcystin LR (MC-LR), and the first antibody is an antibody that can specifically bind to MC-LR.

[0009] Optionally, the aptamer is an aptamer that can specifically bind to microcystin LR, such as 5'-Biotin-GGCGCCAAACAGGACCACCATGACAATTACCCATACCACC TCATTATGCCCCATCTCCGCGAGGTGGTATGGGTA-3' (SEQ ID NO: 1).

[0010] Optionally, the aptamer is modified with streptavidin (BR).

[0011] Optionally, the content of the aptamer in the detection pad is 0.0005-0.001 nmol / mm 2 .

[0012] Optionally, the detection pad includes a base plate and a cellulose acetate membrane fixed to the surface of the base plate and used for chromatographic filtration.

[0013] Optionally, the detection pad stores a second antibody that specifically binds to the first antibody, and the aptamer and the second antibody are arranged at intervals along the chromatography direction, forming a T line and a C line respectively.

[0014] Optionally, the second antibody is goat anti-mouse IgG.

[0015] Optionally, the beacon molecule is at least one of 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid, 4-mercaptobenzonitrile, and 4-mercaptobenzenethiol, preferably 4-ATP.

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

[0017] Optionally, the concentration of the first antibody is 0.8-1.2 mg / mL, preferably 1.0 mg / mL.

[0018] Optionally, the site blocking agent is bovine serum albumin (BSA), and the concentration of the bovine serum albumin is 10 wt %.

[0019] The present application also provides a method for detecting microcystin, comprising the following steps:

[0020] Providing the chromatography test strip;

[0021] The sample to be tested is added to the sample pad, and after the reaction is completed, the detection pad is irradiated with a laser, the Raman signal of the detection pad is collected, a standard curve between the Raman signal and the microcystin concentration is constructed, and the content of microcystin in the sample to be tested is calculated according to the standard curve.

[0022] Compared with the prior art, the present application combines ultra-sensitive SERS technology and highly specific immune technology, and utilizes chromatographic test strips to rapidly and quantitatively detect microcystins in water samples, and the detection has high sensitivity, strong specificity, and good accuracy. 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 UV-visible spectra of Ag / 4-ATP@Au NPs;

[0025] Figure 2B Transmission electron microscopy image of Ag / 4-ATP@Au NPs;

[0026] Figure 2C Characterization diagram of silver and gold elements in Ag / 4-ATP@Au NPs;

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

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

[0029] Figure 2F The Raman spectra of the Ag / 4-ATP@Au NPs test samples, where a is the blank solution and b is the MC-LR positive sample;

[0030] Figure 3 The different substances in Example 1 at 1075 cm -1 Raman signal intensity histogram at characteristic peaks;

[0031] Figure 4 The different concentrations of MC-LR in Example 2 and the concentrations at 1075 cm -1 Standard curve graph between Raman signal intensities at characteristic peaks;

[0032] Figure 5A This is a physical picture of the MC-LR SERS immunochromatography test strip;

[0033] Figure 5BThese are the Raman spectra of water samples and water samples with different spikes. a~d represent spike concentrations of 0, 5.0 μg / L, 50 μg / L, and 500 μg / L, respectively. DETAILED DESCRIPTION

[0034] The technical solution described in this application is further explained below in conjunction with specific implementation methods, but the present application is not limited thereto.

[0035] In this application, MC-LR monoclonal antibody and biotin-modified MC-LR aptamer were purchased from Sangon Biotech (Shanghai) Co., Ltd., and goat anti-mouse IgG was purchased from Changzhou Sino-US Xinxin Biotechnology Co., Ltd.

[0036] Preparation Example 1

[0037] Ag / 4-ATP@Au NPs were prepared by seed growth method and in situ growth method. The specific steps are as follows:

[0038] 1.1 Synthetic Silver Core

[0039] (1) 95 mL of sodium citrate (0.21 wt%) was heated to 70° C., 1.7 mL of 1 wt% silver nitrate solution was added thereto, and then 2.0 mL of freshly prepared glacial NaBH4 (0.1 wt%) was quickly added. After vigorous stirring for 1 h, the mixture was cooled to room temperature for later use;

[0040] (2) 77 mL of sodium citrate solution (0.026 wt %) was heated to 150° C., 10 mL of the solution cooled in step (1) was added thereto, and after mixing evenly, 1.7 mL of silver nitrate (1 wt %) was added under vigorous stirring. After reacting for 1 h, 2.0 mL of sodium citrate (1 wt %) and 1.7 mL of silver nitrate (1 wt %) were added and the reaction was continued for 1 h. Finally, 2 mL of sodium citrate (1 wt %) and 1.7 mL of silver nitrate (1 wt %) were added. After reacting for 1 h, the mixture was centrifuged at 6000 rpm for 20 min. The supernatant was the silver nanoparticle solution.

[0041] 1.2 Synthesis of Ag / 4-ATP

[0042] Take 10mL of silver nanoparticle solution in a glass bottle, add 1.0μL of 4-ATP (1.0mM) solution under stirring, react at room temperature for 2h, centrifuge at 7000rpm for 20min after the reaction is completed, discard the supernatant to obtain the precipitate, and add 10mL of water to re-dissolve to obtain Ag / 4-ATP solution.

[0043] 1.3 Synthesis of Ag / 4-ATP@Au NPs

[0044] (1) Add 0.24 mL of NaOH (0.2 M) and 3 mL of Na2SO3 (0.01 M) to 4.75 mL of HAuCl4 (0.31 μM) solution, and react in the dark for 12 h;

[0045] (2) To 4.0 mL of Ag / 4-ATP solution, 5.0 mL of PVP (5 wt%), 50 μL of Vc (0.5 M), 0.20 mL of NaOH (0.50 M) and 0.20 mL of Na2SO3 (0.1 M) were added in sequence. After ultrasonic treatment for 10 min, 1.0 mL of the mixed solution after the reaction in step (1) was added. After mixing, the mixture was placed in a 65°C water bath and allowed to react for 4 h. After the reaction was completed, the mixture was centrifuged at 9000 rpm for 20 min, the supernatant was discarded to obtain a precipitate, and the precipitate was redissolved in water to obtain an Ag / 4-ATP@Au NPs solution.

[0046] The optical properties of nanoparticles were characterized by UV-visible spectroscopy, such as Figure 2A As shown, Ag / 4-ATP@Au NPs have characteristic absorption at 418 nm, which is consistent with the literature reports, and the half-peak width is approximately equal to 90 nm, proving that the particles have good uniformity.

[0047] In order to further verify the successful synthesis of Ag / 4-ATP@Au NPs, transmission electron microscopy (TEM) was used to characterize the surface morphology of the nanoparticles. Figure 2B As shown, the overall morphology of the nanoparticles is uniform, showing spherical and quasi-spherical shapes; secondly, through mapping element characterization, it can be found that the core of the particles is silver element and the shell is silver element, which can prove the core-shell structure of the nanoparticles, such as Figure 2C to Figure 2E As shown, the particle size of the silver ball nanoparticles is 30-40 nm, and the thickness of the gold layer is 1-2 nm.

[0048] The synthesized Ag / 4-ATP@Au NPs were dropped onto the surface of a silicon wafer and a Raman spectrometer was used to measure the Raman characteristic peak of 4-ATP (1075 cm -1 , which belongs to CS stretching vibration), which proves the successful connection of 4-ATP ( Figure 2F ). The above characterizations can prove the successful synthesis of Ag / 4-ATP@Au NPs.

[0049] Preparation Example 2

[0050] See also Figure 1 The chromatography test strip is provided with a sample pad, a binding pad, a detection pad and a water-absorbing pad on a PVC bottom plate 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.

[0051] The specific preparation steps are as follows:

[0052] (1) 8.0 μL of MC-LR aptamer (10 μM) and 2.0 μL of streptavidin (10 mg / mL) were mixed and reacted at 37° C. for 2 h, and then 1.0 μL of BSA (10 wt %) was added and reacted for 1 h to obtain a mixed solution.

[0053] (2) Using a film streaker, the mixed solution of step (1) and 0.50 mg / mL goat anti-mouse IgG were streaked onto a cellulose acetate membrane to form a test line (T line) and a quality control line (C line), and then placed in a forced air drying oven at 37° C. for 2.5 h.

[0054] (2) 3.0 μL MC-LR monoclonal antibody (1.0 mg / mL) was mixed with 1.0 mL Ag / 4-ATP@Au NPs solution and reacted at room temperature for 30 min. Then, 100 μL BSA (10 wt %) was added. After 1 h, the mixture was centrifuged at 9000 rpm for 10 min at 4 °C. The precipitate was redissolved in 200 μL of redissolving solution (containing 1.0 wt % BSA, 1.0 wt % sucrose, 0.5 wt % Tween-20 and 10 mM Tris-HCl buffer) to obtain a SERS probe.

[0055] (3) 0.10 g BSA and 0.25 g sucrose were added to 10 mL PBS buffer (10 mM, pH 7.4) in sequence, mixed and then 0.1% Tween-20 was added; the conjugate pad was then immersed in the above mixed solution, taken out and placed in a constant temperature shaker at 37°C for drying, and after 2.5 hours, the SERS probe solution was evenly added on its surface, and after infiltration, it was placed in a forced air drying oven at 37°C for drying, and taken out after 2.5 hours to obtain the conjugate pad / SERS probe.

[0056] (4) Attach the cellulose acetate membrane / MC-LR aptamer to the PVC base plate, then attach the conjugate pad to the end of the cellulose acetate membrane close to the T line, with the two overlapping by about 1 mm, and stick the sample pad to the PVC base plate close to the conjugate pad, with the sample pad overlapping by about 2 mm; attach the absorbent pad to the end close to the C line, with the absorbent pad overlapping by 2 mm with the cellulose acetate membrane / MC-LR aptamer; finally, cut the test strip into a 3 mm wide and 6.5 cm long test strip to obtain a SERS immunochromatography test strip.

[0057] Example 1

[0058] Take 60 μL of 1.0 mg / L ciprofloxacin, 100 μg / L MC-YR, 100 μg / L calcium chloride, 5.0 μg / L MC-LR and blank sample dilution and drop them on the sample pad of the chromatography test strip. After 15 minutes, use a portable Raman spectrometer to detect the T line and its Raman signal and record it.

[0059] like Figure 3 As shown, select 1075cm -1 Comparing the Raman signal intensities at the characteristic peaks, the signal intensity of MC-LR is much higher than that of the interferents, which shows that the immunochromatographic test strip provided in the present application has good specificity.

[0060] Example 2

[0061] Tween-20 was added to 1 mM Tris-HCl buffer (pH = 8.5) to obtain a sample diluent. MC-LR standard solutions of 250 μg / L, 50 μg / L, 5.0 μg / L, 0.50 μg / L, and 0.10 μg / L were prepared using the sample diluent. 60 μL of the above MC-LR standard solutions of different concentrations and the blank sample diluent were added dropwise to the sample pad of the SERS immunochromatographic test strip. After 15 minutes, the T line was detected using a portable Raman spectrometer (wavelength of 785 nm).

[0062] like Figure 4 As shown in the figure, with the increasing concentration of MC-LR, the 1075cm -1 The Raman signal intensity at (the characteristic peak of 4-ATP, belonging to CS stretching vibration) also gradually increased. There was a good linear relationship between the signal intensity and the logarithm of the MC-LR concentration. The linear range was 0.10-250 μg / L. The linear equation was: Y = 970.32 LgC MC-LR +1977.77, linear correlation coefficient (R 2 ) is 0.983.

[0063] The above shows that the chromatographic test strip provided in the present application has good detection performance, and the minimum detection limit is lower than the limit value (1.0 μg / L) of the national standard for MC-LR, and can be applied to the quantitative analysis of MC-LR in water samples.

[0064] Example 3

[0065] (1) Take 10 μL of 500 μg / L, 50 μg / L, and 5.0 μg / L MC-LR standard solutions and add them into 90 μL of Nanhu water sample, respectively. Mix thoroughly to obtain three spiked samples with different concentrations.

[0066] (2) 60 μL of the above-mentioned different spiked actual samples and Nanhu water were added to the sample pad of the SERS immunochromatography test strip. After 15 minutes, the T line was detected and the Raman signal was detected and recorded using a portable Raman spectrometer.

[0067] (3) Based on the standard linear curve, the concentration of MC-LR in different samples was obtained, and its recovery rate and relative standard deviation were calculated.

[0068] This example uses South Lake water as the actual sample, and the results are as follows Figure 5A As shown, there is an obvious C line, and the Raman signal of the signal molecule can be detected at the T line ( Figure 5B , a line), at 1075cm -1 Substitute the Raman intensity at into the linear equation obtained in Example 2 to obtain the MC-LR concentration in the sample, see Table 1.

[0069] In order to verify the accuracy of this method, we added MC-LR of different concentrations into the samples to obtain different spiked samples and tested them. The results are shown in Figure 2. Figure 5B As shown in the figure, with the increase of spike concentration, the Raman signal of the signal molecule gradually increases. Similarly, we used three different spiked samples at 1075 cm -1 The Raman intensity at 100 nm was substituted into the obtained linear equation to obtain the MC-LR concentration in different spiked samples. The results are shown in Table 1. The relative standard deviations were all within 10%, and the recoveries were between 82% and 105%, indicating that this method has good stability and accuracy in actual sample determination.

[0070] Table 1 Test results

[0071]

[0072] 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 microcystin, comprising a sample pad, a binding pad and a detection pad arranged in sequence along the chromatographic direction, characterized in that: The conjugate pad stores silver-gold composite nanoparticles with beacon molecules, the surface of the silver-gold composite nanoparticles is coupled with a first antibody that can specifically bind to the microcystin, and the detection pad stores an aptamer that specifically binds to the microcystin.

2. The chromatography test strip according to claim 1, characterized in that The silver-gold composite nanoparticles are a core-shell structure in which a gold layer wraps the surface of a silver ball nanoparticle.

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

4. The chromatography test strip according to claim 1, characterized in that: The microcystin is microcystin LR.

5. The chromatography test strip according to claim 4, characterized in that: The aptamer is a nucleotide sequence as shown in SEQ ID NO:

1.

6. The chromatography test strip according to claim 1, characterized in that: The content of the aptamer in the detection pad is 0.0005-0.001 nmol / mm 2 .

7. 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.

8. The chromatography test strip according to claim 1, characterized in that: The detection pad stores a second antibody that can specifically bind to the first antibody.

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

10. A method for detecting microcystin, characterized in that: The steps include: Providing a chromatography test strip as claimed in any one of claims 1 to 9; The sample to be tested is added to the sample pad, and after the reaction is completed, the detection pad is irradiated with a laser, the Raman signal of the detection pad is collected, a standard curve between the Raman signal and the microcystin concentration is constructed, and the content of microcystin in the sample to be tested is calculated according to the standard curve.

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