High-throughput population-targeted steroid estrogen detection method based on biolayer interferometry

Abstract

The present invention provides a high-throughput population-targeted steroid estrogen detection method based on biolayer interference, which can realize highly sensitive population-targeted identification and detection of steroid estrogens in water. The nucleic acid aptamers of steroid estrogens are screened and obtained by exponential enrichment ligand system evolution technology, and nucleotide-specific editing is performed to construct the first and second aptamers. The first aptamer is fixed to the surface of the streptavidin sensor, and the second aptamer specifically binds to the target steroid estrogen to form a second aptamer-steroid estrogen complex. During the detection, the first aptamer on the streptavidin sensor captures the second aptamer-steroid estrogen complex, resulting in a change in the thickness of the biolayer at the tip of the sensor, and the induced wavelength shift can quantify the concentration of steroid estrogens in water. The method can simultaneously identify estrone, 17β-estradiol and ethinyl estradiol, with a detection concentration of 0.001~1 nM and a detection time of 10 minutes.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection of new pollutants in environmental protection, and develops a high-throughput population-targeted steroid estrogens (SEEs) detection method based on biolayer interferometry (BLI), which is suitable for sensitive and rapid detection of low-concentration steroid estrogens in real water environments. Background Art

[0002] Environmental endocrine disruptors are a typical new class of pollutants. They have low exposure doses and long retention times in nature. They have strong bioaccumulation toxicity, endocrine disruption and even carcinogenicity, and seriously threaten public health. Among them, steroid estrogens (SEEs) include estrone (E1), 17β-estradiol (E2), estriol (E3) and ethinyl estradiol (EE2) and other homologues. They are similar in structure and toxicity, and are prone to produce toxic cumulative mixed effects. This type of pollutant often exists in trace forms in actual water bodies, and coexists with other pollutants and environmental background matrices, making analysis and detection difficult. Although the commonly used chromatographic separation combined with mass spectrometry detection method can achieve nM to pM level detection of SEEs, there are still bottlenecks in practical application. First, the actual sample composition is very complex, and coexisting pollutants with similar carbon skeleton structures to SEEs are easy to interfere with the chromatographic separation effect and affect the accuracy of the analysis results. Secondly, mass spectrometry equipment is expensive, usually requires pre-treatment steps such as extraction and concentration, has a long analysis cycle, and has high requirements for analytical operators, making it difficult to achieve high-throughput in-situ detection. Therefore, it is urgent to develop new analytical detection methods to efficiently identify SEEs pollutants with similar chemical structures and properties, and to achieve high-throughput population targeted analysis of such pollutants. Summary of the invention

[0003] The purpose of the present invention is to provide a high-throughput population-targeted SEEs detection method based on the principle of biolayer interferometry (BLI), which can achieve highly sensitive population-targeted identification and detection of SEEs in complex water environment media and has strong resistance to environmental interference. SEEs nucleic acid aptamers are screened and obtained by exponential enrichment ligand systematic evolution (SELEX) technology, and are fixed on the surface of BLI biosensor. During the detection process, the target SEEs molecules are captured by the nucleic acid aptamers, resulting in changes in the thickness of the biofilm layer at the tip of the BLI sensor. The wavelength shift caused by this can be used to quantify the concentration of SEEs in the water environment medium. This method is fast, sensitive, and resistant to environmental interference.

[0004] The specific technical solution for achieving the purpose of the present invention is:

[0005] A high-throughput population-targeted steroid estrogen detection method based on biolayer interferometry, characterized by: nucleotide editing of the aptamer obtained by SELEX screening to obtain a first aptamer and a second aptamer; then labeling the first aptamer with biotin and immobilizing it on the surface of a streptavidin (SA) sensor; the second aptamer specifically binds to the steroid estrogen SEEs in the detection well plate to form a second aptamer-SEEs complex; the first aptamer immobilized on the surface of the SA sensor captures the second aptamer-SEEs complex, and the concentration of steroid estrogen SEEs in water is quantitatively analyzed by the wavelength shift caused by the increase in optical thickness of the SA sensor tip, specifically including:

[0006] (1) Performing nucleotide editing on the SEEs aptamers obtained by SELEX screening to construct a first aptamer and a second aptamer targeting a SEEs population with a base number; the first aptamer sequence: 5'-CGAAGTCGCCATCTCTTCAGCGGAACGTGGGGAGCTTGCGGGCATACAAACTATGG-3'; the second aptamer sequence: 5'-GAATAGTGAGTCGTATTAAGCTGATCCTATGG-3'; the SEEs include estrone (E1), 17β-estradiol (E2), and ethinyl estradiol (EE2);

[0007] (2) labeling the first aptamer targeting the SEEs population constructed in step (1) with biotin; the concentration of the first aptamer is 125-1000 nM; the concentration of biotin is 4-6 μM;

[0008] (3) The SEEs population-targeting second aptamer constructed in step (1) is mixed with SEEs in a PBS+MgCl2 buffer solution system, wherein the MgCl2 concentration is 1-3 mM, and the reaction is carried out in a 96-well plate. SEEs and the second aptamer undergo a binding reaction to generate a second aptamer-SEEs complex solution, wherein the second aptamer concentration is 125-500 nM, and the binding time is 150-300 s;

[0009] (4) placing the SA sensor in the biotin-labeled targeted first aptamer solution obtained in step (2) to couple the first aptamer to the surface of the SA sensor, thereby preparing a SA sensor with the first aptamer fixed thereon;

[0010] (5) immersing the SA sensor immobilized with the first aptamer obtained in step (4) in a biotin solution to block the avidin sites on the SA sensor that have not yet been bound;

[0011] (6) The biotin-blocked SA sensor in step (5) is immersed in the SEEs population-targeted second aptamer-SEEs complex solution in step (3). The first aptamer immobilized on the surface of the SA sensor captures the second aptamer-SEEs complex in the solution, causing the optical thickness of the SA sensor surface to increase and the interference wavelength to shift. The total SEEs concentration is quantified by detecting the response signal, and the detection concentration range is 0.001 to 1 nM.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The high-throughput population-targeted SEEs detection method based on the BLI principle described in the present invention integrates oligonucleotide editing technology and multi-channel BLI sensing technology, can simultaneously identify SEEs with the same core carbon skeleton, has a detection range of 0.001 to 1 nM, and takes only 10 minutes to detect samples. It can effectively resist the interference of complex background matrices in water environments and is a sensitive, accurate, and high-throughput population-targeted SEEs detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The secondary structure diagram of the first aptamer and the second aptamer targeting the estrogen population obtained in Example 1 of the present invention;

[0015] Figure 2 This is a graph showing the change of instrument response signal with SEEs concentration when the BLI technology is used to quantify the SEEs concentration in water in Experimental Example 1 of the present invention;

[0016] Figure 3 This is a standard curve diagram showing the linear correlation between the instrument response signal and the SEEs concentration when the BLI technology is used to quantify the SEEs concentration in water in Experimental Example 1 of the present invention;

[0017] Figure 4 This is a graph showing the performance of SEEs against background pollutants in complex water bodies measured using the BLI technology in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0019] Example 1

[0020] A high-throughput population-targeted steroid estrogens (SEEs) detection method based on biolayer interferometry (BLI) comprises the following steps:

[0021] (1) The original SEEs-SELEX aptamer was effectively nucleotide edited to construct a first aptamer with a base length of 56 bp and a second aptamer with a base length of 33 bp, see Figure 1, Figure a is a secondary structure diagram of the first aptamer, and Figure b is a secondary structure diagram of the second aptamer;

[0022] (2) biotin-labeling the first aptamer constructed in step (1);

[0023] (3) The second aptamer with a concentration of 125 nM constructed in step (1) was mixed with 10 nM SEEs in different buffer solution systems for 5 min for specific recognition, and the buffer solution used for binding was PBS + 2 mM MgCl2;

[0024] (4) The commercial SA sensor was pre-wetted in PBS buffer solution for 30 min;

[0025] (5) Soak the SA sensor in step (4) in PBS buffer solution for 60 seconds to obtain a stable baseline;

[0026] (6) Soaking the SA sensor in step (5) in the first aptamer in step (2) to couple avidin and biotin to prepare a SA sensor loaded with the first aptamer, wherein the concentration of the first aptamer is 125 nM and the fixation time is 150 s;

[0027] (7) Soaking the SA sensor loaded with the first aptamer in step (6) in a 4 μM biotin solution to block the excess avidin binding sites on the sensor for 60 s;

[0028] (8) Soaking the biotin-blocked SA sensor in step (7) in a PBS buffer solution to obtain a stable baseline;

[0029] (9) The SA sensor in step (8) is immersed in the SEEs solution that has been recognized by the second aptamer in step (3). The first aptamer fixed on the surface of the SA sensor captures the second aptamer-SEEs complex in the solution for 150 seconds. The optical thickness of the SA sensor surface increases, causing the interference wavelength to shift. The response signal is detected and the total SEEs concentration is quantified according to the standard curve.

[0030] Comparative Example 1

[0031] A blank control identification test, the steps are as follows:

[0032] (1) Effectively edit the nucleotides of the original SEEs-SELEX aptamer to construct a first aptamer with a base number of 56 bp and label it with biotin;

[0033] (2) The commercial SA sensor was pre-wetted in PBS buffer solution for 30 min;

[0034] (3) Soak the SA sensor in step (2) in PBS buffer solution for 60 seconds to obtain a stable baseline;

[0035] (4) Soaking the SA sensor in step (3) in the first aptamer in step (2) to couple avidin and biotin to prepare a SA sensor loaded with the first aptamer, wherein the concentration of the first aptamer is 125 nM and the fixation time is 150 s;

[0036] (5) Soaking the SA sensor loaded with the first aptamer in step (4) in a 4 μM biotin solution to block the excess avidin binding sites on the sensor for 60 s;

[0037] (6) Soaking the biotin-blocked SA sensor in step (5) in a PBS buffer solution to obtain a stable baseline;

[0038] (7) The SA sensor in step (6) was immersed in PBS buffer solution as a blank control group, and the capture time was 150 s.

[0039] Comparative Example 2

[0040] A blank control identification test, the steps are as follows:

[0041] (1) Effectively edit the nucleotides of the original SEEs-SELEX aptamer to construct a first aptamer with a base length of 56 bp and a second aptamer with a base length of 33 bp;

[0042] (2) biotin-labeling the first aptamer constructed in step (1);

[0043] (3) The commercial SA sensor was pre-wetted in PBS buffer solution for 30 min;

[0044] (4) Soak the SA sensor in step (3) in PBS buffer solution for 60 seconds to obtain a stable baseline;

[0045] (5) Soaking the SA sensor in step (4) in the first aptamer in step (2) to couple avidin and biotin to prepare a SA sensor loaded with the first aptamer, wherein the concentration of the first aptamer is 125 nM and the fixation time is 150 s;

[0046] (6) Soaking the SA sensor loaded with the first aptamer in step (5) in a 4 μM biotin solution to block the excess avidin binding sites on the sensor for 60 s;

[0047] (7) Soaking the biotin-blocked SA sensor in step (6) in a PBS buffer solution to obtain a stable baseline;

[0048] (8) Soak the SA sensor in step (7) in the second aptamer solution in step (1) for a capture time of 150 s.

[0049] Experimental Example 1

[0050] BLI technology was used to quantify the concentration of SEEs in water.

[0051] First, SEEs of different concentrations were prepared by serial dilution in binding buffer (PBS + 1-3 mM MgCl2). Before the measurement, 200 μL of PBS buffer solution was added to the first column of wells of the 96-well black plate to obtain a stable baseline. A biotinylated first aptamer with a concentration of 125-1000 nM was added to the second column of wells, and the biotinylated first aptamer was coupled with avidin on the surface of the SA sensor to achieve the effect of immobilizing the first aptamer. A 4-6 μM biotin solution was added to the third column of wells to block the excess binding sites on the sensor.

[0052] The second step is the binding step, and 200 μL of PBS buffer solution is added to the fourth column of wells to obtain a stable baseline 2. The first row of the fifth column of wells is the blank control group in step (7) of comparative example 1. The second row is the blank control group in step (8) of comparative example 2. The third to eighth rows are experimental groups. The sixth column is PBS buffer solution for dissociation. Figure 2 This is the BLI response signal change diagram, where ah represents the SEEs concentration range of 0.001-1nM; Figure 3 This is a standard curve diagram showing the linear correlation between the instrument response signal and the SEEs concentration when using BLI technology to quantify the SEEs concentration in water.

[0053] Experimental Example 2

[0054] BLI technology is used to measure the anti-background interference performance of SEEs in complex water bodies.

[0055] First, SEEs of different concentrations were prepared by serial dilution in binding buffer (PBS + 1-3 mM MgCl2). Before the measurement, 200 μL of PBS buffer solution was added to the first column of the 96-well black plate to obtain a stable baseline. The first aptamer with a concentration of 125-1000 nM was added to the second column, and the first aptamer was coupled with the avidin on the sensor surface to immobilize the first aptamer. A 4-6 μM biotin solution was added to the third column to block the excess binding sites on the sensor.

[0056] The second step is the combination step, and 200 μL of PBS buffer solution is added to the fourth column to obtain a stable baseline. The first row of the fifth column of the well plate is the blank control group in step (7) of comparative example 1. The second row is the blank control group in step (8) of comparative example 2. The third to eighth rows are experimental groups, which respectively study the anti-interference performance of the detection method established when the interfering substance is alone or SEEs coexist. The concentration of the interfering substance is 100 times the concentration of SEEs. The interfering substances are divided into sulfamethoxazole (SMZ), norfloxacin (NOR), bisphenol A (BPA), humic acid (HA), Na2SO4, NaCl, and histidine (His). The sixth column is a PBS buffer solution for dissociation. Figure 4 This is a diagram showing the performance of BLI technology in measuring the resistance of SEEs to background pollutant interference in complex water bodies.

Claims

1. A high-throughput population-targeted steroid estrogen detection method based on biolayer interferometry, characterized in that: The aptamers obtained by exponential enrichment ligand system evolution screening are subjected to nucleotide editing to obtain a first aptamer and a second aptamer; the first aptamer is then labeled with biotin and fixed on the surface of a streptavidin sensor; the second aptamer specifically binds to steroid estrogen in a detection well plate to form a second aptamer-steroid estrogen complex; the first aptamer fixed on the surface of the streptavidin sensor captures the second aptamer-steroid estrogen complex, and the concentration of steroid estrogen in water is quantitatively analyzed by wavelength shift caused by the increase in optical thickness of the tip of the streptavidin sensor, specifically including: (1) The steroid estrogen aptamers obtained by exponential enrichment ligand system evolution screening were subjected to nucleotide editing to construct the first aptamer and the second aptamer targeting the steroid estrogen population; the sequence of the first aptamer was: 5'-CGAAGTCGCCATCTCTTCAGCGGAACGTGGGGAGCTTGCGGGCATACAAACTATGG-3'; Second aptamer sequence: 5'-GAATAGTGAGTCGTATTAAGCTGATCCTATGG-3'; the steroid estrogen includes estrone, 17β-estradiol, and ethinyl estradiol; (2) labeling the steroid estrogen population-targeting first aptamer constructed in step (1) with biotin; the concentration of the first aptamer is 125-1000 nM; the concentration of biotin is 4-6 µM; (3) The steroid estrogen population-targeting second aptamer constructed in step (1) is mixed with steroid estrogen in a PBS+MgCl2 buffer solution system, where the MgCl2 concentration is 1-3 mM. The reaction is carried out in a 96-well plate. The steroid estrogen binds to the second aptamer to generate a second aptamer-steroid estrogen complex solution. The second aptamer concentration is 125-500 nM, and the binding time is 150-300 s. (4) placing the streptavidin sensor in the biotin-labeled targeted first aptamer solution obtained in step (2) to couple the first aptamer to the surface of the streptavidin sensor, thereby preparing a streptavidin sensor immobilized with the first aptamer; (5) immersing the streptavidin sensor immobilized with the first aptamer obtained in step (4) in a biotin solution to block the avidin sites on the streptavidin sensor that have not yet been bound; (6) The biotin-blocked streptavidin sensor in step (5) is immersed in the steroid estrogen population-targeted second aptamer-steroid estrogen complex solution in step (3). The first aptamer immobilized on the surface of the streptavidin sensor captures the second aptamer-steroid estrogen complex in the solution, causing the optical thickness of the streptavidin sensor surface to increase, triggering an interference wavelength shift, and the steroid estrogen concentration is quantified by detecting the response signal. The detection concentration range is 0.001 ~ 1 nM.

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