CN114965408A - Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor - Google Patents

Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor Download PDF

Info

Publication number
CN114965408A
CN114965408A CN202210570429.9A CN202210570429A CN114965408A CN 114965408 A CN114965408 A CN 114965408A CN 202210570429 A CN202210570429 A CN 202210570429A CN 114965408 A CN114965408 A CN 114965408A
Authority
CN
China
Prior art keywords
chlorpromazine
aptamer
dye
rho6g
uio
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210570429.9A
Other languages
Chinese (zh)
Inventor
段诺
宋铭倩
吴世嘉
李昌鑫
王周平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN202210570429.9A priority Critical patent/CN114965408A/en
Publication of CN114965408A publication Critical patent/CN114965408A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices

Landscapes

  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for detecting chlorpromazine by using a ratio type fluorescent aptamer sensor, belonging to the technical field of detection. The invention will Uio-66-NH 2 The nanometer material and a Rho6G dye are incubated to obtain MOF-Rho6G, and then chlorpromazine aptamer is added to incubate to obtain a probe MOF-Rho 6G-Apt. The application of the probe can improve the detection sensitivity to the chlorpromazine, and compared with an ELISA kit, the probe has the advantages of good specificity, no need of an antibody, wide detection range and the like.

Description

Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor
Technical Field
The invention relates to a method for detecting chlorpromazine by using a ratio type fluorescent aptamer sensor, belonging to the technical field of detection.
Background
Chlorpromazine is a phenothiazine medicine and is mainly used for enhancing hypnosis, anesthesia, sedation and the like, and the chlorpromazine added into the feed can play an indirect fattening role on animals. GB 31650-2019 states that chlorpromazine is approved for therapeutic use but cannot be detected in animal foods.
At present, more methods are used for measuring chlorpromazine residues. The method for detecting chlorpromazine in animal derived food mainly comprises an enzyme-linked immunosorbent assay, a gas chromatography, a high performance liquid chromatography, a gas chromatography-tandem mass spectrometry, a liquid chromatography-tandem mass spectrometry and the like. Among them, gas chromatography tandem mass spectrometry and liquid chromatography tandem mass spectrometry are the most commonly used, and although chromatography has high detection sensitivity and accurate detection result, expensive instruments and equipment are required, sample pretreatment steps are complicated, and a large amount of environmentally unfriendly organic reagents are required. The enzyme linked immunosorbent assay screen depends on antibodies, and although some enzyme linked immunosorbent assays can achieve rapid and simple detection, the preparation process of the antibodies is complex, and the antibodies in different batches have different differences, which is a defect of the enzyme linked immunosorbent assay.
Aptamer (Aptamer) refers to a DNA or RNA molecule isolated by selection using the ligand system evolution by exponential enrichment (SELEX) technique, which can bind with high affinity and specificity to other targets such as proteins, metal ions, small molecules, polypeptides, and even whole cells. Therefore, the nucleic acid aptamer has wide application prospects in the aspects of biochemical analysis, environmental monitoring, basic medicine, new drug synthesis and the like. Compared with the antibody used by the enzyme-linked immunosorbent assay, the aptamer has the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short preparation period, artificial synthesis and the like, and a series of processes of animal immunization, feeding, protein extraction and purification and the like required by antibody preparation are omitted. Therefore, the aptamer is an ideal molecular probe and can be used as a material for a fluorescence detection method.
Fluorescence sensors with single fluorescence emission are widely used due to their simple design and high selectivity. However, the problems of detecting the influence of the environment and requiring higher sensor sensitivity still remain. The ratio (ratio type) fluorescence sensor has a greater practical value than the conventional fluorescence sensor. Interference caused by detection environment and instrument parameters is reduced through self-calibration dual-emission peak detection.
However, when a ratiometric fluorescent aptamer sensor is used to detect chlorpromazine, the problem still exists in which material is selected as the fluorescence source and how to construct the ratiometric fluorescence emission.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is that the method for detecting chlorpromazine by using a ratio fluorescence method by using a nucleic acid aptamer is lacked in the prior art.
[ solution ]
The invention firstly provides a ratio type fluorescent aptamer sensor for detecting chlorpromazine, and the preparation method comprises the following steps:
(1) Uio-66-NH 2 Dissolving the nanometer material in buffer solution, adding Rho6G dye, mixing, incubating to allow the dye to react with Uio-66-NH 2 Fully contacting the nano materials to obtain MOF-Rho 6G;
(2) adding chlorpromazine aptamer, mixing and incubating such that the chlorpromazine aptamer seals Rho6G dye to Uio-66-NH 2 Obtaining a probe MOF-Rho6G-Apt from the nanometer material;
(3) the probe MOF-Rho6G-Apt was washed to remove free Rho6G dye and chlorpromazine aptamer.
In some embodiments of the present invention, the pH of the buffer solution in step (1) is 7.0 to 7.4, and can be selected from: PBS buffer (1.4mM KH) 2 PO 4 ,4.3mM Na 2 HPO 4 137mM NaCl,2.7mM KCl, pH 7.2-7.4), or, a binding buffer (50mM Tris-HCl,5mM KCl,100mM NaCI,1mM MgCl 2 ,pH 7.4)。
In certain embodiments of the invention, in step (1), Uio-66-NH 2 The ratio of the nano material, the Rho6G dye and the buffer solution can be 3:0.03:1, 3:0.06:1, 3:0.15:1, 3:0.3:1, 3:0.6:1, 3:1.5:1, 3:4.5:1 or 3:0.03: 1.
In certain embodiments of the present invention, in step (1), the incubation is performed at 35-37 ℃ for 10-240 min.
In certain embodiments of the invention, in step (2), the amount of the chlorpromazine aptamer is 1 μ M to 10 μ M.
In certain embodiments of the present invention, in the step (2), the incubation is performed at 25-30 ℃ for 30-180 min.
In certain embodiments of the invention, the sequence of the aptamer may be: 5'-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGAACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3', or alternatively,
5'-AATCAAACGCTAAGGTCGGAGGAAAGTGCACCCATTCTTGCGCCACAAGACACACTGATCGAACGCCGCCAAGCTTGGTACCCGTATCGT-3', or alternatively,
5′-AATCAAACGCTAAGGTAAGGACGAGGTGCACCCATTCTTGGGCACGCGGCACGCGCGAGGACGACCAGGCAAGCTTGGTACCCGTATCGT-3′。
in certain embodiments of the present invention, in step (3), the solution used for washing may be PBS buffer (1.4mM KH) 2 PO 4 ,4.3mM Na 2 HPO 4 137mM NaCl,2.7mM KCl, pH 7.2-7.4), binding buffer (50mM Tris-HCl,5mM KCl,100mM NaCI,1mM MgCl 2 ,pH 7.4)。
In certain embodiments of the invention, in step (3), after washing, the probe is dissolved in a binding buffer and stored at 4 ℃ until use.
In certain embodiments of the present invention, a method for preparing a ratiometric fluorescent aptamer sensor for detecting chlorpromazine comprises the steps of:
(1) 3mg of Uio-66-NH 2 Dissolving the nano material in 500 mu L of PBS buffer solution with pH 7.4, dispersing the nano material by using ultrasound, adding 500 mu L of 3mg/mL Rho6G dye, uniformly mixing, and incubating at room temperature of 200rpm for half an hour to ensure that the dye is fully contacted with the MOF material to obtain MOF-Rho 6G;
(2) adding 80 μ L of 100 μ M chlorpromazine aptamer, mixing, and incubating overnight at room temperature to allow the chlorpromazine aptamer to seal Rho6G to Uio-66-NH 2 Obtaining a probe MOF-Rho6G-Apt from the MOF nano material;
(3) the probe was washed 3 times with PBS solution to remove free dye and aptamers, and then dissolved in binding buffer and stored at 4 ℃ for use.
The invention provides a ratio type fluorescent aptamer sensor for detecting chlorpromazine, which is prepared by applying the preparation method. The sensor is composed of Uio-66-NH 2 The nano material forms a main body framework structure at Uio-66-NH 2 The pores of the nano material are filled with rhodamine 6G dye, and meanwhile, Uio-66-NH 2 The surface of the nano material is combined with a chlorpromazine aptamer single chain through electrostatic interaction so as to seal the rhodamine 6G dye in Uio-66-NH 2 Pores of the nanomaterial.
The invention provides a method for detecting chlorpromazine by using the sensor, which comprises the following steps:
(1) preparation of fluorescence emission spectra F of the ratiometric fluorescent aptamer sensor under 365nm excitation 550 /F 425 A standard curve of the concentration of chlorpromazine,
(2) adding chlorpromazine solution to be detected into the ratio type fluorescent aptamer sensor, fixing the volume with binding buffer solution, incubating, centrifuging to remove supernatant, resuspending the precipitate with binding buffer solution, mixing uniformly, measuring fluorescence emission spectrum under 365nm excitation, and calculating F 550 /F 425 ，
(2) F is to be 550 /F 425 Substituting the standard curve to calculate the concentration of chlorpromazine in the solution to be measured.
[ advantageous effects ]
The ratio type fluorescent aptamer sensor is prepared by utilizing the promethazine aptamer, on one hand, the specificity of the detection method is ensured, and on the other hand, the ratio type fluorescent aptamer sensor is based on the nano material Uio-66-NH 2 And dye rhodamine 6G (Rho 6G) can emit fluorescence near 425nm and 550nm under the excitation of 365nm to establish a dual-emission fluorescent probe, so that the detection sensitivity to chlorpromazine can be improved. When the solution is free of chlorpromazine, the dye is sealed by the aptamer and is Uio-66-NH 2 In the pores of the nano material, the probe has a higher dye emission peak besides the fluorescence emission peak carried by the MOF, F 550 /F 425 The ratio is stable; when chlorpromazine is added, the chlorpromazine aptamer is combined with the chlorpromazine to generate conformational change, the seal is opened, the dye is leaked, the dye emission peak of the probe at 550nm is reduced, the material emission peak at 425nm is almost unchanged, and F 550 /F 425 The ratio is reduced, and the chlorpromazine content is detected by an aptamer fluorescence sensor forming a ratio type.
Compared with an ELISA kit, the ratio-type aptamer fluorescence sensor prepared by the invention has the advantages of good specificity, no need of an antibody, wide detection range and the like.
The fluorescent probe prepared by the invention has high sensitivity, and the detection Limit (LOD) is 0.67 nM; the specificity is strong, and chlorpromazine and analogs thereof (azaperone, haloperidol, promethazine or acepromazine) can be distinguished remarkably; the method is suitable for detecting chlorpromazine residues in food, especially food substrates such as milk and eggs, and has no obvious influence on detection results.
Drawings
FIG. 1 is a schematic diagram of the ratiometric fluorescence detection of chlorpromazine.
FIG. 2 Probe characterization: (a) MOF-Rho and MOF-Rho6G-Apt fluorescence emission spectra with Uio-66-NH in the UV dark box in the upper right corner 2 And MOF-Rho6G-Apt, (b) UV-Vis spectra of supernatant of Rho6G, MOF-Rho6G and MOF-Rho6G-Apt, (c) Uio-66-NH 2 And particle size of MOF-Rho6G-Apt, (d) Uio-66-NH 2 And the potential values of MOF-Rho6G-Apt, (e) Uio-66-NH 2 And the isothermal adsorption curves of MOF-Rho6G-Apt, (f) Uio-66-NH 2 And the infrared pattern of MOF-Rho 6G-Apt.
FIG. 3 optimization of aptamer concentration (a) and probe incubation time with chlorpromazine (b).
FIG. 4(a) is a fluorescence spectrum of chlorpromazine at different concentrations, and (b) is a standard curve for identifying chlorpromazine by CHL-3.
FIG. 5 fluorescence ratios of probes after addition of different targets.
Detailed Description
The terms: Uio-66-NH 2 The MOFs is a Metal-organic framework material.
Example 1 preparation and characterization of the Probe MOF-Rho 6G-DNA
3mg of MOFs (i.e., Uio-66-NH) 2 Nano material) is dissolved in 500 μ L of PBS buffer (pH 7.4) and dispersed with ultrasound, 500 μ L of 3mg/mL Rho6G (rhodamine 6G dye) is added, mixed well and incubated at room temperature at 200rpm for half an hour to allow the dye to fully contact the MOF material, obtaining MOF-Rho 6G. Adding 80 μ L of 100 μ M chlorpromazine aptamer, mixing, and incubating overnight at room temperature to allow the chlorpromazine aptamer to seal Rho6G to Uio-66-NH 2 In the nano material, the nano material is prepared by mixing the raw materials,obtaining the probe MOF-Rho 6G-Apt. The probes were washed 3 times with PBS solution to remove free dye and aptamers, and then dissolved in binding buffer (50mM Tris-HCl,5mM KCl,100mM NaCI,1mM MgCl 2 pH 7.4) at 4 ℃ for further use. The sequence of the chlorpromazine aptamer is as follows: 5'-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGA ACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3' are provided.
Firstly Uio-66-NH 2 And (3) characterizing the fluorescence of the nano material, the dye Rho6G and the probe MOF-Rho 6G-Apt. Uio-66-NH as shown in FIG. 2(a) 2 The nano material and the Rho6G dye can respectively emit fluorescence near 425nm and 550nm at an excitation wavelength of 365 nm. It can be seen that the probe with the clorpromazine aptamer seal has a higher dye fluorescence peak at 550nm and a material fluorescence peak at 425nm, and the appearance of double emission peaks indicates that the fluorescent probe has been successfully prepared. Uio-66-NH as shown in the upper right hand small panel of FIG. 2(a) 2 The nano material and the MOF-Rho6G-Apt can respectively generate blue fluorescence and green fluorescence under 365nm excitation.
And carrying out ultraviolet spectrum scanning on Rho6G dye which is not incubated with the MOFs, centrifugal supernatant obtained after incubation of the MOFs and the Rho6G, and centrifugal supernatant obtained after incubation of MOF-Rho6G and the aptamer. As shown in FIG. 2(b), the original Rho6G dye has a UV peak of about 525nm, and the UV curves of the supernatant MOF-Rho6G and the supernatant MOF-Rho6G-apt are both lower than the original concentration, indicating that the concentration of the dye in the supernatant is lower than the original concentration of the dye, and the dye is successfully adsorbed onto the MOFs. The spectrum of the MOF-Rho6G-Apt supernatant had the lowest value at 525nm, indicating that more dye was retained in the MOFs and that the aptamer had a sealing effect on the dye.
Pair Uio-66-NH 2 The particle sizes of the nanomaterials and the MOF-Rho6G-Apt were measured. FIG. 2(c) is a particle size chart before and after modification of the material, Uio-66-NH 2 The grain diameter of the nano material is 113.3nm, and the grain diameter of the MOF-Rho6G-Apt is 237.5 nm. The hydrated particle size is increased after MOF modification, which indicates that the surface of the material is successfully coated with a new substance. Zeta potential analysis showed the charge change before and after MOF modification, Uio-66-NH, as shown in FIG. 2(d) 2 The nano material is positively charged, and the potential is 32.5 mV. The dye Rho6G is charged to MOFsAfter being sealed by promethazine aptamer, the formed probe potential is greatly changed, and the negative potential of MOF-Rho6G-Apt is-20.5 mV. Uio-66-NH analysis using a fully automated specific surface area analyzer 2 And MOF-Rho 6G-Apt. Calculated, Uio-66-NH, as shown in FIG. 2(e) 2 BET surface area of about 779.36m 2 Per g, pore volume of about 0.320cm 3 Per g, pore size about 1.0197 nm. The material is modified by Rho6G and DNA to form a probe, and the specific surface area is reduced to 431.96m 2 Per g, pore volume was reduced to 0.259cm 3 Per g, pore size about 1.0151 nm. The decrease in specific surface area, pore volume and pore size indicated that the chlorpromazine aptamer had successfully encapsulated the probe and that the Rho6G dye had successfully entered and remained at Uio-66-NH 2 In the pores of (a).
Respectively to Uio-66-NH 2 And scanning the infrared spectrum of the MOF-Rho 6G-Apt. As shown in FIG. 2(f), at 765cm -1 The vibration peak at (A) can be attributed to Uio-66-NH 2 Due to the coordination of the Zr atom with the oxygen atom of the organic ligand. At 1382cm -1 The peak at (a) can be attributed to the aromatic C ═ C bond of the terephthalate aromatic structure. At 1570-1677cm -1 Characteristic absorption peaks in the range are produced by C ═ O vibration in the organic ligand. The probe has unchanged peak position compared with MOF, and proves that the structure of MOFs is not damaged by the introduction of dye and DNA. The above characteristics indicate that the probe MOF-Rho-6G-Apt has been successfully prepared.
Example 2 preparation of fluorescent Probe MOF-Rho 6G-DNA
Dissolving 3mg MOFs in 1mL PBS buffer solution (pH 7.4), dispersing by using ultrasound, adding 500 μ L of 3mg/mL Rho6G, mixing uniformly, and incubating at room temperature and 200rpm for half an hour to ensure that the dye is fully contacted with the MOFs material to obtain MOF-Rho 6G. Then adding chlorpromazine aptamers with different concentrations respectively to make the final concentrations of the chlorpromazine aptamers in the mixture respectively 1 μ M, 2.5 μ M, 5 μ M, 7.5 μ M and 10 μ M, mixing uniformly, and incubating overnight at room temperature to make the chlorpromazine aptamers seal Rho6G to Uio-66-NH 2 Obtaining a probe MOF-Rho6G-Apt from the MOF nano material. Washing the probe with PBS solution for 3 times, standing for 12h, centrifuging, collecting supernatant, and detecting fluorescence of dye leakage in the supernatantCase (F/F) 0 ) Wherein F is the fluorescence of the centrifuged supernatant after addition of aptamer probes at different concentrations, F 0 Fluorescence of the centrifuged supernatant without aptamer probe.
In preparing the probe, enough chlorpromazine aptamer was required for packing the MOF-Rho6G so that Rho6G would not leak freely in its natural state. Therefore, the concentration of the chlorpromazine aptamer is important for preparing the comparative example fluorescent probe. As shown in FIG. 3(a), the sealing effect is almost better and better with increasing aptamer concentration, F/F 0 And are also getting higher. The fluorescence ratio did not differ much when the aptamer concentrations were 7.5. mu.M and 10. mu.M, respectively. This may be due to the fact that the concentration of the aptamer has already tended to saturate. To save costs, an optimal concentration of 7.5. mu.M was chosen as the aptamer in the experiment.
Example 3 method for detecting chlorpromazine Using a ratiometric fluorescent aptamer sensor
Uio-66-NH 2 The nano material can emit blue fluorescence under the excitation wavelength of 365nm, and has a fluorescence emission peak near 425 nm. Uio-66-NH 2 The nano material has a porous structure, rhodamine 6G (Rho 6G) dye can be filled in pores, and the dye has a fluorescence emission peak at 550 nm; at the same time Uio-66-NH 2 The surface of the nano material is positively charged, and can be combined with a chlorpromazine aptamer single chain under the action of static electricity to achieve the sealing effect.
FIG. 1 shows the principle of chlorpromazine detection using ratiometric fluorescent aptamer sensor, Rho6G is filled with Uio-66-NH 2 The pores of the nano material are sealed by chlorpromazine aptamer to prepare the fluorescent probe. When the solution is free of chlorpromazine, the dye is sealed by the aptamer and is Uio-66-NH 2 In the pores of the nano material, the probe has a higher dye emission peak besides the fluorescence emission peak carried by the MOF, F 550 /F 425 The ratio is stable; when chlorpromazine is added, the chlorpromazine aptamer is combined with the chlorpromazine to generate conformational change, the seal is opened, the dye is leaked, the dye emission peak of the probe at 550nm is reduced, the material emission peak at 425nm is almost unchanged, and F 550 /F 425 The ratio is reduced, and the chlorpromazine content is detected by an aptamer fluorescence sensor forming a ratio type.
Example 4 optimization of incubation time for chlorpromazine and aptamer
In the process of detecting chlorpromazine by using MOF-Rho 6G-DNA, time is needed for the combination of chlorpromazine and an aptamer on a probe, so that the incubation time of a fluorescent probe and chlorpromazine needs to be optimized, and the ratio change of the fluorescent probe is more obvious.
200 μ L of the prepared fluorescent probe of example 2 with the optimal aptamer concentration are respectively put into a centrifuge tube of 1.5mL, chlorpromazine with the final concentration of 100nM is added, and the binding buffer is added to make up the volume to 500 μ L. Placing in an incubator at room temperature, incubating for 30min, 1h, 2h, and 3h, centrifuging to remove supernatant, resuspending the precipitate with binding buffer solution, mixing, measuring fluorescence emission spectrum at 365nm excitation position with F-7000 fluorescence spectrometer, and calculating F 550 /F 425 . On the premise of ensuring the combination of chlorpromazine and an aptamer, the time required is reduced as much as possible. F 550 /F 425 The smaller the ratio, the more the dye leaks, and the larger the change in the fluorescence ratio of the probe.
As shown in fig. 3(b), in the initial stage of incubation, more and more aptamers were bound to chlorpromazine and more dye was leaked out as the incubation time was prolonged, and the fluorescence ratio was gradually reduced. The ratio then flattens out, possibly because the binding of the aptamer to chlorpromazine is now stable. The fluorescence ratio of the resuspension was lowest when the incubation time was 1 h. Therefore, 1h was chosen as the optimal incubation time for probe and target in the experiment.
Example 5 detection Limit of the method for detecting chlorpromazine Using a ratiometric fluorescent Adaptor sensor
200 μ L of the prepared fluorescent probe in example 2 with the optimal aptamer concentration are respectively put into a centrifuge tube of 1.5mL, chlorpromazine with different final concentrations is added, and a binding buffer solution is added to make up the volume to 500 μ L. Placing in incubator at room temperature, incubating for 1h, centrifuging to remove supernatant, resuspending the precipitate with binding buffer solution, mixing, measuring fluorescence emission spectrum at 365nm with F-7000 fluorescence spectrometer, and calculating F 550 /F 425 。
As shown in FIG. 4(a), as the concentration of chlorpromazine increases, leakage occursThe dye gradually increases, the fluorescence emission peak of the probe at 550nm becomes weak, F 550 /F 425 The value of (a) becomes smaller and smaller. The fluorescence ratio and the chlorpromazine concentration are in a linear relation in the range of 1 nM-100 nM. The linear regression equation is that y is-0.013 x +2.9326 and R 2 0.9929 (fig. 4 (b)). The limit of detection (LOD) was 0.67nM, calculated using the formula LOD ═ 3S/K, where S is the standard deviation of the blank (without chlorpromazine) and K is the slope of the standard curve.
Example 6 specificity of method for detecting chlorpromazine Using a ratiometric fluorescent aptamer sensor
200 μ L of the prepared fluorescent probe of example 2 with the optimal aptamer concentration was placed in a 1.5mL centrifuge tube, 100nM chlorpromazine, azaperone, haloperidol, promethazine or acepromazine was added, and the volume was made up to 500 μ L with binding buffer. Placing in incubator at room temperature, incubating for 1h, centrifuging to remove supernatant, resuspending the precipitate with binding buffer solution, mixing, measuring fluorescence emission spectrum at 365nm with F-7000 fluorescence spectrometer, and calculating F 550 /F 425 。
As shown in FIG. 5, the green bars represent the effect of each analogue (100nM) on the probe. Only chlorpromazine can cause strong change of fluorescence ratio, and other analogues have small influence on the probe. This difference is mainly due to the strong affinity of chlorpromazine for the aptamer in the probe, resulting in dye leakage after binding. The result shows that the method has good specificity and can be specially used for detecting chlorpromazine.
Example 7 detection of chlorpromazine in milk and eggs Using a ratiometric fluorescent aptamer sensor
The method selects milk and eggs as actual samples to carry out a labeling recovery experiment, and uses a ratio type fluorescence method and a chlorpromazine enzyme linked immunosorbent assay kit to carry out labeling recovery detection on the pretreated milk samples and egg samples so as to verify the practicability of the method.
The method is characterized in that samples of milk and eggs purchased from a supermarket are analyzed by a commercialized chlorpromazine ELISA kit, and the fact that the samples do not contain chlorpromazine is verified. Then, 10mL of a milk sample was taken, centrifuged at 14000rpm at 4 ℃ for 20min, the fat layer was removed, the remaining portion was diluted to 100mL with a binding buffer, filtered through a 0.22. mu.M microfiltration membrane, and the filtrate was collected as a milk extract for detection. Fully mixing and homogenizing eggs, adding 2g of egg samples into a centrifuge tube, adding 4mL of ethyl acetate, shaking for 3min, centrifuging at 6500rpm for 5min at room temperature, drying the supernatant in a water bath at 80 ℃ or nitrogen, and diluting to 500 mu L with a binding buffer solution to obtain the egg extract for detection. And adding 1nM, 10nM and 50nM chlorpromazine into the milk or egg extract to obtain a labeled sample to be detected, detecting the labeled sample by a chlorpromazine ELISA kit and a ratio-type fluorescence method, and calculating the recovery concentration, the recovery rate and the relative standard deviation.
As shown in Table 1, the recovery rate of the method of the invention is 92.2-104.8%, and the recovery rate of the commercial ELISA kit is 92.9-104.6%. The recovery results for both methods are very close. The result shows that the selected food substrate has no obvious influence on the detection of the chlorpromazine content by the method, and can be successfully applied to chlorpromazine analysis in milk and egg samples.
TABLE 1 Chloroprozine spiking recovery in milk and egg samples
Figure BDA0003658904580000081
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A ratiometric fluorescent aptamer sensor for detecting chlorpromazine, characterized by being prepared from Uio-66-NH 2 The nano material forms a main body framework structure at Uio-66-NH 2 The pores of the nano material are filled with rhodamine 6G dye, and meanwhile, Uio-66-NH 2 The surface of the nano material is combined with the chlorpromazine aptamer monomer through electrostatic interactionChain to encapsulate rhodamine 6G dye at Uio-66-NH 2 Pores of the nanomaterial.
2. A ratiometric fluorescent aptamer sensor for detecting chlorpromazine according to claim 1, characterized in that the preparation method comprises the following steps:
(1) Uio-66-NH 2 Dissolving the nanometer material in buffer solution, adding Rho6G dye, mixing, incubating to allow the dye to react with Uio-66-NH 2 Fully contacting the nano materials to obtain MOF-Rho 6G;
(2) adding chlorpromazine aptamer, mixing and incubating such that the chlorpromazine aptamer seals Rho6G dye to Uio-66-NH 2 Obtaining a probe MOF-Rho6G-Apt from the nanometer material;
(3) washing the probe MOF-Rho6G-Apt to remove free Rho6G dye and chlorpromazine aptamer.
3. The ratiometric fluorescent aptamer sensor for detecting chlorpromazine according to claim 2, wherein the pH of the buffer solution in the step (1) is 7.0-7.4.
4. The ratiometric fluorescent aptamer sensor for detecting chlorpromazine according to claim 2 or 3, wherein in step (2), the amount of the chlorpromazine aptamer is 1 μ M to 10 μ M.
5. The ratiometric fluorescent aptamer sensor for detecting chlorpromazine according to claim 1, wherein the sequence of the aptamer is: 5'-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGAACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3', or alternatively,
5'-AATCAAACGCTAAGGTCGGAGGAAAGTGCACCCATTCTTGCGCCACAAGACACACTGATCGAACGCCGCCAAGCTTGGTACCCGTATCGT-3', or alternatively,
5′-AATCAAACGCTAAGGTAAGGACGAGGTGCACCCATTCTTGGGCACGCGGCACGCGCGAGGACGACCAGGCAAGCTTGGTACCCGTATCGT-3′。
6. a ratiometric fluorescent aptamer sensor for detecting chlorpromazine according to claim 2, wherein the preparation method comprises the following steps:
(1) 3mg of Uio-66-NH 2 Dissolving the nano material in 500 mu L of PBS buffer solution with pH 7.4, dispersing the nano material by using ultrasound, adding 500 mu L of 3mg/mL Rho6G dye, uniformly mixing, and incubating at room temperature of 200rpm for half an hour to ensure that the dye is fully contacted with the MOF material to obtain MOF-Rho 6G;
(2) adding 80 μ L of 100 μ M chlorpromazine aptamer, mixing, and incubating overnight at room temperature to allow the chlorpromazine aptamer to seal Rho6G to Uio-66-NH 2 Obtaining a probe MOF-Rho6G-Apt from the MOF nano material;
(3) the probe was washed 3 times with PBS solution to remove free dye and aptamers, and then dissolved in binding buffer and stored at 4 ℃ for use.
7. A method for detecting chlorpromazine by using the sensor of any one of claims 1 to 6, comprising the steps of:
(1) preparation of fluorescence emission spectra F of the ratiometric fluorescent aptamer sensor under 365nm excitation 550 /F 425 A standard curve of the concentration of chlorpromazine,
(2) adding chlorpromazine solution to be detected into the ratio type fluorescent aptamer sensor, fixing the volume with binding buffer solution, incubating, centrifuging to remove supernatant, resuspending the precipitate with binding buffer solution, mixing uniformly, measuring fluorescence emission spectrum under 365nm excitation, and calculating F 550 /F 425 ，
(2) F is to be 550 /F 425 Substituting the standard curve to calculate the concentration of chlorpromazine in the solution to be measured.
8. Kit for the detection of chlorpromazine comprising a sensor according to any of claims 1 to 6.
9. Use of a sensor according to any one of claims 1 to 6 for the detection of chlorpromazine.
10. Use of the kit according to claim 8 for the detection of chlorpromazine.
CN202210570429.9A 2022-05-24 2022-05-24 Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor Pending CN114965408A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210570429.9A CN114965408A (en) 2022-05-24 2022-05-24 Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210570429.9A CN114965408A (en) 2022-05-24 2022-05-24 Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor

Publications (1)

Publication Number Publication Date
CN114965408A true CN114965408A (en) 2022-08-30

Family

ID=82955062

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210570429.9A Pending CN114965408A (en) 2022-05-24 2022-05-24 Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor

Country Status (1)

Country Link
CN (1) CN114965408A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114813862A (en) * 2021-11-12 2022-07-29 重庆大学 Electrochemical biosensor and application thereof
CN117535045A (en) * 2023-07-12 2024-02-09 中国热带农业科学院分析测试中心 Zn-MOF@Au NPS/DNA aptamer fluorescent probe for trace thiamethoxam detection and preparation method thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114813862A (en) * 2021-11-12 2022-07-29 重庆大学 Electrochemical biosensor and application thereof
CN114813862B (en) * 2021-11-12 2024-03-26 重庆大学 Electrochemical biosensor and application thereof
CN117535045A (en) * 2023-07-12 2024-02-09 中国热带农业科学院分析测试中心 Zn-MOF@Au NPS/DNA aptamer fluorescent probe for trace thiamethoxam detection and preparation method thereof
CN117535045B (en) * 2023-07-12 2024-05-31 中国热带农业科学院分析测试中心 Zn-MOF@Au NPS/DNA aptamer fluorescent probe for trace thiamethoxam detection and preparation method thereof

Similar Documents

Publication Publication Date Title
CN114965408A (en) Method for detecting chlorpromazine by using ratio type fluorescent aptamer sensor
Miao et al. Ratiometric electrochemical immunosensor for the detection of procalcitonin based on the ratios of SiO2-Fc–COOH–Au and UiO-66-TB complexes
CN102262125B (en) Electrochemical immune sensor for detecting diethylstilbestrol and preparation method and application of sensor
JP5188706B2 (en) Polydiacetylene supramolecular body and ligand detection method.
JP2017198698A (en) Methods and devices for detection and measurement of analytes
CN107121402B (en) Method for detecting chloramphenicol in water based on metal organic framework compound simulated enzyme catalytic property
CN102262115B (en) Electrochemical immunosensor for melamine content determination, and preparation method and application thereof
CN114594262B (en) Mycotoxin magnetic chemiluminescence immunoassay kit based on bifunctional fusion protein and application thereof
Bottari et al. Electrochemical immunosensor based on ensemble of nanoelectrodes for immunoglobulin IgY detection: Application to identify hen's egg yolk in tempera paintings
CN111693571B (en) Method for detecting GPC3 based on light addressing potential sensor
Li et al. A renewable potentiometric immunosensor based on Fe3O4 nanoparticles immobilized anti‐IgG
CN110308286B (en) Enhanced thyroglobulin electrochemiluminescence immunosensor based on photothermal release signals
CN112940114A (en) Antifungal 1, 3-beta-D-glucan monoclonal antibody and application thereof
Feng et al. A novel strategy for multiplexed immunoassay of tumor markers based on electrochemiluminescence coupled with cyclic voltammetry using graphene-polymer nanotags
CN108841828B (en) Single-stranded DNA aptamer for specifically recognizing tobramycin and application thereof
CN109342420B (en) Fe3O4Application of @ C one-dimensional nanowire
Banciu et al. Optical biosensing of lysozyme
CN114544934A (en) Aspergillus galactomannan detection test strip and application thereof
CN110988325B (en) Blocking agent and kit containing same
CN112710826A (en) Coating and sealing method for improving stability of reagent
CN111693689B (en) Nanoenzyme for enzymatic chemiluminescence detection and application thereof
CN113933281B (en) Exosome detection method based on optical fiber evanescent wave fluorescence biosensor
CN105181956B (en) Application of the fluorescence detection specifically responded based on metal ion in immune detection
CN113406159A (en) Preparation and application of electrochemiluminescence immunosensor for detecting ochratoxin A
CN111024940B (en) Time-resolved fluorescence immunoassay method based on gold magnetic particles

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination