Method for detecting chlorpromazine using ratiometric fluorescent aptasensor

Abstract

The present invention discloses a method for detecting chlorpromazine using a ratio-type fluorescent aptamer sensor, which belongs to the field of detection technology. 2 The nanomaterial was incubated with Rho 6G dye to obtain MOF-Rho 6G, and then the chlorpromazine nucleic acid aptamer was added to incubate to obtain the probe MOF-Rho 6G-Apt. The application of this probe can improve the sensitivity of chlorpromazine detection. Compared with the ELISA kit, it has the advantages of good specificity, no need for antibodies, and a wide detection range.

Description

Technical Field

[0001] The invention relates to a method for detecting chlorpromazine by using a ratio-type fluorescent aptamer sensor, and belongs to the technical field of detection. Background Art

[0002] Chlorpromazine is a phenothiazine drug, mainly used to enhance hypnosis, anesthesia, sedation, etc. Adding chlorpromazine to feed can indirectly fatten animals. GB 31650-2019 stipulates that chlorpromazine is allowed for therapeutic use, but it must not be detected in animal food.

[0003] At present, there are many methods for determining chlorpromazine residues. The main methods for detecting chlorpromazine in animal-derived foods include enzyme-linked immunosorbent assay, gas chromatography, high-performance liquid chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography-tandem mass spectrometry, etc. Among them, gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry are the most commonly used. Although the chromatography method has high detection sensitivity and accurate test results, it requires expensive instruments and equipment, and the sample pretreatment steps are complicated, and a large number of environmentally unfriendly organic reagents are required. Enzyme-linked immunosorbent assay screening relies on antibodies. Although some enzyme-linked immunosorbent assays can achieve fast and simple detection, the preparation process of antibodies is relatively complicated, and there are differences in antibodies from different batches. This is a defect of enzyme-linked immunosorbent assay.

[0004] Aptamers are DNA or RNA molecules separated by systematic evolution of ligands by exponential enrichment (SELEX). They can bind with high affinity and specificity to other targets such as proteins, metal ions, small molecules, peptides, and even whole cells. Therefore, aptamers have shown broad application prospects in biochemical analysis, environmental monitoring, basic medicine, and new drug synthesis. Compared with antibodies used in enzyme-linked immunosorbent assay, aptamers have the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and artificial synthesis, eliminating the series of processes required for the preparation of antibodies, such as animal immunization, feeding, protein extraction, and purification. Therefore, aptamers are an ideal molecular probe that can be used as a material for fluorescence detection methods.

[0005] Fluorescence sensors with single fluorescence emission have been widely used due to their simple design and high selectivity. However, the impact of the detection environment and the need for higher sensor sensitivity still exist. Ratiometric (ratio-type) fluorescence sensors have greater practical value than traditional fluorescence sensors. Through self-calibrated dual emission peak detection, the interference caused by the detection environment and instrument parameters is reduced.

[0006] However, the use of ratiometric fluorescence aptamer sensors for the detection of chlorpromazine still faces the problem of which material to choose as the fluorescence source and how to construct ratiometric fluorescence emission. Summary of the invention

[0007] [Technical issues]

[0008] The technical problem to be solved by the present invention is that the prior art lacks a method for detecting chlorpromazine using a ratio fluorescence method using nucleic acid aptamers.

[0009] [Technical solution]

[0010] The present invention first provides a ratiometric fluorescent aptamer sensor for detecting chlorpromazine, and the preparation method thereof comprises the following steps:

[0011] (1) Dissolving the Uio-66-NH2 nanomaterial in a buffer solution, adding Rho 6G dye, mixing and incubating to allow the dye to fully contact with the Uio-66-NH2 nanomaterial to obtain MOF-Rho 6G;

[0012] (2) adding chlorpromazine aptamer, mixing and incubating so that the chlorpromazine aptamer seals the Rho 6G dye into the Uio-66-NH2 nanomaterial to obtain the probe MOF-Rho 6G-Apt;

[0013] (3) Cleaning the probe MOF-Rho 6G-Apt to remove free Rho 6G dye and chlorpromazine aptamer.

[0014] In certain embodiments of the present invention, the pH of the buffer in step (1) is 7.0-7.4, and can be selected from: PBS buffer (1.4 mM KH2PO4, 4.3 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2-7.4), or, binding buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCI, 1 mM MgCl2, pH 7.4).

[0015] In certain embodiments of the present invention, in step (1), the ratio of Uio-66-NH2 nanomaterial, Rho 6G dye, and buffer 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.

[0016] In certain embodiments of the present invention, in step (1), the incubation is performed at 35-37° C. for 10-240 min.

[0017] In certain embodiments of the present invention, in step (2), the dosage of the chlorpromazine nucleic acid aptamer is 1 μM to 10 μM.

[0018] In certain embodiments of the present invention, in step (2), the incubation is performed at 25-30° C. for 30-180 min.

[0019] In certain embodiments of the present invention, the sequence of the nucleic acid aptamer may be: 5′-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGAACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3′ (SEQ ID NO. 1), or,

[0020] 5′-AATCAAACGCTAAGGTCGGAGGAAAGTGCACCCATTCTTGCGCCACAAGACACACTGATCGAACGCCGCCAAGCTTGGTACCCGTATCGT-3′ (SEQ ID NO. 2), or,

[0021] 5′-AATCAAACGCTAAGGTAAGGACGAGGTGCACCCATTCTTGGGCACGCGGCACGCGAGGACGACCAGGCAAGCTTGGTACCCGTATCGT-3′ (SEQ ID NO. 3).

[0022] In certain embodiments of the present invention, in step (3), the solution used for washing can be PBS buffer (1.4 mM KH2PO4, 4.3 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2-7.4) or binding buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCI, 1 mM MgCl2, pH 7.4).

[0023] In certain embodiments of the present invention, in step (3), after washing, the probe is dissolved in a binding buffer and stored at 4° C. for later use.

[0024] In certain embodiments of the present invention, the method for preparing a ratiometric fluorescent aptasensor for detecting chlorpromazine comprises the following steps:

[0025] (1) Dissolve 3 mg of Uio-66-NH2 nanomaterial in 500 μL of pH 7.4 PBS buffer and disperse it with ultrasound. Add 500 μL of 3 mg / mL Rho 6G dye, mix well, and incubate at room temperature at 200 rpm for half an hour to allow the dye to fully contact with the MOF material to obtain MOF-Rho 6G.

[0026] (2) Add 80 μL of 100 μM chlorpromazine aptamer, mix well, and incubate at room temperature overnight to allow the chlorpromazine aptamer to seal Rho 6G into the Uio-66-NH2MOF nanomaterial to obtain the probe MOF-Rho 6G-Apt;

[0027] (3) Wash the probe three times with PBS solution to remove free dye and aptamer, then dissolve in binding buffer and store at 4°C for future use.

[0028] The present invention provides a ratiometric fluorescent aptamer sensor for detecting chlorpromazine prepared by the above-mentioned preparation method. The sensor is formed of a Uio-66-NH2 nanomaterial to form a main frame structure, and the pores of the Uio-66-NH2 nanomaterial are filled with rhodamine 6G dye. At the same time, the surface of the Uio-66-NH2 nanomaterial is combined with a chlorpromazine nucleic acid aptamer single chain through electrostatic action to seal the rhodamine 6G dye in the pores of the Uio-66-NH2 nanomaterial.

[0029] The present invention provides a method for detecting chlorpromazine using the sensor, comprising the following steps:

[0030] (1) Preparation of the fluorescence emission spectrum of the ratiometric fluorescent aptasensor under 365 nm excitation 550 / F 425 The standard curve with chlorpromazine concentration,

[0031] (2) Add the chlorpromazine solution to be tested to the ratiometric fluorescent aptamer sensor, and adjust the volume with binding buffer. After incubation, centrifuge and remove the supernatant. Resuspend the precipitate with binding buffer and mix well. Then measure the fluorescence emission spectrum under 365 nm excitation and calculate F 550 / F 425 ,

[0032] (2) F 550 / F 425 Substitute into the standard curve and calculate the concentration of chlorpromazine in the test solution.

[0033] [Beneficial Effects]

[0034] The present invention uses chlorpromazine aptamers to prepare ratiometric fluorescent aptamer sensors, which ensure the specificity of the detection method on the one hand, and on the other hand, based on the nanomaterial Uio-66-NH2 and the dye rhodamine 6G (Rho 6G) that can emit fluorescence at 425 nm and 550 nm under 365 nm excitation, a dual-emission fluorescent probe is established to improve the sensitivity of chlorpromazine detection. When there is no chlorpromazine in the solution, the dye is sealed in the pores of the Uio-66-NH2 nanomaterial by the aptamer. In addition to the fluorescence emission peak of the MOF itself, the probe also has a higher dye emission peak. 550 / F 425 The ratio is stable; when chlorpromazine is added, the chlorpromazine aptamer binds to chlorpromazine, undergoes conformational changes, the seal opens, the dye leaks, the dye emission peak of the probe at 550 nm decreases, and the material emission peak at 425 nm remains almost unchanged. 550 / F 425 The ratio decreases, thereby forming a ratiometric aptamer fluorescence sensor to detect chlorpromazine content.

[0035] Compared with ELISA kits, the ratiometric aptamer fluorescent sensor prepared by the present invention has the advantages of good specificity, no need for antibodies, and a wide detection range.

[0036] The fluorescent probe prepared by the present invention has high sensitivity and a detection limit (LOD) of 0.67 nM; it has strong specificity and can significantly distinguish chlorpromazine from its analogs (azaperone, haloperidol, promethazine or acepromazine); it is suitable for detecting chlorpromazine residues in food, especially food matrices such as milk and eggs, and has no significant effect on the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of ratiometric fluorescence detection of chlorpromazine.

[0038] Figure 2 Probe characterization: (a) Fluorescence emission spectra of MOF-Rho and MOF-Rho 6G-Apt, the upper right corner is Uio-66-NH2 and MOF-Rho 6G-Apt in a UV dark box, (b) UV-visible spectra of the supernatants of Rho 6G, MOF-Rho 6G and MOF-Rho 6G-Apt, (c) particle size of Uio-66-NH2 and MOF-Rho 6G-Apt, (d) potential values ​​of Uio-66-NH2 and MOF-Rho 6G-Apt, (e) isothermal adsorption curves of Uio-66-NH2 and MOF-Rho 6G-Apt, (f) infrared images of Uio-66-NH2 and MOF-Rho6G-Apt.

[0039] Figure 3Optimization of aptamer concentration (a) and probe incubation time with chlorpromazine (b).

[0040] Figure 4 (a) Fluorescence spectra of chlorpromazine at different concentrations, (b) standard curve of CHL-3 recognition of chlorpromazine.

[0041] Figure 5 Fluorescence ratio of probes after adding different targets. DETAILED DESCRIPTION

[0042] Terminology: Uio-66-NH2 is a kind of MOFs, MOFs is Metal-organic Frameworks.

[0043] Example 1 Preparation and characterization of probe MOF-Rho 6G-DNA

[0044] 3 mg MOFs (i.e., Uio-66-NH2 nanomaterials) were dissolved in 500 μL PBS buffer (pH 7.4) and dispersed with ultrasound. 500 μL 3 mg / mL Rho 6G (rhodamine 6G dye) was added, mixed and incubated at 200 rpm for half an hour at room temperature to allow the dye to fully contact the MOF material to obtain MOF-Rho 6G. 80 μL 100 μM chlorpromazine aptamer was added, mixed and incubated overnight at room temperature to allow the chlorpromazine aptamer to seal Rho 6G into the Uio-66-NH2 nanomaterial to obtain the probe MOF-Rho 6G-Apt. The probe was washed 3 times with PBS solution to remove free dye and aptamer, and then dissolved in binding buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCI, 1 mM MgCl2, pH 7.4) and stored at 4°C for later use. The sequence of the chlorpromazine aptamer is: 5′-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGAACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3′.

[0045] First, the fluorescence of Uio-66-NH2 nanomaterials, dye Rho 6G, and probe MOF-Rho 6G-Apt was characterized. Figure 2As shown in (a), Uio-66-NH2 nanomaterials and Rho 6G dyes can emit fluorescence at 425 nm and 550 nm respectively under an excitation wavelength of 365 nm. It can be seen that the probe with chlorpromazine aptamer blocking has a higher dye fluorescence peak at 550 nm and a material fluorescence peak at 425 nm. The appearance of double emission peaks indicates that the fluorescent probe has been successfully prepared. Figure 2 (a) As shown in the small figure in the upper right corner, Uio-66-NH2 nanomaterials and MOF-Rho 6G-Apt can produce blue and green fluorescence, respectively, under excitation at 365 nm.

[0046] UV spectra were scanned for Rho 6G dye not incubated with MOFs, MOFs, the centrifugal supernatant after MOFs and Rho 6G were incubated, and the centrifugal supernatant after MOF-Rho 6G and aptamer were incubated. Figure 2 As shown in (b), the UV peak of the original Rho 6G dye is about 525 nm. The UV curves of the MOF-Rho 6G supernatant and MOF-Rho 6G-apt supernatant are lower than the original concentration, indicating that the dye concentration in the supernatant is lower than the initial dye concentration, and the dye is successfully adsorbed on MOFs. The spectrum of the MOF-Rho6G-Apt supernatant has the lowest value at 525 nm, indicating that more dye remains in the MOFs material and the aptamer has a sealing effect on the dye.

[0047] The particle sizes of Uio-66-NH2 nanomaterials and MOF-Rho 6G-Apt were measured. Figure 2 (c) shows the particle size of the material before and after modification. The particle size of the Uio-66-NH2 nanomaterial is 113.3 nm, and the particle size of the MOF-Rho 6G-Apt is 237.5 nm. The hydrated particle size increases after MOF modification, indicating that the new material has been successfully coated on the surface of the material. Zeta potential analysis shows the charge changes before and after MOF modification, such as Figure 2 As shown in (d), the Uio-66-NH2 nanomaterial is positively charged with a potential of 32.5 mV. After the dye Rho 6G is loaded into MOFs and sealed with chlorpromazine aptamers, the potential of the formed probe changes greatly, and the negative potential of MOF-Rho6G-Apt is -20.5 mV. Uio-66-NH2 and MOF-Rho 6G-Apt are analyzed using a fully automatic specific surface area analyzer. Figure 2 As shown in (e), the BET surface area of ​​Uio-66-NH2 is calculated to be about 779.36 m 2 / g, the pore volume is about 0.320cm 3 / g, and the pore size is about 1.0197 nm. After the material is modified with Rho 6G and DNA to form a probe, the specific surface area is reduced to 431.96m 2 / g, the pore volume is reduced to 0.259 cm 3 / g, and the pore size is about 1.0151 nm. The decrease in specific surface area, pore volume, and pore size indicates that the chlorpromazine aptamer has successfully filled the probe, and the Rho 6G dye has successfully entered and remained in the pores of Uio-66-NH2.

[0048] The infrared spectra of Uio-66-NH2 and MOF-Rho 6G-Apt were scanned respectively. Figure 2 (f) at 765cm -1 The vibration peak at 1382 cm-1 can be attributed to the stretching of the Zr-O bond in Uio-66-NH2, which is due to the coordination of the Zr atom with the oxygen atom of the organic ligand. -1 The peaks at 1570–1677 cm -1 The characteristic absorption peak in the range is generated by the C=O vibration in the organic ligand. The peak position of the probe is unchanged compared with MOF, proving that the introduction of dyes and DNA did not destroy the structure of MOFs. The above characteristics show that the probe MOF-Rho-6G-Apt has been successfully prepared.

[0049] Example 2 Preparation of fluorescent probe MOF-Rho 6G-DNA

[0050] 3 mg MOFs were dissolved in 1 mL PBS buffer (pH 7.4) and dispersed with ultrasound. 500 μL 3 mg / mL Rho 6G was added, mixed and incubated at 200 rpm for half an hour at room temperature to allow the dye to fully contact the MOFs material to obtain MOF-Rho 6G. Subsequently, different concentrations of chlorpromazine aptamers were added, so that the final concentrations of chlorpromazine aptamers in the mixture were 1 μM, 2.5 μM, 5 μM, 7.5 μM, and 10 μM, respectively. Mix well and incubate at room temperature overnight to allow the chlorpromazine aptamer to seal Rho 6G into the Uio-66-NH2MOF nanomaterial to obtain the probe MOF-Rho 6G-Apt. The probe was washed three times with PBS solution and placed for 12 h. The supernatant was centrifuged and the fluorescence of dye leakage in the supernatant was detected (F / F0), where F was the fluorescence of the supernatant with different concentrations of aptamer probes added, and F0 was the fluorescence of the supernatant without aptamer probes added.

[0051] When preparing the probe, sufficient chlorpromazine aptamers are needed to seal MOF-Rho 6G so that Rho 6G will not leak randomly in its natural state. Therefore, the concentration of chlorpromazine aptamers is very important for the preparation of proportional fluorescent probes. Figure 3 As shown in (a), as the concentration of the aptamer increases, the sealing effect becomes better and better, and the F / F0 also becomes higher and higher. When the aptamer concentrations are 7.5 μM and 10 μM, the fluorescence ratio is not much different. This may be because the concentration of the aptamer has reached saturation. In order to save costs, 7.5 μM was selected as the optimal concentration of the aptamer in the experiment.

[0052] Example 3 Method for detecting chlorpromazine using ratiometric fluorescent aptasensor

[0053] Uio-66-NH2 nanomaterials can emit blue fluorescence at an excitation wavelength of 365 nm, with a fluorescence emission peak near 425 nm. Uio-66-NH2 nanomaterials have a porous structure, and the pores can be filled with rhodamine 6G (Rho 6G) dye, which has a fluorescence emission peak at 550 nm; at the same time, the surface of Uio-66-NH2 nanomaterials is positively charged, and can rely on electrostatic interaction to bind to the single chain of chlorpromazine aptamer to achieve a sealing effect.

[0054] The principle of using ratiometric fluorescent aptasensor to detect chlorpromazine is as follows Figure 1 As shown in the figure, Rho 6G was filled into the pores of Uio-66-NH2 nanomaterials and sealed with chlorpromazine aptamers to prepare a fluorescent probe. When there was no chlorpromazine in the solution, the dye was sealed in the pores of Uio-66-NH2 nanomaterials by the aptamer. In addition to the fluorescence emission peak of MOF itself, the probe also had a higher dye emission peak. 550 / F 425 The ratio is stable; when chlorpromazine is added, the chlorpromazine aptamer binds to chlorpromazine, undergoes conformational changes, the seal opens, the dye leaks, the dye emission peak of the probe at 550 nm decreases, and the material emission peak at 425 nm remains almost unchanged. 550 / F 425 The ratio decreases, thereby forming a ratiometric aptamer fluorescence sensor to detect chlorpromazine content.

[0055] Example 4 Optimization of the incubation time of chlorpromazine and aptamer

[0056] In the process of using MOF-Rho 6G-DNA to detect chlorpromazine, it takes time for chlorpromazine to bind to the aptamer on the probe. Therefore, it is necessary to optimize the incubation time of the fluorescent probe and chlorpromazine to make the ratio change of the fluorescent probe more obvious.

[0057] Take 200 μL of the fluorescent probe prepared with the optimal aptamer concentration in Example 2 in a 1.5 mL centrifuge tube, add chlorpromazine with a final concentration of 100 nM, and add binding buffer to make up the volume to 500 μL. Place in an incubator at room temperature and incubate for 30 min, 1 h, 2 h, and 3 h, respectively. Centrifuge to remove the supernatant, resuspend the precipitate with binding buffer, mix well, and measure the fluorescence emission spectrum at 365 nm excitation using an F-7000 fluorescence spectrometer to calculate F 550 / F 425 Under the premise of ensuring the binding of chlorpromazine to the aptamer, the required time should be minimized. 550 / F 425 The smaller the ratio, the more dye leakage there is, and the greater the change in the fluorescence ratio of the probe.

[0058] like Figure 3 As shown in (b), at the beginning of the incubation, as the incubation time increases, more and more aptamers bind to chlorpromazine, more and more dyes leak out, and the fluorescence ratio gradually decreases. Then the ratio tends to be flat, probably because the binding of aptamers and chlorpromazine is relatively stable at this time. When the incubation time is 1 h, the resuspended fluorescence ratio is the lowest. Therefore, 1 h was selected as the optimal incubation time for the probe and target in the experiment.

[0059] Example 5 Detection Limit of the Method for Detecting Chlorpromazine Using Ratiometric Fluorescent Aptamer Sensor

[0060] Take 200 μL of the fluorescent probe prepared with the optimal aptamer concentration in Example 2 in a 1.5 mL centrifuge tube, add chlorpromazine at different final concentrations, and add binding buffer to make up the volume to 500 μL. Place in an incubator at room temperature, incubate for 1 h, centrifuge to remove the supernatant, resuspend the precipitate with binding buffer, mix well, measure the fluorescence emission spectrum at 365 nm excitation with an F-7000 fluorescence spectrometer, and calculate F 550 / F 425 .

[0061] like Figure 4 As shown in (a), with the increase of chlorpromazine concentration, the leakage of dye gradually increased, the fluorescence emission peak of the probe at 550nm became weaker, and F 550 / F 425 The value of is getting smaller and smaller. In the range of 1 nM ~ 100nM, the fluorescence ratio is linearly related to the chlorpromazine concentration. The linear regression equation is y = -0.013x + 2.9326 and R 2 = 0.9929 ( Figure 4 (b)). The limit of detection (LOD) was 0.67 nM, calculated using the formula LOD = 3S / K, where S is the standard deviation of the blank sample (without chlorpromazine) and K is the slope of the standard curve.

[0062] Example 6 Specificity of the method for detecting chlorpromazine using a ratiometric fluorescent aptasensor

[0063] Take 200 μL of the fluorescent probe prepared with the optimal aptamer concentration in Example 2 in a 1.5 mL centrifuge tube, add 100 nM chlorpromazine, azaperone, haloperidol, promethazine or acepromazine, and add binding buffer to make up the volume to 500 μL. Place in an incubator at room temperature, incubate for 1 h, centrifuge to remove the supernatant, resuspend the precipitate with binding buffer, mix well, measure the fluorescence emission spectrum at 365 nm excitation with an F-7000 fluorescence spectrometer, and calculate F 550 / F 425 .

[0064] like Figure 5 As shown, the green bar indicates the effect of various analogs (100 nM) on the probe. Only chlorpromazine can cause a strong change in the fluorescence ratio, while the other analogs have a smaller effect 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 results show that this method has good specificity and can be used specifically for the detection of chlorpromazine.

[0065] Example 7 Detection of chlorpromazine in milk and eggs using ratiometric fluorescent aptasensor

[0066] Milk and eggs were selected as actual samples for spike recovery experiments. Ratio fluorescence method and chlorpromazine enzyme-linked immunosorbent assay kit were used to perform spike recovery tests on pre-treated milk and egg samples to verify the practicability of the method of the present invention.

[0067] The milk and egg samples purchased from the supermarket were first analyzed by a commercial chlorpromazine ELISA kit to confirm that they did not contain chlorpromazine. Then, 10 mL of the milk sample was taken and centrifuged at 14000 rpm at 4°C for 20 min. The fat layer was removed and the remaining part was diluted to 100 mL with binding buffer and filtered with a 0.22 μM microporous filter membrane to collect the filtrate as the milk extract for detection. After the eggs were fully mixed and homogenized, 2 g of egg sample was added to the centrifuge tube, and then 4 mL of ethyl acetate was added and shaken for 3 min. After centrifugation at 6500 rpm for 5 min at room temperature, the supernatant was blown dry in an 80°C water bath or blown dry in nitrogen, and diluted to 500 μL with binding buffer to obtain the egg extract for detection. 1 nM, 10 nM, and 50 nM chlorpromazine were spiked into the milk or egg extract to obtain the spiked samples to be detected, and then the spiked samples were detected by the chlorpromazine ELISA kit and the ratiometric fluorescence method, and the recovery concentration, recovery rate, and relative standard deviation were calculated.

[0068] As shown in Table 1, the recovery rate of the method of the present invention is 92.2% to 104.8%, and the recovery rate of the commercial ELISA kit is 92.9% to 104.6%. The recovery results of the two methods are very close. The results show that the selected food matrix has no significant effect on the content of chlorpromazine detected by the method of the present invention, and it can be successfully applied to the analysis of chlorpromazine in milk and egg samples.

[0069] Table 1 Recovery of chlorpromazine spiked in milk and egg samples

[0070]

[0071] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims. SEQUENCE LISTING <110> Jiangnan University <120> Method for detecting chlorpromazine using ratiometric fluorescent aptasensor <130> 2022.5.24 <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 90 <212> DNA <213> synthetic construct <400> 1 aatcaaacgc taaggtcgga gggaagtgca cccattcttg gaaacaggag ctcctgaacc 60 gcccacacgc aagcttggta cccgtatcgt 90 <210> 2 <211> 90 <212> DNA <213> synthetic construct <400> 2 aatcaaacgc taaggtcgga ggaaagtgca cccattcttg cgccacaaga cacactgatc 60 gaacgccgcc aagcttggta cccgtatcgt 90 <210> 3 <211> 90 <212> DNA <213> synthetic construct <400> 3 aatcaaacgc taaggtaagg acgaggtgca cccattcttg ggcacgcggc acgcgcgagg 60 acgaccaggc aagcttggta cccgtatcgt 90

Claims

1. A ratiometric fluorescent aptasensor for detecting chlorpromazine, characterized in that: The main framework structure is formed by Uio-66-NH2 nanomaterials, and the pores of the Uio-66-NH2 nanomaterials are filled with Rho 6G dyes. Meanwhile, the surface of the Uio-66-NH2 nanomaterials is bound to a single-stranded chlorpromazine nucleic acid aptamer by electrostatic interaction, so that the Rho 6G dyes are sealed in the pores of the Uio-66-NH2 nanomaterials. The steps of filling the Rho 6G dye in the pores of the Uio-66-NH2 nanomaterial and electrostatically binding the single-stranded chlorpromazine nucleic acid aptamer on the surface of the Uio-66-NH2 nanomaterial are as follows: (1) Dissolve 3 mg of Uio-66-NH2 nanomaterial in 500 μL of pH 7.4 PBS buffer and disperse it with ultrasound. Add 500 μL of 3 mg / mL Rho 6G dye, mix well, and incubate at room temperature at 200 rpm for half an hour to allow the dye to fully contact with the MOF material to obtain MOF-Rho 6G. (2) Add 80 μL of 100 μM chlorpromazine aptamer, mix well, and incubate at room temperature overnight to allow the chlorpromazine aptamer to seal Rho 6G into the Uio-66-NH2 nanomaterial to obtain the probe MOF-Rho 6G-Apt; (3) The probe was washed three times with PBS solution to remove free dye and aptamer, and then dissolved in binding buffer and stored at 4°C for future use; The sequence of the chlorpromazine nucleic acid aptamer is: 5′-AATCAAACGCTAAGGTCGGAGGGAAGTGCACCCATTCTTGGAAACAGGAGCTCCTGAACCGCCCACACGCAAGCTTGGTACCCGTATCGT-3′, or, 5′-AATCAAACGCTAAGGTCGGAGGAAAGTGCACCCATTCTTGCGCCACAAGACACACTGATCGAACGCCGCCAAGCTTGGTACCCGTATCGT-3′, or, 5′-AATCAAACGCTAAGGTAAGGACGAGGTGCACCCATTCTTGGGCACGCGGCACGCGAGGACGACCAGGCAAGCTTGGTACCCGTATCGT-3′.

2. A method for detecting chlorpromazine using the sensor according to claim 1, characterized in that: The following steps are involved: (1) Preparation of the fluorescence emission spectrum of the ratiometric fluorescent aptasensor under 365 nm excitation 550 / F 425 The standard curve with chlorpromazine concentration, (2) Add the chlorpromazine solution to be tested to the ratiometric fluorescent aptamer sensor, and adjust the volume with binding buffer. After incubation, centrifuge and remove the supernatant. Resuspend the precipitate with binding buffer and mix well. Then measure the fluorescence emission spectrum under 365 nm excitation and calculate F 550 / F 425 , (3) F 550 / F 425 Substitute into the standard curve and calculate the concentration of chlorpromazine in the test solution.

3. A kit for detecting chlorpromazine comprising the sensor according to claim 1.

4. Use of the sensor according to claim 1 in detecting chlorpromazine.

5. Use of the kit according to claim 3 in detecting chlorpromazine.

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