A visual detection method for tetrahydrocannabinol and its metabolites based on ratiometric fluorescence

Abstract

The present invention discloses a method for visual inspection of tetrahydrocannabinol and its metabolites based on ratiometric fluorescence, which belongs to the field of biochemical detection technology. The method utilizes the specific binding ability of G-base-rich THC aptamers with thioflavin T and cresol violet, and realizes label-free ratiometric fluorescence detection analysis of the target THC through competition between the target THC and the two dyes thioflavin T and cresol violet. The method does not require chemical modification and only requires low-cost aptamers, thioflavin T and cresol violet dyes, and the three are stable and easy to preserve in practical applications. Rapid and visual detection of the target THC and its metabolites can be achieved through simple mixing. In the future, it is expected to be combined with a portable fluorescence detector to realize on-site instant detection of the target THC, providing a promising on-site detection strategy for tetrahydrocannabinol regulation. The selected THC aptamer is an oligonucleotide sequence obtained by in vitro screening, which has the advantages of high specificity, easy in vitro synthesis and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical detection, and in particular relates to a visual detection method for tetrahydrocannabinol and its metabolites based on ratiometric fluorescence. Background Art

[0002] Currently, the detection methods for tetrahydrocannabinol (THC) primarily rely on large laboratory instruments such as liquid chromatography / gas chromatography-mass spectrometry and surface-enhanced Raman spectroscopy. Although these methods offer good sensitivity and accurate results for THC detection, they often require high operating costs and complex sample pretreatment processes, making them difficult to use for on-site THC detection. Enzyme-linked immunosorbent assays or fluorescence kits are currently used for on-site THC detection, but their production and synthesis in standard laboratories involves antibody modification, which is relatively costly. Aptamers are oligonucleotide sequences obtained through in vitro screening techniques and have the advantages of high specificity, ease of in vitro synthesis, and stability. They are also known as "chemical antibodies." Currently, only a team from North Carolina State University has published a fluorescence method based on aptamers for the detection of THC. This method involves modifying the fluorophore and quencher used in the aptamer, which, to some extent, weakens the aptamer's binding ability to THC and increases its cost, limiting its applicability in practical applications.

[0003] Enzyme-linked immunosorbent assay (ELISA) or fluorescence-based assays are currently the main technologies for on-site detection of tetrahydrocannabinol (THC). Their production and synthesis in standard laboratories involves antibody modification, which is unstable in actual use and relatively expensive. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for visual inspection of tetrahydrocannabinol and its metabolites based on ratiometric fluorescence. This method utilizes the specific binding ability of G-base-rich THC aptamers to thioflavin T and cresol violet, and through competition between the target THC and the two dyes thioflavin T or cresol violet, achieves label-free ratiometric fluorescence detection and analysis of the target THC, and the analysis results are highly sensitive. This technology does not require chemical modification and only requires low-cost aptamers, thioflavin T and cresol violet dyes. The three are stable and easy to store in practical applications, overcoming the technical problems of the prior art that various group modifications are required, and the applicability is limited, the cost is high, and the instability is high. This technology can achieve rapid and visual detection of the target THC and its metabolites through simple mixing. In the future, it is expected to be combined with a portable fluorescence detector to achieve on-site instant detection of the target THC, providing a promising on-site detection strategy for tetrahydrocannabinol regulation.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for visualizing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence comprises the following steps:

[0007] Thioflavin T and cresyl violet were mixed and added to the THC aptamer to obtain a ratio probe, and then the test solution of tetrahydrocannabinol and its metabolites was added and detected using a molecular fluorescence detector to obtain fluorescence colorimetric data;

[0008] The nucleotide sequence of the THC aptamer is shown in SEQ ID NO.1.

[0009] Preferably, the mixing ratio of Thioflavin T and Cresyl Purple is 1:2-2:1.

[0010] Preferably, the mixing ratio of Thioflavin T and Cresyl Purple is 1:1.5-1.5:1.

[0011] Preferably, the mixing ratio of Thioflavin T and Cresyl Purple is 1:1.

[0012] Preferably, the mixing ratio of Thioflavin T to THC aptamer is 1 μM:1 μM.

[0013] Preferably, the metabolite is tetrahydrocannabinolic acid.

[0014] Preferably, the characteristic fluorescence wavelength of Thioflavin T in the molecular fluorescence detection is 490 nm.

[0015] Preferably, the characteristic fluorescence wavelength of cresyl violet in the molecular fluorescence detection is 620 nm.

[0016] Preferably, the amount of the tetrahydrocannabinol and its metabolites to be tested is 0.6-2 μM.

[0017] Compared with the prior art, the present invention has at least the following technical effects:

[0018] The present invention provides a method for visual inspection of tetrahydrocannabinol and its metabolites based on ratiometric fluorescence. In this method, the specific binding ability of G-base-rich THC aptamers to thioflavin T and cresol violet is utilized to achieve label-free ratiometric fluorescence detection and analysis of THC through competition between THC and two dyes, thioflavin T or cresol violet, and the analysis results are highly sensitive. This technology does not require chemical modification and only requires low-cost aptamers, thioflavin T, and cresol violet dyes. The three are stable and easy to store in practical applications, overcoming the technical problems of the prior art that various group modifications are required, the applicability is limited, the cost is high, and the instability is high. This technology can achieve rapid and visual detection of THC and its metabolites through simple mixing. In the future, it is expected to be combined with a portable fluorescence detector to achieve on-site instant detection of THC, providing a promising on-site detection strategy for tetrahydrocannabinol regulation.

[0019] The THC aptamer selected in this method is an oligonucleotide sequence obtained by in vitro screening technology, which has the advantages of high specificity, easy in vitro synthesis and stability.

[0020] Using this method, the target THC can obtain a fluorescent response within 1 minute, and as the THC concentration increases, the green gradually turns into red. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the fluorescence intensity changes of the target compound THC in Experimental Example 1 when interacting with SYBR Green I (A), Thioflavin T (B), Thiazole Orange (C), Protoporphyrin IX (D), Crystal Violet (E), and Cresyl Violet (F) organic dye molecules, respectively;

[0022] Figure 2 This is a schematic diagram of the fluorescence intensity changes in Experimental Example 2 after Thioflavin T and Cresyl Violet are respectively bound to the THC aptamer and different concentrations of the target substance THC are added;

[0023] Figure 3 In Experiment 3, the fluorescence intensity changes and fluorescence graphs of the ratiometric probe reacting with 0.6 μM and 2 μM of the target substance THC, respectively;

[0024] Figure 4 In Experimental Example 4, the fluorescence and intensity diagrams at different ratios of Thioflavin T and Cresyl Violet are shown;

[0025] Figure 5 In Experimental Example 5, the fluorescence and intensity graphs of the ratio probe and the target THC at different reaction times are shown;

[0026] Figure 6 Fluorescence images of a single dye binding to the target THC and a ratiometric probe binding to the target THC and THC-COOH in Experimental Example 6;

[0027] Figure 7 This is a line chart for detecting the target substance THC in Experiment 6;

[0028] Figure 8 This is a line graph showing the detection of the target compound THC-COOH by the ratio probe in Experiment 6. DETAILED DESCRIPTION

[0029] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0030] The technical solution of a specific embodiment of the present invention is:

[0031] A method for analyzing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence, the method comprises a THC aptamer (specific nucleotide sequence:

[0032] The sensor consists of three parts: THC (5′-CTTACGACCCAGGGGGGTGGACAGGCGGGGGTTAGGGG GGTCGTAAG-3′), thioflavin T, and cresyl violet. The specific sensing steps are as follows: THC, thioflavin T, and cresyl violet with a final concentration of 1 μM, respectively, are prepared in a 200 μL solution. After adding a certain concentration of tetrahydrocannabinol (THC) and its metabolite tetrahydrocannabinolic acid (THC-COOH), the fluorescence intensity changes at thioflavin T (Em=490 nm) and cresyl violet (Em=620 nm) are immediately detected using a molecular fluorescence instrument.

[0033] Screening and test verification:

[0034] Experimental Example 1: Verification and screening of organic dye molecules that specifically interact with THC aptamers

[0035] G-quadruplexes are composed of guanine-rich nucleotide sequences that respond to specific targets or microenvironmental changes by folding into a typical secondary structure. Currently reported organic dyes that can induce or bind to G-quadruplexes include protoporphyrin IX, thiazole orange, thioflavin T, crystal violet, and cresyl violet. These dyes typically interact with G-quadruplexes through π-π stacking and electrostatic interactions, resulting in changes in fluorescence intensity.

[0036] Because THC aptamers are rich in guanine, they form a G-quadruplex-like structure that can bind to the aforementioned dyes and occupy the active site of the THC aptamer, forming a label-free fluorescent probe. Because the target THC has a stronger affinity with the aptamer, it competes with the dye already bound to the THC aptamer, resulting in a change in the dye's fluorescence intensity. This allows the construction of a label-free ratiometric fluorescent probe for THC.

[0037] In the early stage of the experiment, we first tested whether the dyes that often bind to G-quadruplexes also have the ability to bind to THC aptamers, resulting in fluorescence changes.

[0038] like Figure 1 Shown are the fluorescence intensity changes of THC aptamers interacting with organic dye molecules such as SYBR Green I (A), thioflavin T (B), thiazole orange (C), protoporphyrin IX (D), crystal violet (E), and cresyl violet (F).

[0039] The results showed that the fluorescence intensities of SYBR Green I, Thioflavin T, and Thiazole Orange dyes were relatively weak in solution, but their fluorescence was significantly enhanced with the addition of THC aptamers. However, the fluorescence enhancement of Protoporphyrin IX and Crystal Violet dyes after binding to THC aptamers was not obvious, indicating that the above dyes all have certain binding sites with THC aptamers, leading to fluorescence enhancement.

[0040] In contrast to the above dye fluorescence enhancement phenomenon, the fluorescence of cresyl violet dye is very strong in solution, but its fluorescence is significantly quenched after binding with the THC aptamer, indicating that the fluorescence of cresyl violet is weakened to a certain extent after binding with the THC aptamer.

[0041] Experimental Example 2: Verify and screen out a probe for the composition ratio of dye (thioflavin T and cresyl violet) to THC aptamer.

[0042] like Figure 2 In the figure, (a) shows the change in fluorescence intensity when different concentrations of THC are added after thioflavin T is combined with THC aptamer; (b) shows the change in fluorescence intensity when different concentrations of THC are added after cresyl violet is combined with THC aptamer.

[0043] The results showed that after thioflavin T bound to the THC aptamer, fluorescence was evident at 490 nm. The fluorescence decreased with the addition of the target compound, THC. The degree of quenching increased with the concentration of the target compound, indicating that the addition of THC competed with the binding sites of the thioflavin T / THC aptamer complex, causing thioflavin T to re-dissolve into the solution and resulting in fluorescence quenching.

[0044] In contrast, the addition of the target THC after cresyl violet binds to the THC aptamer enhances the fluorescence of the dye. The addition of the target THC can compete with the binding sites of the cresyl violet / THC aptamer complex, causing the cresyl violet to dissolve back into the solution. Since the fluorescence intensity of cresyl violet in the solution is stronger than that of the cresyl violet / THC aptamer, the fluorescence is enhanced.

[0045] Therefore, with the addition of the target substance THC, the fluorescence of thioflavin T weakens and that of cresyl violet strengthens. A ratiometric fluorescent probe is formed by using a weak and a strong signal to achieve a fluorescence change from green to red in response to the target substance THC.

[0046] Experimental Example 3: Verification of the ratiometric probe (THC aptamer + thioflavin T + cresol violet) for THC detection

[0047] like Figure 3 Shown are the fluorescence intensity changes and fluorescence graphs of the probe reacting with 0.6 μM and 2 μM target THC, respectively.

[0048] The results showed that the addition of the target substance THC could significantly quench the fluorescence of thioflavin T and enhance the fluorescence of cresyl violet. It can also be observed from the actual image that the fluorescence of the probe changes from green to red, indicating that the ratio fluorescence method can realize the sensing analysis of the target substance THC.

[0049] Experimental Example 4: Exploring the ratio of thioflavin T to cresyl purple

[0050] like Figure 4 In the figure, (a) is a fluorescence diagram of thioflavin T and cresyl violet at different ratios;

[0051] (b) Schematic diagram of the intensity of thioflavin T and cresyl violet at different ratios.

[0052] The results show that: the result of selecting the probe from green to red and ΔI 620 / 490 The optimal ratio is 1:1.

[0053] Experimental Example 5: Exploring the reaction time between the target substance THC and the ratio probe

[0054] like Figure 5 Shown are the fluorescence and intensity diagrams of the ratio probe reacting with THC at different reaction times.

[0055] The results showed that the ratio probe could react immediately with the target THC, and the fluorescence immediately changed from green to red. The fluorescence did not change much with the increase of reaction time.

[0056] Test Example 6: THC Detection

[0057] like Figure 6 Shown are fluorescence images of a single dye binding target THC and a ratio probe binding target THC and THC-COOH.

[0058] like Figure 7 Shown is a line graph of the ratio probe detecting the target THC;

[0059] like Figure 8 Shown is a line graph of the ratio probe detecting the target compound THC-COOH.

[0060] The results showed that the fluorescence of a single dye did not change significantly with the increase of the target THC concentration. However, the ratio probe composed of two dyes showed obvious fluorescence changes with the increase of the target THC concentration in the presence of the target THC. THC was detected by the ratio of the emission intensity of cresyl violet and thioflavin T.

[0061] Example 1:

[0062] A method for analyzing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence, wherein the specific nucleotide sequence of the THC aptamer in this method is:

[0063] 5′-CTTACGACCCAGGGGGGTGGACAGGCGGGGGTTAGGGG GGTCGTAAG-3′

[0064] The sensor consists of three parts: THC aptamer, thioflavin T, and cresol violet. The specific sensing steps are as follows: THC aptamer, thioflavin T, and cresol violet with a final concentration of 1 μM are prepared in a 200 μL solution. After adding a certain concentration of tetrahydrocannabinol (THC) and its metabolite tetrahydrocannabinolic acid (THC-COOH), the fluorescence intensity changes at thioflavin T (Em=490 nm) and cresol violet (Em=620 nm) are immediately detected using a molecular fluorescence instrument.

[0065] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for visualizing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence, characterized in that: The steps include: Thioflavin T and cresyl violet were mixed and added to the THC aptamer to obtain a ratio probe, and then the test solution of tetrahydrocannabinol and its metabolites was added and detected using a molecular fluorescence detector to obtain fluorescence intensity data; The mixing molar ratio of Thioflavin T and Cresyl Purple is 1:1; The metabolite is tetrahydrocannabinolic acid; The nucleotide sequence of the THC aptamer is shown in SEQ ID NO.

1.

2. The method for visualizing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence according to claim 1, wherein: The characteristic fluorescence wavelength of thioflavin T detected by the molecular fluorescence is 490 nm.

3. The method for visualizing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence according to claim 1, wherein: The characteristic fluorescence wavelength of cresyl violet in the molecular fluorescence detection is 620 nm.

4. The method for visualizing tetrahydrocannabinol and its metabolites based on ratiometric fluorescence according to claim 1, wherein: The amount of the test solution of tetrahydrocannabinol and its metabolites is 0.6-2 μM.

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