Method for instantly detecting various hepatotoxic pyrrolizidine alkaloids

Abstract

The invention relates to the field of chemical detection, and discloses a method for instantly detecting various hepatotoxic pyrrolizidine alkaloids, which comprises the following steps: preparing a reaction base solution containing a combined probe, rhodamine B and heme, the combined probe comprising LFAzyme1 and LFAzyme2; preparing sample solutions containing different concentrations of hepatotoxic pyrrolizidine alkaloids; preparing a substrate solution containing hydrogen peroxide; mixing the sample solution with the reaction base solution, and adding the substrate solution for reaction; and by observing the fluorescence change of the observation liquid, carrying out real-time detection on various hepatotoxic pyrrolizidine alkaloids in the observation liquid. By utilizing the specific binding characteristic of the aptamer structural domain in the combined probe and the HPAs, the mechanism that the probe is disassembled and rhodamine B fluorescence is recovered when the HPAs exist is utilized, and naked eye visual detection without large instruments such as liquid chromatography-mass spectrometry is realized by observing the color change of the fluorescence and combining quantitative analysis of an enzyme-linked immunosorbent assay instrument.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and in particular to a method for instantly detecting multiple hepatotoxic pyrrolizidine alkaloids. Background Art

[0002] Hepatotoxic pyrrolizidine alkaloids (HPAs) are a class of naturally occurring toxic compounds found in over 6,000 flowering plant species, including those in the Asteraceae and Boraginaceae families. Humans can be exposed to these compounds through ingestion of contaminated foods (such as wild vegetables and honey) or herbs (such as Senecio chinensis and Eupatorium truncatum). Due to the limited conjugated bonds in their structure and their weak UV absorption, HPAs require highly sensitive analytical techniques for detection. Furthermore, HPAs are cumulative in toxicity, and long-term ingestion can cause liver fibrosis, cirrhosis, and even liver cancer. Currently, no clear antidote exists. Therefore, establishing efficient and convenient methods for detecting HPAs is of great importance for public health and safety.

[0003] Traditional HPAs detection technology relies primarily on instrumental analysis, exemplified by liquid chromatography-mass spectrometry (LC-MS / MS). This method uses high-performance liquid chromatography to separate the various HPAs components, combined with the high sensitivity of mass spectrometry for qualitative and quantitative analysis. It utilizes the differences in the distribution of HPAs between the stationary and mobile phases for separation, and uses mass spectrometry fragment ion information to determine the molecular structure. However, this method requires the use of large-scale instrumentation (such as a mass spectrometer), which is expensive and leads to high testing costs, making it difficult to popularize in grassroots testing institutions, remote areas, or field scenarios. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a method for the real-time detection of multiple hepatotoxic pyrrolizidine alkaloids, which solves the problem that traditional HPAs detection technology has high equipment costs, resulting in high detection costs and difficulty in popularization in grassroots testing institutions, remote areas or on-site scenarios.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: a method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids, comprising the following steps:

[0006] Preparing a reaction base solution containing a combination probe, rhodamine B, and heme, wherein the combination probe includes LFAzyme1 and LFAzyme2;

[0007] Prepare sample solutions containing different concentrations of hepatotoxic pyrrolizidine alkaloids;

[0008] preparing a substrate solution containing hydrogen peroxide;

[0009] After mixing the sample solution with the reaction base solution, the substrate solution is added to react to obtain the observation solution;

[0010] By observing the fluorescence changes of the observation fluid, multiple hepatotoxic pyrrolizidine alkaloids in the observation fluid can be immediately detected.

[0011] By adopting the above technical solution, the specific binding properties of the aptamer domain in the combination probe to HPAs are utilized, and the mechanism of the probe disintegrating and restoring the fluorescence of rhodamine B in the presence of HPAs is utilized. By observing the fluorescence color change and combining it with quantitative analysis by an enzyme reader, naked-eye visual detection is achieved without the need for large instruments such as liquid chromatography-mass spectrometry. This solves the problem of traditional HPAs detection technology, which has high equipment costs, resulting in high detection costs and difficulty in popularization in grassroots testing institutions, remote areas or on-site scenarios.

[0012] Preferably, the LFAzyme1 is formed by self-assembly of the recognition fragment RS1 and the "auxiliary" DNA HD1, and the LFAzyme2 is formed by self-assembly of the recognition fragment RS2 and the "auxiliary" DNA HD2c.

[0013] Preferably, the nucleotide sequence of the recognition fragment RS1 is 5′-CTTACGACGGCATGCTGGTCGGGTAGGGAATTTTGGGCAGTCGTAAG GG-3′, and the nucleotide sequence of the “helper” DNA HD1 is 5′-TTTGGGTTGGGTTGGGAAACGACTGCCCAAAATTCC-3′.

[0014] Preferably, the nucleotide sequence of the recognition fragment RS2 is 5′-CTTACGGCTGGGTTTGGTGTCCTTCGTGGTGAGGGTTAGTCGTAAGGG-3′, and the nucleotide sequence of the “auxiliary” DNA HD2c is 5′-TGAGGGTTGGGTTGGGAAACGACTAACCCTCACCACG-3′.

[0015] Preferably, the reaction base solution contains 20mM Tris-HCl buffer solution with a pH of 8.0, 50mM NaCl, 20mM KCl, and 0.05% triton-X-100, the concentration of the combination probe is 1μM, the concentrations of LFAzyme1 and LFAzyme2 are both 0.5μM, the concentration of rhodamine B is 30μM, and the concentration of hemoglobin is 5μM.

[0016] Preferably, the substrate solution contains 20 mM Tris-HCl buffer solution with a pH of 8.0, and the concentration of hydrogen peroxide is 4 mM.

[0017] Preferably, the reaction is carried out by adding 10 μL of sample solution to 70 μL of reaction base solution, and then adding 20 μL of substrate solution, and the reaction time is 15 minutes.

[0018] Preferably, the instant detection is performed by irradiating the observation liquid with a handheld laser pen and observing its color change for qualitative analysis. When hepatotoxic pyrrolizidine alkaloids are present, the observation liquid appears bright yellow, and the fluorescence value of the observation liquid at 580 nm is detected by an enzyme marker to quantitatively analyze the content of hepatotoxic pyrrolizidine alkaloids therein.

[0019] The present invention provides a method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids. It has the following beneficial effects:

[0020] 1. The present invention utilizes the specific binding properties of the aptamer domain in the combination probe to HPAs, and the mechanism by which the probe disintegrates and restores the fluorescence of rhodamine B in the presence of HPAs. By observing the fluorescence color change and combining it with quantitative analysis by a microplate reader, it achieves naked-eye visual detection without the need for large instruments such as liquid chromatography-mass spectrometry. This solves the problem that traditional HPAs detection technology has high equipment costs, resulting in high detection costs and difficulty in popularization in grassroots testing institutions, remote areas or on-site scenarios.

[0021] 2. The present invention utilizes the specific binding ability of the aptamer domain to a variety of hepatotoxic pyrrolizidine alkaloids, so that the combined probe disintegrates and restores the fluorescence signal only in the presence of target HPAs, and has no response to interfering substances such as caffeine and matrine. It achieves highly specific recognition of at least 8 HPAs, including senecioine and selenoline, and avoids the problem of false detection caused by structural analogs in traditional detection.

[0022] 3. The present invention uses a peroxidase-like active structure and fluorescence signal amplification mechanism formed by probe self-assembly. In the concentration range of 0-1μM, the HPAs concentration and fluorescence intensity show a strict linear relationship, with a detection limit as low as about 43nM. It can accurately capture trace HPAs, and has improved sensitivity compared to traditional enzyme-linked immunosorbent assays, providing technical support for early risk warning of toxic substance exposure.

[0023] 4. The present invention adopts an extremely simple "mix-react-observe" operation process, which does not require complex pre-treatment such as solid-phase extraction and derivatization. The solution mixing can be completed only by a micropipette, and the color change can be observed with the naked eye using a handheld laser pen within 15 minutes. Non-professionals can operate it independently after training, which completely changes the limitation of traditional LC-MS technology that relies on professional personnel.

[0024] 5. The present invention utilizes the synergistic catalytic effect of the G-4 sequence and heme to trigger fluorescence "on" through conformational changes in the presence of HPAs. No additional fluorescent group labeling or enzyme coupling steps are required, which not only simplifies the operation process but also avoids the interference of the label on the detection system. Compared with the traditional fluorescent probe method, it reduces 2-3 steps and improves detection efficiency.

[0025] 6. The present invention uses a dual verification mechanism of color visualization under laser pen illumination and quantitative detection by an enzyme marker, making the test results both intuitive and ensuring data accuracy. On-site testers can quickly judge risks with the naked eye, and laboratory personnel can accurately quantify the content through fluorescence values, meeting the detection needs of different scenarios.

[0026] 7. The reaction base liquid and substrate solution of the present invention can be prefabricated in advance in the form of freeze-dried reagents or test strips. When used with a portable fluorescence detector, the detection reagents can be transported at room temperature and stored for a long time, solving the dependence of traditional detection technology on cold chain transportation and real-time liquid preparation, and providing convenient conditions for field operations, emergency detection and other scenarios.

[0027] 8. The detection method constructed in the present invention is compatible with the simultaneous detection of multiple HPAs, eliminating the need to develop independent detection systems for each alkaloid. It exhibits significant fluorescence responses to all eight HPAs at a concentration of 200 μM, improving efficiency compared to traditional single-component detection. It is particularly suitable for the rapid screening of samples from multiple contamination sources, providing an efficient tool for food safety supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a flow chart of a method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids proposed by the present invention;

[0029] Figure 2 This is a schematic diagram of the fluorescent probe LFAzyme of the present invention for detecting hepatotoxic pyrrolizidine alkaloids;

[0030] Figure 3 Schematic diagram of the detection performance of the fluorescent probe LFAzyme of the present invention;

[0031] Figure 4 Schematic diagram of the specific detection of HPAs by the fluorescent probe LFAzyme of the present invention;

[0032] Figure 5 Schematic diagram of the quantitative analysis of HPAs in the actual samples of 50% scented tea or 5% honey according to the present invention. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Please see the attached Figure 1 -Attached Figure 5 The present invention provides a method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids, comprising the following steps:

[0035] A reaction base solution containing a combination probe, rhodamine B and heme is prepared. The combination probe includes LFAzyme1 and LFAzyme2; LFAzyme1 is self-assembled by the recognition fragment RS1 and "auxiliary" DNA HD1, and LFAzyme2 is self-assembled by the recognition fragment RS2 and "auxiliary" DNA HD2c.

[0036] The nucleotide sequence of the recognition fragment RS1 is 5′-CTTACGACGGCATGCTGGTCGGGTAGGGAATTTTGGGCAGTCGTAAG GG-3′, and the nucleotide sequence of the “helper” DNA HD1 is TTTGGGTTGGGTTGGGAAACGACTGCCCAAAATTCC-3′.

[0037] The nucleotide sequence of the recognition fragment RS2 is 5′-CTTACGGCTGGGTTTGGTGTCCTTCGTGGTGAGGGTTAGTCGTAAGGG-3′, and the nucleotide sequence of the “helper” DNA HD2c is 5′-TGAGGGTTGGGTTGGGAAACGACTAACCCTCACCACG-3′.

[0038] The reaction base solution contains 20mM Tris-HCl buffer solution with a pH of 8.0, 50mMNaCl, 20mMKCl, and 0.05% triton-X-100. The concentration of the combined probe is 1μM, of which the concentrations of LFAzyme1 and LFAzyme2 are each 0.5μM, the concentration of rhodamine B is 30μM, and the concentration of heme is 5μM.

[0039] Specifically, when preparing a reaction base solution containing a combination probe, rhodamine B, and heme, the combination probe must be prepared first. The combination probe includes LFAzyme1 and LFAzyme2. LFAzyme1 is self-assembled from the recognition fragment RS1 and the "helper" DNA HD1, while LFAzyme2 is self-assembled from the recognition fragment RS2 and the "helper" DNA HD2c. RS1 and RS2 contain the HPAs aptamer domain and a quarter of a G-4 sequence, while HD1 and HD2c consist of a sequence complementary to the aptamer domain and three-quarters of a G-4 sequence. The two can self-assemble at room temperature through complementary base pairing to form a structure with horseradish peroxidase-like activity. Specifically, RS1 and HD1, as well as RS2 and HD2c, were mixed at equimolar concentrations and dissolved in a solution containing 20 mM Tris-HCl buffer (pH 8.0), 50 mM NaCl, and 20 mM KCl. Tris-HCl buffer (pH 8.0) was used to maintain the acid-base environment of the reaction system, while NaCl and KCl stabilized the ionic strength of the solution and promoted the stability of the probe structure. Triton-X-100 increased the compatibility of the solution and prevented probe aggregation. Ultimately, the final concentration of the combined probe was 1 μM, completing the self-assembly of LFAzyme.

[0040] After probe preparation, the reaction base solution was prepared. Rhodamine B (RhB) and hemin were added to the solution containing the combined probe. RhB served as a fluorescent indicator at a final concentration of 30 μM, and hemin at a final concentration of 5 μM. Together with the combined probe, they formed a detection system. In the absence of HPAs in the solution, the self-assembled structure of LFAzyme1 and LFAzyme2, under the action of hemin and hydrogen peroxide, exhibited enzymatic activity, generating hydroxyl radicals (·OH), which quenched RhB fluorescence. However, in the presence of HPAs, HPAs bind to the aptamer domain in the probe, causing the probe to disintegrate and lose its enzymatic activity. This allows the restoration of RhB fluorescence, thus enabling the detection of HPAs.

[0041] To prepare the reaction base solution, the following steps were performed: To a 50 mL centrifuge tube, add a predetermined amount of 200 mM Tris-HCl buffer (pH 8.0). Then, add pre-dissolved 500 mM NaCl and 200 mM KCl, in order, to final concentrations of 20 mM Tris-HCl, 50 mM NaCl, and 20 mM KCl, respectively. Add LFAzyme 1 (RS1 and HD1) and LFAzyme 2 (RS2 and HD2c) solutions to a final concentration of 0.5 μM each in the reaction solution, for a total combined probe concentration of 1 μM. Mix thoroughly. Subsequently, add RhB solution prepared in ultrapure water and a 500 μM hemin solution prepared in DMSO to final concentrations of 30 μM RhB and 5 μM hemin, respectively. Mix thoroughly and store in the dark until ready for use. This reaction base solution preparation procedure ensures accurate concentrations of each component and a stable system, providing a reliable reaction environment for subsequent HPAs detection.

[0042] A sample solution containing different concentrations of hepatotoxic pyrrolizidine alkaloids was prepared; a substrate solution containing hydrogen peroxide was prepared; the substrate solution contained a 20 mM Tris-HCl buffer solution with a pH of 8.0, and the final concentration of hydrogen peroxide was 4 mM.

[0043] Specifically, when preparing sample solutions containing different concentrations of hepatotoxic pyrrolizidine alkaloids (HPAs), DMSO is required as a solvent to ensure that the sample is completely dissolved. The specific operation is to dissolve the HPAs standard (such as senecioine, senecioine, etc.) with DMSO, and prepare a series of sample solutions of different concentrations by gradient dilution. Since HPAs have poor water solubility and are difficult to mix by directly adding to the reaction base solution, HPAs are dissolved in DMSO in advance to form a high-concentration mother solution, and then mixed with triton-X-100 in proportion to increase the compatibility of the solution. During preparation, first weigh a certain amount of HPAs standard, dissolve it in DMSO to make a high-concentration mother solution, and then gradually dilute it with DMSO according to the required concentration gradient, and mix it with triton-X-100 to obtain a sample solution with a final concentration range of 0 to 500 μM, so as to subsequently detect the effects of different concentrations of HPAs on the system.

[0044] When preparing a substrate solution containing hydrogen peroxide, it is important to clarify the mechanism of action of this solution in the detection system. Hydrogen peroxide, as the substrate for the enzyme-catalyzed reaction, together with the combination probe and heme, forms a peroxidase-like catalytic system. In the absence of HPAs, the structure formed by the combination probe self-assembly in the presence of heme catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radicals (·OH), thereby quenching the fluorescence of rhodamine B. In the presence of HPAs, the probe binds to the HPAs, causing structural disintegration and loss of catalytic activity. This reduces hydroxyl radical generation and restores rhodamine B fluorescence. Based on this principle, the preparation of the substrate solution requires strict control of composition and concentration: 20 mM Tris-HCl buffer (pH 8.0) is used as the substrate, and hydrogen peroxide is added to a final concentration of 4 mM. In the specific operation, a certain volume of 200mM Tris-HCl buffer solution (pH 8.0) is first measured. The amount of 30% hydrogen peroxide solution required is then calculated based on the hydrogen peroxide concentration and volume. This solution is accurately pipetted with a micropipette and added to the buffer solution. Mix thoroughly to prepare the substrate solution. The pH value and hydrogen peroxide concentration of this substrate solution must be precisely controlled to ensure the effectiveness of the catalytic reaction and the stability of the detection system, thus ensuring accurate changes in the fluorescence signal in the subsequent reaction.

[0045] After the sample solution and the reaction base solution are mixed, the substrate solution is added to react to obtain the observation solution; the reaction is carried out by adding 10 μL of the sample solution to 70 μL of the reaction base solution, and then adding 20 μL of the substrate solution, and the reaction time is 15 minutes.

[0046] Specifically, when the sample solution is mixed with the reaction base solution and then the substrate solution is added for reaction, a specific operating procedure must be followed to ensure the accuracy and reliability of the detection system. Specifically, a 1.5mL centrifuge tube is used as a reaction container, and 70μL of reaction base solution is accurately pipetted into the tube using a micropipette. The reaction base solution already contains a combination probe (0.5μM each of LFAzyme1 and LFAzyme2), rhodamine B (30μM), heme (5μM) and a buffer system, which can provide a stable environment for subsequent reactions. Subsequently, 10μL of sample solution containing different concentrations of hepatotoxic pyrrolizidine alkaloids (HPAs) is added. The sample solution uses DMSO as a solvent and is mixed with 0.05% triton-X-100 to ensure that the HPAs interact with the combination probe immediately after mixing.

[0047] After mixing thoroughly, 20 μL of substrate solution (20 mM Tris-HCl buffer, pH 8.0, containing 4 mM hydrogen peroxide) must be quickly added to initiate the enzyme-catalyzed reaction. At this point, the final concentrations of each component in the system must meet the detection requirements: the total concentration of the combined probe is 1 μM, the HPAs concentration is determined by the sample dilution ratio, and the hydrogen peroxide concentration is 4 mM. The reaction principle is that when HPAs are present in the sample solution, they specifically bind to the aptamer domains in the combined probe, causing the self-assembled structure of LFAzyme1 and LFAzyme2 to disintegrate, losing their peroxidase-like activity and unable to catalyze the conversion of hydrogen peroxide to hydroxyl radicals (·OH), thereby preserving the fluorescence of rhodamine B. If HPAs are absent from the sample solution, the combined probe remains self-assembled and, under the action of heme and hydrogen peroxide, catalyzes the production of ·OH, quenching the fluorescence of rhodamine B.

[0048] During the reaction, the centrifuge tube needs to be incubated at room temperature (25±2°C) for 15 minutes to ensure that the binding reaction between HPAs and the probe and the enzyme-catalyzed reaction are fully carried out. During the incubation period, the centrifuge tube can be gently shaken to mix the system evenly, but violent shaking should be avoided to prevent the generation of bubbles. After 15 minutes, the reaction system reaches dynamic equilibrium and the fluorescence signal tends to be stable. The observation solution obtained at this time can be used for subsequent fluorescence detection. The setting of this reaction time is based on the binding rate of the combined probe and HPAs and the kinetic characteristics of the enzyme-catalyzed reaction. It can not only ensure the rapidity of the detection, but also ensure the reliability of the fluorescence signal, laying the foundation for the subsequent real-time detection of HPAs by naked eye observation or instrument detection.

[0049] The presence of multiple hepatotoxic pyrrolizidine alkaloids in the observation fluid is immediately detected by observing changes in fluorescence. This immediate test involves illuminating the observation fluid with a handheld laser pen and observing its color change for qualitative analysis. In the presence of hepatotoxic pyrrolizidine alkaloids, the observation fluid turns bright yellow. The fluorescence value of the observation fluid at 580 nm is then measured using a microplate reader to quantitatively analyze the content of hepatotoxic pyrrolizidine alkaloids.

[0050] Specifically, the immediate detection of multiple hepatotoxic pyrrolizidine alkaloids (HPAs) is achieved by observing fluorescence changes in the observation fluid, requiring a combination of qualitative and quantitative analytical methods. For qualitative analysis, a handheld laser pointer (typically 450-550 nm) with a wavelength matching the excitation spectrum of rhodamine B (RhB) is used to illuminate the observation fluid, and the color change is observed with the naked eye in a darkroom environment. The principle is based on the following: when HPAs are present in the observation fluid, the combined probe (LFAzyme1 / LFAzyme2) disintegrates after binding to the HPAs, losing its ability to catalyze hydrogen peroxide to produce hydroxyl radicals (·OH). RhB fluorescence is not quenched, and the solution appears bright yellow under laser pointer excitation. In the absence of HPAs, the probe remains self-assembled, and the catalytically generated ·OH quenches RhB fluorescence, resulting in a green solution under laser pointer excitation. During operation, the laser pointer should be kept approximately 5-10 cm away from the observation fluid surface, with the illumination angle perpendicular to the liquid surface to avoid interference from ambient light. The color difference can be distinguished by the naked eye within 5 minutes.

[0051] Quantitative analysis is achieved by detecting the fluorescence value of the observation liquid at 580nm using a microplate reader. The specific operation is to transfer the observation liquid after 15 minutes of reaction to a 96-well black well plate, set the excitation wavelength of the microplate reader to 540nm and the emission wavelength to 580nm, and read the fluorescence intensity of each well. Since the concentration of HPAs is positively correlated with the degree of fluorescence recovery, it is necessary to first prepare an HPAs standard solution (0-1μM) to construct a standard curve: the HPAs concentration is used as the horizontal axis, the fluorescence intensity (F-F0, F is the detection value, F0 is the fluorescence value of the blank group) is used as the vertical axis, and a linear equation is obtained by fitting. When detecting unknown samples, the HPAs content can be calculated by substituting the measured fluorescence value into the equation. This method utilizes the specific binding of the combined probe to HPAs and the enzyme-catalyzed fluorescence quenching-recovery mechanism to convert molecular recognition events into quantifiable fluorescence signals, realizing a closed-loop detection process from qualitative to quantitative, and the detection process does not require professional instrument operation. The entire process from reaction to result reading can be completed within 15 minutes, which is suitable for on-site rapid screening.

[0052] A combination probe consisting of LFAzyme1 and LFAzyme2 was designed. LFAzyme1 is self-assembled from a recognition fragment RS1 and a "helper" DNA HD1, while LFAzyme2 is self-assembled from a recognition fragment RS2 and a "helper" DNA HD2c. RS1 and RS2 contain the HPAs aptamer domain and a quarter G-4 sequence, while HD1 and HD2c consist of a sequence complementary to the aptamer domain and three-quarter G-4 sequences. These probes self-assemble at room temperature into structures with horseradish peroxidase-like activity. In the presence of HPAs, the aptamer domains in the probes bind to the HPAs, causing the probes to disassemble and lose their enzymatic activity, thereby restoring the fluorescence of rhodamine B. Illuminating with a handheld laser pointer reveals the solution's color change from green to bright yellow, which can be observed with the naked eye. Quantitative analysis is achieved by measuring the fluorescence at 580 nm using a microplate reader.

[0053] A label-free detection system was constructed by leveraging the fluorescence signal conversion mechanism triggered by the specific binding of the combined probe to HPAs. This method requires no complex sample pretreatment steps; simply mixing 10 μL of sample solution with 70 μL of reaction base solution and adding 20 μL of substrate solution completes the reaction within 15 minutes, and results can be obtained by visual observation or simple instrumental testing. This method enables rapid, sensitive, and visual detection of multiple HPAs. The detection process eliminates the need for large, expensive instruments such as liquid chromatography-mass spectrometry (LC-MS / MS), requiring only conventional equipment such as a handheld laser pointer and a microplate reader. The reagent usage is minimal and cost-effective, and the procedure is simple, allowing non-technical personnel to master it with minimal training. This method addresses the challenges of traditional HPAs detection technologies, which rely on large instruments, resulting in high equipment and testing costs, complex operation requiring specialized personnel, and long testing cycles, making them difficult to popularize in grassroots testing institutions, remote areas, or field settings. It provides a practical technical solution for rapid on-site screening and grassroots testing of HPAs, effectively increasing the accessibility and widespread availability of testing.

[0054] The following is further introduced in conjunction with specific embodiments:

[0055] Example 1: Detection of HPAs in Standard Solution

[0056] Experimental material preparation:

[0057] Oligonucleotide probes: RS1 (sequence: 5′-CTTACGACGGCATGCTGGTCGGGTAGGGAATTTTGGGCAGTCGTAAG GG-3′), HD1 (sequence: 5′-TTTGGGTTGGGTTGGGAAACGACTGCCCAAAATTCC-3′), RS2 (sequence: 5′-CTTACGGCTGGGTTTGGTGTCCTTCGTGGTGAGGGTTAGTCGTAAGGG-3′), HD2c (sequence: 5′-TGAGGGTTGGGTTGGGAAACGACTAACCCTCACCACG-3′) were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.

[0058] Chemical reagents: Tris-HCl (analytical grade, Shanghai Sangon Biotechnology Co., Ltd.), NaCl (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), KCl (analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), triton-X-100 (analytical grade, Beijing Solebold Co., Ltd.), rhodamine B (RhB, analytical grade, Aladdin), hemin (heme, analytical grade, Shanghai Maclean Biotechnology Co., Ltd.), 30% H2O2 (analytical grade, Chengdu Kelong Chemical Co., Ltd.).

[0059] Water used for solution preparation: ultrapure water (18.2 MΩ·cm, prepared by Millipore).

[0060] Preparation of combination probes

[0061] Self-assembly of LFAzyme1 and LFAzyme2: Dissolve RS1, HD1, RS2, and HD2c in ultrapure water to prepare a 100 μM stock solution. Dilute with ultrapure water to a 10 μM concentration for each of RS1, HD1, RS2, and HD2c. Add 200 mM Tris-HCl buffer (pH 8.0), 500 mM NaCl, and 200 mM KCl, and gently mix to complete the self-assembly of LFAzyme1 and LFAzyme2.

[0062] Preparation of detection solution

[0063] Solution 1 (reaction base solution): In a 50 mL centrifuge tube, add the following reagents in sequence:

[0064] 200 mM Tris-HCl buffer (pH 8.0): 5 mL

[0065] NaCl (500 mM stock solution): 5 mL

[0066] KCl (200 mM stock solution): 5 mL

[0067] Triton-X-100 (1% w / v stock solution): 5 mL

[0068] RhB (300 μM stock solution, dissolved in water): 5 mL

[0069] Hemin (500 μM stock solution, dissolved in DMSO): 0.5 mL

[0070] LFAzyme 1 (RS1 and HD1) (10 μM): 2.5 mL, 2.5 mL

[0071] LFAzyme2 (RS2 and HD2c) (10 μM): 2.5 mL, 2.5 mL

[0072] Add ultrapure water to 50 mL. After preparation, calibrate the pH to 8.0 using a pH meter. The concentrations of the components in Solution 1 are: 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 20 mM KCl, 0.05% triton-X-100, 30 μM RhB, 5 μM hemin, 0.5 μM LFAzyme 1, and 0.5 μM LFAzyme 2.

[0073] Solution 2 (HPAs standard solution):

[0074] HPAs standard solution: Weigh 33.54 mg of HPAs standard and dissolve in DMSO to 10 mL to obtain a 10 mM stock solution. Prepare a series of standard solutions with concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50, 100, 200, and 500 μM by sequential dilution with DMSO.

[0075] Solution 3 (substrate solution): Take 20.4 μL of 30% H2O2 solution and prepare 2 mL of 100 mM stock solution. Take 4 μL and add it to a centrifuge tube containing 2 μL of 200 mM Tris-HCl buffer (pH 8.0). Add ultrapure water to 20 μL and mix thoroughly to obtain 4 mM H2O2 substrate solution.

[0076] Detection reaction operation

[0077] Take a 1.5 mL centrifuge tube, add 70 μL of solution 1 and 10 μL of solution 2 (HPAs standard solution) of different concentrations in sequence, mix gently, add 20 μL of solution 3, shake quickly to mix, and start the reaction.

[0078] After 15 minutes of reaction, fluorescence was measured at 580 nm using a multifunctional microplate reader (M2), with an excitation wavelength of 540 nm and an emission wavelength of 580 nm. Simultaneously, the reaction system was illuminated with a 450 nm handheld laser pointer. Color changes were recorded in a darkroom using a mobile phone. These changes were then analyzed using RGB values.

[0079] Results and Analysis (see Appendix Figure 2 , Schematic diagram of the fluorescent probe LFAzyme for detecting hepatotoxic pyrrolizidine alkaloids, including (a) LFAzyme construction schematic diagram, and (b) LFAzyme-based detection mechanism. Figure 3 Detection performance of the fluorescent probe LFAzyme, including (a) the change of fluorescence intensity (F-F0) with different concentrations of HPAs, the inset shows the linear range of 0-1μM HPAs; (b) fluorescence images of samples containing various HPAs under the illumination of a handheld laser pointer; (c) R / G values ​​of different concentrations of HPAs, the inset shows the linear range of 0-1μM HPAs)

[0080] Fluorescence quantitative analysis: A standard curve was drawn with HPAs concentration (C, μM) as the horizontal axis, fluorescence intensity (F-F0, F is the detected fluorescence value, F0 is the blank fluorescence value at 0 μM) and R / G value as the vertical axis. The results showed that HPAs showed a good linear relationship in the concentration range of 0-1 μM: A=k·C+b, where k is the slope, b is the intercept, A is the detected fluorescence value or RGB value, and C is the HPAs concentration. The linear equations are:

[0081] F-F0=56.5·C+0.36(R 2 =0.9929)

[0082] R / G=0.86·C+0.02(R 2 =0.9966)

[0083] The limit of detection (LOD) was calculated as 3σ / κ and was 43 nM when fluorescence was used as the analysis value and 63 nM when RGB was used as the analysis value.

[0084] Visualization results: When HPAs concentrations ≥ 0.1 μM, the solution changes from green (RhB fluorescence is quenched in the absence of HPAs) to bright yellow under laser illumination, clearly discernible to the naked eye. At concentrations below 0.1 μM, the color change is less pronounced. A clear color change is observed after 5 minutes of reaction, and the fluorescence signal stabilizes after 15 minutes.

[0085] Example 2: Specific detection of multiple HPAs

[0086] Experimental Materials

[0087] HPAs standards: senecine (Sen), reticulate senecine (Ret), arugula senecine (Er), spring senecine (Sv), seneciopine (Sp), jacobine (Jb), monocrotaline (Mon), heliotropin (He), all with a purity ≥ 98% (Chengdu Pusi Biotechnology Co., Ltd., Shanghai MacLean Biotechnology Co., Ltd.).

[0088] Interfering substances: caffeine (Ca), matrine (Ma), choline (Cho), indigo (Ind), ligustrazine hydrochloride (Lh), and theophylline (The) were all analytically pure (Chengdu Purifa Technology Development Co., Ltd., Chengdu Pusi Biotechnology Co., Ltd.).

[0089] Other materials: same as Example 1.

[0090] Solution preparation

[0091] HPAs sample solution: Each HPAs standard was prepared into a 200 μM solution (solvent: DMSO).

[0092] Interferor sample solution: Each interferor was prepared into a 1000 μM solution (solvent: DMSO).

[0093] Solution 1 and Solution 3: Same as Example 1.

[0094] Testing process

[0095] Take a 1.5 mL centrifuge tube, add 70 μL of solution 1 and 10 μL of different HPAs sample solutions or interfering substance sample solutions, mix well, then add 20 μL of solution 3 and react for 15 minutes.

[0096] Detect the fluorescence value at 580nm and record the color change under laser pen irradiation.

[0097] Results and Analysis (see Appendix Figure 4 Specific detection of HPAs by the fluorescent probe LFAzyme. (a) Fluorescence values ​​(F-F0) and (b) corresponding photographs of different types of HPAs (senecine (Sen), reticulate senecine (Ret), arugula senecine (Er), spring senecine (Sv), seneciophylline (Sp), calycobin (Jb), monocrotaline (Mon), heliotropin (He)) and interfering compounds (caffeine (Ca), matrine (Ma), choline (Cho), indigo (Ind), ligustrazine hydrochloride (Lh), theophylline (The)).

[0098] Fluorescence specificity: All eight HPAs could significantly enhance the fluorescence signal (F-F0>2000), while the fluorescence responses of the six interferents (F-F0<300) had no significant difference from the blank group (F-F0<100), indicating that the probe had good specificity.

[0099] Color visualization: HPAs sample solutions all appeared bright yellow under laser pen illumination, while interfering substance solutions and blank solutions appeared green, which were completely distinguishable to the naked eye, confirming that this method has the specific recognition ability for multiple HPAs.

[0100] Example 3: Specific detection of HPAs in scented tea and honey samples

[0101] Experimental Materials

[0102] Actual samples: commercially available scented tea and Sophora japonica flower honey (purchased from a supermarket).

[0103] HPAs standards and other interfering substances are the same as those in Example 2.

[0104] Other materials: same as Example 1.

[0105] Sample pretreatment

[0106] Preparation of 50% scented tea solution: soak tea bags in ultrapure water, centrifuge at 4000 rpm for 15 minutes, take the supernatant, filter with a 0.22 μm filter membrane to obtain 100% scented tea solution, and dilute to prepare 50% scented tea matrix solution.

[0107] Prepare 5% honey solution: weigh 5 g of Sophora japonica honey, add 95 mL of ultrapure water, vortex mix for 10 minutes, centrifuge at 4000 rpm for 15 minutes, collect the supernatant, and filter with a 0.22 μm filter membrane to obtain a 5% honey matrix solution.

[0108] Results and Analysis (see Appendix Figure 5 Quantification of HPAs in real samples (50% scented tea or 5% honey). (a) Different concentrations of HPAs and (b) 200 μM samples of different types of HPAs (senecine (Sen), reticulate senecine (Ret), rutabaga senecine (Er), spring senecine (Sv), seneciophylline (Sp), caesalpinine (Jb), monocrotaline (Mon), heliotropin (He)) and 1000 μM samples of different interfering substances (caffeine (Ca), matrine (Ma), choline (Cho), indigo (Ind), ligustrazine hydrochloride (Lh), theophylline (The)).

[0109] Color visualization: In 50% floral tea or 5% honey, distinct color was observed when HPAs concentrations were ≥1 μM. Furthermore, as the HPAs concentration increased, a yellow color became increasingly apparent under laser illumination. HPAs sample solutions appeared bright yellow under laser illumination, while interfering substance solutions and blank solutions appeared green, clearly distinguishable to the naked eye. This demonstrates the method's ability to specifically identify multiple HPAs and its strong anti-interference capabilities in complex matrices.

[0110] The nucleotide sequence of RS1 is:

[0111] 5′-CTTACGACGGCATGCTGGTCGGGTAGGGAATTTTGGGCAGTCGTA AGGG-3′

[0112] The nucleotide sequence of HD1 is:

[0113] 5′-TTTGGGTTGGGTTGGGAAACGACTGCCCAAAATTCC-3′

[0114] The nucleotide sequence of RS2 is:

[0115] 5′-CTTACGGCTGGGTTTGGTGTCCTTCGTGGTGAGGGTTAGTCGTAA GGG-3′

[0116] The nucleotide sequence of HD2c is:

[0117] 5′-TGAGGGTTGGGTTGGGAAACGACTAACCCTCACCACG-3′

[0118] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for the instant detection of multiple hepatotoxic pyrrolizidine alkaloids, characterized in that: The following steps are involved: Preparing a reaction base solution containing a combination probe, rhodamine B, and heme, wherein the combination probe includes LFAzyme1 and LFAzyme2; Prepare sample solutions containing different concentrations of hepatotoxic pyrrolizidine alkaloids; preparing a substrate solution containing hydrogen peroxide; After mixing the sample solution with the reaction base solution, the substrate solution is added to react to obtain the observation solution; By observing the fluorescence changes of the observation fluid, multiple hepatotoxic pyrrolizidine alkaloids in the observation fluid can be immediately detected.

2. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The LFAzyme1 is self-assembled from the recognition fragment RS1 and the "auxiliary" DNA HD1, and the LFAzyme2 is self-assembled from the recognition fragment RS2 and the "auxiliary" DNA HD2c.

3. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The nucleotide sequence of the recognition fragment RS1 is 5′-CTTACGACGGCATGCTGGTCGGGTAGGGAATTTTGGGCAGTCGTAAG GG-3′, and the nucleotide sequence of the “auxiliary” DNA HD1 is 5′-TTTGGGTTGGGTTGGGAAACGACTGCCCAAAATTCC-3′.

4. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The nucleotide sequence of the recognition fragment RS2 is 5′-CTTACGGCTGGGTTTGGTGTCCTTCGTGGTGAGGGTTAGTCGTAAGGG-3′, and the nucleotide sequence of the “auxiliary” DNA HD2c is 5′-TGAGGGTTGGGTTGGGAAACGACTAACCCTCACCACG-3′.

5. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The reaction base solution contains 20mM Tris-HCl buffer solution with a pH of 8.0, 50mM NaCl, 20mM KCl, and 0.05% triton-X-100. The concentration of the combined probe is 1μM, wherein the concentrations of LFAzyme1 and LFAzyme2 are both 0.5μM, the concentration of rhodamine B is 30μM, and the concentration of heme is 5μM.

6. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The substrate solution contains a 20 mM Tris-HCl buffer solution with a pH of 8.0, and the concentration of hydrogen peroxide is 4 mM.

7. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The reaction was carried out by adding 10 μL of sample solution to 70 μL of reaction base solution, and then adding 20 μL of substrate solution. The reaction time was 15 minutes.

8. The method for real-time detection of multiple hepatotoxic pyrrolizidine alkaloids according to claim 1, characterized in that: The instant detection is to illuminate the observation liquid with a handheld laser pen and observe its color change for qualitative analysis. When hepatotoxic pyrrolizidine alkaloids are present, the observation liquid turns bright yellow, and the fluorescence value of the observation liquid at 580nm is detected by an enzyme marker to quantitatively analyze the content of hepatotoxic pyrrolizidine alkaloids therein.

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