Biosensor for detecting total amount of pesticide residues as well as detection method and application of biosensor

Abstract

The invention belongs to the technical field of biosensors, and relates to a biosensor for detecting the total amount of pesticide residues as well as a detection method and application of the biosensor. The biosensor comprises alkaline phosphatase, a buffer solution, ascorbic acid-2-phosphate, a double-chain probe, an activation chain, a fluorescence report probe and Cu < 2 + >. The double-stranded probe is formed by hybridizing DNA1 and DNA2, one side of the blunt end of the double-stranded probe is respectively modified with an azide group and an alkynyl group, and one side of the overhanging end of the double-stranded probe is provided with a toehold region and a section of MNAzyme fragment; the activation chain comprises another MNAzyme segment, and the activation chain can be hybridized with the DNA1 to form the MNAzyme. Alkaline phosphatase is used as a broad-spectrum indicator, a collective normalization biosensing strategy for synchronous toxicity detection of multiple pesticide residues is developed based on ALP activity total inhibition rate detection, high sensitivity, operability and multi-component response capability are achieved, and early, rapid and comprehensive monitoring of the pesticide residues is facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensors, and particularly relates to a biosensor for detecting total pesticide residues and a detection method and application thereof. BACKGROUND

[0002] The extensive use of pesticides in modern agriculture is crucial to ensure food production, but the residues of pesticides in agricultural products and their potential synergistic toxicity pose a serious threat to food safety, ecological balance and public health. Especially, the coexistence of multiple pesticide residues can lead to more significant toxicity effects than single compounds. For example, a recent study showed that a mixture of three commonly used pesticides can induce synergistic toxicity and apoptosis in human hepatoma HepG2 cells through the caspase pathway related to reactive oxygen species (ROS). Therefore, the development of a new analysis method that can simultaneously detect multiple pesticide residues is of urgent significance to achieve effective food safety monitoring and accurate risk assessment.

[0003] Currently, pesticide residue detection mainly relies on traditional techniques such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). These methods are considered as standard reference methods due to their high accuracy and sensitivity. However, these traditional techniques require large and precise instruments, complex sample pretreatment, long time consumption and must be operated by professionals, which makes it difficult to meet the actual needs of on-site rapid screening and high-throughput analysis.

[0004] Biosensing technology based on functional nucleic acids (FNAs) has become a promising alternative detection strategy due to its sensitivity, speed, and ease of operation. Functional nucleic acids not only have excellent programmability, predictable Watson-Crick base pairing characteristics, and good chemical stability, but also can be used to rationally construct high-specificity recognition probes and stable sensing interfaces. In particular, with the rapid development of isothermal nucleic acid amplification techniques (such as deoxyribozyme, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), DNA walker, and CRISPR / Cas system), super-sensitive detection can be achieved without a thermal cycler, greatly improving the practicality and accessibility of the method. For example, Zhong et al. constructed an electrochemiluminescence sensor based on CRISPR / Cas12a for acetamiprid detection; Zhu and Song teams used functional nucleic acid-mediated DNA walker to achieve super-sensitive analysis of malathion; Wang et al. developed a nicking enzyme-mediated amplification DNA hydrogel aptamer sensor for identification of chlorpyrifos. Although these advanced methods show excellent performance in detecting single pesticides, their recognition mechanisms are mostly limited to the traditional mode of "one aptamer-one target", which is difficult to cope with the complex scene of coexistence of multiple pesticide residues in actual samples, and cannot meet the early warning needs of broad-spectrum and rapid preliminary screening. SUMMARY

[0005] The application aims to provide a pesticide residue total amount detection biosensor and a detection method and application thereof, so as to overcome the shortcomings of the prior art.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is as follows: In a first aspect, the application provides a pesticide residue total amount detection biosensor, comprising alkaline phosphatase, buffer, ascorbic acid-2-phosphate, double-stranded probe, activation chain, fluorescent reporter probe and Cu 2+ ; The double-stranded probe is formed by hybridization of DNA1 and DNA2, and the blunt end side of the double-stranded probe is modified with azide and alkyne respectively, and the overhanging end side is designed with a toehold region and a segment of MNAzyme fragment. The activation chain comprises another segment of MNAzyme fragment, and the activation chain can hybridize with DNA1 to form MNAzyme.

[0007] In some other embodiments, the fluorescent reporter probe is a DNA molecule with a hairpin structure, which is modified with a fluorescent group at the 5' end and a quenching group at the 3' end, and the loop region sequence of the hairpin structure contains a specific RNA cleavage site. The RNA cleavage site can be specifically recognized and cleaved by MNAzyme. The buffer is rCutSmart buffer.

[0008] In some other embodiments, the nucleotide sequence of DNA1 is shown in SEQ ID NO: 1. The nucleotide sequence of DNA2 is shown in SEQ ID NO: 2. The nucleotide sequence of the activation chain is shown in SEQ ID NO: 3. The nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO: 4.

[0009] In a second aspect, the application provides the application of the pesticide residue total amount detection biosensor in the first aspect in pesticide residue total amount detection.

[0010] In a third aspect, the application provides a pesticide residue total amount detection method, which adopts the pesticide residue total amount detection biosensor in the first aspect, and comprises the following steps: Mixing and incubating the sample extract to be tested with alkaline phosphatase, adding double-stranded probe, Cu2+ The catalytic click reaction is catalyzed by a buffer solution and ascorbic acid-2-phosphate, and then an activated chain and a fluorescent reporter probe are added to perform a signal amplification reaction, and the total amount of pesticide residues is obtained by fluorescence spectrum or gel electrophoresis.

[0011] In some other embodiments, the preparation method of the sample extraction liquid to be tested is as follows: the sample to be tested is added into an extraction agent, vortexed and extracted, centrifuged, filtered, and the filtrate is collected to obtain the sample extraction liquid to be tested. Preferably, 3-5 mL of the extraction agent is added per gram of the sample to be tested, and the extraction agent is one of acetone, acetonitrile and methanol. The extraction time is 8-12 min, and the filter membrane pore size for filtration is 0.2-0.3 μm.

[0012] In some other embodiments, the pesticide is one or more of organophosphorus, pyrethroid and phenoxyacetic acid herbicide. Preferably, the pesticide is one or more of chlorpyrifos, tetramethrin and 2,4-dichlorophenoxyacetic acid.

[0013] In some other embodiments, the temperature for mixing and incubation is 35-38 ℃, and the time is 10-60 min; preferably, the temperature for incubation is 37 ℃, and the time is 40 min. The temperature for the catalytic click reaction is 35-38 ℃, and the time is 30-150 min; preferably, the temperature for the catalytic click reaction is 37 ℃, and the time is 90 min. The temperature for the signal amplification reaction is 35-38 ℃, and the time is 30-300 min; preferably, the temperature for the signal amplification reaction is 37 ℃, and the time is 180 min.

[0014] In some other embodiments, the concentration of alkaline phosphatase is 0.05-5.0 mU·μL -1 , preferably 5.0 mU·μL -1 The concentration of the double-stranded probe is 300-700 nM, preferably 500 nM. The concentration of Cu 2+ is 10-400 μM, preferably 200 μM. The concentration of ascorbic acid-2-phosphate is 20-160 μM, preferably 80 μM. The concentration of the fluorescent reporter probe is 100-800 nM, preferably 400 nM. The buffer solution is one of rCutSmart buffer, Tris-Hydroxymethylaminomethane-sodium chloride-tween 20 buffer, 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate buffer and Tris-Hydroxymethylaminomethane-hydrochloric acid buffer.

[0015] In some other embodiments, the nucleic acid bridge of the activated strand capable of hybridizing with DNA1 to form the MNAzyme has a length of 3 nt-7 nt, preferably 5 nt; The toehold length of the MNAzyme is 5 nt-13 nt, preferably 9 nt; The stem length of the MNAzyme is 1 nt-9 nt, preferably 3 nt; The binding arm length of the MNAzyme is 5-13 nt, preferably 9 nt.

[0016] The beneficial effects of the present application are: (1) The present application develops a collective normalization biosensing strategy for synchronous toxicity detection of multiple pesticide residues based on the total inhibition rate of ALP activity with alkaline phosphatase (ALP) as a broad-spectrum indicator. The method converts the ALP activity inhibition effect into amplified fluorescence signals through MNAzyme assembly. The sensing mechanism starts from the catalysis of ALP to generate ascorbic acid, which then reduces Cu (II) to Cu (I); the generated Cu (I) drives the covalent connection between two specific DNA fragments through click chemistry, and the connection product is the key to the subsequent template-directed active MNAzyme self-assembly; the assembled MNAzyme acts as an efficient catalyst to continuously cut the quencher-labeled hairpin substrate, realizing significant enhancement of fluorescence signals. This cascade design ensures that pesticides with ALP inhibition activity can effectively inhibit the entire amplification pathway, resulting in a dose-dependent decrease in signal.

[0017] (2) The universal and sensitive pesticide residue collective evaluation platform established by the present application provides a powerful tool for preliminary warning and risk assessment in monitoring applications. Among them, chlorpyrifos, tetramethrin and 2,4-dichlorophenoxyacetic acid (2,4-D) show good detection performance, with detection limits of 34.8 nM, 55.0 nM and 26.0 nM, respectively. In addition, the method exhibits excellent performance in actual sample analysis and multiple pesticide residue detection, and the selectivity test confirms its specific response to organophosphorus, pyrethroid and phenoxyacetic acid herbicides. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which form a part of this description, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. They show illustrative embodiments of the application and, together with their description, serve to explain the application.

[0019] Figure 1 This is a schematic diagram of the collective sensing strategy in Embodiment 1 of the present invention. In this diagram, A is a mechanism diagram in which pesticides with ALP inhibitory activity inhibit the catalytic generation of ascorbic acid by ALP, and ascorbic acid reduces Cu(II) to Cu(I). B is a schematic diagram of Cu(I) triggering MNAzyme signal amplification. Figure 2 This is a feasibility verification diagram from Example 1 of the present invention. A is a schematic diagram of Cu (Ⅰ) triggering MNAzyme signal amplification; B is the fluorescence spectrum of the reaction system in the presence of different metal ions; C is a fluorescence intensity bar chart of the reaction system in the presence of different metal ions; D is a schematic diagram of ALP triggering MNAzyme signal amplification; E is the fluorescence spectrum of the reaction system under different ALP conditions; F is a fluorescence intensity bar chart of the reaction system at different ALP concentrations; G is a schematic diagram of pesticide inhibiting MNAzyme signal amplification; H is the fluorescence spectrum of the reaction system in the presence of different pesticides; and I is a polyacrylamide gel electrophoresis (PAGE) image. In I, the numbers 1-8 are lane numbers, and the letter M represents the DNA molecular weight standard. The specific substances added to the lanes are as follows: Lane 1 is DNA4, Lane 2 is DNA3, Lane 3 is DNA1-DNA2, Lane 4 is DNA1-DNA2+DNA3, and Lane 5 is Cu-free. + Positive control, lane 6 is a positive control without AAP, lane 7 is a negative control, and lane 8 is a positive control.

[0020] Figure 3 The diagram shows the optimization of key parameters of MNAzyme in Example 1 of this invention. A is a schematic diagram of the nucleic acid bridge; B is a bar chart of fluorescence intensity for negative and positive controls at different nucleic acid bridge lengths; C is a line graph of the F / F0 ratio at different nucleic acid bridge lengths; D is a schematic diagram of the toehold; E is a bar chart of fluorescence intensity for negative and positive controls at different toehold lengths; F is a line graph of the F / F0 ratio at different toehold lengths; G is a schematic diagram of the stem; H is a bar chart of fluorescence intensity for negative and positive controls at different stem lengths; I is a line graph of the F / F0 ratio at different stem lengths; J is a schematic diagram of the binding arm; K is a bar chart of fluorescence intensity for negative and positive controls at different binding arm lengths; and L is a line graph of the F / F0 ratio at different binding arm lengths. Figure 4Figure for analysis performance of different concentrations of pesticides in Example 1 of the present application, wherein A is a fluorescence spectrum of chlorpyrifos (CPF), B is a fluorescence spectrum of tetramethrin (TTM), C is a fluorescence spectrum of 2,4-D, D is a linear relationship diagram of the concentration of chlorpyrifos and fluorescence intensity, E is a linear relationship diagram of the concentration of tetramethrin and fluorescence intensity, F is a linear relationship diagram of the concentration of 2,4-D and fluorescence intensity, G is a fluorescence intensity column chart of different types of pesticides, and H is a heat map; Figure 5 Figure for actual sample detection and pesticide residue total amount detection performance in Example 1 of the present application, wherein A is a schematic diagram of an actual sample detection process, B is a fluorescence spectrum of a sample with different standard concentrations, C is a concentration-fluorescence relationship diagram of a standard sample (the inset is a calibration curve), and D is a pesticide residue total amount detection performance (three different pesticides are mixed in equimolar, and the total concentration is 10 μM); Figure 6 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different DNA1-DNA2 concentrations in Example 1 of the present application; Figure 7 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different AAP concentrations in Example 1 of the present application; Figure 8 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different Cu² + concentrations in Example 1 of the present application; Figure 9 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different DNA4 concentrations in Example 1 of the present application; Figure 10 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different types of buffer conditions in Example 1 of the present application; Figure 11 Figure for fluorescence intensity change (line chart) under different ALP and pesticide reaction times in Example 1 of the present application; Figure 12 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different click chemistry reaction times in Example 1 of the present application; Figure 13 Figure for fluorescence intensity change (column chart) and fluorescence intensity ratio (F / F0) change (line chart) under different MNAzyme cleavage times in Example 1 of the present application. DETAILED DESCRIPTION

[0021] Those skilled in the art will appreciate that the following examples are included for illustrative purposes only and should not be viewed as limiting the scope of the present application. Unless otherwise indicated, conventional conditions were employed in the examples and the manufacturers' recommendations were followed. Where used, components were conventional and commercially available.

[0022] The existing technologies have excellent detection performance for specific targets, but they mostly rely on the recognition mode of "one aptamer-one target", which cannot meet the simultaneous screening of broad-spectrum potential pesticide residues. In this paper, alkaline phosphatase (ALP) was used as a broad-spectrum indicator, and a collective normalization biosensing strategy for simultaneous toxicity detection of multiple pesticide residues was developed based on the total inhibition rate detection of ALP activity. The principle of the collective sensing strategy is shown in Figure 1 , where A is the mechanism diagram of ALP inhibition activity of pesticides inhibiting the generation of ascorbic acid by ALP, and ascorbic acid reducing Cu (Ⅱ) to Cu (Ⅰ), and B is the schematic diagram of Cu (Ⅰ) triggering MNAzyme signal amplification.

[0023] As shown in Figure 1 , the ALP activity inhibition effect is converted into amplified fluorescence signal through MNAzyme assembly. Four kinds of nucleic acid probes were specifically designed: DNA1, DNA2, DNA3 and DNA4. DNA1 hybridizes with DNA2 to form a double-stranded probe, and the blunt end of one side is modified with azide and alkyne, respectively, and the overhanging end of the other side is designed with a toehold region and an MNAzyme fragment; DNA3 hybridizes with DNA1 and contains another MNAzyme fragment; DNA4 is a hairpin structure DNA, and the two ends are modified with fluorescein (FAM) and quencher (BHQ1), respectively, and the loop region is embedded with a single RNA.

[0024] In the presence of ALP, AAP is converted to ascorbic acid (AA) by removing the phosphate group, and AA reduces Cu (Ⅱ) to Cu (Ⅰ); Cu (Ⅰ) as a key catalytic factor triggers the click chemistry reaction between the azide group and the alkyne group at the end of the double-stranded probe, converting the double-stranded probe into a single-stranded probe; after adding DNA3, it hybridizes with the single-stranded probe, bringing the two MNAzyme fragments close and assembling them into active MNAzyme; MNAzyme recognizes DNA4 through two binding arms, and then catalyzes the hydrolysis of DNA4, resulting in the separation of FAM and BHQ1 at the end of DNA4, and the recovery of fluorescence (B in Figure 1 ).

[0025] When pesticide residues are present, ALP activity is selectively inhibited, thereby hindering the conversion of double-stranded probes to single-stranded probes; at this time, DNA3 is added, which undergoes strand displacement reaction with double-stranded probes to form DNA1-DNA3 hybrid, while DNA2 dissociates from DNA1, and the two MNAzyme fragments are far apart and cannot be assembled to form active MNAzyme. This cascade design ensures that pesticides with ALP inhibitory activity can effectively inhibit the entire amplification pathway, resulting in a dose-dependent decrease in fluorescence signal (A in FIG. 1).

[0026] Example 1 1. Reagents and instruments are as follows: (1) Reagents: rCutSmart buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / mL recombinant albumin, pH 7.9); ammonium persulfate (APS) and tetramethyl ethylenediamine (TEMED) were purchased from Biyun Tian Biotechnology Co., Ltd. (Shanghai, China); DNA molecular weight marker and 6x loading buffer were purchased from Baobioengineering (Dalian) Co., Ltd. (Beijing, China); high performance liquid chromatography (HPLC) purified oligonucleotides (sequences are shown in Table 1) were synthesized by Shengong Biotech (Shanghai) Co., Ltd.; theophylline was purchased from Shengong Biotech (Shanghai) Co., Ltd.; chlorpyrifos (CPF), acep (Acep), piri (Piri), glyphosate (Glyp), Rogor (Rogor), Dipt (Dipt), TTM (TTM), Cyfl (Cyfl), Cype (Cype), DDT (DDT), HCH (HCH), Carb (Carb), Thia (Thia), and 2,4-D (2,4-D) were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China); other reagents were used directly without further purification. The reaction solution was prepared using DEPC-treated water produced by Shengong Biotech (Shanghai) Co., Ltd.

[0027] (2) Instruments: fluorescence detection was performed using an F-320 fluorescence spectrophotometer (Tianjin Gangdong Science and Technology Co., Ltd.); gel electrophoresis experiments were performed using a JY-SCZ9 electrophoresis tank, a HX-105 constant temperature water circulation system, and a DYY-6C electrophoresis power supply (all purchased from Beijing Changliu Scientific Instrument Co., Ltd.); gel imaging was performed using a Tanon 3500-BR ultraviolet imaging system (Shanghai Tian Neng Technology Co., Ltd.).

[0028] Table 1 Nucleic acid sequences

[0029] 2. Experimental protocol First, two single-stranded sequences (DNA1 and DNA2) were annealed in phosphate buffer (PBS, 0.02 M, pH 7.4) to form the DNA1-DNA2 probe. The mixture was heated at 95 °C for 5 min and then slowly cooled to room temperature. The annealed DNA1-DNA2 probe was stored at 4 °C. Other DNA was annealed in a similar way before use and stored at -20 °C. ALP was serially diluted in 1x rCutSmart buffer to a final concentration of 4 U^L -1 .

[0030] A 10 μL reaction system was prepared in 1x rCutSmart buffer, containing 5.00 mU^L -1 ALP, 500 nM DNA1-DNA2 probe, 80 μM ascorbic acid-2-phosphate (AAP), and 200 μM Cu 2+ . The copper-catalyzed click reaction was incubated at 37 °C for 90 min, followed by the addition of 600 nM DNA3 and 400 nM signal probe DNA4, and continued incubation at 37 °C for 3 h in rCutSmart buffer. The fluorescence spectrum was recorded using a fluorescence spectrophotometer, with a photomultiplier tube voltage of 700 V, an excitation and emission slit width of 5 nm, an excitation wavelength of 488 nm, and an emission spectrum range of 500-600 nm. The emission peak at 515 nm was used for quantitative analysis.

[0031] 3. Fluorescent detection of pesticide residues Different concentrations of pesticides were mixed with ALP, Cu 2+ , and DNA1-DNA2 probe in 1x rCutSmart buffer, incubated at 37 °C for 40 min to fully inhibit ALP activity; then AAP was added, and after incubation at 37 °C for 1 h, DNA3, DNA4, and 10x rCutSmart buffer were added, and the total volume was adjusted to 50 μL with water. The fluorescence spectrum was recorded using a fluorescence spectrophotometer.

[0032] 4. Gel characterization A 10 μL reaction system was prepared in 1x rCutSmart buffer, containing 5.00 mU^L -1 ALP, 500 nM DNA1-DNA2 probe, 80 μM AAP, and 200 μM Cu 2+, 37°C for 90 min; then 600 nM DNA3 and 400 nM signal probe DNA4 were added, and incubated in rCutSmart buffer at 37°C for 3 h. After the reaction, 5.0 μL reaction solution was taken, mixed with 1.0 μL 6x loading buffer to prepare the sample for polyacrylamide gel electrophoresis (PAGE), and 3.0 μL was taken for PAGE characterization. The detailed process of gel electrophoresis is as follows: The rationality of the designed sequence was verified by PAGE. The preparation method of 12% PAGE is as follows: mixed acrylamide solution (10 mL, 40%), 5xTBE buffer (5.0 mL, containing 88 mM Tris, 88 mM boric acid, 2.0 mM EDTA disodium, pH=8.3), tetramethyl ethylenediamine (18 μL), ammonium persulfate solution (180 μL, 0.1 g / mL) and DEPC water (10 mL). The electrophoresis was carried out at 37°C under constant current mode (30 mA) for 60 min. After electrophoresis, the gel was stained in SYBR Gold staining solution for 40 min in the dark, and then photographed using a gel imaging system.

[0033] 5 Results and discussion (1) Feasibility verification of collective sensing strategy This method relies on the inhibition of ALP activity by pesticides to control the assembly of MNAzyme by changing the amount of Cu (I) generated, and finally produces amplified fluorescence signal. To verify the feasibility of the method, it was verified in three steps from downstream to upstream, and the results are shown in Figure 2 A is the schematic diagram of Cu (I) triggered MNAzyme signal amplification, B is the fluorescence spectrum of the reaction system in the presence of different metal ions, C is the fluorescence intensity column chart of the reaction system in the presence of different metal ions, D is the schematic diagram of ALP triggered MNAzyme signal amplification, E is the fluorescence spectrum of the reaction system under different ALP conditions, F is the fluorescence intensity column chart of the reaction system under different ALP concentrations, G is the schematic diagram of pesticide inhibition MNAzyme signal amplification, H is the fluorescence spectrum of the reaction system in the presence of different pesticides, I is the polyacrylamide gel electrophoresis (PAGE) image, and the numbers 1-8 in I are the labels of the lanes, and the letter M is the DNA molecular weight marker. The specific substances added in each lane are as follows: lane 1 is DNA4, lane 2 is DNA3, lane 3 is DNA1-DNA2, lane 4 is DNA1-DNA2+DNA3, lane 5 is no Cu 2+The positive control was located in lane 6, which was a positive control without AAP; lane 7 was a negative control; and lane 8 was a positive control. In this validation experiment, the concentrations of the DNA1-DNA2 double-stranded probe were 500 nM, DNA4 was 400 nM, metal ions were 200 μM, AAP was 80 μM, and ALP was 5.00 mU·μL. -1 The pesticide concentration was 10.0 μM, the incubation time between the pesticide and ALP was 40 min, the click chemistry reaction time was 90 min, and the MNAzyme signal amplification time was 3 h. Error bars represent the standard deviation of three parallel measurements.

[0034] First, we will verify whether fluorescence signals can be generated when AA and Cu(II) coexist. For example... Figure 2 As shown in Figure A, the principle is that in the presence of Cu(II), AA reduces it to Cu(I), catalyzing a click chemistry reaction that promotes MNAzyme assembly, which in turn cleaves DNA4 to generate a fluorescent signal. To verify this process, the system was investigated in the presence of Cu(II) and other interfering metal ions (Mg). 2+ Mn 2+ K + Na + Fluorescence properties in the presence of ). Figure 2 Results B in section 1 and C in section 2 show that the system exhibits a strong fluorescence signal in the presence of Cu(II), while the fluorescence intensity decreases significantly in the presence of other interfering metal ions. This indicates that AA and Cu(II) can achieve MNAzyme assembly and signal generation, laying the foundation for the feasibility of the overall principle.

[0035] Based on this, further verification was conducted to determine whether ALP could catalyze the generation of induced fluorescence signals by AA. For example... Figure 2 As shown in D, ALP catalyzes the dephosphorylation of AAP to generate AA, which leads to the assembly of MNAzyme and subsequent fluorescence signal generation in the presence of Cu(II). The results show that the fluorescence signal is weak in the absence of ALP, while a strong signal appears in the presence of ALP; when ALP is co-incubated with theophylline (an ALP activity inhibitor), the fluorescence signal is significantly reduced (…). Figure 2 The presence of E in the figure confirms that ALP can catalyze the formation of AA and trigger fluorescence signal generation. To further support this conclusion, the effect of ALP concentration on the fluorescence intensity of the system was investigated (E). Figure 2 (F in the text): When the ALP concentration increases from 0 to 5.0 mU·μL -1 At that time, the fluorescence signal gradually increased and reached a plateau phase. This result not only supports the conclusion that ALP catalyzes the formation of AA, but also indicates that 5.0 mU·μL -1 The optimal ALP concentration – the initial concentration during the plateau phase – balances signal stability and detection sensitivity.

[0036] In addition to fluorescence experiments, PAGE validation was also performed. For example... Figure 2 As shown in Figure I, in the presence of ALP (lane 8), a low-migration band appears at the top of the lane, the DNA4 band almost disappears, and two high-migration bands appear at the bottom, indicating the formation of a high-molecular-weight MNAzyme that catalyzes the hydrolysis of DNA4 into two short DNA fragments; in contrast, in the absence of ALP (lane 7), AAP (lane 6), or Cu², the bands are significantly different. + At lane 5, the position and intensity of the DNA4 band remained almost unchanged, and no MNAzyme band was observed, confirming that MNAzyme assembly and DNA4 cleavage could not be achieved under these conditions.

[0037] Finally, we verified whether pesticides could reduce fluorescence intensity by inhibiting ALP activity. As shown in G in Figure 2, pesticides selectively bind to ALP and inhibit its activity, leading to inhibited MNAzyme assembly and reduced fluorescence signal. We investigated the effects of three different pesticides (chlorpyrifos, acephate, and methylpyrimidine) on fluorescence signal, and the results showed that all three pesticides significantly reduced fluorescence signal. Figure 2 (H in the original text). In summary, this method has good feasibility.

[0038] (2) The impact of key parameters on collective sensing strategy MNAzyme is the core molecular component that directly generates fluorescence signals; therefore, four key parameters related to MNAzyme assembly were investigated: nucleic acid bridge length, toehold length, stem length, and binding arm length. Fluorescence signals with and without ALP were used as positive and negative controls, respectively, and the ratio of the positive control signal intensity (F) to the negative control signal intensity (F0) (F / F0) was used as the evaluation index. Results are as follows: Figure 3A, B, C, D, E, F, G, H, I, J, K, L. In this validation experiment, the concentration of DNA1-DNA2 double-stranded probe was 500 nM, the concentration of DNA4 was 400 nM, the concentration of metal ions was 200 μM, the concentration of AAP was 80 μM, the concentration of ALP was 5.00 mU·μL -1 ; the concentration of pesticide was 10.0 μM, the incubation time of pesticide and ALP was 40 min, the click chemistry reaction time was 90 min, and the MNAzyme signal amplification time was 3 h. Error bars represent the standard deviation of three parallel measurements.

[0039] The nucleic acid bridge is a single-stranded DNA segment of DNA3 in the MNAzyme, which spans the triazole group formed by click chemistry Figure 3 A in FIG. 1). If the nucleic acid bridge is too long or too short, it will increase the local tension of the molecule and affect the stability of the MNAzyme. The fluorescence intensity of the negative control and the positive control was recorded when the length of the nucleic acid bridge was 3, 4, 5, 6, and 7 nt. The data showed that as the length of the nucleic acid bridge increased, the signal intensity of the negative control remained almost unchanged, and the signal intensity of the positive control first increased and then decreased Figure 3 B in FIG. 1). When the length of the nucleic acid bridge was 5 nt, the F / F0ratio reached a maximum value, indicating that the assembly conditions of the MNAzyme were most suitable at this time, so 5 nt was selected as the optimal length of the nucleic acid bridge.

[0040] The toehold region is the starting region of the hybridization of the DNA1-DNA2 double-stranded probe and DNA3 Figure 3 D in FIG. 1), and its length directly affects the thermodynamic properties of the assembly of the MNAzyme. The signals of the negative control and the positive control were recorded under different toehold lengths Figure 3E in 2 and F in 3), the results showed that: when the toehold length increased from 5 nt to 13 nt, the positive control signal first increased and then tended to be stable, and the F / F0ratio reached the maximum at 9 nt, indicating that the MNAzyme assembly had the optimal thermodynamic performance at this time, so 9 nt was selected as the optimal toehold length.

[0041] MNAzyme was directly assembled by DNA2 and DNA3, and the stem region formed by the hybridization of the two had an important influence on the stability of MNAzyme formation Figure 3 G). By adjusting the sequence of DNA3, the influence of different stem lengths on signal intensity was investigated Figure 3 H in 2 and I in 3): when the stem length increased from 1 nt to 9 nt, the positive control signal slightly increased and then tended to be stable, while the negative control signal significantly increased, which may be due to the transient hybridization of DNA2 and DNA3 leading to the temporary assembly of MNAzyme; in addition, if there is single-stranded DNA2 in the system, too long stem length will enhance the negative signal. When the stem length is 3 nt, the F / F0ratio is the highest, so 3 nt is selected as the optimal stem length.

[0042] The binding arm is the element of MNAzyme to recognize the reaction substrate DNA4 Figure 3 J). Too short binding arm will lead to insufficient affinity with the substrate, and too long binding arm will bind too tightly with the cleavage product, reducing the turnover rate. In the reported research of MNAzyme sensor, the length of the binding arm is generally considered as a key necessary parameter that needs to be optimized. This study investigated the influence of different binding arm lengths (5, 7, 9, 11, 13 nt), the results showed that: with the increase of the binding arm length, the positive control and negative control signals increased to different degrees, and the F / F0ratio reached the maximum at 9 nt Figure 3 K in 2 and L in 3), indicating that the difference between the positive signal and the negative signal is the largest at this time, so 9 nt is selected as the optimal binding arm length.

[0043] In addition to the above MNAzyme assembly related parameters, other parameters also have a significant influence on the performance of the method. The key parameters such as the concentration of DNA1-DNA2 probe, the concentration of AAP, the concentration of Cu 2+ The concentration of DNA4, the type of buffer, the incubation time of pesticide and ALP, the time of click chemistry reaction and the time of MNAzyme reaction were optimized, and the optimization data are shown in Figures 6-13 . Taking the F / F0ratio as the index, the optimal parameters are determined as follows: DNA1-DNA2 probe (500 nM), AAP (80 μM), Cu 2+(200 μM), DNA4 (400 nM), reaction buffer (rCutSmart), pesticide and ALP incubation time (40 min), click chemistry reaction time (90 min), MNAzyme reaction time (180 min). It is worth noting that when Cu 2+ concentration increased to 400 μM, the negative control signal was significantly enhanced ( Figure 8 ), which might be due to the weak reducing property of AAP, which could slightly reduce excess Cu (II) to Cu (I), leading to non-specific MNAzyme assembly. In addition, the investigation of pesticide and ALP incubation time showed that the signal intensity increased significantly after 50 min, indicating that the inhibition of pesticides on ALP was reversible.

[0044] (3) Analysis performance of collective sensing strategy Under the optimal parameter conditions, different concentrations of pesticides were detected. Three different types of pesticides were selected as detection models: chlorpyrifos (CPF, organophosphorus insecticide), tetramethrin (TTM, pyrethroid insecticide), and 2,4-dichlorophenoxyacetic acid (2,4-D, phenoxyacetic acid herbicide), all of which are widely used in the market. The analysis performance of different concentrations of pesticides is shown in Figure 4 Fig. 4, where A is the fluorescence spectrum of chlorpyrifos (CPF), B is the fluorescence spectrum of tetramethrin (TTM), C is the fluorescence spectrum of 2,4-dichlorophenoxyacetic acid (2,4-D), D is the linear relationship between the concentration of chlorpyrifos and the fluorescence intensity, E is the linear relationship between the concentration of tetramethrin and the fluorescence intensity, F is the linear relationship between the concentration of 2,4-dichlorophenoxyacetic acid and the fluorescence intensity, G is the fluorescence intensity column chart of different types of pesticides, and H is the heat map. In the above verification experiment, the concentration of DNA1-DNA2 double-stranded probe was 500 nM, DNA4 was 400 nM, Cu 2+ was 200 μM, AAP was 80 μM, ALP was 5.00 mU·μL -1 ; pesticide and ALP incubation time was 40 min, click chemistry reaction time was 90 min, and MNAzyme signal amplification time was 3 h. Error bars represent the standard deviation of three parallel measurements.

[0045] Figure 4 As shown in Fig. 4A-4C, the fluorescence signal intensity gradually decreased with the increase of the concentration of the three pesticides. Among them, chlorpyrifos showed a good linear relationship with the signal in the concentration range of 0.04-1.40 μM, the linear equation was y = -212.6x + 628.4, and the correlation coefficient R 2=0.990; the linear range of tetramethrin is 1.0-10.0 μM, and the linear equation is y=-39.12x+658.8, R0 2 =0.997; the linear range of 2,4-dichlorophenoxyacetic acid is 2.2-3.2 μM, and the linear equation is y=-217.5x+1099.9, R0 2 =0.994 ( Figure 4 (F in D-4 of the diagram). The limits of detection (LOD) for the three pesticides were calculated using the 3σ method (LOD = 3σ / k, where σ is the standard deviation of three blank samples and k is the slope of the linear calibration curve). The results were: chlorpyrifos 34.8 nM, pyrethroid 55.0 nM, and 2,4-dichlorophenoxyacetic acid 26.0 nM. Compared with previously reported methods, this method has comparable sensitivity and also exhibits multi-component pesticide response capability.

[0046] Table 2 Comparison of this method with other pesticide detection methods

[0047] [1] Chandra S, Bano D, Sahoo K, et al. Synthesis of fluorescentcarbon quantum dots from Jatropha fruits and their application influorometric sensor for the detection of chlorpyrifos. Microchem J. 2022;172:106953. [2] Zhai C, Li Y, Peng Y, et al. Detection of chlorpyrifos in applesusing gold nanoparticles based on surface enhanced Raman spectroscopy. Int JAgric Biol Eng. 2015;8(5):113–120. [3] Zhang M, Chen Z, Liu X, et al. Dual-mode supramolecular fluorescent probe for rapid and on-site detection of chlorpyrifos in the environment. J Hazard Mater. 2023;452:131177. [4] Dong S, Shi Q, He K, Wu J, Zhu Z, Feng J. A Simple Aptamer SERSSensor Based on Mesoporous Silica for the Detection of Chlorpyrifos. Foods.2022;11(21):3331. [5] Vdovenko, M.M.; Stepanova, A.S.; Eremin, S.A.; Van Cuong, N.;Uskova, N.A.; Yu Sakharov, I. Quantification of 2,4-DichlorophenoxyaceticAcid in Oranges and Mandarins by Chemiluminescent ELISA. Food Chem. 2013,141, 865–868. [6] Pan, X.; Xu, X.; Song, S.; Xu, L.; Kuang, H.; Wu, X.; Liu, L.;Xu, C. An Ic-ELISA and Immunochromatographic Strip Assay for the Detection of2,4-Dichlorophenoxyacetic Acid in Bean Sprouts and Cabbage. J. Pharm. Biomed.Anal. 2022, 209, 114524. [7] Mutlu, E.; Senocak, A.;Demirbas, E.; Koca, A.; Akyüz, D.Selective and sensitive molecularly imprinted polymer-based electrochemicalsensor for detection of deltamethrin. Food Chem. 2025, 463, 141121. To evaluate the response of the method to different pesticides, the fluorescence data of 5 categories of 14 pesticides were recorded, including 6 organophosphorus pesticides (OPPs): chlorpyrifos (CPF), acephate (Acep), pirimiphos (Piri), glyphosate (Glyp), Rogor (Rogor), and trichlorfon (Dipt); 3 pyrethroid pesticides (SPs): tetramethrin (TTM), cyfluthrin (Cyfl), and cypermethrin (Cype); 2 organochlorine pesticides (OCPs): DDT and hexachlorocyclohexane (HCH); 2 benzimidazole pesticides (BZM): carbendazim (Carb) and thiabendazole (Thia); and 1 phenoxy carboxylic acid herbicide (PCA): 2,4-dichlorophenoxyacetic acid (2,4-D). Figure 4 As shown in G, the signals of organophosphorus, pyrethroid, and phenoxy carboxylic acid pesticides were significantly lower than those of the blank group, while the signals of organochlorine and benzimidazole pesticides were almost the same as those of the blank group; Figure 4 The values in the heat map of H represent (F0-F) / F0 (F0 is the signal intensity without pesticides, and F is the signal intensity with pesticides), reflecting the reduction of the signal in the presence of pesticides relative to the blank group. It is clear that the method has significant responsiveness to organophosphorus, pyrethroid, and phenoxy carboxylic acid pesticides, while it has almost no response to organochlorine and benzimidazole pesticides.

[0048] (5) Actual sample application Actual sample preparation: 0.50 g of sample was weighed, 2.0 mL of acetone was added, and vortex oscillation was performed for 10 min for extraction. After centrifugation, the supernatant was collected, filtered through a 0.22 μm filter membrane, and used for subsequent detection.

[0049] To evaluate the actual sample detection capability of the method, honeysuckle was selected as the actual sample model, and chlorpyrifos (CPF) was selected as the pesticide model. Honeysuckle is a commonly used traditional Chinese medicine and is widely used in the treatment of viral infections, influenza, and other diseases. Its pesticide residue detection is of great significance for ensuring medication safety. Chlorpyrifos is a commonly used pesticide in honeysuckle cultivation, and its residues can inhibit human cholinesterase activity, damage the nervous system, affect the digestive and respiratory systems, and even endanger life in severe cases. The actual sample detection and pesticide residue total amount detection performance are shown in Figure 5 As shown in A, B, C, and D, the actual sample detection process is shown in A, the fluorescence spectra of samples with different standard concentrations are shown in B, the concentration-fluorescence relationship diagram of the standard sample is shown in C (the inset is the calibration curve), and the pesticide residue total amount detection performance (three different pesticides in equimolar mixture, total concentration 10 μM) is shown in D. In the above verification experiments, the concentrations of DNA1-DNA2 double-stranded probes were 500 nM, the concentration of DNA4 was 400 nM, and the concentration of Cu 2+For 200 μΜ, AAP for 80 μΜ, ALP for 5.00 mU·μL -1 ; pesticide and ALP incubation time 40 min, click chemistry reaction time 90 min, MNAzyme signal amplification time 3 h. Error bars represent the standard deviation of three parallel measurements.

[0050] The actual sample detection process is shown in A of Figure 5 , and the extraction method of honeysuckle samples is shown in the supporting information. Different concentrations of chlorpyrifos were added to the extract, and the fluorescence signal was measured. The results are shown in B of Figure 5 and C of 5: as the concentration of chlorpyrifos increases, the fluorescence signal gradually decreases, and in the concentration range of 0.02-1.00 μΜ, it shows a good linear relationship, the linear equation is y=-331.2x+644.4, R 2 =0.982. In addition, the spiked recovery experiment was carried out at three concentration levels (0.05, 0.50, 1.00 μΜ), and the results are shown in Table 3. The recovery rate was 92%-114%, which was comparable to the results obtained by high performance liquid chromatography-mass spectrometry (HPLC-MS) method, indicating that this method can be used for the determination of pesticide residues in similar samples.

[0051] Table 3 Spiked recovery rate of chlorpyrifos (CPF) in honeysuckle samples

[0052] In practical application scenarios, the simultaneous use and residual probability of multiple pesticides is extremely high, so early warning of the total amount of pesticide residues is crucial. From the five major pesticide categories, one pesticide was selected from each category, and 10 three-component mixed systems were obtained by permutation and combination. Each mixed system mixed three pesticides at the same concentration, and the total concentration was 10 μΜ. The fluorescence signals of the blank sample and the 10 mixed samples were recorded, and the results are shown in D of Figure 5 : compared with the blank sample, the fluorescence signals of the 10 mixed samples were significantly reduced, and the higher the proportion of organophosphorus, phenoxyacetic acid and pyrethroid pesticides, the more obvious the signal reduction. This indicates that this method can successfully respond to mixed samples of multiple pesticides, and has excellent potential in early warning of the total amount of pesticide residues.

[0053] In conclusion, the present application provides a pesticide residue total amount collective sensing detection strategy based on ALP regulation of MNAzyme assembly, which converts the ALP activity inhibition effect into amplified fluorescent signal through MNAzyme assembly. The method normalizes various pesticide residues into ALP activity indicators, establishes a new total toxicity detection collective biosensing strategy, and has important application potential in food safety monitoring and environmental protection. The introduction of the MNAzyme signal amplification mechanism endows the method with high sensitivity detection capability, and does not require complex sample pretreatment and does not depend on precise instruments, which is suitable for on-site detection application. The present study provides a new approach for pesticide residue detection and provides a useful reference for functional nucleic acid-based detection technology.

[0054] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A biosensor for detecting the total amount of pesticide residues, characterized by, comprising alkaline phosphatase, buffer, ascorbate-2-phosphate, double-stranded probe, activated strand, fluorescent reporter probe, and Cu 2+ ; The double-stranded probe is formed by hybridization of DNA1 and DNA2, and the blunt end of the double-stranded probe is modified with azide and alkyne, respectively, and the overhanging end is designed with a toehold region and a MNAzyme fragment; The activation strand comprises another MNAzyme fragment, and the activation strand can hybridize with DNA1 to form a MNAzyme.

2. The biosensor for detecting the total amount of pesticide residues according to claim 1, wherein The fluorescent reporter probe is a DNA molecule with a hairpin structure, the 5' end of which is modified with a fluorescent group, and the 3' end is modified with a quenching group, and the loop region sequence of the hairpin structure comprises a specific RNA cleavage site; The RNA cleavage site can be specifically recognized and cleaved by MNAzyme; The buffer is rCutSmart buffer.

3. The biosensor for detecting the total amount of pesticide residue according to claim 1, wherein The nucleotide sequence of the DNA1 is shown in SEQ ID NO: 1; The nucleotide sequence of the DNA2 is shown in SEQ ID NO: 2; The nucleotide sequence of the activation strand is shown in SEQ ID NO: 3; The nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO:

4.

4. Use of the biosensor for detecting the total amount of pesticide residues according to any one of claims 1-3 in detecting the total amount of pesticide residues.

5. A method for detecting the total amount of pesticide residues, characterized by, The biosensor for detecting the total amount of pesticide residues according to any one of claims 1-3 comprises the following steps: Incubate the sample extract to be tested with alkaline phosphatase, add double-stranded probe, Cu 2+ , buffer solution, ascorbic acid-2-phosphate to catalyze the click reaction, then add the activated chain and fluorescent reporter probe to perform signal amplification reaction, and obtain the total amount of pesticide residues by fluorescence spectrum or gel electrophoresis.

6. The method for detecting the total amount of pesticide residue according to claim 5, characterized by, The preparation method of the sample extraction solution is as follows: the sample to be tested is added to an extraction agent, vortexed and extracted, centrifuged, filtered, and the filtrate is collected to obtain the sample extraction solution; Preferably, 3-5 mL of extraction agent is added per gram of sample to be tested, and the extraction agent is one of acetone, acetonitrile and methanol; The extraction time is 8-12 min, and the filter membrane pore size is 0.2-0.3 μm.

7. The method according to claim 5, wherein The pesticide is one or more of organophosphorus, pyrethroid and phenoxyacetic acid herbicide; Preferably, the pesticide is one or more of chlorpyrifos, tetramethylchrysanthemum and 2,4-dichlorophenoxyacetic acid.

8. The method for detecting the total amount of pesticide residue according to claim 5, characterized by, The temperature of the mixed incubation is 35-38 ℃, and the time is 10-60 min; preferably, the temperature of the incubation is 37 ℃, and the time is 40 min; The temperature of the catalytic click reaction is 35-38 ℃, and the time is 30-150 min; preferably, the temperature of the catalytic click reaction is 37 ℃, and the time is 90 min; The temperature of the signal amplification reaction is 35-38 ℃, and the time is 30-300 min; preferably, the temperature of the signal amplification reaction is 37 ℃, and the time is 180 min.

9. The method of claim 5, wherein the pesticide residue is selected from the group consisting of organophosphates, organochlorines, pyrethroids, carbamates, and organotins. The concentration of the alkaline phosphatase is 0.05-5.0 mU^L -1 , preferably 5.0 mU^L -1 The concentration of the double-stranded probe is 300-700 nM, preferably 500 nM; The Cu 2+ concentration is 10-400 μM, preferably 200 μM; The concentration of ascorbic acid-2-phosphate is 20-160 μM, preferably 80 μM; The concentration of the fluorescent reporter probe is 100-800 nM, preferably 400 nM; The buffer solution is one of rCutSmart buffer, Tris-HCl-EDTA buffer, Tris-HCl buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl-EDTA buffer, Tris-HCl 10. The method of claim 5, wherein the pesticide residue is selected from the group consisting of organophosphates, organochlorines, pyrethroids, carbamates, and organotins. ​ ​ ​ ​

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