Method for detecting pymetrozine based on brown planthopper odor binding protein nlugobp9 and sensor

Abstract

The application discloses a method for detecting pymetrozine based on a brown planthopper odor binding protein NlugOBP9 and a sensor. The method contacts a sample to be detected with separated NlugOBP9 protein, and a detectable signal is generated by specific binding of the NlugOBP9 protein and pymetrozine, and the presence of pymetrozine in the sample or the content of pymetrozine is determined according to the signal intensity. The application also provides a fiber sensor and a lateral flow chromatography test strip based on the detection method. The fiber sensor realizes detection by fixing the NlugOBP9 protein on a sensing area of the fiber sensor, and the lateral flow chromatography test strip realizes detection by spraying NlugOBP9-colloidal gold conjugates on a binding pad. The method has high sensitivity and strong specificity, and can be used for rapid detection of pymetrozine residues in agricultural environmental water samples.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection, and more specifically relates to a method and sensor for detecting pymetrozine based on the brown planthopper odor-binding protein NlugOBP9. Background Technology

[0002] Pymetrozine is a novel pyridine insecticide that interferes with the neuromuscular function of insects by targeting the transient receptor potential vanillic acid channel (TRPV) in the insect's string organ, inhibiting feeding and reproductive behavior, and ultimately leading to insect death. However, with the increasing application of pymetrozine in the field, its environmental residues are becoming a prominent issue, posing a potential threat to aquatic ecosystems and agricultural product safety. Therefore, establishing a rapid, sensitive, and convenient method for detecting pymetrozine residues is of significant practical importance for environmental monitoring and food safety assurance.

[0003] Currently, pymetrozine residue detection mainly employs instrumental analytical methods such as liquid chromatography-tandem mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). While these methods offer high sensitivity and accuracy, they typically require expensive equipment, specialized operators, and complex sample pretreatment processes, resulting in long detection cycles and high costs, making them unsuitable for rapid on-site detection. Immunoassay methods based on antigen-antibody specific recognition (such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography) mitigate these shortcomings to some extent, but antibody preparation is time-consuming and costly, and they are sensitive to chemical environments, with their stability in complex matrices requiring improvement. Therefore, developing novel and stable pymetrozine recognition elements and corresponding rapid detection technologies has significant application value.

[0004] Insects rely on highly sensitive olfactory systems to specifically detect and distinguish thousands of chemical odor molecules, thereby regulating key behaviors such as host localization, mating, and oviposition. This olfactory recognition process depends on the dissolution and transport of odor molecules in sensory lymph, primarily mediated by odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). Recent studies have shown that, in addition to binding to host plant volatiles, OBPs and CSPs can also interact with various chemical pesticides, closely related to the formation of insect resistance. The ability of OBPs and CSPs to bind to structurally diverse compounds not only contributes to the development of resistance but also provides a theoretical basis for their use as protein-based biorecognition elements in pesticide detection platforms.

[0005] Compared to traditional recognition elements (such as antibodies or membrane receptors), OBPs offer several advantages: they can be efficiently and massively produced via prokaryotic expression systems and purified using simple chromatographic methods, offering cost-effectiveness and ease of operation; OBPs exhibit remarkable stability, maintaining their structure and function under variable temperature, pH conditions, and even in the presence of proteases, thus preserving their activity in challenging environments. These robust characteristics make OBPs compatible with a variety of sensor transduction platforms, including electrochemical, optical, and field-effect transistor systems. However, the application of OBPs in pesticide detection remains relatively limited, and further exploration of their potential in agrochemical monitoring is needed. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method and sensor for detecting pymetrozine based on the brown planthopper odor-binding protein NlugOBP9. Based on transcriptome analysis and functional validation, this invention found that NlugOBP9 is closely related to pymetrozine resistance in brown planthoppers; molecular docking, fluorescence quenching, and surface plasmon resonance analysis confirmed that NlugOBP9 has a specific binding ability to pymetrozine. Based on this, this invention constructs an MZI fiber optic sensor and a colloidal gold side-flow chromatography test strip based on NlugOBP9, achieving highly sensitive and selective detection of pymetrozine.

[0007] This invention first provides a method for detecting pymetrozine using brown planthopper odor-binding protein, which includes the following steps: immobilizing brown planthopper odor-binding protein NlugOBP9 on a solid support, bringing the sample to be tested into contact with the brown planthopper odor-binding protein NlugOBP9 on the solid support, detecting the detectable signal generated by the binding of NlugOBP9 with pymetrozine in the sample to be tested, and determining the presence or content of pymetrozine in the sample to be tested based on the intensity of the detectable signal; the amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.2.

[0008] According to a preferred embodiment of the present invention, the solid carrier is an optical fiber, the brown planthopper odor-binding protein NlugOBP9 is fixed on the surface of the sensing area of ​​the optical fiber, and the detectable signal is the change in the optical signal transmitted by the optical fiber.

[0009] According to a preferred embodiment of the present invention, the optical fiber is a Mach-Zehnder interferometer fiber, which is formed by sequentially eccentrically splicing a first single-mode fiber, a second single-mode fiber, and a third single-mode fiber, wherein the second single-mode fiber serves as the sensing region, and the brown planthopper odor-binding protein NlugOBP9 is fixed on the surface of the second single-mode fiber; the change in optical signal is a change in the intensity of the interference spectrum.

[0010] According to a preferred embodiment of the present invention, the solid-phase support is a side-flow chromatography test strip; the side-flow chromatography test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the brown planthopper odor-binding protein NlugOBP9 is coupled with colloidal gold to form an NlugOBP9-colloidal gold conjugate, the NlugOBP9-colloidal gold conjugate is sprayed onto the conjugate pad of the side-flow chromatography test strip, and the detectable signal is the color signal of the test strip detection line.

[0011] According to a preferred embodiment of the present invention, the detection line is coated with a pymetrozine-bovine serum albumin conjugate. During detection, free pymetrozine in the sample competes with the pymetrozine-bovine serum albumin conjugate on the detection line for binding to the NlugOBP9 protein in the NlugOBP9-colloidal gold conjugate. The color intensity of the detection line reflects the pymetrozine content in the sample.

[0012] According to a preferred embodiment of the present invention, the sample to be tested is an agricultural environmental water sample, which may be paddy field water, field runoff, or irrigation water, etc.

[0013] According to a preferred embodiment of the present invention, the detection limit of the method is from 0.01 ng / mL to 5 ng / mL.

[0014] This invention further provides the application of the brown planthopper odor-binding protein NlugOBP9 in the detection of pymetrozine, the amino acid sequence of which is shown in SEQ ID NO.2. Furthermore, the application uses NlugOBP9 as a detection marker. Further, the application utilizes the specific binding ability of NlugOBP9 to pymetrozine, detecting pymetrozine in the sample by detecting changes in the detectable signal caused by the binding of NlugOBP9 to pymetrozine. Preferably, the sample is a liquid sample, such as water. Considering that pymetrozine is mainly used as an insecticide, agricultural environmental water samples can be directly used as the sample.

[0015] This invention further proposes a fiber optic sensor for pymetrozine detection based on the brown planthopper odor-binding protein NlugOBP9. The fiber optic sensor is composed of a first single-mode fiber, a second single-mode fiber, and a third single-mode fiber fused together with their cores. The second single-mode fiber serves as the sensing region. The surface of the second single-mode fiber is subjected to hydroxylation, silanization, and glutaraldehyde cross-linking treatment. The brown planthopper odor-binding protein NlugOBP9 is covalently fixed to the surface of the second single-mode fiber through glutaraldehyde cross-linking. The amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.2.

[0016] This invention also proposes a lateral flow chromatography test strip for pymetrozine detection based on NlugOBP9, comprising a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and fixed on a backing plate, and the conjugate pad is coated with an NlugOBP9-colloidal gold conjugate; the nitrocellulose membrane is provided with a detection line and a control line, the detection line is coated with a pymetrozine-bovine serum albumin conjugate, and the control line is coated with an anti-mouse IgG antibody; the amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.2.

[0017] This invention further provides the application of the aforementioned fiber optic sensor or the aforementioned lateral flow chromatography test strip in detecting pymetrozine residues in agricultural environmental water samples.

[0018] The experimental results of this invention show that:

[0019] (1) This invention is the first to discover that the odor-binding protein NlugOBP9 from brown planthoppers can specifically bind to pymetrozine. SPR binding analysis showed that the binding affinity of NlugOBP9 to pymetrozine (KD = 5.28 × 10⁻⁶) was 5.28 × 10⁻⁶. -6 M) was significantly superior to its affinity for chlorpyrifos (KD = 3.69 × 10⁻⁶). -5 The molecular docking analysis showed that NlugOBP9 has good recognition specificity for pymetrozine. Pymetrozine can occupy the hydrophobic binding pocket of NlugOBP9 and interact with residues such as Leu-24, His-54, Phe-65, Ile-66, Phe-70, and Phe-129. His-54 forms a hydrogen bond with a binding free energy of -6.91 kcal / mol, indicating that NlugOBP9 has a strong predictive affinity for pymetrozine.

[0020] (2) The Mach-Zehnder interferometer fiber optic sensor constructed based on NlugOBP9 protein of the present invention has a detection limit of 0.01 ng / mL for pymetrozine and a detection range of 0.01-100 ng / mL. It also shows no obvious response to common pesticides such as chlorpyrifos, acetamiprid and imidacloprid, and has high selectivity.

[0021] (3) The colloidal gold side-flow chromatography test strip constructed based on NlugOBP9 protein of the present invention has a visual detection limit of 5 ng / mL and a calculated detection limit of 0.82 ng / mL. The signal attenuation at low concentrations is better than that of commercial pymetrozine test strips.

[0022] (4) NlugOBP9 protein can be efficiently prepared through a prokaryotic expression system, with low production cost and good batch consistency, avoiding the problems of long preparation cycle, high cost and poor stability of traditional antibody recognition elements.

[0023] (5) Field water sample testing verification showed that both sensors of the present invention could successfully detect pymetrozine residues in field water samples at 0, 24, 48, and 72 h after application. The MZI sensor maintained stable detection performance even at fiber optic transmission distances exceeding 1 km. The detection results of both sensors on actual field water samples were consistent, verifying the feasibility and reliability of the technical solution of the present invention in practical applications. Attached Figure Description

[0024] Figure 1 This is a diagram showing the molecular docking between NlugOBP9 and pymetrozine.

[0025] Figure 2 The fluorescence quenching effect of NlugOBP9 with pymetrozine and chlorpyrifos is shown in the figure.

[0026] Figure 3 SPR diagrams of NlugOBP9 with pymetrozine and chlorpyrifos;

[0027] Figure 4 Schematic diagram of the construction and detection principle of an off-core fusion spliced ​​MZI fiber optic sensor;

[0028] Figure 5 This is an MZI fiber optic sensor built based on NlugOBP9;

[0029] Figure 6 This is a colloidal gold side-flow chromatography test strip based on NlugOBP9;

[0030] Figure 7 To detect field water samples using an MZI fiber optic sensor based on NlugOBP9 and colloidal gold side-flow chromatography test strips;

[0031] Figure 8 Schematic diagram of the preparation and detection principle of NlugOBP9 functionalized colloidal gold side-flow chromatography test strip;

[0032] Figure 9 The results are for the NlugOBP9 functionalized colloidal gold side-flow chromatography test strip.

[0033] Figure 10 These are the results of field water sample testing. Detailed Implementation

[0034] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0035] Brown planthoppers were collected from the artificial climate chamber of the Zhejiang Provincial Key Laboratory of Biometrics and Quarantine Technology. They were continuously reared with TN1 sensitive rice seedlings under the following conditions: temperature 26 ± 1 ℃, humidity 70% ~ 80%, and photoperiod 16 L: 8D.

[0036] In the early stages of this invention, transcriptomic analysis revealed that the gene corresponding to the NlugOBP9 odor-binding protein, a gene in multiple odor-binding protein families of brown planthoppers, was specifically upregulated in pymetrozine-resistant strains, suggesting a potential interaction between this protein and pymetrozine. Based on this, this invention conducted an in-depth study on the binding ability of the NlugOBP9 protein to pymetrozine and developed it as a biorecognition element for pymetrozine detection. The nucleotide sequence of the NlugOBP9 protein used in this embodiment is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. The nucleotide sequence shown in SEQ ID NO.1 encodes the full-length NlugOBP9 protein containing the signal peptide, and the amino acid sequence shown in SEQ ID NO.2 is the mature peptide sequence after removing the signal peptide. The recombinant NlugOBP9 protein used in the experiment is the mature peptide shown in SEQ ID NO.2.

[0037] Example 1 Recombinant Expression and Purification of NlugOBP9 Protein

[0038] The recombinant plasmid encoding NlugOBP9 and the molecular chaperone plasmid pTf16 were co-transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB agar plates containing 25 μg / mL ampicillin and 25 μg / mL chloramphenicol and incubated overnight at 37°C. Single colonies were inoculated into LB liquid medium and cultured at 37°C until OD500. 600Approximately 0.6-0.8. Induce for 1 h by adding 0.1% (m / v) L-arabinose, cool to 18℃, add 0.5 mM IPTG, and continue culturing at 18℃ for 12-16 h. Collect bacterial cells by centrifugation, resuspend in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), add 1 mM PMSF, and sonicate on ice. Centrifuge at 20,000 × g for 30 min, filter the supernatant through a 0.22 μm membrane, and load onto a Ni-NTA affinity chromatography column. Wash with lysis buffer, then elute with a linear gradient of 50-250 mM imidazole. Collect the eluent containing NlugOBP9, concentrate, and replace with 1× PBS (pH 7.4) using an Amicon Ultra centrifuge filter (10 kDa molecular weight cutoff). Determine protein concentration using NanoDrop, and assess purity using SDS-PAGE. Results: The purified NlugOBP9 had a molecular weight of approximately 14.7 kDa, and its purity met the requirements for subsequent experiments.

[0039] Example 2: Verification of the binding ability of NlugOBP9 to pymetrozine

[0040] The 3D structure of NlugOBP9 was predicted using the Phyre2 server and validated by Ramachandran plot analysis. The 3D structure of pymetrozine was obtained from the PubChem database, converted to PDBQT format using Open Babel, and polar hydrogens and Gasteiger charges were added using AutoDockTools. Docking simulations were performed using AutoDock Vina, with the search space based on the ligand-binding pocket definition identified by CB-DOCK2. Protein-ligand interactions were analyzed using PLIP and visualized in PyMOL. Results are as follows: Figure 1 As shown, pymetrozine is predicted to occupy the hydrophobic binding pocket of NlugOBP9 and interact with residues such as Leu-24, His-54, Phe-65, Ile-66, Phe-70, and Phe-129. His-54 may form a hydrogen bond. The binding free energy ΔG = -6.91 kcal / mol indicates that the two have good binding affinity, providing experimental basis for the subsequent construction of biorecognition elements.

[0041] The purified NlugOBP9 protein was diluted to 5 μM with 1× PBS. Stock solutions of 1 mM pymetrozine and chlorpyrifos were prepared. Measurements were performed using a fluorescence spectrophotometer (RF-5301, Shimadzu, Japan). The maximum excitation wavelength (220-390 nm, emission wavelength 400 nm) of the protein was first determined by excitation spectroscopy scanning. Then, the emission spectrum from 290-450 nm was recorded at the maximum excitation wavelength (282 nm), with excitation and emission slit widths both at 5 nm. For each measurement, 3 mL of protein solution was added to a quartz cuvette, and the initial fluorescence intensity was recorded. The ligand solution was gradually added, gently mixed, and incubated for 2 min, recording the changes in fluorescence intensity. The interaction behavior between NlugOBP9 and the test compounds was analyzed based on the fluorescence quenching response. Results are as follows: Figure 2 As shown, both pymetrozine and chlorpyrifos caused a concentration-dependent decrease in the fluorescence intensity of NlugOBP9. However, pymetrozine produced a more pronounced quenching effect across the entire tested concentration range, with a greater decrease in relative fluorescence intensity than chlorpyrifos. Stern-Volmer analysis showed that the fitting slope of pymetrozine (0.837) was significantly higher than that of chlorpyrifos (0.418), indicating a stronger interaction between NlugOBP9 and pymetrozine.

[0042] Example 3 Surface Plasmon Resonance (SPR) Analysis

[0043] SPR experiments were performed using a Biacore system (GE Healthcare, Sweden) and a CM5 sensor chip. NlugOBP9 protein was immobilized on the CM5 chip surface using a standard amine coupling procedure: the sensor surface was first activated with a mixture of 0.2 M EDC and 0.05 M NHS at a flow rate of 10 μL / min; then 50 μg / mL NlugOBP9 protein (dissolved in 10 mM sodium acetate buffer, pH 4.5) was injected at a flow rate of 10 μL / min for immobilization; finally, the remaining activating groups were blocked with 1 M ethanolamine-HCl (pH 8.5) at a flow rate of 10 μL / min. The reference flow cell used the same activation and blocking procedure, but without immobilizing NlugOBP9 protein. Pymetrozine and chlorpyrifos were dissolved in PBS-P+ running buffer and serially diluted in 96-well plates. Samples were injected sequentially from low to high concentration at a flow rate of 30 μL / min, with the binding phase bound for 60 s, followed by monitoring for dissociation. Regeneration with 10 mM glycine-HCl (pH 2.0) for 5 min after each cycle. Correction was performed by subtracting the reference flow cell signal from the sample flow cell signal. Kinetic parameters (Ka, Kd, ​​KD) were determined by globally fitting the sensor map to a 1:1 Langmuir combined model. Results are as follows. Figure 3As shown, NlugOBP9 exhibits concentration-dependent interactions with both pymetrozine and chlorpyrifos. Compared to chlorpyrifos, NlugOBP9 shows three significant advantages in binding to pymetrozine: faster binding speed (Ka = 1.69 × 10⁻⁶). 4 M -1 s -1 The concentration of chlorpyrifos (Ka = 5.26 × 10⁻⁶) is approximately equal to that of chlorpyrifos. 3 M -1 s -1 The rate of binding of pymetrozine to NlugOBP9 is 3.2 times that of NlugOBP9, indicating that the binding rate of pymetrozine is significantly faster; the dissociation rate is also slower: the dissociation rate constant of pymetrozine (Kd = 8.94 × 10⁻⁶) is 3.2 times that of NlugOBP9. -2 s -1 The concentration was lower than that of chlorpyrifos (Kd = 1.94 × 10⁻⁶). -1 s -1 This indicates that pymetrozine is more difficult to dissociate after binding with NlugOBP9, resulting in a more stable complex; the overall affinity is stronger: pymetrozine's KD value (KD = 5.28 × 10⁻⁶) is significantly higher. -6 M) was significantly lower than that of chlorpyrifos (KD = 3.69 × 10). -5 The smaller the KD value (M), the stronger the affinity, indicating that NlugOBP9's affinity for pymetrozine is approximately 7 times that for chlorpyrifos. In conclusion, NlugOBP9 exhibits preferential binding ability to pymetrozine.

[0044] Example 4: Construction of a NlugOBP9 Functionalized MZI Fiber Optic Sensor

[0045] 4.1 Sensor Fabrication

[0046] like Figure 4 As shown, this embodiment uses single-mode fiber (SMF) to fabricate an off-core fusion spliced ​​MZI fiber sensor. The fiber sensor is composed of a first single-mode fiber SMF1, a second single-mode fiber SMF2, and a third single-mode fiber SMF1 spliced ​​off-core (the core radius of a conventional single-mode fiber is typically 3μm to 10μm, and the offset of the off-core splice is generally set to 2μm to 4μm), with a configuration of SMF1-SMF2-SMF1. The second single-mode fiber serves as the sensing region. When the optical signal passes through the off-core splice point, part of the beam energy is still transmitted through the core, while part leaks into the fiber cladding, exciting the cladding mode. When the core mode and the cladding mode propagate in the sensing fiber, a phase difference occurs due to the different optical path lengths they experience. Interference occurs when they are recoupled at the fiber end, forming the Mach-Zehnder interference effect.

[0047] The middle SMF2 segment is precisely controlled to be 3 cm, serving as the sensing area; the two ends of the SMF1 segment are about 1 cm. Extra lengths of SMF are retained at both ends for connection to the broadband light source and the spectrometer. NlugOBP9 immobilization steps: (1) Hydroxylation treatment: The sensing area is immersed in freshly prepared piranha solution (H2SO4:H2O2 = 3:1, v / v) for 10 min to generate hydroxyl groups on the surface of the optical fiber. After rinsing with ultrapure water, it is dried. (2) Silanization treatment: Immersed in 2% (v / v) 3-aminopropyltriethoxysilane (APTES) solution for 40 min to introduce amino groups. After rinsing with toluene, it is dried. (3) Aldehyde activation: Treated with 2% (v / v) glutaraldehyde for 1 h to generate aldehyde groups. (4) Protein immobilization: The functionalized optical fiber is immersed in NlugOBP9 solution (250 μg / mL, prepared with PBS) and incubated at room temperature for 3 h to achieve covalent protein immobilization.

[0048] 4.2 Sensing Measurement

[0049] An off-center fused MZI fiber optic sensor was integrated into a laboratory-designed microfluidic chip to achieve uniform sample delivery and precise flow rate control in the sensing area. A broadband light source and spectrometer were connected to record spectra in the 1480-1620 nm wavelength range with a resolution of 0.02 nm. Pymetrozine solution was delivered to the microfluidic system using a syringe pump at a flow rate of 0.3 mL / min, and light intensity changes were monitored in real time, recorded every 1 min for 10 min. Selectivity evaluation: Using ultrapure water as a blank control, chlorpyrifos, acetamiprid, and imidacloprid were selected as control compounds (all 100 ng / mL), and light intensity changes were compared. Sensitivity evaluation: Pymetrozine solutions at concentrations ranging from 0.01 to 100 ng / mL were detected, and the spectral response was analyzed to determine the detection range and limit of detection.

[0050] The results are as follows Figure 5 As shown: (1) SEM characterization showed that the bare optical fiber surface was smooth and uniform; after glutaraldehyde crosslinking, a uniformly distributed particle structure appeared; after NlugOBP9 immobilization, the surface particle layer was more dense; after incubation with pymetrozine (100 ng / mL), the surface particles further aggregated and interconnected, confirming the successful construction of the NlugOBP9 functionalized sensing interface. (2) Selectivity evaluation results are as follows: Figure 6 As shown: the light intensity change induced by pymetrozine (0.81049 dB) was significantly higher than that chlorpyrifos (0.35461 dB), acetamiprid (0.16554 dB), imidacloprid (0.26471 dB), and ultrapure water control (0.18729 dB), indicating that the sensor has high selectivity for pymetrozine. (3) Sensitivity evaluation results are as follows Figure 7As shown, the light intensity gradually increased with the increase of pymetrozine concentration. Compared with the ultrapure water control, all concentration groups showed statistically significant light response (P < 0.01), and the detection limit was as low as 0.01 ng / mL.

[0051] Example 5: Construction of NlugOBP9 Functionalized Colloidal Gold Sideflow Chromatography Test Strip

[0052] Figure 8 The diagram illustrates the preparation and detection principle of the NlugOBP9 functionalized colloidal gold lateral flow chromatography test strip, which includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and fixed on a backing plate. The conjugate pad is coated with an NlugOBP9-colloidal gold conjugate. The nitrocellulose membrane has a detection line and a control line. The detection line is coated with a pymetrozine-bovine serum albumin conjugate, and the control line is coated with an anti-mouse IgG antibody.

[0053] This embodiment combines Figure 8 The construction process of the lateral flow chromatography test strip is described below:

[0054] 5.1 Preparation of Colloidal Gold

[0055] Colloidal gold nanoparticles were prepared using the sodium citrate reduction method. 100 mL of 0.01% chloroauric acid solution was heated to boiling, and 1.5 mL of 1% sodium citrate was quickly added. The mixture was stirred continuously and kept boiling for 10-15 min until a stable colloidal gold suspension was formed. The suspension was then cooled to room temperature and stored at 4°C in the dark.

[0056] 5.2 Preparation of NlugOBP9-colloidal gold conjugate

[0057] The pH of the colloidal gold solution and protein labeling conditions were adjusted based on NaCl-induced aggregation behavior. NlugOBP9 protein was added to the pH-adjusted colloidal gold solution and incubated for 30 min, followed by blocking with bovine serum albumin (BSA). The conjugate was collected by centrifugation, unbound protein was removed, and the conjugate was resuspended in storage buffer to obtain the NlugOBP9-colloidal gold conjugate. Optimized parameters: 3.5 μL of 0.1 M K₂CO₃ and 4 μg of NlugOBP9 in 0.5 mL of colloidal gold solution constituted the optimal labeling conditions.

[0058] 5.3 Test strip assembly

[0059] The test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. Pymetrozine antigen (pymetrozine-BSA conjugate) is immobilized on the nitrocellulose membrane to form the detection line (T line) at a concentration of 6 mg / mL; anti-mouse IgG antibody is immobilized on the control line (C line). NlugOBP9-colloidal gold conjugate is sprayed onto the conjugate pad (5 μL / cm), dried, and then the test strip is assembled.

[0060] 5.4 Detection Methods

[0061] The sample solution is added to the sample pad and migrates along the test strip via capillary action. The signal response is evaluated based on the color intensity of the detection line (T line) and control line (C line). A standard inhibition curve is established, and the T / C signal ratio is plotted against the pymetrozine concentration. The limit of detection (cLOD) is calculated as the pymetrozine concentration corresponding to the mean blank signal minus three standard deviations.

[0062] The results are as follows Figure 9 As shown: (1) Sensitivity analysis: The test strip exhibits a typical concentration-dependent competitive inhibition response. As the concentration of pymetrozine increases, the color intensity of the T line gradually decreases, and the T line almost disappears at 5 ng / mL (visual detection limit vLOD), and completely disappears at ≥10 ng / mL. The calculated detection limit (cLOD) obtained by fitting the standard curve is 0.82 ng / mL. (2) Comparison with commercial test strips: Under the same conditions, the NlugOBP9 test strip shows a more obvious signal reduction at low pymetrozine concentrations, while the commercial test strip still retains a visible T line signal under the same conditions, indicating that the NlugOBP9 test strip has better analytical sensitivity.

[0063] Example 6: Application of Field Water Sample Testing

[0064] 6.1 Field Experiment Design

[0065] Apply pymetrozine to paddy fields at the recommended dosage (7.5-10 g active ingredient / mu), and collect field water samples at 0, 24, 48, and 72 h after application.

[0066] 6.2 MZI sensor detection

[0067] Pretreated water samples were introduced into a microfluidic sensing system, and the photoresponse was continuously recorded under identical operating conditions. To simulate a remote monitoring scenario, the sensing area was connected to the detection system via approximately 1 km of single-mode fiber. Results are as follows: Figure 10 As shown, all collected water samples (0, 24, 48, and 72 h) yielded repeatable signal responses, indicating that the sensing platform maintains stable detection performance under kilometer-level signal transmission, verifying the feasibility of deploying this fiber optic platform for long-distance monitoring of pesticide residues in agricultural water environments.

[0068] 6.3 Colloidal gold test strip detection

[0069] The same field water sample was directly added to the test strip for testing. The T-line color intensity was used to evaluate the results, and the C-line was used to verify the effectiveness of the test strip. The results are as follows: Figure 10 As shown, all samples collected within 72 hours exhibited visible competitive inhibition responses, with the T-line gradually weakening, consistent with MZI sensor data. This verifies the feasibility of using the NlugOBP9 fiber optic cable and lateral flow chromatography platform for on-site and remote monitoring of pesticide residues in agricultural aquatic environments.

Claims

1. A method for detecting pymetrozine using brown planthopper odor-binding proteins, characterized in that, The method includes the following steps: immobilizing the brown planthopper odor-binding protein NlugOBP9 on a solid support, bringing the sample to be tested into contact with the brown planthopper odor-binding protein NlugOBP9 on the solid support, detecting the detectable signal generated by the binding of NlugOBP9 with pymetrozine in the sample to be tested, and determining the presence or content of pymetrozine in the sample to be tested based on the intensity of the detectable signal; the amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The solid carrier is an optical fiber, the brown planthopper odor-binding protein NlugOBP9 is fixed on the surface of the sensing area of ​​the optical fiber, and the detectable signal is the change in the optical signal transmitted by the optical fiber.

3. The method according to claim 2, characterized in that, The optical fiber is a Mach-Zehnder interferometer fiber, which is constructed by sequentially eccentrically splicing a first single-mode fiber, a second single-mode fiber, and a third single-mode fiber. The second single-mode fiber serves as the sensing region, and the brown planthopper odor-binding protein NlugOBP9 is fixed on the surface of the second single-mode fiber. The change in optical signal is a change in the intensity of the interference spectrum.

4. The method according to claim 1, characterized in that, The solid-phase support is a side-flow chromatography test strip; the side-flow chromatography test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the brown planthopper odor-binding protein NlugOBP9 is coupled with colloidal gold to form an NlugOBP9-colloidal gold conjugate, which is sprayed onto the conjugate pad of the side-flow chromatography test strip; the detectable signal is the color signal of the test strip's detection line.

5. The method according to claim 4, characterized in that, The detection line is coated with a pymetrozine-bovine serum albumin conjugate. During detection, free pymetrozine in the sample competes with the pymetrozine-bovine serum albumin conjugate on the detection line for binding to the NlugOBP9 protein in the NlugOBP9-colloidal gold conjugate. The color intensity of the detection line reflects the pymetrozine content in the sample.

6. The method according to claim 1, characterized in that, The sample to be tested was an agricultural environmental water sample.

7. Application of brown planthopper odor-binding protein NlugOBP9 in the detection of pymetrozine, wherein the amino acid sequence of brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.

2.

8. A fiber optic sensor for detecting pymetrozine based on the brown planthopper odor-binding protein NlugOBP9, characterized in that, The fiber optic sensor is composed of a first single-mode fiber, a second single-mode fiber, and a third single-mode fiber fused together with a core, with the second single-mode fiber serving as the sensing area. The surface of the second single-mode optical fiber is subjected to hydroxylation, silanization and glutaraldehyde crosslinking treatment. The brown planthopper odor-binding protein NlugOBP9 is covalently fixed to the surface of the second single-mode optical fiber by glutaraldehyde crosslinking. The amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.

2.

9. A side-flow chromatography test strip for detecting pymetrozine based on the brown planthopper odor-binding protein NlugOBP9, characterized in that, The device includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially overlapped and fixed on a backing plate; the conjugate pad is coated with NlugOBP9-colloidal gold conjugate; the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with pymetrozine-bovine serum albumin conjugate, and the control line is coated with anti-mouse IgG antibody; the amino acid sequence of the brown planthopper odor-binding protein NlugOBP9 is shown in SEQ ID NO.

2.

10. The application of the fiber optic sensor of claim 8 or the lateral flow chromatography test strip of claim 9 in the detection of pymetrozine residues in agricultural environmental water samples.

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