Method for detecting inositol hexakisphosphate and application thereof in plant breeding

Abstract

The application discloses a kind of detection methods of inositol hexakisphosphate and its application in plant cultivation, the application utilizes the electrostatic and chelation of nano iron oxide and inositol hexakisphosphate Induce nanoparticle aggregation, cause color change or UV-Vis absorption peak shift (200~220 nm), for the rapid detection of inositol hexakisphosphate concentration in soil or nutrient solution, detection limit 0.1 mg / L, total time <10 minutes.The method includes sample extraction, reaction and optical determination steps, suitable for on-site monitoring of phosphorus fixation and fertilizer quality control.The detection results of the application can guide the application of complex, improve the phosphorus utilization efficiency by 20~30%, and enhance the plant cultivation effect (such as alfalfa yield increase by 18%, rice growth period is shortened by about 7 days).The method of the application is suitable for soil phosphorus fixation monitoring.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural nanotechnology and material analysis, and particularly relates to a method for detecting inositol hexakisphosphate and application thereof in plant cultivation. BACKGROUND

[0002] Phosphorus is a key nutrient for plant growth, but it is easily immobilized or lost in soil or hydroponic systems, resulting in low utilization. Inositol hexakisphosphate (IP6, phytic acid) is the main form of phosphorus storage in plants, which can release phosphate and promote root development through the auxin signaling pathway. In the prior art, although IP6 can be used as a phosphorus source and a growth signal molecule, it is rapidly degraded (85-90% released within 7 days) in soil or hydroponic systems, resulting in low phosphorus utilization (500 mg / L) and the risk of iron overaccumulation leading to plant toxicity, causing oxidative stress and plant toxicity (chlorophyll reduction > 15%). These difficulties have led to the difficulty of existing phosphorus-iron composite materials in simultaneously achieving phosphorus release, iron supplementation, and root signal regulation in greenhouse cultivation, limiting the yield improvement (< 10%) of crops such as alfalfa, tomato, tobacco, and rice.

[0003] IP6 is the main component of organic phosphorus in soil, accounting for about 50-80% of total soil organic phosphorus. It mainly comes from plant residues, seeds, and organic fertilizers, and does not exist stably in free form in soil environment, but tends to combine with soil components to form various complex or adsorbed forms. This combination often leads to immobilization of phosphorus, reducing its bioavailability, affecting plant uptake and soil phosphorus cycling. In the prior art, the detection of inositol hexakisphosphate content in the matrix mainly relies on enzyme colorimetry or ion chromatography, which has the disadvantages of long time (> 1 hour), expensive equipment, and unsuitability for on-site use. The present application uses a complex as an optical nanoprobe, matched with a UV-Vis spectrophotometer or a colorimetric card, to realize rapid and low-cost detection, solving the problem of monitoring phosphorus utilization efficiency. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for detecting inositol hexakisphosphate.

[0005] The technical problem to be solved by the present application is to provide a method for detecting inositol hexakisphosphate.

[0006] The technical problem to be solved by the present application is to provide a low-concentration (100 mg / L) and environmentally friendly IP6-FeNPs complex, which can control the release of phosphorus to 55-60% in 30 days and synergistically promote root development by 20-25%.

[0007] The application also aims to solve the technical problem of providing the application of the inositol hexakisphosphate-nano iron oxide complex in plant cultivation or preparation of plant growth promoters.

[0008] Technical scheme: In order to solve the above technical problems, the application provides a detection method of inositol hexakisphosphate, which utilizes the reaction of nano iron oxide and inositol hexakisphosphate, and detects the concentration of inositol hexakisphosphate in a sample to be tested according to the linear relationship between the absorbance of the reaction solution and the concentration of inositol hexakisphosphate.

[0009] The detection method comprises the following steps:

[0010] (1) adding inositol hexakisphosphate standard solutions with different known concentrations into a nano iron oxide suspension to measure the absorbance, and establishing a standard curve of the absorbance and the concentration of the inositol hexakisphosphate solution;

[0011] (2) adding a sample to be tested containing inositol hexakisphosphate into the nano iron oxide suspension to measure the absorbance, and substituting the absorbance into the standard curve obtained in step (1) to obtain the concentration of inositol hexakisphosphate in the sample to be tested.

[0012] The final concentration of the inositol hexakisphosphate is 0.1-10 mg / L.

[0013] The concentration of the nano iron oxide is 10-100 mg / L.

[0014] The sample to be tested includes soil, nutrient solution or plant tissue.

[0015] The determination of the absorbance is performed by visual color change or UV-Vis.

[0016] The determination of the absorbance of the application quantifies the IP6 concentration in the following two ways:

[0017] Semi-quantitative: the color change of the solution is observed visually, and a standard color card (pre-calibrated for the concentration range of 0.1-10 mg / L IP6) is referred to, and the precision is ±10%.

[0018] Quantitative: the intensity of the 200-220 nm absorption peak is measured by using a portable UV-Vis spectrophotometer, or the optical signal is recorded by using a smartphone App (based on RGB color analysis). The IP6 concentration calculation formula is: C_IP6 = (A_sample - A_blank) / k, wherein A_sample is the absorbance of the sample, A_blank is the absorbance of the blank, and k is a calibration constant (established by using a standard IP6 solution, and the linear range of the mass ratio of FeNPs:IP6 is 2-5:1). The determination time is about 2 minutes.

[0019] The present application also includes the use of the method for detecting inositol hexakisphosphate in detecting phosphorus fixation in soil, controlling fertilizer quality or analyzing hydroponic nutrition.

[0020] The present application also provides a portable detection device, which costs about 50-100 USD / set and is suitable for laboratory or field use. The device comprises the following modules:

[0021] (1) Extraction container: 5 mL centrifuge tube, used for extraction and shaking of matrix sample and acid solution, equipped with a sealing cover to prevent leakage.

[0022] (2) Reaction module: including a dropper (used for accurate addition of FeNPs suspension, 0.5 mL / time) and a small shaker (manual or electric, frequency 100-300 rpm), to ensure uniform reaction.

[0023] (3) Detection module: containing a portable UV-Vis spectrophotometer (wavelength range 190-400 nm, accuracy ±0.01 AU) or a smartphone holder (equipped with an LED light source, wavelength 400-700 nm, for color analysis). The smartphone App captures the RGB value through the camera, combines with the preset calibration curve, and outputs the IP6 concentration.

[0024] (4) Auxiliary components: micro filter (0.45 μm, used for clarification of the extract), reagent bottle (stores FeNPs suspension, light-proof design) and waste liquid collection tank (complies with environmental protection requirements). The device is compact in structure (size about 20 cm x 10 cm x 10 cm), weighs <1 kg, can be powered by a battery, and is suitable for on-site operation. All components are made of corrosion-resistant materials (such as polypropylene, glass), ensuring long-term stability.

[0025] The present application also includes an inositol hexakisphosphate-nano iron oxide complex, which comprises nano iron oxide with a particle size of 10-50 nm and inositol hexakisphosphate loaded on the surface thereof through electrostatic and chelation, and the mass ratio of the nano iron oxide and the inositol hexakisphosphate is 2-5:1, which can ensure sufficient chelation without causing aggregation or reduced stability due to excessive IP6.

[0026] The present application also includes a method for preparing an inositol hexakisphosphate-nano iron oxide complex, comprising the following steps:

[0027] (1) Synthesis of FeNPs: FeNPs with a particle size of 10-50 nm are synthesized by co-precipitation method;

[0028] (2) Functionalization of FeNPs: FeNPs are dispersed in deionized water to obtain a FeNPs solution, ultrasonic treatment, pH adjustment, addition of citric acid, and dispersion in deionized water after centrifugation and washing after reaction;

[0029] (3) IP6 loading: functionalized FeNPs were added into IP6 solution, stirred after ultrasonic, centrifuged and washed, and IP6-FeNPs powder was obtained by freeze-drying.

[0030] In step (1), the specific steps are as follows: FeCl2·4H2O and FeCl3·6H2O were dissolved in deionized water, heated under nitrogen protection, NH4OH was added dropwise, the black Fe3O4 precipitate was collected, washed and vacuum dried to obtain FeNPs with a particle size of 10-50 nm.

[0031] In step (2), the mass ratio of FeNPs solution to citric acid is 8:1-12:1.

[0032] In step (2), the pH is 5.0-6.0.

[0033] In step (3), the mass ratio of IP6 solution to functionalized FeNPs is 0.2:1-0.5:1.

[0034] In step (3), the ultrasonic power is 80-120 W, the ultrasonic frequency is 30-50 kHz, the ultrasonic time is 5-15 min, the freeze-drying temperature is -40 to -60°C, and the freeze-drying time is 20-30 h.

[0035] The content of the application also includes the application of the said inositol hexaphosphate-iron oxide nanoparticle complex in plant cultivation or plant growth promoting agent, and the said plant includes one or several of alfalfa, tomato or tobacco.

[0036] The said cultivation includes soil culture or water culture.

[0037] The mechanism of the application: the six phosphate groups (-PO4 3- ) of IP6 form Fe-O-P bonds (FTIR confirms 600 cm -1 peak) with Fe 3+ / Fe 2+ ions on the surface of functionalized FeNPs through electrostatic adsorption and chelation, construct core-shell structure, and realize nanoscale encapsulation of IP6. Carboxyl groups (-COOH) are introduced by citric acid functionalization, the Zeta potential is adjusted to -25 mV, the dispersibility is improved and oxidation is prevented. In application, the complex slowly hydrolyzes to release phosphorus (35% in 14 days, 55-60% in 30 days) at pH 5.0-6.0, the phosphate activates the PI3K signaling pathway of plants, promotes root elongation and lateral root differentiation (root length +20%, lateral root +25%); at the same time, low-dose Fe 2+ / Fe 3+(<0.1 mg / L) to supplement iron nutrition, enhance photosynthetic enzyme (such as Rubisco) activity (Fv / Fm +10%) and antioxidant enzyme (SOD +15%), and shorten the growth period by 3 days. The synergistic effect is better than single IP6 or FeNPs, and the combination of phosphorus slow release and iron signal regulation improves the utilization efficiency by 20-30%. The detection method described in the application utilizes the electrostatic and chelation of the six phosphate groups (-PO4 3- ) of IP6 and functionalized nano iron oxide (FeNPs, particle size 10-50 nm, surface modified by citric acid, Zeta potential about-25 mV) to form stable Fe-O-P bonds (confirmed by Fourier transform infrared spectroscopy FTIR, characteristic peak 600 cm -1 ). This chelation reaction changes the surface charge of FeNPs, leading to particle aggregation, which in turn triggers optical signal changes, including the solution color changing from brown red to dark brown or the UV-Vis absorption peak intensity at 200-220 nm increasing. These optical changes can be semi-quantitatively or quantitatively analyzed by visual observation, portable UV-Vis spectrophotometer or smartphone application (App). The detection method of the application forms a synergistic effect with plant cultivation applications to improve phosphorus utilization efficiency. The detection method determines the IP6 concentration in the matrix quickly, providing accurate guidance for the application of IP6-FeNPs complex. For example, in soil culture, the IP6 level in the matrix can be detected first to determine whether the complex (100 mg / L) needs to be supplemented to maintain stable phosphorus supply; in water culture, the IP6 concentration in the nutrient solution is detected to optimize the application amount to promote rice root development (root length increased by 20%, lateral root number increased by 25%). Therefore, the detection mechanism can be further applied to prepare FeNPs-IP6 complex, and the chelation mechanism of the complex is: the phosphate groups of IP6 and Fe 3+ / Fe 2+ on the surface of FeNPs form Fe-O-P bonds (FTIR 600 cm -1 peak), change the Zeta potential (shift from-25 mV), cause particle aggregation, and produce measurable optical signals. The signal is positively correlated with the IP6 concentration (UV-Vis absorption peak intensity increases with concentration), and is verified by DLS (particle size increases) and SEM (root adsorption observation).

[0038] Beneficial effects: Compared with the prior art, the application provides a method for detecting inositol hexakisphosphate, which is used for rapidly determining the inositol hexakisphosphate (IP6) concentration in soil, nutrient solution or plant tissue matrix, and has high sensitivity, and the detection limit of IP6 is as low as 0.1 mg / L, which is better than that of an enzyme method (about 40 mg / L), and the total detection time is less than 10 minutes. Compared with the traditional enzyme method colorimetric or ion chromatography method, the method shortens the time by 70% compared with the traditional method (>1 hour), and does not need complex pretreatment or expensive equipment, and uses a UV-Vis spectrophotometer or a colorimetric card, which reduces by 90% compared with an ion chromatography device (>10,000 USD), and is suitable for on-site rapid monitoring; the method is environmentally friendly, non-toxic and environmentally friendly. The detection result of the application can guide the application of the compound, improve the phosphorus utilization efficiency by 20-30%, and enhance the plant cultivation effect (for example, the yield of alfalfa is increased by 18%, and the growth period of rice is shortened by about 7 days). The method of the application is suitable for soil phosphorus fixation monitoring, fertilizer quality control and water culture nutrition analysis, and can be developed into a test strip in the future to further simplify the operation. The compound prepared in the application promotes the fresh weight and yield of alfalfa, tomato and tobacco in soil culture with the addition amount of 100 mg / L inositol hexakisphosphate-nano iron oxide compound, and promotes the root development of rice (root length is increased by 20%, and lateral root number is increased by 25%) in water culture with the addition of 100 mg / L inositol hexakisphosphate-nano iron oxide compound in Kimura B nutrient solution within 14 days, and 35% phosphorus is released and adsorbed by the roots, and the growth period is shortened by 3 days. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 FIG. 1 is a UV-Vis spectrum (graph) for IP6 detection.

[0040] Figure 2 FIG. 3 is a color change graph for IP6 detection.

[0041] Figure 3 FIG. 5 is a soil culture as a seedling stage phenotype graph.

[0042] Figure 4 FIG. 7 is a water culture rice three-week phenotype graph. DETAILED DESCRIPTION

[0043] The application will be further described below in combination with the drawings and examples.

[0044] Example 1: Inositol hexakisphosphate detection method based on FeNPs

[0045] This example describes a method for rapidly detecting the inositol hexakisphosphate (IP6) concentration in a soil matrix. The method uses the chelation reaction of functionalized FeNPs with IP6 to induce particle aggregation and optical changes, so as to realize on-site quantitative analysis.

[0046] 1. Preparation of FeNPs suspension:

[0047] (1) FeNPs synthesis:

[0048] Take 0.02 mol FeCl2·4H2O and 0.04 mol FeCl3·6H2O and dissolve them in 100 mL deionized water (Fe 2+ : Fe 3+ = 1:2). Under nitrogen protection, heat to 80°C, add 10 mL 25% NH4OH dropwise, stir at 800 rpm for 30 minutes. Collect the black Fe3O4 precipitate, wash and dry at 60°C under vacuum, and test the particle size of the FeNPs by DLS (Malvern Zetasizer Nano ZS) to be 10-50 nm.

[0049] (2) FeNPs functionalization:

[0050] Disperse 1 g of FeNPs in 100 mL of deionized water and treat with ultrasonic waves (100 W, 40 kHz, 15 minutes).

[0051] Adjust the pH to 5.5, add 0.1 g of citric acid, and stir at 25°C for 2 hours (300 rpm). Centrifuge (10,000 rpm, 10 minutes), disperse in deionized water after washing, and prepare a FeNPs suspension with a concentration of 100 mg / L (zeta potential -25 mV), and store in the dark for future use.

[0052] 2. Sample extraction:

[0053] Take 1 g of soil matrix (peat soil: vermiculite: perlite = 2:1:1, organic matter 320.13 g / kg, initial phosphorus content 7.28 g / kg), add 5 mL of 0.1 M hydrochloric acid (HCl) solution (pH adjusted to 5.5), and manually shake for 1 minute (300 rpm). Then centrifuge (10,000 rpm, 1 minute) or filter through a 0.45 μm filter to obtain a clear extract. This step has an extraction efficiency of >90% and minimizes interferents.

[0054] 3. Establish a standard curve

[0055] Prepare IP6 solutions of different concentrations (phytate sodium salt, pH 5.5, Macklin Chemical Reagent, Cas No.: 14306-25-3), add 0.5 mL of FeNPs suspension, so that the final concentration of IP6 in the reaction system is 0, 0.1, 1, 5, and 10 mg / L, respectively. Shake manually for 10 seconds to promote the formation of Fe 3+ / Fe 2+ -O-P bonds on the surface of FeNPs (FTIR confirmed at 600 cm -1The solution color changed from brownish red to dark brown, and the UV-Vis absorption peak (200-220 nm) was enhanced due to the aggregation of particles. The absorbance was measured, and the linear equation was A = 0.18 x C IP6 + 0.1 (R = 0.98, A is the absorbance, and C IP6 is the concentration of IP6). C IP6 = (A sample - A blank) / 0.18, where A sample is the sample absorbance, and A blank is the blank absorbance (about 0.1 AU). The results are shown in Table 1 and 2 . Figure 1 .

[0056] Table 1: UV-Vis absorbance and calculated concentration of IP6 at different concentrations

[0057]

[0058] Note: Data are mean ± standard deviation (n = 3 replicates). Accuracy is calculated as (calculated concentration / standard concentration) x 100%. Overall accuracy is 95-105%, and the linear range is 0.1-10 mg / L.

[0059] From Table 1 and Figure 2 , it can be seen that as the concentration of IP6 increases, the absorbance linearly increases (the intensity of the 210 nm peak increases from 0.1 AU to 2.0 AU), and the color changes from light brownish red (RGB 200, 100, 50) to dark brown (RGB 80, 40, 25).

[0060] 4. Actual soil sample detection

[0061] To the 1 mL of the extract obtained in step 2, 0.5 mL of FeNPs suspension was added, and the IP6 phosphate group was promoted to form a Fe-O-P bond with the Fe 3+ / Fe 2+ on the surface of FeNPs (confirmed by FTIR at 600 cm -1 peak). After the reaction, the solution color changed from brownish red to dark brown, and the UV-Vis absorption peak (200-220 nm) was enhanced due to the aggregation of particles. The absorbance of the reaction solution at 200-220 nm was measured using a UV-Vis spectrophotometer. The IP6 concentration was calculated by comparing with the standard curve established in step 3. In the actual soil sample detection, the IP6 concentration of the extract was about 2.5 mg / L, which was consistent with the results of the enzyme colorimetric method (deviation <5%).

[0062] Iron safety: The release of Fe in the reaction was <0.1 mg / L, which was lower than the toxicity threshold (>10 mg / L), and there was no environmental risk.

[0063] In summary, the detection method realizes rapid and accurate analysis through FeNPs-IP6 chelation mechanism, which is superior to the traditional method (time is shortened by 70%, cost is reduced by 20%), and can guide the application of the complex (such as optimizing the addition amount of 100 mg / L in phosphorus fixed soil). The detection limit of this embodiment is 0.1 mg / L, the total time is less than 10 minutes, and it is suitable for monitoring soil phosphorus fixation.

[0064] Example 2 Preparation of IP6-FeNPs complex

[0065] 1. FeNPs synthesis:

[0066] Take 0.02 mol FeCl2·4H2O and 0.04 mol FeCl3·6H2O and dissolve them in 100 mL deionized water (Fe 2+ : Fe 3+ = 1:2). Under nitrogen protection, heat to 80°C, add 10 mL 25% NH4OH dropwise, stir (800 rpm) for 30 minutes. Collect the black Fe3O4 precipitate, wash and dry at 60°C under vacuum, and test the particle size of the FeNPs by DLS (Malvern Zetasizer Nano ZS) to be 10-50 nm.

[0067] 2. FeNPs functionalization:

[0068] Disperse 1 g FeNPs in 100 mL deionized water and treat with ultrasonic wave (100 W, 40 kHz, 15 minutes).

[0069] Adjust the pH to 5.5, add 0.1 g citric acid, and stir at 25°C for 2 hours (300 rpm). Centrifuge (10,000 rpm, 10 minutes), wash and disperse in 50 mL deionized water to obtain a functionalized FeNPs suspension.

[0070] 3. IP6 loading:

[0071] A 50 mL 0.01 M IP6 solution (phytate sodium salt, pH 5.5) was prepared. 1 g of dry weight of functionalized FeNPs suspension (about 45 mL) was added, and after ultrasonication (100 W, 40 kHz, 10 min) it was stirred for 24 h (400 rpm, 25 °C). Centrifugation (10,000 rpm, 15 min), washing, freeze-drying (-50 °C, 24 h) gave IP6-FeNPs powder. Characterization: UV-Vis determined IP6 loading efficiency of 85% (loading efficiency was determined by UV-Vis: initial IP6 concentration (A0, 200-220 nm absorption peak) minus supernatant concentration (A1), efficiency = (A0- A1) / A0x 100% = 85%); FTIR (Thermo Fisher Nicolet iS50) confirmed P-O (1000-1100 cm -1 ) and Fe-O-P (600 cm -1 ) bonds; Zeta potential -25 mV; DLS (Malvern Zetasizer Nano ZS) showed particle size 10-50 nm, uniform coating.

[0072] Example 3 IP6-FeNPs application in soil-grown plants

[0073] 1. Substrate preparation and experimental design:

[0074] The substrate was peat soil: vermiculite: perlite = 2:1:1 (initial organic matter 320.13 g / kg, P 7.28 g / kg, K 21.73 g / kg, N 7.43 g / kg). The plants grown on the substrate were alfalfa (Medicago sativa), tomato (Micro-Tom) and tobacco (NC89), respectively.

[0075] The experimental groups included:

[0076] Experimental group: 100 mg / L IP6-FeNPs solution (IP6 concentration 30 mg / L) was prepared (IP6-FeNPs group), and IP6-FeNPs solution was sprayed on different plants every week, with a spraying concentration of 200 mL / kg substrate.

[0077] Control group: free IP6 (30 mg / L IP6) solution was prepared (IP6 group), and IP6 solution was sprayed on different plants every week, with a spraying concentration of 200 mL / kg substrate; IP6-unloaded nano-iron oxide (FeNPs, 80 mg / kg was sprayed on different plants every week) (FeNPs group) and blank substrate (no additional phosphorus source, CK group).

[0078] Experimental conditions: Greenhouse temperature 22–25°C, humidity 60%, photoperiod 16 hours light / 8 hours dark, light intensity 200 µmol·m -2 ·s -1 Water 200 mL / kg of substrate (pH 6.0) weekly. Growth cycle: alfalfa 5 weeks, tomato and tobacco 8 weeks. See Table 2 for experimental results. Figure 3 .

[0079] Table 2

[0080]

[0081] Note: Data are mean ± standard deviation (n=5 replicates). Different lowercase letters (a, b) indicate significant differences between groups. p <0.05, based on ANOVA test and Tukey post-hoc multiple comparisons). p The value represents the significance level of the difference between groups in the overall population. p Values ​​are rounded to two decimal places, with 0 indicating p < 0.01. The percentages in parentheses represent the relative increase compared to the CK group (for reference only and not included in the statistics).

[0082] From Table 2 and Figure 3 It can be seen that, in terms of the fresh weight of the above-ground parts: alfalfa ( Figure 3 a): The fresh weight of the aboveground parts of the IP6-FeNPs group (2.0g) was significantly higher than that of the control group in all three treatments. p <0.05, no significant difference was found between the IP6, FeNPs group and the blank matrix group. Tomato ( Figure 3 (b) The fresh weight of the aboveground parts of the IP6-FeNPs group (14.4g) was significantly higher than that of the control group in all three treatments. p <0.05), no significant differences were found between the IP6, FeNPs group and the blank matrix group; tobacco ( Figure 3 c): The fresh weight of the aboveground parts of the IP6-FeNPs group (10.3g) was significantly higher than that of the control group in all three treatments. p<0.05), IP6 group, FeNPs group and blank matrix group had no significant difference. In terms of yield: tomato: IP6-FeNPs group fruit yield 115 g, significantly higher than the control group of the three treatments, alfalfa: IP6-FeNPs group dry matter yield 1.18 g, significantly higher than the control group of the three treatments. Tobacco had no fruit yield, IP6-FeNPs group leaf area 48 cm², significantly higher than the control group of the three treatments. Phosphorus content: IP6-FeNPs group significantly increased leaf phosphorus content, iron content: IP6-FeNPs group and FeNPs group increased leaf iron content. Phosphorus use efficiency: IP6-FeNPs group increased by 20~25%; free IP6 and blank matrix group had no significant improvement. Matrix nutrients: IP6-FeNPs group matrix final phosphorus content 4.28 g / kg, free IP6 and blank matrix group phosphorus content <0.01 g / kg, reflecting the rapid release of free IP6 (7 days >85%) leading to phosphorus loss. Iron safety: IP6-FeNPs group matrix solution iron release <0.1 mg / L, lower than the toxicity threshold (>10 mg / L).

[0083] In summary, IP6-FeNPs significantly improved the fresh weight and yield of soil-grown plants by slow-release phosphorus and low-dose iron supplementation. Free IP6 had no difference from the blank matrix due to rapid release.

[0084] Example 4 Application of IP6-FeNPs in Rice Hydroponics

[0085] 1. Experimental design:

[0086] Plant: rice (Nipponbare, cv. Oryza sativa cv. Nipponbare).

[0087] Hydroponics conditions: Kimura B nutrient solution (base phosphorus content 0.36 mM, about 11 mg / L P, Jiangxi Qiyun Biology, product number: QM4003), temperature 25-28°C, humidity 60%, light cycle 16 hours light / 8 hours dark, light intensity 200 µmol·m -2 ·s -1 .

[0088] Treatment groups: Experimental group: nutrient solution added with final concentration of 100 mg / L IP6-FeNPs (final concentration of 30 mg / L IP6). Control group: nutrient solution added with free IP6 (final concentration of 30 mg / L), nano-iron oxide loaded with no IP6 (final concentration of FeNPs, 80 mg / L) and blank (CK, Kimura B nutrient solution only).

[0089] Experimental conditions: nutrient solution was replaced every 2 weeks, maintaining pH 5.0-5.5. Growth period: 6 weeks (vegetative growth period to tillering period). Experimental results are shown in Table 3 andFigure 4 .

[0090] Table 3

[0091]

[0092] Data are mean ± standard deviation (n = 5 replicates). Different lower case letters (a, b, c) indicate significant differences among groups (p < 0.05, based on ANOVA followed by Tukey’s post-hoc multiple comparisons). ANOVA p-value indicates the level of significance of overall differences among groups. p-value is kept to two decimal places, 0.00 means p < 0.01. Percentage in brackets for the indicators is the relative increase value relative to the CK group (for reference only, not involved in statistics).

[0093] From Table 3 and Figure 4 root length increased by 20% (14.3 cm vs. 11.9 cm) and lateral root number increased by 25% (18 vs. 15) compared to CK. Compared to CK, root length in the free IP6 group increased by 10% (13.1 cm) and lateral root number increased by 12% (16). Compared to CK, root length and lateral root number in the FeNPs group did not significantly increase (11.9 cm, 15). In terms of growth period, the tillering period of the IP6-FeNPs group was 7 days earlier than that of CK (26 days vs. 33 days); other treatments did not significantly change compared to CK. In terms of biomass, the fresh weight of the aboveground part of the IP6-FeNPs group increased by 18% (3.8 g vs. 3.2 g) compared to CK; other treatments did not significantly change compared to CK. In terms of phosphorus content, the root phosphorus content of the IP6-FeNPs group increased by 25% (0.34% vs. 0.27%) and the leaf phosphorus content increased by 20% (0.31% vs. 0.26%) compared to CK; other treatments did not significantly change compared to CK. In terms of iron content, the leaf iron content of the IP6-FeNPs group and the FeNPs group increased compared to CK, and the IP6 and CK groups changed significantly. In terms of photosynthetic efficiency, the Fv / Fm value of the IP6-FeNPs group increased by 10% (0.83 vs. 0.75) compared to CK; other treatments did not significantly change compared to CK. In terms of iron safety, the iron release amount of the IP6-FeNPs group was <0.1 mg / L, which was lower than the toxicity threshold (>10 mg / L), and the replacement of the nutrient solution every 2 weeks further reduced the accumulation risk.

[0094] Slow release meaning: IP6-FeNPs group released 35% phosphorus in 14 days, more than 95% of the free IP6 group, providing stable phosphorus supply; SEM observation confirmed that nanoparticles were adsorbed on the root surface, forming a local slow-release microenvironment. Nanoparticle treatment allowed slow release of phosphorus in IP6, improving IP6 utilization efficiency by 20-25% and cost-effectiveness, and reducing phosphorus loss in nutrient solution.

[0095] In summary, IP6-FeNPs significantly outperformed free IP6 and the blank through short-term slow release, root adsorption, and iron supplementation, promoting rice root development and shortening the growth period.

Claims

1. A method for detecting inositol hexakisphosphate, characterized by, The detection method utilizes the reaction of nano-iron oxide and inositol hexakisphosphate, and detects the concentration of inositol hexakisphosphate in the sample to be detected according to the linear relationship between the absorbance of the reaction solution and the concentration of inositol hexakisphosphate; and specifically comprises the following steps: (1) adding inositol hexakisphosphate standard solutions with different known concentrations into a nano-iron oxide suspension to measure the absorbance, and establishing a standard curve of the absorbance and the concentration of the inositol hexakisphosphate solution; (2) adding the sample to be detected containing inositol hexakisphosphate into the nano-iron oxide suspension to measure the absorbance, and substituting the absorbance into the standard curve obtained in step (1) to obtain the concentration of inositol hexakisphosphate in the sample to be detected; The preparation method of the nano-iron oxide suspension is as follows: FeCl2·4H2O and FeCl3·6H2O are dissolved in deionized water, heated under nitrogen protection, NH4OH is added dropwise, stirred, the black Fe3O4 precipitate is collected, washed and vacuum dried to obtain FeNPs with a particle size of 10-50 nm; the FeNPs are dispersed in deionized water to obtain a FeNPs solution, ultrasonic treatment is performed, the pH is adjusted, citric acid is added to modify the surface of the FeNPs, and after reaction, centrifugal washing and dispersion in deionized water are performed; the Zeta potential of the FeNPs suspension is -25 mV; the mass ratio of the FeNPs solution to citric acid is 8:1-12:1, and the pH is 5.0-6.

0.

2. The method for detecting inositol hexakisphosphate according to claim 1, characterized by, The final concentration of the inositol hexakisphosphate is 0.1-10 mg / L.

3. The method for detecting inositol hexakisphosphate according to claim 1, characterized by, The concentration of the nano-iron oxide is 10-100 mg / L.

4. The method of claim 1, wherein the inositol hexakisphosphate is IP6. The sample to be detected includes soil, nutrient solution or plant tissue.

5. The method of claim 1, wherein the inositol hexakisphosphate is IP6. The determination of the absorbance is performed by visual color change or UV-Vis.

6. The application of the detection method of inositol hexakisphosphate in any one of claims 1-5 in plant cultivation, detection of phosphorus fixation in soil, quality control of fertilizer or analysis of water culture nutrition.

7. An inositol-hexakisphosphate-nanoferric oxide complex, characterized in that, The complex comprises nano-iron oxide with a particle size of 10-50 nm and inositol hexakisphosphate loaded on the surface thereof by electrostatic and chelation, and the mass ratio of the nano-iron oxide to the inositol hexakisphosphate is 2-5:1; and the preparation method of the inositol hexakisphosphate-nano-iron oxide complex comprises the following steps: (1) synthesis of FeNPs: FeNPs with a particle size of 10-50 nm are synthesized by co-precipitation; (2) functionalization of FeNPs: the FeNPs are dispersed in deionized water to obtain a FeNPs solution, ultrasonic treatment is performed, the pH is adjusted, and citric acid is added to modify the surface of the FeNPs, and after reaction, centrifugal washing and dispersion in deionized water are performed; (3) IP6 loading: functionalized FeNPs are added into an IP6 solution, stirred after ultrasonic treatment, centrifugal washing is performed, and IP6-FeNPs powder is obtained by freeze drying; The specific steps of step (1) are as follows: FeCl2·4H2O and FeCl3·6H2O are dissolved in deionized water, heated under nitrogen protection, NH4OH is added drop by drop, stirred, the black Fe3O4 precipitate is collected, washed and vacuum dried to obtain FeNPs with a particle size of 10-50 nm; the mass ratio of the FeNPs solution to citric acid in step (2) is 8:1-12:1; the pH in step (2) is 5.0-6.0; the mass ratio of the IP6 solution to functionalized FeNPs in step (3) is 0.2:1-0.5:

1.

8. Use of the inositol hexaphosphate-nano-iron oxide complex of claim 7 in plant cultivation or preparation of a plant growth promoter, the plant including one or more of rice, alfalfa, tomato or tobacco.

9. Use according to claim 8, characterized in that, The cultivation includes soil culture or water culture.

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