Preparation method and application of a low-field NMR homogeneous sensor for detecting Escherichia coli based on click reaction.

Abstract

This invention discloses a method for preparing and applying a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction. The method includes the following steps: 1) preparation of PA-GO@Fe3O4; 2) preparation of the signal unit Gd2O3-Az; 3) preparation of the low-field nuclear magnetic resonance homogeneous test solution. The intensity of the low-field nuclear magnetic resonance signal T1 corresponding to different concentrations of E. coli is measured using a low-field nuclear magnetic resonance instrument, establishing a quantitative relationship between E. coli concentration and the change in T1 signal (ΔT1). Based on this quantitative relationship, the concentration of E. coli in an unknown sample is determined. The advantages are high sensitivity, high precision, and high convenience.

Description

Technical Field

[0001] This invention relates to a low-field nuclear magnetic resonance sensor, and more particularly to a method for preparing and applying a low-field homogeneous nuclear magnetic resonance sensor based on click reaction for detecting Escherichia coli. Background Technology

[0002] Escherichia coli (E. coli) is a Gram-negative bacillus widely found in the intestines of humans and animals, as well as in the surrounding environment. As a foodborne pathogen, E. coli can cause a range of localized tissue and organ infections, including intestinal inflammation and meningitis. Traditional methods for detecting E. coli, such as multi-tube fermentation, membrane filtration, and plate counting, are mostly accurate, reliable, and inexpensive, but time-consuming and cumbersome. Emerging detection methods include fluorescence, surface-enhanced Raman scattering, colorimetry, and electrochemistry, which can achieve rapid and sensitive detection. However, the optical and electrical signals are easily affected by complex background interference, and high requirements are placed on sample pretreatment.

[0003] Low-field NMR has significant advantages in the direct detection of complex samples due to its relatively low background interference. Currently, low-field NMR has been reported for the detection of foodborne pathogens such as Salmonella, Vibrio parahaemolyticus, and Listeria monocytogenes, but there are no reports on its use in E. coli detection. The quantitative strategies in these studies typically involve using antibodies, aptamers, and bacteriophages as biorecognition elements to identify the target analyte, acting as a bridge between the signal unit and the target analyte, and quantifying based on the signal intensity output by the signal unit. However, within this framework, the number of signal units depends entirely on the number of target analytes, making signal amplification difficult and decisively limiting detection sensitivity. Furthermore, the activity of biorecognition elements is unstable and easily affected by environmental factors, impacting detection precision. Finally, these biorecognition elements cannot distinguish between live and dead bacteria and cannot objectively reflect the true extent of bacterial contamination.

[0004] This invention constructs a low-field nuclear magnetic resonance sensing platform based on click chemistry for the detection of *E. coli*. This sensing platform utilizes Cu produced by the copper metabolism of the live target species, *E. coli*. + The catalyst catalyzes the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, triggering the signal unit to leave the sensing system, leading to an increase in the longitudinal relaxation time (T1) and thus enabling quantitative detection. This is due to the Cu produced by the copper metabolism of E. coli. + The number is much greater than that of E. coli, and each Cu + Catalysts can catalyze a large number of signal units to undergo the CuAAC reaction and escape from the sensing system, with each signal unit loaded with a large amount of Gd. 3+This achieves three-stage signal amplification, significantly improving detection sensitivity; the signal generation process is mediated by purely chemical means, avoiding precision issues that might arise from changes in the activity of biorecognition elements; only live bacteria can undergo copper metabolism, eliminating the influence of dead bacteria on detection. Currently, there are no reports of low-field NMR homogeneous sensors based on click reactions for the detection of *E. coli*. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing and applying a low-field nuclear magnetic resonance homogeneous sensor based on click reaction for detecting Escherichia coli, which is highly sensitive, precise and easy to operate.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing and applying a low-field nuclear magnetic resonance homogeneous sensor based on click reaction for detecting Escherichia coli. This method is not for diagnostic or therapeutic purposes and includes the following steps:

[0007] (1) Preparation of PA-GO@Fe3O4

[0008] a. Add 10–30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5–15 mg of N-hydroxysuccinimide (NHS) to 2–16 mL of graphene oxide (GO) dispersion (1–10 mg), and sonicate for 10–100 min to activate the carboxyl groups of GO. Then, add 1–10 mg of amino-functionalized iron(III) oxide (Fe3O4-NH2), and stir at 50–90 °C for 2–8 h. After washing, redisperse the mixture in 5–16 mL of water to obtain the GO@Fe3O4 dispersion.

[0009] b. Add 2–20 mg of potassium hydroxide (KOH) and 0.5–5 mL of 0.1–3 mg / mL propargylamine (PA) aqueous solution to the GO@Fe3O4 dispersion, and stir vigorously at 50–90 °C for 10–48 h. After washing three times with water, redisperse the obtained PA-GO@Fe3O4 in 2–20 mL of water and store at 4 °C for later use.

[0010] (2) Preparation of signal unit Gd2O3-Az

[0011] a. Add 50–600 μL of 3-aminopropyltriethoxysilane (APTES) to 5–50 mL of anhydrous ethanol dispersion of 0.1–1.2 mg / mL Gd₂O₃NRs, sonicate for 10–60 min, and react at 50–80 °C for 1–4 h. After centrifugation and washing, amino-functionalized Gd₂O₃NRs, i.e., Gd₂O₃-NH₂, are obtained.

[0012] b. Disperse Gd₂O₃-NH₂ in 10–50 mL of 2.5 wt% glutaraldehyde aqueous solution and react at room temperature for 1–3 h. After centrifugation and washing, disperse the product in 10–50 mL of 0.01–0.15 mg / mL azide-tetraethylene glycol-aminoAz aqueous solution and stir at room temperature for 1–3 h. Finally, centrifuge, wash, and redisperse in 10–50 mL of water to obtain the signal unit Gd₂O₃-Az, which can be stored at 4 °C for later use.

[0013] (3) Preparation of homogeneous test solution for low-field nuclear magnetic resonance

[0014] a. Add 10–400 μL of Fe3O4@Apt to 1 mL of sample and incubate at 30–40 °C for 10–30 min to capture E. coli. Then, magnetically separate, wash, and disperse in 0.5–1.5 mL of PBS solution. Add 10–100 μL of totipotent nuclease solution and incubate at 30–40 °C for 10–40 min to release the captured E. coli, followed by magnetic separation.

[0015] b. Take 10-400 μL of the supernatant from step (3)a and add it to a sample vial containing 10-400 μL of 2-20 μmol / L copper chloride CuCl2, 10-400 μL of Gd2O3-Az, and 10-400 μL of PA-GO@Fe3O4. Shake at 30-40℃ for 20-60 min to carry out a click chemical reaction and magnetic separation.

[0016] c. Take 100–800 μL of the supernatant from step (3)b and add 10–400 μL of hydrochloric acid (pH=2) solution. Shake at room temperature for 2–20 min to allow Gd₂O₃ NRs to acidify and release Gd. 3+ A homogeneous test solution for low-field nuclear magnetic resonance was obtained.

[0017] Further, the synthesis method of amino-functionalized Fe3O4-NH2 described in step (1) is as follows: 0.49 g FeCl3·6H2O, 0.18–0.54 g sodium citrate, and 0.6–1.8 g urea are added to 15 mL ethylene glycol and stirred vigorously at room temperature for 1 h. The mixture is then transferred to a 25 mL polytetrafluoroethylene-lined stainless steel reactor, heated to 160–200 °C and maintained for 12 h. The product is then collected by magnetic adsorption and washed with ethanol to obtain Fe3O4 nanoparticles (Fe3O4 NPs). The obtained Fe3O4 NPs and 300 μL APTES are then dispersed in 10 mL anhydrous ethanol, sonicated for 0.5 h, and stirred at 60 °C for 2 h. Finally, the mixture is washed with ethanol, collected by magnetic adsorption, and dried at 60 °C to obtain amino-functionalized Fe3O4 NPs, i.e., Fe3O4-NH2.

[0018] Further, the synthesis method of gadolinium oxide nanorods Gd₂O₃ NRs in step (2) is as follows: 10–50 mL of a 0.01–0.20 mol / L aqueous solution of gadolinium nitrate (Gd(NO₃)₃) and 0.5–5 mL of 25% ammonia (NH₃·H₂O) are added to a 50 mL polytetrafluoroethylene-lined stainless steel reactor. After heating at 100–140 °C for 8–16 h, the product is washed and dried at 45–90 °C to obtain gadolinium hydroxide (Gd(OH)₃) powder. Then, the Gd(OH)₃ powder is placed in a tube furnace and calcined at 600–800 °C for 2–6 h to obtain gadolinium oxide nanorods Gd₂O₃ NRs.

[0019] Further, the preparation method of Fe3O4@Apt in step (3) is as follows: Take 3-8 mg of amino-functionalized iron(III) oxide Fe3O4-NH2 and disperse it in 2-10 mL of 2.5 wt% glutaraldehyde aqueous solution. Stir at room temperature for 1-6 h and wash with water. Then redisperse it in 2-9 mL of phosphate buffered PBS. Add 20-200 μL of 2-20 μmol / L aptamer Apt solution and incubate slowly with shaking at 30-40 °C for 2-10 h. Then, magnetically separate and wash to remove unbound Apt. Add 100-600 μL of 0.5-3 wt% bovine serum albumin (BSA) solution and incubate at room temperature for 0.5-3 h to block non-specific binding sites. Finally, after magnetic separation and washing, redisperse it in 2-8 mL of TE-Mg 2+ Fe3O4@Apt dispersion was obtained in buffer solution.

[0020] 2. A method for detecting Escherichia coli using the above-mentioned low-field nuclear magnetic resonance homogeneous sensor. This method is not for diagnosis or treatment purposes and includes the following steps: using the prepared low-field nuclear magnetic resonance homogeneous test solution, measuring the corresponding T1 intensity under different concentrations of Escherichia coli using a low-field nuclear magnetic resonance instrument, and obtaining the concentration of Escherichia coli in the test solution based on the T1 signal change value (ΔT1).

[0021] Furthermore, the test parameters for the low-field nuclear magnetic resonance test are as follows: main frequency 19.00MHz, sampling width 100kHz, radio frequency delay 0.08ms, number of sampling points 2048, waiting time 20000ms, number of inversions 20, number of accumulations 2, digital gain 3, and analog gain 15.0dB.

[0022] Furthermore, the change value of the T1 signal is calculated according to the following formula: ΔT1=T1(positive)-T1(negative), where T1(negative) is the average T1 when E. coli is not present in the sample, and T1(positive) is the average T1 when E. coli is present in the sample.

[0023] Invention Principle: A low-field nuclear magnetic resonance sensor based on click chemistry reaction and its detection principle are shown in Scheme 1. A specific concentration of Cu is present in the sample vial. 2+ A large number of signaling units Gd2O3-Az and PA-GO@Fe3O4, when present with living E. coli, can metabolize Cu through characteristic copper metabolism. 2+ Metabolized into Cu + Cu + As a catalyst, the CuAAC reaction is triggered, causing the signal unit Gd2O3-Az and PA-GO@Fe3O4 to undergo an azide-alkynyl cycloaddition reaction and bind together. The resulting complex Gd2O3-GO@Fe3O4 can be rapidly separated by magnetic force. The remaining signal unit Gd2O3-Az in the flask is then acidified with added hydrochloric acid to generate homogeneously distributed Gd 3+ This generates a stable T1 signal. The higher the number of live E. coli bacteria, the more Cu they produce during metabolism. + The greater the quantity, the more complexes are formed by the click chemical reaction, the less Gd2O3-Az remains in the flask, and the less Gd is generated after hydrochloric acid acid hydrolysis. 3+ The lower the solution concentration, the larger the ΔT1, thus enabling the quantitative detection of live E. coli.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] (1) High sensitivity. First, the target itself can assist in signal amplification; live E. coli continuously amplifies Cu. 2+ Reduced to Cu + This triggers a large-scale CuAAC reaction, which proceeds under mild conditions and with high yields. Secondly, there is a sufficient number of signal tags. Each remaining Gd₂O₃-Az can release a large amount of the signal tag Gd through hydrochloric acid acid hydrolysis. 3+ In addition, GO's large specific surface area and good structural stability provide a site for alkynyl modification and subsequent click reactions.

[0026] (2) High precision. The sensor has high reproducibility, thanks to the stability of the purely chemically modified Gd2O3-Az and PA-GO@Fe3O4 materials.

[0027] (3) High convenience. The sensor detection process is fast and convenient, and is not easily affected by complex samples. Sample detection can be completed in one step of magnetic separation and rapid acid digestion.

[0028] In summary, this invention presents a method for preparing and applying a low-field homogeneous NMR sensor for detecting *E. coli* based on click reactions. Utilizing click chemistry catalyzed by bacterial metabolism, a low-field homogeneous NMR sensor is designed for the rapid detection of foodborne pathogens. By targeting and triggering click chemistry, incorporating functionalized two-dimensional materials, and acid dissolving nanomaterials, a three-stage signal amplification is achieved, significantly enhancing detection sensitivity. Furthermore, the signal generation during detection is entirely chemical, ensuring high precision. In addition, the click reaction catalyzed by bacterial metabolism can distinguish between live and dead bacteria, eliminating the influence of dead bacteria on detection. Therefore, this method offers high sensitivity and precision, requiring only simple pretreatment, providing a feasible strategy for rapid on-site detection of pathogens. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the principle of the low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli according to the present invention.

[0030] Figure 2 T1 signal response diagrams for different concentrations of Escherichia coli;

[0031] Figure 3 The detection sensitivity curve of the low-field nuclear magnetic resonance homogeneous sensor;

[0032] Figure 4 This is specific to low-field homogeneous nuclear magnetic resonance sensors;

[0033] Figure 5 To improve the stability of low-field homogeneous nuclear magnetic resonance sensors. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Specific Implementation Example 1

[0036] Example 1

[0037] Based on Cu + This method, mediated by click-coated chemiluminescence gels, utilizes low-field homogeneous NMR immunoassay for the detection of foodborne pathogens. This method is not intended for diagnosis or treatment. Figure 1 As shown, it includes the following steps:

[0038] (1) Preparation of PA-GO@Fe3O4

[0039] a. 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were added to 6 mL of graphene oxide (GO) dispersion (4 mg), and the mixture was sonicated for 30 min to activate the carboxyl groups of GO. Subsequently, 4 mg of Fe3O4-NH2 was added, and the mixture was stirred at 80 °C for 5 h. After washing, the mixture was redispersed in 8 mL of water to obtain the GO@Fe3O4 dispersion.

[0040] b. Add 10 mg of potassium hydroxide (KOH) and 2 mL of 1 mg / mL propargylamine (PA) aqueous solution to the GO@Fe3O4 dispersion, and stir vigorously at 70 °C for 24 h. After washing three times with water, redisperse the obtained PA-GO@Fe3O4 in 8 mL of water and store at 4 °C for later use.

[0041] (2) Preparation of signal unit Gd2O3-Az

[0042] a. Add 300 μL of 3-aminopropyltriethoxysilane (APTES) to 20 mL of 0.5 mg / mL Gd₂O₃ NRs anhydrous ethanol dispersion, sonicate for 30 min, react at 60 °C for 2 h, and after centrifugation and washing, obtain amino-functionalized Gd₂O₃ NRs, namely Gd₂O₃-NH₂.

[0043] b. Disperse Gd₂O₃-NH₂ in 20 mL of glutaraldehyde aqueous solution and react at room temperature for 2 h. After centrifugation and washing, disperse the product in 20 mL of 0.05 mg / mL azide-tetraethylene glycol-aminoAz aqueous solution and stir at room temperature for 2 h. Finally, centrifuge, wash, and redisperse in 20 mL of water to obtain the signal unit Gd₂O₃-Az, which is stored at 4 °C for later use.

[0044] (3) Preparation of homogeneous test solution for low-field nuclear magnetic resonance

[0045] a. Add 100 μL Fe3O4@Apt and incubate at 37 °C for 20 min to capture E. coli. Subsequently, magnetically separate, wash and disperse in 0.95 mL PBS solution, add 50 μL totipotent nuclease solution, incubate at 37 °C for 20 min to release the captured E. coli, and then magnetically separate.

[0046] b. Take 100 μL of the supernatant from step (3) a, and simultaneously add 100 μL of 10 μmol / L copper chloride CuCl2, 200 μL of Ld2O3-Az, and 100 μL of PA-GO@Fe3O4. Shake at 37℃ for 30 min to carry out click chemical reaction and magnetic separation.

[0047] c. Take 400 μL of the supernatant from step (3)b and add 100 μL of hydrochloric acid (pH=2) solution. Shake at room temperature for 5 min. Gd2O3NRs will acidify and release Gd. 3+ A homogeneous test solution for low-field nuclear magnetic resonance was obtained.

[0048] Example 2

[0049] The difference is the same as in Embodiment 1 above:

[0050] In step (1) of the preparation of PA-GO@Fe3O4:

[0051] a. 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysuccinimide (NHS) were added to 12 mL of graphene oxide (GO) dispersion (8 mg), and the mixture was sonicated for 30 min to activate the carboxyl groups of GO. Subsequently, 8 mg of Fe3O4-NH2 was added, and the mixture was stirred at 80 °C for 5 h. After washing, the mixture was redispersed in 16 mL of water to obtain the GO@Fe3O4 dispersion.

[0052] b. Add 20 mg of potassium hydroxide (KOH) and 4 mL of 2 mg / mL propargylamine (PA) aqueous solution to the GO@Fe3O4 dispersion, and stir vigorously at 70 °C for 24 h. After washing three times with water, redisperse the obtained PA-GO@Fe3O4 in 16 mL of water and store at 4 °C for later use.

[0053] In step (2), during the preparation of the signal unit Gd2O3-Az:

[0054] a. Add 600 μL of 3-aminopropyltriethoxysilane (APTES) to 40 mL of 1 mg / mL Gd₂O₃ NRs anhydrous ethanol dispersion, sonicate for 30 min, react at 60 °C for 2 h, and after centrifugation and washing, obtain amino-functionalized Gd₂O₃ NRs, namely Gd₂O₃-NH₂.

[0055] b. Disperse Gd₂O₃-NH₂ in 40 mL of glutaraldehyde aqueous solution and react at room temperature for 2 h. After centrifugation and washing, disperse the product in 40 mL of 0.1 mg / mL azide-tetraethylene glycol-aminoAz aqueous solution and stir at room temperature for 2 h. Finally, centrifuge, wash, and redisperse in 40 mL of water to obtain the signal unit Gd₂O₃-Az, which is stored at 4 °C for later use.

[0056] In step (3), during the preparation of the low-field NMR homogeneous test solution:

[0057] a. Add 200 μL Fe3O4@Apt and incubate at 37 °C for 20 min to capture E. coli. Subsequently, magnetically separate, wash and disperse in 0.95 mL PBS solution, add 100 μL totipotent nuclease solution, incubate at 37 °C for 20 min to release the captured E. coli, and then magnetically separate.

[0058] b. Take 200 μL of the supernatant from step (3) a, and simultaneously add 200 μL of 20 μmol / L copper chloride CuCl2, 400 μL of Ld2O3-Az, and 200 μL of PA-GO@Fe3O4. Shake at 37℃ for 30 min to carry out click chemical reaction and magnetic separation.

[0059] c. Take 800 μL of the supernatant from step (3)b and add 200 μL of hydrochloric acid (pH=2) solution. Shake at room temperature for 5 min. Gd2O3NRs will be acidified and release Gd. 3+ A homogeneous test solution for low-field nuclear magnetic resonance was obtained.

[0060] Example 3

[0061] The difference is the same as in Embodiment 1 above:

[0062] In step (1) of the preparation of PA-GO@Fe3O4:

[0063] a. 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were added to 3 mL of graphene oxide (GO) dispersion (2 mg), and the mixture was sonicated for 30 min to activate the carboxyl groups of GO. Subsequently, 2 mg of Fe3O4-NH2 was added, and the mixture was stirred at 80 °C for 5 h. After washing, the mixture was redispersed in 4 mL of water to obtain the GO@Fe3O4 dispersion.

[0064] b. Add 5 mg of potassium hydroxide (KOH) and 1 mL of 0.5 mg / mL propargylamine (PA) aqueous solution to the GO@Fe3O4 dispersion, and stir vigorously at 70 °C for 24 h. After washing three times with water, redisperse the obtained PA-GO@Fe3O4 in 4 mL of water and store at 4 °C for later use.

[0065] In step (2), during the preparation of the signal unit Gd2O3-Az:

[0066] a. Add 150 μL of 3-aminopropyltriethoxysilane (APTES) to 10 mL of 0.25 mg / mL Gd₂O₃ NRs anhydrous ethanol dispersion, sonicate for 30 min, react at 60 °C for 2 h, and after centrifugation and washing, amino-functionalized Gd₂O₃ NRs, i.e., Gd₂O₃-NH₂, are obtained.

[0067] b. Disperse Gd₂O₃-NH₂ in 10 mL of glutaraldehyde aqueous solution and react at room temperature for 2 h. After centrifugation and washing, disperse the product in 10 mL of 0.02 mg / mL azide-tetraethylene glycol-aminoAz aqueous solution and stir at room temperature for 2 h. Finally, centrifuge, wash, and redisperse in 10 mL of water to obtain the signal unit Gd₂O₃-Az, which is stored at 4 °C for later use.

[0068] In step (3), during the preparation of the low-field NMR homogeneous test solution:

[0069] a. Add 50 μL Fe3O4@Apt and incubate at 37 °C for 20 min to capture E. coli. Subsequently, magnetically separate, wash and disperse in 0.55 mL PBS solution, add 25 μL totipotent nuclease solution, incubate at 37 °C for 20 min to release the captured E. coli, and then magnetically separate.

[0070] b. Take 50 μL of the supernatant from step (3) a, and simultaneously add 50 μL of 5 μmol / L copper chloride CuCl2, 100 μL of Ld2O3-Az, and 50 μL of PA-GO@Fe3O4. Shake at 37℃ for 30 min to carry out click chemical reaction and magnetic separation.

[0071] c. Take 200 μL of the supernatant from step (3)b and add it to 50 μL of hydrochloric acid (pH=2) solution. Shake at room temperature for 5 min. Gd2O3NRs will be acidified and release Gd. 3+ A homogeneous test solution for low-field nuclear magnetic resonance was obtained. Specific Implementation Example 2

[0073] The low-field homogeneous nuclear magnetic resonance sensor prepared according to the above specific embodiment 1 was placed in a low-field nuclear magnetic resonance instrument for detection. The intensity of the low-field nuclear magnetic resonance signal T1 corresponding to different concentrations of E. coli was measured, and the concentration of E. coli in the test solution was obtained based on the T1 signal value. The detection principle is as follows. Figure 1 As shown.

[0074] like Figure 2 As shown, the T1 signal intensity increases with increasing E. coli concentration.

[0075] like Figure 3 As shown, in the range of 10 to 1.0 × 10 7 Within the CFU / mL range, the change in T1 signal intensity ΔT1(y, ms) showed a good linear relationship with the logarithm of E. coli concentration (x, CFU / mL). The linear regression equation was y = 151.35 * lg x + 19.35, and the correlation coefficient R² was [value missing]. 2=0.993. The noise level is approximately 35ms. Based on the signal-to-noise ratio S / N = 3, the limit of detection (LOD) is calculated to be 3.5 CFU / mL. Specific Implementation Example 3

[0077] Using 1.0×10 4 CFU / mL E. coli, SA, VP standard solutions, blank samples, and mixed samples (containing 1.0 × 10⁻⁶ CFU / mL) 4 The experiment was conducted using CFU / mL E. coli and SA, VP). The results are as follows: Figure 4 As shown, 1.0×10 4 The CFU / mL SA and VP values ​​were close to the ΔT1 produced by the blank sample, while the mixed sample was close to 1.0 × 10⁻⁶. 4 The ΔT1 generated by CFU / mL E. coli standard solution is almost the same, indicating that the sensor has good selectivity for E. coli detection. Specific Implementation Example 4

[0079] The prepared nanomaterials were stored at 4°C, and the long-term storage stability of the sensor was assessed by continuous measurements over 10 days. The results are as follows: Figure 5 As shown, after 10 days, the T1 signal response is still 96.9% of the original value, which indicates that the developed sensor platform has satisfactory stability. Specific Implementation Example 5

[0081] The accuracy and precision of the sensor were evaluated by detecting seawater (S) and milk (M) samples using the standard addition method. The results are shown in Table 1.

[0082] Table 1. Detection of E. coli in seawater and milk spiked samples (x±s, n=5)

[0083]

[0084] Table 1 shows that the recoveries of the spiked seawater samples were 97.2%–104.4%, with RSDs of 1.3%–2.4%; the recoveries of the spiked milk samples were 98.9%–104.7%, with RSDs of 1.5%–2.8%. These results indicate that the sensor has good accuracy and precision and can be used reliably to detect real samples.

[0085] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction, characterized in that... Includes the following steps: (1) Preparation of PA-GO@Fe3O4 a. Add 10–30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5–15 mg of N-hydroxysuccinimide (NHS) to 2–16 mL of graphene oxide (GO) dispersion, and sonicate for 10–100 min to activate the carboxyl groups of GO; then add 1–10 mg of amino-functionalized iron(III) oxide (Fe3O4-NH2), and stir at 50–90 °C for 2–8 h. After washing, redisperse the mixture in 5–16 mL of water to obtain the GO@Fe3O4 dispersion. b. Add 2–20 mg of potassium hydroxide (KOH) and 0.5–5 mL of 0.1–3 mg / mL propargylamine (PA) aqueous solution to the GO@Fe3O4 dispersion, and stir vigorously at 50–90 °C for 10–48 h; after washing three times with water, redisperse the obtained PA-GO@Fe3O4 in 2–20 mL of water and store at 4 °C for later use; (2) Preparation of signal unit Gd2O3-Az a. Add 50-600 μL of 3-aminopropyltriethoxysilane (APTES) to 5-50 mL of 0.1-1.2 mg / mL Gd₂O₃NRs anhydrous ethanol dispersion, sonicate for 10-60 min, and react at 50-80 °C for 1-4 h. After centrifugation and washing, amino-functionalized Gd₂O₃ nanorods, namely Gd₂O₃-NH₂, are obtained. b. Disperse Gd2O3-NH2 in 10-50 mL of 2.5 wt% glutaraldehyde aqueous solution and react at room temperature for 1-3 h; after centrifugation and washing, disperse the product in 10-50 mL of 0.01-0.15 mg / mL azide-tetraethylene glycol-aminoAz aqueous solution and stir at room temperature for 1-3 h; finally, centrifuge, wash, and redisperse in 10-50 mL of water to obtain the signal unit Gd2O3-Az, which can be stored at 4℃ for later use. (3) Preparation of homogeneous test solution for low-field nuclear magnetic resonance a. Add 10–400 μL FFe3O4@Apt to 1 mL of sample and incubate at 30–40 °C for 10–30 min to capture Escherichia coli; then, magnetically separate, wash and disperse in 0.5–1.5 mL PBS solution; Add 10–100 μL of totipotent nuclease solution and incubate at 30–40 °C for 10–40 min to release the captured E. coli, then separate magnetically. b. Take 10-400 μL of the supernatant from step (3)a and add it to a sample vial containing 10-400 μL of 2-20 μmol / L copper chloride CuCl2, 10-400 μL of Gd2O3-Az, and 10-400 μL of PA-GO@Fe3O4. Shake at 30-40℃ for 20-60 min to carry out a click chemical reaction and magnetic separation. c. Take 100–800 μL of the supernatant from step (3)b and add 10–400 μL of pH 2 hydrochloric acid solution. Shake at room temperature for 2–20 min to acidify Gd2O3 NRs and release Gd. 3+ A homogeneous test solution for low-field nuclear magnetic resonance was obtained.

2. The method for preparing a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claim 1, characterized in that... The synthesis method of amino-functionalized Fe3O4-NH2 described in step (1) is as follows: 0.49 g FeCl3·6H2O, 0.18-0.54 g sodium citrate and 0.6-1.8 g urea are added to 15 mL ethylene glycol and stirred vigorously at room temperature for 1 h. The mixture is then transferred to a 25 mL polytetrafluoroethylene-lined stainless steel reactor, heated to 160-200 °C and maintained for 12 h. The product is then collected by magnetic adsorption and washed with ethanol to obtain Fe3O4 nanoparticles Fe3O4NPs. Then, the obtained Fe3O4 NPs and 300 μL LAPTES are dispersed in 10 mL anhydrous ethanol, sonicated for 0.5 h, and stirred at 60 °C for 2 h. Finally, the mixture is washed with ethanol and collected by magnetic adsorption, and dried at 60 °C to obtain amino-functionalized Fe3O4NPs, i.e., Fe3O4-NH2.

3. The method for preparing a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claim 1, characterized in that... The synthesis method of gadolinium oxide nanorods Gd2O3 NRs in step (2) is as follows: 10-50 mL of gadolinium nitrate Gd(NO3)3 aqueous solution with a concentration of 0.01-0.20 mol / L and 0.5-5 mL of 25% ammonia water NH3·H2O are added to a 50 mL polytetrafluoroethylene-lined stainless steel reactor. After heating at 100-140℃ for 8-16 h, the product is washed and dried at 45-90℃ to obtain gadolinium hydroxide Gd(OH)3 powder. Then, the Gd(OH)3 powder is placed in a tube furnace and calcined at 600-800℃ for 2-6 h to obtain gadolinium oxide nanorods Gd2O3 NRs.

4. The method for preparing a low-field homogeneous nuclear magnetic resonance sensor for detecting Escherichia coli based on click reaction according to claim 1, characterized in that... The preparation method of Fe3O4@Apt in step (3) is as follows: Take 3-8 mg of amino-functionalized iron(III) oxide Fe3O4-NH2 and disperse it in 2-10 mL of 2.5 wt% glutaraldehyde aqueous solution. Stir at room temperature for 1-6 h and wash with water. Then redisperse it in 2-9 mL of phosphate buffer PBS. Add 20-200 μL of 2-20 μmol / L aptamer Apt solution and incubate with slow shaking at 30-40 °C for 2-10 h. Then, magnetically separate and wash to remove unbound Apt. Add 100-600 μL of 0.5-3 wt% bovine serum albumin (BSA) solution and incubate at room temperature for 0.5-3 h to block non-specific binding sites. Finally, after magnetic separation and washing, redisperse it in 2-8 mL of LTE-Mg 2+ Fe3O4@Apt dispersion was obtained in buffer solution.

5. The application of a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on a click reaction, obtained by any one of the preparation methods of claims 1-4, characterized in that... Includes the following steps: Using the prepared low-field nuclear magnetic resonance homogeneous test solution, the T1 intensity corresponding to different concentrations of Escherichia coli was measured by a low-field nuclear magnetic resonance instrument, and the concentration of Escherichia coli in the test solution was obtained based on the change value of the T1 signal.

6. The application of the click reaction-based low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli according to claim 5, characterized in that... The low-field nuclear magnetic resonance test parameters are as follows: main frequency 19.00MHz, sampling width 100kHz, radio frequency delay 0.08ms, number of sampling points 2048, waiting time 20000ms, number of inversions 20, number of accumulations 2, digital gain 3, and analog gain 15.0dB.

7. The application of the click reaction-based low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli according to claim 5 or 6, characterized in that... The change value ΔT1 of the T1 signal is calculated according to the following formula: ΔT1=T1(positive)-T1(negative), where T1(negative) is the average T1 when E. coli is not present in the sample, and T1(positive) is the average T1 when E. coli is present in the sample.

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