Preparation method and application of low-field nuclear magnetic resonance homogeneous sensor for detecting escherichia coli based on click reaction

Abstract

The invention discloses a preparation method and application of a low-field nuclear magnetic resonance homogeneous sensor for detecting escherichia coli based on click reaction. The preparation method is characterized by comprising the following steps: 1) preparing PA-GO-coated Fe3O4; (2) preparing a signal unit Gd2O3-Az; (3) preparing a low-field nuclear magnetic resonance homogeneous test solution; the method comprises the following steps: measuring the intensity of a low-field nuclear magnetic resonance signal T1 corresponding to different concentrations under the condition of different concentrations of E.coli by adopting a low-field nuclear magnetic resonance instrument, establishing a quantitative relationship between the E.coli concentration and a T1 signal change value (delta T1), and measuring the concentration of E.coli in an unknown sample according to the quantitative relationship; the method has the advantages of high sensitivity, high precision and high convenience.

Description

Technical Field

[0001] The present invention relates to a low-field nuclear magnetic resonance sensor, and in particular to a preparation method and application of a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on a click reaction. Background Art

[0002] Escherichia coli (E. coli) is a Gram-negative bacillus that is widely present in the intestines of humans and animals and in the surrounding environment. As a foodborne pathogen, E. coli can cause a series of local tissue and organ infections such as intestinal inflammation and meningitis. Traditional E. coli detection methods include multi-tube fermentation, membrane filtration, plate counting, etc. Most of them have accurate and reliable results, low experimental requirements, and low cost, but they are time-consuming and cumbersome to operate. Emerging detection methods include fluorescence, surface-enhanced Raman scattering, colorimetry, and electrochemistry, which can achieve rapid and sensitive detection, but optical and electrical signals are easily interfered by complex backgrounds and have high requirements for sample pretreatment.

[0003] Due to the relatively small background interference of magnetic signals, low-field nuclear magnetic resonance has great advantages in the direct detection of complex samples. At present, low-field nuclear magnetic resonance has been reported to be applied to the detection of foodborne pathogens such as Salmonella, Vibrio parahaemolyticus, Listeria monocytogenes, etc., but there has been no report on the detection of E. coli. The quantitative strategy of the above research is usually: antibodies, aptamers and bacteriophages are used as biorecognition elements to identify targets, act as a bridge between signal units and targets, and quantify according to the signal intensity output by the signal unit. However, under this technical framework, the number of signal units depends entirely on the number of targets, and it is difficult to achieve signal amplification, which decisively limits the detection sensitivity. In addition, the activity of the biorecognition element is unstable and is easily affected by environmental factors, which affects the precision of the detection. Finally, the above-mentioned biorecognition elements cannot distinguish between live and dead bacteria, and it is difficult to objectively reflect the actual contamination of bacteria.

[0004] Based on click chemistry, the present invention constructs a low-field nuclear magnetic resonance sensing platform for E. coli detection. The sensing platform uses the Cu produced by the copper metabolism of the living target E. coli to + The catalyst catalyzes the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, triggering the signal unit to leave the sensing system, resulting in the longitudinal relaxation time (T 1 ) value increases to achieve quantitative detection. + The number is much larger than that of E. coli, and each Cu + The catalyst can catalyze a large number of signal units to undergo CuAAC reaction and leave the sensing system. Each signal unit is loaded with a large amount of Gd 3+, which achieves three-level amplification of the signal and significantly improves the detection sensitivity; the signal generation process is mediated by pure chemical means, and does not involve precision problems that may be caused by changes in the activity of biological recognition elements; only living bacteria can metabolize copper, eliminating the impact of dead bacteria on detection. So far, there has been no report on low-field nuclear magnetic resonance homogeneous sensors based on click reactions for detecting Escherichia coli. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of a low-field nuclear magnetic resonance homogeneous sensor based on click reaction for detecting Escherichia coli with high sensitivity, high precision and convenient operation.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a preparation method and application of a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction. This method is not for the purpose of diagnosis or treatment, and comprises the following steps:

[0007] (1)PA-GO@Fe 3 O 4 Preparation

[0008] a. Add 10-30 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 5-15 mg N-hydroxysuccinimide NHS to 2-16 mL graphene oxide GO dispersion (1-10 mg), and ultrasonicate for 10-100 min to activate the carboxyl groups of GO. Then, add 1-10 mg amino-functionalized ferrosoferric oxide Fe 3 O 4 -NH 2 , and stirred at 50-90 °C for 2-8 h, and then redispersed in 5-16 mL of water after washing to obtain GO@Fe 3 O 4 Dispersion.

[0009] b.To GO@Fe 3 O 4 Add 2-20 mg potassium hydroxide (KOH) and 0.5-5 mL 0.1-3 mg / mL propargylamine (PA) aqueous solution to the dispersion and stir vigorously at 50-90 °C for 10-48 h. Wash with water three times to obtain PA-GO@Fe 3 O 4 Re-disperse in 2-20 mL of water and store at 4°C for later use.

[0010] (2) Signal unit Gd 2 O 3 Preparation of -Az

[0011] a. Add 50-600 μL 3-aminopropyltriethoxysilane APTES to 5-50 mL 0.1-1.2 mg / mL Gd 2 O 3 NRs anhydrous ethanol dispersion, ultrasonic treatment for 10-60 min, and reaction at 50-80 ° C for 1-4 h, centrifugal washing, to obtain amino-functionalized Gd 2 O 3 NRs, namely Gd 2 O 3 -NH 2 .

[0012] b. Gd 2 O 3 -NH 2 Disperse in 10-50 mL 2.5 wt% glutaraldehyde aqueous solution and react at room temperature for 1-3 hours. After centrifugal washing, disperse the product in 10-50 mL 0.01-0.15 mg / mL azide-tetraethylene glycol-amino Az aqueous solution and stir at room temperature for 1-3 hours. Finally, centrifuge and wash and redisperse in 10-50 mL water to obtain the signal unit Gd 2 O 3 -Az, stored at 4℃ until use.

[0013] (3) Preparation of low-field NMR homogeneous test solution

[0014] a. Add 10-400 μL Fe to 1 mL of sample 3 O 4 @Apt and incubate at 30-40℃ for 10-30min to capture E. coli. Then, magnetically separate, wash and disperse in 0.5-1.5mL PBS solution. Then add 10-100μL universal nuclease solution, incubate at 30-40℃ for 10-40min to release the captured E. coli, and magnetically separate.

[0015] b. Take 10-400 μL of the supernatant from step (3) a and add it to 10-400 μL of 2-20 μmol / L copper chloride (CuCl) 2 10~400μL Gd 2 O 3 -Az, 10~400μL PA-GO@Fe 3 O 4 The sample was placed in a sample bottle and shaken at 30-40°C for 20-60 min to perform click chemistry reaction and magnetic separation.

[0016] c. Take 100 to 800 μL of the supernatant from step (3) b and add 10 to 400 μL of hydrochloric acid (pH = 2) solution and shake at room temperature for 2 to 20 minutes. Gd 2O 3 NRs acid hydrolysis and release of Gd 3+ , and obtain a low-field NMR homogeneous test liquid.

[0017] Further, the amino-functionalized ferroferric oxide Fe 3 O 4 -NH 2 The synthesis method is as follows: 0.49gFeCl 3 6H 2 O, 0.18-0.54 g sodium citrate and 0.6-1.8 g urea were added to 15 mL ethylene glycol and stirred vigorously at room temperature for 1 h. The mixed solution was then transferred to a 25 mL polytetrafluoroethylene-lined stainless steel reactor and heated to 160-200 °C and maintained for 12 h. The product was then collected by magnetic adsorption and washed with ethanol to obtain Fe 3 O 4 Nanoparticle Fe 3 O 4 NPs. Then, the prepared Fe 3 O 4 NPs and 300 μL APTES were dispersed in 10 mL of anhydrous ethanol, ultrasonicated for 0.5 h, and stirred at 60 °C for 2 h. Finally, they were washed with ethanol and collected by magnetic adsorption, and then dried at 60 °C to obtain amino-functionalized Fe 3 O 4 NPs, namely Fe 3 O 4 -NH 2 .

[0018] Further, the gadolinium oxide nanorods Gd 2 O 3 The synthesis method of NRs is as follows: 10 to 50 mL of 0.01 to 0.20 mol / L gadolinium nitrate (NO 3 ) 3 Aqueous solution and 0.5-5 mL 25% ammonia NH 3 ·H 2 O was added into a 50 mL polytetrafluoroethylene-lined stainless steel reactor, heated at 100-140°C for 8-16 h, and the product was washed and dried at 45-90°C to obtain gadolinium hydroxide Gd(OH) 3 Then, Gd(OH) 3 The powder is placed in a tube furnace and heated to 600-800°C and calcined for 2-6 hours to obtain gadolinium oxide nanorods Gd 2 O 3 NRs.

[0019] Further, the Fe in step (3) 3 O4 The preparation method of @Apt is as follows: Take 3-8 mg of amino-functionalized ferroferric oxide Fe 3 O 4 -NH 2 Disperse in 2-10 mL 2.5 wt% glutaraldehyde aqueous solution, stir at room temperature for 1-6 h and wash with water, then redisperse in 2-9 mL phosphate buffer PBS, add 20-200 μL 2-20 μmol / L aptamer Apt solution, and incubate at 30-40 ° C with slow shaking for 2-10 h. Then, magnetic separation and washing are performed to remove unbound Apt, and 100-600 μL 0.5-3 wt% bovine serum albumin BSA solution is added, and incubated at room temperature for 0.5-3 h to block nonspecific binding sites. Finally, after magnetic separation and washing, redisperse in 2-8 mL TE-Mg 2+ In buffer solution, Fe 3 O 4 @Apt dispersion.

[0020] 2. A method for detecting Escherichia coli using the above-mentioned low-field nuclear magnetic resonance homogeneous sensor, which is not intended for diagnosis or treatment, and comprises the following steps: using the prepared low-field nuclear magnetic resonance homogeneous test solution, using a low-field nuclear magnetic resonance instrument to measure the corresponding T values ​​under different concentrations of Escherichia coli. 1 Strength, according to T 1 Signal change value (ΔT 1 ) to obtain the concentration of Escherichia coli in the test solution.

[0021] Furthermore, the test parameters of the low-field nuclear magnetic resonance test are: main frequency 19.00 MHz, sampling width 100 kHz, RF delay 0.08 ms, sampling points 2048, waiting time 20000 ms, inversion times 20, accumulation times 2, digital gain 3, and analog gain 15.0 dB.

[0022] Furthermore, the T 1 The signal change value is calculated according to the following formula: ΔT 1 =T 1 (positive)-T 1 (negative), T 1 (negative) is the average T when there is no E. coli in the sample 1 , T 1 (positive) is the average T when E. coli is present in the sample 1 .

[0023] Invention principle: The low-field nuclear magnetic resonance sensor based on click chemistry reaction and its detection principle are shown in Scheme 1. There is a specific concentration of Cu in the sample bottle. 2+, a large number of signal units Gd 2 O 3 -Az and PA-GO@Fe 3 O 4 When live E. coli is present, it can convert Cu 2+ Metabolism to Cu + , Cu + As a catalyst to trigger the CuAAC reaction, the signal unit Gd 2 O 3 -Az and PA-GO@Fe 3 O 4 The azide-alkyne cycloaddition reaction occurs to form a complex Gd 2 O 3 -GO@Fe 3 O 4 The remaining signal unit Gd in the bottle can be quickly separated by magnetic force. 2 O 3 -Az is then acidified by the addition of hydrochloric acid to generate a homogeneously distributed Gd 3+ , resulting in a stable T 1 The more live E. coli cells there are, the more Cu they produce. + The larger the number, the more complexes will be formed by the click chemistry reaction, and the remaining Gd 2 O 3 -The less Az is, the more Gd is generated after hydrochloric acid hydrolysis. 3+ The lower the solution concentration, the more ΔT 1 The larger the value, the quantitative detection of live E. coli bacteria can be achieved.

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

[0025] (1) High sensitivity. First, the target itself can assist in signal amplification. E. coli bacteria continuously convert Cu 2+ Reduced to Cu + , triggering a large number of CuAAC reactions, and the reaction conditions are mild and the yield is high. Secondly, the number of signal tags is sufficient. Each of the remaining Gd 2 O 3 -Az can release a large amount of signal label Gd through hydrochloric acid hydrolysis 3+ In addition, the large specific surface area and good structural stability of GO provide a place for alkyne modification and subsequent click reactions.

[0026] (2) High precision. The sensor has high reproducibility, which is due to the pure chemically modified Gd 2 O 3 -Az and PA-GO@Fe3 O 4 Stability of the material.

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

[0028] In summary, the present invention is based on the preparation method and application of a low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction, and uses the click chemistry reaction catalyzed by bacterial metabolism to design a low-field nuclear magnetic resonance homogeneous sensor for rapid detection of foodborne pathogens. By targeted triggering of click chemistry, the addition of functionalized two-dimensional materials, and acid dissolution of nanomaterials, a three-level amplification of the signal is achieved, which can significantly enhance the sensitivity of the detection. Moreover, during the detection process, the generation of the signal is completely based on pure chemical means, which provides a guarantee for the high precision of the detection. In addition, the click reaction catalyzed by bacterial metabolism can distinguish between the dead and the alive bacteria, eliminating the influence of dead bacteria on the detection. Therefore, the method has high sensitivity, high precision, and only requires simple pretreatment to complete the detection, providing a feasible strategy for rapid on-site detection of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the principle of detecting Escherichia coli using the low-field nuclear magnetic resonance homogeneous sensor of the present invention;

[0030] Figure 2 is the T corresponding to different concentrations of E. coli 1 Signal response diagram;

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

[0032] Figure 4 The specificity of the low-field NMR homogeneous sensor;

[0033] Figure 5 The stability of low-field NMR homogeneous sensors. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the accompanying drawings. Specific embodiment 1

[0036] Example 1

[0037] Based on Cu + A method for detecting foodborne pathogens by low-field nuclear magnetic resonance homogeneous immunoassay using a click chemistry gel system. This method is not intended for diagnosis or treatment. Figure 1 As shown, the following steps are included:

[0038] (1)PA-GO@Fe 3 O 4 Preparation

[0039] a. Add 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 10 mg of N-hydroxysuccinimide (NHS) to 6 mL of graphene oxide (GO) dispersion (4 mg), and ultrasonicate for 30 min to activate the carboxyl groups of GO. Then, add 4 mg of Fe 3 O 4 -NH 2 , and stirred at 80 °C for 5 h, and then redispersed in 8 mL of water after washing to obtain GO@Fe 3 O 4 Dispersion.

[0040] b.To GO@Fe 3 O 4 10 mg potassium hydroxide KOH and 2 mL 1 mg / mL propargylamine PA aqueous solution were added to the dispersion and stirred vigorously at 70 °C for 24 h. After washing with water three times, the obtained PA-GO@Fe 3 O 4 Re-disperse in 8 mL of water and store at 4°C for later use.

[0041] (2) Signal unit Gd 2 O 3 Preparation of -Az

[0042] a. Add 300 μL 3-aminopropyltriethoxysilane APTES to 20 mL 0.5 mg / mL Gd 2 O 3 NRs anhydrous ethanol dispersion was treated with ultrasound for 30 min and reacted at 60 °C for 2 h. After centrifugation and washing, amino-functionalized Gd 2 O 3 NRs, namely Gd 2 O 3 -NH 2 .

[0043] b. Gd 2 O 3 -NH 2 Disperse 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-amino Az aqueous solution and stir at room temperature for 2 h. Finally, centrifuge and wash and redisperse in 20 mL of water to obtain the signal unit Gd 2 O 3 -Az, stored at 4℃ until use.

[0044] (3) Preparation of low-field NMR homogeneous test solution

[0045] a. Add 100 μL Fe 3 O 4 @Apt and incubated at 37°C for 20 min to capture E. coli. Subsequently, magnetic separation, washing and dispersion in 0.95 mL PBS solution, 50 μL universal nuclease solution was added, incubated at 37°C for 20 min to release the captured E. coli, and magnetic separation was performed.

[0046] b. Take 100 μL of the supernatant from step (3) a and add 100 μL of 10 μmol / L copper chloride (CuCl) 2 , 200μLGd 2 O 3 -Az, 100μL PA-GO@Fe 3 O 4 , shaken at 37°C for 30 min to perform click chemistry 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, Gd 2 O 3 NRs acid hydrolysis and release of Gd 3+ , and obtain a low-field NMR homogeneous test liquid.

[0048] Example 2

[0049] The same as the above-mentioned embodiment 1, the difference is:

[0050] Step (1) PA-GO@Fe 3 O 4 In preparation:

[0051] a. Add 40 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mg of N-hydroxysuccinimide (NHS) to 12 mL of graphene oxide (GO) dispersion (8 mg), and ultrasonicate for 30 min to activate the carboxyl groups of GO. Then, add 8 mg of Fe 3 O 4 -NH 2 , and stirred at 80 °C for 5 h, and then redispersed in 16 mL of water after washing to obtain GO@Fe 3 O 4 Dispersion.

[0052] b.To GO@Fe 3 O 4 20 mg of potassium hydroxide KOH and 4 mL of 2 mg / mL propargylamine PA aqueous solution were added to the dispersion and stirred vigorously at 70 °C for 24 h. After washing with water three times, the obtained PA-GO@Fe3 O 4 Re-disperse in 16 mL of water and store at 4 °C for later use.

[0053] Step (2) Signal unit Gd 2 O 3 -Az preparation:

[0054] a. Add 600 μL 3-aminopropyltriethoxysilane APTES to 40 mL 1 mg / mL Gd 2 O 3 NRs anhydrous ethanol dispersion was treated with ultrasound for 30 min and reacted at 60 °C for 2 h. After centrifugation and washing, amino-functionalized Gd 2 O 3 NRs, namely Gd 2 O 3 -NH 2 .

[0055] b. Gd 2 O 3 -NH 2 Disperse 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-amino Az aqueous solution and stir at room temperature for 2 h. Finally, centrifuge and wash and redisperse in 40 mL of water to obtain the signal unit Gd 2 O 3 -Az, stored at 4℃ until use.

[0056] In step (3) preparation of low field nuclear magnetic resonance homogeneous test solution:

[0057] a. Add 200 μL Fe 3 O 4 @Apt and incubated at 37°C for 20 min to capture E. coli. Subsequently, magnetic separation, washing and dispersion in 0.95 mL PBS solution, 100 μL universal nuclease solution was added, incubated at 37°C for 20 min to release the captured E. coli, and magnetic separation was performed.

[0058] b. Take 200 μL of the supernatant from step (3) a and add 200 μL of 20 μmol / L copper chloride (CuCl) 2 、400μLGd 2 O 3 -Az, 200μL PA-GO@Fe 3 O 4 , shaken at 37°C for 30 min to perform click chemistry 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, Gd 2 O 3 NRs acid hydrolysis and release of Gd 3+ , and obtain a low-field NMR homogeneous test liquid.

[0060] Example 3

[0061] The same as the above-mentioned embodiment 1, the difference is:

[0062] Step (1) PA-GO@Fe 3 O 4 In preparation:

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

[0064] b.To GO@Fe 3 O 4 5 mg of potassium hydroxide (KOH) and 1 mL of 0.5 mg / mL propargylamine (PA) aqueous solution were added to the dispersion and stirred vigorously at 70 °C for 24 h. After washing with water three times, the obtained PA-GO@Fe 3 O 4 Re-disperse in 4 mL of water and store at 4°C for later use.

[0065] Step (2) Signal unit Gd 2 O 3 -Az preparation:

[0066] a. Add 150 μL 3-aminopropyltriethoxysilane APTES to 10 mL 0.25 mg / mL Gd 2 O 3 NRs anhydrous ethanol dispersion was treated with ultrasound for 30 min and reacted at 60 °C for 2 h. After centrifugation and washing, amino-functionalized Gd 2 O 3 NRs, namely Gd 2 O 3 -NH 2 .

[0067] b. Gd2 O 3 -NH 2 Disperse 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-amino Az aqueous solution and stir at room temperature for 2 h. Finally, centrifuge and wash and redisperse in 10 mL of water to obtain the signal unit Gd 2 O 3 -Az, stored at 4℃ until use.

[0068] In step (3) preparation of low field nuclear magnetic resonance homogeneous test solution:

[0069] a. Add 50 μL Fe 3 O 4 @Apt and incubated at 37°C for 20 min to capture E. coli. Subsequently, magnetic separation, washing and dispersion in 0.55 mL PBS solution, 25 μL universal nuclease solution was added, incubated at 37°C for 20 min to release the captured E. coli, and magnetic separation was performed.

[0070] b. Take 50 μL of the supernatant from step (3) a and add 50 μL of 5 μmol / L copper chloride (CuCl) 2 、100μLGd 2 O 3 -Az, 50μL PA-GO@Fe 3 O 4 , shaken at 37°C for 30 min to perform click chemistry reaction, and magnetic separation.

[0071] c. Take 200 μL of the supernatant from step (3) b and add 50 μL of hydrochloric acid (pH = 2) solution, shake at room temperature for 5 min, Gd 2 O 3 NRs acid hydrolysis and release of Gd 3+ , and obtain a low-field NMR homogeneous test liquid. Specific embodiment 2

[0073] The low-field nuclear magnetic resonance homogeneous sensor prepared in the above specific embodiment 1 was placed in a low-field nuclear magnetic resonance instrument for detection, and the corresponding low-field nuclear magnetic resonance signal T under different concentrations of E. coli was measured. 1 The strength of T 1 The signal value is used to obtain the concentration of E. coli in the test solution. The detection principle is as follows Figure 1 shown.

[0074] like Figure 2 As shown, T 1 The signal intensity increased with the increase of E. coli concentration.

[0075] like Figure 3 As shown, in 10~1.0×10 7 In the CFU / mL range, T 1 Change in signal intensity ΔT 1 (y, ms) and the logarithm of E. coli concentration (x, CFU / mL) show a good linear relationship. The linear regression equation is y = 151.35*lg x + 19.35, and the square correlation coefficient R 2 =0.993. The noise was about 35 ms, and based on the signal-to-noise ratio S / N=3, the detection limit (LOD) was calculated to be 3.5 CFU / mL. Specific embodiment three

[0077] 1.0×10 4 CFU / mL of E. coli, SA, VP standard solutions, blank samples, and mixed samples (containing 1.0×10 4 CFU / mL of E. coli and SA, VP) were used for the experiment. The results are shown in Figure 4 As shown, 1.0×10 4 ΔT of SA, VP and blank samples with CFU / mL 1 close to that of the mixed sample with 1.0×10 4 ΔT generated by CFU / mL E.coli standard solution 1 The results showed that the sensor has good selectivity for E. coli detection. Specific embodiment 4

[0079] The prepared nanomaterials were stored at 4°C and measured for 10 consecutive days to investigate the long-term storage stability of the sensor. Figure 5 As shown, after 10 days, T 1 The signal response was still 96.9% of the original value, which indicated that the developed sensor platform had satisfactory stability. Specific embodiment five

[0081] The seawater (S) sample and milk sample (M) were tested by the standard addition method to evaluate the accuracy and precision of the sensor, as shown in Table 1.

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

[0083]

[0084] As shown in Table 1, the recovery of the spiked seawater samples was 97.2% to 104.4%, with an RSD of 1.3% to 2.4%; the recovery of the spiked milk samples was 98.9% to 104.7%, with an RSD of 1.5% to 2.8%. The results show that the sensor has good accuracy and precision and can be used to reliably detect actual samples.

[0085] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Preparation method and application of low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction, characterized in that The following steps are involved: (1) Preparation of PA-GO@Fe3O4 a. Add 10-30 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 5-15 mg N-hydroxysuccinimide NHS to 2-16 mL graphene oxide GO dispersion (1-10 mg), and ultrasonicate for 10-100 min to activate the carboxyl groups of GO. Then, add 1-10 mg amino-functionalized ferrosoferric oxide Fe3O4-NH2, stir at 50-90 ° C for 2-8 h, and redisperse in 5-16 mL water after washing to obtain GO@Fe3O4 dispersion. b. Add 2-20 mg potassium hydroxide KOH and 0.5-5 mL 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 with water three times, the obtained PA-GO@Fe3O4 was redispersed in 2-20 mL water and stored at 4°C for later use. (2) Preparation of signal unit Gd2O3-Az a. Add 50-600 μL 3-aminopropyltriethoxysilane APTES to 5-50 mL 0.1-1.2 mg / mL Gd2O3NRs anhydrous ethanol dispersion, ultrasonically treat for 10-60 min, and react at 50-80 °C for 1-4 h. After centrifugation and washing, amino-functionalized Gd2O3 NRs, namely Gd2O3-NH2, are obtained. b. Disperse Gd2O3-NH2 in 10-50mL 2.5wt% glutaraldehyde aqueous solution and react at room temperature for 1-3h. After centrifugal washing, disperse the product in 10-50mL 0.01-0.15mg / mL azide-tetraethylene glycol-amino Az aqueous solution and stir at room temperature for 1-3h. Finally, centrifugal washing and redisperse in 10-50mL water to obtain the signal unit Gd2O3-Az, which is stored at 4℃ for future use. (3) Preparation of low-field NMR homogeneous test solution a. Add 10-400 μL 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 PBS solution. Then add 10-100 μL universal nuclease solution, incubate at 30-40°C for 10-40 min to release captured E. coli and magnetically separate. b. Take 10-400 μL of the supernatant from step (3) a and add it to a sample bottle containing 10-400 μL 2-20 μmol / L copper chloride (CuCl2), 10-400 μL Gd2O3-Az, and 10-400 μL PA-GO@Fe3O4, and shake at 30-40°C for 20-60 min to perform click chemistry reaction and magnetic separation. 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, and the Gd2O3 NRs will acidify and release Gd 3+ , and obtain a low-field NMR homogeneous test liquid.

2. The preparation method and application of the low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The synthesis method of amino-functionalized ferroferric oxide Fe3O4-NH2 described in step (1) is as follows: 0.49g FeCl3·6H2O, 0.18-0.54g sodium citrate and 0.6-1.8g urea are added to 15mL ethylene glycol, and vigorously stirred at room temperature for 1h. Then, the mixed solution is transferred to a 25mL polytetrafluoroethylene-lined stainless steel reactor, heated to 160-200°C and maintained for 12h, and then the product is collected by magnetic adsorption and washed with ethanol to obtain Fe3O4 nanoparticles Fe3O4 NPs. Then, the prepared Fe3O4 NPs and 300μL APTES are dispersed in 10mL anhydrous ethanol, ultrasonically treated for 0.5h, and stirred at 60°C for 2h. Finally, the amino-functionalized Fe3O4 NPs, namely Fe3O4-NH2, are obtained by washing with ethanol and collecting by magnetic adsorption. After drying at 60°C, the Fe3O4 NPs are obtained.

3. The preparation method and application of the low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The synthesis method of the gadolinium oxide nanorods Gd2O3 NRs described in step (2) is as follows: 10 to 50 mL of a 0.01 to 0.20 mol / L gadolinium nitrate Gd(NO3)3 aqueous solution and 0.5 to 5 mL of 25% ammonia NH3·H2O are added to a 50 mL polytetrafluoroethylene-lined stainless steel reactor, heated at 100 to 140° C. for 8 to 16 hours, washed the product and dried at 45 to 90° C. to obtain gadolinium hydroxide Gd(OH)3 powder. Thereafter, the Gd(OH)3 powder is placed in a tube furnace and heated to 600 to 800° C. and calcined for 2 to 6 hours to obtain gadolinium oxide nanorods Gd2O3NRs.

4. The preparation method and application of the low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The preparation method of Fe3O4@Apt described in step (3) is as follows: 3-8 mg of amino-functionalized ferrosoferric oxide Fe3O4-NH2 is dispersed in 2-10 mL of 2.5 wt% glutaraldehyde aqueous solution, stirred at room temperature for 1-6 h and washed with water, then redispersed in 2-9 mL of phosphate buffer PBS, and then 20-200 μL of 2-20 μmol / L aptamer Apt solution is added, and slowly shaken and incubated at 30-40°C for 2-10 h. Then, magnetic separation and washing are performed to remove unbound Apt, and then 100-600 μL of 0.5-3 wt% bovine serum albumin BSA solution is added, and incubated at room temperature for 0.5-3 h to block nonspecific binding sites. Finally, after magnetic separation and washing, it is redispersed in 2-8 mL TE-Mg 2+ In the buffer solution, Fe3O4@Apt dispersion was obtained.

5. A method for preparing a low-field nuclear magnetic resonance homogeneous sensor based on click reaction for detecting Escherichia coli obtained by the preparation method of any one of claims 1 to 4 and its application, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The following steps are involved: The prepared low-field nuclear magnetic resonance homogeneous test solution was used to measure the T1 intensity corresponding to different concentrations of Escherichia coli using a low-field nuclear magnetic resonance instrument, and the concentration of Escherichia coli in the test solution was obtained according to the change value of the T1 signal.

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

7. The preparation method and application of the low-field nuclear magnetic resonance homogeneous sensor for detecting Escherichia coli based on click reaction according to claims 5-6, characterized in that The T1 signal change value (ΔT1) is calculated according to the following formula: ΔT1=T1(positive)-T1(negative), T1(negative) is the average T1 when there is no E. coli in the sample, and T1(positive) is the average T1 when there is E. coli in the sample.

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