D-glucuronic acid modified Eu2O3 nanoparticles, preparation method thereof and application of D-glucuronic acid modified Eu2O3 nanoparticles in cTnT detection

Abstract

The invention discloses D-glucuronic acid modified Eu2O3 nanoparticles, a preparation method of the D-glucuronic acid modified Eu2O3 nanoparticles, and an application of the D-glucuronic acid modified Eu2O3 nanoparticles in cTnT detection. According to the preparation method disclosed by the invention, experimental conditions are optimized, so that the optimal Eu2O3 nanoparticles modified by D-glucuronic acid are obtained. According to the invention, a method for detecting cTnT by using DGA-Eu2O3 nanoparticles through time-resolved fluorescence immunoassay and a method for detecting cTnT through immunochromatography are constructed. The detection method disclosed by the invention has relatively high sensitivity, specificity, precision, accuracy and recovery rate.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and specifically relates to a D-glucuronic acid-modified Eu2O3 nanoparticle, a preparation method thereof, and an application thereof in cTnT detection. Background Art

[0002] Acute myocardial infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries. It is the most common clinical form of coronary syndrome. The number of patients has been increasing year by year and it is one of the leading causes of death in the world today. Detecting the concentration and changes of myocardial markers in peripheral blood during heart damage is an effective means of clinically treating AMI and improving patient survival rates. Among them, cTnT is recognized as the most ideal myocardial marker because it appears early in peripheral blood after myocardial damage, has a high peak value, exists for a long time, and has a high specificity. It has become the gold standard for in vitro diagnosis of AMI.

[0003] Dissociation enhanced time-resolved immunoassay (DELFIA) can be performed using inorganic lanthanide nanoparticles (Ln 3+ -NPs) are used as markers to quantitatively label antigens or antibodies and perform conventional immune responses to form immune complexes. 3+ The number of markers can reach thousands to tens of thousands of Eu 3+ After adding the enhancement solution, a large amount of Ln 3+ From Ln 3+ -NPs and react with the ligands in the enhancement solution to generate a large amount of strongly luminescent Ln 3+ complexes, thereby achieving the purpose of enhancing the detection signal and having high sensitivity. 3+ -NPs must be easily soluble in the acidic enhancement solution, so that the solution of Ln 3+ -NPs solubility has become a hot topic in research, among which the solubility of Ln 3+ The surface modification of NPs has attracted much attention.

[0004] LFIA combines the high specificity of immunoassays with the rapid separation capability of chromatography, and is currently one of the most widely used on-site rapid detection methods. However, LFIA still has some limitations, such as relatively low sensitivity and accuracy, which requires the development of new labeling materials; it is easily affected by the subjective factors of the operator when observing the test results, so the development of detection methods based on portable devices such as smartphones is conducive to obtaining objective and accurate results; in addition, the stability and long-term storage of LIFA may be affected by environmental factors, which requires further improvement of the design and materials of the test strips to improve long-term stability. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a D-glucuronic acid modified Eu2O3 nanoparticle and a preparation method thereof and application in cTnT detection.

[0006] The present invention first provides a method for preparing D-glucuronic acid-modified Eu2O3 nanoparticles, comprising the following steps:

[0007] Step 1: adding europium salt to diol and heating to dissolve, to obtain reaction solution a;

[0008] Step 2: heat and dissolve sodium hydroxide particles in diol, then slowly add the mixture into reaction solution a, heat to 60-100° C. and react for 1-3 hours to obtain reaction solution b;

[0009] Step 3: Slowly drip the H2O2 aqueous solution into the reaction solution b, react at 60-100°C for 1-3h to obtain the reaction solution c;

[0010] Step 4: Add D-glucuronic acid to the reaction solution c, react at 60-100°C for 5-7h, then wash with distilled water and centrifuge to obtain D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3).

[0011] In step 1, the europium salt is europium nitrate; the concentration is 0.2-0.4 mol / L, preferably 0.3 mol / L;

[0012] In step 1, the diol is any one of ethylene glycol (EG), diethylene glycol (DEG) and triethylene glycol (TEG); preferably triethylene glycol (TEG).

[0013] The concentration of sodium hydroxide in step 2 is 0.5-2 mol / L, preferably 1 mol / L;

[0014] The reaction time in step 2 is 1-3h, preferably 2h;

[0015] The molar ratio of europium salt, sodium hydroxide and D-glucuronic acid in step 1, step 2 and step 4 is 1:(2-4):1, preferably 1:3:1;

[0016] In step 3, the concentration of the H2O2 aqueous solution is 35%, and the volume ratio of the added amount to the reaction solution b is 1:(2-3);

[0017] The reaction time in step 3 is 1-3h, preferably 2h;

[0018] The reaction temperature in step 2, step 3 and step 4 is preferably 80°C;

[0019] In step 4, the reaction time is preferably 6 hours; the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 10-15 minutes.

[0020] The present invention also provides D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3) prepared by the above preparation method.

[0021] The present invention also provides a method for using the above-mentioned D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) to detect cTnT by time-resolved fluorescence immunoassay, comprising the following steps:

[0022] (1) The cTnT detection antibody is coupled to the carboxyl group on the surface of DGA-Eu2O3 by the EDC / NHS coupling method to obtain a labeled antibody;

[0023] (2) adding cTnT capture antibody to the polystyrene well plate, and the cTnT capture antibody is bound to the well plate through electrostatic interaction to obtain a solid-phase antibody;

[0024] (3) The sample to be tested is added to the well plate of the solid phase antibody, and then the labeled antibody is further added to the well plate. After incubation and washing, an enhancement solution is added to the well plate, and the fluorescence value of the sample is measured by a time-resolved fluorescence immunoassay analyzer. The cTnT content is quantitatively calculated using a cTnT concentration-fluorescence intensity linear standard curve.

[0025] The coupling buffer in step (1) is 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0026] In step (1), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibody is 10:1:2:1.

[0027] The coating buffer in step (2) is 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0028] The blocking buffer in step (1) and step (2) consists of 50 mmol / L Tris-HCl, 0.9 wt % NaCl, 1 wt % BSA and 0.05 wt % Proclin300.

[0029] The analytical buffer in step (3) consists of 0.05 mol / L Tris-HCl, 0.9 wt % NaCl, 0.2 wt % BSA, 0.01 wt % Tween 20, 20 μM DTPA and 0.05 wt % Proclin 300.

[0030] The enhancement solution in step (3) consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% TritonX-100.

[0031] The present invention also provides a method for using the prepared D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3) to detect cTnT by flow immunochromatography, comprising the following steps:

[0032] (a) cTnT antibody is used as the detection line (T line) and goat anti-mouse IgG antibody is used as the quality control line (C line) on the pretreated nitrocellulose membrane. After the streaking is completed, the coated membrane is dried to obtain the nitrocellulose membrane for immunochromatography;

[0033] (b) assembling the sample pad, the nitrocellulose membrane, and the absorbent pad in sequence to obtain an immunochromatographic test strip;

[0034] (c) The cTnT detection antibody is coupled to the carboxyl group on the surface of DGA-Eu2O3 by the EDC / NHS coupling method to obtain a labeled antibody;

[0035] (d) Mix the labeled antibody with the sample to be tested, draw 80 μL of the mixture and add it to the above-mentioned immunochromatographic test strip. After reacting at room temperature for 20-25 min, add 50 μL of the enhancement solution dropwise. Observe the brightness of the T and C lines under ultraviolet light to obtain qualitative results, and use Color Picker software to determine the R value in the RGB value and calculate the T / C ratio or use a dry fluorescent immunoassay to detect the signal intensity and calculate the T / C value. Quantitatively calculate the cTnT content based on the T / C value and the cTnT concentration linear standard curve.

[0036] The pretreatment in step (a) is performed by soaking the nitrocellulose membrane in a pretreatment solution, wherein the pretreatment solution is a PBS buffer solution with a pH of 7.0, containing 0.5 wt % BSA, 2 wt % sucrose, and 0.1 wt % Tween.

[0037] In step (a), both the cTnT antibody and the goat anti-mouse IgG antibody are diluted with pH=7.4 PBS buffer, and the streaking amount is 1 μl / cm; the concentration of the cTnT antibody is preferably 1.4-1.6 mg / mL, more preferably 1.4 mg / mL; the concentration of the goat anti-mouse IgG antibody is preferably 0.3-0.5 mg / mL, more preferably 0.4 mg / mL.

[0038] The specific steps of step (c) are as follows: dispersing DGA-Eu2O3 in a coupling buffer, then adding EDC and NHS to activate for 20-30 minutes, then centrifuging and washing with a coupling buffer, and ultrasonically resuspending in a coupling buffer; taking a cTnT detection antibody and ultrafiltration and adding it to the above buffer, shaking and reacting at room temperature for 2-3 hours; after the reaction is completed, adding BSA blocking buffer and continuing to incubate at room temperature for 1-1.5 hours, centrifuging the reaction solution to remove the supernatant, and then ultrasonically resuspending it in a blocking buffer.

[0039] In step (c), the coupling buffer is 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0040] In step (c), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibody is 10:1:2:1.

[0041] In step (c), the blocking buffer consists of 50 mmol / L Tris-HCl, 0.9 wt % NaCl, 1 wt % BSA and 0.05 wt % Proclin300.

[0042] In step (d), the enhancement solution consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% TritonX-100.

[0043] In step (d), the concentration of the labeled antibody is 0.1 μg / mL, the dosage is 20 μl, the concentration of the labeled antibody is, the dosage is 16 μl, and the ratio of the labeled antibody to the sample to be detected is 1:4.

[0044] The present invention synthesizes a DGA-Eu2O3 nanoparticle with uniform particle size, which can be well dispersed in water. The nanoparticle has a carboxyl group on the surface and has high stability. It can be quickly coupled with the amino group on the antigen or antibody through the EDC / NHS coupling method. Therefore, it can replace the traditional lanthanide chelate as a marker and can increase the labeling rate of a single antigen or antibody from 30 to thousands, thereby greatly improving the sensitivity of the system.

[0045] The present invention establishes a method for detecting cTnT by time-resolved fluorescence immunoassay using DGA-Eu2O3 nanoparticles. The detection system has a good linear relationship in the linear range of 50-10000ng / L, and the sensitivity reaches 0.45ng / L. In addition, the detection method is not affected by interfering analytes in serum samples, has high specificity, and has good accuracy and repeatability of the detection results. The detection results have good correlation and consistency with clinical measurement values, and have good application prospects in clinical practice.

[0046] The present invention constructs an immunochromatographic cTnT detection method through DGA-Eu2O3 nanoparticles, and the analysis results can be directly observed by naked eyes to achieve qualitative analysis, or use the smartphone RGB software Color Picker for preliminary quantitative detection, or use a dry fluorescent immunoassay analyzer for accurate detection. The sensitivity of the test strip of this method reaches 200ng / L, which is sufficient to support rapid detection of cTnT in various field environments. When a portable fluorescent immunoassay analyzer is used for detection, the sensitivity of the test strip can be further increased to 52ng / L, which makes it possible to perform more accurate cTnT analysis. In addition, the method has high specificity, precision, and accuracy, and the test strip has a high recovery rate and can be reused, reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Scanning electron microscopy images of D-glucuronic acid-modified Eu2O3 dispersed in (A) deionized water and (B) acidic enhancement solution.

[0048] Figure 2 This is the infrared spectra of Eu2O3 and D-glucuronic acid modified Eu2O3.

[0049] Figure 3 Characterization of fluorescence properties. (Figure (A) Fluorescence spectra of Eu2O3, DGA-Eu2O3 and Eu2O3-Abs (λex = 340nm); (B) Comparison of fluorescence signal intensity of DGA-Eu2O3 and Eu2O3-Abs in water, HCl and enhancement solution, the inset is a photo under natural light and 365nm UV light excitation.

[0050] Figure 4 The fluorescence intensity stability results are as follows. (A) DGA-Eu2O3; (B) Eu2O3-Abs fluorescence intensity stability.

[0051] Figure 5 This is the cTnT fluorescence intensity-concentration standard curve.

[0052] Figure 6 Specificity of time-resolved fluorescence immunoassay for cTnT detection (n=3)

[0053] Figure 7 The relationship between the carboxyl content on the surface of DGA-Eu2O3 and the reaction temperature

[0054] Figure 8 The relationship between the carboxyl content on the surface of DGA-Eu2O3 and the reaction time

[0055] Fig. 9 This is the relationship between the surface carboxyl content of DGA-Eu2O3 and the reaction solvent (significant difference analysis P < 0.0001).

[0056] Fig.10 Optimization of experimental conditions for lateral flow immunochromatography. (A) T line concentration; (B) C line concentration; (C) Analysis of significant differences in the amount of Eu2O3-Abs probe used; (D) Reaction time.

[0057] Fig.11 The linear standard curve of T / C value and cTnT concentration: (A) Color Picker detection; (B) dry fluorescent immunoassay detection; (C) gradient concentration test strip detection results (365nm UV light).

[0058] Fig.12 For lateral flow immunochromatography stability testing. DETAILED DESCRIPTION

[0059] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention. The reagents and raw materials used in the present invention can be purchased from the market unless otherwise specified.

[0060] Example 1

[0061] A method for preparing D-glucuronic acid-modified Eu2O3 nanoparticles comprises the following steps:

[0062] In a round-bottom flask, 5 mmol of Eu(NO3)3·5H2O was added to 15 mL of triethylene glycol. The reaction mixture was magnetically stirred at 50°C until the precursor was completely dissolved in triethylene glycol. (The solution became transparent and colorless.)

[0063] Then, 15 mmol of NaOH pellets were heated and dissolved in 15 mL of triethylene glycol, and slowly added to the triethylene glycol reaction solution in which Eu(NO3)3·5H2O was dissolved in the first step, and the temperature was raised to 80°C and reacted for 2 hours. Immediately after the addition of NaOH, the solution initially became white and turbid, and then became transparent again (solution color: yellow).

[0064] 11 mL of H2O2 aqueous solution (35%) was slowly dropped into the reaction mixture via a syringe, and the reaction mixture was magnetically stirred at 80°C for 2 h.

[0065] Weigh 5 mmol of D-glucuronic acid and add it to the above reaction solution, and continue the reaction for more than 6 hours.

[0066] After the reaction was completed, the solution was cooled to room temperature, washed with distilled water three times, and centrifuged at 12000 rpm for 10 min to collect the product DGA-Eu2O3.

[0067] Example 2

[0068] A method for using D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3) for time-resolved fluorescence immunoassay to detect cTnT, wherein the D-glucuronic acid-modified Eu2O3 nanoparticles are from Example 1.

[0069] The buffers used included:

[0070] The coupling buffer was 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0071] The coating buffer was 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0072] The blocking buffer consisted of 50 mmol / L Tris-HCl, 0.9 wt % NaCl, 1 wt % BSA and 0.05 wt % Proclin300.

[0073] The assay buffer consisted of 0.05 mol / L Tris-HCl, 0.9 wt % NaCl, 0.2 wt % BSA, 0.01 wt % Tween 20, 20 μM DTPA and 0.05 wt % Proclin 300.

[0074] The enhancement solution consisted of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% TritonX-100.

[0075] The specific steps are as follows:

[0076] Preparation of Eu2O3-TnT Abs (hereinafter referred to as Eu2O3-Abs): Through a simple EDC / NHS coupling method, the cTnT detection antibody can be combined with the carboxyl group on the surface of DGA-Eu2O3 to achieve the purpose of labeling the antibody. The experimental steps are as follows: Take 100μl of 10mg / mL DGA-Eu2O3 aqueous solution, wash it 3 times with coupling buffer, centrifuge it at 10000rpm for 10min, and ultrasonically disperse it in 1mL coupling buffer. Then add 5μl of 20mg / mL EDC and 10μl of 20mg / mL NHS to activate it for 20min, then centrifuge it at 12000rpm for 10min, discard the supernatant, and wash it twice with coupling buffer. Then ultrasonically resuspend it in 1mL coupling buffer. Take 0.1mg detection antibody and add the above solution after ultrafiltration at 10000rpm, and shake it at room temperature for 2h. After the reaction is completed, add 200μl BSA blocking buffer and continue to incubate at room temperature for 1h. The reaction solution was centrifuged and the supernatant was removed, and then the cells were resuspended in 1 mL of blocking buffer by sonication.

[0077] Preparation of solid-phase antibody: Dilute the ultrafiltered capture antibody to 2 μg / mL with coating buffer. Add 100 μL of diluted capture antibody to each well of a 96-well polystyrene plate. The polystyrene plate and the hydrophobic part of the antibody are tightly bound by strong electrostatic interactions. Then put the plate into a sealed bag and keep it at 4°C overnight. Then discard the coating buffer in the well and rinse it once with a plate washer. Then add 200 μL of blocking buffer (1% BSA) to each well and let it stand at room temperature for 2 hours. Finally, discard the blocking buffer and blow dry, place the plate in a sealed bag containing a desiccant, and seal it and store it at -20°C.

[0078] Add 5 μL of cTnT standard or serum sample from acute myocardial infarction patient to each well of the 96-well plate coated with capture antibody, and then add 95 μL of water-soluble Eu2O3 labeled antibody diluted by analysis buffer (1000 times) to each well. After adding the sample, place it on a shaker, incubate at 37℃ for 10 minutes, and wash it 6 times with a plate washer. Then add 100 μL of enhancement solution to each well and shake at room temperature for 1-5 minutes. Finally, measure the fluorescence value with a time-resolved fluorescence immunoassay analyzer.

[0079] Example 3

[0080] The performance of the product DGA-Eu2O3 was characterized:

[0081] Figure 1 The surface morphology of nanoparticles dispersed in water and in acidic enhancement solution was characterized using scanning electron microscopy. Figure 1 As shown in (A), the synthesized nanoparticles have uniform size and regular morphology. Figure 1 (B) is the surface morphology of the powder obtained after the nanoparticles were dissolved in the enhancement solution for 150 seconds and then centrifuged at 12000 rpm for 10 minutes. The precipitate was collected and freeze-dried. The results show that water-soluble Eu2O3 dissolves rapidly in the enhancement solution. Figure 2 The infrared absorption spectra of Eu2O3 (black line) and D-glucuronic acid-modified Eu2O3 (red line) are shown. Eu2O3 has the following peaks at 1597, 1504 and 1381 cm -1 The peaks at 2934, 1593 and 1082 cm-1 are due to the reaction solvent triethylene glycol binding to the surface of the nanoparticles, while the infrared spectrum of Eu2O3 modified with D-glucuronic acid shows that -1 The characteristic peaks at correspond to CH, C=O and CO, respectively, which proves that the Eu2O3 nanoparticles are modified by D-glucuronic acid and carry carboxyl groups.

[0082] Figure 3 The fluorescence spectra of Eu2O3, DGA-Eu2O3 and Eu2O3-Abs were detected by fluorescence spectrophotometer, such as Figure 3As shown in (A), 10 mg / mL Eu2O3, DGA-Eu2O3 and Eu2O3-Abs showed characteristic peaks around 620 nm under excitation at 340 nm, and the fluorescence intensity gradually decreased with the modification of D-glucuronic acid on the Eu2O3 surface and the coupling of antibodies. Figure 3 (B) shows the signal intensity of 100 mg of DGA-Eu2O3 particles and Eu2O3-Abs particles dispersed in water, hydrochloric acid (pH=2) and acidic enhancement solution. The results show that the signal intensity in the enhancement solution is much greater than the signal intensity dissolved in water and hydrochloric acid. The illustrations show DGA-Eu2O3 dispersed in water, HCl and enhancement solution from left to right. Under natural light, it can be observed that the nanoparticles are easily dissolved under acidic conditions. Under ultraviolet light, they emit strong red fluorescence when dissolved in the enhancement solution, while there is almost no fluorescence when dissolved in water and hydrochloric acid. After the antibody is coupled to the surface of DGA-Eu2O3, the fluorescence performance is not affected. These results show that particles with Eu2O3 as the core can be dissolved in the enhancement solution to generate strong fluorescent complexes to achieve the effect of signal enhancement.

[0083] Figure 4 As shown, the fluorescence intensity of the nanoparticles was continuously detected within 1-8 weeks. The results showed that the fluorescence intensity of the nanoparticles dispersed in water remained basically stable without obvious attenuation. The significant difference analysis showed that there was no significant difference in the fluorescence intensity at each time, which indicated that Eu2O3 nanoparticles can be stably stored in water. In the period of 1-10 days, there was no significant difference in the fluorescence intensity of DGA-Eu2O3 coupled with cTnT, which indicated that the nanoparticles after coupling with antibodies can still be stably stored.

[0084] Example 4

[0085] Performance evaluation of cTnT detection method based on D-glucuronic acid modified Eu2O3

[0086] (1) Linear analysis and sensitivity of standard curve

[0087] like Figure 5 As shown, a standard curve with a linear range of 50-10000 ng / L was established based on water-soluble Eu2O3, the equation was Y=1628.22*X+80172.01, and the linear correlation coefficient R 2The value of the curve is 0.9901, indicating that the curve has a good fit. By adding the mean value (Mean) of the fluorescence value obtained by the 0 concentration standard test (n=10) to 2 times the standard deviation (SD), and substituting the result into the standard curve, the sensitivity of this method is calculated to be 0.45 ng / L. In clinical testing, the normal concentration range of cTnT is <100 ng / L. The method developed in this study can not only accurately measure the cTnT level in the serum of AMI patients, but also has the ability to detect extremely low concentrations of cTnT. This feature enables us to detect the potential risk of myocardial injury in patients early, so as to take timely medical intervention measures to prevent further deterioration of the disease.

[0088] (2) Specificity

[0089] To evaluate the specificity of the assay system, we analyzed the effects of possible interfering analytes in serum samples. + , K + , Ca 2+ The biomarkers cTnI and PSP that may be present in serum samples were used for control experiments. Figure 6 It was observed that the fluorescence intensity of the control group was very weak compared with that of cTnT, which indicated that the interfering analyte did not form a sandwich complex with the detection antibody and capture antibody in the reaction system and undergo cross-reaction, proving the specificity of the detection system for cTnT. This is beneficial for determining low concentration levels of cTnT in serum samples with complex components, so as to facilitate the assessment of whether the patient's serum cTnT level is elevated, which can be used to help clinically determine whether the myocardium is damaged.

[0090] (3) Precision

[0091] Precision refers to the consistency between the test results of multiple tests on the same sample, which reflects the repeatability of the analytical method. As shown in Table 1, the CV within the batch is between 4.96-7.57%, and the CV between batches is between 6.37-8.98%, which shows that the CV within and between batches of this method is less than 10%, and the precision is good. The above results show the good repeatability of this method.

[0092] Table 1 Precision results

[0093]

[0094] (4) Accuracy

[0095] Accuracy refers to the degree of agreement between the test results and the measured values, reflecting the accuracy of the detection system. The accuracy was evaluated by the recovery experiment. In this study, the standard of known concentration was added to the serum sample at a ratio of 1:9 to detect and calculate the recovery rate. Recovery rate = (detection concentration / theoretical concentration) × 100%. As shown in Table 2, the recovery rate is between 97.3% and 103.9%, and the difference between the actual measured concentration and the theoretical concentration is within 10%, indicating that the antigen of this method accurately detects the cTnT concentration in the serum sample and is not affected by other components in the serum.

[0096] Table 2 Accuracy results

[0097]

[0098] Example 5

[0099] Based on Example 1, the optimal reaction conditions of reaction temperature, time and solvent were investigated.

[0100] (1) Reaction temperature

[0101] The reaction temperature affects the content of D-glucuronic acid on the surface of Eu2O3, which in turn affects its dispersibility in water and the surface carboxyl content. Therefore, we screened the reaction temperature, such as Figure 7 As shown in the figure, when the reaction temperature is low, the rate of D-glucuronic acid binding to the surface is slow, and the carboxyl content on the surface of Eu2O3 obtained by the reaction is low. When the temperature rises to 80°C, the reaction rate is accelerated, and the carboxyl content on the surface of Eu2O3 reaches the highest. When the temperature continues to rise, the carboxyl content does not increase significantly. Therefore, 80°C is determined to be the optimal reaction temperature.

[0102] (2) Response time

[0103] In order to determine the optimal reaction time, the reaction time was screened according to the surface carboxyl content. Figure 8 As shown in the figure, with the increase of time, the carboxyl content on the surface of Eu2O3 gradually increases. When the reaction time exceeds 6h, the carboxyl content does not increase significantly. Therefore, the optimal reaction time is determined to be 6h.

[0104] (3) Reaction solvent

[0105] During the experiment, the choice of reaction solvent will affect the surface carboxyl content of Eu2O3 and its biocompatibility. Therefore, we selected ethylene glycol (EG), diethylene glycol (DEG) and triethylene glycol (TEG) as reaction solvents, and used the conductivity method to determine the surface carboxyl content of their products. Fig. 9As shown, the D-glucuronic acid modified Eu2O3 prepared with TEG as the reaction solvent has the highest surface carboxyl content. Therefore, TEG was selected as the best reaction solvent.

[0106] Example 6

[0107] A method for using D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) for flow-through immunochromatography detection of cTnT, comprising the following steps:

[0108] Nitrocellulose membrane pretreatment:

[0109] The hydrophilicity of nitrocellulose membrane is poor and generally has static electricity. Therefore, pretreatment is used to improve the hydrophilicity and eliminate static electricity to improve the effect of immunochromatographic detection. The pretreatment solution is: pH = 7.0 PBS buffer, containing 0.5% BSA, 2% sucrose, and 0.1% Tween. The nitrocellulose membrane is soaked in the treatment solution in the dark for 30 minutes, and then dried in the dark at 37°C.

[0110] Assembly of immunochromatographic test strips:

[0111] (1) Preparation of nitrocellulose membrane: Dilute the detection antibody and quality control antibody with pH 7.4 PBS buffer, and the streaking volume is 1 μl / cm. cTnT antibody is used as the detection line (T line), and goat anti-mouse IgG antibody is used as the quality control line (C line). The distance from the C line to the T line is 5 mm, and the distance from the T line to the lower edge is 9 mm; put the streaked membrane into an oven and dry it in the dark at 37°C for 12 h; seal and store.

[0112] (2) Assembly of the test strip: Assemble the sample pad, nitrocellulose membrane, and absorbent pad in sequence, with an overlap of approximately 2 mm between adjacent structures. Cut into strips 3-4 mm wide, place in a housing, and store in a cool, dry place away from light.

[0113] Detection experimental steps:

[0114] Take different concentrations of standard (diluted with fetal bovine serum) and add Eu2O3 coupled with antibody (preparation method is the same as Example 2), mix thoroughly and then draw 80 μL of the mixture into the prepared test strip, add 50 μL of enhancement solution after reaction at room temperature, observe the brightness of T and C lines under ultraviolet light, and use Color Picker software to determine the R value in the RGB value and calculate the T / C ratio or use a dry fluorescent immunoassay to detect the signal intensity and calculate the T / C value.

[0115] Example 7

[0116] Based on Example 6, the optimal reaction conditions of T-line and C-line concentrations and the amount of Eu2O3 probe were investigated.

[0117] In immunochromatographic testing, if the T-line concentration is too low, the detected fluorescence intensity will be too low, affecting the accuracy of the reading and the stability of the test. If the T-line concentration is too high, it will lead to insensitivity to trace detection and reduce the sensitivity of the test strip. Therefore, the Color Picker software is used for testing to screen the T-line concentration to determine the optimal concentration. Fig.10 As shown in (A), when the streaking concentration is 1.4 mg / mL, the fluorescence intensity of the T line is the strongest. When the streaking concentration exceeds 1.4 mg / mL, the fluorescence intensity does not increase significantly. Therefore, 1.4 mg / mL is selected as the optimal streaking concentration for the T line.

[0118] The C line of the test strip is the control line, and its fluorescence intensity should not be too low, otherwise it will lead to false positives. If the concentration of the C line is too high, even if the T line changes significantly, the T / C value will not change significantly, thus affecting the test results. Therefore, it is necessary to determine the optimal concentration of the C line. Fig.10 As shown in (B), when the C-line concentration is 0.4 mg / mL, the T / C value is closest to 1, so 0.4 mg / mL is selected as the C-line concentration.

[0119] The amount of Eu2O3 probe used is also a key influencing factor. If the amount is too much, the background fluorescence will be too high to interfere with the detection results. If the amount is too little, the required fluorescence intensity cannot be achieved. Fig.10 As shown in (C), through significant difference analysis, when the probe dosage is 20 μl, the T / C value is the best, and as the dosage increases, the T / C value decreases due to the increase in background fluorescence. Therefore, 20 μl is selected as the optimal dosage.

[0120] The reaction time was screened by recording the T / C value that changed with the reaction time, such as Fig.10 As shown in (D), the significant difference analysis showed that when the detection time increased from 20 min to 25 min, the detection results did not change significantly, so the optimal detection time was determined to be 20 min.

[0121] Example 8

[0122] Evaluation of D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) for the detection of cTnT by lateral flow immunochromatography

[0123] (1) Standard curve, sensitivity and precision

[0124] After the sample is added and the reaction is completed, preliminary qualitative detection can be achieved by observing the fluorescence intensity of the T line and C line under ultraviolet light in a dark environment, while quantitative detection can be performed by constructing a standard curve through Color Picker software or dry fluorescent immunoassay analyzer, such as Fig.11 As shown in the figure, regardless of whether it is detected by mobile phone software or fluorescence analyzer, the T / C value will increase with the increase of cTnT antigen concentration, and the R 2 were 0.9971 and 0.9973, respectively, which indicated that there was a good linear relationship between the T / C value and the cTnT concentration. Fig.11 As shown in (C), under UV light, the naked eye can distinguish the chromatography test results of different concentrations. Under the optimal reaction conditions, the sensitivity of the two detection methods is 200ng / L and 52ng / L, and the detection range is 200-5000ng / L and 52-5000ng / L, respectively.

[0125] CV refers to the ratio of the standard deviation to the mean when the detection method repeatedly detects a certain concentration. The smaller the CV, the better the detection precision, and the more small changes in cTnT levels can be observed. In clinical practice, the CV of the quantitative method of POCT detection of cTnT is required to be less than 20%. This is because the main characteristics of POCT are timely and on-site, and in order to better distinguish patients, the detection rate of POCT-cTnT in the normal population is very low. At present, the literature reports that the precision of the low-value area of ​​most POCT cardiac troponin detection is poor, which does not meet the clinical standard of the 2018 version of AMI definition (total CV ≤ 20%). As shown in Table 3, the test strips developed in this study were used to detect three concentrations of high, medium and low samples, and each was repeated 10 times and its precision was calculated. The inter-batch and intra-batch precisions of this method were 6.48%-7.02% and 4.76%-5.37%, respectively, which shows that the repeatability of the test results of this method is good, and the test results of the test strips produced within and between batches are highly consistent.

[0126] Table 3 Precision results of cTnT immunochromatographic detection

[0127]

[0128] (2) Stability test

[0129] The stability of the test strip is crucial to ensure the accuracy and reliability of the test results, and is the basis for its wide application in medical diagnosis and biological testing. This requires that it can maintain consistent performance for a long time under different environmental conditions, such as temperature and humidity changes. Therefore, the prepared test strips are sealed and stored, and their stability is tested. Fig.12 As shown, the test strips were stored in a dry room temperature environment away from light and tested continuously for 6 days using a fluorescent immunoassay analyzer. The obtained T / C values ​​did not change significantly, which proves that the developed immunochromatographic test strips have good stability.

[0130] (3) Specificity

[0131] The antigen-antibody reaction is easily affected by interference factors, resulting in false positives or false negatives. Small concentration changes in the cTnT test results will lead to different diagnostic results, which will have a great impact on clinical treatment and judgment. Therefore, the specificity of the established method needs to be verified. By calculating the cross-reaction rate of the interfering antigen, the specificity of the immunochromatographic test was evaluated using a fluorescent immunoassay analyzer. The T-line fluorescence intensity will only be enhanced when the target antigen is added. As shown in Table 4, when the interfering antigen is added, the T-line fluorescence intensity remains basically unchanged, and the cross-reaction rate is low. These results show that this method has good specificity and strong anti-interference ability when detecting actual serum samples.

[0132] Table 4 Cross-reaction rate of cTnT immunochromatographic detection

[0133]

[0134] (4) Recovery rate

[0135] Recovery rate refers to an indicator determined by recovery experiments in accuracy assessment to evaluate the ability of quantitative detection methods to accurately determine the analyte to be measured. It is a commonly used accuracy assessment method. Three concentrations of antigen standards, high, medium and low, were added to human serum samples at a ratio of 1:9, and the recovery rate was calculated. As can be seen from Table 5, the recovery rates are all between 95% and 105%, which shows that the actual measured concentration and the theoretical concentration differ by less than 10%. This method can accurately quantify the cTnT level in serum samples and is not affected by the remaining components other than the analyte in the serum sample.

[0136] Table 5 Recovery of cTnT immunochromatographic assay

[0137]

[0138] The principles, steps, etc. not explicitly described in the present invention are all obtainable by those skilled in the art through conventional technical means, and therefore will not be described in detail. Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing D-glucuronic acid-modified Eu2O3 nanoparticles, characterized in that: The following steps are involved: Step 1: adding europium salt to diol and heating to dissolve, to obtain reaction solution a; Step 2: heat and dissolve sodium hydroxide particles in diol, then slowly add the mixture into reaction solution a, heat to 60-100° C. and react for 1-3 hours to obtain reaction solution b; Step 3: Slowly drip the H2O2 aqueous solution into the reaction solution b, react at 60-100°C for 1-3h to obtain the reaction solution c; Step 4: Add D-glucuronic acid to the reaction solution c, react at 60-100°C for 5-7h, then wash with distilled water and centrifuge to obtain D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3).

2. The preparation method according to claim 1, characterized in that: The europium salt is europium nitrate; the concentration of the europium nitrate is 0.2-0.4 mol / L; The diol is any one of ethylene glycol (EG), diethylene glycol (DEG) and triethylene glycol (TEG); The concentration of the sodium hydroxide is 0.5-2 mol / L; The molar ratio of the europium salt, sodium hydroxide and D-glucuronic acid is 1:(2-4):1; The concentration of the H2O2 aqueous solution is 35%.

3. The preparation method according to claim 1, characterized in that: The reaction time in step 2 is 1-3h; The reaction time in step 3 is 1-3h; The reaction temperature in step 2, step 3 and step 4 is preferably 80°C; The reaction time in step 4 is preferably 6 hours; the centrifugal speed is 10000-12000 rpm, and the centrifugal time is 10-15 minutes.

4. A D-glucuronic acid-modified Eu2O3 nanoparticle prepared by the preparation method according to any one of claims 1 to 3.

5. A method for using the D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) as claimed in claim 4 to detect cTnT by time-resolved fluorescence immunoassay, characterized in that: The following steps are involved: (1) The cTnT detection antibody is coupled to the carboxyl group on the surface of DGA-Eu2O3 by the EDC / NHS coupling method to obtain a labeled antibody; (2) adding cTnT capture antibody to the polystyrene well plate, and the cTnT capture antibody is bound to the well plate through electrostatic interaction to obtain a solid-phase antibody; (3) The sample to be tested is added to the well plate of the solid phase antibody, and then the labeled antibody is further added to the well plate. After incubation and washing, an enhancement solution is added to the well plate, and the fluorescence value of the sample is measured by a time-resolved fluorescence immunoassay analyzer. The cTnT content is quantitatively calculated using a cTnT concentration-fluorescence intensity linear standard curve.

6. The method according to claim 5, characterized in that The coupling buffer is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer; The coating buffer is 50 mmol / L sodium carbonate-sodium bicarbonate buffer; The blocking buffer consists of 50 mmol / L Tris-HCl, 0.9 wt% NaCl, 1 wt% BSA and 0.05 wt% Proclin300; The assay buffer consists of 0.05 mol / L Tris-HCl, 0.9 wt% NaCl, 0.2 wt% BSA, 0.01 wt% Tween20, 20 μM DTPA and 0.05 wt% Proclin300; The enhancement solution consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% TritonX-100; In the step (1), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibody is 10:1:2:

1.

7. A method for detecting cTnT by flow immunochromatography using the D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) as claimed in claim 4, characterized in that: The following steps are involved: (a) cTnT antibody is used as the detection line (T line) and goat anti-mouse IgG antibody is used as the quality control line (C line) on the pretreated nitrocellulose membrane. After the streaking is completed, the coated membrane is dried to obtain the nitrocellulose membrane for immunochromatography; (b) assembling the sample pad, the nitrocellulose membrane, and the absorbent pad in sequence to obtain an immunochromatographic test strip; (c) The cTnT detection antibody is coupled to the carboxyl group on the surface of DGA-Eu2O3 by the EDC / NHS coupling method to obtain a labeled antibody; (d) Mix the labeled antibody with the sample to be tested, draw 80 μL of the mixture and add it to the above-mentioned immunochromatographic test strip. After reacting at room temperature for 20-25 min, add 50 μL of the enhancement solution dropwise. Observe the brightness of the T and C lines under ultraviolet light to obtain qualitative results, and use Color Picker software to determine the R value in the RGB value and calculate the T / C ratio or use a dry fluorescent immunoassay to detect the signal intensity and calculate the T / C value. Quantitatively calculate the cTnT content based on the T / C value and the cTnT concentration linear standard curve.

8. The method according to claim 7, characterized in that The pretreatment in step (a) is performed by soaking the nitrocellulose membrane in a pretreatment solution, wherein the pretreatment solution is a PBS buffer solution with a pH value of 7.0, containing 0.5 wt % BSA, 2 wt % sucrose, and 0.1 wt % Tween. In the step (a), both the cTnT antibody and the goat anti-mouse IgG antibody were diluted with pH=7.4 PBS buffer, and the streaking volume was 1 μl / cm; the concentration of the cTnT antibody was 1.4-1.6 mg / mL; the concentration of the goat anti-mouse IgG antibody was 0.3-0.5 mg / mL. The specific steps of step (c) are as follows: dispersing DGA-Eu2O3 in a coupling buffer, then adding EDC and NHS to activate for 20-30 minutes, then centrifuging and washing with a coupling buffer, and ultrasonically resuspending in a coupling buffer; taking a cTnT detection antibody and ultrafiltration and adding it to the above buffer, shaking and reacting at room temperature for 2-3 hours; after the reaction is completed, adding BSA blocking buffer and continuing to incubate at room temperature for 1-1.5 hours, centrifuging the reaction solution and removing the supernatant, and then ultrasonically resuspending it in a blocking buffer; In the step (d), the concentration of the labeled antibody is 0.1 μg / mL, the dosage is 20 μl, the concentration of the labeled antibody is, the dosage is 16 μl, and the ratio of the labeled antibody to the sample to be detected is 1:

4.

9. The method according to claim 8, characterized in that The coupling buffer in the step (c) is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer. In the step (c), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibody is 10:1:2:

1. The blocking buffer in step (c) is composed of 50 mmol / L Tris-HCl, 0.9 wt% NaCl, 1 wt% BSA and 0.05 wt% Proclin300. The enhancement solution in step (d) is composed of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% TritonX-100.

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