A D-glucuronic acid-modified Eu2O3 nanoparticle, its preparation method, and its application in cTnT detection.

Abstract

This invention discloses D-glucuronic acid-modified Eu₂O₃ nanoparticles, their preparation method, and their application in cTnT detection. The experimental conditions were optimized to obtain the best D-glucuronic acid-modified Eu₂O₃ nanoparticles. This invention constructs a method for detecting cTnT using time-resolved fluorescence immunoassay and an immunochromatographic method for cTnT detection using DGA-Eu₂O₃ nanoparticles. The detection method of this invention exhibits high sensitivity, specificity, precision, accuracy, and recovery rate.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, specifically relating to a D-glucuronic acid modified Eu2O3 nanoparticle, its preparation method, and its application in cTnT detection. Background Technology

[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, and its incidence is increasing year by year, making it one of the leading causes of death worldwide. Detecting the concentration and changes of myocardial markers in peripheral blood during cardiac injury is an effective means of clinically treating AMI and improving patient survival. Among these, cTnT is recognized as the most ideal myocardial marker due to its early appearance, high peak value, long duration of presence in peripheral blood after myocardial injury, and high specificity, and has become the gold standard for the 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 to initiate routine immune responses, forming immune complexes, and biomolecules. 3+ The number of markers can range from several thousand to tens of thousands of Eu. 3+ After the addition of the enhancement solution, a large amount of Ln 3+ From Ln 3+ The ions dissociate from -NPs and react with ligands in the enhancement solution to generate a large number of strongly luminescent Ln molecules. 3+ Complexes are used to enhance the detection signal, resulting in high sensitivity. However, Ln used in biological detection... 3+ -NPs must be readily soluble in acid-enhancing solutions, therefore, solving Ln 3+ - The solubility of NPs has become a research hotspot, among which the solubility of Ln 3+ Surface modification of -NPs has attracted much attention.

[0004] Fluid-directed immunochromatography (LFIA) combines the high specificity of immunoassay with the rapid separation capability of chromatography, making it one of the most widely used rapid on-site detection methods. However, LFIA also has some limitations, such as relatively low sensitivity and accuracy, necessitating the development of novel labeling materials; the results are easily influenced by operator subjectivity, thus developing detection methods based on portable devices such as smartphones is beneficial for obtaining objective and accurate results; furthermore, the stability and long-term storage of LFIA may be affected by environmental factors, requiring further improvements in test strip design and materials to enhance long-term stability. Summary of the Invention

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

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

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

[0008] Step 2: Dissolve sodium hydroxide granules in diol by heating, then slowly add them to reaction solution a, heat to 60-100℃ and react for 1-3 hours to obtain reaction solution b;

[0009] Step 3: Slowly add the H2O2 aqueous solution dropwise into reaction solution b, and react at 60-100℃ for 1-3 hours to obtain reaction solution c;

[0010] Step 4: Add D-glucuronic acid to reaction solution c, react at 60-100℃ for 5-7 hours, 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, it is triethylene glycol (TEG).

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

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

[0015] In steps 1, 2 and 4, the molar ratio of europium salt, sodium hydroxide and D-glucuronic acid 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 reaction solution b is 1:(2-3).

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

[0018] The preferred reaction temperature in steps 2, 3, and 4 is 80°C.

[0019] In step 4, the preferred reaction time is 6 hours; the centrifugation speed is 10,000-12,000 rpm, and the centrifugation 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] This invention also provides a method for using the above-mentioned D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) for time-resolved fluorescence immunoassay to detect cTnT, comprising the following steps:

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

[0023] (2) Add cTnT capture antibody to polystyrene well plate. The cTnT capture antibody binds to the well plate through electrostatic interaction to obtain solid phase antibody.

[0024] (3) Add the sample to be tested to the well plate of solid phase antibody, and then add the labeled antibody to the well plate. After incubation and washing, add the enhancement solution to the well plate. Measure the fluorescence value of the sample with a time-resolved fluorescence immunoassay analyzer. Quantitatively calculate the content of cTnT by the linear standard curve of cTnT concentration-fluorescence intensity.

[0025] In step (1), the coupling buffer is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

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

[0027] In step (2), the coating buffer is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0028] The blocking buffer in steps (1) and (2) consists of 50 mmol / L Tris-HCl, 0.9 wt% NaCl, 1 wt% BSA and 0.05 wt% Proclin 300.

[0029] The analysis 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] In step (3), the enhancement solution consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% Triton X-100.

[0031] This invention also provides a method for using the D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3) prepared above for the detection of cTnT by flow immunochromatography, comprising the following steps:

[0032] (a) Using cTnT antibody as the detection line (T line) and goat anti-mouse IgG antibody as the quality control line (C line) on the pretreated nitrocellulose membrane, the membrane was dried after the lines were drawn to obtain the immunochromatographic nitrocellulose membrane.

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

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

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

[0036] In step (a), the pretreatment involves soaking the nitrocellulose membrane in a pretreatment solution, which is a PBS buffer with pH 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 were diluted with PBS buffer at pH 7.4, with a streak volume of 1 μl / cm. The concentration of the cTnT antibody was preferably 1.4-1.6 mg / mL, more preferably 1.4 mg / mL, and the concentration of the goat anti-mouse IgG antibody was preferably 0.3-0.5 mg / mL, more preferably 0.4 mg / mL.

[0038] Step (c) involves dispersing DGA-Eu2O3 in a coupling buffer, then adding EDC and NHS for activation for 20-30 min, followed by centrifugation, washing with coupling buffer, and sonication to resuspend in coupling buffer; taking cTnT detection antibody, ultrafiltration, and adding it to the above buffer, and shaking the reaction at room temperature for 2-3 h; after the reaction is complete, adding BSA blocking buffer and continuing incubation at room temperature for 1-1.5 h, centrifuging the reaction solution to remove the supernatant, and then sonication to resuspend in blocking buffer.

[0039] The coupling buffer in step (c) is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer.

[0040] In step (c), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibodies 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% Proclin 300.

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

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

[0044] This invention synthesizes a uniformly sized DGA-Eu2O3 nanoparticle that can be well dispersed in water. These nanoparticles have carboxyl groups on their surface, exhibiting high stability. They can be rapidly coupled to amino groups on antigens or antibodies via EDC / NHS coupling. Therefore, they can replace traditional lanthanide chelates as markers, increasing the labeling rate of a single antigen or antibody from 30 to several thousand, thereby significantly improving the sensitivity of the system.

[0045] This invention establishes a method for detecting cTnT using time-resolved fluorescence immunoassay with DGA-Eu2O3 nanoparticles. The detection system exhibits good linearity within the range of 50-10000 ng / L, with a sensitivity reaching 0.45 ng / L. Furthermore, this method is unaffected by interfering analytes in serum samples, demonstrating high specificity, good accuracy and repeatability, and strong correlation and consistency between the detected results and clinical measurements, indicating promising clinical applications.

[0046] This invention presents an immunochromatographic method for cTnT detection constructed using DGA-Eu2O3 nanoparticles. The analytical results can be qualitatively analyzed by direct visual observation, or preliminarily quantitatively detected using the RGB software Color Picker on a smartphone, or precisely detected using a dry fluorescence immunoassay analyzer. The test strip achieves a sensitivity of 200 ng / L, sufficient for rapid cTnT detection in various field environments. When using a portable fluorescence immunoassay analyzer, the sensitivity can be further increased to 52 ng / L, enabling more precise cTnT analysis. Furthermore, this method exhibits high specificity, precision, and accuracy, with high test strip recovery and reusability, reducing costs. Attached Figure Description

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

[0048] Figure 2 Infrared spectra of Eu2O3 and D-glucuronic acid modified Eu2O3.

[0049] Figure 3 Fluorescence performance was characterized. (Figure (A) Fluorescence spectra of Eu2O3, DGA-Eu2O3 and Eu2O3-Abs (λex = 340 nm); (B) Comparison of fluorescence signal intensity of DGA-Eu2O3 and Eu2O3-Abs in water, HCl and enhancement solution. The inset shows photographs under natural light and 365 nm UV light excitation.)

[0050] Figure 4 Results for fluorescence intensity stability. (A) DGA-Eu2O3; (B) Fluorescence intensity stability of Eu2O3-Abs.

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

[0052] Figure 6 To improve the specificity of time-resolved fluorescence immunoassay for detecting cTnT (n=3)

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

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

[0055] Figure 9 The relationship between the carboxyl group content on the surface of DGA-Eu2O3 and the reaction solvent (significant difference analysis P < 0.0001).

[0056] Figure 10 To optimize the experimental conditions for flow immunochromatography. (A) T-line concentration; (B) C-line concentration; (C) Significant difference analysis of Eu2O3-Abs probe dosage; (D) Reaction time.

[0057] Figure 11 Linear standard curve of T / C value versus cTnT concentration: (A) Color Picker detection; (B) Dry fluorescence immunoassay analyzer detection; (C) Gradient concentration test strip detection results (365nm UV light).

[0058] Figure 12 For stability testing of flow immunochromatography. Detailed Implementation

[0059] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available.

[0060] Example 1

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

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

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

[0064] 11 mL of H2O2 aqueous solution (35%) was slowly added dropwise to the reaction mixture using 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 complete, the solution was cooled to room temperature. The solution was washed three times with distilled water and centrifuged at 12,000 rpm for 10 min to collect the product DGA-Eu2O3.

[0067] Example 2

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

[0069] The buffer solutions used include:

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

[0071] The coating buffer was a 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% Proclin 300.

[0073] The analysis 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% Triton X-100.

[0075] The specific steps are as follows:

[0076] Preparation of Eu2O3-TnT Abs (hereinafter referred to as Eu2O3-Abs for performance characterization): The cTnT detection antibody can be bound to the carboxyl groups on the surface of DGA-Eu2O3 via a simple EDC / NHS coupling method, achieving antibody labeling. The experimental steps are as follows: Take 100 μl of 10 mg / mL DGA-Eu2O3 aqueous solution, wash three times with coupling buffer, centrifuge at 10000 rpm for 10 min, sonicate to disperse in 1 mL coupling buffer, then add 5 μl of 20 mg / mL EDC and 10 μl of 20 mg / mL NHS for activation for 20 min, followed by centrifugation at 12000 rpm for 10 min, discard the supernatant, and wash twice with coupling buffer. Then, sonicate to resuspend in 1 mL coupling buffer. Take 0.1 mg of the detection antibody, ultrafilter at 10000 rpm, and add it to the above solution. Incubate at room temperature with shaking for 2 h. After the reaction is complete, add 200 μl of BSA blocking buffer and continue incubation at room temperature for 1 h. After centrifuging the reaction solution to remove the supernatant, it was then sonicated and resuspended in 1 mL of blocking buffer.

[0077] Preparation of solid-phase antibody: The ultrafiltered capture antibody was diluted to 2 μg / mL with coating buffer. 100 μL of the diluted capture antibody was added to each well of a polystyrene 96-well plate. The hydrophobic portion of the polystyrene plate and the antibody were tightly bound by strong electrostatic interactions. The plate was then placed in a sealed bag and incubated overnight at 4°C. The coating buffer was then discarded, and the plate was washed once with a plate washer. 200 μL of blocking buffer (1% BSA) was then added to each well, and the plate was incubated at room temperature for 2 hours. Finally, the blocking buffer was discarded, the plate was dried, and the plate was placed in a sealed bag containing desiccant and stored at -20°C.

[0078] Add 5 μL of cTnT standard or serum sample from an acute myocardial infarction patient to each well of a 96-well plate coated with capture antibody. Then add 95 μL of water-soluble Eu2O3-labeled antibody (1000-fold dilution) diluted with analytical buffer to each well. After adding the sample, place the plate on a shaker and incubate at 37°C for 10 min, followed by washing 6 times with a plate washer. Then add 100 μL of enhancement buffer to each well and incubate at room temperature for 1–5 min with shaking. Finally, measure the fluorescence value using a time-resolved fluorescence immunoassay analyzer.

[0079] Example 3

[0080] The properties of the product DGA-Eu2O3 were characterized as follows:

[0081] Figure 1 The surface morphology of nanoparticles dispersed in water and acidic enhancement solution was characterized using scanning electron microscopy, such as... Figure 1 As shown in (A), the synthesized nanoparticles are uniform in size and have regular morphology. Figure 1 (B) shows the surface morphology of the powder obtained after dissolving nanoparticles in the reinforcing solution for 150 s, centrifuging at 12000 rpm for 10 min, collecting the precipitate, and freeze-drying. The results indicate that water-soluble Eu2O3 dissolves rapidly in the reinforcing solution. Figure 2 The infrared absorption spectra of Eu₂O₃ (black line) and D-glucuronic acid-modified Eu₂O₃ (red line) are shown. Eu₂O₃ has absorption spectra at 1597, 1504, and 1381 cm⁻¹. -1 The peaks at [values ​​missing] are due to the reaction solvent triethylene glycol binding on the surface of the nanoparticles, while the infrared spectrum of D-glucuronic acid modified Eu2O3 shows peaks at 2934, 1593, and 1082 cm⁻¹. -1 The characteristic peaks at these locations correspond to CH, C=O, and CO, respectively, which proves that Eu2O3 nanoparticles are modified with D-glucuronic acid and thus have carboxyl groups.

[0082] Figure 3 The fluorescence spectra of Eu₂O₃, DGA-Eu₂O₃, and Eu₂O₃-Abs were detected by a fluorescence spectrophotometer, respectively. Figure 3As shown in (A), 10 mg / mL Eu2O3, DGA-Eu2O3 and Eu2O3-Abs exhibited a characteristic peak at around 620 nm under excitation at 340 nm, and the fluorescence intensity gradually decreased with the modification of Eu2O3 surface by D-glucuronic acid and the coupling with antibodies. Figure 3 (B) shows the signal intensities of 100 mg DGA-Eu2O3 particles and Eu2O3-Abs particles dispersed in water, hydrochloric acid (pH=2), and acidic enhancement solution, respectively. The results indicate that the signal intensity in the enhancement solution is significantly greater than that dissolved in water and hydrochloric acid. The inset shows DGA-Eu2O3 dispersed in water, HCl, and the enhancement solution from left to right. Under natural light, it can be observed that the nanoparticles are easily dissolved under acidic conditions. Under UV light, the nanoparticles dissolved in the enhancement solution emit strong red fluorescence, while those dissolved in water and hydrochloric acid show almost no fluorescence. Furthermore, conjugating antibodies to the surface of DGA-Eu2O3 did not affect the fluorescence performance. These results demonstrate that Eu2O3-core particles can dissolve in the enhancement solution to form a strongly fluorescent complex, thereby achieving signal enhancement.

[0083] Figure 4 As shown, the fluorescence intensity of the nanoparticles was continuously measured over 1-8 weeks. The results indicated that the fluorescence intensity of the nanoparticles dispersed in water remained relatively stable without significant attenuation. Significant difference analysis showed no significant difference in fluorescence intensity at different time points, indicating that Eu2O3 nanoparticles can be stably stored in water. Furthermore, the fluorescence intensity of DGA-Eu2O3 conjugated with cTnT also showed no significant difference over 1-10 days, suggesting that the antibody-conjugated nanoparticles can still be stably stored.

[0084] Example 4

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

[0086] (1) Linearity analysis and sensitivity of the 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, with the equation Y = 1628.22*X + 80172.01 and a linear correlation coefficient R. 2The value of 0.9901 indicates a good curve fit. The sensitivity of this method was calculated to be 0.45 ng / L by adding the mean (Mean) of the fluorescence values ​​obtained from the detection of 0 concentration standards (n=10) to twice the standard deviation (SD) and substituting the result into the standard curve. 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 serum cTnT level in AMI patients but also detect extremely low concentrations of cTnT. This characteristic allows us to detect potential myocardial damage risks early, enabling timely medical intervention to prevent further deterioration of the condition.

[0088] (2) Specificity

[0089] To evaluate the specificity of the detection system, we analyzed the influence of potential interfering analytes present in serum samples. This was done under the same conditions using 5000 ng / L Na... + K + Ca 2+ A control experiment was conducted using cTnI and PSP, which may be present in serum samples. Figure 6 It was observed that the fluorescence intensity of the control group was very weak compared to cTnT, indicating that the interfering analyte did not form a sandwich complex with the detection antibody and capture antibody in the reaction system and thus did not cross-react. This demonstrates the specificity of the detection system for cTnT, which is beneficial for measuring low concentrations of cTnT in complex serum samples, so as to assess whether the patient's serum cTnT level is elevated, and to help clinically determine whether there is myocardial damage.

[0090] (3) Precision

[0091] Precision refers to the consistency between the results of multiple tests on the same sample, reflecting the repeatability of the analytical method. As shown in Table 1, the intra-batch CV ranges from 4.96% to 7.57%, and the inter-batch CV ranges from 6.37% to 8.98%. This indicates that the intra-batch and inter-batch CVs of this method are both less than 10%, demonstrating good precision. These results demonstrate 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 detection result and the measured value, reflecting the accuracy of the detection system. Accuracy was evaluated through a recovery experiment. In this study, a standard of known concentration was added to the serum sample at a ratio of 1:9 to calculate the recovery rate. Recovery rate = (detected concentration / theoretical concentration) × 100%. As shown in Table 2, the recovery rate ranged from 97.3% to 103.9%, with the actual measured concentration differing from the theoretical concentration by less than 10%. This indicates that the antigen in this method accurately detects the cTnT concentration in serum samples and is unaffected by other components in the serum.

[0096] Table 2 Accuracy Results

[0097]

[0098] Example 5

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

[0100] (1) Reaction temperature

[0101] The reaction temperature affects the content of D-glucuronic acid on the Eu2O3 surface, which in turn affects its dispersibility in water and the content of surface carboxyl groups. Therefore, we screened the reaction temperature, such as... Figure 7 As shown, when the reaction temperature is low, the rate of D-glucuronic acid binding on the surface is slow, and the carboxyl content on the surface of the resulting Eu2O3 is low. However, when the temperature is increased to 80℃, the reaction rate is accelerated, and the carboxyl content on the surface of the resulting Eu2O3 reaches its maximum. Furthermore, when the temperature continues to rise, the carboxyl content does not increase significantly. Therefore, 80℃ was determined to be the optimal reaction temperature.

[0102] (2) Reaction time

[0103] To determine the optimal reaction time, the reaction time was screened based on the surface carboxyl group content. For example... Figure 8 As shown, the carboxyl content on the surface of Eu2O3 gradually increases with time. When the reaction time exceeds 6 hours, the carboxyl content does not increase significantly. Therefore, the optimal reaction time is determined to be 6 hours.

[0104] (3) Reaction solvent

[0105] During the experiment, the choice of reaction solvent affected the carboxyl content on the surface 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 the products. Figure 9As shown, the D-glucuronic acid-modified Eu2O3 surface prepared with TEG as the reaction solvent has the highest carboxyl content. Therefore, TEG was selected as the best reaction solvent.

[0106] Example 6

[0107] A method for detecting cTnT by flow immunochromatography using D-glucuronic acid-modified Eu2O3 nanoparticles (DGA-Eu2O3) includes the following steps:

[0108] Nitrocellulose membrane pretreatment:

[0109] Nitrocellulose membranes have poor hydrophilicity and are generally statically charged. Therefore, pretreatment is used to improve hydrophilicity and eliminate static electricity, thereby improving the effectiveness of immunochromatographic detection. The pretreatment solution is a pH 7.0 PBS buffer containing 0.5% BSA, 2% sucrose, and 0.1% Tween. The nitrocellulose membrane is immersed in the pretreatment solution in the dark for 30 min, followed by drying at 37°C in the dark.

[0110] Assembly of immunochromatographic test strips:

[0111] (1) Preparation of nitrocellulose membrane: The detection antibody and the quality control antibody were diluted with PBS buffer at pH 7.4, with a streaking volume of 1 μl / cm. cTnT antibody was used as the detection line (T line), and goat anti-mouse IgG antibody was used as the quality control line (C line). The distance between the C line and the T line was 5 mm, and the distance between the T line and the lower edge was 9 mm. The coated membrane with the streaked lines was placed in an oven and dried at 37°C in the dark for 12 h. It was then sealed and stored.

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

[0113] Detection experimental steps:

[0114] Take standards of different concentrations (diluted with fetal bovine serum) and add Eu2O3 conjugated with antibodies (preparation method as in Example 2). After thorough mixing, add 80 μL of the mixture to the prepared test strip. After reacting at room temperature, add 50 μL of enhancement solution. Observe the brightness of the T and C lines under ultraviolet light irradiation. Measure the R value in the RGB values ​​using Color Picker software and calculate the T / C ratio, or calculate the T / C value after detecting the signal intensity using a dry fluorescence immunoassay analyzer.

[0115] Example 7

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

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

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

[0119] The amount of Eu2O3 probe used is also a key influencing factor. Too much probe will result in excessively high background fluorescence, interfering with the detection results; too little probe will fail to achieve the required fluorescence intensity. For example... Figure 10 As shown in (C), the T / C value was optimal when the probe dosage was 20 μl, but the T / C value decreased as the dosage increased due to the increase in background fluorescence. Therefore, 20 μl was selected as the optimal dosage.

[0120] The reaction time was filtered by recording the T / C value as a function of reaction time, such as... Figure 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. Therefore, 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 flow immunochromatography

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

[0124] After the sample reaction is complete, preliminary qualitative detection can be achieved by observing the fluorescence intensity of the T and C lines under ultraviolet light in a dark environment. Quantitative detection can then be performed using Color Picker software or a dry fluorescence immunoassay analyzer, based on a constructed standard curve. Figure 11 As shown, regardless of whether it's detected by mobile software or a fluorescence analyzer, the T / C value increases with increasing cTnT antigen concentration, and the R-value of the obtained standard curve equation... 2 The values ​​were 0.9971 and 0.9973, respectively, indicating a good linear relationship between the T / C value and the cTnT concentration. Meanwhile, as... Figure 11 As shown in (C), under ultraviolet light irradiation, the chromatographic detection results of different concentrations can be distinguished by the naked eye. Under optimal reaction conditions, the sensitivities of the two detection methods are 200 ng / L and 52 ng / L, respectively, and the detection ranges are 200-5000 ng / L and 52-5000 ng / L, respectively.

[0125] CV refers to the ratio of the standard deviation to the mean when a detection method repeatedly measures a certain concentration. The smaller the CV, the better the detection precision and the more easily small changes in cTnT levels can be observed. Clinically, the CV requirement for point-of-care testing (POCT) quantitative methods for cTnT is less than 20%. This is because the main characteristics of POCT are timeliness and on-site detection, and to better differentiate between patients, the detection rate of POCT-cTnT in the normal population is very low. Currently, the literature reports that most POCT methods for cardiac troponin detection have poor precision in the low-value region, failing to meet the clinical standard defined by the 2018 AMI (total CV ≤ 20%). As shown in Table 3, samples with three concentrations (high, medium, and low) were tested using the test strips developed in this study, with each test repeated 10 times and its precision calculated. The inter-batch and intra-batch precision of this method were 6.48%-7.02% and 4.76%-5.37%, respectively, indicating that the test results of this method have good repeatability, and the test results of the test strips produced within and between batches have a high degree of consistency.

[0126] Table 3 Precision results of cTnT immunochromatographic assay

[0127]

[0128] (2) Stability test

[0129] The stability of test strips is crucial for ensuring the accuracy and reliability of test results, and is the foundation for their widespread application in medical diagnostics and biological detection. This requires them to maintain consistent performance over long periods under different environmental conditions, such as changes in temperature and humidity. Therefore, the prepared test strips were sealed and stored to test their stability. Figure 12 As shown, when the test strip was stored in a dry, room-temperature environment protected from light and continuously tested for 6 days using a fluorescence immunoassay analyzer, the T / C value did not change significantly, which proves that the developed immunochromatographic test strip has good stability.

[0130] (3) Specificity

[0131] Antigen-antibody reactions are easily affected by interfering factors, leading to false positives or false negatives. Even small changes in cTnT concentration can result in different diagnostic outcomes, significantly impacting clinical treatment and judgment. Therefore, it is necessary to validate the specificity of the established method. The specificity of the immunochromatographic assay was evaluated using a fluorescence immunoassay analyzer by calculating the cross-reactivity rate of the interfering antigen. The T-line fluorescence intensity only increased when the target antigen was added. As shown in Table 4, the T-line fluorescence intensity remained essentially unchanged when the interfering antigen was added, indicating a low cross-reactivity rate. These results demonstrate that the proposed method has good specificity and strong anti-interference ability when detecting actual serum samples.

[0132] Table 4 Cross-reactivity rates of cTnT immunochromatographic assay

[0133]

[0134] (4) Recovery rate

[0135] Recovery rate, determined through recovery experiments in accuracy assessment, is a commonly used indicator to evaluate the ability of a quantitative detection method to accurately determine the analyte. Three concentrations of antigen standards (high, medium, and low) were added to human serum samples at a 1:9 ratio, and the recovery rates were calculated. Table 5 shows that the recovery rates were all between 95% and 105%, indicating that the actual measured concentration differed from the theoretical concentration by less than 10%. This demonstrates that the method can accurately quantify cTnT levels in serum samples and is unaffected by other components besides the analyte in the serum sample.

[0136] Table 5 Recovery rate of cTnT immunochromatographic assay

[0137]

[0138] Principles and steps not explicitly described in this invention are all obtainable by those skilled in the art through conventional technical means, and therefore will not be elaborated upon. Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing D-glucuronic acid modified Eu2O3 nanoparticles, characterized in that, Includes the following steps: Step 1: Add europium salt to diol and heat to dissolve, to obtain reaction solution a; Step 2: Dissolve sodium hydroxide granules in diol by heating, then slowly add them to reaction solution a, heat to 60-100℃ and react for 1-3 hours to obtain reaction solution b; Step 3: Slowly add the H2O2 aqueous solution dropwise into reaction solution b, and react at 60-100℃ for 1-3 hours to obtain reaction solution c; Step 4: Add D-glucuronic acid to reaction solution c, react at 60-100℃ for 5-7 h, 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 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 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 1-3 hours; The reaction time in step 3 is 1-3 hours; The preferred reaction temperature in steps 2, 3, and 4 is 80°C. The preferred reaction time in step 4 is 6 hours; the centrifugation speed is 10,000-12,000 rpm, and the centrifugation time is 10-15 minutes.

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

5. The application of D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) as described in claim 4 in the preparation of cTnT time-resolved fluorescence immunoassay reagents, characterized in that, Includes the following steps: (1) The cTnT detection antibody was bound to the carboxyl group on the surface of DGA-Eu2O3 by the EDC / NHS coupling method to obtain the labeled antibody; (2) Add cTnT capture antibody to polystyrene plate. The cTnT capture antibody binds to the plate through electrostatic interaction to obtain solid antibody. (3) Add the sample to be tested to the well plate of solid phase antibody, and then add the labeled antibody to the well plate. After incubation and washing, add the enhancement solution to the well plate. Measure the fluorescence value of the sample with a time-resolved fluorescence immunoassay analyzer. Quantitatively calculate the content of cTnT by the linear standard curve of cTnT concentration-fluorescence intensity.

6. The application according to claim 5, characterized in that, In step (1), coupling buffer and blocking buffer are used; in step (2), coating buffer and blocking buffer are used; and in step (3), analysis buffer and enhancement solution are used. The coupling buffer is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer. The coating buffer is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer. The blocking buffer consisted of 50 mmol / L Tris-HCl, 0.9 wt% NaCl, 1 wt% BSA and 0.05 wt% Proclin 300; The analytical buffer solution 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. The enhancement solution consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% Triton X-100; In step (1), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibodies is 10:1:2:

1.

7. The application of D-glucuronic acid modified Eu2O3 nanoparticles (DGA-Eu2O3) as described in claim 4 in the preparation of cTnT analytical immunochromatographic assay reagents, characterized in that, Includes the following steps: (a) Using cTnT antibody as the detection line (T line) and goat anti-mouse IgG antibody as the quality control line (C line) on the pretreated nitrocellulose membrane, the coating membrane was dried after the lines were drawn to obtain the immunochromatographic nitrocellulose membrane. (b) Assemble the sample pad, nitrocellulose membrane, and absorbent pad sequentially to obtain the immunochromatographic test strip; (c) The cTnT detection antibody was bound to the carboxyl group on the surface of DGA-Eu2O3 by EDC / NHS coupling method to obtain the labeled antibody; (d) Mix the labeled antibody with the sample to be tested, add 80 μL of the mixture to the above immunochromatographic test strip, react at room temperature for 20-25 min, and then add 50 μL of enhancement solution. Observe the brightness of the T and C lines under UV light to obtain qualitative results. Use Color Picker software to determine the R value in the RGB values ​​and calculate the T / C ratio, or use a dry fluorescence immunoassay analyzer to detect the signal intensity and calculate the T / C value. Quantitatively calculate the cTnT content based on the linear standard curve of T / C value and cTnT concentration.

8. The application according to claim 7, characterized in that, In step (a), the pretreatment involves soaking the nitrocellulose membrane in a pretreatment solution, which is a PBS buffer with pH=7.0 containing 0.5wt% BSA, 2wt% sucrose, and 0.1wt% Tween. In step (a), both the cTnT antibody and the goat anti-mouse IgG antibody were diluted with PBS buffer at pH 7.4, with a streak volume of 1 μl / cm; the concentration of the cTnT antibody was 1.4-1.6 mg / mL; and 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: DGA-Eu2O3 is dispersed in the coupling buffer, then EDC and NHS are added for activation for 20-30 min, followed by centrifugation, washing with the coupling buffer, and sonication to resuspend in the coupling buffer; cTnT detection antibody is taken, ultrafiltered, and added to the above buffer, and the reaction is carried out with shaking at room temperature for 2-3 h; after the reaction is completed, BSA blocking buffer is added and incubated at room temperature for 1-1.5 h, the reaction solution is centrifuged to remove the supernatant, and then sonicated to resuspend in the blocking buffer; In step (d), the concentration of the labeled antibody is 0.1 μg / mL, the amount used is 20 μl, the concentration of the labeled antibody is 16 μl, and the ratio of the labeled antibody to the sample to be tested is 1:

4.

9. The application according to claim 8, characterized in that, The coupling buffer in step (c) is a 50 mmol / L sodium carbonate-sodium bicarbonate buffer. In step (c), the mass ratio of DGA-Eu2O3, EDC, NHS and cTnT detection antibodies is 10:1:2:1; In step (c), the blocking buffer consists of 50 mmol / L Tris-HCl, 0.9 wt% NaCl, 1 wt% BSA and 0.05 wt% Proclin 300. In step (d), the enhancement solution consists of 15 μmol / L β-NTA, 50 μmol / L TOP, and 0.1% Triton X-100.

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