Fluorescent probe for detecting content of immune globulin in complex biological sample and preparation method thereof

Abstract

The invention provides a fluorescent probe for detecting the content of immune globulin in a complex biological sample, a preparation method of the fluorescent probe and a method for detecting the content of the immune globulin in the complex biological sample by using the fluorescent probe. Fluorescent carbon dots are used as a signal response unit, 4-carboxyphenylboronic acid is used as a recognition unit, and the fluorescent carbon dots and the 4-carboxyphenylboronic acid are chemically combined through a covalent coupling method. The probe has the advantages of storage resistance, good selectivity, good biocompatibility, good chemical stability and light irradiation stability, and can be used for testing and analyzing the content of immune globulin in complex biological samples such as whole blood and living cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and in particular relates to a fluorescent probe for detecting the content of immunoglobulin in complex biological samples and a preparation method thereof. Background Art

[0002] Immunoglobulins make up 20 percent of the total plasma protein mass and are one of the most abundant protein families in human blood. Human serum contains five main types of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. These antibodies are very similar in structure and composition, with IgG being the most abundant. Through its high affinity for foreign structures, IgG proteins establish a link between the effector mechanisms of the adaptive and innate immune systems. Immunoglobulins are a class of glycosylated proteins (also called glycoproteins), whose molecular structure consists of proteins linked by O- and N-linked glycan chains to form glycoproteins. Changes in glycoprotein content and structure are closely linked to diseases such as Alzheimer's disease, breast cancer, gastric cancer, and prostate cancer. Therefore, measuring glycoprotein levels is essential for the early diagnosis of these major diseases.

[0003] Currently, most clinical methods for detecting glycoproteins require antibodies, but this method is expensive and requires demanding experimental conditions. To reduce detection costs, researchers have developed biosensors based on molecularly imprinted polymers, which can specifically recognize peptide and protein target molecules, are simple to operate, and are low-cost. However, these polymers have limitations, such as high relative molecular weight of the associated complexes, poor solubility and stability, complex synthesis steps, and uneven recognition sites due to slow mass transfer in solution. Therefore, the development of new fluorescence analysis methods for glycoprotein detection is essential. Summary of the Invention

[0004] The present invention aims to provide functionalized carbon dots (B-CDs) modified with 4-carboxyphenylboronic acid and the use of B-CDs for immunoglobulin fluorescence detection. The chemical reagents and materials used in the method are non-toxic to the human body, the method is simple to operate, has good detection selectivity, and has strong anti-interference ability. It solves the problems of high detection cost, poor solubility and stability, and complex synthesis steps in existing immunoglobulin detection methods.

[0005] The detection method of the present invention utilizes the selective covalent binding of the boronic acid groups on the surface of B-CDs with the cis-diol in the immunoglobulin to form a stable five-membered cyclic boronate complex in an alkaline medium, thereby quenching fluorescence.

[0006] After immunoglobulin binds to carbon dots, the fluorescence intensity of the carbon dots is significantly quenched, and the immunoglobulin is quantitatively detected based on the change in fluorescence intensity.

[0007] The technical solution adopted in the present invention is:

[0008] A fluorescent probe for detecting the immunoglobulin content in complex biological samples is a functionalized carbon dot modified with 4-carboxyphenylboronic acid.

[0009] Furthermore, the method for detecting the immunoglobulin content in a complex biological sample adopts a fluorescence quenching method.

[0010] Furthermore, the complex biological sample is a whole blood sample or a living cell sample.

[0011] Furthermore, the whole blood sample comes from a healthy organism or an organism infected with Cryptococcus, and the pretreatment method of the whole blood sample is: diluting it 400 times with a phosphate buffer solution with a pH of 7.4.

[0012] Furthermore, the method for using the fluorescent probe to detect the immunoglobulin content in a living cell sample is as follows: the living cell sample is incubated with the fluorescent probe for 0.5 to 4 hours, and the concentration of the fluorescent probe during incubation is 1 mg / mL.

[0013] The fluorescent probe for detecting the immunoglobulin content in complex biological samples described in any of the above items has a particle size of 5.70 to 10.26 nm, and its optimal excitation and emission wavelengths are 460±5 nm and 525±5 nm, respectively. Under the condition of 460 nm laser 300 W irradiation for 60 minutes, the change in fluorescence emission intensity at 525 nm is less than 1%.

[0014] The method for preparing the fluorescent probe for detecting the immunoglobulin content in complex biological samples comprises the following steps:

[0015] 1) Using o-phenylenediamine as a carbon source, fluorescent carbon dots with rich surface amino functional groups, i.e., aminated carbon dots, were prepared;

[0016] 2) Chemically bond 4-carboxyphenylboronic acid to amino carbon dots through covalent coupling to obtain functionalized modified carbon dots.

[0017] Furthermore, in the above-mentioned method for preparing a fluorescent probe for detecting immunoglobulin content in complex biological samples, the amino content on the surface of the amino-modified carbon dots prepared in step 1) is 4 to 10 times the amino content on the surface of the functionalized modified carbon dots prepared in step 2).

[0018] Furthermore, in the above-mentioned method for preparing a fluorescent probe for detecting the content of immunoglobulins in complex biological samples, the specific steps of step 1) are as follows:

[0019] 1.1) Dissolve o-phenylenediamine in anhydrous ethanol to a concentration of 25-35 g / L, ultrasonicate for 5 minutes, add the resulting solution to a polytetrafluoroethylene reactor, place the reactor in an oven, and hydrothermally react at 180°C for 12 hours. Cool to room temperature to obtain a crude product.

[0020] 1.2) Filter the crude product through a 0.22 μm filter membrane. Rotary evaporate the filtrate until the solvent is almost dry. Transfer any remaining material to a 1000 Da molecular weight cutoff regenerated cellulose dialysis bag and dialyze, changing the water every 4-8 hours, until the exudate is colorless and has a pH of 7.

[0021] 1.3) The dialyzed product was dried in a vacuum oven for 24 h to obtain an orange-yellow powder, namely o-phenylenediamine carbon dots, which was stored at 4°C until use.

[0022] Furthermore, in the above-mentioned method for preparing a fluorescent probe for detecting the content of immunoglobulins in complex biological samples, the specific steps of step 2) are as follows:

[0023] 2.1) 60 mg of 4-carboxyphenylboronic acid, 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 24 mg of N-hydroxysuccinimide were placed in 30 mL of methanol and mixed well. After 1 hour, 70 mg of amino-containing carbon dots were added to the mixture and stirred in the dark for 18 hours.

[0024] 2.2) The solution obtained in step 2.1) was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 24 h. After dialysis, the solution was frozen in a refrigerator and freeze-dried for 24 h to obtain a light yellow powder as the final product.

[0025] The present invention has the following beneficial effects: The reagent used to indicate immunoglobulin protein fluorescence intensity is a safe and nontoxic functionalized carbon dot modified with 4-carboxyphenylboronic acid. Under the described conditions, the fluorescence intensity of the B-CDs dots decreases upon addition of immunoglobulin, and the degree of decrease in fluorescence intensity is linearly correlated with the concentration of the added immunoglobulin. The detection method established based on this method has high sensitivity and a detection limit of 4.43 ng / mL. This method successfully detects immunoglobulins in living cells and mouse whole blood, and can distinguish changes in immunoglobulin content in mouse whole blood after infection with Cryptococcus. Therefore, the method used in the present invention has the potential to be applied to the detection of immunoglobulins in complex biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 a is the fluorescence change diagram of phenylboronic acid modified carbon dots after adding different concentrations of IgG.

[0027] Figure 1b is the linear calibration curve between the fluorescence intensity of phenylboronic acid-modified carbon dots and IgG concentration in the range of 0.05–10 μg / mL.

[0028] Figure 1 c is the linear calibration curve between the fluorescence intensity of phenylboronic acid-modified carbon dots and IgG concentration in the range of 2.0 to 6.0 μg / mL.

[0029] Figure 1 d is the linear calibration curve between the fluorescence intensity of phenylboronic acid-modified carbon dots and IgG concentration in the range of 0.05 to 0.8 μg / mL.

[0030] Figure 2 A is a fluorescence image of the cells in Example 4 with the addition of 0 μg / mL IgG.

[0031] Figure 2 B is a fluorescence image of the cells in Example 4 to which 0.5 μg / mL IgG was added.

[0032] Figure 2 C is a fluorescence image of the cells in Example 4 with the addition of 1 μg / mL IgG.

[0033] Figure 2 D is the fluorescence image of the cells in Example 4 with the addition of 5 μg / mL IgG. DETAILED DESCRIPTION

[0034] Example 1

[0035] A method for preparing a fluorescent probe for analyzing and detecting immunoglobulin IgG in complex biological samples comprises the following steps:

[0036] 1) Dissolve 3.75-5.25 g of o-phenylenediamine in 150 mL of anhydrous ethanol and ultrasonicate for 5 min to ensure uniform mixing of the solution. Add the resulting mixed solution to a polytetrafluoroethylene reactor, place the reactor in an oven, and hydrothermally react at 180° C. for 12 h. Cool to room temperature to obtain a crude product.

[0037] 2) Filter the crude product through a 0.22 μm filter membrane and perform rotary evaporation of the filtrate for 20–40 min. After the solvent is almost dry, transfer any remaining material into a 1000 Da molecular weight cutoff regenerated cellulose dialysis bag and dialyze, changing the water every 4–8 h until the exudate is colorless and has a pH of approximately 7.

[0038] 3) Dry the dialyzed product in a vacuum oven for 24 h to obtain an orange-yellow powder, which is o-phenylenediamine carbon dots. Store at 4°C until use.

[0039] 4) 4-Carboxyphenylboronic acid (60 mg), 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 24 mg of N-hydroxysuccinimide (NHS) were placed in 30 mL of methanol and mixed well. After reacting for 1 hour, o-phenylenediamine carbon dots (70 mg) were added to the mixture and stirred in the dark for 18 hours.

[0040] 5) The solution obtained in step 4) was poured into a dialysis bag with MW = 1000 and dialyzed for 24 hours. After dialysis, it was frozen in a refrigerator and then freeze-dried (24 hours) to obtain a light yellow powder, which is the final product, phenylboronic acid-modified carbon dots (B-CDs).

[0041] The prepared o-phenylenediamine carbon dots have a particle size of 3.33±0.71 nm, and their optimal excitation wavelength and emission wavelength are 450±5 nm and 545±5 nm, respectively.

[0042] The prepared phenylboronic acid modified carbon dots have a particle size of 7.98±2.28 nm, and their optimal excitation wavelength and emission wavelength are 460±5 nm and 525±5 nm, respectively.

[0043] The prepared phenylboronic acid-modified carbon dots exhibited a fluorescence emission intensity at 525 nm that varied by less than 1% under 460 nm laser (300 W) irradiation for 60 minutes. Furthermore, after storage of their aqueous dispersion (at a concentration of 0.8 g / L) at room temperature for 30 days, the fluorescence intensity at 460 nm excitation / 525 nm emission varied by less than 1%.

[0044] Example 2

[0045] The detection method includes the following steps:

[0046] 1) Mix 200 μL of 0.8 mg / mL B-CDs solution and 200 μL of IgG solution (0.01 μg / mL to 10 μg / mL) in 2 mL centrifuge tubes, and dilute to 1.5 mL with pH 7.4 PBS buffer.

[0047] 2) using a fluorescence spectrophotometer to measure and record the fluorescence spectrum of the mixture when excited at 460 nm, and to calculate the fluorescence intensity at 525 nm in the emission spectrum;

[0048] 3) Draw a standard curve with fluorescence intensity as the ordinate and IgG concentration as the abscissa;

[0049] Figure 1 The fluorescence changes and standard curve of phenylboronic acid modified carbon dots after adding different concentrations of IgG. Figure 1As shown in a and 1b, as the IgG concentration increased from 0 μg / mL to 10 μg / mL, the fluorescence intensity of the phenylboronic acid-modified carbon dots was gradually quenched. Figure 1 In b, it can be clearly observed that the fluorescence intensity of B-CDs is strongly quenched when the IgG concentration is in the range of 0-1 μg / mL, and the fluorescence intensity decreases slowly when the IgG concentration is in the range of 1-10 μg / mL. According to the statistical results, the IgG concentration and the fluorescence intensity of B-CDs show a good linear correlation in the concentration ranges of 0.05-0.8 μg / mL and 2.0-6.0 μg / mL, respectively ( Figure 1 c and 1d), the linear equations were y = -494x + 907.4 and y = -38.35x + 430.1, respectively, with linear correlation coefficients of 0.99 for both. The limit of detection for IgG was calculated to be 4.43 ng / mL based on the 3Sb / m standard, where m is the slope of the linear equation and Sb is the standard deviation of 11 blank measurements. Notably, the limit of detection of this method is lower than that of most reported fluorescence-based IgG detection methods.

[0050] 4) Following steps 1) and 2), replace the IgG solutions containing different concentrations with the sample solution containing IgG. Observe the fluorescence spectrum at 460 nm excitation and the fluorescence intensity at 525 nm in the emission spectrum using a fluorescence spectrophotometer. Compare the results with a standard curve plotted against known IgG fluorescence intensity and concentration to determine the IgG content.

[0051] Example 3

[0052] The immunoglobulin levels in whole blood samples from healthy mice and mice infected with Cryptococcus were measured. Cryptococcus was isolated from biological samples of clinically infected patients, and 20 different strains were obtained, designated Iso-1, Iso-2, ..., and Iso-20.

[0053] The detection method includes the following steps:

[0054] 1) Take fresh blood samples from healthy mice and 20 infected mice, dilute them 400-fold with PBS buffer solution (pH = 7.4), and store the treated blood samples in a refrigerator at 4°C.

[0055] 2) Mix 200 μL of the diluted supernatant with 200 μL of a 0.8 mg / mL B-CDs solution and dilute to 1.5 mL with a pH 7.4 PBS buffer solution. Measure and record the fluorescence spectrum of the mixture when excited at 460 nm using a fluorescence spectrophotometer. Calculate the fluorescence intensity at 525 nm in the emission spectrum, and calculate the IgG content based on the standard curve.

[0056] 3) Iso-12, Iso-15, and Iso-20 samples were selected for spike recovery assays. Different volumes of IgG standard stock solution (1 mg / mL) were added to mouse blood samples, resulting in spike concentrations of 180, 240, and 300 μg / mL, respectively. Finally, immunoglobulin content was measured using the aforementioned fluorescence analysis method.

[0057] The immunoglobulin detection results of Example 3 are shown in Tables 1 and 2.

[0058] Table 1 Actual sample content of immunoglobulins in blood samples of mice infected with Cryptococcus (parallel determination 3 times)

[0059]

[0060] Table 2 Results of spiked immunoglobulin recovery experiments in blood samples of Cryptococcus-infected mice (determined in triplicate)

[0061]

[0062]

[0063] As shown in the test data in Table 1, the average immunoglobulin concentration in the whole blood of mice uninfected with Cryptococcus was 130.7 μg / mL, while the average concentrations of immunoglobulins in the whole blood of mice infected with different isolates ranged from 133.2 to 191.2 μg / mL. Clearly, the concentration of immunoglobulins in the whole blood of mice infected with Cryptococcus increased significantly compared to before infection. This is consistent with the common biological understanding that immunoglobulins are involved in immune responses against bacterial infections and demonstrates the applicability of this method for measuring immunoglobulin levels in whole blood.

[0064] To verify the accuracy of the method in complex samples, spike recovery tests were conducted. The results are shown in Table 2. Three randomly selected samples (Iso-12, Iso-15, and Iso-20) were spiked with IgG standard solutions at varying concentrations. The spiked immunoglobulin content was then measured. The calculated spike recovery rates ranged from 92.0% to 111.3%, approaching 100%. These spike recovery experiments demonstrated that the complex matrix in whole blood samples did not interfere with the assay, confirming the accuracy and applicability of the assay.

[0065] Example 4

[0066] Imaging analysis of immunoglobulin content in living cells. The specific steps are as follows:

[0067] 1) Using HeLa cells as a model, after the cells have grown to a good state, remove the culture medium and wash the cells twice with PBS buffer solution;

[0068] 2) Add 1 mL of B-CDs solution (1 mg / mL) to the cell culture dish and incubate for 4 hours;

[0069] 3) Add 1 mL of IgG containing different concentrations (0, 0.5, 1 and 5 μg mL -1 ) medium, incubate the cells for 45 min, remove the culture medium, wash the cells with PBS buffer solution, and observe the fluorescence imaging under 460 nm excitation.

[0070] The results are as follows Figure 2 shown. Figure 2 (A) is the fluorescence image of cells with 0 μg / mL IgG added; Figure 2 (B) is the fluorescence image of cells with 0.5 μg / mL IgG added; Figure 2 (C) is the fluorescence image of cells with 1 μg / mL IgG added; Figure 2 (D) is the fluorescence image of cells after adding 5 μg / mL IgG. Figure 2 As shown in the fluorescence images, cells exhibit bright fluorescence without IgG. However, after IgG addition, the fluorescence image dims, and the higher the IgG concentration, the darker the image. This is due to IgG quenching the fluorescence of the carbon dots. Fluorescence imaging experiments demonstrate that phenylboronic acid-modified carbon dots can detect changes in IgG concentration in living cells, demonstrating the feasibility of phenylboronic acid-modified carbon dots for IgG detection in living cells.

Claims

1. A fluorescent probe for detecting the content of immunoglobulins in complex biological samples, characterized in that: The fluorescent probe is a functionalized carbon dot modified with 4-carboxyphenylboronic acid.

2. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 1, characterized in that: The detection method adopts fluorescence quenching method.

3. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 2, characterized in that: The complex biological sample is a whole blood sample or a living cell sample.

4. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 3, characterized in that: The whole blood sample comes from a healthy organism or an organism infected with Cryptococcus. The pretreatment method of the whole blood sample is: diluting it 400 times with a phosphate buffer solution with a pH of 7.

4.

5. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 3, characterized in that: The method for using the fluorescent probe to detect the immunoglobulin content in a living cell sample is as follows: the living cell sample is incubated with the fluorescent probe for 0.5 to 4 hours, and the concentration of the fluorescent probe during incubation is 1 mg / mL.

6. A fluorescent probe for detecting immunoglobulin content in a complex biological sample according to any one of claims 1 to 5, characterized in that: The particle size of the fluorescent probe is 5.70-10.26 nm, and the optimal excitation and emission wavelengths are 460±5 nm and 525±5 nm respectively. Under the condition of 460 nm laser 300 W irradiation for 60 min, the variation of the fluorescence emission intensity at 525 nm is less than 1%.

7. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 6, characterized in that: The preparation method of the fluorescent probe comprises the following steps: 1) Using o-phenylenediamine as a carbon source, fluorescent carbon dots with rich surface amino functional groups, i.e., aminated carbon dots, were prepared; 2) Chemically bond 4-carboxyphenylboronic acid to amino carbon dots through covalent coupling to obtain functionalized modified carbon dots.

8. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 7, characterized in that: The amino content on the surface of the amino-modified carbon dots prepared in step 1) is 4 to 10 times the amino content on the surface of the functionalized modified carbon dots prepared in step 2).

9. A fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 7, characterized in that: Step 1) The specific steps are as follows: 1.1) Dissolve o-phenylenediamine in anhydrous ethanol to a concentration of 25-35 g / L, ultrasonicate for 5 minutes, add the resulting solution to a polytetrafluoroethylene reactor, place the reactor in an oven, and hydrothermally react at 180°C for 12 hours. Cool to room temperature to obtain a crude product. 1.2) Filter the crude product through a 0.22 μm filter membrane. Rotary evaporate the filtrate until the solvent is almost dry. Transfer any remaining material to a 1000 Da molecular weight cutoff regenerated cellulose dialysis bag and dialyze, changing the water every 4-8 hours, until the exudate is colorless and has a pH of 7. 1.3) The dialyzed product was dried in a vacuum oven for 24 h to obtain an orange-yellow powder, namely o-phenylenediamine carbon dots, which was stored at 4°C until use.

10. The fluorescent probe for detecting immunoglobulin content in complex biological samples according to claim 7, characterized in that: Step 2) The specific steps are as follows: 2.1) 60 mg of 4-carboxyphenylboronic acid, 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 24 mg of N-hydroxysuccinimide were placed in 30 mL of methanol and mixed well. After 1 hour, 70 mg of amino-containing carbon dots were added to the mixture and stirred in the dark for 18 hours. 2.2) The solution obtained in step 2.1) was poured into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 24 h. After dialysis, the solution was frozen in a refrigerator and freeze-dried for 24 h to obtain a light yellow powder as the final product.

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