A time-resolved fluorescence immunoassay sensor for glycated hemoglobin

Abstract

The present invention belongs to the field of medical immunology. In particular, it relates to the field of detecting glycated hemoglobin (HbA1c) using a time-resolved fluorescence immunosensor. The method includes the following steps: synthesis of a functionalized Eu<supgt;3+< / supgt; chelate; optimization of the enhancement solution; establishment of an HbA1c standard curve; application of the sensor. The present invention intends to utilize the specific recognition of the glycosylated part of glycated hemoglobin by boric acid to synthesize a functionalized Eu<supgt;3+< / supgt> chelate that can specifically recognize glycated hemoglobin. At the same time, by means of the highly specific affinity of antigen-antibody, a time-resolved fluorescence immunosensor with high sensitivity, strong specificity, convenience, and rapidity is constructed to provide a new method for the quantitative detection of glycated hemoglobin.

Description

Technical Field

[0001] The present invention belongs to the field of medical immunology, and particularly relates to the field of detecting glycated hemoglobin using a time-resolved fluorescence immunosensor. Background Art

[0002] Glycated hemoglobin (HbA1c) can reflect the average blood glucose level over 2 - 3 months, is the most commonly used biomarker for tracking type II diabetes, and is also an important basis for the early diagnosis of diabetes. Due to its important clinical research significance, the quantitative detection of glycated hemoglobin has also received extensive attention from researchers.

[0003] Currently, a variety of detection methods for HbA1c have been reported, including ion exchange chromatography, borate affinity chromatography, electrophoresis, immunoassay, and enzymatic methods. Among them, high-performance liquid chromatography (HPLC) is the standard method for HbA1c.

[0004] Although the HPLC method is relatively mature, the sample volume required by this method is relatively large, and the pretreatment of blood samples is also relatively complex, requiring staff with professional operation experience to complete, and is suitable for large hospitals and research institutions. For remote areas or places where instruments and personnel conditions are not available, the application of this detection method still has certain difficulties.

[0005] The immunoassay based on the specific binding of antigen and antibody has the advantages of high sensitivity, strong specificity, small sample volume, and simple operation, and also has good application in the detection field of HbA1c. And the time-resolved fluorescence immunoassay is a method with higher sensitivity among immunoassays due to its unique detection principle, and is considered to be one of the most promising immunoanalysis methods.

[0006] Based on the above, the present invention intends to synthesize a functionalized Eu3+ chelate that can specifically recognize glycated hemoglobin by using the specific recognition of the glycosylated part of glycated hemoglobin by boric acid, and construct a time-resolved fluorescence immunosensor with high sensitivity, strong specificity, convenience, and rapidity, so as to provide a new method for the quantitative detection of glycated hemoglobin. Summary of the Invention

[0007] The present invention constructs a time-resolved fluorescence immunosensor for glycated hemoglobin based on the principle of the specific recognition of glycated hemoglobin by boric acid. This method is not only sensitive, specific, and accurate, but also has a small sample volume, simple operation, and convenient data reading.

[0008] To achieve the above object, the present invention is realized through the following technical solutions:

[0009] A. Coating: Add the dilution of hemoglobin monoclonal antibody to the black enzyme-labeled wells, with 3 replicate wells, incubate, and wash 3 times.

[0010] B. Blocking: Add blocking solution to the above-mentioned pre-coated enzyme-linked immunosorbent assay (ELISA) plate, incubate, and wash three times.

[0011] C. Sample addition: Add HbA1c dilution to the above-mentioned blocked ELISA wells, incubate, and wash three times.

[0012] D. Addition of functionalized Eu3+ chelate: Add the functionalized Eu 3+ chelate to the above-mentioned ELISA wells, incubate, and wash three times.

[0013] E. Addition of acid enhancement solution: Add acid enhancement solution to the above-mentioned ELISA plate, incubate, and detect the fluorescence signal.

[0014] The concentration of the hemoglobin monoclonal antibody in step A is 1 μg / mL, and the addition amount is 100 μL.

[0015] The incubation conditions of the hemoglobin monoclonal antibody in step A are 37 °C for 2 h.

[0016] The composition of the blocking solution in step B is BSA Tris-HCl buffer.

[0017] The added volume of the blocking solution in step B is 200 μL, and the incubation conditions are 37 °C for 2 h.

[0018] The volume of the sample solution in step C is 100 μL, and the incubation conditions are 37 °C for 0.5 h.

[0019] The functionalized Eu3+ chelate in step D is Eu-DTPA-2APBA, that is, the Eu3+ chelate linked with phenylboronic acid, and its structural formula is shown in formula (1):

[0020]

[0021] Formula (1): Structure of the functionalized Eu3+ chelate

[0022] The synthesis method of the functionalized Eu 3+ chelate in step D is as follows: Weigh aminophenylboronic acid hemisulfonate (APBA) and diethylenetriaminepentaacetic dianhydride (DTPA), dissolve them in anhydrous DMSO, stir at room temperature under nitrogen protection, and purify to obtain DTPA-2APBA after the reaction is complete. Then reflux the reaction product with europium trichloride, and purify after the reaction is complete to obtain it.

[0023] Furthermore, in the synthesis method of the functionalized Eu 3+ chelate in step D, the molar ratio of aminophenylboronic acid hemisulfonate (APBA) to diethylenetriaminepentaacetic dianhydride (DTPA) is 3:1, and the reaction time is 90 min.

[0024] Further, in the synthesis method of the functionalized Eu 3+ chelate, the molar ratio of DTPA-2APBA to europium trichloride is 1:1.2, and the reaction time is 90 min.

[0025] In the step D, the functionalized Eu 3+ chelate solution has a volume of 100 μL, a concentration of 10 μg / mL, and the incubation conditions are 37°C for 0.5 h.

[0026] In the step E, the acid-enhancing solution includes β-thienoyltrifluoroacetone (TTA), trioctylphosphine oxide (TOPO), and Triton X-100.

[0027] Further, the method for screening the concentration of β-thienoyltrifluoroacetone (TTA) in the acid-enhancing solution in the step E is as follows: Add 24 μL of TOPO concentrated stock solution, 40 μL of Triton X-100 concentrated stock solution, 59 μL of glacial acetic acid, and 0.5 mL of potassium hydrogen phthalate concentrated stock solution to each EP tube. Then, add different volumes of TTA concentrated stock solution to form an enhancing solution with different concentrations of TTA. Then, in the EuCl3 solution, add the enhancers with different concentration gradients of TTA in sequence. Let it stand for 10 min, and measure the fluorescence intensity with a fluorescence spectrophotometer. Each sample is repeated in parallel 3 times.

[0028] Further, the method for screening the concentration of trioctylphosphine oxide (TOPO) in the acid-enhancing solution in the step E is as follows: Add 100 μL of TTA concentrated stock solution, 40 μL of Triton X-100 concentrated stock solution, 59 μL of glacial acetic acid, and 0.5 mL of potassium hydrogen phthalate concentrated stock solution to each EP tube. Then, add different volumes of TOPO concentrated stock solution to form an enhancing solution with different concentrations of TOPO. Then, in the EuCl3 solution, add the enhancers with different concentration gradients of TOPO in sequence. Let it stand for 10 min, and measure the fluorescence intensity with a fluorescence spectrophotometer. Each sample is repeated in parallel 3 times.

[0029] Further, the method for screening the concentration of Triton X-100 in the acid-enhancing solution in the step E is as follows: Add 100 μL of TTA concentrated stock solution, 12 μL of TOPO concentrated stock solution, 59 μL of glacial acetic acid, and 0.5 mL of potassium hydrogen phthalate concentrated stock solution to each EP tube. Then, add different volumes of Triton X-100 concentrated stock solution to form an enhancing solution with different concentrations of Triton X-100. Then, in the EuCl3 solution, add the enhancers with different concentration gradients of Triton X-100 in sequence. Let it stand for 10 min, and measure the fluorescence intensity with a fluorescence spectrophotometer. Each sample is repeated in parallel 3 times.

[0030] In step E, the volume of the acid-enhancing solution is 200 μL / well, and the incubation conditions are 37 °C for 15 min.

[0031] In step E, the fluorescence intensity signal is read out at 615 nm by a multi-functional microplate reader, and its excitation wavelength is 340 nm.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1) The present invention provides a time-resolved immunosensor for detecting glycated hemoglobin. The sensor utilizes the specific recognition of boric acid with the glycosylated part of glycated hemoglobin and combines the characteristics of antigen-antibody immune affinity. The constructed sensor has high sensitivity and strong specificity.

[0034] 2) The sensor designed by the present invention requires a small amount of sample, simple sample treatment, convenient operation, rapid detection, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0036] Figure 1 Schematic diagram of the detection of glycated hemoglobin by the TRFIA sensor

[0037] Figure 2 Linear relationship diagram of the fluorescence intensity at a wavelength of 615 nm with the change of HbA1c concentration

[0038] Figure 3 Selectivity of the TRFIA sensor for HbA1c

[0039] Figure 4 Bar chart comparing the detection methods of the TRFIA sensor and a commercially available glycated hemoglobin kit DETAILED DESCRIPTION OF THE INVENTION

[0040] All kinds of instruments and reagents not specifically described in the present invention are well-known commercially available products in the art and can be obtained through commercial channels.

[0041] The following is a further clarification of the present invention in terms of specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art to the present invention fall within the scope defined by the appended claims of this application. To understand the present invention, the following is a further detailed description of the present invention.

[0042] In the implementation cases, if the specific test conditions and test methods are not specified, they shall be implemented according to the conventional conditions and methods or the conditions recommended by the manufacturer.

[0043] The following further illustrates the sensor of the present invention, its preparation method, and application in combination with the accompanying drawings and examples.

[0044] Example 1: Establishment of the HbA1c standard curve, referring to the appendix Figure 1-2 。

[0045] The object of the present invention is to provide a sensor based on boric acid derivatives and time-resolved immunoassay method and its preparation method for the deficiencies of existing glycated hemoglobin detection methods, and a method for rapidly and simply qualitatively or quantitatively detecting glycated hemoglobin by specifically recognizing glycated hemoglobin with this sensor. The standard curve for detecting glycated hemoglobin by this time-resolved immunosensor includes the following steps:

[0046] 1) Dilute the concentrated stock solution of hemoglobin monoclonal antibody to 1 μg / mL, and add 100 μL of it to the black enzyme-labeled wells, repeating 3 replicate wells, and incubate at 37 °C for 2 h. Take out the solution in the wells and wash 3 times repeatedly.

[0047] 2) Add 200 μL of blocking solution to each well in the enzyme-labeled plate coated in step 1), incubate at 37 °C for 2 h, and wash 3 times.

[0048] 3) Add samples to the enzyme-labeled plate blocked in step 2): Dilute the concentrated stock solution of HbA1c in ten gradients of 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000, 1:50000, 1:60000, 1:70000, 1:80000. Add 100 μL of HbA1c dilution solution with different concentrations to each well in the above-mentioned blocked enzyme-labeled wells, incubate at 37 °C for 0.5 h, and wash 3 times.

[0049] 4) Add Eu-DTPA-2APBA to the enzyme-labeled plate with samples added in step 3), incubate at 37 °C for 0.5 h, and wash 3 times.

[0050] 5) Add the acid-enhancing solution to the enzyme-labeled plate with the Eu3+ chelate added in step 4), and incubate at 37 °C for 15 min.

[0051] 6) Fluorescent detection is performed on the well plate processed in 5), with an excitation wavelength of 340 nm and an emission wavelength of 615 nm.

[0052] 7) A standard curve of the fluorescence data measured in 6) against the glycated hemoglobin concentration is made, and the test results are as Figure 2 .

[0053] Example 2: Selectivity experiment of the TRFIA sensor for HbA1c, referring to the appendix Figure 3 .

[0054] 1) The specific operation is similar to that in part of Example 1, only replacing the HbA1c concentrated stock solution with 0.1 mM glucose (Gul), galactose (Gal), fructose (Fru) solutions, 0.01 mM dopamine (DA), ascorbic acid (VC) solutions, as well as 25 μM Hb solution, 1 μM BSA, HbA1c solutions. The added volumes are the same, and other operations are the same. Parallel samples are repeated 3 times.

[0055] 2) The fluorescence signal intensity values at 615 nm obtained are compared to judge the selective specificity of this method for glycated hemoglobin, and the test results are as Figure 3 .

[0056] Example 3: Determination of HbA1c concentration in hemolyzed blood, referring to the appendix Figure 4 .

[0057] 1) Take 10 μL of hemolyzed blood and dilute it 20,000 times as the test sample.

[0058] 2) The specific operation is similar to that in part of Example 1, only replacing the HbA1c concentrated stock solution with the hemolyzed dilution for testing. The added volumes are the same, and other operations are the same. Parallel samples are repeated 3 times.

[0059] 3) Substitute the results measured in 2) into the calculation formula: Calculate the HbA1c concentration in the hemolyzed blood and compare it with the test results of a commercially available HbAlc kit. The test results are as Figure 4 .

Claims

1. A sensor for detecting glycated hemoglobin by time-resolved immunoassay, characterized in that, It includes the following steps: 1) Coating: Add the hemoglobin monoclonal antibody diluent into the black enzyme-labeled wells, with 3 replicate wells, incubate, and wash 3 times; 2) Blocking: Add the blocking solution into the above-mentioned enzyme-labeled plate that has been coated, incubate, and wash 3 times; 3) Sample addition: Add the HbA1c diluent into the above-mentioned enzyme-labeled wells that have been blocked, incubate, and wash 3 times; 4) Functionalized Eu 3+ Chelate: Add the functionalized Eu3+ chelate to the above enzyme-labeled wells, incubate, and wash three times; the functionalized Eu 3+ Chelate is Eu-DTPA-2APBA, that is, Eu chelate linked with phenylboronic acid, and its structural formula is shown in Formula (1): 3+ ​ 5) Add acidic enhancement solution: Add the acidic enhancement solution into the above-mentioned enzyme-labeled plate, incubate, and detect the fluorescence signal.

2. The method for detecting glycated hemoglobin by using a time-resolved immunosensor according to claim 1, wherein The concentration of the hemoglobin monoclonal antibody is 1 μg / mL, and the addition amount is 100 μL.

3. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The incubation conditions of the hemoglobin monoclonal antibody are 37 °C for 2 h.

4. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The composition of the blocking solution is Tris-HCl buffer of BSA.

5. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The added volume of the blocking solution is 200 μL, and the incubation conditions are 37 °C for 2 h.

6. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The volume of the HbA1c diluent is 100 μL, and the incubation conditions are 37 °C for 0.5 h.

7. A method for detecting glycated hemoglobin by using a time-resolved immunosensor according to claim 1, characterized in that, The volume of the functionalized Eu3+ chelate solution is 100 μL, the concentration is 10 μg / mL, and the incubation conditions are 37 °C for 0.5 h.

8. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The acidic enhancement solution includes β-thienoyltrifluoroacetone (TTA), trioctylphosphine oxide (TOPO), and Triton X-100.

9. A method for detecting glycated hemoglobin using a time-resolved immunosensor according to claim 1, characterized in that, The volume of the acidic enhancement solution is 200 μL / well, the incubation conditions are 37 °C for 15 min; the fluorescence intensity signal is read out by a multifunctional microplate reader at 615 nm, and its excitation wavelength is 340 nm.

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