Use of methylene blue cyclopropanecarboxylated derivatives as chromogenic substrate for the detection of hematin
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FOSHAN UNIVERSITY
- Filing Date
- 2026-04-22
- Publication Date
- 2026-07-24
Smart Images
Figure CN122448831A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of quantitative heme detection technology, and in particular to the application of methylene blue cyclopropionyl derivatives as chromogenic substrates for heme detection. Background Technology
[0002] Heme is formed by the complexation of protoporphyrin IX with iron ions (II / III) and serves as a cofactor for hemoglobin and various enzymes. Hemin chloride is the most common small-molecule form of heme and has been used as an iron supplement in the development of anemia-enhancing health products. It is also an important raw material for anti-anemia and anti-tumor drugs. Due to the wide range of applications of heme, its accurate quantification is of significant practical importance. Furthermore, in clinical practice, fecal occult blood testing primarily detects hemoglobin and heme. Hemoglobin is gradually digested and broken down into heme in the intestines and excreted. The amount of free small-molecule heme divided by the total amount of heme + hemoglobin is the heme intestinal conversion rate. The higher the site of gastrointestinal bleeding, the longer the hemoglobin is digested, and the higher the heme intestinal conversion rate. Therefore, the location of intestinal bleeding can be roughly estimated from the intestinal conversion rate. Based on this, the accurate quantification of small-molecule heme with high sensitivity and selectivity is particularly important.
[0003] Currently, the most convenient and widely used method for quantifying heme is colorimetry, with TMB (3,3',5,5'-tetramethylbenzidine) being the most commonly used substrate. The basic principle of TMB detection can be summarized as follows: heme catalyzes the oxidation of colorless TMB by H₂O₂, producing a blue oxidation product. The amount of heme can be reflected by measuring the absorbance at 650 nm. However, the classic TMB test cannot distinguish heme from other iron-containing compounds, and its detection results are affected by the presence of hemoglobin, peroxidase, and Fe in the body. 2+ and Fe 3+ The serious impact.
[0004] Therefore, it is of great significance to develop new detection methods with high selectivity for the heme / H2O2 reaction. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides the application of methylene blue cyclopropionyl derivatives as chromogenic substrates for detecting heme.
[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: The application of methylene blue cyclopropionyl derivatives as chromogenic substrates for the detection of hemin: Methylene blue cyclopropionyl derivative AMB is used as a chromogenic substrate for the detection of hemin (Hemin). The structural formula of the methylene blue cyclopropionyl derivative AMB is as follows: .
[0007] Specifically, excess H2O2 is mixed with 100-400 μM methylene blue cyclopropionyl derivative AMB, and then a sample containing 2-5 μM hemin is added to catalyze the reaction. After the reaction solution is placed at room temperature in the dark for 30 min, a blue oxidation product is generated. The absorbance at 664 nm wavelength is read using a multi-functional microplate reader to achieve quantitative detection of hemin.
[0008] Furthermore, the preparation process of the methylene blue cyclopropionyl derivative AMB is as follows: 1 mmol of methylene blue was dissolved in 15 mL of tetrahydrofuran, and 2 mmol of stannous chloride dihydrate was added. The mixture was stirred at 60 °C for 1 h, and then 3 mmol of triethylamine (TEA) and 3 mmol of cyclopropionyl chloride were added. The mixture was stirred for 15 min and then extracted with water and ethyl acetate to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the methylene blue cyclopropionyl derivative AMB.
[0009] Compared with existing technologies, the methylene blue cyclopropionylated derivative AMB of the present invention exhibits higher sensitivity in the detection of heme (Hemin) compared to the classic substrate TMB. Furthermore, the amino proton of the methylene blue cyclopropionylated derivative AMB of the present invention is fully substituted with a methyl group, inhibiting oxidation involving reactive oxygen species, while still remaining sensitive to the ferric radical oxidation pathway, thus showing better selectivity than TMB. The AMB system can effectively detect heme (Hemin) in serum samples. Therefore, it can be said that AMB is a superior colorimetric substrate than TMB for the detection of heme (Hemin). Attached Figure Description
[0010] Figure 1 The results of the AMB / H2O2 system and the TMB / H2O2 system for the detection of different concentrations of hemin are compared.
[0011] Figure 2 (a) Hemin concentration-dependent absorbance changes in the AMB system, with the inset showing the linear fit in the concentration range of 0.25 μM - 8.0 μM; (b) Hemin concentration-dependent absorbance changes in the TMB system, with the inset showing the linear fit in the concentration range of 0.25 μM - 8.0 μM.
[0012] Figure 3 The results show the selectivity comparison of the AMB and TMB reaction systems for the detection of hemin.
[0013] Figure 4 Comparison of AMB and TMB results for the detection of hemin in serum. Detailed Implementation
[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.
[0015] The chemical structure of the common form of heme (Hemin) is as follows: .
[0016] It has been used as an iron supplement in the development of anemia-enhancing health products, and is also an important raw material for anti-anemia and anti-tumor drugs. The most commonly used colorimetric substrate is TMB (3,3',5,5'-tetramethylbenzidine). The basic principle of its detection can be summarized as follows: Heme catalyzes the oxidation of colorless TMB by H₂O₂, producing a blue oxidation product. The amount of heme can be reflected by measuring the absorbance at 650 nm. The oxidation pathway of TMB in the heme / H₂O₂ system is as follows: .
[0017] As can be seen from the above reaction pathway, the H2O2 reaction of heme can form two types of free radicals: heme ferric free radicals and reactive oxygen species (such as hydroxyl radicals). and superoxide anion These two types of free radicals dominate two different oxidation pathways. The classic substrate TMB can be oxidized via the ferric radical pathway. Furthermore, the amino group of TMB contains both a proton and a lone pair of electrons, making it more susceptible to attack by reactive oxygen species (ROS), resulting in a blue oxidation product. It is well known that ROS can be generated through various pathways, such as Fe... 2+ The Fenton reaction with H2O2 results in poor selectivity of TMB for heme / H2O2. Building upon this, the present invention develops a methylene blue cyclopropionylated derivative, AMB. AMB can be prepared using commercially available and inexpensive methylene blue (MB) as a starting material via the following synthetic route: 284 mg (1 mmol) of methylene blue was dissolved in 15 mL of tetrahydrofuran, and 451.28 mg (2 mmol) of stannous chloride dihydrate was added. The mixture was stirred at 60 °C for about 1 h, and then 417 μL (3 mmol) of triethylamine (TEA) and 272 μL (3 mmol) of cyclopropionyl chloride were added. The mixture was stirred for about 15 min, and then extracted with water and ethyl acetate. The crude product was purified by silica gel column chromatography to obtain AMB in a yield of about 50%.
[0018] ; In this embodiment, AMB / H2O2 and TMB / H2O2 systems were constructed respectively. The system was used to detect different concentrations of hemin. Preliminary experiments were conducted on the detection system, and the detection conditions were optimized to determine the following test conditions: the reaction solution for AMB was 0.04 M borate-sodium hydroxide buffer (BR buffer, pH 7.4), and the buffer for TMB was selected as 0.2 M acetate-sodium acetate buffer with pH 4.5, based on previously reported conditions. H2O2 (8 mM) and AMB (400 μM) solutions were prepared using BR buffer, and H2O2 (8 mM) and AMB (400 μM) solutions were prepared using acetate buffer. Simultaneously, a series of hemin solutions with concentrations ranging from 0.25 μM to 8.0 μM were prepared using both buffers. During testing, 50 μL of substrate solution was mixed with 50 μL of H2O2 first, followed by the addition of 100 μL of hemin solution. After the reaction was carried out at room temperature for 30 min, the absorbance was read using a microplate reader. The AMB reading wavelength was 664 nm, and the TMB reading wavelength was 650 nm. The detection results are as follows: Figure 1 and Figure 2 As shown, Figure 1 The AMB reaction showed a distinct blue color in the solution, with the blue color deepening gradually with increasing heme (Hemin) concentration. Conversely, in the TMB reaction, the blue color gradually intensified at low heme (Hemin) concentrations, and when the heme (Hemin) concentration exceeded 32 μM, the solution exhibited a blue-to-yellow transition, with a significant decrease in absorbance at 650 nm. This suggests that the blue product of TMB may be unstable during the continuous reaction. Figure 2 The linear fitting results showed that in both systems, when the heme concentration was in the range of 0.25 μM–8 μM, there was a good linear correlation between absorbance and heme concentration. The slope of the linear fit for AMB was 0.15, while the slope for TMB was 0.026, with the former being 5.8 times that of the latter. Generally, a larger slope indicates higher sensitivity; therefore, the AMB system exhibits higher sensitivity.
[0019] Furthermore, after obtaining the linear relationship between absorbance and heme concentration, this embodiment selects several interfering substances that may affect the actual test for testing: Heme (Hemin) at a concentration of 5 μM is added to the above AMB / H2O2 and TMB / H2O2 systems, along with substances such as amino acids, peroxidase, hemoglobin, and Fe. 2+ Metal ions, specifically such as Figure 3 As shown, HRP = 10 ng / mL; catalase (CAT) = 8.3 μg / mL, hemoglobin (Hb) = 3.2 μg / mL, H2O2 = 2 mM, and the concentration of all other analytes was 50 μM. The selectivity of the reaction system was determined by measuring the absorbance of the AMB / H2O2 and TMB / H2O2 reaction solutions to assess whether the reaction system responded to these substances. The test results are shown below. Figure 3 As shown. Figure 3 This indicates that TMB exhibits good colorimetric responses to a variety of catalysts, with its response to horseradish peroxidase (HRP) being significantly greater than that to heme (Hemin). Furthermore, the TMB system also responds well to hemoglobin and Fe... 2+ and Fe 3+ It also exhibits a good response. The newly prepared AMB substrate in this embodiment shows the best responsiveness to heme (Hemin), a weak response to high concentrations of Hb, and no response to any other molecules. Therefore, AMB / H2O2 is an excellent colorimetric detection system for heme (Hemin), possessing selectivity unmatched by the classic TMB method. The main principle is that the amino protons of AMB are fully substituted by methyl groups, inhibiting oxidation involving reactive oxygen species, while remaining sensitive to the ferric radical oxidation pathway.
[0020] Finally, the hemin added to the serum was tested using AMB and TMB, respectively. Commercial fetal bovine serum (FBS) was diluted 10-fold with detection buffer, and the reliability of the detection was verified by adding a standard concentration of hemin to the diluted FBS. The recovery rate and the relative standard deviation (RSD) of the three tests were calculated as follows: Figure 4 As shown in Table 1.
[0021] Table 1. Results of spiked recovery experiment of Hemin in serum using AMB / H2O2 (n = 3) ; Depend on Figure 4It was observed that when serum without heme (Hemin) was mixed with TMB / H2O2, the reaction solution exhibited a distinct blue color. This indicates that TMB may be catalyzed and oxidized by certain components in the serum, which could be iron ions or trace amounts of hemoglobin. In the system without the addition of heme (Hemin), AMB showed almost no response; however, upon the addition of 5 μM heme (Hemin), the reaction solution rapidly turned blue.
[0022] As shown in Table 1, this method can effectively quantify Hemin when the spiking concentration of Hemin in the AMB / H2O2 system is greater than 1 μM. However, the recovery rate is low in the detection of 1 μM Hemin.
[0023] The above results indicate that TMB lacks the ability to detect Hemin from complex serum samples, while AMB can effectively detect it. In further experiments, we added different concentrations of Hemin to serum and quantified it using AMB / H₂O₂. Figure 2 The linear equation shown in (a) yielded quantitative results for Hemin and the recovery rate was calculated.
[0024] In summary, AMB exhibits higher sensitivity than the classic substrate TMB in the detection of hemin; moreover, it has better selectivity than TMB. The AMB system can effectively detect hemin in real serum samples.
[0025] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.
Claims
1. The application of methylene blue cyclopropionyl derivatives as chromogenic substrates for the detection of heme, characterized in that, The methylene blue cyclopropionyl derivative AMB was used as a chromogenic substrate for the detection of hemin. The structural formula of the methylene blue cyclopropionyl derivative AMB is as follows: 。 2. The application according to claim 1, characterized in that, Excess H2O2 was mixed with 100-400 μM methylene blue cyclopropionyl derivative AMB, and then the sample to be tested containing 2-5 μM hemin was added to carry out the catalytic reaction. After the reaction solution was placed at room temperature in the dark for 30 min, a blue oxidation product was generated. The absorbance at a wavelength of 664 nm was read using a multi-functional microplate reader to realize the quantitative detection of hemin.
3. The application according to claim 1, characterized in that, The preparation process of the methylene blue cyclopropionyl derivative AMB is as follows: 1 mmol of methylene blue was dissolved in 15 mL of tetrahydrofuran, and 2 mmol of stannous chloride dihydrate was added. The mixture was stirred at 60 °C for 1 h, and then 3 mmol of triethylamine (TEA) and 3 mmol of cyclopropionyl chloride were added. The mixture was stirred for 15 min and then extracted with water and ethyl acetate to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the methylene blue cyclopropionyl derivative AMB.