A method for detecting glucose based on lyotropic liquid crystal droplets

Abstract

The application discloses a glucose detection method based on lyotropic liquid crystal droplets, polyethyleneimine is dissolved in 0.2M NaH2PO4-Na2HPO4 buffer solution with pH value of 7.8 to obtain a polyethyleneimine aqueous solution, cromolyn sodium is added, heated and uniformly mixed to obtain a polyethyleneimine doped cromolyn sodium aqueous solution, glucose oxidase is added to a to-be-detected glucose sample to obtain a mixed solution, the mixed solution is dropped into a liquid crystal cell composed of two parallel glass plates as a test group, meanwhile, blank liquid crystal cells, liquid crystal cells only dropping the to-be-detected glucose sample and liquid crystal cells only dropping a glucose oxidase aqueous solution are used as control groups, the polyethyleneimine doped cromolyn sodium aqueous solution is heated to 60 DEG C and then is dropped into the test group and the control group liquid crystal cells preheated to 60 DEG C, and the droplet morphology is observed under a polarizing microscope. Compared with the prior art, the method is simple in operation, free from current interference, low in cost and high in sensitivity.

Description

Technical Field

[0001] This invention relates to a glucose detection method based on lyotropic liquid crystal droplets, belonging to the fields of optics and biotechnology. Background Technology

[0002] Glucose is an essential component of the human body. Blood glucose levels are called blood sugar. Blood sugar must be maintained at a certain level to meet the needs of various organs and tissues. Normal blood plasma glucose levels are 3.9-7.8 mM, while levels exceeding 11 mM can cause hyperglycemia, leading to a series of complications. Since symptoms of hyperglycemia usually only appear after blood glucose concentrations have significantly increased to 15-20 mM, blood glucose monitoring is of paramount importance in medical diagnosis.

[0003] In existing technologies, there are various methods for glucose detection. Among them, biosensors based on liquid crystal droplet systems have unique advantages due to their high detection sensitivity and specific optical signals. Several methods for biosensoring using liquid crystal droplets have been proposed. For example, Kim et al. prepared thermotropic liquid crystal 5CB droplets using a microfluidic channel method and achieved glucose detection. However, the preparation method is relatively cumbersome and has certain limitations. Furthermore, due to the biotoxicity of thermotropic liquid crystals, it may not be effective in some biological solution environments.

[0004] Sodium cromoglycate is a white, odorless, powdery solid, typically possessing a plate-like aromatic core surrounded by hydrophilic groups. It is soluble in water, dissolving to a transparent, colorless state, exhibiting strong stability while possessing both fluidity and isotropic crystal properties. Within specific temperature and concentration ranges, its molecules stack to form an intermediate phase. With increasing concentration or decreasing temperature, the isotropic (I) phase solution can transform into a nematic (N), columnar (M), or rectangular (O) phase. Due to its biocompatibility, it plays an important role in many biological environments, such as micelles, liposomes, and cell membrane phospholipid bilayers. Detection systems based on sodium cromoglycate bypass the need for complex instruments and avoid electrical interference common in bioanalysis. For example, Shiyanovskii et al. utilized the orientation properties of sodium cromoglycate to detect immune complexes. This method uses the antigen-antibody complex to generate macromolecules that disrupt the parallel orientation of molecules, causing the reaction region to appear in different colors under a polarization microscope, making it observable. This method requires no external power supply, and the substance can remain active within it. Luk et al. found in their experiments that disodium cromoglycate does not disrupt biological membrane structures and used it as a carrier for antigen-antibody recognition detection. At low concentrations, the high birefringence and low viscosity of disodium cromoglycate exhibited excellent properties in immunocomplexation. Therefore, applying lyotropic liquid crystals to biosensing or detection offers advantages such as intuitiveness, no power required, simplicity of use, and minimal additional equipment. Currently, there are no reports on glucose detection based on lyotropic liquid crystal droplets. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of complex preparation methods and biocompatibility in existing liquid crystal sensing methods for glucose detection, and to provide a glucose detection method based on lyotropic liquid crystal droplets. This method involves doping a polyethyleneimine solution into a lyotropic liquid crystal disodium cromoglycate, which is simple to prepare, has good biocompatibility, and can achieve low-cost glucose detection.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] A glucose detection method based on lyotropic liquid crystal droplets includes the following steps:

[0008] (1) Dissolve polyethyleneimine in 0.2M NaH2PO4-Na2HPO4 buffer solution with pH 7.8 to obtain an aqueous solution of polyethyleneimine;

[0009] (2) Add disodium cromoglycate to the aqueous solution of polyethyleneimine, heat and mix evenly to obtain an aqueous solution of disodium cromoglycate doped with polyethyleneimine.

[0010] (3) Add glucose oxidase to the glucose sample to be tested to obtain a mixed solution, and drop it into a liquid crystal cell consisting of two parallel glass plates as the test group. At the same time, use a blank liquid crystal cell, a liquid crystal cell with only the glucose sample to be tested, and a liquid crystal cell with only glucose oxidase aqueous solution as the control group.

[0011] (4) The polyethyleneimine-doped sodium cromoglycate aqueous solution prepared in step (2) was heated to 60°C and then dropped into the test group and control group liquid crystal cells of step (3) that had been preheated to 60°C, respectively, to obtain pH-responsive lyotropic liquid crystal droplets. The droplet morphology in the test group and control group liquid crystal cells was observed under a polarization microscope to detect glucose. In the test group liquid crystal cell, after glucose undergoes an oxidation reaction with oxidase, the droplet morphology changes from bipolar to radial. The higher the proportion of droplets that change to radial, the higher the glucose content.

[0012] Furthermore, in step (1), the mass concentration of the polyethyleneimine aqueous solution is 1-20%.

[0013] Furthermore, in step (2), the mass concentration of the sodium cromoglycate solution doped with polyethyleneimine is 1-20%.

[0014] Furthermore, in step (2), the heating temperature is 70°C.

[0015] Furthermore, in step (3), the mass concentration of glucose oxidase in the mixed solution is 0.001–1%.

[0016] Furthermore, in step (3), the liquid crystal cell has a size of 1.2mm × 1.2mm and a thickness of 10±1μm.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention discloses a glucose detection method based on lyotropic liquid crystal droplets. This method is based on the biocompatible properties of lyotropic liquid crystals. It changes the pH environment in the medium by generating hydrogen ions from the glucose oxidation reaction, thereby achieving morphological changes in polyethyleneimine-sodium cromoglycate droplets and thus realizing glucose detection.

[0019] 2. Compared with existing detection methods, the detection method of the present invention has no current interference, low cost, and high sensitivity, and can achieve the detection of glucose at 1mM.

[0020] 3. This invention provides a new approach for the biosensing detection of glucose using lyotropic liquid crystals. Attached Figure Description

[0021] Figure 1Images of the test and control liquid crystal cells under a 50× polarizing microscope after adding an aqueous solution of disodium cromoglycate doped with polyethyleneimine in Example 1.

[0022] Figure 2 Images of the test and control liquid crystal cells under a 50× polarization microscope after adding an aqueous solution of disodium cromoglycate doped with polyethyleneimine in Example 2. Detailed Implementation

[0023] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the present invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from its essence are within the scope of the invention.

[0024] Example 1

[0025] A glucose detection method based on lyotropic liquid crystal droplets includes the following steps:

[0026] (1) Polyethyleneimine was dissolved in 0.2M NaH2PO4-Na2HPO4 buffer solution with a pH of 7.8 to prepare a 9.1% (w / w) aqueous solution of polyethyleneimine.

[0027] The preparation method of 0.2M NaH2PO4-Na2HPO4 buffer solution with pH value of 7.8 is as follows: Prepare a 0.2M NaH2PO4 solution and a 0.2M Na2HPO4 solution. Mix the NaH2PO4 solution and the Na2HPO4 solution at a volume ratio of 17:183, and then adjust the pH value to 7.8.

[0028] (2) Add 10 mg of disodium cromoglycate to 100 μL (100.81 mg) of polyethyleneimine aqueous solution, heat to 70 °C and vortex fully to obtain a polyethyleneimine-doped disodium cromoglycate aqueous solution with a mass concentration of 9.0%.

[0029] (3) Add glucose oxidase to a 20 mM glucose solution to obtain a mixed solution with a glucose oxidase concentration of 0.01%. Take 120 μL and drop it into a liquid crystal cell consisting of two parallel glass plates (the liquid crystal cell has a size of 1.2 mm × 1.2 mm and a thickness of 10 ± 1 μm) as the test group. At the same time, use a blank liquid crystal cell, a liquid crystal cell with only 120 μL of 20 mM glucose solution and a liquid crystal cell with only 120 μL of glucose oxidase solution with a mass concentration of 0.01% as the control group. All liquid crystal cells are sealed with photopolymer adhesive to prevent evaporation.

[0030] (4) After heating the polyethyleneimine-doped sodium cromoglycate aqueous solution prepared in step (2) to 60°C, drop it into the test group and control group liquid crystal cells of step (3) that have been preheated to 60°C using a TS102XY hot stage to obtain pH-responsive lyotropic liquid crystal droplets. Place the liquid crystal cells horizontally on the TS102XY hot stage, raise the temperature to 60°C first, and then slowly lower the temperature at a rate of 5°C / min. Observe the droplet morphology in the test group and control group liquid crystal cells under the 50× objective lens of a full-wave polarization microscope, and observe and calculate the ratio of radial droplets and bipolar droplets in the test group liquid crystal cell. When the vast majority of bipolar droplets are converted into radial droplets, it indicates that glucose reacts with oxidase. The higher the conversion ratio, the higher the glucose concentration.

[0031] Figure 1 Images of the test and control liquid crystal cells under a 50× polarizing microscope after adding an aqueous solution of disodium cromoglycate doped with polyethyleneimine in Example 1. Figure 1 (a) is the test group with added glucose and glucose oxidase. Figure 1 (b) is the blank liquid crystal cell control group. Figure 1 (c) is the control group that only received 20 mM glucose. Figure 1 (d) is the control group with only 0.01 wt% glucose oxidase added. It can be seen that in the control group without any solution, with only glucose solution, and with only glucose oxidase solution, bipolar droplets account for the vast majority. In the control group with glucose and glucose oxidase added, most of the bipolar droplets are obviously converted into radial droplets. This proves that this conversion is due to the oxidation reaction of glucose mixed with glucose oxidase, which is easy to distinguish and identify.

[0032] Example 2

[0033] A glucose detection method based on lyotropic liquid crystal droplets includes the following steps:

[0034] (1) Polyethyleneimine was dissolved in 0.2M NaH2PO4-Na2HPO4 buffer solution with a pH of 7.8 to prepare a 9.1% (w / w) aqueous solution of polyethyleneimine.

[0035] The preparation method of 0.2M NaH2PO4-Na2HPO4 buffer solution with pH value of 7.8 is as follows: Prepare a 0.2M NaH2PO4 solution and a 0.2M Na2HPO4 solution. Mix the NaH2PO4 solution and the Na2HPO4 solution at a volume ratio of 17:183, and then adjust the pH value to 7.8.

[0036] (2) Add 10 mg of disodium cromoglycate to 100 μL (100.81 mg) of polyethyleneimine aqueous solution, heat to 70 °C and vortex fully to obtain a polyethyleneimine-doped disodium cromoglycate aqueous solution with a mass concentration of 9.0%.

[0037] (3) Add glucose oxidase to a 1 mM glucose solution to obtain a mixed solution with a glucose oxidase concentration of 0.01%. Take 120 μL and drop it into a liquid crystal cell consisting of two parallel glass plates (the liquid crystal cell has a size of 1.2 mm × 1.2 mm and a thickness of 10 ± 1 μm) as the test group. At the same time, use a blank liquid crystal cell, a liquid crystal cell with only 120 μL of 1 mM glucose solution and a liquid crystal cell with only 120 μL of glucose oxidase solution with a mass concentration of 0.01% as the control group. All liquid crystal cells are sealed with photopolymer adhesive to prevent evaporation.

[0038] (4) After heating the polyethyleneimine-doped sodium cromoglycate aqueous solution prepared in step (2) to 60°C, drop it into the test group and control group liquid crystal cells of step (3) that have been preheated to 60°C using a TS102XY hot stage to obtain pH-responsive lyotropic liquid crystal droplets. Place the liquid crystal cells horizontally on the TS102XY hot stage, raise the temperature to 60°C first, and then slowly lower the temperature at a rate of 5°C / min. Observe the droplet morphology in the test group and control group liquid crystal cells under the 50× objective lens of a full-wave polarization microscope, and observe and calculate the ratio of radial droplets and bipolar droplets in the test group liquid crystal cell. When the vast majority of bipolar droplets are converted into radial droplets, it indicates that glucose reacts with oxidase. The higher the conversion ratio, the higher the glucose concentration.

[0039] Figure 2 These are images of the test and control liquid crystal cells under a 50× polarizing microscope after adding an aqueous solution of disodium cromoglycate doped with polyethyleneimine in Example 2. Figure 2 (a) is the test group with added glucose and glucose oxidase. Figure 2 (b) is the blank liquid crystal cell control group. Figure 2 (c) is the control group that only received 1 mM glucose. Figure 2 (d) is the control group with only 0.01 wt% glucose oxidase added. It can be seen that in the test group with both glucose and glucose oxidase, the bipolar droplets in the liquid crystal cell were significantly converted into radial droplets, indicating that polyethyleneimine-doped disodium cromoglycate droplets can achieve glucose detection with a sensitivity of 1 mM. When no significant change in droplet morphology is observed under a polarization microscope, the glucose concentration in the solution can be considered to be below 1 mM.

[0040] It should be noted that the present invention can also experimentally change the volume of the solution dropped into the liquid crystal cell, determine the critical volume corresponding to different glucose concentrations that causes droplet transformation, and use the glucose concentration at this point as a standard to determine the glucose concentration range of the test solution. If the solution does not cause droplet transformation, it can be considered that the glucose concentration in the solution is below this value.

[0041] In summary, this invention achieves pH control of the droplet morphology of polyethyleneimine-doped disodium cromoglycate, and enables specific glucose detection of lyotropic liquid crystals, making them easy to observe with the naked eye. It is expected to provide new assistance for the biosensing detection of lyotropic liquid crystals.

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A method for glucose detection based on lyotropic liquid crystal droplets, characterized in that, Includes the following steps: (1) Dissolve polyethyleneimine in 0.2M NaH2PO4-Na2HPO4 buffer solution with pH 7.8 to obtain an aqueous solution of polyethyleneimine; (2) Add disodium cromoglycate to the aqueous solution of polyethyleneimine, heat and mix evenly to obtain an aqueous solution of disodium cromoglycate doped with polyethyleneimine. (3) Add glucose oxidase to the glucose sample to be tested to obtain a mixed solution, and drop it into a liquid crystal cell consisting of two parallel glass plates as the test group. At the same time, use a blank liquid crystal cell, a liquid crystal cell with only the glucose sample to be tested, and a liquid crystal cell with only glucose oxidase aqueous solution as the control group. (4) After heating the polyethyleneimine-doped sodium cromoglycate aqueous solution prepared in step (2) to 60°C, it was dropped into the test group and control group liquid crystal cells of step (3) which had been preheated to 60°C to obtain pH-responsive lyotropic liquid crystal droplets. The droplet morphology in the test group and control group liquid crystal cells was observed under a polarization microscope to detect glucose.

2. The glucose detection method based on lyotropic liquid crystal droplets as described in claim 1, characterized in that, In step (1), the mass concentration of the polyethyleneimine aqueous solution is 1-20%.

3. The glucose detection method based on lyotropic liquid crystal droplets as described in claim 1, characterized in that, In step (2), the mass concentration of the sodium cromoglycate solution doped with polyethyleneimine is 1-20%.

4. The glucose detection method based on lyotropic liquid crystal droplets as described in claim 1, characterized in that, In step (2), the heating temperature is 70°C.

5. The glucose detection method based on lyotropic liquid crystal droplets as described in claim 1, characterized in that, In step (3), the mass concentration of glucose oxidase in the mixed solution is 0.001-1%.

6. The glucose detection method based on lyotropic liquid crystal droplets as described in any one of claims 1 to 5, characterized in that, In step (3), the liquid crystal cell has a size of 1.2mm × 1.2mm and a thickness of 10±1μm.

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