Method for producing stimulus-responsive hydrogel

Abstract

In a first step S101, the average particle size of a target molecule is measured. The average particle size of the target molecule can be measured by, for example, at least one of measurement by a small-angle X-ray scattering method, measurement by a dynamic light scattering method, and measurement by observation with an electron microscope. Next, in a second step S102, a stimulus-responsive hydrogel is produced using the measured average particle size of the target molecule as the lower limit value of the mesh size of the stimulus-responsive hydrogel.

Description

Method for preparing stimulus-responsive hydrogels

[0001] The present invention relates to a method for producing a stimulus-responsive hydrogel.

[0002] In recent years, the market for ICT devices that acquire one's own vital information has been rapidly expanding, driven by the promotion of home healthcare and self-medication policies (Non-Patent Literature 1). For this reason, there is a growing focus on developing device elements using biocompatible and functional organic materials in biosensors and actuators. For example, research is being conducted on incorporating hydrogels (stimulus-responsive hydrogels) that have properties similar to biological tissue and possess molecular recognition capabilities into chemical sensors (Non-Patent Literature 2). To improve the signal-to-noise ratio when using stimulus-responsive hydrogels as sensor materials, it is crucial to ensure robustness by preventing shape changes other than those related to stimulus response, while simultaneously diffusing the measurement molecules within the gel.

[0003] M. Dautta et al., "Passive and wireless, implantable glucose sensing with phenylboronic acid hydrogel-interlayer RF resonators", Biosensors and Bioelectronics, vol. 151, 112004, 2020.A. Ikeda et al., "Highly sensitive hydrogel-based biosensor with dielectric resonator structure for point-of-care testing", 45th International Conference of the IEEE Engineering in Medicine and Biology Society, vol. 2023.

[0004] So far, the target molecules for the stimulus-responsive hydrogels incorporated into sensors have only been low-molecular-weight molecules with a molecular size on the pm level such as glucose, and there have been no reported examples of macromolecules such as proteins with a molecular size on the order of several tens of nm, which is larger than this. The reason for this is that in order to diffuse large molecules such as proteins, which are the measurement targets, it is necessary to reduce the cross-linking density and widen the size of the network structure, resulting in a decrease in robustness. In this state, the robustness of the stimulus-responsive hydrogel is reduced. When using such a stimulus-responsive hydrogel with low robustness, it is considered that shape changes other than the stimulus response are superimposed on the measured values, resulting in a decrease in the S / N ratio.

[0005] The present invention has been made to solve the above problems, and an object thereof is to provide a highly robust stimulus-responsive hydrogel for measuring macromolecules such as proteins.

[0006] The method for producing a stimulus-responsive hydrogel according to the present invention is a method for producing a stimulus-responsive hydrogel composed of a polymer substance having a three-dimensional network structure and having a probe molecule that binds to a target molecule to be measured on a side chain, and includes a first step of measuring the average particle size of the target molecule, and a second step of producing a stimulus-responsive hydrogel using the measured average particle size of the target molecule as the lower limit value of the mesh size of the stimulus-responsive hydrogel.

[0007] As described above, according to the present invention, since the stimulus-responsive hydrogel is produced using the measured average particle size of the target molecule as the lower limit value of the mesh size of the stimulus-responsive hydrogel, a highly robust stimulus-responsive hydrogel for measuring macromolecules such as proteins can be provided.

[0008] FIG. 1 is a flowchart for explaining the method for producing a stimulus-responsive hydrogel according to an embodiment of the present invention. FIG. 2 is a photograph showing the result of observing the produced stimulus-responsive hydrogel with a confocal laser microscope. FIG. 3 is a characteristic diagram showing the relationship between the position of the observation depth by a confocal laser microscope of the actually produced stimulus-responsive hydrogel according to the embodiment and the intensity of red fluorescence indicating the amount of albumin.

[0009] The following describes a method for producing a stimulus-responsive hydrogel according to an embodiment of the present invention, with reference to Figure 1. This method produces a stimulus-responsive hydrogel composed of a polymer material having a three-dimensional network structure, and having probe molecules in its side chains that bind to the target molecule to be measured. The target molecule is a protein, and the probe molecule is a molecule that binds to proteins.

[0010] First, in the first step S101, the average particle size of the target molecule is measured. The average particle size of the target molecule can be measured by at least one of the following methods: small-angle X-ray scattering, dynamic light scattering, and electron microscopy.

[0011] Next, in the second step S102, a stimulus-responsive hydrogel is prepared using the measured average particle size of the target molecule as the lower limit of the mesh size of the stimulus-responsive hydrogel. For example, the mesh size can be controlled by the concentration of the monomers that make up the polymer material, and a stimulus-responsive hydrogel can be prepared using the average particle size of the target molecule as the lower limit of the mesh size.

[0012] Here, the raw materials for the polymeric substances described above can be monomers containing acrylamide, chitosan, acrylic acid, vinyl, gelatin, and alginic acid. The polymeric substances described above can be synthesized by polymerizing (addition polymerization) these monomers using a crosslinking agent. Alternatively, the polymeric substances described above can be synthesized by polycondensation of these monomers. Furthermore, the raw materials for the probe molecule can be at least one molecule of an amino group, carbonyl group, nitro group, hydroxyl group, or sulfonyl group. In addition, the stimulus-responsive hydrogel can have heteropolar molecules in its side chains that have a different polarity from the probe molecule that reacted with the substance to be measured.

[0013] For example, the polymeric substance constituting the stimulus-responsive hydrogel can be a crosslinked polymer obtained by polymerization (addition polymerization) using acrylamide as the monomer, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate as a photoradical polymerization initiator, and N-N'methyleneacrylamide as a crosslinking agent. The probe molecule can be 3-acrylamidephenylboronic acid. The heteropolar molecule can be N-[(3-dimethylamino)propyl]acrylamide. In this configuration, the mesh size can be controlled by the concentration of the crosslinking agent, and the stimulus-responsive hydrogel can be produced with the average particle size of the target molecule as the lower limit of the mesh size. Alternatively, the mesh size can be controlled by the concentration of the monomer, and the stimulus-responsive hydrogel can be produced with the average particle size of the target molecule as the lower limit of the mesh size.

[0014] [Examples] The following will provide a more detailed explanation using examples. Below, we will describe the observation results of the stimulus-responsive hydrogel that was actually prepared, and the results of measuring glycoalbumin (GA) as the measurement molecule using this stimulus-responsive hydrogel. Note that the GA molecule is of a size that is identical to bovine serum albumin and can be defined as having an elliptical shape of approximately 4.0 nm × 4.0 nm × 14.0 nm (References).

[0015] First, the average particle size of GA was measured using dynamic light scattering (reference). The measurement results showed that the average particle size was approximately 6.9 nm.

[0016] Next, a stimulus-responsive hydrogel was synthesized. Acrylamide was used as the monomer, 3-acrylamidephenylboronic acid as the probe molecule, and N-[(3-dimethylamino)propyl]acrylamide as the heteropolar molecule. This mixture was then polymerized (addition polymerization) by adding lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate as a photoradical polymerization initiator and N-N'methyleneacrylamide as a crosslinking agent.

[0017] By adjusting the concentration of the crosslinking agent, we synthesized a stimulus-responsive hydrogel sample with a mesh size larger than the measured average particle size (approximately 6.9 nm) and smaller than the major axis of GA (approximately 14.0 nm). The mesh size of the prepared sample was estimated by measuring the Young's modulus through compression testing, and it was found to be 12.49 nm, confirming that it was smaller than the major axis of GA. Furthermore, by adjusting the concentration of the crosslinking agent, we synthesized a comparative stimulus-responsive hydrogel sample with a mesh size identical to the major axis of GA (approximately 14.0 nm).

[0018] Figure 2 shows the results of observing the prepared sample and the comparison sample using a confocal laser microscope (CLSM). Figures 2(a) and (b) show the observation results of the prepared sample. Figures 2(c) and (d) show the observation results of the prepared comparison sample. As is clear from the comparison between Figures 2(a) and (b) and Figures 2(c) and (d), the prepared sample is in a more robust state, maintaining its shape better.

[0019] Furthermore, when the prepared stimulus-responsive hydrogel samples were immersed in an albumin solution having a molecular structure equivalent to that of GA, observation using a confocal laser microscope confirmed that albumin had diffused into the interior of the stimulus-responsive hydrogel. Figure 3 shows the relationship between the observation depth of the stimulus-responsive hydrogel and the red fluorescence intensity indicating the amount of albumin.

[0020] As described above, according to the embodiment of the present invention, the average particle size of the measured target molecule is used as the lower limit of the mesh size of the stimulus-responsive hydrogel to produce the stimulus-responsive hydrogel, thereby providing a highly robust stimulus-responsive hydrogel for measuring macromolecules such as proteins.

[0021] It should be noted that the present invention is not limited to the embodiments described above, and it is clear that many modifications and combinations can be implemented within the technical concept of the present invention by those with ordinary skill in the art.

[0022] [References] Tomoe Nakagawa et al., "Development of a biosensing system using a dynamic light scattering device," Research Report of Tokyo Metropolitan Industrial Technology Research Center, No. 6, pp. 108-109, 2011.

Claims

1. A method for producing a stimulus-responsive hydrogel, comprising a polymer material having a three-dimensional network structure and having probe molecules in its side chains that bind to a target molecule to be measured, the method comprising: a first step of measuring the average particle size of the target molecule; and a second step of producing a stimulus-responsive hydrogel using the measured average particle size of the target molecule as the lower limit of the network size of the stimulus-responsive hydrogel.

2. A method for producing a stimulus-responsive hydrogel according to claim 1, wherein the average particle size of the target molecule is measured by at least one of the following methods: measurement by small-angle X-ray scattering, measurement by dynamic light scattering, and measurement by observation with an electron microscope.

3. A method for producing a stimulus-responsive hydrogel according to claim 1, wherein the target molecule is a protein and the probe molecule is a molecule that binds to a protein.

4. A method for producing a stimulus-responsive hydrogel according to any one of claims 1 to 3, wherein the raw material for the polymer substance is a monomer containing acrylamide, chitosan, acrylic acid, vinyl, gelatin, and alginic acid, and the raw material for the probe molecule is at least one molecule of an amino group, a carbonyl group, a nitro group, a hydroxyl group, and a sulfonyl group.

5. A method for producing a stimulus-responsive hydrogel according to claim 4, wherein the second step is to use acrylamide as the raw material for the polymer substance, 3-acrylamidephenylboronic acid as the raw material for the probe molecule, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate as a photopolymerization initiator, and N-N'methyleneacrylamide as a crosslinking agent to produce a stimulus-responsive hydrogel.

6. A method for producing a stimulus-responsive hydrogel according to claim 5, wherein the second step is to control the mesh size by the concentration of the crosslinking agent or the concentration of the polymer substance.

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