Preparation method of hydrogel particles for benthic shellfish behavior observation and application of hydrogel particles in artificial matrix

Abstract

The invention discloses a preparation method of hydrogel particles for benthic shellfish behavior observation and application of the hydrogel particles in an artificial matrix, and the preparation method is characterized by comprising the following steps: adding acrylamide and N, N '-methylenebisacrylamide into deionized water for dissolving, sequentially adding potassium persulfate and tetramethyl diethylamine to obtain a hydrogel solution, and carrying out ultrasonic treatment to obtain hydrogel particles; and transferring the hydrogel solution to a circular mold, standing in a fuming cupboard to form solid particles, washing and drying in a drying oven to obtain the hydrogel particles for behavior observation of the benthic shellfish, the addition concentration of acrylamide in the hydrogel solution is 60-65% of the mass of the deionized water, and the addition concentration of acrylamide in the hydrogel solution is 5-10% of the mass of the deionized water. The invention also provides application of the hydrogel particle in preparation of a benthic shellfish behavior observation artificial matrix, and the hydrogel particle has the advantages of high density, high transparency, no toxicity and the like, and can be effectively suitable for observing behavior tracks of benthic organisms.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for preparing hydrogel particles for observing the behavior of benthic mollusks and their application in artificial matrices. Background Technology

[0002] Burrowing mollusks, such as razor clams ( Sinonovacula constricta ), mud clam ( Tegillarca granosa ) and clams ( Meretrix meretrix Burrowing behavior of burrowing mollusks is an important marine mollusk species that inhabits the intertidal zone or shallow sea ecosystems and has significant economic and nutritional value. The burrowing behavior of burrowing mollusks is of great significance for studying population distribution, energy metabolism, and environmental response mechanisms. It is also an important window for analyzing the adaptive evolution of benthic organisms. However, the observation of the behavior of burrowing mollusks has long faced technical bottlenecks: (1) Visualization is difficult. The activities of mollusks are completely buried in opaque sediments, and traditional photography or visual methods are difficult to directly capture the details of their burrowing, feeding and other behaviors; (2) Microenvironmental disturbance effect. Direct sampling or physical probe insertion can easily damage the burrow structure, resulting in behavioral interruption or data distortion; (3) Lack of spatiotemporal continuity. Behaviors such as burrowing and filter feeding have spatiotemporal continuity, and existing observation methods (such as intermittent sampling or radioactive labeling) are difficult to achieve in-situ, non-destructive real-time monitoring.

[0003] Traditional hydrogel systems have the following key defects in the observation of living organism behavior, which seriously restrict the reliability and application scope of the observation data: (1) The mutual contradiction between mechanical properties and optical transparency, that is, it is difficult to balance mechanical strength and transparency. The light transmittance at high cross-linking strength is reduced. When the degree of cross-linking increases to >5%, such as when the proportion of N,N'-methylenebisacrylamide (MBA) is more than 0.2%, the gel network produces light scattering effect due to the anisotropic structure accumulation, which causes the light transmittance (400-800 nm) to drop sharply to <80% (such as agar-polyacrylamide composite gel), which cannot meet the imaging requirements of visualization or high-speed photography; (2) Insufficient density leads to distortion of the movement trajectory of organisms. The density of conventional hydrogels is significantly lower than the actual density of intertidal sediments. Hydrogels with too low density cannot support benthic shellfish to attach to the upper layer of the hydrogel, which makes it impossible to observe the behavior trajectory of benthic shellfish (razor clams) burrowing into the hydrogel and thereafter. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing high-density, high-transparency, non-toxic hydrogel particles for observing the behavior of benthic mollusks and their application in artificial substrates, which can be well applied to observe the behavioral trajectory of benthic organisms.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing hydrogel particles for observing benthic mollusks' behavior, comprising the following steps: acrylamide (AM) and N,N'-methylenebisacrylamide (MBAA) are dissolved in deionized water, followed by the sequential addition of potassium persulfate (KPS) and tetramethyldiethylamine (TEMED) to obtain a hydrogel solution. The hydrogel solution is transferred to a circular mold, left to stand in a fume hood to form solid particles, then washed with water in an oven, dried, and unreacted AM monomers are removed to obtain hydrogel particles for observing benthic mollusks' behavior. The initiator KPS decomposes to produce SO4-·, which can attack the carbon-carbon double bonds (C=C) of AM monomers, generating acrylamide monomer free radicals (AM·) through homolytic cleavage. Subsequently, AM· forms polyacrylamide free radicals (PAM·) through chain growth. These active chain free radicals further undergo addition reactions with the carbon-carbon double bonds in the crosslinking agent MBA to form crosslinking points. As the reaction proceeds, numerous crosslinking points connect with each other, ultimately constructing a crosslinked polyacrylamide hydrogel with a three-dimensional network structure.

[0006] Furthermore, the concentration of acrylamide added to the hydrogel solution is 60%-65% of the mass of deionized water, forming a dense polymer skeleton to simulate the density of tidal flat sediments.

[0007] Furthermore, the concentration of acrylamide added to the hydrogel solution is 64% of the mass of deionized water.

[0008] Furthermore, the concentration of N,N'-methylenebisacrylamide added is 0.167% of the mass of deionized water. The concentration of crosslinking agent MBAA is 0.167%. A porous three-dimensional network is constructed to balance mechanical strength and light transmittance.

[0009] Furthermore, the concentration of potassium persulfate added is 0.4% of the mass of deionized water, and the concentration of tetramethyldiethylamine added is 0.0417% of the mass of deionized water.

[0010] Furthermore, the circular mold is a 96-hole plate or a 384-hole plate.

[0011] Further, the solid particles are washed with water in an oven at 50-60℃ and dried at 60-80℃, repeated 5-10 times to remove unreacted polyacrylamide monomers.

[0012] The present invention also provides the application of the hydrogel particles prepared by the above method in the preparation of artificial substrates for observing the behavior of benthic mollusks.

[0013] Compared with existing technologies, the advantages of this invention are as follows: It provides a method for preparing hydrogel particles for observing the behavior of benthic mollusks and its application. This hydrogel system is characterized by high density and high transparency, and can support the attachment of benthic organisms such as razor clams. Observing the burrowing behavior and activity trajectory of benthic organisms (razor clams) through an artificially prepared high-transparency, high-density, and non-toxic hydrogel system ensures effective observation and recording of the activity status, behavior patterns, and burrowing behavior of benthic organisms (razor clams). It can realistically present the burrowing and other behavioral characteristics of benthic organisms such as razor clams, providing a novel experimental platform for marine benthic ecology research. Attached Figure Description

[0014] Figure 1 To dynamically capture the drilling behavior of razor clams in a hydrogel system; Figure 2 This is the state of the razor clam after it has burrowed into the hydrogel system; Figure 3 Hydrogel particles prepared using a 96-well plate; Figure 4 Hydrogel particles prepared using 384-well plates; Figure 5 The chromatograms are for the quantitative determination of AM and MBA monomer residues using liquid chromatography-mass spectrometry, where A represents AM and B represents MBA. Figure 6 The images are scanning electron microscope (SEM) images of hydrogel particles, where A is 75× and B is 400×. Figure 7 The value represents the water absorption rate of the hydrogel particles. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0016] Specific Implementation Example 1: A method for preparing hydrogel particles for observing the behavior of benthic mollusks, the specific steps of which are as follows: Acrylamide (AM) and N,N'-methylenebisacrylamide (MBAA) were dissolved in deionized water, and then potassium persulfate (KPS, which initiates coagulation) and tetramethyldiethylamine (TEMED, which catalyzes and accelerates coagulation) were added sequentially to obtain a hydrogel solution. The hydrogel solution was transferred to a circular mold (96-well plate or 384-well plate) and left to stand in a fume hood for 4 hours to form polyacrylamide hydrogel particles. Finally, the polyacrylamide hydrogel particles were washed with water in a 55°C oven and dried at 70°C, repeated 10 times to remove unreacted AM monomers, and hydrogel particles for observation of benthic mollusks were obtained.

[0017] The concentration of acrylamide (AM) in the hydrogel solution is 60%-65% of the mass of deionized water, preferably 64%; the concentration of N,N'-methylenebisacrylamide (MBAA) is 0.167% of the mass of deionized water; the concentration of potassium persulfate (KPS) is 0.4% of the mass of deionized water; and the concentration of tetramethyldiethylamine (TEMED) is 0.0417% of the mass of deionized water.

[0018] Specific Example 2: Screening of the acrylamide ratio in the preparation method of Specific Example 1.

[0019] Acrylamide (AM) solutions were accurately weighed using an electronic balance, with 9.6 g (40%), 11.53 g (48%), 13.44 g (56%), 14.40 g (60%), 15.60 g (65%), and 17.28 g (72%) weighed out, for a total of 6 groups. 0.04 g of MBAA (0.167%) was added to each group and dissolved in 24 mL of deionized water. Then, 0.096 g of KPS (0.4%) was added to each solution system and stirred thoroughly to dissolve. Finally, 10 μL of TEMED (0.0417%) was added and gently mixed. The mixture was then immediately transferred to 100 μL per well of a 96-well plate. The plate was covered and allowed to stand until complete polymerization. After curing, deionized water was added to the pores, and the mixture was washed in a 55℃ oven for 2 hours. After removal, it was dried at 70℃ for 30 minutes, marking one complete washing cycle. This washing was repeated 10 times to ensure thorough removal of unreacted monomers. After the hydrogel particles were washed, razor clams of different sizes (1-5 cm) were placed in each hydrogel system to observe their activity and drilling behavior in different hydrogel systems. Figure 1 and Figure 2 As shown.

[0020] The experimental results are shown in Table 1 below. The behavior of razor clams in hydrogel systems of different concentrations is different. When the AM concentration is less than 60%, the hydrogel density is low and cannot meet the drilling behavior of razor clams with a shell length of about 5 cm. However, when the AM concentration is too high, the mechanical strength will increase, the possibility of monomer residue will increase, and the transparency will decrease. Therefore, the AM concentration in the range of 60%-65% is better.

[0021] Table 1. Activity behavior of razor clams in hydrogels with different acrylamide concentrations.

[0022] Specific Implementation Example 3: Mold compatibility analysis of hydrogel particles prepared by the method in Specific Implementation Example 1.

[0023] Based on the shell length of the razor clam and experimental requirements, a modular mold was used to achieve precise control of the hydrogel structure.

[0024] 1. 96-well plate mold: Hole diameter specifications are 6.75 ± 0.25 mm, depth 10.75 ± 0.25 mm, suitable for adult razor clams (shell length 1-6 cm). Actual hydrogel particles are shown below. Figure 3 As shown.

[0025] 2. 384-hole plate mold: Hole diameter 3.0 ± 0.1 mm, depth 6.0 ± 0.3 mm, suitable for juvenile razor clams (shell length < 1 cm). Actual hydrogel particles are shown below. Figure 4 As shown.

[0026] In summary, regarding the adaptability of the molds, experiments showed that the hydrogel made using the 96-well plate was larger and suitable for the activities of adult razor clams; while the hydrogel made using the 384-well plate was smaller and suitable for the activities of juvenile razor clams.

[0027] Specific Example 4: Toxicity determination of hydrogels prepared by the method of Specific Example 1.

[0028] The hydrogel particles prepared in Specific Example 1 were quantitatively detected using liquid chromatography-mass spectrometry (LC-MS). Specifically, acrylamide and N,N'-methylenebisacrylamide standards were diluted with methanol solution to obtain a series of standard solutions of acrylamide and N,N'-methylenebisacrylamide at different concentrations. 1.0 g of freeze-dried hydrogel particles were taken, homogenized, and 10 mL of extraction solvent (a mixture of methanol and water in a 7:3 volume ratio) was added. The mixture was ultrasonically extracted for 30 min, centrifuged, and the supernatant was collected and loaded onto a membrane. LC conditions: Waters ACQUITY I-Class LC model; ACQUITY UPLC BEH C18 1.7 μm, 100 × 2.1 mm column; mobile phase: A was 0.1% formic acid in water, B was methanol, and the mobile phase gradient was as follows: 0-4 min, A phase 10%, B phase 90%, flow rate 0.3 mL / min. Mass spectrometer model: Sciex Triple Quad 5500+; injection volume: 2 μL; ion source: ESI.

[0029] The results are as follows Figure 5 As shown in Figure A, the acrylamide concentration is 0.92 mg / L, which is lower than the cosmetic standard of 1 mg / L. Figure 5 As shown in Figure B, the concentration of N,N'-methylenebisacrylamide is below the detection threshold of 5 ng / mL, verifying that the hydrogel we prepared is non-toxic after washing with water.

[0030] Specific Example 5: Hardness and resilience test of hydrogel particles prepared by the method of Specific Example 1.

[0031] The hardness and resilience of hydrogel particles were determined using a texture analyzer. The Total Texture Profile Analysis (TPA) method was employed, using a P / 0.5R probe. The pre-test speed was 1.5 mm / s, the test speed was 1.5 mm / s, the return speed was 5 mm / s, the trigger force was 5 g, and the compression degree was 30%. Three consecutive tests were conducted, and the average value was calculated. The standard deviation was then determined. The results showed that the hardness and resilience of the hydrogel particles were 259.2007 ± 8.66 g and 0.902 ± 0.01 g, respectively. This indicates that the hydrogel particles possess a certain degree of rigidity, capable of simulating the mechanical environment of natural sediments (such as intertidal mud and sand). Furthermore, they can rapidly recover their original shape after deformation under stress, exhibiting a "self-healing" characteristic similar to natural sediments. This can reduce environmental disturbance during shellfish movement and ensure the authenticity of behavioral observations.

[0032] Specific Example 6: Scanning electron microscopy characterization of hydrogel particles prepared by the method of Specific Example 1.

[0033] The hydrogel particles prepared in Specific Example 1 were freeze-dried, sputter-coated with gold, and the microstructure of the hydrogel surface was observed using a scanning electron microscope (Hitachi S3400). The results are as follows: Figure 6 China A and Figure 6 As shown in Figure B, the surface of the hydrogel particles has dense, layered pores and a high-density, interconnected network.

[0034] Specific Example 7: Determination of the diameter / height, dry weight, density, and falling speed of hydrogel particles prepared by the method of Specific Example 1.

[0035] A: Diameter / height determination: Use vernier calipers to measure its length / diameter.

[0036] B: Dry weight and density determination. Accurately weigh the freeze-dried hydrogel particles using a calibrated electronic balance, denoted as M1. After the hydrogel particles are saturated with water, weigh them again, denoted as M2. Measure their volume using the water displacement method, denoted as V. Perform three consecutive measurements. Calculate the density and dry weight of the hydrogel particles using the following formulas: Density (ρ), ρ = M2 / V; Dry weight (DW), DW = M1 / V.

[0037] C: Particle falling velocity measurement: Take 1 mL of hydrogel particles and let them fall freely from the top of a water-filled graduated cylinder and time it. Record the height of the graduated cylinder as H and the time when the hydrogel particles fall to the bottom as t1. Repeat the above test three times consecutively and calculate the particle falling rate (m / s) and particle falling rate (PFR) using the following formula: PFR=H / t1.

[0038] D: Breakage rate determination. Take 2.5 g of hydrogel particles after water absorption equilibrium, denoted as M1, and place them in a water flow of 1000 mL / min from bottom to top. After 2 h, observe the breakage of the hydrogel particles and weigh the unbroken hydrogel particles as M2. Calculate the breakage rate using the following formula: Breakage rate (CR), CR = (M1 - M2) / M1 × 100%.

[0039] Table 2. Determination results of hydrogel particles

[0040] Specific Example 8: Determination of water absorption and swelling rate of hydrogel particles prepared by the method of Specific Example 1.

[0041] Weigh 2.5 g of the hydrogel particles prepared by the method in the specific embodiment of freeze drying, denoted as G0, and place them in 100 mL of deionized water. Weigh the hydrogel particles every 12 h until their weight reaches equilibrium and remains constant. Calculate their water absorption rate and swelling rate using the following formula.

[0042] Water absorption rate (W), W=(Gx- G0) / G0, where G0 is the weight of the hydrogel particles after drying, and Gx is the weight of the hydrogel particles after they are saturated with water, in g.

[0043] The swelling ratio (ESR) is calculated as ESR = Gx / G0, where Gx is the weight of the hydrogel particles after swelling equilibrium, and G0 is the dry weight of the hydrogel particles. For example... Figure 7 As shown, the curve represents the water absorption rate of the hydrogel over time, with a final swelling rate of 752.8%.

[0044] In summary, the hydrogel prepared by the method in Specific Embodiment 1 is transparent and non-toxic, and can form a dense polymer skeleton to simulate the density of tidal flat sediments.

[0045] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing hydrogel particles for observing the behavior of benthic mollusks, characterized in that... Includes the following steps: Acrylamide and N,N'-methylenebisacrylamide were dissolved in deionized water, and then potassium persulfate and tetramethyldiethylamine were added sequentially to obtain a hydrogel solution. The hydrogel solution was transferred to a circular mold, left to stand in a fume hood to form solid particles, and then washed with water in an oven and dried to obtain hydrogel particles for observing the behavior of benthic mollusks.

2. The method for preparing hydrogel particles for observing benthic mollusks according to claim 1, characterized in that: The concentration of acrylamide added to the hydrogel solution is 60%-65% of the mass of deionized water.

3. The method for preparing hydrogel particles for observing benthic mollusks according to claim 2, characterized in that: The concentration of acrylamide added to the hydrogel solution is 64% of the mass of deionized water.

4. The method for preparing hydrogel particles for observing benthic mollusks according to claim 1, characterized in that: The concentration of N,N'-methylenebisacrylamide added is 0.167% of the mass of deionized water.

5. The method for preparing hydrogel particles for observing benthic mollusks according to claim 1, characterized in that: The concentration of potassium persulfate added is 0.4% of the mass of deionized water, and the concentration of tetramethyldiethylamine added is 0.0417% of the mass of deionized water.

6. The method for preparing hydrogel particles for observing benthic mollusks according to claim 1, characterized in that: The circular mold is a 96-hole plate or a 384-hole plate.

7. The method for preparing hydrogel particles for observing benthic mollusks according to claim 1, characterized in that: The solid particles are washed with water in an oven at 50-60℃ and dried at 60-80℃, and this process is repeated 5-10 times to remove unreacted polyacrylamide monomers.

8. The use of hydrogel particles prepared by the method of any one of claims 1-7 in the preparation of artificial substrates for observing the behavior of benthic mollusks.

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