Preparation method of hydrogel particles for observing behavior of benthic shellfish and application of the hydrogel particles in artificial substrate

Abstract

The application discloses a preparation method of hydrogel particles for observing behaviors of benthic shellfish and application of the hydrogel particles in an artificial substrate, and has the following characteristics: acrylamide and N,N'-methylene bisacrylamide are dissolved in deionized water, potassium persulfate and tetramethylammonium are sequentially added to obtain a hydrogel solution, the hydrogel solution is transferred to a circular mold, solid particles are formed after standing in a fume hood, and the solid particles are washed and dried in an oven to obtain the hydrogel particles for observing behaviors of benthic shellfish, the addition concentration of acrylamide in the hydrogel solution is 60% to 65% of the mass of deionized water, and the application of the hydrogel particles in preparing the artificial substrate for observing behaviors of benthic shellfish is also provided, and the hydrogel particles have the advantages of high density, high transparency, non-toxicity and the like, and can be effectively applied to observing behavior trajectories 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 scheme adopted by the present application to solve the above technical problems is: a preparation method of hydrogel particles for observing behavior of benthic shellfish, comprising the following steps: acrylamide (AM) and N,N'-methylenebisacrylamide (MBAA) are added to deionized water for dissolution, then potassium persulfate (KPS) and tetramethylammonium ethylamine (TEMED) are sequentially added to obtain a hydrogel solution, the hydrogel solution is transferred to a circular mold, and the solid particles are formed after standing in a fume hood, then the solid particles are washed with water in an oven, dried, and the unreacted AM monomer is removed, thereby obtaining the hydrogel particles for observing the behavior of benthic shellfish. The initiator KPS decomposes to generate SO4-·, and SO4-· can attack the carbon-carbon double bond (C=C) of the AM monomer to generate an acrylamide monomer radical (AM·) through a homolytic reaction. Subsequently, the AM· forms a polyacrylamide radical (PAM·) through a chain growth reaction. These active chain radicals further undergo an addition reaction with the carbon-carbon double bond in the crosslinking agent MBA to form crosslinking points. As the reaction proceeds, the numerous crosslinking points are connected to each other, and finally a crosslinked polyacrylamide hydrogel with a three-dimensional network structure is constructed.

[0006] Further, the addition concentration of acrylamide in the hydrogel solution is 60%-65% of the mass of deionized water. A dense polymer skeleton is formed to simulate the density of a tidal flat deposit.

[0007] Further, the addition concentration of acrylamide in the hydrogel solution is 64% of the mass of deionized water.

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

[0009] Further, the addition concentration of potassium persulfate is 0.4% of the mass of deionized water, and the addition concentration of tetramethylammonium ethylamine is 0.0417% of the mass of deionized water.

[0010] Further, the circular mold is a 96-well plate or a 384-well plate.

[0011] Further, the solid particles are washed with water in an oven at 50-60°C, dried at 60-80°C, and the unreacted polyacrylamide monomer is removed by repeating the above steps for 5-10 times.

[0012] The present application also provides the use of the hydrogel particles prepared by the above method in the preparation of an artificial substrate for observing the behavior of benthic shellfish.

[0013] Compared with the prior art, the invention has the advantages that a preparation method of a water gel particle for observing behavior of benthic shellfish and application thereof are invented, the water gel system has the characteristics of high density and high transparency, and can carry benthic organisms such as Sinonovacula constriceta and the like. The burrowing behavior and activity track of benthic organisms (Sinonovacula constriceta) are observed through the high transparency and high density and non-toxic water gel system prepared artificially, the activity condition, behavior mode and burrowing behavior of benthic organisms (Sinonovacula constriceta) can be effectively observed and recorded, the burrowing behavior characteristics of benthic organisms such as Sinonovacula constriceta can be truly presented, and a new experimental platform is provided for marine benthic organism ecology research. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the dynamic capture of the burrowing behavior of Sinonovacula constriceta in the water gel system;

[0015] Figure 2 It is the state of Sinonovacula constriceta after drilling into the water gel system;

[0016] Figure 3 It is the water gel particle made by using a 96-well plate;

[0017] Figure 4 It is the water gel particle made by using a 384-well plate;

[0018] Figure 5 It is a graph for quantitatively detecting the residual amount of AM and MBA monomers by using liquid chromatography-mass spectrometry, wherein A is AM and B is MBA;

[0019] Figure 6 It is a scanning electron microscope image of the water gel particle, wherein A is 75x and B is 400x;

[0020] Figure 7 It is the water absorption rate of the water gel particle. DETAILED DESCRIPTION

[0021] The invention is further described in detail below in combination with the embodiments of the drawings.

[0022] Specific embodiment one: a preparation method of a water gel particle for observing behavior of benthic shellfish, and the specific steps are as follows:

[0023] The acrylamide (AM) and N,N'-methylene bisacrylamide (MBAA) are dissolved in deionized water, and then potassium persulfate (KPS, initiation combination) and tetramethyl diethylamine (TEMED, catalytic acceleration reaction solidification) are added to obtain a hydrogel solution. The hydrogel solution is transferred to a circular mold (96-well plate or 384-well plate), and placed in a fume hood for 4 h to form polyacrylamide hydrogel particles. Finally, the polyacrylamide hydrogel particles are washed in a 55°C oven, dried at 70°C, and repeated 10 times to remove unreacted AM monomers to obtain hydrogel particles for observing the behavior of benthic shellfish.

[0024] The addition concentration of acrylamide (AM) in the hydrogel solution is 60%-65% of the mass of deionized water, preferably 64%, the addition concentration of N,N'-methylene bisacrylamide (MBAA) is 0.167% of the mass of deionized water, the addition concentration of potassium persulfate (KPS) is 0.4% of the mass of deionized water, and the addition concentration of tetramethyl diethylamine (TEMED) is 0.0417% of the mass of deionized water.

[0025] Specific embodiment two: selection of acrylamide ratio in the preparation method of specific embodiment one.

[0026] An electronic balance is used to accurately weigh acrylamide (AM) solution, and 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%) are weighed, respectively, a total of 6 groups, and 0.04 g of MBAA (0.167%) is added to each group and dissolved in 24 mL of deionized water; then 0.096 g of KPS (0.4%) is added to each solution system, and stirred and dissolved; finally, 10 μL of TEMED (0.0417%) is added, and mixed gently; then the mixed solution is immediately transferred to a 96-well plate, 100 μL per well. Cover the plate and let it stand until it is completely polymerized. After solidification is completed, add deionized water to the wells, and place them in a 55°C oven for 2 h of water washing. After taking them out, dry them at 70°C for 30 min, which is recorded as one complete washing cycle. Repeat the water washing 10 times to ensure that the unreacted monomers are completely removed. After the hydrogel particles are washed, different sizes of Sinonovacula constriceta (1-5 cm) are placed in each hydrogel system, and their activities and drilling behaviors in different hydrogel systems are observed, as shown in Figure 1 and Figure 2 .

[0027] The experimental results are shown in Table 1 below. The behavior of Sinonovacula constricta in different concentrations of hydrogel systems is different. When the AM concentration is less than 60%, the density of the hydrogel is low, and it cannot meet the drilling behavior of the shell length of about 5 cm Sinonovacula constricta. However, when the AM concentration is too high, the mechanical strength increases, the possibility of monomer residue increases, and the transparency decreases. Therefore, the AM concentration in the range of 60%-65% is better.

[0028] Table 1 Activity behavior of Sinonovacula constricta in different acrylamide concentration hydrogels

[0029]

[0030] Specific embodiment three: mold adaptability analysis of the hydrogel particles prepared by the method of specific embodiment one.

[0031] According to the shell length of Sinonovacula constricta and experimental requirements, modular molds are used to realize precise regulation of hydrogel structure.

[0032] 1. 96-well plate mold: hole diameter specification is 6.75 ± 0.25 mm in diameter, depth is 10.75 ± 0.25 mm, and applicable object is adult Sinonovacula constricta (shell length 1-6 cm). The actual hydrogel particles are as shown in Figure 3 .

[0033] 2. 384-well plate mold: hole diameter specification is 3.0 ± 0.1 mm in diameter, depth is 6.0 ± 0.3 mm, and applicable object is Sinonovacula constricta juvenile (shell length <1 cm). The actual hydrogel particles are as shown in Figure 4 .

[0034] In summary, the mold adaptability, the hydrogel made by using 96-well plate is larger, which is suitable for the activity of adult Sinonovacula constricta; the hydrogel made by using 384-well plate is smaller, which is suitable for the activity of Sinonovacula constricta juvenile.

[0035] Specific embodiment four: toxicity determination of the hydrogel prepared by the method of specific embodiment one.

[0036] The hydrogel particles prepared in Specific Embodiment One were quantitatively detected using liquid chromatography-mass spectrometry. Specifically, acrylamide and N,N'-methylenebisacrylamide standards were diluted with a methanol solution to obtain acrylamide and N,N'-methylenebisacrylamide standard series solutions of different concentrations. 1.0 g of the hydrogel particles after freeze-drying were homogenously ground, 10 mL of an extractant (mixed by methanol and water in a volume ratio of 7:3) was added, and ultrasonic extraction was performed for 30 min. The supernatant was obtained by centrifugation and membrane filtration. Liquid phase conditions: liquid phase type, Waters ACQUITY I-Class; chromatographic column, ACQUITY UPLC BEH C18 1.7 μm, 100 x 2.1 mm; mobile phase: A is 0.1% formic acid water, B is methanol, and the mobile phase gradient is as follows: 0-4 min, A phase 10%, B phase 90%, flow rate 0.3 mL / min. Mass spectrometry type: Sciex Triple Quad 5500+; sample size 2 μL; ion source: ESI.

[0037] The results are shown in Table A in Figure 5 The acrylamide concentration was 0.92 mg / L, which was lower than the cosmetic standard of 1 mg / L. As shown in Table B in Figure 5 The N,N'-methylenebisacrylamide concentration was lower than the detection line threshold of 5 ng / mL, verifying that the hydrogel prepared by us was non-toxic after water washing.

[0038] Specific Embodiment Five: Hardness and Recovery Force Test of Hydrogel Particles Prepared by the Method of Specific Embodiment One.

[0039] The hardness and recovery force of the hydrogel particles were determined using a texture analyzer, and the Texture Profile Analysis (TPA) test method was used. The probe was P / 0.5R type, 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, the compression degree was 30%, and the continuous test was performed three times, and the average value was taken and the standard deviation was calculated. The results showed that the hardness and recovery force of the hydrogel particles were 259.2007 ± 8.66 g and 0.902 ± 0.01, respectively, indicating that the hydrogel particles had a certain rigidity, could simulate the mechanical environment of natural sediments (such as intertidal zone sand), and could quickly recover to the original state after deformation, had the "self-repairing" characteristics similar to natural sediments, could reduce the environmental disturbance during the movement of shellfish, and ensured the authenticity of the behavior observation.

[0040] Specific Embodiment Six: Scanning Electron Microscope Characterization of Hydrogel Particles Prepared by the Method of Specific Embodiment One.

[0041] 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.

[0042] Specific Example 7: Determination of the diameter / height, dry weight, density, and particle falling speed of hydrogel particles prepared by the method of Specific Example 1.

[0043] A: Diameter / height determination: Use vernier calipers to measure its length / diameter.

[0044] 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.

[0045] 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.

[0046] 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%.

[0047] Table 2. Determination results of hydrogel particles

[0048]

[0049] Specific Example 8: Determination of water absorption and swelling rate of hydrogel particles prepared by the method of Specific Example 1.

[0050] 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.

[0051] 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.

[0052] 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%.

[0053] 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.

[0054] 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. The application of hydrogel particles in the preparation of artificial substrates for observing the behavior of benthic mollusks, characterized in that... The preparation method of the hydrogel particles includes the following steps: acrylamide and N,N'-methylenebisacrylamide are dissolved in deionized water, potassium persulfate and tetramethyldiethylamine are added sequentially to obtain a hydrogel solution, the hydrogel solution is transferred to a circular mold, and after standing in a fume hood to form solid particles, the particles are washed with water in an oven and dried to obtain hydrogel particles for observing the behavior of benthic mollusks. The concentration of acrylamide in the hydrogel solution is 60%-65% of the mass of deionized water, and the concentration of N,N'-methylenebisacrylamide is 0.167% of the mass of deionized water.

2. The application according to claim 1, characterized in that: The concentration of acrylamide added to the hydrogel solution is 64% of the mass of deionized water.

3. The application 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.

4. The application according to claim 1, characterized in that: The circular mold is a 96-hole plate or a 384-hole plate.

5. The application 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.

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