CN118162001A - Preparation method of biological enzyme catalytic motor water suspension - Google Patents
Preparation method of biological enzyme catalytic motor water suspension Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 27
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000003197 catalytic effect Effects 0.000 title claims description 33
- 239000002077 nanosphere Substances 0.000 claims abstract description 49
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- 238000006555 catalytic reaction Methods 0.000 claims abstract description 18
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- 229910019142 PO4 Inorganic materials 0.000 claims description 22
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 15
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 14
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- 238000000034 method Methods 0.000 claims description 10
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
- 239000012498 ultrapure water Substances 0.000 claims description 9
- OBFQBDOLCADBTP-UHFFFAOYSA-N aminosilicon Chemical compound [Si]N OBFQBDOLCADBTP-UHFFFAOYSA-N 0.000 claims description 8
- WZJVQUUBEVDURL-UHFFFAOYSA-N pentanedial;phosphoric acid Chemical compound OP(O)(O)=O.O=CCCCC=O WZJVQUUBEVDURL-UHFFFAOYSA-N 0.000 claims description 8
- BVUOEDOMUOJKOY-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) benzoate Chemical compound C=1C=CC=CC=1C(=O)ON1C(=O)CCC1=O BVUOEDOMUOJKOY-UHFFFAOYSA-N 0.000 claims description 7
- FHOMNNOYQDOCJQ-VKKIDBQXSA-N (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol hydrate Chemical compound O.O[C@H](CS)[C@H](O)CS FHOMNNOYQDOCJQ-VKKIDBQXSA-N 0.000 claims description 7
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 5
- LLXVXPPXELIDGQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)benzoate Chemical compound C=1C=CC(N2C(C=CC2=O)=O)=CC=1C(=O)ON1C(=O)CCC1=O LLXVXPPXELIDGQ-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 12
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a preparation method of a biological enzyme catalysis type motor water suspension, which belongs to the field of preparation of micro-nano motors, and utilizes chain length difference among different molecular weight high molecular polymers to cover the surface of a glass slide, and places nanospheres on the surface of the glass slide.
Description
Technical Field
The invention belongs to the field of preparation of micro-nano motors, and particularly relates to a preparation method of a biological enzyme catalytic motor water suspension.
Background
For motors, particularly on the nanometer scale, motion rate control is very important. The motor motion behavior is extremely sensitive to small changes of the external environment, and accurate control of the motor motion rate is beneficial to realizing accurate prediction and regulation of the motor motion behavior; the motor movement rate control helps to improve its stability. At high speeds, the motor may be disturbed by the external environment, resulting in deviations in its motion trajectory. By controlling the movement rate of the motor, the interference can be reduced, so that the movement of the motor is more stable; there are many advantages to motors in terms of motion rate control. For example, in the biomedical field, motors are used as drug carriers to achieve accurate delivery of drugs by controlling their rate of movement; in the field of environmental control, the motor can be used as a micro-robot to control the movement rate of the micro-robot, so that the effective removal of pollutants is realized.
Catalytic motors are a unique class of motors that rely on their own catalytic properties to convert chemical energy into kinetic energy through chemical reaction with the surrounding environment, effecting autonomous motion. Among all motors driven by chemical energy, the bubble driving motor is attracting attention due to its high reactivity and moving speed. Common catalytic motors include metal-catalyzed motors and bio-enzyme-catalyzed motors.
In the catalytic motor, the control of the movement rate is usually realized by changing the asymmetric structure of the surface, in other words, the coverage rate of the catalytic material on the surface of the motor is changed, and different coverage rates can generate catalytic power with different sizes and directions, so that the movement rate of the motor is changed, and different movement effects are generated. Thus, by controlling the coverage of the catalytic material on the motor surface, control of the motor movement rate can be achieved.
For a metal catalytic motor, the embedding volume of a micro-nano sphere carrier can be controlled in charged particle liquid through a lifting machine, so that the adsorption area of the charged particles on the surface of the carrier is regulated, but the method has a complex process, and involves the use of the lifting machine, an autoclave and toxic substances, and the method is carried out through the adsorption of the charged surfaces, so that the method is only suitable for the preparation of the metal catalytic motor of the charged particles, and the nano-scale control difficulty is high by using the machine, so that the method is difficult to prepare the nano-scale motor; however, there is no effective way for the bio-enzyme catalysis type motor to realize the fine control of the coverage rate of the catalytic material on the motor surface, especially the nano-sized bio-enzyme catalysis type motor, which limits the use of the bio-enzyme catalysis type motor, so developing a preparation method suitable for controlling the coverage rate of the bio-enzyme catalysis type motor catalytic material has important significance.
Disclosure of Invention
Compared with the prior art, the technical scheme of the invention realizes the fine control of the coverage rate of catalytic substances of the bio-enzyme catalytic motor, further realizes the precise control of the motor movement rate, and is beneficial to realizing the precise prediction and regulation of motor behaviors.
In order to solve the technical problems, the invention adopts the following technical scheme:
S1, immersing a slide in an ethanol solution of sodium hydroxide for 60-120 minutes; taking out the slide, cleaning the slide by ethanol, and then putting the slide into an acetone solution of (3-aminopropyl) trimethoxysilane for soaking for 30-40 minutes; taking out the slide, cleaning the slide by ultrapure water, and then putting the slide into glutaraldehyde phosphate solution, and soaking the slide for 60-120 minutes at 45-55 ℃; taking out the glass slide, and cleaning the glass slide by using phosphate buffer solution to obtain an aldehyde glass slide;
S2, dropwise adding an amino-polyethylene glycol-sulfhydryl water solution on the surface of an aldehyde slide, incubating for 40-50 minutes, and soaking and washing with a phosphate buffer solution; then, the 3-maleimide succinimidyl benzoate aqueous solution and the amino-silicon dioxide nanosphere phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 30 to 40 minutes, and immersed and washed by phosphate buffer; then dripping dithiothreitol water solution on the surface of a glass slide, incubating for 60-120 minutes to separate the glass slide and nanospheres, immersing and washing with phosphate buffer solution, and centrifuging to obtain nanosphere sediment;
S3, adding the nanosphere precipitate into glutaraldehyde phosphate solution, incubating for 60-80 minutes, centrifuging and washing to obtain precipitate, adding the precipitate into biological enzyme aqueous solution, and incubating for 40-60 minutes to obtain biological enzyme catalytic motor aqueous suspension.
Preferably, in the step S1, the volume fraction of the (3-aminopropyl) trimethoxysilane in the acetone solution is 1% -3%; the volume fraction of glutaraldehyde in the glutaraldehyde phosphate solution is 2% -5%.
Preferably, in the step S2, the concentration of the amino-polyethylene glycol-sulfhydryl water solution is 20-40 mM, and the molecular weight of polyethylene glycol in the amino-polyethylene glycol-sulfhydryl is 1K-8K; the concentration of the aqueous solution of 3-maleimidobenzoic acid succinimidyl ester is 40mM; the mass concentration of the amino-silicon dioxide nanospheres in the amino-silicon dioxide nanosphere phosphate suspension is 8-12% W/V, and the diameter of the amino-silicon dioxide nanospheres is 400-600 nm; the concentration of the dithiothreitol aqueous solution is 100-140 mM; the ratio of the dropwise addition amount of the amino-polyethylene glycol-mercapto aqueous solution, the 3-maleimidobenzoic acid succinimidyl ester aqueous solution, the amino-silica nanosphere phosphate suspension and the dithiothreitol aqueous solution to the surface area of the aldehyde slide glass was 300. Mu.L: 300. Mu.L: 200 μL: 300. Mu.L: 2.5cm. Times.2.5 cm.
Preferably, in the step S3, the volume fraction of glutaraldehyde in the glutaraldehyde phosphate solution is 2% -5%; the biological enzyme is urease or catalase, and the concentration of the aqueous solution of the biological enzyme is 4-12 mg/ml.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. According to the invention, the amino-polyethylene glycol-mercapto is utilized for asymmetric modification, the molecular weight of polyethylene glycol in the amino-polyethylene glycol-mercapto is different, the chain length of the amino-polyethylene glycol-mercapto is also different, when nanospheres are combined with the amino-polyethylene glycol-mercapto on the surface of a glass slide, for nanospheres with the same specification, the coverage of polymers with different molecular weights on the surfaces of the nanospheres is different, and the number of combined surface active sites is also different, so that the number of active sites reserved for the bio-enzyme catalytic type is also different, and the number of supported bio-enzyme is different, thereby realizing the regulation and control of the coverage of catalytic substances on the surfaces of the bio-enzyme catalytic type motor;
2. The high molecular polymer refers to high molecular weight formed by repeatedly connecting a plurality of identical and simple structural units through covalent bonds, and the molecular weight is controllable, polyethylene glycol with the molecular weight of 1K-8K is used for experiments, and because the molecular weight of the polyethylene glycol is controllable, the low molecular weight polyethylene glycol can be used for asymmetrically modifying a nano-scale motor, and the high molecular weight can also be used for asymmetrically modifying a micro-scale motor, so that the method has universality and can also be used for asymmetrically modifying the nano-scale motor;
3. According to the invention, polyethylene glycol is customized, functional groups on two sides of the polyethylene glycol are respectively amino and mercapto for chemical connection, disulfide bonds are contained in the polyethylene glycol, the polyethylene glycol is used for connecting nanospheres and slides, dithiothreitol is used for acting on the disulfide bonds to break the polyethylene glycol, and the nanospheres and the slides are separated to obtain nanospheres with surface part of active sites covered.
4. The invention successfully realizes the fine regulation and control of the coverage rate of catalytic substances on the surface of the bio-enzyme catalytic motor, and the surface closed state gradually extends from one end of the sphere to the center of the sphere, thereby realizing the regulation and control of the motor movement rate, meeting the requirements of different fields and expanding the application range of the bio-enzyme catalytic motor.
Drawings
FIG. 1 shows a fluorescence diagram of a catalase catalytic motor prepared in example 1, wherein A is a bright field diagram, and B is a corresponding fluorescence diagram;
FIG. 2 shows a fluorescence diagram of a catalase catalytic motor prepared in example 2, wherein A is a bright field diagram, and B is a corresponding fluorescence diagram;
FIG. 3 shows a fluorescence diagram of a catalase catalytic motor prepared in example 3, wherein A is a bright field diagram, and B is a corresponding fluorescence diagram;
FIG. 4A is a bright field plot and B is a corresponding plot of the urease-catalyzed motor prepared in example 4;
FIG. 5A is a bright field plot and B is a corresponding plot of the urease-catalyzed motor prepared in example 5;
FIG. 6 is a mean square displacement graph of catalase catalyzed motor motion data prepared in example 1;
FIG. 7 is a mean square displacement graph of catalase catalyzed motor motion data prepared in example 2;
FIG. 8 is a mean square displacement graph of catalase catalyzed motor motion data prepared in example 3;
FIG. 9 is a mean square displacement graph of urease-catalyzed motor motion data prepared in example 4;
FIG. 10 is a mean square displacement graph of urease-catalyzed motor motion data prepared in example 5.
Detailed Description
The invention is further described below with reference to the drawings and examples.
Example 1
1. Preparing a biological enzyme catalytic motor water suspension with the coverage rate of catalytic substances being 2-10 percent, comprising the following steps,
S1, putting a glass slide with the specification of 2.5cm multiplied by 2.5cm into an ethanol solution of 1mol/L sodium hydroxide, and soaking for 60 minutes; taking out the slide, cleaning with ethanol, and soaking the slide in an acetone solution with the volume fraction of (3-aminopropyl) trimethoxysilane of 1% for 30 minutes; taking out the slide, cleaning with ultrapure water, putting the slide into phosphate solution with the glutaraldehyde volume fraction of 3%, and soaking for 60 minutes at 50 ℃; taking out the glass slide and cleaning to obtain an aldehyde glass slide;
S2, 300 mu L of 40mM amino-polyethylene glycol-sulfhydryl (the molecular weight of polyethylene glycol is 8K) is dripped on the surface of an aldehyde slide, incubated for 50 minutes at normal temperature, and soaked in phosphate buffer; then 300 mu L of 40mM 3-maleimide succinimidyl benzoate aqueous solution and 200 mu L of 10% W/V amino-silica nanosphere (diameter 500 nm) phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 40 minutes at normal temperature, and immersed and washed by using phosphate buffer; then 300 mu L of 140mM dithiothreitol water solution is dripped on the surface of a glass slide, incubated for 60 minutes, soaked and washed by phosphate buffer solution and centrifuged to obtain nanosphere sediment;
And S3, adding 500 mu L of phosphate solution with the glutaraldehyde volume fraction of 3%, incubating for 60 minutes, centrifuging and washing to obtain a precipitate, adding the precipitate into 4mg/ml fluorescent-labeled catalase aqueous solution, incubating for 40 minutes, centrifuging and washing, and collecting the aqueous suspension of the catalase-modified nanosphere motor.
Example 2
2. Preparing a biological enzyme catalysis type motor water suspension with the coverage rate of the catalysis substance being 10-20 percent, comprising the following steps,
S1, putting a glass slide with the specification of 2.5cm multiplied by 2.5cm into an ethanol solution of 1mol/L sodium hydroxide, and soaking for 60 minutes; taking out the slide, cleaning with ethanol, and soaking the slide in an acetone solution with the volume fraction of (3-aminopropyl) trimethoxysilane of 1% for 30 minutes; taking out the slide, cleaning with ultrapure water, putting the slide into phosphate solution with the glutaraldehyde volume fraction of 3%, and soaking for 60 minutes at 50 ℃; taking out the glass slide and cleaning to obtain an aldehyde glass slide;
S2, 300 mu L of 40mM amino-polyethylene glycol-sulfhydryl (the molecular weight of polyethylene glycol is 7K) is dripped on the surface of an aldehyde slide, incubated for 50 minutes at normal temperature, and soaked in phosphate buffer; then 300 mu L of 40mM 3-maleimide succinimidyl benzoate aqueous solution and 200 mu L of 10% W/V amino-silica nanosphere (diameter 500 nm) phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 40 minutes at normal temperature, and immersed and washed by using phosphate buffer; then 300 mu L of 140mM dithiothreitol water solution is dripped on the surface of a glass slide, incubated for 60 minutes, soaked and washed by phosphate buffer solution and centrifuged to obtain nanosphere sediment;
and S3, adding 500 mu L of phosphate solution with the glutaraldehyde volume fraction of 3%, incubating for 60 minutes, centrifuging and washing to obtain a precipitate, adding the precipitate into a fluorescent-labeled catalase aqueous solution with the concentration of 6mg/ml, incubating for 40 minutes, centrifuging and washing, and collecting the aqueous suspension of the catalase-modified nanosphere motor.
Example 3
3. Preparing a biological enzyme catalysis type motor water suspension with the coverage rate of the catalysis substance being 25-35 percent, comprising the following steps,
S1, putting a glass slide with the specification of 2.5cm multiplied by 2.5cm into an ethanol solution of 1mol/L sodium hydroxide, and soaking for 60 minutes; taking out the slide, cleaning with ethanol, and soaking the slide in an acetone solution with the volume fraction of (3-aminopropyl) trimethoxysilane of 1% for 30 minutes; taking out the slide, cleaning with ultrapure water, putting the slide into phosphate solution with the glutaraldehyde volume fraction of 3%, and soaking for 60 minutes at 50 ℃; taking out the glass slide and cleaning to obtain an aldehyde glass slide;
S2, 300 mu L of 40mM amino-polyethylene glycol-sulfhydryl (the molecular weight of polyethylene glycol is 5K) is dripped on the surface of an aldehyde slide, incubated for 50 minutes at normal temperature, and soaked in phosphate buffer; then 300 mu L of 40mM 3-maleimide succinimidyl benzoate aqueous solution and 200 mu L of 10% W/V amino-silica nanosphere (diameter 500 nm) phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 40 minutes at normal temperature, and immersed and washed by using phosphate buffer; then 300 mu L of 140mM dithiothreitol water solution is dripped on the surface of a glass slide, incubated for 60 minutes, soaked and washed by phosphate buffer solution and centrifuged to obtain nanosphere sediment;
And S3, adding 500 mu L of phosphate solution with the glutaraldehyde volume fraction of 3%, incubating for 60 minutes, centrifuging and washing to obtain a precipitate, adding the precipitate into 8mg/ml fluorescent-labeled catalase aqueous solution, incubating for 40 minutes, centrifuging and washing, and collecting the aqueous suspension of the catalase-modified nanosphere motor.
Example 4
4. Preparing a biological enzyme catalysis type motor water suspension with 50-60% coverage rate of the catalysis material, the steps are as follows,
S1, putting a glass slide with the specification of 2.5cm multiplied by 2.5cm into an ethanol solution of 1mol/L sodium hydroxide to soak for 120 minutes; taking out the slide, cleaning with ethanol, and then putting the slide into an acetone solution with the volume fraction of (3-aminopropyl) trimethoxysilane of 3 percent for soaking for 40 minutes; taking out the slide, cleaning with ultrapure water, putting the slide into phosphate solution with the glutaraldehyde volume fraction of 2%, and soaking for 120 minutes at 45 ℃; taking out the glass slide and cleaning to obtain an aldehyde glass slide;
S2, 300 mu L of 30mM amino-polyethylene glycol-sulfhydryl (the molecular weight of polyethylene glycol is 3K) is dripped on the surface of an aldehyde slide, incubated for 40 minutes at normal temperature, and soaked in phosphate buffer; then 300 mu L of 40mM 3-maleimide succinimidyl benzoate aqueous solution and 200 mu L of 8% W/V amino-silica nanosphere (diameter 400 nm) phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 30 minutes at normal temperature and soaked in phosphate buffer solution; then 300 mu L of 100mM dithiothreitol water solution is dripped on the surface of a glass slide, incubated for 120 minutes, soaked and washed by phosphate buffer solution and centrifuged to obtain nanosphere sediment;
And S3, adding 500 mu L of phosphate solution with the glutaraldehyde volume fraction of 2%, incubating for 70 minutes, centrifuging and washing to obtain a precipitate, adding the precipitate into a fluorescent-labeled urease aqueous solution with the concentration of 10mg/ml, incubating for 50 minutes, centrifuging and washing, and collecting the urease-modified nanosphere motor aqueous suspension.
Example 5
5. Preparing a biological enzyme catalysis type motor water suspension with the coverage rate of the catalysis material being 80-90 percent, comprising the following steps,
S1, putting a glass slide with the specification of 2.5cm multiplied by 2.5cm into an ethanol solution of 1mol/L sodium hydroxide to soak for 120 minutes; taking out the slide, cleaning with ethanol, and then putting the slide into an acetone solution with the volume fraction of (3-aminopropyl) trimethoxysilane of 3 percent for soaking for 40 minutes; taking out the slide, cleaning with ultrapure water, putting the slide into a phosphate solution with the glutaraldehyde volume fraction of 5%, and soaking for 120 minutes at 55 ℃; taking out the glass slide and cleaning to obtain an aldehyde glass slide;
S2, 300 mu L of 20mM amino-polyethylene glycol-sulfhydryl (the molecular weight of polyethylene glycol is 1K) is dripped on the surface of an aldehyde slide, incubated for 40 minutes at normal temperature, and immersed and washed by phosphate buffer; then 300 mu L of 40mM 3-maleimide succinimidyl benzoate aqueous solution and 200 mu L of 12% W/V amino-silica nanosphere (diameter 600 nm) phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 30 minutes at normal temperature, and immersed and washed by using phosphate buffer; then 300 mu L of 100mM dithiothreitol water solution is dripped on the surface of a glass slide, incubated for 120 minutes, soaked and washed by phosphate buffer solution and centrifuged to obtain nanosphere sediment;
And S3, adding 500 mu L of phosphate solution with the glutaraldehyde volume fraction of 5%, incubating for 80 minutes, centrifuging and washing to obtain a precipitate, adding the precipitate into 12mg/ml fluorescent-labeled urease aqueous solution, incubating for 60 minutes, centrifuging and washing, and collecting the urease-modified nanosphere motor aqueous suspension.
Test example 1
To verify the asymmetric structure, observations were made using a fluorescence-modified bio-enzyme, first a fluorescence-modified bio-enzyme was prepared; the catalase/urease is modified by fluorescein isothiocyanate, 0.5 mg and 20mg of catalase are dissolved in 0.1M phosphate (pH=9.2) buffer solution, the reaction is carried out in darkness for 4 hours at room temperature, the product is placed in a pretreated dialysis bag (3.5 KDa) and placed in ultrapure water for reaction for 24 hours, the ultrapure water is replaced every 2 hours in the middle, the unconnected fluorescein isothiocyanate is removed, and finally, the fluorescence-labeled catalase/urease with different concentrations is obtained, and all the biological enzymes used in the examples are the biological enzymes with fluorescence modification.
The asymmetric structure of the nanospheres is observed by using a fluorescence microscope, and as shown in the results of fig. 1-5, the coverage rate of fluorescence modified biological enzymes on the surfaces of the spheres is different, and the coverage rate of the biological enzymes is gradually increased along with the gradual reduction of the molecular weight of the high polymer polyethylene glycol from fig. 1-5, because for nanospheres with the same specification and size, polyethylene glycol and biological enzymes can be attached on the surfaces of the spheres to occupy active sites, the molecular weights of the high polymer polyethylene glycol are different, the chain length can be different, the polyethylene glycol with high molecular weight can occupy more surface active sites, and the number of the surface active sites occupied by the biological enzymes is small, so that the biological enzyme catalytic motors with different coverage rates of the biological enzymes on the surfaces of the nanospheres can be obtained.
The fluorescence data show that the method can prepare bio-enzyme catalytic motors with different coverage rates, can prepare nano-sphere motors with the size in nano scale, successfully loads catalase and urease, and has certain universality for bio-enzymes.
To compare the effect of different biological enzyme coverage on the movement rate of the nanosphere motor, 5 μl of nanosphere motor was mixed with water, 15mM, 30mM catalytic reagent (hydrogen peroxide for catalase and urea for urease), and the movement of the motor was observed and recorded using a microscope, and the video shooting and recording of the movement of the nanosphere motor was performed by an inverted microscope (LeicaDMI 3000B, equipped with a camera PhotometricsEvolve/SC).
The motion data is expressed by mean square displacement as shown in fig. 6 to 10.
It can be seen that for the catalase catalyzed nanosphere motor (fig. 6-8), after hydrogen peroxide is added, the mean square displacement value of the catalase catalyzed nanosphere motor is obviously increased, and the higher the hydrogen peroxide concentration is, the larger the mean square displacement value is, which not only indicates that the catalase is successfully loaded, but also the concentration of the hydrogen peroxide used is not up to the upper limit, the movement rate is still in a room for improvement, and the hydrogen peroxide with higher concentration can be used; by comparing catalase catalysis motors with different coverage rates, the numerical value of mean square displacement is continuously increased along with the improvement of the coverage rate under the catalysis of hydrogen peroxide with the same concentration, which indicates that the asymmetric structure of the motor can have a certain influence on the motion data of the motor, and the motion rate is continuously increased along with the improvement of the coverage rate under the condition that the hemispherical coverage is not achieved.
For the urease catalyzed nanosphere motor (fig. 9-10), after urea is added, the movement rate change is the same as that of the catalase catalyzed motor; for motors with different coverage rates and urease catalysis, the mean square displacement value is reduced along with the continuous increase of coverage rate under the catalysis of urea with the same concentration, because for spherical motors, the coverage rate is usually hemispherical, the movement rate reaches the maximum value when the coverage rate is 45-55%, and the movement rate is usually reduced when the coverage rate exceeds the hemispherical coverage rate.
For motion data, the mean square displacement is maximum with coverage around the hemisphere, and the rate of motion is near the maximum. According to the motion data, the bioenzyme catalytic motors with different motion effects can be prepared in the mode, and the accurate control of the motor motion rate is realized.
Claims (4)
1. A preparation method of a biological enzyme catalysis type motor water suspension is characterized by comprising the following steps,
S1, immersing a slide in an ethanol solution of sodium hydroxide for 60-120 minutes; taking out the slide, cleaning the slide by ethanol, and then putting the slide into an acetone solution of (3-aminopropyl) trimethoxysilane for soaking for 30-40 minutes; taking out the slide, cleaning the slide by ultrapure water, and then putting the slide into glutaraldehyde phosphate solution, and soaking the slide for 60-120 minutes at 45-55 ℃; taking out the glass slide, and cleaning the glass slide by using phosphate buffer solution to obtain an aldehyde glass slide;
S2, dropwise adding an amino-polyethylene glycol-sulfhydryl water solution on the surface of an aldehyde slide, incubating for 40-50 minutes, and soaking and washing with a phosphate buffer solution; then, the 3-maleimide succinimidyl benzoate aqueous solution and the amino-silicon dioxide nanosphere phosphate suspension are sequentially dripped on the surface of a glass slide, incubated for 30 to 40 minutes, and immersed and washed by phosphate buffer; then dripping dithiothreitol water solution on the surface of a slide, incubating for 60-120 minutes to separate the slide and nanospheres, immersing and washing the slide with phosphate buffer solution, and centrifugally collecting to obtain nanosphere precipitate;
S3, adding the nanosphere precipitate into glutaraldehyde phosphate solution, incubating for 60-80 minutes, centrifuging and washing to obtain precipitate, adding the precipitate into biological enzyme aqueous solution, and incubating for 40-60 minutes to obtain biological enzyme catalytic motor aqueous suspension.
2. The method for preparing the aqueous suspension of the bio-enzyme catalyzed motor according to claim 1, wherein in the step S1, the volume fraction of the (3-aminopropyl) trimethoxysilane in the acetone solution is 1% -3%; the volume fraction of glutaraldehyde in the glutaraldehyde phosphate solution is 2% -5%.
3. The method for preparing an aqueous suspension of a bio-enzyme catalyzed motor according to claim 1, wherein in the step S2, the concentration of the amino-polyethylene glycol-mercapto aqueous solution is 20-40 mM, and the molecular weight of the polyethylene glycol in the amino-polyethylene glycol-mercapto is 1K-8K; the concentration of the aqueous solution of 3-maleimidobenzoic acid succinimidyl ester is 40mM; the mass concentration of the amino-silicon dioxide nanospheres in the amino-silicon dioxide nanosphere phosphate suspension is 8-12% W/V, and the diameter of the amino-silicon dioxide nanospheres is 400-600 nm; the concentration of the dithiothreitol aqueous solution is 100-140 mM; the ratio of the dropwise addition amount of the amino-polyethylene glycol-mercapto aqueous solution, the 3-maleimidobenzoic acid succinimidyl ester aqueous solution, the amino-silica nanosphere phosphate suspension and the dithiothreitol aqueous solution to the surface area of the aldehyde slide glass was 300. Mu.L: 300. Mu.L: 200 μL: 300. Mu.L: 2.5cm. Times.2.5 cm.
4. The method for preparing an aqueous suspension of a bio-enzyme catalyzed motor according to claim 1, wherein in the step S3, the volume fraction of glutaraldehyde in the glutaraldehyde phosphate solution is 2% -5%; the biological enzyme is urease or catalase, and the concentration of the aqueous solution of the biological enzyme is 4-12 mg/ml.
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