阅读说明：本技术 一种提高酶驱动微纳米马达驱动力的方法 (Method for improving driving force of enzyme-driven micro-nano motor ) 是由 罗明 李守丽 官建国 于 2019-11-13 设计创作，主要内容包括：本发明公开了一种提高酶驱动微纳米马达驱动力的方法,该方法是通过酶在三维空间上组装扩展以增加酶含量,从而提高酶驱动微纳米马达的驱动力,进一步为通过链霉亲和素与生物素的特异性结合,将生物素化酶组装到所述生物素化粒子上,得到酶驱动微纳米马达。具体为：1)以微纳米粒子为模板,在其表面非对称包覆金属Au层,形成双面神粒子,再利用Au-S键将Biotin-HPDP修饰于双面神粒子表面；2)利用巯基与马来酰亚胺之间的键合,将生物素化试剂修饰于生物酶表面；3)利用链霉亲和素与生物素的特异性结合,将生物素化酶通过层层自组装固定于生物素化双面神粒子表面。该方法普适性强,所制备的马达的酶含量高、驱动力强,能够在模拟血液粘度条件下运动。(The invention discloses a method for improving enzyme-driven micro-nano motor driving force, which is characterized in that enzyme is assembled and expanded on a three-dimensional space to increase enzyme content, so that the driving force of the enzyme-driven micro-nano motor is improved, and further, biotinylated enzyme is assembled on biotinylated particles through specific binding of streptavidin and biotin, so that the enzyme-driven micro-nano motor is obtained. The method specifically comprises the following steps: 1) the method comprises the following steps of (1) asymmetrically coating a metal Au layer on the surface of a micro-nano particle serving as a template to form a double-sided particle, and modifying Biotin-HPDP on the surface of the double-sided particle by utilizing an Au-S bond; 2) modifying a biotinylation reagent on the surface of the biological enzyme by utilizing the bonding between sulfydryl and maleimide; 3) the specific combination of streptavidin and biotin is utilized to fix the biotinylation enzyme on the surface of the biotinylation Shuangshen particle through layer-by-layer self-assembly. The method has strong universality, and the prepared motor has high enzyme content and strong driving force and can move under the condition of simulating blood viscosity.)

1. A method for improving enzyme driving micro-nano motor driving force is characterized in that: the preparation method is that enzyme is assembled and expanded on a three-dimensional space to increase the enzyme content, so that the driving force of the enzyme driving micro-nano motor is improved, and further, biotinylated enzyme is assembled on biotinylated particles through specific binding of streptavidin and biotin, so that the enzyme driving micro-nano motor is obtained.

2. The preparation method for improving the driving force of the enzyme-driven micro-nano motor according to claim 1, which is characterized by comprising the following steps:

1) preparing biotinylated Shuangshen particles: the method comprises the following steps of (1) asymmetrically coating a metal Au layer on the surface of a micro-nano particle serving as a template to form a double-sided particle, and modifying N- (6- [ biotin amine ] hexyl) -3'- (2' -pyridine disulfide) propionamide on the surface of the double-sided particle by utilizing an Au-S bond;

2) preparation of biotinylated enzyme: modifying a biotinylation reagent Na- (3-maleimidopropylpropionyl) biocytin on the surface of the biological enzyme by utilizing the bonding between a sulfhydryl group and maleimide;

3) preparing an enzyme driving micro-nano motor: the specific combination of streptavidin and biotin is utilized to fix the biotinylation enzyme on the surface of the biotinylation Shuangshen particle through layer-by-layer self-assembly.

3. Use of the enzyme-driven micro-nano motor according to claim 1, wherein: the enzyme-driven micro-nano motor can keep autonomous movement under the condition of simulating blood mucus, and can be applied to the field of biomedicine.

Technical Field

The invention relates to the technical field of micro-nano device preparation, in particular to a method for improving enzyme-driven micro-nano motor driving force.

Background

The enzyme-driven micro-nano motor is a micro-nano device for converting chemical energy into mechanical energy through enzyme catalytic reaction. The current study of enzyme driven motors driven by urease/glucose oxidase has received much interest from researchers. Journal of American chemical society (ACS Nano 2016, volume 10, page 3597) reports a motion-controllable urease-driven micro-nano motor, and the motion speed can reach 10 mu m/s. The uk journal of chemical communications (chem. commun.2013, volume 49, page 2397) reports a method of biotinylated protein network assembly amplifying signals. The urease/glucose oxidase driving motor takes urea/glucose as fuel, the biocompatibility is good, and the fuel can be taken from biological media; the enzyme has long service life as a catalyst; and the precise control of the motion direction is realized by introducing a magnetic medium; the urease/glucose oxidase driving motor is expected to be applied to the field of biological medicines. However, the driving force of the enzyme driving micro-nano motor is weak at present, so that the micro-nano motor cannot move under the conditions of high salt and high viscosity. The restriction enzyme drives the micro-nano motor to be applied to the fields of biosensing, targeted drug delivery, minimally invasive surgery and the like.

Therefore, the enzyme is assembled and expanded on a three-dimensional space by utilizing a protein self-assembly technology to prepare the enzyme-driven micro-nano motor, the enzyme content of the motor is high, the driving force is obviously improved, and the motor can autonomously move in a fuel simulating blood viscosity.

Disclosure of Invention

Based on the defects of the prior art, the technical problem solved by the invention is to provide the preparation method for improving the driving force of the enzyme-driven micro-nano motor, the method has strong universality, the prepared micro-nano motor has high enzyme content and strong driving force, the movement speed can reach 21.0 +/-0.8 mu m/s, and the movement speed of the enzyme-driven motor is greatly improved.

In order to solve the technical problems, the invention provides a preparation method for improving the driving force of an enzyme-driven micro-nano motor, wherein the enzyme content is increased by assembling and expanding enzyme in a three-dimensional space, so that the driving force of the enzyme-driven micro-nano motor is improved, and further, biotinylated enzyme is assembled on biotinylated particles through specific binding of streptavidin and biotin, so that the enzyme-driven micro-nano motor is obtained.

As a preferred aspect of the above technical solution, the preparation method for improving the driving force of the enzyme-driven micro-nano motor further includes a part or all of the following technical features:

as an improvement of the technical scheme, the preparation method adopts a three-step method:

1) preparing biotinylated Shuangshen particles: the method comprises the following steps of asymmetrically coating a metal Au layer on the surface of a micro-nano particle serving as a template to form a double-sided particle, and modifying N- (6- [ Biotin amine ] hexyl) -3'- (2' -pyridine disulfide) propionamide (Biotin-HPDP) on the surface of the double-sided particle by utilizing an Au-S bond;

2) preparation of biotinylated enzyme: modifying a biotinylation reagent Na- (3-maleimidopropylpropionyl) biocytin on the surface of the biological enzyme by utilizing the bonding between a sulfhydryl group and maleimide;

3) preparing an enzyme driving micro-nano motor: the specific combination of streptavidin and biotin is utilized to fix the biotinylation enzyme on the surface of the biotinylation Shuangshen particle through layer-by-layer self-assembly.

As an improvement of the technical scheme, the prepared enzyme-driven micro-nano motor can autonomously move under the condition of simulating blood viscosity (4-5 mPa · s), and can be applied to the field of biological medicines.

As an improvement of the above technical solution, the step (1) specifically comprises: dripping ethanol suspension of the micro-nano particles on a glass slide cleaned by oxygen plasma, and naturally evaporating at room temperature to form single-layer micro-nano particles; and moving the glass slide to a magnetron sputtering chamber, sputtering a metal Au layer, collecting the prepared double-sided super-nano particles through ultrasound and centrifugation, and washing with ultrapure water for several times for later use. Dissolving N- (6- [ Biotin amine ] hexyl) -3'- (2' -pyridine disulfide) propionamide (Biotin-HPDP) in N, N-Dimethylformamide (DMF) under the condition of 45 ℃ by ultrasonic waves, then adding tributyl phosphate solution, uniformly mixing, and reacting for 30min under the condition of water bath at 45 ℃; sequentially adding ultrapure water, absolute ethyl alcohol and the Shuangshen micro-nano particles, uniformly mixing, and carrying out oscillation reaction at a constant temperature of 25 ℃ for overnight; washed 2 times with ultrapure water and 3 times with PB buffer solution (10mmol/L, pH 7.4).

As an improvement of the above technical solution, the step (2) specifically comprises: uniformly mixing a biotinylation reagent (Na- (3-maleimidopropylpropionyl) biocytin) and urease, and carrying out oscillation reaction for 2h at a constant temperature of 25 ℃; PB buffer solution (10mmol/L, pH 7.4) was washed and collected by ultrafiltration centrifugation 5 times.

As an improvement of the above technical solution, the step (3) specifically comprises: uniformly mixing the biotinylation double-sided god micro-nano particles, biotinylation enzyme and streptavidin, oscillating and reacting overnight at a constant temperature of 25 ℃, and washing with ultrapure water for 3 times.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the preparation method can realize the spatial assembly and expansion of the enzyme, thereby increasing the enzyme content and obviously improving the driving force and the movement speed of the enzyme-driven micro-nano motor.

2. The enzyme-driven micro-nano motor prepared by the invention has strong driving force, can autonomously move under the condition of simulating blood viscosity, effectively solves the problems that the enzyme-driven micro-nano motor has weak driving force and cannot move under the condition of high viscosity, and provides an effective solution for the medical use of the enzyme-driven micro-nano motor in the field of biological medicine.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood, the present invention may be implemented in accordance with the contents of the description, and in order to make the above and other objects, features, and advantages of the present invention more clearly understood, the following detailed description is given in conjunction with the preferred embodiments.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly described below.

FIG. 1: the invention relates to a process diagram for preparing an enzyme driving motor based on a protein (enzyme) assembly technology;

FIG. 2: the energy dispersion X-ray spectrogram (EDS) of the biotinylated dihedral micron/nano particle is disclosed;

FIG. 3: the optical image of biotin on biotinylated urease is verified by comparing urease with biotinylated urease by using a TMB-HRP colorimetric method;

FIG. 4: the invention relates to bright field and fluorescence field microscope images of an enzyme driving motor after Krypton protein staining;

FIG. 5: the enzyme driving motor of the invention has a movement rate chart under different urea concentrations;

FIG. 6: the enzyme driving motor of the invention moves in 10mM urea and 35 w/v% sucrose solution for 30 s;

Detailed Description

Other aspects, features and advantages of the present invention will become apparent from the following detailed description, which, when taken in conjunction with the drawings, illustrate by way of example the principles of the invention.