阅读说明：本技术 一种蛋白质基体微透镜阵列衍射器件及其制备方法 (Protein matrix microlens array diffraction device and preparation method thereof ) 是由 姜澜 俞嘉晨 闫剑锋 于 2019-09-18 设计创作，主要内容包括：本发明涉及一种蛋白质基体微透镜阵列衍射器件及其制备方法,属于生物检测设备技术领域。该蛋白质基体微透镜阵列衍射器件的基体为蛋白质结晶体,蛋白质结晶体上最大边长L所在的一个表面上加工有微透镜状凸起阵列。本发明d1蛋白质基体微透镜阵列衍射器件,将测量量转化为衍射图样的形状、分布、间距等更容易测量的物理量,提高了检测精度和灵活性。本发明的蛋白质基体微透镜阵列衍射器件制备方法,使用蛋白质单晶作为微透镜阵列衍射器件的基体,在溶液环境中更稳定,使用寿命更长,使用范围更广泛。本发明的微透镜阵列加工的方法为使用飞秒激光加工,直接形成微凸起,加工简便,热损伤小。(The invention relates to a protein matrix microlens array diffraction device and a preparation method thereof, belonging to the technical field of biological detection equipment. The substrate of the protein substrate micro-lens array diffraction device is a protein crystal body, and a micro-lens-shaped convex array is processed on one surface of the protein crystal body where the maximum side length L is located. The d1 protein matrix microlens array diffraction device converts the measured quantity into physical quantity which is easier to measure, such as the shape, distribution, space and the like of a diffraction pattern, and improves the detection precision and flexibility. According to the preparation method of the protein matrix microlens array diffraction device, the protein single crystal is used as the matrix of the microlens array diffraction device, so that the protein matrix microlens array diffraction device is more stable in a solution environment, longer in service life and wider in application range. The method for processing the micro lens array directly forms the micro bulges by femtosecond laser processing, and has simple and convenient processing and small heat damage.)

1. A protein matrix microlens array diffraction device is mainly characterized in that: the matrix of the protein matrix microlens array diffraction device is a protein crystal, and the maximum side length L of the protein crystal is 100-500 mu m; a micro-lens-shaped protrusion array is processed on one surface of the protein crystal body where the maximum side length L is located, and the distance p between every two micro-lens-shaped protrusions in the micro-lens-shaped protrusion array is 10-100 mu m; the diameter d of the lenticular projection is 2-10 μm and the height h of the lenticular projection is 0.05-2 μm.

2. A preparation method of a protein matrix microlens array diffraction device is characterized by comprising the following steps:

(1) the protein crystal is prepared by the following steps:

(1-1) mixing a protein solid and a salt solution with the mass percentage concentration of 2% -5% with each other to obtain a protein solution, wherein the mass fraction of protein in the protein solution is 1-3 times that of the saturated solution, and the pH value of the protein solution is adjusted to 4-6;

(1-2) placing the protein solution prepared in the step (1-1) in a container, standing for 6-24 hours at room temperature, and naturally evaporating water in the protein solution to separate out protein crystals;

(2) and (2) enabling a femtosecond laser to enter the surface of the protein crystal obtained in the step (1), wherein the pulse duration of the femtosecond laser is 35-120 fs, the single pulse energy of the femtosecond laser is 0.5-1 time of the ablation threshold of the protein, and the repetition frequency of the femtosecond laser is 1-1000 Hz.

3. The method of claim 2, wherein the salt is sodium chloride or sodium acetate.

Technical Field

The invention relates to a protein matrix microlens array diffraction device and a preparation method thereof, belonging to the technical field of biological detection equipment.

Background

Protein materials have found widespread use in many fields due to biocompatibility and unique biological activity. Because the protein molecules have a multi-level structure, the structure of the protein molecules is influenced by other molecules or ions in the external temperature and the environment, and the molecular structure, the physical and chemical properties and the like of the protein molecules can be correspondingly changed, the microstructure or the device based on the protein material has unique corresponding characteristics for the environment.

At present, some researches apply the property of the biological material to the field of biosensors to realize the detection of environmental pH and the like, and the main principle is to change the optical property of an optical waveguide through the response of the optical waveguide of a protein matrix to the surrounding environment so as to change the light intensity output by the optical waveguide. The method has the advantages that the measurement parameters are only the light intensity behind the optical waveguide, so that the measurable dimension is less, the requirement on the sensor is higher, the interference of other factors is caused, and the method is not widely applied. In addition, the amorphous protein has poor stability in the environment and is easy to degrade.

A microlens array of optical device features that the microlenses with uniform structure are arranged on a plane periodically for higher optical performance, beam shaping and optical sensing. For example, the patent "microlens array, optical detection device and microlens array preparation method" (application No. 201711429305.4) describes a microlens array integrated in a micro flow channel system, which can realize the detection of nano objects in the micro flow channel. However, the main material of the current microlens array is inorganic material, the response to the environment depends on optical properties such as refractive index, and the capability of biological monitoring is weak.

Therefore, protein-based microarray devices are promising biological detection devices. Because the protein material itself is sensitive to factors such as ambient temperature, the processing of protein-based micro devices presents certain challenges, and the simplification of processing steps is also a major challenge in protein micromachining, while the thermal influence during the processing of protein materials is minimized. At present, some methods utilize femtosecond laser and other processing means to realize micro-nano processing with high precision, small heat affected zone and wide material using range, and are already applied to processing of a plurality of biological materials, transparent materials and the like. For example, the patent "method and system for processing micro-nano structure on single protein crystal by femtosecond laser" (application No. 2019103009814) in the application proposes a method and system for processing protein single crystal by femtosecond laser, which can ablate microstructure with the precision of 1-10 μm on the protein single crystal, but the method mainly utilizes femtosecond laser pulse higher than the ablation threshold of protein, and has no related method for realizing micro-convex structure preparation by femtosecond laser modification lower than the ablation threshold.

Disclosure of Invention

The invention provides a protein matrix microlens array diffraction device and a preparation method thereof, which improve the existing method for processing protein single crystals by femtosecond laser, realize the preparation of microlenses by a microprotrusion structure by using the femtosecond laser modification below an ablation threshold value, and process a device formed by periodically arranged microlens arrays on the surface of protein crystals.

The protein matrix microlens array diffraction device provided by the invention has the advantages that the matrix of the protein matrix microlens array diffraction device is a protein crystal, and the maximum side length L of the protein crystal is 100-; a micro-lens-shaped protrusion array is processed on one surface of the protein crystal body where the maximum side length L is located, and the distance p between every two micro-lens-shaped protrusions in the micro-lens-shaped protrusion array is 10-100 mu m; the diameter d of the lenticular projection is 2-10 μm and the height h of the lenticular projection is 0.05-2 μm.

The invention provides a preparation method of a protein matrix microlens array diffraction device, which comprises the following steps:

(1) the protein crystal is prepared by the following steps:

(1-1) mixing a protein solid and a salt solution with the mass percentage concentration of 2% -5% with each other to obtain a protein solution, wherein the mass fraction of protein in the protein solution is 1-3 times that of the saturated solution, and the pH value of the protein solution is adjusted to 4-6;

(1-2) placing the protein solution prepared in the step (1-1) in a container, standing for 6-24 hours at room temperature, and naturally evaporating water in the protein solution to separate out protein crystals;

(2) and (2) enabling a femtosecond laser to enter the surface of the protein crystal obtained in the step (1), wherein the pulse duration of the femtosecond laser is 35-120 fs, the single pulse energy of the femtosecond laser is 0.5-1 time of the ablation threshold of the protein, and the repetition frequency of the femtosecond laser is 1-1000 Hz.

The protein matrix micro-lens array diffraction device and the preparation method thereof provided by the invention have the advantages that:

1. the protein matrix micro-lens array diffraction device prepared by the method is used as a biological detection device and has the characteristics of unique response characteristic and good biocompatibility; compared with the existing detection device using biological materials, such as a method for detecting the change of the light intensity output by the light waveguide by using the protein matrix optical waveguide to realize biological detection, the protein matrix microlens array diffraction device converts the measured quantity into the physical quantity which is easier to measure, such as the shape, distribution, space and the like of the diffraction pattern, and improves the detection precision and flexibility.

2. According to the preparation method of the protein matrix microlens array diffraction device, the protein single crystal is used as the matrix of the microlens array diffraction device, so that the protein matrix microlens array diffraction device is more stable in a solution environment, longer in service life and wider in application range.

3. According to the preparation method of the protein matrix microlens array diffraction device, the microlens array is processed by femtosecond laser, so that the microprotrusions are directly formed, the processing is simple and convenient, and the thermal damage is small.

Drawings

FIG. 1 is a schematic structural diagram of a protein-based microlens array diffraction device prepared by the method of the present invention.

Fig. 2 is a schematic side view of each microlens in the microlens array.

FIG. 3 is a diagram of the state of the protein-based microlens array diffraction device prepared according to the embodiment of the present invention.

Fig. 4 is a structural diagram and an effect diagram of a protein-based microlens array diffraction device prepared according to an embodiment of the present invention, wherein (a) is a schematic diagram of a microlens array in a micro-convex shape, and (b) is a diffraction pattern formed by the microlens array diffraction device in a solution environment to be tested.

In fig. 3, 1 is a continuous laser, 2 is a protein matrix microlens array diffraction device, 3 is a transparent cuvette, 4 is a back focal plane of a protein matrix microlens, 5 is a lens barrel, and 6 is an imaging system.

Detailed Description

The structure of the protein matrix microlens array diffraction device provided by the invention is shown in figure 1, the matrix of the protein matrix microlens array diffraction device is a protein crystal, and the maximum side length L of the protein crystal is 100-; a micro-lens-shaped protrusion array is processed on one surface of the protein crystal body where the maximum side length L is located, and the distance p between every two micro-lens-shaped protrusions in the micro-lens-shaped protrusion array is 10-100 mu m; the diameter d of the lenticular projection is 2-10 μm and the height h of the lenticular projection is 0.05-2 μm.

The invention provides a preparation method of a protein matrix microlens array diffraction device, which comprises the following steps:

(1) the protein crystal is prepared by the following steps:

(1-1) mixing a protein solid and a salt solution with the mass percentage concentration of 2% -5% with each other to obtain a protein solution, wherein the mass fraction of protein in the protein solution is 1-3 times that of the saturated solution, and the pH value of the protein solution is adjusted to 4-6;

(1-2) placing the protein solution prepared in the step (1-1) in a container, standing for 6-24 hours at room temperature, and naturally evaporating water in the protein solution to separate out protein crystals;

(2) and (2) enabling femtosecond laser to enter the surface of the protein crystal obtained in the step (1), wherein the pulse duration of the femtosecond laser is 35-120 fs, the single pulse energy of the femtosecond laser is lower than the ablation threshold of the protein, but the femtosecond laser can modify the protein and form a micro-bulge, the micro-bulge is 0.5-1 time of the ablation threshold of the protein, and the repetition frequency of the femtosecond laser is 1-1000 Hz. The femtosecond laser is focused at the positions of periodically arranged microlenses to be processed by controlling the attenuation sheet in the processing light path to control the single pulse energy of the femtosecond laser and controlling the shutter and the translation stage in the processing light path, and micro-convex structures are processed one by one to form a microlens array diffraction device.

The invention is described in detail below with reference to the accompanying drawings:

FIG. 1 is a schematic diagram of a microlens array diffraction device, in which a substrate of the protein-based microlens array diffraction device is a natural protein crystal, and the maximum side length L of the natural protein crystal is 100-500 μm; processing a micro-lens-shaped protrusion array on the surface of the maximum side length L of the natural crystal body, wherein the distance p between every two micro-lens-shaped protrusions in the micro-lens-shaped protrusion array is 10-100 mu m; as shown in FIG. 2, which is a schematic side view of each microlens in the array, the shaded area is protein, the diameter d of the microlens-like protrusions is 2-10 μm, and the height h of the microlens-like protrusions is 0.05-2 μm. The arrow is the direction of light rays, the light incident in parallel can be focused through a curved surface, and the focal length f of the micro lens can be obtained according to the geometrical relation by the following formula, wherein n is the refractive index of the protein crystal:

the verification light path of the protein matrix microlens array diffraction device is shown in fig. 3, and after a beam of laser is vertically incident to the crystal surface processed with the microlens array, a diffraction pattern is formed on the back focal plane of the microlens. The position, shape and intensity distribution of diffraction patterns of different environmental solutions are related to shape parameters and optical properties of proteins and surrounding environments, and the parameters are influenced by the concentrations of molecules and ions in the solutions in the environments. By the principle, the detection of the molecular and ionic concentrations of different solutions can be realized.

The invention also provides a preparation method of the protein matrix micro-lens array diffraction device, which comprises the following steps:

(1) the protein crystal is prepared by the following steps:

(1-1) mixing a protein solid and a salt solution with the mass percentage concentration of 2% -5% with each other to obtain a protein solution, wherein the mass fraction of protein in the protein solution is 1-3 times that of the saturated solution, and the pH value of the protein solution is adjusted to 4-6;

(1-2) placing the protein solution prepared in the step (1-1) in a container, standing for 6-24 hours at room temperature, and naturally evaporating water in the protein solution to separate out protein crystals;

(2) and (2) enabling femtosecond laser to enter the surface of the protein crystal obtained in the step (1), wherein the pulse duration of the femtosecond laser is 35-120 fs, the single pulse energy is lower than the ablation threshold of the protein but can modify the protein and form a microprotrusion, the single pulse energy is 0.5-1 time of the ablation threshold of the protein, and the repetition frequency is 1-1000 Hz. The femtosecond laser is focused at the positions of the periodically arranged microlenses to be processed each time by controlling the attenuation sheet in the processing light path to control the single pulse energy of the femtosecond laser and controlling the shutter and the translation stage in the processing light path, and the micro-convex structures are processed one by one to form the microlens array diffraction device.

The salts in the above process are sodium chloride and sodium acetate.

The invention is further described with reference to the following examples:

in this embodiment, taking chicken egg white lysozyme crystal as an example, a microlens array is processed on the surface of the protein crystal, and the device structure is shown in fig. 1, wherein the protein crystal is a chicken egg white lysozyme crystal cultured by a general crystallization method and having a maximum side length L of 100-; the pitch of the microlens array on the crystal surface is 10 μm; the radius and height of the microlens, under the designed processing parameters, were characterized by atomic force microscopy to give d 3 μm and h 1 μm.

The femtosecond laser used in the method is a titanium sapphire femtosecond laser produced by Coherent company, the central wavelength is 800nm, and the pulse width is less than 35 fs.