EXPERIMENT SYSTEM AND METHOD FOR ACCURATE CONTROLLING OF MACROMOLECULAR CRYSTALLIZATION PROCESS

Abstract

An experiment system and method for accurate controlling of macromolecular crystallization process. The system has a platform-equipped horizontal moving slot and channel dedicated backwash module, a droplet adding control module, an observing module, a user observation computer system, and an experimental condition control module. A high-precision movement knob of the x-axis platform and the y-axis platform of the system and the accurate position control of a syringe needle are used to ensure that the macromolecular solution can be added into the correct positions of convex or concave. The crystallization induction period of the target crystal faun is determined by the real-time data of the high-speed microcamera, and the crystal cultivation environment is adjusted in real time. This is simple and easy to operate, high in productivity, can be applied to the conventional experimental replication.

Claims

1. An experiment system for accurate controlling of macromolecular crystallization process, wherein the experiment system is a closed space encircled by a shell at ordinary pressure and is mainly composed of a platform-equipped horizontal moving slot and channel dedicated backwash module, a droplet adding control module, an observing module, a user observation computer system, and an experiment condition control module; the platform-equipped horizontal moving slot and channel dedicated backwash module, the droplet adding control module, the observing module and the experiment condition control module are positioned inside the closed space, and the user observation computer system is positioned outside the closed space; the platform-equipped horizontal moving slot and channel dedicated backwash module comprises a platform horizontal moving slot, an x-axis horizontal adjusting mechanism, a y-axis horizontal adjusting mechanism and a high-throughput macromolecular crystal cultivation platform; the high-throughput macromolecular crystal cultivation platform is placed on the platform horizontal moving slot; the horizontal position of the platform horizontal moving slot is adjusted through the x-axis horizontal adjusting mechanism and the vertical height of the same is adjusted through the y-axis horizontal adjusting mechanism, so as to ensure accurate movement of the high-throughput macromolecular crystal cultivation platform; the droplet adding control module comprises a sample injector, a piston propulsion adjusting mechanism and a sample injector height adjusting mechanism, the piston propulsion adjusting mechanism is used for accurately controlling the volume of a droplet added by the sample injector, the sample injector height adjusting mechanism is used for controlling the position of the sample injector; the observing module comprises a high-speed microcamera and a camera adjusting unit, the high-speed microcamera is fixed to the camera adjusting unit, a state of crystal inside the droplet on the high-throughput macromolecular crystal cultivation platform is observed by controlling the angle, brightness and magnification factor of the high-speed microcamera; the experiment condition control module comprises a temperature and humidity measurement and control apparatus for controlling humidity and temperature required in the closed space; the user observation computer system comprises a data export line and a user observing computer, and is used as an external extension of the observing system for facilitating the user to observe crystal growth under a camera lens of the high-speed microcamera using computer software; the user observing computer is connected with the camera adjusting unit through the data export line; the high-throughput macromolecular crystal cultivation platform comprises a backwash liquor inlet, a backwash liquor flow channel and a backwash liquor outlet, backwash liquor enters from the backwash liquor inlet, and after washing the crystal in the backwash liquor flow channel, the backwash liquor flows out of the backwash liquor outlet.

2. The experiment system for accurate controlling of macromolecular crystallization process according to claim 1, wherein the backwash liquor flow channel is a convex or concave structure of micron scale structure with regular patterns; a crystal platform formed by convex structures and concave structures with different layout forms is selected according to actual needs, and there are two layout forms; the first layout form is a lattice layout, with the convex structures on one side of the crystal platform according to the lattice layout, and with the concave structures on the other side of the crystal platform according to the lattice layout; the second layout form is a tunnel layout, with the convex structures and the concave structures alternately arranged to form a crystal platform with tunnel type.

3. The experiment system for accurate controlling of macromolecular crystallization process according to claim 2, wherein according to the morphology of a target crystal system, the sizes of the convex structures and the concave structures are determined, and the height of the convex structures or the concave structures is 10 m to 500 m.

4. The experiment system for accurate controlling of macromolecular crystallization process according to claim 2, wherein if the experiment system is used for preparing high-throughput crystal, the crystal platform is designed as the second tunnel layout form to facilitate the washing of anti-solvent and the centralized collection of crystal products, and the connected sample injector is selected to improve the sample introduction efficiency; if the experiment system is used for screening crystal features, the crystal platform is designed as the first lattice layout form to improve the repeatability of crystal screening, and the crystal platform is made of material that does not react with the target crystal system.

5. The experiment system for accurate controlling of macromolecular crystallization process according to claim 2, wherein for the temperature and humidity measurement and control apparatus, the control range of humidity is 10% to 100% and the control range of temperature is 50 C. to 200 C.; the errors of the x-axis horizontal adjusting mechanism, the y-axis horizontal adjusting mechanism and the sample injector height adjusting mechanism are one thousandth per 25.4 mm; and the magnification factor of the high-speed microcamera is 10 to 600 times.

6. The experiment system for accurate controlling of macromolecular crystallization process according to claim 4, wherein for the temperature and humidity measurement and control apparatus, the control range of humidity is 10% to 100% and the control range of temperature is 50 C. to 200 C.; the errors of the x-axis horizontal adjusting mechanism, the y-axis horizontal adjusting mechanism and the sample injector height adjusting mechanism are one thousandth per 25.4 mm; and the magnification factor of the high-speed microcamera is 10 to 600 times.

7. The experiment system for accurate controlling of macromolecular crystallization process according to claim 4, wherein the connected sample injector improves the sample introduction efficiency through the following operation: adjusting the x-axis horizontal adjusting mechanism and the y-axis horizontal adjusting mechanism, aligning a target site for adding a droplet on the platform with the sample injector of a connected structure, adjusting the sample injector height adjusting mechanism according to the height of the high-throughput macromolecular crystal cultivation platform, when ready, selecting a sample injector control system to extrude the droplet and the volume of which is indicated by the graduations on the sample injector of the connected structure, each sample injector being controlled by the sample injector control system to ensure that the amount of the droplets extruded by each sample injector of the sample injectors of the connected structure are identical, and processing capacities are consistent; the method uses the connected structure to connect a plurality of sample injectors in series or in parallel to increase output efficiency.

8. An experiment method for accurate controlling of macromolecular crystallization process, comprising the following steps: (1) placing crystal culture solution prepared with target crystal system and solvent at a temperature of 20-30 C. for standby; disinfecting the crystal platform; adding the prepared crystal culture solution into the sample injector; placing the high-throughput macromolecular crystal cultivation platform on the platform horizontal moving slot, to complete preparation before crystal cultivation; (2) adjusting the temperature and humidity measurement and control apparatus so that temperature and humidity conditions in the closed space reach the values set during crystallization, wherein temperature and humidity throughout the experiment are controlled by the experiment condition control module; (3) adjusting the x-axis horizontal adjusting mechanism and the y-axis horizontal adjusting mechanism during the experiment, aligning a target site for adding a droplet on the high-throughput macromolecular crystal cultivation platform with the sample injector, adjusting the sample injector height adjusting mechanism according to the height of the high-throughput macromolecular crystal cultivation platform, when ready, rotating a piston propulsion mechanism to extrude the droplet, the volume of which is indicated by the graduations on the sample injector; (4) observing the state of the crystal droplet through the high-speed microcamera of the observing module, converting the observed content into an electrical signal and presenting on the user observing computer through the data export line, and collecting and storing crystallization pictures or videos; and (5) taking out the high-throughput macromolecular crystal cultivation platform from the platform horizontal moving slot when crystal cultivation is completed, connecting a pipeline of the anti-solvent to the backwash liquor inlet to start washing, washing liquid passing through the backwash liquor flow channel and being collected at the backwash liquor outlet, and backwashing out the crystal to complete the operation of high-throughput crystal cultivation.

Description

DESCRIPTION OF DRAWINGS

[0027] FIG. 1 is a side view of the system.

[0028] FIG. 2 is a local amplified schematic diagram of a high-throughput macromolecular crystal cultivation platform of the system.

[0029] FIG. 3 is a top view of design of four crystal cultivation platforms applicable to the system.

[0030] FIG. 4 is an operation schematic diagram of a high-throughput connected sample injector.

[0031] In FIG. 1: 1 shell; 2 platform horizontal moving slot; 3 x-axis horizontal adjusting mechanism;

[0032] 4 y-axis horizontal adjusting mechanism; 5 high-throughput macromolecular crystal cultivation platform; 6 sample injector;

[0033] 7 piston propulsion adjusting mechanism; 8 sample injector height adjusting mechanism; 9 high-power camera;

[0034] 10 data export line; 11 camera adjusting unit; 12 user observing computer; 13 temperature and humidity measurement and control apparatus; 14 backwash liquor inlet; 15 backwash liquor flow channel;

[0035] 16 backwash liquor outlet; I platform-equipped horizontal moving slot and channel dedicated backwash module;

[0036] II droplet adding control module; III observing module; IV user observation computer system; and

[0037] V experiment condition control module.

[0038] In FIG. 3: each picture is part of a high-precision crystal platform and the feature size of each structure is 0.01 mm to 0.5 mm, wherein (1) square structure; (2) circular structure; (3) hexagonal structure; and (4) diamond structure; each feature structure may be a convex structure or a concave structure as needed.

[0039] In FIG. 4: 17 sample injector control system; 18 connected structure (a plurality of 6 sample injectors are attached); 19 micron-scale crystal platform (10 micron to 500 micron); and 20 droplet adding groove (anti-solvent washing channel).

DETAILED DESCRIPTION

[0040] Specific embodiments of the present invention are further described below in combination with the drawings and the technical solution.

[0041] The use method of the present invention is elaborated with the examples of lysozyme crystal cultivation.

Example 1 Crystal Screening of Lysozyme Using Sample Injector 6

[0042] Lysozyme crystal culture solution was prepared and put into the sample injector 6. The high-throughput macromolecular crystal cultivation platform 5 was placed on the platform horizontal moving slot 2, so that the preparation before crystal cultivation was completed.

[0043] The temperature and humidity measurement and control apparatus 13 was adjusted so that the temperature and humidity conditions in the closed space of the shell 1 reached the values set during lysozyme crystallization. Temperature and humidity throughout the experiment were controlled by the experiment condition control module V.

[0044] The x-axis horizontal adjusting mechanism 3 and the y-axis horizontal adjusting mechanism 4 were adjusted during the experiment, a target site for adding a droplet on the platform was aligned with the sample injector 6, the sample injector height adjusting mechanism 8 was adjusted according to the height of the high-throughput macromolecular crystal cultivation platform 5, when ready, a piston propulsion mechanism 7 was rotated to extrude the droplet, and the volume of which was indicated by the graduations on the sample injector 6.

[0045] The state of the crystal droplet was observed through the high-speed microcamera 9 of the observing module III, the observed content was converted into an electrical signal and presented on the user observing computer 12 through the data export line 10, and crystallization pictures or videos were collected and stored.

[0046] The high-throughput macromolecular crystal cultivation platform 5 was taken out from the platform horizontal moving slot 2 when lysozyme crystal cultivation was completed, a pipeline of the anti-solvent was connected to the backwash liquor inlet 14 to start washing, washing liquid passed through the backwash liquor flow channel 15 and was collected at the backwash liquor outlet 16, and the crystal was backwashed out to complete the operation of high-throughput crystal cultivation.

Example 2 High-Throughput Output of Lysozyme Using Sample Injector 6 of Connected Structure 18

[0047] Lysozyme crystal culture solution was prepared and put into the sample injector 6 of the connected structure 18 for standby. The high-throughput macromolecular crystal cultivation platform 5 was placed on the platform horizontal moving slot 2, so that the preparation before crystal cultivation was complete.

[0048] The method of adjusting the temperature and the humidity was in accordance with that of Example 1. The x-axis horizontal adjusting mechanism 3 and the y-axis horizontal adjusting mechanism 4 were adjusted, a target site for adding a droplet on the platform was aligned with the sample injector 6 of the connected structure 18, the sample injector height adjusting mechanism 8 was adjusted according to the height of the high-throughput macromolecular crystal cultivation platform 5, when ready, a sample injector control system 17 was selected to extrude the droplet and the volume of which was indicated by the graduations on the sample injector 6 of the connected structure 18, each sample injector was controlled by the sample injector control system 17 to ensure that the amount of the droplets extruded by each sample injector of the sample injectors 6 of the connected structure 18 were identical and processing capacities were consistent. As shown in FIG. 4, the same repeatable droplet morphology 20 can be obtained on a micron-scale crystal platform 19.

[0049] The means of observing crystal morphology was in accordance with that of Example 1. The high-throughput macromolecular crystal cultivation platform 5 was taken out from the platform horizontal moving slot 2 when lysozyme crystal cultivation was completed, a pipeline of the anti-solvent was connected to the backwash liquor inlet 14 to start washing, washing liquid passed through the backwash liquor flow channel 15 and was collected at the backwash liquor outlet 16; and the crystal was backwashed out to complete the operation of high-throughput crystal cultivation.

[0050] The method uses the connected structure (18) to connect a plurality of sample injectors (6) in series or in parallel to improve the output efficiency. The method is simple and easy to operate, high in productivity, can be applied to the conventional experiment replication as well as exploration of limiting crystallization conditions. The above description is only simple embodiments of the present invention and is not limited to the present invention. Equivalent replacement, name amendment, apparatus improvement, etc., which are similar to the present invention shall be included within the protection scope of the present invention.