WO2019128766A1 - 一种适用于大分子结晶过程精确调控的实验系统和方法 - Google Patents

一种适用于大分子结晶过程精确调控的实验系统和方法 Download PDF

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WO2019128766A1
WO2019128766A1 PCT/CN2018/121637 CN2018121637W WO2019128766A1 WO 2019128766 A1 WO2019128766 A1 WO 2019128766A1 CN 2018121637 W CN2018121637 W CN 2018121637W WO 2019128766 A1 WO2019128766 A1 WO 2019128766A1
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platform
crystal
injector
macromolecular
crystallization
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PCT/CN2018/121637
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English (en)
French (fr)
Inventor
姜晓滨
韩明光
贺高红
李津
李祥村
肖武
吴雪梅
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大连理工大学
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Priority claimed from CN201711443611.3A external-priority patent/CN108165486B/zh
Priority claimed from CN201711443978.5A external-priority patent/CN108159730B/zh
Application filed by 大连理工大学 filed Critical 大连理工大学
Priority to JP2019551564A priority Critical patent/JP6742624B2/ja
Priority to US16/349,572 priority patent/US11452948B2/en
Priority to EP18871811.8A priority patent/EP3530726A4/en
Publication of WO2019128766A1 publication Critical patent/WO2019128766A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/0063—Control or regulation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/004—Fractional crystallisation; Fractionating or rectifying columns
    • B01D9/0045—Washing of crystals, e.g. in wash columns
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/005—Selection of auxiliary, e.g. for control of crystallisation nuclei, of crystal growth, of adherence to walls; Arrangements for introduction thereof
    • B01D9/0054—Use of anti-solvent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/0077—Screening for crystallisation conditions or for crystal forms
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B35/00—Apparatus not otherwise provided for, specially adapted for the growth, production or after-treatment of single crystals or of a homogeneous polycrystalline material with defined structure
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D2009/0086—Processes or apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N2021/8477—Investigating crystals, e.g. liquid crystals

Definitions

  • the invention belongs to the technical field of crystallization and relates to an experimental system and method suitable for precise regulation of macromolecular crystallization processes.
  • CN 103254274A discloses a regulation optimization device for protein crystals for determining whether a substance is a target protein in a crystalline state.
  • the method since it is limited to biological macromolecules capable of analyzing structures using X-single-crystal diffraction technology, the method has no universality and is not significant for industrial production and control.
  • the precise regulation of macromolecular crystals and the related devices for crystallization observation have not been reported in the literature.
  • the macromolecular crystals in the confined space can precisely improve the crystal crystal form regulation due to the surrounding environment limitation, and obtain the target crystal.
  • the shape of the confinement space has a positive effect on the crystal growth crystal plane. This conclusion can be extended not only to biomacromolecules, but also to the problem of crystallization of macromolecular drugs.
  • the confined space is not conducive to the crystal structure related crystal morphology observation, crystal induction period measurement and crystal extraction. Therefore, the existing traditional experimental systems and monitoring methods need to be improved, and a crystallization experiment system integrating observation, control environment and precise injection is needed.
  • the limited structure of the high-precision micron-scale crystallization platform can be any pattern of rules, using the high-precision moving mechanism of the x, y-axis platform of the system itself, and the needle Precise position control ensures that the macromolecular solution can be dropped into the correct position of the protrusion or depression, and can ensure the precise operation of the crystal crystallization micro-environment and the post-crystal backwash.
  • the invention mainly designs an experimental system for providing crystal culture environment, droplet drop position control, crystal growth process observation and crystal screening for high-precision macromolecular crystals, and the system can quantitatively drop target macromolecular solution and observe in real time.
  • the actual growth of the crystal
  • the macromolecular crystal culture crystallizing platform uses a micro-original convex structure having a specific micron-scale surface shape (a lattice structure or a convex structure of a groove is included), or a flat structure having a groove structure of a specific size is processed. Use the micro component platform.
  • the droplets are batchly dropped onto the micro-origin by a matrix dropping device, and the temperature and humidity conditions around the micro-component platform are controlled, and the droplet shape and the evaporation rate are controlled to ensure the output of the target crystal form target.
  • the method can provide a crystallization place for all crystallizable substances, especially a small amount of rare drugs which have been extracted, has simple operation, low requirements on surrounding environment and less loss of medicine, and is a simple and environmentally friendly crystallization technique which can be repeated in large quantities.
  • the experimental system is a closed space surrounded by the outer casing 1 under normal pressure, and the horizontal moving groove and the special backwashing module of the flow channel are mainly arranged by the platform, and the droplets are added dropwise.
  • the control module II, the observation module III, the user observation computer system IV and the experimental condition control module V are composed; the platform arranges the horizontal movement groove and the flow channel special backwash module I, the droplet drop control module II, the observation module III And the experimental condition control module V is located inside the confined space, and the user observation computer system IV is located outside the confined space;
  • the platform-arranged horizontal moving tank and the runner-specific backwashing module I comprise a platform horizontal moving tank 2, an x-axis horizontal adjusting mechanism 3, a y-axis horizontal adjusting mechanism 4 and a high-flux macromolecular crystal culture platform 5;
  • the high-throughput macromolecular crystallization culture platform 5 is placed on the platform horizontal moving groove 2, the horizontal moving groove 2 of the platform is adjusted by the x-axis horizontal adjusting mechanism 3, and the vertical height is adjusted by the y-axis horizontal adjusting mechanism 4 to ensure high The flux macromolecular crystal culture platform 5 moves accurately;
  • the droplet dropping control module II comprises an injector 6, a piston propulsion adjusting mechanism 7 and an injector height adjusting mechanism 8, and the piston propulsion adjusting mechanism 7 is used for the droplet volume dropped by the precision injector 6. a sample height adjustment mechanism 8 for controlling the position of the injector 6;
  • the observation module III includes a high-powered camera 9 and a camera adjustment unit 11.
  • the high-power camera 9 is fixed on the camera adjustment unit 11, and the high-throughput is observed by controlling the angle, brightness and magnification of the high-power camera 9.
  • the experimental condition control module V includes a temperature and humidity measuring and controlling device 13 for regulating the humidity and temperature required in the sealed space;
  • the user observation computer system IV includes a data export line 10 and a user observation computer 12 as an external extension of the observation system to facilitate the user to observe the crystal growth under the lens of the high-power camera 9 by using computer software; the user observes the computer 12 connected to the camera adjustment unit 11 through the data export line 10;
  • the high-throughput macromolecular crystal growth platform 5 includes a backwash inlet 14, a backwash channel 15, and a backwash outlet 16, the backwash enters from the backwash inlet 14, and the backwash is in the backwash. After the crystals are rinsed in the flow path 15, the backwash liquid flows out from the backwash liquid outlet 16.
  • the backwashing flow passage 15 is a regular pattern of convex or concave structures of a micron-scale structure. According to the actual needs, the crystal platform formed by the convex structure and the concave structure with different layout forms is selected, and there are two layout forms;
  • the first layout form is a dot matrix layout, the convex structure is located on one side of the crystallization platform, according to the lattice layout; the concave structure is located on the other side of the crystallization platform, according to the lattice layout;
  • the second layout form is a tunnel layout, and the convex structure and the concave structure are alternately arranged to form a tunnel-type crystal platform;
  • the height of the convex structure or the concave structure is 10 ⁇ m ⁇ 500 ⁇ m;
  • the temperature and humidity measuring and controlling device 13 has a humidity control range of 10% to 100%, and a temperature control range of -50 ° C to 200 ° C.
  • the x-axis horizontal adjustment mechanism 3, the y-axis horizontal adjustment mechanism 4 and the injector height adjustment mechanism 8 have an error of one thousandth of every 25.4 mm error.
  • the scale on 6 is given, and the injector control system 17 of each injector is controlled to ensure that each injector of the injector 6 of the row structure 18 has the same amount of droplets extruded and the throughput is the same.
  • the method can utilize a series arrangement 18 to connect a plurality of injectors 6 in series or in parallel to increase production efficiency.
  • the high-powered camera 9 has a magnification of 10 to 600 times.
  • the high-flux macromolecular crystal growth platform 5 is taken out from the platform horizontal moving tank 2, the anti-solvent line is connected to the backwash inlet 14 to start rinsing, and the rinsing liquid is passed through the backwash.
  • the flow path 15 is collected at the backwash liquid outlet 16 to backwash the crystals to complete the operation of crystal high-flux culture.
  • the invention has the beneficial effects of using the high-precision moving mechanism of the x, y-axis platform of the system itself and the precise position control of the needle to ensure that the macromolecular solution can be dropped to the correct position of the protrusion or the recess, and the crystal microscopic environment can be ensured. And precise operation of back-stage crystal backwashing; the crystallization induction period of the target crystal form is determined by real-time data of high-power camera, and the crystal culture environment is adjusted in real time according to the experimental conditions to improve the crystallization efficiency and success rate. For high-throughput and large-output crystallization production requirements, the use of side-by-side injection can ensure accuracy and high-throughput crystal production.
  • Figure 1 is a side elevational view of the system.
  • FIG. 2 is a partially enlarged schematic view of a high-throughput macromolecular crystal growth platform of the present system.
  • Figure 3 is a top plan view of four crystallizing platforms designed for use in the present system.
  • Figure 4 is a schematic diagram of the operation of a high-throughput tandem injector
  • Figure 1 1 outer casing; 2 platform horizontal moving groove; 3x axis horizontal adjustment mechanism;
  • each of the figures is part of a high-precision crystallization platform, and each feature has a feature size of 0.01 mm to 0.5 mm, of which (1), square structure, (2), circular structure, (3), Hexagonal structure, (4), diamond-shaped structure; each of the characteristic structures may be either a convex structure or a concave structure as needed.
  • Figure 4 17 injector control system; 18 row structure (with multiple 6 injectors); 19 micron crystal platform (10 microns to 500 microns); 20 droplet drop grooves (anti-solvent cleaning channel) ).
  • Example 1 completes crystal screening of lysozyme using injector 6
  • the lysozyme crystal culture solution is disposed, placed in the sampler 6 for use, and the high-flux macromolecular crystal growth platform 5 is placed on the platform horizontal moving tank 2 to complete the preparation work before the crystal culture;
  • the temperature and humidity measuring and controlling device 13 adjusts the temperature and humidity conditions in the sealed space casing 1 to the values set by the lysozyme crystallization process, and the temperature and humidity of the whole experimental process are controlled by the experimental condition control module V;
  • the x-axis horizontal adjustment mechanism 3 and the y-axis horizontal adjustment mechanism 4 are adjusted, and the target site of the droplets on the platform is aligned with the injector 6, while according to the high-throughput macromolecular crystal culture platform 5
  • the height adjustment injector height adjustment mechanism 8 is ready to rotate the piston advancement mechanism 7 to extrude the droplets, and the volume of the droplets is given by the scale on the injector 6;
  • the high-flux macromolecular crystal growth platform 5 is taken out from the platform horizontal moving tank 2, the anti-solvent line is connected to the backwash inlet 14 to start the flushing, and the flushing liquid passes through the backwash stream. Lane 15, and collected at the backwash outlet 16, the crystal is backwashed to complete the operation of the crystal high-flux culture.
  • Example 2 uses the injector 6 of the row structure 18 to achieve high throughput of lysozyme
  • the lysozyme crystal culture solution is disposed, placed in the sampler 6 of the row structure 18 for use, and the high-flux macromolecular crystal growth platform 5 is placed on the platform horizontal moving tank 2 to complete the preparation work before the crystal culture;
  • the x-axis level adjusting mechanism 3 and the y-axis level adjusting mechanism 4 are adjusted to align the target point of the drip drop on the platform with the injector 6 of the row structure 18, and according to The height-adjusting injector height adjustment mechanism 8 of the high-throughput macromolecular crystal growth platform 5, when ready, selects the injector control system 17 to extrude droplets, the volume of which passes through the injector 6 of the bank structure 18.
  • the upper scale is given, and the injector control system 17 of each injector is controlled to ensure that each injector of the injector 6 of the bank structure 18 has the same amount of droplets to be extruded, and the throughput is uniform.
  • the same repeatable droplet topography 20 can be obtained on the micron-scale crystallization platform 19;
  • the means for observing the crystal morphology was the same as in Example 1.
  • the high-flux macromolecular crystal growth platform 5 was taken out from the platform horizontal moving tank 2, and the anti-solvent line was connected to the backwash inlet. 14.
  • the rinsing is started.
  • the rinsing liquid passes through the backwashing liquid passage 15 and is collected at the backwashing liquid outlet 16, and the crystal is backwashed to complete the operation of crystal high-flux culture.
  • the method can utilize the parallel structure (18) to connect a plurality of injectors (6) in series or in parallel, thereby improving output efficiency, being simple and easy to perform, and having high yield, which can be applied to repeated routine experiments or to limit crystallization. Conditional exploration.
  • the above description is only a simple embodiment of the present invention, and is not limited to the present invention, and equivalent replacements, naming modifications, device improvements, and the like, which are similar to the present invention, are included in the scope of the present invention.

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  • Chemical & Material Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Metallurgy (AREA)
  • Organic Chemistry (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)
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Abstract

一种适用于大分子结晶过程精确调控的实验系统和方法。该实验系统为常压下的密闭空间，包括平台布置水平移动槽及流道专用反洗模块（I）、液滴滴加控制模块（II）、观察模块（III）、用户观察用电脑系统（IV）和实验条件控制模块（V）。利用系统本身x，y轴水平调节机构（3、4），以及针头精确的位置控制，保证大分子溶液能够滴加到凸起或凹陷的正确位置，并能保证晶体结晶微观环境和后期晶体反洗的精确操作；通过高倍摄像仪（9）实时数据测定目标晶型的结晶诱导期，并根据实验情况实时调整晶体的培养环境，提高结晶效率和成功率。

Description

一种适用于大分子结晶过程精确调控的实验系统和方法 技术领域
本发明属于结晶技术领域，涉及一种适用于大分子结晶过程精确调控的实验系统和方法。
 背景技术
以大分子药物为主的药物的需求日益增长，推动相关大分子药物结晶的发展。从发现大分子药物的药效作用到药物结构的设计，稳定的生产流程设计和制备工艺开发已成为新药物能否商业化的重要研究方向。其中，大分子药结晶过程是生产流程设计的重点，调控药物目标晶型，缩短结晶诱导周期，晶体大批量的制备等相关研究极大影响药物制备过程中成本及成产可行性。由于病原体变异较快，对药物的适应能力增强，需要药物频繁的更新来应对变异的病原体。因此，需要大量制备目标晶体，并通过精确控制的制备工艺保证结晶体系的晶型、粒度、形貌、纯度等特性。以此为目标的大分子结晶过程精确调控的实验系统和方法开发也显得尤为重要。CN 103254274A公开了一种蛋白质晶体的调控优化装置，用以确定结晶状态下物质是否为目标蛋白质。但是由于只局限在能够利用X单晶衍射技术分析结构的生物大分子，该方法没有普适性，对应用于工业生产和控制意义不显著。目前，具有普适性的大分子晶体的精确调控研究和结晶观测的相关装置还未见文献报道。
研究表明，限域空间下的大分子晶体由于周围环境限制作用可以精确提高晶体晶型的调控，获得目标的晶体，同时限域空间的形状对晶体生长晶面的调控具有积极的作用。该结论不仅可以推广到生物大分子，也可以解决大分子药物结晶过程的问题。然而，限域空间不利于结晶体系相关的晶型观察，晶体诱导期测定和晶体采出等。因此，现有的传统的实验系统和监测方法亟待改进，需要集观察、控制环境、精确注样为一体的结晶实验系统。该实验系统需要满足准确滴加目标体系液滴的条件；同时，高精度微米级结晶平台的限域结构可以是规则的任何图形，利用系统本身x，y轴平台的高精度移动机构，以及针头精确的位置控制，保证大分子溶液能够滴加到凸起或凹陷的正确位置，并能保证晶体结晶微观环境和后期晶体反洗的精确操作。此外，还需方便在实验的过程中及时筛选并调控目标晶型，观察晶体，通过高倍摄像仪实时数据测定目标晶型的结晶诱导期，并根据实验情况实时调整晶体的培养环境，提高结晶效率和成功率。
 技术问题
本发明主要是设计一种为高精度的大分子晶体提供晶体培养环境，液滴滴加位置控制，晶体生长过程观察，晶体筛选的实验系统，该系统可以定量滴加目标大分子溶液并实时观察晶体的实际生长情况。其中，大分子晶体培养结晶平台用微原件具有特定微米级表面形状的凸起结构（点阵结构或凹槽的凸起结构均包含在内），或者加工出具有特定尺寸的凹槽结构的平用微元件平台。通过矩阵式滴加装置，将液滴批量地滴在微原件上，通过控制微元件平台周围温湿度条件，控制液滴形态及蒸发的速率保证目标物系目标晶型的产出。该方法能对所有可结晶物系，尤其是已提取的少量稀有药物提供结晶场所，操作简单，周围环境要求低，药品流失量少，是一种简单环保，可大批量重复的结晶技术。
 技术解决方案
本发明的技术方案：
一种适用于大分子结晶过程精确调控的实验系统，该实验系统为常压下由外壳1围成的密闭空间，主要由平台布置水平移动槽及流道专用反洗模块Ⅰ、液滴滴加控制模块Ⅱ、观察模块Ⅲ、用户观察用电脑系统Ⅳ和实验条件控制模块Ⅴ组成；所述的平台布置水平移动槽及流道专用反洗模块Ⅰ、液滴滴加控制模块Ⅱ、观察模块Ⅲ和实验条件控制模块Ⅴ位于密闭空间内部，用户观察用电脑系统Ⅳ位于密闭空间外部；
所述的平台布置水平移动槽及流道专用反洗模块Ⅰ包括平台水平移动槽2、x轴水平调节机构3、y轴水平调节机构4和高通量大分子结晶培养平台5；所述的高通量大分子结晶培养平台5置于平台水平移动槽2上，所述的平台水平移动槽2通过x轴水平调节机构3调节水平位置，通过y轴水平调节机构4调节垂直高度，保证高通量大分子结晶培养平台5精准移动；
所述的液滴滴加控制模块Ⅱ包括进样器6、活塞推进调节机构7和进样器高度调节机构8，活塞推进调节机构7用于精准进样器6滴加的液滴体积，进样器高度调节机构8用于控制进样器6的位置；
所述的观察模块Ⅲ包括高倍摄像仪9和摄像仪调节单元11，高倍摄像仪9固定在摄像仪调节单元11上，通过控制高倍摄像仪9的角度、亮度及放大倍数来观察高通量大分子结晶培养平台5上液滴内部晶体的状态；
所述的实验条件控制模块Ⅴ包括温度湿度测量与控制装置13，调控密闭空间内所需的湿度和温度；
所述的用户观察用电脑系统Ⅳ包括数据导出线10和用户观察电脑12，作为观察系统的外部延伸以方便用户利用电脑软件观察高倍摄像仪9镜头下的晶体生长情况；所述的用户观察电脑12通过数据导出线10连接摄像仪调节单元11；
所述的高通量大分子结晶培养平台5包括反洗液入口14、反洗液流道15和反洗液出口16，反洗液从反洗液入口14进入，反洗液在反洗液流道15内冲洗晶体后，反洗液从反洗液出口16流出。
所述的反洗液流道15，是微米级结构的有规则图形的凸起或凹陷结构。根据实际需求，选择布局形式不同的凸起结构和凹陷结构形成的晶体平台，有两种布局形式；
第一种布局形式为点阵布局，凸起结构位于结晶平台的一侧，按照点阵布局；凹陷结构位于结晶平台的另一侧，按照点阵布局；
第二种布局形式为坑道布局，凸起结构和凹陷结构交替布局，形成坑道式的结晶平台；
根据目标晶体体系的形貌，确定凸起结构和凹陷结构的尺寸，凸起结构或凹陷结构的高度为10μm~500μm；
所述的温度湿度测量与控制装置13，湿度的控制范围为10%~100%，温度的控制范围为-50℃~200℃。
所述的x轴水平调节机构3、y轴水平调节机构4和进样器高度调节机构8，调节机构的误差均为每25.4mm误差千分之一。
所述精准进样器6，若是高通量晶体的制备和生产，晶体平台设计成坑道形式，方便反溶剂的清洗和产品集中收集，并选择联排精准进样器6提高进样效率；若是晶体的筛选，晶体平台设计成点阵形式，提高晶体筛选的可重复性；所述的结晶平台的材料为对目标结晶体系不发生反应的材料；所述联排的进样器6提高进样效率，利用以下操作方式实现：调节x轴水平调节机构3和y轴水平调节机构4，将平台上的滴加液滴的目标位点对准联排结构18的进样器6，同时根据高通量大分子结晶培养平台5的高度调整进样器高度调节机构（8），准备就绪后，选择注样器控制系统17挤出液滴，液滴的体积通过联排结构18的进样器6上的刻度给出，每一个进样器的注样器控制系统17控制，保证联排结构18的进样器6的每一个进样器挤出的液滴含量相同，处理量一致。该方法可以利用联排结构18串联或者并联多个进样器6，提高产出效率。
所述的高倍摄像仪9，放大倍数为10~600倍。
一种适用于大分子结晶过程精确调控的实验方法，步骤如下：
（1）将目标结晶体系与溶剂配置晶体培养液，置于20~30℃温度条件下备用；对结晶平台进行消毒处理；其中，所述的溶剂为蒸馏水或无水乙醇；将配置好的晶体培养液加入至进样器6中，将高通量大分子结晶培养平台5放在平台水平移动槽2上，完成晶体培养前的准备工作；
（2）调节温度湿度测量与控制装置13使密闭空间内的温度和湿度条件达到结晶过程设定的值，整个实验过程的温度和湿度均由实验条件控制模块Ⅴ控制；
（3）实验过程中，调节x轴水平调节机构3和y轴水平调节机构4，将高通量大分子结晶培养平台5上的滴加液滴的目标位点对准进样器6，同时根据高通量大分子结晶培养平台5的高度调整进样器高度调节机构8，准备就绪后旋转活塞推进机构7挤出液滴，液滴的体积通过进样器6上的刻度给出；
（4）利用观察模块Ⅲ的高倍摄像仪9观察结晶液滴状态，观察的内容转换为电信号通过数据导出线10呈现在用户观察电脑12上，收集并保存结晶状态图片或视频；
（5）当晶体培养完成，将高通量大分子结晶培养平台5从平台水平移动槽2中拿出，将反溶剂的管线接在反洗液入口14，开始冲洗，冲洗液体通过反洗液流道15，并在反洗液出口16收集，将晶体反洗出来，完成晶体高通量培养的操作。
 有益效果
本发明的有益效果：利用系统本身x，y轴平台的高精度移动机构，以及针头精确的位置控制，保证大分子溶液能够滴加到凸起或凹陷的正确位置，并能保证晶体结晶微观环境和后期晶体反洗的精确操作；通过高倍摄像仪实时数据测定目标晶型的结晶诱导期，并根据实验情况实时调整晶体的培养环境，提高结晶效率和成功率。对于高通量大产出的结晶生产要求，可以利用联排注射的方式，既可以保证精确性，又可以实现高通量晶体的生产。
 附图说明
图1是本系统的侧面示意图。
图2是本系统的高通量大分子结晶培养平台的局部放大示意图。
图3是适用本系统的四种结晶培养平台设计俯视图。
图4是高通量联排的进样器操作示意图
图1中：1外壳；2平台水平移动槽；3x轴水平调节机构；
4y轴水平调节机构；5高通量大分子结晶培养平台；6进样器；
7活塞推进调节机构；8进样器高度调节机构；9高倍摄像仪；
10数据导出线；11摄像仪调节单元；12用户观察电脑；
13温度湿度测量与控制装置；14反洗液入口；15反洗液流道；
16反洗液出口；Ⅰ平台布置水平移动槽及流道专用反洗模块；
Ⅱ液滴滴加控制模块；Ⅲ观察模块；Ⅳ用户观察用电脑系统；
Ⅴ实验条件控制模块。
图3中：每一个图形均为高精度结晶平台的一部分，且各个结构的特征尺寸均为0.01mm~0.5mm，其中（1）、方形结构，（2）、圆形结构，（3）、六边形结构，（4）、菱形结构；每一个特征结构根据需要既可以是凸起结构，也可以是凹陷结构。
图4中：17进样器控制系统；18联排结构（附带多个6进样器）；19微米级结晶平台（10微米到500微米）；20液滴滴加凹槽（反溶剂清洗通道）。
 本发明的实施方式
以下结合附图和技术方案，进一步说明本发明的具体实施方式。
以溶菌酶晶体培养的案例，详细阐明该发明的使用方法。
实施例1使用进样器6完成溶菌酶的晶体筛选
将溶菌酶晶体培养液配置好，放入进样器6中备用，将高通量大分子结晶培养平台5放在平台水平移动槽2上，完成晶体培养前的准备工作；
调节温度湿度测量与控制装置13使密闭空间外壳1内的温度和湿度条件达到溶菌酶结晶过程设定的值，整个实验过程的温度和湿度均由实验条件控制模块Ⅴ控制；
实验过程中，调节x轴水平调节机构3和y轴水平调节机构4，将平台上的滴加液滴的目标位点对准进样器6，同时根据高通量大分子结晶培养平台5的高度调整进样器高度调节机构8，准备就绪后旋转活塞推进机构7挤出液滴，液滴的体积通过进样器6上的刻度给出；
利用观察模块Ⅲ的高倍摄像仪9观察结晶液滴状态，观察的内容转换为电信号通过数据导出线10呈现在用户观察电脑12上，收集并保存结晶状态图片或视频；
当溶菌酶晶体培养完成，将高通量大分子结晶培养平台5从平台水平移动槽2中拿出，将反溶剂的管线接在反洗液入口14，开始冲洗，冲洗液体通过反洗液流道15，并在反洗液出口16收集，将晶体反洗出来，完成晶体高通量培养的操作。
实施例2使用联排结构18的进样器6完成溶菌酶高通量产出
将溶菌酶晶体培养液配置好，放入联排结构18的进样器6中备用，将高通量大分子结晶培养平台5放在平台水平移动槽2上，完成晶体培养前的准备工作；
调节温度湿度方法遵照实施例1，调节x轴水平调节机构3和y轴水平调节机构4，将平台上的滴加液滴的目标位点对准联排结构18的进样器6，同时根据高通量大分子结晶培养平台5的高度调整进样器高度调节机构8，准备就绪后，选择进样器控制系统17挤出液滴，液滴的体积通过联排结构18的进样器6上的刻度给出，每一个进样器的进样器控制系统17控制，保证联排结构18的进样器6的每一个进样器挤出的液滴含量相同，处理量一致。如图4所示在微米级结晶平台19可以获得相同的可重复的液滴形貌20；
观察晶体形貌的手段与实施例1相同，当溶菌酶晶体培养完成，将高通量大分子结晶培养平台5从平台水平移动槽2中拿出，将反溶剂的管线接在反洗液入口14，开始冲洗，冲洗液体通过反洗液流道15，并在反洗液出口16收集，将晶体反洗出来，完成晶体高通量培养的操作。
该方法可以利用联排结构（18）串联或者并联多个进样器（6），提高产出效率，简单易行，产率较高，既可适用于常规实验重复，也可利用在极限结晶条件探索。以上所述仅为本发明的简单的实施案例而已，并不仅仅局限于本发明，凡与本发明类似的等同替换，命名修改和装置改进等，均应包含在本发明的保护范围之内。

Claims (8)

  1. 一种适用于大分子结晶过程精确调控的实验系统，其特征在于，该实验系统为常压下由外壳（1）围成的密闭空间，主要由平台布置水平移动槽及流道专用反洗模块（Ⅰ）、液滴滴加控制模块（Ⅱ）、观察模块（Ⅲ）、用户观察用电脑系统（Ⅳ）和实验条件控制模块（Ⅴ）组成；所述的平台布置水平移动槽及流道专用反洗模块（Ⅰ）、液滴滴加控制模块（Ⅱ）、观察模块Ⅲ和实验条件控制模块（Ⅴ）位于密闭空间内部，用户观察用电脑系统（Ⅳ）位于密闭空间外部；
    所述的平台布置水平移动槽及流道专用反洗模块（Ⅰ）包括平台水平移动槽（2）、x轴水平调节机构（3）、y轴水平调节机构（4）和高通量大分子结晶培养平台（5）；所述的高通量大分子结晶培养平台（5）置于平台水平移动槽（2）上，所述的平台水平移动槽（2）通过x轴水平调节机构（3）调节水平位置，通过y轴水平调节机构（4）调节垂直高度，保证高通量大分子结晶培养平台（5）精准移动；
    所述的液滴滴加控制模块（Ⅱ）包括进样器（6）、活塞推进调节机构（7）和进样器高度调节机构（8），活塞推进调节机构（7）用于精准控制进样器（6）滴加的液滴体积，进样器高度调节机构（8）用于控制进样器（6）的位置；
    所述的观察模块Ⅲ包括高倍摄像仪（9）和摄像仪调节单元（11），高倍摄像仪（9）固定在摄像仪调节单元（11）上，通过控制高倍摄像仪（9）的角度、亮度及放大倍数来观察高通量大分子结晶培养平台（5）上液滴内部晶体的状态；
    所述的实验条件控制模块（Ⅴ）包括温度湿度测量与控制装置（13），调控密闭空间内所需的湿度和温度；
    所述的用户观察用电脑系统（Ⅳ）包括数据导出线（10）和用户观察电脑（12），作为观察系统的外部延伸以方便用户利用电脑软件观察高倍摄像仪（9）镜头下的晶体生长情况；所述的用户观察电脑（12）通过数据导出线（10）连接摄像仪调节单元（11）；
    所述的高通量大分子结晶培养平台（5）包括反洗液入口（14）、反洗液流道（15）和反洗液出口（16），反洗液从反洗液入口（14）进入，反洗液在反洗液流道（15）内冲洗晶体后，反洗液从反洗液出口（16）流出。
  2. 根据权利要求1所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，所述的反洗液流道（15），是微米级结构的有规则图形的凸起或凹陷结构；根据实际需求，选择布局形式不同的凸起结构和凹陷结构形成的晶体平台，有两种布局形式；
    第一种布局形式为点阵布局，凸起结构位于结晶平台的一侧，按照点阵布局；凹陷结构位于结晶平台的另一侧，按照点阵布局；
    第二种布局形式为坑道布局，凸起结构和凹陷结构交替布局，形成坑道式的结晶平台。
  3. 根据权利要求2所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，根据目标晶体体系的形貌，确定凸起结构和凹陷结构的尺寸，凸起结构或凹陷结构的高度为10μm~500μm。
  4. 根据权利要求2或3所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，若是用于高通量晶体的制备，晶体平台设计成第二种坑道布局形式，方便反溶剂的清洗和集中收集晶体产品，并选择联排的进样器（6）提高进样效率；若是晶体的特征筛选，晶体平台设计成第一种点阵布局形式，提高晶体筛选的可重复性，且所述的结晶平台的材料为不与目标结晶体系发生反应的材料。
  5. 根据权利要求2或3所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，所述的温度湿度测量与控制装置（13），湿度的控制范围为10%~100%，温度的控制范围为-50℃~200℃；
    所述的x轴水平调节机构（3）、y轴水平调节机构（4）和进样器高度调节机构（8），调节机构的误差均为每25.4mm误差千分之一；
    所述的高倍摄像仪（9），放大倍数为10~600倍。
  6. 根据权利要求4所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，所述的温度湿度测量与控制装置（13），湿度的控制范围为10%~100%，温度的控制范围为-50℃~200℃；
    所述的x轴水平调节机构（3）、y轴水平调节机构（4）和进样器高度调节机构（8），调节机构的误差均为每25.4mm误差千分之一；
    所述的高倍摄像仪（9），放大倍数为10~600倍。
  7. 根据权利要求4或6所述的一种适用于大分子结晶过程精确调控的实验系统，其特征在于，所述联排的进样器（6）提高进样效率，利用以下操作方式实现：调节x轴水平调节机构（3）和y轴水平调节机构（4），将平台上的滴加液滴的目标位点对准联排结构（18）的进样器（6），同时根据高通量大分子结晶培养平台（5）的高度调整进样器高度调节机构（8），准备就绪后，选择注样器控制系统（17）挤出液滴，液滴的体积通过联排结构（18）的进样器（6）上的刻度给出，每一个进样器的注样器控制系统（17）控制，保证联排结构（18）的进样器（6）的每一个进样器挤出的液滴含量相同，处理量一致；该方法利用联排结构（18）串联或者并联多个进样器（6），提高产出效率。
  8. 一种适用于大分子结晶过程精确调控的实验方法，其特征在于，步骤如下：
    （1）将目标结晶体系与溶剂配置晶体培养液，置于20~30℃温度条件下备用；对结晶平台进行消毒处理；将配置好的晶体培养液加入至注样器（6）中，将高通量大分子结晶培养平台（5）放在平台水平移动槽（2）上，完成晶体培养前的准备工作；
    （2）调节温度湿度测量与控制装置（13）使密闭空间内的温度和湿度条件达到结晶过程设定的值，整个实验过程的温度和湿度均由实验条件控制模块（Ⅴ）控制；
    （3）实验过程中，调节x轴水平调节机构（3）和y轴水平调节机构（4），将高通量大分子结晶培养平台（5）上的滴加液滴的目标位点对准进样器（6），同时根据高通量大分子结晶培养平台（5）的高度调整进样器高度调节机构（8），准备就绪后旋转活塞推进机构（7）挤出液滴，液滴的体积通过进样器（6）上的刻度给出；
    （4）利用观察模块（Ⅲ）的高倍摄像仪（9）观察结晶液滴状态，观察的内容转换为电信号通过数据导出线（10）呈现在用户观察电脑（12）上，收集并保存结晶状态图片或视频；
    （5）当晶体培养完成，将高通量大分子结晶培养平台（5）从平台水平移动槽（2）中拿出，将反溶剂的管线接在反洗液入口（14），开始冲洗，冲洗液体通过反洗液流道（15），并在反洗液出口（16）收集，将晶体反洗出来，完成晶体高通量培养的操作。
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