WO2021114789A1 - 一种基于生物膜力学探针系统的超准力钳实验方法 - Google Patents

一种基于生物膜力学探针系统的超准力钳实验方法 Download PDF

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WO2021114789A1
WO2021114789A1 PCT/CN2020/115248 CN2020115248W WO2021114789A1 WO 2021114789 A1 WO2021114789 A1 WO 2021114789A1 CN 2020115248 W CN2020115248 W CN 2020115248W WO 2021114789 A1 WO2021114789 A1 WO 2021114789A1
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relative distance
probe
force
ball
red blood
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PCT/CN2020/115248
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French (fr)
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陈伟
安宸毅
胡炜
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浙江大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
    • G01Q60/38—Probes, their manufacture, or their related instrumentation, e.g. holders

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  • the invention relates to a quantitative detection method for detecting the dissociation rate of the interaction between biological macromolecules, in particular to an ultra-quasi force clamp experimental method based on a biofilm mechanics probe system.
  • BFP Biomembrane Mechanics Probe
  • the "force clamp” experiment quickly loads the force acting on the molecular bond to the set value, observes and collects the binding time of the single molecular bond under the set force value, thereby
  • the force regulation law of the molecular bond can be directly obtained without model fitting, and the dissociation rate (k off ) of the molecular bond under the condition of "0" force can be obtained according to the "Bell” model fitting. Therefore, the "force clamp” experiment based on the biomembrane mechanics probe system is especially suitable for in situ detection of the dissociation rate of membrane protein-related molecular bonds.
  • the biomechanical force clamp experiment has detected a variety of transient molecular interactions with a fast dissociation rate, it still cannot be applied to the study of molecular interactions with a slow dissociation rate. This is because the force feedback control function is lacking in the detection process, and various disturbances during the long bonding time of the molecular bond will cause the force value acting on the molecular bond to continuously shift; in addition, in the ultra-long molecular bond bonding time During the recording process, the probe end will also drift as a whole. Even if the position of the probe end ball is maintained at the set position, the actual force acting on the molecular bond may also have a large deviation, which will eventually lead to poor accuracy of the measurement result. , And even come to the wrong conclusion.
  • the purpose of the present invention is to provide an ultra-quasi force clamp experiment method based on a biomembrane mechanics probe system.
  • the present invention corrects the overall drift of the probe end by adding an additional reference ball in the "force clamp” experiment to improve the accuracy of the recorded force value, and is embedded in the "record molecular bond binding time” stage of the "force clamp” experiment control
  • the PID feedback control algorithm is used to stably “clamp” the force value recorded during the "record molecular bond binding time” stage at the set position, and finally achieve a stable and accurate clamping of the force acting on the molecular bond.
  • the probe end includes a probe ball and red blood cells.
  • a reference ball is added to the probe end.
  • the reference ball passes through the interaction between protein molecules. It is used to paste on the end of the first micropipette that sucks red blood cells, and track the relative distance between the reference ball located on the side border of the red blood cell and the probe ball located on the side border of the red blood cell in real time to control the movement of the target end and force clamp experiment.
  • the piezoelectric motion platform controls the target end to be far away from the probe end in the initial position and at rest.
  • the red blood cells are in the initial state, with the reference ball located at the boundary near the red blood cell side and the probe ball located at the boundary near the red blood cell side.
  • the distance between the two is regarded as the relative distance, and the relative distance is tracked in real time through high-speed addition shooting, and the average value of the relative distance recorded in 0.2 seconds is used as the initial relative distance, and then the next stage is entered;
  • the piezoelectric motion platform drives the cells/balls at the target end to move closer to the red blood cells at a preset speed through the second micropipette, and push the probe ball to the center to compress the red blood cells until the relative distance is reduced. Reduce the preset compression distance and enter the next stage;
  • the piezoelectric motion platform drives the cells/balls at the target end to withdraw away from the red blood cells at a preset speed through the second micropipette;
  • the piezoelectric motion platform During the withdrawal process, monitor the relative distance. If the relative distance is longer than the initial relative distance by the length corresponding to the preset force value, the force value is the value of the force between the molecular bonds, that is, the red blood cells are stretched to the set length. Then this experiment is an adhesion event, the piezoelectric motion platform immediately stops the current withdrawal motion, and enters the binding time recording stage;
  • the piezoelectric motion platform implements feedback compensation according to the deviation between the relative distance corresponding to the preset force value and the actual relative distance, until the relative distance is restored to the initial relative distance again. That is, the molecular bond is automatically broken and enters the reset stage;
  • the relative distance corresponding to the preset force value is the initial relative distance plus the length corresponding to the preset force value.
  • the end of the first micropipette will deviate from the offset, making the actual relative distance deviate from the preset force Value corresponds to the relative distance, so the relative distance is stabilized at the relative distance corresponding to the set force value through real-time feedback compensation.
  • Reset phase real-time monitoring of the relative distance, the piezoelectric motion platform has been withdrawn to the initial position, ending the current cycle, saving the relative distance, time and other data of the current experiment and preparing for the next cycle.
  • the red blood cell functions as a force sensor, which is similar to a linear spring within a certain force range.
  • the relative distance change can be converted into the real-time force acting on the molecular bond according to the elastic coefficient of the red blood cell.
  • the present invention adds a reference ball in the "force clamp” experiment, uses the interaction between protein molecules to stick to the tip of the micropipette sucking red blood cells, and accurately tracks the force acting on the molecular bonds in the "double boundary” tracking mode. And the force feedback control function is added, which can implement stable and accurate clamping of the force acting on the molecular bond.
  • the real-time monitored relative distance is taken as the actual relative distance, and after the actual relative distance is filtered by the filter of the PID controller, the reference relative distance corresponding to the preset force value is subtracted to obtain the deviation ,
  • the deviation is input to the PID controller, and the deviation control value output by the PID controller is input to the motion module that controls the piezoelectric motion platform, and the feedback compensation motion is executed.
  • the surface of the first micropipette is adsorbed with Spy-tag protein molecules
  • the surface of the reference pellet is coated with Spy-catcher protein molecules
  • the reference pellet is fixed and adhered through the interaction between the Spy-tag and Spy-catcher protein molecules On the side of the end of the first micropipette.
  • the diameter of the reference ball is larger than the diameter of the probe ball, which can be visually distinguished by the size of the ball during the experiment.
  • the reference ball is a glass ball.
  • the force value data is sampled and recorded in the register with 3 times the initial data sampling time interval as the data recording time interval.
  • the data recording time interval Increased to 20 times the sampling time interval; when the number of recorded data exceeds 5000 times, the data recording time interval is further increased by 5 times.
  • the present invention significantly improves the accuracy of the force acting on the molecular bond by adding reference balls and developing a "double boundary" force tracking mode based on the biofilm mechanics probe system; in the "force clamp” experiment control "record molecular bond”
  • the feedback control of the embedding force in the "binding time” stage realizes the stable “clamping” of the force acting on the molecular bond during the molecular bond bonding time in the "force clamp” experiment; and the ability to dynamically adjust the recording time interval, significantly extending the molecular bond
  • the recording time limit of the combined time is provided.
  • the present invention is mainly aimed at the force regulation experiment of the dissociation rate of the stronger interaction between molecules in the field of life sciences, and can more accurately and effectively obtain the change of the binding time of the single molecular bond under the action of biological force.
  • the "force clamp” experiment involved in the present invention has the following advantages:
  • the recording time limit of the molecular bond binding time is extended to more than 200 seconds.
  • the present invention can apply an accurate and stable force to molecular bonds with a slow dissociation rate, and record the binding time of molecular bonds for a long time, and is especially suitable for the regulation law of stronger intermolecular interaction dissociation rate. In situ detection.
  • Figure 1 is a schematic diagram of the biofilm-based mechanics probe system involved in the present invention.
  • Fig. 2 is a schematic diagram of the feedback control involved in the present invention.
  • Figure 3 is an example of dynamically adjusting the data recording time involved in the present invention.
  • Figure 4 is an example of the experimental record data of the original biofilm mechanics probe.
  • the first micropipette for sucking red blood cells 2. Red blood cells, 3. Probe beads coated with the target protein molecule on the surface, 4. Cells/beads expressing/coating another target protein molecule on the surface, 5 , The second micropipette for sucking the target cell/ball, 6.
  • the piezoelectric motion platform to control the movement of the target end 7.
  • the reference sphere added in the "super-precision" force clamp experiment, 8.
  • the boundary of the probe ball that is tracked in real time by the high-speed camera, 9.
  • the reference ball boundary that is tracked in real time by the high-speed camera in the biofilm mechanics probe system, and the relative distance that is tracked in real time by the high-speed camera in the biofilm mechanics probe system 11.
  • Data example of “force-time” of super-precision "force clamp” technology 12.
  • the implemented biofilm mechanics probe system includes a probe end and a target end.
  • the probe end includes a first micropipette 1, red blood cells 2, a probe ball 3, and a reference ball 7, and the target end includes Cells/balloons 4 and second micropipette 5; the end of the first micropipette 1 sucks red blood cells 2, and the end side of the first micropipette 1 is stably connected with the reference ball 7 through the strong interaction between protein molecules, and the second micropipette
  • the end of the pipette 5 sucks the cell/bead 4, the cell/bead 4 expresses/the surface is coated with a target protein molecule, if it is the cell 4, it expresses a target protein molecule, if it is the bead 4, the surface is coated with a target protein molecule
  • the surface of the probe bead 3 is coated with another target protein molecule, and the probe bead 3 is connected to the front end of the red blood cell 2 through a
  • the first micropipette 1, the red blood cell 2, the probe ball 3 and the reference ball 7 constitute the probe end.
  • the probe end remains fixed, but it is easily affected by environmental disturbances and will move slightly; the cell/ball 4 and the first Two micro-pipettes 5 constitute the target end, and the piezoelectric motion platform 6 drives and controls the movement of the target end.
  • Spy-tag protein molecules are adsorbed on the surface of the first micropipette 1, and Spy-catcher protein molecules are attached to the surface of the reference pellet 7, and the reference pellet 7 is fixed and adhered to by the interaction between the Spy-tag and Spy-catcher protein molecules.
  • the diameter of the reference ball 7 is larger than the diameter of the probe ball 3.
  • the diameter of the reference ball 7 is about 5 microns, which is significantly larger than the diameter of the probe ball 3 by about 2 microns.
  • the specific reference ball 7 is a glass ball, and the material is borosilicate.
  • the first micropipette 1 at the probe end is immersed in a solution containing Spy-tag protein molecules in advance, so that the first micropipette 1 physically adsorbs a large number of Spy-tag protein molecules.
  • the present invention finds that in the existing force clamp experiment, the probe end remains fixed, but the first micropipette component of the probe end is easily affected by environmental disturbances and will move slightly. Simply tracking the position information of the boundary of the probe ball 3 cannot accurately measure red blood cells. The actual deformation of 3.
  • the position of the right boundary 9 of the reference ball 7 and the left boundary 8 of the probe ball 3 is tracked in real time and synchronously by using a high-speed camera to take pictures.
  • the change in the relative distance between the two boundaries is used to characterize the red blood cells.
  • Deformation of 3 Since the reference ball 7 is stably attached to the side of the first micropipette 1 at the probe end and moves synchronously with the first micropipette 1, the position change of the reference ball 7 can be considered as the overall position of the probe end of the first micropipette 1. Variety. Even in the case of the overall drift of the probe ends 1, 2, 3, and 7 caused by environmental disturbance, the relative distance between the two boundaries can still accurately characterize the deformation of the red blood cell 3.
  • the force acting on the molecular bond is stably and accurately used for clamp production, which enables the binding time of the molecular bond under the force to be detected for a long time, stably and accurately.
  • the present invention adopts the technical scheme of automatically adjusting the data recording time interval according to the length of the molecular bond binding time, and the data collection time can be greatly extended, which solves the problem of the limited register capacity of the computer memory during the experiment process of the biofilm mechanics probe system and cannot be effectively stored.
  • the piezoelectric motion platform 6 controls the target ends 4, 5 to be far away from the probe ends 1, 2, 3, 7, and are in the initial position and stationary.
  • the red blood cell 2 is in the initial state, and the reference ball 7 is located on the border of the side close to the red blood cell 2 9
  • the distance between the probe ball 3 and the boundary 8 on the side close to the red blood cell 2 is taken as the relative distance 10.
  • the relative distance is tracked in real time through high-speed addition shooting, and the average value of the relative distance recorded in 0.2 seconds is taken as the initial relative distance. Enter the next stage;
  • the piezoelectric motion platform 6 drives the cells/balls 4 at the target end through the second micropipette 5 to move in the direction close to the red blood cells 2 at a preset speed, and push the probe balls 3 toward the center Compress the red blood cell 2 until the relative distance is reduced by the preset compression distance, and enter the next stage;
  • Withdrawal stage the piezoelectric motion platform 6 drives the cells/balls 4 at the target end to withdraw movement away from the red blood cells 2 at a preset speed through the second micropipette 5;
  • the piezoelectric motion platform 6 During the withdrawal process, monitor the relative distance. If the relative distance is longer than the initial relative distance by the length corresponding to the preset force value, the force value is the value of the force between the molecular bonds, that is, the red blood cell 2 is stretched to the set length , This experiment is an adhesion event, and the piezoelectric motion platform 6 immediately stops the current withdrawal motion and enters the next stage, that is, the combined time recording stage;
  • the piezoelectric motion platform 6 implements feedback compensation according to the deviation between the relative distance corresponding to the preset force value and the actual relative distance, until the relative distance returns to the initial relative distance again , That is, the molecular bond is automatically broken and enters the reset stage;
  • step 5 the real-time monitored relative distance is taken as the actual relative distance.
  • the actual relative distance is filtered by the filter of the PID control module of the Labview platform, it is a reference corresponding to the preset force value.
  • the relative distance is subtracted to obtain the deviation, and the deviation is input to the PID controller, and the deviation control value output by the PID controller is input to the motion module that controls the piezoelectric motion platform 6 to perform feedback compensation motion.
  • the specific PID controller adopts pure proportional feedback control, and the proportional parameter is 1.
  • the sampling frequency of the biofilm mechanics probe system is higher to 600Hz.
  • the piezoelectric motion platform is controlled to perform feedback compensation motion, which may cause the piezoelectric motion platform to fail. Normal operation and loss of recorded data due to occupation of host computer resources. Therefore, in specific implementation, the above-mentioned compensation is performed after every 10 sampling experiments.
  • the present invention uses the high-speed camera of the biofilm mechanics probe system to collect the relative distance 10 between the probe ball and the reference ball in real time, and passes through the piezoelectric motion platform at the target end according to the actual relative distance and the preset reference relative distance.
  • -753.1CD implements feedback compensation movement to stably maintain the force acting on the molecular bond at the set force value.
  • the preset reference relative distance is the initial relative distance plus the red blood cell shape variable corresponding to the preset force value.
  • Reset phase real-time monitoring of the relative distance, the piezoelectric motion platform 6 has been withdrawn to the initial position, ending the current cycle, by measuring the force value data and saving the data, and preparing for the next cycle.
  • the force value data is sampled and recorded in the register with 3 times the initial data sampling time interval as the data recording time interval.
  • the data recording time interval is increased to sampling 20 times the time interval; when the number of recorded data exceeds 5000 times, the data recording time interval is further increased by 5 times.
  • the invention can stably record the molecular bond bonding time exceeding 200 seconds.
  • the present invention can dynamically adjust the data recording time interval by referring to the arrangement of the small balls, and extend the recording time limit of the molecular bond binding time.
  • the invention dynamically increases the time interval of data recording according to the length of the combined time that has been recorded on the basis of ensuring the highest sampling frequency ⁇ 0.6kHz, thereby avoiding computer memory overflow caused by excessively large recorded data.

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Abstract

一种基于生物膜力学探针系统的超准力钳实验方法。力钳实验涉及有探针端(1、2、3、7)和目标端(4、5)，探针端(1、2、3、7)包括有探针小球(3)、红细胞(2)，在生物膜力学探针力钳实验中，在探针端(1、2、3、7)增加了参考小球(7)，参考小球(7)通过蛋白质分子间的强相互作用粘贴在吸取红细胞(2)的第一微吸管(1)端部，并实时追踪参考小球(7)位于靠近红细胞(2)一侧边界(9)和探针小球(3)位于靠近红细胞(2)一侧边界(8)之间的相对距离(10)，通过反馈算法控制目标端(4、5)的移动而进行力钳实验。通过增加参考小球(7)并开发基于生物膜力学探针系统的"双边界"力追踪模式，显著提升了作用在分子键上的力的准确性，能够对解离速率较慢的分子键施加准确、稳定的作用力，显著延长了分子键结合时间的记录时限，进行长时间记录。

Description

一种基于生物膜力学探针系统的超准力钳实验方法 技术领域
本发明涉及检测生物大分子间相互作用解离速率的定量检测方法，尤其是涉及一种基于生物膜力学探针系统的超准力钳实验方法。
背景技术
随着单分子力谱技术的发展，原子力显微镜、光镊、磁镊、生物膜力学探针等技术已经在单分子水平解析了多种生物力对单分子键的调控规律。生物膜力学探针(BFP)系统兼具弹性系数小、操作难度低、易于对活细胞操作等优势，尤其适用于对活细胞表面生物大分子相互作用的原位检测。作为动态力谱实验的重要组成部分，“力钳”实验将作用在分子键上的作用力快速加载到设定值，观测并收集单分子键在该设定力值作用下的结合时间，从而可以不经过模型拟合直接得到分子键的受力调控规律，并可以根据“贝尔”模型拟合得出“0”力条件下分子键的解离速率(dissociation rate，k off)。因此，基于生物膜力学探针系统的“力钳”实验尤其适用于膜蛋白相关分子键解离速率的原位检测。
尽管生物力学力钳实验已经检测了多种解离速率较快的瞬态分子间相互作用，但是它仍然无法应用于解离速率较慢的分子间相互作用的研究。这是因为在检测过程中缺少力的反馈控制功能，在分子键的较长结合时间内各种扰动都会导致作用在分子键上的力值不断偏移；此外，在超长分子键结合时间的记录过程中，探针端也会产生整体漂移，即便探针端小球的位置被维持在设定位置，实际作用在分子键上的力也可能产生较大偏差，最终造成测量结果准确性较差，甚至得出错误结论。
因此，提升作用在分子键上力的稳定性、准确性是应用基于生物膜力学探针系统的“力钳”技术准确检测较强分子间相互作用解离速率的首要条件。
发明内容
为了解决背景技术中存在的问题，本发明目的在于提供一种基于生物膜力学探针系统的超准力钳实验方法。
本发明通过在“力钳”实验中额外添加一个参考小球校正探针端的整体漂移，提升记录力值的准确性，并在“力钳”实验控制中的“记录分子键结合时间”阶段嵌入了PID反馈控制算法，将“记录分子键结合时间”阶段内记录的力值稳定“钳制”在设定位置，最终实现对作用在分子键上的力稳定而准确的 钳制。
为实现上述发明目的，本发明采用的技术方案是：
在生物膜力学探针力钳实验中，有探针端和目标端，探针端包括有探针小球、红细胞，在探针端增加了参考小球，参考小球通过蛋白质分子间的相互作用粘贴在吸取红细胞的第一微吸管端部，并实时追踪参考小球位于靠近红细胞一侧边界和探针小球位于靠近红细胞一侧边界之间的相对距离来控制目标端的移动而进行力钳实验。
方法具体为：
1)初始化阶段：
压电运动平台控制目标端远离探针端处于初始位置并静止，此时红细胞处于初始状态，以参考小球位于靠近红细胞一侧边界和探针小球位于靠近红细胞一侧边界的两个边界之间的距离作为相对距离，通过高速相加拍摄实时追踪相对距离，以0.2秒内记录的相对距离的平均值作为初始相对距离，随后进入下一阶段；
2)撞击阶段：实时监测相对距离，压电运动平台经第二微吸管带动目标端的细胞/小球按照预先设定速度向靠近红细胞运动，对心推动探针小球压缩红细胞，直至相对距离减小了预先设定的压缩距离，进入下一阶段；
3)接触阶段：实时监测相对距离，压电运动平台保持静止，探针小球与细胞/小球稳定接触，经过预先设定接触时间后，进入下一阶段；
4)撤回阶段：压电运动平台经第二微吸管带动目标端的细胞/小球按照预先设定速度向远离红细胞撤回运动；
在撤回过程中，实施监测相对距离，若该相对距离比初始相对距离增加了预先设定力值对应的长度，力值为分子键间的作用力的值，即红细胞被拉伸设定长度，则本次实验为粘附事件，压电运动平台立即停止当前撤回运动，进入结合时间记录阶段；
若该相对距离恢复到初始相对距离而未被拉伸，则直接进入6)重置阶段；
5)结合时间记录阶段：实时监测相对距离，与此同时，压电运动平台根据预先设定力值对应的相对距离与实际相对距离的偏差实施反馈补偿，直至相对距离再次恢复到初始相对距离，即分子键自动断裂，进入重置阶段；
预先设定力值对应的相对距离即为初始相对距离加上预先设定力值对应的长度，但由于环境扰动导致第一微吸管的末端会偏离偏移，使得实际相对距离偏离预先设定力值对应的相对距离，因此通过实时反馈补偿将相对距离稳定在设定力值对应的相对距离。
6)重置阶段：实时监测相对距离，压电运动平台已撤回至初始位置，结束当前循环，保存当前次实验的相对距离、时间等数据并准备下一次循环。
生物膜力学探针系统中，红细胞的作用是力传感器，在一定力的范围内类似于一种线性弹簧，相对距离的变化可以根据红细胞的弹性系数换算为实时作用在分子键上的力值。
本发明在“力钳”实验中增加了参考小球，利用蛋白质分子间的相互作用粘贴在吸取红细胞的微吸管尖端，以“双边界”追踪模式对作用在分子键上的力进行准确追踪，并增加了力的反馈控制功能，可以对作用在分子键上的力实施稳定、准确的钳制。
所述步骤5)中，将实时监测的相对距离作为实际相对距离，将实际相对距离经PID控制器的滤波器滤波处理后，和预先设定的力值对应的参考相对距离作相减获得偏差，将该偏差输入到PID控制器，PID控制器输出的偏差控制量输入到控制压电运动平台的运动模块，执行反馈补偿运动。
所述的第一微吸管表面吸附有Spy-tag蛋白质分子，参考小球表面包被了Spy-catcher蛋白质分子，通过Spy-tag、Spy-catcher蛋白质分子间的相互作用将参考小球固定粘附于第一微吸管端部侧面。
所述的参考小球的直径大于探针小球的直径，可以在实验过程中通过小球的尺寸直观地进行区分。
所述的参考小球为玻璃球。
所述的力钳实验中，开始以初始的数据采样时间间隔的3倍作为数据记录时间间隔对力值数据进行采样并记录于寄存器中，当分子键结合时间超过5秒后，数据记录时间间隔增加为采样时间间隔的20倍；当记录数据次数超过5000次后，数据记录时间间隔进一步增大5倍。
本发明具有的有益效果是：
本发明通过增加参考小球并开发基于生物膜力学探针系统的“双边界”力追踪模式，显著提升了作用在分子键上的力的准确性；在“力钳”实验控制“记录分子键结合时间”阶段中嵌入力的反馈控制，实现了对“力钳”实验中分子键结合时间内作用在分子键上力的稳定“钳制”；并能够动态调整记录时间间隔，显著延长了分子键结合时间的记录时限。
本发明主要针对生命科学领域中较强分子间相互作用解离速率受力调控实验，能够更加准确、有效地获得单分子键在生物力作用下结合时间的变化情况。相比于原“力钳”实验，本发明所涉及的“力钳”实验具有以下优势：
1)结合时间内作用在分子键上的力更加准确、稳定地被“钳制”在设定力 值；
2)分子键结合时间的记录时限延长至200秒以上。
因此，本发明能够对解离速率较慢的分子键施加准确、稳定的作用力，并对分子键的结合时间进行长时间记录，尤其适用于较强分子间相互作用解离速率受力调控规律的原位检测。
附图说明
图1是本发明所涉及的基于生物膜力学探针系统原理图。
图2是本发明所涉及的反馈控制原理图。
图3是本发明所涉及的动态调整数据记录时间实例。
图4是原生物膜力学探针实验记录数据示例。
图中：1、吸取红细胞的第一微吸管，2、红细胞，3、表面包被目标蛋白质分子的探针小球，4、表达/表面包被另一目标蛋白质分子的细胞/小球，5、吸取目标细胞/小球的第二微吸管，6、控制目标端运动的压电运动平台，7、“超准”力钳实验中加入的参考小球，8、生物膜力学探针系统中通过高速相机实时追踪的探针小球边界，9、生物膜力学探针系统中通过高速相机实时追踪的参考小球边界，10、生物膜力学探针系统中通过高速相机实时追踪的相对距离，11、超准“力钳”技术“力—时间”数据实例，12、超准“力钳”技术“记录时间间隔—记录时间”数据实例。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
如图1所示，具体实施的生物膜力学探针系统包括探针端和目标端，探针端包括第一微吸管1、红细胞2、探针小球3和参考小球7，目标端包括细胞/小球4和第二微吸管5；第一微吸管1端部吸取红细胞2，第一微吸管1端部侧面通过蛋白质分子间的强相互作用稳定连接有参考小球7，第二微吸管5端部吸取细胞/小球4，细胞/小球4表达/表面包被一目标蛋白质分子，若为细胞4则表达有一目标蛋白质分子，若为小球4则表面包被有一目标蛋白质分子；探针小球3表面包被有另一目标蛋白质分子，探针小球3通过分子间的强相互作用(例如链霉亲和素、生物素之间的相互作用)连接到红细胞2前端；第二微吸管5吸取细胞/小球4并连接到压电运动平台6上，由压电运动平台6带动第二微吸管5移动而带动细胞/小球4远离或者靠近探针端的探针小球3而进行力钳实验。
由第一微吸管1、红细胞2、探针小球3和参考小球7构成了探针端，探针端保持固定，但容易受环境扰动影响会微动；由细胞/小球4和第二微吸管5构 成了目标端，压电运动平台6带动控制目标端运动。
第一微吸管1表面吸附有Spy-tag蛋白质分子，参考小球7表面连接了Spy-catcher蛋白质分子，通过Spy-tag、Spy-catcher蛋白质分子间的相互作用将参考小球7固定粘附于第一微吸管1端部侧面。
参考小球7的直径大于探针小球3的直径。具体实施中，参考小球7直径约5微米，明显大于探针小球3的直径约2微米。
具体实施的参考小球7为玻璃球，材质为硼硅酸盐。
具体实施中，探针端的第一微吸管1提前在包含Spy-tag蛋白质分子的溶液中浸泡，使得第一微吸管1物理吸附大量带有Spy-tag的蛋白质分子。
本发明发现，在现有力钳实验中，探针端保持固定，但探针端的第一微吸管部件容易受环境扰动影响会微动，单纯追踪探针小球3边界的位置信息无法准确测量红细胞3的实际形变量。
本发明在图1中，通过高速相机拍摄，参考小球7右侧边界9及探针小球3左侧边界8的位置被实时、同步追踪，利用两边界之间的相对距离的变化表征红细胞3的形变。由于参考小球7稳定粘贴在探针端的第一微吸管1侧面，与第一微吸管1同步运动，参考小球7的位置变化即可以认为是第一微吸管1的探针端整体的位置变化。即便在环境扰动导致的探针端1、2、3、7整体漂移情况中，两边界之间的相对距离仍能准确表征红细胞3的形变量。
本发明中作用分子键上的力受到稳定而准确的钳制作用，这使得在受力情况下的分子键的结合时间能够被长时间、稳定、准确地检测。同时，本发明采用了根据分子键结合时间长度自动调整数据记录时间间隔的技术方案，数据采集时间能够大大延长，解决了生物膜力学探针系统的实验过程计算机内存的寄存器容量有限、无法有效存储长时间实验数据的技术问题。
本发明的具体实施例及其实施过程为：
1)初始化阶段：
压电运动平台6控制目标端4、5远离探针端1、2、3、7，处于初始位置并静止，此时红细胞2处于初始状态，以参考小球7位于靠近红细胞2一侧边界9和探针小球3位于靠近红细胞2一侧边界8之间的距离作为相对距离10，通过高速相加拍摄实时追踪相对距离，以0.2秒内记录的相对距离的平均值作为初始相对距离，随后进入下一阶段；
2)撞击阶段：实时监测相对距离，压电运动平台6经第二微吸管5带动目标端的细胞/小球4按照预先设定速度向靠近红细胞2的方向运动，对心推动探针小球3压缩红细胞2，直至相对距离减小了预先设定的压缩距离，进入下一阶段；
3)接触阶段：实时监测相对距离，压电运动平台6保持静止，探针小球3与细胞/小球4稳定接触，经过预先设定接触时间后，进入下一阶段；
4)撤回阶段：压电运动平台6经第二微吸管5带动目标端的细胞/小球4按照预先设定速度向远离红细胞2撤回运动；
在撤回过程中，实施监测相对距离，若该相对距离比初始相对距离增加了预先设定力值对应的长度，力值为分子键间的作用力的值，即红细胞2被拉伸设定长度，则本次实验为粘附事件，压电运动平台6立即停止当前撤回运动，进入下一阶段，即结合时间记录阶段；
若该相对距离仅仅恢复到初始相对距离而未被拉伸，则直接进入6)重置阶段；
5)结合时间记录阶段：实时监测相对距离，与此同时，压电运动平台6根据预先设定力值对应的相对距离与实际相对距离的偏差实施反馈补偿，直至相对距离再次恢复到初始相对距离，即分子键自动断裂，进入重置阶段；
如图2所示，步骤5)中，将实时监测的相对距离作为实际相对距离，将实际相对距离经Labview平台的PID控制模块的滤波器滤波处理后，和预先设定的力值对应的参考相对距离相减获得偏差，将该偏差输入到PID控制器，PID控制器输出的偏差控制量输入到控制压电运动平台6的运动模块，执行反馈补偿运动。
具体的PID控制器采用纯比例反馈控制，比例参数为1。
在本发明“双边界”追踪模式下，生物膜力学探针系统采样频率较高～600Hz,根据每一次采样计算的偏差值控制压电运动平台执行反馈补偿运动将可能会导致压电运动平台无法正常运行，并因占用计算机主机资源导致记录数据丢失。因此在具体实施中，每10次采样实验后进行上述的一次补偿。
这样，本发明利用生物膜力学探针系统高速相机实时采集探针小球、参考小球的相对距离10，根据该实际相对距离与预设的参考相对距离通过目标端的压电运动平台Physik Instrument P-753.1CD执行反馈补偿运动，将作用在分子键上的力稳定维持在设定力值。预设的参考相对距离为初始相对距离加上预先设定力值对应的红细胞形变量。
6)重置阶段：实时监测相对距离，压电运动平台6已经撤回至初始位置，结束当前循环，通过测量力值数据并保存数据并准备下一次循环。
力钳实验中，开始以初始的数据采样时间间隔的3倍作为数据记录时间间隔对力值数据进行采样并记录于寄存器中，当分子键结合时间超过5秒后，数据记录时间间隔增加为采样时间间隔的20倍；当记录数据次数超过5000次后， 数据记录时间间隔进一步增大5倍。本发明可以稳定记录超过200秒的分子键结合时间。
实验记录到超长结合时间(约185s)的数据结果如图3所示，稳定、准确力钳作用下“力—时间”数据实例(10)和“记录时间间隔—记录时间”数据实例(11)。以未增加参考小球(7)并且未加入力的反馈控制情况下的原生物膜力学探针系统进行同样上述实验，实施数次仅能存储15秒的数据结果，其数据结果如图4所示。对比可见，本发明所测得的结果非常稳定、准确，并且能在同样寄存器容量的情况下存储获得200s结合时间内的数据，这就反映了本发明能对红细胞(2)的形变量准确测量，克服了环境扰动导致的探针端整体漂移带来的数据测量不准确的问题和存储有效性问题。
由此，本发明通过参考小球的布置，能实现动态调整数据记录时间间隔，延长分子键结合时间的记录时限。
本发明在保证最高采样频率～0.6kHz的基础上根据已经记录的结合时间的长度动态增加数据记录的时间间隔，从而避免记录数据过大导致的计算机内存溢出。
上述具体实施方式用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。

Claims (7)

  1. 一种基于生物膜力学探针系统的超准力钳实验方法，力钳实验涉及有探针端和目标端，探针端包括有探针小球(3)、红细胞(2)，其特征在于：在生物膜力学探针力钳实验中，在探针端增加了参考小球(7)，参考小球(7)通过蛋白质分子间的相互作用粘贴在吸取红细胞(2)的第一微吸管(1)端部，并实时追踪参考小球(7)位于靠近红细胞(2)一侧边界和探针小球(3)位于靠近红细胞(2)一侧边界之间的相对距离来控制目标端的移动而进行力钳实验。
  2. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：方法具体为：
    1)初始化阶段：
    压电运动平台(6)控制目标端(4、5)远离探针端(1、2、3、7)处于初始位置并静止，此时红细胞(2)处于初始状态，以参考小球(7)位于靠近红细胞(2)一侧边界(9)和探针小球(3)位于靠近红细胞(2)一侧边界(8)的两个边界之间的距离作为相对距离(10)，通过高速相加拍摄实时追踪相对距离，以0.2秒内记录的相对距离的平均值作为初始相对距离，随后进入下一阶段；
    2)撞击阶段：实时监测相对距离，压电运动平台(6)经第二微吸管(5)带动目标端的细胞/小球(4)按照预先设定速度向靠近红细胞(2)运动，对心推动探针小球(3)压缩红细胞(2)，直至相对距离减小了预先设定的压缩距离，进入下一阶段；
    3)接触阶段：实时监测相对距离，压电运动平台(6)保持静止，探针小球(3)与细胞/小球(4)稳定接触，经过预先设定接触时间后，进入下一阶段；
    4)撤回阶段：压电运动平台(6)经第二微吸管(5)带动目标端的细胞/小球(4)按照预先设定速度向远离红细胞(2)撤回运动；
    在撤回过程中，实施监测相对距离，若该相对距离比初始相对距离增加了预先设定力值对应的长度，则本次实验为粘附事件，压电运动平台(6)立即停止当前撤回运动，进入结合时间记录阶段；
    若该相对距离恢复到初始相对距离而未被拉伸，则直接进入6)重置阶段；
    5)结合时间记录阶段：实时监测相对距离，与此同时，压电运动平台(6)根据预先设定力值对应的相对距离与实际相对距离的偏差实施反馈补偿，直至相对距离再次恢复到初始相对距离，即分子键自动断裂，进入重置阶段；
    6)重置阶段：实时监测相对距离，压电运动平台(6)已撤回至初始位置， 结束当前循环，保存当前次实验的相对距离、时间等数据并准备下一次循环。
  3. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：所述步骤5)中，将实时监测的相对距离作为实际相对距离，将实际相对距离经PID控制器的滤波器滤波处理后，和预先设定的力值对应的参考相对距离作相减获得偏差，将该偏差输入到PID控制器，PID控制器输出的偏差控制量输入到控制压电运动平台(6)的运动模块，执行反馈补偿运动。
  4. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：所述的第一微吸管(1)表面吸附有Spy-tag蛋白质分子，参考小球(7)表面包被了Spy-catcher蛋白质分子，通过Spy-tag、Spy-catcher蛋白质分子间的相互作用将参考小球(7)固定粘附于第一微吸管(1)端部侧面。
  5. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：所述的参考小球(7)的直径大于探针小球(3)的直径，可以在实验过程中通过小球的尺寸直观地进行区分。
  6. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：所述的参考小球(7)为玻璃球。
  7. 根据权利要求1所述的一种基于生物膜力学探针系统的超准力钳实验方法，其特征在于：所述的力钳实验中，开始以初始的数据采样时间间隔的3倍作为数据记录时间间隔对力值数据进行采样并记录于寄存器中，当分子键结合时间超过5秒后，数据记录时间间隔增加为采样时间间隔的20倍；当记录数据次数超过5000次后，数据记录时间间隔进一步增大5倍。
PCT/CN2020/115248 2019-12-11 2020-09-15 一种基于生物膜力学探针系统的超准力钳实验方法 WO2021114789A1 (zh)

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