CN102151997A - Method for processing micropore of patch clamp chip - Google Patents
Method for processing micropore of patch clamp chip Download PDFInfo
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- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种膜片钳芯片激光微孔加工方法，包括1)将紫外激光器发出的功率为1～7瓦的紫外高斯激光束，通过扩束镜扩束；2)将扩束镜扩束后的紫外高斯激光束通过调节光圈取出孔径范围为1.2～1.8mm的紫外高斯激光束的中心光斑；3)将所述紫外高斯激光束的中心光斑通过聚焦镜聚焦到玻璃膜片钳芯片表面进行打孔，打孔过程中通过控制步骤2)中调节光圈的中心直径，来使玻璃膜片钳芯片上形成漏斗状微孔。本发明使得聚焦后的紫外激光的高能量在极端的时间内能破坏钳芯片夹具上玻璃材料的分子结构，并在玻璃材料上形成均匀的孔洞。
The invention discloses a patch clamp chip laser microhole processing method, which comprises 1) expanding the ultraviolet Gaussian laser beam with a power of 1 to 7 watts emitted by an ultraviolet laser through a beam expander; 2) expanding the beam through the beam expander; The ultraviolet Gaussian laser beam after the beam is adjusted to take out the central spot of the ultraviolet Gaussian laser beam with an aperture ranging from 1.2 to 1.8 mm; 3) focusing the central spot of the ultraviolet Gaussian laser beam on the surface of the glass patch clamp chip through a focusing mirror Punching is carried out. During the punching process, the central diameter of the aperture is controlled in step 2) to form a funnel-shaped microhole on the glass patch clamp chip. The invention enables the high energy of the focused ultraviolet laser to destroy the molecular structure of the glass material on the clamp chip holder within an extreme time and form uniform holes on the glass material.
Description
技术领域technical field
本发明涉及激光微孔加工技术领域，具体涉及一种膜片钳芯片微孔加工方法。The invention relates to the technical field of laser microhole processing, in particular to a microhole processing method for a patch clamp chip.
技术背景technical background
膜片钳技术是离子通道研究中的重要技术。经典膜片钳虽然具有技术信息含量大、分辨率高的优点，但是通量低、操作烦琐、实验条件要求高等缺点限制了它在细胞生理和药物发现等研究中的应用。因此膜片钳芯片技术应运而生。膜片钳芯片技术能够进行高通量的电生理测量、自动形成高阻抗封接，并能与光学测量手段相结合，它促进了离子通道和高通量药物筛选的研究。膜片钳芯片技术采用带有微米级孔洞的平面结构代替玻璃微电极(如图1所示)与细胞形成封接，只需将细胞悬液滴在孔洞上，施加一个负压或是一个静电场，将细胞引导到孔洞上，再施加一个负压即可使细胞与芯片间形成高阻抗封接。这样操作更加方便、快捷。并且可将芯片制作为电极阵列对多个细胞同时进行记录。上述膜片嵌芯片的微孔呈漏洞状(如图2所示)，微孔的入口孔径大于出口孔径，微孔入口孔径的范围为2～10μm，微孔出口孔径的范围为1～2μm，这种结构有利于细胞悬液的灌注。Patch clamp technique is an important technique in ion channel research. Although the classic patch clamp has the advantages of large technical information content and high resolution, its disadvantages such as low throughput, cumbersome operation, and high requirements for experimental conditions limit its application in researches such as cell physiology and drug discovery. Therefore, patch clamp chip technology came into being. Patch clamp chip technology can perform high-throughput electrophysiological measurements, automatically form high-impedance seals, and can be combined with optical measurement methods, which promotes the research of ion channels and high-throughput drug screening. The patch clamp chip technology uses a planar structure with micron-scale holes instead of glass microelectrodes (as shown in Figure 1) to form a seal with the cells. Just drop the cell suspension on the holes and apply a negative pressure or an electrostatic charge. The field guides the cells to the hole, and then applies a negative pressure to form a high-impedance seal between the cells and the chip. This operation is more convenient and faster. And the chip can be made as an electrode array to record multiple cells simultaneously. The micropores of the above-mentioned membrane-embedded chip are in the form of holes (as shown in Figure 2), the inlet aperture of the micropore is larger than the outlet aperture, the range of the inlet aperture of the micropore is 2-10 μm, and the range of the outlet aperture of the micropore is 1-2 μm. This structure facilitates the perfusion of the cell suspension.
目前，已经采用的制作膜片钳芯片的材料有硅、石英晶体或玻璃和有机聚合体。采用基于硅材料基底制作的膜片钳芯片，在打孔实验中存在一系列的问题，难以形成上述漏洞状微孔；利用石英晶体良好的绝缘性质制作的膜片钳芯片，可以用标准的平面加工技术，得到微米到亚微米级的孔洞，实验中发现这种膜片钳芯片的性能参数很好，但是打出的孔洞的形状总是三角形，使得细胞与芯片间很难形成高阻抗的封接；玻璃和有机聚合体也是很好的绝缘材料，而且还具有透明，方便观察以及可与其他光学检测手段如荧光检测技术相结合的优点，玻璃膜片钳芯片与细胞能较好地形成高阻抗封接。另外，玻璃具有很好的电和机械性质、表面具有亲水性，是比较理想的膜片嵌芯片材料。At present, the materials used to make patch clamp chips include silicon, quartz crystal or glass and organic polymers. The patch clamp chip made based on the silicon material substrate has a series of problems in the hole punching experiment, and it is difficult to form the above-mentioned loophole-shaped microholes; the patch clamp chip made of the good insulating properties of the quartz crystal can be used on a standard plane. Processing technology, to obtain micron to submicron holes, the experiment found that the performance parameters of this patch clamp chip are very good, but the shape of the hole is always triangular, making it difficult to form a high-impedance seal between the cell and the chip ; Glass and organic polymers are also good insulating materials, and they also have the advantages of being transparent, convenient for observation, and can be combined with other optical detection methods such as fluorescence detection technology. Glass patch clamp chips and cells can form high impedance well. seal. In addition, glass has good electrical and mechanical properties, and the surface is hydrophilic, so it is an ideal material for diaphragm embedded chips.
目前在玻璃上加工微孔的方法通常采用单个离子追踪刻蚀技术，在芯片上加工的孔洞直径可达1μm，甚至更小，且孔洞圆润、光滑。但是该方法效率比较低，推广难度较大。因此如何在玻璃上加工出1～10μm孔径的漏洞状微孔是大规模应用膜片钳芯片技术的关键。At present, the method of processing microholes on glass usually adopts single ion tracking etching technology, and the diameter of the holes processed on the chip can reach 1 μm or even smaller, and the holes are round and smooth. However, the efficiency of this method is relatively low, and it is difficult to popularize. Therefore, how to process hole-like micropores with a diameter of 1-10 μm on glass is the key to the large-scale application of patch clamp chip technology.
众所周知，激光打孔加工是激光加工技术的重要组成部分，激光打孔与其他打孔方法相比，最大区别是它具有高速、高精度和高适应性的特点。除此之外，它还具有打孔均匀、热影响区小、加工时无噪声、切缝边缘垂直度好、切边光滑、加工过程容易实现自动化控制等优点，但是目前的激光打孔技术最小加工的孔径在100μm左右，且难以打出漏洞状微孔。另外，传统的YAG激光和CO2激光很难作用于玻璃材料，加工的效果差，而且所加工的玻璃很容易产生裂纹和崩边，不能满足膜片嵌芯片加工的要求。因此如何将激光打孔技术应用到膜片嵌芯片的加工是本发明要解决的关键问题。As we all know, laser drilling is an important part of laser processing technology. Compared with other drilling methods, laser drilling has the characteristics of high speed, high precision and high adaptability. In addition, it also has the advantages of uniform drilling, small heat-affected zone, no noise during processing, good verticality of slit edge, smooth cutting edge, easy automatic control of the processing process, etc., but the current laser drilling technology has the smallest The processed pore diameter is about 100 μm, and it is difficult to punch hole-like micropores. In addition, the traditional YAG laser and CO 2 laser are difficult to act on glass materials, and the processing effect is poor, and the processed glass is prone to cracks and edge chipping, which cannot meet the requirements of film-embedded chip processing. Therefore, how to apply the laser drilling technology to the processing of the membrane-embedded chip is the key problem to be solved by the present invention.
发明内容Contents of the invention
本发明的目的是针对上述技术问题，提供一种能实现在玻璃上加工微米级孔洞的膜片钳芯片微孔加工方法，使其能适用于工业化大规模生产。The object of the present invention is to solve the above technical problems and provide a patch clamp chip microhole processing method capable of processing micron-scale holes on glass, so that it can be applied to large-scale industrial production.
为实现此目的，本发明所设计的一种膜片钳芯片微孔加工方法，其特征在于，它包括如下步骤：To achieve this goal, a kind of patch clamp chip micropore processing method designed by the present invention is characterized in that it comprises the steps:
步骤1)将紫外激光器发出的功率为1～7瓦的紫外高斯激光束，通过扩束镜扩束；Step 1) expanding the ultraviolet Gaussian laser beam with a power of 1 to 7 watts emitted by the ultraviolet laser through a beam expander;
步骤2)将扩束镜扩束后的紫外高斯激光束通过调节光圈取出光束直径范围为1.2～1.8mm的紫外高斯激光束的中心光斑；Step 2) taking out the central spot of the ultraviolet Gaussian laser beam whose beam diameter ranges from 1.2 to 1.8mm by adjusting the aperture of the ultraviolet Gaussian laser beam expanded by the beam expander;
步骤3)将所述紫外高斯激光束的中心光斑通过聚焦镜聚焦到玻璃膜片钳芯片表面进行打孔，打孔过程中通过控制步骤1)中紫外激光器发出的紫外高斯激光束功率的大小和步骤2)中调节光圈的中心直径，来使玻璃膜片钳芯片上形成漏斗状微孔。Step 3) focusing the central spot of the ultraviolet Gaussian laser beam on the surface of the glass patch clamp chip through a focusing mirror to punch holes, and controlling the power of the ultraviolet Gaussian laser beam emitted by the ultraviolet laser in step 1) and In step 2), the central diameter of the aperture is adjusted to form a funnel-shaped microhole on the glass patch clamp chip.
在上述步骤3)中，所述紫外高斯激光束的中心光斑可以先通过反射镜反射后再进入所述聚焦镜。In the above step 3), the central spot of the ultraviolet Gaussian laser beam can be reflected by a mirror before entering the focusing mirror.
在上述方案中，还可以采用紫外高斯激光束不动，通过水平移动玻璃膜片钳芯片的方法，完成整个玻璃膜片钳芯片上阵列式排列微孔的加工。In the above solution, the ultraviolet Gaussian laser beam can also be used to move the glass patch clamp chip horizontally to complete the processing of the arrayed microholes on the entire glass patch clamp chip.
优选地，所述玻璃膜片钳芯片置于运动控制平台上，运动控制平台通过计算机数控系统控制水平移动，并带动玻璃膜片钳芯片水平移动；计算机数控系统还控制紫外激光器的工作状态。Preferably, the glass patch clamp chip is placed on a motion control platform, and the motion control platform moves horizontally through a computer numerical control system, and drives the glass patch clamp chip to move horizontally; the computer numerical control system also controls the working state of the ultraviolet laser.
优选地，所述紫外激光器发出的紫外高斯激光束的波长范围为266～355nm，频率范围为10～100KHz，脉宽范围为15～50ns；所述调节光圈的光圈直径可调范围为1.2～1.8mm；所述扩束镜将紫外高斯激光束进行8～10倍的扩束。Preferably, the wavelength range of the ultraviolet Gaussian laser beam emitted by the ultraviolet laser is 266-355nm, the frequency range is 10-100KHz, and the pulse width range is 15-50ns; the adjustable range of the aperture diameter of the adjustment aperture is 1.2-1.8 mm; the beam expander expands the ultraviolet Gaussian laser beam by 8 to 10 times.
优选地，所述微孔的入口孔径大于出口孔径，微孔入口孔径的范围为2～10μm，微孔出口孔径的范围为1～2μm。Preferably, the inlet diameter of the micropores is larger than the outlet diameter, the inlet diameter of the micropores ranges from 2 to 10 μm, and the outlet diameter of the micropores ranges from 1 to 2 μm.
优选地，所述玻璃膜片钳芯片厚度为0.1mm～0.15mm；所述聚焦镜为远心透镜，紫外高斯激光束经过该透镜聚焦后的最小光斑直径为0.7μm。Preferably, the thickness of the glass patch clamp chip is 0.1 mm to 0.15 mm; the focusing mirror is a telecentric lens, and the minimum spot diameter of the ultraviolet Gaussian laser beam after being focused by the lens is 0.7 μm.
下面介绍本发明的激光微孔加工原理。The laser microhole processing principle of the present invention is introduced below.
1)紫外激光与玻璃无机材料的相互作用机理：1) The interaction mechanism between ultraviolet laser and glass inorganic materials:
激光微孔加工中依据被加工材料的不同特性及对不同波长的激光的吸收程度不同，往往选择某一特定波长的激光对材料进行加工，以期达到最好的加工效果。考虑到玻璃材料的特性，申请人选择输出波长范围为266nm～355nm的紫外激光器来对玻璃膜片钳芯片的微孔进行加工。该波长的激光非常适合于无机材料。由于光束的衍射现象是限制加工部件最小尺寸的主要因素，激光可达到的聚焦点的最小光斑直径随着波长的增加而线性增加，因此较短波长的激光能够加工出更小的部件。另外，紫外激光微处理过程从本质上来说不是“热”处理过程，紫外激光高能量的光子可以直接破坏玻璃材料的化学键，这一加工过程称为“光蚀”效应，紫外激光加工是实质上是冷处理过程，热影响微乎其微，高能量的紫外光直接破坏玻璃材料的化学键，使得这种“冷”加工出来的孔径直径可控并具有光滑均匀的边缘。In laser microhole processing, according to the different characteristics of the processed material and the different degrees of absorption of different wavelengths of laser light, a specific wavelength of laser light is often selected to process the material in order to achieve the best processing effect. Considering the characteristics of the glass material, the applicant selected an ultraviolet laser with an output wavelength range of 266nm-355nm to process the micropores of the glass patch clamp chip. Lasers of this wavelength are very suitable for inorganic materials. Since the diffraction phenomenon of the beam is the main factor limiting the minimum size of the processed part, the minimum spot diameter of the focus point that the laser can reach increases linearly with the increase of the wavelength, so the shorter wavelength laser can process smaller parts. In addition, the ultraviolet laser microprocessing process is not a "thermal" treatment process in essence. The high-energy photons of the ultraviolet laser can directly destroy the chemical bonds of the glass material. This process is called the "photoetching" effect. It is a cold treatment process with minimal thermal influence, and the high-energy ultraviolet light directly destroys the chemical bonds of the glass material, making the aperture diameter controllable and having smooth and uniform edges produced by this "cold" processing.
2)微孔成型机理：2) Microporous forming mechanism:
激光器发出的激光理论上是高斯光束，理想的高斯光束有利于图2所示的漏洞状微孔的成型，但是实际上激光不可能达到这种理想状态。为了能够使激光尽可能的接近理想高斯光束模式，本发明将扩束镜和调节光圈组合起来起到筛选光束的作用(如图3所示)。首先，紫外激光器发出的紫外高斯激光经过扩束镜扩束，扩束镜扩束后的光束比较粗，该光束到达调节光圈后只有光轴中间部分可以通过调节光圈。如图4所示，通过控制调节光圈中心孔直径的大小可以起到选择光束的作用，光圈中心孔直径越小透过去的激光越接近高斯光束，也确保了得到更小的聚焦光斑，从调节光圈出来的激光束经过远心透镜聚焦后焦点的激光功率密度分布也是高斯分布，靠近紫外高斯激光束光轴的激光经过聚焦后功率密度更高，在极短的时间内破坏玻璃材料的分子结构形成微孔，远离紫外高斯激光束光轴的激光功率密度要小很多，所以在短时间内难以穿透玻璃材料，远离紫外高斯激光束光轴的激光打在玻璃膜片钳芯片上形成微孔的锥面。通过控制调节光圈的中心直径，可使玻璃膜片钳芯片上形成漏斗状微孔。The laser emitted by the laser is theoretically a Gaussian beam, and the ideal Gaussian beam is conducive to the formation of the hole-shaped microhole shown in Figure 2, but in practice the laser cannot achieve this ideal state. In order to make the laser as close as possible to the ideal Gaussian beam mode, the present invention combines a beam expander and an adjustable aperture to screen the beam (as shown in FIG. 3 ). First of all, the ultraviolet Gaussian laser emitted by the ultraviolet laser is expanded by the beam expander. The beam expanded by the beam expander is relatively thick. After the beam reaches the adjusting aperture, only the middle part of the optical axis can pass through the adjusting aperture. As shown in Figure 4, adjusting the diameter of the central hole of the aperture can play a role in selecting the beam. The smaller the diameter of the central hole of the aperture, the closer the transmitted laser beam is to the Gaussian beam, which also ensures a smaller focused spot. From the adjustment After the laser beam from the aperture is focused by the telecentric lens, the laser power density distribution at the focal point is also a Gaussian distribution. The laser beam close to the optical axis of the ultraviolet Gaussian laser beam has a higher power density after focusing, which destroys the molecular structure of the glass material in a very short time. Forming microholes, the laser power density away from the optical axis of the ultraviolet Gaussian laser beam is much smaller, so it is difficult to penetrate the glass material in a short time, and the laser far away from the optical axis of the ultraviolet Gaussian laser beam forms micropores on the glass patch clamp chip of the cone. By controlling and adjusting the central diameter of the aperture, a funnel-shaped microhole can be formed on the glass patch clamp chip.
本发明采用紫外激光加工玻璃膜片钳芯片，紫外高斯激光束通过扩束、调节光圈、反射、聚焦后作用于玻璃膜片钳芯片上，直接破坏玻璃材料的化学键，来形成入口孔径的范围为2～10μm、出口孔径的范围为1～2μm的微孔，通过调节光圈的直径大小间接可以调节所加工微孔的直径大小，通过调节紫外高斯激光束的功率和调节光圈的中心直径来调节玻璃膜片钳芯片上形成的微孔的锥度。通过计算机数控系统和运动控制平台可以实现整个玻璃膜片钳芯片的大批量工业化加工。本发明为在玻璃膜片钳上加工出微米级的孔洞以及使芯片进行大规模生产开辟了一条新途径。The present invention uses ultraviolet laser to process the glass patch clamp chip, and the ultraviolet Gaussian laser beam acts on the glass patch clamp chip after expanding the beam, adjusting the aperture, reflecting and focusing, directly destroying the chemical bonds of the glass material to form the range of the entrance aperture. 2～10μm, the range of the exit aperture is 1～2μm, the diameter of the processed microhole can be adjusted indirectly by adjusting the diameter of the aperture, and the glass can be adjusted by adjusting the power of the ultraviolet Gaussian laser beam and the central diameter of the aperture. Taper of the microwells formed on the patch clamp chip. Mass industrial processing of the entire glass patch clamp chip can be realized through a computer numerical control system and a motion control platform. The invention opens up a new way for processing micron-scale holes on the glass patch clamp and making the chip go into mass production.
附图说明Description of drawings
图1为单个膜片钳芯片的工作原理图；Fig. 1 is a working principle diagram of a single patch clamp chip;
图2为膜片钳芯片微孔的结构示意图；Fig. 2 is the schematic diagram of the structure of the micropore of the patch clamp chip;
图3为本发明的膜片钳芯片微孔加工装置的结构示意图；Fig. 3 is the structural representation of patch clamp chip microhole processing device of the present invention;
图4为高斯光束调节的原理图；Fig. 4 is the schematic diagram of Gaussian beam adjustment;
其中，1-紫外激光器、2-扩束镜、3-调节光圈、4-反射镜、5-聚焦镜、6-膜片钳芯片夹具、7-运动控制平台、8-计算机数控系统。Among them, 1-ultraviolet laser, 2-beam expander, 3-adjusting aperture, 4-mirror, 5-focusing mirror, 6-patch clamp chip fixture, 7-motion control platform, 8-computer numerical control system.
具体实施方式Detailed ways
以下结合附图和实施例对本发明作进一步的详细说明：Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail:
本实施例所述的一种膜片钳芯片微孔加工方法，采用如图3所示的一种膜片钳芯片微孔加工装置，该装置包括紫外激光器1、扩束镜2、调节光圈3、反射镜4、聚焦镜5、玻璃膜片钳芯片夹具6、计算机数控系统8和运动控制平台7，其中，紫外激光器1的输出光路上设置扩束镜2，扩束镜2的输出光路上设置调节光圈3，调节光圈3的输出光路上设置反射镜4，反射镜4的反射输出光路上设置聚焦镜5，聚焦镜5的输出光路上设置玻璃膜片钳芯片夹具6。计算机数控系统8的激光控制信号输出端连接紫外激光器1的控制接口，计算机数控系统8的位置控制信号输出端连接运动控制平台7的控制接口，玻璃膜片钳芯片夹具6置于运动控制平台7上。A kind of patch clamp chip microhole processing method described in this embodiment adopts a kind of patch clamp chip microhole processing device as shown in Figure 3, and this device comprises
本装置的扩束镜2的激光扩束倍数为8～10倍；调节光圈3的光圈直径可调范围为1.2mm～1.8mm；紫外激光器1发出的紫外高斯激光束的波长为266nm～355nm；紫外激光器1发出的紫外高斯激光束的频率为10KHz～100KHz，紫外激光器1发出的紫外高斯激光束的功率为1～7瓦。The laser beam expansion factor of the
本发明的工作过程为：将待加工的玻璃膜片嵌芯片放置在运动控制平台上，通过红光指示找到第一个待加工孔的位置，打开紫外激光器1，设置紫外激光器1相应的工作参数(包括电流20A、频率为15-20KHz)，使激光器工作在额定电流和温度下，紫外激光器1的模式设置为MODE_1(首脉冲抑制模式)，使输出的激光能量处于比较稳定的状态。紫外高斯激光束通过扩束镜2扩束后再由调节光圈3取出孔径范围为1.2～1.8mm的紫外高斯激光束的中心光斑，然后由反射镜4改变紫外高斯激光束的角度，最后通过聚焦镜5对紫外激光聚焦，聚焦后到达玻璃膜片钳钳芯片夹具6夹住的玻璃材料上的光功率为1～7W之间，并在玻璃材料上钻出微孔洞，调节聚焦镜5将使紫外高斯激光束的焦点刚好落在待加工玻璃膜片嵌芯片的上表面。通过控制调节光圈的中心直径，来使玻璃膜片钳芯片上形成入口孔径范围为2～10μm，微孔出口孔径范围为1～2μm的漏斗状微孔。另外，在加工过程中，采用激光束不动，通过计算机数控系统8控制运动控制平台7，进行水平移动玻璃膜片钳芯片的方法，完成整个玻璃膜片钳芯片上阵列式排列微孔的加工，实现了整个加工流程的自动化。The working process of the present invention is as follows: place the glass film embedded chip to be processed on the motion control platform, find the position of the first hole to be processed through the red light indicator, turn on the
另外，本实施例实施时，调节光圈直径大小为1.5mm，开激光并延时1秒钟后，取下玻璃膜片钳芯片夹具6在光学显微镜下测量孔径大小；如果玻璃材料没有被打穿，则无法测量其大小，表明激光能量还不够，需要将激光能量适度调大直到刚好能打穿为止，反之，如果出光孔孔径大于了2μm，则需要将激光能量适度调小直到满足要求为止；如果入光孔孔径偏大则将调节光圈的直径调小，反之亦然。通过反复的调节测试最终确保加工的孔径达到所要求的值为止。根据第一个孔的加工参数，通过数控软件完成批量加工。In addition, during the implementation of this embodiment, adjust the diameter of the aperture to 1.5 mm, turn on the laser and delay for 1 second, remove the glass patch
本发明采用紫外激光加工玻璃材料的膜片钳芯片，高峰值功率的紫外激光在极短时间内破坏材料的微观结构形成大小均匀孔洞，为在玻璃上加工出微米级的孔洞以及使芯片进行大规模生产开辟了一条新途径。The invention adopts ultraviolet laser to process the patch clamp chip of glass material, and the ultraviolet laser with high peak power destroys the microstructure of the material in a very short time to form holes of uniform size, in order to process micron-sized holes on the glass and make the chip large Mass production opens up a new avenue.
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。The content not described in detail in this specification belongs to the prior art known to those skilled in the art.
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