技术领域technical field
本发明涉及生物传感器、电分析化学微纳制备技术领域，是一种可以同时检测细胞电生理和细胞多突触位点的多种电化学神经递质信号的超微阵列传感器及其制备方法。The invention relates to the technical field of micro-nano preparation of biosensors and electroanalytical chemistry, and relates to an ultramicro array sensor capable of simultaneously detecting various electrochemical neurotransmitter signals of cell electrophysiology and cell multi-synaptic sites and a preparation method thereof.
背景技术Background technique
神经细胞轴突末梢囊泡储存的神经递质是相当稳定的，以囊泡为单位倾囊释放，被称为量子式释放。一次动作电位的到达，能使大约200～300个囊泡的内容排放。神经细胞量子化神经递质释放进行实时记录是了解神经信号传导、作用机制的重要直接方式。监测细胞量子释放必须具备超高灵敏度、高选择性、高时间分辨、高空问分辨、超小体积的分析技术。传统检测神经细胞量子释放的方法基本为膜片钳配合碳纤维电极技术，通常仅能获得少量1—2个通道的数据，而且电极定位困难、操作繁琐，无法长期监测，很大程度上限制了对细胞量子释放的检测。The neurotransmitters stored in vesicles at the axon terminal of nerve cells are quite stable, and they are released in units of vesicles, which is called quantum release. The arrival of an action potential can discharge the contents of about 200 to 300 vesicles. Real-time recording of quantized neurotransmitter release by nerve cells is an important and direct way to understand nerve signal transduction and mechanism of action. To monitor quantum release from cells, analytical techniques with ultra-high sensitivity, high selectivity, high time-resolution, high-space resolution, and ultra-small volume must be possessed. The traditional method of detecting quantum release of nerve cells is basically patch clamp combined with carbon fiber electrode technology, usually only a small number of 1-2 channel data can be obtained, and the electrode positioning is difficult, the operation is cumbersome, and long-term monitoring is impossible, which greatly limits the ability to detect Detection of cellular quantum release.
随着微机电系统(MEMS)加工技术的发展，微电极阵列(microelectrode，MEA)提供了一种长期、多位点监测细胞的方法，常规的MEA电极直径在10～50μm，远大于囊泡和突触的量子释放尺寸，虽然可以定位到单个细胞，但是对于定位于检测突触囊泡的量子释放，在空间和时间分辨率上还是不足，不能区分单个电极上细胞不同位点释放的空间差异，难以测到实时的量子释放信号。因此需要研制尺寸更小的电极，以实现单细胞释放时空分辨监测以及深入到细胞突触间隙进行研究，在更深层次上探讨细胞释放机制。With the development of microelectromechanical system (MEMS) processing technology, microelectrode array (microelectrode, MEA) provides a long-term, multi-site monitoring method for cells. The diameter of conventional MEA electrodes is 10-50 μm, which is much larger than that of vesicles and vesicles. Although the quantum release size of the synapse can be localized to a single cell, the spatial and temporal resolution is still insufficient for the quantum release of the detection of synaptic vesicles, and the spatial difference of the release at different positions of the cell on a single electrode cannot be distinguished , it is difficult to detect the real-time quantum release signal. Therefore, it is necessary to develop electrodes with smaller size to realize the time-spatial resolution monitoring of single-cell release and to conduct research in the synaptic cleft of cells, so as to explore the mechanism of cell release at a deeper level.
发明内容Contents of the invention
本发明针对细胞及突触囊泡量子释放微量物质的实时检测，解决目前问题：只能检测单个细胞或囊泡，一次只能检测一种神经递质、电极尺寸过大，难以在突触尺寸内进行检测；设计制备一种位点小、记录点多、对神经细胞无损伤、可以在二维尺度上同时检测多个记录点(同时检测多个细胞、多种神经递质)的超微平面电极阵列传感器。The present invention is aimed at the real-time detection of micro-substances released by cells and synaptic vesicles, and solves the current problems: only a single cell or vesicle can be detected, and only one neurotransmitter can be detected at a time. In-house detection; design and prepare a small site, many recording points, no damage to nerve cells, and can simultaneously detect multiple recording points on a two-dimensional scale (simultaneous detection of multiple cells, multiple neurotransmitters) Planar electrode array sensor.
根据本发明一方面，其提供了一种超微平面电极阵列传感器，其特征在于：包括超微平面电极阵列和复合功能膜层，其中所述超微平面电极阵列由绝缘基底、微电极阵列、对电极、参比电极、电极引线以及触点构成，所述微电极阵列包括多个，分布在绝缘基底上，多个微电极阵列可共用参比电极和对电极；所述复合功能膜层包括纳米修饰、生物兼容修饰、生物特异层修饰相结合的修饰层，其分区域修饰在超微平面电极阵列。According to one aspect of the present invention, it provides an ultra-micro-planar electrode array sensor, which is characterized in that it includes an ultra-micro-planar electrode array and a composite functional film layer, wherein the ultra-micro-planar electrode array is composed of an insulating substrate, a micro-electrode array, Composed of counter electrode, reference electrode, electrode leads and contacts, the microelectrode array includes multiple, distributed on the insulating substrate, multiple microelectrode arrays can share the reference electrode and the counter electrode; the composite functional film layer includes A modified layer combining nanometer modification, biocompatible modification, and biospecific layer modification, which is modified in sub-regions on the ultra-microplanar electrode array.
根据本发明另一方面，其提供了一种超微平面电极阵列传感器的制备方法，其特征在于，包括如下步骤：According to another aspect of the present invention, it provides a method for preparing an ultramicroplanar electrode array sensor, which is characterized in that it includes the following steps:
步骤1、在经过表面清洗的绝缘基底上旋涂一层光刻胶，光刻显影后形成引线和微电极阵列图案；Step 1. Spin-coat a layer of photoresist on the surface-cleaned insulating substrate, and form lead wires and micro-electrode array patterns after photolithography and development;
步骤2、在微电极阵列图案表面溅射一层微电极导电薄膜层；Step 2, sputtering a layer of microelectrode conductive film layer on the surface of the microelectrode array pattern;
步骤3、采用剥离工艺去除多余微电极导电薄膜层，留下所需电极、引线和触点，形成基础金属电极阵列；Step 3, using a stripping process to remove the redundant micro-electrode conductive film layer, leaving the required electrodes, leads and contacts to form a basic metal electrode array;
步骤4、在基础金属电极阵列表面通过等离子体增强化学气相沉积绝缘层；Step 4, depositing an insulating layer on the surface of the basic metal electrode array by plasma enhanced chemical vapor phase;
步骤5、在沉积有绝缘层的基础电极阵列的第一区域，采用双层布线的第一层版图进行二次光刻，采用步进光刻工艺形成超微电极阵列中心图形，然后采用等离子刻蚀的方法，暴露出第一超微电极阵列及触点，保留所有引线表面覆盖的绝缘层；Step 5. In the first area of the basic electrode array deposited with an insulating layer, the first layer layout of the double-layer wiring is used to carry out secondary photolithography, and the stepping photolithography process is used to form the central pattern of the ultra-micro electrode array, and then plasma etching is used. The method of etching exposes the first ultra-micro electrode array and contacts, and retains the insulating layer covered on the surface of all leads;
步骤6、在沉积有绝缘层的基础电极阵列的第二区域，重复步骤5刻蚀暴露出第二超微电极阵列及相应触点；Step 6. In the second area of the basic electrode array deposited with an insulating layer, repeat step 5 to etch to expose the second ultramicro electrode array and corresponding contacts;
步骤7、采用SU-8、PDMS或聚酰亚胺制备有超微电极阵列的芯片上通过普通光刻形成坝型分区结构。Step 7, using SU-8, PDMS or polyimide to prepare a chip with an ultra-micro electrode array to form a dam-type partition structure by ordinary photolithography.
本发明的超微平面阵列传感器与现有棒状超微电极传感器比较具有如下优势：Compared with the existing rod-shaped ultra-micro electrode sensor, the ultra-micro planar array sensor of the present invention has the following advantages:
细胞在微平面阵列传感器上生长附着后检测，贴附更好，完全原位、无创检测。无需费时费力去定位。Cells grow and attach to the microplane array sensor for detection, better adhesion, complete in situ and non-invasive detection. No need to waste time and effort to locate.
超微平面阵列传感器可以同时检测多个细胞、多个突触位点囊泡释放。Nanoplanar array sensors can simultaneously detect vesicle release from multiple cells and multiple synaptic sites.
超微平面阵列传感器一个传感器可检测多种神经递质。Nanoplanar array sensor can detect multiple neurotransmitters with one sensor.
细胞在微平面阵列传感器采用微纳工艺制备，可批量制备，性能更均一、稳定。The cell-in-microplane array sensor is prepared by a micro-nano process, which can be prepared in batches, and has more uniform and stable performance.
本超微平面阵列传感器与现有阵列微电极比较具有如下优势：Compared with the existing array microelectrode, the ultra-microplanar array sensor has the following advantages:
1、超微平面阵列传感器电极位点更小，尺寸范围0.5μm-5μm，更接近于细胞突触囊泡的尺寸范围，扩散迅速，利于囊泡量子释放的瞬态检测。1. The electrode site of the ultra-microplanar array sensor is smaller, and the size range is 0.5 μm-5 μm, which is closer to the size range of cell synaptic vesicles, and the diffusion is rapid, which is conducive to the transient detection of vesicle quantum release.
2、超微平面阵列传感器采用隔离区设计技术和特异性修饰技术，可实现多种神经递质检测。2. The ultra-microplanar array sensor adopts isolation region design technology and specific modification technology, which can realize the detection of various neurotransmitters.
本发明的超微平面阵列传感器采用微机电加工技术制备结合纳米修饰技术，可实现对细胞的无损实时检测，可用于多个位点的单细胞和单细胞多突触囊泡量子释放的多种神经递质的原位实时检测，并且可以实现对同一种细胞进行不同刺激的同时检测，进行同时对比检测和统计分析。The ultra-microplanar array sensor of the present invention is prepared by micro-electromechanical processing technology combined with nano-modification technology, which can realize non-destructive real-time detection of cells, and can be used for various single-cell and single-cell polysynaptic vesicle quantum releases at multiple sites. In situ real-time detection of neurotransmitters, and simultaneous detection of different stimuli for the same cell, simultaneous comparative detection and statistical analysis.
本发明的这种高密度超微平面电极阵列具有面积小(0.5μm～2μm电极位点直径)、记录点多、对神经细胞无损伤、可以在二维尺度上同时检测多达上百个记录点的神经细胞囊泡的神经递质检测。由于电极阵列的高密特性，还可确定单个神经细胞分泌的空间位置信息。进行了分区结构设计，在同一个传感器阵列上可以同时检测多种神经递质。The high-density ultra-microplanar electrode array of the present invention has small area (0.5 μm-2 μm electrode site diameter), many recording points, no damage to nerve cells, and can simultaneously detect up to hundreds of recordings on a two-dimensional scale. Neurotransmitter detection of dot neuronal vesicles. Due to the high-density characteristics of the electrode array, the spatial location information secreted by a single nerve cell can also be determined. The partition structure design is carried out, and multiple neurotransmitters can be detected simultaneously on the same sensor array.
本发明的上述各种目的、方法、特点和优点，通过下面结合附图和实施例可以得到更加详细的说明。The various purposes, methods, features and advantages of the present invention can be described in more detail below in conjunction with the accompanying drawings and embodiments.
附图说明Description of drawings
图1是本发明中超微平面电极阵列传感器的制备工艺流程图；Fig. 1 is the preparation process flowchart of ultramicro planar electrode array sensor among the present invention;
图2是本发明中超微平面电极阵列传感器的双层布板局部图；Fig. 2 is a partial diagram of the double-layer layout of the ultramicroplanar electrode array sensor in the present invention;
图3是本发明中超微电极阵列传感器的单层布板局部平面图；Fig. 3 is a partial plan view of a single-layer layout of an ultramicroelectrode array sensor in the present invention;
图4是本发明实施例中铂黑修饰的超微平面电极显微图。Fig. 4 is a micrograph of a platinum black-modified ultramicroplanar electrode in an embodiment of the present invention.
具体实施方式Detailed ways
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明进一步详细说明。In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings.
本发明提出的超微平面电极阵列传感器包括：超微电极阵列和复合膜层，其中超微电极阵列由绝缘基底、微电极阵列、对电极、参比电极、电极引线以及触点构成。所述绝缘基底是整个芯片的载体，在绝缘基底表面的中心位置，分布了若干个以矩阵形式排布的、由导电薄膜材料制成的圆形超微电极，构成微电极阵列。所述微电极阵列包括多个，分布在绝缘基底上，多个微电极阵列可共用参比电极和对电极。复合膜层是纳米修饰层、生物兼容修饰层、生物特异层修饰层有机组合而成，其分区域修饰在超微电极阵列表面。超微电极为平面是由于贵金属导电层溅射厚度仅为100～300nm。这种厚度形成的电极是平面薄膜的。电极阵列在中间位置，触点在四周，对电极和参比电极根据设计在电极阵列的一侧；引线是连接触点和电极阵列的。The ultra-micro-planar electrode array sensor proposed by the present invention includes: an ultra-micro-electrode array and a composite film layer, wherein the ultra-micro-electrode array is composed of an insulating substrate, a micro-electrode array, a counter electrode, a reference electrode, electrode leads and contacts. The insulating base is the carrier of the whole chip. On the central position of the surface of the insulating base, several circular ultra-micro electrodes made of conductive thin film materials arranged in a matrix form are distributed to form a micro electrode array. The micro-electrode array includes multiple micro-electrode arrays distributed on the insulating base, and the multiple micro-electrode arrays can share reference electrodes and counter electrodes. The composite film layer is an organic combination of a nano-modified layer, a bio-compatible modified layer, and a bio-specific modified layer, which are regionally modified on the surface of the ultramicroelectrode array. The reason why the ultramicro electrode is plane is that the sputtering thickness of the noble metal conductive layer is only 100-300nm. Electrodes formed at this thickness are planar thin films. The electrode array is in the middle, the contacts are around, the counter electrode and the reference electrode are on one side of the electrode array according to the design; the leads are connected to the contacts and the electrode array.
其中，所述微电极阵列选用的导电薄膜材料为生物相容性好的金属或金属化合物；微电极阵列包含60～128个微电极，其中微电极根据其表面修饰的复合功能膜层的不同能够用于神经电生理信号的检测，用于神经电生理信号检测的微电极直径0.5μm～5μm，还能够用于神经递质电化学信号检测，以及用于施加电刺激，施加电刺激的微电极直径0.5μm～5μm，微电极间距5μm～200μm。Wherein, the conductive film material selected by the microelectrode array is a metal or a metal compound with good biocompatibility; the microelectrode array includes 60 to 128 microelectrodes, wherein the microelectrodes can For the detection of nerve electrophysiological signals, the diameter of the microelectrode used for the detection of nerve electrophysiological signals is 0.5 μm to 5 μm, and it can also be used for the detection of neurotransmitter electrochemical signals, as well as for applying electrical stimulation, microelectrodes for applying electrical stimulation The diameter is 0.5 μm to 5 μm, and the distance between the micro-electrodes is 5 μm to 200 μm.
引线及触点的导电薄膜材料与微电极相同，厚度大于300nm，保证其机械强度能够承受标准电子元器件中弹性金属探针所造成的压力，并保证多次细胞培养检测使用清洗后仍具有良好的导电性。The conductive film material of the leads and contacts is the same as that of the micro-electrode, and the thickness is greater than 300nm, so as to ensure that its mechanical strength can withstand the pressure caused by the elastic metal probe in standard electronic components, and to ensure that it still has good performance after repeated cell culture testing and cleaning. conductivity.
所述超微平面阵列传感器，可检测神经细胞的电生理信号和电化学信号，可同时检测多个神经细胞多突触位点的神经递质量子释放信号；可同时检测单个神经细胞多种神经递质信号。The ultra-microplanar array sensor can detect electrophysiological signals and electrochemical signals of nerve cells, and can simultaneously detect neurotransmitter proton release signals at multiple synaptic sites of multiple nerve cells; it can simultaneously detect multiple neurons of a single nerve cell Transmitter signal.
本发明中所述绝缘基底材料是由从玻璃、耐热玻璃、硅片等组成的组中选择出来的一种制成。该基材硬度与厚度适中，可溅射金属层，耐受一定高温，为下一步工艺奠定基础。The insulating base material in the present invention is made of one selected from the group consisting of glass, heat-resistant glass, silicon wafer and the like. The substrate has moderate hardness and thickness, can sputter a metal layer, can withstand a certain high temperature, and lays the foundation for the next step of the process.
图1示出了本发明提出的一种超微平面电极阵列传感器的制备方法工艺流程图。如图1所示，该方法包括：Fig. 1 shows a process flow chart of a preparation method of an ultramicroplanar electrode array sensor proposed by the present invention. As shown in Figure 1, the method includes:
步骤1、将绝缘基底进行清洗处理后，在基片上涂覆光刻胶；可以采用正胶，也可以采用负胶，根据不同制版方式可采用不同光刻胶，采用普通光刻工艺在光刻胶上光刻显影后形成所需的引线和电极，构成微电极阵列图案。Step 1. After cleaning the insulating substrate, apply photoresist on the substrate; positive or negative resist can be used, and different photoresists can be used according to different plate making methods. After photolithography and development on the glue, the required leads and electrodes are formed to form a micro-electrode array pattern.
步骤2、在整片基材上溅射微电极导电薄膜层，所述微电极导电薄膜层为一层电极金属层，剥离光刻胶留下所需电极、引线和触点，形成基础金属电极阵列。电极是由两层结构组成，包括金属的下层种子层钛(Ti)或铬(Cr)和由从金、铂和钯中构成的组中选择出来的一种贵金属制成的上层。绝缘层上预溅射的钛或铬下金属层利于金或铂与电极表面的有效粘结。贵金属金和铂适合作电极，因为它们在电极表面区域的稳定性、电化学的还原性、抗氧化性等方面的电化学性质都非常好；并且加工简单，与玻璃／硅的粘接性好且导电率高，而且具有生物兼容性。本发明的铂或金层薄至200nm～500nm。Step 2, sputtering the micro-electrode conductive film layer on the entire substrate, the micro-electrode conductive film layer is a layer of electrode metal layer, peeling off the photoresist to leave the required electrodes, leads and contacts to form the basic metal electrode array. The electrodes are composed of a two-layer structure comprising a lower seed layer of metal titanium (Ti) or chromium (Cr) and an upper layer made of a noble metal selected from the group consisting of gold, platinum and palladium. A pre-sputtered titanium or chromium undermetal layer on the insulating layer facilitates effective bonding of gold or platinum to the electrode surface. The noble metals gold and platinum are suitable as electrodes because of their excellent electrochemical properties in terms of electrode surface area stability, electrochemical reducibility, oxidation resistance, etc.; and simple processing and good adhesion to glass/silicon And high conductivity, and has biocompatibility. The platinum or gold layer of the present invention is as thin as 200nm to 500nm.
步骤3、在基础电极阵列表面上，通过等离子体增强化学气相沉积(PECVD)二氧化硅、氮化硅、氧化硅等中的一种作为绝缘层，其厚度在250nm～500nm之间。Step 3. On the surface of the basic electrode array, one of silicon dioxide, silicon nitride, and silicon oxide is deposited as an insulating layer by plasma enhanced chemical vapor deposition (PECVD), and its thickness is between 250nm and 500nm.
步骤4、在沉积有绝缘层的基础电极阵列的第一区域，采用双层布线的第一层版图进行二次光刻，采用步进光刻工艺进行超微电极阵列中心图形的形成(尤其针对0.5μm～2μm电极位点直径)，然后采用等离子刻蚀的方法，暴露出基础电极阵列的第一区域中的超微电极阵列A及相应触点，保留所有引线表面覆盖的绝缘层；该步骤目的是为了使用绝缘层覆盖基础电极阵列中的引线，并刻蚀暴露出位于第一区域中的超微电极阵列A及相应触点；Step 4. In the first area of the basic electrode array deposited with an insulating layer, the first layer layout of the double-layer wiring is used to carry out secondary photolithography, and the stepping photolithography process is used to form the central pattern of the ultra-micro electrode array (especially for 0.5 μm ~ 2 μm electrode site diameter), and then adopt the plasma etching method to expose the ultra-micro electrode array A and the corresponding contacts in the first area of the basic electrode array, and retain the insulating layer covered on the surface of all the leads; this step The purpose is to use an insulating layer to cover the leads in the basic electrode array, and etch to expose the ultra-micro electrode array A and corresponding contacts located in the first region;
步骤5、在沉积有绝缘层的基础电极阵列的第二区域，重复步骤4刻蚀暴露出超微电极阵列B、对电极、参比电极及相应触点B。其中，双层布线设计可以在第一层形成参比电极、对电极也可以在第二层形成参比电极、对电极，超微电极阵列A和B可以共用参比电极和对电极。双层布线在保证良好的导线情况下，便于在有限的平面上形成更密集的超微电极位点，利于捕获细胞突触位点的量子释放。Step 5. In the second area of the basic electrode array deposited with an insulating layer, repeat step 4 to etch to expose the ultramicro electrode array B, the counter electrode, the reference electrode and the corresponding contact B. Among them, the double-layer wiring design can form a reference electrode and a counter electrode on the first layer, or can form a reference electrode and a counter electrode on the second layer, and the ultramicro electrode arrays A and B can share the reference electrode and the counter electrode. Under the condition of ensuring a good wire, the double-layer wiring facilitates the formation of denser ultra-micro electrode sites on a limited plane, which is conducive to capturing the quantum release of cell synaptic sites.
步骤6、分区结构：采用PDMS、聚酰亚胺、SU-8等形成分区结构，不同结构间用高度为2μm～2000μm的聚合物材料形成隔离坝，将多个电极位点分隔成多个区域，便于不同修饰和多种神经递质检测，在各个区域进行不同特异性修饰和多种神经递质检测。聚合物隔离材料选用PDMS、聚酰亚胺、SU-8其中的一种或两种的组合结构。Step 6. Partition structure: Use PDMS, polyimide, SU-8, etc. to form a partition structure, and use polymer materials with a height of 2 μm to 2000 μm to form isolation dams between different structures to separate multiple electrode sites into multiple regions , to facilitate the detection of different modifications and multiple neurotransmitters, and perform different specific modifications and multiple neurotransmitter detections in various regions. The polymer isolation material is selected from PDMS, polyimide, SU-8, or a combination of two of them.
步骤6、在电极表面修饰形成复合功能膜层，其修饰按以下先后顺序进行：纳米修饰、生物特异层修饰、生物兼容修饰，对于不同区域可以根据需要修饰上述三层中的一层、两层或三层。Step 6. Modify the surface of the electrode to form a composite functional film layer. The modification is carried out in the following order: nano-modification, bio-specific layer modification, and bio-compatible modification. For different regions, one or two layers of the above three layers can be modified as required or three layers.
纳米修饰层：不同分区固定纳米材料，用以增加电极性能。纳米修饰层采用金属或其它优良导电物质进行定点修饰。修饰金属材料包括铂、金、钛、铑、锇等，其中优选铂黑、氮化钛。其它优良导电物质包括碳、聚吡咯等，优选碳纳米材料。为了获得高效电化学活性的电极表面用等离子清洗机对电极阵列进行活化处理。电极经过表面活化处理后，不仅可以去除电极表面的污染物，同时还能起到活化电极表面的作用，增加电极表面的亲水性，刻蚀的条件可根据电极表面的情况做适当的调整。纳米修饰层定点修饰可采用电镀、电聚合、共聚合和微涂敷等。Nano-modified layer: Different partitions fix nano-materials to increase electrode performance. The nano-modified layer uses metal or other excellent conductive substances for fixed-point modification. Modified metal materials include platinum, gold, titanium, rhodium, osmium, etc., among which platinum black and titanium nitride are preferred. Other excellent conductive substances include carbon, polypyrrole, etc., preferably carbon nanomaterials. In order to obtain high-efficiency electrochemically active electrode surfaces, the electrode arrays were activated with a plasma cleaner. After the electrode is surface activated, it can not only remove the pollutants on the electrode surface, but also activate the surface of the electrode and increase the hydrophilicity of the electrode surface. The etching conditions can be adjusted according to the condition of the electrode surface. Electroplating, electropolymerization, copolymerization and micro-coating can be used for the fixed-point modification of the nano-modified layer.
特异选择反应修饰层：固定不同的特异性材料(如结构选择性修饰材料、酶等生物特异性修饰材料等)，用于对应检测不同的神经递质。修饰组装成为超微平面阵列传感器。特异选择反应修饰层包括酶或抗体或抗原或高分子定向选择试剂和电子受体、辅助试剂。酶试剂包括氧化酶、还原酶等；抗体包括单抗、多抗等；电子受体包括电子接受体由从铁氰酸盐、亚甲基蓝、二茂铁及其衍生物和铑／锇离子聚合物组成的组中选择出的一种；辅助试剂是由耦联试剂、酶／抗体激活剂、缓冲液和表面活性剂等组成。Specific selection reaction modification layer: immobilize different specific materials (such as structure selective modification materials, enzymes and other biological specific modification materials, etc.), for corresponding detection of different neurotransmitters. Modified and assembled into ultra-microplanar array sensors. The specific selection reaction modification layer includes enzymes or antibodies or antigens or macromolecule directional selection reagents, electron acceptors and auxiliary reagents. Enzyme reagents include oxidase, reductase, etc.; antibodies include monoclonal antibody, polyclonal antibody, etc.; electron acceptors include electron acceptors composed of ferricyanate, methylene blue, ferrocene and its derivatives, and rhodium/osmium ion polymers One selected from the group; auxiliary reagents are composed of coupling reagents, enzyme/antibody activators, buffers and surfactants.
生物兼容性选择性修饰，包括超微电极位点区域局部亲和修饰，采用聚赖氨酸(polylysine)、巯基丙酸、聚乙烯亚胺(PEI)、昆布氨酸(laminin)中的一种或组合。引线区域采用PEG、Teflon等进行排斥蛋白物质的修饰。其中，纳米修饰层、特异选择反应修饰层、生物兼容性修饰层是分先后顺序修饰的。可以在同一区域先后修饰，也可以在不同区域分别修饰。Selective modification of biocompatibility, including local affinity modification of ultramicroelectrode sites, using one of polylysine, mercaptopropionic acid, polyethyleneimine (PEI), and laminin or a combination. The lead area is modified with PEG, Teflon, etc. to repel protein substances. Among them, the nano-modified layer, the specific selective reaction modified layer, and the biocompatible modified layer are modified sequentially. Modifications can be done sequentially in the same area, or separately in different areas.
本发明实施例提供了一种在电极表面进行纳米修饰层的方法。该实施例中，通过在电极表面组装一层纳米颗粒进行纳米修饰。具体的，通过在电极表面电镀铂黑或电聚合聚吡咯，可获得具有不同孔径的电极纳米层。该方法进一步加强了电极表面的亲水性，增大了电极的有效表面积。不仅有利于酶试剂的固定化，而且增加了电极的电化学活性。The embodiment of the present invention provides a method for forming a nano-modified layer on the surface of an electrode. In this embodiment, nano-modification is performed by assembling a layer of nanoparticles on the electrode surface. Specifically, electrode nanolayers with different pore sizes can be obtained by electroplating platinum black or electropolymerized polypyrrole on the electrode surface. The method further strengthens the hydrophilicity of the electrode surface and increases the effective surface area of the electrode. It is not only beneficial to the immobilization of enzyme reagents, but also increases the electrochemical activity of the electrode.
本发明将反应试剂固定在电极纳米层上形成试剂层，固定在电极纳米层上的反应试剂包括电子接受体，与被分析物起反应且能产生与被分析物浓度相对应的电流的耦联反应的组合试剂，同时还包括电子媒介体、酶试剂、缓冲液和表面活性剂等。In the present invention, the reaction reagent is fixed on the electrode nano layer to form a reagent layer, and the reaction reagent fixed on the electrode nano layer includes an electron acceptor, which reacts with the analyte and can generate a coupling current corresponding to the concentration of the analyte. The combined reagents for the reaction also include electron mediators, enzyme reagents, buffers, and surfactants.
本发明试剂组合所依据的反应原理如下：The reaction principle on which the reagent combination of the present invention is based is as follows:
其中Ach乙酰胆碱；AchE乙酰胆碱脂解酶；acetate乳酸；choline胆碱；ChOx胆碱氧化酶，betaine aldehyde三甲胺乙醛。Among them, Ach acetylcholine; AchE acetylcholine lipase; acetate lactic acid; choline choline; ChOx choline oxidase, betaine aldehyde trimethylamine acetaldehyde.
本发明的实施例，提供了在纳米颗粒上固定反应试剂的方法。An embodiment of the present invention provides a method for immobilizing reaction reagents on nanoparticles.
首先在电极表面修饰锇聚合物导电媒介体聚合物，常温放置12小时以上。然后将适当浓度的乙酰胆碱氧化酶、乙酰胆碱脂解酶、戊二醛和牛血清白蛋白等混合后，立即涂覆在电极表面，在35～37℃干燥箱中干燥20～25分钟后，去离子水冲洗去除未交联的戊二醛，常温干燥后，密封低温储藏。First, modify the osmium polymer conductive medium polymer on the surface of the electrode, and place it at room temperature for more than 12 hours. Then mix appropriate concentrations of acetylcholine oxidase, acetylcholine lipase, glutaraldehyde and bovine serum albumin, etc., and immediately coat the surface of the electrode, dry in a 35-37°C drying oven for 20-25 minutes, and then deionized water Rinse to remove uncrosslinked glutaraldehyde, dry at room temperature, seal and store at low temperature.
起氧化还原作用的电子接受体由从氧化还原聚合物(Os、铑等)、铁氰酸盐、亚甲基蓝、二茂铁及其衍生物、对笨醌、吩嗪硫酸甲酯、靛酚及其衍生物和β-萘醌-4-磺酸钾组成的组中选择出的一种；通过电子介体将酶反应过程中产生的电子从酶反应中心转移到电极表面，使得电流型酶传感器的响应速度和检测灵敏度得到了提高，同时降低了反应的电压。所述电子接受体为Os氧化还原聚合物，可有效降低检测限，使工作电位降至0V左右，减少血液中其它活性物质的干扰。The electron acceptor that plays a redox role is composed of redox polymers (Os, rhodium, etc.), ferricyanate, methylene blue, ferrocene and its derivatives, p-benzoquinone, phenazine methyl sulfate, indophenol and its derivatives. One selected from the group consisting of derivatives and potassium β-naphthoquinone-4-sulfonate; the electrons generated during the enzyme reaction are transferred from the enzyme reaction center to the electrode surface through the electron mediator, so that the current-type enzyme sensor Response speed and detection sensitivity are improved while reducing the voltage of the reaction. The electron acceptor is an Os redox polymer, which can effectively reduce the detection limit, reduce the working potential to about 0V, and reduce the interference of other active substances in the blood.
缓冲液为磷酸缓冲液、TRIS缓冲液、MES缓冲液和生理盐水组成的组中选择出的一种；所述缓冲液为磷酸缓冲液。缓冲液用于提供一个pH值稳定的反应环境，最佳pH反应为6～8。The buffer is one selected from the group consisting of phosphate buffer, TRIS buffer, MES buffer and physiological saline; the buffer is phosphate buffer. The buffer is used to provide a reaction environment with a stable pH value, and the optimal pH response is 6-8.
所述表面活性剂为TritonX-100。添加0.01～1％非离子型的表面活性剂，提高了混合液与试条的亲和率，使混合试剂更容易迅速均匀涂覆在电极表面，形成的涂覆层薄而均匀，利于提高检测时电子传递速率。当表面活性剂浓度高于0.5％时，酶活性受到抑制和影响。因此选择0.01～0.1％浓度表面活性剂。The surfactant is TritonX-100. Adding 0.01-1% non-ionic surfactant improves the affinity between the mixed solution and the test strip, making it easier for the mixed reagent to be quickly and evenly coated on the electrode surface, forming a thin and uniform coating layer, which is beneficial to improve detection electron transfer rate. When the surfactant concentration is higher than 0.5%, the enzyme activity is inhibited and affected. Therefore, a surfactant with a concentration of 0.01 to 0.1% is selected.
本发明的上述各种目的、方法、特点和优点，通过下面结合附图和实施例可以得到更加详细的说明。The various purposes, methods, features and advantages of the present invention can be described in more detail below in conjunction with the accompanying drawings and embodiments.
图1、图2是本发明设计的超微电极传感器的制备工艺流程图和局部平面放大图。首先清洗绝缘基材玻璃片(厚度约1mm)，先后在玻璃上涂覆光刻胶(正胶6130或2840或AZ1500)，普通光刻显影形成电极和引线触点图形，然后溅射贵金属电极薄膜Ti基底层(厚20～30nm)和Pt(200～300nm)，剥离光刻胶，形成基础电极阵列；PECVD沉积绝缘层Si3N4(300～500nm)；采用步进光刻工艺在基础电极阵列表面的第一区域进行二次光刻(采用双层布线的第一层版图进行)，光刻胶上形成所需的超微电极阵列图形；采用反应离子刻蚀工艺进行刻蚀超微电极阵列图形去除绝缘层，在绝缘层和电极材料薄膜上形成超微电极阵列图形A和触点(15个×4行电极阵列：电极位点直径分别为2μm、3μm、4μm、5μm(如图3所示)；其中越小的电极越利于囊泡释放检测)；在基础电极阵列表面的第二区域重复光刻、贵金属溅射、覆盖绝缘层、采用双层布线的第二层版图进行二次光刻以及等离子刻蚀步骤，制备出超微电极阵列B、对电极、参比电极及触点B(15个×4行电极阵列：电极位点直径分别为2μm、3μm、4μm、5μm(如图3所示))。旋涂SU-8胶，普通光刻显影，形成分割区域(10～20μm高度分割坝)的电极阵列。Fig. 1 and Fig. 2 are the preparation process flow chart and partial plane enlarged view of the ultra-micro electrode sensor designed by the present invention. First, clean the insulating substrate glass sheet (thickness is about 1mm), and then apply photoresist (positive resist 6130 or 2840 or AZ1500) on the glass successively, and develop with ordinary photolithography to form electrode and lead contact patterns, and then sputter noble metal electrode film Ti base layer (thickness 20-30nm) and Pt (200-300nm), peel off the photoresist to form the basic electrode array; PECVD deposits the insulating layer Si3N4 (300-500nm); use stepping photolithography process on the surface of the basic electrode array The second photolithography is carried out in the first area (using the first layer layout of double-layer wiring), and the required ultra-micro electrode array pattern is formed on the photoresist; the ultra-micro electrode array pattern is etched and removed by reactive ion etching process Insulating layer, forming ultra-micro electrode array pattern A and contacts on the insulating layer and electrode material film (15 x 4 rows of electrode arrays: electrode site diameters are 2 μm, 3 μm, 4 μm, 5 μm (as shown in Figure 3) ; wherein the smaller electrode is more conducive to vesicle release detection); repeat photolithography, noble metal sputtering, covering insulating layer, second layer layout using double-layer wiring for secondary photolithography on the second area of the basic electrode array surface and In the plasma etching step, the ultramicro electrode array B, the counter electrode, the reference electrode and the contact B (15 × 4 rows of electrode arrays: the diameters of the electrode sites are 2 μm, 3 μm, 4 μm, and 5 μm respectively (as shown in Figure 3 Show)). Spin-coat SU-8 glue, develop with ordinary photolithography, and form an electrode array of segmented regions (10-20 μm height segmented dams).
超微电极阵列通过电镀形成纳米层(如图4所示)。电镀液为0.1M的吡咯，0.1M的KCl，采用0.5～0.7V的电镀工作电位进行恒压电镀，或者采用0.01～0.05mA的工作电流进行恒流电镀，电镀时间为30秒～3分钟，也可以采用-0.1V～0.8V进行循环伏安电镀，扫描20～40圈。也可以选择Au、Pt进行纳米金属材料的电镀。然后修饰酶层，例如乙酰胆碱检测区域：乙酰胆碱脂解酶100U／ml；胆碱氧化酶：100U／ml；Triton X-100：0.01％，戊二醛：0.1％，牛血清白蛋白：5％。多巴胺检测区域则修饰0.1～1％Nafion，干燥形成Nafion膜。不同递质检测区修饰不同的酶或特异检测物质。修饰完成后，密封低温保存。使用前，首先进行紫外灭菌，然后在电极位点区进行生物兼容性物质的选择性修饰，选用聚赖氨酸进行涂敷或模印。然后再接种神经细胞进行培养。The ultramicroelectrode array forms a nanometer layer by electroplating (as shown in Figure 4). The electroplating solution is 0.1M pyrrole and 0.1M KCl, and the electroplating working potential of 0.5-0.7V is used for constant-voltage electroplating, or the working current of 0.01-0.05mA is used for constant-current electroplating, and the electroplating time is 30 seconds to 3 minutes. You can also use -0.1V～0.8V for cyclic voltammetry plating, and scan 20～40 circles. Au and Pt can also be selected for electroplating of nano metal materials. Then modify the enzyme layer, such as acetylcholine detection area: acetylcholine lipase 100U/ml; choline oxidase: 100U/ml; Triton X-100: 0.01%, glutaraldehyde: 0.1%, bovine serum albumin: 5%. The dopamine detection area was modified with 0.1-1% Nafion, and dried to form a Nafion film. Different transmitter detection regions modify different enzymes or specific detection substances. After the modification is completed, it is sealed and stored at low temperature. Before use, ultraviolet sterilization is carried out first, and then selective modification of biocompatible substances is carried out in the electrode site area, and polylysine is selected for coating or stamping. Then inoculate neurons for culture.
本发明用了优选实施例进行说明，优选实施例只是为了说明的目的，而不是对本发明的限制。在上述说明的基础上可以对本发明作许多改进和改变。因此，在所附权利要求书的范围内，本发明可以有不是上述的其它实现方式。例如：电极形状的不同、其它纳米颗粒和非纳米材料修饰的电极反应区、不同的试剂组合形式等。The present invention is described using preferred embodiments, which are for the purpose of illustration only, not limitations of the present invention. Many modifications and variations of the present invention are possible on the basis of the above description. Therefore, within the scope of the appended claims, the invention may be practiced in other ways than those described above. For example: different electrode shapes, electrode reaction areas modified by other nanoparticles and non-nanomaterials, different reagent combinations, etc.
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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