CN103558368B - A kind of biological cell feature measurement nano electrode array structure and manufacture method thereof - Google Patents
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Abstract
本发明涉及一种生物细胞特性测量纳米电极阵列结构及其制造方法，该电极阵列主要用于细胞电特性测量。克服现有技术电极尺度较大的不足，提出一种纳米电极阵列方案，并利用激光干涉光刻技术方法通过直接或间接制造，提供了该纳米电极阵列的制造方法。以及利用该纳米电极阵列测量细胞电信号特性的方法，该纳米电极阵列可获得在亚细胞水平上揭示细胞细节信号的变化，有利于对细胞生物特性进行更好的研究，获得比以往更精细的结果，提高细胞电信号测量分辨率。同时本发明纳米电极阵列制造方法性能可靠，具有制造成本较低的优势。
The invention relates to a nano-electrode array structure for measuring biological cell characteristics and a manufacturing method thereof. The electrode array is mainly used for measuring cell electrical characteristics. To overcome the disadvantages of relatively large electrodes in the prior art, a nano-electrode array scheme is proposed, and a method for manufacturing the nano-electrode array is provided through direct or indirect manufacturing using laser interference lithography. As well as the method of using the nano-electrode array to measure the characteristics of cell electrical signals, the nano-electrode array can reveal the changes of cell details at the subcellular level, which is conducive to better research on the biological characteristics of cells and obtains finer than before. As a result, cell electrical signal measurement resolution is improved. At the same time, the nanometer electrode array manufacturing method of the present invention has reliable performance and has the advantage of lower manufacturing cost.
Description
Technical field
The present invention relates to a kind of biological cell feature measurement nano electrode array structure and manufacture method thereof, this electrod-array is mainly used in cell electrical characteristics and measures.
Background technology
Biomedicine, the particularly the most stem-winding new development of Neurobiology are single channel recording or Patch-clamp techniques.This technology utilizes glass micro-attraction electrode that area is only several mm
2cell patch seal up, to 10
-12a level, records the movable electric current of single or several passage, thus epoch-making molecular level electrophysiology technology being brought up to record and research single protein.Inventor Neher and Sakman of this technology wins Nobel prize's soul in 1991 for this reason.The discovery of patch clamp technique, people on a molecular scale for single channel-active provides many information, thus by the understanding of people to channel-active, can directly bring up to molecular level.Recently only in diaphragm record glass-tube, put into ATP class material causes pierceability diaphragm technology, also can record the voltage of cell, electric current and electric capacity, and without the need to transfixation cell membrane.
In recent years, adopt advanced microelectronic integrated circuit manufacturing technology, be that many measuring point microelectrode array technology of material develops rapidly with semiconductor silicon, success ratio and the repeated problem of product obtain good solution.This electrode has the features such as volume is little, measuring point is many, version is diversified, stable and reliable for performance.Nowadays, commercialization the semiconductor integrated silicon microelectrode array (Siliconmicroelectrodearray be widely used, SMEA) two large classes are mainly contained, the aciculiform microelectrode array (Needlemicroelectrodearray) of one Lei Shi Univ Utah USA exploitation, is called as Utah electrode or plane electrode; The another kind of linear microelectrode array (Linearsiliconmicroelectrodearray, LMSEA) being Univ Michigan-Ann Arbor USA and developing, is called as Michigan electrode or lineation electrode.Utah electrode monoblock silicon wafer to manufacture, does substrate with N-type silicon, and form multiple P-type silicon passage with thermomigration wherein, be penetrated into another side from the one side of substrate, these P-type silicon passages are insulated from each other; Remove unnecessary N-type silicon, only stay skim to be wrapped in after around P-type silicon, form multiple fine needle, produce square electrode pin array.The signals collecting position at electrode needle tip is platinum coating, and its length is about 35-75 μm, and most advanced and sophisticated exposed area is about 0.005mm
2.Michigan electrodes belongs to film microelectrode (thin-filmmicroelectrode), similar to IC manufacturing, adopting traditional microelectronic manufacturing technology, is on the thin slice of substrate at silicon or stupalith, according to the electrode circuit designed, conducting metal in spraying plating; Or be coated with in the printed board of conductive metal layer whole, unwanted part is removed in etching, and leave the electrode circuit of needs, conducting metal can be nickel, stainless steel, tungsten, gold or platinum.Then, except measuring point, the conducting wire of all the other linkage record points and output terminal covers insulation course, and conventional insulating material is silicon nitride.In order to strengthen electric conductivity and biocompatibility, iridium or gold on measuring point plated surface.The specification of Michigan electrod-array is a lot, measuring point contact area has the specification to thousands of square micron up to a hundred, on same root record bar, measuring point number has 4-16 (single-row), even 64 (multiple row), and the spacing between measuring point has 25-200 μm; On same electrode, recording bar number has 1-8 root, and the spacing between recording bar has 125-500 μm of .(to seal continent swallow, 2009)
Along with the development of nanometer technology, development in recent years has gone out new nano-electrode array.Electrode dimensions can be accomplished less, more crypto set.Nano-electrode array is as a kind of nanostucture system of man-made assembly, there is high transfer rate, low electrostatic double layer charging current, small time constant, the advantage such as little IR falls and high s/n ratio, workable and measurement sensistivity be high, thus have broad application prospects in electrochemical theory research, biology sensor, electrocatalysis material and high energy chemisty power electrode material etc.At present, people adopt multiple material design to prepare the nano-electrode array comprising discoid, cylindrical, spherical, conical, the slotting various shape such as finger-like and well shape, its method for making mainly comprises template, etching method and self-assembly method etc., and the sign of electrode mainly adopts electron microscope technique and electrochemical method.The electrode of the method processing of traditional machining and P.e.c. is difficult to accomplish hundreds of nanometer or even tens nanometers, common commercial microelectrode array is mostly at micro-meter scale, nano-electrode array of the present invention utilizes laser interference photolithography technology, the striated pattern ablation machined material surface produced by double laser beams exposure, thus generation litho pattern, form raster-like nano-electrode array.
Laser interference manufacturing technology is the interference and diffraction characteristic utilizing light, by specific light beam array mode, regulate and control the light intensity distributions in interference field, directly can be processed to form litho pattern to material surface, or utilize photosensitive material to carry out record, carry out development exposure again, chemistry or physical method etching, produce litho pattern at material surface.Because laser interference photolithography technology does not need expensive projection optical system, the area of exposure field is only limited to the clear aperature of system, therefore it is particularly suitable for some needs and produces figure in wide area, and because its large and thin size causes the device manufacture that the flatness of substrate surface cannot be precisely controlled, be emerging a kind of means of photolithography, and have and reach the high-resolution potentiality in about λ/10.There is very strong comparative benefits in acquisition micro/nano-scale periodic surface configuration aspects, have broad application prospects.
Summary of the invention
The technology of the present invention is dealt with problems: overcome the deficiencies in the prior art, reduce electrode dimensions, improve cell electrical characteristics Measurement Resolution, obtain the change disclosing cell detail signal on subcellsular level, a kind of nano-electrode array and manufacture method thereof are provided, the electrode dimensions obtained is less, more intensive, and resolution improves; And simple and reliable, have cost advantage, yardstick is convenient to be controlled.
The technology of the present invention solution: a kind of biological cell feature measurement nano electrode array structure, comprising: substrate and the periodicity nano-electrode array etched on substrate; Described periodicity nano-electrode array is one group or two groups of independence wiregratings, and often organizing wiregrating width is 20nm-500nm, and be highly 5nm-200nm, the cycle is 200nm-1000nm, and described two groups of wiregratings are at a distance of 200nm-500nm.
Described often group wiregrating width is 20nm-500nm, and be highly 20nm-200nm, the cycle is 200nm-1000nm.Described two groups of wiregratings are at a distance of 200nm-500nm, and this scale electrode array can effectively detect unicellular electric signal, cell local can be caused to change to medicine etc. and analyze.And specific cells region operated and encourages, monitor the stress reaction of cell simultaneously.
Described substrate is silicon, glass, quartz or PMMA material.
A manufacture method for biological cell feature measurement nano electrode array structure, its feature is: even coated layer of conductive film on substrate, recycling interference lithography technique, and etch conductive film, prepare nano-electrode array, concrete steps are as follows:
Step 1: conductive material painting is invested on baseplate material by physical method, form uniform monolayers dispensing of conductive film, described film thickness is 20nm-200nm;
Step 2: utilize interference lithography technique to etch conductive film, baseplate material is formed nano-electrode array.
Physical method described in described step 1, comprises spin coating, plating or sputtering, forms uniform monolayers dispensing of conductive film.
Conductive material described in described step 1, comprises the metal of good conductivity of ITO or Ag, Au, Pt.
Interference lithography technique described in described step 2, comprises and utilizes interference lithography system to produce nanostructured optical field distribution, directly process or indirectly process conductive film.
Described direct processing utilizes interference lithography system to produce nanostructured optical field distribution, directly carries out etching form corresponding periodically nano-electrode array to conductive film.
Described indirect processing utilizes interference lithography system to produce nanostructured optical field distribution, first photoresists are exposed, form periodic nano-structure graphic mask, the method for recycling chemical etching or reactive ion etching, periodically nano-electrode array is formed to conductive film etching.
The present invention's beneficial effect is compared with prior art:
(1) utilize laser interference photolithography technology to realize nanoscale grating electrode array, comparing classic method has machining precision high with other manufacturing technology methods, and manufacture method is convenient, is easy to realize large area manufacture, the advantage that manufacturing system is simple and reliable.Adopt the method that microprobe detects, avoid traditional electrode array cannot carry out signal extraction difficulty at nanoscale, the present invention can obtain cell each local electric signal distributions feature, be conducive to better studying cell biological characteristic, obtain result meticulousr than ever, electrode dimensions is less, more intensive, improves cell electric signal measurement resolution;
(2) this manufacture method and system simple and reliable, have low-cost advantage, yardstick is convenient to be controlled;
(3) contact conductor of the present invention is the difficult point limiting traditional microelectrode array processing and manufacturing, and the yardstick limiting electrod-array reduces, and this nano-electrode array does not need to draw signal wire, utilizes Single probe or double probe system to carry out signal-obtaining.
Accompanying drawing explanation
Fig. 1 is a nanometer grating electrode structure schematic diagram;
Fig. 2 is a kind of nanometer grating electrode group structure schematic diagram;
In Fig. 1 and Fig. 2, be 1. nano-electrode array, 2. electrode array substrate.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further described.
Technical solution of the present invention, can adopt direct method and indirect method to realize.Adopt direct method first to even coated layer of conductive film on glass, quartz or PMMA material substrate, then laser interference lithography manufacturing system is utilized, control the laser wavelength of incidence of laser interference lithography, the incident angle of laser beam in laser interference lithographic system, forms periodic optical grating periodically optical field distribution.Control interference optical field energy, direct etching is carried out to conductive film, form nano-electrode array as shown in Figure 1.Periodically the wiregrating width of nano-electrode array is 20nm-500nm, and be highly 20nm-200nm, the cycle is 200nm-1000nm.Also twice etching can be adopted to obtain group independent lines gate electrode array of two shown in Fig. 2, first control laser interference lithography manufacturing system to etch electrode half region, form nanometer grating electrode as shown in figure, then precision displacement table control electrode is utilized to be displaced to other half region, shift length can be 200nm-500nm, etching formation second group of nanometer grating electrod-array, as shown in Figure 2.Adopt indirect method, to after even coated layer of conductive film on glass, quartz or PMMA material substrate, spin coating one deck photoresist again, utilize laser interference lithography manufacturing system, control the laser wavelength of incidence of laser interference lithography, the incident angle of laser beam in laser interference lithographic system, the formation cycle is the grating periodic optical field distribution of 200nm-1000nm.Control interference optical field energy, photoresist is exposed, form nano-electrode array photoetching offset plate figure as shown in Figure 1.The method of the physical etchings such as the method for recycling chemical corrosion or ICP is carried out etching to conductive film and is formed periodically nano-electrode array, and its wiregrating width is 20nm-500nm, is highly 20nm-200nm, and the cycle is 200nm-1000nm.
Laser interference manufacturing system comprises high power laser system, laser beam beam splitting and closes beam control light path, accurate sample displacement control system composition.High power laser system is mainly for generation of the laser pulse of specific wavelength, high monochromaticity, high-energy-density; Laser beam beam splitting and and beam control light path be mainly used in that beam splitting is carried out to laser beam and form required interfering beam, then control each road interfering beam and carry out conjunction bundle with specific polarization state, incident angle and Space Angle at sample surfaces to be processed, directly form periodically interference optical field, directly litho pattern is processed to form to material surface, or utilize photosensitive material to carry out record, carry out development exposure again, chemistry or physical method etching, produce litho pattern at material surface.
This nanometer grating array can coordinate two probe micro-nano control system to measure cell electric signal.Method is as follows, cell is placed on electrode, usual cell dimensions is all several micro-to some tens of pm magnitude, cell can contact with tens of grating electrodes simultaneously, the probe controlling double probe system is connected with the electrode of cells contacting, another probe is connected with another electrode of cells contacting, flow through cell membrane and produce transmembrane current formation loop, the electric signal measurement system of two probe micro-nano control system now can be utilized to measure cell electric signal, then respectively electric signal measurement is carried out to the electrode of every bar and cells contacting successively, cell each local electric signal distributions feature can be obtained by scanning technique fast, be conducive to better studying cell biological characteristic, obtain result meticulousr than ever.
Shown in Fig. 1, in embodiment, first to even coated layer of conductive film on glass, quartz or PMMA material substrate, then laser interference lithography manufacturing system is utilized, control the laser wavelength of incidence 266nm of laser interference lithography, the incident angle 30 ° of laser beam in laser interference lithographic system, the formation cycle is the grating periodic optical field distribution of 266nm.Control interference optical field energy, direct etching is carried out to conductive film, form nano-electrode array as shown in Figure 1.Periodically the wiregrating width of nano-electrode array is 20nm-100nm, and be highly 20nm-200nm, the cycle is 266nm.
In the embodiment depicted in figure 2, two groups of independent lines gate electrode arrays can adopt twice etching to obtain, control laser interference lithography manufacturing system to etch electrode half region, form nanometer grating electrode as shown in figure, then precision displacement table control electrode is utilized to be displaced to other half region, shift length can be 200nm-500nm, etching formation second group of nanometer grating electrod-array.
Shown in Fig. 1, in embodiment, first to even coated layer of conductive film on glass, quartz or PMMA material substrate, spin coating one deck photoresist again, adopt indirect mode, utilize laser interference lithography manufacturing system, control the laser wavelength of incidence 355nm of laser interference lithography, the incident angle 15 ° of laser beam in laser interference lithographic system, the formation cycle is the grating periodic optical field distribution of 686nm.Control interference optical field energy, photoresist is exposed, form nano-electrode array photoetching offset plate figure as shown in Figure 1.The method of the physical etchings such as the method for recycling chemical corrosion or ICP is carried out etching to conductive film and is formed periodically nano-electrode array, and its wiregrating width is 20nm-300nm, is highly 20nm-200nm, and the cycle is 686nm.Applicable nano-electrode array dimension can be selected for cell dimensions and surface appearance feature, be conducive to obtaining best test signal.
Non-elaborated part of the present invention belongs to techniques well known.
The above; be only part embodiment of the present invention, but protection scope of the present invention is not limited thereto, any those skilled in the art are in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.
Claims (8)
1. a biological cell feature measurement nano electrode array structure, is characterized in that: the periodicity nano-electrode array comprising substrate and etch on substrate; Described periodicity nano-electrode array is one group or two groups of independence wiregratings, often organizing wiregrating width is 20nm-300nm, be highly 20nm-200nm, cycle is 686nm, or, often organizing wiregrating width is 20nm-300nm, be highly 20nm-200nm, cycle is 266nm, described two groups of wiregratings are at a distance of 200nm-500nm, and this scale electrode array can effectively detect unicellular electric signal, cause cell local to change analyze medicine etc., and cell specific region operated and encourages, monitor the stress reaction of cell simultaneously.
2. biological cell feature measurement nano electrode array structure according to claim 1, is characterized in that: described substrate is silicon, glass, quartz or PMMA material.
3. a manufacture method for biological cell feature measurement nano electrode array structure, is characterized in that: even coated layer of conductive film on substrate, recycling interference lithography technique, and etch conductive film, prepare nano-electrode array, concrete steps are as follows:
Step 1: conductive material painting is invested on baseplate material by physical method, form uniform monolayers dispensing of conductive film, described film thickness is 20nm-200nm;
Step 2: utilize interference lithography technique to etch conductive film, baseplate material is formed periodically nano-electrode array; Described periodicity nano-electrode array is one group or two groups of independence wiregratings, often organizing wiregrating width is 20nm-300nm, be highly 20nm-200nm, cycle is 686nm, or often organizing wiregrating width is 20nm-300nm, is highly 20nm-200nm, cycle is 266nm, and described two groups of wiregratings are at a distance of 200nm-500nm.
4. the manufacture method of a kind of biological cell feature measurement nano electrode array structure according to claim 3, is characterized in that: physical method described in described step 1, comprises spin coating, plating or sputtering, forms uniform monolayers dispensing of conductive film.
5. the manufacture method of a kind of biological cell feature measurement nano electrode array structure according to claim 3, is characterized in that: conductive material described in described step 1, comprises the metal of ITO or Ag, Au, Pt good conductivity.
6. the manufacture method of a kind of biological cell feature measurement nano electrode array structure according to claim 5, it is characterized in that: interference lithography technique described in described step 2, comprise and utilize interference lithography system to produce nanostructured optical field distribution, conductive film is directly processed or indirectly processes.
7. the manufacture method of a kind of biological cell feature measurement nano electrode array structure according to claim 6, it is characterized in that: described direct processing utilizes interference lithography system to produce nanostructured optical field distribution, direct etching is carried out to conductive film and forms corresponding periodically nano-electrode array.
8. the manufacture method of a kind of biological cell feature measurement nano electrode array structure according to claim 6, it is characterized in that: described indirect processing utilizes interference lithography system to produce nanostructured optical field distribution, first photoresists are exposed, form periodic nano-structure graphic mask, the method of recycling chemical etching or reactive ion etching, forms periodically nano-electrode array to conductive film etching.
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| 纳米阵列电极研究;曹立新等;《化学进展》;20080930;第20卷(第9期);正文2.2.2节 * |
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