CN1242260C - Single cell sensor for ectocytic action potential detection and its preparing method - Google Patents
Single cell sensor for ectocytic action potential detection and its preparing method Download PDFInfo
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- CN1242260C CN1242260C CN 200310122949 CN200310122949A CN1242260C CN 1242260 C CN1242260 C CN 1242260C CN 200310122949 CN200310122949 CN 200310122949 CN 200310122949 A CN200310122949 A CN 200310122949A CN 1242260 C CN1242260 C CN 1242260C
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Abstract
The present invention discloses a single cell sensor used for measuring ectocytic action potential of cells and a preparation method thereof. On the basis of an early multiple light source cell micro physiological gauge, a technology of light addressing potentiometric sensor (LAPS) is utilized to overcome the limit of geometric characteristics of the sensor on cell culture, and light source moving addressing is adopted to track and position randomly cultured single cells. A light source system of the present invention is also improved, precision and stability are enhanced, the measurement can be positioned on single cells or even single ion channels, and thus, the action of drugs on single cells and action target spots are exactly measured. The single cell sensor can realize quick and real-time monitoring on outer action potential of neural cells, qualitative analysis of exciting and inhibiting action of difference drugs on difference concentration can be realized, and the single cell sensor is mainly used for drug screening, analysis and evaluation.
Description
Technical field
The present invention relates to a kind of single-cell sensor that is used for the outer action potential measurement of cell born of the same parents and preparation method thereof.
Background technology
U.S. molecular device company in 1992 proposes the Light Addressable Potentiometric Sensor of living cells with EIS (electrolyte/insulation course/silicon) structure combined first, constitute responsive testing tool, detect the variation of the outer microenvironment potential of hydrogen of born of the same parents, on this basis, there is the researchist to improve sensor construction and data processing method again, makes such sensor can detect the variation of several ions simultaneously.But, what these cell sensors detected all is the metabolic product of cell mass, so be difficult to determine the mechanism of action of medicine to individual cells, simultaneously, also be difficult to measurement is accurately navigated to ion channel, can't determine the action target spot of medicine pair cell, be a unavoidable difficult point for the quantification that realizes drug screening.
Summary of the invention
The object of the present invention is to provide a kind of single-cell sensor that is used for action potentials of cells detection and Pharmaceutical Analysis measurement and preparation method thereof, can carry out qualitative and detection by quantitative the outer action potential of individual cells.
In order to achieve the above object, the technical solution used in the present invention is as follows:
1, system architecture of the present invention:
Fixing copper clad plate on aluminium sheet adheres on the copper clad plate and has SiO
2The Si sheet of the polishing of layer is drawn working electrode by Ohmic contact from copper clad plate, wherein, and the SiO of Si sheet
2Laminar surface is made the coating of enhancing attaching property or is changed its surperficial roughness by photoetching technique with coating process on its surface, described coating process process is as follows: the phosphate buffer of 100 μ g/ml poly-ornithines and the phosphate buffer of 8 μ g/ml laminins were mixed by 1: 1, miillpore filter bacteriological filtration with 0.22 μ m will be stained with SiO
2The copper clad plate of the Si sheet of the polishing of layer immerses in this mixed solution, and 37 ℃, 5%CO
2Under the environment, soaked 24 hours, with distilled water flushing twice, freezing standby again; Change the SiO of Si sheet
2The process of the roughness of laminar surface is as follows: be 2 * 2mm with photoetching technique on its surface
2The microcavity figure, change surfaceness, and then carry out the processing of coating, obtain being stained with the treated SiO in surface
2The copper clad plate of the Si sheet of the polishing of layer, the miniature measurement chamber that dimethyl silicone polymer is made is fixed on SiO
2On the layer, cover one deck bio-compatibility glue on the miniature measurement chamber, bonding with the optical glass of the gold-plated film reference electrode of lower surface, two opening parts on glass are connected the feed liquor emulsion tube and the fluid emulsion tube of medicine and nutrient solution respectively, and electrode is linked to each other with gold-plated film reference electrode.Said miniature measurement chamber is 1～2.
2, preparation method of the present invention:
(1) preparation of Light Addressable Potentiometric Sensor
Select for use＜100〉n type single crystalline Si sheets are as the substrate of Light Addressable Potentiometric Sensor, and the Si sheet is put into 1000 ℃ of high temperature furnaces and was carried out thermal oxide 20 minutes after polished and cleaned, makes the positive growth of Si sheet one layer thickness be about the SiO of 30nm
2Film, from the back side with the polishing of the thickness of Si sheet to 100 μ m, with conducting resinl the Si sheet is bonded on the copper clad plate then, 160 ℃ of hot settings 2 hours are drawn working electrode by Ohmic contact from copper clad plate;
(2) SiO of Si sheet
2Laminar surface is handled
The scheme of two kinds of cell fixation: coating process: promptly do the coating that strengthens attaching property on Si sheet surface; By changing the roughness on Si sheet surface, do the figure of inducing cell growth;
The coating process process is as follows: the phosphate buffer of 100 μ g/ml poly-ornithines and the phosphate buffer of 8 μ g/ml laminins were mixed by 1: 1, and the miillpore filter bacteriological filtration with 0.22 μ m will be stained with SiO
2The copper clad plate of the Si sheet of the polishing of layer immerses in this mixed solution, and 37 ℃, 5%CO
2Under the environment, soaked 24 hours, with distilled water flushing twice, freezing standby again;
The process of the roughness on change Si sheet surface is as follows: be 2 * 2mm with photoetching technique on Si sheet surface
2The microcavity figure, change the surfaceness of Si sheet, and then carry out the processing of coating;
(3) reference electrode preparation
Hot acetone cleans anti-reflection optical glass, and 80 ℃ of oven dry at glass surface, are made mask with coordinate paper, adopts magnetron sputtering technique to press the gold-plated film of mask shape at glass surface, and with conducting resinl that lead and golden film reference electrode is bonding, reference electrode is connected again with to electrode;
(4) settle miniature measurement chamber
With dimethyl silicone polymer preparation 0.2 * 5 * 5cm
3Membrane structure, carve two and measure chambeies last with mould, the chamber is of a size of 0.2 * 1.4 * 0.5cm
3, with bio-compatibility glue this structure is adhered to the Light Addressable Potentiometric Sensor surface, cultured cell in this chamber, during measurement, sticking above optical glass is loam cake, forms the miniature measurement chamber of sealing.
The advantage that the present invention has is:
This single-cell sensor has improved precision and degree of stability, and it is good that cell attaches upgrowth situation, and measurement and positioning in individual cells, can accurately be measured under the medicine irritation, and the outer action potential of single celled born of the same parents changes.The present invention has improved light source design, has improved precision and degree of stability, can be with measurement and positioning in individual cells even single ion channel, thus will accurately measure effect and the action target spot of medicine to individual cells.This single-cell sensor can be realized quick, the monitoring in real time of the outer action potential of neurocyte, and the excitement and the inhibiting effect of qualitative analysis variable concentrations different pharmaceutical pair cell are mainly used in drug screening, analysis and evaluation.
Description of drawings
The present invention is further illustrated below in conjunction with drawings and Examples.
Fig. 1 is the I-I directional profile structural drawing of single-cell sensor along Fig. 2;
Fig. 2 is the single-cell sensor vertical view;
Fig. 3 adopts coating process to improve the sensitivity of device;
Fig. 4 is a cell growth condition after the silicon device surface treatment;
Fig. 5 is the micro pattern that changes the silicon device surfaceness;
Fig. 6 is that single-cell sensor detects the instance system synoptic diagram;
Fig. 7 is the family curve synoptic diagram of n type silicon substrate LAPS;
When Fig. 8 is no medicine irritation, the baseline of single-cell sensor;
The response of single-cell sensor when Fig. 9 is the stimulation of low concentration acetylcholine;
Figure 10 is the response of middle concentration acetylcholine single-cell sensor when stimulating;
The response of single-cell sensor when Figure 11 is the stimulation of high concentration acetylcholine.
Among the figure, 1. aluminium sheet, 2. copper clad plate, 3. smooth addressable potentiometric sensor, the Si sheet after the 3.1. polishing is thin, 3.2.SiO
2Layer, 4. optical glass, 5. gold film reference electrode, 6. red laser, 7. dimethyl silicone polymer microlaser cavity, 8. bio-compatibility glue, 9. poly bird amino acid and laminin layer, 10. working electrode, 11. pairs of electrodes, 12. feed liquor emulsion tubes, 13. fluid emulsion tubes.
Embodiment
1, the detection architecture of sensor
As shown in Figure 1 and Figure 2, the present invention's fixing copper clad plate 2 on aluminium sheet 1 adheres on the copper clad plate 2 and has SiO
2The Si sheet 3.1 that the polishing of layer 3.2 is thin, the miniature measurement chamber 7 that dimethyl silicone polymer is done is fixed on SiO
2On the layer 3.2, cover one deck bio-compatibility glue 8 on the miniature measurement of the dimethyl silicone polymer chamber 7, bonding with the optical glass 4 of the gold-plated film reference electrode 5 of lower surface, coating one deck poly bird amino acid and laminin layer 9 in the miniature measurement chamber 7, two opening parts on glass are connected the feed liquor emulsion tube 12 and the fluid emulsion tube 13 of medicine and nutrient solution respectively, draw working electrode 10 on the copper clad plate, electrode 11 is linked to each other with golden film reference electrode 5.During measurement, sticking optical glass is loam cake above the miniature measurement chamber, forms the plugging meter chamber.Red laser 6 is selected directly over a certain cell of irradiation, the feed liquor emulsion tube is connected with peristaltic pump, break-make control medicine irritation by pump, links to each other with constant potential/electric current instrument to electrode at working electrode, reference electrode, and constant potential/electric current instrument links to each other with digital compensation circuit by lock-in amplifier, insert computing machine, with the computer realization both-way communication, the control temperature also realizes data acquisition process, and computing machine links to each other with digital/analog converter, control peristaltic pump break-make, the detection system block diagram as shown in Figure 6.
2, the preparation of sensor
(1) preparation of Light Addressable Potentiometric Sensor
Select for use＜100〉n type single crystalline Si sheets are as the substrate of Light Addressable Potentiometric Sensor, and the Si sheet is put into 1000 ℃ of high temperature furnaces and was carried out thermal oxide 20 minutes after polished and cleaned, makes the positive growth of Si sheet one layer thickness be about the SiO of 30nm
2Film, from the back side with the polishing of the thickness of Si sheet to 100 μ m, with conducting resinl the Si sheet is bonded on the copper clad plate then, 160 ℃ of hot settings 2 hours are drawn working electrode by Ohmic contact from copper clad plate;
(2) SiO of Si sheet
2Layer 3.2 surface treatment
The present invention has designed the scheme of two kinds of cell fixation: one, and coating process is promptly done the coating that strengthens attaching property on Si sheet surface; Two, by changing the roughness on Si sheet surface, do the figure of inducing cell growth.
The coating process process is as follows: the phosphate buffer of 100 μ g/ml poly-ornithines and the phosphate buffer of 8 μ g/ml laminins mixed by 1: 1,, the Si sheet immersed in this mixed solution with the miillpore filter bacteriological filtration of 0.22 μ m, and 37 ℃, 5%CO
2Under the environment, soaked 24 hours, with distilled water flushing twice, freezing standby again.The experimental result proof has been done the single-cell sensor after the surface treatment, and slope obviously increases, so the device sensitivity raising, thereby has improved the sensitivity of measuring, and as shown in Figure 3, and cellular morphology is normal, and cynapse is obvious, and growing state is good, as shown in Figure 4.
The process of the roughness on change Si sheet surface is as follows: be 2 * 2mm with photoetching technique on the LAPS surface
2The microcavity figure, change the surfaceness of Si sheet, and then carry out the processing of coating, as shown in Figure 5.
(3) reference electrode preparation
Hot acetone cleans anti-reflection optical glass, 80 ℃ of oven dry.At glass surface, make mask with coordinate paper, adopt magnetron sputtering technique to press the gold-plated film of mask shape at glass surface, with conducting resinl that lead and golden film is bonding, draw reference electrode.
(4) miniature measurement chamber design
Prepare one 0.2 * 5 * 5cm with dimethyl silicone polymer
3Membrane structure, carve and measure chambeies last with mould with reference to similar two of gold electrode, the chamber is of a size of 0.2 * 1.4 * 0.5cm
3With bio-compatibility glue this structure is adhered to the LAPS sensor surface, cultured cell in this chamber, during measurement, sticking above optical glass is loam cake, forms the plugging meter chamber.
Principle of sensors
Because what will measure is the physiological parameter of extracellular liquid environment, so employing is the LAPS system with EIS structure.Its ultimate principle is semi-conductive inner photoeffect, and promptly when semiconductor was subjected to the rayed of certain wavelength, semiconductor absorbed photon, and the transition of forbidden band to conduction band takes place, and promptly produces electron hole pair.In the ordinary course of things, electron hole pair is very fast compound, does not detect electric current in external circuit.If (n type silicon adds negative pressure, and P type silicon adds malleation) produces depletion layer when adding reverse bias voltage to LAPS in the semiconductor, at this moment the electron hole pair near depletion layer draws back with regard to depleted layer.When fixing light intensity, will produce photovoltage, LAPS adopts the rayed of intensity modulated at the front or the back side of device, just can measure electric current in external circuit.The size of electric current, relevant with thickness (promptly outer bias voltage) of light intensity, depletion layer etc.
Be the family curve synoptic diagram of n type silicon substrate Light Addressable Potentiometric Sensor as shown in Figure 7, this curve can be divided into cut-off region, zone of transition (workspace) and saturation region, and this is by the decision of the characteristic of silicon.Family curve is exactly the film response along the translation of bias voltage axle, selected element, LAPS sensitivity, the film response that family curve has determined bias voltage and the corresponding relation of measured value etc.
Single-cell sensor detects action potentials of cells embodiment:
The acetylcholine (Ach) of having selected 1: 10000,1: 100000,1: 1000000 (g/ml) three kinds of concentration is as neuronic stimulating drug, and experimental result shows that acetylcholine is excitation for corticocerebral neuron.
As shown in Figure 8, when not adding any medicine irritation, the signal that cell sensor records, it is the baseline of signal analysis, as can be seen, very little irregular disturbance is arranged among the figure, mainly be since the measuring system coaxial cable to fail to play the small vibrations of excellent shielding effect and environment caused.As shown in Figure 9, be under 1: 1000000 low concentration Ach effect, the response of cell sensor, curve is similar to baseline among the figure, does not almost change, and illustrates under this concentration, and Ach fails also to cause that cell produces action potential.As shown in figure 10, be in 1: 100000 under the concentration Ach effect, the response of cell sensor, figure as can be seen thus, outer current potential frequency and the amplitude of born of the same parents all changes, and detects a frequency and is about the electric signal that the 1Hz amplitude is about 1.8mV, and preliminary judgement is the action potential of cell.As shown in figure 11, be under 1: 10000 high concentration Ach effect, the response of cell sensor, this signal frequency and amplitude all have very big variation.
This presentation of results when Ach solution changes, can cause the change of born of the same parents' volta potential of cell in the finite concentration scope, single-cell sensor can detect electric signal frequency and changes in amplitude, and, increasing with Ach concentration, the amplitude of current potential increases.
Claims (3)
1, a kind of single-cell sensor that is used for the outer action potential measurement of cell born of the same parents is characterized in that: go up fixedly copper clad plate (2) at aluminium sheet (1), copper clad plate (2) is gone up to adhere to has SiO
2The Si sheet of the polishing of layer (3.2) is drawn working electrode by Ohmic contact from copper clad plate, wherein, and the SiO of Si sheet
2Layer (3.2) surface is made the coating of enhancing attaching property or is changed its surperficial roughness by photoetching technique with coating process on its surface, described coating process process is as follows: the phosphate buffer of 100 μ g/ml poly-ornithines and the phosphate buffer of 8 μ g/ml laminins were mixed by 1: 1, miillpore filter bacteriological filtration with 0.22 μ m will be stained with SiO
2The copper clad plate of the Si sheet of the polishing of layer (3.2) immerses in this mixed solution, and 37 ℃, 5%CO
2Under the environment, soaked 24 hours, with distilled water flushing twice, freezing standby again; Change the SiO of Si sheet
2The process of the roughness of laminar surface is as follows: be 2 * 2mm with photoetching technique on its surface
2The microcavity figure, change surfaceness, and then carry out the processing of coating, obtain being stained with the treated SiO in surface
2The copper clad plate of the Si sheet of the polishing of layer (3.2), the miniature measurement chamber (7) that dimethyl silicone polymer is made is fixed on SiO
2On the layer (3.2), miniature measurement chamber (7) is gone up and is covered one deck bio-compatibility glue (8), bonding with the optical glass (4) of the gold-plated film reference electrode of lower surface (5), two opening parts on glass are connected the feed liquor emulsion tube (12) and the fluid emulsion tube (13) of medicine and nutrient solution respectively, and electrode (11) is linked to each other with gold-plated film reference electrode (5).
2, a kind of single-cell sensor that the outer action potential of cell born of the same parents is measured that is used for according to claim 1, it is characterized in that: said miniature measurement chamber (7) is 1～2.
3, a kind of preparation method who is used for the single-cell sensor of the outer action potential measurement of cell born of the same parents is characterized in that:
(1) select for use＜100〉n type single crystalline Si sheets are as the substrate of Light Addressable Potentiometric Sensor, and the Si sheet is put into 1000 ℃ of high temperature furnaces and was carried out thermal oxide 20 minutes after polished and cleaned, and making the positive growth of Si sheet one layer thickness is the SiO of 30nm
2Film, from the back side with the polishing of the thickness of Si sheet to 100 μ m, with conducting resinl the Si sheet is bonded on the copper clad plate then, 160 ℃ of hot settings 2 hours are drawn working electrode by Ohmic contact from copper clad plate;
(2) SiO of Si sheet
2Layer (3.2) surface treatment
The scheme of two kinds of cell fixation: coating process: promptly do the coating that strengthens attaching property on its surface; By changing its surperficial roughness, do the figure of inducing cell growth;
The coating process process is as follows: the phosphate buffer of 100 μ g/ml poly-ornithines and the phosphate buffer of 8 μ g/ml laminins were mixed by 1: 1, and the miillpore filter bacteriological filtration with 0.22 μ m will be stained with SiO
2The copper clad plate of the Si sheet of the polishing of layer (3.2) immerses in this mixed solution, and 37 ℃, 5%CO
2Under the environment, soaked 24 hours, with distilled water flushing twice, freezing standby again;
The process that changes Si sheet surfaceness is as follows: with the SiO of photoetching technique at the Si sheet
22 * 2mm is on layer (3.2) surface
2The microcavity figure, change Si sheet surfaceness, and then carry out the processing of coating;
(3) reference electrode preparation
Hot acetone cleans anti-reflection optical glass, and 80 ℃ of oven dry at glass surface, are made mask with coordinate paper, adopts magnetron sputtering technique to press the gold-plated film of mask shape at glass surface, and with conducting resinl that lead and golden film reference electrode is bonding, reference electrode is connected again with to electrode;
(4) settle miniature measurement chamber
With dimethyl silicone polymer preparation 0.2 * 5 * 5cm
3Membrane structure, carve two and measure chambeies last with mould, the chamber is of a size of 0.2 * 1.4 * 0.5cm
3, with bio-compatibility glue this structure is adhered to the Light Addressable Potentiometric Sensor surface, cultured cell in this chamber, during measurement, sticking above optical glass is loam cake, forms the miniature measurement chamber of sealing.
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| CN 200310122949 CN1242260C (en) | 2003-12-25 | 2003-12-25 | Single cell sensor for ectocytic action potential detection and its preparing method |
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| CN102279255B (en) * | 2010-10-20 | 2013-08-28 | 重庆工商大学 | Mechanical dynamic regulation and control chip for single cell total environment |
| CN102360007A (en) * | 2011-06-24 | 2012-02-22 | 中国人民解放军军事医学科学院基础医学研究所 | Well-type neurochip and preparation method thereof |
| CN103901089B (en) * | 2014-04-16 | 2016-08-24 | 国家纳米科学中心 | The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method |
| CN105300412B (en) * | 2015-07-27 | 2017-09-15 | 桂林电子科技大学 | A kind of temperature self-compensation method for LAPS |
| CN105823817B (en) * | 2016-01-04 | 2018-04-20 | 浙江农林大学 | Cancer therapy drug vinorelbine Composition analyzed device and detection method |
| CN105866217B (en) * | 2016-03-21 | 2018-04-10 | 杭州市红十字会医院 | Harringtonine treatment of cancer effect detection device and method |
| CN105866210B (en) * | 2016-03-21 | 2018-04-13 | 杭州市红十字会医院 | A kind of Composition analyzed device and detection method for anti-cancer agent vincristine |
| CN105695329B (en) * | 2016-04-08 | 2018-08-17 | 西南交通大学 | High-flux electric stimulates the experimental provision and method of Cell differentiation inducing activity and medicine controlled releasing |
| CN112098464B (en) * | 2020-08-25 | 2023-12-26 | 天津海星辉科技有限公司 | Microarray carbohydrate metabolism analysis detection device and method |
| CN111735867B (en) * | 2020-08-25 | 2020-11-27 | 天津海星辉科技有限公司 | Carbohydrate lactobacillus metabolism analysis and detection device and method |
| CN114002296B (en) * | 2022-01-04 | 2022-03-29 | 苏州大学 | Bioactive substance transient photovoltage measurement assembly, device and method |
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