Etd 0712105 122345

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Etd 0712105 122345

  1. 1. Biochip fabrication of action potential measurement for single cells
  2. 2. (Patch Clamp) (pipette) (Voltage clamp) (action potential) (Soft lithography) ICP( ) I
  3. 3. ABSTRACT Patch clamp is a well-developed lelectrophysiological recording technique used to study ion channel function and regulation. The conventional method of performing patch clamp technique employs a glass micropipette onto the cell by manual manipulation. Despite this technique is extremely sensitive and information-rich, but requires a highly-skilled operator and is limited in throughput. Voltage clamp that determine the behavior of the ion channel conductances responsible for the generation of the action potential is the another method to record the flow of ionic current across the cell membrance. The method is held a constant membrane potential while the ionic current flowing through the membrane is measured. The thesis presents cell-platforms and etcing pores on silicon by using soft lithography and etching technique insteading of conventional glass micropipette recording method. we successfully demonstrated practicability of the process by the cells adhered on the platform and the etching pores on the silicon chip by using ICP. The less laborious manipulation time saving and high sampling throughput will be expected .In the future, we can automatically real time controlled and changed in response to the cell’s physiologic characteristics measured by ionic channel activities. Keyword MEMS Patch clamp Voltage Clamp Soft lithography ICP II
  4. 4. 奈 福 III
  5. 5. 奈 IV
  6. 6. ……………………………………………………………………I …………………………………………………………..………. ……………………………………………………………………..…. ………………………………………………………………..………. ……………………………………………………………………. …………………………………………………………………...… …………………………….…………………………………..1 1-1 ………………………………..………………………………….1 1-2 ………………………………………………..…….1 1-3 …………………………..……………………….2 1-4 ………………….…………………………………………..3 1-5 ……………………………………………………………...4 1-6 ………………………………………………………..…….9 …………………………...10 2-1 ………………………………………….10 2-1-1 ………………………………………….……….10 2-1-2 …………………………………………………..13 2-1-3 …………………………..………15 2-1-4 (Voltage Clamp)……………………….……………17 2-2 ………………………………19 2-2-1 (HTS) ………………………..20 V
  7. 7. 2-2-1-1 AVIVA Biosciences Corp Axon Instruments,Inc…………..20 2-2-1-2 Cytocentrics CCS(Reutlingen ,Germany)…………………22 2-2-1-3 Flyion GmbH (Tubingen , Germany)……………………...24 2-2-1-4 Ionworks HT –Molecular Devices corp.(Sunnyvale ,USA).25 2-2-1-5 Sophion Biosciences(Ballerup,Demark)…………………..26 ……………….….……………………….30 3-1 ………………….….……………………………..30 3-2 ………………………….….…………………………..31 3-2-1 (Clean)…………………………….……………………...31 3-2-2 (Oxidation)…………………….…….……………………34 3-2-3 ( Photolithography)……………………………………….35 3-3 (Soft lithography)……………………………………………39 3-3-1 …………………………………………………..39 3-3-2 ………………………………………………………39 3-3-3 …………………………………………………43 3-4 (TMAH )………………………………………...46 3-4-1 …………………………………………………46 3-4-2 ……………………………………………………46 3-5 ( ,ICP)…………………………..47 3-5-1 ………………………………………………………48 3-5-2 ………………………..………………………..50 3-5-3 ……………………………………………………52 VI
  8. 8. ……………………………………………………….55 4-1 ………………………………………………………………….55 4-2 TMAH ………………………………………………………58 4-3 ICP ……………………………………………………...…...61 ……………………………………………….…67 5-1 ………………………………………………………………….…67 5-2 ……………………………………………………………….67 …………………………………………………………………...69 ……………………………………………………………………… VII
  9. 9. 1 ……………………………………………….….28 2 …………………………………………………29 3 …………………………………………………………54 VIII
  10. 10. 1.1 [2] …………………………………...…..………3 1.2 Nanion 5 1.3 PDMS 6 1.4 PDMS 6 1.5 7 1.6 MCA 7 1.7 …………………………………………8 1.8 sidewall 8 2.1 [15] ……………………………...………………..11 2.2 2.3 ………..…………12 2.4 …………………………………………………….13 2.5 …………………………………….14 2.6 - …………………………………………15 2.7 (Patch Clamp)[6,18,19] ………………………………..16 2.8 …………………………………....………18 2.9 [20] ………………………….………18 2.10 (A) (B) [21] ………..20 2.11 SealChip16…………………………………………………...……20 TM 2.12 PatchXpress ………………………………………………21 2.13 CytoPatchTM 23 IX
  11. 11. 2.14 CytoPatchTM SEM 23 2.15 CytoPatch Site 23 2.16 Flyion 24 2.17 Flyscreen 8500………………………………………………….24 2.18 Ionworks HT ………………………….26 2.19 QPatch 16TM QPatch HT…………………………………….27 3.1 ……………………………………….30 3.2 ………………………………………..31 3.3 NDL (Wet bench)………………………33 3.4 (a) (Oxidation Furnace) (b) …………………………….35 3.5 (a) (b) (c) ……36 3.6 NDL(a) (Track) (b) (Aligner) ……………………………….38 3.7 ………………………………………41 3.8 OM (a) (b) 42 3.9 (a) 16µm 1.6µm (b) 20µm 1.6µm © 30µm 1.6µm …………42 3.10 DSPC 45 3.11 (a) SH-(CH2)11COOH PDMS substrate (b)substrate SH-(CH2)11COOH (c) substrate DSPC (d) 12 substrate DSPC X
  12. 12. DSPC …………………………………………45 3.12 ………………………………47 3.13 TMAH Si ……………………………………………..47 3.14 NDL (a) (ICP) (b) (Sputter) ………………………………………48 3.15 51 3.16 53 3.17 ICP ………………………………54 4.1 4.2 µm µm ……………………… 4.3 µm ………………………… 4.4 (a) (b) 18µm x36µm ……………………57 4.5 18x18µm 2 ……………………………...……….57 4.6 (a) 27µm x54µm (b) 37umx74um (c) 38µm XI
  13. 13. (d) 38µm ……………………………………57 4.7 TMAH (5wt%) (a) (b) BOE SiO2 ( 760µm) ………58 4.8 TMAH (25wt%) (a) (b) BOE SiO2 ( 760µm) ………59 4.9 ……………………………… 4.10 ………………………… 4.11 …………………………………… 4.12 (a) (b) …………………………………………62 4.12 (c) (d) ……………………63 4.13 ……………………..63 4.14 IC (dummy) ………………...………64 4.15 (a) (b) …………………………………………………..64 4.16 …………………………………………………………65 4.17 SEM ……………………………………..…..65 4.18 …………………………...……………….66 XII
  14. 14. (Micro – Electro – Mechanical – Systems MEMS ) (Bio-MEMS) (Biochip) 1959 · (Richard Feynmann,1918~1988) “There is plenty of room at the bottom”[1] (Micro-Electro-Mechanical-System, MEMS) (Batch) 1
  15. 15. MEMS - ? (Silicon chip) (glass) DNA resting membrane potential (Polarization) depolarize 2
  16. 16. 2.1 [2] (Patch clamp) 3
  17. 17. (Patch-on-a-chip)[3] Patch-on-a-chip 1. 2. 3. 4. 5. 1940 (Gerard) 4
  18. 18. [4] 1952 (Hodgkin) (Huxley) (Katz) (voltage-clamp) [5] 1976 Erwin Neher Bert Sakmann single channel recording or patch clamprecording 1991 [6] 2002 Sophion APATCHI-1 [7] 2002 (Munich University) 奈 (Center of Nanoscience) Niels Fertig (Planar micro structured quartz chip) 1.2 (Xenopus oocytes) (cell-attached mode) (mammalian) (whole-cell mode) Nanion [8] 1.2 Nanion 5
  19. 19. 2002 (Yale) Kathryn PDMS (Polydimethysiloxane)( 3-3-3 ) (pipette) 10 (GigaSeal) 1.3 Axon Instruments [9] 1.3 PDMS PDMS 2004 (University of California) Rigo Pantoja PDMS 1.4 [10] 1.4 PDMS 6
  20. 20. 2002 (Swiss Federal Institute of Technology) (Institute of Microelectronics and Microsystems) Lehnert (Hamburg) Evotec OAI AG Netzer (SiO2) (micronozzle) 1.5 [11] 1.5 2003 Masato TANABE MCA (Micro Channel Array) 1.6 [12] 1.6 MCA 7
  21. 21. 2004 (Lehigh University) Santosh Pandey (current transport model) 1.7[13] 1.7 2004 , (Sensor and Actuator) J.Seo PDMS (sidewall) (lateral) 1.8 [14] 1.8 sidewall 8
  22. 22. (Voltage clamp) - (Voltage Clamp) (BioMEMS) (Patch-on-a-chip) (Soft lithography) 9
  23. 23. (Patch Clamp) (Lipid) (lipid bilayer) 2.1 [15] 1~2 2~6 nm 12 nm 10
  24. 24. 2.2 [15] (glycoprotein) (integral protein) (peripheral protein) 2.2 [16] 2.2 11
  25. 25. (channels) (pores) 2.3 [17] (1) (Transport protein) (2) (Channel protein) (3) (Carrier protein) (Channel protein) (Carrier proteins) Energy (Simple diffusion) (Diffusuion) (Active transport) 2.3 12
  26. 26. diffusion passive transport active transport 1. voltage gating Outside Na+ (Gate closed) (Gate open) Inside 2.4 13
  27. 27. 2. ligand gating ligand acetylcholine acetylcholine channel 0.65 2.5 acetylcholine 2.5 (2) ATP 14
  28. 28. (sodium - potassium pump 2.6 + 3-Na Outside 2-k+ ATPase Inside 3-Na+ + 2-K ATP ADP+Pi 2.6 1976 ‧ (Erwin Neher) ‧ (Bert Sakmann) single channel recording or patch clamp recording pipette 1~2 2.7 15
  29. 29. [6,18,19] rectangular voltage-clamp steps 2.7 (Patch clamp)[6,18,19] (Patch clamp recording) (1) (2) (3) (cell attached) (4) (whole cell recording) (Inside-out patch) (secondary messengers) (5) intracellular recording (6) (Fluorescence microscope) Ca++ 16
  30. 30. 2-1-4 (Voltage Clamp) R ohms, Ω (Conductance G) siemens S (G=1/R) 17
  31. 31. 2.8 2.9 [20] 18
  32. 32. 19
  33. 33. 2.10 (A) (B) [21] 2-2-1 (HTS) (High - throughput screening) 2-2-1-1 AVIVA Biosciences Corp Axon Instruments,Inc SealChip16 AVIVA 2.11 [22] 90% 10 ( gigaseal) 75% (whole cell patch-clamp ) whole-cell 15 15 MΩ (membrane resistance) 200MΩ 2.11 SealChip16 20
  34. 34. SealChip16 Axon Instrument PatchXpress TM 2.12 16 SealChip16 16 1µm 4.5mm TM 2.12 PatchXpress PatchXpress SealChip 1. whole cell patch-clamp 2. 16 3. 16 gigaseal whole cell patch-clamp (access resistence) 4. 50µl 5. 16 2000 6. 21
  35. 35. 2-2-1-2 Cytocentrics CCS(Reutlingen ,Germany) Cytocentrics CCS CytoPatchTM (focused ion beam) CytoPatchTM 200 [21] Cytocentering gigaseal 2.13 (SiO2) CytoPatchTM chip 5 2 .14 CytoPatchTM chip SEM CytoPatchTM cytocentring CytoPatchTM CytoPatch Site 2.15 CytoPatch Automat 1~50 CytoPatch sites Multi Channel Systems npi electronic 22
  36. 36. 2.13 CytoPatchTM 2.14 CytoPatchTM SEM 2.15 CytoPatch Site 23
  37. 37. 2-2-1-3 Flyion GmbH (Tubingen , Germany) Flyion Gmbh 2.16 [23] Flyscreen 8500 1~6 2.17 Flyion 2.16 Flyion 2.17 Flyscreen 8500 24
  38. 38. 2-2-1-4 Ionworks HT –Molecular Devices corp. (Sunnyvale , USA) Ionwork HT 48 8 384 (well) voltage gating 2.18 [24] 96 384 12 HTS Amphotericin B ( perforated patch) (co-factor) whole-cell 1GΩ (seal resistance) 100MΩ HT 48 headstage headstage 25
  39. 39. state-dependent block desensitising ligand gating 2.18 Ionworks HT 2-2-1-5 Sophion Biosciences(Ballerup,Demark) Sophion Biosciences Apatchi-1TM QPatch 16TM QPatch HT 2.19 [25] 16 48 gigaseal 26
  40. 40. 2.19 QPatch 16TM QPatch HT N/A Not available 27
  41. 41. Whole-cell Whole-cell Institution Chip Cell positioning Gigaseal rate Status access recording rate Affymax Galss,laser drilling Suction N/A N/A N/A N/A Patch AVIVA >75% , 15min Galss with modified >90% , >1GΩ Suction Xpress and Biosciences , Suction Ra<15MΩ; surface dielectrophoresis (loss<1%) Seal chip Axon Instruments Rm>200MΩ available Axon Instruments PDMS,molding Suction ~50% Suction ~50% N/A Various substrates , laser CeNeS drilling , and other, Suction N/A N/A N/A N/A methods Under Cytocentrics CCS Quartz glass Suction N/A N/A N/A development Si3N4 membrane Under Cytion Electrophoresis N/A N/A N/A supported by a Si Chip development IonWork HT Plastic chip laser 60-80% , 6min Essen Instrument, Perforated and drilling, with a glass Suction 20-250MΩ Ra=10-15MΩ Molecular Devices patch PatchPlate coating Rm=10MΩ available Glass chip, gold NPC 16 Nanion ion-etched track Suction 30-50% Suction 30-50% , 30min under Technologies followed by wet etching development Qpatch 16 Silicon chip, etching Fluid channel and 96 Sophion Biosciences with biocompatible N/A Suction N/A ,Suction under coating development PDMS, molding Xenopus occyte Yale University Suction N/A N/A N/A 13% , gigaseal 28
  42. 42. Man u f a c t u r e r Produts Substrate/Seal Description/Throughput Amplifiers/Source Costs per chip per Status partners website data point r e f e r e n c ee Axon Instruments Inc PatchXpressTM planar patch 16 channel Sealchip, 16 channel range$140-180<$3. available/Aviva Union City, CA,USA 7000A glass chip/ 16 channel washout head, parallel/Axon 8/dp BioSciences G Seal 1 channel dispenser 20000 dp/day Cytocentrics CCS CytoPatchTM planar patch modular instrument, with up to 20 $8<$3.2/dp β-test inQ3 03/ GmbH Reutlingen , Automat quartz chip/ up to 20 patch calmp channels,switched BionChip, MultiChannel Germany G Seal sites mode parallel Systems, 2000 dp/stie/day(if fast operation/npi NMI, npi electronic, no acting compounds) electronic and Multi sales/ 40000 dp/day with 20 Channel Systems commercialization sites/unit partner lFlyion GmbH FlyScreen glass micropipette single channel Flip Tip® up to 6 independent Fliptips®A-$3 available in Europe ® Tubingen, Germany 8500 embedded in plastic dispenser with 2 channels channels/HEKA Fliptips®L-$4 /HEKA,Tecan, Manz jacket(Flip Tip)/ (functions) 3-6Flip Tip <$1.6/dp Automation no sales / G Seal recordings positions commercializtion partner (scalable) 300-1000 dp/day Molecular Devices IonWorksTMHT planar patch 384 PatchPlate at 1536 48 channel range $ 145-200 per available/ lower Corp Sunnyvale, plastic chip/ spacing(~14 L/well),12 parallel/proprietary patch throughut CA,USA Avg.~150MΩ seal channel dispenser 3000 development plate,<$2.0/dp IonworksTMAPC target dp/day launch Q1 04 Nanion Technologies NPC©1(Port-a- planar patch 1, or 16 channel chips 1 channel © NPC 1-$10 NPC©in -test,target © GmbH Munich , Patch) glass chip perfusion (scalable) 1 channel sequential/HEKA 16 NPC 16-$100 launch Q4 03/ Germany NPC©16s(sequ cartidge/G seal dispenser channel parallel <$2.5/dp for HEKA,Dagan, Tecan, ential) NPC©1-50 dp/day (asynchronous)/Dagan NPC©16 Bruxton, NPC©16p(paral NPC©16s-200 dp/day No sales/ lel) NPC© 16p-2000 dp/day commercializtion partner TM Sophion Bioscience QPatch 16 planar patch QpatchTM16-16channel- 16 and 96 channel QPatchTM16-$100< QPatchTM16 in -test A/S Ballerup , QPatchTM96 silicon chip/ 250-1200 dp/day parallel $2.5/dp targen Denmark G seal QpatchTM96-96channel- (asynchronous)/propri launch Q3 03/CRL,no 1500-7000dp/day 4/8 etary development sales/ commercialization channel dispenser partner 29
  43. 43. (Patch-on-a-chip) (Photomask) 3µm 3.1 3.1 30
  44. 44. 30µm 710µm 3.2 3.2 (AutoCAD 2004) (Clean) 31
  45. 45. 奈 ( NDL) NDL (Clean room) ( Wet Bench ) RCA 3.3 RCA 1965 RCA 1970 RCA RCA CARO DHF SC-1 SC-2 (1)CARO RCA 若 hydrophobic 3 硫 1 120- 130 硫 (2)DHF hydrophobic (D.I.water) (rinse) 32
  46. 46. (3)SC-1 Standard clean 1 SC-1 5 1 30 1 29 70 - 80 SC-1 (4)SC-2 Standard clean 2 SC-2 6 1 30 1 37 70 - 80 SC-2 留 3.3 NDL (Wet bench) 33
  47. 47. (Oxidation) (oxidation) (SiO2) (mask) SiO2 10~20Å SiO2 600 (thermal oxidation) SiO2 56 SiO2 Wet Oxidation Dry Oxidation Si s + O2(g →SiO2 s ( 3.1) Si s +2H2O g →SiO2 s + 2H2 g ( 3.2) 34
  48. 48. SiO2 SiO2 Si SiO2 3.4(a) ( ) 1092 3.4(b) 50Å 度 (a) (b) 3.4 (a) (Oxidation Furnace) (b) ( Photolithography) 35
  49. 49. (Exposure) (Development) (a) (b) (c) 3.5 (a) (b) (c) (DehydrationBake) Priming (Soft bake) (Hard Bake) (1) (2) (Hexamethyldisilazane, HMDS) 36
  50. 50. (3) (Spincoating) (5) (6) (Standing wave) (7) 37
  51. 51. (8) NDL (Track) (Aligner) 3.6 (a) (b) 3.6 NDL(a) (Track) (b) (Aligner) 38
  52. 52. (surface coating) (plasma treatment) (self-assembly monolayer SAM) LB (Langmuir-Blodgett film) microcontact printing CP replica molding REM microtransfer molding TM micromolding in capillaries MIMIC solvent-assisted micromolding SAMIM phase-shift photolitography [28] (microcontact printing) , 留 (substrate) ( 3.7) 39
  53. 53. (a) RCA (b) 1092 2 0.9µm SiO2 (c) (d) BOE (Buffer Oxide Etch) 50nm/min 20 SiO2 (e) 90 TMAH 0.9µm/min 0.5~4 (f)BOE . (g) (E-Beam) (Cr) (Au) 500Å 1500Å 40
  54. 54. (a) (e) TMAH (b) (f) (c) (g) (d) 3.7 3.8(a) 3.7(b) (OM) 3.9(a~c) (Alpha-step) 1.6 m 16 m 20 m 30 m 41
  55. 55. (a) (b) 3.8 OM (a) (b) 度 度 度 度 (a) (b) 度 度 (c) 3.9 (a) 16µm 1.6µm (b) 20µm 1.6µm (c) 30µm 1.6µm 42
  56. 56. 3-3-3 PDMS (Stamp) (substrate) PDMS(Polydimethysiloxane) Dow Corning (Sylgard 184 silicone elastomer) (Curing agent) 10:1 PDMS (1) (2) 22 dyne/cm2 PDMS PDMS 3.10 SH-(CH2)11COOH PDMS PDMS PDMS 60 PDMS SH-(CH2)11COOH SH-(CH2)11COOH 硫 硫 (Van der Waals force) self-assembled monolayers, SAM COOH 43
  57. 57. (a) (b) 3.10 (a) PDMS (b) PDMS ( ) DSPC 12 DSPC PC(phosphorylcholine) [29] SH-(CH2)11COOH 硫-硫 undecyl cholinyl COOH 3.10 ( 3.11) 44
  58. 58. 3.10 DSPC PDMS PDMS SH-(CH2)11COOH DSPC DSPC 3.11 (a) SH-(CH2)11COOH PDMS substrate (b)substrate SH-(CH2)11COOH (c) substrate DSPC (d) 12 substrate DSPC DSPC 45
  59. 59. (TMAH ) TMAH (Tetramethyl ammonium hydroxide (CH3)4NOH )) (Etch mask) (SiO2) TMAH 110°C IC 3-4-1 TMAH SiO2 (111) (100) 54.74° [30] (Etching Stop) TMAH SiO2 3µm 46
  60. 60. 3.12 3.13 54.74 ゚ 3um 3.12 (µm) Si TMAH Si (hr) 3.13 TMAH Si Dry Etching Plasma Etching NDL Inductively Coupled Plasma ICP 3.14(a) 47
  61. 61. (a) (b) 3.14 NDL (a) (ICP) (b) (Sputter) 3-5-1 (Plasma) (Radical) (Partially Ionized Gas) 48
  62. 62. 若 (DC) (RF) (1) (2) (Secondary Electron) (DC) (RF) 49
  63. 63. 3-5-2 Physical Chemical (undercut) SF6 (g)+e- → Sx Fy(g)+ Sx Fy + F-(g)+e- ………………..….……………..(3-1 ) Si+ F-1 → Si- nF………………………………..…….……………….(3-2 ) Si-nF→SiFx (adsorb)…………………………………….………...……..(3-3 ) SiFx(adsorb) →SiF4(g)………………………………...……………….….(3-4 ) 50
  64. 64. 3-15 ICP load-lock 1000 W 13.56 MHz RF 30W 13.56 MHz RF (backside helium cooling) [31] 3.15 51
  65. 65. 3-5-3 NDL (Sputter)( 3.14(b)) 3 3.17 (a) (b) (c) 52
  66. 66. (d) (e) ICP (f) (g) (h) (i) , ICP (j) 3.16 53
  67. 67. 參數 Forward Power (W) 6 RF Generator Reflect Power (W) 1 Forward Power (W) 1200 ICP Generator Reflect Power (W) 10 DC Bias (V) 35 SF6 flow rate (sccm) 100 O2 flow rate (sccm) 9 C4F8 flow rate (sccm) 0 Ar flow rate (sccm) 0 Chamber Pressure (mtorr) 9.8 APC Controller Valve position (Deg) 61.7 CRYO Temperature ) -100 Pressure (Torr) 15 Helium cooling Flow Meter (sccm) 11.3 (µm/min) Si ICP 3.17 ICP 54
  68. 68. human CD34+ Progenitor Cell 0.1M PBS (Phosphate Buffered Saline) 10.9 Na2HPO4 3.2 NaH2PO4 90 NaCl 1000 Distilled water pH 7.2 Trypan blue 4.1 ( 1.6 m) 4.2(a) 4.2(b) 4.2(c) 7 m 8 m 4.3-5 55
  69. 69. (a) (b) (c) 4.2 (a) (b) 7x7µm 2 (c) 8x8µm 2 ( ) (a) (b) µm 56
  70. 70. (a) (b) 4.4 (a) (b) 18µm x36µm 4.5 18x18µm 2 (a) (b) (c) (d) 4.6 (a) 27µm x54µm (b) 37umx74um (c) 38µm (d) 38µm 57
  71. 71. 若 TMAH TMAH (5wt% 25wt%) ( ) 4.7(a) 4.8(a) 4.7(b) 4.8(b) BOE (Buffer Oxide Etch) SiO2 (Etch mask) (a) (b) 4.7 TMAH (5wt%) (a) (b) BOE SiO2 ( 760µm) 58
  72. 72. (a) (b) 4.8 TMAH (25wt%) (a) (b) BOE SiO2 ( 760µm) Wm Wo z Wo 3 m Wm 710 m Wo=Wm- 2 z ………………………………………… Wm z Wo 59
  73. 73. Wo 60
  74. 74. 4-3 ICP ICP ICP (RF) 留 留 留 61
  75. 75. (1) 4.12(a) 4 m ICP 4.12(b) 4 m 15 m 4.12(c-d) (a) (b) 4.12 (a) (b) 62
  76. 76. (c) (d) 4.12 (c) (d) 4.13 63
  77. 77. 4.14 IC (dummy) (2) (a) (b) 4.15 (a) (b) 64
  78. 78. (3) 4.16 4.17 4.18 3.19 m 4.17 SEM 65
  79. 79. 4.18 66
  80. 80. 若 (series resistance) (electrical couple) ; Bosch ICP seal resistance 67
  81. 81. ICP (Micro-pump)( ) (Micro-mixer) ( ) 68
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