非损伤性扫描离子选择电极技术及其在后基因...（十一）

4. 总结及展望

SIET在借鉴众多科学家工作经验的基础上，经过多年的改进和完善，为科研人员提供了一个较为友好的软硬件环境，在数据的生成，采集以及校准等方面，极大地方便了研究人员。特别是应用SIET强大的3维立体测量方式，研究人员可以获得其他电生理技术无法测到的被测样品某些点的特异活性 ^[10]。

对于较为熟悉膜片钳技术的科研人员来讲，SIET是一个与膜片钳无论在时间分辨率还是在空间分辨率上不同的技术（如图1b），但由于膜片钳技术似乎在研究离子通道之外，作为十分有限，两者在研究过程中可以极好的相互补充。

由于SIET技术的出现，人们对于生物体特异离子转运系统的研究，在灵敏度上，时间和空间分辨率上已经被大大地提高了，并已成熟地与细胞和分子生物学技术、其他电生理技术和显微荧光成像技术配合使用(见3.5和4.10)。大家普遍认为SIET技术将在主动运输离子或分子泵和协同运输载体的研究方面发挥重大的作用。

分子遗传学的进展使得我们能够对这些运输载体分子加以确定，克隆和进行可控制的表达。当这些运输载体在分子水平方面通过在酵母，卵细胞等系统中的表达予以鉴定，或者某些细胞成份的物理结构和生理功能阐明之后^{[41,42,43,44]}，SIET技术的非损伤性，多离子/分子同时测量及灵活的空间测量方式将在细胞和组织水平上的功能鉴定方面发挥重要的，甚至是无法替代的作用。

5. 致谢

特别感谢匡廷云院士在身患重病的情况下对SIET技术的关心与支持。感谢杨福愉院士和林克椿教授给予的指导性建议。

同时感谢与裴真明教授(Department of Biology, Duke University, Durham, NC 27708)、王兆一教授(Creighton Cancer Center,1912 California St,Omaha, NE 68178)、许华曦教授(Center for Neuroscience and Aging, The Burnham Institute, La Jolla, California)、刘平生教授(Univ. of Texas Southwestern Medical Center at Dallas)、公衍道教授和隋森芳教授(清华大学生物科学与技术系)、徐涛教授，赫荣乔教授，沈钧贤教授，黄有国教授(中国科学院生物物理所)、武维华教授(中国农业大学生物学院植物生理学与生物化学国家重点实验室)、崔宗杰教授(北京师范大学细胞生物学研究所)、曾争主任医师(北京大学第一医院感染疾病科)、 黄海长研究员(北京大学第一医院肾脏内科-北京大学肾脏病研究所)、高天明教授(南方医科大学)、高永雄教授(香港中文大学)、林澧仪(台湾中央研究動物所)有关SIET技术所作的有益的探讨。

对Applicable Electronics Inc.和Science Wares Inc.的资金和技术支持表示感谢。

6. 参考文献

ADDIN EN.REFLIST

1    Kühtreiber W M, Jaffe L F. Detection of  extracellular calcium gradients with a calcium-specific vibrating  electrode. J. Cell Biol. 1990, 110(5): 1565~1573
2    许越, 邱泽生. 膜片钳技术及其在高等植物细胞研究中的应用与展望. 植物生理学通讯 1993, 29(3): 169~174
Xu  Y, Qiu Z S.  Patch-Clamp Technique and Its Applications to and  Prospects for the Studies of Higher Plant Cells. Plant Physiology  Communications, 1993, 29(3): 169~174
3    周专, 康华光. 国产膜片钳仪研制成功. 生物化学与生物物理进展 1990, 11(2): 128~137
Zhou Z, Kang H G.  Domestic Pacth-Clamp System Developed.  Prog. Biochem. Biophys. 1990, 11(2): 128~137
4    曹忠升, 康华光, 邹寿彬, 等. 应用膜片钳技术研究细胞分泌. 生物化学与生物物理进展 1992, 19(1): 14~19
Cao Z S, Kang H G, Zou S B, et al.  Study Cell Secretion with Pacth-Clamp Technique. Prog. Biochem. Biophys. 1992, 19(1): 14~19
5    Ammann D. Ion Selective Micro-electrodes. New York: Springer-Verlag; 1986
6    Kochian L V, Shaff J E, Kühtreiber W M, et al. Use of an Extracellular, Ion-Selective, Vibrating Microelectrode System For the Quantification of K⁺, H⁺, and Ca²⁺ Fluxes in Maize Roots and Maize Suspension Cells. Planta 1992, 188(4): 601~610
7    Kunkel J, Lin L-Y, Xu Y, et al.  The strategic use of Good buffers to measure proton gradients about  growing pollen tubes. In: al. AGe (ed.) Cell Biology of Plant and Fungal  Tip Growth. Amsterdam: IOS Press; 2001: 81~94
8    Cardenas L, Feijo J A, Kunkel J G, et al.  Rhizobium Nod factors induce increases in intracellular free calcium  and extracellular calcium influxes in bean root hairs. Plant J 1999,  19(3): 347~352
9    Messerli M A, Smith P J S, Lewis R C, et al.  Chloride fluxes in lily pollen tubes: a critical reevaluation. Plant J 2004, 40(5): 799~812
10  Xu Y, Kunkel J G, Brewer S et al.   MageFlux: A Web-Interfaced 3D Plotting System to Facilitate  Bio-currents Data Conversion and Visualization. In preparation. 2005
11  Degenhardt J, Larsen P B, Howell S H, et al.  Aluminum resistance in the Arabidopsis mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiology 1998, 117(1): 19~27
12  Huang J W W, Shaff J E, Grunes D L, et al.   Aluminum Effects On Calcium Fluxes At the Root Apex of  Aluminum-Tolerant and Aluminum-Sensitive Wheat Cultivars. Plant  Physiology 1992, 98(1): 230~237
13  Feijo J A, Sainhas J, Hackett G R, et al.   Growing pollen tubes possess a constitutive alkaline band in the clear  zone and a growth-dependent acidic tip. J of Cell Biol. 1999, 144(3):  483~496
14  Felle H H, Hepler P K. The cytosolic Ca²⁺ concentration gradient of Sinapis alba root hairs as revealed by Ca²⁺-selective microelectrode tests and fura-dextran ratio imaging. Plant Physiology 1997, 114(1): 39~45
15  Holdaway-Clarke T L, Hackett G A, Kunkel J G, et al.   Oscillations of cell expansion rate, cytoplasmic calcium, and calcium  influx in the pollen tube. J of General Physiology 1998, 112(1): 35A~35A
16  HoldawayClarke T L, Feijo J A, Hackett G R, et al.   Pollen tube growth and the intracellular cytosolic calcium gradient  oscillate in phase while extracellular calcium influx is delayed. Plant  Cell 1997, 9(11): 1999~2010
17           Miller D D, Callaham D A, Gross D J, et al.  Free Ca²⁺ Gradient in Growing Pollen Tubes of Lilium. Journal of Cell Science 1992, 101: 7~12
18   Messerli M A, Robinson K R. Cytoplasmic acidification and current  influx follow growth pulses of Lilium longiflorum pollen tubes. Plant J  1998, 16(1): 87~91
19  Trimarchi J R, Liu, L., Smith, P J S et al.   Non-invasive measurement of potassium efflux as an early indicator of  cell death in mouse embryos. Biol. Reproduct 2000, 63: 851~857
20  Trimarchi J R, Liu L, Porterfield D M, et al.   Oxidative phosphorylation-dependent and -independent oxygen  consumption by individual preimplantation mouse embryos. Biol. Reproduct  2000, 62: 1866~1874
21  Trimarchi J R, Liu L, Porterfield D M, et al.  A non-invasive method for measuring pre-implantation embryo physiology. Zygote 2000, 8: 15~24
22            Pelc R, Smith P J S, Ashley C C. In vivo recording of calcium  fluxes accompanying ''''catch'''' contraction of molluscan smooth  muscle. J. Physiol. Physiological Society Meeting, Leeds. 1996, 497: 41P
23   Pelc R, Smith P J S, Ashley C C.  Non-invasive recording of calcium  fluxes in the molluscan ''''catch'''' muscle using the  calcium-selective, self-referencing microelectrode (''''vibrating''''  probe). EMBO/HHMI Central European Scientists Meeting. 2004
24  Devlin C L. 5-Hydroxytryptamine stimulates Ca²⁺ flux in the ventricular muscle of mollusk (Busycon canaliculatum) during cardioexcitation. Biol. Bull 2001, 200(3): 344~350
25  Devlin C L, William A, Shawn A, et al.  Muscarinic acetylcholine receptor compounds alter net Ca²⁺ flux and contractility in an invertebrate smooth muscle. Invertebrate Neuroscience 2003, 5(1): 9~17
26   Devlin C L, Amole W, Shea K. Calcium stores used during muscarinic  receptor activation in a holothurian smooth muscle. Society for  Experimental Biology Annual Meeting, (Neuropharmacology session, #A13.6  Poster). 2003
27  Berridge M J, Bootman M D, Lipp P. Calcium - a life and death signal. Nature 1998, 395(6703): 645~648
28  Godell C M, Smyers M E, Eddleman C S, et al.  Calpain activity promotes the sealing of severed giant Axons. PNAS 1997, 94(9): 4751~4756
29  Eddleman C S, Ballinger M L, Smyers M E, et al. Repair of plasmalemmal lesions by vesicles. PNAS 1997, 94(9): 4745~4750
30  Malchow R P, Molina A J A, Hammar K, et al.   Proton fluxes of retinal horizontal cells of the skate: modulation by  glutamate. In: Invest. Opthalmol. Vis. Science Annual Meeting,  Association for Research in Vision and Opthalmology, 2002
31  Molina A J A, Birnbaum A, Smith P J S, et al.  Calcium modulation of H⁺ fluxes from retinal horizontal cells. In: Annual Meeting, Society for Neuroscience, 2002
32   Malchow R P, Verzi M P, Smith P J S. Extracellular pH gradients  measured from isolated retinal cells. Biol Bull. 1998, 195(2):203-4
33  Malchow R P, Bodily J, Hammar K, et al.   Calcium-dependent regulation of intracellular and extracellular  hydrogen ion levels of retinal horizontal cells. Association for  Research in Vision and Opthalmology Annual Meeting 2003
34   Malchow R P, Molina A, Bodily J. Regulation of proton flux from  isolated retinal horizontal cells of the skate. General Scientific  Meetings, Biol. Bull. 2002, 203: 268
35            Malchow R P, Ramsey D J. Responses of retinal Muller cells to  neurotransmitter candidates:  a comparative survey. Biol. Bull. 1999,  197(2): 229~230
36  Magoski N S, Knox R J, Kaczmarek L K. Activation of a Ca²⁺-permeable cation channel produces a prolonged attenuation of intracellular Ca²⁺ release in Aplysia bag cell neurones. J Physiol (Lond) 2000, 522(2): 271~283
37  Knox R J, Jonas E A, Kao L S, et al.  Kaczmarek LK. Ca²⁺ influx and activation of a cation current are coupled to intracellular Ca²⁺ release in peptidergic neurons of Aplysia californica. J Physiol (Lond) 1996, 494(3): 627~639
38  Molina A J A, Verzi M P, Birnbaum A D, et al.  Neurotransmitter modulation of extracellular H⁺ fluxes from isolated retinal horizontal cells of the skate. J Physiol (Lond) 2004, 560(3): 639~657
39  Jenkins G N. The influence of environmental fluids on enamel solubility. J. Dent. Res. 1966, 45: 662~669
40  Vincent P, Chua M, Nogue F, et al.  A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs. J. Cell Biol. 2005, 168(5): 801~812
41  Liu Z, Yan H, Wang K, et al.  Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 2004, 428(6980): 287~292
42  He Y, Tang R H, Hao Y, et al.  Nitric Oxide Represses the Arabidopsis Floral Transition. Science 2004, 305(5692): 1968~1971
43  Han S, Tang R, Anderson L K, et al.  A cell surface receptor mediates extracellular Ca²⁺ sensing in guard cells. Nature 2003, 425(6954): 196 ~ 200
44  Wang S Q, Song L S, Lakatta E G, et al. Ca²⁺ signaling between single L-type Ca²⁺ channels and ryanodine receptors in heart cells. Nature 2001, 410(6828): 592~596
　

Non-Invasive Scanning Ion-selective Electrode Technique and

Its Applications in the Post Genomic Era

Yue Xu^1,2,3)   Joseph G. Kunkel³⁾   Alan Shipley⁴⁾   Ping Zhang⁵⁾    WANG Shi-Qiang⁶⁾

ZHANG Xu-Jia⁷⁾   HE Yi-Kun⁸⁾   YIN Li-Ping⁸⁾   YANG Huang-Tian⁹⁾   SHANGGUAN Yu¹⁾  

YE Xin-Sheng¹⁰⁾

(   ¹⁾YoungerUSA Company, P.O. Box 37106, Raleigh, NC 27627 USA
²⁾Department of Botany, North Carolina State University, Raleigh, NC 27695 USA
³⁾Vibrating Probe Facility, University of Massachusetts at Amherst, Amherst, MA 01003 USA
⁴⁾Applicable Electronics Inc., P.O. Box 589, Forestdale, MA 02644 USA
⁵⁾Department of Physiology, Yale University, New Haven, CT 06520 USA
⁶⁾Department of Physiology and Biophysics, State Key Lab of Biomembrane and membrane Biotech,
 Peking University College of Life Sciences, Beijing 100871
⁷⁾National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences,
 Beijing 100101
⁸⁾College of Life Science, Capital Normal University, Beijing 100037
⁹⁾Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of
 Sciences & Shanghai Second Medical University, Shanghai 200025
¹⁰⁾Department of Life Sciences, National Natural Science Foundation of China, Beijing 100085 )

 Abstract The SIET non-invasively measures both ionic concentrations and  ionic fluxes in aqueous media with a spatial resolution of less than 10  micrometers with picomolar sensitivity. The SIET measures the  extracellular fluxes in and out of living biological membranes in-vitro.  Ca²⁺, H⁺, Cl^-, K⁺, Cd²⁺, Al³⁺, Mg²⁺, O₂  and NO are some of the ions/molecules that can be measured at present,  and more ion and molecular species will be available in the near future.  Some of the many applications of this technique for example, ionic flux  measurements in both animal and plant cells demonstrate the unique  advantages of the SIET compared to other techniques. The SIET can  measure extra-cellular ion movement in such a way that it provides  information that has been only theoretical.

Keywords non-invasive electrophysiology technique, ion-selective electrode, ionic transmemberane transportation, SIET
Corresponding Author. Yue (Jeff) Xu, YoungerUSA Co., PO Box 37106, Raleigh, NC 27627 USA,