CAJ | 학술논문

　　SIET（self-referencing ion electrode technique，自参考离子选择性电极技术）是电生理学研究的新手段，可以在植物抗逆研究中无损地获得植物细胞、组织、器官微区内离子流动态变化信息，而离子选择性微电极的制备及性能测试的标准化是 SIET 系统对植物活细胞、活体组织原位离子流测试的前提。该文以钾离子选择性微电极为例，详细讨论了离子选择性微电极的拉制、硅烷化、灌充等制备过程，研究了微电极内阻等电极参数的测量方法，测试了微电极的能斯特响应斜率、检测范围、响应时间等参数，讨论了制备过程中微电极性能的影响因素。离子选择性微电极使用WD-2型微电极拉制仪由无导液丝的TW150-3型硼硅酸盐玻璃毛细管拉制成形，其尖端直径为1～9μm，干燥后用5%硅烷试剂在150℃温度下做硅烷化处理，再灌充入内充液与LIX（liquid ion exchanger，液态离子交换剂）而制成。研究表明：LIX成分是影响微电极内阻的重要因素，灌充LIX后的钾离子选择微电极（LIX长度为150～210μm）内阻达到108～109?，明显高于灌充LIX前；微电极在0.01～500 mmol/L K+浓度范围内具有很好的线性关系，R2=0.9998，能斯特斜率为53.095 mV/dec；微电极对1和100 mmol/L KCl溶液的平均响应时间t95%小于1 s。研究结果表明，离子选择性玻璃微电极的制备过程是影响微电极性能的关键，微电极尖端尺寸、内阻、响应时间等参数对微电极的应用影响显著。该研究可为离子选择性微电极的制备及其在 SIET 系统中的应用提供参考。
　　SIET（self-referencing ion electrode technique，자삼고리자선택성전겁기술）시전생이학연구적신수단，가이재식물항역연구중무손지획득식물세포、조직、기관미구내리자류동태변화신식，이리자선택성미전겁적제비급성능측시적표준화시 SIET 계통대식물활세포、활체조직원위리자류측시적전제。해문이갑리자선택성미전겁위례，상세토론료리자선택성미전겁적랍제、규완화、관충등제비과정，연구료미전겁내조등전겁삼수적측량방법，측시료미전겁적능사특향응사솔、검측범위、향응시간등삼수，토론료제비과정중미전겁성능적영향인소。리자선택성미전겁사용WD-2형미전겁랍제의유무도액사적TW150-3형붕규산염파리모세관랍제성형，기첨단직경위1～9μm，간조후용5%규완시제재150℃온도하주규완화처리，재관충입내충액여LIX（liquid ion exchanger，액태리자교환제）이제성。연구표명：LIX성분시영향미전겁내조적중요인소，관충LIX후적갑리자선택미전겁（LIX장도위150～210μm）내조체도108～109?，명현고우관충LIX전；미전겁재0.01～500 mmol/L K+농도범위내구유흔호적선성관계，R2=0.9998，능사특사솔위53.095 mV/dec；미전겁대1화100 mmol/L KCl용액적평균향응시간t95%소우1 s。연구결과표명，리자선택성파리미전겁적제비과정시영향미전겁성능적관건，미전겁첨단척촌、내조、향응시간등삼수대미전겁적응용영향현저。해연구가위리자선택성미전겁적제비급기재 SIET 계통중적응용제공삼고。
An self-referencing ion electrode technique provides a novel electrophysiological tool which can non-invasively measure the dynamic influxes and effluxes of ions from cells and organs in vivo. In fact, the foundation of this technique is the fabrication and performance test of an ion selective microelectrode (ISME). In this paper, the K+ISMEs with good performances were obtained. We elaborated the procedure to prepare the glass micropipettes and to fill the pipettes with internal filling solution and liquid ion exchangers (LIX) of potassium, and then estimated the performance of these ion selective microelectrodes. Measurement of tip size, measuring method of resistance, testing of detection range, Nernstian slope, and response time, were described in detail. Ion selective microelectrodes were calibrated before and after experiments using two or more different kinds of concentrations of K+within its operating range based directly on the potentiometric analysis. The procedure for ion selective microelectrodes fabrication is strict. The electrodes (the diameter of the apex of the tip was 1~9μm) were pulled from non-filamented borosilicate glass capillaries (TW150-3, World Precision Instruments, USA) on a vertical micropipette puller (WD-2, Chengdu Instrument Factory, Chengdu), oven dried, and then silanized by injecting 2 mL of 5%dimethyldichlorosilane (Sinopharm Chemcial Reagent Co. Ltd, Beijing) with n-hexane as the solvent in a glass preparation chamber at 150℃. Afterwards, dried and cooled electrode blanks were back-filled with a 100 mmol/L KCl solution. Immediately after back-filling, the microelectrode tips were front-filled with liquid ion exchangers of potassium (60031, Sigma, USA). Furthermore, some of the factors that affect the performance of the microelectrode in the preparation were discussed in detail. The ISME’s resistance reaches to 108~109?filling with LIX (length is 150~210μm), much higher than that which occurs without LIX. The detection range obtained by the K+ISME is linear within a wide range of 0.01~500 mM KCl solutions with the slope of 53.095 mV per decade, and R2 of 0.9998, which means the K+ISME response is in accordance with the Nernst equation. Besides the attainable Nernstian response range, the response time t95%, from the beginning of that ISE immersed into the K+ion standard solutions with two concentrations, 1 and 100 mmol/L KCl, with 8 assays each concentration, to the 95%of stable potentials, is less than 1s. These measurements were made at room temperature (20~25℃). The results show that the fabrication of the ion selective glass microelectrodes is the key to obtaining the high performance of the microelectrode. The parameters of microelectrodes, i.e. tip size, resistance and response time, etc. are very important in practical application. This work can provide a reference basis for the fabrication and application in SIET of ion selective microelectrodes. Moreover, the standardized fabrication is the precondition to measure the dynamic influxes and effluxes of ions from cells and organs in vivo using the SIET.