纳米光纤传感系统的研制及在单细胞检测中的应用
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摘要
常规的细胞生物化学分析方法一般以大量细胞为研究对象,只能用平均结果反映单个细胞的化学信息。利用纳米光纤传感技术可以实现单个细胞水平的研究,可以获得反映细胞生理状态和过程的更准确、更全面的信息。
集倒置荧光显微镜、单光子计数器和100 ps的数据采集卡等,我们搭建了适合单细胞分析的纳米光纤传感平台,并使用labview编写了采集、处理程序。整机调试表明:系统稳定可靠,时间累加后可以探测到单个细胞(导入荧光素)的荧光强度。
基于纳米光纤探针和荧光探针CFDA,使用荧光强度比值法研究了单个酵母细胞内pH值的测量。对比使用FLS920全功能稳态/瞬态荧光光谱仪测得的群体细胞的pH标准曲线,极大地提高了探测的灵敏度(约3.8倍)。
我们测量了在相同的环境条件下的单个细胞之间的pH值。结果表明个体细胞pH是存在差别的,这种差别最大可以达到0.2/pH。因此传统的用平均的结果并不能反映单细胞之间化学信息的差异。
我们研究了细胞外pH值对单个细胞内pH值影响,发现没有基因敲除的酵母细胞比基因敲除的细胞(醇脱氢酶基因ADH2和ADH2等位基因同时敲除,或只敲除醇脱氢酶基因ADH2)具有更强的维持细胞内pH值的能力。特别是外部pH大于9.5的时候,这种差别更加突出。
The conventional analytical methods which generally choose a mass of cells as the research object could only get an average results of the chosen cells.So,it is often inaccurate or even wrong.But the research based on the nano-fiber optical sensing technology could give the information of a single cell.With the research at a single cell level,more accurate and comprehensive information which indicates the physiological state and the process of the cell could also be obtained.
We have built a system which suits single cell analysis.The system mainly involves an inverted fluorescence microscope,a single photon counter and a 100 ps data acquisition card.And we also have written a labview based acquisition procedure for the data acquisition card.Overall debugging indicates that our system is very stable and it can distinguish the fluorescence intensity of a single cell(cell was injected fluorescein).
With nano-fiber probes and fluorescence probes,we have measured intracellular pH value of a single yeast cell using the ratio method of the fluorescence intensity.Contrast with the result obtained from multitudinous cells by using the FLS920 combined fluorescence lifetime & steady state spectrometer,we have greatly improved the detection sensitivity(approximately 3.8 times).
We have measured the pH values of different single cells under the same environment.The results showed that different single cells have different pH values,even though they are under the same condition.The maximal divergence between them could reach 0.2/pH.So,the average results obtained by the conventional analytical method can't indicate the chemical information differences between the cells.
We have studied the effect of the extracellular pH on the intracellular pH value of a single cell.By comparing the effects of external pH_o on pH_i of both natural yeast cell and its heterozygote strains(one with ADH2 gene removed,the other with both ADH2 and its allele removed),a different behavior was observed between these strains,and the natural yeast cells have stronger power to regulate and maintain pH_i as a response to the pH_o changing.This difference will be more outstanding especially when the pH_o is above 9.5.
集倒置荧光显微镜、单光子计数器和100 ps的数据采集卡等,我们搭建了适合单细胞分析的纳米光纤传感平台,并使用labview编写了采集、处理程序。整机调试表明:系统稳定可靠,时间累加后可以探测到单个细胞(导入荧光素)的荧光强度。
基于纳米光纤探针和荧光探针CFDA,使用荧光强度比值法研究了单个酵母细胞内pH值的测量。对比使用FLS920全功能稳态/瞬态荧光光谱仪测得的群体细胞的pH标准曲线,极大地提高了探测的灵敏度(约3.8倍)。
我们测量了在相同的环境条件下的单个细胞之间的pH值。结果表明个体细胞pH是存在差别的,这种差别最大可以达到0.2/pH。因此传统的用平均的结果并不能反映单细胞之间化学信息的差异。
我们研究了细胞外pH值对单个细胞内pH值影响,发现没有基因敲除的酵母细胞比基因敲除的细胞(醇脱氢酶基因ADH2和ADH2等位基因同时敲除,或只敲除醇脱氢酶基因ADH2)具有更强的维持细胞内pH值的能力。特别是外部pH大于9.5的时候,这种差别更加突出。
The conventional analytical methods which generally choose a mass of cells as the research object could only get an average results of the chosen cells.So,it is often inaccurate or even wrong.But the research based on the nano-fiber optical sensing technology could give the information of a single cell.With the research at a single cell level,more accurate and comprehensive information which indicates the physiological state and the process of the cell could also be obtained.
We have built a system which suits single cell analysis.The system mainly involves an inverted fluorescence microscope,a single photon counter and a 100 ps data acquisition card.And we also have written a labview based acquisition procedure for the data acquisition card.Overall debugging indicates that our system is very stable and it can distinguish the fluorescence intensity of a single cell(cell was injected fluorescein).
With nano-fiber probes and fluorescence probes,we have measured intracellular pH value of a single yeast cell using the ratio method of the fluorescence intensity.Contrast with the result obtained from multitudinous cells by using the FLS920 combined fluorescence lifetime & steady state spectrometer,we have greatly improved the detection sensitivity(approximately 3.8 times).
We have measured the pH values of different single cells under the same environment.The results showed that different single cells have different pH values,even though they are under the same condition.The maximal divergence between them could reach 0.2/pH.So,the average results obtained by the conventional analytical method can't indicate the chemical information differences between the cells.
We have studied the effect of the extracellular pH on the intracellular pH value of a single cell.By comparing the effects of external pH_o on pH_i of both natural yeast cell and its heterozygote strains(one with ADH2 gene removed,the other with both ADH2 and its allele removed),a different behavior was observed between these strains,and the natural yeast cells have stronger power to regulate and maintain pH_i as a response to the pH_o changing.This difference will be more outstanding especially when the pH_o is above 9.5.
引文
1.O.S.Wolfbeis.Fiber-optic chemical sensors and biosensors.Anal.Chem.,2004,76:3269-3284.
2.W.Tan,Z.Y.Shi,S.Smith,D.Birnbaum,and R.Kopelman.Submicrometer intracellular chemical optical fiber sensors,science,1992,258:778-781.
3.W.Tan,Z.Y.Shi,and R.Kopelman.Development of submicron chemical fiber optic sensors.Anal.Chem.,1992,64:2985-2990.
4.T.Vo-Dinh,G.D.Griffin,J.P.Alarie,et al.Advanced nanosensors and nanoprobes[R].USA:foresight Institute,1998,143-148.
5.T.Vo-Dinh,J.P.Alarie,B.M.Cullum,G.D.Griffin.Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell,Nat.Biotechnol.,2000,18:764-767.
6.P.M.Kasili,J.M.Song,T.Vo-Dinh.Optical sensor for the detection of caspase-9 activity in a single cell.J.Am.Chem.Soc.,2004,126(9):2799-2806.
7.周继明,江世明.传感技术与应用.长沙:中南大学出版社,2005.
8.吴礼光,刘茉娥.生物传感器研究进展.化学进展,1995,7(4):285-301.
9.许改霞,王平,李蓉等.纳米传感技术及其在生物医学中的应用.国外医学生物医学工程分册,2002,25(2):49-54.
10.王玉田.光电子学与光纤传感器技术.北京:国防工业出版社,2003.
11.程介克.单细胞分析.北京:科学出版社,2005.
12.胡深,庞代文,王宗礼等.毛细管电泳安培测定鼠单个交感神经细胞中儿茶酚胺神经递质.分析化学,1998,26(6):752-756.
13.E.S.Christopher,D.M.Gavin,B.K.Tantiana,et al.Anal.Chem.,1998,70:4570-4577.
14.H.T.Chang,E.S.Yeung.Determination of Catecholamines in Single Adrenal Medullary Cells by Capillary Electrophoresis and Laser-Dnduced Native Fluorescence.Anal.Chem.,1995,67:1079-1083.
15.袁倬斌,赵红.单细胞分析方法的现状与展望.自然杂志,22(6):315-320.
16.宋玲,杨军民.中国药学杂志,1999,34(3):172-174.
17.徐修容,唐琴梅,陈生弟等.以D-樟脑-β-磺酸离子对试剂高效液相色谱-电化检测法测定帕金森症患猴脑组织多巴胺和代谢产物.色谱,1998,16(5).
18.S.C.Taylor,p.Chris.Biochem.Biophys.Res.Commun.,1998.248(1):13-17.
19.彭图志,杨丽菊,李惠萍等.碳纤维电极对大鼠的活体伏安分析.高等学校化学学报,1995.12(12):1847.
20.田臻锋,郭茂田,陈兴科等.纳米级PEBBLE传感器的研究进展.纳米器件与技术,2005.1:7-12.
21.B.Culshaw,J.Dakin,Fiber optical sensor,李少惠等译.武汉:华中理工大学出版社,1997.
22.廖廷彪.光纤光学,北京:清华大学出版社,2000.
23.P.M.Kasili.The Development of Optical Nanosensor Technology for Single Cell Analysis.[The dissertation of the university of Tennessee],2004.35-36.
24.陈智浩,顾东华.彭江得等.熔烧温度对双锥光纤光学特性的影响.中国激光,1998,25(3):257-260.
25.S.Anand,and R.Prasad.Rise in intracellular pH is concurrent with 'start' rogression of Saccharomyces cerevisiae.J.Gen.Microbiol.,1989,135:2173-2179.
26.I.H.Madshus.Regulation of intracellular pH in eukaryotic cells.J.Biochem..1988,250:1-8.
27.W.B.Busa,and R.Nuccitelli.Metabolic regulation via intracellular pH.Am.J.Physiol.,1984,246:409-438.
28.A.Kotyk.Intracellular pH of baker's yeast.Folia Microbiol(Praha).,1963,8:27-31.
29.E.P.Bakker.The role of alkali-cation transport in energy coupling of neutrophilic and acidophilic bacteria:an assessment of methods and concepts.FEMS Microbiol.Rev.,1990,75:319-334.
30.J.M.Salhany,T.Yamane,R.G.Shulman,and S.Ogawa.High resolution ~(31)P nuclear magnetic resonance studies of intact yeast cells.Proc.Natl.Acad.Sci.,1975,72:4966-4970.
31.R.B.Moon,and J.H.Richards.Determination of Intracellular pH by ~(31)P Magnetic Resonance.J.Biol.Chem.,1973,248:7276-7278.
32.J.Karagiannis,and P.G.Young.Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe.J,Cell Sci.,2001,114:2929-2941.
33.T.Imai,1.Nakajima,and T.Ohno.Development of a new method for evaluation of yeast vitality by measuring intracellular pH.J.Am,Soc.Brew.Chem.,1994,52:5-8.
34.P.Cimprich,J.Slavik,and A.Kotyk.Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae.FEMS Microbiol.Lett.,1995,130:245-252.
35.郭晓贤,宋浩雷,江贤章等.ADH2等位基因缺失的酿酒酵母杂合子的构建.中国医学研究与临床.2006,9(4):7-11.
36.董建军,史媛英,贾士儒等.胞内pH法评价酵母活性的研究.酿酒,2004.31(4):67-68.
37.U.Lutsdorf,and R.Megnet.Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae.Arch.Biochem.Biophys.,1968,126:933-944.
38.E.Racker.Alcohol dehydrogenase from bakers yeast.Method.Enzymol.,1955,1:500-503.
39.M.Ciriacy.Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae,Ⅱ.Two loci controlling synthesis of the glucose repressible ADH Ⅱ.Mol.Gen.Genet.,1975,138:157-164.
40.C.L.Denis,M.Ciriacy,and E.T.Young.A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae.J.Mol.Biol.,1981,148:355-368.
41.R.Serrano.Structure and function of proton translocating ATPase in plasma membranes of plants and fungi.Biochim.Biophys.Acta.,1988,947(1):1-28.
42.M.Ghislain,A.Goffeau.The pmal and pma2 H~+-ATPase from Schizosaccharomyces pombe are functionally interchangeable.J.Biol.Chem.,1991,266(27):18276-18279.
43.R.Serrano.Plasma membrane ATPase of fungi and plants as a novel type of proton pump.Curr.Top.Cell Reg.,1984,23:87-126.
44.J.Delhez,J.P.Dufour,D.Thines,A.Goffeau.Comparison of the properties of plasma membrane-bound and mitochondria-bound ATPases in the yeast Schizosaccharomyces pombe.Eur.J.Biochem.,1977,79(1):319-328.
2.W.Tan,Z.Y.Shi,S.Smith,D.Birnbaum,and R.Kopelman.Submicrometer intracellular chemical optical fiber sensors,science,1992,258:778-781.
3.W.Tan,Z.Y.Shi,and R.Kopelman.Development of submicron chemical fiber optic sensors.Anal.Chem.,1992,64:2985-2990.
4.T.Vo-Dinh,G.D.Griffin,J.P.Alarie,et al.Advanced nanosensors and nanoprobes[R].USA:foresight Institute,1998,143-148.
5.T.Vo-Dinh,J.P.Alarie,B.M.Cullum,G.D.Griffin.Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell,Nat.Biotechnol.,2000,18:764-767.
6.P.M.Kasili,J.M.Song,T.Vo-Dinh.Optical sensor for the detection of caspase-9 activity in a single cell.J.Am.Chem.Soc.,2004,126(9):2799-2806.
7.周继明,江世明.传感技术与应用.长沙:中南大学出版社,2005.
8.吴礼光,刘茉娥.生物传感器研究进展.化学进展,1995,7(4):285-301.
9.许改霞,王平,李蓉等.纳米传感技术及其在生物医学中的应用.国外医学生物医学工程分册,2002,25(2):49-54.
10.王玉田.光电子学与光纤传感器技术.北京:国防工业出版社,2003.
11.程介克.单细胞分析.北京:科学出版社,2005.
12.胡深,庞代文,王宗礼等.毛细管电泳安培测定鼠单个交感神经细胞中儿茶酚胺神经递质.分析化学,1998,26(6):752-756.
13.E.S.Christopher,D.M.Gavin,B.K.Tantiana,et al.Anal.Chem.,1998,70:4570-4577.
14.H.T.Chang,E.S.Yeung.Determination of Catecholamines in Single Adrenal Medullary Cells by Capillary Electrophoresis and Laser-Dnduced Native Fluorescence.Anal.Chem.,1995,67:1079-1083.
15.袁倬斌,赵红.单细胞分析方法的现状与展望.自然杂志,22(6):315-320.
16.宋玲,杨军民.中国药学杂志,1999,34(3):172-174.
17.徐修容,唐琴梅,陈生弟等.以D-樟脑-β-磺酸离子对试剂高效液相色谱-电化检测法测定帕金森症患猴脑组织多巴胺和代谢产物.色谱,1998,16(5).
18.S.C.Taylor,p.Chris.Biochem.Biophys.Res.Commun.,1998.248(1):13-17.
19.彭图志,杨丽菊,李惠萍等.碳纤维电极对大鼠的活体伏安分析.高等学校化学学报,1995.12(12):1847.
20.田臻锋,郭茂田,陈兴科等.纳米级PEBBLE传感器的研究进展.纳米器件与技术,2005.1:7-12.
21.B.Culshaw,J.Dakin,Fiber optical sensor,李少惠等译.武汉:华中理工大学出版社,1997.
22.廖廷彪.光纤光学,北京:清华大学出版社,2000.
23.P.M.Kasili.The Development of Optical Nanosensor Technology for Single Cell Analysis.[The dissertation of the university of Tennessee],2004.35-36.
24.陈智浩,顾东华.彭江得等.熔烧温度对双锥光纤光学特性的影响.中国激光,1998,25(3):257-260.
25.S.Anand,and R.Prasad.Rise in intracellular pH is concurrent with 'start' rogression of Saccharomyces cerevisiae.J.Gen.Microbiol.,1989,135:2173-2179.
26.I.H.Madshus.Regulation of intracellular pH in eukaryotic cells.J.Biochem..1988,250:1-8.
27.W.B.Busa,and R.Nuccitelli.Metabolic regulation via intracellular pH.Am.J.Physiol.,1984,246:409-438.
28.A.Kotyk.Intracellular pH of baker's yeast.Folia Microbiol(Praha).,1963,8:27-31.
29.E.P.Bakker.The role of alkali-cation transport in energy coupling of neutrophilic and acidophilic bacteria:an assessment of methods and concepts.FEMS Microbiol.Rev.,1990,75:319-334.
30.J.M.Salhany,T.Yamane,R.G.Shulman,and S.Ogawa.High resolution ~(31)P nuclear magnetic resonance studies of intact yeast cells.Proc.Natl.Acad.Sci.,1975,72:4966-4970.
31.R.B.Moon,and J.H.Richards.Determination of Intracellular pH by ~(31)P Magnetic Resonance.J.Biol.Chem.,1973,248:7276-7278.
32.J.Karagiannis,and P.G.Young.Intracellular pH homeostasis during cell-cycle progression and growth state transition in Schizosaccharomyces pombe.J,Cell Sci.,2001,114:2929-2941.
33.T.Imai,1.Nakajima,and T.Ohno.Development of a new method for evaluation of yeast vitality by measuring intracellular pH.J.Am,Soc.Brew.Chem.,1994,52:5-8.
34.P.Cimprich,J.Slavik,and A.Kotyk.Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae.FEMS Microbiol.Lett.,1995,130:245-252.
35.郭晓贤,宋浩雷,江贤章等.ADH2等位基因缺失的酿酒酵母杂合子的构建.中国医学研究与临床.2006,9(4):7-11.
36.董建军,史媛英,贾士儒等.胞内pH法评价酵母活性的研究.酿酒,2004.31(4):67-68.
37.U.Lutsdorf,and R.Megnet.Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae.Arch.Biochem.Biophys.,1968,126:933-944.
38.E.Racker.Alcohol dehydrogenase from bakers yeast.Method.Enzymol.,1955,1:500-503.
39.M.Ciriacy.Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae,Ⅱ.Two loci controlling synthesis of the glucose repressible ADH Ⅱ.Mol.Gen.Genet.,1975,138:157-164.
40.C.L.Denis,M.Ciriacy,and E.T.Young.A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae.J.Mol.Biol.,1981,148:355-368.
41.R.Serrano.Structure and function of proton translocating ATPase in plasma membranes of plants and fungi.Biochim.Biophys.Acta.,1988,947(1):1-28.
42.M.Ghislain,A.Goffeau.The pmal and pma2 H~+-ATPase from Schizosaccharomyces pombe are functionally interchangeable.J.Biol.Chem.,1991,266(27):18276-18279.
43.R.Serrano.Plasma membrane ATPase of fungi and plants as a novel type of proton pump.Curr.Top.Cell Reg.,1984,23:87-126.
44.J.Delhez,J.P.Dufour,D.Thines,A.Goffeau.Comparison of the properties of plasma membrane-bound and mitochondria-bound ATPases in the yeast Schizosaccharomyces pombe.Eur.J.Biochem.,1977,79(1):319-328.