实验操作
1.Buffer:取100mL EtOH与400mL Tris-HCl(4mM pH=6)混合均匀后备用;
2.铁离子标液:取氯化铁用水溶解后定容,配置成2mM 氯化铁水溶液;
3.Sensor:取探针用乙醇溶解定容,配置成100uM 铁离子探针乙醇溶液;
4.制作标准曲线:取400uL Buffer,加入50uL Sensor,一定量的铁离子标液,用纯水补齐至500uL,混匀后,以空白样(#0)校零后,测试590nm处的荧光值.根据铁离子浓度和对应荧光值制作标准曲线.
测试Fe3+ | Buffer | Sensor | Fe3+ | H2O |
#0 | 400uL | 50uL | 0 | 50uL |
#1 | 400uL | 50uL | 5uL | 45uL |
#2 | 400uL | 50uL | 10uL | 40uL |
#3 | 400uL | 50uL | 15uL | 35uL |
#4 | 400uL | 50uL | 20uL | 30uL |
4.测试样品:取400uL Buffer,加入50uL Sensor,50uL水样,混匀后,测试590nm处的荧光值.根据标准曲线计算水样中铁离子的含量.
注意事项
1.水样如果浑浊需过滤处理后才能进行测试;
2.如果水样中铁离子含量较高,请稀释一定倍数后再进行测试;
3.探针避光低温保存,若没用完的Sensor溶液请低温避光保存,以免变质影响检测;
4.在以上测试条件下,Hg离子会有一定的影响,如果测试样品中含有Hg离子,可加入1% 二巯基丙磺酸钠对Hg离子进行掩蔽.
五 附录
Fig1.(a) Fluorescence spectra of derivative 1 (10 uM) in an ethanol–water (1:4, v:v, Tris–HCl pH 6) solution in the presence of different metal ions (20 equiv). Metal ions include Fe3+, K+, Na+, Mg2+, Ca2+, Mn2+, Co2+, Fe2+, Ni2+, Cu2+, Zn2+, Sr2+, Ag+, Cd2+, Hg2+ and Pb2+. Excitation/emission was performed at 570/593 nm.
