室温离子液体在电分析化学中的应用研究

1、室温离子液体在电分析化室温离子液体在电分析化学中的应用研究学中的应用研究 第三届全国离子液体研究开发与应用技术研讨会第三届全国离子液体研究开发与应用技术研讨会王升富 熊华玉 陈婷 陈苗苗 王薇 海南省海口市2016.1.17-19湖北大学化学化工学院传统的非水介质有机溶剂体系缺点：传统的非水介质有机溶剂体系缺点：u易挥发，有毒，对环境污染大；易挥发，有毒，对环境污染大；u对一些无机底物不溶或溶解度小；对一些无机底物不溶或溶解度小；u需引入支持电解质，可能导致反应体系相对复杂；需引入支持电解质，可能导致反应体系相对复杂；u电化学窗口较窄，不利于一些反应的进行等。电化学窗口较窄，不利于一些反应的进

2、行等。 这些不足在一定程度上限制了生物大分子在非这些不足在一定程度上限制了生物大分子在非水介质中的研究应用水介质中的研究应用1.1.室温离子液体作为非水介质室温离子液体作为非水介质 室温离子液体优点：室温离子液体优点：u是一类新型的非水介质，被认为是传统有机溶剂的是一类新型的非水介质，被认为是传统有机溶剂的理想替代物，它不仅可减小合成工业中产生的污染，理想替代物，它不仅可减小合成工业中产生的污染，还可降低生产成本；还可降低生产成本；u具有溶剂和催化剂的双重功能；具有溶剂和催化剂的双重功能；u具有电位窗口宽，化学和热力学稳定性好，蒸汽压具有电位窗口宽，化学和热力学稳定性好，蒸汽压低，导电性能好等

3、特点；低，导电性能好等特点；u作为非水介质，受作为非水介质，受O2、pH等的影响小，可简化试等的影响小，可简化试验条件。验条件。 离子液体在电化学领域开始引起人们的极大兴趣离子液体在电化学领域开始引起人们的极大兴趣1.1 1.1 蛋白质在蛋白质在ILIL中的电化学及电催化中的电化学及电催化研究表明蛋白质在IL中具有更好的活性及稳定性离子液体作为非水介质，无需添加额外的电解质离子液体具有较好的催化作用 疏水性离子液体 亲水性离子液体1.1.1 固定在琼脂糖膜中血红素蛋白质在疏水性IL bmimPF6 中的电化学行为光谱表征Fig. 1.1.1. FTIR spectra and UV-vis s

4、pectra of (a) Hb-agarose film, (b) Hb film, and (c) Hb in the agarose film containing DMF. 外形表征Fig 1.1.2. AFM images of (A) agarose, (B) agarose-Hb, and (C) agarose-Hb-DMF on HOPG surfaces. Fig 1.1.3. (A) CVs in bmimPF6 for (a) agarose film, (b) Cat-agarose film, (c) Hb-agarose film, and (d) HRP-aga

5、rose film. (B) CVs in bmimPF6 for (a) agarose film, (b) Cyt c-agarose film, and (c) Mb-agarose film.蛋白质在bmimPF6中的电化学表征扫速对电化学行为的影响Fig 1.1.4. (A) CVs of Hb-agarose film in bmimPF6 over a range of scan rates (B) plots of cathodic and anodic ip vs v不同离子液体对蛋白质电化学行为的影响Fig 1.1.5. CVs of Hb-agarose film at

6、0.1 V s-1 in (a) bmim-PF6, (b) emimBF4, and (c) bmimBF4蛋白质在离子液体中对TCA ，t-BuOOH 的电催化行为Fig.1.1. 6. CVs of an HRP agarose/GC electrode in bmimPF6 containing TCA (left), t-BuOOH (right)Table 1.1. Kinetics Parameters of Different Substrates Electrocatalyzed by Protein-Agarose FilmsLangmuir 2005, 21, 9260-

7、92661.1.2 固定在单壁碳纳米管中血红素蛋白质在疏水性IL bmimPF6 中的电化学行为Fig. 1.1.7. CVs in bmimPF6 for (a) SWCNTs-CTABfilm, (b) HRP-SWCNTs-CTAB film, (c) Hb-SWCNTs-CTAB film and (d) Mb-SWCNTs-CTAB film.蛋白质的电催化行为Fig. 1.1.8. I-T curves of the Mb-SWCNTs-CTAB film on successive injection of t-BuOOH into bmimPF6. Table 1.2 Elec

8、trocatalytic parameters and relationship between catalytic current and concentrations of peroxides.Electrochemistry Communications 11 (2009) 2862891.1.3 固定在琼脂糖膜中蛋白质在亲水性ILbmimBF4 中的电化学行为Fig. 1.1.9. CVs in bmimBF4 containing 6.10%, 6.10%, 5.21%, 5.88 % (v/v) of H2O for (a) agarose, (b) Mbagarose, (c)

9、Hbagarose, and (d) Catagarose.离子液体中水含量的影响Fig. 1.1.10. (A) CVs of Catagarose/GC in (a) dry bmimBF4, (b) bmimBF4 containing 5.88 % H2O; (B) Dependence ofcathodic currents of Catagarose/GC on water contents.蛋白质在离子液体中的稳定性Fig. 1.1.11. Thermal stability and prolonged immersion time Stability of HRP in bmi

10、mBF4 with 4.53% (v/v) water.血红素蛋白质对H2O2的催化Fig. 1.1.12. A. CVs of an HRP-AG/GC in bmimBF4 with 4.53% (v/v) water and different H2O2; B. Plots of ipc vs. concentration of H2O2.相关参数的计算Electrochemistry Communications 9 (2007) 13371342Electrochemistry Communications 9 (2007) 17091714Table 1.3 Electrocata

11、lytic parameters and relationship between catalytic current and H2O2 concentrations1.1.4 固定在壳聚糖膜中血红素蛋白质在亲水性IL bmimBF4 中的电化学行为Fig. 1.1.13. AFM images of (a) chitosan; (b) chitosanDMF; (c) chitosanHRP; (d) chitosanHRPDMF on HOPG surfaces. Fig. 1.1.14. CVs in bmimBF4 for (a) chitosanfilm, (b) Cyt c, (c

12、) Cat, (d)HRP, (e) Hb, and (f) Mb.5种蛋白质的直接电化学行为蛋白质对H2O2的电催化Fig. 1.1.15. (A) CVs in bmimBF4 for HRP organohydrogel in different H2O2, and chitosan in different H2O2. (B) Plots of ip vs. the concentration of H2O2. 相关参数的计算Table 1.4 Electrocatalytic parameters and relationship between ip and H2O2 concen

13、trationsElectrochemistry Communications 9 (2007) 164816541.1.5固定在碳包镍-壳聚糖膜中蛋白质在ILbmimBF4中的电化学Fig. 1.1.16. SEM images of (a) CNNCS, (b) HbCNNCS, (c) HbCNNCSDMF.蛋白质的直接电化学Fig. 1.1.17. CVs of (a) CNNCSDMF, (b) MbCNNCSDMF, (c) MbCSDMF and (d) MbCNNCS in IL. CVs of CNNCSDMF without protein (a) and with HRP

14、 (b), Hb (c), Mb (d) or Cyt-c (e) in IL蛋白质对H2O2的催化Fig. 1.1.18. CVs and Amperometric response for HbCNNCSDMF/GCE in IL containing different H2O2电催化参数的计算Bioelectrochemistry 94 (2013) 9499Table 1.5 Electrocatalytic parameters and relationship between catalytic current and H2O2 concentrations.1.1.6 DNA修

15、饰蛋白质在两种IL中的电化学行为Fig. 1.1.19. AFM images of (A) Mb, (B) DNA, and (C) MbDNA直接电化学Fig. 1.1.20. CVs for (a) DNA film, (b) Mb film, (c) MbDNA film in (A) bmimPF6, and (B) bmimBF4 containing 6.9% H2O.条件优化Fig. 1.1.21. CVs of MbDNA after immersion in bmimPF6 for 0 -4 h. The relationship between Ip of MbDNA a

16、nd the water content in bmimBF4 solution.四种蛋白质的直接电化学Fig. 1.1.22. CVs for (a) DNA film, (b) HRPDNA film, (c) HbDNA film,(d) MbDNA film, and (e) CatDNA film in (A) bmimPF6 and (B) bmimBF4+ 6.9% H2O.Fig. 1.1.23. SWV and reverse current voltammograms for HbDNA film at different frequencies in (A) bmimPF

17、6; (B) bmimBF4 containing 6.98% water. 方波模拟图参数的计算Bioelectrochemistry 91 (2013) 814Table 1.6 The electrochemical parameters of the proteinDNA films in bmimPF6 and bmimBF4.本章小结：n1、亲水或疏水性离子液体都能作为蛋白质催化底物的溶剂n2、在离子液体中的蛋白质具有较好的稳定性及催化活性1.2 1.2 离子液体中自由基的产生及对生物离子液体中自由基的产生及对生物大分子损伤的电化学研究大分子损伤的电化学研究 研究背景：l 离子液体

18、粘度较大，自由基的扩散受到一定限制，导致自由基的寿命增加，可以帮助我们更详细地研究自由基损伤生物大分子的过程及机理 l 离子液体对大部分有机及无机物具有较好的溶解性，可以直接研究抗氧化剂小分子等对损伤的抑制作用。自由基产生的方法 经典Fenton试剂 酶催化1.2.1 离子液体中Fenton自由基的产生以及对DNA的损伤Scheme 1.2.1 Schematic diagram of DNA damage.在离子液体中产生自由基的证明Fig. 1.2.1. ESR spectra were obtained with FeSO4, H2O2 and DMPO in 1 mL BMIMPF6.

19、Fig.1.2.2. Ipt/Ip0 of Co(bpy)33+ for DNA after and before incubation with: (a) BMIMPF6 +FeSO4+H2O2, (b) IL, (c) IL + FeSO4, (d)IL+H2O2, and (e) PBS + FeSO4+ H2O2.Fig. 1.2.3. Dependence of Ipt/Ip0 in Co(bpy)33+ for DNA after and before incubation with IL+: (a)FeSO4 for 10 min, and then H2O2 for 30 mi

20、n, (b) FeSO4 for 10 min, 10 mM EDTA, and then H2O2, (c) FeSO4, H2O2 and AA, (d) FeSO4 and AA, and (e) H2O2 and AA.Fig. 1.2.4. (A) Ip of Co(bpy)33+ for DNA after incubation with BMIMPF6 + FeSO4+ H2O2 at different times. (B) Dependence of Ipt/Ip0 of Co(bpy)33+ on incubation time for DNA in IL+ (a) FeS

21、O4, H2O2; (b) FeSO4,H2O2 and AA.不同抗氧化剂的影响Fig. 1.2.5 Ipt/Ip0 of Co(bpy)33+for DNA film after and before incubation with IL+ (a) FeSO4, H2O2, (b)a+AA, (c) a+AE, (d) a+rutin, (e) FeSO4, AA, (f) H2O2, AA.抗氧化剂浓度的影响Fig. 1.2.6 Influence of concentrations of antioxidants on Ipt/Ip0 in Co(bpy)33+ for DNA aft

22、er incubated in BMIMPF6 containing FeSO4, H2O2 and () AE, () AA, () rutinFig. 1.2.7 Dependence of Ipt/Ip0 in Co(bpy)33+ for DNA film in BMIMPF6 containing: (a) FeSO4, H2O2, and catalase, (b) FeSO4, H2O2 and inactivated catalase, (c) FeSO4 and catalase, (d) H2O2 and catalaseSensors and Actuators B 16

23、1 (2012) 274 278Microchim Acta 176 (2012) 4794841.2.2 离子液体中酶催化葡萄糖产生H2O2及自由基的产生及其对DNA的损伤Scheme 1.2.2 Schematic diagram for working principle of SWV detection of in situ DNA damage for DNA-GOx film损伤条件的对比Fig. 1.2.8 Dependence of Ipt/Ip0 inCo(bpy)33+ for DNA-GOx (a) after incubation inBMIMPF6 + FeSO4 +

24、 glucose;(b) FeSO4 in IL; (c) glucose in IL; for DNA (d) FeSO4 in IL; (e) FeSO4 and glucose in IL; (f) glucose in IL; for GOx film (g) FeSO4 in IL; (h) FeSO4 and glucose inIL; (i) glucose in ILFig. 1.2.9 Ipt/Ip0 of Co(bpy)33+for DNA-GOx film after and before incubation with: (a) PBS (4.0)+ FeSO4, gl

25、ucose; (b) PBS (7.0)+FeSO4, glucose; (c) BMIMPF6 +FeSO4,+ glucose阻抗对比图Fig. 1.2.10 Nyquist plots in impedance measurements of electrodes: bare GCE (a); the DNA-GOx/GCE before (b) and after (c) incubation with FeSO4 and glucose in BMIMPF6. 损伤条件的优化Fig. 1.2.11 Dependence of Ipt/Ip0 in Co(bpy)33+ on （A）i

26、ncubation time for DNA-GOx film after incubated with (a) FeSO4 and glucose; (b) FeSO4; (c) glucose in ILdifferent concentration of (B)FeSO4 and (C) glucose.ABC抗氧化剂的影响Fig. 1.2.12 Ipt/Ip0 of DNA-GOx film in Co(bpy)33+ after incubation (a) FeSO4, glucose in IL; (b) AA+a; (c) AE+a; (d) Rutin +a抗氧化剂浓度和时间

27、的影响Fig. 1.2.13 Influence of (A) concentrations of different antioxidant and (B) incubation time on Ipt/Ip0 for AA (a), AE (b), and Rutin(c).Microchim Acta 178 (2012) 45511.2.3 离子液体中多巴胺催化Fe3+产生自由基对DNA的损伤Scheme 1.2.3. Schematic diagram about DNA damage initiated by Fe3+ catalyzed oxidation of dopamine

28、 in IL.损伤条件的对比图Fig. 1.2.14 Dependence of It/I0 in Co(bpy)33+for DNA incubation with (a) dopamine, Fe3+in bmimPF6; (b) FeSO4, H2O2 in IL, (c) dopamine, Fe3+ in PBS; (d) Fe3+ in IL;(e) dopamine in IL; (f) blank.Fig. 1.2.15. EIS spectra of the bare GCE (a); DNA/GCE before (b) and after (c) incubation w

29、ith IL+ dopamine + Fe3+. Inset: Randles equivalent circuit used to model impedance data损伤条件的优化Fig. 1.2.16. It/I0 of Co(bpy)33+on (A) incubation time and (B) different molar ratio of dopamine/Fe3+ for the DNA/GCE incubated with bmimPF6 +dopamine + Fe3+. 抗氧化剂的影响Fig. 1.2.17. (A) DPV curves of DNA film

30、in Co(bpy)33+ in IL before (a) and after incu-bation with (b) d+AA; (c) d+rutin; (d) dopamine + Fe3+. (B) Effect of antioxidant concentrations on It/I0 of Co(bpy)33+ for DNA.Electrochimica Acta 114 (2013) 265 270 1.2.4 亲水性离子液体中Fenton自由基的产生以及对BSA的损伤Fig. 1.2.18. (A) Six consecutive CVs recorded at nan

31、o CNi/Au kept in PoPD solution. (B) The corresponding EQCM frequency change observed for 6 CVs as that of (A). (C) EQCM frequency change observed for BSA at the PoPD/CNi/Au surface.Fig. 1.2.19 (A) SWVs of PoPD/CNi (a and b), BSA/PoPD/CNi (c and d), CNi (e) before (solid line) and after (dashed line)

32、 incubation with bmimBF4+ Fe2+ +H2O2 (B) EIS of BSA/PoPD/CNi before (a) and after (b) incubation with bmimBF4 +Fe2+ + H2O2.Fig. 1.2.20. SWV oxidation peak current ratio of BSA/PoPD/CNi films after and before incubation in bmimBF4 containing: (a) Fe2+ + H2O2, (b) blank,(c) Fe2+, (d) H2O2.PBS与IL作为介质对B

33、SA损伤的对比Fig. 1.2.21. Ipt/Ip0 of BSA/PoPD/CNi after and before incubation in (a) PBS +12.5mM Fe2+ + H2O2, (b) bmimBF4+ 12.5mMFe2+ +H2O2 (c) PBS+ 50mM Fe2+ + H2O2, (d) IL+50mM Fe2+ + H2O2.抗氧化剂浓度的影响Fig. 1.2.22. Influence of concentrations of different antioxidant (AE , AA and catechin) on Ipt/Ip0 for BS

34、A/PoPD/CNi films.AE吸附在BSA的证明Fig. 1.2.23. Ipt/Ip0 of BSA/PoPD/CNi films after and before incubation in: (a) bmimBF4 + Fe2+ +H2O2, (b)AE + (a), (c) bmimBF4 + AE, (d) bmimBF4 + AE for 30 min, water rinse, dry in air, then bmimBF4 + Fe2+ + H2O2.过氧化氢酶的影响Fig. 1.2.24. Ipt/Ip0 of BSA/PoPD/CNi films after an

35、d before incubation with: (a) bmimBF4 + Fe2+ + H2O2, (b)Cat + (a), (c) IL+ Cat, (d) IL+ Cat and + Fe2+, (e) IL + Cat + H2O2, (f) IL +inCat, (g) inCat + (a).Sensors and Actuators B 169 (2012) 368 3731.2.5 疏水性离子液体中Fenton自由基的产生以及对BSA的损伤Scheme 1.2.4. Schematic diagram about BSA damage. Fig. 1.2.25. (A)

36、I/I0 of Co(bpy)33+for BSA after incubated with Fenton reagents or control in BMIMPF6. (B) I/I0 of BSA after and beforeincubated with: PBS containing Fenton for 20 (a) or 40 min (b); IL containing Fenton for 20 (c) or 40 min (d).交流阻抗Fig. 1.2.26. EIS spectra of BSA before (a)and after (b) incubation w

37、ith FeSO4 and H2O2. Inset: Randles equiva-lent circuit条件的优化Fig. 1.2.27. Effect of I/I0 in Co(bpy)33+for BSA in BMIMPF6 on the molar ratio of Fe2+and H2O2 at (A)8mM H2O2.(B) 0.5 mM Fe2+. (c)DPV of Co(bpy)33+for BSA on the incubation time after incubated with Fenton.抗氧化剂的影响Fig. 1.2.28. (A) DPVs of Co(

38、bpy)33+for BSA in IL (a) before, (d) after incubated with Fe2+H2O2, (b) (d) + AA, (c) (d) + catechin. (B) Influence of antioxidant concentrations on I/I0 of Co(bpy)33+for BSA after incubated in IL+ FeSO4, H2O2and (a) AA, (b) catechin.Sensors and Actuators B 188 (2013) 741 746 本章小结n1、离子液体作为溶剂，研究自由基对生

39、物大分子的损伤是可行的，而且对大分子的损伤程度大于水体系中。n2、亲疏水性离子液体均可作为溶剂，两者对自由基损伤生物大分子的研究结果未见明显区别。2. 室温离子液体作为非水电解质室温离子液体作为非水电解质n无毒，对环境污染小；n电化学窗口宽，有利于一些反应的进行，可以帮助我们更详细地研究某些反应的机理等。n离子液体的种类繁多，可以根据需要选择合适的离子液体作为电解质不同支持电解质的对比图Fig. 2.1.1 CVs of BR in DMF containing (b) KClO4, (c) 1 vol.% of bmimPF6, absence of BR containing (a) 1

40、vol.% of bmimPF6 as the supporting electrolyte .不同浓度BR叠加CVFig. 2.1.2 CVs in DMF containing 1 vol.% of bmimPF6 for (a) blank solvent only, (b) - (e) different concentrations of BR. 不同电解时间后，BR的紫外可见光谱图Fig. 2.1.3 (A)Spectra and (B) CVs in DMF containing 1 vol.% of bmimPF6 for 2.210-4 M BR solution elect

41、rolyzed at 0.65 V vs. SCE. (a) Initial solution, (bf): 12 - 36 h passed. Fig. 2.1.4 CVs in DMF containing 1 vol.% of bmimPF6 for BR at different concentrations (A)(af: 0 - 4.510-4 M).(B) (af: 5.410-4 - 1.610-3 M). 不同浓度BR的叠加图BR电化学氧化路径Am. J. Biomed. Sci. 2011, 3(3), 191-198 SCHEME 2.1: Main electroche

42、mical oxidation pathways of BR in (left) neutral form (right) basic form 3. 3. 离子液体作为电极修饰剂离子液体作为电极修饰剂 离子液体粘度大，导电性能好，可以用作碳糊电极或玻碳电极的黏粘剂，比不导电的黏粘剂的效果好碳糊电极玻碳电极3.1 离子液体修饰蛋白质碳糊电极 SEMFig. 3.1.1. SEM micrographies of CPE (a) and CPIE (b).条件的优化Fig. 3.1.2. Effect of varying the quantity of IL (carbon + paraffi

43、n oil + Mb 79:14:7, 73:14:6:7; . 64:12:17:7; 57:11:25:7) on the amperometric response to H2O2. HRP对H2O2的催化Fig. 3.1.3. Currenttime curves for successive H2O2 additions to phosphate buffer at the HRPCPIE (a), the HRPCPE (b), and the CPE (c). 稳定性Fig. 3.1.4. (A)Amperometric response of the HRPCPIE in th

44、e range of pH 3.59.0 to H2O2. (B) Effect of varying the temperature of (a) HRPCPE, (b) HRPCPIE on the current response to H2O2. 时间稳定性Fig. 3.1.5. Variation of i-t to H2O2 of ILcontained Mb-sensor with time. Electrochemistry Communications 9 (2007) 267126753.2 离子液体修饰碳糊电极对小分子的测定Fig. 3.1.6 SEM micrograp

45、hies of (A) CPE and (B) CPIE不同修饰电极的对比图Fig. 3.1.7 CVs of rutin in B-R (pH 3.0) buffer solution, at (a) the DNA-CPE, (b) the CPIE and (c) the DNA-CPIE扫速的影响Fig. 3.1.8 CVs of rutin in B-R buffer solution (pH 3.0) at the DNA-CPIE at different scan rates. Inset: Plot of Ip vs. scan rate. 离子液体含量的影响Fig. 3.1

46、.9 Influence of content of IL (carbon powder: paraffin oil: IL a=84:16:0, b=82:15:3; c=80:15:5; d=78:14:8)Rutin浓度的测定Fig. 3.1.10 DPV curves of rutin in B-R solution at the DNA-CPIE at different concentrations Inset: Calibration plot of peak current vs. concentration of rutin回收率的测定Table 3.1 Recovery test of rutinMicrochim Acta 170 (2010) 2732离子液体修饰碳糊电极对AE的测定Fig. 3.1.11 CVs of AE at b the CPIE and c the CPE in buffer; Also shown CVs of a the CPIE in the absence of AE.pH和扫速的影响Fig. 3.1.12 (A)CVs of AE in Tris-HCl buffer at different pHs. (B)