室溫離子液體在電分析化學中的應用研究

1、室溫離子液體在電分析化室溫離子液體在電分析化學中的應用研究學中的應用研究 第三屆全國離子液體研究開發(fā)與應用技術(shù)研討會第三屆全國離子液體研究開發(fā)與應用技術(shù)研討會王升富 熊華玉 陳婷 陳苗苗 王薇 海南省?？谑?016.1.17-19湖北大學化學化工學院傳統(tǒng)的非水介質(zhì)有機溶劑體系缺點：傳統(tǒng)的非水介質(zhì)有機溶劑體系缺點：u易揮發(fā)，有毒，對環(huán)境污染大；易揮發(fā)，有毒，對環(huán)境污染大；u對一些無機底物不溶或溶解度??；對一些無機底物不溶或溶解度小；u需引入支持電解質(zhì)，可能導致反應體系相對復雜；需引入支持電解質(zhì)，可能導致反應體系相對復雜；u電化學窗口較窄，不利于一些反應的進行等。電化學窗口較窄，不利于一些反應的進

2、行等。 這些不足在一定程度上限制了生物大分子在非這些不足在一定程度上限制了生物大分子在非水介質(zhì)中的研究應用水介質(zhì)中的研究應用1.1.室溫離子液體作為非水介質(zhì)室溫離子液體作為非水介質(zhì) 室溫離子液體優(yōu)點：室溫離子液體優(yōu)點：u是一類新型的非水介質(zhì)，被認為是傳統(tǒng)有機溶劑的是一類新型的非水介質(zhì)，被認為是傳統(tǒng)有機溶劑的理想替代物，它不僅可減小合成工業(yè)中產(chǎn)生的污染，理想替代物，它不僅可減小合成工業(yè)中產(chǎn)生的污染，還可降低生產(chǎn)成本；還可降低生產(chǎn)成本；u具有溶劑和催化劑的雙重功能；具有溶劑和催化劑的雙重功能；u具有電位窗口寬，化學和熱力學穩(wěn)定性好，蒸汽壓具有電位窗口寬，化學和熱力學穩(wěn)定性好，蒸汽壓低，導電性能好等

3、特點；低，導電性能好等特點；u作為非水介質(zhì)，受作為非水介質(zhì)，受O2、pH等的影響小，可簡化試等的影響小，可簡化試驗條件。驗條件。 離子液體在電化學領(lǐng)域開始引起人們的極大興趣離子液體在電化學領(lǐng)域開始引起人們的極大興趣1.1 1.1 蛋白質(zhì)在蛋白質(zhì)在ILIL中的電化學及電催化中的電化學及電催化研究表明蛋白質(zhì)在IL中具有更好的活性及穩(wěn)定性離子液體作為非水介質(zhì)，無需添加額外的電解質(zhì)離子液體具有較好的催化作用 疏水性離子液體 親水性離子液體1.1.1 固定在瓊脂糖膜中血紅素蛋白質(zhì)在疏水性IL bmimPF6 中的電化學行為光譜表征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.蛋白質(zhì)在bmimPF6中的電化學表征掃速對電化學行為的影響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不同離子液體對蛋白質(zhì)電化學行為的影響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蛋白質(zhì)在離子液體中對TCA ，t-BuOOH 的電催化行為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 固定在單壁碳納米管中血紅素蛋白質(zhì)在疏水性IL bmimPF6 中的電化學行為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.蛋白質(zhì)的電催化行為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 固定在瓊脂糖膜中蛋白質(zhì)在親水性ILbmimBF4 中的電化學行為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.離子液體中水含量的影響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.蛋白質(zhì)在離子液體中的穩(wěn)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.血紅素蛋白質(zhì)對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.相關(guān)參數(shù)的計算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 固定在殼聚糖膜中血紅素蛋白質(zhì)在親水性IL bmimBF4 中的電化學行為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種蛋白質(zhì)的直接電化學行為蛋白質(zhì)對H2O2的電催化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. 相關(guān)參數(shù)的計算Table 1.4 Electrocatalytic parameters and relationship between ip and H2O2 concen

13、trationsElectrochemistry Communications 9 (2007) 164816541.1.5固定在碳包鎳-殼聚糖膜中蛋白質(zhì)在ILbmimBF4中的電化學Fig. 1.1.16. SEM images of (a) CNNCS, (b) HbCNNCS, (c) HbCNNCSDMF.蛋白質(zhì)的直接電化學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蛋白質(zhì)對H2O2的催化Fig. 1.1.18. CVs and Amperometric response for HbCNNCSDMF/GCE in IL containing different H2O2電催化參數(shù)的計算Bioelectrochemistry 94 (2013) 9499Table 1.5 Electrocatalytic parameters and relationship between catalytic current and H2O2 concentrations.1.1.6 DNA修

15、飾蛋白質(zhì)在兩種IL中的電化學行為Fig. 1.1.19. AFM images of (A) Mb, (B) DNA, and (C) MbDNA直接電化學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.條件優(yōu)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.四種蛋白質(zhì)的直接電化學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. 方波模擬圖參數(shù)的計算Bioelectrochemistry 91 (2013) 814Table 1.6 The electrochemical parameters of the proteinDNA films in bmimPF6 and bmimBF4.本章小結(jié)：n1、親水或疏水性離子液體都能作為蛋白質(zhì)催化底物的溶劑n2、在離子液體中的蛋白質(zhì)具有較好的穩(wěn)定性及催化活性1.2 1.2 離子液體中自由基的產(chǎn)生及對生物離子液體中自由基的產(chǎn)生及對生物大分子損傷的電化學研究大分子損傷的電化學研究 研究背景：l 離子液體

18、粘度較大，自由基的擴散受到一定限制，導致自由基的壽命增加，可以幫助我們更詳細地研究自由基損傷生物大分子的過程及機理 l 離子液體對大部分有機及無機物具有較好的溶解性，可以直接研究抗氧化劑小分子等對損傷的抑制作用。自由基產(chǎn)生的方法 經(jīng)典Fenton試劑 酶催化1.2.1 離子液體中Fenton自由基的產(chǎn)生以及對DNA的損傷Scheme 1.2.1 Schematic diagram of DNA damage.在離子液體中產(chǎn)生自由基的證明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.不同抗氧化劑的影響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.抗氧化劑濃度的影響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 離子液體中酶催化葡萄糖產(chǎn)生H2O2及自由基的產(chǎn)生及其對DNA的損傷Scheme 1.2.2 Schematic diagram for working principle of SWV detection of in situ DNA damage for DNA-GOx film損傷條件的對比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阻抗對比圖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. 損傷條件的優(yōu)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抗氧化劑的影響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抗氧化劑濃度和時間

27、的影響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 離子液體中多巴胺催化Fe3+產(chǎn)生自由基對DNA的損傷Scheme 1.2.3. Schematic diagram about DNA damage initiated by Fe3+ catalyzed oxidation of dopamine

28、 in IL.損傷條件的對比圖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損傷條件的優(yōu)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+. 抗氧化劑的影響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 親水性離子液體中Fenton自由基的產(chǎn)生以及對BSA的損傷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與IL作為介質(zhì)對B

33、SA損傷的對比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.抗氧化劑濃度的影響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的證明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.過氧化氫酶的影響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 疏水性離子液體中Fenton自由基的產(chǎn)生以及對BSA的損傷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條件的優(yōu)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.抗氧化劑的影響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 本章小結(jié)n1、離子液體作為溶劑，研究自由基對生

39、物大分子的損傷是可行的，而且對大分子的損傷程度大于水體系中。n2、親疏水性離子液體均可作為溶劑，兩者對自由基損傷生物大分子的研究結(jié)果未見明顯區(qū)別。2. 室溫離子液體作為非水電解質(zhì)室溫離子液體作為非水電解質(zhì)n無毒，對環(huán)境污染??；n電化學窗口寬，有利于一些反應的進行，可以幫助我們更詳細地研究某些反應的機理等。n離子液體的種類繁多，可以根據(jù)需要選擇合適的離子液體作為電解質(zhì)不同支持電解質(zhì)的對比圖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 .不同濃度BR疊加CVFig. 2.1.2 CVs in DMF containing 1 vol.% of bmimPF6 for (a) blank solvent only, (b) - (e) different concentrations of BR. 不同電解時間后，BR的紫外可見光譜圖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). 不同濃度BR的疊加圖BR電化學氧化路徑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. 離子液體作為電極修飾劑離子液體作為電極修飾劑 離子液體粘度大，導電性能好，可以用作碳糊電極或玻碳電極的黏粘劑，比不導電的黏粘劑的效果好碳糊電極玻碳電極3.1 離子液體修飾蛋白質(zhì)碳糊電極 SEMFig. 3.1.1. SEM micrographies of CPE (a) and CPIE (b).條件的優(yōu)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對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). 穩(wěn)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. 時間穩(wěn)定性Fig. 3.1.5. Variation of i-t to H2O2 of ILcontained Mb-sensor with time. Electrochemistry Communications 9 (2007) 267126753.2 離子液體修飾碳糊電極對小分子的測定Fig. 3.1.6 SEM micrograp

45、hies of (A) CPE and (B) CPIE不同修飾電極的對比圖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掃速的影響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. 離子液體含量的影響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濃度的測定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回收率的測定Table 3.1 Recovery test of rutinMicrochim Acta 170 (2010) 2732離子液體修飾碳糊電極對AE的測定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和掃速的影響Fig. 3.1.12 (A)CVs of AE in Tris-HCl buffer at different pHs. (B)