Ionic liquids designed for advanced applications in bioelectrochemistry

Abstract

Recent applications of ionic liquids (ILs) as sustainable media in biochemistry and bioelectrochemistry are reviewed. The use of ILs as solvents for biopolymers such as proteins is particularly important in bioelectrochemistry. Maintenance of the higher-ordered structure of proteins after dissolution in IL is essential for the applications envisaged. The affinity between ILs and proteins is discussed in relation to the design of new solvents. IL-modified electrodes are another important topic for bioelectrochemistry. Some bio-related facets of ILs are also reviewed for the understanding of the usefulness of ILs for this field. The use of ILs in bioscience has led to the development of new types of biodevices, and has also been mentioned.

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Kyoko Fujita (back, left), Miyuki Masuda (back, middle), Kenichi Murata (back, right) Nobuhumi Nakamura (front, left), Hiroyuki Ohno (front, right)

Kyoko Fujita received her PhD degree (2004) from Tokyo University of Agriculture and Technology (TUAT; Japan). During her PhD studies in Prof. Ohno’s group, she received a Research Fellowship for Young Scientists at the Japan Society for the Promotion of Science (JSPS). Before working at TUAT, she worked as a postdoctoral research fellow with Prof. D. R. MacFarlane and Prof. M. Forsyth at Monash University (Australia) until 2006. She is a postdoctoral research fellow in TUAT. Her research interests are bioelectrochemistry especially ionic liquids as protein solvents and the effective water state for bioscience.

Kenichi Murata received his BEng (2006), MEng (2007), and PhD (2009) degrees in biotechnology all from TUAT. This was followed by a JSPS postdoctoral research fellow under the direction of Prof. N. Nakamura until 2010. His thesis was focused on enzymatic biofuel cells. In 2010, he started research at Sony Corporation (Tokyo, Japan). His scientific interests are focused on bioelectronic devices.

Miyuki Masuda received her BEng (2008) and MEng (2010) degrees from TUAT. She is currently a PhD candidate in the Department of Biotechnology in TUAT under the direction of Prof. N. Nakamura. Her PhD research is focused on ethanol–O2 enzymatic biofuel cells.

Nobuhumi Nakamura received his BSc (1988) and MSc (1990) degrees in Chemistry from Tokyo University of Science (Japan) and his PhD degree from Osaka University (Japan) in 1993. He spent 5 years as a postdoctoral research fellow (at Osaka University as a JSPS postdoctoral fellow, at Oregon Graduate Institute (USA), and at Kyushu University (Fukuoka, Japan)). In 1998 he moved to TUAT as a lecturer and then he was promoted to an Associate Professor in 2003. His research interests are focused on electron transfer of enzymes and biodevices.

Hiroyuki Ohno received a PhD degree in 1981 from Waseda University, Tokyo, Japan. After working at Waseda University and Case Western Reserve University (USA), he moved to TUAT as an Associate Professor in 1988. He was promoted to professor in 1997. His recent research activities are concentrated on the science of ionic liquids, especially the design of functional ionic liquids. He has published more than 100 papers and reviews on ionic liquids in this decade. He served as director of the university library, Vice Dean and Dean of his university. FRSC since 2008.

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1. Introduction

Chemists are now familiar with the words “ionic liquid” as these compounds have totally different properties from molecular liquids. The key point of ILs is their non-volatile nature. Reports on ILs are still increasing. The research areas with ILs gradually shifted from electrochemistry to biochemistry and bioelectrochemistry. The performance of bioelectrochemical devices can be improved significantly by using ILs instead of volatile molecular liquids. We also envisage ILs as superior alternatives to volatile organic solvents. In the early stages, research on ILs focused on the two fields of organic synthesis and electrochemical technology. Many interesting findings have already been summarised in various books and reviews.^1–6 In recent years, research on ILs has diversified into various areas, including biotechnology and nanotechnology. In the field of biotechnology, extensive studies have been made on biotransformation,^7–13 two-phase extraction systems,¹⁴ and biomass treatment.^15–19 Electrodeposition of metals^20–23 and synthesis of metal nanoparticles^24,25 are major topics in nanotechnology. As the boundary between electrochemistry and bioscience is another important field for ILs, this review concentrates on research related to both bioscience and electrochemistry.

2. Advanced properties of ILs

2.1 ILs for electrochemical devices

ILs are melts of organic salts that exist in the liquid state over a wide temperature range, and sometimes below room temperature. These compounds are generally composed of an organic cation and an organic or inorganic anion. The presence of a bulky and structurally asymmetric cation gives rise to lower cohesive energies for crystalline forms than in inorganic salts such as NaCl, and guarantees the ionic state of the liquid phase. The interaction between the ions that make up the IL are strong, long-range Coulomb forces and are responsible for the remarkable physicochemical properties of ILs, such as low melting point, almost zero pressure of saturated vapour, non-combustibility, and a wide electrochemical potential window. These properties make it possible to improve the durability and safety of electrochemical devices, extending the range of operating temperature and enabling improvements in power and energy density.⁶ Therefore, ILs are superior alternatives for electrolyte solutions used in electrochemical devices such as electric double layer capacitors, fuel cells, lithium batteries and solar cells.

ILs are generally redox-robust. They typically have a wide potential window, which is one of the most important advantages of ILs as electrolytes.²⁶ Potential windows of 4.5–5.0 V have been reported for ILs, and even an electrochemical potential window of 7.0 V for 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim]BF₄) and 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim]PF₆).²⁷ These values are not reached by ionic liquids that contain water, however. Addition of more than 3 wt% water to [C4mim]BF₄ and [C4mim]PF₆ decreased the electrochemical window by at least 2.0 V,²⁸ although water reduced the viscosity and thereby increased the conductivity. In bioelectrochemistry, a small amount of water is often added to ILs. In particular, the electrodes described below in the sub-section “ILs for modified electrodes” operate in contact with aqueous solution.²⁹ When using ILs with water, the resulting change in physicochemical properties should be taken into account.

2.2 ILs for biosciences

The use of ILs as a protein matrix is the main spur for the development of ILs for application in the biosciences. ILs are currently being studied as a novel matrix, so that proteins can be used in a wider range of conditions,^8,30 to improve reaction efficiency,³¹ in new devices,³² and for the preservation of biomaterials.^33,34 Proteins generally are handled in a buffer solution. Concentrated salt solutions are not used for dissolving proteins, because the concentration of ions leads to denaturation of the protein. In fact, proteins do not dissolve readily in general ILs.^30,35,36 When proteins dissolve in ILs, structural changes have been observed.^36–38 A homogeneous solution of proteins in ILs is not essential for the study of ILs as protein solvents, because it has been reported that enzymes dispersed in ILs exhibit much greater activity than in buffer.^30,39 Protein dissolution is attractive from the viewpoint of reaction efficiency and application. It is important to understand the properties of ILs which make them capable of dissolving proteins in a homogeneous manner. This would further the capability and durability of bioelectrochemical devices in which ILs are used as an alternative electrolyte solution.

Table 1 Chemical structure of ILs described in this article

Cations		Anions
a Welton et al. described that the abbreviation of methyl groups on both imidazolium and pyrrolidinium cations should be “C1”, not “m”. We referred to a methyl group only here, so the methyl group was abbreviated to “m”.
	1-Alkyl-3-methylimidazolium^a	BF4^−	Tetrafluoroborate
	[Cnmim] (n = 2–12)		BF4
	1-(2-Ethoxyethanol)-3-methylimidazolium^a	PF6^−	Hexafluorophosphate
	[et2mim]		PF6
	1-[2-(2-Ethoxy)-ethoxyethanol]-3-methylimidazolium^a		Ethylsulfate
	[et3mim]		[C2SO4]
	N-Alkyl-N-methylpyrrolidinium^a	Br^−	Bromide
	[Cnmpyrr] (n = 3, 4)		Br
	Cholinium	Cl^−	Chloride
	[ch]		Cl
	1-Allyl-3-methylimidazolium		Dihydrogen phosphate
	[amim]		[dhp]
	1-Methyl-3-(2,6-(S)-dimethylocten-2-yl)-imidazolium		Bis(trifluoromethanesulfonyl)imide
	[mdim]		[NTf2]
	1-Octylpyridinium	HCOO^−	Formate
	[C8pyr]		HCOO
	1-Allyl-3-ethylimidazolium		Methylsulfate
	[aeim]		[CH3OSO3]
	1-Ethylimidazolium
	[C2im]

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This section looks at the design of ILs; chemical modification of proteins is introduced by focusing on the dissolving of proteins.

Pure ILs as protein solvents. Hoagland et al. reported that cytochrome c (cyt c) dissolved in a neat hydrophilic IL.⁴⁰ Cyt c was heated at 60 °C in a vacuum for 1–5 days, during which it dissolved fully in 1-ethyl-3-methylimidazolium ethylsulfate ([C2mim][C2SO₄]). Various optical analyses show that the tertiary structure changes after dissolution, but the secondary structure remains almost intact. This change was not a typical protein denaturation occurring in solution, such as aggregation and precipitation. In fact, the iron–sulphur bond of the Met 80 ligand of the heme group, was disrupted (see Fig. 1). The ligand disturbance and molecular expansion of cyt c lead to an enhancement in peroxidase activity. This study concluded that the dissolution of enzymes in neat ILs commonly required molecular interactions strong enough to disturb the higher ordered structure of enzymes. The detailed mechanism of dissolution of cyt c in neat [C2mim][C2SO₄] is not yet clear.

Fig. 1 Soret region CD spectrum of cyt c in 0.01 M, pH 7 phosphate buffer, and IL.⁴⁰

Polarity is an important factor in the dissolving of various materials in ILs. The polarity of an IL is generally estimated via solvatochromism of some organic dyes.^41–43 The hydrogen bond donor ability (α value), hydrogen bond acceptor ability (β value) and the dipolarity–polarisability (π* value) were calculated from the affinity between an IL and several dyes, inferred from observations of the maximum absorption wavelength. Anions in ILs giving high β values are reported to facilitate the dissolution of cellulose; this would be an important practical breakthrough.⁴⁴ ILs with a high β value have been recognised to break intra- and inter-molecular hydrogen bonds in cellulose fibres. Such highly polar ILs also dissolved silk and other biopolymers.⁴⁵ The maintenance of protein structure after dissolution is important in retaining function, yet the change to the higher ordered structure generally causes loss of function when proteins dissolve in such highly polar solvents.

It has been reported that dried polar ILs having a chloride anion dissolve cyt c.⁴⁶ Cyt c was mixed in several ILs and was stirred for 30 min at 80 °C. Cyt c was dissolved in 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) at a final concentration of 10 mM, and in 1-butyl-3-methylimidazolium bromide ([C4mim]Br) at concentrations of 10 mM and 0.5 mM. The solubility of cyt c has been found to depend on the cation as well as the anion. To investigate the factors influencing solubility, the polarity of ILs was estimated in terms of the Kamlet–Taft parameters. Polar ILs having β > 0.7 proved effective in dissolving cyt c. (Fig. 2). No clear correlation between the value of α and the solubility of cyt c was found. Against this, correlation between the values of π* and β and the solubility of cyt c was observed. These results suggest that β and π* are significant parameters for the dissolution of cyt c. A Soret band was observed at 409 nm for cyt c in these ILs by UV-vis spectroscopy. The charge transfer (CT) band at 695 nm was not confirmed. This data suggests that the electronic state of the heme was not changed, but that there is a perturbation or loss of the axial sulphur ligand of the heme. Tuning of the polarity is a valuable procedure for selecting ILs to dissolve proteins.

Fig. 2 Solubility dependence of cyt c on the π* and β values of the ILs (X, insoluble; △, < 0.05 mM; ○, 0.5 mM; and ⊚, 10 mM).⁴⁶

Hydrated ILs as potential solvents for native proteins. The effect of polarity on the dissolution of protein in IL has been described above. Is there any occasion to use pure ILs as a solvent for proteins? In fact, even dry proteins have some adsorbed water molecules. Furthermore, it is difficult to maintain anhydrous IL in practical applications. This suggests the deliberate addition of a small amount of water to the IL to improve its solvent properties without changing its fundamental properties. A mixture of IL and water provides a promising way to dissolve proteins without structural change.⁴⁷ The major component of such a mixture would be the IL, not water. There are many papers about IL–water mixtures, but most involve a concentrated aqueous salt solution. In these concentrated aqueous salt (IL) solutions, the high ionic concentration has unfavourable effects on proteins. Nevertheless, there is considerable improvement in the solubility of proteins upon adding a small amount of water to ILs.⁴⁷ To investigate hydrated ILs as solvents for proteins, some cations and anions were studied and cyt c was used as the test protein. The solubility of cyt c in hydrated ILs depends on the component ions even in the presence of the same amount of water. Cyt c tended not to dissolve in the most hydrated ILs examined. The correlation between ion structure and solubility suggests that ILs anions possessing oxo-acid residues further the dissolution of proteins.⁴⁸ The electron transfer ability of cyt c dissolved in differing hydrated ILs was not the same. One effect of the component ions on protein activity after dissolution is known as the kosmotropicity, which is ordered in value similar to the Hofmeister series.⁴⁹ The kosmotropicity is related to the structural formation of water around ions. Zhao studied the effect of kosmotropicity on the enzymatic reaction in a mixture of water and ILs.³¹ Kosmotropic anions stabilise proteins and kosmotropic cations destabilise them.

Improved stability of protein was observed in hydrated ILs; this is impossible in an aqueous phase. Long-term stability of cyt c stored in hydrated IL containing 20 wt% water (three water molecules per ion pair) has been confirmed. The IL was composed of cholinium ([ch]) cations and dihydrogen phosphate ([dhp]) anions. Upon keeping cyt c in hydrated form [ch][dhp] (Hy[ch][dhp]) for three weeks at room temperature, about 90% of redox activity for the freshly prepared buffer solution was observed.⁴⁸ The secondary and tertiary structures of cyt c dissolved in several hydrated ILs were investigated using circular dichroism (CD) spectroscopy and Raman spectroscopy. The resulting spectra suggested that the vicinity of the active site was slightly changed in most hydrated ILs, such as N-butyl-N-methyl pyrrolidinium dihydrogen phosphate ([C4mpyrr][dhp]). Cyt c dissolved in Hy[ch][dhp] maintained a higher ordered structure, as in buffer (Fig. 3). Cyt c dissolved in Hy[ch][dhp] retained 70% of activity after storage at room temperature for 18 months.⁴⁸ The thermal stability of cyt c was also improved, unlike in buffer solution.

Fig. 3 CD spectra of dissolved cyt c in (a) [C4mpyrr][dhp], (b) Hy[ch][dhp] and (c) buffer.⁵¹

The solubility in Hy[ch][dhp] of proteins other than cyt c has also been investigated. Horseradish peroxidase (HRP), hemoglobin (Hb), myoglobin (Mb), azurin, pseudoazurin and ascorbate oxidase were added to Hy[ch][dhp] containing 20 wt% water. Hemoglobin and myoglobin dissolved only slightly in Hy[ch][dhp]; the other proteins dissolved readily.^50,51 Albumin, with a high helix content of about 70%, was almost completely insoluble. The reason is still unclear. Although it is difficult to dissolve any proteins in Hy[ch][dhp], dissolution of target proteins should be possible by suitable design of the ion structure and by controlling the water content.

Solubilisation methods for general proteins in various ILs. When proteins will not dissolve in hydrated ILs, chemical modification is an effective method. Polyethylene oxide (PEO) modification⁵² or comb-shaped polyethylene glycol (PEG) modification⁵³ on the protein surface has proved effective. The effect of the length of the PEO chain on the solubilisation of proteins in ILs has been analysed.⁵⁴ Furthermore, a comb-shaped PEG was more effective than a linear chain PEG in modifying enzymatic activity in ILs.⁵³ Unfortunately, this chemical modification is complicated and it is difficult to control the modification state. In another method, solubilisation has been confirmed by mixing protein and organic materials. For example, a mixture of crown ether in IL and surface coating of the protein surface by ether structures,⁵⁵ IL,⁵⁶ PEG⁵⁷ and IL-based gel⁵⁸ has been reported. Some ILs are favourably interacted with PEO chains, according to the hard and soft acids and bases (HSAB) theory.⁵⁹

3. Application of ILs in biodevices

3.1 ILs as a matrix for proteins

ILs should be capable of acting as a device matrix such as a battery electrolyte, since they are thermally and chemically stable compared with traditional matrices.^1,3,60 In regard to biodevices,^61,62 the availability of ILs should improve the conductivity and the thermal and long-term stability of proteins. In this section, recent studies of the electrochemistry of proteins in IL solvents will be reviewed.

For homogeneously dissolved proteins. Electrochemical analysis of proteins dissolved in ILs is difficult, because of the high viscosity of the ILs. Optical measurement to characterise the electron transfer reaction in ILs is then preferred to general electrochemical methods.³⁰ This is because optical data such as spectral changes are integrated data, and it is easy to detect changes, in contrast to data such as current changes. The inter- and intra-electron transfer reactions of cellobiose dehydrogenase (CDH) dissolved in Hy[ch][dhp] has been investigated by UV-vis spectroscopy.⁵⁰ CDH is a monomer comprising two separate domains, containing flavin and heme respectively. These domains work as an electron acceptor and an electron donor. The electrochemical analysis clearly revealed electron transfer reactions from cellobiose to CDH and from CDH to dissolved cyt c, acting as an electron acceptor in Hy[ch][dhp].

The redox reaction of PEO-modified cyt c dissolved in 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4mim][NTf₂]) has been analysed by optoelectrochemistry.⁵² Optical waveguide spectroscopy is an advanced method for detecting and analysing redox species dissolved in ILs.² Progress in the electron transfer reaction of PEO-modified cyt c dissolved in [C4mim][NTf₂] was observed only after addition of KCl. The role of the chloride anion has been estimated as a charge compensator for heme.⁵⁴

The redox reaction of cyt c dissolved in a pure polar IL was also analysed by optical waveguide spectroscopy.⁴⁶ Cyt c was dissolved in 1-allyl-3-methylimidazolium chloride ([amim]Cl), which is a highly polar IL having low viscosity among the chloride salts. After dissolution of cyt c at 80 °C, the redox response was analysed at room temperature by optical waveguide spectroscopy. A spectral shift of the Soret band was observed with an applied potential. A similar spectral shift was observed even at 140 °C. This is a typical example of the fact that ILs provide a thermally stable matrix for proteins due to their relatively high viscosity; the thermal motion of molecules is less vigorous than in ordinary liquids with lower viscosity.

The redox reaction has been studied of protein dissolved in a mixture of IL and water using an electrochemical method, thin layer cyclic voltammetry.⁶³ Direct and rapid redox reaction of Hb or Mb was observed in an aqueous solution containing 1 M [C4mim]BF₄. The IL was observed to play a key role in the electron transfer. In general, no redox reaction of Hb was observed in aqueous KCl solution containing low concentrations of [C4mim]BF₄. A higher [C4mim]BF₄ concentration gives a better signal-to-noise ratio, up to 2 M. A good redox response was observed by using some IL, although it is difficult to use IL as a solvent.

ILs for immobilised proteins. Electrochemical analysis of dissolved biomaterials having large molecular weight is difficult in high-viscosity ILs, because of the very small diffusion coefficient. To get around this problem, electrochemical analysis in ILs of immobilised heme proteins on the electrode have been reported.³² Compton et al. reported the redox responses of cyt c immobilised with an alkanethiol self-assembled monolayer (SAM) having carboxyl terminal.⁶⁴ Cyt c does not retain redox activity in dry [C4mim]PF₆ or dry [C4mim][NTf₂]. Reversible redox couples were observed after the electrode was transferred from IL to buffer solution. The redox responses also failed in water-saturated ILs. A small amount of water is clearly important.

Wang et al. reported the redox responses of heme proteins immobilised on the electrode with agarose hydrogel films in [C4mim]PF₆ and [C4mim]BF₄.^65–67 Direct electron transfer (DET) reactions were analysed for Hb, Mb, peroxidase, cyt c and catalase.⁶⁵ The electrocatalytic reaction of these heme proteins entrapped in agarose hydrogel on the electrode was also investigated using trichloroacetic acid and tert-butyl hydroperoxide in [C4mim]PF₆. Electrocatalytic reduction of H₂O₂ by heme proteins entrapped in agarose hydrogel films was also observed. A small amount of water in [C4mim]BF₄ is necessary to maintain the electrochemical activities of these heme proteins.⁶⁶ The authors concluded that addition of water to the hydrophilic IL was necessary to realise the activity of proteins entrapped in agarose hydrogel. However, as mentioned by Gorton,⁶⁸ it is important to check the redox potential and the state of proteins immobilised on the electrode in order to gain accurate insight into the redox activity. A desorption of heme from the protein might occur during immobilisation of proteins on the electrode.

DET reaction of heme proteins and catalytic activity has also been investigated using cetyltrimethylammonium bromide films containing single-wall carbon nanotubes (SWCNTs–CTAB).⁶⁹ A pair of well defined and nearly reversible responses was observed in [C4mim]PF₆. The electrode was prepared by casting the mixture of protein solution and SWCNTs–CTAB, followed by drying at room temperature. The resulting electrode was expected to retain the water molecules in the SWCNTs–CTAB film. As a result of the hydrophobicity of [C4mim]PF₆, water molecules were believed to be trapped at the electrode surface, and contributed to the enzymatic reaction. When proteinase K–SWCNTs conjugates were introduced into ILs containing 5.0% (v/v) water, greater enzymatic activity was observed in hydrophobic ILs.⁷⁰ The enzymatic activity in highly hydrophobic ILs was about 10 times greater than in hydrophilic ILs.

A similar effect of hydrophobicity of ILs on the electron transfer reaction of cyt c₃ has been reported.⁷¹ The redox reaction of cyt c₃ immobilised on pyrolytic graphite by casting was investigated in [C2mim]BF₄, [C4mim]BF₄, and [C2mim][NTf₂]. A better response was observed in hydrophobic [C2mim][NTf₂], and the current was almost 30 times greater than that in 4-(2-hydroxyethyl)piperazine-1-(ethanesulfonic acid) (HEPES) buffer, as seen in Fig. 4. This result suggests that a hydration layer of some sort was formed around proteins in hydrophobic ILs. No increase in the peak current and potential shift was observed when an electrode modified with cyt c₃ was directly transferred from HEPES buffer to [C2mim][NTf₂]. However, no such increase of redox current observed in [C2mim][NTf₂] was observed in [C2mim]BF₄ or [C4mim]BF₄ even though the redox response was confirmed. The signal in [C4mim]BF₄ was not stable with time, and it shifted gradually toward the positive potential side. Based on these results, hydrophilic ILs are considered to absorb water and destroy hydrate shells around proteins, as hydrophilic organic solvents did. This might explain the denaturation of proteins. These results are closely related to the kosmotropicity. The redox response of cyt c₃ in [C4mim]BF₄ was greater than that in [C2mim]BF₄, because the [C4mim] cation was less chaotropic than [C2mim]. Although the direct electron transfer reaction of cyt c₃ was confirmed in ILs, H₂ oxidation of hydrogenase was not observed in the three ILs described above, [C2mim]BF₄, [C4mim]BF₄ and [C2mim][NTf₂]. A small amount of water was believed to exist so as to realise the direct electron transfer reaction of proteins immobilised on the electrode. More hydrophobic ILs are suitable as the matrix for electron transfer reaction of proteins. Water saturated hydrophobic ILs have the capability to act as a matrix for protein electrochemistry.

Fig. 4 Cyclic voltammograms at 20 mV s⁻¹ of cyt c₃ adsorbed onto the PG electrode in RTILs: [C4mim]BF₄ (black line), [C2mim][NTf₂] (dark grey line), [C2mim]BF₄ (fine grey line).⁷¹

In terms of water removal from proteins, hydrophobic ILs are preferable for electrochemistry. Proteins should be active when sufficient (but not excess) water molecules are maintained around them. ILs containing a certain amount of water, so-called hydrated ILs, have accordingly been analysed as solvents for proteins, as mentioned above. A well-defined redox response was observed in hydrated ILs. Hy[ch][dhp] exhibited good affinity with some proteins, and an improvement in thermal and long-term stability of the dissolved proteins was confirmed.^48,51 In Hy[ch][dhp], a redox response of enzymes was also observed.⁷²

A fructose dehydrogenase (FDH)-immobilised gold nanoparticle electrode was studied in Hy[ch][dhp]. The electron transfer reaction, based on the electrochemically catalysed oxidation of D-fructose via direct electron transfer between the active center of FDH and the electrodes, was clearly detected in Hy[ch][dhp] (Fig. 5 (a)). No redox response of FDH was observed in the absence of fructose (see Fig. 5 (b)). Similarly, a CDH-immobilised nanoparticle electrode gave rise to a catalytic current in the presence of cellobiose. CDH certainly catalysed the oxidation of cellobiose through a DET reaction between the active site of CDH domains and the electrode in Hy[ch][dhp]. Furthermore, substrate inhibition was observed, the same as that in the water phase. The long term stability of the bioelectroactivity of the CDH immobilised electrode in Hy[ch][dhp] was investigated. The activity of CDH immobilised on the electrode was maintained after three weeks at room temperature in Hy[ch][dhp], although no reaction was detected in the water phase. These results indicate that Hy[ch][dhp] is a candidate for the construction of durable biodevices.

Fig. 5 Cyclic voltammograms of FDH-modified MET–AuNP electrode in the presence of (89 mM) (a) and absence (b) of D-fructose in Hy[ch][dhp].⁷²

In the protein-immobilised system, many parameters were not controlled well, such as the protein orientation on the electrode surface. Selection of the mediator for the DET reaction of proteins was a further issue. In the fixing system, however, the effect of diffusion was eliminated even in ILs having high viscosity. The construction of a stable device should prove possible

3.2 ILs for modified electrodes

ILs can be used not only as the supporting electrolyte for the electrochemical process, but also as the modifier for chemically modified electrodes. The high ionic conductivity of ILs makes them preferable for electrode materials in sensing devices. Microdroplets and thin film deposits of 1-methyl-3-(2,6-(S)-dimethyloctene-2-yl)-imidazolium tetrafluoroborate ([mdim]BF₄) on a pyrolytic graphite electrode surface were first studied by Wadhawan et al.⁷³ Subsequently, many papers have been published about IL-modified electrodes for sensing devices.

IL-type carbon paste electrodes for electrochemical sensing. ILs have been successfully applied to the fabrication of carbon paste electrodes (CPEs).^74–76 CPEs are widely used in electrochemical and bioanalytical applications, because of their advantages over other electrodes of easy preparation and renewal processes.⁷⁷ Traditional CPEs have been made from a mixture of conducting graphite powder and a non-conductive liquid binder, such as paraffin oil. ILs can act as an alternative to the binder of CPE as a result of their suitable physicochemical properties. Unlike the classic binder for CPE, IL-binder is composed of charged species, and exhibits ionic conductivity. In addition, the use of ILs can overcome the problem of impurities in these oils (such as sulphides) which may have undesirable effects on detection and analysis. The high viscosity of ILs is considered to be a valuable property in the fabrication process of CPEs. Liu et al.⁷⁴ reported the first IL-based carbon paste electrode (IL-CPE), using hydrophobic [C4mim]PF₆, and showed that the IL causes an increase in the sensitivity of the response toward potassium ferricyanide. Unfortunately, the very high background current of this electrode has limited its application. This high background current is probably due to the ordering of the cations and anions comprising the IL in response to a change in the applied potential at the electrode–electrolyte interface. To overcome this problem, Maleki and co-workers⁷⁶ adopted a new strategy for preparing paste electrodes based on the use of n-octylpyridinium hexafluorophosphate ([C8pyr]PF₆) as an IL binder. The resulting electrode provides very low background current and also gives a surprisingly high electrochemical performance after the electrode is compressed and heated to the melting point of the IL. Fig. 6 compares the typical morphological features of CPE and IL([C8pyr]PF₆)-CPE. A scanning electron microscopy (SEM) image of the IL-CPE shows a uniform surface, although CPE have irregularly shaped micrometre-sized flakes of graphite.⁷⁶ The uniformity of the surface indicates good adherence of [C8pyr]PF₆ to the graphite. The [C8pyr]PF₆ both lowers the background current and improves the electrochemical response. The IL-CPE provides a remarkable increase in the rate of electron transfer of different organic and inorganic electroactive compounds, and offers a marked decrease in the overvoltage for the electrochemical detection of biomolecules such as reduced nicotinamide adenine dinucleotide (NADH), dopamine, and ascorbic acid.⁷⁶ The electrochemical behaviour of IL-CPE based on [C8pyr]PF₆ is better than that of IL-CPE using imidazolium based IL, 1-octyl-3-methylimidazolium hexafluorophosphate ([C8mim]PF₆), because of the difference in the inherent catalytic activity of the IL.⁷⁸ The heating process required for IL-CPE with [C8pyr]PF₆ is not suitable for biosensors, however, because of the possible inactivation of biomacromolecules at high temperatures. Compton and co-workers⁷⁹ reported that the use of multi-walled carbon nanotubes (MWCNTs) instead of graphite can prevent the heating process, and an IL-CPE composed of MWCNT and [C8pyr]PF₆ has a very low background current, high sensitivity and stability.

Fig. 6 SEM images of (a) CPE and (b) IL-CPE 50 : 50 of graphite–[C8pyr]PF₆ electrodes.⁷⁶

IL-modified electrodes for enzymatic electrochemical reactions. Several authors have reviewed IL-modified electrodes for bioelectronic devices using proteins or enzymes.^29,32,80 Here, pioneering work and noteworthy recent reports on IL-modified electrodes will be selected and reviewed.

Both biocompatibility and a large surface area-to-volume ratio for high enzyme loading are necessary for the electrode material to immobilise enzymes. To realise the stable immobilisation of enzymes in biocompatible polymers, nanoparticles and/or other supporting materials have been added, although the immobilisation of enzymes without any modifier except for IL has also been reported.^81,82 Many biocompatible materials immobilise enzymes when incorporated in IL-modified electrodes; for instance, polymers,^83–91 polymerised ILs,^92–95 sol–gel derived glass,⁹⁶ sol–gel ILs,⁹⁷ mesoporous molecular sieve MCM-41,⁹⁸ CaCO₃ nanoparticles,⁹⁹ and clay.^100–103 These films can retain the native structure of enzymes and enhance the electrochemical response at electrodes. The biocompatibility of these composite films is most commonly assessed by UV-vis absorption spectroscopy and FT-IR spectroscopy. Lu et al.⁸³ showed that a chitosan–[C4mim]BF₄–Hb composite is more thermally stable than a chitosan–Hb mixture, according to thermogravimetric analysis data. This is a good example of an IL which provides high ionic conductivity and further benefits to a composite film.

It is possible to realise a large surface area for high enzyme loading by using electro-conductive nanomaterials. Single-walled carbon nanotubes (SWCNTs) can be dispersed in imidazolium-based ILs by mechanical milling so as to form a thermally stable gel,¹⁰⁴ although SWCNTs are easily entangled with each other to form agglomerates. The SWCNTs in the gel exist as a three-dimensional network of considerably untangled and much finer bundles that are physically cross-linked due to cation–π and π–π interactions between the imidazolium cations and CNTs.^104,105 In bioelectrochemistry, SWCNT,^106–108 MWCNT^93,109–118 and graphene^95,119–122 are commonly used to fabricate such composites. Furthermore, metal nanoparticles^123–128 and several inorganic semiconductive nanomaterials, such as NiO,¹²⁹ MnO₂,¹³⁰ CoO₂,¹³¹ V₂O₅,¹³² Fe₂O₃,¹³³ and CdS,¹³⁴ have also been used to form IL-nanomaterial composites. The extremely low volatility of ILs makes it possible to study their morphology by SEM; this is not possible for other molecular liquid-modified electrodes. It has been reported that IL–carbon nanomaterial composites at electrodes show a unique structure and form a relatively uniform surface.^115,135

IL-modified electrodes used in an aqueous solution have been considered. Since the solubility of hydrophobic ILs in water is much smaller than that of water in the IL, the IL-modified electrodes function stably. Hydrophilic ILs have also been used, and the resulting electrodes are stable, at least on a voltammetric timescale. This may be a result of the adsorption of ILs on the electrode. The stability of IL-modified electrodes should therefore be discussed carefully. To prevent leaking of ILs, polymerised IL-wrapped CNTs were designed and fabricated by direct polymerisation of IL monomers in the presence of CNTs.⁹³ The resulting stable IL-functionalised composites are considered to be based on the strong π–π stacking interaction between the imidazolium ion moieties and the CNTs.¹³⁶

Biodevices, such as biosensors and biofuel cells based on enzymatic reactions, consist of two components: a bioreceptor and a transducer. The bioreceptor is an enzyme that recognises the target analyte, whereas the transducer converts the recognition at the molecular level into a measurable signal. The transducer system should be selected and designed by taking into account aspects of the enzymatic reaction. In enzymatic biosensors based on an IL-modified electrode, the amperometric technique is commonly used to measure the signal, although a transistor-type sensing system has recently been reported.¹³⁷

For some enzymes, such as hydrolase (including organophosphorus hydrolase,¹¹³ lipase,¹¹² acetylcholine esterase¹¹⁶) and some oxidases (including laccase^{102,103,125,138} and polyphenol oxidase¹⁰¹), the products are redox active and can easily be detected at the substrate electrode. In particular, organophosphorus hydrolase catalyses hydrolysis of p-nitrophenolphosphate to p-nitrophenol, and the resulting p-nitrophenol, is electrochemically detectable.¹¹³

It has been reported that IL–nanomaterial composites electrochemically catalyse the electrochemical reaction of biomolecules with low overpotentials. The lowered overpotential is attributed to the properties of the nanoparticles, but the IL also has inherent catalytic activity.⁷⁸ NADH and H₂O₂ can be detected with a low overpotential at IL-modified electrodes. Since NADH and H₂O₂ are involved in many enzymatic reactions, it is important to detect them with a lower overpotential. The combination of H₂O₂-producing oxidases and IL–nanomaterial composites provides a biosensor for the corresponding substrate of the oxidases. Many IL-modified electrodes based on materials that reduce H₂O₂ have been reported for the detection of glucose,^{92,93,95,107,110,119} cholesterol,¹²⁶ and glutamate¹²⁷ by applying the corresponding oxidases.

Many oxidoreductases can communicate electrochemically with the electrode via a reversible redox active molecule known as an “electron transfer mediator”. In this system, mediators and/or cofactors should be immobilised at the electrode surface with the enzyme. HRP was immobilised with ferrocene as a mediator in a IL-based sol–gel matrix.⁹⁶ The efficient mediated bioelectrocatalysis of NADH-dependent dehydrogenase is difficult. This is because it is not easy to immobilise NADH on the electrodes together with enzymes, and NADH often has to be added to the analyte solution.¹¹⁵ Yu et al. successfully immobilised NADH by developing a NADH-based IL and forming a SWCNT–IL bucky gel as shown in Fig. 7.¹⁰⁸

Fig. 7 (A) Bioelectrocatalytic oxidation of glucose at the multifunctional gel based biosensor in 0.1 M phosphate buffer (pH 7.0) in the absence (black current) or presence (red curve) of 40 mM glucose. Scan rate, 1 mV s⁻¹. Inset, digital picture of the prepared multifunctional gel. (B) Illustration of the reaction schemes involved in the bioelectrocatalytic oxidation of glucose at the multifunctional gel based biosensor.¹⁰⁸ Methylene green (MG) was used together with NADH as transducers.

DET reactions, in which enzymes directly exchange electrons with electrodes without a mediator, can be performed using some oxidoreductases and redox proteins. However, the DET reaction between the enzymes and the traditional bare working electrode is problematic, because of deeply buried electroactive prosthetic groups in the apoprotein domain. Modified electrodes have therefore been designed to provide a suitable microenvironment for enzymes and facilitate DET reactions. In the last few years, ILs have also been used to fabricate DET-type electrodes as components for modified electrodes. IL-modified electrodes for Hb,^83,99,111 Mb,^82,106,139 cyt c,¹⁰⁶ and HRP^109,132 have all been reported to improve the DET reaction toward biosensors. Multicopper oxidases such as laccase and bilirubin oxidase, which oxidise a wide variety of compounds with concomitant oxygen-reduction to H₂O, have also been incorporated into IL-modified electrodes to facilitate the DET reaction.^97,117,140 Electrodes with multicopper oxidase can be used both as a sensing component for their substrate, as phenols (electron donor) and oxygen (electron acceptor), and as a catalyst of oxygen reduction for biofuel cells. IL–MWCNT bucky-paper (BP) facilitates the diffusion of oxygen more readily than agglomerated MWCNT, and the composite facilitates the DET reaction of laccase as shown in Fig. 8.¹¹⁸

Fig. 8 Galvanostatic polarisation curves of different electrodes in O₂-saturated citrate buffer. (a) 30 μM thick bucky-paper cathodes without laccase (black squares), with adsorbed laccase (DET, black circles), and 2,2′-azinobis(3-ethylebenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (mediated electron transfer, gray triangles). (b) Comparison of different MWCNT-based electrodes with the current density normalised to the mass of carbon nanotubes (note the logarithmic scale). Electrodes fabricated as bucky-paper (black circles) and by the conventional technique (gray triangles) were investigated with adsorbed laccase in the absence of a mediator.¹¹⁸

The study of IL-modified electrodes for biofuel cells is still in its early stages.^61,141 The design of ILs for biofuel cell is interesting from the viewpoint of their high ionic conductivity. ILs have tremendous potential as solvents for biofuel cells.⁶¹

As stated, IL-modified electrodes for biodevices have been developed by hybridising enzymes, ILs, and polymers or nanomaterials. Because of the synergetic effects of electro-conductive materials together with biocompatible polymers and ionic conductive ILs, biosensors developed with a composite of these materials have fast responses, high sensitivity, low detection limits, and high stability. The value of ILs for electrode modification has been recognised, and led to a rapid increase in the number of papers on this subject.²⁹

4. Application of ILs in electrochemical devices using biopolymers

Biopolymers, such as enzymes and proteins, have been widely studied as transducers for amperometric biosensors and biofuel cells. Some biopolymers have also been regarded as functional materials. There are many reports on the preparation of composites of ILs and biopolymers. This section reviews recent reports of composites as polymer electrolytes for electrodevices.

To prepare polymer electrolytes composed of ILs and biopolymers, many biopolymers have been utilised, including gelatin,¹⁴² DNA,^143–148 and several polysaccharides such as cellulose,^149–151 chitosan,^152,153 chitin,¹⁵⁴ and agarose.^155,156 These biopolymer-based composites possess high mechanical strength, flexibility, and biodegradability. In addition, the added ILs gave high ionic conductivity to the composites. These properties are necessary for electrolyte-substituent membranes in electrodevices such as actuators, lithium ion batteries, and solar cells.¹⁵⁷ Most of these polymer electrolytes were prepared by simple mixing of biopolymers with ILs. Another method includes preparing IL-like domains in biopolymers through the neutralisation of charged groups in the biopolymers.^145,148 These ionic liquidised biopolymers provide novel applications as ion conducting materials.

4.1 Composites comprised of ILs and saccharides

In the field of electrodevices, cellulose has been regarded as a passive element and has been used as a separator in solid-state batteries.¹⁵⁸ As cellulose does not dissolve in conventional molecular liquids, it is difficult to prepare cellulose-based composites. Against this, it is known that ILs having chloride anions are good solvents of cellulose.¹⁵⁹ As mentioned above, some ILs are expected to dissolve cellulose, based on their high polarity. Recently, a few polar ILs have been designed to dissolve cellulose under mild conditions.^44,160 These experiments are furthering the preparation of cellulose-based composites using ILs as solvents.¹⁶¹

A supercapacitor has been prepared based on a polymer electrolyte composed of cellulose and MWCNTs, using [C4mim]Cl.¹⁴⁹ This IL was used both as a solvent for cellulose and as an electrolyte in the resulting composite. The composite has high mechanical strength and flexibility. According to electrochemical measurement, the specific capacitance of this composite is 22 F g⁻¹. The power density of this supercapacitor is 1.5 kW kg⁻¹, which is comparable to commercially available ones (0.01–10 kW kg⁻¹). This supercapacitor could be operated from −78 to +150 °C, a much wider range of temperatures than for existing capacitors (from −50 to +85 °C).

The incorporation of an organoboron unit improves the selectivity of target cation transport in polymer electrolytes, and/or enhances the ionic conductivity of the matrix.¹⁶² It is also known that polysaccharides, including cellulose, bind boric acid via the condensation reaction.¹⁶³ Incorporation of a boric acid unit into cellulose using this reaction is a useful way to prepare highly ion conductive materials.¹⁵⁰ As shown schematically in Fig. 9, an organoboron gel composed of 1-allyl-3-ethylimidazolium formate ([aeim]HCOO), boric acid, and lithium hydroxide had high ionic conductivity, of 1.98 × 10⁻³ S cm⁻¹ at 30 °C, comparable to that of pure IL (1.74 × 10⁻³ S cm⁻¹).¹⁵⁰

Fig. 9 Synthesis of an organoboron ion gel.¹⁵⁰

As another way to improve the lithium ion conduction of cellulose based-composites, cellulose triacetate (CTA) was used as a supporting polymer electrolyte.¹⁵¹ ILs composed of cations having an ester group have been reported to improve lithium ion conduction.¹⁶⁴ The use of CTA as a supporting polymer for the preparation of polymer electrolyte affects the lithium ion conduction because CTA contains functional ester groups. The resulting polymer electrolyte composed of CTA, lithium bis(trifluoromethanesulfonyl)imide (Li[NTf₂]), and N-methyl-N′-propylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([C3mpyrr][NTf₂]) exhibited high ionic conductivity (10⁻³–10⁻⁴ S cm⁻¹). FT-IR and conductivity measurements confirm that the ester group of CTA is responsible for enhancing the dissociation of Li[NTf₂].

Other biopolymers have been used as supporting scaffolds of polymer electrolytes containing ILs. ILs generally have high intrinsic ionic conductivity. An ion gel prepared with agarose was reported to have ionic conductivity almost as high as the neat ILs.^155,156 This is because the agarose-based ion gel could be prepared by adding only a very small amount of agarose to ILs. The addition of a small amount of gelator facilitates the preparation of highly ion conductive gels with ILs.

4.2 ILs and DNA composites

There is interest in using DNA, which is a representative biopolymer, to create functional materials for electronics. Using metallointercalator-tethered DNA, rapid photo-induced electron transfer was demonstrated over a distance of at least 40 Å between a ruthenium complex as a donor, and a rhodium complex as an acceptor.¹⁶⁵ This finding encouraged the development of DNA-based electron-conducting nanowires.¹⁶⁶ Apart from its notable role in genetics, DNA is a useful biopolymer, like polysaccharides or other polypeptides. By combining DNA with ILs, novel functional materials have been prepared for electrochemical devices. The dissolution of DNA in ILs and/or the interaction between DNA and ILs has also been investigated.^167–170 Since DNA has a large number of heteroaromatic bases, it should have a high affinity with ILs. Accordingly, DNA films containing ILs were prepared by mixing DNA with ILs.¹⁴⁴ The ionic conductivity of a DNA film containing 93.9 wt% 1-ethylimidazolium tetrafluoroborate ([C2im]BF₄) was 5.05 × 10⁻³ S cm⁻¹ at 50 °C, and is comparable to that of bulk [C2im]BF₄ (2.02 × 10⁻² S cm⁻¹). The [C2im]BF₄ used here was easily prepared by the neutralisation of ethylimidazole with tetrafluoroboric acid, which was sometimes expected to show proton conduction due to relatively labile protons.

Ohno et al. prepared ionic liquidised DNA and applied it to ion conductive materials.^144,148 Ionic liquidised DNA was synthesised by neutralisation of DNA with tetrafluoroboric acid.¹⁴⁸ The resulting ionic liquidised DNA has low ionic conductivity. Of the four nucleic acid bases, only adenine and cytosine formed salts with BF₄⁻. For this reason, a continuous ionic conduction path could not be formed in the DNA chains. To prepare a film with higher ionic conductivity, ionic liquidised DNA was mixed with [C2im]BF₄. The ionic conductivity of the resulting film was improved by increasing the concentration of the added [C2im]BF₄. The hydrogen bonding between complementary base pairs was broken by the neutralisation, however, and the double stranded helix structure of DNA was not maintained in the film. An attempt to maintain the double stranded helix structure of DNA after ionic liquidisation was made by neutralising phosphate anion groups with imidazolium cations.¹⁴⁵ Additionally, the hydrophobicity of ionic liquidised DNA can be controlled by changing the alkyl chain length of the imidazolium cation.

A serious problem with organic dye-based electrochromic systems is the irreversible dimerisation of organic dyes. It is well known that π-conjugated heteroaromatic compounds form a complex with DNA by a process called intercalation or groove-binding.¹⁷¹ To realise the reversible redox reaction of organic dyes without dimerisation, DNA was used as a host for the dyes.¹⁴³ However, N,N′-diheptyl-4,4′-bipyridinium dibromide (HV), which is a kind of dye, is considered to be not only groove-bound but also freely dissolved, and free HVs formed dimers, lessening the reversibility. To improve the binding stability between HVs and DNA, ionic liquidised DNA was used as a host matrix.^146,147 Since ionic liquidised DNA needs to retain the double stranded-helical structure for use as a host for dyes, ionic liquidised DNA was prepared by exchanging the Na⁺, counter cation of DNA, with a alkylimidazolium cation.¹⁴⁵ DNA having 1-dodecyl-3-methylimidazolium cations ([C12mim]-DNA) is water-insoluble. HVs form a stable complex with [C12mim]-DNA through groove-binding. HVs were complexed with ionic liquidised DNA on an indium-tin oxide coated glass electrode. Upon combining N,N,N′,N′-tetramethyl-p-phenylenediamine as an active material at the counter electrode, and the IL copolymer as ion sources to the ionic liquidised DNA, an electrochromic cell was constructed based on DNA. Upon applying potentials of just ±1.0 V, reversible colour changes were observed accompanied with the redox reaction of the complexed organic dyes.¹⁴⁶ There was little decrease in the performance on the colour change with time due to compartmented dyes in the DNA.

5. Conclusion and future aspects

This review has summarised applications of ILs in certain fields of bioscience. ILs are widely used as solvents in many fields. In some cases ILs have simply been used to replace organic solvents. In bioscience, IL studies are starting to dominate the classical research fields of reaction solvents and/or electrolyte materials. Water is believed to be the best solvent for biomolecules, but we are confident that water will eventually yield to ILs in many areas of bioscience as a result of their remarkable characteristics. Further research should be based on what ILs alone can do that water or other solvents cannot, as more is learned about them.

Acknowledgements

The authors were supported partly by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 21225007).

References

1. Ionic liquids in synthesis, ed. P. Wasserscheid, T. Welton, Wiley-VCH, Weinheim, Germany, 2007.
2. Electrochemical aspects of ionic liquids, 2nd edn, ed. H. Ohno, John Wiley & Sons, Hoboken, NJ, USA, 2011.
3. T. Welton, Chem. Rev., 1999, 99, 2071–2083.
4. N. Jain, A. Kumar, S. Chauhan and S. M. S. Chauhan, Tetrahedron, 2005, 61, 1015–1060.
5. D. R. Macfarlane, M. Forsyth, P. C. Howlett, J. M. Pringle, J. Sun, G. Annat, W. Neil and E. I. Izgorodina, Acc. Chem. Res., 2007, 40, 1165–1173.
6. P. Hapiot and C. Lagrost, Chem. Rev., 2008, 108, 2238–2264.
7. U. Kragl, M. Eckstein and N. Kaftzik, Curr. Opin. Biotechnol., 2002, 13, 565–571.
8. R. A. Sheldon, R. M. Lau, M. J. Sorgedrager, F. van Rantwijk and K. R. Seddon, Green Chem., 2002, 4, 147–151.
9. F. van Rantwijk, R. M. Lau and R. A. Sheldon, Trends Biotechnol., 2003, 21, 131–138.
10. S. Park and R. J. Kazlauskas, Curr. Opin. Biotechnol., 2003, 14, 432–437.
11. P. C. A. G. Pinto, M. L. M. F. S. Saraiva and J. L. F. C. Lima, Anal. Sci., 2008, 24, 1231–1238.
12. C. Roosen, P. Mueller and L. Greiner, Appl. Microbiol. Biotechnol., 2008, 81, 607–614.
13. P. D. de Maria and Z. Maugeri, Curr. Opin. Chem. Biol., 2011, 15, 220–225.
14. S. Oppermann, F. Stein and U. Kragl, Appl. Microbiol. Biotechnol., 2011, 89, 493–499.
15. S. D. Zhu, Y. X. Wu, Q. M. Chen, Z. N. Yu, C. W. Wang, S. W. Jin, Y. G. Ding and G. Wu, Green Chem., 2006, 8, 325–327.
16. L. Feng and Z.-I. Chen, J. Mol. Liq., 2008, 142, 1–5.
17. H. Ohno and Y. Fukaya, Chem. Lett., 2009, 38, 2–7.
18. A. Pinkert, K. N. Marsh, S. Pang and M. P. Staiger, Chem. Rev., 2009, 109, 6712–6728.
19. M. Mora-Pale, L. Meli, T. V. Doherty, R. J. Linhardt and J. S. Dordick, Biotechnol. Bioeng., 2011, 108, 1229–1245.
20. F. Endres, ChemPhysChem, 2002, 3, 144–154.
21. A. P. Abbott and K. J. McKenzie, Phys. Chem. Chem. Phys., 2006, 8, 4265–4279.
22. S. Z. El Abedin, E. M. Moustafa, R. Hempelmann, H. Natter and F. Endres, ChemPhysChem, 2006, 7, 1535–1543.
23. Y.-Z. Su, Y.-C. Fu, Y.-M. Wei, J.-W. Yan and B.-W. Mao, ChemPhysChem, 2010, 11, 2764–2778.
24. T. Torimoto, T. Tsuda, K.-I. Okazaki and S. Kuwabata, Adv. Mater., 2010, 22, 1196–1221.
25. C. Vollmer and C. Janiak, Coord. Chem. Rev., 2011, 255, 2039–2057.
26. M. C. Buzzeo, R. G. Evans and R. G. Compton, ChemPhysChem, 2004, 5, 1106–1120.
27. P. A. Z. Suarez, V. M. Selbach, J. E. L. Dullius, S. Einloft, C. M. S. Piatnicki, D. S. Azambuja, R. F. de Souza and J. Dupont, Electrochim. Acta, 1997, 42, 2533–2535.
28. U. Schroder, J. D. Wadhawan, R. G. Compton, F. Marken, P. A. Z. Suarez, C. S. Consorti, R. F. de Souza and J. Dupont, New J. Chem., 2000, 24, 1009–1015.
29. M. Opallo and A. Lesniewski, J. Electroanal. Chem., 2011, 656, 2–16.
30. S. V. Muginova, A. Z. Galimova, A. E. Polyakov and T. N. Shekhovtsova, J. Anal. Chem., 2010, 65, 331–351.
31. H. Zhao, J. Chem. Technol. Biotechnol., 2010, 85, 891–907.
32. M. J. A. Shiddiky and A. A. J. Torriero, Biosens. Bioelectron., 2011, 26, 1775–1787.
33. P. Majewski, A. Pernak, M. Grzymislawski, K. Iwanik and J. Pernak, Acta Histochem., 2003, 105, 135–142.
34. K. Fujita, M. Forsyth, D. R. MacFarlane, R. W. Reid and G. D. Elliott, Biotechnol. Bioeng., 2006, 94, 1209–1213.
35. N. Kimizuka and T. Nakashima, Langmuir, 2001, 17, 6759–6761.
36. R. Madeira Lau, M. J. Sorgedrager, G. Carrea, F. van Rantwijk, F. Secundo and R. A. Sheldon, Green Chem., 2004, 6, 483–487.
37. M. Erbeldinger, A. J. Mesiano and A. J. Russell, Biotechnol. Prog., 2000, 16, 1131–1133.
38. T. de Diego, P. Lozano, S. Gmouh, M. Vaultier and J. L. Iborra, Biomacromolecules, 2005, 6, 1457–1464.
39. T. Itoh, J. Synth. Org. Chem. Jpn., 2009, 67, 143–155.
40. M. Bihari, T. P. Russell and D. A. Hoagland, Biomacromolecules, 2010, 11, 2944–2948.
41. C. Reichardt, Green Chem., 2005, 7, 339–351.
42. M. A. Ab Rani, A. Brant, L. Crowhurst, A. Dolan, M. Lui, N. H. Hassan, J. P. Hallett, P. A. Hunt, H. Niedermeyer, J. M. Perez-Arlandis, M. Schrems, T. Welton and R. Wilding, Phys. Chem. Chem. Phys., 2011, 13, 16831–16840.
43. L. Crowhurst, P. R. Mawdsley, J. M. Perez-Arlandis, P. A. Salter and T. Welton, Phys. Chem. Chem. Phys., 2003, 5, 2790–2794.
44. Y. Fukaya, K. Hayashi, M. Wada and H. Ohno, Green Chem., 2008, 10, 44–46.
45. D. M. Phillips, L. F. Drummy, D. G. Conrady, D. M. Fox, R. R. Naik, M. O. Stone, P. C. Trulove, H. C. de Long and R. A. Mantz, J. Am. Chem. Soc., 2004, 126, 14350–14351.
46. K. Tamura, N. Nakamura and H. Ohno, Biotechnol. Bioeng., 2012, 109, 729–735.
47. K. Fujita, D. R. MacFarlane and M. Forsyth, Chem. Commun., 2005, 4804–4806.
48. K. Fujita, D. R. MacFarlane, M. Forsyth, M. Yoshizawa-Fujita, K. Murata, N. Nakamura and H. Ohno, Biomacromolecules, 2007, 8, 2080–2086.
49. H. Zhao, J. Mol. Catal. B: Enzym., 2005, 37, 16–25.
50. K. Fujita, N. Nakamura, K. Igarashi, M. Samejima and H. Ohno, Green Chem., 2009, 11, 351–354.
51. K. Fujita and H. Ohno, Biopolymers, 2010, 93, 1093–1099.
52. H. Ohno, C. Suzuki, K. Fukumoto, M. Yoshizawa and K. Fujita, Chem. Lett., 2003, 32, 450–451.
53. K. Nakashima, T. Maruyama, N. Kamiya and M. Goto, Chem. Commun., 2005, 4297–4299.
54. H. Ohno, C. Suzuki and K. Fujita, Electrochim. Acta, 2006, 51, 3685–3691.
55. K. Shimojo, K. Nakashima, N. Kamiya and M. Goto, Biomacromolecules, 2006, 7, 2–5.
56. T. Itoh, Y. Matsushita, Y. Abe, S.-H. Han, S. Wada, S. Hayase, M. Kawatsura, S. Takai, M. Morimoto and Y. Hirose, Chem.–Eur. J., 2006, 12, 9228–9237.
57. T. Maruyama, S. Nagasawa and M. Goto, Biotechnol. Lett., 2002, 24, 1341–1345.
58. K. Nakashima, N. Kamiya, D. Koda, T. Maruyama and M. Goto, Org. Biomol. Chem., 2009, 7, 2353–2358.
59. A. Tsurumaki, J. Kagimoto and H. Ohno, Polym. Adv. Technol., 2011, 22, 1223–1228.
60. K. R. Seddon, J. Chem. Technol. Biotechnol., 1997, 68, 351–356.
61. M. Armand, F. Endres, D. R. MacFarlane, H. Ohno and B. Scrosati, Nat. Mater., 2009, 8, 621–629.
62. Ionic liquids: the front and future of material development, ed. H. Ohno, CMC Press, Tokyo, 2003.
63. G. Loget, S. Chevance, C. Poriel, G. Simonneaux, C. Lagrost and J. Rault-Berthelot, ChemPhysChem, 2011, 12, 411–418.
64. D. L. Compton and J. A. Laszlo, J. Electroanal. Chem., 2003, 553, 187–190.
65. S. F. Wang, T. Chen, Z. L. Zhang, X. C. Shen, Z. X. Lu, D. W. Pang and K. Y. Wong, Langmuir, 2005, 21, 9260–9266.
66. S. F. Wang, T. Chen, Z. L. Zhang, D. W. Pang and K. Y. Wong, Electrochem. Commun., 2007, 9, 1709–1714.
67. S. Wang, Z. Guo and H. Zhang, Bioelectrochemistry, 2011, 82, 55–62.
68. Z. Brusova, L. Gorton and E. Magner, Langmuir, 2006, 22, 11453–11455.
69. H. Xu, H.-Y. Xiong, Q.-X. Zeng, L. Jia, Y. Wang and S.-F. Wang, Electrochem. Commun., 2009, 11, 286–289.
70. B. Eker, P. Asuri, S. Murugesan, R. J. Linhardt and J. S. Dordick, Appl. Biochem. Biotechnol., 2007, 143, 153–163.
71. A. Ciaccafava, M. Alberola, S. Hameury, P. Infossi, M. T. Giudici-Orticoni and E. Lojou, Electrochim. Acta, 2011, 56, 3359–3368.
72. K. Fujita, N. Nakamura, K. Murata, K. Igarashi, M. Samejima and H. Ohno, Electrochim. Acta, 2011, 56, 7224–7227.
73. J. D. Wadhawan, U. Schroder, A. Neudeck, S. J. Wilkins, R. G. Compton, F. Marken, C. S. Consorti, R. F. de Souza and J. Dupont, J. Electroanal. Chem., 2000, 493, 75–83.
74. H. T. Liu, P. He, Z. Y. Li, C. Y. Sun, L. H. Shi, Y. Liu, G. Y. Zhu and J. H. Li, Electrochem. Commun., 2005, 7, 1357–1363.
75. G. Shul, J. Sirieix-Plenet, L. Gaillon and M. Opallo, Electrochem. Commun., 2006, 8, 1111–1114.
76. N. Maleki, A. Safavi and F. Tajabadi, Anal. Chem., 2006, 78, 3820–3826.
77. I. Svancara, K. Vytras, K. Kalcher, A. Walcarius and J. Wang, Electroanalysis, 2009, 21, 7–28.
78. N. Maleki, A. Safavi and F. Tajabadi, Electroanalysis, 2007, 19, 2247–2250.
79. R. T. Kachoosangi, M. M. Musameh, I. Abu-Yousef, J. M. Yousef, S. M. Kanan, L. Xiao, S. G. Davies, A. Russell and R. G. Compton, Anal. Chem., 2009, 81, 435–442.
80. D. Wei and A. Ivaska, Anal. Chim. Acta, 2008, 607, 126–135.
81. P. Yu, Y. Q. Lin, L. Xiang, L. Su, J. Zhang and L. Q. Mao, Langmuir, 2005, 21, 9000–9006.
82. X. D. Shangguan and J. B. Zheng, Electroanalysis, 2009, 21, 881–886.
83. X. B. Lu, J. Q. Hu, X. Yao, Z. P. Wang and J. H. Li, Biomacromolecules, 2006, 7, 975–980.
84. Y. X. Xu, C. G. Hu and S. S. Hu, Anal. Chim. Acta, 2010, 663, 19–26.
85. A. Cao, H. Ai, Y. Ding, C. Dai and J. Fei, Sens. Actuators, B, 2011, 155, 632–638.
86. X. D. Shangguan, J. B. Zheng and Q. L. Sheng, Electroanalysis, 2009, 21, 1469–1474.
87. R. Gao and J. Zheng, Electrochem. Commun., 2009, 11, 1527–1529.
88. Z. Zhu, Z. Sun, Y. Wang, Y. Zeng, W. Sun and X. Huang, J. Electroanal. Chem., 2010, 650, 31–35.
89. S. K. Moccelini, A. C. Franzoi, I. C. Vieira, J. Dupont and C. W. Scheeren, Biosens. Bioelectron., 2011, 26, 3549–3554.
90. R. Gao, J. Zheng and L. Qiao, Electroanalysis, 2010, 22, 1084–1089.
91. A. Safavi and F. Farjami, Anal. Biochem., 2010, 402, 20–25.
92. M. S. P. Lopez, D. Mecerreyes, E. Lopez-Cabarcos and B. Lopez-Ruiz, Biosens. Bioelectron., 2006, 21, 2320–2328.
93. C. Xiao, X. Chu, B. Wu, H. Pang, X. Zhang and J. Chen, Talanta, 2010, 80, 1719–1724.
94. Q. Zhang, X. Lv, Y. Qiao, L. Zhang, D. L. Liu, W. Zhang, G. X. Han and X. M. Song, Electroanalysis, 2010, 22, 1000–1004.
95. Q. Zhang, S. Y. Wu, L. Zhang, J. Lu, F. Verproot, Y. Liu, Z. Q. Xing, J. H. Li and X. M. Song, Biosens. Bioelectron., 2011, 26, 2632–2637.
96. Y. Liu, L. H. Shi, M. J. Wang, Z. Y. Li, H. T. Liu and J. H. Li, Green Chem., 2005, 7, 655–658.
97. K. Szot, R. P. Lynch, A. Lesniewski, E. Majewska, J. Sirieix-Plenet, L. Gaillon and M. Opallo, Electrochim. Acta, 2011, 56, 10306–10312.
98. Y. H. Li, X. D. Zeng, X. Y. Liu, X. S. Liu, W. Z. Wei and S. L. Luo, Colloids Surf., B, 2010, 79, 241–245.
99. W. Sun, R. F. Gao and K. Jiao, J. Phys. Chem. B, 2007, 111, 4560–4567.
100. Z. Dai, Y. Xiao, X. Yu, Z. Mai, X. Zhao and X. Zou, Biosens. Bioelectron., 2009, 24, 1629–1634.
101. E. Zapp, D. Brondani, I. C. Vieira, J. Dupont and C. W. Scheerenb, Electroanalysis, 2011, 23, 1124–1133.
102. E. Zapp, D. Brondani, I. C. Vieira, J. Dupont and C. W. Scheeren, Electroanalysis, 2011, 23, 764–776.
103. E. Zapp, D. Brondani, I. C. Vieira, C. W. Scheeren, J. Dupont, A. M. J. Barbosa and V. S. Ferreira, Sens. Actuators, B, 2011, 155, 331–339.
104. T. Fukushima, A. Kosaka, Y. Ishimura, T. Yamamoto, T. Takigawa, N. Ishii and T. Aida, Science, 2003, 300, 2072–2074.
105. J. Wang, H. Chu and Y. Li, ACS Nano, 2008, 2, 2540–2546.
106. P. Du, S. N. Liu, P. Wu and C. X. Cai, Electrochim. Acta, 2007, 52, 6534–6547.
107. Y. Zhang, Y. Shen, D. Han, Z. Wang, J. Song, F. Li and L. Niu, Biosens. Bioelectron., 2007, 23, 438–443.
108. P. Yu, H. Zhou, H. Cheng, Q. Qian and L. Mao, Anal. Chem., 2011, 83, 5715–5720.
109. F. Zhao, X. Wu, M. K. Wang, Y. Liu, L. X. Gao and S. J. Dong, Anal. Chem., 2004, 76, 4960–4967.
110. Y. Liu, X. Zou and S. Dong, Electrochem. Commun., 2006, 8, 1429–1434.
111. W. Sun, X. Q. Li, Y. Wang, R. J. Zhao and K. Jiao, Electrochim. Acta, 2009, 54, 4141–4148.
112. R. Pauliukaite, A. P. Doherty, K. D. Murnaghan and C. M. A. Brett, J. Electroanal. Chem., 2011, 656, 96–101.
113. B. G. Choi, H. Park, T. J. Park, D. H. Kim, S. Y. Lee and W. H. Hong, Electrochem. Commun., 2009, 11, 672–675.
114. Z. Zhu, L. Qu, X. Li, Y. Zeng, W. Sun and X. Huang, Electrochim. Acta, 2010, 55, 5959–5965.
115. A. Salimi, S. Lasghari and A. Noorbakhash, Electroanalysis, 2010, 22, 1707–1716.
116. L. G. Zamfir, L. Rotariu and C. Bala, Biosens. Bioelectron., 2011, 26, 3692–3695.
117. Y. Liu, L. Huang and S. Dong, Biosens. Bioelectron., 2007, 23, 35–41.
118. L. Hussein, S. Rubenwolf, F. von Stetten, G. Urban, R. Zengerle, M. Krueger and S. Kerzenmacher, Biosens. Bioelectron., 2011, 26, 4133–4138.
119. C. Shan, H. Yang, J. Song, D. Han, A. Ivaska and L. Niu, Anal. Chem., 2009, 81, 2378–2382.
120. H. G. Liao, H. Wu, J. Wang, J. Liu, Y. X. Jiang, S. G. Sun and Y. H. Lin, Electroanalysis, 2010, 22, 2297–2302.
121. L. Wang, X. H. Zhang, H. Y. Xiong and S. F. Wang, Biosens. Bioelectron., 2010, 26, 991–995.
122. Y. Qiu, X. Qu, J. Dong, S. Ai and R. Han, J. Hazard. Mater., 2011, 190, 480–485.
123. W.-L. Zhu, Y. Zhou and J.-R. Zhang, Talanta, 2009, 80, 224–230.
124. Y. Zhang, R. Yan, F. Zhao and B. Zeng, Colloids Surf., B, 2009, 71, 288–292.
125. A. C. Franzoi, I. C. Vieira, C. W. Scheeren and J. Dupont, Electroanalysis, 2010, 22, 1376–1385.
126. A. Safavi and F. Farjami, Biosens. Bioelectron., 2011, 26, 2547–2552.
127. Y. Yu, X. Liu, D. Jiang, Q. Sun, T. Zhou, M. Zhu, L. Jin and G. Shi, Biosens. Bioelectron., 2011, 26, 3227–3232.
128. J. Huang, J. Zheng and Q. Sheng, Microchim. Acta, 2011, 173, 157–163.
129. S. Dong, P. Zhang, H. Liu, N. Li and T. Huang, Biosens. Bioelectron., 2011, 26, 4082–4087.
130. Z. Zhu, L. Qu, Q. Niu, Y. Zeng, W. Sun and X. Huang, Biosens. Bioelectron., 2011, 26, 2119–2124.
131. Z. Zhu, X. Li, Y. Zeng, W. Sun, W. Zhu and X. Huang, J. Phys. Chem. C, 2011, 115, 12547–12553.
132. Z. Zhu, X. Sun, Y. Wang, Y. Zeng, W. Sun and X. Huang, Mater. Chem. Phys., 2010, 124, 488–492.
133. Q. Shi, J. Zhang, C. Zhang, W. Nie, B. Zhang and H. Zhang, J. Colloid Interface Sci., 2010, 343, 188–193.
134. Z. Zhu, X. Li, Y. Wang, Y. Zeng, W. Sun and X. Huang, Anal. Chim. Acta, 2010, 670, 51–56.
135. Y. Xu, C. Hu and S. Hu, Anal. Chim. Acta, 2010, 663, 19–26.
136. T. Fukushima and T. Aida, Chem.–Eur. J., 2007, 13, 5048–5058.
137. S. Y. Yang, F. Cicoira, R. Byrne, F. Benito-Lopez, D. Diamond, R. M. Owens and G. G. Malliaras, Chem. Commun., 2010, 46, 7972–7974.
138. S. K. Moccelini, A. C. Franzoi, I. C. Vieira, J. Dupont and C. W. Scheeren, Biosens. Bioelectron., 2011, 26, 3549–3554.
139. T. Zhan, M. Xi, Y. Wang, W. Sun and W. Hou, J. Colloid Interface Sci., 2010, 346, 188–193.
140. L. Hussein, S. Rubenwolf, F. von Stetten, G. Urban, R. Zengerle, M. Krueger and S. Kerzenmacher, Biosens. Bioelectron., 2011, 26, 4133–4138.
141. X. Wu, F. Zhao, J. R. Varcoe, A. E. Thumser, C. Avignone-Rossa and R. C. T. Slade, Biosens. Bioelectron., 2009, 25, 326–331.
142. P. Vidinha, N. M. T. Lourenco, C. Pinheiro, A. R. Bras, T. Carvalho, T. Santos-Silva, A. Mukhopadhyay, M. J. Romao, J. Parola, M. Dionisio, J. M. S. Cabral, C. A. M. Afonso and S. Barreiros, Chem. Commun., 2008, 5842–5844.
143. T. Kakibe and H. Ohno, Chem. Commun., 2008, 377–379.
144. H. Ohno and N. Nishimura, J. Electrochem. Soc., 2001, 148, E168–E170.
145. N. Nishimura, Y. Nomura, N. Nakamura and H. Ohno, Biomaterials, 2005, 26, 5558–5563.
146. T. Kakibe and H. Ohno, J. Mater. Chem., 2009, 19, 4960–4964.
147. T. Kakibe, R. Kurihara and H. Ohno, Chem. Lett., 2009, 38, 494–495.
148. N. Nishimura and H. Ohno, J. Mater. Chem., 2002, 12, 2299–2304.
149. V. L. Pushparaj, M. M. Shaijumon, A. Kumar, S. Murugesan, L. Ci, R. Vajtai, R. J. Linhardt, O. Nalamasu and P. M. Ajayan, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 13574–13577.
150. N. Matsumi, Y. Nakamura, K. Aoi, T. Watanabe, T. Mizumo and H. Ohno, Polym. J., 2009, 41, 437–441.
151. J. M. Lee, D. Q. Nguyen, S. B. Lee, H. Kim, B. S. Ahn, H. Lee and H. S. Kim, J. Appl. Polym. Sci., 2010, 115, 32–36.
152. P. K. Singh, B. Bhattacharya, R. K. Nagarale, K.-W. Kim and H.-W. Rhee, Synth. Met., 2010, 160, 139–142.
153. L. H. Lu and W. Chen, Adv. Mater., 2010, 22, 3745–3748.
154. S. Yamazaki, A. Takegawa, Y. Kaneko, J. Kadokawa, M. Yamagata and M. Ishikawa, Electrochem. Commun., 2009, 11, 68–70.
155. K. Suzuki, M. Yamaguchi, M. Kumagai, N. Tanabe and S. Yanagida, C. R. Chim., 2006, 9, 611–616.
156. T. Singh, T. J. Trivedi and A. Kumar, Green Chem., 2010, 12, 1029–1035.
157. J. Le Bideau, L. Viau and A. Vioux, Chem. Soc. Rev., 2011, 40, 907–925.
158. I. Ferreira, B. Bras, J. I. Martins, N. Correia, P. Barquinha, E. Fortunato and R. Martins, Electrochim. Acta, 2011, 56, 1099–1105.
159. R. P. Swatloski, S. K. Spear, J. D. Holbrey and R. D. Rogers, J. Am. Chem. Soc., 2002, 124, 4974–4975.
160. M. Abe, Y. Fukaya and H. Ohno, Green Chem., 2010, 12, 1274–1280.
161. M. B. Turner, S. K. Spear, J. D. Holbrey and R. D. Rogers, Biomacromolecules, 2004, 5, 1379–1384.
162. M. A. Mehta and T. Fujinami, Chem. Lett., 1997, 915–916.
163. T. Sato, Y. Tsujii, T. Fukuda and T. Miyamoto, Macromolecules, 1992, 25, 3890–3895.
164. D. Q. Nguyen, J. Hwang, J. S. Lee, H. Kim, H. Lee, M. Cheong, B. Lee and H. S. Kim, Electrochem. Commun., 2007, 9, 109–114.
165. C. J. Murphy, M. R. Arkin, Y. Jenkins, N. D. Ghatlia, S. H. Bossmann, N. J. Turro and J. K. Barton, Science, 1993, 262, 1025–1029.
166. V. D. Lakhno, Int. J. Quantum Chem., 2008, 108, 1970–1981.
167. J. H. Wang, D. H. Cheng, X. W. Chen, Z. Du and Z. L. Fang, Anal. Chem., 2007, 79, 620–625.
168. R. Vijayaraghavan, A. Izgorodin, V. Ganesh, M. Surianarayanan and D. R. MacFarlane, Angew. Chem., Int. Ed., 2010, 49, 1631–1633.
169. Y. Ding, L. Zhang, J. Xie and R. Guo, J. Phys. Chem. B, 2010, 114, 2033–2043.
170. L. Cardoso and N. M. Micaelo, ChemPhysChem, 2011, 12, 275–277.
171. S. Takenaka, H. Sato, T. Ihara and M. Takagi, J. Heterocycl. Chem., 1997, 34, 123–127.

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