Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices

Apr 6, 2016 - Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural character...
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Letter pubs.acs.org/JPCL

Lyotropic Liquid Crystalline Cubic Phases as Versatile Host Matrices for Membrane-Bound Enzymes Wenjie Sun,† Jijo J. Vallooran,† Wye-Khay Fong,†,‡ and Raffaele Mezzenga*,† †

Food and Soft Materials Science, Department of Health Science and Technology, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland ‡ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia S Supporting Information *

ABSTRACT: Lyotropic liquid crystalline cubic mesophases can function as host matrices for enzymes because of their biomimetic structural characteristics, optical transparency, and capability to coexist with water. This study demonstrates that the in meso immobilized membrane-bound enzyme D-fructose dehydrogenase (FDH) preserves its full activity, follows ideal Michaelis−Menten kinetics, and shows improved stability compared to its behavior in solution. Even after 5 days, the immobilized FDH retained its full activity in meso, whereas a model hydrophilic enzyme, horseradish peroxidase, maintained only 21% of its original activity. We reason that the lipidic bilayers in the three-dimensional structures of cubic mesophases provide an ideal environment for the reconstitution of a membrane-bound enzyme. The preserved activity, long-term stability, and reusability demonstrate that these hybrid nanomaterials are ideal matrices for biosensing and biocatalytic fuel cell applications.

I

structures (Pn3m, Ia3d, and Im3m) and the capability of the gel to coexist with excess water (Pn3m and Im3m). The nanostructure of lipid-based bicontinuous cubic phases is mathematically described by two sets of three-dimensional (3D) periodic interpenetrating but noncommunicating water channels separated by a set of lipidic bilayers which, taking triply periodical minimal surfaces as a model, have a mean curvature close to zero at each point. It has however been recently demonstrated by cryo-electron tomography that triply periodic minimal surfaces reproduce faithfully the topology of dispersed cubic phases.15 This characteristic structure allows these cubic phases to be widely utilized as host matrices for hydrophilic,16−18 hydrophobic,19,20 and amphiphilic molecules;21−24 for the delivery of drugs and nutrients;16−24 and as matrices for material synthesis,25,26 protein crystallization,27−29 and biosensing.9,10,30−36 These materials with adaptable nanostructures can be formed via the self-assembly of specific amphiphilic lipids such as phytantriol37−39 and some monoglycerides.40−42 Highly specific and sensitive biosensors can be constructed with a bicontinuous cubic phase through the incorporation of specific enzymes, for instance the detection of glucose (glucose oxidase)30−33 and the detection of toxic phenolic compounds and hydrogen peroxide (horseradish peroxidase).9,10 Most

n general, enzyme activity decreases when immobilized, no matter whether it is physically entrapped1,2 or covalently bonded.3−5 Thus, research has focused upon improving enzyme activity within the immobilization barriers.6−8 Our previous studies demonstrated that the immobilization of the hydrophilic enzyme, horseradish peroxidase (HRP), within lyotropic liquid crystalline mesophases (cubic Pn3m and Ia3d, lamellar and reversed hexagonal) resulted in a large activity loss of the enzyme.9,10 However, we were able to manipulate the physical features of the lyotropic liquid crystalline mesophases to mitigate the activity loss in two ways: first, by swelling the water channel diameter of the cubic Pn3m mesophase by the addition of the selected hydration enhancing surfactant sucrose stearate;9 and second, by increasing the topological water connectivity of the host mesophases,10 where more free space was provided for the hydrophilic enzyme to approach its natural conformation, resulting in a better performance of the enzyme. It is proposed that membrane-bound enzymes may be better immobilized in cubic mesophases than hydrophilic enzymes. Though successful reconstitution of membrane proteins has been demonstrated in liposomes,11 sponge phase,12 and nanodiscs13,14 by the support of the native-like lipid membrane bilayer, there is little known about the kinetics of membranebound enzymes in biomimetic matrices. Apart from biomimetic structural characteristics, the bicontinuous cubic mesophases have two other advantages which make them stand out from other membrane mimetic materials as host matrices for biosensing and biofuel cells: optically transparent gel-like © XXXX American Chemical Society

Received: February 23, 2016 Accepted: April 6, 2016

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DOI: 10.1021/acs.jpclett.6b00416 J. Phys. Chem. Lett. 2016, 7, 1507−1512

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Figure 1. SAXS spectra of the lipidic mesophase with a cubic Pn3m space group obtained by mixing monolinolein with the indicated amount of water (a); influence of enzyme (1 mg/mL FDH) incorporation and the enzyme reaction on the Pn3m phase constructed by monoglyceride with 30% water (b).

Scheme 1. Illustration of Sample Preparation Procedurea

a

(a) One syringe was loaded with lipid and a small amount of enzyme solution (FDH); the second syringe was loaded with the required amount of substrates (D-fructose, PMS, and MTT). After homogenization with the coupled syringe setup,51 the bulk mesophase was then transferred to a demountable cuvette where the reaction can be followed colorimetrically. (b) The reaction is initiated only when mixing occurs. In the presence of FDH, an electron was removed from D-fructose. MTT was then used as electron acceptor in the presence of PMS, an electron mediator, and converted to the hydrophobic MTT formazan.52

be greatly extended in the mesophase. The mechanism behind the preservation of activity and stability is also discussed. The preserved enzyme activity and extended stability of the in meso immobilized enzyme when compared to the nonimmobilized form in solution are significant for the future applications of the in meso immobilized enzyme in biosensing. Furthermore, the lipidic cubic mesophase where the enzyme can retain its activity and stability has great potential in constructing biofuel cells with higher efficiency and longer lifetime.46−48 In the present study, the activity of in meso immobilized enzyme was investigated in a bulk cubic Pn3m mesophase composed of monolinolein+30% water (Figure 1b) with a water channel size calculated to be 3.2 nm in diameter. Both the enzyme and substrates were homogenized in bulk mesophase (Scheme 1a). The entire reaction (Scheme 1b) occurs within the mesophase itself. Liquid crystalline mesophases are readily identified using small-angle X-ray scattering (SAXS) by their specific Bragg peak positions. The bicontinuous cubic structure utilized in this study was the double-diamond bicontinuous cubic phase (Pn3m) as shown by the Bragg reflections at relative positions in q at √2:√3:√4:√6:√8:√9 (Figure 1). To calculate the diameter of the water channel for the cubic Pn3m phase, TPMS

recently, cubic phases were exploited for the rapid immobilization and detection of pathogenic microorganisms, specifically HIV, Ebola, and malaria, where signal changes in birefringence within lyotropic liquid crystalline cubic mesophases during the enzymatic reaction were simply followed optically.36 In this study, we have utilized the membrane-bound enzyme D-fructose dehydrogenase (FDH) from Gluconobacter industrius, which has been used for the quantitative microdetermination of 43,44 D-fructose in clinical and food applications. In its native environment, FDH is bound to the cellular membrane. It is composed of one large, hydrophilic dehydrogenase subunit and cytochrome c subunits, joined by a smaller hydrophobic transmembrane binding subunit. Researchers have successfully constructed fructose biosensors using FDH.35,45 However, there is little known on the kinetics of the membrane-bound enzyme in biomimetic matrices. In the present study, the enzymatic kinetics of the in meso immobilized FDH was investigated in bulk lyotropic liquid crystalline cubic Pn3m mesophases, along with its long-term stability and reusability in meso. For the first time, it has been shown that the enzymatic activity of FDH was preserved in bulk mesophase and that the enzyme kinetics in the mesophase followed the Michaelis− Menten model. Moreover, enzyme stability was also found to 1508

DOI: 10.1021/acs.jpclett.6b00416 J. Phys. Chem. Lett. 2016, 7, 1507−1512

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Figure 2. FDH saturation curve between substrate concentration and reaction rate in bulk Pn3m (monolinolein: 30% water) (a) and in solution (b). The initial velocity at each substrate concentration was extracted from the respective enzyme reaction progress curve.

retained. As can be seen, lipidic mesophases are not the most ideal immobilization matrices for HRP. The present study utilizes the membrane-bound enzyme FDH. The activity of the immobilized FDH in bulk cubic Pn3m mesophase was demonstrated colorimetrically by monitoring the conversion of a hydrophilic electron acceptor MTT to its reduced form, hydrophobic MTT formazan (which mainly partitioned into the oil phase, seen in Figure S1), as the result of the acceptance of an electron from D-fructose catalyzed by FDH, as shown in Scheme 1b. The enzymatic saturation curve between the substrate concentration and the enzyme reaction rate in meso is shown in Figure 2a, together with that in water (Figure 2b). As can be seen, the in meso immobilized FDH was found to follow Michaelis−Menten kinetics (Figure 2a) as per the FDH in solution. While for the immobilized hydrophilic enzyme, HRP, a huge deviation from normal Michaelis− Menten kinetics was previously observed, in this case, the more generalized Hill model (eq 6) with an introduced Hill coefficient, n, had to be applied to extract the kinetic constant.9,10

arguments were used, and the following equation from Turner et al.49 was applied: 3 ⎛ L lip ⎞ 4 ⎛ L lip ⎞ Φ = 2A 0⎜ ⎟ + χπ ⎜ ⎟ 3 ⎝ a ⎠ ⎝ a ⎠

(1)

where a is the lattice parameter as measured by SAXS and Φ is the lipid volume fraction (ρmonolinolein = 1.05 g/cm3); A0 and χ are the ratio of the area of the minimal surface in a unit cell to (unit cell volume)2/3 and the Euler−Poincaré characteristic, respectively, depending on the specific cubic phase: A0 = 1.919 and χ = −2 for Pn3m. The lattice parameter, a, of the bicontinuous cubic phase is a = dQ

(2)

d = 2π /q

(3)

with Q = √2, √3, √4, √6, √8, and √9 for the Pn3m corresponding to these reflection peaks. Following Briggs et al.,50 the radius of the water channel for Pn3m can be derived using r = 0.391a − L lip

v= (4)

Vmax [s] K m + [s]

(6)

The enzymatic kinetics parameter Km of FDH was extracted using the Michaelis−Menten kinetics (eq 5) and is shown in Table 1, where it can be seen that the Km in bulk mesophase (1.7 mM) is only slightly larger than Km in water (1.3 mM), indicating a greatly preserved activity in the mesophase.

Previously, we demonstrated that in meso immobilization of the hydrophilic enzyme HRP resulted in a decreased activity and a drastic deviation from its Michaelis−Menten kinetics (eq 5) in solution. v=

Vmax n n [s] K m + [s] n

Table 1. Enzymatic Kinetics Parameter, Km, of FDH as Obtained from Michaelis−Menten Model (eq 5) in Solution and Bulk Lipidic Cubic Phase, Compared to the Km of HRP Adapted from Ref 9

(5)

where Km and Vmax are the Michaelis−Menten constant and the maximum velocity, respectively, both of which can be obtained by fitting the enzymatic kinetics curves. When the Km value for an enzyme is low, its affinity is considered high for its substrate. The lipidic cubic phase formed by monolinolein has a water channel diameter (D) with a maximum 3.5 nm, which was too confined for the hydrophilic enzyme HRP (∼6 nm) to properly function. Due to the constriction of the enzyme, the activity of HRP decreased significantly, as shown by the differences in the Michaelis−Menten constant, Km, which increased from 1.4 mM in water to 7.5 mM in meso.9 This enzyme activity loss was greatly relieved in a swollen mesophase formed by the addition of the hydration enhancer sucrose stearate, where D was increased to 7.2 nm. This resulted in a Km of 3.0 mM, approaching the Km (HRP) = 1.4 mM in solution, but still only half of the binding affinity between HRP and substrates was

kinetic parameters

in meso

in solution

Km,FDH (mM) Km,HRP (mM)

1.7 7.5

1.3 1.4

In general, the activity of enzymes immobilized on different matrices has been observed to drop,1−5 the opposite of what was observed in the present study. The preserved enzyme activity of the in meso immobilized FDH may be attributed to two unique features of the hosting mesophase. First, because FDH is a membrane-bound enzyme found in bacterial membranes, it is better reconstituted in the lipidic cubic mesophase because the bicontinuous lipid bilayers and water 1509

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Figure 3. Stability of FDH and HRP incorporated into the cubic Pn3m mesophase over 5 days (a) and 10 days in buffer solution (b) at T = 4 °C. The enzymatic activity tests were performed at T = 37 °C.

Figure 4. Reusability of (a) FDH and (b) HRP immobilized in lipidic cubic phases (monoglyceride-35% water) versus test cycles at T = 37 °C. The data are n = 3 ± σ, normalized to the first cycle.

diameter (Figure S2); however, further swelling of the water channel size of the host cubic Pn3m mesophase to 5.7 nm in diameter (Figure S2), or the incorporation of excess water into the mesophase (Figure S3), resulted in a lower stability of the enzyme. This dramatic difference gives us a clear indication that lipidic cubic mesophases are excellent host matrices for the membrane-bound enzyme, FDH, as shown by the preserved activity and extended stability of the enzyme. These factors are essential for the construction of the membrane-bound enzymebased biosensor and biofuel cell, where higher activity can improve the sensitivity of the detection and preserved stability can enable a much longer shelf time of these biomimetic devices. In addition to extended enzyme stability, the intrinsic capability of mesophases to retain nanoscale structural features at thermodynamic equilibrium with the surrounding water enables the in meso immobilized FDH to be reused over multiple cycles.53,54 This was demonstrated by encapsulating FDH in meso and subsequently applying substrate solution to the immobilized enzyme (Scheme S1a), whereby the immobilized enzyme was easily reused by simply removing the exhausted substrate solution and replacing it with a fresh dose for detection. Figure 4 demonstrates the reusability of the in meso FDH (panel a), as the matrix retains 63% of the activity after 10 cycles. In contrast, the in meso immobilized HRP lost 50% of function at the second cycle (panel b). The massive increase of FDH activity at the second cycle was attributed to the time required for the diffusion of the substrate (XTT) to get into the mesophase in order to saturate the enzyme at the first cycle, leading to a maximum normalized relative activity. In comparison, the drop in activity of HRP was already significant

channels mimic the native environment of FDH. Second, the preserved activity can also be attributed to the compatibility of the hydrophobic converted product (MTT formazan) with the lipidic cubic mesophase. The hydrophobic product can quickly diffuse away through the lipidic channels, leaving the active site of the enzyme free to be accessed by the new substrate. As the membrane-bound enzyme FDH was better reconstituted in the lipidic mesophase than the hydrophilic enzyme HRP, a much longer lifetime of the enzyme was expected in addition to the preserved activity. The long-term storage stability of the membrane-bound enzyme FDH and the hydrophilic enzyme HRP in mesophase and in solution are shown in Figure 3. Dramatic differences were observed here. The membrane-bound enzyme, FDH, stayed stable in meso for 5 days (100% activity), while the hydrophilic enzyme HRP retained only 21% activity after 5 days. The opposite was observed in water; FDH lost over half of its activity within 10 days while HRP retained more than 90%. From Figure 3, it is understood that the hydrophilic enzyme HRP is more stable in solution as it remains in its original conformation; however, when it is immobilized in meso, the enzyme in the confined space of the water channels can more easily result in its partial denaturation and/or inaccessibility of active sites. On the other hand, FDH is more stable when immobilized in the mesophase because the amphiphilic nature of the lipidic cubic phase can better reconstitute the native conformation of the membrane-bound enzyme. Though the amphiphilic nature of the lipidic cubic phase facilitates the stability of the membrane-bound enzyme, the incorporation of more water into the host mesophase can lower the enzyme stability. The membrane-bound enzyme remained stable in the swollen Pn3m mesophase, which had a 4.7 nm 1510

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ACKNOWLEDGMENTS The authors gratefully acknowledge the China Scholarship Council and ETH Zurich for financial support of this work.

at the second cycle; thus, such a substrate saturation effect was not detectable. The reusability of the in meso immobilized enzyme depends on the stability of the enzyme and the release rate of the enzyme from within the matrices. As shown above, the membrane-bound enzyme can remain 100% stable in meso for at least 5 days. It was also found that the release of FDH from mesophase is much slower compared to the hydrophilic HRP (Figure S4), which can be easily understood in terms of both its higher hydrophobicity and larger molecule size (∼140 kDa). Thus, the matching of the physicochemical properties of the membrane-bound enzyme with the cubic mesophase facilitates the reusability of this hybrid nanomaterial for Dfructose biosensing. This study demonstrates the preservation of the enzymatic activity for the membrane-bound enzyme, FDH, when immobilized in a bulk lipidic cubic phase, where the enzyme kinetics follows the Michaelis−Menten model. This differs from our previous reports where the model hydrophilic enzyme HRP was utilized. In that case, a large decrease in activity of the in meso immobilized enzyme with substantial deviations from Michaelis−Menten behavior was observed. This improved enzyme performance can be attributed to both the improved reconstitution of the membrane-bound enzyme, FDH, within the lipidic cubic mesophase and the faster diffusion of the hydrophobic converted product through the mesophase. The improved reconstitution also resulted in the greatly extended stability of the immobilized membrane-bound enzyme, demonstrated by remaining the 100% activity in meso after 5 days compared to the reduction of activity to 50% in solution. Due to the longer-term stability of the membrane-bound enzyme, the in meso encapsulated enzyme has been demonstrated to be highly reusable for biosensing applications. These findings provide us with a deeper understanding of enzyme immobilization within lipidic mesophases, with potential direct implications for the design of biosensors and biofuel cells based on in meso enzymatic reactions.





REFERENCES

(1) Taqieddin, E.; Amiji, M. Enzyme Immobilization in Novel Alginate-Chitosan Core/Shell Microcapsules. Biomaterials 2004, 25, 1937−1945. (2) Altstein, M.; Segev, G.; Aharonson, N.; Ben-Aziz, O.; Turniansky, A.; Avnir, D. Sol-GelEntrapped Cholinesterases: a Microtiter Plate Method for Monitoring Anti-Cholinesterase Compounds. J. Agric. Food Chem. 1998, 46, 3318−3324. (3) Dyal, A.; Loos, K.; Noto, M.; Chang, S. W.; Spagnoli, C.; Shafi, K. V.; Ulman, A.; Cowman, M.; Gross, R. A. Activity of Candida Rugosa Lipase Immobilized on γ-Fe2O3Magnetic Nanoparticles. J. Am. Chem. Soc. 2003, 125, 1684−1685. (4) Wu, J. C.; Hutchings, C. H.; Lindsay, M. J.; Werner, C. J.; Bundy, B. C. Enhanced Enzyme Stability through Site-Directed Covalent Immobilization. J. Biotechnol. 2015, 193, 83−90. (5) Wahba, M. I.; Hassan, M. E. Novel Grafted Agar Disks for the Covalent Immobilization of β-D-Galactosidase. Biopolymers 2015, 103, 675−684. (6) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Enzyme Immobilization in a Biomimetic Silica Support. Nat. Biotechnol. 2004, 22, 211−213. (7) Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernández-Lafuente, R. Modifying Enzyme Activity and Selectivity by Immobilization. Chem. Soc. Rev. 2013, 42, 6290−6307. (8) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of Enzyme Activity, Stability and Selectivity via Immobilization Techniques. Enzyme Microb. Technol. 2007, 40, 1451−1463. (9) Sun, W.; Vallooran, J. J.; Zabara, A.; Mezzenga, R. Controlling Enzymatic Activity and Kinetics in Swollen Mesophases by Physical Nano-Confinement. Nanoscale 2014, 6, 6853−5859. (10) Sun, W.; Vallooran, J. J.; Mezzenga, R. Enzyme Kinetics in Liquid Crystalline Mesophases: Size Matter, But Also Topology. Langmuir 2015, 31, 4558−4565. (11) Rigaud, J.-L.; Lévy, D. Reconstitution of Membrane Proteins into Liposomes. Methods Enzymol. 2003, 372, 65−86. (12) Wadsten, P.; Wöhri, A. B.; Snijder, A.; Katona, G.; Gardiner, A. T.; Cogdell, R. J.; Neutze, R.; Engström, S. Lipidic Sponge Phase Crystallization of Membrane Proteins. J. Mol. Biol. 2006, 364, 44−53. (13) Mi, L. Z.; Grey, M. J.; Nishida, N.; Walz, T.; Lu, C.; Springer, T. A. Functional and Structural Stability of the Epidermal Growth Factor Receptor in Detergent Micelles and Phospholipid Nanodiscs. Biochemistry 2008, 47, 10314−10323. (14) Bayburt, T. H.; Sligar, S. G. Membrane Protein Assembly into Nanodiscs. FEBS Lett. 2010, 584, 1721−1727. (15) Demurtas, D.; Guichard, P.; Martiel, I.; Mezzenga, R.; Hébert, C.; Sagalowicz, L. Direct Visualization of Dispersed Lipid Bicontinuous Cubic Phases by Cryo-Electron Tomography. Nat. Commun. 2015, 6, 8915. (16) Fong, W.; Hanley, T.; Boyd, B. Stimuli Responsive Liquid Crystals Provide ‘on-demand’ Drug Delivery in vitro and in vivo. J. Controlled Release 2009, 135, 218−226. (17) Negrini, R.; Mezzenga, R. pH-Responsive Lyotropic Liquid Crystals for Controlled Drug Delivery. Langmuir 2011, 27, 5296− 5303. (18) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyté, Z.; Monduzzi, M.; Larsson, K. Structural Effects, Mobility, and Redox Behavior of Vitamin K1 Hosted in the Monoolein/Water Liquid Crystalline Phases. Langmuir 1997, 13, 5476−5483. (19) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Lyotropic Liquid Crystalline Phases Formed from a Glycerate Surfactant as Sustained Release Drug Delivery System. Int. J. Pharm. 2006, 309, 218−226.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b00416. Details on materials and experimental methods, partitioning of the MTT formazan product into the oil−water two phases (Figure S1), SAXS spectra of the lipidic cubic Pn3m mesophases with swelling water channel size and the respective storage stability of the membrane-bound enzyme FDH (Figure S2), influence of the amount of water incorporated in the host mesophase on the storage stability of the in meso immobilized FDH (Figure S3), comparison of the release rate of FDH and HRP from mesophase (Figure S4), and the enzyme kinetics of the in meso FDH in excess water (Figure S5) (PDF)



Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: raff[email protected]. Notes

The authors declare no competing financial interest. 1511

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The Journal of Physical Chemistry Letters (20) Amar-Yuli, I.; Libster, D.; Aserin, A.; Garti, N. Solubilization of Food Bioactives within Lyotropic Liquid Crystalline Mesophases. Curr. Opin. Colloid Interface Sci. 2009, 14, 21−32. (21) Clogston, J.; Caffrey, M. Controlling Release from the Lipidic Cubic Phase. Amino Acids, Peptides, Proteins and Nucleic Acids. J. Controlled Release 2005, 107, 97−111. (22) Angelov, B.; Angelova, A.; Filippov, S. K.; Drechsler, M.; Štěpánek, P.; Lesieur, S. Multicompartment Lipid Cubic Nanoparticles with High Protein Upload: Millisecond Dynamics of Formation. ACS Nano 2014, 8, 5216−5226. (23) Angelov, B.; Angelova, A.; Filippov, S. K.; Narayanan, T.; Drechsler, M.; Štěpánek, P.; Couvreur, P.; Lesieur, S. DNA/Fusogenic Lipid Nanocarrier Assembly: Millisecond Structural Dynamics. J. Phys. Chem. Lett. 2013, 4, 1959−1964. (24) Angelova, A.; Angelov, B.; Drechsler, M.; Garamus, V. M.; Lesieur, S. Protein Entrapment in PEGylated Lipid Nanoparticles. Int. J. Pharm. 2013, 454, 625−632. (25) Braun, V. P.; Stupp, I. S. CdS Mineralization of Hexagonal, Lamellar, and Cubic Lyotropic Liquid Crystals. Mater. Res. Bull. 1999, 34, 463−469. (26) Wang, D.; Kou, R.; Choi, D.; Yang, Y.; Nie, Y.; Li, J.; Saraf, L. V.; Hu, D.; Zhang, J.; Graff, G. L.; Liu, J.; Pope, M. A.; Aksay, I. A. Ternary Self-assembly of Ordered Metal Oxide−Graphene Nanocomposites for Electrochemical Energy Storage. ACS Nano 2010, 4, 1587−1595. (27) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C. High-Resolution Crystal Structure of an Engineered Human 2-Adrenergic G Protein-Coupled Receptor. Science 2007, 318, 1258−1265. (28) Caffrey, M.; Cherezov, V. Crystallizing Membrane Proteins Using Lipidic Mesophases. Nat. Protoc. 2009, 4, 706−731. (29) Zabara, A.; Mezzenga, R. Plenty of Room to Crystallize: Swollen Lipidic Mesophases for Improved and Controlled In-Meso Protein Crystallization. Soft Matter 2012, 8, 6535−6541. (30) Razumas, V.; Kanapieniené, J.; Nylander, T.; Engström, S.; Larsson, K. Electrochemical Biosensors for Glucose, Lactate, Urea and Creatinine Based on Enzymes Entrapped in a Cubic Liquid Crystalline Phase. Anal. Chim. Acta 1994, 289, 155−162. (31) Nylander, T.; Mattisson, C.; Razumas, V.; Miezis, Y.; Håkansson, B. A Study of Entrapped Enzyme Stability and Substrate Diffusion in a Monoglyceride-Based Cubic Liquid Crystalline Phase. Colloids Surf., A 1996, 114, 311−320. (32) Nazaruk, E.; Bilewicz, R. Catalytic Activity of Oxidases Hosted in Lipidic Cubic Phases on Electrodes. Bioelectrochemistry 2007, 71, 8− 14. (33) Nazaruk, E.; Bilewicz, R.; Lindblom, G.; Lindholm-Sethson, B. Cubic Phases in Biosensing Systems. Anal. Bioanal. Chem. 2008, 391, 1569−1578. (34) Li, D.; Caffrey, M. Lipid Cubic Phase as a Membrane Mimetic for Integral Membrane Protein Enzymes. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 8639−8644. (35) Nazaruk, E.; Landau, E. M.; Bilewicz, R. Membrane Bound Enzyme Hosted in Liquid Crystalline Cubic Phase for Sensing and Fuel Cells. Electrochim. Acta 2014, 140, 96−100. (36) Vallooran, J. J.; Handschin, S.; Pillai, S. M.; Vetter, B. N.; Rusch, S.; Beck, H.-P.; Mezzenga, R. Lipidic Cubic Phases as Versatile Platform for the Rapid Detection of Biomarkers, Viruses, Bacteria and Parasites. Adv. Funct. Mater. 2016, 26, 181−190. (37) Barauskas, J.; Landh, T. Phase Behavior of the Phytantriol/ Water System. Langmuir 2003, 19, 9562−9565. (38) Dong, Y.-D.; Larson, I.; Hanley, T.; Boyd, B. J. Bulk and Dispersed Aqueous Phase Behaviors of Phytantriol: Effect of Vitamin E Acetate and F127 Polymer on Liquid Crystal Nanostructure. Langmuir 2006, 22, 9512−9518. (39) Dong, Y.-D.; Dong, W. A.; Larson, I.; Rappolt, M.; Amenitsch, H.; Hanley, T.; Boyd, B. J. Impurities in Commercial Phytantriol Significantly Alter Its Lyotropic Liquid-Crystalline Phase Behavior. Langmuir 2008, 24, 6998−7003.

(40) Kulkarni, C. V.; Wachter, W.; Iglesias-Salto, G.; Engelskirchen, S.; Ahualli, S. Monoolein: a Magic Lipid? Phys. Chem. Chem. Phys. 2011, 13, 3004−3021. (41) Qiu, H.; Caffrey, M. The Phase Diagram of the Monoolein/ Water System: Metastability and Equilibrium Aspects. Biomaterials 2000, 21, 223−234. (42) Mezzenga, R.; Meyer, C.; Servais, C.; Romoscanu, A.; Sagalowicz, L.; Hayward, R. Shear Rheology of Lyotropic Liquid Crystals: a Case Study. Langmuir 2005, 21, 3322−3333. (43) Yamada, Y.; Aida, K.; Uemura, T. Enzymatic Studies on the Oxidation of Sugar and Sugar Alcohol. I. Purification and Properties of Article-Bound Fructose Dehydrogenase. J. Biochem. 1967, 61, 636− 646. (44) Ameyama, M.; Shinagawa, E.; Matsushita, K.; Adachi, O. DFructose Dehydrogenase of Gluconobacter Industrius: Purification, Characterization, and Application to Enzymatic Microdetermination of D-Fructose. J. Bacteriol. 1981, 145, 814−23. (45) Kato, K.; Walde, P.; Mitsui, H.; Higashi, N. Enzymatic and Stability of D-Fructose Dehydrogenase and Sarcosine Dehydrogenase Immobilized onto Giant Vesicles. Biotechnol. Bioeng. 2003, 84, 415− 423. (46) Minteer, S. D.; Liaw, B. Y.; Cooney, M. J. Enzyme-Based Biofuel Cells. Curr. Opin. Biotechnol. 2007, 18, 228−234. (47) Moehlenbrock, M. J.; Minteer, S. D. Extended Lifetime Biofuel Cells. Chem. Soc. Rev. 2008, 37, 1188−1196. (48) Kim, J.; Jia, H.; Wang, P. Challenges in Biocatalysis for EnzymeBased Biofuel Cells. Biotechnol. Adv. 2006, 24, 296−308. (49) Turner, D. C.; Wang, Z. G.; Gruner, S. M.; Mannock, D. A.; McElhaney, R. N. Structural Study of the Inverted Cubic Phases of DiDodecyl Alkyl-Beta-D-Glucopyranosyl-Rac-Glycerol. J. Phys. II 1992, 2, 2039−2063. (50) Briggs, J.; Chung, H.; Caffrey, M. The TemperatureComposition Phase Diagram and Mesophase Structure Characterization of the Monoolein/Water System. J. Phys. II 1996, 6, 723−751. (51) Cheng, A.; Hummel, B.; Qiu, H.; Caffrey, M. A Simple Mechanical Mixer for Small Viscous Lipid-Containing Samples. Chem. Phys. Lipids 1998, 95, 11−21. (52) Amine, A.; Moscone, D.; Bernardo, R. A.; Marconi, E.; Palleschi, G. A New Enzymatic Spectrophotometric Assay for the Determination of Lactulose in Milk. Anal. Chim. Acta 2000, 406, 217−224. (53) Angelova, A.; Ollivon, M.; Campitelli, A.; Bourgaux, C. Lipid Cubic Phases as Stable Nanochannel Network Structures for Protein Biochip Development: X-ray Diffraction Study. Langmuir 2003, 19, 6928−6935. (54) Nazaruk, E.; Górecka, E.; Bilewicz, R. Enzymes and Mediators Hosted Together in Lipidic Mesophases for the Construction of Biodevices. J. Colloid Interface Sci. 2012, 385, 130−136.

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DOI: 10.1021/acs.jpclett.6b00416 J. Phys. Chem. Lett. 2016, 7, 1507−1512