Proton diffusion and hydrolysis enzymatic reaction in 100 nm scale biomimetic nanochannels

INTRODUCTION

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ChooseTop of pageABSTRACTINTRODUCTION <<MATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONCONCLUSIONSREFERENCESPrevious sectionNext sectionBiological membranes are self-assembled bilayers of lipid molecules that provide a basic platform for the expression of biological functions and the separation of biomolecules and liquids in the range of 10 nm (e.g., endoplasmic reticulum) to 10 μm (e.g., animal cells). The phospholipid bilayer is a major component of a cell membrane, and there has been significant interest in the structure and dynamics of water interacting with phospholipid bilayers.11. J.-X. Cheng, S. Pautot, D. A. Weitz, and X. S. Xie, Proc. Natl. Acad. Sci. U.S.A. 100, 9826–9830 (2003). https://doi.org/10.1073/pnas.1732202100 For this purpose, various measurement techniques have been applied to reveal hydration with water and the dynamics inside/near lipid bilayers, such as nuclear magnetic resonance (NMR) spectroscopy,22. E. G. Finer and A. Darke, Chem. Phys. Lipids 12, 1–16 (1974). https://doi.org/10.1016/0009-3084(74)90064-4 neutron scattering,33. J. Fitter, R. E. Lechner, and N. A. Dencher, J. Phys. Chem. B 103, 8036–8050 (1999). https://doi.org/10.1021/jp9912410 x-ray scattering,44. M. C. Wiener, G. I. King, and S. H. White, Biophys. J. 60, 568–576 (1991). https://doi.org/10.1016/S0006-3495(91)82086-0 molecular dynamics simulation,55. S. König, E. Sackmann, D. Richter, R. Zorn, C. Carlile, and T. M. Bayerl, J. Chem. Phys. 100, 3307–3316 (1994). https://doi.org/10.1063/1.466422 and infrared absorption spectroscopy.66. X. Chen, W. Hua, Z. Huang, and H. C. Allen, J. Am. Chem. Soc. 132, 11336–11342 (2010). https://doi.org/10.1021/ja1048237 Proton transport along a lipid bilayer was also investigated, and fast transport was revealed.7–97. N. Amdursky, Y. Lin, N. Aho, and G. Groenhof, Proc. Natl. Acad. Sci. U.S.A. 116, 2443–2451 (2019). https://doi.org/10.1073/pnas.18123511168. S. Serowy, S. M. Saparov, Y. N. Antonenko, W. Kozlovsky, V. Hagen, and P. Pohl, Biophys. J. 84, 1031–1037 (2003). https://doi.org/10.1016/S0006-3495(03)74919-49. A. Springer, V. Hagen, D. A. Cherepanov, Y. N. Antonenko, and P. Pohl, Proc. Natl. Acad. Sci. U.S.A. 108, 14461–14466 (2011). https://doi.org/10.1073/pnas.1107476108 These studies indicated that water in the lipid bilayer shows ordering due to interaction with lipid molecules. In addition, water molecules adjacent to lipid bilayers (ca. 1 nm from the surface) form strong hydrogen bonds with the polar heads of lipid phosphate groups.1010. W. Zhao, D. E. Moilanen, E. E. Fenn, and M. D. Fayer, “Water at the surfaces of aligned phospholipid multibilayer model membranes probed with ultrafast vibrational spectroscopy,” J. Am Chem. Soc. 130(42), 13927–13937 (2008). https://doi.org/10.1021/ja803252y The size of the space studied was typically several nanometers. So, it is difficult to judge whether the water molecules behave as a liquid.Liquids bounded by lipid bilayers with sizes on the scale of 10–100 nm are also important for understanding biological activity because the spaces can generally be observed in biological systems such as organelles (e.g., mitochondria), synaptic clefts, and intercellular spaces in tissues. For example, chemical synapses have junctions with a 10–100 nm scale gap, through which signals are transduced. Due to the large size compared with a water molecule, the water molecules behave as a liquid, while the liquid properties might be different from those in normal bulk water. Many specific liquid properties and long-range interactions have been suggested in this small gap.1111. P. Ball, Chem. Rev. 108, 74–108 (2008). https://doi.org/10.1021/cr068037a For example, recent cell biology has focused on nanodroplet formation (10–100 nm scale) in aqueous solutions (phase separation) as a mechanism for selective and efficient chemical reactions in cells.1212. A. S. Lyon, W. B. Peeples, and M. K. Rosen, Nat. Rev. Mol. Cell Biol. 22, 215–235 (2021). https://doi.org/10.1038/s41580-020-00303-z The liquid properties and chemical reactivity are the most important issues. However, investigation of the liquid properties and cellular activity (e.g., the effect of proton conduction in proton channels) has been difficult due to the difficulty of in vivo measurements and the lack of in vitro measurement tools. As an example, the surface force apparatus (SFA) was utilized to measure the friction between a particle covered with lipid molecules and a glass surface coated with lipids,1313. S. Leroy, A. Steinberger, C. Cottin-Bizonne, A.-M. Trunfio-Sfarghiu, and E. Charlaix, Soft Matter 5, 4997–5002 (2009). https://doi.org/10.1039/b914543e whereas it is difficult to measure the liquid properties in a confined space. Lipid nanotubes with sizes of 100–300 nm have been used for molecular and particle transport.1414. Y. Zhou and T. Shimizu, Chem. Mater. 20, 625–633 (2008). https://doi.org/10.1021/cm701999m However, it is still difficult to investigate the liquid properties and biological functions. Engineering tools to mimic 10–100 nm cellular spaces and integrate functions to investigate the liquid property and chemical reactions are thus of significant interest for studying the effects of such confinements.Microfluidics is a promising technology to treat very small spaces by top-down fabrication and perform controlled experiments with respect to flow rate, surface chemistry, integration of functions, and precise detection. Target sizes of microfluidic technology have recently been downscaled to the range of 10–100 nm.1515. K. Mawatari, Y. Kazoe, H. Shimizu, Y. Pihosh, and T. Kitamori, Anal. Chem. 86, 4068–4077 (2014). https://doi.org/10.1021/ac4026303 The space is a transition region that bridges individual molecules and normal fluid in bulk space, and unique liquid properties can be expected. Many unique properties of water and the important parameters have been reported by realizing nanofluidic circuits on a glass substrate to investigate liquid properties. For example, enhancement of the proton transfer rate was observed in nanochannels fabricated on fused-silica substrates.1616. H. Chinen, K. Mawatari, Y. Pihosh, K. Morikawa, Y. Kazoe, T. Tsukahara, and T. Kitamori, Angew. Chem. Int. Ed. 51, 3573–3577 (2012). https://doi.org/10.1002/anie.201104883,1717. T. Tsukahara, A. Hibara, Y. Ikeda, and T. Kitamori, Angew. Chem. Ed. 46, 1180–1183 (2007). https://doi.org/10.1002/anie.200604502 The liquid properties changed when the space size was 100 nm scale, which was much larger than the molecular size. A protic solvent plays an important role because of the capacity of hydrogen bonding.1818. T. Tsukahara, W. Mizutani, K. Mawatari, and T. Kitamori, J. Phys. Chem. B 113, 10808–10816 (2009). https://doi.org/10.1021/jp903275t Surface Si–OH groups (hydrogen-bonding formation group) is necessary and act as proton donors, leading to the formation of a hydrogen-bonding network. Recent experiments have verified that H2O and D2O show a large isotope effect;1919. K. Mawatari, K. Isogai, K. Morikawa, H. Ushiyama, and T. Kitamori, J. Phys. Chem. B 125, 3178–3183 (2021). https://doi.org/10.1021/acs.jpcb.1c00780 the proton diffusion was enhanced and the activation energy was reduced by 25% for H2O when the channel size was reduced to 180 nm. However, D2O did not show the liquid property change. All of the results suggest that consideration of a long-range hydrogen-bonding network at the 10–100 nm scale is required to explain the phenomena, which is a novel view of confined liquids revealed by nanofluidic engineering. Anomalous ionic and molecular transport in nano-scale or even sub-nanometer channels were also reported.2020. M. Wang, Y. Hou, L. Yu, and X. Hou, Nano Lett. 20, 6937–6946 (2020). https://doi.org/10.1021/acs.nanolett.0c02999,2121. Y. Hou and X. Hou, Science 373, 628–629 (2021). https://doi.org/10.1126/science.abj0437These results suggest that surface protic functional groups and a high surface-to-volume (S/V) ratio are important for changes to the liquid properties. The dissociation of SiOH to SiO– and H+ can be characterized by the pKa, which is 5.8 in bulk.2222. P. J. Scales, F. Grieser, T. W. Healy, L. R. White, and D. Y. C. Chan, Langmuir 8, 965–974 (1992). https://doi.org/10.1021/la00039a037 The lipid bilayers have a stronger proton donor group (e.g., phosphate acid) than SiOH and will also contribute to the formation of a strong hydrogen bonding network through phosphate groups in the size range of 10–100 nm. Modification of the surface of the nanochannels with lipid bilayers will provide confined spaces (biomimetic nanochannels) that can be used as an in vitro analytical tool for biological fluids and chemical reactions in the size range of 10–100 nm. For this, our group has already reported the modification of lipid bilayers in nanochannels using a vesicle fusion method.2323. L. Li, Y. Kazoe, K. Mawatari, Y. Sugii, and T. Kitamori, J. Phys. Chem. Lett. 3(17), 2447–2452 (2012). https://doi.org/10.1021/jz3009198,2424. H. Emon, K. Mawatari, T. Tsukahara, and T. Kitamori, in Proceedings of Micro Total Analysis System (Chemical and Biological Microsystems Society, 2009), p. 1524–1526.

In the present study, we develop biomimetic nanochannels to investigate proton diffusion coefficient because protons play an important role in biological systems (e.g., energy transduction in mitochondria). In addition, the proton-related enzymatic reaction is investigated, and the chemical equilibrium and kinetics are measured by changing the channel size and shape.

MATERIALS AND METHODS

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ChooseTop of pageABSTRACTINTRODUCTIONMATERIALS AND METHODS <<RESULTS AND DISCUSSIONDISCUSSIONCONCLUSIONSREFERENCESPrevious sectionNext sectionThe basic concept of this study is illustrated in Fig. 1. The lipid bilayer was modified on the glass nanochannels, and the proton diffusion and enzymatic reaction were investigated by designing an integrated micro- and nanofluidic circuit on a glass substrate. A vesicle fusion method2525. P. S. Cremer and S. G. Boxer, J. Phys. Chem. B 103, 2554–2559 (1999). https://doi.org/10.1021/jp983996x was applied to modify the lipid bilayers in closed small nanochannels. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was utilized as a representative lipid bilayer because it is usually the main component in the lipid bilayers of animal cells. DOPC contains phosphoric acid with a low pKa of around 1,2626. T. J. Mcintosh and S. A. Simon, Annu. Rev. Biophys. Biomol. Struct. 23, 27–51 (1994). https://doi.org/10.1146/annurev.bb.23.060194.000331 which is much smaller than 5.8 in SiOH, so that a strong hydrogen bonding network can be expected. A Texas Red DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was mixed with DOPC to evaluate the modification with a fluorescence microscope. The preparation procedure was reported in a previous paper.2323. L. Li, Y. Kazoe, K. Mawatari, Y. Sugii, and T. Kitamori, J. Phys. Chem. Lett. 3(17), 2447–2452 (2012). https://doi.org/10.1021/jz3009198 Lipid molecules (DOPC:Texas-Red-DHPE = 99:1 by weight) in CHCl3 were dried using a rotary evaporator. A NaCl solution was added to the dried lipids, and various size vesicles were formed. At this point, in addition to the size variations, multilamellar vesicles were also observed. A freeze-and-thaw process was applied with liquid nitrogen to make unilamellar vesicles, and the process was repeated five times. To prepare small vesicles to protect from clogging of the nanochannels, the vesicle solution was filtered 10 times using a 100 nm filter and a 30 nm filter using a Mini-Extruder (Avanti Polar Lipid, Inc.). The vesicle solutions were also immersed in an ultrasonic bath for 3 h to produce smaller vesicles. Finally, the vesicle solution was diluted with 100 mM NaCl solution to produce a 0.2 mg/ml vesicle solution. The size distribution of vesicles was measured by the dynamic light scattering method, and an average diameter as small as 52 nm was confirmed, which is important to modify the nanochannels without clogging. The vesicle solution was finally introduced into the nanochannels with a pressure controller (PC-20, Nagano Keiki Co., Ltd., Japan). To confirm unilamellar lipid-bilayer formation, the substrates were bonded at low temperatures2727. R. Ohta, K. Mawatari, T. Takeuchi, K. Morikawa, and T. Kitamori, Biomicrofluidics 13, 024104 (2019). https://doi.org/10.1063/1.5087003 and peeled off after lipid modification. The height of the lipid bilayer was analyzed using fluid atomic force microscopy (AFM); the height was almost 5 nm (the thickness of a lipid bilayer is approximately 5–6 nm), and formation of the unilamellar lipid bilayers was confirmed.2828. Y. Kazoe, K. Mawatari, L. Li, H. Emon, N. Miyawaki, H. Chinen, K. Morikawa, A. Yoshizaki, P. S. Dittrich, and T. Kitamori, J. Phys. Chem. Lett. 11, 5756–5762 (2020). https://doi.org/10.1021/acs.jpclett.0c01084Proton diffusion coefficients were measured using a reported method with fluorescein solution as a pH probe [Fig. 2(a)].1616. H. Chinen, K. Mawatari, Y. Pihosh, K. Morikawa, Y. Kazoe, T. Tsukahara, and T. Kitamori, Angew. Chem. Int. Ed. 51, 3573–3577 (2012). https://doi.org/10.1002/anie.201104883 The microchannels were 500 μm wide and 30 μm deep. A 300 kPa hydrostatic pressure was applied to control the flow velocity in the microchannels. After forming a pH gradient between the microchannels, proton diffusion was measured under a stopped-flow condition. In this experiment, phosphate-buffered saline (PBS) solutions were prepared to investigate the salt effect (breaking hydrogen-bonding network) on the liquid property in nanochannels. PBS contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. HCl solution was added to the PBS solution to prepare a pH = 2 solution. The concentration of fluorescein was kept at 10−4 M for the pH = 2 and pH = 7 solutions.An enzymatic reaction was measured with another microfluidic device as shown in Fig. 2(b). Here, a hydrolysis enzymatic reaction between Tokyo Green-β-galactoside and β-galactosidase was selected because the products can be quantitatively measured with a fluorescence microscope. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was used as a buffer at pH = 7.4. Two microchannels (100 μm wide and 6 μm deep) were utilized to introduce the substrate and enzyme solutions by the application of a hydrostatic pressure at 200 kPa. The two solutions were mixed in a microchannel and introduced to the nanochannels. The pressure was then changed to zero to stop the flow in the nanochannels. The enzymatic reaction was monitored with a fluorescent microscope, and the fluorescence intensity was recorded with a CCD camera.

DISCUSSION

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ChooseTop of pageABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSION <<CONCLUSIONSREFERENCESPrevious sectionNext sectionThe change in the liquid properties in bare glass nanochannels could be discussed by the interaction between SiOH and protic solvents such as water.1818. T. Tsukahara, W. Mizutani, K. Mawatari, and T. Kitamori, J. Phys. Chem. B 113, 10808–10816 (2009). https://doi.org/10.1021/jp903275t SiOH on glass is dissociated into SiO− and H+, and hydrogen bonding is formed with water molecules. The density of SiOH is important for inducing changes in the liquid properties.3030. K. Ikeda, Y. Kazoe, T. Tsukahara, K. Mawatari, and T. Kitamori, in Proceedings of Micro Total Analysis System (Chemical and Biological Microsystems Society, 2014), p. 61–63. Also, the formation of hydrogen bonds is closely related to pKa of SiOH, which is 5.8 for the dissociation of SiOH.2222. P. J. Scales, F. Grieser, T. W. Healy, L. R. White, and D. Y. C. Chan, Langmuir 8, 965–974 (1992). https://doi.org/10.1021/la00039a037 DOPC also contains acidic POH groups as proton-releasing functional groups that form a hydrogen-bonding network, as shown in Fig. 7. pKa for POH is around 1, which is much smaller than that for SiOH. Therefore, DOPC can form a stronger hydrogen-bonding network than SiOH. The densities of DOPC and SiOH are 1 molecule/72 Å2 (Ref. 3122. P. J. Scales, F. Grieser, T. W. Healy, L. R. White, and D. Y. C. Chan, Langmuir 8, 965–974 (1992). https://doi.org/10.1021/la00039a037) and 1 molecule/20 Å2,2222. P. J. Scales, F. Grieser, T. W. Healy, L. R. White, and D. Y. C. Chan, Langmuir 8, 965–974 (1992). https://doi.org/10.1021/la00039a037 respectively. The density is not so different, although a detailed discussion is required on the effects of pKa and density. Based on experimental results, pKa might be dominant. Even in the PBS solution, enhancement of proton diffusion was observed. A difference between the square- and plate-type nanochannels was also observed, which can be explained by the S/V ratio.Both Km and Vmax were enhanced in the enzymatic reaction within the biomimetic channel, which is most likely due to the enhancement of k2 (rate constant in the hydrolysis reaction). It is currently difficult to discuss the detailed mechanism. However, the results coincide with the trend in the change of liquid properties shown in this study (size and shape). This suggests that the enhanced enzymatic activity can be attributed to a change in the liquid properties in the nanochannels. This is probably related to the proton-related properties. From the viewpoint of kinetics, the proton diffusion coefficient was enhanced due to the strong hydrogen bonding network formed by DOPC. About the equilibrium, our previous results showed that the dissociation constant for SiOH was more than 10 times higher than the bulk value (pKa was decreased from the bulk value).3232. K. Morikawa, K. Mawatari, Y. Kazoe, T. Tsukahara, and T. Kitamori, Appl. Phys. Lett. 99, 123115 (2011). https://doi.org/10.1063/1.3644481 This suggests that the liquid in the nanochannels behaves like an acidic solution from the viewpoint of pKa, even when the pH is controlled by the addition of HCl. In this experiment, the pH was controlled at pH = 7.5 with a HEPES buffer solution, which is an optimum pH of this enzyme.2929. T. Tsukahara, K. Mawatari, A. Hibara, and T. Kitamori, Anal. Bioanal. Chem. 391, 2745–2752 (2008). https://doi.org/10.1007/s00216-008-2198-2 The carboxyl and amino groups of amino acids at the reaction sites of an enzyme generally act as acid and base catalysts, respectively. Therefore, control of pKa for the functional groups is generally important for accelerating the enzymatic reaction, although a direct change in the solution pH sometimes changes the three-dimensional structure of the enzyme, which leads to a loss of activity. Possibly, aqueous solutions in the nanochannels change the equilibrium and kinetics of protons due to the interaction between the surface protonic functional group and water through the hydrogen-bonding network, even if the pH and temperature are constant.To clarify the mechanism, a systematic study using different enzymes and conditions (pH, composition of lipid bilayer, temperature, etc.) is further needed. For structural analysis, x-ray diffractometry measurements of liquids in nanochannels recently became possible with a nano x-ray diffractometry device.3333. K. Mawatari, H. Koreeda, K. Ohara, S. Kohara, K. Yoshida, T. Yamaguchi, and T. Kitamori, Lab Chip 18, 1259–1264 (2018). https://doi.org/10.1039/C8LC00077H,34 The water structure in the confined space has attracted much attention, especially with respect to biological functions. The origin of the long-range interaction and interaction with enzymes is expected to be clarified with this technique. Although the detailed mechanism cannot be clarified in this study, the usefulness of the biomimetic nanochannels was confirmed, and the change of liquid properties and enzyme reaction kinetics and equilibrium in cell-mimic environments were observed for the first time.

CONCLUSIONS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONDISCUSSIONCONCLUSIONS <<REFERENCESPrevious sectionNext section

Proton diffusion and enzymatic activity in biomimetic nanochannels were investigated, where the glass nanochannels were modified with a lipid bilayer. The proton diffusion coefficient was increased by 2–5 times, which was larger than that in bare glass nanochannels. Hydrolysis enzymatic reactions were enhanced, and the size dependence and effect of the S/V ratio were confirmed, which was consistent with previous reports on changes in the liquid properties within 100 nm glass nanochannels. These results indicate that the liquid properties should be considered in discussions on biological functions and chemical equilibrium/kinetics. The use of microfluidics as novel tools to investigate chemistry and biology in confined spaces will become a basic experimental platform for controlled environments, which is important for scientific investigations.

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