Effective Brownian Ratchet Separation by a Combination of Molecular

Article pubs.acs.org/Langmuir

Effective Brownian Ratchet Separation by a Combination of Molecular Filtering and a Self-Spreading Lipid Bilayer System Toshinori Motegi,†,§ Hideki Nabika,†,∥ Yingqiang Fu,‡ Lili Chen,‡ Yinlu Sun,‡ Jianwei Zhao,‡ and Kei Murakoshi*,† †

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210008, China

‡

ABSTRACT: A new molecular manipulation method in the self-spreading lipid bilayer membrane by combining Brownian ratchet and molecular filtering effects is reported. The newly designed ratchet obstacle was developed to effectively separate dye−lipid molecules. The self-spreading lipid bilayer acted as both a molecular transport system and a manipulation medium. By controlling the size and shape of ratchet obstacles, we achieved a significant increase in the separation angle for dye− lipid molecules compared to that with the previous ratchet obstacle. A clear difference was observed between the experimental results and the simple random walk simulation that takes into consideration only the geometrical effect of the ratchet obstacles. This difference was explained by considering an obstacle-dependent local decrease in molecular diffusivity near the obstacles, known as the molecular filtering effect at nanospace. Our experimental findings open up a novel controlling factor in the Brownian ratchet manipulation that allow the efficient separation of molecules in the lipid bilayer based on the combination of Brownian ratchet and molecular filtering effects.

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INTRODUCTION

Therefore, energy supply systems utilizing electricity or water flow is used for continuous operation. One possible approach to solving these limitations is the use of a self-spreading lipid bilayer as a substitute energy supply system.21−24 Our previous study has shown that the selfspreading lipid bilayer can transport molecules in the bilayer toward any desired direction without the application of an external energy system such as electricity or water flow.25 The self-spreading of this membrane can be used as a thermodynamic energy gradient that develops when the membrane moves from a metastable aggregate state to a stable bilayer formed on the hydrated solid substrate. There is no need to supply external energy to induce this molecular flow. Thus, our previous study has proposed a new twodimensional ratchet separation method by combining a selfspreading lipid bilayer and a substrate with metallic ratchet nano-obstacles.25 This bilayer membrane incorporated aggregates between cholera toxin subunit B and various numbers of ganglioside GM1, showing different drift directions even in cases in which the diffusion coefficient is the same. It is believed that this phenomenon is caused by the local affinity difference between the molecules and the membrane. Using this hypothesis, we developed a new strategy for enhanced Brownian ratchet separation.

A particle suspended in a fluid usually shows Brownian motion, which is a random, i.e., nondirectional, motion. However, in systems possessing both broken time-reversal and broken spatial reflection symmetries, the nonequilibrium effects (the so-called “Brownian ratchet”) make the particle motion directional.1−7 The particle affected by the Brownian ratchet can be spatially separated because the direction of this particle drift is different from that of other particles. The particle separation is often achieved by utilizing the difference in the size-dependent drift or diffusion.8−10 Because the particles in Brownian systems move with different velocities, each particle fraction can be extracted at different distances from the point where the particles are injected.11−15 In contrast, in the systems based on the Brownian ratchet mechanism, the drift direction of the particles depends on the particle sizes and the combination of continuous energy flow and asymmetric obstacles.16,17 This separation basis can be used to fabricate smaller devices that show continuous operation. A numerical model and the earlier experimental results on the Brownian ratchet separation system suggest that the drift direction of molecules depends on the molecular diffusivity.18−20 In other words, the ratchet system presently used is not appropriate for the separation of molecules with the same diffusivity, even if they have different molecular structures or show intermolecular interactions with the surrounding medium such as water or a lipid membrane. Additionally, the earlier Brownian ratchet separation systems show a high level of energy consumption. © 2014 American Chemical Society

Received: March 13, 2014 Revised: June 10, 2014 Published: June 10, 2014 7496

dx.doi.org/10.1021/la500943k | Langmuir 2014, 30, 7496−7501

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Article

A few studies have already reported a novel molecular filtering effect on a self-spreading lipid bilayer through the nanogap between the metallic obstacles.26−28 The local compression of the spreading lipid bilayer in the nanogap increases the local lipid density and changes the solubility of membrane-incorporated molecules. When using the common dye−lipid molecules, Texas Red-labeled DHPE (TR-DHPE), the retarded drift velocity of TR-DHPE at the nanogap appeared. This retardation effect on molecular transport acts as a filter for the molecules that drift through the nanogap. The degree of the retardation effect strongly depends on the gap width and shape. A narrower and longer gap is suggested for a strong retardation effect on the membrane-incorporated TRDHPE. Moreover, the solubility in the compressed lipid bilayer phase is not determined by a specific molecular parameter; instead, it is determined by wide-ranging parameters such as size, charge, polarity, hydrophilicity, steric configuration, and chirality, irrespective of diffusivity. The molecular selectivity complements our previous Brownian ratchet system with the self-spreading lipid bilayer that recognizes molecules only by diffusivity. Therefore, the incorporation of a self-spreading molecular filtering effect into a conventional ratchet system will lead to a comprehensive molecular recognition and help in the design of a two-dimensional molecular separation method. In this study, a new strategy for the enhanced Brownian ratchet was introduced by using uniquely shaped ratchet obstacles. Using the commonly used Brownian ratchet obstacles that are composed of simple rectangle-shaped obstacles (normal ratchet), the deformed obstacles with narrower gaps (horn-shaped ratchet) and longer gaps (channel-shaped ratchet) were employed as the separation substrates. A small amount of TR-DHPE was incorporated into the self-spreading lipid bilayer, which was formed on the glass substrate containing each type of metallic ratchet obstacle. The diffusion of single TR-DHPE molecules was tracked by total internal reflection fluorescence microscopy (TIRFM).29,30 Moreover, in combination with random walk simulation, we evaluated the simple geometrical effect of ratchet obstacles with different shapes on the diffusion properties of lipid molecules.

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Figure 1. Schematic illustrations of three different ratchet obstacles (top) and AFM images of each ratchet obstacles (bottom). (a) Normal ratchet obstacles. Rectangles with an aspect ratio of 1:4 are tilted 45° from the flow direction. (b) Horn-shaped ratchet obstacles. The additional triangle part for normal ratchet obstacles is colored red. The narrowest gap width between obstacles is 100 nm. (c) Channel-shaped ratchet obstacles. The additional part for normal ratchet obstacles is also colored red. The channel width is 175 nm. was left on the substrate. When the substrate was immersed in the phosphate buffer solution (pH 6.8) containing 25 mM KH2PO4 and 25 mM Na2HPO4, the lipid bilayer spread spontaneously from the lipid lump on the substrate. Under this electrolyte condition, singlelipid bilayers, and not multilamellar structures, spontaneously spread from the lipid aggregate to the array region.27 Determination of Diffusivity of a Single Molecule. The singlemolecule observation for TR-DHPE was conducted using objectivetype total internal fluorescence microscopy (TIRFM) with an IX71 inverted microscope (Olympus).32 An excitation laser (532 nm, 10 mW) was delivered through an objective lens (100× NA = 1.45). Emission from the fluorescent TR-DHPE was detected using a CCD camera (C9018, Hamamatsu Photonics). The images were recorded at 30 frames/s. The molecular trajectories were obtained by recording the centers of masses of the bright spots using Image-Pro Plus version 5.1 (Media Cybernetics). The trajectories were recorded only for molecules that maintained a constant fluorescence for more than 60 frames. Within a series of images, trajectories with diffusion coefficients of horn-shaped > normal. The result that the horn-shaped geometry (gap size of 100 nm) caused a ratchet effect stronger than that of the normal geometry (gap size of 250 nm) is reasonable, because the molecular filtering effect at the nanogap increases with a decreasing gap size around the size region of interest.26,27 Furthermore, the molecular filtering effect is known to be enhanced when using metallic channel-shaped gaps.42 In our ratchet experiment, the filtering effect should be more obvious on the channel-shaped ratchet obstacles than on the hornshaped ratchet obstacles. Thus, the molecular filtering effect results in an increase in the retention time at the gaps and the consequent largest separation angle when using channel-shaped obstacles. On the basis of these considerations, it is suggested that the experimentally obtained separation angle results from the molecular filtering effect in addition to the geometrical effect, which is almost equivalent among the three different obstacles under our experimental conditions. This result demonstrated the importance of molecular filtering effects in the local region near the obstacles. Thus, the combination of the self-spreading lipid bilayer and the Brownian ratchet mechanism is expected to emerge as a new, effective molecular separation technique.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-11-706-2704. Fax: +81-11-706-4810. E-mail: [email protected]. Present Addresses §

T.M.: Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi 441-8580, Japan. ∥ H.N.: Department of Chemistry, Faculty of Material and Biological Science, Yamagata University, Yamagata 990-8560, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Grant-in-Aid for Scientific Research 21350001 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. T.M. also thanks the Japan Society for the Promotion of Science (JSPS) for a Research Fellowship for Young Scientists.

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REFERENCES

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CONCLUSION Effective ratchet separation of dye−lipid molecules in a selfspreading lipid bilayer was achieved with minute changes in the ratchet obstacle shapes. This improvement in ratchet separation is associated with the local retention of molecular diffusion at 7500

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