Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Summary

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Abstract

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

Introduction

By focusing on the rigid, rod-shaped structures of biopolymers, dynamic soft materials have been used for various applications, including polysaccharide biofilm matrices¹, "active gels" composed of cytoskeletal proteins², and "DNA origami" of desired shapes³. To clarify the structural properties, many strategies have been explored, such as transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and confocal fluorescence microscopy. However, because these methods are mostly undertaken in a dried or static state, it is difficult to explain the dynamic behaviors in macroscopic scales, as seen in actual living systems. Recently, we successfully observed the dynamic behavior of biopolymers on the aqueous air-LC interface through polarized light⁴. During the visualization of the oriented structure while drying the biopolymer solution, the temporal changes indicated self-integration of biopolymers on the unstable air-LC interface.

Here, we describe a protocol for the drying of LC biopolymer solutions at the air-LC interface using polarized instruments. As opposed to other analyses of the LC phase that do not consider drying^5,6, the LC dynamics during the drying process were investigated here by evaluating the orientational order parameter  in the lateral view of the fluid phase in a one-side-open cell. The combination of the cell evaporation and the use of polarized instruments allowed for macroscopic monitoring with a controlled evaporation direction. In addition, it was possible to validate the drying records by focusing on the crystalline structures of the adsorbed microdomains, which were affected by molecular weight, concentration, etc. To demonstrate the effectiveness of the method, the drying processes of basic biopolymers with rigid rod shapes, such as polysaccharides, microtubules (MTs), and DNA, were investigated. We chose these biopolymers because they are typical examples of hierarchical macromolecules with megamolecular weights, and their intermolecular interactions enable them to form LC states.

Protocol

1. Instruments

1. Polarization device
   1. To construct a polarization device, provide a light source with a halogen lamp, a light guide, polarizers, a sample stage, an optical rail, rod stands, and a digital single-lens reflex camera (see Figure 1C and Materials List for the polarization device parts).

2. Preparation of the Biopolymer Solution

1. Polysaccharide solutions
   1. Dissolve sacran⁷ (0.5 g) in pure water (100 mL) by stirring at ~80 °C for more than 12 h. During the dissolution, cover the container with plastic wrap to prevent evaporation. Prepare an aqueous solution of xanthan gum in the same manner.
   2. Cool the solutions at ~25 °C to obtain 0.5 wt% aqueous solutions.
   3. Centrifuge the sacran solution to remove impurities (48,400 x g, 4 °C, 1 h, 3 times).
2. MT solution
   1. Prepare a 0.5 wt% tubulin solution (1 mL) in a Britton-Robinson buffer (80 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES); 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); and 5 mM MgCl₂, pH 6.8) on ice⁸.
   2. Use 0.5 wt% tubulin solution (50 µL) and guanosine-5'-[(α,β)-methyleno]triphosphate (GpCpp) (5 µL) to prepare a GpCpp-containing tubulin solution (50 µL). Incubate at 37 °C for 3 h to obtain a stable MT nucleus.
      NOTE: The role of GpCpp is to support MT formation and to completely suppress the depolymerization of MT to tubulin.
   3. Mix the 0.5 wt% tubulin solution (950 µL) and the GpCpp-containing tubulin solution (50 µL) at ~25 °C for 1 day to obtain a stable 0.5 wt% MT solution.
3. DNA solution
   1. Prepare a 0.5 wt% DNA solution (1 mL) in Tris-EDTA buffer solution (10 mM Tris, pH 8.0, with 1 mM EDTA).
4. Keep the 0.5 wt% biopolymer sample solutions at 25 °C for the drying experiment.

3. Drying Experiments and Observation under Cross-polarized Light

1. Solutions in a one-side-open cell (Figure 1A)
   1. Cut a silicon sheet (see Materials List) into an appropriate shape with a thickness of 1 mm (inner dimension of 5-15 mm, 1 mm, and ~20 mm; Figure 1A).
      1. Assemble a one-side-open cell composed of a silicon spacer with inner dimension of 5-15 mm, 1 mm, and ~20 mm and two non-modified glass slides (76 mm × 1 mm × 26 mm). Fix both sides of the cell with double clips in advance to keep the sample solution from leaking out.
   2. Slowly add each of the 0.5 wt% biopolymer solution (100-300 µL) using a ~1-mm pore-size pipette tip to each cell at ~25 °C. Remove air bubbles from the cells using a syringe needle.
   3. Place the cells in an oven with an air circulator at 60 °C under atmospheric pressure for evaporation; the evaporation direction is opposite to that of gravity.
2. Observations under cross-polarized light (Figure 1B-1C)
   1. Provide straight visible light via a 100 W halogen lamp with a flat-surface light source over a wide area (80 mm x 80 mm). Adjust the polarizers to 45° and 135° using the holders (Figure 1C).
   2. Fix the positions of the light source, polarizers, sample stage, and camera using an optical rail and rod stands (Figure 1C). Place the sample stage between the two polarizers (the distance between the polarizers should be ~5 cm). Place the camera ~20 cm from the sample stage to allow focusing.
   3. At given times, place the samples from step 3.1.3 between the polarizers on the stage parallel to the XZ-plane and cover the device with a black curtain; the actual device is shown in Figure 1C.
   4. Photograph the samples through linear crossed polarizers using a digital single-lens reflex camera with a standard zoom lens (see Materials List). Control the camera settings, such as focal distance, using computer software (see Materials List).
3. Spatio-temporal analysis of the transmitted light intensity (Figure 1C)
   1. To evaluate the change of the orientational order parameter in the drying process, collect photographs hourly for 24 h.
   2. Measure the transmitted light intensity along the centerline in the Z-direction as a gray value using an image processing program (e.g., ImageJ).
   3. Plot a graph of the gray value as a function of the distance from the upper open side.
4. Microscopic observations under cross-polarized light (Figure 1D)
   1. To check the methods, make microscopic observations with a polarization microscope equipped with a CCD camera⁹. Keep a first-order retardation plate in the light path. Control the conditions for the photos using a PC software.

Results

Using the device as shown in Figure 1, the self-integration from microdomain to macrodomain on a drying air-LC interface was evaluated (Figure 2A). As the first demonstration of the drying experiment, two kinds of megamolecular polysaccharides, sacran (M_w = 1.9 x 10⁷ g mol^-1) and xanthan gum (4.7 x 10⁶ g mol^-1), were compared. Figure 2B shows photographs of the solutions in t...

Discussion

It was sometimes difficult for the camera to focus on the sample due to the transmitted light intensity being too low. In such cases, placing an extended transparent plastic film on the stage helped to arrange the focus. The limitation of the observable resolution was dependent on the camera lens, ~10 µm in this case. The observable limitation of the sample thickness, Δy, was dependent on the maximum light intensity of the lamp, ~10 mm in this case.

The advantage of the device shown ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
sacran	Green Science Materials Inc., Japan		From Aphanothece sacrum. Mw = 1.9 × 107 g mol-1
xanthan gum	Taiyo Kagaku Co., Japan	Neosoft XC	From Xanthomonas campestris. Mw = 4.7 × 106 g mol-1
tubulin	Cytoskeleton, Inc., USA	T240	From porcine brain.
GpCpp	Jena Bioscience, Germany	NU405L
piperazine-N,N′-bis(2-ethanesulfonic acid)	Sigma-Aldrichi, Co. LLC.	P6757-500G	PIPES
ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid	Dojindo Molecular Technologies, Inc.	342-01314	EGTA
MgCl2-6H2O	Wako Pure Chemical Industries, Ltd.	135-15055
KOH	Wako Pure Chemical Industries, Ltd.	162-21813	pellet
DNA	Sigma-Aldrich, Co. LLC.	D1626	From salmon testes. Mw = 1.3 × 106 Da (~2,000 bp)
Tris-EDTA buffer solution	Sigma-Aldrich, Co. LLC.	T9285-100ML	10 mM Tris, pH 8.0, with 1 mM EDTA
slide glass	Matsunami Glass Ind., Ltd., Japan	S1111
silicon rubber sheet	Asone Co.	6-611-32	Thickness: 1 mm
centrifuge	Beckman-Coulter, Inc., USA	Avanti J-25	equipped with a JA-20 rotor
light source	Sumita Optical Glass, Inc., Japan	LS-LHA
light guide	Sumita Optical Glass, Inc., Japan	GF7.2-1-L1500R-M80 (AAAR-015M)	80 mm × 80 mm
halogen lamp	Ushio Inc., Japan	JCR 15V150WBN
holder	Sigmakoki, Co.,Ltd.	KMH-80
sample stage	Sigmakoki, Co.,Ltd.	TARW-25503L
sample holder	Sigmakoki, Co.,Ltd.	SHA-25RO
rod	Sigmakoki, Co.,Ltd.	ROU-12-40
posts holder	Sigmakoki, Co.,Ltd.	RS-6-40
posts holder	Sigmakoki, Co.,Ltd.	RS-12-60
posts holder	Sigmakoki, Co.,Ltd.	RS-12-80
posts holder	Sigmakoki, Co.,Ltd.	RS-12-130
carrier	Sigmakoki, Co.,Ltd.	CAA-25LS
camera holder	Sigmakoki, Co.,Ltd.	CMH-2
medium optical rail	Sigmakoki, Co.,Ltd.	OBA-500SH
lenstube	Tomytech, BORG	lenstube BK	80φ, L25 mm
lenstube	Tomytech, BORG	lenstube BK	80φ, L50 mm
multiband	Tomytech, BORG		80φ
V plate	Tomytech, BORG	V plate 60S
plate holder	Viexen, Co.,Ltd.	plate holder SX
EOS Kiss X7i	Canon Inc., Japan	8594B001	with a standard zoom lens,  EFP 18-55 mm
photographic software	Canon Inc., Japan	EOS Utility
PC	Microsoft	Surface
polarization microscope	Olympus	BX51
first order retardation plate	Olympus	U-TP530	λ = 530 nm
CCD camera	Olympus	DP80
photographic software	Olympus	cellSens Standard
Java-based image processing program	the National Institutes of Health	ImageJ