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....

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, cove....

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