3D Atomic Force Microscopy Provides Insight Into Chitin Nanocrystals

Chitin is a renewable biopolymer that is abundant in nature. Despite its importance as a basic building block in various biological materials, its surface crystalline structure remains unexplored.

Study: Investigating the structural details of chitin nanocrystal-water interfaces by three-dimensional atomic force microscopy. Image Credit: Malpolon/Shutterstock.com

In a recently published article in the magazine Small Methodsthe researchers used atomic force microscopy (AFM) and molecular dynamics (MD) simulations to reveal the structural details of the chitin nanocrystal (chitin NC) water interface at the molecular level.

Composition of chitin

β (1-4) linked N-acetyl-d-glucosamine residues are the building blocks in the chitin chain commonly found in crustacean shells. Chitin shares many structural and functional similarities with cellulose. Chitins such as cellulose are found in nature in the form of ordered crystalline microfibrils along with some proteins and minerals. Pure chitin NCs are obtained from crab and shrimp shells through mechanical and chemical treatments. Depending on the source, chitin exists in three types of polymorphs, α-, β- and -chitins. However, the most stable form is α-chitin.

The morphological and structural details and chemistry of chitin determine its properties. However, the molecular-scale details of the surface structure of chitin NC at the interface in an aqueous medium are limited, and it is imperative to understand the three-dimensional (3D) structural organization of water molecules at the interface on the surface of chitin in order to understand the kinetics. to determine enzymatic hydrolysis.

Structural details of chitin NC water interface

In the present work, the chitin NCs were isolated from shrimp shells in the water, and their molecular-level structural details were realized by combining MD simulations with frequency modulation 3D atomic force microscopy (FM-3D-AFM). The structural details of the individual chitin chain arrangements of NC were realized from a highly resolved AFM image, which showed a well-ordered surface without many structural defects. The 3D interface hydration layers of structured water molecules on the chitin NC surface were visualized with sub-nanometer resolution at the single-chain level.

On the chitin NC surface, the water molecules are stable molecularly ordered hydration layer structures, which encapsulate the chitin NC surface inhomogeneously. This suggests that the amphiphilic surface character of the chitin NC consists of multiple crystalline planes, different chain arrangements and the ability to form hydrogen bonds.

MD simulations helped to confirm these findings and the results were consistent with those of the AFM. The results obtained can serve a crucial purpose in investigating the enzymatic and chemical degradation at the aqueous chitin interface and understanding the structure-property relationships on chitin NC surfaces.

Research results

The chitin NCs were prepared from chitin powder via hydrochloric acid hydrolysis and their morphology and size distribution were assessed by transmission electron microscope (TEM) and FM-AFM measurements.

The TEM images of chitin NCs showed needle-shaped nanostructures with a diameter of 10-20 nanometers and a length of 100-600 nanometers on chitin fragments. Furthermore, the images obtained from AFM confirmed the homogeneous morphologies with laterally aggregated and isolated NCs. The less charged surface or incomplete fibrillation of NCs resulted in the formation of chitin-NC aggregates.

The diameter of individual NCs was found to be 10 to 40 nanometers and 100-300 nanometers in length. The observed diameter of chitin NCs was larger than their actual size due to tip convolution effects. In addition, the histogram analysis performed on several samples confirmed that the height distribution was 4-15 nanometers, which is consistent with the mean size of the chitin NC derived from shrimp.

The FM-AFM images revealed that the surface of the individual chitin NC had an extreme chain arrangement covering the entire crystal surface. The high degree of crystallinity observed was free of structural defects, indicating that the amorphous domains are mainly composed of proteins.

The minerals were removed by acid treatment while preserving the crystalline structures. Structural characterization of several individual chitin NC surfaces at the molecular level confirmed that the structural features at the interface are best accessed when the axis of chitin NC is parallel to the probe or at an angle of 45 degrees.

At the solid-liquid interface, the AFM tip interacts with surface features and an ordered layer of solvent molecules on the surface. The MD simulations and AFM measurements confirmed that the observed hydration structures are equivalent to the underlying structures of chitin NCs at the molecular level.

The top half of the AFM image revealed a zigzag arrangement of chitin chains and the bottom half of the image showed a row-like pattern. The functional surface groups with different chain orientations resulted in two types of surface arrangement in chitin NCs. In addition, the researcher hypothesized that water molecules could rearrange the interface to maximize interaction with the surface of chitin NC. High-resolution AFM showed different contrast patterns due to the hydrogen bonds formed by the exposed amide and hydroxyl groups with the neighboring water molecules.

In conclusion, the researchers characterized the chitin NC surface at the atomic and molecular level. They expect the study results could help to gain a deeper understanding of the surface chemistry and structure-activity relationships of chitin NC. The present study could pave a path to assess the enzymatic and chemical activities on the surface of chitin NCs.

Reference

Yurtsever, A., Wang, P.-X., Priante, F., Morais, Y., Miyata, K., MacLachlan, MJ, Foster, AS, Fukuma, T., (2022) Investigating the structural details of Chitin Nanocrystal-Water Interfaces by Three-Dimensional Atomic Force Microscopy. Small Methods2200320.https://onlinelibrary.wiley.com/doi/full/10.1002/smtd.202200320

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