3D atomic force microscopy offers insight into chitin nanocrystals

Chitin is a renewable biopolymer abundantly available in nature. Despite its importance as a basic building block in various biological materials, its surface crystal structure remains unexplored.


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

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

Composition of Chitin

β(1-4) bound NOT-Acetyl-d-glucosamine residues are the building blocks of the chitin chain commonly found on shellfish shells. Chitin has many structural and functional similarities with cellulose. Chitins like cellulose are found in nature as ordered crystalline microfibrils along with some proteins and minerals. Pure chitin NCs are obtained from crab and shrimp shells by mechanical and chemical treatments. According to the source, chitin exists in three types of polymorphs, α, β and γ chitins. However, the most stable form is α-chitin.

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

Structural details of the chitin NC-water interface

In the present work, chitin NCs were isolated from shrimp shells in water, and their molecular-level structural details were achieved by combining MD simulations with frequency-modulated 3D atomic force microscopy ( FM-3D-AFM). Structural details of the individual chitin chain arrangements of NC were made 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 surface of chitin NC were visualized with sub-nanometer resolution at the single chain level.

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

MD simulations helped corroborate these findings and the results aligned with those obtained from AFM. The obtained results can serve a crucial purpose in exploring the enzymatic and chemical degradation at the aqueous interface of chitin and understanding the structure-property relationships at chitin NC surfaces.

search results

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

TEM images of chitin NCs showed needle-like nanostructures 10–20 nanometers in diameter and 100–600 nanometers in length on chitin fragments. Moreover, 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 has been observed to be 10–40 nanometers and 100–300 nanometers in length. The observed diameter of the chitin NCs was larger than their actual size due to tip convolution effects. Moreover, histogram analysis performed on different samples confirmed that the height distribution was 4–15 nanometers, which is consistent with the average height of chitin NC, derived from shrimp.

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

The minerals have been removed by acid treatment while retaining the crystal structures. Structural characterization of different individual chitin NC surfaces at the molecular level confirmed that structural features at the interface are best accessed when the chitin NC axis is parallel to the probe or at a 45 degree angle.

At the solid-liquid interface, the AFM tip interacts with surface features and an ordered layer of solvent molecules on the surface. 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 upper half of the AFM image revealed a zigzag arrangement of chitin chains, and the lower half of the image displayed a row-like pattern. Surface functional groups with different chain orientations resulted in two types of surface arrangement in chitin NCs. Additionally, the researcher hypothesized that the water molecules could reorganize the interface to maximize the interaction with the surface of the chitin NC. High-resolution AFM showed different contrast patterns due to hydrogen bonds formed by exposed amide and hydroxyl groups with nearby water molecules.

In conclusion, the researchers characterized the NC surface of chitin at the atomic and molecular levels. They anticipate that the research results may help to better understand the surface chemistry of chitin NC and structure-activity relationships. The present study could pave the way for the evaluation of enzymatic and chemical activities on the surface of chitin NCs.


Yurtsever, A., Wang, P.-X., Priante, F., Morais, Y., Miyata, K., MacLachlan, MJ, Foster, AS, Fukuma, T., (2022) Probing 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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