Self-Assembly of Peptides

Maghesree Chakraborty

University of Rochester, Department of Chemical Engineering

1. Introduction

Peptides are chains of amino acids which are typically shorter than proteins. It has been found that some peptides, as short as two residues long, are capable of self-assembling into a variety of structures like rods, tubes, vesicles, etc. This self-assembling propensity of peptides has made them popular candidates for functional material design. Peptide self-assemblies have been used for different biomedical applications like drug delivery, bio-sensing and tissue engineering.

In this project the self-assembled structures of a two residue long peptide, diphenylalanine (FF) as shown in Figure 1, were studied. Previous studies have shown that FF displays diverse self assembled structures (like nanofibers, microtubes, nanorods) based on different self-assembly conditions. Effects of solvent and surface were studied.

Figure 1: Diphenylalanine peptide.

2. Sample Preparation

Solutions of FF, with concentration 1mg/ml, were prepared using three different solvents: deionized (DI) water, 1:1 mixture of DI water : methanol and 100% methanol. The solutions were left undisturbed for 22 hours allowing self-assembly of FF. 10µl of each of the three solutions was placed on two different surfaces: glass and silicon wafer. The surfaces were allowed to dry down completely under ambient temperature before further analysis. For characterization using a scanning electron microscope (SEM), the samples were were sputter coated with gold for 1 minute using 15mA current. For characterization using a transmission electron microscope(TEM), 5µl of each of the three solutions were placed on glow dischared copper TEM grids with lacey carbon film. After 3 minutes, excess fluid was removed and the grids were stained with 2% uranyl acetate solution. After resting the grids for 3 additional minutes, the excess stain was removed and the TEM samples were left to dry under ambient conditions for 1 minute. Fourier-transform infrared spectroscopy (FTIR) analysis was conducted on the FF in 100% methanol sample. The solution was placed between two KBr windows.

3. Results and Discussion

A. Scanning Electron Microscopy:

All micrographs were obtained using the Zeiss Auriga Scanning Electron Microscope.


Figure 2: FF in 100% DI water on glass at different magnifications.


Figure 3: FF in 100% DI water on silicon wafer at different magnifications.


Figure 4: FF in 1:1 DI water and methanol on glass at different magnifications.


Figure 5: FF in 1:1 DI water and methanol on silicon wafer at different magnifications.


Figure 6: FF in 100% methanol on glass at different magnifications.


Figure 7: FF in 100% methanol on silicon wafer at different magnifications.

As seen from the micrographs, FF peptides mostly forms hollow needle like structures in 100% DI water on both the glass and silicon wafer. FF 1:1 DI water and methanol solution assembled into hollow needle like stuctures as well as solid tubular stuctures. FF formed mesh like network in methanol as observed in both the samples: FF on silicon wafer and FF on glass.

B. Transmission Electron Microscopy:

All images were obtained using the FEI Tecnai F20 G2 Transmission Electron Microscope.


Figure 8: TEM images of FF in 100% DI water. The diffraction pattern and the image following it shows the single crystalline nature of the FF assembly.


Figure 9: TEM images of FF in 1:1 DI water : methanol. A large heterogeneity in the sizes of the FF self-assemblies can be observed.


Figure 10: TEM images of FF in 100% methanol. Complementing the SEM images, network like assemblies with distributed nodules can be observed.

C. Atomic Force Microscopy (AFM):

All results were collected using the NTMDT AFM Microscope. FF in 100% water on glass sample was used as the AFM sample.


Figure 11: AFM images of FF in 100% DI water on glass.

The 3D image along with the height profile and the corresponding reference 2D image of the sample shows that the self-assembled structure has distinct facades. Based on the AFM results and SEM micrographs, it can be concluded that the self-assembled hollow needle like structures of FF in 100% DI water has almost a hexagonal cross-section. The maximum height from the profile is approximately 0.9µm and the width is approximately 4µm.

D. X-Ray Analysis:

In order to confirm that the SEM micrographs obtained indeed show self-assembled FF, characteristic X-ray analysis was conducted. I have included only the results for the FF in 100% DI water on glass sample.

Figure 12: X-ray spectrum of FF assemblies in water on glass.


Figure 13: The top panel from the left shows the reference SEM micrograph and the carbon, oxygen and sodium x-ray maps. The bottom panel from the left shows the magnesium, aluminium, silicon and calcium x-ray maps.

Unlike other x-ray maps, the carbon signal is concentrated around the region where the self-assembled structure is present. This confirms that the stucture observed is FF self-assemblies and not salts of sodium or other minerals.

E. Fourier-Transform Infrared Spectroscopy:

The FF solution in 100% methanol was used for the FTIR spectroscopy.

Figure 14: FTIR spectrum of the FF sample in 100% methanol.

5. Conclusions

It was observed that the FF dipeptide self-assembled into hollow hexagonal needles in 100% DI water. Besides the needles, solid tubular self-assemblies were also observed in the 1:1 DI water : methanol solution of FF. In methanol, FF self-assembled into mesh like network structure with nodules. The surface on which the solutions were placed did not play significant role in determining the self-assembly structures since prominent differences were not observed in the glass and silicon wafer samples using the same FF solution.


I would like to acknowledge Prof. Andrew D. White for providing all the materials and work space for this project. I sincerely thank Brian McIntyre for helping me throughout this project. Thank you Brian for being available to answer all the questions that I had and for assisting me with the AFM, TEM and FTIR instruments. I would also like to thank Karen Bentley for helping me out the the TEM sample preparation.

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1. X. Yan et al., Self-assembly and application of diphenylalanine-based nanostructures, Chem. Soc. Rev., 1861, 39(6), 2010.

2. A. N. Rissanou et al., Effect of Solvent on the Self-Assembly of Dialanine and Diphenylalanine Peptides, J. Phys. Chem. B., 3962, 117, 2013.

3. Q. Xiong et al., Conformation Dependence of Diphenylalanine Self-Assembly Structures and Dynamics: Insights from Hybrid-Resolution Simulations, ACS Nano, 4455, 13(4), 2019.