Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

Summary

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

Abstract

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Introduction

The interaction between proteins and inorganic minerals leads to the construction of composite materials with distinctive properties. This includes materials with high mechanical strength or unique optical properties.^1,2 For example, the combination of the protein collagen with the mineral hydroxyapatite generates either soft or hard bones for different functionalities. ³ Short peptides can also bind inorganic materials with high specificity. ^4,5,6 The specificity of these....

Protocol

1. Tip Modification

1. Purchase silicon nitride (Si₃N₄) AFM cantilevers with silicon tips (nominal cantilever radius of ~2 nm).
2. Clean each AFM cantilever by dipping in anhydrous ethanol for 20 min. Dry at room temperature. Then treat the cantilevers by exposing them to O₂ plasma for 5 min.
3. Suspend the clean tips above (3 cm) a solution containing methyltriethoxysilane and 3-(aminopropyl) triethoxysilane in a ratio of 15:1 (v/v) in a desiccator under an inert atmosphere (either nitrogen or argon) and connect the desiccator to a vacuum pump. Vacuum for 2 h to form a monolayer of these two types of mixed silan....

Results

Figure 1 exhibits the tip modification procedure. In the first step, a plasma treatment changes the surface of the silicon nitride tip. The tip presents OH groups. These groups will then react with the silanes. At the end of this step, the surface of the tip will be covered by free -NH₂ groups. These free amines will then react with Fmoc -PEG-NHS, a covalent linker. The Fmoc group of the PEG linker is removed by pipyridine, a deprotecting reagent. Finally, the .......

Discussion

Steps 1.3, 1.4 and 1.7 in the protocol should be carried out with extensive care and in a very gentle manner. In step 1.3, the tip should not be in contact with the silane mixture and the silanization process should be carried out in an inert atmosphere (moisture free).⁴⁵ This is done in order to prevent multilayer formation and because silane molecules readily undergo hydrolysis in the presence of moisture.⁴⁵

In step 1.4, the temperature and tim.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon nitride (Si3N4) AFM cantilevers with silicon tips	Bruker (Camarilo, CA, USA)		MSNL10, nominal cantilevers radius ~2 nm
Methyltriethoxysilane	Acros Organics (New Jersey, USA)		For Silaylation of the AFM tip
3-(Aminopropyl) triethoxysilane	Sigma-Aldrich (Jerusalem, Israel)		For Silaylation of the AFM tip
Triisopropylsilane	Sigma-Aldrich (Jerusalem, Israel)		Used for peptide deprotection
N-Ethyldiisopropylamine	Alfa-Aesar (Lancashire, UK)		Used for tip modification
Triethylamine	Alfa-Aesar (Lancashire, UK)		Used for tip modification
Piperidine	Alfa-Aesar (Lancashire, UK)		Used for tip Fmoc deprotection
Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide  (Fmoc-PEG-NHS)	Iris Biotech GmbH (Deutschland, Germany)		Used as the covalent flexible linker  (MW = 5,000 Da)
2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU)	Alfa Aser (Heysham, England)		Used as a coupling reagent.
N-methyl-2-pyrrolidone (NMP)	Acros Organics (New Jersey, USA)		Used as Solvent in Tip modification procedure
DMF (dimethylformamide)	Merck (Darmstadt, Germany)		Used as Solvent in Tip modification procedure
Chloroform	Bio-Lab (Jerusalem, Israel)		Used as Solvent in Tip modification procedure
Ethanol (Anhydrous)	Gadot (Netanya, Israel)		Used as Solvent in Tip modification procedure
Trifluoro acetic acid (TFA)	Merck (Darmstadt, Germany)
Acetic anhydride	Merck (Darmstadt, Germany)
Peptides	GL Biochem (Shanghai, China).
Phenylalanine and Tyrosine	Biochem (Darmstadt, Germany)
30% TiO2 dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)	Applied Vision Laboratories (Jerusalem, Israel)		(30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP). Used for TiO2 substrate preparation
Mica substrates	TED PELLA, INC. (Redding, California, USA)		9.9 mm diameter