Study of Short Peptide Adsorption on Solution Dispersed Inorganic Nanoparticles Using Depletion Method

Summary

The first step in comprehending biomolecule-inorganic solid phase interaction is revealing fundamental physicochemical constants that may be evaluated by establishing adsorption isotherms. Adsorption from the liquid phase is restricted by kinetics, surface capacity, pH, and competitive adsorption, which all should be cautiously considered before setting an adsorption experiment.

Abstract

Fundamentals of inorganic-organic interactions are critically important in the discovery and development of novel biointerfaces amenable for utilization in biotechnology and medicine. Recent studies indicate that proteins interact with surfaces through limited adsorption sites. Protein fragments such as amino acids and peptides can be used for interaction modeling between complex biological macromolecules and inorganic surfaces. During the last three decades, many valid and sensitive methods have been developed to measure the physical chemistry fundamentals of those interactions: isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), quartz crystal microbalance (QCM), total internal reflection fluorescence (TIRF), and attenuated total reflectance spectroscopy (ATR).

The simplest and most affordable technique for the measurement of adsorption is the depletion method, where the change in sorbate concentration (depletion) after contact with solution-dispersed sorbent is calculated and assumed to be adsorbed. Adsorption isotherms based on depletion data provide all basic physicochemical data. However, adsorption from solutions requires longer equilibration times due to kinetic restrictions and sorbents with a high specific surface area, making it almost inapplicable to macroscopic fixed plane surfaces. Moreover, factors such as the instability of sols, nanoparticle aggregates, sorbent crystallinity, nanoparticle size distribution, pH of the solution, and competition for adsorption, should be considered while studying adsorbing peptides. Depletion data isotherm construction provides comprehensive physical chemistry data for literally every soluble sorbate yet remains the most accessible methodology, as it does not require expensive setups. This article describes a basic protocol for the experimental study of peptide adsorption on inorganic oxide and covers all critical points that affect the process.

Introduction

For the last 50 years the interaction between inorganic surfaces and peptides has drawn a lot of attention due to its high importance in material science and medicine. Biomedical research is focused on the compatibility and stability of bioinorganic surfaces, which have direct implications for regenerative medicine, tissue engineering^1,2,3, and implantation^4,5,6,7. Contemporary bioresponsive devices, such as sensors and actuators, are based on funct....

Protocol

1. Preparation of dipeptide stock solutions and dilutions

1. Preparation of 16 mM dipeptide solution
   1. Place 0.183 g of a dipeptide (Ile-His) (see Table of Materials) in a sterile polymeric test tube, dilute to 35 mL with double-distilled water (DDW), and dissolve at room temperature (RT) under vigorous stirring.
      NOTE: If the dipeptide does not dissolve in DDW while stirring, place the dipeptide solution into an ultrasonic bath and sonicate for a few minutes.
   2. Prepare.......

Discussion

Adsorption from solutions for isotherm construction requires a longer time for equilibration due to kinetic restrictions and sorbents with a high specific surface area. Moreover, instability of sols, nanoparticle aggregates, crystallinity, nanoparticle size distribution, pH of the solution, and competition for adsorption should be considered while adsorbing amino acids. However, adsorption isotherm construction using the depletion method remains the most available methodology, because it does not require expensive setups.......

Materials

Name	Company	Catalog Number	Comments
2-(N-Morpholino)ethanesulfonic acid	TCI Chemicals	4432-31-9	MES, >98%
Acetonitrile	Panreac AppliChem		HPLC grade
Chromatography vials			glass
Dipeptide Ile-His	Bachem	4000894
Double-distilled water			DDW was obtained on spot
Heating cleaning bath "Ultrasons-HD"	J.P. Selecta	3000865	5 L, 40 kHz, 120 Watts
High-performance liquid chromatograph system equipped with a UV−vis detector	Shimadzu, LC-20 Prominence		HPLC
Isopropanol	Sigma-Aldrich (Merck)	67-63-0	99.70%
LabSolutions Lite	Shimadzu	223-60410	Software for high-performance liquid chromatography system
Nanocrystalline TiO2			Pure anatase with at least 99% crystallinity. Average particle size 10.62 ± 3.31 nm. Specific surface 131.9 m2/g (BET). See Langmuir 2019, 35, 538−550, for details.
Phenyl isothiocyanate	Acros Organics	103-72-0	PITC, 98%
Reversed-phase Zorbax column	ZORBAX LC		150×2.5 mm i.d. with a mean particle size of 5 μm
Syringe filter	Vladfilter		25 mm, 0.2 μm pore, cellulose acetate
Test sterile polymeric tube			polypropylene
Thermostat TC-502	Brookfield		Refrigerating/heating circulating bath with the programmable controller for the sample derivatization
Triethylamine	Sigma-Aldrich (Merck)	121-44-8	TEA; 99%
Trifluoroacetic acid	Panreac AppliChem	163317	TFA, 99%