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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 engineering1,2,3, and implantation4,5,6,7. 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.......

Representative Results

Adsorption of a dipeptide on nanocrystalline titanium dioxide was studied at the biocompatible conditions in a temperature range of 0−40 °C. Experimental dipeptide adsorption (A, mmol/g) on the surface of a titanium dioxide was evaluated as

figure-representative results-371

Where C0 and Ce are the dipe.......

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.......

Acknowledgements

This work was financially supported by the Russian Foundation for Basic Research (Grant No. 15-03-07834-a).

....

Materials

NameCompanyCatalog NumberComments
2-(N-Morpholino)ethanesulfonic acidTCI Chemicals4432-31-9MES, >98%
AcetonitrilePanreac AppliChemHPLC grade
Chromatography vialsglass
Dipeptide Ile-HisBachem4000894
Double-distilled waterDDW was obtained on spot
Heating cleaning bath "Ultrasons-HD"J.P. Selecta30008655 L, 40 kHz, 120 Watts
High-performance liquid chromatograph system equipped with a UV−vis detectorShimadzu, LC-20 ProminenceHPLC
IsopropanolSigma-Aldrich (Merck)67-63-099.70%
LabSolutions LiteShimadzu223-60410Software for high-performance liquid chromatography system
Nanocrystalline TiO2Pure 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 isothiocyanateAcros Organics103-72-0PITC, 98%
Reversed-phase Zorbax columnZORBAX LC150×2.5 mm i.d. with a mean particle size of 5 μm
Syringe filterVladfilter25 mm, 0.2 μm pore, cellulose acetate
Test sterile polymeric tubepolypropylene
Thermostat TC-502BrookfieldRefrigerating/heating circulating bath with the programmable controller for the sample derivatization
TriethylamineSigma-Aldrich (Merck)121-44-8TEA; 99%
Trifluoroacetic acidPanreac AppliChem163317TFA, 99%

References

  1. Garcia, A. J. Interfaces to Control Cell-Biomaterial Adhesive Interactions. Polymers for Regenerative Medicine. 203, 171-190 (2006).
  2. Mahmood, T. A., et al. Modulation of chondrocyte....

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Peptide AdsorptionInorganic NanoparticlesDepletion MethodAdsorption IsothermsDipeptide AdsorptionTitanium DioxidePH AdjustmentMES BufferSolution PreparationConcentration Dilution

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