Name: study of short peptide adsorption on solution dispersed inorganic nanoparticles using depletion method
Uploaded: 2020-04-11T13:00:00
Description: Watch this Scientific Journal Video about Study of Short Peptide Adsorption on Solution Dispersed Inorganic Nanoparticles Using Depletion Method at JoVE.com

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

1. Preparation of dipeptide stock solutions and dilutions
<ol>
	<li>Preparation of 16 mM dipeptide solution
	<ol>
		<li>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.</li>
		<li>Prepare...

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...

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 functional proteins immobilized on oxide semiconducting surfaces8,9,10,11,12,13. Modern purification practices for protein production often rely on biomolecule interaction properties in downstream purification and separation14.
Among multiple inorganic oxides, titanium dioxide remains the most utilized in combination with biologically relevant substrates15,16. Research in the area of TiO2-based biointerfaces has concentrated on establishing strong and specific binding of proteins and peptides without changing their biological and structural properties. Ultimately, the major objective is a high surface density layer of biomolecules with high stability and increased functionality that will advance the creation of titanium-based biotechnological and medical applications17.
Titanium and its alloys have been used extensively as a surgical implant material for at least six decades because a surface TiO2 layer with a thickness of a few nanometers is corrosion resistant and exhibits a high level of biocompatibility in many in vivo applications18,19,20. Titanium dioxide is also widely considered an inorganic substrate produced in biomineralization, where nucleation and inorganic phase growth accompanied by proteins and peptides may provide materials with promising catalytic and optical properties21,22,23,24.
Given the high relevance of the interaction between inorganic materials and biomolecules in general and protein-TiO2 interactions in particular, there has been a lot of research to address the manipulation and control of the adsorption of proteins on TiO2. Due to these studies, some fundamental properties of this interaction have been revealed, such as adsorption kinetics, surface coverage, and biomolecule conformation, giving substantial support for further advances in biointerfaces5,13.
However, protein complexity adds considerable restrictions on full determination and understanding of a protein&#39;s molecular level interaction with inorganic surfaces. Assuming that the biomolecules interact with the inorganic surfaces through limited sites, some proteins with known structures and amino acid sequences have been reduced to their components-peptides and amino acids-which are studied separately. Some of these peptides have demonstrated significant activity, making them a unique subject of adsorption studies without the need for previous protein separation25,26,27,28,29,30.
Quantitative characterization of peptide adsorption on TiO2 or other inorganic surfaces can be accomplished by means of physical methods that have been adapted specifically for biomolecules for the past few decades. These methods include isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), quartz crystal microbalance (QCM), total internal reflection fluorescence (TIRF), and attenuated total reflectance spectroscopy (ATR), all of which allow for the detection of the adsorption strength by providing key thermodynamic data: The binding constant, Gibbs free energy, enthalpy, and entropy31.
The adsorption of biomolecules to the inorganic material may be accomplished in two ways: 1) ITC as well as the depletion method use particles dispersed in a solution binding to fixed macroscopic surfaces; 2) SPR, QCM, TIRF, and ATR use macroscopic surfaces modified with inorganic material, such as gold-coated glass or metal chips, quartz crystals, zinc sulfide crystals, and PMMA chips, respectively.
Isothermal titration calorimetry (ITC) is a label-free physical method that measures the heat produced or consumed upon titration of solutions or heterogeneous mixtures. Sensitive calorimetric cells detect heat effects as small as 100 nanojoules, making the measurement of adsorption heat on nanoparticle surfaces possible. Thermal behavior of the sorbate during continuous addition- titration, provides a full thermodynamic profile of the interaction revealing enthalpy, binding constant, and entropy at a given temperature32,33,34,35,36.
Surface plasmon resonance (SPR) spectroscopy is a surface-sensitive optical technique based on the measurement of the refractive index of the media in close proximity to the studied surface. It is a real-time and label-free method for monitoring reversible adsorption and adsorbed layer thickness. The binding constant can be calculated from the association and dissociation rates. Adsorption experiments performed at different temperatures may provide information about the temperature dependence of the activation energy and sequentially other thermodynamic parameters37,38,39.
The quartz crystal microbalance (QCM) method measures the change in the oscillating frequency of piezoelectric crystals during the adsorption and desorption processes. The binding constant may be evaluated from the ratio of the adsorption and desorption rate constants. QCM is used for relative mass measurements and therefore, needs no calibration25,27,40. QCM is used for adsorption from both gas and liquid. The liquid technique allows QCM to be used as an analysis tool to describe deposition on variously modified surfaces41.
Total internal reflection fluorescence (TIRF) is a sensitive optical interfacial technique based on the measurement of the fluorescence of adsorbed fluorophores excited with internally reflected evanescent waves. The method allows for the detection of fluorescent molecules covering the surface with thicknesses on the order of tens of nanometers, which is why it is used in the study of macromolecular adsorption on various surfaces42,43. In situ monitoring of the fluorescence dynamics upon adsorption and desorption provide the adsorption kinetics and hence thermodynamic data42,43.
Attenuated total reflectance (ATR) was used by Roddick-Lanzilotta to establish lysine adsorption isotherms based on the lysine spectral bands at 1,600 and 1,525 cm-1. This is the first time that the binding constant for a peptide on TiO2 was determined using an in situ infrared method44. This technique was effective in establishing adsorption isotherms for polylysine peptides45 and acidic amino acids46.
Unlike the abovementioned methods, where the adsorption parameter is measured in situ, in a conventional experiment the amount of the adsorbed biomolecules is measured by the concentration change after the surface contacted the solution. Because the concentration of a sorbate decays in a vast majority of adsorption cases, this method is referred to as the depletion method. Concentration measurements require a validated analytical assay, which may be based on an intrinsic analytical property of the sorbate or based on the labeling47,48,49,50 or derivatization51,52 thereof.
Adsorption experiments using QCM, SPR, TIRF, or ATR require special surface preparation of the chips and sensors used for adsorption studies. Prepared surfaces should be used once and require change upon switching the adsorbate, due to the inevitable hydration of the oxide surface or possible chemisorption of a sorbate. Only one sample at a time can be run using ITC, QCM, SPR, TIRF, or ATR, whereas in the depletion method one can run dozens of samples, for which the quantity is only limited by the thermostat capacity and sorbent availability. This is especially important when processing large sample batches or libraries of bioactive molecules. Importantly, the depletion method does not require costly equipment but solely a thermostat.
However, despite its obvious advantages the depletion method requires complex procedural features that may seem cumbersome. This article presents how to perform a comprehensive physicochemical study of dipeptide adsorption on TiO2 using the depletion method and addresses issues that researchers may face when performing relevant experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid</td>
			<td>TCI Chemicals</td>
			<td>4432-31-9</td>
			<td>MES, &#62;98%</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Panreac AppliChem</td>
			<td></td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Chromatography vials</td>
			<td></td>
			<td></td>
			<td>glass</td>
		</tr>
		<tr>
			<td>Dipeptide Ile-His</td>
			<td>Bachem</td>
			<td>4000894</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-distilled water</td>
			<td></td>
			<td></td>
			<td>DDW was obtained on spot</td>
		</tr>
		<tr>
			<td>Heating cleaning bath &#34;Ultrasons-HD&#34;</td>
			<td>J.P. Selecta</td>
			<td>3000865</td>
			<td>5 L, 40 kHz, 120 Watts</td>
		</tr>
		<tr>
			<td>High-performance liquid chromatograph system equipped with a UV&#8722;vis detector</td>
			<td>Shimadzu, LC-20 Prominence</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td><a href="https://www.sigmaaldrich.com/catalog/search?term=67-63-0&amp;interface=CAS%20No.&amp;lang=en&amp;region=US&amp;focus=product" target="_blank">67-63-0</a></td>
			<td>99.70%</td>
		</tr>
		<tr>
			<td>LabSolutions Lite</td>
			<td>Shimadzu</td>
			<td>223-60410</td>
			<td>Software for high-performance liquid chromatography system</td>
		</tr>
		<tr>
			<td>Nanocrystalline TiO2</td>
			<td></td>
			<td></td>
			<td>Pure anatase with at least 99% crystallinity. Average particle size 10.62 &#177; 3.31 nm. Specific surface 131.9 m2/g (BET). See Langmuir 2019, 35, 538&#8722;550, for details.</td>
		</tr>
		<tr>
			<td>Phenyl isothiocyanate</td>
			<td>Acros Organics</td>
			<td>103-72-0</td>
			<td>PITC, 98%</td>
		</tr>
		<tr>
			<td>Reversed-phase Zorbax column</td>
			<td>ZORBAX LC</td>
			<td></td>
			<td>150&#215;2.5 mm i.d. with a mean particle size of 5 &#956;m</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Vladfilter</td>
			<td></td>
			<td>25 mm, 0.2 &#956;m pore, cellulose acetate</td>
		</tr>
		<tr>
			<td>Test sterile polymeric tube</td>
			<td></td>
			<td></td>
			<td>polypropylene</td>
		</tr>
		<tr>
			<td>Thermostat TC-502</td>
			<td>Brookfield</td>
			<td></td>
			<td>Refrigerating/heating circulating bath with the programmable controller for the sample derivatization</td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td><a href="https://www.sigmaaldrich.com/catalog/search?term=121-44-8&amp;interface=CAS%20No.&amp;N=0&amp;mode=partialmax&amp;lang=en&amp;region=RU&amp;focus=product" target="_blank">121-44-8</a></td>
			<td>TEA; 99%</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Panreac AppliChem</td>
			<td>163317</td>
			<td>TFA, 99%</td>
		</tr>
	</tbody>
</table>

study of short peptide adsorption on solution dispersed inorganic nanoparticles using depletion method

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.

Adsorption of a dipeptide on nanocrystalline titanium dioxide was studied at the biocompatible conditions in a temperature range of 0&#8722;40 &#176;C. Experimental dipeptide adsorption (A, mmol/g) on the surface of a titanium dioxide was evaluated as
<img src="/files/ftp_upload/60526/60526eq1.jpg" />
Where C0 and Ce are the dipe...

Watch this Scientific Journal Video about Study of Short Peptide Adsorption on Solution Dispersed Inorganic Nanoparticles Using Depletion Method at JoVE.com

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

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.

Fundamentals of inorganic-organic interactions are critically important in the discovery and development of novel biointerfaces amenable for utilization ...

The interplay of proteins and peptides with inorganic material is a fundamental phenomenon with implications for nanotechnology, biomaterials and biotechnology. The first step in comprehending of such phenomenon is revealing the fundamental physico-chemical constants such as adsorption constant, Gibbs free energy, enthalpy, entropy, and limited adsorption which may be evaluated through establishing adsorption isotherms. However, adsorption from liquid phase is restricted with kinetics, surface capacity, pH, and comparative adsorption which all should be consciously considered before setting the experiment.In this video, my colleagues Elena Korina and Sergei Neifert will present the physico-chemical study of dipeptide adsorption on solution disperse titanium dioxide covering preparation features that will help avoid hidden risks which researchers may face while performing relevant experiments. Place 183 milligrams of dipeptide into the sterile polymeric test tube and dilute up to approximately 35 milliliters with double distilled water and dissolve at room temperature under vigorous stirring. If the dipeptide doesn't dissolve in double distilled water and stirring, place the dipeptide solution into the ultrasonic bath and sonicate for a few minutes.Adjust the pH of pre-dissolved solution of a peptide to 7.4 by cautiously adding solution of MES or sodium hydroxide to the dipeptide solution upon stirring at room temperature and monitoring the pH with a pH meter. After adjusting the pH, pour the solution into measuring cylinder. Rinse the test tube and fill the measuring cylinder with the double distilled water up to 40 milliliters to make the final concentration of 16 millimolar.Prepare dipeptide dilutions with concentrations between 0.4 and 12 millimolar by diluting 16 millimolar dipeptide solution with double distilled water. For example, in order to prepare eight millimolar dipeptide solution, add seven milliliters of double distilled water to 10 milliliter of 16 millimolar dipeptide solution. After dilution, adjust the pH to 7.4 by adding drop-wise solution of MES or sodium hydroxide to the dipeptide solution.After adjusting the pH, pour the solution into the measuring cylinder. Rinse the test tube and fill the measuring cylinder up to 20 milliliters with double distilled water to make a concentration of eight millimolar. Other dilutions of dipeptide stock solution are prepared accordingly.In the end, we get a row of dipeptide dilutions ready for adsorption studies. Prepare 10 millimolar MES buffer solution. Adjust the pH to 7.4 with trisodium hydroxide upon stirring and monitoring the pH with a pH meter.This solution will be used for the sole preparation. Grind approximately 200 milligrams of nanocrystalline titanium oxide in a mortar for at least five minutes. Weigh 40 milligrams of grind titanium dioxide nanoparticles.Put the flask into the sonication bath using the laboratory stand. Add the grind titanium dioxide in 20 milliliters of MES buffer into the flask using the glass funnel and sonicate in an ultrasonic bath for 20 minutes. Set the thermostat to the desired temperature.Add one milliliter of the sonicated sole of titanium dioxide to the marked adsorption vials. Place the marked adsorption vials against corresponding dilutions in an improvised floating device made of styrofoam and put it into the thermostat to equilibrate the temperature for at least five minutes. After that, add one milliliter of dipeptide dilution to the corresponding marked adsorption vial making sure all mixing solutions have same temperature.Keep the series of obtained adsorption samples to the thermostat for 24 hours to achieve the adsorption equilibrium. Occasionally agitate titanium oxide dispersions during thermostating. In order to avoid temperature-induced re-equilibrium, take out one sample from the thermostat for filtration at a time.Take a sample of the dipeptide solution from each glass vial with a syringe. Remove the needle from the syringe and put on the syringe filter to filter the dipeptide solution into the glass vial. Repeat the filtration with samples of other concentrations.Now these samples are ready for analysis. Make the 50 milliliter solution of TFA in acetonitrile. Spike 0.34 milliliters of TFA in the measuring cylinder and adjust the volume of solution to 50 milliliters with acetonitrile at room temperature.Prepare the derivatization solution. Spike 299 microliters of phenylisothiocyanate and 347 microliters of triethylamine in the measuring cylinder and fill the cylinder up to 50 milliliters with acetonitrile. Prior to the high performance liquid chromatography analysis, derivatize the samples with Edman's reagents in the chromatography vials.Mix the 400 microliters of the sample with 400 microliters of derivatization reagent. Keep the samples at 60 degrees for 15 minutes. After heating, neutralize the samples with 225 microliters of TFA solution and wait for a few minutes to cool sample to room temperature.Use HPLC analysis to determine the concentration of the dipeptide solution before and after the adsorption. Start the analysis of the samples with the necessary conditions which are set by the software. The dependencies of adsorption from the equilibrium dipeptide concentration after adsorption, adsorption isotherms were plotted accordingly to the obtained experimental data.The measurements of dipeptide adsorption were data processed using the Henry model. The equilibrium binding constant was obtained from the slope of the dependence of dipeptide adsorption on the dipeptide equilibrium concentration. The van't Hoff equation was used for determining the standard Gibbs free energy for each temperature.Plot in a graph of free energy versus temperature, we determined the enthalpy as interception of the linear graph with a free energy axis for dipeptide. The change in entropy for each temperature was determined from the fundamental equation. The calculated values of equilibrium binding constant, standard Gibbs free energy, enthalpy and entropy for dipeptide are presented in table one.Adsorption isotherm construction from depletion data remains to be the most available methodology that does not require expensive setups, providing exhaustive physico-chemical data for literally every soluble sorbate. Combined with spectroscopic or computer-based data, it may reveal fundamental structural features of complex behavior of biomolecules upon contacting the inorganic nanoparticles.

Research

Education

JoVE Journal

JoVE Core

Chemistry

Solutions and Colloids

SCIENTISTS IN ACTION

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium Under Mild Reaction Conditions

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-phosphinetriyltripiperidine]}palladium Under Mild Reaction Conditions

Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family ...

Preparation of Macroporous Epitaxial Quartz Films on Silicon by Chemical Solution Deposition

This work describes the detailed protocol for preparing piezoelectric macroporous epitaxial quartz films on silicon(100) substrates. This is a three-step ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

The Synthesis of [Sn10(Si(SiMe3)3)4]2- Using a Metastable Sn(I) Halide Solution Synthesized via a Co-condensation Technique

The Synthesis of [Sn10SiSiMe334]2- Using a Metastable SnI Halide Solution Synthesized via a Co-condensation Technique

The number of well-characterized metalloid tin clusters, synthesized by applying the disproportionation of a metastable Sn(I) halide in the presence of a ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

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

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...