Name: study of short peptide adsorption on solution dispersed inorganic nanoparticles using depletion method
Uploaded: 2020-04-11T13:00:00.000+00:00
Duration: 9 min 43 s
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 a 50 mM solution of 2-(N-morpholino)ethanesulfonic acid (MES) buffer by dissolving 0.533 g of dry 2-(N-morpholino)ethanesulfonic acid in 50 mL of DDW in the sterile test tube. Prepare a 50 mM sodium hydroxide solution by dissolving 200 mg of sodium hydroxide in 100 mL of DDW.</li>
		<li>Adjust the pH of the predissolved dipeptide solution to 7.4 by carefully adding (microliter titration) 50 mM MES, or 50 mM sodium hydroxide to the 16 mM dipeptide solution, stirring at RT and monitoring the pH with a pH meter. After adjusting the pH, pour the solution into a measuring cylinder, rinse the test tube, and fill the measuring cylinder with DDW to 40 mL to make a final concentration of 16 mM.</li>
	</ol>
	</li>
	<li>Preparation of dipeptide dilutions from 16 mM stock solution
	<ol>
		<li>Prepare peptide dilutions with concentrations between 0.4 and 12.0 mM by diluting the 16 mM dipeptide solution with DDW. For example, in order to prepare an 8 mM dipeptide solution, add 7 mL of DDW to 10 mL of the 16 mM dipeptide solution. After dilution, adjust the pH to 7.4 by adding 50 mM MES or 50 mM NaOH drop by drop to the dipeptide solution (see step 1.1.3). After adjusting the pH, pour the solution into a measuring cylinder, rinse the test tube, and fill the measuring cylinder up to 20 mL with DDW to make the dipeptide concentration 8 mM. 
		NOTE: Other dilutions of 16 mM dipeptide stock solution with concentrations of 0.4, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0, 8.0, and 12.0 mM, are prepared in accordance with Figure 1. The adjustment of each dipeptide solution pH to 7.4 is described in step 1.1.3.</li>
	</ol>
	</li>
</ol>
2. Preparation of titania sol
<ol>
	<li>Prepare a 10 mM solution of MES buffer by dissolving 1.066 g of MES in 500 mL of DDW. Adjust the pH to 7.4 with dry sodium hydroxide upon stirring and monitoring the pH with the pH meter.</li>
	<li>Grind 200 mg of nanocrystalline TiO2 in a mortar for at least 5 min (see Table of Materials).</li>
	<li>Weigh 40 mg of the ground titanium dioxide nanoparticles into a laboratory flask. Put the flask into the sonication bath (see Table of Materials) using the laboratory stand.</li>
	<li>Add 20 mL of 10 mM MES buffer into the flask with TiO2 and sonicate in an ultrasonic bath (5 L, 40 kHz, 120 W) for 20 min.</li>
</ol>
3. Mixing and thermostating
<ol>
	<li>Set the thermostat (see Table of Materials) to the desired temperatures (i.e., 0.00, 10.00, 20.00, 30.00, or 40.00 &#176;C).</li>
	<li>Add 1 mL of the sonicated sol of TiO2 to the marked adsorption vials. Place the marked adsorption vials against corresponding dipeptide dilution in a makeshift flotation device made of extruded polystyrene foam. Place the flotation device with the marked vials and corresponding dipeptide dilutions into the thermostat for at least 5 min.</li>
	<li>Add 1 mL of each dipeptide dilution to the corresponding marked adsorption vial, making sure all mixing solutions have the same temperature. Keep the series of obtained adsorption samples on the thermostat at 0.00, 10.00, 20.00, 30.00, or 40.00 &#176;C for 24 h to achieve the adsorption equilibrium. 
	NOTE: Cautiously shake all the samples of obtained dispersions prior to putting them into the thermostat.</li>
	<li>Occasionally mix the TiO2 dispersions by manually shaking them during thermostating.</li>
</ol>
4. Filtration of the thermostated samples
<ol>
	<li>In order to avoid temperature-induced reequilibration take out one sample at a time from the thermostat for filtration.</li>
	<li>Take a sample of the dipeptide solution from each glass vial with a syringe, through a syringe needle. Remove the needle from the syringe and put on the syringe filter (see Table of Materials) to filter the dipeptide solution into the glass vial. Repeat the filtration with the other samples.</li>
	<li>Analyze the filtrate in accordance with section 5. 
	NOTE: Do not centrifuge the samples, because it takes a few minutes and may cause a change in the concentration equilibrium.</li>
</ol>
5. Derivatization and HPLC analysis
<ol>
	<li>Make a 50 mL solution of trifluoroacetic acid (TFA) in acetonitrile. Add 0.34 mL of TFA in the measuring cylinder and adjust the volume of the solution to 50 mL with acetonitrile at RT. 
	CAUTION: Work with TFA under a fume hood with exhaust ventilation, because trifluoroacetic acid is harmful when inhaled, causes severe skin burns, and is toxic for aquatic organisms even at low concentrations53.</li>
	<li>Prepare the derivatization solution (i.e., Edman reagent54) by placing 299 &#181;L of phenyl isothiocyanate and 347 &#181;L of triethylamine in a graduated cylinder and adjusting the volume of solution to 50 mL with acetonitrile at RT.</li>
	<li>Prior to the high-performance liquid chromatography (HPLC) analysis, derivatize the samples with Edman&#39;s reagent in the chromatography vials. Mix 400 &#181;L of the sample with 400 &#181;L of Edman&#39;s reagent. Heat the sample at 60 &#176;C for 15 min. After heating, neutralize the sample with 225 &#181;L of the TFA solution and wait for a few minutes to cool the sample to RT.</li>
	<li>Use HPLC analysis (see Table of Materials) to determine the concentration of the dipeptide solution before and after adsorption. Place the chromatography vials with the analyzed solutions into the HPLC autosampler and start analyzing the samples with the necessary conditions, which are set by the software (see Table of Materials). 
	NOTE: The mobile phase consists of 0.1% TFA in deionized water and pure acetonitrile, with acetonitrile gradients from 20-90% at 286 nm for 13 min. Analyze each sample in triplicate. Measure the dipeptide solution concentration using the previously established calibration curve (Figure 2). For chromatography specifications see Shchelokov et al.55.</li>
</ol>

The authors have nothing to disclose.

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, and yet it provides exhaustive physical chemistry data for literally every soluble sorbate.
A distinction has to be made between adsorption modes (i.e., solution dispersed particles or on a fixed surface) when a crystalline material is used as a sorbent. One should expect a substantial difference in the distribution of crystalline faces on macroscopically flat surfaces and on particles. Resulting thermodynamic parameters determined from adsorption of peptides on nanoparticles may not correspond to the thermodynamic parameters of peptide adsorption to macroscopically flat surfaces.
The average amount of peptides adsorbed on the inorganic surfaces is extremely low. At room temperature, this value is about several hundreds of micrograms per square meter28. This small amount of adsorbate requires accurate measurement methods and solids with well-developed surfaces. Therefore, small particle substances with a large specific surface (hundreds of square meters) should be used for adsorption experiments43,56,57,58,59,60.
Peptides are, like proteins, unstable, and retain their functionality at a narrow range of conditions. Adsorption experiments were performed on nanocrystalline titanium dioxide at biocompatible temperatures of 0 &#176;C-40 &#176;C (273.15 K-313.15 K), which are similar to those of a normal, functioning, living organism. Adsorption at higher or lower temperatures are irrelevant and should not be considered for the experiment.
Multifunctional biologically active compounds also exhibit high susceptibility to pH of the media, as it affects the surface charge and therefore Coulomb interactions between charged functional groups61,62,63. The sorbent charge of oxide materials is also pH-dependent due to active proton exchange at the hydrated surface64. In order to establish pH stable conditions for adsorption equilibrium use of a buffer is required. In this study, MES buffer is used for its noncoordinating property65, so it would not compete with the peptide for adsorption on the metal oxide surface, unlike phosphate buffers66.
This recent test of amino acid adsorption shows that the major binding site on the nanoparticle is the surface defect55. Defect distribution on the surface is one of the least controllable features of the nanocrystalline substrates, hence one should use sorbent from the same batch in order to maintain consistency in adsorption studies.
QCM, plasmon resonance, and ITC are genuine methods with subtle sensitivity that in a combination of spectroscopic methods reveal structural peculiarities of the adsorbate during interplay with the surface. However, they do not overcome kinetic restrictions and still require considerable time to achieve adsorption equilibration. Furthermore, only one sample at a time can be processed, which makes batch sample analysis challenging. On the other hand, the depletion method presented is simple and only restricted to the thermostat capacity, making the processing of a large number of samples possible.
The thermostated samples should be filtered as soon as they are removed from the thermostat in order to avoid temperature-induced reequilibration. Although equilibration at a new temperature may take up to a few hours, keeping the adsorption samples at a different temperature should be minimized. Centrifugation of the samples for supernatant separation is also not recommended, because it takes up to a few minutes and may cause a change in the concentration equilibrium. The choice of the filter material depends on the sorbate nature and should reduce possible filter-binding for maximum recovery. It is best to follow vendor instructions and recommendations when choosing specific filters.
Additionally, one should bear in mind that concentration change in the adsorption studies should be monitored using validated quantification method using mass spectrometry, radio-spectroscopy, or UV-visible spectroscopy. The analysis is easy if the adsorbate is spectroscopically active, otherwise additional labeling or derivatization of the adsorbate is required.

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><tbody><tr><td>2-(&lt;em&gt;N&lt;/em&gt;-Morpholino)ethanesulfonic acid</td><td>TCI Chemicals</td><td>4432-31-9</td><td>MES, &gt;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 &quot;Ultrasons-HD&quot;</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&amp;minus;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>&lt;a href=&quot;https://www.sigmaaldrich.com/catalog/search?term=67-63-0&amp;amp;interface=CAS%20No.&amp;amp;lang=en&amp;amp;region=US&amp;amp;focus=product&quot; target=&quot;_blank&quot;&gt;67-63-0&lt;/a&gt;</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 TiO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>Pure anatase with at least 99% crystallinity. Average particle size 10.62 &amp;plusmn; 3.31 nm. Specific surface 131.9 m2/g (BET). See &lt;em&gt;Langmuir&lt;/em&gt; 2019, 35, 538&amp;minus;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&amp;times;2.5 mm i.d. with a mean particle size of 5 &amp;mu;m</td></tr><tr><td>Syringe filter</td><td>Vladfilter</td><td></td><td>25 mm, 0.2 &amp;mu;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>&lt;a href=&quot;https://www.sigmaaldrich.com/catalog/search?term=121-44-8&amp;amp;interface=CAS%20No.&amp;amp;N=0&amp;amp;mode=partialmax&amp;amp;lang=en&amp;amp;region=RU&amp;amp;focus=product&quot; target=&quot;_blank&quot;&gt;121-44-8&lt;/a&gt;</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 dipeptide starting and equilibrium concentrations in millimoles, respectively; V is the volume of a dipeptide solution in liters; and m is the weight of the sorbent in grams.
The measurements of the dipeptide adsorption were data processed using the Henry model. This isotherm model assumes adsorption at relatively low concentrations with the sorbate molecules isolated from each other on a sorbent surface and is suitable for describing the experimental data (Figure 3). Note, however, that this model can only be applied in the case of reversible adsorption, which should be confirmed as well. IR-spectroscopy of the material rinsed multiple times is suitable for this purpose. The obtained equilibrium peptide amounts on the TiO2 and solution are related in accordance with the linear equation:
<img src="/files/ftp_upload/60526/60526eq2.jpg" />
where KH is Henry&#39;s adsorption constant.
The equilibrium binding constant KH was obtained from the slope of the dependence of dipeptide adsorption (A) on the dipeptide equilibrium concentration (Ce). The standard Gibbs free energy (&#916;G, kJ/mol) for each temperature T was determined through the Van&#39;t Hoff equation:
<img src="/files/ftp_upload/60526/60526eq3.jpg" />
where R is the ideal gas constant in J/mol*K, and T is the temperature of the adsorption process in Kelvin.
Dipeptide Gibbs free energies determined at each temperature (Figure 4) disclosed enthalpy (&#916;H) as an interception of the linear regression with the axis. The regression variable, the entropy of the process (&#916;S), was derived from the fundamental equation:
<img src="/files/ftp_upload/60526/60526eq4.jpg" />
The calculated values of the equilibrium binding constant (KH), standard Gibbs energy (&#916;G), enthalpy (&#916;H), and entropy (&#916;S) for Ile-His are presented in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60526/60526fig1.jpg" /> 
Figure 1: Dilution of the 16 mM dipeptide stock solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60526/60526fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60526/60526fig2.jpg" /> 
Figure 2: Calibration curve at different dipeptide concentration. The dipeptide concentrations were between 0.4-16.0 mM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60526/60526fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60526/60526fig3.jpg" /> 
Figure 3: The dipeptide adsorption isotherms calculated by the Henry model for each temperature. Dipeptide adsorption isotherms at (A) 0 &#176;C (B) 10 &#176;C (C) 20 &#176;C (D) 30 &#176;C, and (E) 40 &#176;C, respectively. The calculated correlation coefficients (R2) fell into a 0.96&#8722;0.99 range for all obtained Henry model isotherms. Error bars represent the 95% confidence interval for each sample concentration measured in triplicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60526/60526fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60526/60526fig4.jpg" /> 
Figure 4: Dependence of the standard Gibbs free energy of the dipeptide adsorption on temperature. Error bars represent the 95% confidence interval for Gibbs free energy as indirect measurement based on Henry Model. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60526/60526fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>T, K</td>
			<td>KH</td>
			<td>&#916;G0, kJ/mol</td>
			<td>&#916;H0, kJ/mol</td>
			<td>&#916;S0, kJ/mol K</td>
		</tr>
		<tr>
			<td>273.15</td>
			<td>0.32 &#177; 0.01</td>
			<td>2.6 &#177; 0.0</td>
			<td rowspan="5">- 41 &#177; 9</td>
			<td rowspan="5">- 0.16 &#177; 0.03</td>
		</tr>
		<tr>
			<td>283.15</td>
			<td>0.25 &#177; 0.01</td>
			<td>3.2 &#177; 0.1</td>
		</tr>
		<tr>
			<td>293.15</td>
			<td>0.17 &#177; 0.06</td>
			<td>4.3 &#177; 0.9</td>
		</tr>
		<tr>
			<td>303.15</td>
			<td>0.050 &#177; 0.002</td>
			<td>7.6 &#177; 0.1</td>
		</tr>
		<tr>
			<td>313.15</td>
			<td>0.037 &#177; 0.002</td>
			<td>8.3 &#177; 0.1</td>
		</tr>
	</tbody>
</table>
Table 1: Thermodynamic parameters of dipeptide adsorption.

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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 ...

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6.5K Views.  South Ural State University. Het samenspel van eiwitten en peptiden met anorganisch materiaal is een fundamenteel fenomeen met implicaties voor nanotechnologie, biomaterialen en biotechnologie. De eerste stap in het begrijpen van een dergelijk fenomeen is het onthullen van de fundamentele fysisch-chemische constanten zoals adsorptieconstante, Gibbs vrije energie, enthalpy, entropie, en beperkte adsorptie die kunnen worden geëvalueerd door de vaststelling van adsorptie isothermen. Adsorptie uit vloeibare fase is echter b...

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

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Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Bioengineering

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.

Formulation of stable, functional inks is critical to expanding the applications of additive manufacturing. In turn, knowledge of the mechanisms of dispersant/particle bonding is required for effective ink formulation. Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) is presented as a simple, inexpensive way to gain insight into these mechanisms.

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Engineering

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

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 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.

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.

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

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

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

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