The authors acknowledge (1) financial supports from Department of Nanobiomedical Science &#38; BK21 PLUS NBM Global Research Center for Regenerative Medicine in Dankook University, and from the Priority Research Centers Program (No. 2009-0093829) funded by NRF, Republic of Korea, (2) the facilities, and the scientific and technical assistance, of the Australian Centre for Microscopy and Microanalysis at the University of Sydney. HKC is grateful to the Australian Research Council for the financial supports through a Discovery Project grant (DP0985367&#38; DP120102778). WCh is grateful to the Australian Research Council for the financial supports through a linkage Project grant (LP120200489, LP110200316).

1. Preparing the Colloidal Probe and AFM Substrate
<ol>
	<li>To prepare colloidal probes, use a method developed previously by the authors.19
	<ol>
		<li>In brief, use a 45&#176; angle holder to affix a tipless cantilever at the specific angle of 45&#176; (Figure 1A).</li>
		<li>Prepare an epoxy slide by smearing a thin layer of epoxy onto a microscope slide. Use a clean spatula or a slow stream of nitrogen to ensure that the layer of epoxy added to the microscope slide is of minimal height.</li>
		<li>Affix the epoxy slide to a 40X optical zoom microscope lens using a custom designed holder (Figure 1B). Then use the cantilever to approach the epoxy slide and acquire a small amount of epoxy on the cantilever.</li>
		<li>Repeat these steps to also attach a single particle of interest at the apex of the cantilever (Figure 1C).</li>
	</ol>
	</li>
	<li>Prepare the AFM substrate by affixing colloidal particles onto an AFM coverslip using a thermoplastic mounting adhesive.
	<ol>
		<li>Heat a 35 mm round cover slip to 120 &#176;C, and apply a small amount of the adhesive to the coverslip. The high temperature is necessary to melt the thermoplastic adhesive for application.</li>
		<li>Then cool, the coverslip to 40 &#176;C before dusting the colloidal particles onto the glue. NOTE: At 40 &#176;C the glue is sufficiently set that the particles will not become embedded into the glue, but the glue is sticky enough to ensure that the particles will adhere to the substrate.</li>
		<li>Further cool the coverslip to RT and use a gentle stream of nitrogen to blow off any excess unattached particles.</li>
		<li>Wash the substrate several times with the liquid medium that will be used for colloidal probe measurements to ensure that all unattached particles are removed from the substrate. NOTE: This is important to reduce the effects of free flowing particles during the measurement, which can interact with the cantilever and introduce errors in the results.</li>
	</ol>
	</li>
</ol>
2. Mounting the Colloidal Probe, Aligning Laser, and Equilibrating System
<ol>
	<li>Mount the coverslip with the colloidal particles into the bottom half of a liquid cell, making sure that the O-ring is seated properly to prevent any leaking.</li>
	<li>Place a hydrophobic transparent sheet onto the microscope stage to guard against any liquid that may leak during the experiment, especially if only using the bottom half of a liquid cell for the measurement, and place the liquid cell onto the microscope stage. NOTE: For simplicity one can use only the bottom half of a liquid cell, given that the system can be equilibrated adequately; tip &#8211; the evaporation changes the condition of the measurement and impacts the results/reading.</li>
	<li>Attach the colloidal probe to the AFM scanning head and assemble onto the AFM. With the AFM instrument software on, use the knobs on the scanning head to bring the cantilever tip into focus. NOTE: All procedural steps and measurements were completed using an MFP-3D-Bio AFM with Asylum Research software.</li>
	<li>To maximize intensity, align the laser onto the tip of the cantilever using the appropriate adjustment knobs on the scanning head.</li>
	<li>Allow the system to equilibrate for 5-10 min or until the deflection value stabilizes. Use the deflection adjustment knob to bring the deflection to zero or slightly negative.</li>
	<li>After the system has equilibrated in air, use the AFM software (Thermal Panel in the Master Panel window) to thermally calculate the InvOLS (sensitivity) and spring constant of the colloidal probe. NOTE: This sensitivity will be temporarily used until the true sensitivity is measured at the completion of the measurement (see step 4).
	<ol>
		<li>Select either &#8220;Cal Spring Constant&#8221; or &#8220;Cal InvOLS&#8221; and then click on &#8220;Capture Thermal Data&#8221;.</li>
		<li>Once a prominent peak is apparent, stop capturing data, and click to zoom over the main peak.</li>
		<li>Click on &#8220;Initialize Fit&#8221; followed by &#8220;Fit Thermal Data,&#8221; to obtain the automatically calculated spring constant or InvOLS values.</li>
	</ol>
	</li>
	<li>Slowly add 2 ml of the liquid medium to the liquid cell using a syringe and ensure that no bubbles are present around the cantilever. Re-align the laser, since the refractive index of the medium has now changed, and once again equilibrate the system allowing the deflection value to stabilize before adjusting the deflection back to zero. NOTE: If a large temperature difference exists between the environment and liquid, equilibration will take longer.</li>
</ol>
3. Imaging and Data Acquisition
<ol>
	<li>Set the initial scan size to 20 &#181;m, scan rate to 1 Hz, scan angle to 90&#176;, set point to 0.2 V and obtain a scan of the sample. Adjust the gain as needed to obtain overlapped trace and retrace curves.</li>
	<li>Once a particle of interest is found, immediately zoom onto that particle to limit extended probe interactions with the substrate prior to obtaining force volume measurements.</li>
	<li>Once zoomed in, acquire a sufficient image of a single particle or portion of a single particle. Then switch to the Force Panel in the software. Bring the red position bar to the highest position, set the force distance to 5 &#181;m, scan rate to 0.1 Hz, trigger channel to none and conduct a single force measurement. Make sure the probe does not contact the substrate.</li>
	<li>From the single measurement graph obtained, calculate the virtual deflection line by right clicking on the graph window, and selecting the &#8220;Calculate Virtual Def Line&#8221; option. This will automatically calculate the virtual deflection and update the value as needed within the software.</li>
	<li>Change the trigger channel to deflection and set the trigger point to 20 nm. Set the force distance to 1 &#181;m and adjust the scan velocity as desired depending on the measured forces of interest.</li>
	<li>Manually adjust the value for the deflection Inverse Optical Lever Sensitivity (InvOLS) in the Review Force Panel after conducting 2-3 consecutive preliminary single force measurements.
	<ol>
		<li>Conduct a single force measurement, then click on the &#8220;Review&#8221; button on the Force Panel which opens up a Master Force Panel.</li>
		<li>Highlight the most recently completed force measurement. Under the &#8220;Axis&#8221; heading ensure that only &#8220;DeflV&#8221; is checked. Change the &#8220;X-Axis&#8221; input field to &#8220;Sep&#8221; using the dropdown menu and click on &#8220;make graph.&#8221;</li>
		<li>Click on the &#8220;Parm&#8221; tab on the Master Force Panel and adjust the value of &#8220;InvOLS&#8221; until the contact region of the graph is completely vertical. Then populate this value in the &#8220;Defl InvOLS&#8221; field located under the Cal&#160;sub-tab in the Force tab located on the main Master Panel window.</li>
		<li>Repeat this 2-3 times to ensure that the InvOLS value does not change significantly.</li>
	</ol>
	</li>
	<li>Now that all parameters have been set up, ensure that the liquid medium level is still sufficient and that the deflection is still stable. NOTE: At this time, single force curves or force maps can be obtained. If dwell force measurements are desired, the dwell options can be accessed in the Force Panel.</li>
</ol>
4. Post-tuning of Sensitivity for Analysis
<ol>
	<li>After the completion of measurement acquisition, measure the true sensitivity of the colloidal probe. To do this, conduct a force measurement using a relatively large deflection/force with the colloidal probe in the same liquid medium against an &#8220;infinitely&#8221; hard surface such as mica. NOTE: Sensitivity was obtained after completion of the experiments because the large deflection/force may damage colloidal probes prepared with porous or fragile colloids.</li>
	<li>The slope of the contact region is used by the software to automatically calculate the sensitivity (Figure 2). Use this true value of sensitivity during data analysis of all the curves obtained using that particular colloidal probe.</li>
</ol>

<ol>
	<li>Sindel, U., Zimmermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+interaction+forces+between+individual+powder+particles+using+an+atomic+force+microscope.">Measurement of interaction forces between individual powder particles using an atomic force microscope.</a> Powder Technology. 117, 247-254 (2001).</li><li>Ducker, W. A., Senden, T. J., Pashley, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+colloidal+forces+using+an+atomic+force+microscope.">Direct measurement of colloidal forces using an atomic force microscope.</a> Nature. 353, 239-241 (1991).</li><li>Israelachvili, J. N., Adams, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+forces+between+two+mica+surfaces+in+aqueous+electrolyte+solutions+in+the+range+0&#8211;100+nm.">Measurement of forces between two mica surfaces in aqueous electrolyte solutions in the range 0&#8211;100 nm.</a> Journal of the Chemical Society, Faraday Transactions. 1, 975-1001 (1978).</li><li>Upadhyay, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetised+thermo+responsive+lipid+vehicles+for+targeted+and+controlled+lung+drug+delivery.">Magnetised thermo responsive lipid vehicles for targeted and controlled lung drug delivery.</a> Pharmaceutical Research. 29, 2456-2467 (2012).</li><li>Chrzanowski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biointerface:+protein+enhanced+stem+cells+binding+to+implant+surface.">Biointerface: protein enhanced stem cells binding to implant surface.</a> Journal of Materials Science: Materials in Medicine. 23, 2203-2215 (2012).</li><li>Chrzanowski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+evaluation+of+nickel&#8211;titanium+surface+properties+after+alkali+and+electrochemical+treatments.">Nanomechanical evaluation of nickel&#8211;titanium surface properties after alkali and electrochemical treatments.</a> Journal of The Royal Society Interface. 5, 1009-1022 (2008).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. 104, 145-152 (2013).</li><li>Page, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+adhesion+of+Staphylococcus+aureus+to+coated+glass+substrates.">Study of the adhesion of Staphylococcus aureus to coated glass substrates.</a> Journal of materials science. 46, 6355-6363 (2011).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Physical Review Letters. 56, 930-933 (1103).</li><li>Butt, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+electrostatic,+van+der+Waals,+and+hydration+forces+in+electrolyte+solutions+with+an+atomic+force+microscope.">Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope.</a> Biophysical Journal. 60, 1438-1444 (1991).</li><li>Johnson, K., Kendall, K., Roberts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+energy+and+the+contact+of+elastic+solids.">Surface energy and the contact of elastic solids.</a> Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences. 324, 301-313 (1971).</li><li>Deraguin, B., Landau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+strongly+charged+lyophobic+sols+and+of+the+adhesion+of+strongly+charged+particles+in+solution+of+electrolytes.">Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes.</a> Acta Physicochim: USSR. 14, 633-662 (1941).</li><li>Derjaguin, B., Muller, V., Toporov, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+deformations+on+the+adhesion+of+particles.">Effect of contact deformations on the adhesion of particles.</a> Journal of Colloid and Interface Science. 53, 314-326 (1975).</li><li>Verwey, E. J. W., Overbeek, J. T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+lyophobic+colloids.">Theory of the stability of lyophobic colloids.</a> DoverPublications.com, doi:10.1021/j150453a001. , (1999).</li><li>Kappl, M., Butt, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colloidal+probe+technique+and+its+application+to+adhesion+force+measurements.">The colloidal probe technique and its application to adhesion force measurements.</a> Particle &amp; Particle Systems Characterization. 19, 129-143 (2002).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. , (2012).</li><li>Sa, D. J., de Juan Pardo, E. M., de Las Rivas Astiz, R., Sen, S., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+indentational+elasticity+measurements+of+hydrogel+extracellular+matrix+substrates.">High-throughput indentational elasticity measurements of hydrogel extracellular matrix substrates.</a> Applied Physics Letters. 95, 063701-063701 (2009).</li><li>Zauscher, S., Klingenberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+between+cellulose+surfaces+measured+with+colloidal+probe+microscopy.">Friction between cellulose surfaces measured with colloidal probe microscopy.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 178, 213-229 (2001).</li><li>Sa, D., Chan, H. -. K., Chrzanowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Micro-+and+Nano-particles+on+Tipless+Cantilevers+for+Colloidal+Probe+Microscopy.">Attachment of Micro- and Nano-particles on Tipless Cantilevers for Colloidal Probe Microscopy.</a> International Journal of Colloid and Interface. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

Several sources of system instability present during liquid colloidal probe nanoscopy can easily be mitigated through proper equilibration procedures. Instabilities as discussed previously result in erroneous results and force curves that are more difficult to analyze objectively. If all sources of instability have been tended and graphs similar to that shown in Figure 4 are still present, another measurement parameter may be the reason. Other measurement parameters that are important to consider during ...

Atomic force microscopy (AFM) is a technique that enables qualitative and quantitative imaging and probing of a material surface.4-6 Traditionally, AFM is used for the evaluation of surface topography, morphology and structure of multi-phasic materials. AFM has the capability to quantitatively evaluate nano-scale interactions, such as charge, attraction, repulsion and adhesion forces between a specific probe and substrate in both air and liquid mediums.7,8 The AFM originally developed by Binning, Quate and Gerber9 uses a probe of known/determined sensitivity and spring constant to approach and/or scan a sample. Due to the physical interactions between the probe and the sample, the cantilever is deflected during contact or proximity and depending on the mode of operation, this deflection can be translated to acquire the topography of the sample or measure forces present between the probe and sample. Modifications to the AFM technique, such as colloidal probe nanoscopy,10 have allowed scientist to directly evaluate the nano-force interactions between two materials present in a colloidal system of interest.
In colloidal probe nanoscopy, a spherical particle of choice is attached to the apex of a cantilever, replacing the traditional conical and pyramidal tips. A spherical particle is ideal to allow comparison with theoretical models such as the Johnson, Kendal, Roberts (JKR)11 and Derjaguin, Landau, Vervwey, Overbeek (DLVO)12-14 theories and to minimize the influence of surface roughness on the measurement.15 These theories are used to define the contact mechanics and inter-particle forces expected within a colloidal system. The DLVO theory combines the attractive van der Waal forces and repulsive electrostatic forces (due to electrical double layers) to quantitatively explain the aggregation behavior of aqueous colloidal systems, while the JKR theory incorporates the effect of contact pressure and adhesion to model elastic contact between two components. Once an appropriate probe is produced, it is used to approach any other material/particle to evaluate the forces between the two components. Using a standard manufactured tip one will be able to measure interactive forces between that tip and a material of choice, but the benefit of using a custom made colloidal probe allows the measurement of forces present between materials present within the studied system. Measurable interactions include: adhesive, attractive, repulsive, charge, and even electrostatic forces present between the particles.16 Additionally, the colloidal probe technique can be used to explore tangential forces present between particles and material elasticity.17,18
The ability to conduct measurements in various media is one of the major advantages of colloidal probe nanoscopy. Ambient conditions, liquid media, or humidity-controlled conditions can all be used to mimic environmental conditions of the system studied. The ability to conduct measurements in a liquid environment enables the study of colloidal systems in an environment that it naturally occurs; thus, being able to quantitatively acquire data that is directly translatable to the system in its natural state. For example, particle interactions present within metered dose inhalers (MDI) can be studied using a model liquid propellant with similar properties to the propellant used in MDIs. The same interactions measured in air would not be representative of the system existent in the inhaler. Furthermore, the liquid medium can be modified to evaluate the effect of moisture ingress, a secondary surfactant, or temperature on the particle interactions in an MDI. The ability to control temperature can be used to mimic certain steps in the manufacturing of colloidal systems to evaluate how temperature either in the manufacturing of or storage of colloidal systems may have an impact on particle interactions.
Measurements that can be obtained using colloidal probes include; Topography scanning, individual force-distance curves, force-distance adhesion maps, and dwell force-distance measurements. Key parameters that are measured using the colloidal probe nanoscopy method presented in this paper include the snap-in, max load, and separation energy values. Snap-in is a measurement of the attractive forces, max load the value of the maximum adhesion force, and the separation energy conveys the energy required to withdraw the particle from contact. These values can be measured through instantaneous or dwell force measurements. Two different types of dwell measurements include deflection and indentation. The length and type of dwell measurement can be specifically chosen to mimic specific interactions that are present within a system of interest. An example is using deflection dwell - which holds the samples in contact at a desired deflection value &#8211; to evaluate the adhesive bonds that develop in aggregates formed in dispersions. The adhesive bonds formed can be measured as a function of time and can provide insight into the forces required to redisperse the aggregates after prolonged storage. The plethora of data that can be obtained using this method is a testament to the versatility of the method.

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Double-Bubble Epoxy</td>
			<td style="width:179px;">Hardman</td>
			<td style="width:119px;">4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Veeco Tipless Probes</td>
			<td style="width:179px;">Veeco</td>
			<td style="width:119px;">NP-O10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Porous Particles</td>
			<td style="width:179px;">Pearl Therapeutics</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Atomic Force Microscope (MFP)</td>
			<td style="width:179px;">Asylum&#160;</td>
			<td style="width:119px;">MFP-3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SPIP Scanning Probe Image Processor Software</td>
			<td style="width:179px;">NanoScience&#160; Instruments</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">35 mm Coverslips</td>
			<td style="width:179px;">Asylum</td>
			<td style="width:119px;">504.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tempfix</td>
			<td style="width:179px;">Ted Pella. Inc.</td>
			<td style="width:119px;">16030</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Liquid colloidal systems are used for several pharmaceutical drug delivery systems. For inhalation drug delivery, a common colloidal system is the suspension pressurized metered dose inhaler (pMDI). Particle interactions present within the pMDI play a vital role in formulation physical stability, storage, and drug delivery uniformity. In this manuscript, inter-particle forces between porous lipid-based particles (~2 &#181;m optical mean particle diameter) in a model propellant (2H,3H-perfluoropentane) were evaluated at R...

Watch this Scientific Journal Video about Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy at JoVE.com

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

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 ...

quantitative-qualitative-examination-particle-particle-interactions

Research

JoVE Journal

Chemistry

11.0K visualizações.  University of Sydney. 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. 

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

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

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 ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Photodeposition of Pd onto Colloidal Au Nanorods by Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot electrons upon SPR irradiation drive reductive deposition of Pd on colloidal AuNR in the presence of [PdCl4]2-. Plasmon-driven reduction of secondary metals potentiates covalent, sub-wavelength deposition at targeted locations coinciding with electric field &#8220;hot-spots&#8221; of the plasmonic substrate using an external field (e.g., laser). The process described herein details a solution-phase deposition of a catalytically-active noble metal (Pd) from a transition metal halide salt (H2PdCl4) onto aqueously-suspended, anisotropic plasmonic structures (AuNR). The solution-phase process is amenable to making other bimetallic architectures. Transmission UV-vis monitoring of the photochemical reaction, coupled with ex situ XPS and statistical TEM analysis, provide immediate experimental feedback to evaluate properties of the bimetallic structures as they evolve during the photocatalytic reaction. Resonant plasmon irradiation of AuNR in the presence of [PdCl4]2- creates a thin, covalently-bound Pd0 shell without any significant dampening effect on its plasmonic behavior in this representative experiment/batch. Overall, plasmonic photodeposition offers an alternative route for high-volume, economical synthesis of optoelectronic materials with sub-5 nm features (e.g., heterometallic photocatalysts or optoelectronic interconnects).

A protocol for anisotropic photodeposition of Pd onto aqueously-suspended Au nanorods via localized surface plasmon excitation is presented.

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot ...

Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation

In this work, we demonstrate a facile method for colloidal lead halide perovskite nanoplatelet synthesis (Chemical formula: L2[ABX3]n-1BX4, L: butylammonium and octylammonium, A: methylammonium or formamidinium, B: lead, X: bromide and iodide, n: number of [BX6]4- octahedral layers in the direction of nanoplatelet thickness) via ligand-assisted reprecipitation. Individual perovskite precursor solutions are prepared by dissolving each nanoplatelet constituent salt in N,N-dimethylformamide (DMF), which is a polar organic solvent, and then mixing in specific ratios for targeted nanoplatelet thickness and composition. Once the mixed precursor solution is dropped into nonpolar toluene, the abrupt change in the solubility induces the instantaneous crystallization of nanoplatelets with surface-bound alkylammonium halide ligands providing colloidal stability. Photoluminescence and absorption spectra reveal emissive and strongly quantum-confined features. X-ray diffraction and transmission electron microscopy confirm the two-dimensional structure of the nanoplatelets. Furthermore, we demonstrate that the band gap of perovskite nanoplatelets can be continuously tuned in the visible range by varying the stoichiometry of the halide ion(s). Lastly, we demonstrate the flexibility of the ligand-assisted reprecipitation method by introducing multiple species as surface-capping ligands. This methodology represents a simple procedure for preparing dispersions of emissive 2D colloidal semiconductors.

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.