This work was funded by the National Institutes of Health (NIH) grant R35GM142920. The research reported here made use of shared facilities of the UCSB MRSEC (NSF DMR 1720256), a member of the Materials Research Facilities Network (www.mrfn.org). We thank Phoebe Hertler for contributing to the original article this work references. We thank Dr. Claire Chisholm for assisting in the acquisition of the scanning electron microscopy images.

1. Establishing a low-noise system
NOTE: The relevant experiments require a potentiostat capable of achieving the highly time-resolved measurement of low currents. To achieve this, employ a research-grade commercial potentiostat capable of a 1 &#181;s time resolution that can quantify currents at the femtoampere level. To further reduce electronic interference from the environment, conduct experiments within two nesting Faraday cages. Ensure that the setup is capable of a root-mean-square deviation of less than 100 fA for a chronoamperometry experiment sampled at 10 Hz in 0.1 M potassium chloride.
<ol>
	<li>Obtain and set up the equipment, including the potentiostat and Faraday cages. 
	&#8203;NOTE: Faraday cages may be obtained commercially or custom-fabricated using conductive metals (e.g., copper or aluminum). Custom-fabricated aluminum Faraday cages were used for the study described here (see Table of Materials).</li>
</ol>
2. Experimental preparation
<ol>
	<li>Use commercially available 2 &#181;m diameter carboxylate-modified polystyrene beads (see Table of Materials). 
	NOTE: While this system can be generalized to other electro-inactive species23,33, it is critical to remember that the impacts rely on electrophoretic migration in addition to Brownian motion. Thus, apply a potential attractive to the species of interest, and maintain low salt concentrations17.</li>
	<li>Prepare the following solutions, which can be stored at room temperature for at least 1 month.
	<ol>
		<li>Prepare a 50 mM carbonate solution, and titrate to pH 12.0 using 1 M NaOH. Monitor the pH weekly.</li>
		<li>Prepare a 1 M sodium perchlorate solution.</li>
	</ol>
	</li>
	<li>Prepare the following solutions fresh each day, discarding them at the end of the day.
	<ol>
		<li>Prepare a 10 mM TEMPO in 50 mM carbonate solution, pH 12.0.</li>
		<li>Prepare a 500 mM maltose stock in 50 mM carbonate solution, pH 12.0.</li>
	</ol>
	</li>
	<li>Select the working electrode. For consistent results, select an ultramicroelectrode (see Table of Materials) such that the radius of the species to be characterized is no less than 10%-15% of the electrode radius17,21,23,33,34. 
	NOTE: This ratio can be minimized after determining the magnitude of the impact-associated current steps for the optimal detection of a particular species of interest. The electrode material selected must catalyze the background redox reaction.</li>
	<li>Prepare two electrochemical cells of 5 mL each.
	<ol>
		<li>Prepare a control cell containing 1 mM TEMPO and 5 mM sodium perchlorate (see Table of Materials) in carbonate buffer at pH 12.0.</li>
		<li>Prepare a test cell containing a solution of 1 mM TEMPO, 5 mM sodium perchlorate, and 120 mM maltose in carbonate buffer at pH 12.0. 
		NOTE: The ratio of the redox mediator to the substrate (here, TEMPO to maltose) can be varied to explore the effects of the reaction rate20. This value provides insight into the homogeneous chemical reaction.</li>
		<li>After preparing these cells, set them aside for later electrochemical measurements.</li>
	</ol>
	</li>
</ol>
3. Electrode polishing
<ol>
	<li>Prior to each experimental run, polish the electrode sequentially for 2 min each with a 1 &#181;m, 0.3 &#181;m, and 0.5 &#181;m alumina slurry (see Table of Materials) on polishing pads.</li>
	<li>Move the electrode in a &#34;figure 8&#34; pattern to ensure an even polish35,36. Liberally rinse with deionized water, and dry with a laboratory wipe. 
	&#8203;NOTE: Do not sonicate the ultramicroelectrodes, as this may damage them.</li>
</ol>
4. Electrochemical measurements
NOTE: See Figure 2 for the results.
<ol>
	<li>Utilize a three-electrode setup for the electrochemical measurements. For the experiments described here, employ an 11 &#181;m carbon-fiber ultramicroelectrode, a platinum wire counter electrode, and a saturated calomel reference electrode (SCE) (see Table of Materials). 
	NOTE: Electrochemical potential windows for the experiments are noted below; for reference, TEMPO has a formal potential of 0.49 V versus SCE, and maltose is not electroactive in the potential&#160;window used in these experiments. Any leaking from the reference electrode may affect the total salt concentration37, thus reducing the electrophoretic driving of the particles to the ultramicroelectrode and reducing the counting efficiency17. If the experiments yield a low impact frequency, consider switching to a leakless reference electrode38,39.</li>
	<li>Set the control cell in the Faraday cages, and connect the electrodes to the appropriate cables. Collect the first set of electrochemical measurements for each cell. This will consist of a cyclic voltammetry experiment and a chronoamperometry experiment, as detailed below.
	<ol>
		<li>Collect the cyclic voltammetry data using a potential window from 0.2 V to 0.8 V versus SCE at scan rates of 10 mV&#183;s&#8722;1, 20 mV&#183;s&#8722;1, 30 mV&#183;s&#8722;1, 40 mV&#183;s&#8722;1, and 50 mV&#183;s&#8722;1.</li>
		<li>Collect the chronoamperometry data by applying 0.8 V versus SCE for 10 min, and record at a 10 Hz sampling rate. 
		NOTE: A 10 min sampling period is recommended for chronoamperometry experiments to obtain 10-15 individual impacts at later steps. No impacts are expected at this juncture.</li>
	</ol>
	</li>
	<li>Next, spike a dilute solution of polystyrene beads to a final concentration of 0.66 pM17 into each of the electrochemical cells. After the addition of the polystyrene beads, collect the second set of electrochemical measurements for each cell. 
	NOTE: The impact frequency will be a function of this concentration and requires optimization to collect sufficient data for statistical analysis without saturating the chronoamperogram with overlapping impacts40,41,42,43.
	<ol>
		<li>Collect chronoamperometry data by applying 0.8 V versus SCE for 10 min, and record at a 10 Hz sampling rate.</li>
		<li>Repeat the chronoamperometric measurements until sufficient data points have been collected for statistical analysis. To detect differences between the multiple sizing methods with a confidence level of 95% and a power of 80%, select a sample size of approximately 200 individual impact events.</li>
	</ol>
	</li>
</ol>
5. Scanning electron microscopy (SEM)
NOTE: Use scanning electron microscopy as a &#34;gold standard&#34; technique to confirm the nanoparticle sizes and sample heterogeneity19,44.
<ol>
	<li>Sample preparation
	<ol>
		<li>To prepare a sample for imaging, dilute a carboxyl latex bead suspension&#160;(see Table of Materials)&#160;1:20 in water. Drop-cast 10 &#181;L onto a glass slide, dry under a nitrogen stream, and sputter-coat the sample with a conductive layer of gold-palladium under argon.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Using an accelerating beam voltage of 5 kV and a current of 0.4 nA, collect images as appropriate for statistical analysis. Use ImageJ45,46 or an equivalent image analysis software to determine the particle sizes.</li>
	</ol>
	</li>
</ol>
6. Electrochemical data analysis
<ol>
	<li>Record the electrochemical data using the potentiostat&#39;s software, and analyze these results using a written script20 that can extract the current magnitudes from the detected changes in the steady-state current (current steps) resulting from nanoimpact events20. 
	NOTE: This script is included as part of the supplemental information in our previously published report20.</li>
	<li>Convert the amplitudes of the current steps to bead radii using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/65116/65116eq01.jpg" style="margin: auto;" /> 
	NOTE: Here, rb is the bead radius, rel is the electrode radius,&#160;&#916;Iss/Iss is the ratio between the change in the current produced by a particle&#39;s adsorption and the initial steady-state current observed prior to the adsorption of that particle, and f(MT,Geom) = 0.067 is an empirical scaling factor that depends on both geometric and mass transport considerations20,25.</li>
	<li>Plot the frequency of detecting a given bead radius versus the radius (Figure 3) to quantify the distribution metrics.</li>
</ol>
7. Modeling
NOTE: If desired, the mechanism by which electrocatalytic interruption works can be validated by confirming the shift from the diffusion-limited current generation to the reaction rate-limited current generation. To describe and visualize, use two different numerical simulation programs: a voltammogram fitting software, such as DigiSim, to determine the homogeneous rate constant, and a multiphysics modeling platform, such as COMSOL Multiphysics, to visualize the local changes to the diffusion profile at the ultramicroelectrode surface (see Table of Materials).
<ol>
	<li>Voltammogram fitting 
	NOTE: Use the voltammogram fitting software to determine the homogeneous rate constant (Figure 4).
	<ol>
		<li>Collect cyclic voltammograms in a solution containing 1 mM TEMPO (only) and 1 mM TEMPO plus 120 mM maltose. For each condition, collect data at various scan rates, and use this data for numerical fittings of these experimental datasets.
		<ol>
			<li>Fit the voltammograms collected from the TEMPO-only experiment using an E mechanism, which describes a reaction process driven solely by the electrode47,48. This will yield the electrode parameters.</li>
			<li>Using the electrode parameters obtained from step 7.1.1.1, fit the resulting voltammograms from the 1 mM TEMPO plus 120 maltose solution to an EC&#8242; mechanism47,48, which describes an electrode process that is followed by a homogeneous chemical reaction that regenerates the redox mediator. This will yield the homogeneous rate constant.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Multiphysics modeling
	<ol>
		<li>Use a multiphysics modeling platform to visualize the changes in the diffusion profiles at the ultramicroelectrode surface for both the control and electrocatalytic interruption systems20,49,50,51,52 (Figure 5). Use the electrode parameters and homogeneous rate constant obtained from the voltammogram fitting as initial conditions. A broad overview of the workflow, which can be adapted to different software, is provided below.</li>
		<li>Input the global parameters. These consist of fixed values such as (but not limited to) the concentration values, diffusion coefficients, electrode radius, and temperature.</li>
		<li>Build the simulation space. This is a set of geometries that include the electrode, the insulating sheath, the surrounding solution space representing the region of interest, and an infinite element domain representing the bulk surroundings.</li>
		<li>Introduce physics packages to define the simulation.
		<ol>
			<li>Associate the electrode space with an electroanalysis study. Define the initial values and electrode reaction of interest here.</li>
			<li>Associate the surrounding solution space with a chemistry study. Define the homogeneous chemical reaction that follows the electrode reaction here.</li>
			<li>Introduce a mesh over the entire simulation space. This defines how the geometry is divided to solve the model. To achieve high-quality results, use a finer mesh near the electrode.</li>
		</ol>
		</li>
		<li>To observe changes as a result of the homogeneous rate constant, vary the value of this parameter using a parametric study to solve the model.</li>
		<li>To observe changes as a function of time, vary the value of this parameter using a time-dependent study to solve the model.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Electrocatalytic interruption is easy to implement and reduces the imprecision associated with nanoimpact electrochemistry by an order of magnitude. This enhanced precision directly enables researchers to discriminate between differently sized particles in a mixed solution20. It also enhances the ability to reliably detect redox-inactive particles smaller than the historically reported limit of 15%-20% of the radius of the electrode17,21,23,34.
While electrochemical interruption can accommodate various redox systems for the detection of nanoparticles of various electro-inactive materials, identifying such redox systems has remained a major challenge. The main barrier to implementing electrochemical interruption is identifying a chemical reaction that is rapid enough to significantly reduce the confounding contributions of edge effects. Specifically, while some examples of EC&#8242; reactions, wherein an electrode reaction is followed by a chemical reaction that regenerates the electrode reactant, are well characterized in the literature29,32,53,54,55, few are sufficiently fast to improve the measurement precision. In this study, from those reactions that are sufficiently fast, a TEMPO-maltose system was chosen, and this yielded an observed rate constant of 2,200 M-1&#183;s-1. This, in conjunction with multiphysics simulations that demonstrate that faster reaction rates lead to more homogeneous flux at the electrode edge, supports the conclusion that only fast chemical reactions yield several-fold current enhancements at ultramicroelectrodes.
Catalytic interruption does not require data manipulation or modifications to commercially available ultramicroelectrodes. To explain the heterogeneous current magnitudes characteristic of nanoimpact data, Bonezzi and Boika introduced a theoretical model that relates the current step magnitude to particle size25. This analysis, however, heavily relies on averaging the current magnitudes as a function of the collisional frequency. Not only does this preclude insight into the properties of individual particles, but this technique also remains reliant on the flux of the redox reporter to the electrode and does not remove the problem of edge effects, thus resulting in reduced precision. Deng et al. introduced the first experimental approach to addressing edge effects, utilizing a hemispherical ultramicroelectrode fabricated from mercury51. Mercury droplet electrodes, however, are toxic, mechanically unstable, and stable over only a limited potential window56. Furthermore, fabricating (and maintaining) perfectly hemispherical microelectrodes using other materials remains challenging51,52. More recently, Moazzenzade et al. proposed ring ultramicroelectrodes for nanoimpact characterization52. This geometry is promising but requires nanofabrication capabilities. In contrast, catalytic interruption enables nanoimpact experiments with materials universally found in an electrochemistry laboratory.

Nanoimpact electrochemistry is an electrochemical technique that enables the time-resolved detection of individual particles in situ in a sample1,2,3,4,5,6,7. The individual particles that can be characterized by this approach span a wide range of materials6,8,9,10,11,12,13 and encompass dimensions from individual atoms to whole cells7,8,14,15,16. To accommodate the detection and characterization of such small materials, the technique utilizes micron- and submicron-scale disk ultramicroelectrodes. The impact of an electroactive nanoparticle on such an electrode produces an easily quantifiable current change as the nanoparticle undergoes a redox reaction. To expand this to the detection of electro-inactive materials, a background electrochemical reaction is used to produce a steady-state current that is reduced in a step-wise fashion as the adsorption of the nanoparticles changes the surface area of the electrode17. In this scheme, ultramicroelectrodes are employed to increase the relative change produced by each nanoimpact. The radial diffusion layer that such microelectrodes produce, however, reduces the measurement precision due to &#34;edge effects&#34;18. These occur because the flux of the redox species to the electrode is greater at the edges of the electrode than at its center19. Thus, when a single nanoparticle lands at the edge of the electrode surface, the resulting current event is larger than that seen for an identical particle landing at the center of the electrode19, and this effect is more significant for ultramicroelectrodes due to their small area-to-circumference ratio. These edge effects detract significantly from the precision of nanoimpact electrochemistry; due to their presence, the estimated particle size distributions produced by nanoimpact sizing are 20 times wider than those obtained using &#34;gold standard&#34; microscopy techniques20. This reduced precision detracts from the use of nanoimpact electrochemistry as an analytical technique for evaluating the heterogeneity of redox inactive materials at the nanoscale4,17,19,21,22,23,24,25,26.
We have recently introduced a method (Figure 1) that mitigates edge effects in nanoimpact approaches20. In this method, the introduction of a substrate regenerates the redox species near the ultramicroelectrode surface. This shifts the rate-limiting step in current generation from diffusion to the rate of the homogeneous chemical reaction of the redox species in solution27,28, thus reducing the extent to which the radial diffusion field contributes to heterogeneous currents. Specifically, the oxidization of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) provides the background redox reaction at the ultramicroelectrode29. The addition of maltose to this regenerates the reduced form of TEMPO30,31. This regeneration is rapid32, and it compresses the diffusion layer and reduces the current heterogeneity associated with spatial landing20. As a result, the &#34;electrocatalytic interruption&#34; approach improves the precision of nanoimpact particle sizing by an order of magnitude.

<table><tbody><tr><td>0.05 &amp;micro;m microalumina polish</td><td>Buehler</td><td>4010075</td><td></td></tr><tr><td>0.3 &amp;micro;m microalumina polish</td><td>Buehler</td><td>4010077</td><td></td></tr><tr><td>1 &amp;micro;m microalumina polish</td><td>Buehler</td><td>4010079</td><td></td></tr><tr><td>20 mL scintillation vials</td><td>Fisher Sci</td><td>03-339-26C</td><td></td></tr><tr><td>Analytical balance</td><td>Ohaus</td><td></td><td></td></tr><tr><td>Apreo C LoVac FEG SEM</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Carbon fiber microelectrode</td><td>ALS</td><td>002007</td><td>Working electrode; purchased from CH Instruments</td></tr><tr><td>Carboxyl Latex Beads, 4% w/v, 2 &amp;micro;m</td><td>ThermoFisher Scientific</td><td>C37278</td><td></td></tr><tr><td>COMSOL Multiphysics</td><td>COMSOL Multiphysics</td><td>v6.0</td><td></td></tr><tr><td>D-(+)-Maltose monohydrate</td><td>Sigma Aldrich</td><td>M5885</td><td></td></tr><tr><td>DigiSim</td><td>Bioanalytical Systems, Inc.</td><td>v3.03b</td><td>Discontinued; comparable software is available commercially through the same vendor</td></tr><tr><td>EC-Lab</td><td>BioLogic</td><td>v11.27</td><td></td></tr><tr><td>Faraday cages</td><td></td><td></td><td>Custom; analogous equipment can be commercially purchased or fabriated of conductive sheet metals (e.g., copper or aluminum)</td></tr><tr><td>Hummer Sputter Coater</td><td>Anatech USA</td><td></td><td></td></tr><tr><td>OriginPro</td><td>OriginLab</td><td>v2022b</td><td></td></tr><tr><td>P1000 micropipette</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>P2 micropipette</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>P20 micropipette</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>P200 micropipette</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Platinum Wire Electrode</td><td>CH Instruments</td><td>CHI115</td><td>Counter electrode</td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>PSA-backed MicroCloth</td><td>Buehler</td><td>407218</td><td></td></tr><tr><td>Saturated Calomel Electrode</td><td>CH Instruments</td><td>CHI150</td><td>Reference electrode</td></tr><tr><td>Sodium carbonate</td><td>Fisher Chemical</td><td>S263</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma Aldrich</td><td>S8045</td><td></td></tr><tr><td>Sodium perchlorate</td><td>EM Science</td><td>SX0692</td><td></td></tr><tr><td>SP-300</td><td>BioLogic</td><td></td><td></td></tr><tr><td>TEMPO</td><td>Oakwood Chemical</td><td>013714</td><td></td></tr><tr><td>Ultra Low Current module</td><td>BioLogic</td><td></td><td></td></tr></tbody></table>

precise electrochemical sizing of individual electro-inactive particles

Nanoimpact electrochemistry enables the time-resolved in situ characterization (e.g., size, catalytic activity) of single nanomaterial units, providing a means of elucidating heterogeneities that would be masked in ensemble studies. To implement this technique with redox inactive particles, a solution-phase redox reaction is used to produce a steady-state background current on a disk ultramicroelectrode. When a particle adsorbs onto the electrode, it produces a stepwise reduction in the exposed electrode area, which produces, in turn, a stepwise decrease in the current commensurate with the size of the adsorbing species. Historically, however, nanoimpact electrochemistry has suffered from &#34;edge effects,&#34; in which the radial diffusion layer formed at the circumference of the ultramicroelectrodes renders the step size dependent not only on the size of the particle but also on where it lands on the electrode. The introduction of electrocatalytic current generation, however, mitigates the heterogeneity caused by edge effects, thus improving the measurement precision. In this approach, termed &#34;electrocatalytic interruption,&#34; a substrate that regenerates the redox probe at the diffusion layer is introduced. This shifts the rate-limiting step of the current generation from diffusion to the homogeneous reaction rate constant, thus reducing flux heterogeneity and increasing the precision of particle sizing by an order of magnitude. The protocol described here explains the set-up and data collection employed in nanoimpact experiments implementing this effect for improved precision in the sizing of redox in-active materials.

Electrocatalytic interruption mitigates edge effects by shifting the primary current generation mechanism from diffusion-limited (i.e., limited by the transport of a redox probe to the electrode) to kinetically limited (i.e., limited by a rapid, solution-phase reaction)20. This method is modular, meaning it allows a mix-and-match approach to choosing the electrode material, redox probe, and substrate, and this renders electrocatalytic interruption amenable to the detection of many nano- and bio-materials6,7,8,9,10,11,12,13,14,15,22. Implementing this technique on a 5.5 &#181;m radius carbon-fiber electrode produced a 10-fold improvement in the precision associated with the electrochemical sizing of a model system (polystyrene beads) in a solution containing TEMPO as a redox probe and maltose as a substrate.
Following this protocol, the data sets required to validate this mechanism and its ability to restore analytical precision when sizing electro-inactive nanoparticles can be obtained. First, the cyclic voltammogram data collected in the absence of polystyrene beads showed a reversible redox event in control experiments involving TEMPO alone. From here, the addition of maltose resulted in an increase in the oxidative peak and a concurrent loss in the reductive peak as the oxidized TEMPO was regenerated by maltose. Second, the chronoamperograms collected under these conditions demonstrated that the steady-state currents at an oxidative potential were higher, consistent with the catalytic amplification observed in the cyclic voltammetry results. This step also suggests that the bulk chemical reaction is maintained by the electrode reaction; thus, any improvements over the control method will last over the measurement duration. However, this alone is insufficient for assessing any improvements to the measurement precision; to do so, chronoamperometry data must be collected in the presence of polystyrene beads.
To assess the sizing precision, chronoamperogram data were collected using 2 &#181;m carboxylated polystyrene beads. Upon their addition, step-wise changes in the chronoamperogram current were observed as individual particles impacted and absorbed (Figure 2A control, Figure 2B electrocatalytic interruption). Each step-wise change in the steady-state current magnitude was converted to particle radii, and the data were visualized as histograms to compare the distribution from these electrochemical techniques to that of a gold standard technique, such as scanning electron microscopy (Figure 3). This comparison then allowed the characterization of precision metrics associated with each sizing approach.
Modeling was utilized to support these experimental observations. Specifically, fitting the cyclic voltammograms from before yielded parameters that characterized both the electrode reaction and the solution-phase chemical reaction (Figure 4). From the control solution, some sample parameters obtained were Ef&#952; = 0.49 V, k0 = 0.02 cm&#183;s-1, and &#957; = 10 mV&#183;s-1 at T = 25&#176; C. From the test solution, the kinetic parameters that limited current generation could be obtained; specifically, as Keq approaches infinity, kobs = 2,200 M-1&#183;s-1. The numerical simulations could then use these values as the initial conditions for generating a concentration profile of the redox probe (Figure 5). In the absence of maltose, the resultant diffusion profile was radial, leading to heterogeneous material flux; specifically, more material diffused to the electrode at the edges. The introduction of maltose compressed the diffusion profile, producing, in turn, more homogeneous currents across the electrode surface.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65116/65116fig01.jpg" /> 
Figure 1: Schematic of the experimental protocol. Polish the electrodes prior to each experimental run. Collect a baseline set of electrochemical measurements (cyclic voltammetry and chronoamperometry) in the absence of beads with and without implementing electrocatalytic interruption to observe the current enhancement with the addition of the substrate. Spike in the beads, and collect a second set of electrochemical measurements for the size determination of the impacting nanoparticles. Validate the mechanism of action using numerical simulations. <a href="https://www.jove.com/files/ftp_upload/65116/65116fig01large.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/65116/65116fig02.jpg" /> 
Figure 2: Chronoamperograms collected using an 11 &#181;m diameter carbon-fiber ultramicroelectrode demonstrating the improvement in measurement precision achieved using electrocatalytic interruption. Specifically, when measuring the current versus time in a solution of 1 mM TEMPO in the (A) absence (control) and (B) presence of 120 mM maltose (electrocatalytic interruption), the steps observed in the latter case were more homogeneous. Reprinted with permission from Chung et al.20. <a href="https://www.jove.com/files/ftp_upload/65116/65116fig02large.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/65116/65116fig03.jpg" /> 
Figure 3: Precise electrochemical sizing data when using electrocatalytic interruption compared to the conventional, diffusion-limited electrochemical approach. To represent this data, prepare histograms comparing the size distributions determined using scanning electron microscopy (light grey) and electrochemistry (electrocatalytic interruption, pink; control, dark grey). Conventional nanoimpact studies, in which the current is limited by the mass transport of the mediator, produce artificially broad estimated size distributions (dark grey). In contrast, implementing electrocatalytic interruption leads to narrower, more precise size estimations (pink). Reprinted with permission from Chung et al.20. <a href="https://www.jove.com/files/ftp_upload/65116/65116fig03large.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/65116/65116fig04.jpg" /> 
Figure 4: Modeling the electrode kinetics to characterize the new reaction scheme. Using cyclic voltammogram fitting software, extract the electrode reaction parameters from the experimental data. (A) Data with 1 mM TEMPO. (B) Data with 1 mM TEMPO plus 120 mM maltose. Reprinted with permission from Chung et al.20. <a href="https://www.jove.com/files/ftp_upload/65116/65116fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65116/65116fig05.jpg" /> 
Figure 5: Changes in material flux at the electrode surface upon introducing electrocatalytic interruption visualized by numerical simulations. (A) The addition of maltose compresses the diffusion layer in a concentration-dependent manner. (B) The addition of maltose depresses the heterogeneous flux at the electrode edges. Reprinted with permission from Chung et al.20. <a href="https://www.jove.com/files/ftp_upload/65116/65116fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Precise Electrochemical Sizing of Individual Electro-Inactive Particles at JoVE.com

Precise Electrochemical Sizing of Individual Electro-Inactive Particles

As an analytical technique, nanoimpact electrochemistry, an increasingly important approach to counting and characterizing nanometer-scale, electro-inactive particles, suffers from poor precision due to the heterogeneous current distributions that arise from its use of ultramicroelectrodes. Outlined here is a generalized approach, termed "electrocatalytic interruption," that enhances precision in such measurements.

Nanoimpact electrochemistry enables the time-resolved in situ characterization (e.g., size, catalytic activity) of single nanomaterial units, providing a ...

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A large variety of analytical methods are available to characterize particles in aerosols and suspensions. The choice of the appropriate technique depends ...

Preparation of Janus Particles and Alternating Current Electrokinetic Measurements with a Rapidly Fabricated Indium Tin Oxide Electrode Array

This article provides a simple method to prepare partially or fully coated metallic particles and to perform the rapid fabrication of electrode arrays, which can facilitate electrical experiments in microfluidic devices. Janus particles are asymmetric particles that contain two different surface properties on their two sides. To prepare Janus particles, a monolayer of silica particles is prepared by a drying process. Gold (Au) is deposited on one side of each particle using a sputtering device. The fully coated metallic particles are completed after the second coating process. To analyze the electrical surface properties of Janus particles, alternating current (AC) electrokinetic measurements, such as dielectrophoresis (DEP) and electrorotation (EROT)- which require specifically designed electrode arrays in the experimental device- are performed. However, traditional methods to fabricate electrode arrays, such as the photolithographic technique, require a series of complicated procedures. Here, we introduce a flexible method to fabricate a designed electrode array. An indium tin oxide (ITO) glass is patterned by a fiber laser marking machine (1,064 nm, 20 W, 90 to 120 ns pulse-width, and 20 to 80 kHz pulse repetition frequency) to create a four-phase electrode array. To generate the four-phase electric field, the electrodes are connected to a 2-channel function generator and to two invertors. The phase shift between the adjacent electrodes is set at either 90&#176; (for EROT) or 180&#176; (for DEP). Representative results of AC electrokinetic measurements with a four-phase ITO electrode array are presented.

In this article, a simple method to prepare partially or fully coated metallic particles and to perform AC electrokinetic property measurements with a rapidly fabricated indium tin oxide (ITO) electrode array is demonstrated.

This article provides a simple method to prepare partially or fully coated metallic particles and to perform the rapid fabrication of electrode arrays, ...

Engineering

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Bioengineering

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

Electrochemical Roughening of Thin-Film Platinum Macro and Microelectrodes

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries of the metal. Using this method, a crack free, thin-film macroelectrode surface with up to 40 times increase in active surface area was obtained. The roughening is easy to do in a standard electrochemical characterization laboratory and incudes the application of voltage pulses followed by extended application of a reductive voltage in a perchloric acid solution. The protocol includes the chemical and electrochemical preparation of both a macroscale (1.2 mm diameter) and microscale (20 &#181;m diameter) platinum disc electrode surface, roughening the electrode surface and characterizing the effects of surface roughening on electrode active surface area. This electrochemical characterization includes cyclic voltammetry and impedance spectroscopy and is demonstrated for both the macroelectrodes and the microelectrodes. Roughening increases electrode active surface area, decreases electrode impedance, increases platinum charge injection limits to those of titanium nitride electrodes of same geometry and improves substrates for adhesion of electrochemically deposited films.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries. The electrochemical techniques of cyclic voltammetry and impedance spectroscopy are demonstrated to characterize these electrode surfaces.

This protocol demonstrates a method for electrochemical roughening of thin-film platinum electrodes without preferential dissolution at grain boundaries ...

Versatile Dual-Inlet Sample Introduction System for Multi-Mode Single Particle Inductively Coupled Plasma Mass Spectrometry Analysis and Validation

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number concentration by using the single-particle mode of the instrument (spICP-MS). The accuracy of measurement depends on the setup, operational conditions of the instrument and specific parameters that are set by the user. The transport efficiency of the ICP-MS is crucial for the quantification of the NP and usually requires a reference material with homogenous size distribution and a known particle number concentration.
Currently, NP reference materials are available for only a few metals and in limited sizes. If particles are characterized without a reference standard, the results of both size and particle number may be biased. Therefore, a dual-inlet setup for characterizing nanoparticles with spICP-MS was developed to overcome this problem. This setup is based on a conventional introduction system consisting of a pneumatic nebulizer (PN) for nanoparticle solutions and a microdroplet generator (&#181;DG) for ionic calibration solutions. A new and flexible interface was developed to facilitate the coupling of &#181;DG, PN and the ICP-MS system. The interface consists of available laboratory components and allows for the calibration, nanoparticle (NP) characterization and cleaning of the arrangement, while the ICP-MS instrument is still running.
Three independent analysis modes are available for determining particle size and number concentration. Each mode is based on a different calibration principle. While mode I (counting) and mode III (&#181;DG) are known from the literature, mode II (sensitivity), is used to determine the transport efficiency by inorganic ionic standard solutions only. It is independent of NP reference materials. The &#181;DG based inlet system described here guarantees superior analyte sensitivities and, therefore, lower detection limits (LOD). The size dependent LODs achieved are less than 15 nm for all NP (Au, Ag, CeO2) investigated.

Here we provide a protocol for the use of a dual-inlet system for single particle inductively coupled mass spectrometry which allows for a standard independent nanoparticle characterization.

Metal-containing nanoparticles (NP) can be characterized with inductively coupled plasma mass spectrometers (ICP-MS) in terms of their size and number ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

Author Spotlight: Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy  

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Precise Electrochemical Sizing of Individual Electro-Inactive Particles

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