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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05 &#181;m microalumina polish</td>
			<td>Buehler</td>
			<td>4010075</td>
			<td></td>
		</tr>
		<tr>
			<td>0.3 &#181;m microalumina polish</td>
			<td>Buehler</td>
			<td>4010077</td>
			<td></td>
		</tr>
		<tr>
			<td>1 &#181;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 &#181;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>

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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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Universidad San Sebastian

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Chemistry

1.1K Views.  University of California at Santa Barbara. 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.

Video: Precise Electrochemical Sizing of Individual Electro-Inactive Particles

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

A Protocol for Electrochemical Evaluations and State of Charge Diagnostics of a Symmetric Organic Redox Flow Battery

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid and deployment of renewable energy technologies. Among the many flow battery chemistries, non-aqueous flow batteries have the potential to achieve high energy density because of the broad voltage windows of non-aqueous electrolytes. However, significant technical hurdles exist currently limiting non-aqueous flow batteries to demonstrate their full potential, such as low redox concentrations, low operating currents, under-explored battery status monitoring, etc. In an attempt to address these limitations, we recently reported a non-aqueous flow battery based on a highly soluble, redox-active organic nitronyl nitroxide radical compound, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). This redox material exhibits an ambipolar electrochemical property, and therefore can serve as both anolyte and catholyte redox materials to form a symmetric flow battery chemistry. Moreover, we demonstrated that Fourier transform infrared (FTIR) spectroscopy could measure the PTIO concentrations during the PTIO flow battery cycling and offer reasonably accurate detection of the battery state of charge (SOC), as cross-validated by electron spin resonance (ESR) measurements. Herein we present a video protocol for the electrochemical evaluation and SOC diagnosis of the PTIO symmetric flow battery. With a detailed description, we experimentally demonstrated the route to achieve such purposes. This protocol aims to spark more interests and insights on the safety and reliability in the field of non-aqueous redox flow batteries.

We present the protocols for electrochemically evaluating a symmetric non-aqueous organic redox flow battery and for diagnosing its state of charge using FTIR.

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

This protocol presents both the synthesis method of the Ni single atom catalyst, and the electrochemical testing of its catalytic activity and selectivity in aqueous CO2 reduction. Different from traditional metal nanocrystals, the synthesis of metal single atoms involves a matrix material that can confine those single atoms and prevent them from aggregation. We report an electrospinning and thermal annealing method to prepare Ni single atoms dispersed and coordinated in a graphene shell, as active centers for CO2 reduction to CO. During the synthesis, N dopants play a critical role in generating graphene vacancies to trap Ni atoms. Aberration-corrected scanning transmission electron microscopy and three-dimensional atom probe tomography were employed to identify the single Ni atomic sites in graphene vacancies. Detailed setup of electrochemical CO2 reduction apparatus coupled with an on-line gas chromatography is also demonstrated. Compared to metallic Ni, Ni single atom catalyst exhibit dramatically improved CO2 reduction and suppressed H2 evolution side reaction.

Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction

Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions.

This protocol presents both the synthesis method of the Ni single atom catalyst, and the electrochemical testing of its catalytic activity and selectivity ...