This work is funded by the national science foundation (Award Number: 1756444) via Biological &#38; Environmental Interfaces of Nano Materials, the USDA National Institute of Food and Agriculture, AFRI project [2018-07549] and Assistance Agreement No. 83945101-0 awarded by the U.S. Environmental Protection Agency to New Jersey Institute of Technology. It has not been formally reviewed by EPA. The views expressed in this document are solely those of authors and do not necessarily reflect those of the Agency. EPA does not endorse any products or commercial services mentioned in this publication. The authors also thank Undergraduate Research and Innovation program (URI) Phase-1 &#38; Phase-2 at New Jersey Institute of Technology.

1. Sample preparation
<ol>
	<li>Preparation of faceted Cu2O nanoparticles and deposition on silicon substrate
	<ol>
		<li>Dissolve 0.175 g of CuCl2&#8729;2H2O (99.9%) into 100 mL of deionized (DI) water to generate an aqueous solution of 10 mM CuCl2.</li>
		<li>Add 10.0 mL of 2.0 M NaOH and 10 mL of 0.6 M ascorbic acid dropwise into the CuCl2 solution.</li>
		<li>Heat the solution in a 250 mL round-bottom flask under constant stirring in a 55 &#176;C water bath for 3 h.</li>
		<li>Collect the resulting precipitate by centrifugation (5,000 x g for 15 min), followed by washing with DI water 3 times and ethanol twice to remove the residual inorganic ions and polymers.</li>
		<li>Dry precipitate in vacuum at 60 &#176;C for 5 h53.</li>
		<li>Use the prepared silicon wafer as the substrate to deposit Cu2O nanoparticles as illustrated in Figure 1A using epoxy to ensure the testing. 
		Caution: The silicon wafer (&#216;3&#8221; Silicon wafer, Type P/&#60;111&#62;) was cut into a single piece of 38 mm x 38 mm, followed by washing using ethanol, methanol and DI water to remove organic and inorganic contaminants.</li>
		<li>Directly deposit 10 &#181;L of epoxy on the cleaned silicon wafer using a pipette tip and tile with a clean glass slide. After about 5 min, drop 10 &#181;L of the nanoparticles/water suspension (10 mg L-1) on different epoxy-coated silicon wafer substrates, separately. The four different red spots shown in Figure 1B indicate the potential position of the deposited nanoparticles.</li>
		<li>Vacuum dry the substrate at 40 &#176;C for 6 h.</li>
		<li>Place the sample substrate into the EC sample cell (Figure 4) to be filled with 1.8 mL of a 0.1 M KCl containing 10 mM Ru(NH3)6Cl3(98%).</li>
	</ol>
	</li>
	<li>Preparation of NBs
	<ol>
		<li>Generate oxygen nanobubbles by direct injection of compressed oxygen (purity 99.999%) through a tubular ceramic membrane (100 nm pore size, WFA0.1) into DI water. 
		NOTE: The gas was injected continuously under a pressure of 414 kPa and a flow of 0.45 L&#183;m-1 until reaching stable bubble size distribution as reported elsewhere54.</li>
		<li>Add 1.8 mL of the water suspension of NBs on a gold substrate in the EC sample cell and stabilize for 10 min. 
		NOTE: Fresh 40 mm x 40 mm gold plates (Au on Si) were used as the substrate to immobilize NBs.</li>
		<li>Decant 0.9 mL of NB suspension and replace with 0.9 mL of a 10 mM Ru(NH3)6Cl3 solution in 0.1 M KCl.</li>
	</ol>
	</li>
</ol>
2. Setup of AFM-SECM
NOTE: AFM was used in the presented AFM-SECM measurements. To perform the EC analyses, the AFM was equipped with a bipotentiostat and SECM accessories. As shown in Figure S1, the bipotentiostat was connected to the AFM controller and both the potentiostat and AFM were connected to the same computer. The accessories include an SECM chuck, an SECM probe holder with protective boot, and a strain-release module with a resistance selector (10 M&#937; resistance was used) to limit the maximum current flow55. As shown in Figure 2, the AFM-SECM probes have a tip radius of 25 nm and a tip height of 215 nm. The sample acted as a working electrode, which shares the same pseudo-reference using the Ag wire electrode (25 mm diameter) and the counter electrode of a Pt wire (25 mm in diameter). The probe and the sample could be biased at different potentials (vs the Ag wire pseudo-reference electrode) to enable different redox reactions. In the presented work, the tip reduces the [Ru(NH3)6]3+ to [Ru(NH3)6]2+ at -400 mV versus an Ag wire pseudo-reference electrode.
<ol>
	<li>Replace the existing sample chuck with SECM chuck and screw the chuck in place using two M3 x 6 mm socket head cap screws and a 2.5 mm hex wrench (Figure 3A).</li>
	<li>Connect the temperature control cable to the SECM chuck, and connect the low-noise SECM cables to the spring connector block (color to color) and switch block (Figure 3B). 
	NOTE: The switch needs to be kept on the right side during SECM testing.</li>
	<li>Install the strain-release module onto the AFM scanner and also connect it to the working electrode connector on the spring connector block with extension cable (Figure 3C).</li>
	<li>Assemble the EC sample cell.
	<ol>
		<li>Put the insert onto the top ring (Figure 4A).</li>
		<li>Assemble two O-rings onto the bottom groove and top groove of the insert, respectively (Figure 4B and Figure 4C).</li>
		<li>Put a glass cover onto the top ring top and then tighten by four screws lightly and diagonally (Figure 4D).</li>
		<li>Use a hard sharp wire with a 24 mm diameter (Figure 4E) to poke two holes in the O-ring through two channels of plastic part on top ring (Figure 4F).</li>
		<li>Insert Ag wire and Pt wire through the hole on the O-ring, and curve the Pt wire to a circle in EC sample cell as shown in Figure 4G.</li>
		<li>To seal the EC sample cell top part, press the assembled EC sample cell down on the EC sample cell bottom to make the O-ring fully contact the glass cover (Figure 4H).</li>
		<li>Place the top part of the EC sample cell upside-down and face the test sample (or substrate) downward so that the spring-loaded pins (pogo pins) touch the sample surface as shown in Figure 4I and Figure 4J. The test sample should be covering the O-ring to make EC sample cell bottom part seal.</li>
		<li>Put the EC sample cell bottom on and tighten diagonally with right length screw (Figure 4K).</li>
	</ol>
	</li>
</ol>
3. Operation of AFM-SECM
<ol>
	<li>Initialization of the AFM and bipotentiostat instruments
	<ol>
		<li>Double click the two software icons to initialize the AFM system and the bipotentiostat control interface.</li>
	</ol>
	</li>
	<li>Loading SECM Probe
	<ol>
		<li>Prepare the ESD field service package including antistatic pad, electrostatic discharge (ESD) protective probe stand, wearable anti-static gloves and wrist strap (Figure 5A). Figure 5B shows the connection of the ESD monitor with wrist strap. 
		NOTE: The ESD monitor beeps when the red pad is connected with ground. The beep will stop when user wears the wrist strap.</li>
		<li>To prevent AFM scanner from exposure to liquid, use a protective boot (Figure 6A) during AFM-SECM testing. Put the probe holder onto the ESD protective probe stand (Figure 6B). Use a pair of plastic tweezers to attach the protective boot to the tip holder (Figure 6C). Then, align the small cut in the protective boot to the notch in the probe holder as illustrated in Figure 6D.</li>
		<li>Open the box of AFM-SECM probes (Figure 7A) using a tip tweezer (green color) to grab the probe from both sides of the grooves (Figure 7B). While using the disk gripper (silver color) to hold the probe holder on the stand, put the probe wire into the hole of the stand, and then slide the probe into the slot of the probe holder (Figure 7C). After the probe is inside the slot, use the flat end of the tweezer to push it in. Make sure the probe is completely in the tip holder (Figure 7D).</li>
		<li>As shown in Figure 8A, attach the whole probe holder (including the holder-boot) to the scanner.</li>
		<li>Use the Teflon tip tweezer to grab the wire right below the copper ring and connect it to the module (Figure 8B).</li>
		<li>Put the scanner back to the dovetail.</li>
	</ol>
	</li>
	<li>Loading the sample cell
	<ol>
		<li>After assembling the test sample (or substrate) in the EC sample cell, which was mentioned in Section 2.4, put the EC sample cell on the central point of the SECM chuck and the pseudo-reference electrode (Ag wire) and connect the counterelectrode (Pt wire) to the spring connector block (Figure 3). The EC sample cell is magnetically attached to the chuck.</li>
	</ol>
	</li>
	<li>SECM software preparation before imaging
	<ol>
		<li>In the AFM-SECM software, select SECM- PeakForce QNM to load the workspace (Figure S2).</li>
		<li>In Setup, load the SECM probe, and then align a laser on the tip using an alignment station.</li>
		<li>Go to Navigation (Figure S3). Move the scanner downwards slowly to focus on the sample surface. Adjust the position of EC sample cell slightly to make sure the scanner would not touch the glass cover of EC sample cell while moving. After focusing on the sample, click Update Blind Engage Position. 
		Caution: Different samples have different heights, so it is necessary to update the blind engage position after changing a sample.</li>
		<li>Click Move to Add Fluid Position.</li>
		<li>Add ~1.8 mL of the buffer solution into the EC sample cell, to make sure the level of the solution is lower than the glass cover. If the water level is over the glass cover, water can creep to the scanner and cause an electric short and break the scanner. Wait for another 5 minutes and use a pipette to agitate the solution to remove bubbles. 
		NOTE: The buffer solution (10 mM [Ru(NH3)6]3+ with supporting electrolyte of 0.1 M KCl) should be constantly stored in a refrigerator after preparation. Use a syringe with filter (no larger than 1 &#181;m pore size) to filter the solution before using it.</li>
		<li>Click Move to Blind Engage Position. The tip will move back into the buffer solution. Adjust the laser slightly to make sure the laser is aligned on the tip.</li>
		<li>Open CHI software. As shown in Figure S4, click on the Technique command on the toolbar to open up the tech selector and select Open Circuit Potential &#8211; Time. Use the default setting (Run time as 400 s) for OCP measurement and run the OCP measurement. 
		NOTE: The potential showed in OCP test should be near zero stably.</li>
		<li>Click the Technique command again and run Cyclic Voltammetry (CV), as shown in Figure S5 and Figure S6. 
		NOTE: Set up the parameters as below. Set &#8220;sweep segments&#8221; to a larger number if needed. The &#8220;init E/Final E&#8221; should be as the same as the potential value from OCP measurement and &#8220;High E&#8221; and &#8220;Low E&#8221; could be +0.3 V or &#8722;0.3 V of &#8220; init E/Final E&#8221;, respectively. Here we use 0 V as initial and high E and -0.4 V as Low and Final E. The scan rate was 0.05 V/s and the sensitivity was 1 e-009. Run the CV test, the highest current (i) measured here should be 0.3-1.2 nA for 10 mM [Ru(NH3)6]3+.</li>
	</ol>
	</li>
	<li>SECM Imaging
	<ol>
		<li>Go back to the AFM-SECM software. Since the tip is already in the liquid, click Engage.</li>
		<li>After scanning, turn on lift mode (Lift by Sensor) with a lift height of 100 nm and adjust the lift height based on the sample roughness.</li>
		<li>In CHI software, run a chronoamperometry with parameters shown in Figure S7. Set the initial E as -0.4 V, the pulse width as 1000 seconds (which is the maximum number accepted by the system), and the sensitivity the same with CV scan. 
		NOTE: The chronoamperometry technique was chosen because of the absence of amperometric i-t technique in the presented bi-potentiostat.</li>
		<li>With the CHI program running, go back to AFM-SECM software, check the real-time reading on the strip chart and click on Start (Figure S8). The reading will be updated in real-time. Then both topography imaging and current imaging process will begin. Save images in the AFM-SECM software.</li>
	</ol>
	</li>
	<li>Check approach curve
	<ol>
		<li>Engage the tip on sample or substrate region with a scan size of 1 &#181;m.</li>
		<li>Run the Chronoamperometry as mentioned in 3.5.3.</li>
		<li>Go back to AFM-SECM Software and select the command Go To Ramp.</li>
		<li>Click Ramp. An approach curve would be recorded in the AFM-SECM software.</li>
	</ol>
	</li>
	<li>Tip cleaning
	<ol>
		<li>Using the EC sample cell as a clean water container. Move the tip in and out of the liquid using blind engage functions in the navigation panel. Change the clean water three times. After this three-time cleaning, using clean wipes to carefully remove residual water from the probe holder and put the probe back in the probe box. 
		Caution: After imaging, the AFM-SECM probe needs to be carefully cleaned. Never use water coming out from the wash bottle to clean the probe as the electrostatic charge might damage the probe.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

A combined AFM-SECM technique that enables high-resolution multimodal imaging has been described in this protocol. This technique allows for topography to be mapped simultaneously with the SECM current collected or mapped on single nanoparticles or nanobubbles. Experiments were performed using commercial probes. These probes were designed to provide chemical compatibility with a wide range of electrochemical environments, electrochemical performance, mechanical stability, and multiple-cycle handling18</...

Characterization of electrochemical (EC) behavior can provide critical insights into the kinetics and mechanisms of interfacial reactions in diverse fields, such as biology1,2, energy3,4, material synthesis5,6,7, and chemical process8,9. Traditional EC measurements including electrochemical impedance spectroscopy10, electrochemical noise methods11, galvanostatic intermittent titration12, and cyclic voltammetry13 are usually performed at macroscopic scale and provide a surface-average response. Thus, it is difficult to extract information on how electrochemical-activity is distributed across a surface, but local scale surface properties in nanoscale are especially important where nanomaterials are widely used. Therefore, new techniques capable of simultaneously capturing both nanoscale multidimensional information and electrochemistry are highly desirable.
Scanning electrochemical microscopy (SECM) is a widely used technique for measuring the localized electrochemical activity of materials at micro- and nanoscales14. Typically, SECM uses an ultra-microelectrode as a probe for detecting electroactive chemical species as it scans a sample surface to spatially resolve local electrochemical properties15. The measured current at the probe is produced by reduction (or oxidation) of the mediator species, and this current is an indicator of the electrochemical reactivity at the surface of the sample. SECM has evolved significantly after its first inception in 198916,17 but it is still challenged by two main limitations. Since EC signals are typically sensitive to tip-substrate interaction characteristics, one limitation of SECM is that keeping the probe at a constant height prevents a direct correlation of electrochemical activity with the surface landscape, due to the convolution of topography with the collected EC information18. Second, it is difficult for a commercial SECM system to obtain sub-micrometer (&#181;m) image resolution as the spatial resolution is partially determined by the probe dimensions, which is on the micrometer scale19. Therefore, nanoelectrodes, the electrodes with a diameter in the nanometer range, are increasingly used in SECM to achieve a resolution below the sub-micrometer scale20,21,22,23.
To provide a constant tip-substrate distance control and obtain a higher spatial electrochemical resolution, several hybrid techniques of SECM have been used, such as ion conductance positioning24, shear force positioning25, alternating current SECM26, and atomic force microscopy (AFM) positioning. Among these instrumentations, SECM integrating AFM positioning (AFM-SECM) has become a highly promising approach. As AFM can provide fixed tip-substrate distances, the integrated AFM-SECM technique enables simultaneous acquisition of nanoscale surface structural and electrochemical information through mapping or sample sweeping with the sharp AFM tips. Since the first successful operation of AFM-SECM by MacPherson and Unwin in 199627, significant improvements have been achieved on probe design and fabrication, as well as its applications in various research fields such as electrochemistry in chemical and biological processes. For example, AFM-SECM has been implemented for imaging composite material surfaces, such as noble metal nanoparticles28, functionalized or dimensionally stable electrodes29,30, and electronic devices31. AFM-SECM can map the electrochemically active sites from the tip current image.
Simultaneous topographical and electrochemical measurements could also be achieved by other techniques such as conductive AFM32,33,34,35, electrochemical AFM (EC-AFM)36,37,38,39, scanning ion conductance microscopy-scanning electrochemical microscopy (SICM-SECM)24,40, and scanning electrochemical cell microscopy (SECCM)41,42 The comparison between these techniques has been discussed in a review paper1. The aim of the present work was to employ SECM-AFM to demonstrate the electrochemical mapping and measurement on faceted crystalline cuprous oxide nanomaterials and nanobubbles in water. Faceted nanomaterials are widely synthesized for metal oxide catalysts in clean energy applications because the facets with distinctive crystallographic features have distinctive surface atomic structures and further dominate their catalytic properties. Moreover, we also measured and compared the electrochemical behavior at the liquid/gas interfaces for surface nanobubbles (NBs) on gold substrates. NBs are bubbles with a diameter of &#60;1 &#956;m (also known as ultrafine bubbles)43, and they elicit many intriguing properties44,45, including long residence times in the solutions46,47 and high&#160;efficiency of gas mass transfer46,48. Furthermore, the collapse of NBs creates shock waves and the formation of hydroxyl radicals (&#8226;OH)49,50,51,52. We measured the electrochemical reactivity of oxygen NBs in the solution to better understand the fundamental chemical properties of NBs.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic force microsopy</td>
			<td>Bruker, CA</td>
			<td>Dimenison Icon</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipotentiostat</td>
			<td>CH Instruments, Inc.</td>
			<td>CHI 700E</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>TED PELLA, Inc.</td>
			<td>16013</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh gold plates</td>
			<td>Bruker, CA</td>
			<td>model 119-017-307</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM-AFM probes</td>
			<td>Bruker, CA</td>
			<td>990-050138</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM strain-release module</td>
			<td>Bruker, CA</td>
			<td>840-012-724</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM Probe Holder</td>
			<td>Bruker, CA</td>
			<td>900-050121</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM Chuck</td>
			<td>Bruker, CA</td>
			<td>PF-SECM Chuck</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM O-ring</td>
			<td>Bruker, CA</td>
			<td>598-000-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM cover glass, SECM Cell</td>
			<td>Bruker, CA</td>
			<td>900-050137</td>
			<td></td>
		</tr>
		<tr>
			<td>EC Cell Assy</td>
			<td>Bruker, CA</td>
			<td>932-017-300</td>
			<td></td>
		</tr>
		<tr>
			<td>ESD Field Service</td>
			<td>Bruker, CA</td>
			<td>490-000-066</td>
			<td></td>
		</tr>
		<tr>
			<td>PF-SECM Boot</td>
			<td>Bruker, CA</td>
			<td>900-050136</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring connector block</td>
			<td>Bruker, CA</td>
			<td>900-050524</td>
			<td></td>
		</tr>
		<tr>
			<td>PFSECM Tweezers</td>
			<td>Bruker, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cable, SECM Tip module</td>
			<td>Bruker, CA</td>
			<td>468-050171</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag wire</td>
			<td>Bruker, CA</td>
			<td>249-000-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Pt wire</td>
			<td>Bruker, CA</td>
			<td>248-000-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard sharp wire</td>
			<td>Bruker, CA</td>
			<td>TT-ECM10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubular ceramic membrane</td>
			<td>Refracton</td>
			<td>WFA0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) chloride dihydrate</td>
			<td>ACROS Organics</td>
			<td>AC315281000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Chemical</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Fisher Chemical</td>
			<td>A61-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Loctite</td>
			<td>Instant Mix</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Chemical</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexaammineruthenium(III) chloride</td>
			<td>ACROS Organics</td>
			<td>AC363342500</td>
			<td></td>
		</tr>
	</tbody>
</table>

probing surface electrochemical activity of nanomaterials using a hybrid atomic force microscope-scanning electrochemical microscope (afm-secm)

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. Atomic force microscopy (AFM) is a versatile tool to characterize micro- and nanostructure in terms of topography and mechanical properties. However, conventional SECM or AFM provides limited laterally resolved information on electrical or electrochemical properties at nanoscale. For instance, the activity of a nanomaterial surface at crystal facet levels is difficult to resolve by conventional electrochemistry methods. This paper reports the application of a combination of AFM and SECM, namely, AFM-SECM, to probe nanoscale surface electrochemical activity while acquiring high-resolution topographical data. Such measurements are critical to understanding the relationship between nanostructure and reaction activity, which is relevant to a wide range of applications in material science, life science and chemical processes. The versatility of the combined AFM-SECM is demonstrated by mapping topographical and electrochemical properties of faceted nanoparticles (NPs) and nanobubbles (NBs), respectively. Compared to previously reported SECM imaging of nanostructures, this AFM-SECM enables quantitative assessment of local surface activity or reactivity with higher resolution of surface mapping.

Topography and current imaging of ONBs by AFM-SECM
Previous studies that characterized NBs with AFM only reported topography images to reveal the size and distribution of NBs immobilized on a solid substrate56,57. The experiments here revealed both morphological and electrochemical information. Individual oxygen nanobubbles (ONBs) can be clearly identi&#64257;ed in Figure 9, which provides...

Watch this Scientific Journal Video about Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope AFM-SECM at JoVE.com

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope AFM-SECM

Atomic force microscopy (AFM) combined with scanning electrochemical microscopy (SECM), namely, AFM-SECM, can be used to simultaneously acquire high-resolution topographical and electrochemical information on material surfaces at nanoscale. Such information is critical to understanding heterogeneous properties (e.g., reactivity, defects, and reaction sites) on local surfaces of nanomaterials, electrodes and biomaterials.

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope (AFM-SECM)

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. ...

probing-surface-electrochemical-activity-nanomaterials-using-hybrid

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

6.7K Views.  New Jersey Institute of Technology. Atomic force microscopy (AFM) combined with scanning electrochemical microscopy (SECM), namely, AFM-SECM, can be used to simultaneously acquire high-resolution topographical and electrochemical information on material surfaces at nanoscale. Such information is critical to understanding heterogeneous properties (e.g., reactivity, defects, and reaction sites) on local surfaces of nanomaterials, electrodes and biomaterials.

Video: Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope AFM-SECM

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 ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

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 ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

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 ...

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The cholesteric LC is composed of a nematic LC 4&#39;-pentyloxy-4-cyanobiphenyl (5OCB), redox-responsive chiral dopant (FcD), and a supporting electrolyte 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMIm-OTf). The most important component is FcD. This molecule changes its helical twisting power (HTP) value in response to redox reactions. Therefore, in situ electrochemical redox reactions in the LC mixture allow for the device to change its reflection color in response to electrical stimuli. The LC mixture was introduced, by a capillary action, into a sandwich-type ITO glass cell comprising two glass slides with patterned indium tin oxide (ITO) electrodes, one of which was coated with poly(3,4-ethylenedioxythiophene)-co-poly(ethylene glycol) doped with perchlorate (PEDOT+). Upon application of +1.5 V, the reflection color of the device changed from blue (467 nm) to green (485 nm) in 0.4 s. Subsequent application of 0 V made the device recover the original blue color in 2.7 s. This device is characterized by its fastest electrical response and lowest operating voltage among any previously reported cholesteric LC device. This device could pave the way for the development of next generation reflective displays with low energy consumption rates.

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The ...

Imine Metathesis by Silica-Supported Catalysts Using the Methodology of Surface Organometallic Chemistry

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared according to the methodology developed by surface organometallic chemistry (SOMC). In this framework, catalytic cycles can be determined by isolating crucial intermediates. All air-sensitive materials are handled under inert atmosphere (using gloveboxes or a Schlenk line) or high vacuum lines (HVLs, &#60;10-5 mbar). The preparation of SiO2-700 (silica dehydroxylated at 700 &#176;C) and subsequent applications (the grafting of complexes and catalytic runs) requires the use of HVLs and double-Schlenk techniques. Several well-known characterization methods are used, such as Fourier-transform infrared spectroscopy (FTIR), elemental microanalysis, solid-state nuclear magnetic resonance spectroscopy (SSNMR), and state-of-the-art dynamic nuclear polarization surface enhanced NMR spectroscopy (DNP-SENS). FTIR and elemental microanalysis permit scientists to establish the grafting and its stoichiometry. 1H and 13C SSNMR allows the structural determination of the hydrocarbon ligands coordination sphere. DNP SENS is an emerging powerful technique in solid characterization for the detection of poorly sensitive nuclei (15N, in our case). SiO2-700 is treated with about one equivalent of the metal precursor compared to the amount of surface silanol (0.30 mmol&#183;g-1) in pentane at room temperature. Then, volatiles are removed, and the powder samples are dried under dynamic high vacuum to afford the desired materials [(&#8801;Si-O-)Hf(&#951;2&#960;-MeNCH2)(&#951;1-NMe2)(&#951;1-HNMe2)]. After a thermal treatment under high vacuum, the grafted complex is converted into metal imido silica complex [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)]. [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] effectively promotes the metathesis of imines, using the combination of two imine substrates, N-(4-phenylbenzylidene)benzylamine, or N-(4-fluorobenzylidene)-4-fluoroaniline, with N-benzylidenetert-butylamine as substrates. A significantly lower conversion is observed with the blank runs; thus, the presence of the imido group in [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is correlated to the catalytic performance.

A new group IV metal catalyst for imine metathesis is prepared by grafting amine metal complex onto dehydroxylated silica. Surface metal fragments are characterized using FT-IR, elemental microanalysis, and solid-state NMR spectroscopy. Further dynamic nuclear polarization surface enhanced NMR spectroscopy experiments complement the determination of the coordination sphere.

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared ...

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 ...

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope (AFM-SECM)

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