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...

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. ...

This protocol employees a powerful and innovative technology so called Atomic Force Microscope coupled with Electro-chemicals Scanning Microscope which is AFM-SECM to scan the morphological and the electrochemical information on faceted nanomaterials and nanobubbles in water. AFM-SECM is capable of mapping electrochemically reactive surface based on tip current images and also enable simultaneous acquisition of nanoscale surface structures and electrochemistry information on sample materials. The sample preparation for this method require that the solid particles are immobilized on the substrate completely and the bindings between samples and substrates ensures electrical conductivity.The choice of the Redox Mediator is critical as well. More so, demonstration is necessary to really show that it held under a dedicated, separate operation in this protocol such as sample preparation systems that happen under the imaging process. Deposit 10 microliters of epoxy on a cleaned silicon wafer using a pipette tip and tile it with a clean glass slide.After about five minutes, drop 10 microliters of the copper oxide nanoparticle suspension on the epoxy coated silicon wafers substrates. Then vacuum dry the substrate at 40 degrees Celsius or six hours. To prepare oxygen nanobubbles directly inject compressed oxygen through a tubular ceramic membrane into deionized water.Deposit 1.8 milliliters of the nanobubble suspension on a gold substrate in the electrochemical sample cell and stabilize it for 10 minutes. Replace the existing sample chunk with the SECM chunk. Then screw it in place using two M3, 6 mm Socket Head Cap Screws and a 2.5 millimeter hex wrench.Install the strain release module onto the AFM scanner and connect it to the working electrode connector on the spring connector block with an extension cable. Double-click the two software icons to initialize the AFM system and the by potentiostat control interface. Prepare the electrostatic discharge field, surface package including an anti-static pad, electrostatic discharge protective probe stand, wearable anti-static gloves and wrist strap.To prevent the AFM scanner from exposure to liquid, use a protective boot during AFM-SECM testing. Put the probe holder onto the electrostatic discharge protective probe stand and use a pair of plastic tweezers to attach the protective boot to the tip holder. Then align the small cut in the protective boot to the notch in the probe holder.Open the box of AFM-SECM probes with a tip tweezer and grab the probe from both sides of the grooves. Use the disc gripper to hold the probe holder on the stand and put the probe wire into the hole of the stand. Then slide the probe into the slot of the probe holder.After the probe is inside the slot, use the flat end of the tweezer to push it in. Attach the whole probe holder to the scanner and use the PTFE tip tweezer to grab the wire right below the copper ring and connect it to the module. Then put the scanner back to the dovetail.Place the previously assembled electrochemical sample cell with the test sample on the central point of the SECM chunk. Then connect the pseudo reference electrode and the counter electrode to the spring connector block. In the AFM-SECM software, select SECM PeakForce QNM to load the workspace.In setup, load the SECM probe and then align a laser to the tip using an alignment station. Go to Navigation and move the scanner downwards slowly to focus on the sample surface. Adjust the position of the electrochemical sample cells slightly to make sure the scanner doesn't touch the glass cover of the sample cell while moving.After focusing on the sample, click Update Blind Engage Position. Click, Move to Add Fluid Position and add approximately 1.8 milliliters of the buffer solution into the sample cell. Making sure that the level of the solution is lower than the glass cover and that the wires are immersed in the solution, use a pipette to agitate the solution to remove any bubbles and wait for five minutes.Click, Move to Blind Engage Position which will make the tip move back into the buffer solution. Adjust the laser slightly to make sure the laser is aligned on the tip. Open the electrochemical workstation software and click on the Technique Command on the toolbar to open the tech selector.Select Open Circuit Potential Time and use the default setting to run the OCP measurement which should be almost zero and stable. Click the Technique Command again and select Cyclic Voltammetry then enter the cyclic voltammetry parameters and proceed with SECM imaging. Go back to the AFM-SECM software and click, Engage.Next select chronoamperometry and set the chronoamperometry parameters with the initially as minus 0.4 volts the pulse width as 1000 seconds and the same sensitivity as the CV scan. With the program running, go back to the AFM-SECM software check the real time reading on the strip chart and click on start. Save the images in the AFM-SECM software.Using the electric chemical sample cell as a clean water container, move the tip in and out of the liquid with the Blind Engage Functions in the navigation panel, changing the water three times. Then use clean wipes to carefully remove residual water from the probe holder and put the probe back in the probe box. This protocol was used to characterize individual oxygen nanobubbles, revealing both morphological and electrochemical information.The comparison of the topography and the current image demonstrates the correlation between the locations of the nanobubbles and the current spots. The topography and current images of copper oxide nanoparticles are shown here. The tip current image indicates that the nanoparticle visible in the topography image is associated with an evident electric current spot.Whereas the background current corresponds to the flat silicon substrate. Here are five representative cyclic voltammetry curves of the AFM-SECM tip at approximately one millimeter from the substrate. The diffusion limited tip current did not decrease with time.The changes of the tip current as the tip approaches the sample surface are plotted here. The AFM-SECM tip, approached the substrate surface in the Z direction until it reached a set point indicating the physical tip substrate contact or bending. When this protocol make sure that the solid particle are immobilized as the substrate they're completely with electrical conductivity and there are no bubbles in the solution in the sample cell.The sample preparation method are relevant to a wider range of applications that involves nanomaterials, especially for nano material characterization. The AFM-SECM technique can be used to acquire simultaneous topography and the electrochemistry image at the nanoscale which is important in the development and application of nanomaterials in research fields such as material science, chemistry and life science.

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

Abstract