This research was supported by the National Natural Science Foundation of China (61871183&#65292;21834001), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023), the &#8220;Chen Guang&#8221; Project from the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).

1. Preparation of Solutions
NOTE: Pay attention to general safety precautions for all chemicals. Dispose of chemicals in a fume hood, and wear gloves, goggles, and a lab coat. Keep flammable liquids away from fire or sparks. All aqueous solutions were prepared using ultrapure water (18.2 M&#937; cm at 25 &#176;C). The prepared solutions were filtered using a 0.22 &#956;m pore-size filter.
<ol>
	<li>Preparation of KCl solution
	<ol>
		<li>Dissolve 0.074 g of potassium chloride in 100 mL of deionized water.</li>
	</ol>
	</li>
	<li>Preparation of NaBH4 solution 
	<ol>
		<li>Dissolve 0.018 g of sodium borohydride in 10 mL of ethanol.</li>
	</ol>
	</li>
	<li>Preparation of HAuCl4 solution
	<ol>
		<li>Dissolve 0.010 g of potassium chloride in 1 mL of 1% chloroauric acid solution.</li>
	</ol>
	</li>
	<li>Preparation of silicone rubber
	<ol>
		<li>Mix silicone rubber containing Part A and Part B (see Table of Materials) at a ratio of 1:1 by volume.</li>
		<li>Use the mixed silicone rubber to paint a reaction area at the slide immediately during the pot time of 1 min.</li>
		<li>Cure the prepared silicone rubber on the slide for 5 min.</li>
	</ol>
	</li>
	<li>Preparation of gold nanoparticles30
	<ol>
		<li>Add 4.8 mL of chloroauric acid with a mass fraction of 1% into 40 mL of deionized water with vigorous stirring.</li>
		<li>Heat the solution to a boil.</li>
		<li>Quickly add 10 mL of a trisodium citrate solution with a mass fraction of 1% into the solution.</li>
		<li>Heat the solution for an additional 15 min until the final solution is red in color. 
		NOTE: In our case, the chloroauric acid solution was rapidly reduced by trisodium citrate, and it was observed that the solution rapidly changed from clear-yellow to dark-black.</li>
	</ol>
	</li>
</ol>
2. Preparation of Experimental Setup
<ol>
	<li>Preparation of current measurement system
	<ol>
		<li>Turn on the current measurement system containing the current amplifier (see Table of Materials) and low-noise data acquisition system (see Table of Materials)</li>
		<li>Switch on the voltage-clamp mode.</li>
		<li>Set the filter bandwidth to 10 kHz and sampling rate to 100 kHz.</li>
		<li>Assemble a self-designed specific homemade copper cage to shield external noise for experimental cells and the pre-amplifier on the inverted microscope (see Table of Materials).</li>
		<li>Ground the shell of the Faraday cage, shells of the amplifier, and inverted microscope system.</li>
	</ol>
	</li>
	<li>Setup of the dark-field detection system 
	<ol>
		<li>Generation of the gold nanotip inside the nanopipette is monitored by the dark-field microscope. 
		NOTE: An inverted microscope system (see Table of Materials) is used to take images and scatter spectra. A true-color digital CCD camera is employed to take images of the nanopipette and nanopore electrode. A dark-field condenser [numerical aperture (NA) = 0.8&#8211;0.95)] is utilized to form a dark-field illumination. 10X (NA = 0.3), 20X (NA = 0.45), and 40X (NA = 0.6) objectives are used to collect images of the closed-type WNE. Fluorescence detection is used to further verify whether there are gaps between the nanotip and inner wall of the nanopipette. This experiment is performed by another EMCCD (see Table of Materials) also integrated on the inverted microscope, and the excitation light is a built-in mercury lamp with a band-pass filter of 450&#8211;490 nm.</li>
	</ol>
	</li>
</ol>
3. Fabrication of Closed-Type WNE
<ol>
	<li>Fabrication of nanopipettes
	<ol>
		<li>Put the quartz capillaries (see Table of Materials) in a 15 mL centrifuge tube filled with acetone for 10 min of ultrasonic cleaning.</li>
		<li>Pour off&#160;acetone, then add ethanol into the same centrifuge tube.</li>
		<li>Put the centrifuge tube in an ultrasonic cleaner for 10 min of cleaning.</li>
		<li>Put the capillaries into another 15 mL centrifuge tube with deionized water for removal of the ethanol, with 10 min of ultrasonic cleaning.</li>
		<li>Continually ultrasonic clean the capillaries three times with deionized water to remove the residual ethanol.</li>
		<li>Dry the capillaries using a nitrogen gas stream.</li>
		<li>Keep the capillaries in a new, clean centrifuge tube.</li>
		<li>Turn on the CO2 laser puller (see Table of Materials)</li>
		<li>Preheat the puller for 15-20 min to ensure a steady laser power.</li>
		<li>Install the cleaned capillary in the puller.</li>
		<li>Set the pulling parameters of heat, filament, velocity, delay, and pulling force on the panel of the CO2 laser puller for a specific diameter. The detailed parameter for pulling a 30 nm diameter nanopipette in this protocol is shown in Table 1 (Figure 1).</li>
		<li>Fix the prepared nanopipette on a Petri dish with the reusable adhesive (see Table of Materials) for further characterization.</li>
	</ol>
	</li>
	<li>Fabrication of closed-type WNE
	<ol>
		<li>Inject 10 &#956;L of the prepared HAuCl4 solution into the nanopipette with a microloader.</li>
		<li>Centrifuge the nanopipette for 5 min at around 1878 x g for the removal of air bubbles in the nanopipette. 
		NOTE: For this step, we placed the nanopipette with the tip facing down into a homemade holder within a 2 mL centrifuge tube.</li>
		<li>Fix the nanopipette on a coverslip with the prepared silicone rubber (see step 1.4) and define the area inside the nanopipette as the &#8220;cis&#8221; side and outside as the &#8220;trans&#8221; side.</li>
		<li>Wait 5 min until the rubber is cured.</li>
		<li>Put the integrated ensemble on the objective table of the inverted microscope.</li>
		<li>Turn on and adjust the dark-field illumination to focus the nanopipette tip under a 10X microscope objective.</li>
		<li>Change to 20X and 40X objectives for a higher spatial resolution.</li>
		<li>Place one Ag/AgCl electrode inside the nanopipette.</li>
		<li>Place the other grounded Ag/AgCl electrode into the trans side.</li>
		<li>Connect a pair of Ag/AgCl electrodes to the pre-amplifier.</li>
		<li>Turn on the current measurement system and the corresponding software (see Table of Materials) for ionic current recording.</li>
		<li>Set the applied potential to 300 mV.</li>
		<li>Slowly add 150 &#956;L of NaBH4 solution into the trans side to trigger the reaction between HAuCl4 and NaBH4 (Figure 2). 
		NOTE: The reduction of NaBH4 in aqueous solution takes place at a violent reaction rate. Therefore, the generation of H2 from the reduction of NaBH4 may induce a defective structure of the nanotip by the generation of cavities during the gold nanotip growth.</li>
		<li>Simultaneously, electrically and optically record the current trace and the dark-field image/scattering spectra using the current measurement and dark-field detection systems (Figure 3). 
		NOTE: The ethanol solution is volatile under dark-field illumination. Pay attention to the volume of ethanol during the fabrication process.</li>
		<li>Turn off the applied potential after ionic current tracing back to 0 pA.</li>
		<li>Wash the prepared closed-type WNE with flowing deionized water from the bottom to the tip.</li>
	</ol>
	</li>
	<li>Characterization of closed-type WNE
	<ol>
		<li>Characterize closed-type WNE with a scanning electron microscope (SEM), which is a general method for characterization of nanopipettes22,31,32,33,34.</li>
		<li>Use a calcium ion fluorescent experiment to verify the sealing condition of the gold nanotip inside the nanopipette.
		<ol>
			<li>Inject 10 &#956;L of CaCl2 solution into the cis side of the closed-type WNE and Fluo-8 solution into the trans side.</li>
			<li>Connect the Ag/AgCl electrodes to the headstage.</li>
		</ol>
		</li>
		<li>Apply a 400 mV bias potential and use the EMCCD (see Table of Materials) to monitor the fluorescence response at the tip area. Use focus ions beam (FIB) to sculpt the closed-type WNE from the tip to the bottom, then determine the length of the interior metal layer or nanotip with SEM characterization.</li>
	</ol>
	</li>
	<li>Single nanoparticle collision with closed-type WNE
	<ol>
		<li>Change the solution in the trans and cis sides to a KCl solution after fabrication of the closed-type WNE.</li>
		<li>Transfer 50 &#956;L of 30 nm gold nanoparticle solution into the trans side. Record the current signal of single nanoparticle collision events at a potential of 300 mV (Figure 5).</li>
		<li>Change the applied voltage to monitor the frequency, amplitude, and shape change of the current signal.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interests.

Fabrication of a well-defined nanopipette is the first step in the closed-type WNE fabrication process. By focusing a CO2 laser onto the center of the capillary, one capillary separates into two symmetrical nanopipettes with nanoscale conical tips. The diameter is easily controlled, ranging from 30-200 nm, by adjusting the parameters of the laser puller. It is noted that the parameters for pulling can vary for different pipette pullers. The environmental temperature and humidity can also influence the final di...

Nanoparticles have attracted tremendous attention due to diverse features such as their catalytic ability, particular optical features, electroactivity, and high surface-to-volume ratios1,2,3,4. Electrochemical analysis of single nanoparticles is a direct method for understanding the intrinsic chemical and electrochemical processes at the nanoscale level. To achieve highly sensitive measurements of single nanoparticles, two electrochemical approaches have been previously applied to read out nanoparticle information from current responses5,6,7. One of these approaches involves immobilizing or capturing an individual nanoparticle on the interface of the nanoelectrode for the study of electrocatalysis8,9. The other strategy is driven by single nanoparticle collision with the surface of an electrode, which generates a transient current fluctuation from the dynamic redox process.
Both of these methods require a nanoscale ultrasensitive sensing interface that matches the diameter of single nanoparticles. However, traditional fabrication of nanoelectrodes has mainly incorporated the micro-electromechanical systems (MEMS) or laser pulling techniques, which are tedious and undisciplinable10,11,12,13. For example, MEMS-based fabrication of nanoelectrodes is expensive and requires the use of a clean room, restricting the massive production and popularization of nanoelectrodes. On the other hand, laser pulling fabrication of nanoelectrodes relies heavily on experience of the operators during the sealing and pulling of a metal wire inside the capillary. If the metal wire is not well-sealed in the capillary, the gap between the inner wall of the nanopipette and wire can dramatically introduce excess background current noise and enlarge the electroactive sensing area. These drawbacks largely decrease the sensitivity of the nanoelectrode. On the other hand, the existence of a gap can enlarge the electrode area and reduce sensitivity of the nanoelectrode. As a consequence, it is hard to guarantee a reproducible performance due to the uncontrollable electrode morphologies in each fabrication process14,15. Therefore, a general fabrication method of nanoelectrodes with excellent reproducibility is urgently needed to facilitate electrochemical exploration of the intrinsic features of single nanoparticles.
Recently, the nanopore technique has been developed as an elegant and label-free approach for single molecule analysis16,17,18,19,20. Owing to its controllable fabrication, the nanopipette provides a nanoscale confinement, with a uniform diameter ranging from 30-200 nm by a laser capillary puller21,22,23,24. Moreover, this simple and reproducible fabrication procedure ensures the generalization of the nanopipette. Recently, we proposed a wireless nanopore electrode (WNE), which does not require the sealing of a metal wire inside the nanopipette. Through a facile and reproducible fabrication process, the WNE possesses a nanoscale metal deposition within the nanopipette to form an electroactive interface25,26,27,28. Since the WNE possesses a well-defined structure and uniform morphology of its confinements, it achieves high current resolution as well as low resistance-capacitance (RC) time constant for performing high temporal resolution. We previously reported two types of WNEs, open-type and closed-type, for realizing single entity analysis. The open-type WNE employs a nanometal layer deposited on the inner wall of a nanopipette, which converts the faradic current of a single entity to the ionic current response26. Usually, the diameter of an open-type WNE is around 100 nm. To further decrease the diameter of WNE, we presented the closed-type WNE, in which a solid metal nanotip fully occupies the nanopipette tip through a chemical-electrochemical approach. This method can rapidly generate a 30 nm gold nanotip inside a nanopore confinement. The well-defined interface at the tip area of a closed-type WNE ensures a high signal-to-noise ratio for electrochemical measurements of single nanoparticles. As a charged gold nanoparticle collides with the closed-type WNE, an ultrafast charging-discharging process at the tip interface induces a capacitive feedback response (CFR) in the ionic current trace. Compared to a previous single nanoparticle collision study via a nanoelectrode with metal wire inside29, the closed-type WNE showed a higher current resolution of 0.6 pA &#177; 0.1 pA (RMS) and higher temporal resolution of 0.01 ms.
Herein, we describe a detailed fabrication procedure for a closed-type WNE that has highly controlled dimensions and outstanding reproducibility. In this protocol, a simple reaction between AuCl4- and BH4- is designed to generate a gold nanotip that completely blocks the orifice of a nanopipette. Then, bipolar electrochemistry is adopted for continuous growth of a gold nanotip that reaches the length of several micrometers inside the nanopipette. This simple procedure enables the implementation of this nanoelectrode fabrication, which can be carried out in any general chemistry laboratory without a clean room and expensive equipment. To determine the size, morphology, and inner structure of a closed-type WNE, this protocol provides a detailed characterization procedure with use of a scanning electron microscope (SEM) and fluorescence spectroscopy. One recent example is highlighted, which directly measures the intrinsic and dynamic interactions of gold nanoparticles (AuNPs) colliding towards the nanointerface of a closed-type WNE. We believe that the closed-type WNE may pave a new path for future electrochemical studies of living cells, nanomaterials, and sensors at single-entity levels.

<table>
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>650501</td>
			<td>Highly flammable and volatile</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler Toledo</td>
			<td>ME104E</td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blu-Tack reusable adhesive</td>
			<td>Bostik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Corning Inc.</td>
			<td></td>
			<td>Centrifuge Tubes with CentriStar Cap, 15 ml</td>
		</tr>
		<tr>
			<td>Chloroauric acid</td>
			<td>Energy Chemical</td>
			<td>E0601760010</td>
			<td>HAuCl4</td>
		</tr>
		<tr>
			<td>Clampfit 10.4 software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550A digitizer</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DS Fi1c true-color CCD camera</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoflex 5 Addtion cure silicone rubber</td>
			<td>Smooth-On</td>
			<td>17050377</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Reference 2 pipettes</td>
			<td>Eppendorf</td>
			<td>492000904</td>
			<td>10, 100 and 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>24102</td>
			<td>Highly flammable and volatile</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td></td>
			<td></td>
			<td>Copper</td>
		</tr>
		<tr>
			<td>iXon 888 EMCCD</td>
			<td>Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Axygen Scientific</td>
			<td></td>
			<td>0.6, 1.5 and 2.0 mL</td>
		</tr>
		<tr>
			<td>Microloader</td>
			<td>Eppendorf</td>
			<td>5242 956.003</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>LOT 16938</td>
			<td>20 mm*60 mm-1 mm thick</td>
		</tr>
		<tr>
			<td>Milli-Q water purifier</td>
			<td>Millipore</td>
			<td>SIMS00000</td>
			<td>Denton Electron Beam Evaporator</td>
		</tr>
		<tr>
			<td>P-2000 laser puller</td>
			<td>Sutter Instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Axygen Scientific</td>
			<td></td>
			<td>10, 200 and 1,000 &#181;L</td>
		</tr>
		<tr>
			<td>Potassium chloride,+D25+A2:F2+A2:F25</td>
			<td>Sigma Aldrich</td>
			<td>P9333-500G</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Quartz pipettes</td>
			<td>Sutter</td>
			<td>QF100-50-7.5</td>
			<td>O.D.:1.0 mm, I.D.:0.5 mm, 75 mm length</td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone thinner</td>
			<td>Smooth-On</td>
			<td>1506330</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Alfa Aesar</td>
			<td>11466</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride,</td>
			<td>Tianlian Chem. Tech.</td>
			<td>71320</td>
			<td>NaBH4</td>
		</tr>
		<tr>
			<td>Ti-U inverted dark-field microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a closed-type wireless nanopore electrode for analyzing single nanoparticles

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

We demonstrate a facile approach to fabricate a well-defined 30 nm wireless nanopore electrode based on a quartz conical nanopipette. The fabrication of a nanopipette is demonstrated in Figure 1, which includes three main steps. A microcapillary with an inner diameter of 0.5 mm and outer diameter of 1.0 mm is fixed in the puller, then a laser is focused on the center of the capillary to melt the quartz. By applying forces to the terminals of the capillary, it...

Watch this Scientific Journal Video about A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles at JoVE.com

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

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

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

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Chemistry

7.4K Views.  East China University of Science and Technology. Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

Video: A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

In Situ Lithiated Reference Electrode: Four Electrode Design for In-operando Impedance Spectroscopy

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate multiple processes responsible for long-term performance decay. Given the complexity of physical processes occurring inside the cell, it is often challenging to achieve a full understanding of the root causes of this performance degradation. This difficulty arises in part from the fact that any electrochemical measurement of a battery will return the combined contributions of all components in the cell. Incorporation of a reference electrode can solve part of the problem, as it allows the electrochemical reactions of the cathode and the anode to be individually probed. A variation in the voltage range experienced by the cathode, for example, can indicate alterations in the pool of cyclable lithium ions in the full-cell. The structural evolution of the many interphases existing in the battery can also be monitored, by measuring the contributions of each electrode to the overall cell impedance. Such wealth of information amplifies the reach of diagnostic analysis in Li-ion batteries and provides valuable input to the optimization of individual cell components. In this work, we introduce the design of a test cell able to accommodate multiple reference electrodes, and present reference electrodes that are appropriate for each specific type of measurement, detailing the assembly process in order to maximize the accuracy of the experimental results.

The incorporation of reference electrodes in a Li-ion battery provides valuable information to elucidate degradation mechanisms at high voltages. In this article, we present a cell design that accommodates multiple reference electrodes, along with the assembly steps to assure maximum accuracy of the data obtained in electrochemical measurements.

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.