We acknowledge support by the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Ontario Research Fund. We thank Y. Liu for aid in nanopore fabrication and characterization, L. Andrzejewski for valuable discussions and technical support, and A. Marziali for help with nanopore software and instrumentation design.

1. Nanopore Fabrication and Cleaning
Note: Once a nanopore exists in an insulating membrane, it can be directly mounted in the liquid cell without further processing or cleaning, as described in step 2. However, if it is necessary to remove traces of contaminants between experiments, nanopore chips can be cleaned using either piranha solution3,15,16 (3:1 H2SO4:H2O2) or by exposure to oxygen plasma2. As such, steps 1.2 to 1.9 in the following protocol are optional if precleaning by exposure to piranha solution is not necessary.
<ol>
	<li>Degas filtered deionized (DI) water by placing under vacuum in a sonicator for 30 min&#160;at 40 &#176;C.</li>
	<li>Prepare piranha solution in a 10 ml beaker by carefully adding 3 ml sulfuric acid followed by 1 ml hydrogen peroxide. Mix thoroughly by refluxing in the pipette. CAUTION: Piranha solution is extremely hazardous. Please take all precautions.</li>
	<li>Using acid-resistant tweezers, carefully insert the nanopore-containing membrane chip edge-first into the piranha solution to completely submerge the chip and avoid it floating on the surface.</li>
	<li>Rinse tweezers thoroughly in filtered water.</li>
	<li>Place the beaker on a hot-plate preset to 90 &#176;C and allow it to clean for at least 30 min.</li>
	<li>Carefully remove the piranha solution from the beaker using a clean glass pipette and discard in copious amounts of water.</li>
	<li>Using a clean glass pipette add 5 ml of the degassed deionized water from step 1.1 into the beaker to rinse. Remove the water and repeat at least 5x.</li>
	<li>Carefully remove the nanopore chip from the beaker using clean sharp-tip tweezers. Handle with extreme care as the nanopore membrane is very fragile.</li>
	<li>Dry the chip by gently applying suction to its edge using an aspirator. Store the chip in a clean Petri dish until ready for use.</li>
</ol>
2. Mounting the Nanopore
<ol>
	<li>Clean the Teflon nanopore cell (Figure 1) by placing in 20% nitric acid solution and boiling for 10 minutes. CAUTION: Use all necessary personal protective equipment and handle acids with care.</li>
	<li>Carefully remove the cell from nitric acid and place in boiling DI water for 10 min.</li>
	<li>Boil the cell in DI water for an additional 10 min&#160;to ensure complete removal of nitric acid. Remove the beaker from the hot plate and allow it to cool to room temperature.</li>
	<li>Remove the cell from the beaker and blow dry with filtered air or N2. Store the cell in a clean Petri dish.</li>
	<li>Degas filtered KCl solution (buffered with HEPES at pH 8) by placing under vacuum in a sonicator for 30 min&#160;at 40 &#176;C.</li>
	<li>Clean two silicone elastomer gaskets for each nanopore chip by sonicating in ethanol for at least 10 min.</li>
	<li>Place the nanopore chip on a clean elastomer gasket being careful to align the membrane window with the gasket opening. Place and align a second gasket atop the chip.</li>
	<li>Place the chip and gaskets on the reservoir inlet of one half of the cleaned nanopore cell. Assemble the cell by screwing the other half in place. An exploded view of the nanopore cell components is shown in Figure 1.</li>
	<li>Wet the nanopore chip by pipetting ethanol into the cell reservoirs and placing in a vacuum chamber until a few bubbles are seen to exit the inlets.</li>
	<li>Remove ethanol by flushing the reservoirs with at least 3 ml degassed filtered KCl solution. Take care to remove overflow using an aspirator.</li>
</ol>
3. Nanopore Characterization
<ol>
	<li>Place the nanopore cell in the electrically shielded experimental setup and place Ag/AgCl electrodes in each reservoir. This setup is similar to that shown in Figure 2 with the exception of the external power supply and current amplifier which are replaced with a low-noise resistive feedback amplifier.</li>
	<li>Using the low-noise amplifier in voltage-clamp mode, apply potentials sweeping from -200 mV to +200 mV and record the I-V characteristics.</li>
	<li>Fit the I-V curve to obtain nanopore conductance, which can be used to calculate its diameter in solution17. If the calculated diameter is much smaller than expected from TEM imaging, the pore is likely not completely wetted and/or contains debris or contamination.</li>
	<li>Apply a 200 mV potential across the nanopore and record the ionic current for 30 sec.</li>
	<li>Perform a power spectral density (PSD) analysis of the ionic current and integrate to quantify the electrical noise characteristics of the nanopore. If the noise is above 15 pA RMS at 5 kHz bandwidth, then the pore is likely not completely wetted and/or contains contamination and cannot be reliably used in experiment.</li>
</ol>
4. Conditioning Nanopores Using High Electric Fields
Note: If the I-V curve generated exhibited asymmetry or less-than-expected conductance, or the current trace showed instability and high noise levels at low-frequencies, it is necessary to condition the nanopore with high electric fields to remove any contamination on the pore surface and/or wet the pore. While this method does not affect the high-frequency noise caused by the membrane capacitance or any parasitic capacitance coupled to the input of the current amplifier used in measurements, low-frequency noise (also called 1/f noise)18 can be greatly reduced. A schematic of the setup used to perform this conditioning is shown in Figure 2.
<ol>
	<li>Disconnect the electrodes from the patch-clamp amplifier.</li>
	<li>Connect one of the electrodes to a computer-controlled power supply capable of generating &#62;6 V (&#62;0.2 V/nm electric field strength for the 30-nm thick membranes used here) and the other to an external current amplifier that can be monitored in real time. Note: The application of high electric fields can be used to condition nanopores in various membrane materials and thicknesses. While both 30-nm and 10-nm membranes are discussed here, voltages described refer to those used for 30-nm thick membranes unless otherwise stated. </li>
	<li>Apply a potential difference of 400 mV (measurement voltage) across the nanopore for at least 5 sec.</li>
	<li>Calculate the mean current value from the final 1 sec of data to determine the conductance of the nanopore. Calculate the diameter of the nanopore based on this conductance, which should be done automatically using the software and the nanopore conductance model of choice based on the most likely geometry. It should correspond to the diameter measured from the I-V curve.</li>
	<li>Apply a 200 msec pulse of 6 V (wetting voltage) across the nanopore to produce an electric field of 0.2 V/nm followed by a 5 sec measurement period at 400 mV. Again, calculate a diameter of the nanopore using the final 1 sec of data and compare with the value expected from TEM measurements to ensure that the nanopore is fully wet. If necessary, repeat several times.</li>
	<li>If necessary, repeat the application of high electric field pulses with increasing voltage until the current signal during the measurement period is stable and showing the expected conductance. It is not recommended to exceed 10 V (i.e. &#62;0.3 V/nm), as this can significantly enlarge or damage the nanopore rapidly.</li>
</ol>
5. Enlarging Nanopores Using High Electric Fields
Note: The diameter of the nanopore is crucial in determining its functionality for a particular biomolecular sensing application. To this end, a nanopore created using a TEM can be enlarged to a desired size by applying high electric fields until the appropriate diameter is achieved with the same setup used to clean and wet the nanopore (Figure 2).
<ol>
	<li>Using the same electronic configuration as in part 4, apply a 200-500 mV bias across the pore to obtain a diameter measurement. While less precise than fitting an I-V curve, a single point measurement can be used to roughly estimate the nanopore size rapidly.</li>
	<li>Apply a 2 sec pulse of 8 V across the nanopore followed by a measurement period of at least 5 sec at 400 mV. Calculation of the new diameter will typically show a very small increase in nanopore size (&#60;0.1 nm).</li>
	<li>Repeat this process cyclically, alternating between enlargement and measurement voltages to obtain in situ and real-time measurements of increasing nanopore diameter.</li>
	<li>If faster growth rate is desirable, increase the magnitude of the voltage applied incrementally up to 10 V. Growth will typically accelerate as the pore enlarges with the rate of increase in conductance ranging from 0.03 nS/sec&#160;to 10 nS/sec, depending on the size of the nanopore, strength of the electric field and electrolyte solution properties.</li>
	<li>When the desired diameter is reached, stop the application of high electric fields. This can be done automatically using the computer program.</li>
	<li>Reconnect the patch-clamp amplifier to the electrodes.</li>
	<li>Acquire new I-V and current trace data at 200 mV to confirm the diameter of the nanopore and verify low-noise current signals as in steps 3.2 - 3.5 above. If necessary, repeat conditioning and enlarging protocol (steps 4.1 - 5.5).</li>
</ol>
6. DNA Translocation
<ol>
	<li>Prior to adding a biomolecular sample, perform a control experiment to ensure that there is no contamination in the reservoir. Acquire a current trace under an applied potential of +150 to +300 mV in the absence of any sample to verify that no current blockades are detected after 2 min.</li>
	<li>Add &#955; DNA (48.5 kbp double-stranded) to the cis reservoir for a final concentration of 0.5-2 ng/&#181;l. Reflux gently by pipette for at least 10 sec to ensure homogeneous distribution of the sample throughout the reservoir.</li>
	<li>For a 30-nm thick nanopore, apply a potential bias of +150 to +300 mV to the trans reservoir and measure the ionic current passing through the nanopore. For very short translocation events, it is desirable to sample at a high frequency (250 kHz or greater) with a relatively high low-pass filtering frequency (100 kHz).</li>
	<li>Monitor the ionic current using software to detect transient current blockades as molecules translocate through the nanopore. The ionic current traces of molecular translocation can be analyzed to determine blockage depth, duration and frequency to infer information about the sample of interest. Conversely, if information about the translocating molecules is known, this data can be used to investigate properties of the nanopore itself.</li>
</ol>

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The authors have nothing to disclose.

Control of nanopore size is of fundamental importance in biomolecular sensing applications. Nanopore diameters must be on the order of the size of the molecules being probed; they must be large enough to accommodate the sample but small enough to achieve optimal signal-to-noise. While the control of size using the method presented of applying high electric fields is unidirectional in that nanopore diameters are only increased throughout the process, nanopores with diameters between 3-100 nm can be fashioned, with subnanometer precision. As 3-4 nm pores can be readily fabricated using a TEM23,&#160;this allows for the reliable fabrication of solid-state nanopores for a wide range of applications from probing ssDNA structure to the interaction of bulky protein-ligand complexes. While nanopore growth above 100 nm can be very fast and less precise, more moderate enlarging conditions can be employed to achieve better control over the process. As such, the most important step for achieving effective size control is the choice of pulse strength and duration in order to balance enlarging efficiency and the level of precision required in achieving a desired pore diameter. This is further highlighted by the enlargement of thinner nanopores (10-nm thickness), where enlargement is observed a lower bias but comparable electric field strength. Depending on the final size, it is generally possible to enlarge a nanopore to sub-100-nm diameters in a few minutes.
Similarly, large low-frequency current fluctuations preclude single-molecule studies as it is nearly impossible to differentiate translocation signals from background noise. Incomplete wetting24, the presence of carbonaceous residues remaining after initial fabrication25 and adsorption of debris on the nanopore wall13 can degrade signal quality, requiring additional cleaning with harsh chemical treatments that are often inefficacious. Interestingly, it is common for solid-state nanopore protocols to emphasize the importance of cleaning the nanopore in piranha solution or with oxygen plasma before mounting to aid wetting or remove any contamination left over from the drilling, imaging and handling processes. Even with this treatment, however, nanopores often do not wet or continue to exhibit high noise, and the suggested solution for failed attempts is to perform additional cleaning which can be extremely time consuming14. With the application of high electric fields, these lengthy protocols may not be necessary depending on the application. It was found that most devices could be reconditioned in situ using the method described herein, consequently reducing preparation time and the need to deal with harsh chemicals. The most important steps in mitigating electrical noise is a simple increase in voltage and/or pulse duration to completely wet the pore and remove loosely-bound debris. Nanopores treated in this fashion can reliably be used in biomolecule translocation experiments, such as the passage of DNA and proteins. If these molecules adhere to the pore wall leading to a clogged and noisy electrical signal, high electric field pulses can be reapplied to remove the obstruction and regain low-noise properties for further experimentation, without unmounting of the nanopore chip from the fluidic cell.
The application of high electric fields using the setup described is limited by the requirement of an external power supply that can apply up to 10 V and current amplifier, which lack the sensitivity and low-noise properties at high-bandwidth (&#62;1 kHz) for single molecule sensing. While typical biomolecular experiments rely on a low-noise current amplifier that is limited to &#177;1 V, it is straightforward to design a single system that could accomplish both high electric field conditioning and sensitive current measurement with an adjustable gain. Despite this limitation, the transition from one setup to the other is quick and straightforward. In comparison with existing techniques for controlling nanopore size such as the use of SEM5, thermal oxidation and membrane reshaping8,&#160;high electric fields offer a faster, more precise and less expensive methodology that can be performed on the lab bench using standard equipment and provide a broader range of nanopore sizes. The ability to rapidly and reproducibly reduce low-frequency noise also makes initial fabrication more reliable and prolongs the lifetime of solid-state nanopores, as previously used pores can be rejuvenated for further experiments. Altogether, over 95% of nanopores of varying thicknesses conditioned with high electric fields exhibited very little low-frequency noise characteristic, rendering them suitable for biomolecule sensing. Fabrication is thus easier and more reliable, making solid-state nanopore experiments more accessible to researchers and potentially allowing for a path towards commercialization of nanopore technologies through more robust fabrication processes.

Biological and solid-state nanopores provide a means of sensing biomolecular analytes at the single molecule level1. Individual nanopores are typically embedded in thin insulating membranes, providing the only conduit for ionic current to pass between two liquid reservoirs. Utilizing the principles of larger-scale Coulter counters, nanopore experiments relate changes in ionic current to determine the length, size, charge and conformation of charged biomolecules as they are electrophoretically driven through a nanopore in the presence of an external electric field.
While biological nanopores such as &#945;-hemolysin typically offer greater sensitivity and low-noise properties3, the supporting lipid bilayer is fragile and of fixed size, limiting their applicability. Solid-state nanopores, on the other hand, are fabricated in thin (10-50 nm) silicon nitride or silicon oxide membranes and can be made of different sizes, be readily integrated with wafer-scale technologies6,7, and are more robust, allowing for a wider range of experimental conditions. Despite these advantages, solid-state nanopore technologies suffer from several practical drawbacks that limit their usefulness for biomolecular studies. While control of nanopore size is possible, it is typically expensive and laborious to achieve, requiring specialized equipment and skilled personnel. For example, nanopores drilled by focused-ion beam have been recently shown to shrink under specific experimental conditions in a scanning electron microscope (SEM)5. In other approaches, nanopores drilled by transmission electron microscopy (TEM) can expand or shrink depending on the beam conditions and subsequent exposure to aqueous solvents8. In these cases, the achievable range of nanopore sizes is limited, difficult to control and even unreliable as the size of the nanopore can change following chemical treatment or when immersed in a particular liquid environment9.
The ionic current through solid-state nanopores can also suffer from high noise, the sources of which are an intensely investigated topic in nanopore literature2,3,10,11. While various methods have been proposed to reduce electrical noise, the yield of reliable, stable low-noise nanopores is typically low. Deposition of carbonaceous residues during drilling and imaging can have detrimental effects on the electrical signal quality, often making complete wetting a challenge and causing the formation of nanobubbles that can be difficult to remove12. Furthermore, clogging of the nanopore by analyte molecules degrades signal quality rendering pores unusable for further experiment13,14. Altogether, these effects greatly reduce yield of functional nanopore devices and increase the cost associated with solid-state nanopore research.
The application of a voltage with Ag/AgCl electrodes to produce high electric fields in the range of 0.15-0.3 V/nm presents a surprisingly simple solution to these challenges. Through the cyclic application of short voltage pulses, a clean, low-noise nanopore surface ideal for single-molecule studies is produced. Prolonged exposure to high electric fields initiates the removal of the membrane material constituting the pore wall, resulting in an increase in nanopore diameter. This growth can be precisely controlled by tuning the pulse strength and duration. As current traces degrade over the course of an experiment due to clogging of the nanopore as molecules adsorb to the nanopore surface, this process can be repeated to recover clogged devices that would have otherwise been discarded. As such, the yield of functional nanopores is further increased by the ability to use the same device multiple times. This method provides several advantages as it is rapidly performed in liquid under experimental conditions, requires only standard laboratory equipment, can be automated with software, and produces functional high-quality nanopores with a yield of over 95%.

<table border="0" cellpadding="0" cellspacing="0" width="516">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JEM-2100F TEM</td>
			<td style="width:136px;">JEOL</td>
			<td style="width:81px;"></td>
			<td style="width:83px;">Drilling requires 200 kV accelerating voltage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axon Axopatch 200B patch-clamp amplifier</td>
			<td style="width:136px;">Molecular Devices</td>
			<td style="width:81px;"></td>
			<td>Low-noise voltage and current amplifier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">X-Series data acquisition card</td>
			<td style="width:136px;">National Instruments</td>
			<td style="width:81px;">PCI-6351</td>
			<td>Interfacing with setup, apply of high electric fields</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LabVIEW 2012 software</td>
			<td style="width:136px;">National Instruments</td>
			<td style="width:81px;"></td>
			<td>Apply voltages, record current, data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Current amplifier</td>
			<td style="width:136px;">Keithley</td>
			<td style="width:81px;"></td>
			<td>Current amplification during high electric field pulses</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30-nm thick silicon nitride TEM membrane windows</td>
			<td style="width:136px;">Norcada Inc.</td>
			<td style="width:81px;">NT005X</td>
			<td>Substrate in which nanopores are created</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10-nm thick silicon nitride TEM membrane windows</td>
			<td style="width:136px;">Norcada Inc.</td>
			<td style="width:81px;">NT005Z</td>
			<td>Substrate in which nanopores are created</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicone elastomer O-rings</td>
			<td style="width:136px;">Marian Chicago</td>
			<td style="width:81px;">HT6135</td>
			<td>Punched for sealing the nanopore chip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ag/AgCl electrodes</td>
			<td style="width:136px;">In Vivo Metric</td>
			<td style="width:81px;">E255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric acid</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:81px;">52004P</td>
			<td>Used for cleaning cells - handle with caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:81px;">H323</td>
			<td>Used for piranha solution - handle with caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric acid</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:81px;">A300</td>
			<td>Used for piranha solution - handle with caution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:81px;">P335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:81px;">BP310</td>
			<td>Buffering KCl solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary Faraday cage</td>
			<td style="width:136px;"></td>
			<td style="width:81px;"></td>
			<td>Shielding nanopore cell, electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Secondary Faraday cage</td>
			<td style="width:136px;"></td>
			<td style="width:81px;"></td>
			<td>Shielding headstage, electrode wires</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Teflon cell</td>
			<td style="width:136px;"></td>
			<td style="width:81px;"></td>
			<td>To hold nanopore chip and reservoirs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hot plate</td>
			<td style="width:136px;">VWR</td>
			<td style="width:81px;"></td>
			<td>Heating piranha solution</td>
		</tr>
	</tbody>
</table>

fine-tuning the size and minimizing the noise of solid-state nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

The nanopores used in this study were drilled in 30-nm or 10-nm thick silicon nitride membrane windows. While the protocol described can be applied to solid-state nanopores of various material fabricated using any method, they are commonly drilled by TEM using previously established protocols11,14. Nanopores drilled by TEM are typically between 4-8&#160;nm in diameter (Figure 2). While both 30-nm and 10-nm thick membranes can be mounted and conditioned using the above protocol, voltage biases described refer those required for 30-nm thick membranes unless otherwise stated. For membranes of different size, the applied voltage should be adjusted to generate an electric field in the range of 0.15-0.3 V/nm inside the nanopore.
Figure 3a shows two typical conductance traces of a 10-nm nanopore in a 30-nm thick membrane before and after treatment with high electric fields. Upon mounting a newly drilled nanopore, the likelihood of obtaining an unstable and noisy ionic current signal, exhibiting a high degree of low-frequency fluctuation, is usually high. The nanopore shown in Figure 3a highlights this behavior. Its conductance is considerably less than expected for a nanopore of its size, most likely due to incomplete wetting. Upon the application of high electric fields of 0.27 V/nm in magnitude produced by 8 V pulses (90 pulses of 2 sec duration), the nanopore becomes fully wet and is subsequently enlarged to 21 nm in diameter. At this point, the pore exhibits a stable conductance with low-noise properties. Quantitative analysis of noise in similar nanopores is shown as power spectral density plots in Figure 3b. The low-frequency noise amplitude of unwet and/or clogged pores is very high (&#62;20 pA RMS), rendering them unusable in experiment. Upon conditioning with high electric fields, noise power at low frequencies (&#60;10 kHz) is diminished by up to 3 orders of magnitude and ready for low-noise experiments.
Figure 4a shows a typical current measurement as the potential applied is pulsed between high electric fields for enlarging and low electric field measurement periods. After each subsequent pulse, the resultant ionic current through the nanopore at the measurement voltage (i.e. the nanopore conductance) increases by a finite amount. This demonstrates that the nanopore is increased in size, as the diameter d can be inferred from its conductance G in a solution of conductivity &#963;, approximating the nanopore as having cylindrical geometry of effective length leff. While various other models exist for relating nanopore conductance to its geometry17,19-21, the following relation, which incorporates a geometric term and an access resistance term, has been proven valid for TEM-drilled nanopores in high salt concentrations, over a wide range of diameters of interest for dsDNA translocation17,22.
<img alt="" src="/files/ftp_upload/51081/51081eqn.jpg" width="150px" />
Once the desired diameter is reached, the process is automatically stopped by the software. The resulting nanopore diameter can then be confirmed using precise I-V measurements, as shown in Figure 4b.
It is important to note that nanopores treated using high electric fields are fully functional. This is validated by the detection of &#955; DNA translocation, as shown in the conductance traces presented in Figure 5a. In this figure, dsDNA is driven through two nanopores that were enlarged to 11-nm and 32-nm using the described method. In each case, the baseline conductance is extremely stable and clear blockades are observed as dsDNA molecules translocate through the nanopore, displaying high signal-to-noise single-molecule translocation events as compared to untreated pores that exhibit high noise. As shown in the insets of Figure 5a, multiple discrete blockage levels are observed as individual folded molecules translocate, as expected for nanopores of these sizes. Histograms of the nanopore conductance during translocation events through each pore are shown in Figure 5b. The low-noise properties of the nanopores reveal distinct, easily resolvable peaks corresponding to the baseline (no DNA), single (one DNA strand - unfolded) and double blockage states (two DNA strands - folded). Of note is the fact that the change in conductance corresponding to a single dsDNA molecule occupying the pore is different for the large and small nanopores. This provides indirect evidence that the application of high electric fields is in fact enlarging existing nanopores, as the same blockage amplitude would be observed if other pores or cracks were being created in the membrane during the process17.
Similarly, Figure 6 illustrates the effectiveness of high electric fields for enlarging nanopores fabricated in membranes of different thickness. Here, a nanopore created in a 10-nm SiNx membrane is initially partially unwet, displaying unstable and relatively small conductance. Upon the application of alternating &#177;3 V (&#177;0.3 V/nm) pulses of 4 sec duration (30 total), the nanopore becomes wet and exhibits ideal I-V characteristics for a 3 nm pore. The methodology was then repeated for 400 subsequent pulses and the nanopore was enlarged to 8 nm. This enlargement, performed at comparable electric fields but lower applied voltage bias than for nanopores fabricated in 30-nm membranes, shows that the process is primarily electric field driven. As the current blockade produced by translocation through a thinner membrane is larger than that produced in thicker pores, nanopores in thin membranes treated in this fashion can be used to study shorter molecules such as proteins with increased sensitivity.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig1.jpg" width="600px" /> 
Figure 1. Nanopore cell assembly. A silicon nitride membrane containing a nanopore is placed between silicone elastomer gaskets, which are in turn compressed by two Teflon half-cells containing electrolyte reservoirs. <a href="https://www.jove.com/files/ftp_upload/51081/51081fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig2.jpg" width="600px" /> 
Figure 2. Nanopore conditioning and enlarging setup. A nanopore drilled in a 30 nm thick silicon nitride membrane (left) connects two electrolyte reservoirs. A computer is used to control either a patch-clamp amplifier or external power supply (DAQ card) which applies a potential bias across the nanopore via Ag/AgCl electrodes immersed in the electrolyte reservoirs. The current amplifier relays the ionic current measured to be monitored in real time using computer software. This figure has been modified from [11]. <a href="https://www.jove.com/files/ftp_upload/51081/51081fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig3.jpg" width="600px" /> 
Figure 3. Current traces before and after application of high electric fields. (a) Upon mounting, and even following cleaning with piranha solution, the conductance of the nanopore is unstable and less than expected for a cylindrical 10-nm pore (blue). After the application of 2 sec pulses of 8 V, the nanopore is fully wetted and enlarged, exhibiting a stable conductance and can be used for biomolecular sensing experiments (green). (b) Power spectral density plots of an incompletely wetted and clogged nanopore (blue and orange, respectively). Upon application of 200 msec pulses of 8 V, the nanopores were wetted and debris removed (green and red, respectively). This figure has been modified from [11]. <a href="https://www.jove.com/files/ftp_upload/51081/51081fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig4.jpg" width="600px" /> 
Figure 4. Nanopore enlarging using high electric fields. (a) Alternating between enlargement and measurement potential biases (red) reveals that the ionic current through the nanopore (blue) increases in finite steps. The resulting conductance measurement can be used to infer nanopore diameter. Once the desired diameter has been achieved, the process is stopped. (b) Precise I-V measurements of conductance confirm that nanopore sizes have increased. Such plots provide a better estimate of the pore size than single-point current values as they can be fit and their symmetric and Ohmic behavior can be confirmed. This figure has been modified from [11]. <a href="https://www.jove.com/files/ftp_upload/51081/51081fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig5.jpg" width="600px" /> 
Figure 5. DNA translocation through conditioned nanopores. (a) The addition of dsDNA (48.5 kbp) to one side of the nanopore at a bias of 150 mV produces transient blockades in the conductance traces of 11-nm (blue) and 32-nm pores (red). (b) Histograms of the conductance of each of the nanopores show discrete peaks corresponding to the baseline, single and double translocation events. This figure has been modified from [11]. <a href="https://www.jove.com/files/ftp_upload/51081/51081fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/51081/51081fig6.jpg" width="600px" /> 
Figure 6. Enlargement of nanopores in 10 nm membranes. A nanopore in a 10 nm membrane originally exhibits very little conductance and asymmetric I-V characteristics (orange). Upon the application of 30 pulses of alternating between &#177;3 V (4 sec duration), the nanopore wets and exhibits ideal I-V properties with a conductance consistent with that expected for a 3 nm pore (blue). A further 400 pulses of &#177;3 V enlarges the nanopore to a diameter of 8 nm (green). <a href="https://www.jove.com/files/ftp_upload/51081/51081fig6highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores at JoVE.com

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

fine-tuning-the-size-and-minimizing-the-noise-of-solid-state-nanopores

Research

JoVE Journal

Engineering

13.4K vistas.  University of Ottawa.  Se presenta una metodología para la preparación de nanoporos en estado sólido en solución para los experimentos de translocación biomoleculares. Mediante la aplicación de pulsos cortos de campos eléctricos elevados, el diámetro de nanoporos puede ser controlable ampliada con precisión subnanometer y sus características de ruido eléctrico mejorado significativamente. Este procedimiento se lleva a cabo in situ utilizando el equipo de laboratorio estándar bajo c...

Video: Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 &deg;C), flammable, and volatile organic electrolytes. These organic based electrolyte systems are viable at ambient temperatures, but require a cooling system to ensure that temperatures do not exceed 80 &deg;C. These cooling systems tend to increase battery costs and can malfunction which can lead to battery malfunction and explosions, thus endangering human life. Increases in petroleum prices lead to a huge demand for safe, electric hybrid vehicles that are more economically viable to operate as oil prices continue to rise. Existing organic based electrolytes used in lithium ion batteries are not applicable to high temperature automotive applications. A safer alternative to organic electrolytes is solid polymer electrolytes. This work will highlight the synthesis for a graft copolymer electrolyte (GCE) poly(oxyethylene) methacrylate (POEM) to a block with a lower glass transition temperature (Tg) poly(oxyethylene) acrylate (POEA). The conduction mechanism has been discussed and it has been demonstrated the relationship between polymer segmental motion and ionic conductivity indeed has a Vogel-Tammann-Fulcher (VTF) dependence. Batteries containing commercially available LP30 organic (LiPF6 in ethylene carbonate (EC):dimethyl carbonate (DMC) at a 1:1 ratio) and GCE were cycled at ambient temperature. It was found that at ambient temperature, the batteries containing GCE showed a greater overpotential when compared to LP30 electrolyte. However at temperatures greater than 60 &deg;C, the GCE cell exhibited much lower overpotential due to fast polymer electrolyte conductivity and nearly the full theoretical specific capacity of 170 mAh/g was accessed.

Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (&gt;120 &deg;C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes.

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 ...

Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures1. MLCD results in the formation of highly controlled, ultra shallow and sharp doping profiles at the nanometer scale. In MLCD process the dopant source is a monolayer containing dopant atoms.
In this article a detailed procedure for surface doping of silicon substrate as well as silicon nanowires is demonstrated. Phosphorus dopant source was formed using tetraethyl methylenediphosphonate monolayer on a silicon substrate. This monolayer containing substrate was brought to contact with a pristine intrinsic silicon target substrate and annealed while in contact. Sheet resistance of the target substrate was measured using 4 point probe. Intrinsic silicon nanowires were synthesized by chemical vapor deposition (CVD) process using a vapor-liquid-solid (VLS) mechanism; gold nanoparticles were used as catalyst for nanowire growth. The nanowires were suspended in ethanol by mild sonication. This suspension was used to dropcast the nanowires on silicon substrate with a silicon nitride dielectric top layer. These nanowires were doped with phosphorus in similar manner as used for the intrinsic silicon wafer. Standard photolithography process was used to fabricate metal electrodes for the formation of nanowire based field effect transistor (NW-FET). The electrical properties of a representative nanowire device were measured by a semiconductor device analyzer and a probe station.

A detailed procedure for surface doping of Silicon interfaces is provided. The ultra-shallow surface doping is demonstrated by using phosphorus containing monolayers and rapid annealing process. The method can be used for doping of macroscopic area surfaces as well as nanostructures.

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures1. MLCD results in the formation of highly controlled, ultra ...

Casting Protocols for the Production of Open Cell Aluminum Foams by the Replication Technique and the Effect on Porosity

Metal foams are interesting materials from both a fundamental understanding and practical applications point of view. Uses have been proposed, and in many cases validated experimentally, for light weight or impact energy absorbing structures, as high surface area heat exchangers or electrodes, as implants to the body, and many more. Although great progress has been made in understanding their structure-properties relationships, the large number of different processing techniques, each producing material with different characteristics and structure, means that understanding of the individual effects of all aspects of structure is not complete. The replication process, where molten metal is infiltrated between grains of a removable preform material, allows a markedly high degree of control and has been used to good effect to elucidate some of these relationships. Nevertheless, the process has many steps that are dependent on individual &#8220;know-how&#8221;, and this paper aims to provide a detailed description of all stages of one embodiment of this processing method, using materials and equipment that would be relatively easy to set up in a research environment. The goal of this protocol and its variants is to produce metal foams in an effective and simple way, giving the possibility to tailor the outcome of the samples by modifying certain steps within the process. By following this, open cell aluminum foams with pore sizes of 1&#8211;2.36 mm diameter and 61% to 77% porosity can be obtained.

Replication is one of the processing techniques used for the production of porous metal sponges. In this paper one implementation of the method for the production of open celled porous aluminum is shown in detail.

Metal foams are interesting materials from both a fundamental understanding and practical applications point of view. Uses have been proposed, and in many ...

Patterning via Optical Saturable Transitions - Fabrication and Characterization

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical Saturable Transitions (POST). In this technique the chemical properties of organic photochromic molecules that undergo single-photon reactions are exploited, enabling rapid top-down nanopatterning over large areas at low light intensities, thereby, allowing for the circumvention of the far-field diffraction barrier.4 Simple, cost-effective, high throughput and resolution alternatives to nanopatterning are being explored, such as, two-photon polymerization5,6, beam pen lithography (BPL)7, scanning electron beam lithography (SEBL), and focused ion beam (FIB) patterning. However, multi-photon approaches require high light intensities, which limit their potential for high throughput and offer low image contrast. Although, electron and ion beam lithographic processes offer increased resolution, the serial nature of the process is limited to slow writing speeds, which also prevents patterning of features over large areas. Beam-pen lithography is an approach towards parallel near-field optical lithography. However, the gap between the source of the beam and the surface of the photoresist needs to be controlled extremely precisely for good pattern uniformity and this is very challenging to accomplish for large arrays of beams. Patterning via Optical Saturable Transitions (POST) is an alternative optical nanopatterning technique for patterning sub-wavelength features1-3. Since this technique uses single photons instead of electrons, it is extremely fast and does not require high light intensities1-3, opening the door to massive parallelization.

We report that the diffraction limit of conventional optical lithography can be overcome by exploiting the transitions of organic photochromic derivatives induced by their photoisomerization at low light intensities.1-3 This paper outlines our fabrication technique and two locking mechanisms, namely: dissolution of one photoisomer and electrochemical oxidation.

This protocol describes the fabrication and characterization of nanostructures using a novel nanolithographic technique called Patterning via Optical ...

Quantification of Hydrogen Concentrations in Surface and Interface Layers and Bulk Materials through Depth Profiling with Nuclear Reaction Analysis

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and non-destructively reveals the hydrogen density distribution at surfaces, at interfaces, and in the volume of solid materials with high depth resolution. The technique applies a 15N ion beam of 6.385 MeV provided by an electrostatic accelerator and specifically detects the 1H isotope in depths up to about 2 &#956;m from the target surface. Surface H coverages are measured with a sensitivity in the order of ~1013 cm-2 (~1% of a typical atomic monolayer density) and H volume concentrations with a detection limit of ~1018 cm-3 (~100 at. ppm). The near-surface depth resolution is 2-5 nm for surface-normal 15N ion incidence onto the target and can be enhanced to values below 1 nm for very flat targets by adopting a surface-grazing incidence geometry. The method is versatile and readily applied to any high vacuum compatible homogeneous material with a smooth surface (no pores). Electrically conductive targets usually tolerate the ion beam irradiation with negligible degradation. Hydrogen quantitation and correct depth analysis require knowledge of the elementary composition (besides hydrogen) and mass density of the target material. Especially in combination with ultra-high vacuum methods for in-situ target preparation and characterization, 1H(15N,&#945;&#947;)12C NRA is ideally suited for hydrogen analysis at atomically controlled surfaces and nanostructured interfaces. We exemplarily demonstrate here the application of 15N NRA at the MALT Tandem accelerator facility of the University of Tokyo to (1) quantitatively measure the surface coverage and the bulk concentration of hydrogen in the near-surface region of a H2 exposed Pd(110) single crystal, and (2) to determine the depth location and layer density of hydrogen near the interfaces of thin SiO2 films on Si(100).

We illustrate the application of 1H(15N,&#945;&#947;)12C resonant nuclear reaction analysis (NRA) to quantitatively evaluate the density of hydrogen atoms on the surface, in the volume, and at an interfacial layer of solid materials. The near-surface hydrogen depth profiling of a Pd(110) single crystal and of SiO2/Si(100) stacks is described.

Nuclear reaction analysis (NRA) via the resonant 1H(15N,&#945;&#947;)12C reaction is a highly effective method of depth profiling that quantitatively and ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Use of Sacrificial Nanoparticles to Remove the Effects of Shot-noise in Contact Holes Fabricated by E-beam Lithography

Nano-patterns fabricated with extreme ultraviolet (EUV) or electron-beam (E-beam) lithography exhibit unexpected variations in size. This variation has been attributed to statistical fluctuations in the number of photons/electrons arriving at a given nano-region arising from shot-noise (SN). The SN varies inversely to the square root of a number of photons/electrons. For a fixed dosage, the SN is larger in EUV and E-beam lithographies than for traditional (193 nm) optical lithography. Bottom-up and top-down patterning approaches are combined to minimize the effects of shot noise in nano-hole patterning. Specifically, an amino-silane surfactant self-assembles on a silicon wafer that is subsequently spin-coated with a 100 nm film of a PMMA-based E-beam photoresist. Exposure to the E-beam and the subsequent development uncover the underlying surfactant film at the bottoms of the holes. Dipping the wafer in a suspension of negatively charged, citrate-capped, 20 nm gold nanoparticles (GNP) deposits one particle per hole. The exposed positively charged surfactant film in the hole electrostatically funnels the negatively charged nanoparticle to the center of an exposed hole, which permanently fixes the positional registry. Next, by heating near the glass transition temperature of the photoresist polymer, the photoresist film reflows and engulfs the nanoparticles. This process erases the holes affected by SN but leaves the deposited GNPs locked in place by strong electrostatic binding. Treatment with oxygen plasma exposes the GNPs by etching a thin layer of the photoresist. Wet-etching the exposed GNPs with a solution of I2/KI yields uniform holes located at the center of indentations patterned by E-beam lithography. The experiments presented show that the approach reduces the variation in the size of the holes caused by SN from 35% to below 10%. The method extends the patterning limits of transistor contact holes to below 20 nm.

Uniformly sized nanoparticles can remove fluctuations in contact hole dimensions patterned in poly(methyl methacrylate) (PMMA) photoresist films by electron beam (E-beam) lithography. The process involves electrostatic funneling to center and deposit nanoparticles in contact holes, followed by photoresist reflow and plasma- and wet-etching steps.

Nano-patterns fabricated with extreme ultraviolet (EUV) or electron-beam (E-beam) lithography exhibit unexpected variations in size. This variation has ...

Preparation of Liquid-exfoliated Transition Metal Dichalcogenide Nanosheets with Controlled Size and Thickness: A State of the Art Protocol

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. Layered crystals of molybdenum disulphide (MoS2) and tungsten disulphide (WS2) are exfoliated in aqueous surfactant solution by sonication. This yields highly polydisperse mixtures containing nanosheets with broad size and thickness distributions. However, for most purposes, specific sizes (in terms of both lateral dimension and thickness) are required. For example, large and thin nanosheets are desired for (opto) electronic applications, while laterally small nanosheets are interesting for catalytic applications. Therefore, post-exfoliation size selection is an important step that we address here. We provide a detailed protocol on the efficient size selection in large quantities by liquid cascade centrifugation and the size and thickness quantification by statistical microscopic analysis (atomic force microscopy and transmission electron microscopy). The comparison of MoS2 and WS2 shows that both materials are size-selected in a similar way by the same procedure. Importantly, the dispersions of size-selected nanosheets show systematic changes in their optical extinction spectra with size due to edge and confinement effects. We show how these optical changes are related quantitatively to the nanosheets dimensions and describe how mean nanosheets length and layer number can be extracted reliably from the extinction spectra. The exfoliation and size selection protocol can be applied to a broad range of layered crystals as we have previously demonstrated for graphene, gallium sulphide (GaS) and black phosphorus.

A protocol for the liquid exfoliation of layered materials to nanosheets, their size selection and size measurement by microscopic and spectroscopic techniques is presented.

We summarize recent advances in the production of liquid-exfoliated transition metal dichalcogenide (TMD) nanosheets with controlled size and thickness. ...

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of lithium-ion (Li-ion) batteries. However, a number of setbacks prevent their integration into commercial devices. The main limiting factor is due to nanoscale phenomena occurring at the electrode/electrolyte interfaces, ultimately leading to degradation of battery operation. These key problems are highly challenging to observe and characterize as these batteries contain multiple buried interfaces. One approach for direct observation of interfacial phenomena in thin film batteries is through the fabrication of electrochemically active nanobatteries by a focused ion beam (FIB). As such, a reliable technique to fabricate nanobatteries was developed and demonstrated in recent work. Herein, a detailed protocol with a step-by-step process is presented to enable the reproduction of this nanobattery fabrication process. In particular, this technique was applied to a thin film battery consisting of LiCoO2/LiPON/a-Si, and has further been previously demonstrated by in situ cycling within a transmission electron microscope.

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

A protocol for the fabrication of electrochemically active LiPON-based solid-state lithium-ion nanobatteries using a focused ion beam is presented.

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of ...