The authors thank Izon Science Ltd for their support. The work was supported by the European Commission for Research (PCIG11-GA-2012-321836 Nano4Bio).&#160;

1. Making the Phosphate Buffered Saline with Tween-20 (PBST) Buffer
<ol>
	<li>Dissolve one PBS tablet (0.01 M phosphate buffer, 0.0027 M Potassium Chloride, 0.137 M Sodium Chloride, pH 7.4) in 200 ml deionized water (18.2 M&#8486; cm).</li>
	<li>Add 100 &#181;l (0.05 (v/v)%) Tween-20 to the 200 ml buffer solution as a surfactant.</li>
</ol>
2. Preparing the Carboxyl Polystyrene Particle Standards
<ol>
	<li>Vortex the calibration particles for 30 sec before sonication for 2 min at 80 watts to create monodispersity of the particles.</li>
	<li>Dilute the calibration particles 1 in 100 to a concentration of 1x1010 particles/ml in PBST buffer and vortex for 30 sec.</li>
</ol>
3. Preparing Streptavidin Coated Particles
<ol>
	<li>Vortex the particles for 30 sec before sonication for 2 min at 80 watts to ensure monodispersity.</li>
	<li>Dilute the streptavidin coated particles 1 in 100 in PBST buffer to achieve a resulting concentration of 1x109 particles/ml and vortex for 30 sec. 
	Note: A typical sample volume is 200 &#181;l. For example, if investigating five DNA concentrations prepare 1 ml of diluted streptavidin coated particles.</li>
</ol>
4. Preparation of Oligonucleotides
<ol>
	<li>Reconstitute oligonucleotides with deionized water to a resulting concentration of 100 &#181;M.</li>
</ol>
5. Addition of Capture Probe (CP) DNA to the Streptavidin Coated Particles
<ol>
	<li>Prior to DNA binding, vortex the streptavidin coated particles (200 &#181;l sample volume) for 30 sec followed by a 2 min sonication at 80 watts.</li>
	<li>Based on the binding capacity provided by the supplier (4,352 pmol/mg), add the appropriate concentration of DNA to the particles for resulting concentrations of 10, 20, 30, 40, 47, 95, 140, and 210 nM DNA.</li>
	<li>Vortex the samples for 10 sec and place on a rotary wheel at room temperature for 30 min to allow for the DNA to bind to the particle surfaces via a streptavidin-biotin interaction.</li>
	<li>Once the capture DNA has been added and incubated with the streptavidin coated particles, remove the excess DNA in solution via magnetic separation by placing the samples onto a magnetic rack for 30 min.</li>
	<li>Remove the supernatant, taking care not to disturb the newly formed cluster of particles closest to the magnet, and replace with the same volume of new PBST buffer.</li>
</ol>
6. Hybridizing Complementary DNA to the CP-particles
<ol>
	<li>Add the required amount of target DNA (in excess at 500 nM) to ensure the maximum possible target binding was reached.</li>
	<li>Vortex the samples for 10 sec and place on a rotary wheel at room temperature for 30 min.</li>
	<li>Once the hybridization is complete, remove the excess target DNA via magnetic separation by placing the samples onto a magnetic rack for 30 min.</li>
	<li>Remove the supernatant, taking care not to disturb the newly formed cluster of particles closest to the magnet, and replace with the same volume of new PBST buffer.</li>
	<li>Repeat steps 6.1 to 6.4 for duplicate samples and place these samples on a rotary wheel at room temperature for 16 hours to investigate DNA hybridization times.</li>
</ol>
7. TRPS Setup
<ol>
	<li>Plug in the instrument into a computer system with software in place.</li>
	<li>Calibrate the initial stretch using a caliper.
	<ol>
		<li>Measure the distance between the outside of two parallel jaws.</li>
		<li>Input into the software by typing the stretch in the &#39;stretch&#39; field in the &#39;Instrument Settings&#39; tab and clicking &#39;Calibrate stretch&#39; underneath the tab.</li>
	</ol>
	</li>
	<li>Laterally fit a polyurethane nanopore membrane of appropriate sizing for analysis onto the jaws with the nanopore ID number facing upward. Then, stretch the jaws to the stretch required for analysis using the stretch adjustment handle on the side of the instrument. Stretch the jaws between 43 and 48 mm. 
	Note: The exact value of the stretch is determined alongside applied voltage so that calibration particle blockades are at least 0.3 nA in size. The stretch is already inputted into the software in step 7.2 and will automatically adjust as the jaws are stretched.</li>
	<li>Place 80 &#181;l of PBST buffer in the lower fluid cell, beneath the nanopore, ensuring there are no bubbles present that may affect the measurement. If there are bubbles seen, remove and replace the buffer.</li>
	<li>Click the upper fluid cell into place and place 40 &#181;l of buffer into it, again ensuring there are no bubbles present. If bubbles are present in the upper fluid cell, remove them by replacing the liquid.</li>
	<li>When a reproducible baseline current has been reached from replacing the upper fluid cell with buffer, add 40 &#181;l of the sample to the upper fluid cell and measure by clicking &#39;start&#39; in the &#39;Data Acquisition&#39; tab on the software screen. 
	Note: The data acquisition is completed at a frequency of 50 kHz with a blockade magnitude lower limit of 0.05 nA, although this can be altered using the software via the &#39;Analyse Data&#39; tab (under &#39;Analysis Settings&#39; and &#39;Resistive Blockades&#39;).</li>
	<li>Place a Faraday cage over the top of the fluid cell system to reduce electrical background noise on the measurements.</li>
	<li>Use a variable pressure module (VPM) to apply a pressure or vacuum to the samples.
	<ol>
		<li>To apply an external pressure connect the nozzle to the upper fluid cell, then rotate the pressure arm and click into place (depending on whether a positive pressure (PRE) or a vacuum (VAC) will be applied).</li>
		<li>Apply pressure in a &#39;cm&#39; or &#39;mm&#39; scale using the pressure stage knob situated on the top of the VPM. Press the knob down to apply pressure on the &#39;cm&#39; scale and pull it upward to apply pressure on the &#39;mm&#39; scale.</li>
	</ol>
	</li>
</ol>
8. Preparing Samples for TRPS Analysis
<ol>
	<li>Vortex samples for 30 sec and sonicate for 2 min at 80 watts prior to TRPS analysis.</li>
</ol>
9. Calibrating the Nanopore for Zeta Analysis
<ol>
	<li>After placing 40 &#181;l calibration particles (1x1010 particles/ml) into the upper fluid cell, complete a TRPS measurement (setup as in section 7) at 3 applied voltages. Alter the voltage by clicking on the &#39;+&#39; and &#39;-&#39; buttons on the voltage scale in the &#39;Instrument Settings&#39; tab on the software.</li>
	<li>Check that the 3 voltages return background currents of approximately 140, 110, and 80 nA. Ensure that at the medium voltage the calibration particles produce an average blockade magnitude of at least 0.3 nA.</li>
	<li>Apply a pressure so the average full width half maximum (FWHM) durations of the calibration particles are at least 0.15 msec. Do this manually using the pressure arm attached to the variable pressure module. Select pressure (PRE) or vacuum (VAC) by rotating the arm until it clicks in the desired position and apply accordingly following set up instructions in step 7.8.2. Once these conditions have been achieved, start the run by clicking &#39;start&#39; on the software in the &#39;Data Acquisition&#39; tab.</li>
	<li>Complete the run by pressing &#39;stop&#39; in the &#39;Data Acquisition&#39; tab when at least 500 particles have been measured (see &#39;Particle Count&#39; at the bottom of the software screen during the measurement) and the run has exceeded 30 sec (see &#39;Run Time&#39; also toward the bottom of the screen).</li>
	<li>Calibrate the system by completing a calibration run as described every time a new nanopore is introduced or for each new day of analysis by completing step 9.1-9.4.</li>
</ol>
10. Running a Sample
<ol>
	<li>Run the samples at the highest or second highest voltage as the calibration samples at ensuring a similar (&#177;10 nA), if not the same, baseline current.
	<ol>
		<li>Once the appropriate baseline current is achieved, replace the electrolyte in the upper fluid cell with 40 &#181;l of sample. When a sample is introduced, blockades will be seen on the signal trace. Start the sample run by clicking &#39;start&#39; in the &#39;Data Acquisition&#39; tab and record a minimum of 500 particles (check &#39;Particle Count&#39; situated under the signal trace) and ensure the run time is a minimum of 30 sec (see &#39;Run Time&#39; also situated below the signal trace).</li>
		<li>To complete the measurement, click &#39;stop&#39; in the &#39;Data Acquisition tab&#39; and save the data file.</li>
	</ol>
	</li>
	<li>To save the file, input the file information in the following format; &#39;Investigation&#39; is the folder the file will be saved in, &#39;Nanopore ID&#39; is the serial number of the pore being used, &#39;Part #&#39; is the type of pore (i.e., NP150/NP200), &#39;Sample ID&#39; is the name of the sample, &#39;Calibration or sample&#39; details whether it is a calibration or sample measurement, &#39;Dilution&#39; is used if the sample was diluted (type 100 if the sample was diluted 100-fold), &#39;Pressure&#39; is the applied pressure to the sample (in cm - see section 7.8), &#39;Electrolyte ID&#39; is the name of the buffer the sample is made up in, and &#39;Notes&#39; are any personal notes about the sample or run.</li>
	<li>Between each sample run, wash the system by placing 40 &#181;l of PBST buffer into the upper fluid cell several times and applying various pressures (usually at -10, -5 cm (vacuum), and 5 and 10 cm (positive pressure)) until no more blockade events are present, ensuring there are no residual particles remaining in the system and therefore no cross contamination between samples. Run samples in triplicate with this wash step completed between each repeat sample run as well as between different samples.</li>
</ol>

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E.L.C.J.B. is supported by Izon Science Ltd.

The calculation for the zeta potential used a calibration based method related to work by Arjmandi et al.21. The duration of the translocation of particles as they traverse a nanopore is measured as a function of applied voltage, using an average electric field and particle velocities over the entirety of a regular conical pore. &#160;The electrophoretic mobility is the derivative of 1/T (where T is the blockade duration) with respect to voltage, multiplied&#160;by the square of the sensing zone lengt...

Functionalized nanoparticles are becoming increasingly popular as biosensors in both medical and environmental fields. The ability to alter a nanoparticle&#39;s surface chemistry, with DNA, for example, is proving useful for targeted drug delivery systems1 and monitoring DNA-protein interactions2-4. An increasingly common nanoparticle property being utilized in bioassays and in the delivery of therapeutics is superparamagnetism5. Superparamagnetic particles (SPPs) are extremely useful in identifying and removing specific analytes from complex mixtures and can do so with the simple use of a single magnet. Once removed, the analyte-bound particles can be characterized and analyzed fit for purpose.
Previous methods used for the detection and characterization of nanoparticles include optical techniques such as dynamic light scattering (DLS), otherwise known as photon correlation spectroscopy. Although a high throughput technique, DLS is limited to being an averaging based technique and when analyzing multimodal samples without the addition of specialist software, the larger particles will produce a much more dominant signal, leaving some of the smaller particles completely unnoticed6,7. Particle-by-particle characterization techniques are therefore much more favorable to analyze nanoparticle and functionalized nanoparticle systems.
RPS based technologies are based around applying an electric field to a sample and monitoring the transportation mechanism of the particles through a synthetic or biological nanopore. A relatively recent nanoparticle detection and characterization technique based on RPS is tunable resistive pulse sensing (TRPS)8-16. TRPS is a two-electrode system separated by an elastomeric, tunable pore membrane. A tunable pore method allows for analytes of a range of shape17 and size to be measured via their transport mechanisms through the pore. Tunable pores have previously been used for the detection of small particles (70-95 nm diameter) producing comparable results to other techniques such as transmission electron spectroscopy (TEM)10. When an electric field is applied, an ionic current is observed and as particles/molecules pass through the pore, they temporarily block the pore, causing a reduction in the current that can be defined as a &#39;blockade event&#39;. Each blockade event is representative of a single particle so that each particle within a sample can be characterized individually based on the blockade magnitude, &#916;<img alt="Equation 1" src="/files/ftp_upload/54577/54577eq1.jpg" />, and full width half-maximum, FWHM, as well as other blockade properties. Analyzing individual particles as they pass through a nanopore is advantageous for multimodal samples as TRPS can successfully and effectively distinguish a range of particle sizes amongst a single sample. Tunable resistive pulse sensing completes size10, zeta potential12,18 and concentration15 measurements simultaneously in a single run and can therefore still differentiate samples of similar, if not the same&#160;size by their surface charge19; an advantage over alternative sizing techniques.
Zeta potential is defined as the electrostatic potential at the plane of shear20, and is calculated from particle velocities as they traverse a pore19. Zeta potential measurements of individual particles thus gives insight into the translocation mechanisms and behavior of nanoparticle systems in solution, valuable information for the future of nanoparticle assay designs for a range of applications. Particle-by-particle analysis of such nature also allows for the spread and distribution of zeta potential values amongst a sample population to be explored, allowing for more information on reaction kinetics (single-stranded to double-stranded DNA, for example) and particle stabilities in solution to be attained.
Here, we describe a technique that detects and characterizes both unmodified and DNA-modified SPP surfaces. The protocol described herein is applicable to a range of inorganic and biological nanoparticles, but we demonstrate the procedure using DNA-modified surfaces due to their wide range of applications. The technique allows the user to distinguish between single-stranded and double-stranded DNA targets on a nanoparticle surface, based on particle translocation velocities through a pore system and thus their zeta potentials.

<table><tbody><tr><td>Phosphate buffered Saline (PBS)</td><td>Sigma Aldrich, UK</td><td>P4417</td><td>1 tablet dissolved in 200&amp;nbsp;ml deionized water to make buffer solution.</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich, UK</td><td>P1379</td><td>0.05% (v/v) in PBS buffer as a surfactant</td></tr><tr><td>Carboxyl polystyrene nanoparticles</td><td>Bangs Laboratories, US</td><td>CPC200</td><td>Nominal diamter of 220&amp;nbsp;nm, raw concentration of 1 x 10&lt;sup&gt;12&lt;/sup&gt; particles/ml, specific surface charge of 86 &amp;micro;eq/g (equivalent to a surface charge density of 3.2 x 10&lt;sup&gt;19&lt;/sup&gt; C/nm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>Streptavidin coated nanoparticles</td><td>Ademtech, France</td><td>3121</td><td>Batch had binding capacity of 4,352 pmol/mg (188 nM theoretical DNA binding capacity) at a raw concentration of 1.1 x 10&lt;sup&gt;11&lt;/sup&gt; particles/ml.</td></tr><tr><td>Biotinylated oligonucleotides</td><td>Sigma Aldrich, UK</td><td>VC00001</td><td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Biotin modification at 3&#039; end; Lyophilized powders reconstituted to 100&amp;nbsp;&amp;micro;M using deionized water, and diluted as required. Sequences: CP 5&#039;ATGGTTAAACCTCACTAC
GCGTGGC[Btn]3&#039;</td></tr><tr><td>Standard olignonucleotides</td><td>Sigma Aldrich, UK</td><td>VC00001</td><td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Lyophilized powders reconstituted to 100 &amp;micro;M using deionized water, and diluted as required. Sequences of DNA targets: Fully complementary - 5&#039;GCCACGCGTAGTGA
GGTTTAACCAT3&#039;, Middle binding - 5&#039;GTAGTGAGGT3&#039;, End binding - 5&#039;GTTTAACCAT3&#039;, Partially complementary overhanging - 5&#039;GTGAGGTTTAACCAT
TTTTTTTTTTTTTTT3&#039;.</td></tr><tr><td>Izon qNano</td><td>Izon Science, NZ</td><td></td><td>Inherent pressure on system of 47&amp;nbsp;Pa</td></tr><tr><td>Izon Variable Pressure Module (VPM)</td><td>Izon Science, NZ</td><td></td><td>Each &#039;cm&#039; of pressure is equivalent to approximately 100 Pa.</td></tr><tr><td>Polyurethane nanopore membranes</td><td>Izon Science, NZ</td><td>NP150</td><td>Analyte size range 60-480 nm, pore diameter of calculated to be 799 nm at a 45 mm stretch.&amp;nbsp;</td></tr><tr><td>Magrack 6</td><td>GE Healthcare, UK</td><td>28-9489-64</td><td></td></tr><tr><td>Sonic Bath</td><td>Fisher Scientific, UK</td><td>10692353</td><td>80 Watts</td></tr><tr><td>Vortexer</td><td>IKA, Germany</td><td>0003365000</td><td></td></tr><tr><td>Rotary Wheel&amp;nbsp;</td><td>Labnet International, US</td><td>H5500-230 V</td><td></td></tr></tbody></table>

determination of zeta potential via nanoparticle translocation velocities through a tunable nanopore: using dna-modified particles as an example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

<img alt="Figure 1" src="/files/ftp_upload/54577/54577fig1.jpg" /> 
Figure 1. Schematic representation of the processes of magnetic purification and a TRPS measurement. A) Example of magnetic purification of sample starting with a sample containing excess, unbound capture probe DNA. B) TRPS measurement example i) Particle passing through the nanopore and ii) Blockade event produced from particle temporarily occluding ions in the pore...

Watch this Scientific Journal Video about Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example at JoVE.com

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

determination-zeta-potential-via-nanoparticle-translocation

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.1K Views.  Loughborough University. Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Video: Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

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.

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 ...

Engineering

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Chemistry

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

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.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

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.

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 ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Biochemistry

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...