Name: nanoparticle tracking analysis of gold nanoparticles in aqueous media through an inter-laboratory comparison
Uploaded: 2020-10-20T13:30:00.000+00:00
Duration: 7 min 8 s
Description: Watch this Scientific Journal Video about Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison at JoVE.com

The authors acknowledge financial support from H2020 funded project: ACEnano (Grant Agreement no 720952). This work was also partially supported by the International Cooperative R&#38;D Program funded by the Ministry of Trade, Industry, and Energy of Korea (grant number N053100009, &#8220;Horizon2020 Kor-EU collaborative R&#38;BD on ACEnano Toolbox&#8221;) which enabled participation of the Korean partners in the consortium of Horizon 2020 ACEnano Project.

The methodology described here was used for the third round of the inter-laboratory comparisons.
1. Sample Preparation
<ol>
	<li>Filter water through a 0.02 &#181;m syringe filter. Water filtration is necessary to remove any contamination particles before using it for sample dilution.</li>
	<li>To analyze a freshly prepared sample, dilute a sample of 60 nm gold colloid dispersion volumetrically by a factor of 50 in filtered ultrapure water. The suggested concentration for NTA analysis is 1 x 107 &#8211; 1 x 109 particles per mL.</li>
</ol>
2. Performing the measurement
<ol>
	<li>Switching on the system
	<ol>
		<li>Connect the NTA instrument, syringe pump and the computer. Switch on the hardware and software. The associated software (see Table of Materials) ensures all hardware communications are running and that a live temperature readout is displayed.</li>
		<li>Remove the laser module from the NTA and using a tissue and compressed air completely dry the glass surfaces and the low volume flow cell (LVFC) internal channels, tubing, and fluidic ports.</li>
	</ol>
	</li>
	<li>Priming the tubing
	<ol>
		<li>Rinse the inlet fluidic tubing with ultrapure water to remove any particles and reduce the likelihood of air bubbles that would interfere with measurements. For rinsing, the end of the inlet tubing inside the instrument casing is placed in a waste container.</li>
		<li>Insert a 1 mL syringe (without needle) of filtered water into the Luer port and push ~900 &#956;L of liquid through the inlet tubing as fast as the back pressure allows. Leave the syringe containing the remaining liquid attached to prevent any syphoning.</li>
	</ol>
	</li>
	<li>Syringe pump tubing connection
	<ol>
		<li>Assemble the LVFC onto the laser module to create the sample chamber as seen in Figure 1. Attach the outlet tubing to the right-hand side port of the LVFC. 
		NOTE: The inlet and outlet tubing are different in diameters, with the inlet being smaller in diameter than the outlet. Swapping the inlet-outlet tubing connection may cause over pressuring the flow cell and leaking.</li>
		<li>Disconnect the syringe from the inlet tubing and exchange for a new syringe containing 1 mL of filtered water, ensuring liquid-to-liquid contact. Connect the inlet tubing to the left port of the LVFC. Slowly introduce ~500 &#181;L of fluid into the sample chamber. Take care to ensure no air bubbles are introduced during loading. The final tubing configuration is shown in Figure 2.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61741/61741fig1v2.jpg" /> 
Figure 1: Low Volume Flow Cell assembly mounted on laser module.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61741/61741fig02.jpg" /> 
Figure 2: Low Volume Flow Cell tubing configuration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61741/61741fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>Laser module loading and system check
	<ol>
		<li>Insert the laser module with the water filled LVFC into the instrument and lock into place.</li>
		<li>Place the syringe into the syringe pump cradle and secure. Initialize the camera by clicking on Start Camera in the software interface. In the Hardware tab of the interface, click on Scatter to move the reference position.</li>
		<li>Set the camera level to 16 and adjust the focus manually to check the diluent for any particles. Adjust the field of view position by left clicking on the main viewing window and using the mouse to drag up and down to check for any particles. If there are more than three particles in the field of view, this implies a problem with the water purity or the cleaning process and, therefore, the cleaning process needs to be repeated or the water needs to be replaced or filtered.</li>
		<li>Remove the laser module from the instrument.</li>
		<li>Disconnect the syringe from the inlet tubing and replace it with a syringe full of air only. Slowly introduce the air into the sample chamber to remove the liquid inside. Remove the LVFC from the laser module and disconnect the tubings. Clean the glass surfaces of the LVFC and optical glass of the laser module with water and dry with a tissue and compressed air. Dry the tubing with compressed air. Reassemble the LVFC onto the laser module and connect the tubing, ready for sample loading. 
		NOTE: This step is not always required, however, in this case it was added as an extra precaution to further reduce any possible variation.</li>
	</ol>
	</li>
	<li>Loading sample
	<ol>
		<li>Repeat step 2.2.2. Connect a syringe containing 1 mL of the 60 nm gold nanoparticles dispersion made in step 1.1 to the Luer port. Slowly inject 750 &#181;L of the sample into the LVFC via the inlet tubing with the laser module viewed outside the instrument to ensure no bubbles are introduced.</li>
		<li>Load the laser module back to the NTA instrument and initialize the camera by clicking on Start Camera in the software interface. In the Hardware tab of the interface, click Scatter to move to the reference focus position, check that this is set correctly to give a clear image of the particles.</li>
		<li>Check that the field of view is set centrally with respect to the laser beam position. Adjust accordingly by left clicking on the main viewing window in the software and mouse dragging up and down.</li>
		<li>Run the AutoSetup function to automatically optimize the focus and camera level ensuring that the optimal image quality is achieved. 
		NOTE: The automatic camera and focus parameters allow for more consistency amongst the different labs since this is user independent.</li>
	</ol>
	</li>
	<li>Sample analysis
	<ol>
		<li>Create a measurement script in Standard measurement, SOP tab, to obtain 5 repeat videos of 60 s under slow (particles should be passing across from one side of the screen to the other in approximately 10 s) and constant flow (Supplementary File 1). 
		NOTE: Flow is recommended to ensure a better representation of the overall sample is presented for measurement. Precision and repeatability of concentration measurements are significantly improved when a slow flow is imparted on the sample to ensure that a greater number of new particles flow through the measurement zone and are analyzed during an experiment. The video length depends on the profile distribution and how variable it is over the analysis time. 5 videos of 60 s are considered as a typical measurement duration.</li>
		<li>Set the experiment file name and location for the data and start the run. The analysis following the outlined procedure was carried out by the seven laboratories of the Horizon 2020 ACEnano project17.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
NOTE: All data analysis was done within the v 3.4 software (see Table of Materials), no additional manual conversions or calculations are used. The particle sizing data is presented in raw form as a histogram distribution and is calculated from the measured change in position of the particle using the Stokes-Einstein equation. The software determines the average distance moved by each particle in the x and y planes. This value allows the particle diffusion coefficient (D) to be determined from which, if the sample temperature T and solvent viscosity &#951; are known, the equivalent spherical hydrodynamic radius, RH, of the particles can be calculated. The temperature of the sample is automatically recorded by the NTA. The default sample viscosity used by the software is for water and is included in the measurement script shown above, though viscosity can be amended by the user when different sample diluents are used, either before or after the measurement is taken.
<ol>
	<li>Set the detection threshold (DT) by dragging the slider bar or clicking the + and - buttons in the software under Detection Threshold, which is the analysis parameter for optimal tracking of the visualized particles, between 2 and 20. Ensure that the DT value chosen identify and track as many visible particles as possible (marked automatically as red crosses on the software image screen).
	<ol>
		<li>As guidance for setting the detection threshold, the number of identified particles in an image should be in the range of approximately 30-80 where no more than 10 red crosses should correspond to sites not considered to be particles by the observer. There should be no more than 5 blue crosses (indicative of noise) observed.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61741/61741fig03.jpg" /> 
Figure 3: Threshold setting observations.&#160;A bad (left) and good (right) detection threshold setting observation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61741/61741fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Automatically process the particle tracking analysis videos by pressing the Process button in the software. Leave all the processing parameters set to automatic and export the data as a .csv format results file with the full particle size distribution and additional metadata describing the measurement setup. To verify the measurement quality, look at the Analyze tab in the software or check the .csv output file for any warnings message or alerts. An example of the PDF results report is shown in Supplementary File 2.</li>
	<li>Read the mode size results and the associated standard deviation from the PDF report. 
	NOTE: The mode size results were used to compare the sizes obtained amongst the seven laboratories and are shown and discussed in Section 5.</li>
</ol>
4. Cleaning and drying
<ol>
	<li>After use, flush the system thoroughly with clean water to remove all traces of sample from the tubing and optical surfaces. Cleaning effectiveness can be monitored by observation of the amount of particles in the field of view.&#160;</li>
	<li>Remove the laser module from the NTA instrument.&#160;</li>
	<li>Load a syringe of air through the system to empty the tubing and the LVFC.</li>
</ol>

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The author Jo Sullivan, Agnieszka Siupa, Pauline Carnell-Morris and Michele Carboni are employees at Malvern Panalytical Ltd. that manufactures instruments used in this article.

The inconsistent results obtained from the Round 1 ILC highlighted the need for instrument health checks for older systems as well as the development of a more detailed SOP, the need for hands-on training and a better understanding of measurement and analysis settings so as to ensure more consistent results across the different labs. In fact, Hole et al. found that the absence of shared guidelines on how to use an NTA system and prepare samples resulted in variability across laboratories even for relatively monodispersed samples16. Therefore, all ILC participants attended a training workshop covering the best practices for the system operation and measurement conditions, as well as cleaning and maintenance guidance for the specific NTA instrument. All participants also performed measurements on the same instrument in their own labs for the subsequent ILC rounds. The procedure first involved a round that tested the system locally in each laboratory by running an ILC on latex standard samples (ILC Round 2), before being used by the partners to repeat the gold measurements (ILC Round 3). The aim of measuring these gold samples by means of NTA was to introduce confidence and clarity into nanomaterial risk assessment methods and practices needed to impact nanosafety guidance protocols.
NTA is a technique that can measure the hydrodynamic spherical equivalent diameter of particles and can be used for particle by particle, real-time visual analysis of polydispersed systems ranging from 10 nm &#8211; 50 nm, to approximately 1000 nm in size (depending on the sample properties and instrument configuration). Minimal sample preparation is required. Despite minimal sample preparation, this step is critical for the protocol and great care should be taken when diluting a sample and choosing a diluent. Shape can be a limiting factor with respect to NTA as spherical equivalent size measurements are obtained and non-spherical particles will have a less accurate size value.
For NTA technology, some result variation is always to be expected as only a representative sample is observed from the whole sample. Regardless, all results meet the ISO 19430 standard for particle sizing. The optimal concentration to provide is typically around 108 particles/ml within a 30-60 second analysis time. For samples with lower particle concentrations, longer analysis times will be required to ensure reproducible results. For samples containing a concentration of particles greater than 109 particles/mL, there is a greater likelihood of tracking problems and samples will need to be diluted down to a suitable range for NTA measurement.
Overall the results from the 3rd&#160;Round ILC show good reproducibility of gold nanoparticle measurements with NTA with increased accuracy and repeatability. All NTA measurements were carried out using the automatic camera level and focus settings to adjust the image, as selected by the Auto-Setup feature in the software. The camera level set by the software was very consistent, with a camera level of 10 or 11 being set in all cases showing that as expected, the more automation a process includes the more consistency is achieved. The sizing results were comparable to those obtained by the manufacturer by means of TEM indicating that the results were reproducible, however minimal differences are to be expected from different techniques since TEM does not determine the hydrodynamical diameter. The significant improvement in the consistency of results show the importance and benefits of instrument maintenance, detailed SOPs, best practice updates, user knowledge and applied measurement automation for NTA. In conclusion the ILC validated this specific NTA SOP and hence introduced confidence and clarity into this specific nanomaterial risk assessment method.

Nanomaterials (NMs) can vary in both physical and chemical characteristics that in turn influence their behavior, stability, and toxicity1,2,3,4,5. One of the major difficulties, when developing a thorough understanding of NM properties, hazards, and behaviors, is the ability to obtain reproducible information about physical and chemical nanomaterial characteristics. Examples of such physical properties include particle size and size distribution6,7,8. These are important parameters as they are a key aspect of the European Commission&#8217;s (EC) definition of the term &#8216;nano&#8217;9.
Achieving precise particle size measurements is also critical for many different industrial and research applications and processes in addition to understanding the fate and toxicity effects of NMs6,10. It is important to have well established methods capable of measuring accurately, reliably, and reproducibly the size of NMs. Furthermore, reported information should provide deep understanding of the technique used e.g., indicate the type of size parameter (e.g., actual size or hydrodynamic size) as well as the sample condition e.g., the specific medium in which the NM is present, and for the method to perform reliably in different media. In order to measure size, a number of techniques can be used, including electron microscopy (EM), dynamic light scattering (DLS), single particle inductively coupled plasma mass spectrometry (spICP-MS), differential centrifugal sedimentation (DCS), scanning probe microscopy (SPM), small-angle X-ray scattering (SAXS) and nanoparticle tracking analysis (NTA).
NTA is a relatively new technology which has been well advanced in recent years and has been shown to reliably measure the hydrodynamic diameter of spherical NMs in complex aqueous media such as those with environmental relevance, e.g., freshwater systems. The hydrodynamic diameter is &#8216;the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured&#8217;11; in practical terms and in aqueous media this describes a diameter larger than that of the particle itself, which also includes a layer of molecules (mostly water) held at the surface of the particle by weak electrostatic forces. The hydrodynamic diameter of a particle will vary in different media, getting smaller as the ionic strength of the media in which it is measured gets higher.
An additional important feature of the NTA technique is that it allows the analyst to achieve number-weighted size measurements, which are required in the context of the EC nanomaterial definition. High resolution, particle-by-particle analysis makes this technique less prone to interference caused by agglomerates or larger particles when present in a heterogeneous test sample with a high rate of particle throughput10,12.
The measurement procedure consists of preparing a suitable suspension of the sample, which often requires sample dilution, followed by video recording of the particles&#8217; Brownian motion behavior and video analysis. From the sample chamber, a laser beam is passed, and the suspension particles in the path of the laser beam scatter light leading to their visualization using an optical microscope with a mounted camera. The camera captures a video file of the scattered laser light from the particles moving under Brownian motion. Many particles can be tracked individually to determine their diffusion coefficients and their hydrodynamic diameters can be calculated using the Stokes-Einstein equation: d = kT/3&#960;&#951;D where d is the hydrodynamic diameter, k is the Boltzmann constant, T is the temperature, &#951; is the viscosity and D is the diffusion coefficient10. NTA can also be used to track the aggregation behavior of particles that are generally colloidally unstable (the particles must, however, be colloidally stable over the measurement time scale)13,14. NTA is an absolute method and no system calibration is required on the instrument used in this work. If users want to check the system performance this can be easily done by measuring size standard materials as frequently as wanted.
The NTA instrument is easy to operate with quick analysis time (under 10 min per sample). For high quality measurements with good data repeatability and reproducibility, a number of factors should be considered in both sample preparation as well as in instrument operation. If such factors are not carefully considered, measurements on the same material across different laboratories and operators can be subject to unknown or poorly quantified uncertainties. During NP characterization, using best practice in-house developed SOPs does not always guarantee consistency with other laboratories, as shown by Roebben et al. for the DLS technique15.
In fact, an early (first round) NTA ILC between different laboratories, users and instruments revealed inconsistent results. One of the main issues was with the use of various older legacy instruments which had not had regular services or calibration checks, as well as differences in method interpretation. An NTA ILC study by Hole et al. found that with the absence of shared guidelines on how to use a system and prepare samples, variability across laboratories can be large even for relatively monodispersed samples16. This along with the results from the first round of the ILC highlights the need for good instrument maintenance as well as method training and well-developed standard operating procedures (SOPs). The latter act as a powerful tool to describe and document compliance with good practice. If well detailed, standard operating procedures (SOPs) can offer clarity, explanation, understanding, standardization, and quality assurance.
The recommendation for adopting an ILC study is, therefore, ideal for both developing and testing protocols16. The ILC exercise acted to validate this specific NTA SOP and hence introduced confidence and clarity into this specific nanomaterial risk assessment method. It involved three rounds. Round 1 analyzed 60 nm gold nanoparticles on each participant&#8217;s own instruments before training. Round 2 involved analyzing 100 nm latex using a new instrument with common configuration as a simple test in order to determine that the instrument was set up correctly and the users had a good knowledge on how to use the instrument. Round 3 involved the analysis of 60 nm gold nanoparticles on the new instrument with common configuration, after training. Participants in the ILC came from seven different labs, all consortium members of the Horizon 2020 ACEnano project17.
The aim of this article is to discuss the method and results from a third round of benchmarking for the NTA technology where 60 nm gold NPs were re-analyzed by seven partners following detailed training and SOP development. Comparison and reference to the results obtained in the first round of the ILC will also be made. All analyses from round 3 of ILC were carried out using the same instrument (see Table of Materials) of identical configuration supplied with a 405 nm laser and a high sensitivity sCMOS camera. Benchmarking assesses the performance of the technology on samples and hence leads to the development of &#8216;best practice&#8217; protocols. Thus, this article also shares and makes the NTA method for the instrument used in this ILC available for the scientific community as it has been harmonized via conducting and evaluating the ILCs according to international standards.

<table><tbody><tr><td>60 nm gold colloid dispersion</td><td>BBI Solutions OEM Ltd.</td><td>Product EM. GC60, Batch number 024650</td><td></td></tr><tr><td>0.02 &amp;micro;m syringe filter - Whatman Anotop 25 Sterile Syringe Filters</td><td>Sigma Aldrich</td><td>WHA68092102</td><td></td></tr><tr><td>NanoSight</td><td>Malvern Panalytical Ltd.</td><td>NS300</td><td></td></tr><tr><td>NanoSight NTA Software v3.4</td><td>Malvern Panalytical Ltd.</td><td>v3.4</td><td></td></tr><tr><td>Syringe PP/PE without needle luer slip tip, centered, capacity 1&amp;nbsp;mL, graduated, 0.01&amp;nbsp;mL, sterile</td><td>Sigma Aldrich</td><td>Z230723</td><td></td></tr></tbody></table>

nanoparticle tracking analysis of gold nanoparticles in aqueous media through an inter-laboratory comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The Round 1 ILC results using various NTA instrument configurations are shown in Figure 4. With the exception of Lab 6, the repeatability between the 5 capture repeats was good but several labs recorded a mode size higher than expected. Lab 6 results showed poor repeatability and a much higher mode size measured. After the investigation, it was found that the systems reporting biggest size variations were either not maintained as recommended or the analysis was affected by inconsistency in sample preparation whereby the dilution step can create variation caused by different pipetting equipment, user operation and technique, and/or measurement set up including the flow cell not being clean, the wrong camera level being used, the image not being focused properly, and setting the analysis Detection Threshold incorrectly.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61741/61741fig4v2.jpg" /> 
Figure 4: ILC Round 1 Mode size results.&#160;Mode size results from all NTA benchmarking partners for Round 1 60 nm gold nanoparticle dispersion carried out on different NTA instruments (as abbreviated in the x axis).&#160;
The NTA result accuracy from Round 3 was improved by all the laboratories implementing the same SOP and instrument settings. The mode size results obtained for this ILC Round 3 can be seen in Figure 5. The average mode across all labs was 62.02 &#177; 1.97 nm. All measured results from Round 3 were more consistent than the first stage results with the results falling well within 10% of the 60.5 nm mean size for the batch as stated by the manufacturer. The coefficient of variation for the gold samples stated by the manufacturer was &#8804;8%.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61741/61741fig5v2.jpg" /> 
Figure 5: ILC Round 3 Mode size results.&#160;Mode size results from all NTA benchmarking partners for 60 nm gold ILC Round 3 analyzed on the same NTA instrument. The average mode across all labs was 62.02 &#177; 1.97 nm.&#160;
To verify the particle size, as provided by the manufacturer, a small number (N=82) of particles were analyzed by Transmission Electron Microscopy (TEM). Approximately 10 &#181;l of the undiluted dispersion was drop cast on a carbon coated Cu TEM grid and dried in air before imaging in an analytical TEM at 200 kV. Images like Supplementary Figure 1 were taken from areas with minimal particle overlap and analyzed using a semi-automatic image analyses process. An automatic watershed method was applied to separate particles and artefacts of this process were excluded as well as on edge particles18. The mean diameter was calculated either as average from the major and minor axis (61 &#177; 7 nm) or as a conversion from the measured area (62 &#177; 6 nm) assuming spherical particles. Particles appear to be mostly spherical with an average aspect ratio of 1.1. The TEM results show a slightly higher diameter than the manufacturer value (60.5 nm) but are within the tolerance level. Additionally, there is a very good agreement with the NTA derived value of hydrodynamic diameter.
Supplementary Figure 1: TEM image of 60 nm Gold Nanoparticles. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61741/61741supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary File 1: Measurement script. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61741/Measurement_script.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 2: Example of PDF results report. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61741/PDF_results_report.pdf" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison at JoVE.com

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

nanoparticle-tracking-analysis-gold-nanoparticles-aqueous-media

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.3K Views.  University of Birmingham. Het hier beschreven protocol heeft tot doel de hydrodynamische diameter van bolvormige nanodeeltjes, meer bepaald gouden nanodeeltjes, in waterige media te meten door middel van Nanoparticle Tracking Analysis (NTA). Dit laatste omvat het volgen van de beweging van deeltjes als gevolg van Browniaanse beweging en het implementeren van de Stokes-Einstein-vergelijking om de hydrodynamische diameter te verkrijgen.

Video: Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

Engineering

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 ...

Bioengineering

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Biochemistry

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Biology

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

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