Orthogonal sample measurement
How can I trust my data? This is a common concern for many researchers using particle measurement instrumentation. Is the data affected in any way by the measurement technology, or alternatively by the user during sample preparation or by the analysis parameters employed? To answer this question we need to consider the use of independent and orthogonal measurement techniques in order to provide confidence and measurement validation.
Two such complementary technologies are Nanoparticle Tracking Analysis and Dynamic Light Scattering. This technical note will describe both technologies and the complementary data they produce as well as explore how their unique capabilities, when used in conjunction, provide the most comprehensive suite of measurement parameters available to those interested in the characterization of nanoscale materials.
Nanoparticle Tracking Analysis (NTA)
Nanoparticle Tracking Analysis (NTA) is a high resolution, single particle technique which visualizes the light scattered from nanoparticles in liquid suspension. It is a 3-step process, which begins with capturing a video of the particles, followed by tracking their motion, and finally analyzing the data to generate a size distribution profile:
Figure 1: 3-step process involved in NTA measurements
Through tracking the Brownian motion of each individual particle and subsequent application of the Stokes-Einstein equation, particle size can be calculated on a particle-by-particle basis.
Key features of NTA technology are:
- High resolution size distributions: through single particle measurement
- Size range: from 10 nm – 2 µm
- Fluorescence mode: measuring fluorescently-labeled particles only (range of excitation wavelengths available)
- Measurement of particle concentration: number of particles per mL
- Single particle Zeta Potential measurement and simultaneous measurement of particle size and concentration
- Multi-parameter analysis: the ability to measure particle size, particle concentration, fluorescence and zeta potential, concurrently, in real time, on individual particles
- Unique visual validation of sample: seeing is believing!
Dynamic Light Scattering (DLS)
Dynamic Light Scattering (DLS) is used to measure particle and molecule size. DLS is an ensemble technique which, like NTA, relates the diffusion of particles moving under Brownian motion to particle size. As both technologies are based on the same underlying principle (i.e. measurement of Brownian motion), they represent ideal methodologies to provide orthogonal measurements. Both technologies offer unique measurement parameters and have application-specific advantages, but when used in conjunction provide a comprehensive and powerful measurement suite.
Key features of DLS technology are:
- Non-Invasive Back Scatter technology (NIBS): laser path is varied according to light intensity (in terms of both particle size and sample concentration). Wide dynamic range of sizes and concentrations, so more samples can be measured in their native state
- Size range: from 0.3 nm – 10 µm
- Laser doppler micro-electrophoresis: used to measure zeta potential
- Surface zeta potential: using tracer particles to measure the zeta potential of a surface
- Static Light Scattering: used to determine the molecular weight of proteins and polymers
- Microrheology: using tracer particles to probe the structure of dilute polymer and protein solutions
- Automation: sample screening through application of nanosampler
- Flexibility: ability to connect to separation technologies, such as SEC or FFF
- Molecular weight determination
- Ease of use and industry standard measurement technology
Measuring a heterogeneous sample
A common challenge in particle characterization is how to measure a polydisperse sample. Sample heterogeneity in terms of different material types and different particle sizes, along with differences in the relative concentrations of those species, represents a characterization challenge. However, given the complementary advantages of NTA and DLS, most eventualities can be covered by using both technologies in conjunction.
Dealing with a range of particle sizes
There are two very common characterization challenges in samples which contain a range of particle sizes.
The first issue arises when the user wishes to measure a sample containing a population of small particles, yet is also interested in the presence of a few larger or over-sized particles. In many of these applications, the smaller particles represent the functional aspect of the product or sample and the large particles represent the unwanted or damaging aspect of the sample. There are numerous applications where this is seen; for example: inkjet inks where aggregates may block the inkjet nozzle; in therapeutic proteins, aggregates may initiate adverse immune responses in the patient; in semiconductor wafer fabrication, large particles in the CMP polishing slurries may scratch the wafer. In all of these examples the sample can be described as containing a high concentration of small particles with a low concentration of larger or over-sized particles.
In such cases, the Zetasizer Nano is an ideal tool. The sample can be often be measured in undiluted form, so any event-associated sample dilution can be avoided. Figure 2 shows a sample of hemoglobin in PBS and the effect of sample heating on the degree of sample aggregation. Figure 3 shows the time-dependent aggregation of the sample at two different temperatures, demonstrating the ability of the Zetasizer Nano to monitor the dynamic changes in aggregation over time. In this example, the trend-spotting capabilities of the Zetasizer Nano provide quick, robust and understandable data.
Figure 2. Dynamic light scattering size distribution by intensity data for hemoglobin, showing the change in particle size associated with sample heating from 37°C to 44°C
Figure 3. Dynamic light scattering data for hemoglobin showing the change in particle size over time associated with sample heating at 41°C and 44°C
The second common issue arises in situations where the user wishes to accurately measure a sample which exhibits a wide range of particle sizes, perhaps to understand how differently sized particles affect sample performance. In such cases, the single particle, high resolution sizing capabilities of NTA represent an ideal tool for accurately measuring the range of particles present within the sample.
To demonstrate the resolution of NTA across a range of particle sizes, a test sample was prepared containing a mixture of 100 nm, 200 nm, 300 nm and 400 nm reference beads, as shown in Figure 4. By resolving these four particle size populations within a single sample, confidence is gained in the measurement of real-life polydispersed samples. The ability to measure particle size distributions at high resolution is one of the key strengths of NTA technology and perfectly complements the broad range of particle sizes measurable through DLS.
Figure 4. NanoSight NTA size distribution data for a mixture of 100 nm, 200 nm, 300 nm and 400 nm reference standards measured simultaneously, demonstrating the high resolution sizing capabilities of the technology
Dealing with a range of particle concentrations
The ability to measure samples over a wide dynamic concentration range is critically important as many facilities may have disparate uses for their analytical instrumentation. Many applications require that samples are measured in their native form without dilution, as the process of dilution may cause a change in sample characteristics. Many DLS instruments employ backscatter detection to extend the upper concentration limit. When measuring at 173°, the laser does not pass through the whole of the sample, therefore the chance of multiple scattering is reduced, allowing more concentrated samples to be measured. In addition, because larger particles scatter light more efficiently in the forward angles, measuring at 173° reduces the likelihood that larger contaminant particles will perturb the measurement.
The Malvern Panalytical Zetasizer is the only DLS instrument which takes this capability one step further through the application of Non-Invasive Back Scatter (NIBS) technology. NIBS technology features movable optics (schematic shown in Figure 5) combined with measurement at 173°, resulting in the ability to alter path length to perfectly optimize sample analysis irrespective of sample concentration and size. To demonstrate the power of NIBS technology, a 200 nm calibration standard was measured at a range of sample concentrations - Figure 6 shows the measured particle size versus sample dilution. This demonstrates that the Zetasizer Nano is able to generate a straight line response of particle size versus sample dilution from a dilution of x3 to x100,000. This is crucially important for users wanting to measure their samples without dilution or for institutions which require the broadest range in concentration measurement capabilities across disparate applications.
Figure 5. A schematic of the measurement position for DLS for (a) weakly scattering or low concentration samples and (b) concentrated or strongly scattering samples
Figure 6. Dynamic light scattering size data for 200 nm polystyrene reference standards measured at a range of sample dilutions using the Zetasizer Nano, showing a linear response in particle sizing irrespective of sample dilution
Measurement of fluorescent particles
NanoSight instruments have the ability to measure particle size, particle concentration and zeta potential when operating in fluorescence mode, such that only fluorescent particles are detected and measured. The instrument comes with a choice of excitation wavelengths (405 nm, 488 nm, 532 nm and 642 nm) providing flexibility in the choice of fluorophore measured.
Users may wish to measure fluorescent nanoparticles for two reasons. Firstly, it is a common requirement to understand the fate of nanoparticles in biologically-relevant conditions. When measuring drug delivery vectors for example, it is important to understand the size of the vectors to understand where in the body these particles may end up, what the rate of delivery may be, and how aggregation may affect the biological performance. However, in most cases particle size is measured in vitro, removing the analysis from the real biological environment. The use of fluorescence measurement means that suitably labeled drug delivery vectors can be measured in biologically-relevant conditions and therefore the degree of aggregation or vector destruction, for example, can be measured in biologically-relevant conditions. This information can be used to help develop the efficiency of a delivery vector. Figure 7 shows a sample of liposomes which have been labeled using a lipophilic fluorescent dye and then suspended in FBS growth medium. When operating in light scatter mode (Figure 7A), all components of the medium are visualized and it is impossible to specifically detect and measure the liposomes of interest. Fig 7B shows the same sample at the same concentration; however, operating in fluorescence mode. In fluorescence mode, only fluorescent light is detected by the camera and as such only the fluorescent liposomes are measured. This offers the ability to specifically measure target particles in complex biological media, taking understanding of the fate of the delivery vectors one step closer to reality.
Figure 7. NanoSight instrument screen shots, showing fluorescently labeled liposomes suspended in fetal bovine serum, operating in light scatter mode (A) and fluorescence mode (B)
The second area where fluorescence measurement commonly adds value is in the identification of specific particles within an unknown sample as is the case in diagnostic applications. Here, fluorescently labeled antibodies can be used to bind specifically to surface antigens on a particle which the user wishes to identify within a sample containing a range of particle types.
Measuring zeta potential
Aggregation can be an issue in colloidal systems, and understanding the zeta potential of a system provides insight into the likelihood that aggregation will occur. Most colloidal dispersions carry a surface charge originating from the nature of the particles and the surrounding environment of the particles in dispersion. The stability of a colloidal system can be determined by the sum of the attractive Van der Waals forces and the repulsive electrical double layer forces. Factors affecting zeta potential include pH, conductivity and concentration of formulation components, and it is generally accepted that systems that have a zeta potential between +30 mV and -30 mV will be unstable long term and aggregation will occur.
The Malvern Panalytical Zetasizer Nano instrument utilizes laser doppler micro-electrophoresis to measure zeta potential. An electric field is applied to a solution of molecules or a dispersion of particles, which then move with a velocity related to their zeta potential. This velocity is measured using a patented laser interferometric technique called M3-PALS (Phase Analysis Light Scattering). This enables the calculation of electrophoretic mobility and, from this, the zeta potential and also the zeta potential distribution, even in samples of very low mobility. A surface zeta potential accessory uses tracer particles to measure electro-osmosis close to a sample surface to calculate the zeta potential of the surface. The instrument can be supplied with an autotitrator which allows the user to study the effects of changes in pH or conductivity on the stability of a system.
The ability to measure zeta potential on the Zetasizer Nano instrument is complemented by the capacity to measure single particle zeta potential by NTA using an instrument from the NanoSight range. The single particle nature of the latter technique results in high resolution zeta potential distributions. Using NTA, zeta potential is calculated from measurements of the velocity of a particle in suspension when an electric field is applied (electrophoretic velocity and electro-osmosis). The NanoSight NS500, for example, records the total drift velocity for each tracked particle, which is composed of elements of both types of movement. By observing the total velocity at different depths within the closed sample chamber, and assuming a zero net flow over the entire chamber depth, it is possible to separate the two components. The electrophoretic velocity (due to the electrical force on the particles) can therefore be measured for every particle tracked by the NS500. This can then be used to calculate zeta potential on a single particle basis. NTA provides the ability to measure zeta potential at the same time as particle size, thus providing the user with simultaneous multi-parameter particle characterization.
Figure 8: NanoSight particle by particle zeta potential distribution versus particle concentration data (A) and particle size versus particle zeta potential plot (B)
Measuring particle concentration
For many users, measurement of particle size is not the only important variable which needs to be measured. For many applications, an understanding of particle concentration is critical. In vaccines, understanding the titer (concentration) of viruses or virus-like particles (VLPs) is required to understand the dosage of a vaccine. In drug delivery applications, understanding the concentration of delivery vectors may be linked to the dosage of delivered drug. In exosome applications, understanding the concentration of vesicles may be critically important in determining the onset of disease as the concentration of specific vesicles may spike when compared with control patient samples. Lastly, European regulations are being developed which cover the definition and regulation of particles that are known as nanomaterials. This European definition states that a material which contains 50% of its population with one aspect between 1 nm and 100 nm is a nanomaterial. This definition is number-based, and therefore the ability to count particles in this size range will become increasingly important as regulations develop and materials classed as nanomaterials are identified and regulated as such in terms of safety. In all of these cases, NanoSight nanoparticle tracking analysis instruments provide the ability to supplement high resolution particle size measurements with the capability to determine the concentration of particles in terms of particles per mL within each given size class.
Users may wish to screen a set of samples to determine the effects of changing sample variables across a range of conditions. This may result in a large number of samples to measure as well the requirement to measure them in a standardized and repeatable fashion such that potentially subtle differences in results can be detected and analyzed. This represents a challenge from a resource perspective and also from a repeatability perspective. To avoid this issue, the Zetasizer Automatic Plate Sampler (APS) instrument has been designed. This instrument can measure small volumes of samples in industry standard 96 or 384 well plates, providing advanced light scattering analysis on each sample, a schematic of which is shown in Figure 9. Standard operating procedures can be run to ensure repeatability of measurement between samples. Data analysis takes the form of a color coding plate navigator (Figure 10) where specific trends are color coded to allow for easy-to-interpret data.
Figure 9. Schematic of the Malvern Panalytical Zetasizer APS instrument
Figure 10. Malvern Panalytical Zetasizer APS Plate Navigator software allows for powerful yet simple data analysis. Trends in data can be color coded to assist interpretation of results
The Zetasizer Nano dynamic light scattering instrument family and the NanoSight Nanoparticle Tracking Analysis instrument range complement each other perfectly, providing a suite of measurement capabilities useful to many researchers interested in characterizing nanoparticle and bioparticulate systems. Both instruments measure particle size and zeta potential. The broad particle sizing range of the Zetasizer Nano is complemented by the high resolution single particle sizing of the NanoSight NTA. Routine, industry-standard zeta potential measurements as provided by the Zetasizer Nano are complemented by the high resolution zeta potential measurements of the NanoSight NTA which also allow for simultaneous measurement of particle size. The Zetasizer APS instrument supplements these measurement parameters with the ability to measure particle size distribution in samples within 96 and 384 well plates, allowing for both routine and automated sample screening. The NIBS technology implemented in the Zetasizer Nano family allows for the measurement of samples across a broad range of sample concentrations, providing flexibility and the capacity to measure samples in an undiluted form. In addition, the NanoSight NTA range has the capability to measure sample concentration in applications where particle concentration may be important; for example, drug delivery, vaccine development or exosome/microvesicle applications. Lastly, the NanoSight NTA range provides the ability to measure fluorescence, allowing the user to monitor the fate of fluorescently labeled particles in biological media, or through antibody labeling determine surface markers on biological particles.
This broad range of measurement capabilities is both complementary in nature, providing a wealth of valuable information, but also orthogonal, in that data from one technology can be validated and supported by the second technology. In addition, as with all Malvern Panalytical products, the instrument is accompanied by the world’s most extensive team of technical experts ready and willing to support your characterization challenges.
Please visit our website at www.malvernpanalytical.com or contact your local representative for more information.