Tunable Resistive Pulse Sensing:
Essential information about
nanoparticles is revealed quickly
and accurately
Introduction
High quality research on the nano-scale demands
highly accurate characterisation techniques 
significant breakthroughs are often achieved by
increased
knowledge
through
improved
measurement capabilities. Greater accuracy and
reliability in characterisation measurements allow
an increased level of detail for researchers, and
reduce the risk of misconstrued conclusions. This
paper explores the key aspects for nanoparticle
characterisation and compares a number of
available techniques. Tunable Resistive Pulse
Sensing (TRPS) clearly is an essential
measurement technique for nanoparticle analysis
(see Table 1).
Table 1. Unique features of TRPS
Features
High-throughput (3000
particles/min) single particle
analysis
Benefits
Comparison
Other single particle analysis
techniques, such as NTA suffer
from limited statistics; or are very
time consuming (TEM).
Excellent size resolution
Measured pulses are proportional Only DSC has a comparable
to particle volume. Cubic
resolution. Poor resolution leads
relationship guarantees high
to obscuring of characteristics in
resolution.
the population.
Large dynamic range:
Further dynamic range increase,
Currently this is comparable with
40 nm –10 µm
through improvements of pore
most other techniques.
technology and higher
signal/noise.
Simultaneous size and zeta
Important for a range of
No other method can
potential capability
applications such as colloidal
simultaneously measure size and
stability testing, surface
zeta potential on a particle by
modification monitoring and
particle basis.
phenotyping.
5
Concentration range: 10 High precision concentration
Other techniques lack high
12
10 /ml
analysis.
precision in concentration
measurement.
Fraction analysis of
TRPS software assistants guide
This is only possible for high
concentration stated for specific settings to produce standardised resolution techniques that can
size ranges
concentration measurements,
measure concentration in small
independent of sample.
size ranges.
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Sufficient data gathered to give
accurate high resolution
measurements.
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Real time monitoring of particle
interactions and chemical
reactions
Real time monitoring is enabled
through high accuracy in size and
zeta potential.
Wide range of applications.
Other techniques that lack
accuracy make real time
monitoring difficult.
Versatility and tunability
Increase of sensitivity in particle
size.
Precise control of electrokinetic
and convective particle velocities,
enabling accurate zeta potential
measurements.
Preserves valuable samples and
allows multiple repeats without
wasting sample.
TRPS can operate under
physiological electrolyte
conditions.
In a direct comparison regular
coulter counters are more limited
in their use.
Small sample volume, (30-40 µl)
Physiological conditions
- Osmolarity
Most other techniques require
significantly larger volumes.
Some techniques, such as PALS
suffer from artefacts due to
sample heating at physiological
conditions.
NTA - Nanoparticle Tracking Analysis, TEM - Transmission Electron Microscopy, DCS - Differential Centrifugal
Sedimentation, PALS - Phase Analysis Light Scattering
Critical aspects of nanoparticle
analysis
To understand the detailed characteristics of
nanoparticles in suspension, measurements need
to be accurate, high resolution, non-biased, and
high throughput to ensure a statistically relevant
observation. The most useful will be those which
measure and collate the properties of individual
particles, as opposed to ensembles. It is well
known that particle size distribution (PSD)
measurements carried out using different
techniques, or even machines from different
manufacturers, can show considerable variability.1
Institutions such as the National Institute of
Standards and Technology (NIST) have begun to
realise the necessity for guidelines to control the
potential sources of error.2
Accuracy and precision
When working with a measurement system, it is
vital that the user has an appreciation of the
influence of any user-defined settings or inputs,
and any inherent biases or assumptions within the
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technology which may affect the results. The
measurement of standardised nanoparticle
samples (e.g. from NIST) by users from different
laboratories, using independent instruments, is
the most relevant assessment of accuracy and
precision; an example of this is shown in Figure 1.
Figure 1. Repeat measurements of the same sample show
high reproducibility: A bimodal sample was distributed to
and measured by three users in different groups across the
world, without prior communication between the groups.
The size distributions overlay very well, indicating the
reliability of the data.
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Resolution and dynamic range
Resolution refers to the ability to resolve the PSD
of nanoparticle samples, many of which are
polydisperse or multi-modal (consist of two or
more distinct populations). High PSD resolution is
important in many applications, for example
determining subpopulations within a polydisperse
extracellular vesicle (EV) sample. In this case,
accurate measurement of the polydispersity –
more so than simply the mean size – is essential as
this can be indicative of the origin of the vesicles,
and has potential as a diagnostic marker.3,4,5
Fundamental technological limitations result in
varying
resolution
between
common
characterisation techniques, as demonstrated in
Figure 2. In this example Tunable Resistive Pulse
Sensing (TRPS) and differential centrifugal
sedimentation (DCS) were shown to be the only
techniques capable of resolving the three distinct
particle populations (220 nm, 330 nm, and 410 nm
particles) within the sample.6 Dynamic particle
size range, which refers to the size range of
particles that can be resolved by the instrument,
needs to be considered alongside particle
resolution.
Figure 2. Comparison of resolution of various common nanoparticle measurement systems: Dynamic Light Scattering (DLS),
Tunable Resistive Pulse Sensing (TRPS), Particle Tracking Analysis (PTA or NTA) and Differential Centrifugal Sedimentation (DCS).6
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Overview of key technologies
method for PSD determination
development in the 1960s.
Current techniques for characterising
nanomaterials
A range of techniques are currently available to
characterise nanoparticle suspensions. These can
be broadly categorised as ensemble or single
particle techniques.7 Ensemble techniques
observe the bulk dispersion and report an
averaged result of the particle properties. Because
of the specific qualities of nanoparticles, a
population comprising predominantly of small
particles with a few large agglomerates could have
considerably different properties than a
homogenous sample of medium-sized particles.
This averaging can lead to low resolution and
potential bias or information exclusion. A further
limitation of ensemble techniques for
nanomedicine applications is the inability to
directly provide number-based PSD. PSD can only
be calculated indirectly and this lack of a direct
measurement can lead to significant uncertainties
in obtaining the true particle distribution.
Single particle techniques, such as TRPS and NTA,
are able to measure and report the properties of
each individual particle, and the resulting numberbased PSD provides more accurate information
about the sample properties. However, some
single particle techniques (such as NTA) sample
only hundreds of particles, and may require
sufficient sample volume to ensure low
concentration populations are accurately
represented.
since
its
Due to the intensity-weighted approach, DLS
suffers from a number of limitations which render
it unsuitable for many applications. High quality
data can only be achieved when the refractive
index of the solvent is accurately known and the
concentration of particles and electrolyte are
low.8 The major limitation of DLS is that it skews
the PSD in the presence of large particles.9,10 A
small number of large particles can significantly
bias the result, swamping the scattering of the
smaller particles and overestimating the average
diameter. For these reasons DLS is not suitable for
polydisperse samples.
Differential Centrifugal Sedimentation (DCS)
Sedimentation through centrifugation (DCS) is an
ensemble technique for the separation of particles
based on size. In DCS a density gradient is set up
inside a rotating disc, with the sample being
introduced to the centre. The velocity of the
particle away from the axis of rotation is
dependent on three forces – centrifugal, buoyant,
and frictional forces – as well as the density of the
particle and fluid, and the volume of the particle.
The particles will sediment until the forces
balance, resulting in distinct bands.
Although DCS has shown to have very high
resolution in size, its accuracy is highly dependent
on the precise knowledge of the solution viscosity
and density, meaning it is only suited for
homogenous samples.
Ensemble measurement techniques
Single particle techniques
Dynamic light scattering (DLS)
Light scattering-based techniques are used for the
measurement of nanoparticle size and zeta
potential.8 DLS measures the size of particles in
solution by analysing the intensity fluctuations of
light scattered by the sample. The ease of use,
sample recovery and applicability to a wide range
of particle and solvent types made DLS a popular
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Electron microscopy (EM)
Transmission electron microscopy (TEM) has long
been used for the characterisation of
nanoparticles. The advantages of this microscopy
technique lie in its high size resolution and its
ability to collect detailed information about
particle shape and composition. TEM imaging
relies on the density of the sample, therefore
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many commonly used nanoparticles, including
metallic or oxide particles, are easily imaged with
TEM.
defined adjustments to the detection parameters
can affect the detection limit, therefore biasing
the sample.10
Hollow or less electron dense particles, such as
liposomes and other biological vesicles, show less
contrast relative to the background and require
negative staining in a heavy metal film. Negative
stain TEM brings the potential for artefacts, such
as flattening or collapse of hollow particles, giving
misleading results. TEM is very labour intensive for
obtaining a statistically significant measurement
of PSD.6 The technique is still largely qualitative as
the analysis of large data sets is prohibitively time
consuming, and user-defined parameters for
automation can result in information bias.
Furthermore, the high vacuum environment
prevents in-situ analysis, and the high energy
electron beam can damage biological and polymer
based samples.11
Tunable Resistive Pulse Sensing (TRPS)
Tunable Resistive Pulse Sensing (TRPS) of
nanoparticles using Coulter-type counters has
been shown to be a fast and accurate alternative
to traditional sizing methods, and is becoming
accepted as the preferred method in the field of
nanomedicines.16,17 This technique provides a
direct measure of particle concentration, and high
resolution analysis of particle size and surface
charge.18–21 Resistive pulse sensing was historically
used for measuring microparticles, but advances
in the fabrication of pores have led to the
technique being used for single particle
characterisation of nanoparticles.22 A voltage is
applied across a pore which is filled with
electrolyte, resulting in an ionic current. Particles
traverse the pore with a velocity that is dependent
on the particles zeta potential, causing a transient
blockage in the ionic current for each particle
(Figure 3). Measurement of these blockade events
allows high-throughput, single particle analysis of
colloidal samples with very high resolution due to
its cubic relationship with diameter.
Nanoparticle Tracking Analysis (NTA)
Similarly to DLS, NTA calculates the hydrodynamic
radius of individual particles via the EinsteinStokes equation.12,13 The Brownian motion of
single particles is tracked independently using the
light scattered from an incident laser light source.
In this way the diffusion constant is directly
measured, and particles with diameters below the
wavelength of the incident light can be detected
due to their Rayleigh scattering.14
The tracking of Brownian motion through the
mean square particle displacement with time is
inherently leading to a lack of size resolution.
Another disadvantage of NTA is that the refractive
index of the sample must be sufficiently different
from the buffer, and prior knowledge of the
refractive index is required.15 Instrument set up
can bias the results – the ultramicroscope must be
isolated from mechanical vibration, and the user-
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Tunable pore size increases the dynamic range of
TRPS, making it suitable for analysis of extremely
polydisperse samples.23 It also increases the
analytical sensitivity by tuning the pore diameter
to the particulate system at hand. The tunability in
applied voltage and pressure lends the system
precise control over electrokinetic and convective
particle speeds, enabling highly accurate single
particle size and charge measurements. As a
consequence real time monitoring of particle
interactions and chemical reactions become
possible (see Table 1).
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Ionic current “pulses” as seen in real time,
The close up view of a single current pulse
generated by individual particles passing
shows the characteristic pulse shape
through the pore
Figure 3. Typical current pulses in TRPS.
A historical disadvantage of TRPS is the potential
for pore blockages to occur due to large or
adhesive, pore-binding particles, however this has
been addressed by the release of qEV size
exclusion columns and the Izon Science coating
reagent, which have greatly improved measured
data quality and reduced the occurrence of pore
blockages, particularly for biological samples.
Recent improvements in software have also
greatly improved the ease of use and precision of
the instrument through the inclusion of
measurement assistants, which guide the user to
a stable, calculated system setup, optimised for
the particles and nanopore being used.
Izon Science have released an improved version of
its flagship TRPS instrument called “qNano Gold”.
This instrument has the protocols and the
reagents to treat the pore preventing biological
molecules altering its properties, as well as
improved limit of detection that allows smaller
particles to be analysed with larger pores, giving
greater system stability.
TRPS for accurate particle-byparticle measurement
Size measurements
The relationship between particle volume and
blockade magnitude of the resistive pulse ΔR
generated by a TRPS instrument is linear, and
hence the particle diameter can be determined
with extremely high accuracy (equation 1).22 For
example doubling the particle diameter means an
eight fold increase in resistive pulse magnitude,
resulting in high sensitivity to differences in
particle size.
Equation 1
∆𝑅
𝑅
=
𝑑3
𝐷2 𝐿
R is the pore resistance, D is the pore diameter, d
is the particle diameter and L is the pore length.
This represents the simplest case of a spherical
particle and a cylindrical pore. For more complex
particle and pore geometries other factors must
be taken into account.22
In order to guarantee accurate and reliable
measurements of particle size, the pore is
calibrated with a calibration standard of known
size. This becomes particularly important when
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analysing samples which contain multimodal or
aggregated populations.6 It also can be beneficial
in monitoring the processing of nanoparticles such
as the reduction in polydispersity of a sample
through additional filtering.21
Concentration measurements
In TRPS particle concentrations are calculated
using equation 2.
Equation 2
𝐽 = 𝐶𝑄
where 𝐽 is the particle count rate, 𝐶 is the particle
concentration and 𝑄 is the fluid flow rate.16 𝑄 is
proportional to pressure and hence the particle
count rate is proportional to both, the particle
concentration and the applied pressure.24 Hence,
a plot of particle count rate vs applied pressure
gives a gradient proportional to the particle
concentration. For a pore of unknown length and
diameter, the use of a calibration sample of known
concentration allows the unknown concentration
of a different sample to be calculated.
In order to standardise measurements, in
particular of biological samples, TRPS determines
particle concentrations within a clearly defined
particle size range (denoted as fractions). Often
concentration measurement techniques only
measure the ‘total’ particle concentration, which
will crucially depend on the dynamic size range of
the technique used. Hence concentration
measurements for specific size ranges are
beneficial in order to compare different samples
from possibly different research groups and
various techniques. Figure 4 shows an example of
liposome samples, for which concentrations are
evaluated within the exosome diameter range of
80 -180 nm.
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Figure 4. Liposome concentration fraction measurements
(over the size range of 80 -180 nm) using TRPS.
Charge measurements
A unique feature of TRPS is its potential to
measure individual particle charge and zeta
potential, based on the duration of the resistive
pulse.21 The zeta potential of each particle can be
calculated from the measured electrophoretic
mobility, using the Smoluchowski equation.21 The
magnitude of the pulse is independent of the
respective particle zeta potential, allowing
simultaneous and decoupled size and charge
measurements to be carried out. This enables a
truly unique approach for investigating particle
properties.
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The single particle nature of TRPS means that subpopulations (with different charge and/or size)
within a sample can be discriminated (Figure 5 and
6). Figure 5 displays an example of a penta-modal
population of various polystyrene standards with
different charges and diameters, measured with
TRPS. Figure 6 shows that a mixed bimodal sample
of nanoparticles with equivalent sizes but
0
50
100
different zeta potentials was not able to be
resolved by an ensemble technique (phase
analysis light scattering, PALS). The same sample
when analysed using TRPS showed clearly defined
populations. The zeta potentials of the unmixed
samples, as determined with PALS and TRPS were
in very good agreement.
Diameter [nm]
150
200
250
300
350
400
450
0
CPC200
Zeta Potential [mV]
-5
CPN150
-10
CPN180
-15
CPC340
-20
CPN280
-25
-30
-35
-40
Figure 5. Simultaneous size and zeta potential measurements of five different polystyrene standards. Please note, each dot
represents a single particle.
The potential to link the charge and size
information of nanoparticles will be useful for a
range of applications such as phenotyping or
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determining the success and degree of a particular
particle surface modification.
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Figure 6. Comparison of TRPS and PALS analyses of bimodal charged samples: Analysis of the two samples of near neutral
polystyrene particles (CPN400) and negatively charged carboxylated (CPC400) particles (traces as labelled for PALS data; blue
and green data points for TRPS data respectively) show good agreement between the two techniques. However, when the two
populations were mixed to give a bimodal sample, PALS was unable to resolve the two populations, due to it being an averaging
technique (bottom trace). In contrast, TRPS shows two distinct populations (red data points), which agree well with the zeta
potential values of unmixed samples.
Conclusion
Accurate and reliable measurement of particle
characteristics is essential for the development of
nanoparticles for industrial or medical
applications. As research into biological
nanoparticles such as exosomes grows,
dependable techniques will need to be adopted,
so that results from different research groups can
be compared with confidence.
While a number of techniques are traditionally
used to characterise nanoparticles, it is realised
that without the use of bespoke equipment, or
multiple techniques – which greatly increase the
expense of the experiment – current
measurements often lack the resolution or
accuracy that is required.
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TRPS is recognized as the most accurate system for
the simultaneous characterisation of size,
concentration and charge properties of
nanoparticles. TRPS provides high throughput
particle-by-particle information, and has been
repeatedly shown to surpass other particle
analysis techniques, when resolving polydisperse
or multi-modal populations with high accuracy
and precision. Furthermore, the small and
affordable nature of the instrument means it is
accessible for many research groups, and the
wealth of information from a single measurement
makes it an economical choice.
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