Tunable Resistive Pulse Sensing: Essential information ... · Dynamic light scattering (DLS) Light...

IZON SCIENCE LIMITED April 2015 pg. 1 [email protected] www.izon.com

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

Benefits

Comparison

High-throughput (3000

particles/min) single particle

analysis

Sufficient data gathered to give

accurate high resolution

measurements.

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

to particle volume. Cubic

relationship guarantees high

resolution.

Only DSC has a comparable

resolution. Poor resolution leads

to obscuring of characteristics in

the population.

Large dynamic range:

40 nm –10 µm

Further dynamic range increase,

through improvements of pore

technology and higher

signal/noise.

Currently this is comparable with

most other techniques.

Simultaneous size and zeta

potential capability

Important for a range of

applications such as colloidal

stability testing, surface

modification monitoring and

phenotyping.

No other method can

simultaneously measure size and

zeta potential on a particle by

particle basis.

Concentration range: 105-

1012/ml

High precision concentration

analysis.

Other techniques lack high

precision in concentration

measurement.

Fraction analysis of

concentration stated for specific

size ranges

TRPS software assistants guide

settings to produce standardised

concentration measurements,

independent of sample.

This is only possible for high

resolution techniques that can

measure concentration in small

size ranges.

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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.

In a direct comparison regular

coulter counters are more limited

in their use.

Small sample volume, (30-40 µl)

Preserves valuable samples and

allows multiple repeats without

wasting sample.

Most other techniques require

significantly larger volumes.

Physiological conditions

- Osmolarity

TRPS can operate under

physiological electrolyte

conditions.

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

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.




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




Overview of key technologies

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 number-

based 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.

Ensemble measurement 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

method for PSD determination since its

development in the 1960s.

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.

Single particle techniques

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




many commonly used nanoparticles, including

metallic or oxide particles, are easily imaged with

TEM.

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

Nanoparticle Tracking Analysis (NTA)

Similarly to DLS, NTA calculates the hydrodynamic

radius of individual particles via the Einstein-

Stokes 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-

defined adjustments to the detection parameters

can affect the detection limit, therefore biasing

the sample.10

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.

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).




Ionic current “pulses” as seen in real time,

generated by individual particles passing

through the pore

The close up view of a single current pulse

shows the characteristic pulse shape

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-by-

particle 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

𝐷2𝐿

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




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.

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.




The single particle nature of TRPS means that sub-

populations (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

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.

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

determining the success and degree of a particular

particle surface modification.

-40

-35

-30

-25

-20

-15

-10

-5

0

0 50 100 150 200 250 300 350 400 450

Ze

ta P

ote

ntia

l [m

V]

Diameter [nm]

CPC200

CPN150

CPN180

CPC340

CPN280




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.

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.




Contact Izon Science at [email protected] to discuss how we can support you.

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