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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
A sticky measurement problem: number concentration of agglomerated nanoparticles Caterina Minelli, Dorota Bartczak, Ruud Peters, Jenny Rissler, Anna Undas, Aneta Sikora, Eva Sjostrom, Heidi Goenaga-Infante, and Alexander G Shard Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04209 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 14, 2019
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A sticky measurement problem: number concentration of agglomerated nanoparticles Caterina Minelli,1* Dorota Bartczak,2 Ruud Peters,3 Jenny Rissler,4 Anna Undas,3 Aneta Sikora,1† Eva Sjöström,4 Heidi Goenaga-Infante2 and Alexander G. Shard1. 1
Chemical, Medical and Environmental Science Department, National Physical laboratory,
Hampton road, Teddington TW11 0LW (UK) 2
LGC Limited, Queens Road, Teddington TW11 0LY (UK)
3
RIKILT – Wageningen University & Research, 6700 AE Wageningen, The Netherlands
4
Bioscience and Materials, RISE Research Institutes of Sweden, Scheelevägen 27, Lund,
Sweden. KEYWORDS concentration, nanoparticles, particle number, colloids, agglomerates, gold.
ABSTRACT Measuring the number concentration of colloidal nanoparticles is critical for assessing reproducibility, compliance with regulation and performing risk assessments of nanoparticle-enabled products. For nanomedicines, their number concentration directly relates to their dose. However, the lack of relevant reference materials and established traceable measurement approaches make the validation of methods for nanoparticle number concentration
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difficult. Furthermore, commercial products often exhibit agglomeration, but guidelines for dealing with non-ideal samples are scarce. We have compared the performance of five benchtop measurement methods for the measurement of colloidal number concentration in presence of different levels of agglomeration. The methods are: UV-Visible spectroscopy, differential centrifugal sedimentation (DCS), dynamic light scattering (DLS), particle tracking analysis (PTA) and single particle inductively coupled plasma mass spectrometry (spICP-MS). We find that both ensemble and particle-by-particle methods are in close agreement for monodisperse nanoparticle samples and three methods are within 20% agreement for agglomerated samples. We discuss the sources of measurement uncertainties, including how particle agglomeration affects measurement results. This work is a first step towards validation and expansion of the toolbox of methods available for the measurement of real-world nanoparticle products.
Introduction Nanoparticles are increasingly used in innovative products manufactured by advanced industries and provide enhanced and unique properties of great commercial and societal value. The demand for high performance materials places increasingly stringent tolerances on the properties of nanoparticles and it is critical to establish methods to measure them. There now exist many methods to produce and measure nanoparticles that have excellent consistency in particle size and shape.1, 2, 3 The low variance of these properties across the constituent particle population, i.e. morphologically identifiable particles inside an agglomerate (or aggregate), is important for generating useful materials. Measuring the number concentration of particles in colloidal suspension is a major commercial interest for a large range of industries as it enables
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them to optimise and reproduce formulations and products. It enables compliance with present and future regulation, linked for example to the EC Recommendation for the definition of a nanomaterial (2011/696/EU),4 and underpins any claim to reliability, performance and lifetime in the formulation of products containing nanoparticles. In most cases, this number concentration is not known but is calculated upon the basis of assumptions and mass-balance considerations, generally without accounting for the agglomerated fraction. Although innovative methods5, 6, 7, 8, 9 are emerging for the measurement of colloidal concentrations, no certified reference materials are currently available for their calibration and validation and there is a lack of suitable documentary standards. Nanoparticles utilised in industrial applications are far from being ideal systems and methods need to be adapted to deal with sample agglomeration. A review authored by industry experts including DuPont, BASF, Dow and Bayer highlighted the need for methods to accurately count industrially relevant nano-objects.10 In almost all cases, nanoparticles need to be appropriately dispersed either in, or on a matrix. Usually, it is desirable for the particles to be separated from each other and evenly distributed. However, this is not easily achieved and particles have a tendency to stick together and form agglomerates. This occurs especially in the presence of complex matrices of real-life commercial products. It becomes a particular challenge when the surface of the nanoparticles is modified with different chemistries, such as organic coatings and biological ligands. In these cases, even where the monodisperse constituent particle population could be separated, there is then no general method to establish how many particles have been lost through the process and therefore reproducibility of manufacture and performance is adversely affected. Whilst commonly used methods, such as electron microscopy and X-ray diffraction provide useful information on the size, shape and variability of the constituent particle
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population, it is much more difficult to establish how many of the particles have stuck together as agglomerates due to artefacts arising from sample preparation. Currently, limited methods are available for the measurement of nanoparticle aggregates in colloidal suspension. It is a recognised problem for magnetic nanoparticles,11 toxicological studies,12 coatings,13 drug delivery vehicles14 and particles for bioanalysis,15 but reliable measurement solutions have not been proposed so far. Furthermore, it is important to understand the contribution that agglomerates or aggregates make to the measured value of number concentration, since some methods will count agglomerates as one particle, others as many particles and few have the size resolution to resolve them from the constituent particle population.16 In this work we evaluate 5 techniques that can in principle be used for the measurement of the concentration of gold nanoparticles and discuss their performance when dealing with monodisperse and agglomerated samples. We consider UV-visible spectroscopy, which has been studied in great detail for monodisperse gold nanoparticles of various shapes;9, 17, 18 dynamic light scattering (DLS), which is commonly employed to estimate particle size but also provides an estimate of the relative particle volume concentration;19, 20 differential centrifugal sedimentation (DCS), which can separate particle sub-populations with high resolution and has been shown to provide useful measurements of relative mass concentrations for these subpopulations;21 particle tracking analysis (PTA) and single particle inductively coupled plasma mass spectrometry (spICP-MS) which count particles within a given volume of liquid and therefore ostensibly provide a direct measure of particle number concentration. The model particle system used in this work are a set of 4 samples of 80 nm gold nanoparticles agglomerated to different degrees using a biotin-avidin binding strategy. The size of the particles was chosen to be well above the detection limit of all of the methods used in this work. Gold
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nanoparticles were chosen because of the facile and robust chemistry of their surface functionalisation and the possibility to quantify the total mass of gold of each sample by UV-Vis spectroscopy.9 Furthermore, the system has previously been used as a demonstration of the applicability of DCS for the relative mass measurement of particle populations.21 This makes it a useful system to establish the sensitivity of other methods to agglomeration and provide some comparative data, which will help the selection and implementation of the methods for complex real-world nanoparticle systems.
Materials and Methods Gold nanoparticles with nominal size of 80 nm were purchased from BBI International (Cardiff, UK). The particle manufacturer declared a size of the particles of 78.8 nm, as measured by transmission electron microscopy (TEM), and a concentration of 1.10·1010 nanoparticles/mL (NPs/mL). The agglomeration of the particles was induced by using a surface biotin-avidin strategy. One batch of the particles, Batch 1, was incubated in a 28.5 μM solution of 2,5,8,11,14,17,20Heptaoxadocosane-22-thiol (mPEG-thiol, Mr 356.5, Polypure, Oslo, Norway) in 1 mM 3-[4-(2Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS, Sigma, MO). Another batch of the particles, Batch 2, was incubated in a 1 mM EPPS containing 22.8 μM mPEG-thiol and 6.42 μM biotinPEG-thiol (Mr 788.0, Polypure, Oslo, Norway). Both particle batches were incubated for 2 hours under gentle shaking. The excess ligands were removed via centrifugation. 4 cycles of 1 hour centrifugation of the samples at 500 rcf were followed by gentle removal of the supernatant and redispersion of the particles in fresh 1 mM EPPS buffer at pH 7.8.
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Batch 2 was subsequently incubated in Neutralised Chimeric Avidin (NCAvd, Mr 14324) to induce agglomeration of the particles. The NCAvd was based on a previously described thermostable avidin form,22 which was developed by applying charge-neutralized mutations.23, 24 The production and characterization of the NCAvd are detailed elsewhere.25, 26 For the agglomeration to take place, the particles were centrifuged for 1 hour at 500 rcf in presence of 0.87 μM NCAvd. After centrifugation, the sample was gently shaken to redisperse the particles. Four samples exhibiting increasing level of agglomeration were produced by mixing the two batches of particles in EPPS buffer in different ratios according to Table 1. The final volume of the particles was the same as the initial volume of the unmodified particles. The total particle concentration is therefore expected to be close to the nominal particle concentration of 1.10·1010 NPs/mL, although the centrifugation steps for the removal of excess PEG molecules may have introduced a significant loss (~10%) of particles. This was quantified by comparing the UV-Vis absorption at 450 nm of the particle solution before and after the centrifugation steps. The excess NCAvd was left in solution. DCS was performed using a CPS 24000 disc centrifuge (CPS Instruments Inc., Stuart, Florida, USA) equipped with an LED laser emitting light with wavelength between 385 nm and 425 nm and with spectral intensity peak at 405 nm. The instrument was operated at 20 000 rpm with a 14.4 mL 8% to 24% (m/m) sucrose gradient in water (average gradient density ρf = 1.064 g/ cm3). This was generated by a series of injections of decreasing in sucrose concentration, followed by a final addition of 0.5 mL dodecane as an evaporation barrier. A period of 30 min was allowed prior to measurement acquisition to facilitate thermal equilibrium. A calibration of the instrument was performed before each sample injection by using polyvinyl chloride (PVC) calibration particles with nominal Stokes’ modal diameter of 237 nm and density of 1.385 g/cm3
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provided by the instrument manufacturer. The uncertainties in the size and density of the calibrant were set at 5% and 3.5% respectively, according to the work by Kestens et al.27 The injected sample volume was measured by weighing the syringe containing the sample before and after each injection and assuming a density of the sample solution of 1 g/cm3. For the data analysis, recalling that the spectral intensity peak of the laser instrument is at 405 nm, the refractive index and absorption of the gold particles were set to 1.62 and 1.95 respectively and the average refractive index and viscosity of the gradient at 1.357 and 1.3 mPa·s respectively. These values are needed to convert the extinction intensity based size distribution measured by the instrument into a mass-weighted size distribution. This conversion was performed by the instrument software through application of Stokes28 and Mie light scattering theory29 for solid spheres. To measure the particle number concentration, the mass-weighted size distributions were fitted with a multimodal function to identify and separate the populations of agglomerates. The concentration of the non-agglomerated particles was measured by integrating the corresponding peak and dividing the resulting mass by the average mass of a single particle, taking into account the measured injected volume. The relative particle number concentration of the other populations of agglomerated with respect to the population of non-agglomerated particles was measured as described elsewhere.21 UV-visible spectra were acquired in quartz cuvettes using a LAMBDA 850 spectrophotometer (Perkin Elmer Inc., MA, USA). Samples were analysed over the wavelength range 250 nm to 800 nm. The molar extinction coefficient at 450 nm was calculated according to Haiss et al.17 by using the size derived from the localized surface plasmon resonance peak of sample Agg118 and resulted in 3.96·1010 M-1cm-1.
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DLS measurements were performed using a Zetasizer Nano ZS 3600 (Malvern Panalyticals Ltd., Malvern, UK) equipped with a max. 4 mW He-Ne laser, emitting at 633 nm and measuring the scattered light at an angle of 173°. The software used is Malvern Zetasizer Software v7.11. The measurements were performed in disposable capillary cuvettes (DTS0012, Malvern Panalytical Ltd., UK) at 25 °C and preceded by a 2 min equilibration time. The laser and the optical system of the instrument was regularly tested throughout this work based on the scattered light intensity of pure toluene and no significant change was observed. The measurement position was fixed to the centre of the sample (4.65 mm). To estimate concentration from the DLS measurements multiple scattering events were reduced by diluting of the samples. This also reduced the uncertainty introduced by the attenuation of the signal. The scattered-light intensityweighted size distributions were expressed in terms of their Z-average (Z-ave) and the polydispersity index (PI) calculated by the instrument software by applying the cumulant method. The Z-average is defined as the scattered light intensity-weighted harmonic mean diameter.30 The volume weighted and number weighted median size was also provided by the program, as well as derived count rates and estimated volume concentration in the sample. The relative volume concentration was estimated by the software, and the derivation is based on the scattering intensity (number of scattering events), refractive index, absorption and particle size distributions, assuming spherical particles. The refractive index and absorption values used for the gold nanoparticles were 0.183 and 3.43 respectively. From the relative volume concentrations we estimated the particle number concentration by dividing by the volume of the single particles, calculated using the Z-ave value as the particle diameter. PTA measurements and data analysis were performed with NS500, manufactured by Malvern Panalytical and equipped with a violet diode laser as a light source (405 nm CW, max power
