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This content was downloaded from IP address 158.109.58.130 on 22/12/2021 at 14:43 2D Mater. 8 (2021) 035044 https://doi.org/10.1088/2053-1583/abfe01 PAPER A method for the measurement of mass and number of graphene OPEN ACCESS oxide sheets in suspension based on non-spherical RECEIVED 11 January 2021 approximations REVISED 26 April 2021 Livia Elena Crica1, Thomas James Dennison2, Elise Andrea Guerini2 and Kostas Kostarelos1,3,∗ ACCEPTED FOR PUBLICATION 1 4 May 2021 Nanomedicine Lab, National Graphene Institute and Faculty of Biology, Medicine & Health, The University of Manchester, Manchester M13 9PT, United Kingdom PUBLISHED 2 Malvern Panalytical Ltd, Grovewood Road, Malvern, Worcestershire WR14 1XZ, United Kingdom 2 June 2021 3 Catalan Institute of Nanoscience and Nanotechnology (ICN2), UAB Campus Bellaterra, Barcelona, Spain ∗ Author to whom any correspondence should be addressed. Original content from this work may be used E-mail: kostas.kostarelos@manchester.ac.uk under the terms of the Creative Commons Keywords: RMM, buoyant mass, particle, 2D materials Attribution 4.0 licence. Supplementary material for this article is available online Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal Abstract citation and DOI. Currently, particle analysis of 2D materials in suspension is commonly restricted to microscopic techniques in the dry state, and thus does not permit an accurate investigation of colloidal suspensions. Colloids in bulk can be assessed by light scattering and diffraction to investigate features such as their hydrodynamic size, charge and concentration. However, the main drawback of such techniques lies in the application of analytical and computational methods based on models assuming particle sphericity which are not representative for 2D materials. Resonance mass measurement (RMM) is a technique which can enable the analysis of 2D materials in suspension without the assumptions of spherical models. Here, we report the application of RMM to measure particle mass and concentration for three types of graphene oxide (GO) aqueous dispersions. Using micro- and nano-suspended resonating sensors, we were able to decipher gravimetric differences between GO and graphitic materials. Our results support the urge for proper definitions and standardisations of graphene based materials, and offer a new method of characterisation for 2D material colloids in liquid suspension. 1. Introduction 2013, Bøggild 2018). For this reason, good practice guidelines and standards are under development by Two-dimensional (2D) materials, starting with various institutions (Bianco et al 2013, ISO 2017, graphene-based materials (GBMs), are expected to be Pollard and Clifford 2017, Kauling et al 2018, Pollard incorporated into a vast range of applications from 2017). electronics to materials science and from aerospace Currently, microscopic techniques, such as optical engineering to biomedicine (Novoselov et al 2012, microscopy (OM), scanning and transmission elec- Dimitrios et al 2013, Randviir et al 2014). However tron microscopy (SEM and TEM) and atomic force GBMs come in various forms, with different proper- microscopy (AFM) are the most commonly used ties: different lateral size and thickness distribution, methodologies for the analysis of individual GBM oxidation degree and purity, often leading to con- sheets (Pollard 2017). However, when 2D sheets are in tradictions within the scientific community (Bianco colloidal suspension, microscopic analysis of GBMs 2013, Wick et al 2014, Kauling et al 2018). There in a dry state is not ideal. This is because sheets is an urgent need for proper definitions and stand- can fold, wrinkle, bundle or stack to adopt differ- ardisation for GBMs which are often described with ent conformations, either when freely floating in dif- similar product names despite several and significant ferent fluids or during the drying processes applied, deviations in physicochemical characteristics, thereby thereby affecting various properties, such as their presenting an obstacle to the industrial adoption and size, thickness and density or even mass (Whitby translation of these innovative materials (Bianco et al et al 2012). Common methods to analyse particles © 2021 The Author(s). Published by IOP Publishing Ltd 2D Mater. 8 (2021) 035044 L E Crica et al Figure 1. Illustration of the RMM principle: particles flow through the microfluidic channel and change the resonant frequency of the sensor. The frequency shift caused by each particle is translated to buoyant mass. As particles are measured individually in a known volume, the number of counts per millilitre is approximated. The limit of detection (LOD) separates the background noise and undetectable particles from the particle count. in suspension are dynamic light scattering (DLS) and GBMs by mass in this way could be more valuable nanoparticle tracking analysis (NTA), which explore than reporting a spherical size by light scattering tech- the light scattered by particles in Brownian motion niques, since these materials are two-dimensional. to approximate their size. However, both techniques So far, RMM has proved useful in the analysis rely on spherical models which are unsuitable for of mammalian and bacterial cells, in identifying pro- flat and irregularly shaped nanomaterials such as tein aggregates, and characterising nanomaterials and GBMs. nanobubbles (Nittayacharn et al 2018, Krueger et al An alternative technique for the analysis of sus- 2019, Pollo et al 2019). Son et al measured the change pended particles is resonance mass measurement in volume and density of cells undergoing mitosis to (RMM). RMM is an innovative and emerging tech- discover that cells swell and reduce their density as nique used to characterise and count particles within a result of intracellular water regulation (Son et al the subvisible and submicron range (Weinbuch et al 2015). Lewis et al used RMM to compare the live 2013, Höldrich et al 2017). At the heart of the tech- and dead bacterial populations and monitor bacterial nology lies a microelectromechanical sensor compris- growth using real time monitoring of cell concen- ing a resonating cantilever with a microfluidic chan- tration (2014). The change in mass of nanomateri- nel, through which suspended particles are measured als upon surface modification was also assessed by under flow (figure 1). When a particle between 50 nm RMM. Höldrich et al used this technique to determ- and 5 µm flows through the cantilever it will cause a ine the number of pepsin molecules adsorbed on gold shift in the cantilever’s resonant frequency, should the nanoparticles, the results of which were in agreement density of the particle differ to the fluid in which it is with commercial protein quantification kits (2017). dispersed. This frequency shift indicates the buoyant Similarly, Nejadnik et al studied the interaction of mass of the particle being measured; denser materials bovine serum albumin and monoclonal human anti- will cause a negative frequency shift and have a pos- body with polystyrene nanoparticles and showed a itive buoyant mass, whereas less dense materials will shift in particle mass which could not be determined cause a positive frequency shift and have a negative with other techniques (Nejadnik and Jiskoot 2015). buoyant mass. This can be exploited to discriminate RMM can also be used to differentiate particles based between positively and negatively buoyant particles on their buoyancy where size-dependent techniques within a sample. The extent of the frequency shift fail. In pharmaceutical sciences for instance, RMM directly relates to the particle’s buoyant mass, from can be used to detect positively buoyant contamin- which the dry mass can be inferred as well as particle ants such as silicon oil in drug formulations that con- volume and diameter (assuming a spherical shape). tain negatively buoyant particles (e.g. protein aggreg- Since particles flow through the cantilever individu- ates) (Weinbuch et al 2013, Pollo et al 2019). In a ally and are counted in a known volume, RMM can similar way, ultrafine bubbles can be distinguished accurately determine particle concentration. Further- from other types of particles in suspensions by more, given the ability to measure individual particles measuring their positively buoyant mass (Kobayashi and the extreme sensitivity of this technique (RMM is et al 2014). able to measure particle mass as low as 10−18 g), high- However, due to its relative novelty, RMM is resolution particle mass distribution information is currently an insufficiently explored technique which inherently obtained. We foresee that characterising could serve sectors where the particle number and 2 2D Mater. 8 (2021) 035044 L E Crica et al mass in suspension is of interest but difficult to obtain with conventional techniques. For the characterisa- tion of GBMs in aqueous dispersions in particular, we theorised that RMM could be more precise in meas- uring the particle mass and concentration of GBMs than other more traditional techniques. Indeed, the uniqueness of this technique stands in its ability to measure individual particles and clusters/aggregates in fluids without using spherical models, a major drawback of many alternative techniques for 2D materials. Herein, we characterised the physicochem- ical properties, including the particle mass and con- centration by RMM, of three types of graphene oxide (GO) aqueous dispersions, including one prepared in-house and two commercial products. To the best of our knowledge, these are the first mass measurements of GO sheets in an aqueous dispersion. We report the added value of measuring buoyant mass using RMM as an extremely sensitive technique that complements conventional microscopy. This new characterisation parameter could be of interest in the undergoing development of standards for the production and quality control of available or emerging 2D materi- als, where the same product name still serves as an umbrella for several materials despite their different physicochemical properties. Figure 2. GO material spectroscopic characterisation: structural differences between the lab prepared and 2. Results and discussion commercial GO suspensions, as determined by (A) Raman spectroscopy and (B) UV–VIS spectroscopy. I D /I G values represent the mean value ±SD of three measurements per To briefly check the origin of the three materials, sample. we evaluated their physicochemical, structural and morphological properties. Lab GO and Commer- cial GO-A presented the typical Raman fingerprints, whereas the spectra of Commercial GO-B indic- Colloidal stability is of great importance for RMM ated the existence of graphitic domains by the split- measurements: at high sedimentation rates, a fraction ting of the G band to G + D’ and by the pres- of the sheet population might be inaccurately meas- ence of a sharp 2D peak (figure 2(A)) (Pimenta et al ured, leading to poor measurements. 2007). This is also reflected by the reduced metric Microscopy analysis using TEM and AFM showed of disorder (I D /I G ratio) found for the Commer- predominantly small and thin layers within the Lab cial GO-B (I D /I G = 1.00 ± 0.01) in comparison GO (figure 3(A)) and larger thin sheets in Com- to the other two samples (Lab GO = 1.23 ± 0.03; mercial GO-A (figure 3(D)), whereas Commercial Commercial GO-A = 1.42 ± 0.01). Furthermore, GO-B contained mostly large and thick agglomerates the UV–VIS absorbance spectra of Commercial GO- (figure 3(G)). Overall, Lab GO contained monolay- B (figure 2(B)) suggests a more graphitic struc- ers with lateral sizes up to 1 µm (figures 3(B) and ture, free from functional groups, rather than a (C)). Single-layers also dominated in the Commer- GO-specific one (Alam et al 2018). These indica- cial GO-A sample, however their lateral size spanned tions correlate with our observations of the poor from ∼0.5 to a few tens of µm (figures 3(E) and (F)), colloidal stability of Commercial GO-B in water while Commercial GO-B contained mainly aggreg- (figure S1 (available online at stacks.iop.org/2DM/8/ ates with >100 nm thickness and micrometric sizes 035044/mmedia)). In contrast, the absorbance bands (figures 3(H) and (I)). at 230 nm (π → π ∗ transition of C=C bonds) and To assess the mass via RMM and because the lat- 310 nm (n → π ∗ electron transitions in the C=O eral size of the two commercial GO samples exceeded groups) shape the fingerprint of highly exfoliated and 1 µm (size limit for the nanosensor), we opted to use oxidised GO (Paredes et al 2008, Dhifaf et al 2016), only the microsensor, while the Lab GO was analysed for both Lab GO and Commercial GO-A samples. As using both sensors. Figure 4(A) shows the buoyant a consequence of the oxidised nature, which inhib- mass distribution of the three types of GO measured its platelet re-stacking via electrostatic repulsions with the micro-sensor. (Dreyer et al 2010), both Lab GO and Commercial Despite all materials having their maximum loc- GO are stable colloids in MilliQ water (figure S1). ated at a similar buoyant mass, the percentage of the 3 2D Mater. 8 (2021) 035044 L E Crica et al Figure 3. GO material morphology: aspect, lateral size and thickness determined by TEM and AFM for (A)–(C) Lab GO, (D)–(F) Commercial GO-A and (G)–(I) Commercial GO-B. Figures (C), (F) and (I) show the size and thickness of the flakes sectioned within the AFM images (B), (E) and (H), respectively. total counts was higher for the Lab GO. Moreover, contained thin particles with a lateral size down to Lab GO spanned from 6.18 fg (LOD micro) to a few tens of nm (20–50 nm by AFM). A fraction 84 fg, while the commercial GO-A and -B contained of these sheets would lie below the detection limit particles with buoyant mass up to 968 and 12 400 fg, of both the micro- and nano-sensors of the RMM respectively. The narrower mass distribution of Lab instrument. In agreement with this, the low particle GO suggested a lower sample polydispersity. When count of Lab GO measured using the microsensor measured with the nanosensor, the mass distribution suggested that the highest fraction of sheets was of Lab GO ranged from 0.75 to 12.3 fg (figure 4(B)). indeed below the detection limit. This was then con- In these gravimetric ranges, the average buoyant mass firmed using the nanosensor, which is more sensitive of the particles was 11.2 and 1.2 fg for Lab GO (micro- to lighter sheets. As expected, when using this sensor, sensor vs nanosensor), 24 fg for Commercial GO-A Lab GO contained a higher number of particles in and 28 fg for Commercial GO-B (figure 4(C)). 1 mg compared to Commercial GO-A and GO-B. All Next, the particle number n (#/mg) reported RMM results correlated with the AFM and TEM data, in figure 4(D) was calculated using the following where Lab GO contained thin small sheets, Commer- formula: cial GO-A had thin large sheets and Commercial GO- ( ) B contained larger and thicker graphitic aggregates. # Cp ml Based on these results, the authors disagree with n= ( ) Cm mg ml−1 defining the Commercial B sample as ‘graphene oxide’ and would highlight the importance in con- where Cp (#/ml) is the particle concentration and Cm sulting the good practice guides and ISO standard (mg ml−1 ) is the mass concentration of the GO stock. referenced here before labelling such materials (Pol- By using this formula instead of simply dividing 1 mg lard and Clifford 2017, Pollard 2017, ISO 2017). We by the average dry mass, we avoid an overestimation, believe that the average mass per sheet and total i.e. assuming that all particles in 1 mg of GO would number of sheets in dispersion are complementary be detectable by the instrument (figure S3). information that can be used for a more compre- Based upon previous work (Dhifaf et al 2016, hensive characterisation of GBMs. RMM could also Artur Filipe et al 2018), we expected that Lab GO serve as a quality control technique in the production 4 2D Mater. 8 (2021) 035044 L E Crica et al Figure 4. RMM analysis: (A) and (B) buoyant mass distribution of the three types of GO measured with the microsensor and nanosensor, respectively. The nanosensor provides better resolution for measuring the Lab GO as compared with the microsensor. (C) and (D) Average buoyant mass (±SD) and counts/mg (±SD) of Lab GO compared with Commercial GO-A and -B. Each sample was measured in triplicate, with 1500 counts per replicate. Statistical significance was assessed using one-way ANOVA, with ∗ <0.01. Figure 5. Impact of the LOD on particle measurement: the illustrations exemplify, in an oversimplified manner, why (A) the Lab-GO was poorly measured with the microsensor (high fraction of the flakes below the LOD), while (B) the nanosensor improved the measurement. of different batches of materials. This tool could be The development of suspended resonating sensors also used to evaluate the mass changes in GBMs with higher sensitivity towards less dense and smal- upon reduction, functionalisation and adsorption, ler sheets would be of great benefit for more accurate or to investigate the behaviour of GBMs in differ- measurements of the mass and number of graphene- ent solvents. The main advantages that RMM brings based platelets in dispersions. are the possibility to perform measurements directly Examples where RMM might be useful are in the desired solvents (as opposed to microscopy nanomedicine and nanosafety, where, similar to the in dry state) and applying non-spherical models (in lateral size (Ma et al 2015, Vranic et al 2018), the comparison to DLS or NTA). The limitations of the mass or number of sheets are complementary inform- technique rely on the dependence of detection limit ation for understanding the effects of 2D materials on primarily on the density and size of the material to be biological systems. Although most often the effects of tested. The graphic in figure 5 illustrates how the LOD GBMs in cell culture and animal models are expressed impacted on the RMM measurement of our Lab-GO. as a function of mass concentration, we have seen 5 2D Mater. 8 (2021) 035044 L E Crica et al here that, when comparing different materials, the MilliQ water and the excess was removed using fil- same mass can equate to different numbers of sheets. ter paper (WhatmanTM cat. no. 1001-070, grade no. The buoyant mass and particle count could be used 1). Next, GO suspension (100 µg ml−1 ) was added to better bridge the gap between the physicochemical and left to adsorb for 1–2 min, followed again by properties of 2D materials and their effects on living a washing step and removal of the excess. The ared systems. samples were then dried overnight at 37 ◦ C before imaging. The measurements were done in ScanAsyst 3. Conclusions mode using ScanAsyst Air probes on a Multimode 8 instrument (Bruker, UK). Data post-processing was Firstly, this study emphasises the potential of RMM done with Nanoscope Analysis version 1.4 (Bruker) as a novel technique for the characterisation of 2D dedicated software. materials, providing unique information such as the Transmission electron microscopy (TEM): mass and number of individual particles or aggreg- samples for TEM were prepared on 300-mesh carbon- ates in suspension. RMM offers an alternative for the coated copper grids (C300Cu, EM resolutions, UK). quantification of 2D materials in dispersion when A FEI Tecnai 12 BioTwin microscope (FEI, Eind- their optical properties cannot be explored. hoven, NL), operating at 100 kV and equipped with Secondly, this paper highlights the importance an AMT digital camera (Gatan, UK) was used. of thoroughly describing and understanding differ- Resonant mass measurements (RMM): RMM was ent types of GBMs defined by similar nomenclature. performed on an Archimedes instrument (Malvern Here, we compared a lab-prepared GO with two dif- Panalytical, UK) operating with Archimedes software ferent commercially available GO aqueous disper- version 1.21. Based on the specifications of the instru- sions. We report for the first time that the mass and ment, the target concentration for best measurements concentration of GO sheets can be measured using would be 8 × 106 particles ml−1 for the microsensor suspended resonating sensors. Significant differences and 2 × 108 particles ml−1 for the nano-sensor. The in terms of average buoyant mass and particle counts LOD was optimised to 0.04 Hz for the microsensor were identified between the three different types of and to 0.016 Hz for the nanosensor. The rationale GO, results which were in agreement with particle size in setting the LOD was to enable the measurement and thickness by AFM and TEM. of lightest possible GO particles without significant background noise. A low background noise would 4. Experimental normally be seen as zero to few particle counts in the Materials: an aqueous dispersion of small GO sheets water reference sample, which was run before each prepared as previously described (Dhifaf et al 2016, measurement. In addition, the coincidence factor, Artur Filipe et al 2018) was compared with two which is a measurement indicator referring to meas- commercially available GO dispersions. Samples were urement duplicates (e.g. particle clusters) was mon- labelled as Lab GO, Commercial GO-A and Commer- itored to be maintained below 3%, where possible. cial GO-B. The particle density was fixed to 2.26 g cc−1 , i.e. the Raman spectroscopy: Raman spectroscopy was density of crystal graphite (Dismukes et al 1993), and performed on drop-casted GO (100 µg ml−1 ) on glass samples were measured in water until 1500 counts slides using a calibrated micro-Raman spectrometer were reached. Results are reported as particle mass (Thermo Scientific, UK), with laser of 633 nm and distribution, particle average mass and counts per mg. 0.4 mW. The objective and pinhole aperture were Statistical analysis: the buoyant mass and number 50× (NA 0.75) and 50 µm, respectively. Three meas- counts per milligram were expressed as means with urements of different spots were acquired for each standard deviation (±SD) and evaluated for statist- sample. The raw data was processed by fluorescence ical differences by one-way ANOVA using OriginPro correction and further normalised by the intensity of SR2 (b9.4.2.380, Academic). the G band. UV–Visible (UV–VIS) spectroscopy: UV–VIS Data availability statement absorbance spectra were recorded using a Cary 50 Bio UV/Vis spectrophotometer (Varian Inc., Agi- All data that support the findings of this study are lent Technologies, UK). Serial dilutions of GO (2.5– included within the article (and any supplementary 20 µg ml−1 ) were prepared and their absorbance at files). 200–800 nm was recorded. All measurements were performed at room temperature, using MilliQ water Acknowledgments (Merck-Millipore, UK) as blank. Atomic force microscopy (AFM): AFM samples This work was supported through a studentship were prepared on 0.01% poly-L-lysine (PLL, Sigma offered by the EPSRC Centre for Doctoral Train- Aldrich, UK) coated mica substrates (Agar Scientific, ing in the Science and Applications of Graphene UK). PLL was adsorbed on freshly cleaved mica for and Related Nanomaterials (GrapheneNOWNANO 1–2 min, then the substrate was gently washed with CDT; EP/L01548X/1) within the Nanomedicine Lab 6 2D Mater. 8 (2021) 035044 L E Crica et al at the Faculty of Biology, Medicine and Health Kauling A P, Seefeldt A T, Pisoni D P, Pradeep R C, Bentini R, (FBMH) and the National Graphene Institute, The Oliveira R V B, Novoselov K S and Castro Neto A H 2018 The worldwide graphene flake production Adv. Mater. University of Manchester, UK. The microscopy facil- 30 1803784 ities at the FBMH EM Core Facility and the Bioima- Kobayashi H, Maeda S, Kashiwa M and Fujita T 2014 ging Facility and the Wellcome Trust were accessed in Measurement and identification of ultrafine bubbles by this study. The authors would also like to acknow- resonant mass measurement method 9232 Int. Conf. on Optical Particle Characterization (OPC 2014) (SPIE) p 5 ledge Artur Filipe Rodrigues and Cyrill Bussy for Krueger A B, Hadley J, Cheney P P, Markova N, Carpenter J F and their scientific advice and for critically reading this Fradkin A H 2019 Application of a best practice approach manuscript. Malvern Panalytical UK funded and using resonant mass measurement for biotherapeutic offered technical support for all resonance mass product characterization J. Pharm. Sci. 108 1675–85 Lewis C L, Craig C C and Senecal A G 2014 Mass and density measurements. measurements of live and dead gram-negative and gram-positive bacterial populations Appl. Environ. Authors contribution Microbiol. 80 3622–31 Ma J, Liu R, Wang X, Liu Q, Chen Y, Valle R P, Zuo Y Y, Xia T L E C synthesized the GO, characterised all samples, and Liu S 2015 Crucial role of lateral size for graphene oxide in activating macrophages and stimulating designed the study and wrote the manuscript. T J D pro-inflammatory responses in cells and animals ACS Nano and E A G offered support and advice for Archimedes 9 10498–515 use and RMM data interpretation. K K designed and Nejadnik M R and Jiskoot W 2015 Measurement of the average directed the study, offered intellectual support and mass of proteins adsorbed to a nanoparticle by using a suspended microchannel resonator J. Pharm. Sci. contributed to writing the manuscript. 104 698–704 Nittayacharn P, Leon A D, Abenojar E C and Exner A A 2018 The ORCID iDs effect of lipid solubilization on the performance of doxorubicin-loaded nanobubbles 2018 IEEE Int. Ultrasonics Livia Elena Crica  https://orcid.org/0000-0002- Symp. 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