Principles around Accurate Blood Volume Collection Using Capillary

Nov 21, 2017 - The authors thank Trajan Scientific and Medical for providing technical support. The authors would like to acknowledge funding from the...
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Principles around accurate blood volume collection using capillary action Florian Lapierre, Andrew A. Gooley, and Michael Breadmore Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02825 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 25, 2017

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Principles around accurate blood volume collection using capillary action Florian Lapierre1,2, Andrew Gooley1,3, Michael Breadmore1,2 1

ASTech, ARC Training Centre for Portable Analytical Separation Technologies, Australia

2

Australian Centre for Research on Separation Science, School of Physical Sciences, University of Tasmania, Private Bag 75, Hobart, Tasmania 7001, Australia

3

Trajan Scientific and Medical, 7 Argent Place, Ringwood, Victoria 3134, Australia

Abstract Capillary action is one mechanism microfluidics uses to draw liquid autonomously in a substrate without the need of external energy. This behaviour can be exploited to collect accurate volumes of liquids such as blood in narrow columns known as capillary tubes and help the development of inexpensive, user-friendly personalised biomedical tools. Precision bore glass capillaries demonstrate the ‘state of the art’ for volume accuracy and precision but height and radius must be carefully chosen in order to exploit the capillary action behaviour efficiently. This article investigates the influence of surface glass aging, due to prolonged exposure to humid air, and hematocrit level, on the blood capillary rise. It provides also the tools to correctly define the optimum capillary dimensions to collect an accurate volume of blood in a glass capillary tube.

Introduction Affordable health care is one of the most significant problems facing both developed and developing countries around the world today. The significant drive towards improved health care in the next few decades will rely extensively on personalised medicine which by its very nature will require the collection and rapid analysis of individual samples with regularity, at a cost that is affordable and with simplicity to be operated by untrained users 1,2. The development of inexpensive, easy to use personalised diagnostic tools is a challenge ideally addressed by microfluidic technology platforms 3. Microfluidics offers the advantages of using small volumes of sample and reagent, faster processing, and the ability to integrate multiple chemical and biochemical processes in a single device for ease of automation and user-friendliness, allowing widespread use. However, while the literature and market seem to focus mainly on how to process these liquids efficiently for analysis 4, there has been little focus on how to collect a volume of fluid with high precision and accuracy; a critical aspect to perform quantitative analysis of drugs/biomarkers in small liquid samples (i.e. using LC-MS). A few tools have been developed (i.e. syringes, pipettes) that are omnipresent for larger volumes (10 µL or greater), but the collection of smaller volumes has so far been restricted to laboratory settings with equipment operated by experienced users. One microfluidic solution that can provide a simple way to collect fluid relies on the use of “capillary driven flow” or “capillary action”. Capillary action is defined by the movement of fluid via surface tension and adhesive forces and is easily seen when handling blotting paper 5. The same mechanism also applies in narrow columns, namely capillaries, where the surface tension force is greater than the gravitational and viscous forces hereby filling the capillary tube with the solution without the use of pumps or any other external energy 6. The process is entirely autonomous, relies only on the properties of the liquid, surface and size of the capillary, is spontaneous and works with any inclination of the capillary. The volume collected is defined by the capillary dimensions and it does not overfill. Thus, when the capillary tube is filled with the liquid from one edge to another, the filling

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stops automatically, leaving the desired volume of fluid inside the capillary tube. The accuracy and precision of the volume collected is therefore defined by the tolerances with which the precision bore capillary can be made. Capillaries solely dependent on capillary action for filling are not commonly used for blood collection and most phlebotomists generally collect blood with capillary tubes which do not rely on such behaviour. The consequence of using capillaries not optimised for capillary action collection, is that the phlebotomist needs to negatively incline the capillary tube to enable the blood to flow into it. Breaking the contact between blood and the capillary for an instant increases the risk of introducing an air bubble inside the capillary, leading to inaccurate volume blood collection. With such problems, it is impossible to rely on the inexperienced patient being able to collect the blood sample themselves and guarantee the quality of the sample. However, precision glass capillaries have the capability of collecting fixed accurate volumes of liquid (up to 99.5% for the commercially available Vitrex capillary) via capillary action with very high precision (%RSD < 0.75%) from one edge of the tube to the other. Nevertheless, there is a requirement to determine the appropriate dimension. One limitation of such technology is that one capillary can collect only one defined volume of liquid. Such performance has not been overcome by any other laboratory technologies (i.e. pipette) and a precision bore glass capillary represents today the state-of-the-art for precision liquid collection. This work examines the limit and influence of surface and blood properties on the volume of blood that can be accurately collected through capillary action.

Theory The dynamics of capillary flow has been studied since the 17th century and is applied in a myriad of applications, especially in the medical field 7. The most common law describing capillary action in a cylindrical tube is defined by Equation 1 8. The height h reached by the liquid via capillary action is dependent on the liquid-air surface tension γ, the contact angle θ between the liquid and the surface of the tube, the density ρ of the liquid, the local acceleration g due to gravity and the radius r of the capillary tube. Thus, for a column of a specified radius, there is a limit to the height with which it can be filled, which in turn through Equation 2 defines the volume V of fluid collected inside the cylindrical capillary tube. Equation 1: height h of a liquid filling a column of radius r, surface tension γ, contact angle θ and liquid density ρ.

ℎ=

2ߛ cos ߠ ߩ݃‫ݎ‬

Equation 2: Volume V of a cylinder of radius r and height h

ܸ = ߨ‫ ݎ‬ଶ ℎ Equation 1 shows that the capillary rise is dependent on the fluid properties and on the surface chemistry of the capillary bore. The capillary action is therefore limited to a range of capillary dimensions for specific liquids and capillary material properties; in other words, the liquid to be collected and the nature of the capillary will influence the volume of liquid collected. To understand this limitation, the Washburn-Lucas equation was developed in 1921 9. The equation describes the capillary flow of Newtonian liquids in cylindrical tubes to determine the distance travelled by a liquid in a narrow column via capillary action over time 10. The Washburn Equation 3 is defined as: Equation 3: Washburn equation from 11

ℎ ℎ ‫ݐ‬ሺℎሻ = −ܶ ൤ + cos ߠ ∗ lnሺ1 − ሻ൨ ‫ܮ‬ ‫ ∗ ܮ‬cos ߠ

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ଶఊ

ଵ଺ఊఎ

Where ‫ = ܮ‬ఘ௚௥ and ܶ = ఘమ ௚మ ௥ య are characteristic length and time scales that depend on the radius r of

the capillary and on the liquid properties (viscosity η, density ρ, and surface tension γ), and t(h), the time to fill the capillary to height h 11.

Even though the law is based on Newtonian liquids, recent publications have validated such law for non-Newtonian liquids such as blood for specific conditions. Indeed, whole blood exhibits a Newtonian behaviour with a constant viscosity at high shear stress – i.e. when the blood flows rapidly into a tube – and blood capillary rise was well predicted through the use of such law 12,13. Therefore, from this equation, knowing the dimension of the capillary tube, the liquid characteristics and the contact angle, it is possible to predict whether or not a narrow column can be completely filled with the desired liquid, as well as the time required to fill. Conversely, knowing the capillary dimensions and fluid properties, it is possible to determine the contact angle by measuring the time required to fill the column 14.

Methods Blood preparation (with different hematocrit levels) Capillary blood was collected from a healthy male volunteer using a standard finger lancet blood collection protocol: the puncture site was cleaned with an isopropanol wipe and allowed to dry. A lancing device (UniStik3, Owen Mumford, UK) pricked the finger and the first expressed volume of blood (~ 30 µL) was removed. The finger was then gently massaged to promote blood flow and to collect up to 500 µL of blood into an EDTA coated tube (BD Microtainer K2E tubes, US). Hematocrit (HCT) level was adjusted from 35 % to 53 % by the addition or withdraw of the plasma fraction in whole blood. A blood sample was centrifuged at 10000 g for 2 min and a volume of plasma (upper fraction) was collected and redistributed to other blood samples, thus increasing or reducing the red blood cell concentration. Each sample was assessed using the hemoCue® Hb 201+ System (hemoCue AB, SE) and samples of blood were used within one hour. The volunteer signed a consent form stating their understanding of the experiment and protecting their privacy. Ethical approval was given by ethical committee at the University of Tasmania (application number: H0015476) and any remaining blood volume was safely discarded. Borosilicate glass capillaries Precision bore borosilicate glass capillaries with a hydrolytic resistance Class HGB1 (intended for pharmaceutical use) were purchased from Vitrex with a fixed volume of 3 µL. The inner radius was measured at 181 µm ± 0.005 µm with an average height of 28.977 mm ± 0.047 µm (%RSD = 0.16 %). A dual dye ratiometric photometry volume measurement system (VMS, Artel NH) was used to validate the volume of fluid collected. Volumes were measured at 2.994 µL ± 0.020 µL (accuracy = 99.81 %) with a %RSD of 0.68 % (see Supporting Information Table S1) and are in correlation with the manufacturer’s specification. These high quality medical tubes are inert and specialised for the handling and transporting of liquids while preserving their intrinsic integrity. Cover glass Borosilicate cover slips with the same hydrolytic resistance Class HGB1 to the capillary tubes were provided by Trajan Scientific & Medical. Contact angle measurements with 5 µL of blood (HCT measured at 44 %) were performed on the cover slip before the annealing process, and after being exposed to humid air for a defined time (0 to 4320 min). Annealing process Capillaries and cover slips were annealed with a ramping temperature up to 600 °C for ½ hour and slowly cooled down to room temperature for another ½ hour. Following the annealing process, the

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capillaries and cover slips were stored in a tropical aluminium packaging containing desiccant. The latter avoids moisture accumulation on the glass surface and light exposure from the environment. It is to be noted that no washing steps were performed at any time. The capillary tubes used in this study are certified high quality medical tubes for handling and transporting fluids. Exposure time After the annealing process, sets of capillaries and cover slips were left in an air-conditioned room with relative humidity (RH) and temperature set at 45 % and 23 °C, respectively. Capillaries were then used for blood collection at defined times (0 to 4470 min). Capillary blood collection For each blood sample at a defined HCT level and each capillary exposure time, 10 capillaries were used for collection. The capillaries were held in a vertical position against gravity on a custom-made X-Y-Z stage incorporating a support with engraved grooves ensuring the vertical inclination of the capillary tubes. The collection of blood from the bottom edge to the top was performed by bringing a volume of blood at the vicinity of the lower edge of each capillary tube. A video camera recorded the filling of the 10 capillaries at 24 frames per seconds (see video S1). Video processing Using ImageJ software, the video of 4 capillaries was processed with the following procedure: a line was drawn inside the capillary where the blood flowed via capillary action (Figure 1.a). This line represents the length of the capillary tube (h = 28.977 mm) from one edge to the other. Using the “reslice” function, a time space plot image was generated (Figure 1.b). This image illustrates the position of the blood inside the capillary (Y-axis) in time (X-axis). By adjusting the colour threshold, the edge detection of the interface blood/air in the capillary can be extrapolated (Figure 1.c). The image was finally converted as XY coordinates where Y-axis is equal to the length of the capillary (h = 28.977 mm) and X-axis to the time (1 pixel = 41.708ms).

Figure 1: (a) Picture from the video source at 24 frames per seconds. 10 capillaries (h = 29mm | r = 0.181µm) are held vertically. EDTA blood contained in an Eppendorf vial is brought in contact with the bottom edge of the capillary tube until the capillary is filled with blood. (b) Time space plot of the filling of one capillary. The arrow points from the original frame to the corresponding time space plot location. (c) Resulting image after red colour threshold and edge detection. X and Y axis are converted from pixels to seconds and millimetres, respectively.

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Results and discussions Capillary action model The purpose of this article is to give the reader the tools to choose the optimum capillary dimensions for collecting accurately a defined micro-volume of liquid (e.g. blood) via capillary action. We have correlated the study based on the theoretical and experimental analysis of precision bore borosilicate glass capillaries (exposed to humid air for a defined time) with blood (at different HCT level) using contact angle measurements and capillary rise behaviour. A preliminary study explored the significance of borosilicate glass exposure to humid air as determined by contact angle measurements using glass cover slips with 5 µL drops of whole blood at constant HCT. Annealed cover slips exposed to a clean humid environment showed an increase in contact angle, modulating from ~10° (annealed coverslip) to 36° within 72 hr. The contact angle was measured at 70° for the non-annealed cover slip (See table S2 in Supporting Information). This indicates that there is an aging process of the borosilicate glass surface chemistry which depends on its storage condition, and most likely its humidity environment 15,16. Moreover, the minimum contact angle of whole blood with annealed borosilicate glass never reaches values below 6°; no superhydrophilic behaviour was observed. No hydrophobic behaviour (≥ 90°) was measured either with exposed borosilicate glass and whole blood. This means that borosilicate glass will neither exhibit a behaviour where it absorbs completely the total volume of blood on its surface nor repels the water molecules in such a case that the capillary action would not operate. These defining contact angles were inserted in the capillary action Equation 1 to predict the possible capillary tube radius and height where capillary action would overcome gravitational force. For blood, the average density is estimated at ρ = 1060 kg.m-3 17, and the surface tension is at γ = 0.058 N/m 18. The yellow dotted line in Figure 2 corresponds to the capillary height and radius that can be filled by capillary action when the contact angle between blood and borosilicate glass is 10°. All the dimensions below this curve can be filled via capillary action if the contact angle stays at 10°. Following the same principle, the green dotted line represents the capillary dimension limit when the contact angle is 70°. Again, all the dimension below this curve enable capillary action when the contact angle is of 70°.

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Figure 2 : Limit of capillary tube diameter and height modelled with the capillary action Equation 1 for contact angle between blood and borosilicate glass of 10° (yellow dot line) and 70° (green dot line). The blue dash line represents the possible dimensions for a fixed volume of 3 µL. The bold section of the line represents the possible dimensions of a 3 µL capillary when the contact angle between blood and borosilicate reaches its maximum value of 70°; h70 and 2r70 are the maximum height and minimum diameter at such condition. The red dots represent the defined capillary tubes provided by Vitrex with volumes of 3, 5, and 10 µL. Each dot intersects the capillary action Equation 1 when θ = 61.5° (3 µL), θ = 52.5° (5 µL), θ = 30.5° (10 µL).

From these curves, there exists a range of diameters and heights where the capillary force overcomes gravitational force, and where the defined capillary tube will be entirely filled. For instance, the blue dash line represents all the capillary dimensions that will give a fixed volume of 3 µL. The intersection of the blue line with that calculated with a contact angle of 70° defines the maximum height (h70 = 15.2 mm) and minimum diameter (2r70 = 0.502 mm) that can be filled by capillary action under these conditions (bold section of the blue dash line). If the height of the capillary is larger than 15.2 mm, then the capillary will not completely fill and the volume will not be accurately collected. Following the same reasoning, if the contact angle between blood and borosilicate is only 10°, the maximum capillary height of h10 = 126.3 mm and minimum diameter of r10 = 0.087 mm are calculated. The same logic can be applied for volumes of 5 µL and 10 µL, with the calculated results summarised in Table 1.

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Table 1: Maximum capillary height and minimum capillary diameter for their corresponding volumes when the contact angle between blood and borosilicate glass is maximum at 70° and minimum at 10°. Using the Vitrex capillaries, the capillary tube dimensions are fixed for each volume and the maximum contact angle between blood and borosilicate θmax can be estimated. If the contact angle between blood and borosilicate glass goes beyond that value, the capillary action is overcome by gravitational force and the capillary tube will not be filled entirely, losing its volume accuracy capability.

Volume (µL)

θ = 70° h70 (mm) 2r70 (mm)

θ = 10° h10 (mm) 2r10 (mm)

3

15.2

0.502

126.3

0.174

5

9.1

0.836

75.8

0.290

10

4.5

1.682

37.9

0.580

Vitrex capillary hcap = 29 mm 2rcap = 0.363 mm hcap = 29 mm 2rcap = 0.468 mm hcap = 29 mm 2rcap = 0.663 mm

θmax (°) 61.5 52.5 30.5

Alternatively, Equation 1 can also be used to determine the maximum contact angle θmax until which the capillary force will not be sufficient to fill a defined capillary tube entirely with blood. For instance, Vitrex borosilicate capillaries have defined height and radius with their respective volume of 3, 5, 10 µL, and represented as a red dot in Figure 2 and in Table 1. θmax is estimated from Equation 1 at 61.5° for a 3 µL volume capacity, 50.5° and 30.5° for volumes of 5 and 10 µL, respectively. These contact angles are all below the maximum contact angle measured between blood and borosilicate glass θ = 70°. This means that there exist some conditions where the blood will not fill entirely the capillary tube with capillary action. In other words, if the contact angle between blood and the bore of the capillary passes beyond the limit value θmax, then, the capillary tube will not be able to be filled entirely and the volume accuracy of blood collection will be lost. Knowing that the maximum contact angle between blood and borosilicate glass is 70°, the range of capillary dimensions for which a volume of 3 µL can be accurately collected is h ≤ 15.2 mm and with a radius of ≥ 0.502 mm. Capillary height and radius for 5 µL and 10 µL volumes are summarised in Table 1. With capillaries of these dimensions, theoretically, no matter the condition of storage of the borosilicate glass, the capillary action will always be effective and the entire volume will be filled with blood. Washburn model & experimental data The capillary action model described above provides whether or not the volume can be collected, but not the time required to do so. This can be estimated from the Lucas-Washburn equation which characterises the height h travelled by a liquid - in this study, blood - via capillary action for a given time t. Figure 3 shows the distance travelled by blood in time inside borosilicate capillaries exposed for different periods of time to humid air, and of h = 29 mm and 2r = 0.363 mm (V = 3 µL). For the black square values, the capillary tube was freshly annealed while for the red circle, blue triangle and purple triangle, the filling measurements were performed 240, 360 and 480 min after the annealing process. It is to be noted that the same source of blood (hematocrit level measured at 35 %) was used for each of these experiments. The Washburn equation was then fitted on each curve by setting the viscosity at 0.0035 Pa.s, the density of blood (HCT 35 %) at 1044.3 kg.m-3 17, the surface tension at 0.058 N/m and radius at 0.181 mm. The resulting contact angle for each experimental data was then calculated from the fitted curves. The coefficient of regression R2 is modulating between 0.95 to 0.99 showing good correlation between experimental data and the Washburn equation. It is clear from these data that the speed at which the capillary tube is filled decreases with the time of capillary exposition to air and humidity, taking 1.2 sec with dry/fresh capillary to 2.6 sec after 480 min exposition to humid air. Given that the same source of blood was used with constant HCT, this

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reflects a change in contact angle. From a freshly annealed capillary tube, the contact angle is estimated at 33.62° and increases up to 52.26° after exposition to humid air for 480 min.

Figure 3: Whole blood at HCT 35 % capillary rise (height) versus time for 3 µL volume borosilicate tube (h = 29 mm – r = 0.181 µm) with (black square) 0 min, (red circle) 240 min, (blue triangle) 360 min, and (pink triangle) 480 min exposure to humid environment (RH @ 45 %). Dash lines correspond to the Washburn equation fitting of the experimental data to extrapolate the resulting contact angle between blood and the borosilicate bore (R2 > 0.9). The contact angle increases with the increasing exposure time to humid air.

In order to understand the impact of blood HCT level, contact angle and time to fill the 3 µL capillary tubes were measured by modulating the HCT concentration from 35 % to 53 % (see Table S3 for corresponding blood physical properties) and exposition to humid environment from 0 to 4470 min. Figure 4 compares the experimental data (red square) of capillary filling time and contact angle with the theoretical Washburn Equation 3 (black dash line) given for a 3 µL borosilicate capillary tube with h = 29 mm and r = 0.181 mm. From the theoretical curve, one can notice that for contact angle below 30°, the time to fill the capillary is stabilised at around 1 s. However, from contact angle greater that 55°, the time to fill the capillary is greater than 2.5 s. This means that for the latter range, a small change in contact angle will result in a large change in the filling time, which will extend even further as the contact angle gets closer to 61.5°. Beyond this contact angle, the 3 µL capillary tube cannot be filled entirely. Also, the model and experimental data show that if the time to fill the capillary tube is greater than 12.05 sec, then the capillary is not entirely filled. If the contact angle between blood and the glass surface increases beyond 61.5°, then the capillary tube will never fill entirely. This was experimentally verified using capillaries left 6 months after annealing (contact angle of 64.69°), and the blood reached a maximum height of 27 mm in 16 sec ± 4 sec. This is less than the full height of 29 mm, preventing the complete filling of the capillary. Theoretical curves for volumes of 5 µL (purple

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dash line) and 10 µL (green dash line) show the same behaviour while their limits are estimated at 52.5° and 30.5° - identical to that predicted by the capillary action Equation 1.

Figure 4: Experimental data (squares) of contact angle θ and filling time t for different time of exposure to humid air (gradient of red) and HCT levels with the corresponding theoretical prediction (black dash line) for a capillary tube of 3 µL (r = 0.181 mm – h = 29 mm) from Equation 3. The limit at 61.5° is also predicted from the Washburn equation with such capillary. Purple and green dash lines correspond to the theoretical predictions from Equation 3 for capillary tubes of volumes 5 µL and 10 µL, respectively. Limits at 52.5° and 30.5° are also calculated from such glass capillary dimensions with whole blood.

Experimental data (22 different conditions of HCT level and exposure time, repeated 4 times) fits with the theoretical Washburn-Lucas curve. Comparing the different hematocrit samples, the contact angle and filling time increase with the increasing HCT level. Moreover, for low HCT level and low exposure time, the standard deviation is greater on the contact angle than on the filling time, while for high HCT level and long exposure time, the standard deviation is greater on the filling time than on the contact angle. This behaviour is in correlation with the theoretical curve where a small change in the filling time implies a large contact angle variation in the 0-30° contact angle zone. On the contrary, in the 55-61.5° contact angle zone, a small change in contact angle variation implies larger filling time variation. In both cases, the Washburn equation’s prediction is accurate with experimental data and that the filling time depends on both the aging time of the capillary, as well as the HCT level in the sample. Knowing that the contact angle between different sources of blood and borosilicate glass modulates from 30° to ~60°, the 5 µL volume Vitrex capillary tube can only be filled when the contact angle is below 52.5° (purple dash line in Figure 4). Hence, only 54 % of the experimental hematocrit and capillary combinations will fill this volume. For the 10 µL Vitrex capillary tube (green dash line), only a freshly annealed capillary tube sampled with low HCT blood can entirely fill the capillary.

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From a practical perspective, knowing that the surface chemistry of the glass capillary can change over time, it is possible to select the optimum capillary height and diameter in order to collect a desired volume of blood accurately – even in the worst storage condition. For instance, by selecting a capillary of height of 15.2 mm and diameter of 0.502 mm for a total volume of 3 µL, even with a 70° contact angle, the capillary action will be effective. Based on the Washburn equation, it would take 0.148 sec to fill the capillary entirely with a contact angle of 10° with blood while it would take 5.925 sec at 70°, thus providing robust conditions under which an accurate volume of blood can be collected quickly by capillary action.

Conclusion Capillary action is a simple approach for the collection of small volumes of liquid that can bring effortless and accurate blood collection directly to the patient. However, capillary action must come with the understanding of its advantages (i.e. no generation of air bubble, fast, no external energy required) and limitations (i.e. small volumes, limited dimensions, liquids and surface chemistry) in order to develop the adequate tool. In this article, we show through theoretical models and experimental data that capillary action is dependent on the liquids used and the properties of the capillary (i.e. dimension, and nature of material). The annealing process of the borosilicate glass resets the surface chemistry to its original state and the use of tropical packaging (i.e. aluminium container with desiccant cap) enables the storage of the capillaries for months without affecting the glass surface chemistry. Keeping the glass in its natural inert state is primordial to preserve the intrinsic quality of the collected sample for clinical analysis. We have shown that blood exhibits Newtonian behaviour when filling the capillary and that this behaviour and affirmations are in correlation with models and experiments found in the literature. This study also shows that the surface chemistry of borosilicate glass changes when exposed to a humid environment. We also showed that a greater HCT level in blood leads to greater contact angle in the capillary, limiting the capillary action behaviour. Based on the capillary and Lucas-Washburn equations, it is possible to predict the behaviour of liquids such as blood in defined capillary tubes and calculate the optimum capillary height and radius to enable the collection of 3, 5 and 10 µL of blood in any situation, by anyone. The integration of these capillaries in an embodiment such as the hemaPEN® will enable blood microsampling from finger prick. The accuracy of blood volume is defined by the quality of the glass capillary and its dimension, and blood collection should be totally independent of the user. Blood collection would happen within seconds while avoiding the generation of air bubbles during the collection.

Acknowledgement The authors thank Trajan Scientific and Medical for providing technical support. The authors would like to acknowledge funding from the Australian Research Council through the Training Centre for Portable Analytical Separation Technologies (IC140100022). MCB is the recipient of an ARC Future Fellowship (FT130100101).

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