Pushparani Micheal Raj*a,
Laurent Barbeb,
Martin Anderssonb,
Milena De Albuquerque Moreirab,
Dörthe Haasea,
James Woottona,
Susan Nehzatia,
Ann E. Terrya,
Ross J. Frielc,
Maria Tenjeb and
Kajsa G. V. Sigfridsson Clauss*a
aMAX IV Laboratory, Lund University, Lund, Sweden. E-mail: pushpa.micheal@gmail.com
bDept. Materials Science and Engineering, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
cSchool of Information Technology, Halmstad University, Halmstad, Sweden
First published on 7th September 2021
Some of the most fundamental chemical building blocks of life on Earth are the metal elements. X-ray absorption spectroscopy (XAS) is an element-specific technique that can analyse the local atomic and electronic structure of, for example, the active sites in catalysts and energy materials and allow the metal sites in biological samples to be identified and understood. A microfluidic device capable of withstanding the intense hard X-ray beams of a 4th generation synchrotron and harsh chemical sample conditions is presented in this work. The device is evaluated at the K-edges of iron and bromine and the L3-edge of lead, in both transmission and fluorescence mode detection and in a wide range of sample concentrations, as low as 0.001 M. The device is fabricated in silicon and glass with plasma etched microchannels defined in the silicon wafer before anodic bonding of the glass wafer into a complete device. The device is supported with a well-designed printed chip holder that made the microfluidic device portable and easy to handle. The chip holder plays a pivotal role in mounting the delicate microfluidic device on the beamline stage. Testing validated that the device was sufficiently robust to contain and flow through harsh acids and toxic samples. There was also no significant radiation damage to the device observed, despite focusing with intense X-ray beams for multiple hours. The quality of X-ray spectra collected is comparable to that from standard methods; hence we present a robust microfluidic device to analyse liquid samples using synchrotron XAS.
The high brilliance synchrotron light facilities worldwide provide high-intensity X-ray beams to determine the molecular structure, fingerprinting and study of many previously invisible dynamics and interactions lying in multiple layers of an atom.7 Synchrotron facilities offer X-rays in a wide range of energies (from a few hundred eV to tens of keV), and the energy matches orbitals of the atoms (ionising radiation). The radiation interacts with matter (transmitted, scattered or absorbed). It is used to study samples in all physical forms from gas, liquid and solid (crystalline as well as amorphous) using a range of X-ray techniques (scattering, diffraction, 2D–3D-imaging and spectroscopy). However, in addition to providing spatial and/or structural and electronic information, the ionising X-ray beam can damage the samples during analysis. Water-containing samples are sensitive to X-ray induced radiolysis of the water, which forms both gas (bubbles) and aqueous free electrons, that readily reduce high valence metal ions (radiation-induced reduction). For a liquid sample, this effect can be avoided/minimised by continuously exposing a fresh sample to the beam by, for example, using a flow cell.
X-ray spectroscopic techniques are based on the photoelectric effect where an inner shell electron absorbs the X-ray, and the photoelectron is ejected from the atom creating a core hole. X-ray absorption spectroscopy (XAS) is an element-specific probe to determine the local atomic and/or electronic structure of matter. It has been a key tool to collect spectroscopic information about atomic energy level structure for decades. This information is obtained by tuning the X-ray photon beam energy to match the core electron excitation energy range. The edges possibly determined are K-, L- and M-, named by which core electron is excited (quantum numbers n = 1, 2 and 3 correspond to K-, L- and M-edges, respectively).8 The broad range of the X-ray sources' tunability makes this synchrotron-based analyses a powerful tool to determine oscillatory structure above the absorption edge, furnishing information about interatomic distances, near neighbour coordination numbers and e.g., identity. This is provided by Extended X-ray Absorption Fine Structure (EXAFS).
Similarly, X-ray Absorption Near Edge Structure (XANES) gives information on the unoccupied valence orbitals, oxidation state, and coordination geometry (bond angles). Thus, it is compelling to combine the best of microfluidics and X-ray spectroscopy; so that one may study samples without damaging and whilst consuming low sample quantities (typically a few tens to hundreds of microliters). The small footprint and modular nature of a microfluidic platform eases integration into the X-ray detecting setup and can meet requirements such as, for example, glove box containment, which reduces the risk of exposure to hazardous samples. Thus, microfluidics are potentially invaluable when combined with analytical methods like synchrotrons or X-ray Free-Electron Lasers (XFELs). Several microfluidic devices are presented in the literature for various studies involving diffraction, small-angle scattering, serial crystallography and others.9–12 Successful experimentation and data collections for protein structure determination, protein crystallisation,13 studies on hydrated cells, characterisation of cells,9 production of hexosomes,14 and other in situ studies15–17 have been reported. The devices have widely been used in scattering and diffraction studies using synchrotron facilities. Microfluidic devices used for analysing liquids using XAS reported in literature are limited to specific applications, such as vacuum studies,18 soft X-ray XAS (energy range 0.1–1 keV)19 or hard XAS (>1 keV) at lower energy levels (e.g. 4 keV).20
The microfluidic device reported here permits continuous screening and determination of the K-edge (and L-edges) of various metals in solutions at different concentrations and allows atomic structure determination of the metal of interest. The main challenge was to create a microfluidic chip capable of handling fluids with negligible sample damage and deposition of exposed material on the microchannel walls during beam scans over the same position for extended experiment durations. Optical transparency was considered a significant asset to view the sample movement during the X-ray scans through a visible light camera integrated into the beamline sample stage. Without this feature, it is not easy to confirm the correct sample flow through the device. The device presented here has high potential to be used for various hard X-ray absorption spectroscopy studies, as the materials used are X-ray robust, biocompatible and chemically compatible with several substances. The energy range in the synchrotron facilities is high and tuned to specific energies of interest. The SiG device performed with full functionality for more than 24 h of data collection. X-ray transparent materials are not well suited to the microfluidic chip processing as they do not etch well and so glass is superior in this instance for channel accuracy and thereby, microfluidic device discussed here could be a satisfactory alternative to study chemicals, metalloproteins, cells, etc., with a high energy source like synchrotron.
Microfluidic platforms are fabricated using various materials such as paper, thermoplastics, hydrogels, silicon, and glass. The materials' surface properties are vital in choosing the appropriate one for the application.12,17,21,22 Hence, material choice is considered vital in any microfluidic research based on the experimental method and samples involved. The microfabrication technique is also a dominant consideration for spectroscopy techniques, such as XAS, as the surface is required to be smooth to avoid/reduce any undesirable scattering effects from a materials surface roughness.23 The material and fabrication technique considerations needed for the XAS analysis method led to the present work. A silicon–glass plasma-etched and anodically bonded microfluidic device to flow samples and study sample behaviours by interacting with high energy X-rays was developed and validated in this work.
Once the layers were bonded tightly, the inlets and outlets were connected to fused silica capillaries and PTFE tubing. The active microfluidic system has two kinds of fluidic resistances – external (tubing and fittings) and internal (resistance created within the channel design).24 External resistance is a powerful tool to enhance system performance and adjust flow control. In this device, a fused silica capillary of relatively smaller diameter was intentionally connected to the SiG chip to increase the entire system's fluidic resistance (eqn (1) and (2)25). Different capillary and chip lengths were tested to identify appropriate fluidic resistance and flow rate. It was efficient to modify the external resistance through the capillary length, and hence the desired flow rate of >40 μl min−1 was obtained using a channel and capillary length of 60 mm and 20 mm, respectively. The chip was tested to withstand a continuous flow using a pressure range between 10 mbar and 2 bar (the controller system capability allowed a 2 bar maximum pressure at the channel outlet). Using the Hagen–Poiseuille's law25 and given the channel width w, height h, length of the channel section l, diameter d and the viscosity of the fluid flowing η:
(1) |
(2) |
The fluid flow in the chip was monitored and controlled externally by a pressure controller (OB1 – MK3+ with four channel outlets from Elveflow, France) and a flow sensor (Microfluidic Flow Sensor (MFS) from Elveflow, France). The pressure controller was connected to 3 bar N2 gas with constant pressurised gas supplied via standard laboratory gas lines. The channel features were visualised under a customised microscope (Nikon Eclipse Ci – NIS-Elements BR 5.21.01) to imitate the chips' position at the beamline; thus, the devices were mounted horizontally to the perpendicular direction of the beam.
Plasma etching creates a very smooth surface for channels that are less likely to disturb the desired laminar flow. The surface smoothness and dicing quality were confirmed using optical microscope imaging. The channel walls were free of any residues from the fabrication process, so there was no potential blockage of fluid flow (Fig. 2). For spectroscopic techniques, the path length (distance travelled by the X-rays through the sample medium) is an essential consideration to exploit the maximum X-ray scattering intensity.26,27 Increased path lengths are a consequence of wider and deeper channels, which leads to an increased sample consumption rate. Thus, appropriate calculations are made to attain optimal path length via the dimensions of the microchannels. The beam size should also be smaller than the exposed channel to avoid ‘undesired’ scattering from the channel edges.28 Beam to channel edge interaction may also result in sample deposition to the channel walls and/or device damage.
All the samples used in this study worked well at ambient temperature with no external control system for temperature or humidity or CO2, and thus no specific arrangement was made for the microenvironment of the samples.
Considering the beam and beamline requirements at Balder, MAX IV, a chip holder was developed that provides strong support to side connect fluidic holders and avoid any strain on the inlet and outlet of the microfluidic device (Fig. 1). The side connects are placed at an angle with respect to the hexapod arrangement at Balder and thereby providing efficient data collection in fluorescence mode. The chip holder is compatible with the SiG device presented in this paper. The chip holder has a larger open section to ensure minimal X-ray beam impedance and make it easier to perform online and offline setup. The chip holder incorporates guided capillary paths, that prevents cracking of the junctions between the capillary and the chip.
Fig. 1 CAD image of the chip holder that supports the SiG chip mounting and angled coupling holders to prevent strain on the chip and fixings during usage. |
The design was developed using SolidWorks software and fabricated using a Fused Deposition Modelling (FDM) 3D Printer with Acrylonitrile Butadiene Styrene (ABS) filament. The chip holder was made as multiple parts and glued together as a single piece. The SiG chip was removable and clamped in place by the two chip clamps (Fig. 1d) and four hex nuts and bolts (Fig. 1i).
The sample mounting at Balder is equipped with translational stages: vertical 102 mm linear PI L-406.40SD00 is holding a hexapod (PI H-811.I2) upside down, equipped with a manual full circle rotation plate, in turn, holding the lateral 103 mm linear piezo stage (SLC-24150, SmarAct). This facilitates sample positioning at 90° (transmission mode) or 45° (fluorescence mode, Fig. 2a) to the beam path. This assembly is holding the sample mounting pins from above, with a chip holder with the SiG chip mounted on the pins and secured with a metal latch (Fig. 2d). The chip holder supports the inlet and outlet ports for the three separate channels on each side (Fig. 2b). A small endoscope microscope (Supereyes N015-2) is mounted facing the sample (when in 45°) for visual sample alignment to the beam with in-house software, Orthoviewer (not shown).
The SiG device was utilised to flow samples with an element of interest at >13 keV X-ray energy (atomic number 31 and above). Some of these elements, especially in combination with organic solvents have potentially increased toxicity and hazard levels. Thus, a moderate-risk glove bag was prepared around the entire setup that covered the chip, connectors, flow sensors, and reservoirs, including waste, to avoid any splashing of potentially hazardous liquids on the detectors and chambers (see Fig. 2d). The glove bag had appropriate Kapton® (polyimide, low X-ray background material) windows on the beam path and fluorescence detector side to withstand the beam and reduce unwanted X-ray signals to a minimum, see Fig. 2d.
A requirement of high importance was the flow rate control, as lower flow rates might result in constrained sample replenishment and therefore radiation damage of the sample because of prolonged exposure (in the range of millisecond) to the beam. To control the sample flow through the SiG device, a pressure-driven OB1 Elveflow controller (MK3, Elvesys, France) was placed on the experimental table and connected to the sample reservoir. The device was optimised to have a flow rate range from 40 μl min−1 to 1200 μl min−1, which assured sample replenishment required for the beam characteristics, determined based on tests conducted off-beamline and beamline. The fluid flow was remotely controlled outside the beamline hutch (the hutch door is interlocked during X-ray experiments to avoid exposure to dangerous levels of ionising radiation) to start/stop the flow and continuously monitor the applied pressure and resulting flow.
Lead di(acetate) trihydrate, PbAc2 (C4H6O4Pb·3H2O) (CAS 6080-56-4) from Sigma Aldrich was dissolved in MQ water to a concentration of 1 and 0.1 M. Lead dichloride, PbCl2 (CAS 7758-95-4) from Sigma Aldrich was dissolved in MQ water to a concentration of 0.01 and 0.001 M. Before filling the sample reservoir in the flow set up, all solutions were degassed by ultrasonic bath and filtered through a syringe filter (cut off 2 μm) to remove air and any particles, respectively.
The sample solution was flowed through the fluidic channel at rates between 40 μl min−1 to 80 μl min−1. At an energy above the absorption edge of interest, the fluorescence counts per second were checked, and the region of interest (roi) was set around the emission line of interest (Fe Kα, Br Kα or Pb Lα). The distance between the sample and the fluorescence detector was optimised to be in the linear response range. If needed, a Z-1 filter (Mn for Fe and Se for Br/Pb) was used in front of the detector to decrease the elastic scattering peak.
Continuous fly scans30 to record EXAFS (settings: dt 10 ms, energy step 0.25 eV, −200 to +560 eV around E0, 100 s per scan) or only XANES (settings: dt 10 ms, energy step 0.2 eV, −50 to +70 eV around E0, 25 s per scan) were started when the sample was confirmed in the channel by checking the increase in fluorescence count. The number of scans is indicated in each figure (Fig. 3, 5 and 6). E0 in this case was the tabulated edge energy of the element (Fe K 7112 eV, Br K 13474 eV, Pb L3 13035 eV).
Samples were changed by changing the sample reservoir with a new solution, first water to clean the microfluidic device, and then a new sample (fluorescence counts in the roi was used to confirm the presence or absence of sample in the channel). Different SiG devices were used for the four different salt solutions tested, even though the SiG chip is fully reusable for the same samples after proper cleaning. Post experiment, the SiG devices were scrutinised under the microscope for any sign of radiation damage to the device material.
The multiple scan repeats of each sample solution were first examined for changes in the edge region; if no changes were observed, they were merged into an averaged spectrum. The spectrum normalisation and extraction of EXAFS oscillations and conversion of the energy scale to the wave-vector (k) scale were performed as previously described.31,32 Simulation of k3-weighted EXAFS (S02 = 1.0) was done using phase functions calculated with FEFF7.33 Fourier transforms of EXAFS spectra were calculated using in-house software and cos2 windows extending over 10% at both k-range ends (k = 2.1–10.9 Å−1). E0 (13474 eV) was refined in the fit procedure to a zero-energy shift of 3.8 eV. The goodness of fit was judged by calculation of the Fourier-filtered R-factor (RF).32
Demeter program,34 Hephaestus, was used to calculate the attenuation length and transmitted fraction of X-rays through the device windows of borosilicate glass (formula (SiO2)0.9(B2O3)0.1, density 2.23 g cm−3) at the X-ray energies (edge and Kα emission) of Fe (7.1 and 6.4 keV) and Br (13.5 and 11.9 keV). It was also used for calculations of the sample molar concentration for transmission of unit edge step.
Fig. 4 The chipholder and mounted SiG chip with three connected channels. (a) Side view facing the beam and detector; (b) off-beamside view; (c) top view observing the angled holders for connectors. |
The angled coupling holders were inserted to the main holder body. Chip holder was printed in parts as the main chip window and side connect holders. The angle avoided collision with other equipment during horizontal translations. The chip holder is light weight and stiff which makes the whole microfluidic set up to be portable into and out of the Balder experimental station and allows for offline setup. As the SiG devices and attaching capillaries are delicate the chip holder is a vital component in the microfluidic setup. The chip holder allows the use of three channels in parallel with different samples, potentially, being used in each.
The microfluidic device was visually inspected for any radiation damage (RD), as this is considered one of the most common side-effect results when operating with intense X-ray beams at synchrotron facilities. High-intensity ionising radiation can have multiple effects on materials in a relatively short time, often causing the material to physically, mechanically, optically and chemically alter or degrade.36,37 RD provides a physical limit to X-ray analysis on certain fragile samples, materials used in sample holders and, in many cases, the duration of a test at a single local point of interest. Inelastic scattering converts photon energy into lattice vibrations, causing heating, which results in physical damage to the material, such as melting or deformation.38 The results from this work show that the SiG device was able to withstand the high energies of X-ray beams at the Balder beamline and provide good optical transparency to aid correct experimental setup. The SiG chip appeared sufficiently X-ray inert, to the intense beam focused on a single position for more than 24 h to maintain full microfluidic and XAS functionality.
Post-experiments, the chip was imaged to observe any physical changes that might have occurred due to RD or any deformations or depositions to understand the result of beam–material interaction. The devices, pre- and post-experiments at beamline, were microscopically compared (Fig. 6). There was slight discolouration in the epoxy glue material at the tubing to chip junction, that has interacted with the iron chloride sample, but with little effects on device functionalities (Fig. 6b). Moreover, the SiG chip, when used with NaBr solution, resulted in no discolouration, and no other forms of damage, even when the chip was scanned and focused with an intense X-ray beam on the same spot for a longer time, e.g., more than 48 h. Hence, the SiG device was successfully used as a sample delivery system for XAS experiments of higher energy ranges since no deviations physically, optically, or chemically were observed. The materials of choice, silicon, and borosilicate were chemically inert and resistive to X-rays and, therefore, optimal for use with the focused beam at prolonged exposure times with harsh, toxic, and hazardous samples at high-energy ranges.
In Fig. 7, the XANES scans show the rise of the edge in the μ(E) absorption coefficient, detected in fluorescence geometry (sample at 45°) by either the fluorescence detector (If/I0s – blue/green line), or as the transmitted signal detected in I1 (log(I0s/I1) – dark grey line). The properties of the SDD fluorescence detector limits fluorescence detection: too high concentrations suffer from non-linearity by the detector due to dead time in the detector elements. The deadtime problem, which suppresses the signal intensity, is handled by limiting the total photon count number hitting the detector by either moving the detector away from the sample or attenuating the signal. Too low a concentration would have a problem with the signal-to-noise ratio. As the range of concentrations was varied from 1 M to 1 mM, there is an evident decrease in the signal-to-noise ratio as the concentrations reduced. The SiG device can be used to record quality data down to 1 mM concentrations by increasing the number of scans. In Fig. 7b, each spectrum is an average of 5 scans, which easily can be increased without consuming too large sample volumes (5 scans á 25 s, at 40 μl min−1 = 2 min would spend 80 μl).
The second problem is that the fluorescence signal's proportionality to μ(E) depends on the sample's thickness and concentration. The linear proportionality breaks down in thicker and more concentrated samples, where the penetration depth decreases while the absorption coefficient increases over the edge, resulting in fewer atoms of interest contributing to the detected signal.39 In the absorption spectrum, structures are suppressed; for example, the white line (top of the edge) and the EXAFS oscillations; this is called self-absorption. The transmission signal is not distorted by the dead time in the fluorescence detector and would not suffer from the same self-absorption. Therefore, it would represent the “true” signal shape of the edges. However, at too low absorber concentrations, the edge step is too small to be detected. Comparing the simultaneously recorded fluorescence and transmission spectra for each concentration, an optimal detection range for the SiG device can be defined for the 13–14 keV X-rays.
The optimal concentration of the PbAc2 and NaBr water solutions for a transmission sample to give an absorption edge step of ∼1 can be calculated. For the SiG chip with the sample channel depth of 525 μm and an X-ray pathlength in 45° of 742 μm would the concentrations be ∼1.35 M NaBr at the Br K-edge and ∼0.69 M PbAc2 at the Pb L3-edge. Thus, transmission detection is close to optimal for the 1 M samples in the SiG device. However, in the concentration range tested for the Pb L3-edge, transmission detection can be used down to 10 mM, while the 1 mM spectrum is too noisy with an artefact from a monochromator crystal glitch visible as a sharp peak in Fig. 7a – top spectrum dark grey. The optimal range of fluorescence detected data would be >1 to <100 mM concentration (Fig. 7a). At 1 M the self-absorption starts to be visible as a suppressed peak in the fluorescence spectrum (blue) compared to the transmission spectrum (dark grey). At the Br K-edge, similar ranges apply, but the self-absorption in fluorescence is stronger and appear to some extent already in the 100 mM range and is evident in the 1 M spectrum; see Fig. 7b – bottom spectrum in green. Transmission detection was feasible down to 1 mM, even if the fluorescence spectrum, in this case, was less noisy, see Fig. 7b top spectra. The signal-to-noise ratio was overall better in the Br data set than the Pb data set (Fig. 7b versus 7a), which can be explained firstly by the fact that the fluorescence yield is higher for K-edge than L-edge (=more fluorescence generated/absorption event). Secondly, that the ∼450 eV higher excitation energy of the Br K-edge gave ∼60 μm longer attenuation length in borosilicate glass for the incoming beam and the ∼1400 eV higher emission energy (Br Kα 11.92 keV, Pb Lα 10.55 keV) gave ∼130 μm longer attenuation length for the signal going to the fluorescence detector (=less absorption of X-rays in the SiG device glass windows for Br K-edge).
To obtain information on the local atomic structure around the absorbing atom, one would typically extend the energy range to include the extended X-ray absorption fine structure (EXAFS) region 50–1000 eV above the absorption edge. To evaluate the data quality for EXAFS measurements from the microfluidic device, longer energy scans were repeated ten times on the bromide solutions (total 17 min scan time). Bromide in water solution is a difficult test object since the EXAFS oscillations are rapidly dampened with very low amplitude k > 9 Å−1.40,41 The week oscillations at higher k will suffer significantly from noise. In the concentration series, the 1 and 0.1 M solutions gave good enough EXAFS data in transmission after 10 scans (Fig. 7a) to attempt to fit a simple model of one oxygen shell (Br–O) simulating the coordinating water molecules around the bromide. In the Fourier transform of EXAFS (Fig. 8b), only one main peak is visible around the reduced distance of 2.7 Å, indicating one main distance of the coordinating waters. A best fit was achieved for both concentrations when fixing the Br–O coordination number to 8 and let the interatomic distance (R) and Debye-Waller factor (σ2) be fitted to ∼3.25 Å and ∼0.026 Å2, respectively, see the green lines in Fig. 8. In the aqueous NaBr solution, each Br− would then be surrounded by ∼8 water molecules at a distance of ∼3.25 Å, which is in good agreement with literature data on aqueous RbBr solution40 (8–10 water at ∼3.26 Å from EXAFS and MXAN analysis). By increasing the number of scans, the signal-to-noise could, of course, be improved, but in this case, it would also increase the sample consumption in the fluidic setup.
Further research with this microfluidic setup is being carried out with a wider range of samples and integration of the device with additional analysis techniques. As a robust, well-characterised system, this platform opens capabilities to seek answers to novel scientific questions at synchrotron light sources.
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