Structured surface wetting of a PTFE flow-cell for terahertz spectroscopy of proteins
Introduction
Terahertz (THz) spectroscopy is a promising candidate for biomedical sensing, as large biological molecules such as proteins and nucleic acids have vibration modes on the picosecond timescale [1]. A major obstacle arises when trying to investigate these samples in their native, aqueous environment, where the highly absorbing water attenuates the THz signal. The water can be removed from the sample, either partially or entirely [2], [3], or the absorption can be effectively reduced by freezing the water, shifting resonance of the molecular vibration outside of the THz range [4]. By reducing the effective thickness of the sample, however, the liquid water can be retained, representing an authentic in vivo protein model. In particular, the hydrogen-bond network of the surrounding water itself can be probed with THz radiation [5], [6]. In this case, the influence of the protein on the surrounding solvent can be detected over greater distances than other techniques [7], [8], [9], [10] such as nuclear magnetic resonance (NMR), with such influences on the network of bonds correlating to the macromolecules’ function [11], [12].
Implementing a THz compatible microfluidic device for transmission spectroscopy is an ideal solution to overcome water’s strong signal attenuation, as the effective beam path-length is dramatically shortened, with added benefits such as reduced error from sample exchange and the possibility to expand such device’s functionality in combination with real-time THz spectroscopy, leading to the commonly quoted term: Lab-on-chip [13], [14]. A gas-liquid boundary in a THz compatible cell could assist in the investigation of biological processes. For example, while THz spectra of single concentrations of oxy and deoxy-hemoglobin do not show any fine spectral features in absorption in the THz band [15], changes in structure of globular proteins are detectable with THz spectroscopy by studying the effect of the macromolecule on the surrounding hydrogen bond network [16]. Differences in hydration dynamics between the two states of hemoglobin have been observed with microwave radiation [17], however, by using THz radiation instead, and by flowing oxygen and nitrogen gases adjacent to the protein solution (oxygenating and de-oxygenating the protein respectively [18], [19]), the investigation of longer-range influences can be made with protein modification occurring in situ. Hemoglobin’s function to flexibly bind and relinquish oxygen is coupled with the surrounding water network, and further investigation into its mechanism may provide greater insight into a wide range of hemoglobin disorders [20]. Furthermore, with the addition of a gas-liquid interface, such microfluidic devices can be used for the detection of airborne analytes such as drugs [21] or hazardous contaminants (ammonia) [22] by diffusion into the controlled liquid medium.
Such devices are often comprised of silicon or quartz [23], [24], which have good spectroscopic properties at THz frequencies whilst benefiting from mechanical rigidity. However, specialized manufacturing processes are required to fabricate such devices, and prototyping can be slow. A faster and cheaper pathway is to use polymer based devices. Polydimethylsiloxane (PDMS) is a commonly used material in microfluidic devices; however its THz absorption characteristics are significantly dependent on its preparation methods [25]. Instead, here, we propose the use of plasma treated and machined polytetrafluoroethylene (PTFE). It has desirable properties for both use in a THz spectrometer and as a basis for a microfluidic device. Primarily, these are its low, non-dispersive refractive index between 300 GHz and 3 THz, negligible absorption over a broad THz spectrum, and its well-known chemical resistance and bio-compatibility. Whilst these attributes are appealing, two key problems emerge when trying to use PTFE for microfluidics: its lack of rigidity and the incompatibility with adhesives, complicating encapsulation methods. We aim to resolve this by producing a superhydrophobic PTFE surface which acts as a barrier to an incorporated hydrophilic channel. We show that the surface free energy contrast will enable sample confinement within the channel, bypassing the need for bonding or firmly clamping an upper substrate.
The hydrophobicity of native PTFE (θc ≈ 120°) can be both increased and decreased through plasma treatments of various powers and ionic species. High power, directional argon and oxygen plasmas can modify PTFE’s surface to be superhydrophobic (contact angle, θc > 150°) whereas a low power, ambient plasma exposure can increase the hydrophilicity of the surface [26], [27], with reported contact angles as low as 40 degrees [28]. X-ray photoelectron scattering measurements have revealed that in the case of a low power exposure, it is the surface chemistry that is modified [29]. In contrast, exposure to higher energy plasmas, more specifically the accelerated particles found in reactive ion etching (RIE), changes the topology of the PTFE surface by creating significant roughness at the micro and nanoscales such that its roughness supersedes chemical effects to define a superhydrophobic surface [30].
In this paper, we exploit the surface modification of PTFE to both increase and decrease its wettability to produce a quasi-open microfluidic flow-cell, whereby confinement of aqueous samples occurs due to surface tension forces, and not from direct bonding of the substrate. We demonstrate the novel combination of RIE, machining and plasma ashing to achieve this effect, outlined in Fig. 1, allowing PTFE to be used in a sandwich scheme whereby manufacturing tolerance can be relaxed. The figure illustrates the straightforward three-step process implemented to produce a microfluidic channel with a stark contrast in contact angles between the milled channel and the surrounding substrate surface. Lastly, we show spectroscopic measurements of water and bovine serum albumin (BSA) for different concentrations in the fabricated microfluidic prototype device with a Terahertz Time-Domain Spectrometer (THz-TDS). The approach investigated exhibits the advantages of microfluidic devices where different solutions can be measured, sweeping concentrations and easily obtaining reference scans with the addition of a liquid-gas interface to enable in situ liquid-gas reactions to be monitored, using a material system with the chemical resistance, THz transparency, biocompatibility and hydrophobicity of PTFE. The device presented is unique in its combination of THz compatibility and provision of a liquid-gas interface, allowing application to monitoring the reaction of biological substances with gases in situ, as previously outlined. Additionally, this design inherently reduces the issues of bubble trapping disrupting flow and measurement, but further adds capability of the device to accept samples with dissolved gases such as carbonated drinks; the spectroscopy of which has only been achieved in unique self-referencing geometries [31] or with the implementation of nano-antennae [32].
Section snippets
Balancing pressures for surface tension confinement
It is possible to exploit contrasts in surface wetting characteristics to make microfluidic devices that rely on capillary forces alone and no physical boundary or walls to control flow paths [33], [34], [35], [36]. Such a scheme negates the need for problematic encapsulation by relaxing the requirement for alignment and bonding, the latter of which is particularly difficult with PTFE. Additionally, having an easily accessible liquid-gas interface allows the application of gases that may react
Surface modification of PTFE
To modify the surface chemically, such that it becomes hydrophilic, PTFE discs parted from an extruded rod were treated with oxygen plasma in a plasma asher for three minutes at a 50 W power setting, at maximum flow-rate, selected from a parameter sweep where higher or lower values reduced the wetting effect. The contact angle of the treated surface reduced from a measured 100.1 ± 2.1° to 68.7 ± 7.5° as can be seen in Fig. 4(b) and (c) respectively, with (a) showing a scanning electron
Conclusion
A PTFE flow-cell for terahertz spectroscopy of biological media has been demonstrated in this work. PTFE offers low absorption at THz frequencies and the low surface free energy characteristic of PTFE produces a non-fouling and bio-compatible surface, desirable for many microfluidic applications. However, this characteristic hampers efforts for bonding and encapsulating the microfluidic channels. New encapsulation approaches are therefore required for the effective implementation of PTFE in
Funding
This work was funded by EPSRC via a Ph.D. studentship for N.T. Klokkou. Additionally, Bethany Bowden thanks the Defence Science and Technology Laboratory (Contract no. DSTLX-1000128554) for supporting an EPSRC industrial CASE student award.
CRediT authorship contribution statement
Nicholas T. Klokkou: Conceptualization, Investigation, Methodology, Software, Formal analysis, Validation, Writing – original draft, Visualization. David J. Rowe: Conceptualization, Writing – review & editing. Bethany M. Bowden: Investigation, Formal analysis, Writing – review & editing. Neil P. Sessions: Investigation. Jonathan J. West: Investigation, Writing – review & editing. James S. Wilkinson: Supervision, Writing – review & editing. Vasilis Apostolopoulos: Supervision, Writing – review &
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Nicholas Klokkou received his MPhys in Physics at the University of Southampton in 2016, where he is now pursuing a Ph.D. with the Terahertz Laboratories and the Integrated Photonic Devices group. His research interests are in combining microfluidic devices with terahertz time-domain spectroscopy, to investigate protein hydration dynamics, along with further developing frequency dependent complex refractive index extraction techniques through iterative algorithms and machine learning, for
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2022, Applied Surface ScienceCitation Excerpt :The disadvantages of chemical etching include toxicity to humans, ecological hazard, malodor, and PTFE surface coloration [7,11,12]. Other methods for modification of PTFE surface properties are ion irradiation [3], corona treatment [13], and plasma treatment [14]. An environmentally friendlier and safer alternative for the improvement of adhesion and wettability of polymers is treatment with various gaseous plasmas.
Nicholas Klokkou received his MPhys in Physics at the University of Southampton in 2016, where he is now pursuing a Ph.D. with the Terahertz Laboratories and the Integrated Photonic Devices group. His research interests are in combining microfluidic devices with terahertz time-domain spectroscopy, to investigate protein hydration dynamics, along with further developing frequency dependent complex refractive index extraction techniques through iterative algorithms and machine learning, for sample characterisation.
David Rowe is a research fellow with the Mid-Infrared Silicon Photonics Group as part of the Zepler Institute at the University of Southampton. His research interests are in translating novel photonics into lab-on-a-chip devices for biomedical applications, particularly mid-infrared spectroscopy for point-of-care diagnostics. He received his BEng in Electronic Engineering from Cardiff University in 2009 and later completed his Ph.D. ('Microfluidic microwave resonant sensors') in 2012. He has received various awards including the 2011 Institute of Physics Mansel Davies award and was nominated for a Vice Chancellor's Award in 2019 for his work on equality and diversity.
Bethany Bowden received her MChem in Chemistry from Cardiff University in 2018 and is now pursuing a Ph.D. as part of the Electrochemistry group at the University of Southampton. Her research interests are in waveguide enhanced Raman spectroscopy and the modification of tantalum pentoxide surfaces to optimise their functionality and stability, such as to promote binding of specific species in the analyte medium.
Neil Sessions graduated in Chemistry from the University of Exeter, UK, in 1998. He joined the Optoelectronics Research Centre at Southampton University in 2000 working on soft glass optical fibre fabrication and on integrated photonic materials and devices. He now manages the Zepler Institute’s Integrated Photonics Cleanroom.
Jonathan West received a Ph.D. in Biosensors and Microfluidics in 2003 from the National Microelectronics Research Centre (now Tyndall Institute) in Cork, Ireland, then worked on technology commercialization at INEX in Newcastle. He moved to ISAS, Dortmund, Germany, to establish his microfluidics for cell biology research theme and is now a lecturer in Biomedical Microfluidics at the University of Southampton. His lab is focused on microfluidic innovation for biological discovery en route to clinical translation.
James Wilkinson is Professor of Optoelectronics in Southampton University’s Optoelectronics Research Centre, of which he is a founder-member. He took his BSc and Ph.D., both in electronic engineering, from University College London in 1977 and 1985. He conducted research into optical fibre systems at the GEC Hirst Research Centre, London, from 1977 to 1979, and into optical monitoring and control for haemodialysis procedures at St Bartholomew’s Hospital, London, between 1983 and 1985. His research focusses on optical materials and devices for sensing and telecommunications. James is a Fellow of the IEEE, IET and the IoP and was a cofounder of UoS spin-out Mesophotonics Ltd, which commercialised Klarite SERS chips. From 2014 to 2018 he was Associate Dean, Planning and Strategy, for the Faculty of Physical Sciences and Engineering at Southampton.
Vasilis Apostolopoulos received his Ph.D. degree at the University of Southampton in 2003 where he is now an associate professor. He has worked at the University of Cambridge and the Ecole Polytechnique de Lausanne. He is interested in developing THz time domain spectroscopy and its interface with optical systems such as integrated optics, metamaterial devices and microfluidics. He is also interested in Laser sources, specifically vertical external surface emitting lasers and their applications for nonlinear processes such as generation of Kerr micro-combs.