On-demand radiosynthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) on an electrowetting-on-dielectric microfluidic chip for 18F-labeling of protein.

An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric (EWOD) mechanism was developed for on-demand synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), the most commonly used 18F-prosthetic group for biomolecule labeling. In order to facilitate the development of peptides, and proteins as new diagnostic and therapeutic agents, we have diversified the compact EWOD microfluidic platform to perform the three-step radiosynthesis of [18F]SFB starting from the no carrier added [18F]fluoride ion. In this report, we established an optimal microliter droplet reaction condition to obtain reliable yields and synthesized [18F]SFB with sufficient radioactivity for subsequent conjugation to the anti-PSCA cys-diabody (A2cDb) and for small animal imaging. The three-step, one-pot radiosynthesis of [18F]SFB radiochemistry was adapted to a batch microfluidic platform with a reaction droplet sandwiched between two parallel plates of an EWOD chip, and optimized. Specifically, the ratio of precursor to base, droplet volume, reagent concentration, reaction time, and evaporation time were found be to be critical parameters. [18F]SFB was successfully synthesized on the EWOD chip in 39 ± 7% (n = 4) radiochemical yield in a total synthesis time of ∼120 min ([18F]fluoride activation, [18F]fluorination, hydrolysis, and coupling reaction, HPLC purification, drying and reformulation). The reformulation and stabilization step for [18F]SFB was important to obtain a high protein labeling efficiency of 33.1 ± 12.5% (n = 3). A small-animal immunoPET pilot study demonstrated that the [18F]SFB-PSCA diabody conjugate showed specific uptake in the PSCA-positive human prostate cancer xenograft. The successful development of a compact footprint of the EWOD radiosynthesizer has the potential to empower biologists to produce PET probes of interest themselves in a standard laboratory.

Two types of Cassie-to-Wenzel wetting transitions on superhydrophobic surfaces during drop impact.

Despite the fact that superhydrophobic surfaces possess useful and unique properties, their practical application has remained limited by durability issues. Among those, the wetting transition, whereby a surface gets impregnated by the liquid and permanently loses its superhydrophobicity, certainly constitutes the most limiting aspect under many realistic conditions. In this study, we revisit this so-called Cassie-to-Wenzel transition (CWT) under the broadly encountered situation of liquid drop impact. Using model hydrophobic micropillar surfaces of various geometrical characteristics and high speed imaging, we identify that CWT can occur through different mechanisms, and at different impact stages. At early impact stages, right after contact, CWT occurs through the well established dynamic pressure scenario of which we provide here a fully quantitative description. Comparing the critical wetting pressure of surfaces and the theoretical pressure distribution inside the liquid drop, we provide not only the CWT threshold but also the hardly reported wetted area which directly affects the surface spoiling. At a later stage, we report for the first time to our knowledge, a new CWT which occurs during the drop recoil toward bouncing. With the help of numerical simulations, we discuss the mechanism underlying this new transition and provide a simple model based on impulse conservation which successfully captures the transition threshold. By shedding light on the complex interaction between impacting water drops and surface structures, the present study will facilitate designing superhydrophobic surfaces with a desirable wetting state during drop impact.

Repellent surfaces. Turning a surface superrepellent even to completely wetting liquids.

Superhydrophobic and superoleophobic surfaces have so far been made by roughening a hydrophobic material. However, no surfaces were able to repel extremely-low-energy liquids such as fluorinated solvents, which completely wet even the most hydrophobic material. We show how roughness alone, if made of a specific doubly reentrant structure that enables very low liquid-solid contact fraction, can render the surface of any material superrepellent. Starting from a completely wettable material (silica), we micro- and nanostructure its surface to make it superomniphobic and bounce off all available liquids, including perfluorohexane. The same superomniphobicity is further confirmed with identical surfaces of a metal and a polymer. Free of any hydrophobic coating, the superomniphobic silica surface also withstands temperatures over 1000°C and resists biofouling.

EWOD (electrowetting on dielectric) digital microfluidics powered by finger actuation.

We report finger-actuated digital microfluidics (F-DMF) based on the manipulation of discrete droplets via the electrowetting on dielectric (EWOD) phenomenon. Instead of requiring an external power supply, our F-DMF uses piezoelectric elements to convert mechanical energy produced by human fingers to electric voltage pulses for droplet actuation. Voltage outputs of over 40 V are provided by single piezoelectric elements, which is necessary for oil-free EWOD devices with thin (typically <1 µm) dielectric layers. Higher actuation voltages can be provided using multiple piezoelectric elements connected in series when needed. Using this energy conversion scheme, we confirmed basic modes of EWOD droplet operation, such as droplet transport, splitting and merging. Using two piezoelectric elements in series, we also successfully demonstrated applications of F-DMF for glucose detection and immunoassay. Not requiring power sources, F-DMF offers intriguing paths for various portable and other microfluidic applications.

Radiolabelling diverse positron emission tomography (PET) tracers using a single digital microfluidic reactor chip.

Radiotracer synthesis is an ideal application for microfluidics because only nanogram quantities are needed for positron emission tomography (PET) imaging. Thousands of radiotracers have been developed in research settings but only a few are readily available, severely limiting the biological problems that can be studied in vivo via PET. We report the development of an electrowetting-on-dielectric (EWOD) digital microfluidic chip that can synthesize a variety of (18)F-labeled tracers targeting a range of biological processes by confirming complete syntheses of four radiotracers: a sugar, a DNA nucleoside, a protein labelling compound, and a neurotransmitter. The chip employs concentric multifunctional electrodes that are used for heating, temperature sensing, and EWOD actuation. All of the key synthesis steps for each of the four (18)F-labeled tracers are demonstrated and characterized with the chip: concentration of fluoride ion, solvent exchange, and chemical reactions. The obtained fluorination efficiencies of 90-95% are comparable to, or greater than, those achieved by conventional approaches.

Efficient radiosynthesis of 3'-deoxy-3'-18F-fluorothymidine using electrowetting-on-dielectric digital microfluidic chip.

UNLABELLED: Access to diverse PET tracers for preclinical and clinical research remains a major obstacle to research in cancer and other disease research. The prohibitive cost and limited availability of tracers could be alleviated by microfluidic radiosynthesis technologies combined with a high-yield microscale radiosynthetic method. In this report, we demonstrate the multistep synthesis of 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) with high yield on an electrowetting-on-dielectric (EWOD) microfluidic radiosynthesizer, previously developed in our group. We have identified and established several parameters that are most critical in the microscale radiosynthesis, such as the reaction time, reagent concentration, and molar ratios, to successfully synthesize (18)F-FLT in this compact platform. METHODS: (18)F-FLT was synthesized from the 3-N-Boc-1-[5-O-(4,4'-dimethoxytrityl)-3-O-nosyl-2-deoxy-ß-D-lyxofuranosyl] thymine precursor on the EWOD chip starting from the first solvent exchange and (18)F-fluoride ion activation step to the final deprotection step. The fluorination reaction was performed in a mixture of thexyl alcohol and dimethyl sulfoxide. The crude product after deprotection was collected from the chip and purified on a custom-made solid-phase extraction cartridge and subjected to quality control testing. The purified (18)F-FLT was suitable for small-animal PET studies in multiple nude mice xenografted with the A431 carcinoma cell line. RESULTS: (18)F-FLT was successfully synthesized on the EWOD microdevice coupled with an off-chip solid-phase extraction purification with a decayed-corrected radiochemical yield of 63% ± 5% (n = 5) and passed all of the quality control tests required by the U.S. Pharmacopeia for radiotracers to be injected into humans. We have successfully demonstrated the synthesis of several batches of (18)F-FLT on EWOD, starting with approximately 333 MBq of radioactivity and obtained up to 52 MBq (non-decay-corrected) of (18)F-FLT on cartridge purification. The specific activity of 2 representative preparations of (18)F-FLT synthesized on the EWOD chip were measured to be 1,800 and 2,400 GBq/µmol. CONCLUSION: The EWOD microchip and optimized synthesis method in combination represent an effective platform for synthesizing (18)F-FLT with high yield and of good quality for imaging. This compact platform, with configurable synthesis steps, could potentially form the basis of a stand-alone system that decouples PET probe production from the cyclotron and specialized radiochemistry facilities and increases diversity and flexibility in probe production.

Optimization of microfluidic PET tracer synthesis with Cerenkov imaging.

Microfluidic technologies provide an attractive platform for the synthesis of radiolabeled compounds. Visualization of radioisotopes on chip is critical for synthesis optimization and technological development. With Cerenkov imaging, beta particle emitting isotopes can be localized with a sensitive CCD camera. In order for Cerenkov imaging to also serve as a quantitative tool, it is necessary to understand how material properties relevant to Cerenkov emission, namely, index of refraction and beta particle stopping power, affect Cerenkov light output. In this report, we investigate the fundamental physical characteristics of Cerenkov photon yield at different stages of [(18)F]FDG synthesis on the electrowetting on dielectric (EWOD) microfluidic platform. We also demonstrate how Cerenkov imaging has enabled synthesis optimization. Geant4, a Monte Carlo program applied extensively in high energy physics, is used to simulate Cerenkov photon yield from (18)F beta particles traversing materials of interest during [(18)F]FDG synthesis on chip. Our simulations show that the majority (approximately two-thirds) of the (18)F beta particle energy available to produce Cerenkov photons is deposited on the glass plates of the EWOD chip. This result suggests the possibility of using a single calibration factor to convert Cerenkov signal to radioactivity, independent of droplet composition. We validate our simulations with a controlled measurement examining varying ratios of [(18)O]H2O, dimethyl sulfoxide (DMSO), and acetonitrile (MeCN), and find a consistent calibration independent of solvent composition. However, the calibration factor may underestimate the radioactivity in actual synthesis due to discoloration of the droplet during certain steps of probe synthesis. In addition to the attractive quantitative potential of Cerenkov imaging, this imaging strategy provides indispensable qualitative data to guide synthesis optimization. We are able to use this imaging technique to optimize the mixing protocol as well as identify and correct for loss of radioactivity due to the migration of radioactive vapor outside of the EWOD heater, enabling an overall increase in the crude radiochemical yield from 50 ± 3% (n = 3) to 72 ± 13% (n = 5).

On-demand droplet loading for automated organic chemistry on digital microfluidics.

Organic chemistry applications on digital microfluidic devices often involve reagents that are volatile or sensitive and must be introduced to the chip immediately before use. We present a new technique for automated, on-demand loading of ~1 µL droplets from large (~1 mL), sealed, off-chip reservoirs to a digital microfluidic chip in order to address this challenge. Unlike aqueous liquids which generally are non-wetting to the hydrophobic surface and must be actively drawn into the electrowetting-on-dielectric (EWOD) chip by electrode activation, organic liquids tend to be wetting and can spontaneously flood the chip, and hence require a retracting force for controlled liquid delivery. Using a combination of compressed inert gas and gravity to exert driving and retracting forces on the liquid, the simple loading technique enables precise loading of droplets of both wetting and non-wetting liquids in a reliable manner. A key feature from a practical point of view is that all of the wetted parts are inexpensive and potentially disposable, thus avoiding cross-contamination in chemical and biochemical applications. We provide a theoretical treatment of the underlying physics, discuss the effect of geometry and liquid properties on its performance, and show repeatable reagent loading using the technique. Its versatility is demonstrated with the loading of several aqueous and non-aqueous liquids on an EWOD digital microfluidic device.

Accurate dispensing of volatile reagents on demand for chemical reactions in EWOD chips.

Digital microfluidic chips provide a new platform for manipulating chemicals for multi-step chemical synthesis or assays at the microscale. The organic solvents and reagents needed for these applications are often volatile, sensitive to contamination, and wetting, i.e. have contact angles of <90° even on the highly hydrophobic surfaces (e.g., Teflon® or Cytop®) typically used on digital microfluidic chips. Furthermore, often the applications dictate that the processes are performed in a gas environment, not allowing the use of a filler liquid (e.g., oil). These properties pose challenges for delivering controlled volumes of liquid to the chip. An automated, simple, accurate and reliable method of delivering reagents from sealed, off-chip reservoirs is presented here. This platform overcomes the issues of evaporative losses of volatile solvents, cross-contamination, and flooding of the chip by combining a syringe pump, a simple on-chip liquid detector and a robust interface design. The impedance-based liquid detection requires only minimal added hardware to provide a feedback signal to ensure accurate volumes of volatile solvents are introduced to the chip, independent of time delays between dispensing operations. On-demand dispensing of multiple droplets of acetonitrile, a frequently used but difficult to handle solvent due to its wetting properties and volatility, was demonstrated and used to synthesize the positron emission tomography (PET) probe [(18)F]FDG reliably.

Dynamic contact angles and hysteresis under electrowetting-on-dielectric.

By designing and implementing a new experimental method, we have measured the dynamic advancing and receding contact angles and the resulting hysteresis of droplets under electrowetting-on-dielectric (EWOD). Measurements were obtained over wide ranges of applied EWOD voltages, or electrowetting numbers (0 ≤ Ew ≤ 0.9), and droplet sliding speeds, or capillary numbers (1.4 × 10(-5) ≤ Ca ≤ 6.9 × 10(-3)). If Ew or Ca is low, dynamic contact angle hysteresis is not affected much by the EWOD voltage or the sliding speed; that is, the hysteresis increases by less than 50% with a 2 order-of-magnitude increase in sliding speed when Ca < 10(-3). If both Ew and Ca are high, however, the hysteresis increases with either the EWOD voltage or the sliding speed. Stick-slip oscillations were observed at Ew > 0.4. Data are interpreted with simplified hydrodynamic (Cox-Voinov) and molecular-kinetic theory (MKT) models; the Cox-Voinov model captures the trend of the data, but it yields unreasonable fitting parameters. MKT fitting parameters associated with the advancing contact line are reasonable, but a lack of symmetry indicates that a more intricate model is required.

A miniature capillary breakup extensional rheometer by electrostatically assisted generation of liquid filaments.

A micromachined chip capable of generating liquid microfilaments has been developed for a miniature version of the Capillary Breakup Extensional Rheometer (CaBER®). The proposed system is exceptionally simple and compact because liquid samples are actuated by voltages administered on-chip, which therefore requires only electrical connections (rather than a linear motor, an integral part of the CaBER®). Since chip features are photolithographically defined, the miniature rheometer can handle sub-microlitre samples. Following the CaBER®, we show that a commercial LED micrometer effectively measures diameters of filaments generated by the electrowetting-on-dielectric (EWOD) forces. Since negligible electric fields are sustained within the liquid far away from the measurement region, the applied EWOD voltage does not influence tested material properties. Through breakup experiments using a wide range of Newtonian and complex fluids (e.g., glycerol, xanthan gum, dilute polystyrene, and dilute solutions of various molecular weight polyethylene oxide) we demonstrate a versatile testing platform for scarce and precious samples such as biochemical fluids and novel materials. Measured Newtonian and complex dynamics agree well with published theories and experiments.

Influence of surface hierarchy of superhydrophobic surfaces on liquid slip.

We investigated how the surface hierarchy of superhydrophobic (SHPo) surfaces influences liquid slip by testing well-defined microposts that have nanoposts only on their top. Contrary to the commonly held belief, our results show that such hierarchical surfaces do not always lead to an increase of slip length despite their reduced solid fraction and enhanced hydrophobicity compared to single-scale surfaces. Adding nanoposts on top of the microposts resulted in an increase of slip length only if the original microposts had a solid fraction above a threshold value. For solid fractions below this threshold, adding nanoposts decreased the slip length. We propose that there were not enough nanoposts on the top surface of very thin microposts to support the liquid pressure, allowing the liquid to intrude down to the top corners of the microposts.

Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS.

Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in dithiothreitol (DTT) over a range of heater temperatures (22-70 °C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130 °C in dimethyl sulfoxide (DMSO) to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 °C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air.

Maximizing the giant liquid slip on superhydrophobic microstructures by nanostructuring their sidewalls.

In an effort to maximize the liquid slip on superhydrophobic surfaces, we investigate the role of the nanoscale roughness on microscale structures by developing well-defined micro-nano hierarchical structures. The nonwetting stability and slip length on the dual-scale micro-nano structures are measured and compared with those on single-scale micro-smooth structures. A force balance between a liquid pressure and a surface tension indicates that hydrophobic nanostructures on the sidewall of microposts or microgrates would expand the range of the nonwetted state. When a higher gas fraction or a larger pitch can be tested without wetting, a larger slip length is expected on the microstructures. An ideal dual-scale structure is described that isolates the role of the nanostructures, and a fabrication technique is developed to achieve such a microstructure-smooth tops and nanostructured sidewalls. The tests confirm such micro-nano structures allow a nonwetted state at a higher gas fraction or a larger pitch than the previous micro-smooth structures. As a result, we achieve the maximum slip length of approximately 400 microm on the dual-scale structures, an increase of approximately 100% over the previous maximum reported on the single-scale (i.e., micro-smooth) structures. The study ameliorates our understanding of the role of each scale on hierarchical structures for a wetting transition and a liquid slip. The resulting giant slip is large enough to influence many fluidic applications, even in macroscale.

EWOD-driven droplet microfluidic device integrated with optoelectronic tweezers as an automated platform for cellular isolation and analysis.

We report the integration of two technologies: droplet microfluidics using electrowetting-on-dielectric (EWOD) and individual particle manipulation using optoelectronic tweezers (OET)-in one microfluidic device. The integrated device successfully demonstrates a sequence involving both EWOD and OET operations. We encountered various challenges during integration of the two different technologies and present how they are addressed. To show the applicability of the device in cellular biology, live HeLa cells are used in the experiments. The unique advantages of EWOD and OET make their integration a significant step towards a powerful tool for many applications, such as single cell studies involving multiplexed environmental stimuli.

Droplet evaporation of pure water and protein solution on nanostructured superhydrophobic surfaces of varying heights.

Evaporation of liquids on substrates is important for many applications including lab-on-a-chip, especially when they are in droplets. Unlike on planar substrates, droplet evaporation on micropatterned substrates has been studied only recently and none so far on nanopatterns. Driven by the applicability of nanostructured surfaces to biomaterials and tissue engineering, we report on the evaporative process of sessile droplets of pure water and a protein solution on superhydrophobic surfaces of sharp-tip post structures in a submicrometer pitch (230 nm) and varying heights (100-500 nm). We find that the nanotopographical three-dimensionalities such as structural height and sidewall profile affect the surface superhydrophobicity in such a way that only tall and slender nanostructures provide the surface with great superhydrophobicity (a contact angle more than 170 degrees). The evaporation process was different between the pure water and the protein solution; unlike pure water, a significant contact-line spreading and pinning effect was observed in a droplet of a protein solution with an intermediate transition from a dewetting (Cassie) to a wetting (Wenzel) state. Enabled by well-defined nanostructures, our results highlight that the surface superhydrophobicity and the droplet evaporation are significantly affected by the three-dimensional nanometric topography and the surface fouling such as protein adsorption.

Capillary spreading dynamics of electrowetted sessile droplets in air.

We report the contact line dynamics of sessile water droplets, 1.1-1.6 mm in radius, spread by electrowetting in air. Coplanar electrodes patterned on the substrate allow a true sessile condition with no wire into the droplet. The frequency response of the droplets is studied using 25 VAC ranging from 10 to 205 Hz. The effect of contact angle hysteresis is seen in form of stick-slip motion. A model developed provides a good match to the experimental result. Step response is studied with voltages in the range of 20-80 VDC. Two regimes of motion are observed. In the first regime, local flows cause the contact line speed to increase and reach a maximum while the contact angle is still changing. Global flows in the second regime cause the contact line to move with a reduced speed and attain the spherical shape pertaining to the new equilibrium contact angle. A model is used to describe the motion.

All-electronic droplet generation on-chip with real-time feedback control for EWOD digital microfluidics.

Electrowetting-on-dielectric (EWOD) actuation enables digital (or droplet) microfluidics where small packets of liquids are manipulated on a two-dimensional surface. Due to its mechanical simplicity and low energy consumption, EWOD holds particular promise for portable systems. To improve volume precision of the droplets, which is desired for quantitative applications such as biochemical assays, existing practices would require near-perfect device fabrication and operation conditions unless the droplets are generated under feedback control by an extra pump setup off of the chip. In this paper, we develop an all-electronic (i.e., no ancillary pumping) real-time feedback control of on-chip droplet generation. A fast voltage modulation, capacitance sensing, and discrete-time PID feedback controller are integrated on the operating electronic board. A significant improvement is obtained in the droplet volume uniformity, compared with an open loop control as well as the previous feedback control employing an external pump. Furthermore, this new capability empowers users to prescribe the droplet volume even below the previously considered minimum, allowing, for example, 1 : x (x < 1) mixing, in comparison to the previously considered n : m mixing (i.e., n and m unit droplets).

Micropumping of liquid by directional growth and selective venting of gas bubbles.

We introduce a new mechanism to pump liquid in microchannels based on the directional growth and displacement of gas bubbles in conjunction with the non-directional and selective removal of the bubbles. A majority of the existing bubble-driven micropumps employs boiling despite the unfavorable scaling of energy consumption for miniaturization because the vapor bubbles can be easily removed by condensation. Other gas generation methods are rarely suitable for micropumping applications because it is difficult to remove the gas bubbles promptly from a pump loop. In order to eradicate this limitation, the rapid removal of insoluble gas bubbles without liquid leakage is achieved with hydrophobic nanopores, allowing the use of virtually any kind of bubbles. In this paper, electrolysis and gas injection are demonstrated as two distinctively different gas sources. The proposed mechanism is first proved by circulating water in a looped microchannel. Using H(2) and O(2) gas bubbles continuously generated by electrolysis, a prototype device with a looped channel shows a volumetric flow rate of 4.5-13.5 nL s(-1) with a direct current (DC) power input of 2-85 mW. A similar device with an open-ended microchannel gives a maximum flow rate of approximately 65 nL s(-1) and a maximum pressure head of approximately 195 Pa with 14 mW input. The electrolytic-bubble-driven micropump operates with a 10-100 times higher power efficiency than its thermal-bubble-driven counterparts and exhibits better controllability. The pumping mechanism is then implemented by injecting nitrogen gas bubbles to demonstrate the flexibility of bubble sources, which would allow one to choose them for specific needs (e.g., energy efficiency, thermal sensitivity, biocompatibility, and adjustable flow rate), making the proposed mechanism attractive for many applications including micro total analysis systems (microTAS) and micro fuel cells.

A study of EWOD-driven droplets by PIV investigation.

Despite the recent interest in droplet-based microfluidics using electrowetting-on-dielectric (EWOD), fundamental understanding of the fluid dynamics remains limited to two-dimensional (2D) reduction of the Navier-Stokes equation. Experimental data are in dire need to verify the predictions and advance the field. We report an investigation of the flow inside droplets actuated by EWOD in air using micro particle image velocimetry (micro-PIV). Using the continuity equation, we reconstruct the 3D velocity field from the 2D PIV experimental data. We present some fundamental findings and build valuable insights that will help design sophisticated EWOD microfluidic devices. For example, the results confirm that efficient mixing in a droplet may be achieved by moving the droplet along an irreversible pattern that breaks the symmetry of the two circulating inner flows.