Pei Yuin Keng

Micro-chemical synthesis of molecular probes on an electronic microfluidic device

We have developed an all-electronic digital microfluidic device for microscale chemical synthesis in organic solvents, operated by electrowetting-on-dielectric (EWOD). As an example of the principles, we demonstrate the multistep synthesis of [18F]FDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of [18F]FTAG (88 ± 7%, n = 11) and quantitative hydrolysis to [18F]FDG (> 95%, n = 11). We furthermore show that batches of purified [18F]FDG can successfully be used for PET imaging in mice and that they pass typical quality control requirements for human use (including radiochemical purity, residual solvents, Kryptofix, chemical purity, and pH). We report statistical repeatability of the radiosynthesis rather than best-case results, demonstrating the robustness of the EWOD microfluidic platform. Exhibiting high compatibility with organic solvents and the ability to carry out sophisticated actuation and sensing of reaction droplets, EWOD is a unique platform for performing diverse microscale chemical syntheses in small volumes, including multistep processes with intermediate solvent-exchange steps.

On Chip Droplet Characterization: A Practical, High-Sensitivity Measurement of Droplet Impedance in Digital Microfluidics

We demonstrate a new approach to impedance measurement on digital microfluidics chips for the purpose of simple, sensitive, and accurate volume and liquid composition measurement. Adding only a single series resistor to existing AC droplet actuation circuits, the platform is simple to implement and has negligible effect on actuation voltage. To accurately measure the complex voltage across the resistor (and hence current through the device and droplet), the designed system is based on software-implemented lock-in amplification detection of the voltage drop across the resistor which filters out noise, enabling high-resolution and low-limit signal recovery. We observe picoliter sensitivity with linear correlation of voltage to volume extending to the microliter volumes that can be handled by digital microfluidic devices. Due to the minimal hardware, the system is robust and measurements are highly repeatable. The detection technique provides both phase and magnitude information of the real-time current flowing through the droplet for a full impedance measurement. The sensitivity and resolution of this platform enables it to distinguish between various liquids which, as demonstrated in this paper, could potentially be extended to quantify solute concentrations, liquid mixtures, and presence of analytes.

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.

Emerging Technologies for Decentralized Production of PET Tracers

The use of Positron Emission Tomography (PET) to monitor biological processes in vivo (Michael E. Phelps 2000) has seen dramatic growth and acceptance in the research, pharmaceutical, and medical communities over the last few decades, with clinical PET growing from ~900,000 scans in 2004 to over 1.74 million in 2010 in the United States alone; growth in foreign markets is comparable (Muschlitz 2011). These scans are conducted in ~2,200 clinical PET centers, all providing molecular imaging diagnostics of the biology of various diseases, including cancer, Alzheimer’s, and Parkinson’s. Additionally, PET is a powerful tool in the drug discovery and development process, providing in vivo pharmacokinetics and pharmacodynamics using radiolabeled versions of new drugs. A portion of clinical PET centers support drug trials carried out by pharmaceutical and biotech companies by synthesizing these molecules. PET biomarkers can also be used to select the best treatment for individual patients. Patient stratification via PET is anticipated to increase the quality of therapeutics available to patients with a concomitant decrease in the cost of bringing these therapeutics to market. (In the current randomized approach to patient selection, ~75% of patients do not have an efficacious response to treatment.) Furthermore, PET has been widely used in preclinical research and its use in cell cultures (Vu et al. 2011) and animal models is growing dramatically due to the recent advent of preclinical PET imaging systems that are easy-to-use, compact, and affordable (Zhang et al. 2010). Coupled with the Critical Path Initiative of the FDA to partner a biomarker with each drug in clinical trials, as well as the ongoing technetium (99mTc) shortage affecting single photo emission computed tomography (SPECT) imaging, there is an even greater demand for PET, especially so given its superior sensitivity and image quality.

High yield and high specific activity synthesis of [18F]fallypride in a batch microfluidic reactor for micro-PET imaging

[(18)F]fallypride was synthesized in a batch microfluidic chip with a radiochemical yield of 65 ± 6% (n = 7) and an average specific activity of 730 GBq μmol(-1) (20 Ci μmol(-1)) (n = 4). Specific activity was ~2-fold higher than [(18)F]fallypride synthesized in a macroscale radiosynthesizer, despite starting with significantly less radioactivity, and thus safer conditions, in the microchip.

The separation and detection of PET tracers via capillary electrophoresis for chemical identity and purity analysis.

CE coupled with UV detection was assessed as a possible platform for the chemical identity and purity analysis of positron emission tomography (PET) tracers using [(18)F]FAC and [(18)F]FLT as examples. Representative samples containing mixtures of the tracers plus well-known impurities, as well as real radioactive samples (formulated for injection), were analyzed. Using MEKC with SDS in a neutral phosphate buffer, the separation of all compounds in the samples was achieved with baseline resolutions in less than 4.5min and 3min for FLT and FAC samples, respectively. In comparison to the gold-standard for chemical analysis (i.e. HPLC/UV), we have demonstrated improvements in analysis times, and comparable LOD. Although the reproducibility in migration time is slightly lower than that of the HPLC, identification of the compounds was still possible due to good peak separation. In addition, we show that CE can be used to identify and quantify Krytofix2.2.2 (a toxic and commonly used phase transfer catalyst) in less than 2min and with a LOD of 45μg/mL (non-optimized). These results demonstrate adequate performance for chemical identity and purity analysis. Combined with the potential for miniaturization into a microchip format, these results suggest the potential of CE as an integral part of a miniaturized quality control system for PET tracers.

Cationic imidazolium polymer monoliths for efficient solvent exchange, activation and fluorination on a continuous flow system

Polystyrene-imidazolium (PS-Im+Cl−) monolith was synthesized within a flow-through microfluidic chip and Teflon tubing for activating [18F]fluoride ions. The [18F]fluoride ions were trapped on the PS-Im+ monolith and were subsequently released with various phase-transfer catalysts (PTC) and carbonate or bicarbonate bases in microliter volumes of organic solvents containing 0.5% of water. The activated [18F]fluoride complex released from the PS-Im+ monolith was used to fluorinate various known PET probe precursors with diverse reactivity in a subsequent flow-through microfluidic chip without performing additional azeotropic distillation. The fluorination yields under the optimized condition for the protected [18F]FDG, protected [18F]FLT, 4-[18F]fluoroethylbenzoate, and [18F]fallypride were 93%, 96%, 77% and 73%, respectively. This method, utilizing the PS-Im+ monolith on a flow through microfluidic platform, enables the entire fluorine-18 radiochemistry to be performed on a flow-through microfluidic device within a shorter synthesis time and with fluorination efficiency that is comparable to or higher than conventional means.

Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography

Positron emission tomography (PET) is a molecular diagnostic imaging technology to quantitatively visualize biological processes in vivo. For many applications, including imaging of low-tissue density targets (e.g., neuroreceptors), imaging in small animals, and evaluation of novel tracers, the injected PET tracer must be produced with high molar activity to ensure low occupancy of biological targets and avoid pharmacologic effects. Additionally, high molar activity is essential for tracers with lengthy syntheses or tracers transported to distant imaging sites. Here we show that radiosynthesis of PET tracers in microliter volumes instead of conventional milliliter volumes results in substantially increased molar activity, and we identify the most relevant variables affecting this parameter. Furthermore, using the PET tracer [18F]fallypride, we illustrate that molar activity can have a significant impact on biodistribution. With full automation, microdroplet platforms could provide a means for radiochemists to routinely, conveniently, and safely produce PET tracers with high molar activity.For many applications, positron emission tomography tracers must be produced with high specific activity. Here the authors identify variables leading to increased specific activity when tracers are synthesized in microliter volumes, and show that specific activity can influence tracer biodistribution.

High pressure EWOD digital microfluidics

We are developing new electrowetting-on-dielectric (EWOD) digital microfluidic systems for operating at non-atmospheric conditions. The first generation is a compact pressure chamber with an electric feed-through, enabling EWOD operation within a gaseous medium of well-controlled pressure and composition. EWOD performance is insensitive to chamber pressure because the chip is of open-channel architecture. We demonstrate two different types of previously unachievable processes - (i) controlling evaporation rates of common solvents (water, methanol, acetonitrile) by adjusting the pressure of an inert gaseous medium (N2), and (ii) controlling the reaction rate of a solid-liquid-gas-phase reaction by adjusting the pressure of a gas-phase reagent (H2).