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

Abstract

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 ½ 18 FFDG, the most common radiotracer for positron emission tomography (PET), with high and reliable radio-fluorination efficiency of ½ 18 FFTAG (88 AE 7%, n ¼ 11) and quantitative hydrolysis to ½ 18 FFDG (>95%, n ¼ 11). We furthermore show that batches of purified ½ 18 FFDG 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 bestcase 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. molecular imaging | PET probes | synthetic chemistry | lab on a chip | on-chip chemistry T he use of micro-reaction technology in chemistry has grown tremendously over the past several years (1), due primarily to the highly precise control of reaction conditions that is possible through rapid mixing and heat transport, leading to improved reaction speeds and selectivity compared to macroscale approaches (2). Additional advantages include straightforward scale-up of production without changing conditions, and increased safety in dangerous syntheses due to the minute amounts of reagents within the reactor at any given time. A further advantage of microfluidics is the ability to perform reactions in extremely small volumes, which is valuable for many applications, especially when working with scarce reagents, such as isolated proteins or natural products, products of long synthetic pathways, or short-lived radiolabeled radioisotopes where the needed mass quantities are extremely low (3).Myriad microfluidic platforms have been explored for chemical reactions that can be classified into three basic formats: (i) flow-through (or continuous flow), (ii) droplet or slug, or (iii) batch. In flow-through systems, streams of two or more reagents are mixed and reacted by flowing through a residence time unit held at a constant temperature or immersed in a fixed microwave field. Continuous liquid-liquid extraction and other processes have been developed to enable multistep reactions where different solvents are required in different steps (4). Droplet and slug systems are a variant of flow-through systems, in which individual droplets or slugs (with volumes down to tens of nanoliters) are separated by an immiscible carrier fluid, each acting as an isolated batch microreactor and enabling vastly reduced reaction volumes. Screening assays and optimization studies...

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

“…The hydrophobic coating consisted of Cytop® (glass transition temperature T g = 108˚C) in early devices [21,26], but was switched to Teflon® AF 2400 (T g = 240˚C) in the current devices for its higher thermal stability. Teflon® AF 2400 was considered even more stable than Teflon® AF 1600 (T g = 160˚C) used in the work preliminary to this report [22,25].…”

“…Past reports have demonstrated a range of droplet functions (i.e., generating, moving, splitting, and merging) [17], precise control of droplet volumes [18], on chip liquid composition measurement [19], and suitability for multiple reagent chemical synthesis [20]. Our group has recently reported the use of EWOD for the synthesis of the most commonly used PET radiotracer, [ 18 F]FDG [21].…”

Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics

“…Key features we found to enable successful panning were: 1) capturing site‐specifically biotinylated SpyCatcher bait in solution, rather than attaching SpyCatcher to beads, 2) TEV protease cleavage to elute phage specifically from beads, and 3) washes harsh enough to dissociate non‐covalent interactions, but retaining phage infectivity (1× glycine pH 2 and 4× Tween‐20). For model selection, we incubated M13 phage displaying SpyTag on pIII with either reactive bait (SpyCatcher) or the negative control SpyCatcher EQ 2a. Using this panning procedure we obtained 4 orders of magnitude enrichment for the specific covalently reacting partner (Figure 1 b).…”

“…Since mutating central residues in SpyTag abolished SpyCatcher reactivity,2a we made two different libraries, randomizing at the N‐terminal or C‐terminal ends of SpyTag (Figure 1 c,d). After panning, NLib1 (PPVPTIVMVDAYKPTK) gave the fastest reaction, with the first two residues able to be removed without affecting the rate (Figure S1a in the Supporting Information).…”

“…Additional features important for successful SpyCatcher selection were: 1) a DsbA signal sequence for co‐translational translocation of SpyCatcher‐pIII7 and 2) growing in the XL‐1 Blue E. coli strain at 18 °C. For model selection, the bait was biotinylated Avitag‐SpyTag‐MBP (Figure 2 a), which showed an approximately 1000‐fold enhanced capture of WT SpyTag bait compared to non‐reactive SpyTag DA bait2a (Figure 2 b). The sequence of selected clones is indicated in Figure 2 c. Mutations were widely distributed over the structure, with many mutated residues distant from the SpyTag binding site (Figure 2 d).…”