On-demand radiosynthesis of -succinimidyl-4-[F]fluorobenzoate ([F]SFB) on an electrowetting-on-dielectric microfluidic chip for F-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 -succinimidyl-4-[F]fluorobenzoate ([F]SFB), the most commonly used F-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 [F]SFB starting from the no carrier added [F]fluoride ion. In this report, we established an optimal microliter droplet reaction condition to obtain reliable yields and synthesized [F]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 [F]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. [F]SFB was successfully synthesized on the EWOD chip in 39 ± 7% ( = 4) radiochemical yield in a total synthesis time of ∼120 min ([F]fluoride activation, [F]fluorination, hydrolysis, and coupling reaction, HPLC purification, drying and reformulation). The reformulation and stabilization step for [F]SFB was important to obtain a high protein labeling efficiency of 33.1 ± 12.5% ( = 3). A small-animal immunoPET pilot study demonstrated that the [F]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.