Performing multi-step chemical reactions in microliter-sized droplets by leveraging a simple passive transport mechanism

Abstract

Despite the increasing importance of positron emission tomography (PET) imaging in research and clinical management of disease, access to myriad new radioactive tracers is severely limited due to their short half-lives (which requires daily production) and the high cost and complexity of tracer production. The application of droplet microfluidics based on electrowetting-on-dielectric (EWOD) to the field of radiochemistry can significantly reduce the amount of radiation shielding necessary for safety and the amount of precursor and other reagents needed for the synthesis. Furthermore, significant improvements in the molar activity of the tracers have been observed. However, widespread use of this technology is currently hindered in part by the high cost of prototype chips and the operating complexity. To address these issues, we developed a novel microfluidic device based on patterned wettability for multi-step radiochemical reactions in microliter droplets and implemented automated systems for reagent loading and collection of the crude product after synthesis. In this paper, we describe a simple and inexpensive method for fabricating the chips, demonstrate the feasibility of prototype chips for performing multi-step radiochemical reactions to produce the PET tracers [¹⁸F]fallypride and [¹⁸F]FDG, and further show that synthesized [¹⁸F]fallypride can be used for in vivo mouse imaging.

Figure 1.

(A) Photograph of fabricated passive microfluidic chip (top view). The star pattern is a hydrophilic surface (silicon); the remainder is hydrophobic (Teflon). The diameter of the central circular reaction zone is 3.0 mm. The taper angle α of each delivery channel is 5°, and length from the narrow end to center is 9.7 mm. The width of the narrow end of each delivery channel is 0.17 mm. (B) Illustration of passive transport mechanism of a droplet on a wedge-shaped pathway. F_net is the net force due to the larger contact line at the leading (right) edge of the liquid footprint compared to the trailing (left) edge, driving the liquid in the direction of the wider track.

Figure 2.

(A) Schematic of droplet microreactor system, including reagent dispensing system, crude product collection system, and heating and cooling system. (B) Schematic showing configuration for product collection, i.e. with collection tubing lowered into the droplet. The pneumatic cylinder used to lower the tubing is omitted for clarity. (C) Photograph of the microfluidic platform.

Figure 3.

Synthesis schemes for the example PET radiotracers. (A) Radiosynthesis of [¹⁸F]fallypride, illustrating [¹⁸F]fluoride drying step followed by radiofluorination of precursor. (B) Radiosynthesis of [¹⁸F]FDG, showing the [¹⁸F]fluoride drying step, followed by radiofluorination of the precursor and the deprotection (hydrolysis) reaction.

Figure 4.

Schematic of [¹⁸F]fallypride synthesis on the passive microfluidic chip. (A) [¹⁸F]fluoride solution is loaded and dried. (B) Precursor solution is loaded and fluorination reaction is performed. (C) Collection solution is loaded to dilute the crude product, which is then collected. Note that each reagent is loaded from a dedicated dispenser and reagent pathway. The synthesis of [¹⁸F]FDG is quite similar but there is an additional reaction step between steps B and C. After the fluorination reaction, the deprotection agent (NaOH) is added, transported to the center, and the room temperature hydrolysis reaction is performed.

Figure 5.

Examples of radio-HPLC chromatograms of [¹⁸F]fallypride synthesis on the microfluidic reaction chip. (A) Analysis of crude product. Note that the apparent double peak of [¹⁸F]Fallypride is an artifact due to saturation of the radiation detector. (B) Analysis of formulated product. The RCP was 99%.

Figure 6.

Examples of radio-TLC chromatograms of [¹⁸F]FDG synthesis on the microfluidic reaction chip. (A) Analysis of crude product using 95:5 MeCN/water mobile phase. (B) Analysis of the same sample but using the 50:50 EtOAc/hexane mobile phase. The peak represents both unreacted [¹⁸F]fluoride and [¹⁸F]FDG, and the absence of a second peak indicates no residual [¹⁸F]FTAG or partially hydrolyzed [¹⁸F]FTAG. (C) Analysis of purified [¹⁸F]FDG using 95:5 MeCN/water mobile phase. The RCP was >99%. (D) Analysis of the same sample, but using the 50:50 EtOAc/hexane mobile phase.

Figure 7.

Sequence of photographs of the microfluidic chip during the mock synthesis of [¹⁸F]fallypride. (A) A DI water droplet (2μL, dyed yellow) containing TBAHCO₃ (77mM) was loaded, spontaneously transported to the reaction site, and then the chip was heated to 105°C to remove the solvent. (B) Next, two droplets of a 1:1 v/v mixture of MeCN and thexyl alcohol (1μL, dyed red) were loaded from a separate inlet and transported to the reaction site in sequence, after which the droplet was heated to 110°C to simulate fluorination reaction. Note that loading in two separate portions instead of a single larger droplet helped to prevent over-flowing of the reaction site. (C) Next, two droplets of collection solution (9:1 v/v MeOH/water) (5 μL each, dyed blue) were loaded from a third inlet and transported to the center to dilute the reaction mixture. Finally the collection tubing was lowered and the droplet was collected into a vial with the aid of vacuum. Very little residue was apparent on the chip after collection.

Figure 8.

Distribution of radioactivity visualized using Cerenkov imaging after different steps of radiosyntheses. (Top) [¹⁸F]Fallypride: (A) after [¹⁸F]fluoride drying step; (B) after fluorination reaction; (C) residual radioactivity on chip after collection of product. (Bottom) [¹⁸F]FDG: (D) after [¹⁸F]fluoride drying step; (E) after fluorination step; (F) residual radioactivity on chip after collection of product. Images are corrected for radioactive decay.

Figure 9.

Small-animal PET/CT images from the static scan after 60 min uptake of [¹⁸F]Fallypride. (A) Maximum intensity projection (MIP) image of whole mouse; (B) Transverse slice highlighting uptake in striata in the brain.