Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography

Abstract

Methods for tagging biomolecules with fluorine 18 as immuno-positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.

Figure 1

Schematic illustrations of the detailed architecture of a digital microfluidic droplet generation (DMDG) chip, working principle and chip-based screening. (A) A setup of DMDG chip in which food dyes were used to aid visualization of different components; (B) Schematic of a DMDG chip. The DMDG chip is composed of three functional parts: (1) droplet generation core, where droplets with specific compositions can be generated digitally; (2) on-chip peristaltic pump, which precisely produces serial compressed nitrogen pulses that can reliably deliver intact droplets to the desired location and (3) mixing channel. Inlets 1–8 are for cleaning, vacuum, cleaning/buffer 1, [¹⁸F]SFB, A2 Db, buffer 2, pH control buffer, and waste respectively. Section I is specifically used for [¹⁸F]SFB, section II for A2 Db and buffer 2, section III for pH control buffer, respectively. The fluidic channel width is ca 200 µm and height is ca 40 µm. (C) The reaction scheme of ¹⁸F-labeling diabody using [¹⁸F]SFB. (D) Schematic of adjusting pH in each droplet. (E) Schematic of adjusting the concentration of A2 Db in each droplet.

Figure 2

(A) The effect of pH on RLY (n=3). (B) The effect of diabody concentration on RLY at pH 8.7 (n=3). (C) Unlabeled A2 Db, and [¹⁸F]FB-A2 Db produced from bench-scale vial and microfluidic chip were run on SDS-PAGE and analyzed by autoradiography. Proteins were later revealed by Coomasie blue staining. (D) Compare RLY by using conventional bench-scale method and on-chip approach (n=3).

Figure 3

Biochemical, in vitro and in vivo characterizations of [¹⁸F]FB-A2 Db. (A) Immunoreactivity of [¹⁸F]FB-A2 Db produced from vial or chip to PSCA-expressing SKW 6.4 and SKW 6.4 cells (control) (n=2). (B) Co-registered microPET/CT dynamic scan images (coronal [top] and transverse [bottom] slices) of nude mice bearing LAPC-9 (PSCA-positive human prostate cancer) xenografts. Tumor locations are indicated by yellow arrows. (C) A co-registered microPET/CT image (coronal projections) 4h p.i. of [¹⁸F]FB-A2 Db. (D) Graph of biodistribution study of [¹⁸F]FB-A2 Db (4 h p.i.) (n=3).