An oligonucleotide synthesizer based on a microreactor chip and an inkjet printer

Abstract

Synthetic oligonucleotides (oligos) are important tools in the fields of molecular biology and genetic engineering. For applications requiring a large number of oligos with high concentration, it is critical to perform high throughput oligo synthesis and achieve high yield of each oligo. This study reports a microreactor chip for oligo synthesis. By incorporating silica beads in the microreactors, the surface area of the solid substrate for oligo synthesis increases significantly in each microreactor. These beads are fixed in the microreactors to withstand the flushing step in oligo synthesis. Compared to conventional synthesis methods, this design is able to avoid protocols to hold the beads and integrate more microreactors on a chip. An inkjet printer is utilized to deliver chemical reagents in the microreactors. To evaluate the feasibility of oligo synthesis using this proof-of-concept synthesizer, an oligo with six nucleotide units is successfully synthesized.

Figure 1

The schematic view of the oligonucleotide synthesis on our platform. (a) The synthesis processes involve designing target sequence, delivering chemical reagents through the inkjet printer, and oligonucleotide synthesis in the microreactor chip. (b) The single microreactor is filled with silica beads which enhance the surface area for the following synthesis. The beads are inherently fixed in the microreactor using sintering process. (c) The oligonucleotide synthesis on the silica beads follows the four-step with phosphoramidite strategy: deprotection, coupling, capping and oxidation.

Figure 2

The microreactor array chip fabrication and characterization. (a) The fabrication processes to prepare the microreactor array chip: microwells were fabricated on substrate (1–2); a layer of dry film was bonded and patterned on the microwells (3); silica beads were self-assembled and fixed within the microwells and extra beads among microwells were removed with the dry film (4–6). (b) The photograph of a microreactor array chip. (c) SEM image of a single microreactor. (d) SEM image showing the physical bonding among silica beads. Scale bars in (b), (c), and (d) are 2 mm, 100 μm, and 2 μm, respectively. (e)The setup to test the mechanical strength of the beads in the microreactor. (f) The physical model of the packed beads and (g) the distributed microchannel network among the beads.

Figure 3

The reagent delivery system utilizing a commercial inkjet printer. (a) The photograph of the reagent delivery system. The cartridges were loaded with chemical reagents which can be delivered to the microreactor chip under the printer heads. A vacuum waste collection was employed to collect waste reagents in the microreactor. The system was stored in a nitrogen dry glove box. (b) The projection between the target pattern in Microsoft PowerPoint (1) and the printed reagents on an immobilized substrate (2–3). The position of the reagents on the substrate can be tuned and different reagents can be printed to the same position. (c) Pigment ink pattern on an immobilized paper after 40 repeat printing cycles on each point. (d) Acetonitrile pattern on an immobilized cover slide after 40 repeat printing cycles on each point. Scale bars in (c) and (d), 2 mm.

Figure 4

Oligonucleotide synthesis using the microreactor chip and the printer-based reagent delivery system. (a) The oligonucleotide synthesis includes surface treatment of the beads in the microreactors (1), the 4-step oligo synthesis (2), product collection (3), and product detection (4). (b) The respective chemical reactions on a single bead corresponding to (a). (c) Mass spectrometry of the synthesized oligo on the platform. There are six oligos with 1-6 nucleotide units and the sequence is determined from the mass difference of two adjacent peaks, respectively.