Bioinspired multivalent DNA network for capture and release of cells

Abstract

Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.

Fig. 1.

Isolation and detection of cancer cells in whole blood using long multivalent DNA aptamer-based microfluidic device. The zoomed-in box illustrates a captured cell is bound by several long DNA molecules via multiple aptamer domains (red color).

Fig. 2.

Single-cell force measurement revealed that the RCA-aptamer binds target cells more effectively than Unit-aptamers. (A) Representative images of individual CCRF-CEM cells held in a piezo-controlled micropipette that were brought (Left) into direct contact with an optically trapped Unit-aptamer coated bead or (Right) at a 1-μm distance from an RCA-aptamer coated bead. RCA-aptamers formed stronger bonds to the target cell than the maximum trap force (>40 pN and a force loading rate of ∼58 pN/s) as evidenced by the micropipette/cell pulling the bead out of the optical trap, whereas the Unit-aptamer system was unable to achieve this. Images were obtained with 40× oil immersion objective. (B) RCA-aptamer beads exhibited a much higher frequency of binding to target cells (100% for direct contact and 100% for 1-µm distance) than Unit-aptamer beads (29% for direct contact and 0% for a 1-µm distance).

Fig. 3.

(A) RCA-aptamer device captured cells specifically and exhibited significantly greater cell capture efficiency compared with Unit-aptamers. Cell-capture performance is shown as a function of (B) graft density and (C) length of RCA products.

Fig. 4.

Microfluidic RCA-HB chip for cell capture. (A) Design of the HB-chip with grooves shown in the blowout. (B) Fluorescent image (false colored) of CCRF-CEM cells captured in the 3D DNA network on the herringbone surface. The RCA-product was stained with Sybr Green II (Green) and the cells were fluorescently tagged with CMTPX (Red). (Scale bar, 20 µm.) (C) Purity of captured CCRF-CEM cells spiked at a concentration of 1,000 cells/mL of whole blood and flowed at 120 µL/h (nominal shear stress of 1.5 dyn/cm²) through a HB-chip functionalized with RCA-aptamer or PTK7-antibody. (D) Capture efficiency of CEM-CCRF cells (1,000 cells/mL of whole blood) at different flow rates for HB-chip modified with RCA product, Unit-aptamer, PTK7-antibody, and scrambled RCA-aptamer (control). True mass balance was used to calculate efficiency values. The values shown are mean ± SD for n = 5 (RCA-aptamer) or n = 3 (other groups) independent experiments. (E) Comparison between the current RCA system and other major cell capture devices in terms of efficiency vs. specific throughput (flow rate over device footprint area). RCA HB-chip, PTK7 antibody HB-chip, EpCAM antibody HB-chip (12), CTC chip (6), and EpCAM antibody functionalized silicon nanowires incorporated in a microfluidic mixer (10) are shown.