Capture, isolation and release of cancer cells with aptamer-functionalized glass bead array

Abstract

Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.

Figure 1

Schematics showing steps of fabrication and experiments. SU-8 photoresist is spin-cast on silicon wafer, exposed and wells are developed to form the desired pattern. PDMS is poured on SU-8 master, baked, and peeled off. 50 μm diameter GBs are loaded into 25 μm deep pits and the substrate is covered with a flat PDMS slab. Cancer cell suspension is flowed through the device, and cells are captured by aptamer functionalized GBs. Captured cells are finally released from the GB surface after GBs are collected from the device.

Figure 2

Schematics showing cells exposed to anti-EGFR aptamer complex, binding between cells and aptamer-DNA-biotin-SAPE complex and introduction of RELease molecule. Fluorescence came from SAPE bound to anti-EGFR aptamer. After washing, cells show fluorescence due to selective binding with aptamer. The addition of RELease RNA agent competitively hybridizes with anti-EGFR aptamer, opening up its hairpin structure. It can release 76% of anti-EGFR aptamers from cellular surfaces.

Figure 3

(a) Velocity patterns in the device at 10 μm height; (b) Stream lines for the velocity field at 10 μm height. The flow rate is set as 1 ml/h. (c) Velocity pattern along the inlet flow. The direction and weight of each arrow depict fluid movement and amplitude of the force, respectively; (d) Velocity pattern perpendicular to the flow with arrow size depicting the amplitude of the velocity vector. The positions of the three slices are at the central plane, and 15 μm before and after the central plane of beads. (e) Average number ± standard deviation of captured hGBM cells at specific distances from inlet on anti-EGFR and mutant aptamer functionalized plain Hele-Shaw devices.

Figure 4

SEM micrographs of Hele-Shaw microfluidic device with pits and loaded glass beads. (a) Shows the equilateral triangle array of pits (50 μm diameter, 25 μm depth, 20 μm spacing); (b) Shows the glass beads loaded in the arrayed pits made in PDMS. The scale bars are 100 μm in both micrographs. The inset to (b) shows a close-up view of a re-suspended glass bead with hGBM cell. Flow rate of cell suspension was set as 1 ml/h.

Figure 5

(a) Native polyacrylamide gel electrophoresis (PAGE) shows the release effect of the RELease sequence. The FACS extension binds to the capture probe and when RELease sequence is used the band for anti-EGFR aptamer shifts showing structure changes in the aptamer. The structure change looses binding capability with the cellular EGFR. (b) Fluorescence intensity of FACS flow cytometer data shows the shift in signal from just cells to the cell bound with anti-EGFR aptamer construct and then upon introduction of RELease molecule. RELease agent significantly affects the fluorescence intensity before and after removing the anti-EGFR aptamer.