Specific capture and temperature-mediated release of cells in an aptamer-based microfluidic device

Abstract

Isolation of cells from heterogeneous mixtures is critically important in both basic cell biology studies and clinical diagnostics. Cell isolation can be realized based on physical properties such as size, density and electrical properties. Alternatively, affinity binding of target cells by surface-immobilized ligands, such as antibodies, can be used to achieve specific cell isolation. Microfluidics technology has recently been used in conjunction with antibody-based affinity isolation methods to capture, purify and isolate cells with higher yield rates, better efficiencies and lower costs. However, a method that allows easy release and collection of live cells from affinity surfaces for subsequent analysis and detection has yet to be developed. This paper presents a microfluidic device that not only achieves specific affinity capture and enrichment, but also enables non-destructive, temperature-mediated release and retrieval of cells. Specific cell capture is achieved using surface-immobilized aptamers in a microchamber. Release of the captured cells is realized by a moderate temperature change, effected via integrated heaters and a temperature sensor, to reversibly disrupt the cell-aptamer interaction. Experimental results with CCRF-CEM cells have demonstrated that the device is capable of specific capture and temperature-mediated release of cells, that the released cells remain viable and that the aptamer-functionalized surface is regenerable.

Fig. 1

Principle of specific cell capture and temperature-mediated release. (A) Cell capture at room temperature. (B) D-PBS wash after capture. (C) Cell release at a moderately higher temperature.

Fig. 2

(A) Schematic of the microfluidic device for the specific capture and temperature-mediated release of CCRF-CEM cells. (B) Deposition, patterning and passivation of gold sensor and heaters. (C) Fabrication of SU-8 mold. (D) Demolding of PDMS microchamber. (E) Treatment with chlorotrimethylsilane and PDMS spin-coating. (F) Bonding of PDMS microchamber to PDMS-coated temperature control chip. (G) Insertion of inlet and outlet capillary tubes. (H) Photograph of a fabricated device. (I) Micrograph of the temperature sensor and heaters in the device. (J) Experimental setup for specific capture and temperature-mediated release of cells. Dimensions are given in micrometers.

Fig. 3

(A) Image of the chamber after the introduction of a cell sample. (B) Image of the chamber after the introduction of 10 cell samples and D-PBS washing. (C) Time response of cell capture: percentage of cells captured (η) versus incubation duration (t). The solid line represents an exponential fit to the experimental data with a regression equation: η = 1 − e^−t/24 (R² = 0.982, n = 3). (D) Concentration response of cell capture: density of captured cells (ρ_capture) as a function of the cell suspension concentration (c_cell). The solid line represents a linear fit to the experimental data with a regression equation: ρ_capture = 0.3874c_cell (R² = 0.995, n = 3).

Fig. 4

(A) Percentage of captured cells remaining on the substrate as a function of time while rinsing at constant temperature (48 °C and room temperature) and flow rate (5 µL min⁻¹). (B) Captured cell density versus the number of cell suspension samples introduced while the temperature was maintained at either 48 °C or room temperature. (C) Effect of temperature on cell release efficiency while rinsing at 5 µL min⁻¹. (D) Effect of flow rate on cell release efficiency while the chamber temperature was maintained at 48 °C. (E) Cell capture and re-capture on the regenerated aptamer-functionalized surface: the normalized percentage of remaining cells after the first, second and third capture and regeneration cycle.

Fig. 5

Image of PI stained cells (A) and JC-1 stained cells (B) following cell capture and release experiment, generated by a combination of phase contrast and fluorescent micrographs. (C) Concentrations of normal cells and heat-treated cells as a function of culture duration.