A novel dual-well array chip for efficiently trapping single-cell in large isolated micro-well without complicated accessory equipment

Abstract

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 μm in diameter) with reaction-wells (100 μm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

FIG. 1.

Schematic representation of the dual-well array structure and working principles. (a)–(i) Schematic representation of single-cell analysis by the dual-well array chip. (a) PBS buffer was first added to the dual-well and then the bubbles in the micro-wells were degassed by vacuum chamber (b). Cell suspension solution (PBS as solvent) was added to the dual-well (c). PBS buffer was used to wash out the cells outside the capture-cells (d) and finally forming single-cell array (e). (f) Drying out the array by nitrogen. (g) The prepared reagents were transferred onto one end of the dual-well array by a pipette. (h) The blade was dragged slowly across the chip in a smooth motion to load reagent and then sealing oil was added to cover the micro-wells. (i) Packing the chip as sandwich structure. (j) Schematic diagram of the movement of cells in the dual-well according to (d). Cells were loaded into both reaction-wells and capture-wells in the dual well array. Cells that are outside the capture-well are cleared away by the momentum of the liquid. (k) A 3D microscopic image of an individual dual-well.

FIG. 2.

Velocity profile and streamlines of the dual-well structure. (a) Velocity profile of the model with an inlet velocity of 0.1 m/s. The highest velocity is at the middle of the main channel and it decreases to zero at the bottom of the capture-wells. The ordinate origin (X-axis) was set in the middle of model and the (Y-axis) on the basal surface of the channel, as shown in the picture. (b) The velocity profile and streamlines of an individual dual-well. (c) and (d) Graphs showing the magnitude of velocity at the location [line (O-N)] marked in (a) and [line (O-S)] and (points: O, P, Q, R, and S) marked in (b) with four inlet flow rates: 0.1, 0.05, 0.01, and 0.005 m/s.

FIG. 3.

(a) Simulated contours of the flow velocity and streamlines of dual-well and schematic representation of multi-cell in wells. The points (A–F) represent the locations where cell may stay. (b) Graph indicates the X-axis velocity at the cells location with inlet flow rate of 0.1 m/s.

FIG. 4.

(a) and (b) demonstrate the velocity profile and wall shear stress for micro-well with 100 μm in diameter and 20 μm in deepness, respectively. (c) and (d) are for micro-well with 20 μm in diameter and 20 μm in deepness. (e) and (f) are for dual-well insists of a capture-well (20 μm in diameter and 20 μm in deepness) and a reaction-well (100 μm in diameter and 20 μm in deepness). All simulated processes were carried out at inlet flow rate of 0.1 m/s.

FIG. 5.

(a) 3D image of a positive model made of silicon material. (b) Dual-well array made of PDMS material. (c) Cells introduced into a dual-well and (d) cells in the dual-wells after the washing step. Bright-field and corresponding fluorescence images (e) of multiple cells in an individual dual-well before washing step. Fluorescence images show single-cell (f) and multi-cell (g) in an individual dual-well after washing step.

FIG. 6.

(a) The detection principle of intracellular enzymatic assay for β-galactosidase. (b) Merged photo of bright-field image of the enzymatic assay and fluorescence images of the single-cell array. The numbers point out the dual-well which has trapped cells. (c) Kinetics of the average fluorescence intensity from the wells containing a cell. (d)–(g) Post-lysis fluorescence images of the wells for monitoring the presence of the fluorescent product (fluorescein) from FDG hydrolysis by intracellular β-gal.