Chip Design

Our Different designs

Introduction and first idea

One of the biggest challenges of circulating tumor cells is their scarcity in the blood of patients. To overcome this problem, our first idea was to develop a microfluidic chip in order to perform single cell analysis. The biggest advantage of using a microfluidic chip is its ability to perform high-throughput cell biology. In order to do so, we wanted to produce water-in-oil emulsion droplets, that can then be sorted by a machine analogous to FACS, (inspired from [Chiu 2015]). In the droplets, a mixture of bacteria and mammalian cells would be present. And the bacteria would express the green fluorescent protein only in the presence of cancer cells, exhibiting both increased lactate production rate and sensitivity to sTRAIL.

First Design

However due to the complexity of this setup, we decided to first explore another design consisting of valves and chambers. Instead of having droplets to isolate single cells, we wanted to have a two-layer microfluidic chip. One of the layer would have been the flow of cells and the other layer, valves controlled by pressure that are able to close the chambers. We drew the designs using Autocad. On the figure, the orange layer represents the pressure control of the valves and the red layer represents the flow of the liquid.

Realistic and Final Design

Because of time constraints, we did not make the previous chip but instead we designed a "nano-well" plate which represents our proof of principle. Here, there is no flow going through the chip. Characteristics of the chip

• The volume of every well is 1nL.
• There are 4992 wells in our chip.

Fabrication and handling of the chip

Fabrication of the chip

In the following, the sequence of steps to fabricate the chip are detailed

1. The design was drawn thanks to the Autocad software.
2. The subsequent mask was made by a company.
3. The glass wafer was created using photolithography (positive or negative photoresist ?? ).
4. PDMS was electrospun on the glass wafer.

Plasma treatment

At first, the chip was hydrophobic. Plasma treatment of the chip has been shown to make chips hydrophilic (ref). We used this technique to treat our chip. The protocol is described in the following.The freshly made chips were then treated at 40% power (100% = 50W at ~14 MHz) for 50s.

Coating

In order to make the cells attach to chip, the chip was incubated two hours in BSA or Fibronectin solutions.

Loading of the cells

Number of cells per well

First of all, we need to know which concentration of cells we need to have 1 cell per nanoLiter (equivalent to \(10^{6} \) per mL. However, if we apply Poisson Distribution to this result.

\begin{align*} \lambda &=1 \\ P(X=0) &= 36\%\\ P(X=1) &=36\%\\ P(X=2) &= 18\%\\ \end{align*}

So 36 % of the wells will be empty. To solve this problem we applied a concentration of 2 cells per nanoLiter.

\begin{align*} \lambda &=2 \\ P(X=0) &=13\%\\ P(X=1) &= 27\%\\ P(X=2) &= 27\%\\ P(X=3) &= 18\%\\ \end{align*}

This time only 13% of the wells are empty.