Difference between revisions of "Team:ETH Zurich/Chip"

 
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<h1>Chip Design</h1>
 
<h1>Chip Design</h1>
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<h2>Our Different designs</h2>
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<h2>Our chip designs</h2>
 
<h3>Introduction and first idea</h3>
 
<h3>Introduction and first idea</h3>
 
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<p>Idea design of microfluidic chip</p>
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<p><b>Figure 1.</b> First concept of microfluidic chip</p>
 
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<p>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.  
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<p>One of the biggest challenges of detecting 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.  
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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 [<a href="https://2015.igem.org/Team:ETH_Zurich/References#Chiu2015">Chiu 2015</a>]).  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.  </p>
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In order to do so, we wanted to produce water-in-oil emulsion droplets, that can then be sorted or analyzed by a machine analogous to FACS, (inspired from [<a href="https://2015.igem.org/Team:ETH_Zurich/References#Chiu2015">Chiu 2015</a>]).  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.  </p>
 
<h3>First Design</h3>
 
<h3>First Design</h3>
 
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<p>First design of the microfluidic chip</p>
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<p><b>Figure 2.</b> First design of microfluidic chip: On the figure, the orange layer represents the pressure control of the valves and the red layer represents the flow layer. </p>
 
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<p> 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.
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<p> 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 be the flow layer, where cells are flushed in. The other layer would consist of microfluidic valves, controlled by an external pressure source, and capable of occluding the flow layer. Thus, small chambers separating single cells could be formed.
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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. </p>
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We drew the designs using AutoCAD. </p>
 
<h3>Realistic and Final Design</h3>
 
<h3>Realistic and Final Design</h3>
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<p> 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.
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<b> Characteristics of the chip </b>
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<li>The volume of every well is 1nL.</li>
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<li>There are 4992 wells in our chip.</li>
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<p>Final design of the microfluidic chip</p>
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<p><b>Figure 3.</b> Final design of the microfluidic chip</p>
 
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<p> 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.
 
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<b> Characteristics of the chip </b>
 
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<ul>
 
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<li>The volume of every well is 1nL.</li>
 
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<li>There are 4992 wells in our chip.</li>
 
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<h2> Fabrication and handling of the chip </h2>
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<h2>Results and accomplishments</h2>
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<h3> Fabrication of the chip </h3>
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<p> In the following, the sequence of steps to fabricate the chip are detailed </p>
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<p> We implemented a testing system for single cell analysis in a nanowellplate and were able to <a href="https://2015.igem.org/Team:ETH_Zurich/Results#Towards_a_more_sensitive_lactate_dependent_system">detect lactate produced by singel mammalian cells</a>. Also, we co-cultured bacteria and mammalian cells in the chip successfully.<p>  
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<li> The design was drawn thanks to the Autocad software.</li>
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<li> The subsequent mask was made by a company.</li>
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<li> The glass wafer was created using photolithography (positive or negative photoresist ?? ).</li>
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<p>Thanks to this experiment, we could show that we can introduce a coherent number of bacteria and mammalian cells into the chip. The aim was to obtain single mammalian cells in the chip with 1000 times more bacteria. The loading of the bacteria and mammalian cells is described <a href="https://2015.igem.org/Team:ETH_Zurich/Experiments#Loading_of_Mammalian_Cells_and_bacteria">elsewhere</a>.</p>
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<li> PDMS was electrospun on the glass wafer.</li>
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<p> To visualize the mixture of cells, we used bacteria expressing RFP and 3T3 mouse fibroblast cells expressing GFP as a nuclear marker.</p>
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<h4>Results</h4>
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<h3> Plasma treatment</h3>
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<p> We achieved to seed the cells in the desired ratio per chamber. We can see that 3T3s grow in the chip, even in the presence of bacteria (even over a couple of hours).</p>
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<p> 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. </p>
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<h3> Coating</h3>
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<p> In order to make the cells attach to chip, the chip was incubated two hours in BSA or Fibronectin solutions. </p>
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<h3> Loading of the cells</h3>
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<h4> Number of cells per well </h4>
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<p><b>Figure 4.</b> Picture of 3T3 (green fluorescence) co-cultured with bacteria (red fluorescence). </p>
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<p> 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. </p>
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P(X=0) &= 36\%\\
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P(X=1) &=36\%\\
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P(X=2) &= 18\%\\
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<p>So 36 % of the wells will be empty.
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To solve this problem we applied a concentration of 2 cells per nanoLiter.</p>
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\lambda &=2 \\
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P(X=0) &=13\%\\
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P(X=1) &= 27\%\\
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P(X=2) &=  27\%\\
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P(X=3) &=  18\%\\
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<p> This time only 13% of the wells are empty. </p>
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Latest revision as of 01:34, 19 September 2015

"What I cannot create I do not understand."
- Richard Feynmann

Chip Design

Our chip designs

Introduction and first idea

Figure 1. First concept of microfluidic chip

One of the biggest challenges of detecting 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 or analyzed 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

Figure 2. First design of microfluidic chip: On the figure, the orange layer represents the pressure control of the valves and the red layer represents the flow layer.

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 be the flow layer, where cells are flushed in. The other layer would consist of microfluidic valves, controlled by an external pressure source, and capable of occluding the flow layer. Thus, small chambers separating single cells could be formed. We drew the designs using AutoCAD.

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.

Figure 3. Final design of the microfluidic chip

Results and accomplishments

We implemented a testing system for single cell analysis in a nanowellplate and were able to detect lactate produced by singel mammalian cells. Also, we co-cultured bacteria and mammalian cells in the chip successfully.

Thanks to this experiment, we could show that we can introduce a coherent number of bacteria and mammalian cells into the chip. The aim was to obtain single mammalian cells in the chip with 1000 times more bacteria. The loading of the bacteria and mammalian cells is described elsewhere.

To visualize the mixture of cells, we used bacteria expressing RFP and 3T3 mouse fibroblast cells expressing GFP as a nuclear marker.

Results

We achieved to seed the cells in the desired ratio per chamber. We can see that 3T3s grow in the chip, even in the presence of bacteria (even over a couple of hours).

Figure 4. Picture of 3T3 (green fluorescence) co-cultured with bacteria (red fluorescence).

We would like to thank our sponsors