Difference between revisions of "Team:BostonU HW/Demonstrate"

Line 197: Line 197:
 
                 </a></p>
 
                 </a></p>
  
−
                 To test the model we plated a GFP gradient on a 96-well plate with TERRA using a single output microfluidic chip. The fluorescent reading of the 4x6 well gradient of three, two, and one 0.047 mL GFP droplets provides a quantitative measure of sample volume in wells. </p>
+
To prove that TERRA is compatible with the benchtop analytical tools, we ran a basic experiment that demonstrated the ability to detect GFP. A smiley face was dispensed onto a 96-well plate using the user interface and TERRA. The final plate was then read using the lab’s plate reader, the results demonstrate that the outputs of TERRA can be analyzed using benchtop analytics.
 +
 
 +
<img src="https://static.igem.org/mediawiki/2018/d/d1/T--BostonU_HW--terra_smile.jpeg" width="100%" class="img-fluid">
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2018/e/e7/T--BostonU_HW--spreadsheet.jpeg" width="100%" class="img-fluid">
 +
<br>
 +
 
 +
Now that we verified that TERRA is compatible with benchtop analytical tool, we can use a plate reader to prove that we can accurately dispense sample volumes.
 +
                  
 +
To test the model we plated a GFP gradient on a 96-well plate with TERRA using a single output microfluidic chip. The fluorescent reading of the 4x6 well gradient of three, two, and one 0.047 mL GFP droplets provides a quantitative measure of sample volume in wells. </p>
  
 
                 <div class="row">
 
                 <div class="row">

Revision as of 03:18, 18 October 2018

Argon Design System - Free Design System for Bootstrap 4

Argon Design System - Free Design System for Bootstrap 4

Demonstrate

In order to fulfill the goal of creating an automated system that selectively dispenses the output of a microfluidic chip, TERRA is required to have certain functionality.

Functional Requirements:
  1. Application agnostic - treats the user’s microfluidic chip as a black box system so that TERRA can handle a variety of different applications and microfluidic chips

  2. Selectively dispense microfluidic output - uses a UI and XY translational stage to move a well plate so microfluidic outputs are dispensed into user selected wells

  3. Fully Automated - output selection is facilitated by automating valve actuation for each output

  4. Integration with benchtop analytics - Dispense the sample volume inputted by the user into selected

  5. Cost effective - Less than current options and accessible to researchers; >$1000

We have designed TERRA to fulfill all the requirements.


Application Agnostic

Creating an application agnostic system ensures that TERRA can handle a variety of different applications and third party microfluidic chips. In order to make TERRA application agnostic, the system has to treat the user’s microfluidic chip as a black box and work solely with the outputs of the user’s microfluidic chip. In addition, treating the microfluidic chip as a black box offloads the burden of microfluidic expertise off the user, making TERRA accessible to the greater synthetic biology community.

To make TERRA application agnostic, we designed the TERRA Adapter. The TERRA Adapter is a microfluidic chip that connects the user’s microfluidic chip to TERRA, mediating selection and redirection of outputs. The TERRA Adapter is currently a feature available on 3DuF so that users can manufacture and assemble their own TERRA Adapters for their experiments.

Click here to learn more about the TERRA Adapter:


Selectively Dispense Microfluidic Output

To export the outputs of microfluidic chip onto standard lab vessels, users need control over output selection and dispense locations. To accomplish this we designed TERRA to include a UI that takes user input and a XY translational stage that moves the well plate to select wells.

Click here to find more about the user interface:

Click here to find more about the XY translational stage:

To verify this we ran a variety of different single output configurations on a 96 well plate, using color and fluorescence as markers.

We created two initial configurations on the 96-well plate using colored water as a preliminary run. The first is of two simple blue circles and the second is the phrase “igem”. The sample volume in each well was 0.0479 mL.



After testing the system with colored water, we ran a single output microfluidic chip with GFP to obtain quantitative measurements. We created an output design of a smiley-face and verified the selection locations by running the plate in a plate reader. As shown in the diagram, the GFP mixture is appropriately dispensed onto the well-plate.

Fully Automated

Automation for TERRA is key because it reduces human error and frees up time for the researcher. Manual selection between outputs on the TERRA Adapter reduces the efficiency of the overall run because it not only requires the user to be on standby but introduces variable pressure differentials on chip during actuation. To automate TERRA, a control syringe system is used to select and redirect outputs on the TERRA Adapter, which is especially important for multi-output chips. To verify that TERRA is fully automated, we ran multi-output microfluidic chips. However, due to manufacturing issues with our microfluidic chips and lack of time, we could not run a multi-output chip on TERRA.

Automation for TERRA is key because it reduces human error and frees up time for the researcher. Manual selection between outputs on the TERRA Adapter reduces the efficiency of the overall run because it not only requires the user to be on standby but introduces variable pressure differentials on chip during actuation. To automate TERRA, a control syringe system is used to select and redirect outputs on the TERRA Adapter, which is especially important for multi-output chips. To verify that TERRA is fully automated, we ran multi-output microfluidic chips. However, due to manufacturing issues with our microfluidic chips and lack of time, we could not run a multi-output chip on TERRA.

For a multi-output chip, each output on the TERRA Adapter will have a set of valves for output selection. By actuating these valves in certain combinations, the TERRA Adapter selects for certain outputs while directing all others to waste.

Visit the TERRA Adapter page to learn more about how TERRA handles multi-output selection:

To also handle multi-output chips, TERRA implements a flush protocol in between outputs to clean the nozzle tube with and prime it with the next output. To clean the nozzle tube, the TERRA must first move the plate to a corner of the plane after an output is finished dispensing so that ethanol does not contaminate the plate. A visual below demonstrates TERRA executing this protocol.

Integration with Benchtop Analytics

To accurately dispense user selected sample volumes, TERRA uses a droplet formation model to calculate dispensing times. Given a certain flow rate and tubing properties, the system calculates the dispense time per droplet and multiplies this value by the number of droplets required to fill the sample volume. Our model is located here:

To prove that TERRA is compatible with the benchtop analytical tools, we ran a basic experiment that demonstrated the ability to detect GFP. A smiley face was dispensed onto a 96-well plate using the user interface and TERRA. The final plate was then read using the lab’s plate reader, the results demonstrate that the outputs of TERRA can be analyzed using benchtop analytics.

Now that we verified that TERRA is compatible with benchtop analytical tool, we can use a plate reader to prove that we can accurately dispense sample volumes. To test the model we plated a GFP gradient on a 96-well plate with TERRA using a single output microfluidic chip. The fluorescent reading of the 4x6 well gradient of three, two, and one 0.047 mL GFP droplets provides a quantitative measure of sample volume in wells.


In order to dispense different amounts of fluid into different wells, we needed to input the protocol into the UI individually per fluid volume. The resulting well-plate was analyzed in a plate reader to evaluate the accuracy of the dispensing system.



In the gradient, we observed a general decrease in fluorescence as droplets decreased. This proportional decrease, alongside video analysis, demonstrates that each third of the gradient had different sample volumes. However, the data the relative fluorescence of individual wells were inconsistent compared to the general trend. This can be attributed to GFP not diluted properly or spread evenly within the well. For further verification we require a test that directly measures the sample volume in wells on a plate.


Cost and DIY Approach

Our goal from the beginning has been to make TERRA accessible and therefore we took a DIY approach to our design. This would keep costs low, but would require more assembly of the system. For that reason we have made all the CAD, STL, and additionally all the code available on the wiki and also our GitHub. This is a bill of materials and the cost breakdown of our entire system.

For the XY translational stage:

Part Quantity Unit Price
Stepper Motors 2 $14.95
Timing Belt + Pulley System 1 $13.98
Bearings 2 $10.00
Steel Rods 3 $15.72
Electrical Components 1 $20.00
Arduino + Adafruit 16 Servo Shield 1 $56.00
Total: $187.04

For the one syringe pump unit:

Part Quantity Unit Price
Servo Motors 1 $12.95
Control Syringe 3D Printed Parts 4 Approx. $10.00 (according to material)
Total for per syringe pump: $22.95