Team:UC Davis/Electrochemistry

From 2014.igem.org

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   <h2>Coupling Enzymes</h2>
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   <h2>Enzyme Dependent Actvity</h2>
   <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
   <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
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     <span><h2>Coupling Enzymes</h2></span>
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     <span><h2>Enzyme Dependent Actvity</h2></span>
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<img src="https://static.igem.org/mediawiki/2014/2/27/Echem_cell_UCD_iGEM_2014.jpg"  class="genpicfloatright" width="380px"></img>
<img src="https://static.igem.org/mediawiki/2014/2/27/Echem_cell_UCD_iGEM_2014.jpg"  class="genpicfloatright" width="380px"></img>
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Sensitive: have  a low limit of detection for NADH
Sensitive: have  a low limit of detection for NADH
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</li>
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<li>
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<li><p>
Reactive: Detect NADH with high linear range
Reactive: Detect NADH with high linear range
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</li>
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<li>
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<li><p>
Selective: Be robust to any possible solution components
Selective: Be robust to any possible solution components
</li>
</li>
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<li><p>
Affordable: Cost accessible to the average consumer
Affordable: Cost accessible to the average consumer
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<li><p>
Efficient: Use a low sample volume
Efficient: Use a low sample volume
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Compatible: Be compatible with our, as well as other potentiostats
Compatible: Be compatible with our, as well as other potentiostats
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Portable
Portable
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</ul></p><p>
<br>
<br>
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We tested three screen printed base electrode types, and five different working electrode modification schemes in order to achieve the requisite sensitivity for our system. We settled on Dropsens screen printed #610 Electrodes, depicted above. To find out more about our electrode selection process, <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Electrode_Choice">click here</a>.  
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We tested three screen printed base electrode types, and five different working electrode modification schemes in order to achieve the requisite sensitivity for our system. We settled on Dropsens screen printed #610 Electrodes, depicted above. To find out more about our electrode selection process, <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Electrode_Choice" class="brightlink">click here</a>.  
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<p> Once the electrode type was chosen on the basis of its sensitivity, the analytical solution components were optimized to maintain maximum selectivity for NADH. Any solution component introduced in the original Olive Oil Sample, aldehyde extraction processes, or the enzyme purification step could have theoretically impacted the electrode’s sensitivity for NADH. Collaboration with the protein engineering team led to the development of a solution mix that enabled selective detection of enzyme generated NADH in a solution with a large background  of  various solution components.  
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<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/3f/System_Optimization.jpg" width="600px" style="border:3px solid black" class="genpic"></p>
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These interference is often shown in our graph as “NOISE”. In order to examine each and individual noise factor, we have divided our experiment into two parts:
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Once the electrode type was chosen on the basis of its sensitivity, the analytical solution components were optimized to maintain maximum selectivity for NADH. Electrochemical systems are inherently sensitive to the addition of electroactive compounds to the solution being analyzed. In our biosensor, we needed to be able to combine an olive oil extraction solution containing aldehydes, purified protein, and buffer for our system to function properly. All of these system components had the potential to introduce electroactive solution components that would increase noise, or inhibit our system’s ability to see a signal related to NADH generation. Thus, it was necessary to optimize the protein purification as well as extraction buffers to allow unhindered electrode function. Additionally, we optimized the system to maintain a proper NADH signal to system noise ratio so as to allow for effective NADH detection and system function.  
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<u>Optimizing solution</u><br><br>
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Optimizing solution includes: <br><br>
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1) Testing how each and every chemical in the testing solution affects the signals collected from electrode.<br>
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2) Deciding on concentration of each chemical which would minimizing the noise level.<br><br>
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<u>Optimizing system</u><br><br>
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This part of experiment deals with many internal and external possibilities that may or may not created the “noise” in our system. Some of our solutions has not shown any consistent improvement of decreasing these noise; however,few displays some improvement in getting rid of these noise.  <br><br>
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To find more about our system optimization, <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_System_Optimization">click here</a>.  
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<p>To find more about our system optimization,  
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_System_Optimization" class="brightlink">
 +
click here
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</a>.  
</p>
</p>
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</div>
</div>
<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">
<div class="mainTitleHeader">
<div class="mainTitleHeader">
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<p>Coupling Enzymes</p>
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<p>Enzyme Dependent Activity</p>
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</a>
</a>
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<p>Once enzymes have been engineered and successfully displayed their specificity, our mission is to come up with the most suitable solution and enzyme ratio to get the most clear signals from our electrode. This includes: <br><br>
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<p align="center"><img src="https://static.igem.org/mediawiki/2014/b/bb/EnzymeTESTTTT.png" width="650px" style="border:3px solid black" class="genpic"></p>
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<u>Measuring enzyme activity in our system:</u><br><br>
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Having optimized our electrochemical setup, we were ready to measure enzyme activity. In order to establish system function, we tested a single enzyme and substrate over time. After corroborating system function with on enzyme, we tested our three engineered enzymes with twelve different substrates. Our electrochemical sensor was able to detect differences in enzyme activity at high levels of aldehyde, proving the viability of our system. Looking forward, we plan to further increase sensitivity of our electrochemical system in order to enhance our sensor’s capability.  
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Searching for satisfactory range of concentration for enzyme to be added to our solution in order to demonstrate the signals that are coherent to the enzyme kinetics data.  
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</p>
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<br><br>
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<p>
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<u>Demonstrate enzyme specificity measured from our system device:</u><br><br>
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  To find out more about our enzyme testing,  
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Prove that our system and device display the same specificity of each enzyme as we previously seen on the enzyme kinetics data. <br><br>
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<a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests" class="brightlink">
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click here
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  To find out more about our enzyme testing, <a href="https://2014.igem.org/Team:UC_Davis/Electrochemistry_Enzyme_Tests">click here</a>.  
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</a>.  
</p>
</p>

Latest revision as of 02:51, 18 October 2014

UC Davis iGEM 2014

Electrode Choice

Electrode Choice

System Optimization

System Optimization

Enzyme Dependent Actvity

Enzyme Dependent Actvity

Having settled on NAD+ dependent Aldehyde Dehydrogenases as our method of differentiating between aldehydes, we needed to develop an efficient electrode system to detect enzyme activity via NADH. We acquired, selected, and optimized an electrode setup for the detection of NADH at low concentrations in a complex solution. Additionally, we demonstrated the ability of the electrode setup to detect enzyme generated NADH over time, and thereby functionally deconvolute aldehyde profiles within a sample.

Electrode Choice

We developed our Electrode System to be:

  • Sensitive: have a low limit of detection for NADH

  • Reactive: Detect NADH with high linear range

  • Selective: Be robust to any possible solution components

  • Affordable: Cost accessible to the average consumer

  • Efficient: Use a low sample volume

  • Compatible: Be compatible with our, as well as other potentiostats

  • Portable


We tested three screen printed base electrode types, and five different working electrode modification schemes in order to achieve the requisite sensitivity for our system. We settled on Dropsens screen printed #610 Electrodes, depicted above. To find out more about our electrode selection process, click here.

System Optimization

Once the electrode type was chosen on the basis of its sensitivity, the analytical solution components were optimized to maintain maximum selectivity for NADH. Electrochemical systems are inherently sensitive to the addition of electroactive compounds to the solution being analyzed. In our biosensor, we needed to be able to combine an olive oil extraction solution containing aldehydes, purified protein, and buffer for our system to function properly. All of these system components had the potential to introduce electroactive solution components that would increase noise, or inhibit our system’s ability to see a signal related to NADH generation. Thus, it was necessary to optimize the protein purification as well as extraction buffers to allow unhindered electrode function. Additionally, we optimized the system to maintain a proper NADH signal to system noise ratio so as to allow for effective NADH detection and system function.

To find more about our system optimization, click here .

Enzyme Dependent Activity

Having optimized our electrochemical setup, we were ready to measure enzyme activity. In order to establish system function, we tested a single enzyme and substrate over time. After corroborating system function with on enzyme, we tested our three engineered enzymes with twelve different substrates. Our electrochemical sensor was able to detect differences in enzyme activity at high levels of aldehyde, proving the viability of our system. Looking forward, we plan to further increase sensitivity of our electrochemical system in order to enhance our sensor’s capability.

To find out more about our enzyme testing, click here .

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