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Team:UC Davis/Electrochemistry System Optimization
From 2014.igem.org
Electrode Choice
Electrode Choice
System Optimization
System Optimization
Enzyme Dependent Activity
Enzyme Dependent Activity
Overview
As discussed under “Electrode Choice,” our electrode system utilizes the oxidation of NADH at the working electrode to generate a current that is interpreted as a signal.
Detection of NADH on Dropsens Electrodes, 1 µM to 100 µM.
(Flow carrier: 0.1M phosphate buffer and 0.1M KCl) - image from "DropSens.com"
Consequently, an ideal electrode system would feature NADH as the only compound undergoing electron transfer at the electrode. However, electrochemical systems like our own that depend on surface interactions are susceptible to the interfering effects of extraneous compounds in solution, often compounds not directly related to quantification of a choice analyte. This can lead to obfuscating noise, as electro-active compounds that oxidize at the electrode shed electrodes that contribute a signal. To this end, noise was a perennial issue for the electrode system, mostly because too much noise could overshadow the small electrical signals produced by small concentrations of analytes, making quantification impossible.
Figure 1. Electrode electrochemical mechanism: Electrode, potentiostat and computer (left); Dropsens Electrode: Each strip has three electrodes for electrochemical testing (right)
To address the issues of noise in our signal and efficient NADH oxidation, various mediator polymerization schemes were investigated, and system parameters optimized. In addition, the solution components of our enzyme buffer solution were investigated to ascertain their compatibility with our system. This section discusses the experimental groundwork that laid the foundation for enzyme testing.
Solution Optimization
Enzyme Buffer
Many electrode systems feature enzyme immobilized onto the electrode either directly or through binding to a crosslinking mediator like glutaraldehyde. Due to our time constraint, we chose not to undergo complex enzyme immobilization experimentation, but rather to simply suspend our enzymes in a suitable aqueous environment. This buffer solution was tailored to stabilize the engineered aldehyde dehydrogenase (ALDH) enzymes, and to promote advantageous kinetics. the grafting of extraneous solutes on the electrode--all of which lead to difficulties in accurately detecting a signal.
There are many chemicals that are crucial for enzyme survival. This portion of experiment discusses the tests that uncovered 1. what chemicals were compatible with the electrode at various concentrations and 2. what chemicals would introduce the least noise to the signal. The creation of NADH standard curves was employed to demonstrate if the addition of a particular solute would obfuscate signal linearity, or our ability to distinguish signal at all.
Buffer solution components include:
Phosphate Buffer + salts (0.1M)
After reviewing multiple literature and research in the electrode that we decided to use(dropsens), we decided to use 0.1M KCl with phosphate buffer. A salt concentration of 0.1M in a buffer of pH 7.4 is a very common solution parameter in electrochemical experiments. This setups allows current to be passed through solution, and is thus crucial to our system. In addition, these ions are not oxidized at the electrode and thus do not contribute noise to the signal. (see figure(2)-referring to Dropsens NADH conductivity experimental data stated 0.1M salt concentration.)
Reducing agents
These reactive chemical species are crucial enzyme stability and ultimately survival, and therefore must be included in the buffer solution. We tested two different reducing agents to see which would be compatible. Their description and compatibility is discussed below.
DTT
Dithiothreitol (DTT) is a reducing agent that is used to stabilize enzymes that contain free sulfhydryl groups by reducing the amount of disulfide bonds formed. To determine the concentration of DTT that would be optimal for our system we generated 3 NADH standard curves. Each curve represents the how 3 different concentrations of DTT (0µM, 1 µM, and 10 µM) affect the linearity of the NADH standard curve. As seen in the graph below, we were able to generate linear NADH curves with all three concentrations of DTT. Therefore, for future experiments we decided to use the highest concentration, 10 µM DTT, for enzyme stabilization.
Assessment of DTT Linearity:
Download Protocols:
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(XX MB)
TCEP
TCEP (tris(2-carboxyethyl)phosphine is one of the reducing agents that we tested within our buffer system.
As shown in the figure, TCEP contributes much noise to the system, making harder to detect the signals from enzyme. Therefore, we decided to exclude TCEP in our buffer.
Assessment of TCEP Linearity:
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Download Full Dataset:
(XX MB)
Glycerol
Glycerol is a hardy perennial in any enzyme solution due to its important role in enzyme stability. Therefore, similar protocols as those above were conducted to examine its contribution to noise.
Extraction Buffer
An objective of the wider project was the extraction of aldehydes from hydrophobic olive oil. This would allow the aldehydes to interact with enzymes and ultimately NADH to transmit a signal at the electrode. To make these aldehydes accessible, an extraction protocol was developed. This technique required the use of a number of solvents--in particular detergents, all of which needed to be tested for possible interfering effects with the electrode.
Thus we had to examine which detergents, such as Tween and Isopropyl alcohol gave less noise and less interference with other chemicals in the final buffer solution.
Isopropyl alcohol
We wanted to test the effect of IPA in the buffer solution. We made a solution of 0M NADH with 1x PBKCl and varied the concentrations of IPA (10 µM, 50 µM, and 100 µM). All three solutions with the varying IPA concentrations gave the same signal. From this we can conclude that IPA within the buffer solution will not affect our ability to detect NADH.
Tween 20
One of the easiest methods we found for extracting aldehydes from olive oil is to add Tween 20 in the bugger. However, as shown in the figure, after Tween 20 has been added to our standard NADH control solution, we can clearly see the noise and interference in our electrode.
Inhibition
To more definitively correlate aldehyde concentration to output current, we needed to examine the possibility of substrate or product inhibition. Such effects could negate NADH generation via substrate conversion, and thus yield an incorrectly lower signal.
Substrate inhibition
The compound capable of substrate inhibition was the aldehyde. We tested the aldehyde pentanal to ascertain possible potential inhibitory effects. We conducted 3 NADH standard curves with NADH concentrations of 0, 250, 500 and 1000µM. The curves generated were conducted as follows:
NADH curve without pentanal in solution
NADH curve with 100µM pentanal in solution
NADH curve without pentanal in solution
After completing each run we saw there was little to no effect on the detection of NADH. The graphs below show these results:
Product inhibition(NADH inhibition)
The effect of the product NADH on the forward reaction was assayed on a plate reader be preparing solutions with a constant concentration of NADH, pentanal, and enzyme, and variable concentrations of NAD+. If the enzyme velocity was approximately the same for all these combinations, then we could conclude that production inhibition would not feature.
As shown below, the notion of production inhibition was dispelled because the velocities were found to all be approximately the same.
Electrical Optimization
Voltage
Hedging(current infusion-spontaneous current infusion) went away as we changes the current from 0.6 -> 0.7 V
Used higher voltage to reduce noise (picture shows that at 0.3v has tons of noise for support)
Needed enough voltage to oxidize NADH(0.33mV) as minimum
Cover slip
Possible diffusion limited situation
Shaking to promote homogeneity of solution (or diffusion)
No net benefit unnecessarily complicated the ergonomics
(this protocol was excluded in our established experimental procedure)
Electrical Noise
Tin foil cage to remove 60Hz noise -> didn't help
Although some of our attempts to reduce electrical noise were not successful, at the end, we were able to generate a linear curve for the detection of NADH. The graph below shows that with the optimization of all components in solution, a linear curve for the detection of NADH can be generated.
Detailed protocols and data summaries
