Team:UC Davis/Electrochemistry Enzyme Tests

Once set up our electrochemical system to be comparable to our home-made detection device, testing our engineered enzymes in our system is the next step. Aldehyde dehydrogenase has been successfully engineered to have specificity in different chain length of aldehyde group or saturity of aldehyde groups. The data that proves these specificity has been collected through plate reader, which is the device measures the enzyme kinetics. However, unlike the plate reader, our system contains more complex and sensitive issue that closely related to electrochemistry. Therefore, planning out experiments that can measure current in our necessity level of detection, which will also be suitable for out system was the first part of enzyme testing; and the second part was to carry out the experimental data which can be comparable to plate reader data.

This portion of experiment is necessary to determine a maximum and minimum concentration of detecting enzyme activity. Since the necessary detection range for substrate concentration was limited to micromolar range(based on aldehyde concentration on rancid olive oil data), determining the amount of enzyme spiked into the substrate was a key. Our protocol was to mix different concentration of pentanal(as a substrate for this experiment) and enzyme to observe each response.

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 three 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.
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• TCEP

TCEP (tris(2-carboxyethyl)phosphine is one of the reducing agents that we tested within our buffer system.
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• 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.
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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.

Tween

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One of the easiest and most common method for extracting aldehyde from olive oil is to add tween in the bugger. However, as shown in the figure, after tween has been added to our standard NADH control solution, we can clearly see the noise and interference in our electrode.

Isopropyl alcohol

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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.

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:

1. NADH curve without pentanal in solution
2. NADH curve with 100µM pentanal in solution
3. 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:
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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.
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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