Team:UC Davis/Electrochemistry System Optimization

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.

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.