Team:UC Davis/Electrochemistry Electrode Choice

From 2014.igem.org

UC Davis iGEM 2014

Electrode Choice

Electrode Choice

System Optimization

System Optimization

Enzyme Dependent Activity

Enzyme Dependent Activity

Overview

The first step in building an electrochemical sensor of any kind is appropriate selection of electrodes. Choosing the right electrodes influences both the device’s sensitivity for the analyte of interest, and also the device’s selectivity against other possible interfering agents. We had a variety of choices, from manufacturing our own electrodes with their own mediators, to purchasing pre-manufactured electrodes. We prepared and validated function for several of these electrode types to determine which one was the best for our purpose.

Which electrode to use?

Selecting the electrode was the first step in building our electrochemical system. In the selection process, many features were considered. Our electrodes had 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


Based on our design requirements, we needed an electrode that was specifically designed to detect NADH. That is, it had to be sensitive enough to detect the minute amounts of NADH produced by our enzymes over time and reactive enough to give a wide range of detection for NADH. At the same time, all the sensitivity in the world means little if interfering agents from our protein buffers or extraction solutions interfered with the electrical signal developed from NADH oxidation. We needed a selective electrode. Lastly, to make our sensor usable and accessible, it had to be portable, affordable, and efficient. Satisfying all these design constraints by the end of the summer would mean our device had the potential to be a small, accessible, and powerfully quantitative device for detection of rancidity in olive oil.

Our initial idea was to custom build an electrode that suited our specific purposes. However, after reviewing the literature and conducting several electrode preparations, we concluded that purchasing an electrode prepared for NADH detection was the wisest choice with our time constraint.

After deciding to go with a screen printed, pre-manufactured electrode, we had to decide between several screen printed electrode types. A multitude of firms produce screen printed electrodes, all with different dimensions, electrode substrates, and mediators. Two of these electrode types are shown below. The electrodes are from Pine Instrument’s and DropSens. Both electrodes gave us the advantage of a built-in counter, reference, and working electrode.

Both electrode types were inexpensive, and portable. However, they differed in their efficiency. Pine’s electrode setup used approximately 200 uL, whereas DropSens used 70 uL. Additionally, though Pine Instrument’s electrode offered us more freedom to apply our own mediators, the dropsens electrode came precoated with Meldola’s Blue, a mediator effective in catalyzing NADH oxidation.

Importance of Mediator

The critical compound allowing for NADH oxidation in the Dropsens #610 electrode is the electrochemical mediator Meldola’s Blue. An electrochemical mediator is a compound that catalyzes the oxidation or reduction of a particular species at an electrode surface. Having a mediator present allows one to lower the cell potential necessary to oxidize a particular species while maintaining the same or higher level of oxidative current. This is critical as lowering the running cell potential increases electrode lifetime, lowers the oxidation of extraneous species (greater selectivity), and in many cases increases the amount of signal output for every unit of analyte (greater sensitivity).

The electrode system consists of a screen-printed chip embedded with three electrodes: the counter, working, and reference electrodes. Though the potential of the reference electrode is kept constant, a voltage bias is applied across the working and counter electrodes to facilitate a buildup of excess positive charge on the working electrode. The working electrode consists of a carbon ink infused with polymer. Since some polymers have selective oxidative affinity for NADH, we decided to test two polymers that would work the best for our electrode. We chose to polymerize Azure A and Meldola’s blue onto the surface of Pine Instruments electrodes.

Azure A

  1. Showed selective oxidative affinity for NADH

  2. Dissolved and activated in hydrochloric acid and sodium nitrate

  3. Electrografted onto surface


Meldola's Blue

  1. Showed selective oxidative affinity for NADH

  2. Dissolved and activated in Phosphate buffer

  3. Electropolymerized onto surface

  4. Displayed greater electron deposition and flow

  5. Demonstrated more reliable signals
    (NO indication of major chemical interference at the electrode surface)


Comparison experimental result:

Polymerization with both compounds, Azure A and Meldola’s Blue both gives some selective oxidative affinity for NADH by lowering the potential required to oxidize NADH at the electrode; therefore, expanding the voltage range favorable for us to conduct experiments at. Both AA and MB has a selective oxidative affinity for NADH, reducing the over potential necessary for NADH oxidation at the working electrode. Although both are advantageous in terms of selectivity for detecting NADH, we have decided to use a polycyclic aromatic monomer dye, Meldola’s Blue (MB) for the following reason:

  • MB facilitates greater electron deposition and flow.

  • Azure A demonstrated second order responses, which can be possible indicative of non-trivial chemical interactions at the electrode surface.

  • MB gave us more reliable readings, especially showed no second order responses.

  • We can specifically ordered MB-infused electrodes from Dropsens.


The Dropsens #610 electrode satisfied our design requirements most effectively, and most importantly demonstrated the most sensitivity towards NADH oxidation.

Electrode Schema and Diagram

The dropsens #610 electrode consists of three electrodes screen printed onto a 3.4 x 1.0 x .05 mm substrate. These electrodes consist of the working electrode, the counter electrode, and the reference electrode. The working electrode serves to act as the main electroactive surface for NADH oxidation, and is embedded with a catalytic mediator called, “Meldola’s Blue. The counter electrode is present to provide a sink or source of electrons to maintain the voltage bias between the counter and working electrode, and the reference electrode provides a reference voltage to which the counter and working electrode voltage can be pinned to. The dropsens #610 electrode utilizes a small sample volume (50-70 μL), is coupleable to a standard USB output via cable connector, and costs just $2.00 per electrode. The electrode immediately satisfied our requirements of portability, affordability, and efficiency.

Now that we had our electrodes, our first objective was to prove that the biosensor system could detect NADH. We also needed to determine the upper and lower limits of detection. To do so, a standard curve was created by testing multiple concentrations of NADH, yielding a linear range from 50 – 500 uM with a resolution of +/- 10 uM.

Assessment of NADH Linearity: Download Protocols: Download Full Dataset: (XX MB)


Detailed protocols and data summaries

Experiment #1234 Example: Download Protocols: Download Full Dataset: (XX MB)