RESULTS

Our Experimental Question:

If pyocyanin is present, PQQ-Glucose dehydrogenase (PQQGDH) can utilize pyocyanin as an electron acceptor which will produce an electrochemical current response in a concentration dependent manner. If the pyocyanin is absent from the system, there will not be adequate electron transfer and no current will be produced in the system.

Enzyme Fuel Cells

In an enzyme fuel cell there are two electrodes - an anode and a cathode - that are placed in a solution. The anode and cathode create a complementary electrode pair that each have immobilized enzymes on their surface. At the anode, the immobilized enzyme oxidizes the substrate in the solution. With the assistance of redox mediators, the electron is transferred to the surface of the anode, creating flow of current. Meanwhile, the enzyme at the cathode causes the reduction of surrounding oxygen molecules and transfers electrons back into the system to maintain the Law of Conservation of Mass. When current from the enzyme fuel cell is connected to a resistor, which generates a voltage. With increasing current and constant resistance, an increased voltage differential between the anode and cathode becomes more prominent, as represented by Ohm’s Law:

V (voltage) = I(current) x R(resistance)

In this closed electrochemical cell, we employ PQQGDH as the anode, which catalyzes the oxidation of glucose to gluconic acid, and MultiCopper Oxidase (MCO) as the cathode to reduce oxygen. We use a potentiostat to measure the voltage drop to quantify the current being generated by the reaction, indicating the electrons are being transferred to the surface of the electrode by pyocyanin. To evaluate the efficacy of this closed electrochemical cell, we utilize a potentiostat to quantify the voltage drop, and therefore the resulting current dampening, generated by this reaction which confirms our hypothesis that pyocyanin may be a sufficient electron mediator of this system.

PQQGDH anode and mCOP cathode demo

Test Results

Ferricyanide and PQQGDH Power Test

Goal: Verify the concentration dependency of PQQGDH by utilizing ferricyanide as a known electron mediator.

Figure 1. Concentration dependent power generation of ferricyanide utilizing PQQGDH.
Due to shipping delays in our pyocyanin, the first weeks of wet lab were spent using ferricyanide as an alternative electron acceptor. The results display our proof-of-concept that increasing concentrations of ferricyanide at clinically relevant pyocyanin concentrations, 0-100 μM [1], there is a voltage drop across the anode and cathode, and therefore power is generated. These results guarantee that electron acceptor concentration can be monitored by the change of open circuit potential, in the presence of an enzyme with its substrate.

Pyocyanin and PQQGDH Power Test

Goal: Validate that pyocyanin is able to accept an electron from PQQGDH.

See Dry Lab section on our Experiments page for details on data cleaning and analysis.
Three pairs of anodic electrodes (PQQGDH) and cathodic electrode (MCO) enzyme fuel cell pairs were tested with and without glucose at 100 μM pyocyanin concentrations and at three different resistances It is expected that without glucose, the PQQGDH cannot perform an enzymatic reaction and no voltage drop will occur.

Figure 1. Mean Voltage for Electrode Pair 1.
Enzyme fuel cells with PQQGDH on the anode and mCOP on the cathode were placed in a 10 mM PPB buffer and 100μM pyocyanin solution. The power generation without glucose appears to be higher than with the 20 mM glucose present, especially for R3 at 1 MΩ resistance.


Figure 3. Mean Voltage for Electrode Pair 3.
Enzyme fuel cells with a bare gold disc electrode on the anode and mCOP on the cathode were placed in 10 mM PPB buffer solution and 100μM pyocyanin. The power generation without glucose appears to be higher than with glucose present for all three resistance values. This electrode pair had the highest voltage reached at 70 mV.

Figure 2. Mean Voltage for Electrode Pair 2
Enzyme fuel cells with PQQGDH on the anode and mCOP on the cathode were placed in a 10 mM PPB buffer and 100μM pyocyanin solution. The power generation without glucose appears to be roughly the same at the R1 and R3 values, and significantly higher with glucose at the R2 value.


Figure 4. Mean Voltage for Control Group.
Enzyme fuel cells with no enzyme on the anode and mCOP on the cathode were placed in 10 mM PPB buffer solution and 100μM pyocyanin. The glucose-absent cells showed no reaction occurring, while the glucose-present cells showed a voltage drop despite no PQQGDH in the solution.

Table 1. Summary Table of PQQGDH and Pyocyanin Power Test for 1Ω resistance.
Means calculated from Dry Lab analysis as explained in Experiments. Each “pair” refers to an anode and cathode in one enzyme fuel cell. Table 1 highlights the mean values calculated from our highest resistance value at 1MΩ. This resistance value was chosen for each anode/cathode pair because it displayed the clearest distinction between enzyme fuel cells with and without glucose present.

Discussion

In our pyocyanin power test experiments, the expected result was to detect a higher voltage drop when the glucose is present due to the electron transfer reactions that occur with PQQGDH and pyocyanin. At present, our results are inconclusive due to the increase in voltage for the fuel cells without glucose compared to those with glucose, as seen for electrode pair 2 and 3 in Table 1. There are a few points of consideration that lead us to think this data is unreliable.

Firstly, the enzymes on the anode were crude enzymes, due to our difficulty in purifying the PQQGDH enzyme. Because this was crude enzyme sample, we cannot be sure how much of this solution matrix is comprised of our target PQQGDH enzyme and that other unwanted proteins may have obscured our results. Secondly, the PQQGDH was not incubated with Ca+ ions to improve the metal-ion coordinate binding between the GDH and PQQ cofactor. Future experiments will incorporate this step. Finally, the control was a clean, bare gold disc electrode. However, a more representative control would be an electrode without enzyme but with Ketjen black. Since ketjen black creates a conductive carbon matrix to immobilize the enzyme onto the surface of the electrode, it limits the electrode surface for electron transfer. It is also interesting that voltage measurements were often higher when the PQQGDH had no glucose to oxidize. This indicates an alternative current source that is generating the power and not the PQQGDH.

These results cannot yet confirm whether or not pyocyanin can act as an electron acceptor for PQQGDH. However, further testing with pyocyanin and PQQGDH is required to understand the thermodyanamic and electrochemical properties of this reaction.

Cyclic Voltammetry (CV) Activity Testing

Goal: Determine if the transfer of an electron from PQQGDH to pyocyanin is thermodynamically possible.

Figure 5. PQQ and Pyocyanin CV.
Standard Cyclic Voltammetry is a fundamental electrochemical technique that is able to perform two alternating potential sweeps in a triangular waveform, designated the anodic and cathodic sweeps. The forward oxidative sweep from negative to positive potential creates a positive current, while the reverse reductive sweep creates a negative current. In order for PQQGDH to spontaneously reduce pyocyanin, the oxidation peak of PQQ must have a higher numeric potential than the reduction peak of pyocyanin. Here, we have a CV scan of 100 μM pyocyanin and 50 μM PQQ that displays a higher oxidation peak for PQQ, around -0.15 V, than the pyocyanin reduction peak at -0.25V. These results indicate that it is thermodynamically possible for pyocyanin to be an electron acceptor, without considering factors such as mediator accessibility.

Chitosan Hydrogels

Goal: Demonstrate proof-of-concept to incorporate a chitosan hydrogel matrix to the electrode fabrication.

The chitosan hydrogels with dye were successfully deposited onto the surface of a gold disc electrode. Our pH dependency testing was unsuccessful because the concentration of dye release was too low. Further testing was dissuaded to focus on the PQQGDH-pyocyanin electron transfer mechanism.

Final Conclusion

While we were not able to test and validate our sense-and-treat design altogether, we took significant steps in building our P. aeruginosa biosensor. Initial tests of PQQGDH and pyocyanin were uncertain in showing that pyocyanin could be an electron acceptor of PQQGDH. However, the cyclic voltammetry scan proved that this reaction is still possible, and, given more trials with the discussed improvements, results may prove to be more successful. Overall, we completed the beginning stages of building our design and hope to continue this project past the iGEM season to obtain more concrete results.

Reference

[1] Alatraktchi FA, Andersen SB, Johansen HK, Molin S, Svendsen WE. Fast Selective Detection of Pyocyanin Using Cyclic Voltammetry. Sensors (Basel). 2016 Mar 19;16(3):408. doi: 10.3390/s16030408. PMID: 27007376; PMCID: PMC4813983.