Experiments | Paris_Bettencourt

Description - Rationale

Potentiostats are standard equipment in electro-chemistry laboratories, but the price generally ranges from 1,000 to 10,000 €. As Electro-Genetics is a nascent field, rare are the biology labs equipped with such devices and their cost is a barrier to entry for most iGEM projects. The IO Rodeo potentiostat is a well-characterised and published in peer-reviewed scientific journals[1-5] open-source project comprised of a main Printed Circuit Board (PCB) harbouring the Operational Amplifiers (OP Amps) central to potentiostat operation and extension capabilities allowing for multiplexed measurements of multiple electrodes.

If the assembled devices could (in regular times) be bought from the website for 200$, thus incredibly lowering the barrier to entry to electro-chemistry, the product was out of stock during the period of iGEM 2022 because of an international shortage of microchips

After inspection of the PCB Design files, we understood that the missing components were the Quad SPST CMOS Analogue Switches DG411DY-T1-E3 and the Drift Voltage Reference REF3330AIDBZR. We replaced both of them with available Though hole parts. The new PCB (Available on our Gitlab) can be ordered for manufacturing and assembly of all surface-mount parts in online PCB Manufacturers. We ordered 5 of them for the price of 45$ thus massively lowering the price of these devices.

We characterised this device through cyclic voltammetry and constant voltage voltammetry experiments and compared our results with commercially available PalmSense Sensit BT potentiostat, graciously lended to us by Prof. Vincent Noel of the university Paris Cite.

We used it in:

Calibration of Potentiostat

We prepared a common reference redox solution of ferri/ferrocyanide (K3[Fe(CN)6]) to calibrate the IO Rodeo potentiostat to the commercial Palm Sens ⟨™⟩ Sensit BT potentiostat. We performed cyclic voltammetry, a method often used in electrochemistry to investigate redox systems. It is a technique for qualitative analysis method of reaction rate and mechanism. By reproducing the same results in our open-source potentiostat as the commercial one, we demonstrate that our potentiostat detects the necessary range of redox reactions.

Electrochemical gene induction

We used this device in the context of the characterisation of the Input parts of our Electro-genetic toolkit to induce gene expression with an electrical potential (See pSoxS Experiments) and in the context of the lysis based Output part of our Electro-Genetic Toolkit.

Materials and Methods

Calibration of potentiostat

The commercial potentiostat Palm Sens ⟨™⟩ Sensit BT was lent to us by Vincent Noel, professor at the UFR Chimie (Université Paris Cité). IO-Rodeo was designed on a PCB board and ordered. A stock solution of 2 mM Potassium ferrocyanide (K4[Fe(CN)6]) in 1 M Potassium nitrate (KNO3) was prepared following the protocol on this link ([https://www.als-japan.com/1898.html#defaultTab11](https://www.als-japan.com/1898.html#defaultTab11)). 3 mL of this solution were transferred into a culture tube. DropSens electrodes were immersed in the solution and connected to the potentiostats.

Cyclic voltammetry was performed first with the Palm Sens ⟨™⟩ Sensit BT, and then with the IO-Rodeo potentiostat. The settings of the commercial potentiostat were selected through the provider software PSTrace, whereas the IO-rodeo cyclic-voltammetry parameters were coded in python. The code for the IO-Rodeo cyclic voltammetry and chronoamperometry was imported from the IO-rodeo potentiostat documentation ([http://stuff.iorodeo.com/docs/potentiostat/examples.html#cyclic-voltammetry](http://stuff.iorodeo.com/docs/potentiostat/examples.html#cyclic-voltammetry)) and the parameters adjusted.

Electronic induction of gene expression

The IO Rodeo potentiostat, connected to the DropSens electrodes, was used in the pSoxS experiment (https://2022.igem.wiki/paris-bettencourt/template_experiment) involving the redox species pyocyanin. Cyclic voltammetry was performed in a LB solution with 10uM of pyocyanin, to find the reduction peak. The reduction voltage potential was applied by chronoamperometry (also known as constant voltage voltammetry) for 16 hours to reduce pyocyanin, and activate expression of fluorescence in cells.

Results

Comparison of cyclic voltammetry between the commercial and the IO-Rodeo potentiostats

The Cyclic voltammetry graph of the commercial potentiostat (left) shows an oxydation peak at 0.1 V and a reduction peak at -0.05V. On the IO-Rodeo we notice a slight shift in the peak values with the oxydation peak at 0.25V and the reduction peak at 0.12V. These values are not absolute and represent a qualitative measure of redox activity.

Pyocyanin cyclic voltammetry and chronoamperometry

In the pyocyanin cyclic voltammetry graph we notice a oxydation peak at 0.3V and a reduction peak at -0.5 V. This relative value was used for the chronoamperometry experiment to reduce pyocyanin.

An applied voltage potential of -0.5V was applied for 16 hours.

References

1. Bullen JC, Dworsky LN, Eikelboom M, Carriere M, Alvarez A, Salaün P (2022) **Low-cost electrochemical detection of arsenic in the groundwater of Guanajuato state, central Mexico using an open-source potentiostat**. *PLoS ONE* 17(1): e0262124. [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262124](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262124)

2. Fatoni A, Widanarto W, Anggraeni MD, Dwiasi DW (2022) **Glucose biosensor based on activated carbon – NiFe2O4 nanoparticles composite modified carbon paste electrode** *Results in Chemistry*, Volume 4, 100433 [https://www.sciencedirect.com/science/article/pii/S2211715622001527](https://www.sciencedirect.com/science/article/pii/S2211715622001527)

3. Bogoslowski, S., Geng, F., Gao, Z., Rajabzadeh, A.R., Srinivasan, S. (2021). **Integrated Thinking - A Cross-Disciplinary Project-Based Engineering Education. ****In: Auer, M.E., Centea, D. (eds) Visions and Concepts for Education 4.0. ICBL 2020. Advances in Intelligent Systems and Computing, vol 1314. Springer, Cham. [https://doi.org/10.1007/978-3-030-67209-6_28](https://doi.org/10.1007/978-3-030-67209-6_28)

4. Fatoni A, Wijonarko A, Anggraeni MD, Hermawan D, Diastuti H, Zusfahair (2021) **Alginate NiFe2O4 Nanoparticles Cryogel for Electrochemical Glucose Biosensor Development.** *Gels*. 2021 Dec 17;7(4):272. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8701366/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8701366/)

5. Guillem P, Bustos RH, Garzon V, Munoz A, Juez G (2021) **A low-cost electrochemical biosensor platform for C-reactive protein detection.** *Sensing and Bio-Sensing Research* 31 (2021) 100402. [https://doi.org/10.1016/j.sbsr.2021.100402](https://doi.org/10.1016/j.sbsr.2021.100402)

6. Cordova-Huaman, A.V., Jauja-Ccana, V.R. and La Rosa-Toro, A. (2021) ‘Low-cost smartphone-controlled potentiostat based on Arduino for teaching electrochemistry fundamentals and applications’, *Heliyon*, 7(2), p. e06259. Available at: [https://doi.org/10.1016/j.heliyon.2021.e06259](https://doi.org/10.1016/j.heliyon.2021.e06259).

7. Crespo, J.R. *et al.* (no date) ‘Development of a low-cost Arduino-based potentiostat’, p. 21.

8. Tahernia, M. *et al.* (2020) ‘A Disposable, Papertronic Three-Electrode Potentiostat for Monitoring Bacterial Electrochemical Activity’, *ACS Omega*, 5(38), pp. 24717–24723. Available at: [https://doi.org/10.1021/acsomega.0c03299](https://doi.org/10.1021/acsomega.0c03299)

IO Rodeo Potentiostat

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