iGEM 2022 — Team iiser-mohali

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# Notebook

Following is the journal containing information regarding our weekly activities from the beginning to the end of the cycle:

We ordered our DNA aptamers from IDT as two µmole DNA oligo in standard desalting conditions. We resuspended the lyophilized aptamer with 0.1 molar PBS. Then we measured the Cyclic voltammetry and impedance of the bare Glassy Carbon electrode before modifying it with GO/PANI solution.

Ideation Stage

* January 9 - January 15

1. Introductory session overcoming iGEM session by advisors.
2. Getting to know about the iGEM competition.

* January 16 - January 22

1. Another meeting with advisors after the first meeting to discuss queries raised in the first meeting.

* January 23 - January 29

1. Break due to Midsemester exams.

* January 30 - February 5

1. Open call for assignment submissions to get selected for the team.

* February 6 - February 12

1. Meeting with advisors to discuss the assignments and further the team selection process.

* February 13 - February 19

1. Brainstorming for ideas started, and further discussion with advisors to refine the ideas put forward.

* February 20 - February 26

1. Another Brainstorming meeting to discuss the ideas put forward in the previous session and brainstorm for new ideas.
2. An open call for submission of 2nd assignment was sent out.
3. Individuals suggested teaming up in a group of a minimum of 3 to present their idea for the coming iGEM season.

* February 27 - March 5

1. Meeting with advisors to discuss more refined ideas and submission of 2nd assignment.

* March 6 - March 12

1. Break due to Midsemester exams.

* March 13 - March 19

1. Presentation of the idea to the iGEM selection committee of the institute.

* March 20 – March 26

1. Meeting with Prof. Satyajit Jena, our Primary PI, to discuss our project to seek his guidance and support.
2. Meeting with Prof. Ananth Venkatesan to help us with plasma ion sputtering of our neural chip.
3. Meeting with Prof. Samarjit Bhattacharyya to seek his guidance and support for neural chip development.

* March 27 - April 2

1. Another meeting with Prof. Satyajit Jena, our Primary PI, to discuss neural chip development and training process.

* April 3- April 9

1. Meeting with Prof. Ramendra Sunder Dey to discuss the project's aptamer aspect and seek his support in developing the aptasensor.

* April 10 – April 16

1. In preparation for the final meeting with the institutional iGEM committee, we met our mentors and instructors to refine our project idea further.

* April 17-April 23

1. Final meeting with the iGEM committee to discuss the chosen idea.
2. Our idea got approved to work with, but only if we made a small modification to our initial plan.

* April 24- April 30

1. Break due to Midsemester exams.

Getting Started

* May 1 – May 7

1. Formation of the team and assignment of team roles.
2. Meeting with mentors, advisors, and instructors to discuss our project idea and initial plans.
3. Discussing general logistics and expectations.
4. Everyone joins sub-teams to begin working on the project.
5. Human Practices and Outreach teams decided their long-term goals and the list of professionals and events they will be managing the whole season.
6. The Wet Lab team determined the aptamer sequences against the respective microbes and an initial protocol for neural chip development.

* May 8 – May 14

1. Reaching out to the IISER, Mohali community to see what we can do to have a local impact.
2. The hardware and dry lab teams started setting their initial goals.

* May 15- May 21

1. The fundraising team started looking for sponsorship.
2. The team started discussions for Team-Logo and the name of the Project.
3. We started our discussion for collaboration, and we collaborated with Team iGEM Patras to develop a video discussing iGEM, Synthetic Biology, Wet Lab and Dry Lab, and the Survey on GMO and Syn-Bio.
4. The Wet Lab team started looking for alternatives to thiol-modified aptamers and did an extensive literature search and discussion with experts to attach unmodified aptamers to GO.
5. The dry lab Team started the discussion to perform GROMACS simulations of the proteins against our aptamer sequences.

* May 22 – May 28

1. The team decided to work with a colorblind-friendly palette throughout the season and decided on our logo.
2. The team started working on our collaborations with iGEM Team-Patras and finalized our application for Promega Grant.
3. The team started out working on Outreach and Human practices activities.

* May 29 – June 4

1. The team finalized the Logo for the project and submitted the survey questionnaire for review to Institutional Ethics committee member Anu Sabhlok.
2. The team applied for Beckman Coulter free reagent kits.
3. Team also started having discussions with other Indian Teams regarding the All India iGEM Meet 2022.

Summer

* June 5 – June 11

1. The team discussed with Science communicator Ipsa Jain and received valuable feedback from her regarding our Communication and Education work.
2. The team had another meeting to finalize our Logo and discussed starting out sharing our work on social media.
3. We discussed having a potential meeting with Team iGEM Berhampur who was also working with Aptamers this year.
4. We also finalized ordering our custom DNA oligo from IDT for our Aptamer experiments.

* June 12 – June 18

1. The team had its meeting with iGEM Team IISER Berhampur and discussed potential collaborations with them.
2. Wet Lab Co-head met with Anca from iGEM 2021 Team- Rochester to gain insights into attaching unmodified aptamers over GO and perform the electrochemical discussion method.
3. We started discussions regarding Collaboration with other iGEM Teams.
4. The Wet Lab team was notified about an issue we could face while using a neural chip to detect the initially chosen microbes. The Wet Lab team decided to work with Penicillin to sort out the problems that could have arisen.
5. The social media team had a meeting to kick off the season over our social media handles officially.
6. Team also decided on our mascot Monsieur Neurapurr.
7. The team met to finalize our application for Impact Grant Award 2022.

* June 19 – June 25

1. The team had its initial meeting with Team AFCM Egypt and Team Linkoping Sweden and discussed potential collaboration.
2. The team decided on the goals for fulfilling medal criteria and planned work accordingly.
3. The team finalized the preliminary safety form for submission and met with advisors and mentors over it.
4. The team finalized their plan to host the Syn-Bio camp and discussed it with volunteers.

* June 26 – July 2

1. The team met with Volunteers, brainstormed together for the conduction of the Syn-Bio camp, and also launched posters for registration.
2. The team discussed our participation in AIIM 2022 and got two Researchers on board for the Virtual Talk Series.
3. The team again met to discuss the Impact Grant Applications.
4. Syn-Bio Workshop plans finalized.
5. The team had an initial collaboration meeting with High School Team ASIJ Tokyo.
6. The marketing and fundraising team started a massive campaign to raise funds.
7. The outreach Team collaborated with Team- IISER TVM to develop a lab tour video.

* July 3 – July 9

1. Brochures and infographics designed by the design team.
2. The team had review meetings to analyze the work and plan for the goals.
3. The human Practices and Wet Lab team met with Dr. Richa Sharma and Dr. Raghavarao to discuss some of the electrochemical sensing aspects of the aptasensor.

* July 10 – July 16

1. The team met with volunteers to prepare for Syn-Bio workshops.
2. The hardware team finalized the design to sputter for the neural chip.
3. The marketing and Fundraising team reached out to sponsors
4. The team decided on track selection for the project.
5. The team received the distribution kit.

* July 17 – July 23

1. The neural chip development team placed an order for Retinoic acid.
2. Initial designs for the Neural chip got sputtered and tested out.
3. The team had a meeting and finalized the presentation for AIIM 2022.
4. The design team got done with the 3D rendering of our prototype.
5. The team successfully conducted the Syn-Bio workshop for high school students.
6. The human Practices team met Prof. Anu Sabhlok to discuss the social and ethical aspects of the Project.
7. The team started the survey on GMOs and Syn-bio to understand the general public's perspective on GMOs and Syn-Bio.

* July 24 – July 30

1. The team attended the AIIM 2022.
2. Aptamers received from IDT.
3. We had a meeting with iGEM Team Montpellier to discuss our collaboration.
4. The dry Lab team finalized its goals for docking and figured out its work.

* July 31 – Aug 6

1. The team received Impact Grant Award.
2. We had our first meeting with our collaborator iGEM Thessaloniki, Greece.
3. The wet lab team met to finalize the protocol and start the experiments related to Aptasensors.
4. The team visited Verka Milk Plant, Chandigarh.
5. The Wet Lab team started InterLab Calibration Experiments.
6. The team placed an order for neural cell lines.
7. The team scheduled the meeting with engineers from BLACKROCK NEUROTECH.
8. The team successfully concluded its virtual talk series event.
9. The team submitted IBSC Application to get BSL-2 clearance from Insitute’s BioSafety Committee.
10. The team decided on the script for Project's Promotional Video.
11. InterLab study calibration done.

* Aug 7 – Aug 13

1. The team received sponsorship from INDUS PROTECH.
2. The team completed the Inter-Lab study measurements.
3. The team completed shooting clips for Project's Promotional
4. The wet Team successfully modified Glassy Carbon electrodes with GO/PANI mixture.
5. The human Practices team met Prof. Indranil Banerjee to discuss the project.
6. The human Practices team met Prof. TVR from IISc to discuss the sustainability aspect of the project.
7. The comic Strip development team finalized the story to work upon.

* Aug 14- Aug 20

1. The human Practices Team visited PBTI, Chandigarh.
2. The Design Team completed the animation for Project's Promotional Video.
3. Wiki Team started designing the basic templates for the Wiki.
4. The team met Nanjiang-WorldShaper High School students and discussed a potential collaboration.
5. Wet Lab Team successfully attached Aptamer over GO/PANI modified GC.
6. The team got BSL-2 clearance from Insitute’s BioSafety Committee to work with cell lines and S. typhimurium.
7. The human Practices team visited Dr. Archana at PGIMER to discuss some of the aspects of the project.
8. The dry Lab team finalized the parameters for running MDS.

* Aug 21- Aug 27

1. The wet Lab team successfully tested aptamer for E.coli.
2. The human Practices and Dry lab team met with the CEO of Cortical Labs to discuss some aspects of neural chip development.
3. The dry Lab team finalized its plans to develop our software-” KAMI.”

Closing the loop

* Aug 28- Sept 3

1. The wet Lab Team placed an order for Penicillium chrysogenum and S. typhimurium.
2. The dry Lab Team, under the guidance of our Instructor Soumadeep, started the development of the software -” KAMI.”

* Sept 4 – Sept 10

1. The wet Lab Team received N2A and SH-SY5Y cell lines.
2. The human Practices team visited KVK, Mohali.
3. The team received sponsorship of $1250 from Blue Copper Technology Pvt. Ltd.

* Sept 11- Sept 17

1. The wet Lab Team started to culture N2A and SH-SY5Y cell lines.
2. The team delivered student talk over iGEM and our Project to the BS-MS community of IACS, Kolkata.
3. The team finalized the development of “Book on Aptamers” with Team Linkoping Sweden and others.

* Sept 18- Sept 24

1. The wet Lab Team received Penicillium chrysogenum and S. typhimurium.
2. Wet Lab Team tested aptamers for Penicillin G as well.
3. The wiki team finalized their plans regarding the web design and started working on it.
4. Comic strip development finalized the illustrations for the comic.
5. The dry Lab team finalized the Docking and almost completed it.

* Sept 25 – Oct 1

1. The wet lab team tested the sensitivity of aptamers against E. coli and S. typhimrium.
2. The wet lab team started two different cell lines.
3. The dry Lab team performed MDS and finalized modeling work related to the project.
4. The outreach team conducted Synthetic Biology based demonstration for middle and High school students on the occasion of 16th Foundation Day.
5. The wiki Team tested uploading and extensively worked on Wiki development.
6. The comic strip development team started collating the panels for the comic and did some final editing.
7. Gold medal criteria of long-term partnership achieved and discussed with iGEM Team Montpellier.
8. The dry Lab Team wrapped up the development of the software-” KAMI” successfully.

* Oct 2 – Oct 8

1. The dry Lab team wrapped up their work.
2. The comic strip got published on our social media handles and sent out to high school students we met during Syn-Bio Workshop.
3. Boi–Safety video final editing is done and released over our YouTube channel and social media pages.
4. The team received a sponsorship offer of $500 from ACS Publication.
5. The team extensively documented the work, and Wiki Team worked on Wiki development and uploading.

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[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

The neural chip is a highly sensitive device detection and processing device. We faced some challenges along the way in the development of the hardware. We wanted to reduce the noise generated by the device by random firing of neurons, and to make the device as robust as possible. We initially took design inspiration from MEA60 device, for reading data from cortical neurons. This ran into the issue of increased noise due to high number of output electrodes. We decided to keep the number of electrodes to a minimum to reduce on noise. We also increased the surface area of the output electrodes and increased the number of input electrodes, by designing two input electrodes for the same signal, to overcome the issue of one of the electrodes breaking. With each iteration of our chip design we managed to reduce the noise generated by our system and achieved better and more accurate predictions.

## First Iteration

![](https://static.igem.wiki/teams/4391/wiki/eng/eng1.png)

Circuit design after first iteration

![](https://static.igem.wiki/teams/4391/wiki/eng/des0.png)

From this first step, we decided that we needed to use vinyl masks for sputtering and had to increase the number of input electrodes to 6, although the number of input parameters was 3. We kept 2 electrodes for the same input signal.

## Second Iteration

![](https://static.igem.wiki/teams/4391/wiki/eng/eng2.png)

Design 1

![](https://static.igem.wiki/teams/4391/wiki/eng/des2.png)

Design 2

![](https://static.igem.wiki/teams/4391/wiki/eng/des1.png)

Design 1 worked better than design 2, both in the quality of sputtering and neural connectivity.

## Third Iteration

![](https://static.igem.wiki/teams/4391/wiki/eng/eng3.png)

This worked and in the next round of testing, we reported fewer than 10% cell death after a week of incubation.

# References

1. West, Jonathan & Dinh, Ngoc-Duy & Hardelauf, Heike & Chiang, Ya-Yu & Newman, Tracey & Vargas-Caballero, Mariana & Asuni, Ayodeji & Deinhardt, Katrin & Arundell, Martin. (2015). Bridging Two Cultures: Minimalistic Networks Prepared by Microfluidic Arraying, and Open Access Compartments for Electrophysiology. 10.1007/978-1-4939-2510-0\_3.
2. Ying HS, Gottron FJ, Choi DW. Assessment of cell viability in primary neuronal cultures. Curr Protoc Neurosci. 2001 May;Chapter 7:Unit7.18. doi: 10.1002/0471142301.ns0718s13. PMID: 18428528.
3. [Growing Human Neurons Connected to a Computer](https://www.youtube.com/watch?v=V2YDApNRK3g&t=98s)
4. T. B. DeMarse and K. P. Dockendorf, "Adaptive flight control with living neuronal networks on microelectrode arrays," Proceedings. 2005 IEEE International Joint Conference on Neural Networks, 2005., 2005, pp. 1548-1551 vol. 3, doi: 10.1109/IJCNN.2005.1556108.
5. [Inkscape](https://inkscape.org/)

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Our project aimed to develop a technology that envisions using neural as a detector and an aptamer as a biosensor to detect microbes quickly and reliably in a sample. These neural chips are created using N2A and SH-SY5Y cell lines and can be trained using the Hebbian learning principle to compute and make decisions.

On the other hand, we designed a biosensor that uses aptamers (single-stranded oligonucleotide sequences) to sense the presence of microbes. These aptamers are attached to Graphene oxide -Polyaniline solution and the conformational change resulting from aptamer binding alters the electrical properties of the modified electrodes.

By measuring the changes in these electrochemical properties, we can detect the presence of microbes with our device. To maximize our efficiency, our wet lab team split into two sub-teams: one to design and test the aptamer and another to engineer the neural chip.

Click on the buttons to learn more about the experiments carried out by each sub-team for these parts of our project:

Aptamers
Neural Chip

We ordered our DNA aptamers from IDT as two µmole DNA oligo in standard desalting conditions. We resuspended the lyophilized aptamer with 0.1 molar PBS. Then we measured the Cyclic voltammetry and impedance of the bare Glassy Carbon electrode before modifying it with GO/PANI solution.

Protocol to measure CV and impedance of the electrodes

1. The cyclic voltammetric and Electrochemical impedance spectroscopy (EIS) response of GC was measured in 1 mM Fe3+/Fe2+ solution in 0.1M PBS in a three-electrode setup.
2. Ag/AgCl in 3.5M KCl was taken as a reference electrode and Pt as a counter electrode. EIS was measured in the frequency range from 100 kHz to 10 MHz at ∆E value of the CV curve.
3. CV was measured between potential -0.1V to 0.5V at a 20 mVs-1 scan rate.

Preparation of Fe 2+/Fe 3+ solution

Potassium ferri cyanide (K3[Fe(CN)6]) and Potassium Ferro cyanide (K4[Fe(CN)6]) solution 5mM concentration was taken in 0.1PBS solution. Ferro/Ferri ions provide reversible reactions in defined electrochemical potential, and PBS with a pH of 7.4 gives suitable conditions for biological analysis.

Once we took the CV and Impedance of the bare Glassy Carbon, we modified Glassy Carbon using a GO/PANI mixture.

Protocol to perform GO/PANI modification over Glassy Carbon electrode

1. GO was diluted to 3mg mL-1 in 0.5M perchloric acid HClO4. GO and a conducting polymer Polyaniline (PANI) was mixed into GO to improve its functionalities and charge conduction value, forming a suitable interaction with the aptamer molecule.
2. GO and aniline was taken in the ratio of 4:1, respectively, by weight. Suspension solution was stirred for half an hour and sonicated for a few minutes for even spreading of Aniline onto GO.
3. For electrodeposition chronoamperometry of GO/PANI in 3 electrode assembly, a negative potential of -1.1V was applied for 180 sec in GC working electrode, leading to the deposition of GO and, at the same time, polymerization of aniline to PANI.

Preparation of GO

Graphene oxide was oxidized from graphite powder using a modified Hummer method. Briefly, it is a two-step process:

1. The first step involves the oxidation of graphite using P2O5 and K2S2O8 as oxidizing agents to prepare pre-oxidized graphite.
2. The second step involves pre-oxidized graphite was further oxidized using KMnO4 and exfoliated in DI water. The exfoliated suspension solution was collected and kept in the dark for further use.

After modifying the Glassy carbon electrode with GO/PANI mixture, we performed Electrochemical Impedance Spectroscopy (EIS). We analyzed the change in CV and Impedance curve to detect the modification of the Glassy carbon electrode.

The cyclic voltammetric and Electrochemical impedance spectroscopy (EIS) response of GC was measured in 1 mM Fe3+/Fe2+ solution in 0.1M PBS in a three-electrode setup. Ag/AgCl in 3.5M KCl was taken as a reference electrode and Pt as a counter electrode. EIS was measured in the frequency range from 100 kHz to 10 MHz at ∆E value of the CV curve. CV was measured between potential -0.1V to 0.5V at a 20 mVs-1 scan rate. The modification was successful.

After the glassy carbon electrode was modified, an aptamer was attached.

Protocol to immobilize Aptamer over GC modified with GO-Aniline

1. The lyophilized aptamer received from IDT was resuspended in a 0.1M PBS solution.
2. Further, the aptamer solution was diluted to a working concentration of 250 nM.
3. This was used for Self-assembled monolayer (SAM) formation. 8µL of aptamer solution was drop cast on the modified GC and was kept for drying overnight (12h) at 4ºC inside a vacuum desiccator.

Note- The remaining aptamer solution was stored at -20 ºC.

After the Aptamer solution drop cast over GC dries up, Cyclic Voltammetry and Impedance were taken to analyze the electrochemical changes, indicating the successful immobilization of aptamers.

Further, to test out the working of our aptamer, we cultured *E. coli* (MTCC 443) and *Penicillium chrysogenum* (MTCC 1348).

Protocol to culture *E. coli* (MTCC 443)

1. *E. coli* ampule from MTCC was heated for 10-15 minutes and was broken with the help of a knife.
2. The knife was sterilized before use. Once the ampule was broken, the *E. coli* was resuspended with 1ml LB media.
3. After resuspension, the Media was plated over the LB-Agar plate using a spreading technique and was left overnight for around 16 hrs to grow at 37º C.
4. After 16hrs, pick out the colony from the plate and inoculate 10 ml LB media with it as the primary culture of the bacteria. Leave the culture to grow at 37º C and 200 RPM overnight for around 16 hrs.

Preparation of LB media

1. Take 12.5 gm of pre-mixed LB media powder to prepare 500 ml broth.
2. After adding 12.5 gm of powder, make it up to the volume of 500ml with deionized water.
3. Once that is done, cover the flask with the foil and put the autoclave tape over the flask.
4. Keep it in the autoclave; once that is done, take it out and keep it at room temperature.

Preparation of LB-Agar plates

1. Add 25g of pre-mixed LB broth powder per Litre to an appropriately sized flask.
2. Add 1L deionized water and stir it until clumps are gone.
3. Add 15 g Agar per Litre. This will not dissolve.
4. Autoclave for at least a 20minute liquid cycle.
5. Once the liquid is cool to the touch, pour into plates, covering the surface and avoiding bubbles.
6. Burn off bubbles with a bunsen burner or pop with a sterile tip
7. Let plates cool with the lids ajar.
8. Invert plates, and store them by labeling LB plates over them.

Composition of LB powder

1. 10 g of peptone
2. 5 g of yeast extract
3. 10 g of NaCl

Protocol to culture *Penicillium chrysogenum* (MTCC 1348)

1. *Penicillium chrysogenum* (MTCC 1348) ampule from MTCC was heated for 10-15 minutes and was broken with the help of a knife.
2. The knife was sterilized before use. Once the ampule was broken, the *Penicillium chrysogenum* (MTCC 1348) was resuspended with 1ml YPD media.
3. After resuspension, the Media was plated over the YPD-Agar plate using the spreading technique and was left for two days to grow at 30º C.
4. After two days, pick out the colony from the plate and inoculate 10 ml YPD media with it as the primary culture of the bacteria. Leave the culture to grow at 30º C and 220 RPM overnight for around two days.

Preparation of YPD media

1. Take 25 gm of pre-mixed YPD media powder to prepare 500 ml broth.
2. After adding 25 gm of YPD powder, make it up to the volume of 500ml with deionized water.
3. Once that is done, cover the flask with the foil and put the autoclave tape over the flask.
4. Keep it in the autoclave; once that is done, take it out and keep it at room temperature.

Preparation of YPD-Agar plates

1. 20g of peptic digest of animal tissue.
2. 10g of yeast extract.
3. 20g of dextrose.

After, we cultured the microbes. We would test our aptamer.

Protocol to test respective aptamers

1. The GC immobilized with Aptamers is taken out from 4ºC, and its impedance and CV were taken to analyze whether aptamers were successfully deposited.
2. Then, after CV and Impedance are taken, the GC surface is gently washed with deionized water.
3. The washed GC is left to dry at room temperature.
4. The GC is placed over a holder inside the vacuum desiccator and the laminar flow hood.
5. 8µL of the respective culture specific to the aptamer is dropped and cast over the aptamer surface.
6. After drop-casting, GC was left to dry up at 40ºC in an oven for around 25 minutes.
7. Once the GC dries up, CV and Impedance are taken, and an Electrochemical study is performed.

Once we tested that our designed aptamers were working, we decided to check the sensitivity of our aptamer. Hence, we decided to analyze the response of our aptamer against the different concentrations of the microbes.

Protocol to determine the concentration of microbes

1. The culture was diluted 20 times, made up to 1ml volume, and the O. D was taken at 650nm wavelength.
2. 1 O.D of cells equals 108, and 0.1 O. D equals 104.
3. The O.D, when measured, was 0.8 since it was diluted 20 times the actual O.D was 1.6. Hence the concentration of the microbes in the sample was 16 x 108 bacterial cells/ml.

After determining the conc. Of the microbe in the sample. It was further diluted to a conc. of 1.6 x 108 bacterial cells/ml. Then it was tested with our aptamer to understand the changes in varying concentration ranges.

Protocol to measure the sensitivity of the aptamer against *E. coli* (MTCC 443)

1. Two different GCs were taken and modified with GO/PANI mixture. Further, they were deposited with the aptamer specific to E. coli (MTCC 443)
2. We performed EIS after the drop-casted solution dried up when left at 40ºC for 25 minutes.
3. After successful immobilization of the aptamer, CV and Impedance was taken, then we drop-casted one of the aptamers attached over modified GC with 8µL of the sample containing microbe of conc. 16 x 108 bacterial cells/ml and another with concentration of 1.6 x 108 bacterial cells/ml.

The CV and Impedance curve will help understand the effect of concentration on the designed aptamer's sensitivity.

Protocol to measure the sensitivity of the aptamer against *Penicillium chrysogenum* (MTCC 1348)

1. Two different GCs were taken and modified with GO/PANI mixture. Further, they were deposited with the aptamer specific to the *Penicillium chrysogenum* (MTCC 1348).
2. After successful immobilization of the aptamer, CV and Impedance was taken, then we drop-casted one of the aptamers attached over modified GC with 8µL of the sample containing microbe of 16 x 108 bacterial cells/ml conc. And another with conc. of 1.6 x 108 bacterial cells/ml
3. We performed EIS after the drop-casted solution dried up when left at 40ºC for 25 minutes.

The CV and Impedance curve will help understand the effect of concentration on the designed aptamer's sensitivity.

# References

1. Chergui, S., Rhili, K., Poorahong, S., & Siaj, M. (2020). Graphene Oxide Membrane Immobilized Aptamer as a Highly Selective Hormone Removal. Membranes, 10(9). <https://doi.org/10.3390/membranes10090229>
2. Subramanian, P., Lesniewski, A., Kaminska, I., Vlandas, A., Vasilescu, A., Niedziolka-Jonsson, J., . . . Szunerits, S. (2013). Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces. Biosensors and Bioelectronics, 50, 239-243. [https://doi.org/https://doi.org/10.1016/j.bios.2013.06.026](https://doi.org/https%3A//doi.org/10.1016/j.bios.2013.06.026)
3. <https://www.thermofisher.com/in/en/home/life-science/cell-culture/microbiological-culture/bacterial-growth-media/lb-broth-and-lb-agar.html#:~:text=LB%20Agar%20(Lennox)&text=per%20one%20liter%3A-,10%20g%20SELECT%20Peptone%20140,12%20g%20SELECT%20Agar>
4. <https://www.addgene.org/protocols/pouring-lb-agar-plates/>
5. <https://himedialabs.com/TD/M1363.pdf>
6. <https://med.wmich.edu/sites/default/files/Pioli_Lab_LB_Media_Recipe.pdf>
7. [https://2021.igem.org/Team:Rochester/Experiments](https://2021.igem.org/Team%3ARochester/Experiments)
8. Gupta, Ritika, et al., Naked eye colorimetric detection of Escherichia coli using aptamer conjugated graphene oxide enclosed Gold nanoparticles, Sensors and Actuators B: Chemical. <https://doi.org/10.1016/j.snb.2020.129100>

Protocol to develop ECAD design

Cad (Computer Aided Design) is used to prepare engineering drawings. Computer-aided drafting helps in preparing drawings without the use of the usual drawing instruments. This way far less time is required to prepare a drawing. The output can also be printed. For our purpose, we used “Inkscape” an open-source vector graphics editor. It uses Scalable Vector Graphics (SVG) as the native format.

To install Inkscape on your device:

1. Go to inkscape.org,
2. And download the version that suits your device.
3. Open the Inkscape setup
4. Click next and read the license agreement.
5. Click next, customize all the features required, and confirm the installation location.
6. Click next and then click install to install it on your device.

Now, the installation of Inkscape is done on your computer. You can use Inkscape to create your designs and can also get them printed.

To prepare design on inkscape:

1. Open the Inkscape software.
2. Click on “new document” after specifying the size of the document you need (you can do it later also).
3. Now you can see the Inkscape window, and toolbar on all four sides of the window.
4. On the left, there are different tools to draw (like pencil, shapes, measuring tools, etc). on the right, there are different options to edit your document. Below, there are fill and stroke options.
5. In the working space (of white background), we can see a page boundary. All our design needs to be inside of it.

For our purpose, we required our ecad design to be printed on a glass slide (75mm x 25 mm). We edited the document properties and made the page size the size of the glass slide so that the design looks the same when we get it printed.

Protocol for using the vacuum deposition chamber

Fabrication Details

* The sample is fabricated by depositing a 60-70nm thick layer of Titanium in an electron beam evaporator on a glass slide covered with a (insert details of stencil).

Electron beam evaporation

* Electron beam evaporation is used to deposit extremely thin films of materials, ranging from angstroms to microns. A target boat with the desired material is bombarded with an electron beam which converts the material to a gaseous state which then precipitates into very thin layers.

Setup

* The setup consists of a vacuum chamber connected to turbo and roughing pumps to decrease the pressure of the chamber. At the base of the chamber is the grounding of the electron beam source filament, a magnetic source which controls the electron beam and the anode basket to load the graphite boat with material inside. At the top, directly above the boat is a metallic substrate disc shielded by two movable shutters to stabilise the rate before deposition. There are two gauges to measure the pressure, APGX and AIMX gauge and a quartz crystal microbalance which measures the thickness of the deposited materials. Next to the chamber is a TT power supply to control the electron beam.

Deposition specifications

* The vacuum chamber is brought to a pressure of about 10^-6 Torr for the deposition. The deposition is done at a voltage of 7.63kV and emission current of about 51mA. The emission current is increased till the deposition rate of about 1-1.5A/s is reached to ensure even coating. The pressure of the chamber is measured using an Edwards inverted magnetron Gauge(AIMX) and the rate of deposition is measured by a quartz crystal microbalance.

Protocol to measure resistance of neural chip

1. Insert the black ground probe into COM socket on a multimeter.
2. Insert the red probe (positive) on the V socket (can also be marked by omega or a diode symbol)
3. Set the range of voltage to 10 mV.
4. If the meter is autoranging, turn the dial to the 'V' setting with the symbol for AC or DC.
5. The multimeter is connected in parallel across an electrode in order to measure voltage.
6. The black probe is placed at the beginning of the input electrode
7. Switch on the multimeter
8. Complete the circuit by touching the red probe on the other end
9. The reading will be available on the LCD display

Protocol to culture cell lines

1. N2a and SH-SY 5Y cell lines were received from NCCS Pune.
2. Upon receiving the cell lines, they were immediately placed in an incubator at 37॰C, 5% CO2 and 95% relative humidity.
3. Growth media was prepared by mixing 10ml FBS (Fetal Bovine Serum) in 90ml DMEM (Dulbeco’s Modified Eagle Media). This was supplemented with non-essential Amino Acid mix (1gm/20ml), henceforth referred to as growth media.
4. Both the cell culture flasks were then transferred inside the Laminar AIr flow hood and the contents were pipetted into a Falcon tube.
5. The Falcon tube was centrifuged at 1000rpm for 10 mins to pelletise the cells.
6. The pellet was resuspended in 30ml of growth media for individual cell lines
7. The cells were then transferred to cell culture flasks and kept in the incubator at 35॰C, 5% CO2 and 95% relative humidity
8. The cells were allowed to grow for a period of 3 days and steps 4 to 7 were repeated for multiplying the cells.

Protocol to perform trypan blue test

To check the viability of our cells we performed Trypan blue assay. We took the SH-SY 5Y and N2a culture flask respectively and peletised them at 1000rpm for 10 minutes.

1. Supernantant is discarded.
2. The pellets were resuspended in FBS free DMEM (2ml).
3. 1 ml of 0.4% Trypan Blue, in PBS (Phosphate buffered saline) is then added to the cell suspension.
4. The cells are incubated in the incubator for 3 minutes.
5. 100μL of the cell suspension with Trypan Blue is added to the hemocytometer plate groove
6. Both the chambers are filled in gently under the coverslip and capillary action pulls in the suspension
7. Place the slide under microscope and focus on the grid at 10X magnification.
8. Count the number of viable (translucent) cells and the number of dead cells (blue) seperately

The percentage of viable cells is calculated as:

V= number of viable cells/Total number of cells \* 100

Protocol to perform cell differentiation of N2a cells

1. The cells after the 2 round of growth (one cycle is 3days), the cells were pelletised
2. A differenciation solution was prepared by mixing 0.5ml FBS in 49.5ml of DMEM. Henceforth called differentiation media.
3. The pellet was resuspended in the differentiation media
4. The resuspended cells were pipetted (5ml) in each neural chip.
5. The filter cover was screwed on.
6. The neural chips were placed in a tissue incubator (35॰C, 5% CO2 and 95% relative humidity).
7. The media was pipeted and replaced with fresh media after every 3 days.
8. One of the neural chips was Trypsinised (0.05% Trypsin in EDTA, 2ml) for 3 minutes, quenched using Basic culture Media and taken for Trypan Blue assay on Day 3, to check the viability of the cells post differentiation.

Protocol to perform cell differentiation of SH-SY 5Y cells

We modifed the differentiation protocol submitted to iGEM by team Tuebingen 2018.

The differentiation protocol used by us was simpler and used retinol from day 13 rather than retinoic acid to create a less harsh condition for differentiation.

We report sucess by using our modified method of differentiation.

We performed the differentiation steps in the neural chip.

* Day 1

1. Undiffrenciated cells are washed with Serum free DMEM, and Trypsinised using (0.05% trypsin EDTA), and quenched with DMEM, serum free (10ml).
2. The cells are then transferred into 15ml Falcon and centrifuged at 1000rpm for 10 minutes
3. The pellet is resuspended in 15 ml of Differentiation media 1 (1% FBS in DMEM (with non essential amino acid) + 1% Retinoic acid). Retinoic acid was made from a stock of 99% pure Retinoic acid diluted to 50mM concentration in DMEM.
4. The cells are pipetted into the neural chip (5ml per chip)
5. Cells are incubated at 37॰C, 5% CO2 and 95% Relative Humidity.

* Day 3

1. We gently aspirate off the media and refill the well with differentiation media 1.
2. The filter cover is refitted and the chips are placed in the Incubator

* Day 7

1. Gently aspirate off old media and refill the well with Differntiation media 2 (0.5% FBS in DMEM (with non essential amino acid) + 1% Retinoic acid solution)
2. Place in the incubator

* Day 9

1. Repeat step from Day 3 and 7

* Day 13

1. One of the neural chip is taken for microscopy of differentiating cells
2. Old media is aspirated and Differenciation media 3 (supplimented DMEM (with KCl, Glutamax, non essential amino acids, db-cAMP) is added with(200μL) 1% retinol)) is added
3. Neurite growth is confirmed with microscopy images
4. Chips are placed back in the incubator

* Day 17

1. One of the neural chips is taken for microscopy
2. Old media is aspirate and Differenciation media 3 is added.
3. Microscopy images confirm fully differentiated neuron like cells.

The chips were then used for detection and interpretation of signals from an oscilloscope, all inside a LAF(Laminar Air flow hood). We kept 3 chips on our lab bench to test their viability outside the incubator. We report no significant cell death upto 5 days outside the incubator and LAF. The chips showed signs of contamination on Day6 in such condition. We suspect that this is due to defects in the hardware used. Cells that were kept in the incubator survived for 10-18 days.

Protocol to perform measurement of the chip

1. An oscilloscope was setup inside a LAF and the measurement probes were placed across the neural chip in a parallel connection.
2. Input voltage of 150mV was kept in 3 of the input by using a battery powered arduino setup
3. The voltage was lowered across two of the input electrodes to simulate binding of the Aptamer to target bacteria and increase in it’s resistance.
4. Initially both the output electrodes show a positive current, but upon decreasing the current in the first two input electrodes (that is correlated to output A) we observe a lowering of voltage in Output B. This is indicative of the neuron like cells detecting, processing and finally giving a output.

# References

1. [Inkscape](https://inkscape.org/)
2. <https://telemark.com/wp-content/uploads/Manual-TT-Series-E-Beam-Source-Power-Supply.pdf>
3. <https://www.electronicshub.org/how-to-use-a-multimeter/>
4. <https://nccs.res.in/>
5. [https://doi.org/10.1002%2F0471142735.ima03bs111](https://doi.org/10.1002/0471142735.ima03bs111)
6. Roger G. Tremblay, Marianna Sikorska, Jagdeep K. Sandhu, Patricia Lanthier, Maria Ribecco-Lutkiewicz, Mahmud Bani-Yaghoub, Differentiation of mouse Neuro 2A cells into dopamine neurons, Journal of Neuroscience Methods, Volume 186, Issue 1, 2010, Pages 60-67, ISSN 0165-0270,
   <https://doi.org/10.1016/j.jneumeth.2009.11.004>.
7. <http://2018.igem.org/wiki/images/1/1d/T--Tuebingen--DifferentiationProtocol.pdf>

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# Segments

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[Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)

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# Results

Background information

## Microbes Chosen:

Initially, as team we were looking out to develop a device which can work as one of the best point of care technology for detecting diseases. We soon realised that the technology we envisioned to develop can not only detect the microbes commonly responsible for causing sepsis like diseases but rather can detect any target molecule against which we can generate a highly specific and sensitive aptamer.

Our institutional iGEM committee’s head Prof. Anand Bachchawat also suggested to us that since our technology could detect any target molecule if an aptamer could be generated against it then why don’t we develop a “ Proof of Concept” that it could detect microbes which are more commonly available around us and are easier to work with.

We considered these aspects and decided to work with E. coli, S. typhimurium and Pseudomonas aeruginosa. Through the literature search, we found that these common microbes have already aptamers developed against them using whole cell-SELEX which are highly sensitive and specific.We decided to choose the microbes that had already available aptamer sequences in the literature as we understood that whole cell-SELEX process takes time to model an aptamer sequence and given the time frame of the competition we won’t be able to complete everything we wanted to exhibit if we are to begin with SELEX.

Our team wanted to make a system that would be better than the existing fluorescent-based detection methods due to its high false positive rate. We also wanted to quickly expand to the sample's multiple, simultaneous, multi-cohort detection. This was not possible with existing technologies with individual detection capabilities and data with differing error margins, which are later processed for a complete analysis of the disease. We solved all the above issues by implementing the processing system within the detection system using a neural chip that simultaneously detects and processes signals. It is also cheap, easy to mass manufacture, and accessible with minimal electrical equipment (in the proposed implementation a battery-powered reading box would suffice). The system would also have an error margin which would be easy to calculate against accurate world data, unlike data derived from individual detection, which have errors that cannot be easily calculated. Neurons are also susceptible to changes in the electrical fluctuations at the 70-100mV range, thus reducing the need for high-powered devices like spectrophotometers that also increase measurement and maintenance costs.

Further, our team member figured out later that the technology we envisioned developing the device and the neural chip we were developing for bio-computation could not perform the computation without dimension reduction. We could not have three inputs and three outputs if we had to use a neural chip to perform bio-computation and analyze the electrochemical changes that would happen upon the binding of microbes to the aptamer. The dimension reduction property would prove our chip's biocomputation ability and help reduce stray signals that would be generated and measured. This is why the chip was planned to have 3input signals corresponding to three different organisms but only two output signals. The interpretation of these signals would be based on the current output of the chip. The chip would present an overall signal lowering in both outputs when detecting the third organism. This ensured that the number of outputs and the interpreting device's involvement were minimized.

Hence, we further, our team member figured out later that the technology we were envisioning to develop the device and the neural chip we were developing for bio-computation cannot perform the computation without the dimension reduction. We cannot have three inputs and three outputs if we had to use neural chip to perform bio-computation anad analyse the electrochemical changes that would happen upon the binding of microbes to the aptamer. This would act as a biomarker for the fungi Penicillium chrysogenum.

Thus, we decided to work with E.coli MTCC 443, S. typhimurium MTCC , Penicillium chrysogenum as our chosen microbes.

Why Aptamers, Neural Chips and Electrochemical sensing?

Aptamers have emerged as a great tool in the recent years in the development of point of care diagnostics, drug delivery and therapeutics. Aptamers could be either ss DNA or RNA oligonucleotide sequences that adapt a secondary structure able to specifically bind to a target molecule. They are mainly generated by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which is a reiterative process starting with a large DNA or RNA library, and at each step, only high binding affinity sequences are separated from the library. One example is Silver Enhanced Fluorescence-Activated Cell Sorting. In this process, the target protein or the control/negative proteins (which are used to ensure specificity of the aptamer) are immobilized on polystyrene microspheres and added to the large DNA/RNA initial library, modified with a fluorophore. Silver decahedral nanoparticles conjugated to a fluorophore are also added, and then these NPs will interact with the labelled DNA and clump together. Through fluorescence-associated cell sorting, one can then choose the cells that had DNA clones that tightly bound to the protein.

We can observe that only aptamers that bind to the cell or protein of interest are selected from the initial library. This is the first round, where enrichment occurs. All other rounds are negative selection, where the protein or cell of interest reacts with silver nanoparticles, and the non-binding DNA sequences are directed to the waste container via an electric current. The collection container sequence is then amplified via PCR, and ssDNA or RNA aptamers have been generated as a result. They can then be sequenced via standard sequencing methods. Aptamers have the advantage that any molecules can be targeted, and there are no immunizations of animals required. This results in a high affinity and high specificity receptor that can be selected for and then synthesized completely in vitro. Moreover, since aptamers are just a DNA sequence, they can be ordered from DNA synthesis companies.

Fortunately, the sequences we wanted to work with were highly specific and sensitive with KD value in nM range and were available under IDT’s sponsorship programme.

Fluorescence detection using Aptamers was initially considered, but our team soon realized a number of problems with this method. Aptamer being a small molecule is prone to more molecular motion than traditional antigen-antibody detection systems. This makes the fluophore and the quencher interaction more prone to misfiring. This increases the false positive rate in such systems. We also considered the problems faced by the use of spectroscopes and image processing of biological data. Spectroscopes have a error percentage of 5% at best condition. This makes the data highly unsuitable for further processing as additional processing will further add error to the measurement.

Also most spectroscopes in a developing country like ours, do not have regular maintenance. We were made aware of this issue by food scientists working in Verka, during our vist. This has made the food industry trust less on spectroscopic data for bacteria detection and prefer culturing the bacteria to check for its presence. This is a time consuming process that is prone to environmental contamination.

To solve all of these problems we decided to use a property of the aptamer which is linear, easy to measure, shows large range changes, scalable and devoid of further processing steps. We hence decided to use the impedance change property of Aptamers and interpret out results using a neural chip, which solves all the above problems. The chip processes the data from the aptamer and directly outputs a easily quantifiable signal, generated from an Ensemble of neurons. The neurons are also more sensitive than traditional current detection systems, firing at 70-100mV range. The trainability of the network also allowed us to build the compuational system within the detection system rather than taking the data separately and processing it. This reduces dependency on big and expensive machinery and is easily deployable in the field. The results are instantaneous and can be monitored in real time. The neural chip itself is reusable, cheap to manufacture and easy to dispose. It contains no heavy metal contaminants, other than the wiring of the chip, which itself can be reused to make more chips. It also requires no maintenance, other than a normal temperature of 37\*C, and once a supply chain can be established will be a perfect tool for rapid, cheap and sustainable multicohort detection.

For E. coli ATCC generic strain 25922 (MTCC 443) , according to the studies, whole bacterial cell is used as a target and P12-31 is highly specific and sensitive aptamer towards it. Its Kd value is 87.03± 17.32, Bmax value is 0.56±0.04 and around 10113 no. of binding sites are available for it. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli.

Sequence of P12-31 aptamer is given below which is obtained by Whole-Cell SELEX:

5'CCCTCCGGGGGGGGGGGTCATCGGGATACCTGGTAAGGATACCCTCCGGGGGG
GTCATCGGGATACCTGGTAAGGATA 3'

Structure:

![Image of structure](https://static.igem.wiki/teams/4391/wiki/results-img-one.png)

Aptamers
Neural Chip

## Results and Conclusions

EIS measurement is a powerful technique for analyzing the physiochemical changes at the interface (i.e., between the bulk solution and solid electrode) arising from the formation of insulating film incurred by the interaction of the analytes with their probing molecules immobilized on electrode surfaces. Measurement of charge transfer resistance RcT the impedance increases across the interfacial layer EIS provides a rapid, sensitive, and nondestructive technique for detecting various analytes, including biomolecules that would otherwise be difficult to quantify.

Impedance is usually expressed as a Nyquist plot. A typical Nyquist plot consists of a semicircle in the high-frequency region representative of the electron transfer limited process and a straight line in the low-frequency region associated with the diffusion-controlled process. In fig:3, The bare GC showed a small semicircle in the high-frequency region with the calculated RCT value of 3300Ω. After the electrochemical deposition of GO/PANI, the Nyquist plot for EIS spectra shows the decrease in RCT value showing the reduction in the surface resistance, therefore providing a more effective platform for developing aptasensor.3 The Nyquist plot of the electrode where the aptamer molecule shows an increase in the RCT value to 11000, showing the successful assembly of the aptamer layer on the modified GC. Aptamer acts as a barrier to the anion electrolyte's restriction [Fe(CN)6]-3/-4 to the electrode surface.4

The Nyquist response shows a vast increase in the RCT value to 40000 Ω showing the complete coverage of E. coli by the biosensor.

Similar observation can be observed for CV (fig:5) and EIS response (fig:6) for developing the Penicillin aptasensor. On another GC, a Change in the RCT from 1400 Ω to 1100 Ω after deposition shows the GO/PANI layer formation on the GC surface. EIS curves also show the change in RCT value for penicillin aptasensor and penicillin, which is seen by the RCT value or the semicircle size of the EIS response. The RCT values for penicillin aptasensor of around 1500 Ω shows the SAM of aptamer onto the deposited GO/PANI surface, the RCT value changes to 3600 Ω after dropcasting of penicillin onto modified aptasensor GC. Hence, the sensitivity of the developed aptasensor towards penicillin is good.

![](https://static.igem.wiki/teams/4391/wiki/results/rs1.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs2.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs3.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs4.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs5.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs6.png)

## Sensitivity measurement:

For the measurement of the sensitivity of the E.Coli aptasensor for the given E.coli strain, the concentration of the E.coli was varied in two different ratios in terms of optical density, i.e., 0.7OD and 0.07OD.

The experiment was performed in two different GCs with two different E.coli concentrations. The previous experiments for the development of aptasensor were the same, and the GCs CV (Figures 7 and 8 ) and EIS (Figures 9 and 10) responses were recorded.

For GC with E.coli concentration of 0.7OD, the CV responses were recorded (Figure: 7). The change in CV curves area with the going from bare GC to the E.coli assembled form was observed. As seen in figure: 7, the CV responses show a decrease in the current value with the given potential window. Figure 8 shows the CV of the 0.07 OD E.coli, showing a similar response as that of 0.7OD E.coli. Still, the CV curve for the 0.7 OD E.coli assembled aptasensor is more horizontal than the other concentration of 0.07 OD E.coli. This shows the current is hindered more in 0.7OD E.coli than the other concentration ratio.

Nyquist plot for 0.7OD (figure: 9) and 0.07OD (figure: 10) concentration of E.coli shows the immense RCT value for 0.7OD concentration than 0.07OD concentration, i.e., 5000 Ω in former and 13800 Ω in later, this is the inference of the increase in bacterial assembly on the electrode surface.

![](https://static.igem.wiki/teams/4391/wiki/results/rs7.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs8.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs9.png)

![](https://static.igem.wiki/teams/4391/wiki/results/rs10.png)

## Results for the hardware resistance measurements

To ensure that the physical hardware provided as little resistance to the incoming signals, to the neural chip we use Ti-Au layered input and output electrodes. Titanium provides structural support to the electrode and the Gold layer makes the electrode bio-safe and highly conductive. We however suspected that the electrode may show slight variability in its resistance, due to oxidation of exposed titanium. To ensure that the change in resistance was minimal, we kept a record of the resistance values over 8 days for the input electrodes of a selected chip, plated without liquid media. We report that the change in the electrode resistance was minimal, and stabilised over the span of 8days. This indicates that the sputtering was successful and the gold layer was preventing oxidation of Titanium underneath.

![](https://static.igem.wiki/teams/4391/wiki/results/nr1.png)

![](https://static.igem.wiki/teams/4391/wiki/results/nr2.png)

Measuring resistance of electrodes

![](https://static.igem.wiki/teams/4391/wiki/results/nr3.png)

Resistance values

## Taking a reading from the chip

We modelled our neural chip to be most sensitive around a current range of 150mA. A current drop below 100mA would virtually cause the neurons around the input electrodes to stop firing. We simulated these conditions using Arduino, where initially all input of the electrodes were kept at 160mA and then selectively switched off inputs by lowering their current strength to 50mA.

We used a bifocal fluorescence microscope to image the neuron-like cells. We used it in bright field mode and captured the pictures using an attached camera.

![](https://static.igem.wiki/teams/4391/wiki/results/nr4.png)

# References

1. Marton S et. al (2016) Isolation of an Aptamer that Binds Specifically to E. coli. PLoS ONE 11(4): e0153637. doi:10.1371/ journal.pone.0153637
2. Purkait, T., Singh, G., Kumar, D., Singh, M. & Dey, R. S. High-performance flexible supercapacitors based on electrochemically tailored three-dimensional reduced graphene oxide networks. Sci. Reports 2018 81 8, 1–13 (2018).
3. Duan, Nuo & Wu et. al (2013). Selection and Characterization of Aptamers against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX). Journal of agricultural and food chemistry. 61. 10.1021/jf400767d
4. Qaanei, M., Taheri, R. A. & Eskandari, K. Electrochemical aptasensor for Escherichia coli O157:H7 bacteria detection using a nanocomposite of reduced graphene oxide, gold nanoparticles, and polyvinyl alcohol. Anal. Methods 13, 3101–3109 (2021).
5. Paniel Nathalie, Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetricsensor(2016),
   <http://dx.doi.org/10.1016/j.talanta.2016.09.058>
6. Kaur, H., Shorie, M. & Sabherwal, P. Electrochemical aptasensor using boron-carbon nanorods decorated by nickel nanoparticles to detect E. coli O157:H7. Microchim. Acta 187, 1–10 (2020).
7. Gupta, A., Bhardwaj, S. K., Sharma, A. L., Kim, K. H. & Deep, A. Development of an advanced electrochemical biosensing platform for E. coli using hybrid metal-organic framework/polyaniline composite. Environ. Res. 171, 395–402 (2019).
8. <https://research.ufl.edu/publications/explore/v10n1/extract2.html>
9. <https://www.multichannelsystems.com/products/meas-60-electrodes>
10. [Long-term culture of SH-SY5Y neuroblastoma cells in the absence of neurotrophins: A novel model of neuronal ageing](https://doi.org/10.1016/j.jneumeth.2021.109301)

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