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:
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
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.
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
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.
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.
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:
The first step involves the oxidation of graphite using P2O5 and K2S2O8 as oxidizing agents to prepare pre-oxidized graphite.
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
The lyophilized aptamer received from IDT was resuspended in a 0.1M PBS solution.
Further, the aptamer solution was diluted to a working concentration of 250 nM.
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)
E. coli ampule from MTCC was heated for 10-15 minutes and was broken with the help of a knife.
The knife was sterilized before use. Once the ampule was broken, the E. coli was resuspended with 1ml LB media.
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.
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
Take 12.5 gm of pre-mixed LB media powder to prepare 500 ml broth.
After adding 12.5 gm of powder, make it up to the volume of 500ml with deionized water.
Once that is done, cover the flask with the foil and put the autoclave tape over the flask.
Keep it in the autoclave; once that is done, take it out and keep it at room temperature.
Preparation of LB-Agar plates
Add 25g of pre-mixed LB broth powder per Litre to an appropriately sized flask.
Add 1L deionized water and stir it until clumps are gone.
Add 15 g Agar per Litre. This will not dissolve.
Autoclave for at least a 20minute liquid cycle.
Once the liquid is cool to the touch, pour into plates, covering the surface and avoiding bubbles.
Burn off bubbles with a bunsen burner or pop with a sterile tip
Let plates cool with the lids ajar.
Invert plates, and store them by labeling LB plates over them.
Composition of LB powder
10 g of peptone
5 g of yeast extract
10 g of NaCl
Protocol to culture Penicillium chrysogenum (MTCC 1348)
Penicillium chrysogenum (MTCC 1348) ampule from MTCC was heated for 10-15 minutes and was broken with the help of a knife.
The knife was sterilized before use. Once the ampule was broken, the Penicillium chrysogenum (MTCC 1348) was resuspended with 1ml YPD media.
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.
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
Take 25 gm of pre-mixed YPD media powder to prepare 500 ml broth.
After adding 25 gm of YPD powder, make it up to the volume of 500ml with deionized water.
Once that is done, cover the flask with the foil and put the autoclave tape over the flask.
Keep it in the autoclave; once that is done, take it out and keep it at room temperature.
Preparation of YPD-Agar plates
20g of peptic digest of animal tissue.
10g of yeast extract.
20g of dextrose.
After, we cultured the microbes. We would test our aptamer.
Protocol to test respective aptamers
The GC immobilized with Aptamers is taken out from 4ºC, and its impedance and CV were taken to analyze whether aptamers were successfully deposited.
Then, after CV and Impedance are taken, the GC surface is gently washed with deionized water.
The washed GC is left to dry at room temperature.
The GC is placed over a holder inside the vacuum desiccator and the laminar flow hood.
8µL of the respective culture specific to the aptamer is dropped and cast over the aptamer surface.
After drop-casting, GC was left to dry up at 40ºC in an oven for around 25 minutes.
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
The culture was diluted 20 times, made up to 1ml volume, and the O. D was taken at 650nm wavelength.
1 O.D of cells equals 108, and 0.1 O. D equals 104.
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)
Two different GCs were taken and modified with GO/PANI mixture. Further, they were deposited with the aptamer specific to E. coli (MTCC 443)
We performed EIS after the drop-casted solution dried up when left at 40ºC for 25 minutes.
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)
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).
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
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
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
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
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:
Go to inkscape.org,
And download the version that suits your device.
Open the Inkscape setup
Click next and read the license agreement.
Click next, customize all the features required, and confirm the installation location.
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:
Open the Inkscape software.
Click on “new document” after specifying the size of the document you need (you can do it later also).
Now you can see the Inkscape window, and toolbar on all four sides of the window.
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.
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
Insert the black ground probe into COM socket on a multimeter.
Insert the red probe (positive) on the V socket (can also be marked by omega or a diode symbol)
Set the range of voltage to 10 mV.
If the meter is autoranging, turn the dial to the 'V' setting with the symbol for AC or DC.
The multimeter is connected in parallel across an electrode in order to measure voltage.
The black probe is placed at the beginning of the input electrode
Switch on the multimeter
Complete the circuit by touching the red probe on the other end
The reading will be available on the LCD display
Protocol to culture cell lines
N2a and SH-SY 5Y cell lines were received from NCCS Pune.
Upon receiving the cell lines, they were immediately placed in an incubator at 37॰C, 5% CO2 and 95% relative humidity.
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.
Both the cell culture flasks were then transferred inside the Laminar AIr flow hood and the contents were pipetted into a Falcon tube.
The Falcon tube was centrifuged at 1000rpm for 10 mins to pelletise the cells.
The pellet was resuspended in 30ml of growth media for individual cell lines
The cells were then transferred to cell culture flasks and kept in the incubator at 35॰C, 5% CO2 and 95% relative humidity
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.
Supernantant is discarded.
The pellets were resuspended in FBS free DMEM (2ml).
1 ml of 0.4% Trypan Blue, in PBS (Phosphate buffered saline) is then added to the cell suspension.
The cells are incubated in the incubator for 3 minutes.
100μL of the cell suspension with Trypan Blue is added to the hemocytometer plate groove
Both the chambers are filled in gently under the coverslip and capillary action pulls in the suspension
Place the slide under microscope and focus on the grid at 10X magnification.
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
The cells after the 2 round of growth (one cycle is 3days), the cells were pelletised
A differenciation solution was prepared by mixing 0.5ml FBS in 49.5ml of DMEM. Henceforth called differentiation media.
The pellet was resuspended in the differentiation media
The resuspended cells were pipetted (5ml) in each neural chip.
The filter cover was screwed on.
The neural chips were placed in a tissue incubator (35॰C, 5% CO2 and 95% relative humidity).
The media was pipeted and replaced with fresh media after every 3 days.
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
Undiffrenciated cells are washed with Serum free DMEM, and Trypsinised using (0.05% trypsin EDTA), and quenched with DMEM, serum free (10ml).
The cells are then transferred into 15ml Falcon and centrifuged at 1000rpm for 10 minutes
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.
The cells are pipetted into the neural chip (5ml per chip)
Cells are incubated at 37॰C, 5% CO2 and 95% Relative Humidity.
Day 3
We gently aspirate off the media and refill the well with differentiation media 1.
The filter cover is refitted and the chips are placed in the Incubator
Day 7
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)
Place in the incubator
Day 9
Repeat step from Day 3 and 7
Day 13
One of the neural chip is taken for microscopy of differentiating cells
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
Neurite growth is confirmed with microscopy images
Chips are placed back in the incubator
Day 17
One of the neural chips is taken for microscopy
Old media is aspirate and Differenciation media 3 is added.
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
An oscilloscope was setup inside a LAF and the measurement probes were placed across the neural chip in a parallel connection.
Input voltage of 150mV was kept in 3 of the input by using a battery powered arduino setup
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.
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.
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.
