Our project envisions developing a working device that could detect any target molecule reliably, efficiently, accurately, and quickly using a Neural chip and Aptamer. As a Proof of Concept, we decided to detect the presence of microbes in any given sample.

We decided to develop a device incorporating Aptamer, Neural chip, and Micro-controller as its main components.

Through the literature search, we identified the aptamer sequences for the commonly found microbes E. coli (MTCC 443), Penicillium chrysogenum (MTCC 1348), and Salmonella typhimurium that were highly specific to these species. Then, we ordered the DNA oligo from IDT's sponsorship. The ordered aptamers, when received, were resuspended in 0.1 M PBS, and these aptamers were further diluted to a working concentration of 250nM.

Then, we modified the Glassy Carbon electrode with GO/PANI mixture since direct binding of an unmodified aptamer over the glassy carbon electrode doesn't occur. Hence, the modification helps attach Aptamer to the GC and forms a strong chemical bond.

GO/PANI improves functionalities and charges conduction values that can form a suitable interaction with the aptamer molecule. Small electrochemical changes occur when microbes bind to the aptamers attached over modified GC, which helps detect the presence of the microbes in the sample.

The GO was diluted to 3mg m/L in 0.5M perchloric acid HClO4. GO and aniline was taken in the ratio of 4:1, respectively, by weight. The 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.

Further, maximizing the biosensor performance formation of a high-density layer with few defects is essential. Therefore, the lyophilized Aptamer was diluted with 0.1M PBS solution to 250nM Aptamer and 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 (12hr) at 4^oC.

Our designed aptasensor would be further connected to the neural chip, which can perform bio-computation to detect electrical changes, and the neural chip was further connected to the micro-controller, a device that could relay the output and would let us know whether the microbe is present in the sample.

Initially, the Graphene oxide was prepared using the Hummer method reported from the previously available literature. After that, conducting polymer polyaniline was mixed into the 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. The 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. GO/PANI was electrodeposited onto a glassy carbon electrode.

Maximizing the biosensor performance formation of a high-density layer with few defects is essential. Therefore, the lyophilized Aptamer was diluted with 0.1M PBS solution to 250nM Aptamer and 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 (12hr) at 4^oC.

The SAM aptasensor was then dropped cast with an 8µL microbe and kept to dry, and its electrochemical response was measured.

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.

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 ∆the E value of the CV curve. CV was measured between potential -0.1V to 0.5V at a 20 mV/s scan rate.

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.

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. 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.

The Nyquist response shows a massive 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:4) and EIS response (fig:5) 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 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.

Thus, our designed aptasensors developed against their respective microbes can successfully detect them from their sample.

Once we tested our aptamers successfully, we decided to test out the sensitivity of our aptansensor. 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., 0.8 OD and 0.08 OD. But, as it was diluted 20 times before the dilution, the actual O.D was 16 and 1.6, respectively.

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 16 x 10⁸ cells/ml, 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 the decrease in the current value with the given potential window. Figure 8 shows the CV of the 1.6 x 10⁸ cells/ml E.coli showing a similar response as that of 16 OD E.coli. Still, the CV curve for the 1.6 x 10⁸ cells/ml E.coli assembled aptasensor is more horizontal than the other concentration 1.6 x 10⁸ cells/ml E.coli. This shows the current is hindered more in 16 x 10⁸ cells/ml E.coli than the other concentration ratio.

Nyquist plot for 16 x 10⁸ cells/ml (figure: 9) and 1.6 x 10⁸ cells/ml OD (figure: 10) concentration of E.coli shows the large RCT value for 16 x 10⁸ cells/ml concentration than 1.6 x 10⁸ cells/ml 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.

Developing the neural chip

Ti-Au layered input and output electrodes are used with the neural chip to guarantee that there is minimal physical hardware resistance to the incoming impulses. The electrode is structurally supported by titanium, and its gold covering makes it biocompatible and extremely conductive. But we had a sneaking suspicion that the electrode's resistance would vary slightly as a result of the titanium's exposed surface oxidising. We recorded the resistance values for the input electrodes of a chosen chip throughout the course of 8 days while it was plated without a liquid medium in order to make sure that the resistance changed as little as possible. We state that the electrode resistance only slightly changed over the course of 8 days before stabilising.

Prototype with vinyl negative coating

Variation of resistance over 8 days

Our neural chip was designed to be most responsive in the 150 mA current band. The neurons near the input electrodes would essentially stop firing if the current dropped below 100 mA. We used Arduino to recreate these conditions, keeping all electrode inputs at 160 mA at first, then turning off some of them by reducing their current to 20 mA. We plotted the current values that were observed in the two outputs A and B at different input conditions. This gave us definitive proof that the neural chip was able to distinguish between the input stimuli and perform dimension reduction.

We conducted this experiment to prove the sensitivity of the neural chip to electrical inputs and to verify that these signals could be used to distinguish between different input parameters, ie. used for detection.

Trypan Blue assessment of cells

For both growing and differentiation of cells, we used a slightly modified protocol than that was documented by iGEM Tuebingen. We had replaced the use of Retinoic acid in the last steps to rely on the bioconversion of retinol into retinoic acid by the cells themselves to support their differentiation. In this process, we hoped to be able to provide a gentle environment for the differentiation of the cells. This was also essential for us as we were conducting differentiation protocol in the neural chip, that was sputtered with Gold-Titanium electrodes. We were apprehensive that the Retinoic acid used in the Differentiation media if used for a prolonged period may react with the Titanium under the Gold. This would increase the crack in the gold layer if any developed and would change the resistance of the electrodes over time.

To verify that the differentiation steps worked with this new method and that cell death was minimum, we conducted regular Trypan blue assessments on the cells.

Microscopy and verification of differentiated cells

To verify that our modified protocol for differentiation of SH-SY 5Y cells and N2a cells worked we conducted a microscopic analysis of the growth plates. The results of these experiments were positive and confirmed that we indeed had cells with neuron-like morphology.

Microscopic image of neuron