Binding the Aptamers to the Electrode
Once the aptamer has been successfully discovered, improved and tested, we’ll need a way to integrate it into an IoT device. IoT has multiple requirements and we could extrapolate some of them into the measurement system; The system has to be affordable, automatic and the limit of detection must be under the range of the protein’s concentration in the air.
Although our first idea includes an an optical to measure the protein present in the sample, this approach has three main limitations:
The detection limit of this system is over the protein concentration that we need to measure.
The optically systems are fine for working at the lab, but the unstable street conditions introduce so much noise in the system.
Is complicated to maintain an optical system stable outside a laboratory. The mechanics of the optics tend to decalibrate.
The electrochemical system has been conceived to solve these difficulties.
The main part of the system is the electrodes above which we bound the aptamers, the called “Working electrodes”. Additionally, for taking the measurements with a potentiostat, we need another 2 electrodes, the “Reference electrode”, and the “Auxiliary electrode”.
From now on, the word “electrode” will represent this 3 electrode system.
This system of three electrodes its sold together in ceramic boards. We have ordered them to DropSens, which is a Spanish business and a global referent in the field. Across the large repertoire, we have chosen electrodes with the following characteristics:
The working electrode is made of carbon with gold nanoparticles. The carbon has a better electrochemical window than gold or silver (check this post for more information) and gold are the ideal substrate to join DNA (It only have to be thiolated).
The auxiliary electrode is also made of carbon.
The reference electrode is made of silver.
The DropSens reference of our electrode is 110GNP and you could see the complete datasheet here.
Testing the electrodes
The project is too wide to be waiting for the complete development of aptamers before developing an integration into electrode method.
In parallel to aptamer discovery, we have adjusted the protocol for TBA, the most studied aptamer since it was discovered in 1992.
You could see the complete protocol for aptamer design, order, and integration in our protocols page.
To test the system, firstly we have adjusted the correct Ferricyanide quantity. We have tried two first concentration 1mM and 5mM with raw electrodes with a Cyclic Voltammetry (CV) test. The results have the typical duck shape of a CV test. As it’s logical, the current improve with higher ferricyanide concentrations.
From now on, the parameters which we have used to perform the cyclic voltammetry methods are the same:
- Voltage range from -0.3 V to 0.3 V.
- A current limit of 1000 uA.
- A sample rate of 100 Hz.
- A scan rate of 0.05 mV/s.
Figure 1: The current improve significantly with 5mM of ferricyanide concentration compared to 1mM. Further assays increasing the ferricyanide concentration more than 5 mM have no effect (not shown) meaning that the system gets saturated by electrons at this concentration.
After the ferricyanide concentration has been optimized, the next step implies testing the correct aptamer bounding and the capacity of the system to detect the protein of interest. So, for the next testing we have measure 4 different electrodes:
A raw electrode.
An electrode with bonded aptamer but without thrombin incubation.
An electrode with bonded aptamer after 1h incubated on a 0,05mg/mL thrombin solution.
An electrode with bonded aptamer after 1h incubated on a 0,5mg/mL thrombin solution.
The results demonstrate the capacity of our electrode to change the voltage proportionally to the bound thrombin. We could establish a mathematical model which correlates the voltage drop (compared to a negative control) with the thrombin concentration in the sample.
Figure 2:(a) The results show the capacity of our sensor to measure the correct aptamer binding as well as the thrombin concentrations in the range of 0.5 mg/mL to 0.05 mg/mL. (b) We could use just the current peak as an indicator of thrombin concentration for further correlations between signal and concentration.
Future improvements of the system
Our system has been proved but lacks some important points that we are planning to improve in the future months and for which we are looking for funding partners:
Minimize the measurement hardware: Although we haven’t given a lot of credits, we have used RodeoStat as our potentiostat. It’s based on an open potentiostat called CheapStat, which has inspired previous iGEM teams in earlier years. Although the platform is expensive compared to the original CheapStat, the advantage of RodeoStat is that it comes fully assembled. The main issue on RodeoStat are the dimensions of the PCB. It is very poor optimized, and the PCB is an order the magnitude bigger than what we will eventually achieve. This is key because as our device is oriented to be commercially produced and oriented to IoT applications, the size could determine the handling of the device.
Improve the N: The number of trials that we have achieved is not enough to make some statistics and a good modeling which correlates signal with concentration. We are also going to need more measurements with more environmental conditions. As we need to establish a replicable system into the streets we need to be confident about the reproducibility of the measurements.
Try more sensible voltammetry methods: We are also going to need more measurements with more environmental conditions. As we need to establish a replicable system into the streets we need to be confident about the reproducibility of the measurements.