Objective

Once the aptamer has been successfully discovered, improved and tested, we will need a way of integrating it into an IoT device. IoT has multiple requirements and we need to consider some of them for developing the measurement system that we require. 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 optical system to measure the protein in the sample, this approach has three main limitations:

1. The detection limit of this system is over the protein concentration that we need to measure.

2. Optical systems are suitable for working at the lab, but the unstable street conditions introduce too much noise in the system.

3. It is complicated to maintain a stable optical system outside a laboratory. The mechanics of the optics tends to decalibrate.

The electrochemical system has been conceived to solve these difficulties.

The Electrodes

The main part of the system is constituted by the electrodes that we bind the aptamers to: the so called “Working electrodes”. Additionally, for taking the measurements with a potentiostat, we need another two electrodes, the “Reference electrode”, and the “Auxiliary electrode”.

From now on the word “electrode” will represent this three electrode system.

This system of three electrodes is distributed comercially embedded in a ceramic board. We have ordered them to DropSens, which is a Spanish company and a global referent in the field. Across the large repertoire, we have chosen electrodes with the following characteristics:

1. The working electrode is made of carbon with gold nanoparticles. The carbon has a better electrochemical window than gold or silver (check this postfor more information) and gold is the ideal substrate for binding DNA (It only has to be thiolated).

2. The auxiliary electrode is also made of carbon.

3. 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 discovery of aptamers before developing an integration method.

In parallel to aptamer discovery, we have adjusted the protocol for TBA, the most studied aptamer since its discovery in 1992.

You could see the complete protocol for aptamer design, ordering, and integration in our protocols page.

To test the system, we initially have adjusted the correct Ferricyanide quantity. We have tried two different concentrations 1mM and 5mM in raw electrodes with a Cyclic Voltammetry (CV) test. The results have the typical duck shape of a CV test. As expected, the current increases with higher ferricyanide concentrations.

After this point, the parameters that we used to perform the cyclic voltammetry were 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 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.

Table 2. Nmol of aptamers traped in the membrane and the % of sequences retain by unspecifically interactions for each round of Selex with Ole-E1 protein.

After the ferricyanide concentration was optimized, the next step implied testing the correct aptamer binding and the capacity of the system to detect the protein of interest. So, for the next test we measured 4 different electrodes:

1. A raw electrode.

2. An electrode with bound aptamer and without thrombin incubation.

3. An electrode with bound aptamer after 1h incubation on a 0,05mg/mL thrombin solution.

4. An electrode with bound aptamer after 1h incubation 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) to 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 upcoming months, and therefore we are looking for funding partners at the moment:

1. Scale down the measurement hardware: Although we have not given a lot of credit to Rodeostat, we have used it as our potentiostat. It is 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 is the size of the PCB. It is poorly optimized, and the PCB is an order of magnitude bigger than what we will eventually achieve. This is key because as our device is oriented to be commercially produced and to IoT applications, the size could determine the ease of handling of our device.

2. Improve the N: The number of trials that we have achieved is not enough to an accurate statistical model that correlates signal to concentration. We are also going to need more measurements with more environmental conditions. As we need to establish a replicable system outside the lab we need to be confident about the reproducibility of the measurements.

3. Try more sensible voltammetry methods: Although cyclic voltammetry has given good results, there are some available methods that are described to be more accurate. One example is the Square Wave Voltammetry method which doesn’t require any kind of further hardware modification.