Since no cowboy is fully dressed without his lasso we decided to create a device that would go hand in hand with The Lung Ranger. We needed to create a detector that was able to utilise the light given off by the engineered E.coli. The device would need to be able to show the user the presence of a type of bacteria and the amount of present. So we developed the Light Amplifying Signal Sensing Object (L.A.S.S.O.). The L.A.S.S.O. involves entering sputum samples combined with the synthetically modified E. coli into a structure which houses an electronic circuit that produces a voltage signal dependent on the bioluminescence produced by the sample. The signal is passed to an Arduino Uno to convert it to a digital value, which is sent to a computer application. The application can then display the results to the user. The team’s vision is that the L.A.S.S.O. will be used in clinics to quickly determine the bacterial load that a cystic fibrosis patient is infected with. Compared to the the current laborious culturing procedures that are used for detection, it would allow medical staff to react quicker to bacterial infections with eradication therapy. It could then move to home visits to allow for mobile testing, and possibly become a self monitoring medical device for patients.

The L.A.S.S.O. is more than just the development of an idea; we wanted to see whether this device could be realistically implemented into society. To create a product that had a future in the real world we decided it would be best to talk to people we envisioned using it. People who understand the daily struggles and realities of treating cystic fibrosis. So we took a customer based approach by meeting with patients and medical staff to hear their view on how the L.A.S.S.O. should be designed to best meet their needs. We combined this with discussions we had with people in the medical device industry to find out what standards we would need to meet when developing the L.A.S.S.O. This would provide a holistic view of the users needs.

The main point that was brought to our attention from the patients and medical staff was that the L.A.S.S.O. should be portable. Several patients need to travel long distances to reach their local clinic to give sputum samples to be tested. By making the device small and easy to use we could make it possible for the tests to be done at home. This would ease the pressure on the patients and make it more feasible for them to be tested more frequently than the current 3 months. A possible issue could arise with breakdown in communication. As the tests are not being carried out in the clinic there is a greater risk for information not to be sent to the relevant people. To prevent this we added the functionality for results to be automatically sent to the relevant medical staff to ensure they had a copy of a patients results.

.
. More examples / information
.

By taking this approach we involved our future users from the start. Allowing them to become invested in the L.A.S.S.O. and understand how the synthetic biology used is helpful.

From talking to members of the industry we found that the main body for creating procedures in which medical devices should be built is the International Organisation of Standards (ISO). As we live in a global community where such standards are important to ensure that “products and services are safe, reliable and of good quality”. During the development of the L.A.S.S.O. we attempted to apply as many of the points given in the quality management standards 9001 and 13845. (9001 is for general quality management; giving standards a company should follow when developing a product. While 13485 expands on 9001 to specialise on how a medical device should be developed.) This lead to us creating documentation to show we were meeting the users needs, a requirements document. This gave us the final aims of how the L.A.S.S.O. would operate and the functionality involved. This became an iterative process with the documentation being updated after feedback from our supervisors, patients and medical staff.

At the start of the project our aim was to pick up fluorescent light changes in order to quantify bacteria in the sputum. Through further discussions we decided to move onto detecting bioluminescence instead. This gave us the advantages of the bioluminescent light being more stable and it would not require an excitation light source emitted which could interfere with our readings.

To trial a photodetector circuit we initially started of one made up an LED light source, picked up by a photodiode in connection to an operational amplifier. Due to the photodiodes high impedance, the current produced in the presence of light was extremely small thus the operational amplifier was needed in parallel connection to a resistor to ensure the voltage was increased to a level where our microcontroller, an Arduino Uno, could record the value produced.

The initial setup involved a non-inverting amplifier taking the signal from the photodiode and creating a set gain. The photodiode was connected to the inverting input of the operational amplifier with a resistor going through to ground. This meant that there was a certain offset value for the photodiode in darkness and once it was exposed to light it would produce current in a linear fashion.

Through further work on our circuit combined advice from Steve Reynolds, a physics lecturer here at The University of Dundee, we moved to a different circuit setup. This was based on the photodiode working in zero bias mode: one side of the diode was grounded therefore only a positive current could be produced and the problem of dark current was made minimal. The second major change implemented into the circuitry was the use of low width bandpass filters. These are able to reduce the 50 Hz ambient noise created by mains electricity in the UK. Initially our results were very oscillatory in their nature, thus it was hard to compare readings from different light levels based on results as they were not steady. We used two 1nF capacitors together with 1MΩ resistors to create a filter with a time constant of 6.3s which provided a decrease in the amplified noise and thus our readings became more steady.

After several tests and trials we were able to decide what combination of components would work best in our system. We soldered our circuit design onto veroboards as opposed to connecting our components to solderless breadboards as this made our circuitry compact and ensured that noise which was added to the signal through additional wiring was cut out. The bioluminescent output from the E.coli is picked up by the photodiode working in zero bias mode. This was fed through an operational amplifier being used as an inverting amplifier with a 100MΩ resistor. The gain in this type of circuit is the current multiplied by the value of the resistor in parallel. This ensures that a higher difference can be seen in the bioluminescent samples of various light levels as stronger light produces higher current. We developed the idea of suppressing the 50 Hz of ambient noise that was interfering with our results further, by reducing it by a factor of 20 000. To minimize in this way we placed three low pass bandwidth filters into the circuit consisting of 100kΩ resistors and 1μF capacitors, which would provide a time constant delay to the signal of 0.62s (a lower time delay to our previous construct). This would be accounted for when taking the readings into the microcontroller. After this noise reduction the signal was passed into the second gain circuit with the operational amplifier. This was a non-inverting gain circuit that comprised of a 100kΩ and a 1kΩ resistor, resulting in a gain of 101. Through this whole process the signal is amplified from a miniscule current to one that can be read by the Arduino Uno. For a safety precaution; to ensure our circuitry is not overloaded, we placed a split voltage setup using 1kΩ resistors. The operational amplifier is powered by 9 volts therefore it cannot produce a gain that goes over that supply voltage. Splitting this maximum possible voltage in two ensures that the reading going into the Arduino will never be above 4.5 volts, hence the pin being used to read the signal cannot be damaged.