Introduction

Our device went through several changes throughout the project which can be seen elsewhere on our site (check out our design process here). This section will cover the final operation and workings of the device. The device houses the diagnostic capsule and electronics. The device has been designed to be used in a GP’s office with minimal interaction; doctors will be busy and cannot spend all their time watching the device. Our goal with the device is to meet the specifications set out by the experts we interviewed. We believe we have created this with the design and prototype. It is suggested to read the biological operation of the device first. You can read up on this here.

The page has been split into two sections as there are two main components of the device:

1. Capsule – The capsule covers the three biological stages of the operation where the reagents react. This is the replaceable part of the device which needs a new capsule for each test.
2. Chamber – The measurement stage of the device where the GFP levels are measured and quantified to give the result. This part of the device can be used for an unlimited number of times.

Freeze-Drying Bacteria

We looked at freeze-drying bacteria as an alternative to building a capsule containing a bacterial solution. The idea of freeze dried cells comes from the tardigrade mechanism. Tardigrades are microscopic eight-limbed animals that can survive drought and other harsh conditions by shrivelling up and losing water in a process called desiccation. Just as the yeast used for baking comes in the form of a freeze-dried powder and becomes activated when hydrated, bacterial cells can also be freeze-dried to attain dormancy for later use. When yeast is hydrated in baking, it usually takes 15-20 minutes for it to completely activate and cause the dough to rise. Similarly, mammalian cells take approximately an hour to fully activate once hydrated from their freeze-dried state.

E. coli would be expected to activate relatively faster than mammalian cells. Freeze-drying would be useful to us as the cells could remain dormant until needed, meaning storage would be more reliable and the dormant bacteria could be better accepted as being “safe”. We came up with the following three ideas as scenarios where this technique could be used:

1. Activate E. coli using a smaple of bodily fluid.
2. Prime the paper using water or a buffer solution for a few minutes before testing the bodily fluid.
3. Design an all-in-one microfluidic unit that contains an internal reservoir which will pre-wet the freeze-dried bacteria to activate them before being exposed to the bodily fluid. Figure 1 demonstrates this design idea.

Figure 1. An all-in-one paper microfluidic unit. Freeze-dried bacteria is incorporated into the device and will be activated upon pressing the buffer reservoir. Before the infected blood is sampled, the cells are activated by mixing with a buffer solution. Everything is encapsulated in a light casing that protects the technology from manual handling.

However, due to a lack of resources, we were unable to investigate this idea further and decided our current final design was a better bet!

Design

The capsule contains all the reagents for the three biological reactions (covered in the biology section), housed within an inner and outer tube (figure 1). The inner tube contains our engineered machine, the modified E. coli , and the outer tube contains the siderophores and iron. At one end of the capsule is a simple one-way valve which liquids can be injected into. The tube would be designed and manufactured like a glow stick with the outer-tube being very flexible and the inner tube being brittle. Hence, when bent, the inner tube would break and spill its contents. The capsule is a one use item; it has been designed to be kept affordable for each test.

Figure 1. The capsule design.

Operation

The operation of the capsule follows (figure 2):

1. Take a blood sample from the patient. Inject the blood with a syringe through the one-way valve (figure 2.1). The one-way valve will keep everything contained and in the tube.
2. The blood sample mixes with the siderophores and iron in the chamber (figure 2.2). Shake the tube for around a minute which should give the lipocalin in the blood plenty of time to mix. If lipocalin is present, it will bind to the siderephore-iron complexes.
3. Break the inner tube. This can be done by simply bending the tube which will break the inner tube containing the E.coli (figure 2.3). Any siderophore-iron complexes not bound to lipocalin will be taken up by the E. coli.
4. Give the tube another shake for good measure. It is now ready to be placed into the measurement chamber.

Figure 2. Images showing the operation of the capsule.

Design

This is the second part of the device. This is a much larger device that houses all the measurement electronics and the stand for the capsule. The schematic design can be seen below in figure 3. On the right is the measurement chamber. This is where the capsule would sit in a darkened chamber, which allows for the optimal conditions to measure any levels of GFP fluorescence. On the left are the electronics. This mainly consists of a micro-processing board.

Figure 3. Schematic design of the chamber. Left: basic schematic of the front view of the chamber; right: basic schematic of the top view of the chamber.

In reference to figure 3, the parts of the measurement chamber are:

1. Arduino board – This is a micro-processor mounted onto a board that can take in and send out electronic signals. This will take in data from the sensor measurements to decide if the GFP levels are high enough or not for a bacterial infection.
2. Capsule mount – This is where the capsule would sit in the measurement chamber.
3. Back mounted LED’s – two LED’s sit under the mount to activate the GFP and RFP. The GFP LED emits at 400nm and the RFP LED emits at 570nm.
4. Light dependent resisters (LDR's) – These are electronic components that change resistance based on light levels received.
5. Coloured filters – These sit by the LDR’s and remove any light that is not emitted from the GFP or the RFP in order to get the best readings.

Operation

The operation of the device follows (figure 4):

1. Place the capsule onto the mount inside the right side of the device. Close the chamber door to ensure the chamber is in full darkness.
2. Hit button to start operation. The device waits for 1 hour while GFP and RFP production is induced.
3. Device will activate the LED for the GFP in the capsule. The E.coli should now have mixed with the lipocalin and the GFP & RFP will be at optimal levels. The GFP will activate and fluoresce at a specific wavelength.
4. The light waves pass through a coloured filter that matches the GFP wavelength. This will eliminate noise in the form of light not from the GFP.
5. The light wave will then contact the LDR. This is placed into a potential divider combination of resisters that essentially gives a signal out if the light level is high enough.
6. The signal will go back to the Arduino, which will then process the signal. If the light level falls below the programmed threshold then the Arduino will determine that there was little GFP production, due to the high levels of lipocalin, and will give the result that the person likely has a bacterial infection.
7. As a backup, the device will repeat steps 3-6 for the RFP in the capsule as well. This is to check that the E.coli were not just dead and therefore not giving off any GFP fluorescence.

Figure 4. Images showing the operation of the device.