Team:UCAS-China/Hardware Details
Purification of CkTcs and Cdh in factory
Our ultimate purpose is to achieve massive production of the DeCaffi cup. For this reason, it is inevitable for us to design a method that can be utilized to purify the enzyme we need (namely, CkTcs and Cdh) in factory. Taking into account much more complicated factors compared with the purification in laboratory, the approach is different from the one in “design” part.
From complete cells to pure enzymes, we need to do three steps:
Step1: Cytoclasis.
We first plan to use ultrasonic cell pulverizer, but it will cause the temperature rising rapidly. When doing big-scale production, it is difficult to release the heat and sound energy. Therefore, we choose to break dry yeast using high pressure homogenizer (HPH) when doing massive production. HPH can be used for crushing yeast, E. coli, pseudomonas, bacillus and even black beetle. What’s more, the size of valve of the high-pressure homogenizer is another important factor affecting the rate of cell fragmentation. Some filamentous bacteria can clog the valves of homogenizers, especially when the concentration of bacteria in solution is high. E. coli that grows on rich media is more difficult to break than E. coli that grows on synthetic media.
Step2: centrifugation and isolation
After we crush the cells, we get a mixture of cell structure, metabolites, all kinds of enzyme. Now we need to isolate the enzymes we want by centrifugation.
Centrifugal separation process can be divided into three parts: centrifugal filtration, centrifugal precipitation and centrifugal separation. There are mainly two kinds of equipment for us to choose：Filter centrifuges and settling centrifuges. The first kind of machine is mainly used in the condition that suspended solid particles are of high concentration and large in size. And the later one is more effective when dealing with the solution in which the concentration of solid is relatively low.
Since we can gain dry yeast from fermentation tank. After doing step1, the high concentration of solid particles means filter centrifuges will be more suitable.
Then we began to find out the best centrifugation time and rotate speed. (the equipment we use )
Step3：precipitation
At last，we need to draw out the enzymes. Considering that the products will be used to process food, we avoid organic solvent precipitation for most organic solvent is harmful to human. Salting out is a better choice. The material we used is ammonium sulfate, which is soluble and inexpensive. Ammonium sulfate has a strong ability to precipitate proteins, and its saturated solution allows most proteins to settle with no damaging effect on them .
Next, we did an experiment to find out the most suitable temperature and pH when doing salting out.
Enzyme electrodes and caffeine detector on the cup
Purpose
To detect the caffeine concentration of the original and processed coffee, we designed an enzyme electrode. With the selectivity of enzyme, we want to detect caffeine in a complex solution with different kinds of chemicals.
Choice of electrode
Because the cup must be easy to take, it shouldn’t be heavy. What’s more, we shouldn’t waste too much coffee for detection, because the user wants to drink enough coffee. And the electrode must be cheap. Finally, we use screen-printed electrode for our detection.
Figure 1. Screen-printed electrodes
Modification of electrodes
We need to immobilize the enzyme on the electrode. We decided to use a mixture of Cdh which is produced by our engineering bacteria and coenzyme Q0 to transmit the electrons of from the enzyme and the electrode.
We choose to modify our enzyme electrode according to the acticle.(Gulnara Safina, 2010)
Figure 2. 3 chemicals for electrode modifying
Three drops of chemicals and the enzyme are put on the electrodes. Oxidized graphene is used to improve conductivity. And two cross linkers are used for enzyme immobilization. Because Cdh is not enough, we use Glucose oxidase for our test first.
Figure 3. 5 different concentrations of enzymes (GOx) to modify the electrodes
With 150U, 50U, 10U, 5U, 1U of enzymes GOx.
The electrodes are similar to sugar test papers in the market, which are light and easy to take. Disposable electrodes are also clean, preventing the deviance of detection resulting from reuse and the potential unclean things in the second use.
Potentiostat: A portable one
Commonly, the electrodes are tested by electrochemical workstation.
Figure 4. Common electrodes connected to a chemical workstation, which is heavy and expensive
But they are too big and expensive. We need a portable one to be installed on our cup. Thanks to the cheap and powerful electronic components and the power of open source hardware and software(Allison V. Cordova-Huaman, 2021), we are able to make a little potentiostat which can support the use of electrodes. A combination of Arduino and hand-made circuit board make it possible to put a potentiostat on the cup.
Figure 5. Arduino and the potentiostat, little, cheap but useful
We use the standard electronic sockets, which perfectly apply to the electrodes and is also very cheap.
Figure 6. screen-printed electrodes connected to the potentiostat
With hardware support, we can use some software to control the voltage and time. We can apply some common detecting methods, such as cyclic voltammetry, linear voltammetry and chronoamperometry to the drop of solution on the electrode. By calculating the mathematical relationship between caffeine concentration and peak current and peak voltage, we will be able to know how much caffeine is in the cup and whether our de-caffeine filter is useful. This will give users a better feeling.
Future improvements
According to the references, the enzyme electrode detecting methods showed good selectivity and sensitivity for lactose detection. But we haven’t found prior experience of electrode modification by caffeine dehydrogenase. More experiments are needed to show the optimal pH, temperature and amount of chemicals for Cdh modification. Linear area and reaction model should also be found with more efforts. As for the circuit boards, new integrated designs can be used to minimize the weight of the boards, making it easier for us to put it on the cup.
If we go further, we may edit the genes of our bacteria and make the modification easier, which means we can reduce the labor and process needed to put Cdh from the bacteria to the electrode. Even new engineering bacteria can be designed to serve as caffeine detector.
De-caffeine filter
Purpose
To use the enzyme CkTcs and Cdh efficiently and prevent them from leaking into the coffee, we need to immobilize these enzymes on the de-caffeine filter.
We design a solid filter, a cylinder with plenty of holes which can let the coffee flow through.
It can either be put directly into any coffee cup or be installed at the center of the coffee cup designed by us. The filter’s structure is shown as below.
Figure 7. The cap of the cylinder with holes and a tiny magnet is omitted to show the inner structure of the de-caffeine filter. Layers are made of paper and enzymes.
Figure 8. Filter Sample
Structure of the filter
The filter can be opened. And we can see four layers inside the filter. Layers on the top and at the bottom are CkTcs and Cdh layers respectively. We immobilize the enzyme on a base according to a method described by the article.(Dattatray K. Bedade, 2019) But instead of using the calcium alginate beads, we change the immobilization base into Cellulose Acetate membrane filter. We also use some other kinds of paper such as weighing paper and regular filter paper to immobilize the enzymes.
Figure 9,10. Activation of different papers by cross linkers
Figure 11. Immobilization of enzymes of different concentrations and different papers.
Using Glucose Oxidase for a test.
Advantages and extensions
The papers have many advantages, including big reacting surface and convenience of modifying its shape. Also, two kinds of reactors are can be put together simply by putting one paper on another. The transformation from caffeine to theophylline is a two-step reaction which requires two kinds of enzymes and two kinds of coenzymes. By putting two piece of paper containing different kinds of enzymes, we hope to transform the product of the first reaction instantly it is produced. Between the two different enzyme papers are normal filter papers which have been immersed in different coenzymes solutions.
Apart from paper containing enzymes, users can also put other things in the filter, such as other paper with different immobilized enzymes to transform various kinds of ingredients of their coffee. Thus, the function of the filter can be extended.
Reference
(1) Dattatray K. Bedade, Y. B. S., Rekha S. Singhal. (2019). Chitosan coated calcium alginate beads for covalent immobilization of acrylamidase: Process parameters and removal of acrylamide from coffee. Food Chemistry, 275, 95-104. https://doi.org/10.1016/j.foodchem.2018.09.090
(2) Allison V. Cordova-Huaman, V. R. J.-C., Adolfo La Rosa-Toro,. (2021). Low-cost smartphone-controlled potentiostat based on Arduino for teaching electrochemistry fundamentals and applications, . 7(2). https://doi.org/10.1016/j.heliyon.2021.e06259
(3) Gulnara Safina, R. L., Lo Gorton,. (2010). A simple and sensitive method for lactose detection based on direct electron transfer between immobilised cellobiose dehydrogenase and screen-printed carbon electrodes, . Electrochimica Acta,, 55(26), 7690-7695. https://doi.org/10.1016/j.electacta.2009.10.052
