We believe that everyone has the right to ensure food security, so we design a product which contains E. coli with the gene fragments to detect toxins. We embed E.coli into microcapsules and put them into centrifuge tubes. We have biological safety system. Our product will be kept in P1 lab*. People can send oil samples to the lab, which fastens the inspection and provides scientific clues for the government when inspecting illegal industries. The embedding of E. coli saves cost and time and has high-efficiency detection mechanisms. *125 universities in Taiwan have P1 labs, and 66 universities in northern Taiwan have P1 labs.
Product Design
The tube is divided into the left side (red) and the right side (blue) by a partition. The red side is a straw for effectiveness test. Press the red straw so that the test copper ion is added into microcapsules. Green fluorescence means the E.coli survives; if not, we can know that the product is unavailable. The blue side is a tube for piercing. Press the blue straw to pierce chitosan so that the oil can go into the layer of microcapsules.
Microcapsule (micro-powder-like): Microcapsule is the most important part of this product, which is made by freeze drying so that the water is reduced. As long as it exposes to any substances such as heavy metals, benzopyrene and aflatoxin, green fluorescence will soon express and from that we know there are toxins in the edible oil. Without tough inspection process, we can easily test the oil samples with very low cost.
Chitosan: both layers of chitosan are exceedingly easy to dissolve in acetic acid, which has a big effect in attach to cooper(II) ion.
The bottom layer of the tube is sodium percarbonate, an oxidizing agent. Sodium percarbonate is an ingredient in a number of home and laundry cleaning products, including non-chlorine bleach products such as OxiClean, Tide laundry detergent, and Vanish. Dissolved in water, it yields a mixture of hydrogen peroxide (which eventually decomposes to water and oxygen) and sodium carbonate ("soda ash").
Safety-Triple biosafety certificated mechanisms
The first and foremost risk that our final product: microcapsules with genetic-modified E-coli will pose is that E-coli might leak out of the tube and further contaminate the environment. Our work on biosafety issue is to reduce the chance that the E-coli will leak out and contaminate the environment. We have developed triple biosafety certificated mechanisms that will reduce the risk.
Microcapsule
We embed E.coli in the microcapsule by immobilization, which prevent E.coli from polluting environment.
Chitosan
We solidify chitosan to form a film and put it on the upper and the lower layer of microcapsules, so microcapsules won’t not leak or be destroyed.
Sodium percarbonate
Sterilizing procedure: By using acetic acid to dissolve chitosan and produce water, the water flows to the lower part and triggers precarbonate, thus activates the sterilizing process.
Experiment Design
1. Immobilization
1.1 Compare the effect of different immobilization methods on microcapsules’ patterns
We chose three methods to immobilize E.coli. Then we made a pre-test to ensure the feasibility of each method, including immobilization materials and equipment.
1.1.1Procedure
PVA(Polyvinyl alcohol)-SA(Alginate)
Pour some solution into beakers.
Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
SA(Alginate)
Pour some solution into beakers.
Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
ACA(Alginate & Chitosan)
Pour some solution into beakers.
Draw certain amount of 1.5%SA and add it into 100mmol/LCaCl2
Throw the microcapsule into 0.3%Chitosan.
Throw the microcapsule into 0.15%SA.
Use syringe to inject sodium citrate in the microcapsule.
1.1.2 Result
1. Different materials of immobilization
We decided to apply only SA and ACA materials in our experiment, and we deleted step e. of ACA because it’s infeasible.
PVA-SA
White, Teardrop-shaped
SA
colorless (bluish), sphere
ACA
Bluish, sphere
2. Comparison of syringe and pipet
We decide to apply syringe in our experiment because it has a better effect on microcapsules’ patterns than pipet.
syringe
Consistent size, faster, without bubble
pipet
Vary in size, slow, with bubble
1.2 Compare the effect of different preserving temperatures of microcapsules on E.colis' survival
We chose three common preserving temperatures to carry out our experiment, including room temperature 25℃, cooler compartment 4℃, and freezer compartment -18℃.
1.2.1 Procedure
Embed the bacterial liquid and preserve it in -18/4/25℃
Break and dissolve cell-loaded microcapsules with microcapsule-broken solution every 48 hours.
Draw bacterial liquid and culture on the solid medium.
Put them in 37℃ for 48 hours.
Count the amount of E.coli
1.2.2 Result
According to the data, it achieved the highest amount of CFU(Colony-forming unit) at fifth day and decreased at seventh day.
The higher the temperature is, the higher the amount of CFU.
The results show no differences between immobilized and non-immobilized group.
1.3 The test of whether toxin can enter microcapsule to activate the biosensor.
We chose three common preserving temperatures to carry out our experiment, including room temperature 25℃, cooler compartment 4℃, and freezer compartment -18℃.
1.3.1 Procedure
Embed cloning E.coli into microcapsules. (Bacterial liquid:immobilizing solution=1:4)
Separate microcapsules into five centrifuge tubes.
Dilute CuSO4 by serial dilution. ( Concentration: 4000/400/40/4ppm )
Add different concentration of CuSO4 into its corresponding centrifuge tube. (CuSO4:microcapsules=1:1 )
Take a picture of each tube under UV light every five minutes.
1.3.2 Result
Our microcapsule can detect each solution of CuSO4 immediately in five minutes.
2. Freeze-dried & Heavy metal absorption of chitosan
2.1 Compare the effect of freeze-dried and oven-dried chitosan on heavy metal absorption
There are many kinds of heavy metal, and here we take Cu(II) ion for example.
2.1.1 Procedure
prepare 1.5 g of chitosan
add in 0.4 M 50 ml of citric acid
use the magnetic mixer at the speed of 100 rpm to mix the mixture for 12~24 hr
use the peristaltic pump to drop the mixture into 0.9 M NaOH mixture
wait for 72 hr after the reaction
after been washed with DI water, put the chitosan-citrate beads into the oven and dry at 35℃ for 24 hr
2.1.2 Result
The freeze-dried beads have better adsorption to copper
▼Table 1: The effect of freeze-dried and oven-dried chitosan on Cu(II) ion adsorption
Chitosan
Adsorption(mg/g)
Adsorption(mg/g)
Adsorption(mg/g)
Adsorption(mg/g)
temperature
30℃
40℃
50℃
60℃
Freeze dried
123
129
131
141
oven dried
113
115
120
127
Figure 1: Explore different effect of freeze-dried and oven-dried chitosan on Cu(II) ion adsorption
Figure 2: The surface of the freeze-dried chitosan
Figure 3: The cross section of the freeze-dried chitosan
Figure 4: The surface of the oven-dried chitosan
Figure 5: The cross section of the oven-dried chitosan
2.2 Compare the effect of different initial concentrations on chitosan for heavy metal adsorption
2.2.1 Procedure
prepare 3.929 g of copper sulphate.
add DI water to 1000 ml
take out 200 ml of the mixture, and add DI water to 1000 ml
add 1 g of freeze dryer-dried chitosan-citrate beads into the different concentration Cu(II) ions mixture, and mix at the speed of 100 rpm 100 mg/L 250 mg/L 500 mg/L 750 mg/L 1000 mg/L
take samples at 0 hr, 1 hr, 2 hr, 4 hr, 8 hr, 24 hr, 48hr, 72 hr
2.2.2 Result
The higher the concentration, the better the chitosan-citrate beads’ adsorption; but the percentage of adsorption will be lower
▼Table 2: The effect of different initial concentrations on chitosan-citrate beads for Cu(II) ion adsorption.
Chitosan-citrate
Initial concentration of copper(ppm)
Adsorption(mg/g)
Percentage of adsorption(%)
100
83
83.07%
200
123
65.455%
500
196
39.272%
750
270
36%
1000
308
30.857%
Figure 6: The effect of different initial concentrations on chitosan-citrate beads for Cu(II) ion adsorption.
(adsorption value)
Figure 7: The effect of different initial concentrations on chitosan-citrate beads for Cu(II) ion adsorption. (percentage of absorption)
Figure 8: The SEM picture of the surface of the freeze dried beads
Figure 9: The EDS picture of the surface of the freeze dried beads
Figure 10: The EDS picture of the cross the section of freeze-dried beads
Figure 11: The EDS picture of the cross
Figure 12: The SEM picture of the surface of the oven-dried beads
Figure 13: The EDS picture of the surface of the oven-dried beads
Figure 14: The EDS picture of the cross section of the oven-dried beads
Figure 15: The EDS picture of the cross section of the oven-dried beads
3.Biosafety
In order to avoid polluting the environment , we design a quick and easy mechanism to sterilize.
3.1 Procedure
Add sodium carbonate 6.6g into centrifuge tube.
Add 240 microcapsules on the layer of sodium carbonate.
Add water 20mL and trigger sterilization.
3.2 Result
▲Add 10ml Acetic acid into our product, and trigger the sterilization.
Reference
Craig W. Jones (1999). Applications of hydrogen peroxide and its derivatives. Royal Society of Chemistry. ISBN 0-85404-536-8.
"Oxygen-based bleaches", The Royal Society of Chemistry, and Reckitt Benckiser (the manufacturers of Vanish)
McKillop, A (1995). "Sodium perborate and sodium percarbonate: Cheap, safe and versatile oxidising agents for organic synthesis". Tetrahedron51 (22): 6145. doi:10.1016/0040-4020(95)00304-Q.
