Team:HSNU-TAIPEI/Design

Product

Introduction

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

  1. 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.
  2. 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.
  3. Chitosan: both layers of chitosan are exceedingly easy to dissolve in acetic acid, which has a big effect in attach to cooper(II) ion.
  4. 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)
  1. Pour some solution into beakers.
  2. Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
SA(Alginate)
  1. Pour some solution into beakers.
  2. Draw certain amount of 8%PVA-1%SA and add it into 3%BA(Boric acid)-1%CaCl2.
ACA(Alginate & Chitosan)
  1. Pour some solution into beakers.
  2. Draw certain amount of 1.5%SA and add it into 100mmol/LCaCl2
  3. Throw the microcapsule into 0.3%Chitosan.
  4. Throw the microcapsule into 0.15%SA.
  5. 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
  1. Embed the bacterial liquid and preserve it in -18/4/25℃
  2. Break and dissolve cell-loaded microcapsules with microcapsule-broken solution every 48 hours.
  3. Draw bacterial liquid and culture on the solid medium.
  4. Put them in 37℃ for 48 hours.
  5. Count the amount of E.coli
1.2.2 Result

WE HAVEN’T FINISHED

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
  1. Embed cloning E.coli into microcapsules. (Bacterial liquid:immobilizing solution=1:4)
  2. Separate microcapsules into five centrifuge tubes.
  3. Dilute CuSO4 by serial dilution. ( Concentration: 4000/400/40/4ppm )
  4. Add different concentration of CuSO4 into its corresponding centrifuge tube. (CuSO4:microcapsules=1:1 )
  5. 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
  1. prepare 1.5 g of chitosan
  2. add in 0.4 M 50 ml of citric acid
  3. use the magnetic mixer at the speed of 100 rpm to mix the mixture for 12~24 hr
  4. use the peristaltic pump to drop the mixture into 0.9 M NaOH mixture
  5. wait for 72 hr after the reaction
  6. 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
  1. prepare 3.929 g of copper sulphate.
  2. add DI water to 1000 ml
  3. take out 200 ml of the mixture, and add DI water to 1000 ml
  4. 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
  5. 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

Reference

  1. Craig W. Jones (1999). Applications of hydrogen peroxide and its derivatives. Royal Society of Chemistry. ISBN 0-85404-536-8.
  2. "Oxygen-based bleaches", The Royal Society of Chemistry, and Reckitt Benckiser (the manufacturers of Vanish)
  3. 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.