BXMU-LABBOOK

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Background


Risk of Tradtional Tattoo

Tattoos have been developed over years as a decorative art for personalization. They are frequently used by some to celebrate events, to memorialize an important one, to customize individuals and etc. However, traditional tattoo techniques always come with serious side effects including potential health risks, instant pains, and removal problems.
Allergic reactions and skin infection are most common undesired outcome of performing tattoos. It is already known that tattoo inks may contain certain chemicals which are mutagenic (capable of causing mutations), teratogenic (capable of causing birth defects), and carcinogenic (capable of causing cancer). Additionally, there are certain chances that people might be infected with fatal diseases such as AIDS if tattoo pins are not replaced after each tattoo operation. Instant pains are almost inevitable during tattoo processes. Traditional tattoo processes always involves tattoo pins which carry different inks. These pins penetrate through human cuticular layer and stimulate the nerve cells in the dermis, causing instant dramatic pains. Tattoo removal has always been a problem over years. Tattoo was even considered permanent since it's difficult to remove. Therefore, our team is targeting at building a method to perform harmless biotattoos.


Introduction to DHA

DHA, the ingredient for self tanning, has been used successfully for about 40 years now. Its unique self tanning ability, skin substantivity and remarkable safety make DHA the most important active substance for sunless tanning. DHA induces a concentration-dependent formation of brown color complexes through an irreversible non-enzymatic glycosylation of amines or amino groups in skin proteins. This process is known as the Maillard reaction.It was demonstrated by Microscopic studies that DHA-pigment masses distributed irregularly in the keratin layer.Highly concentrated DHA (around 10%) is generally required to for effective tanning. However, DHA at this concentration range might bring servere dermatitis and other negative side effects to human health. Therefore, in situ synthesis of DHA through enzymatic reactions methods were utilized to continuously produce DHA on desired areas while the DHA concentration remains within a safe range. Here a multi-enzyme system GDH-NOX was employed to effectively produce DHA. To make this process commercially easily accessible, the regeneration of NAD+ was achieved by couplled GDH-NOX multi-enzyme system.

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Overview


Our Overview

The method used to form the GUVs is “Vesicle Formation from emulsion”. This method consists in a controlled hydration of dried or nearly dried films of lipids deposited on a solid surface, in this case electrodes.

  1. Preparation of lipids solution
  2. The lipid mixture contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) in a 9:1 proportion in 1 mg/ml concentration. The solution is prepared with the organic solvent chloroform.

  3. Lipids spreading
  4. .

  5. Solvent evaporation

Expecting Results

The method employed consists of rehydratation and extrusion, whose detailed procedures are summarized as following:

  1. Preparation of lipids solution
  2. The lipid mixture of 1 mg/ml concentration contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) with 0%-10% DOPS volume. The solution is prepared with the organic solvent chloroform.

  3. Evaporation
  4. 25 µl of sample solution are distributed as evenly as possible in the interior surface of a crystal meanwhile it is blown-dry using nitrogen. To totally evacuate the solvent of chloroform, the vial is left during one hour inside a drier.

  5. Hydratation
  6. 500 µl of the working buffer (HEPES, NaCl and w/o Mg2+) is used to hydrate the dry lipid.

  7. Incubation
  8. The vials are left for 2 hours of incubation; mix the sample using a vortexer.

  9. Freeze/thaw
  10. The sample is immersed in liquid nitrogen followed by boiling water for 5 cycles totally.

  11. Extrusion
  12. Assemble the membrane inside the extruder as it follows:

    1. The Teflon piece is wetted with buffer.
    2. The 2 pieces of cellulose membrane are placed in a support in the center of the Teflon piece.
    3. Put the PC membrane with a pore size of 50-100 µm on the taller piece of the holder.
    4. A drop of buffer is placed on top of the membrane.
    5. The 2 pieces of Teflon holder are placed together.
    6. The syringe is filled with the lipid solution, and extrude at least 11 times.
    7. The extruded sample from the acceptor syringe is withdrawn. The solution should look clearer than it was before extrusion.
  13. Cleaning
  14. The Teflon piece, o-ring and syringe should be rinsed with copious 2-propanol and deionized water after use. Otherwise the residues will contaminate the next sample.

References

    1. Tsui-Fen Chou, Christopher So, Brian R. White, Jonathan C. T. Carlson, Mehmet Sarikaya, Carston R. Wagner. Enzyme Nanorings[J]. ACS Nano. 2008, 2 (12),2519–2525.
    2. Adrian Fegan, Brian White, Jonathan C. T. Carlson, Carston R. Wagner. Chemically controlled protein assembly: techniques and applications[J]. Chem Rev. 2010, 110, 3315-3336.
    3. Brian R. White, Qing Li, Carston R. Wagner. Chemically Induced Self-Assembly of Enzyme Nanorings[J]. Methods Mol Biol. 2011,743, 17-26.
    4. Sonja Billerbeck, Johannes H?rle, Sven Panke.The good of two worlds: increasing complexity in cell-free systems[J]. Current Opinion in Biotechnology. 2013, In Press.
    5. Wei Zhang, Quan Luo, Lu Miao, Chunxi Hou, Yushi Bai, Zeyuan Dong, Jiayun Xu, Junqiu Liu. Self-assembly of glutathione S-transferase into nanowires.Nanoscale[J]. 2012, 4, 5847-5851.
    6. N Benamar, A F Laplante, F Lahjomri and R M Leblanc. Modulated photoacoustic spectroscopy study of an artificial tanning on human skin induced by dihydroxyacetone[J]. Physiol. Meas. 25, 1199, doi:10.1088/0967-3334/25/5/010.
    7. Anita B Petersena, Hans Christian Wulfa, Robert Gniadeckia, Barbara Gajkowskab. Dihydroxyacetone, the active browning ingredient in sunless tanning lotions, induces DNA damage, cell-cycle block and apoptosis in cultured HaCaT keratinocytes[J]. Mutation Research/Genetic Toxicology and Environmental Mutagenesis.2004, 560(2),173–186.
    8.Adrian Fegan, Brian White, Jonathan C. T. Carlson, Carston R. Wagner. Chemically controlled protein assembly: techniques and applications[J]. Chem Rev. 2010, 110, 3315-3336.

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