Team:HUST-China/results group3

results_group3

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The Experiment Standard

We measured the standard curves of three pigments before using them for hair dyeing experiment. We also found that the amount of melanin contained in hair can have a significant effect on hair dyeing outcomes. Therefore, we define different colors of hair based on bleaching.

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result1

For the convenience of dyeing, we buy a series of bleached hair. The lightest color is the 9°hair, and the darkest is the 4 ° hair.

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The Best Condition of hair dye

We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair to get a series of dyeing discs. And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.

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result2

Chart of the best condition of hair dye |Dye/Condition|temperature|Dyeing aid ingredients|concentration（g/L）|comment| |indigo 2min|Room temperature|none|2|The color deepens significantly while dyeing for multiple times |curcumin 30min|50℃|Ethyl alcohol|0.5| |lycopene 30min|Room temperature|alum|2| The best dyeing condition

The exhibition of hair dyed on the best condition

Under the best conditions, we dyed the hair from 4 degree to 9 degree, and got a series of colors. It is found that it only needed to be bleached to 8 degree so that the hair would show a bright color for all three kinds of dye.

As to lycopene hair, 8 or 9 degree hair was red, 7 degree(or below) hair was brownish, and the longer the hair was dyed, the redder it would become.

As to indigo hair, 7 to 9 degree hair would become blue. As the dyeing time goes, the color would turn blue from an indigo color; 5 to 6 degree hair would be dyed to celadon, and 4 degree hair was still brown.

The dyeing results of lycopene（room temperature，adding alum，2g/L）。fron left to right: 9°（5,10,30min），8°（5,10,30min）, 7°（5,10,30min），6°（5,10,30min）, 5°（10,30min），4°（5,10,30min）

The dyeing results of indigo (room temperature，2g/L）. From left to right: 9°（0.5,2,6min），8°（0.5,2,6min）, 7°（0.5,2,6min），6°（0.5,2,6min）, 5°（0.5,2,6min），4°（0.5,2,6min）

The dyeing results of indigo（room temperature, ethanol added，0.5g/L）. From left to right: 10min（9-4°），30min（9-4°）8°

Problems occurred during the research:

Lycopene

Problem: No literature on coloring fabrics or hair with lycopene

Solution: We conducted a gradient experiment (0.5, 1, 2, 5 g/L) to explore the effective concentration of lycopene for hair dyeing. Finally, 2g/L of lycopene is selected, at which concentration the dye fluid will not be too viscous, and has a better dyeing effect as the picture below shows.

(From left to right: 0.5(30 min), 1(5, 10, 30 min), 2(5, 10, 30min), 5(5, 10, 30min)g/L of lycopene)

Problem: Lycopene dye the hair with a low efficiency, a low color fastness, and a constantly discoloring process when the hair is showered by water.

Solution: We looked up the data and selected three eco-friendly color aids (alum, potassium tartrate, citric acid). Through direct color comparison and elution experiments, we found that alum can significantly improve the coloration rate and color fastness of lycopene.

（From left to right, 1st-4th groups: alum（30min）、potassium tartrate（40min）、citric acid（40 min）、no color aids（40min）; 5th-8th groups: the 1st-4th hair after washing 7 times）

Curcumin

Problem: The coloration rate of the curcumin aqueduct solution is low

Solution: We carried out the same dye addition and elution experiments as lycopene, and found that alum, potassium tartarate and citric acid cannot improve the coloring effect of curcumin. The data showed that curcumin is soluble in ethanol, and the optimal coloring temperature is 50 degree, so we adjusted the solvent to 33% ethanol solution. The process of coloring was conducted in the oven at 50 degrees, and the results showed that the coloring effect significantly improved.

Dyeing result of curcumin made in different solvents(30 min, 50°C). Left: water solution, Right: 33% ethanol alcohol solution

Indigo Problem: At the beginning of the experiment, we found that indigo could not be successfully adsorbed by the hair.

Solution: We looked up the data and found that in the printing and dyeing industry, it is necessary to restore the low solubility of indigo to water-soluble leucoindigo, which is used to color. We used the textile reducing agent - sodium sulfate to reduce indigo dye, and it came out to be successful.

Considering the safety of hair dyeing, we tried to use glucose as a reducing agent, by which we also obtained effective dyeing fluids. The method of using is indigo: glucose: caustic soda: 1:6:1, react for 15 min under 50°C. In the final product, we expected that yeast could conduct oxygen-free fermentation to reduce indigo with ethanol.

Problem: After dyeing your hair indigo, your hair becomes noticeably brittle.

Solution: Testing the average pull limit of a single hair, we found that the hair pull limit for indigo treatment was indeed significantly lower than that of normal hair. We assumed that this is related to the destruction of hair by alkaline substances. Therefore, we shorten the processing time of indigo dye, and finally designed to dye 2min at a time, repeated the procedure five times to achieve a saturation effect.

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Hair stress experiment

We used a micro scale experimental instrument to quantitatively evaluate the changes in mechanical properties of hair after hair dyeing. The results show that Indigo and lycopene as dyes have no obvious damage to hair. Curcumin has a repairing effect on hair.

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result3

After hair dyeing, the mechanical properties of hair in each group changed to varying degrees. For example, the hair softens after a long treatment of the indigo solution becomes soft, elastic but easy to break. So we borrowed the micro scale experimental instrument independently developed by Professor Liu.

During the pre-experiment, we found there is no significant difference in ultimate stress between traditional dyeing hair and the untreated one, and we believe it’s the added hair repair component causes the slug. Indigo and lycopene as dyes also have no obvious damage to hair. What’s more, the yang's modulus of curcumin treatment hair is significantly higher. So we believe it keeps hair tough.

Indigo dyed hair being treated with micro tensile / micro torsional mechanical tester

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Compound hair dye paste

After finishing the solution experiment, we try to mix the natural pigment into a dye that can be applied directly to the hair. At present, lycopene dye and curcumin dye with NO.1 cream matrix as carrier are obtained, and natural essence is added to improve the odor of dye paste. Indigo is an oxidizing dye with special properties, so we designed a timely fermenter. In this way, we can use our product right now when indigo is produced and reduced to indigo white.

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result4

Lycopene

Difficulty: when we use lycopene paste to dye hair, it is not red but orange

Solution: by analyzing the cream formula, we think what is causing this problem is sodium sulfite. We add sodium sulfite to prevent further oxidation of the pigment, but it may also reduce and fade the pigment. The solution experiment proved our conjecture. A new lycopene dye that doesn’t contain sodium sulfite found its way to red hair.

Lycopene dying cream

Ingredient	Content
Cream matrix	100g
Sodium sulfite	0.2g
Absolute ethanol	1mL
pH 6.8 phosphate buffer	1mL
Solid paraffin	1 drop or not
Essence	1 drop
20% Lycopene	5g
Alum	0.6g

Curcumin EDTA is added to the curcumin paste to prevent metal ions from interfering with the effect -- for example, Fe3 + producing a reddish-brown tint.

Curcumin dying cream

Ingredient	Content
Cream matrix	100g
EDTA	0.2g
Sodium sulfite	0.1g
Absolute ethanol	1mL
pH 6.8 phosphate buffer	1mL
Isopropyl alcohol	2mL
Solid paraffin	1 drop or not
Essence	1 drop
curcumin	0.5g

The result of curcumin staining cream. 9 ° from left to right. Hair was dyed at room temperature for 30min, 60min and 0min; Dyeing at 50 ℃ for 10min and 30min

Indigo:

Difficult: we can make indigo paste, but the hair does not dye well.

Solution: Indigo is a water-soluble component, and need to be oxidized to indigo after fixing to the hair. With water-in-oil paste as the matrix, indigo white can not fully enter the interior of the hair, and the oily substances in the matrix and excessive reductant prevent indigo white from oxidation in the hair, resulting in no effective coloring.

So we decided to design a timely manner in which indigo could be produced and used at the same time. Therefore, we consider that indigo dye can be produced and used in time -- the direct production of indigo by yeast, and the production of indigo solution as a dye in time.

For this idea, we dye indigo solution directly on the hair and find that it can be painted, but it can not color the hair evenly. So we designed a hair dye comb to make it possible to evenly smear indigo cryptosomes on the hair. The matching device is a timely fermentation tank, which can meet the needs of users with our engineering bacteria as raw materials, timely production and timely use of indigo white. For detailed information, please refer to Hardware part.

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Color fastness test

Color fastness is an important aspect to measure the effect of dye, so we design a set of elution scheme and test the color fastness of three kinds of natural pigment dye products and the same color traditional dye paste. The results showed that the color fastness of the natural pigment dyes was better than that of the traditional dyes.

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result5

The experiment of color fastness:

Compound 25g/L shampoo solution. Add 10 ML shampoo solution and a bunch of hair in a 15 ML centrifuge tube for 5 min at 100 r/min. The experiment was repeated seven times. The absorbance of the eluent at the maximum absorption band of the corresponding color was measured and the curve was drawn. Results as shown in figure, the single elution amount of three kinds of natural pigment was less than that of the same color traditional dyes, and the color change after 7 times elution was also less than that of the traditional dyes. Furthermore, curcumin dyeing cream treatment of hair is almost non-decolorization.

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Toxicological experiment of broad bean root tip

In order to test the safety of our dye paste, we carried out a toxicology experiment of broad bean root tip. The rate of micronucleus in root tip cells on broad bean was measured. The higher the micronucleus rate was, the higher the teratogenic rate was. The results showed that the teratogenicity of our product was significantly lower than that of the two traditional hair dyes on the market, indicating that our product was less toxic.

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Micronucleus (MCN) is an abnormal structure in eukaryotic cells, which is often produced by the action of radiation or chemical drugs. In the intercellular phase, the Micronuclei were round or oval, free from the main nucleus, the size should be less than 1/3 of the main nucleus. Only the eucaryotic test system can directly predict the genetic damage of mutagens to human beings and other higher organisms. In this respect, the micronucleus test is an ideal method. And some studies show that the plant micronucleus test and the animal test have a coincidence rate of up to 99%. Micronucleus test using broad bean root tip as experimental material can accurately show the effect of various treatments on induced distortion.

Specific experimental process:

1. We bought 2 catties of sun-dried broad bean seeds, put them in a wet porcelain tray after soaking in water overnight. After 2 days, about 95% of the bean seeds grew roots about 1-2 cm long.
2. We divided 10 broad beans into 1 group. Each group of root tips were immersed in the following concentrations of dye paste solution for 24 hours.
3. We took the broad beans soaked in the dye solution, washed them with distilled water, then put them into distilled water and cultured for 24 hours.
4. The root tips of Vicia Faba were cut off and fixed in 15-fold Kano Solution (anhydrous ethanol: Glacial Acetic acid = 3:1) for 24 hours.
5. We took out the root tip, washed it with distilled water, put it into 1 m hydrochloric acid solution, and reacted at 60 °C for 20 minutes to soften the root tip. The root tip was cut 1 mm, smashed on the slide, stained with the modified carbolic acid red dye for 15 min, covered with the slide, then flattened and observed under the microscope.
6. We first find the meristematic cells, which are closely arranged and square, look at 1000 cells, and record the number of cells with micronucleus. Micronucleus rate = micronucleus cell number/1000 × 1000%

Indigo	Traditional blue	Lycopene	Traditional red	curcumin	Negative control:Distilled water	Positive control:1g/L CuSO4
9g/L	9g/L	9g/L	9g/L	9g/L
6g/L	6g/L	6g/L	6g/L	6g/L
4.5g/L	4.5g/L	4.5g/L	4.5g/L	4.5g/L
3g/L	3g/L	3g/L	3g/L	3g/L
1.5g/L	1.5g/L	1.5g/L	1.5g/L	1.5g/L

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Due to the huge workload of counting micronucleus, we tried to develop counting algorithm. We use the algorithm framework of Yolov5 to implement what is available on the author’s Github or Model section, which is not covered here. We selected the typical normal nuclei and Micronuclei from the frame of the microscope. There were about 200 chromatin images and 50 micronuclei images in the normal nuclei. We trained a recognition model by using these images as training set. With this model, a large number of imaging results can be analyzed in a short time. We use this model to count micronuclei and measure the cytotoxicity of various substances. The results show that this model is in good agreement with the commonly used counting standards under the experimental conditions. It can be used to compare the difference in toxicity between different substances. For detailed information, please refer to Model part. #Color stability testing We would expose our hair dyed with our dye paste and two chemical dyes (red and blue) to constant bright light, take pictures before and after lighting, and calculate the degree of discoloration. The result shows that the light stability of our dye paste is slightly less than that of the chemical dye paste, but the difference is invisible to the naked eye in the actual application scene, so the color of our dye paste can exist stably in the natural light, it’s still very practical.

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result7

What we did: We cut a bunch of hair in half, pasted half of it onto a piece of cardboard, and exposed it to a constant, bright light for 11 days. Our lights are LED lights and sodium lamps, cycle for 22 hours of light and 2 hours of darkness alternately. At the end of the illumination, half of the hair without illumination was pasted on, and the fade index was analyzed by the algorithm. Considering the change of RGB values, it is difficult to determine the model parameters, and there may be multi-value based on the prediction results given by the neural network. We choose the linear combination of RGB to calculate the gray value, and take the gray value as the index of the concentration. It can be used as an index to evaluate the degree of fading by calculating the change of gray difference between the two groups. This indicator is not linear and therefore should not be understood as an indicator in a percentage sense. On a scale of one to one, the higher the score, the deeper the discoloration. There was no significant difference between indigo and chemical blue, the scores of indigo were 0.178 and 0.161, respectively. Chemical red faded more slowly than lycopene, with a score of 0.186 and 0.313, respectively.

The process of exploration: When we first carried out the strong light experiment, we irradiated single hair and only treated it for 2 days. The results showed that the dyeing of each hair in the same strand was very different due to the uneven dyeing, and the treatment time is so short that almost all the hair remains the same. In the second experiment, we took a photo of the hair in front of the light and compared it with the photo after the light exposure. The light exposure was extended to 7 days. Although the discoloration was detectable this time, it varied widely between the groups, suggesting that one hair’s discoloration was highly accidental. So on the third occasion, we decided to reduce the chance by increasing the amount of hair, using a single strand of hair, and extending the light again to get the difference.

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Color matching experiment and Sumu experiment

By adding color aids, mixing two dyes or secondary coloring, we get more hair colors. Now, we have different brightness of the rainbow seven colors and gray, red, brown, tawny and other colors. At the same time, we also used hematoxylin as a dye to explore a series of dyeing. Hematoxylin itself can be brown, add alum can be red, add alkali can be purple. Hematoxylin 's synthetic biology production will be a future extension project.

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A coloring list of the three pigments

Picture	Color	Coloring method
	Red	8 ° hair in the room temperature treated with lycopene for 30 minutes
	Orange	9 ° hair in the room temperature treated with curcumin for 30 minutes then treated with lycopene for another 30 minutes
	Light orange	9 ° hair in the room temperature treated with curcumin mixed with lycopene (2:1) for 30 minutes
	Yellow	9 ° hair in 50 ℃ treated with curcumin for 30 minutes
	Green	7 ° hair in the room temperature treated with indigo for 2 minutes
	Cyan	9 ° hair in the room temperature treated with indigo for 30 seconds
	Blue	9 ° hair in the room temperature treated with indigo for 2 minutes
	Purple	9 ° hair in the room temperature treated with indigo for 5 minutes then treated with lycopene for another 10 minutes
	Gray	9 ° hair in the room temperature treated with lycopene for 5 minutes then treated with indigo for another 30 seconds
	Reddish brown	6 ° hair in the room temperature treated with lycopene for 10 minutes
	Tan	9 ° hair in the room temperature added tannic acid treated with curcumin for 10 minutes

the coloring effect of hematoxylin under various conditions

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Experiment of short peptide perm

Short peptides perming hair experiment

We used 0.1% and 0.01% short peptides for perm experiment, and set chemical perm and up water perm as control group. The perming effect of short peptide is more obvious than that of ultra-pure water, which shows that in alkaline and 50° conditions, the hair cuticle is opened and the longer react time it takes, the better perming result we get. And after a series experiment, we found that hair dealt with SDS can have more obvious curl effect.

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result9

Chemical perm agent(bought online)	Ultra-pure water	0.001% short peptide solution	0.1% short peptide solution
500ul
Immerse hair(washed and dried) 20min

The experimental results showed that the Chemical perm agent had the most obvious effect, while others didn’t have any obvious perming effect. We’ve considered 3 reasons why short peptides have no obvious effect:

(1) Maybe the peptides didn’t get into hair cuticle

(2) The time for short peptides to form disulfide bonds wasn’t enough

(3) The reaction temperature is not enough.

So, we get another exploring experiment.

Time and Temperature:

（1）Drop a drop of 3M NaOH into 0.01% short peptides solution. [PH test paper: PH=10]

（2）Control experiment: drop a drop of 3M NaOH into UP water

（3）Both react at 50° for 1h.

Re-explore a better condition for short peptides’perming hair

1）immerse 10 hairs in SDS 3 times to remove charges on hair surface.

2）Wash and put them in 0.01% short peptides solution

3）Control experiment: Up water, the subsequent processing steps are the same as 1)2)

4）both:50°，1h

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Perming hair stress experiment-9

We used different kinds of perms for hairs with 2 groups of different hair, and the hair strains will be damaged after perming. So we designed and conducted stress experiments to test the mechanics of the hair after perming. The figure below shows the experiment data, by which we can know that perming by short peptides will have very little damage to the hair, and short peptide perm damage is less than chemical perm. Moreover, the results of the damaged hair groups showed that the damage of short peptide perm is much smaller than that of chemical perm. Hence, we speculated that perming procedure would cause some damage to hair, but the use of short peptide perm could be used to repair the damaged hair.

Figure I: Healthy hair stress test results (Horizontal: the deformation length; Ordinate: pull force)

It is shown that the hair of the short peptide perm group changes in shape under constantly increasing pull force, and most hair strains can withstand more pull than hair permed by chemical perms. This suggests that short peptide perms do less damage to hair performance than chemical perms. According to the experimental results, we judge that the concentration of short peptides has little effect on the results.

Figure II: Damaged hair stress test results（Horizontal：the maximum length; Ordinate: pull force）

This set of data comes from a pulling test of damaged hair. It is shown in this figure that both sets of hairs that use short peptides for perm can withstand more pull than the hairs after chemical perm. And there isn’t much difference between the results of short peptide group and that of the water group. We speculate that short peptides can by some means repair the damaged hair, which also portrays the excellent performance of short peptide perm that it can protect the hair and maintain the hair quality.

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