Team:Paris Bettencourt/Notebook/Assay
Week 4th - 10th July
Press wax on cellulose paper
We used candle wax (vanilla scented) from Franprix. Wax was melted at 95 degrees. The 96 well plate template was used to create wax impressions on the cotton. Since wax is hydrophobic and the cotton we have in the lab is hydrophobic, it spreaded quickly, the diffusion was fast which makes the cotton unusable to carry out experiments or assay development.
Cellulose paper for chromatography was used to carry out further experiments for assay developement. The template dipped in melted wax is pressed against the paper for sometime. We created successful impressions of wax on the paper where the wax penetrates vertically into the paper and creating specific boundaries. At times, we had to reheat the template pressed against the paper to further improve the penetration (but no longer than 10 secs at 100degrees). We could successfully recreate the assay on the paper. The wells are not even given the impression and dipping the template were done manually.
Anthocyanin drops of volume 20ùl, 10ùl, 2ùl and 1ùl were used to test the wells. The Anthocyanin drops did not spread and they were contained in the wells. We think 10ul can be used to perform experiments on cellulose paper. For experiments on assay, we need cotton which is neutral in nature.
Week 18th -24th July
Pressing wax on cotton fabric
We are still continuing research on wax, we have sent emails to the Musée Grévin, wax specialist, and iMakr that seemed to have printed candles in the shape of celebrities.
Scandles, 3D printed candles
In the meantime, we used the CNC (Computer Numeric Control) on a block of wax we've found in the Openlab, C RI-Paris we work in, to make 96 holes. The aim is to heat it and press it against cotton fabric to create wax impression of wells in which we could pour liquid without having crosstalk between wells. We made the model in 3D for the CNC and it really worked well, wax is soft so it was easy but still quite long, approximately 3 hours. The diffusion of wax on chemically untreated cotton is very fast even though cotton is hydrophilic and wax is hydrophobic.
A mould to pour wax on frabric
We also decided to make a mould to poor wax in so that it would create circles of wax on cotton. We created a mould in solidworks to 3D print it. We first did it in one part and the 3D print was not possible because some parts didn't have support and hangings are not supported by 3D printer. Therefore, we then divided it into two parts that we 3D printed separately. The way 3D printing works is lateral deposition. It is does not have longitudinal strength. The pillars in the model were very tall, and consequently very fragile, two of them broke easily as one of our teammate played with it. The base of the pillars against which we press the fabric are not uniform. Therefore, we cannot get uniform wells and avoid crosstalk when we pour wax through the mold.
Week 25th -31th July
This week we had a good talk with our advisor, Edwin, and realised together it would be much more interesting if we created an actual 96-well plate, that would be cheap and standardised to test products directly on fabric. So we are going to change our way of thinking from 2D to 3D.
Using 3D printed wax on fabric
First we still are looking options with wax as we saw wax can be 3D printed, indeed we've found a wax filament. To know more about it we've planned a skype call with Arthur Dalaise, one of our teammate's cousin that is founding a 3D printer service startup. The good news is that he agreed to try out printing with this wax filament for free and gave us really good advice !
Our only concern with 3D printing is that we don't know if wax will stick to the fabric, not burn it and make real wells that don't allow liquid to spread.
Using PDMS to create our own 96-well plate
Teja had a revelation and thought of using PDMS, a silicone-based polymer, to create a 96-well plate where we would sandwich cotton fabric between two layers of PDMS with holes. The bottom side of PDMS is then bonded to glass using a plasma cleaner so that the fabric can be scanned using a flat bed scanner.
Our first try shown in the above figure looked promising. The top layer of PDMS is prepared with 10:1 ratio of silicone polymer to curing agent. The bottom layer of PDMS is prepared with 5:1 ratio of silicone polymer to curing agent. Once the mixture is prepared, it is degassed twice to remove bubbles and then heated at 75 degrees for two hours in a convective heater for curing. PDMS is punched to create the wells using a 6mm diameter puncher. The punching went well but not the spacing between the wells. Manual punching resulted in non uniform spacing. The bottom layer of PDMS is first bonded to the glass using a plasma cleaner. A small layer of 1:5 mixture that was first prepared is applied to the bottom layer of PDMS. A The cotton fabric is then sandwiched between two layers of PDMS with holes. The alignment of holes is not precise with the fabric sandwiched between PDMS layers. Then the whole sandwich is heated at 75 degrees for one hours in a convective heater for curing. Although there is a strong bond between cotton and PDMS, the PDMS got diffused into the wells which hinders the use of fabric in the microplate. We are in pursuit of finding new ways to have a precise result.
A 3D printed mould for PDMS
We decided to 3D print a mould with pillars to pour PDMS on it and have a layer of PDMS with holes in it. The pillars are 1cm high but half of it is slightly conic so that we can remove the PDMS easier. We use microbot 2x 3D printer to print our Moulds. The temperature of the platform of 3D printer is 120 degrees and the temperature of the extruder is 220. We used ABL filament for 3D printing.
So this mould didn't work as well as planned ... The PDMS layer really hard to remove from the mould and we broke it and the mould while removing it. But the wells created on the parts that were not broken are really nice so we will do another try with a different mould with pillars that won't be as high.
Week 1st - 7th August
A new try for PDMS' 3D printed mould
On this picture you can see our new 3D model with smaller pillars the total height is around 6mm. The perfect cylindrical part is 6mm in diameter and 2mm in height. The height of the conical part is 4mm and the top diameter is 4mm. We are very confident with this model.
And this is the result of the 3D printing that went really well this time, we did a 80% infill so that the pillars don't break this time. The result of the PDMS is on the left, it is really what we expected and we are really happy with this. We created a 96 well PDMS!!
Lasercutting Cotton Fabric
We also decided to laser-cut cotton, our research on internet have shown it is already done and seems very efficient. We want to have circles of cotton that are linked so we don't have to put each circles of cotton into each wells. It worked really well and we managed to lasercut multiple layers at the same time (around 8).
The borders of the lasercut cotton is then a bit burnt, which was totally expected but we fear it may interfere so we just soaked it in water and it disapeared instantly but the fabric shreds slightly after being wet.
Combining the different layers
The cotton seems to fit really well, but we tried to stick the PDMS, cotton and glass layers on a small scale and it was not very good yet, the liquid spreads because the cotton is creating a small gap between the glass and the PDMS.
3D printed wax on cotton
Thanks to Arthur Dalaise who helped us we have a functional assay using 3D printed wax filament on cotton. The first layers were printed with heated platform (100°C) with a 190°C temperature for the extruder and then the sheet of fabric was laid on, and finally the rest of the layers were printed on top of this with a unheated platform and a very hot extruder (240°C). The first step lasts for 30 mins and the second one for 6 hours. So the result is exactly what we want but the time it takes is really too long, so we will still continue the PDMS experiments.
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