Re: [DIYbio] Plant Protoplast Microfluidics

Oh, I found this too, which shows printing toner on transparency
plastic then embossing with PDMS can achieve 6-12 microns (STL, single
tone layer or DTL, double tone layer)

Toner and paper-based fabrication techniques for microfluidic
applications (Review)
http://diyhpl.us/~nmz787/pdf/Toner_and_paper-based_fabrication_techniques_for_microfluidic_applications.pdf

I guess the link (in my previous email) was on toner-transfer with
etching, so there
was also diffusion going on in that to reduce resolution.

There's this too (which is ref 17 of the last link)
http://diyhpl.us/~bryan/papers2/microfluidics/Rapid%20fabrication%20of%20microfluidic%20devices%20in%20poly(dimethylsiloxane)%20by%20photocopying.pdf

and ref 18 (which is where it get's the STL and DTL data)
http://diyhpl.us/~bryan/papers2/microfluidics/A%20Dry%20Process%20for%20Production%20of%20Microfluidic%20Devices%20Based%20on%20the%20Lamination%20of%20Laser-Printed%20Polyester%20Films.pdf

So seems like your idea is feasible and is worth finding someone with
a laser printer and laminator!



On Thu, Dec 5, 2013 at 6:49 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
> On Thu, Dec 5, 2013 at 11:13 AM, Sebastian Cocioba <scocioba@gmail.com> wrote:
>> Hi Everyone!
>>
>> I've been toying with some designs for a protoplast culture and PEG
>> transformation chip and had a few questions:
>>
>> 1. Some of the only papers citing tobacco protoplast cultures and
>> microfluidics have a design that consists of a wide channel which at one end
>> have a sieve of posts a few microns in diameter and a few micros apart. This
>> collects the cells, which range from 40-70um while replacing the media. This
>> would cause an aggregate as the sieve fills and would damage cells...i
>> think.
>
> Maybe, how did the paper(s) get around aggregation and/or damage?
> Simply not loading so many cells so an aggregate won't be possible?
>
>> Do you think an laser printer set to 1200 DPI can print a grid 20-30
>> microns apart? The post size won't matter as long as the sieving space is
>> tight.
>
> First some basic arithmetic, let's say 1 inch is 25400 microns, then
> 25400/1200 = 21.16666
>
> So that's 1 dot per 21.66 microns.
>
> Then you have to figure out your minimum and maximum dot sizes. For a
> laser printer this is the toner particle size, from wiki:
> "Originally, the particle size of toner averaged 14–16 micrometres[1]
> or greater. To improve image resolution, particle size was reduced,
> eventually reaching about 8–10 micrometers for 600 dots per inch
> resolution."
> and
> "Toner has traditionally been made by compounding the ingredients and
> creating a slab which was broken or pelletized, then turned into a
> fine powder with a controlled particle size range by air jet milling.
> This process results in toner granules with varying sizes and
> aspherical shapes. To get a finer print, some companies are using a
> chemical process to grow toner particles from molecular reagents. This
> results in more uniform size and shapes of toner particles. The
> smaller, uniform shapes permit more accurate colour reproduction and
> more efficient toner use."
>
> But there are other factors that limit the resolution: how many
> particles the printer calls 'light' or 'dark' in its image settings;
> how light or dark the pixel is in the data being sent to the printer;
> the odds that you can actually place (1) 5 micron particle in the 21.6
> micron pixel; are the particles round, oblong, random spiky looking
> crystals?
>
> See one method here:
> Ohttp://diyhpl.us/~nmz787/pdf/Rapid_and_inexpensive_fabrication_of_polymeric_microfluidic_devices_via_toner_transfer_masking.pdf
> http://diyhpl.us/~nmz787/pdf/Rapid_and_inexpensive_fabrication_of_polymeric_microfluidic_devices_via_toner_transfer_masking__Supplement.pdf
>
>>
>> 2. How is oxygenation and media recirculation done in bacterial culture
>> chips? Do they just circulate the media without losing cells somehow?
>
> You could do that, with say a nanoporous membrane:
> http://www.simpore.com/products.html
>
> or maybe a decent field of posts to block cells if they're big enough,
> otherwise I've seen the gas permeability of PDMS used, so it would be
> a parallel-plate membrane setup, cross-flow counter-flow or cocurrent
> flow:
> https://www.housing.ou.edu/content/dam/CoE/CBME/Undergraduate_Lab/CHE3432/PDF_files/Chapter13MembraneSeparation.pdf
>
> A practical guide to microfluidic perfusion culture of adherent mammalian cells
> http://www.rle.mit.edu/biomicro/documents/lykim_LOC2007.pdf
>
>> 3. Lets say I get protoplasts to make microcolonies, how (aside from cutting
>> open the PDMS culture chamber) would I get them out to replate?
>
> Open a big valve that was previously shut? Backflush?
>
>> The whole idea of this chip is to repeatedly perform PEG fusions and naked
>> plasmid uptake experiments on protoplasts and regenerate them. There is a
>> lot of manual skill and tricks involved in PEG transformation so I want to
>> take luck and dexterity out of the equation. Any advice would be fantastic.
>
> Get a 1080p webcam fitted to your microscope. Logitech makes the C615
> which is ~$50, any dSLR that feature electronic shutters (aka 'full HD
> video', where the physical shutter doesn't actuate) would be even
> better. Connect them to a laptop or raspberryPi (since you need some
> GPIO anyway for controlling valves and pumps and lights/filterwheels,
> etc), then you can at least monitor your cells easily and record
> images/videos, and later maybe start to automate with machine vision
> (i.e. rinse with PEG until cells reach detected diameter).
>
>> 4. Know any literature on standardized or good design practices for general
>> microfluidics fabrication? I'm rockin PDMS-glass chips made using shrinky
>> dinks and if it works Polyolefin shrink wrap chips too. I found a book that
>> covers all the theory and fluid dynamics behind the curtain which is great
>> but little on channel layout theory/advice/style.
>
> These are pretty good:
> www.kni.caltech.edu/foundry/basic_rules.html
>
>> 5. Has anyone made valves using the shrinky dink method? The channels end up
>> rounded which isn't the ideal geometry for the control layer.
>
> The toner-transfer paper I linked earlier says that the horizontal is
> traded off for vertical, so channel volume remains constant.



--
-Nathan

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