Hi everyone!
It is time for me to introduce to you the new DropArrayTM platform that my company has developed.
DropArrayTM is a platform aimed to rival the 96-well plate. The 96-well plate is the most common platform used to run assays, especially those that require numerous washing steps. One example of such assays is Enzyme-Linked Immunosorbent Assay (ELISA).
However, running assays on a 96-well plate has been shown to consume relatively large amounts of reagents, samples, and assay time. As for the washing steps, there is the risk of cross-contamination and/or carry-over of liquids across the different wells.
This new platform has been designed and developed to overcome the problems mentioned above.
I will briefly describe the components of the DropArrayTM that I most oftenly use:
S48/S96 Plate
The plates are actually microscope glass slides patterned with Teflon coat. The coat acts as a hydrophobic surface. The S48 plate has 48 wells (12x4), and the S96, 96 wells (12x8). The diameter of the wells is 2mm.
LT100 Wash Station
This station comprises of a chamber and control buttons to set the speed, duration and volume needed for washing. There is an inlet tubing to allow wash buffers to flow into the washing chamber, and an outlet tubing to drain it out of the chamber. The plate is loaded into the chamber and the washing (or rinsing) process can then start: drain any oil > fill chamber with wash buffer > shake > drain wash buffer.
The shaking step is to remove any media or liquid from the wells.
Incubation Oil
For cell seeding. After the cells are seeded into the wells, the wells are covered with incubation oil to prevent evaporation of media during the incubation period inside the CO2 incubator.
Rinsing Oil
Rinsing oil also prevents total evaporation of liquid from the wells when running the assay. What makes the rinsing oil different from the incubation oil is that the former has lower boiling point. So, the incubation oil is more suitable for the CO2 incubator environment. The rinsing oil is also applied to prevent carry-over between wash steps.
There are few other components, such as: Plate Adapter, Dispensing Guide, Tip Cassette, and Lid. But since I seldom/do not use them, I will not explain them here. Feel free to click here if you would like to know more.
The DropArrayTM platform is also used for cell-based assays. I will describe one such assay that I had done in week 9 in the next entry. So, stay tuned!
Nor Liyana
0607927A
TG 02- Group 8
Week 10 sharings
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12 comments:
September 1, 2008 at 10:27 PM
Hi Liyana,
I have some questions for you,
1)Regarding the S48/S96 plate, what is the significance of having the coat being hydrophobic?
2)What is the composition of the incubation oil? Will it affect the composition of cell media and subsequently affect cell growth?
3)How does the rinsing oil prevent carry-over between wash steps?
4)What are other examples of assays (besides ELISA) which consume relatively large amounts of reagents, samples, and assay time?
5)How does the new invention (DropArray™) prevent or minimize the above (qn 4) downsides of assays performed with conventional 96-well plates?
Thankz!
Han Yang
TG01
September 6, 2008 at 1:07 AM
hey hey
regarding ur incubation/rinsing oil, won't it affect ur sample? is there any other alternatives, say example, using a cover instead of incubation oil to prevent the evaporation of the media?
thx thx
=)
Mayafirhana
TG02
September 7, 2008 at 3:17 PM
hello liy! i hope youre doing well =)
haven't been bumping into you ehh. haha anyway, what is a teflon coat? how does it provide a hydrophobic effect?
thanks!
Nurathirah
0606561I
September 7, 2008 at 10:59 PM
Hello Liyana
For this machine, are there any quality check done? How often?
Is the components in the rinsing oil same with that in the incubation oil?
Thanks!!
Amir
TG02
September 8, 2008 at 12:11 AM
Hello Liyana
Both the incubation and rinsing oil need to be included in the test? or just one of it will do?
Thanks!
LeeJin
TG02
September 11, 2008 at 10:19 AM
hey Liyana,
during the draining of the chamber via the outle tubing, is there any risk of draining out the actual sample as well? and during the shaking at the wash station, isn't it also causing the wells to be susceptible to cross contamination? or is there any special way of doing it? and lastly, whats the use of the coat being hydrophobic and not hydrophilic? actually i have more questions to ask you, but HY has asked most of it. haha. Thanks. =)
Debbie
TG02
September 15, 2008 at 12:45 AM
to Han Yang:
1. the hydrophobic coat is like a wall that separates the wells. unlike the conventional 96-well plate, the S48/S96 plate is just a flat surface.
imagine, if there is no coat, then the plate is just like a normal microscope slide; your dispensed sample will be all over the slide.
water is the most common solvent for any reagent/media/samples, so the small round surfaces of the slide that are exposed (i.e. not covered with the Teflon coat) are where the droplets of sample will be. water will not stay on hydrophobic surface, rather it will 'be attracted and move' to the hydrophilic surface.
2. i do not know the composition of the incubation oil. it is supposed to be proprietary. so yeah, trade secret!
and no, it will not affect the cell media or cell growth.
Come to think of it, the cells are not in contact with the oil, because the cells are attached to the bottom of the hydrophilic well, and covered with medium.
i will post some pictures next time to give you and our friends a better idea =)
3. i will get back to you regarding how rinsing oil prevents carry-over.
4. two examples that i found: Human Leptin Receptor
Enzyme Immunoassay (http://www.caymanchem.com/pdfs/10007608.pdf) and Functional Immunoassay Technology (FITTM)
Glomerular Filtration Rate (GFR) (http://www.biopal.com/images/FIT-GFR%20Gd-DTPA%20kit%20manual.pdf)
my company is focusing on the ELISA market. apparently, there are many kinds of tests using the ELISA method, such as Nucleosome ELISA, Fas Ligand ELISA and Bcl-2 ELISA (http://www.merckbiosciences.co.uk/html/cbc/kitresource_apoptosis_activity_detection_kits.htm).
5. one thing for sure, the DropArray plate uses only 2-3 ul of sample/reagents per well, as compared to 96-well plate (usually 100ul per well for ELISA). when you have lower amount of sample/reagent, you also need lesser time to wait for the reaction to occur (think incubation times). also, on normal 96-well plate, one washing step usually means washing 3-5 times (i.e. 3-5 washes per wash step). the LT100 wash station allows just one-time wash per wash step.
Nor Liyana
0607927A
September 15, 2008 at 1:25 AM
to Maya:
No, the rinsing oil will not affect the sample.
the incubation oil floods the plate, i.e. it immersing all wells, all droplets. so, this is like a 'full protection' against evaporation of liquid while inside the incubator.
using a cover, this is more like a secondary protection (the S48/S96 plate does have a lid). the thing is, even after covering the wells with the cover, there is still gap/opening at the sides. so yeah, full immersion is better. the oil can be recycled too.
September 15, 2008 at 9:27 AM
Teflon is a trademark brand from DuPont. it is actually polytetrafluoroethene or polytetrafluoroethylene (PTFE), a synthetic fluoropolymer.
(fluoropolymer is a polymer that contains fluorine)
PTFE is hydrophobic, so water and water containing subtances do not stick to it. one familiar example is the inside surface of our cooking pans. if you put water, you notice that the surface is not wet. the water easily slides off the pan.
PTFE is resistant to chemical and can withstand high temperature (melting point is 327 degree Celcius). it is also non-reactive, which means, it can be used with any chemicals without resulting in corrosive or harmful byproducts that affect the processes.
i recommend you this webpage to understand more on the properties of PTFE: http://www2.dupont.com/Teflon_Industrial/en_US/uses_apps/pharmaceutical/index.html
hope this information helps. oh, and PTFE was found by accident =) http://en.wikipedia.org/wiki/PTFE
Nor Liyana
0607927A
September 15, 2008 at 9:46 AM
to Amir:
the DropArray platform is still new (my company started in April this year). so, we are still doing QC tests and optimisation on the platform.
a colleague of mine runs ELISA tests on the platform, while my supervisor and i do cell -seeding and -assay(s). other than that, what i had done so far are 'dispensing tests' and 'rinsing tests'.
examples of optimisation: we want to optimise the design of the wash station or even the lid for the plate, or optimise the parameters for washing steps in cell assays (e.g. we do not want the cells to be washed off due to high shaking speed), or find ways to automate the whole platform.
(currently, only the washing step is automated)
oh, and i just got to know (like, few minutes ago) that the composition of the two oils are the same. but they have different viscosity and boiling points.
Nor Liyana
0607927A
September 15, 2008 at 10:20 AM
to Lee Jin:
the incubation oil used for cell-assays only. the first step before doing a cell assay is to seed the cells onto the wells of the S48 or S96 plate. then, incubate the cells in the CO2 cylinder for several hours, depending on the requirements stated in the assay protocol.
the sample used is very little, 2-3ul per well, so it will quickly evaporate if exposed to the 37 degree Celcius. thus, the incubation oil (with its relatively higher viscosity and boiling point as compared to the rinsing oil) serves to prevent evaporation of medium during the incubation period. the oil is dispensed onto the plate, immersing the droplets of sample.
after, say, 24 hours of incubation, the incubation oil will be drained off from the plate and replaced with rinsing oil.
The rinsing oil is used to prevent evaporation of reagents/media for a short period of time (minutes). after every washing step, i put 0.6ml of the oil on the plate. then, i dispense the required reagents onto the wells.
basically, the rinsing oil is used in both cell assays and ELISA.
Nor Liyana
0607927A
September 15, 2008 at 3:16 PM
to Debbie:
for cell-based assay, the cells will not be drained off in the draining step. this is because the cells are already attached to the surface of the glass slide. same goes to the antibodies/proteins in ELISA. they are already bound to the surface, so they will not be washed away.
basically, samples will stay where they are (in the wells) while the oil or wash buffer is drained (since the Teflon coat is hydrophobic, the oil/wash buffer simply slides off the plate). but, if you flick or shake the plate hard, the droplets of liquid/reagent will come off.
if the sample/reagents are the same across all wells, then there is no need to worry about cross-contamination.
if they are different, e.g. different concentration of a particular reagent in each well, then 2 factors are considered: 1. the dilution effect - the volume of the reagent/medium is only 2-3ul per well. when the wash buffer is added, that volume becomes diluted many times. so, the chance of cross-contamination is greatly reduced. the 2nd factor also helps: duration of shaking - i cannot disclose how long it is, but it is too quick for any reaction or adhesion to happen.
the coat acts like a wall, to separate the samples.
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