Dispensing Aqueous Solutions with the ATS
An acoustic beam is focused on the surface of a liquid and the reflection of that energy causes the surface to rise. As more energy is applied the surface rises enough so that the surface wraps around a bolus of liquid and then momentum takes over allowing a droplet to form and be projected out of the surface. It is important to note here that the surface of the liquid pulls back on the droplet as it moves. And, most importantly, if the trajectory is not perpendicular to the surface the droplet will be pulled in the direction of the smallest angle with the surface.
The typical shape of this meniscus resembles a bowl, higher on the edges and lower in the center. Lower surface tension will allow this bowl to become deeper. And, how the liquid clings to the walls of the wellplate will influence the shape of this bowl. The optimal shape of the meniscus is for the edge of the fluid to cling at the same height along the well wall continuously around the entire well. In this case the lowest point of the meniscus should coincide with the center of the well. Therefore, aligning with the center of the well would also allow the acoustic beam to generate a droplet perpendicular to the surface, and therefore straight out of the well.
A hydrophobic surface is one where the contact angle for a drop of water on the surface is greater than 90 degrees. A hydrophilic surface is defined as a surface in which the contact angle for a drop of water on the surface is less than 90 degrees. There are many ways in which a surface may be modified to change the contact angle for a drop of water. However, for wellplates that are being used to contain chemically reactant solutions adding chemicals to the surface will most likely contaminate any chemical reactions intended to be performed with these compounds.
This is the typical surface of a wellplate. This hydrophobicity causes water to “stick” on the walls of a well creating a non-uniform edge of the liquid and likely offsetting the meniscus from the center of the well.
This is because the micro pattern is a groove in which water is attracted by capillary action. Once the surface is wetted, the surface contact angle is lowered. For wellplates this micro patterning may be created by exposing the plastic surface to plasma. This plasma etches tiny grooves in the surface that the water is attracted to.
The first which has been discussed is the meniscus. As the liquid drains then the surface of the liquid slides down the walls of the well. In figure 1 liquid is continuously dispensed from the same well as the plate is moved. The time sequence is from top to bottom and from right to left. So, the second consideration is that each 50nl dispense consists of 5 10nl droplets dispensed on each other. Even though the meniscus is gradually moving and creating a fairly large displacement from the center of the grid the series of droplets are all dispensed from approximately the same meniscus shape. Therefore, the surface shape does not change very fast.
Since the meniscus does not change very fast and using treated plates minimize how much the meniscus changes there is one more important consideration when dispensing aqueous solutions and that is the initial condition of the filled plate. It is important to consider how all of the wells can be made to have the same meniscus shape. One might assume that the centrifuge is a good method to create a uniform meniscus shape, but unless your centrifuge has more than a meter radius you will quickly notice that the wells in the center will have a more centered meniscus than the wells further from the center of the plate. This is due to the actual vector force of the centrifuge pushing the meniscus further away from the center. This effect will be more dramatic in untreated plates, but it is also seen in treated plates. So, after using the centrifuge it is recommended to use a vortexer at a reasonable speed in order to allow the surface to find a more natural and centered location. By performing this action on the plate it alleviates the non-uniform menisci created by the dispensing or centrifuging of the wellplate.
The key to this application is the use of plasma treated plates. In this particular application the Greiner 788 890 wellplate was used. This is a low profile, low volume, plasma treated 384 plate that allows the fluid surface to be very close to the arraying surface. This plate happens to be a COP plate, but the Greiner 781 091 cell culture plate is a plasma treated polystyrene plate used to illustrate the drop placement in figure 1.
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