Measuring Volume Changes as a Result of Environment

By Gigante, B., Knaide, T. | Application Note

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Introduction

As the demand for high throughput drug screening increases, modern drug discovery and research labs are utilizing complex automation systems to assist in screening novel drug candidates. 96 and 384 well plates are routinely used to achieve the necessary throughput required. As sample density increases, humans become more removed from the screening process and proper sample care can occasionally be overlooked. One of the primary sources of random error can be attributed to environmental factors such as humidity and temperature within the laboratory. Sample plates are commonly unsealed or de-lidded and stacked within a carousel on a robotic platform or loaded onto the deck of a high speed liquid handling robot. In many cases, these plates are left exposed to the laboratory environment for hours at a time. Many experiments rely on accurate and precise volumes within the wells to ensure concentrations of samples are correct. In certain cases, the last plate processed on a robotic platform has been exposed to the environment much longer than the first plate. Depending on the nature of the solute, liquid samples can either evaporate or absorb water volume based on laboratory conditions. If absorption or evaporation is studied and understood, a scientist can take necessary precautions to reduce volume and concentration errors due to the laboratory environment.

Requirements

A quick and easy way to understand the severity of absorption and evaporation on plates is to monitor small volume changes over the course of a normal day using the VMS (Volume Measuring System) from Artel.  The VMS has the ability to quickly report individual volumes of sample in each well of 96 or 384 well plates.  The VMS uses a pressure based volume measurement that enables the scientist to get individual volumes of a 384 well plate in less than 2 minutes. The VMS can be integrated directly into an automated process and used as a volume sensor to monitor pipetting steps.  In the case of this tutorial, the VMS was used as a stand-alone device to obtain volume measurements from each of the test scenarios.

Materials

  1. Two 384 well polypropylene plates (Greiner 781201)
  2. One plastic plate cover (VWR 28317-488)
  3. Dimethyl Sulfoxide (DMSO – Sigma Aldrich 276855)
  4. Tissue Culture grade water (Sigma W3500)
  5. VMS-Volume Measuring System from Artel

Method

To demonstrate an application of the VMS technology, an experiment was conducted using two 384 well plates (Greiner 781201) to measure volume change due to environmental effects. Each plate was filled with 50µL of tissue culture grade water (Sigma W3500) and 50µL of Dimethyl Sulfoxide (DMSO, Sigma Aldrich 276855) using a calibrated hand pipettor as illustrated in Figure 1. The initial starting volumes of each well were measured by the VMS and recorded as Time 0.  Both plates were then left on the lab bench for the remainder of the day (8 hours). Plate 1 was left open to the environment while Plate 2 was protected using a plastic cover (VWR 28317-488).  Every hour the volume in each well of each plate was measured using the VMS (Time 1 through Time 8). The cover on Plate 2 was only removed briefly each hour to take measurements. The temperature and humidity of the laboratory were recorded for each time point.

Figure 1. 384-well plate map

Figure-1-384-well-plate-map

Results

Table 1 summarizes the results of the experiment.  The microliter values at each time point represent the average amount of water either absorbed or evaporated from each well at a given time point compared to the starting volume.  Positive values represent an increase in volume while negative values represent a loss of volume.  The starting volume of each well was approximately 50 µL.

Table 1. Measured volume change

Using the Artel VMS, small changes in volume were measured over the course of the 8-hour experiment as illustrated in Figure 1.  The water-filled wells in the covered plate lost an average of 4.2%, while the uncovered side lost 14.8%.  The covered, DMSO-filled side gained 3.2%, and the uncovered, DMSO-filled side gained 16.2%, due to the hygroscopic nature of the solvent.  There were also two other observations worth noting: The first is an obvious “edge-effect” on each side of the uncovered plate.  More water was lost around the edge wells than in the center – over 2%.  The second observation involved the interface columns between the DMSO and Water-filled wells (columns 12 and 13).  Average water gain in column 12 (DMSO) was 3% greater than the mean average of the entire DMSO side, while average water loss in column 13 (Water) was 5% greater than the mean average of the entire water side.  Interestingly, this effect was even more pronounced in the covered plate.  Column 12 (DMSO) lost 9% more volume while column 13 (Water) gained 9% more volume that the mean averages of their respective sides.   These observations are illustrated in Figure 3.  The proximity of the water to the DMSO appears to increase evaporation and absorption.   The plastic cover used during the experiment did not produce the airtight seal that a heat or adhesive seal provides.  Instead, the plastic cover seemed to provide a microenvironment wherein the DMSO in column 12 was able to act as a desiccating chamber for the water-filled wells in column 13.  Some condensation formed on the underside of the cover over the entire water section, but seemed to be more prevalent over the two middle columns.    This also seems to show that the proximity of the hygroscopic DMSO in column 12 to the water in column 13 affected the evaporation of the water.  The evaporated water did not all end up being absorbed by the DMSO; some also condensed on the cover.

Figure 2. Change in measured volume over time

Figure 3. Heat map of measured volume change

Conclusions

The laboratory environment can be a significant source of error when dealing with volume sensitive samples.  Evaporation in aqueous solutions due to dry conditions can cause increases in sample concentration.  Almost all assays performed within microplates are concentration-dependent, and microplate wells that have a higher volume due to water uptake will result in unknown compound concentration.  If you don’t know the concentration of your compound, you cannot draw an accurate conclusion about its activity or potency.1

Applying what was learned from this experiment, a starting solution of 10 millimolar dissolved in water would be over 12 millimolar by the end of the day if left open to the environment.  Samples stored in solvents that are hygroscopic (such as DMSO) can also absorb moisture from the air, causing a decrease in the final concentration which can lead to poor results.   One way to mitigate these risks is to have a controlled and monitored laboratory environment.  A good example is the case of a sample storage laboratory that maintains collections stored in DMSO.  Ideally, the laboratory should have a dry, stable environment.  The use of plate covers, adhesive seals, and heat seals are also ways to reduce the effects of environment.  This is not always possible when using robotic systems.  Proper protocol design and even environmental modifications such as an inert gas (Argon) blanket can be extremely useful when plates need to be exposed for long periods of time.  As observed by the experimental results, a lot can happen in 8 hours especially if precautions are not taken to protect your samples.


References

  1. http://www.artel-usa.com/understanding-dmso-hydration-in-stored-compounds/

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