One of the most desired aspects of a liquid handling platform is minimizing the liquid dead volume, especially when it comes to valuable scarce samples or expensive reagents.
Reducing the dead volume can save a considerable amount of costs that are otherwise spent on samples and reagents and can also provide more number of assays for each unit volume, thereby further reducing the associated labor costs and enabling more data for each cycle.
This article shows how Fluent Laboratory Automation Solutions integrated with Adaptive Signal Technology™ (AST) advanced liquid level detection are extremely useful in reducing dead volume without affecting the accuracy or precision of liquid transfers. The article also describes how new capabilities in Fluent enable more of the liquid volume to ‘stay alive’.
What is dead volume?
In this context, the term ‘dead’ indicates ‘unusable’ and is typically referred to the residual volume of liquid in the source labware following the transfer of liquid. Moreover, it cannot be shifted to the destination labware without the inherent danger of partial air aspiration. A rule of thumb is that in order to ensure accurate transfer, a small amount of surplus liquid is needed in all types of labware.
Tilting the tube is one way to minimize the dead volume; however, this poses some restrictions in the case of a vertical pipette. Aspirating the liquid at a specified height without liquid level detection is another way to reduce the dead volume, but this is feasible only when all the wells had the same known volume of liquid.
The role of liquid level detection
Liquid level detection is a vital process control function in any automated liquid handling system. It occurs in the source labware before the initiation of liquid transfer. The volume available is measured automatically and then evaluated against the volume needed for the transfer.
As soon as the run is initiated, the programmed available volume threshold is what governs the decision on whether aspiration of the preferred liquid volume has to be continued or not. In case, the volume identified is less than this threshold, then a ‘not enough liquid’ alert is produced, and aspiration is eventually stopped. Therefore, the liquid remaining in the source labware turns into a dead volume.
The needed volume threshold in a standard system is set to comprise of a large security margin, which is governed by the tolerances in the surface tension, labware geometry, and tolerances of the liquid level detection and movement and positioning of the pipetting tip. The function of this security margin is to make sure that even in the worst situation partial air aspiration and liquid shortage do not occur.
The tip submerges to a programmed depth as soon as the liquid is identified, and when the tip aspirates, the liquid level goes back while the submerge depth continues to remain stable. This process ensures that the tip is exposed only to minimum contamination without any inherent risk of tip dip-out occurring during aspiration.
Here, the submerge depth’s dead volume serves as a liquid volume reserve when the tip aspirates down to z-max to compensate for the tolerance of the liquid level detection. Figure 1 shows a visual representation of the dead volume.
Figure 1. Visual representation of the dead volume
How much dead volume is there?
One way to determine the dead volume is to use a known liquid volume (100 μl) and introduce it into a sample tube, followed by setting the liquid handling system to transport a small quantity (10 μl) into another tube numerous times.
A ‘not enough liquid’ alert then emerges following a specific number of aspiration and dispense cycles. At this point, the liquid remaining in the sample tube represents the dead volume. In this case, if a liquid handling system with standard liquid level detection is used, approximately 60 μl continues to remain in a 1.5 ml microtube.
The same method can be employed to assess the dead volume in a reagent reservoir where at least 20 ml is needed to span the ridged base of a low dead volume reservoir for accurate aspiration. Moreover, in the case of multi-dispensing modes, a surplus volume is aspirated together with the transfer volume and then subsequently discarded.
This generates the same pipette tip conditions for each aliquot, right from the beginning to the end. This extra dead volume is not dependent of the liquid level detection and is needed to counteract the impact of the hydrostatic pressure on the tip.
Therefore, if there are concerns regarding the dead volume, opting for a single dispense mode is better than settling for multidispensing. Though pipetting will be slower, the amount of a single aliquot will be saved for each aspiration into the tip.
What is the solution to reducing the dead volume?
Dead volume always happens to be a part of the liquid handling system owing to many tolerance factors that affect liquid detection as well as the associated volume measurement. Conversely, AST can be used to reduce these tolerances. It is an advanced liquid level detection integrated into the Fluent Laboratory Automation Solutions.
Three key attributes for reducing the dead volume
AST offers the following advantages for reducing dead volume.
Sensitivity of the liquid level detection
Very low volumes as small as 2 μl in a 96-well PCR plate and10 μl in a 1.5 ml microfuge tube can be reliably detected with AST. However, a standard liquid level detection technology will fail to identify less than 45 μl in a 96-well PCR plate. This high level of sensitivity proves more advantageous in reducing the dead volume whenever extremely small volumes are transferred from incredibly low liquid levels.
Accurate volume determination
A labware library is included in the FluentControl™ application software with correct definitions of well/tube, thus facilitating accurate measurement of the volume content on the basis of the detected liquid level (Figure 2).
Additionally, with the help of the labware editor in the FluentControl™, users can easily define the shape of plate wells and tubes that are not part of the library. In certain cases, labware manufacturers may provide drawings with true dimensions.
Figure 2. Accurate volume determination
In the case of complicated labware shapes, rather than using the default calculation, a dual-column table that provides the detected liquid level and the associated detected volume can be used.
Together with Fluent’s reduced tolerance stack, the accurate measurement of volume content provides the required volume threshold for setting the liquid detection lower than is actually feasible.
Highly repeatable liquid level detection
The successive detection of the liquid level in the same well was used to assess the repeatability of liquid level detection in FluentControl™. The detection was performed six times using all the eight channels with a 10 μl disposable tip for individual detections in a trough; +/-0.2 mm was the highest deviation to be observed. This is indeed extraordinary considering the fact that performance solely depends on the following factors:
- Flatness of the labware
- Flatness of the nest and segment accommodating the labware
- Precision of the tip positioning of the Flexible Channel Arm
- Exact fit of the disposable tip on the tip adapter
In addition, the ensuing deviation of the detected volume relies on the labware diameter at the defined detection level: +/-4 μl for of 2.5 mm diameter or +/-16 μl for 10 mm diameter in the tapered part of a microtube. The following benefits are realized from the excellent repeatability of the liquid level detection:
- It is possible to set the required volume threshold for liquid detection lower than usually possible
- It is also possible to set the tip movement end point proximal to the well bottom without the inherent risk of pushing the tip against the bottom of the well. Such a condition would result in inadequate volume transfer. If labware geometry tolerances allow, it is possible to set the clearance down to 0.3 mm while at least 1 mm clearance is proposed for the majority of other liquid handling platforms. This is quite advantageous when transferring exceptionally small volumes from low liquid levels.
Managing dead volumes is a simple matter with fluent
There are five steps to reduce the dead volume. In FluentControl™, the default settings are configured to give reliable security against air aspiration for a wide range of liquids.
If there is a concern regarding the dead volume, then the settings can be improved for a certain type of liquid, labware, and volume of interest. Figure 3 shows the various steps for managing dead volumes.
Figure 3. Simple steps for managing dead volumes
Achieving a low dead volume
For instance, if 10 μl of liquid has to be transferred from the source labware to the destination labware, it is not known how much excess volume is actually required in the source labware to realize precise volume transfer, without activating ‘not enough liquid’ alerts. This is because such alerts will tend to obstruct the run.
Required transfer volume (μl)
Volume to be dispensed in the destination labware.
Detected volume range (μl)
Volume range measured experimentally for a defined volume present in the source well between the minimum and the maximum detected volume.
Needed volume threshold (μl)
Recommended threshold setting to ensure that, even if the detected volume is higher than the actual volume present in the well, there is enough liquid in the well for accurate transfer of the required transfer volume.
Minimum volume in source (μl)
Recommended volume that needs to be physically present in the source well to ensure that, even if the detected volume is lower than the actual volume present in the well, it will still be above the needed volume threshold, ensuring that ‘not enough liquid’ exceptions do not occur during the run.
Dead volume per well (μl)
Excess volume required in the source well, in addition to the required transfer volume.
Determining the minimum volume in source well
In this example, the aim was to measure the least volume in source well of a 96-well PCR plate in order to guarantee accurate transfer of 10 μl tap water through the use of 0 μl disposable tips.
1) A colorimetric method was employed to experimentally measure the minimum volume required in the source for a precise, accurate required transfer volume of 10 μl, using a range of known volumes.
The least amount of volume required in this case was found to be 12.5 μl. This condition is due to a thin layer of liquid that remains in the tip following dispensing. The FluentControl liquid class has a programmed correction that compensates the volume lost in the tip with a slight over-aspiration.
2) The detected volume ranges were determined experimentally for volumes – 12.5, 15, 17.5 and 20 μl – in the wells of a 96-well PCR plate.
In Figure 4, the vertical bars spanning from the least to the highest detected volumes indicate the detected volume ranges acquired in the experiment.
Figure 4. Detected volume ranges obtained in the experiment.
3) The needed volume threshold must ensure that at least 12.5 μl is always present in the well.
In this case, the series of liquid level values acquired in wells packed with 12.5 μl spanned from 9.4 to 15.6 μl. Therefore, the needed volume threshold was eventually set to 15.7 μl, as indicated with a red circle.
4) The minimum volume in the source must ensure that the volume determined during the run is always above the needed volume threshold.
When transferring samples, at least 20 μl is required in the source to ensure that the identified volume always remains above 15.7 μl. This probably ensures that the run is not disturbed with ‘not enough liquid’ exceptions, and as a result aspiration can always be continued.
For reagent transfer, consecutive aspirations from the same vial are employed frequently, and ‘not enough liquid’ exceptions are handled automatically by turning to a ‘refill’ prompt or the subsequent vial. In this manner, the vial will have just 12.5 to 19.9 μl.
5) Conclusion for the maximum dead volume
In this case, 10 μl was the highest dead volume for each well and is less than one-third of the dead volume needed for standard liquid level detection technologies.
Dead volume is always a component of automated liquid handling systems. Both costs and performance can be considerably affected if dead volumes are not optimized. In fact, scientists can exploit the remarkable capabilities of the Fluent Laboratory Automation Solution to reduce dead volumes for costly reagents and valuable samples in their automated processes.
This article has shown how Fluent provides as much as threefold reduction in dead volume as opposed to a standard laboratory automation platform when used for a general application. This advancement can be attributed to Tecan’s Adaptive Signal Technology, an advanced liquid sensing technology that allows sophisticated and more efficient evaluation of the liquid volume accessible in the source labware.
Improved process security in combination with high mechanical precision allows for greater liquid recovery and at the same time, maintains excellent pipetting precision and accuracy.
Produced from materials originally authored by Florence Collins, Tecan.
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