An automated liquid handling system is typically procured to ensure excellent speed and throughput, but for the majority of users, the reliability of results appears to be a more important factor.
Liquid level detection is an unobtrusive feature that is responsible for ensuring the consistency and reliability of the pipetting procedures. However, all liquid level sensing technologies are not developed in the same manner.
The Adaptive Signal Technology (AST) from Tecan is an advanced capacitive liquid level sensing which, apart from detecting the liquid level in a microplate well, differentiates between bubbles and the actual liquid level, prevents wells over-flowing, identifies tip occlusions, and provides innovative opportunities for in-process error handling.
What is capacitive liquid level detection?
The first type of performance monitoring to emerge on automated liquid handling robots is capacitive liquid level detection (cLLD) (Figure 1). cLLD is still frequently used today and involves applying a small amount of AC voltage to the apex of individual pipetting channels.
Detection of the liquid level inside a well is made possible by comparing the capacitance change between the grounded worktable and the conductive tip as the tip shifts from the reference environment, i.e., air into the liquid.
Figure 1. Capacitive Liquid Level detection (cLLD) principle
Users can employ certain liquid handling software applications to set a threshold value for the capacitance change or signal determined when the tip penetrates the liquid.
This signal relies on the relative static permittivity and liquid conductivity, and it also relies on the carrier and labware configuration and the distance of the tip to the liquid surface, worktable, or labware, even in the absence of direct contact.
To ensure consistent detection of the liquid level, the change in signal as the tip moves across the air-liquid boundary has to be greater than all the non-contact capacitance changes. Therefore, establishing a suitable signal threshold for improved cLLD performance is all about striking a balance between insensitivity to the environment and sensitivity to the liquid:
- While liquid detection is made more sensitive by lowering the threshold, environmental interference may increase considerably which would lead to false positives
- While liquid detection is made less sensitive by raising the threshold, the tip may become immersed before reaching the threshold. In the worst situation, extremely weak conductivity liquids may not be detected at all, which may lead to well overflowing, over-submergence of the tip, and cross-contamination
What is Adaptive Signal Technology?
Based on the same principle as cLLD, the AST includes three major enhancements such as:
- Adaptive threshold settings
- Refined split signal evaluation
- Aspiration monitoring
These improvements provide excellent sensitivity, reduce the possibility of over- or under-sensitivity, and enable automated and intelligent error handling, thus allowing liquid level detection:
- Rugged and insensitive to interference
- Fully intelligent and automated, without any need for user adjustments
- Can detect very small volumes
Refined split signal evaluation
The capacitance change signal is then filtered into slow and fast output frequencies so as to improve process security. Validation of liquid detection is done only when both signals are activated under a specified period of time. Let us consider the following cases:
Fast signal trigger – defines liquid level
- The liquid level is established by the position of the tip at the moment of signal activation
- The downward movement of the tip is halted, thus reducing the amount of tip immersed in the liquid
- The slow signal is assessed after triggering the fast signal. However, the system will automatically retry if the slow signal is not validated
Figure 2. Simplified schematic representation of the two signals (fast and slow), and their detection thresholds (not all detection parameters are represented).
Figure 2 shows the graphic representation of the two signals and their detection thresholds.
Slow signal trigger – provides confirmation of liquid detection
- Assessed following fast signal trigger to validate liquid level detection
- Can be activated even without a fast signal trigger if signal goes beyond a specified slope threshold, suggesting an unusual drift in the signal intensity
- A slow signal trigger in the absence of a fast signal trigger creates a ‘safety stop’ of the downward tip movement and the ensuing assessment of the fast signal. The system will automatically retry if the fast signal is not validated
Automated and adaptive threshold settings
Although a number of factors affect the intensity of capacitance change, most cLLD technologies depend on a straightforward signal intensity threshold. This means, the ‘one-size-fits-all’ method can create over-sensitivity (false positive detection) or under-sensitivity (missing detection) problems for specific combinations of liquid and labware.
In the case of AST, an extremely sensitive and consistent liquid level detection can be accomplished by producing a constantly re-evaluated dynamic threshold, which is adjusted according to the tip position and the volume of liquid remaining in the well during aspiration. This dynamic threshold setting enables the system to identify smaller volumes, and at the same time, reduces the sensitivity to interference.
To ensure better process security, AST comes with two optional functions to track aspiration following a successful liquid level detection. These options help prevent inaccurate results owing to foam aspiration, tip blockage, or unpredicted tip tracking.
During aspiration, the capacitance change between the worktable and the tip can be monitored by Aspiration Supervision, but provided the tip is 5 mm over the base of the well. During aspiration, if the tip moves across the liquid-air boundary, an Aspiration Supervision exception will be triggered by the resulting capacitance change.
The capacitance change can be monitored by Retract Supervision as the tip pulls back from the liquid following aspiration, but provided the tip is 5 mm over the base of the well. While retracting, if the tip fails to move across the liquid-air boundary within the predicted height range, a Retract Supervision exception will be triggered by the lack of capacitance change.
How does AST enhance process reliability?
Protection against electrostatic discharge interference
During the downward movement of the tip, high frequency interference like an electrostatic discharge can trigger a surge, which, in turn, drives the fast signal intensity across the threshold for detection. This type of surge has a short interval and therefore, is not likely to cause a signal trigger.
However, if there is an incorrect fast signal trigger, the slow signal will not be affected at all. This means, there is no way of confirming the false liquid level detection (Figure 3).
Figure 3. Schematic representation of the sensitivity profile concept used in AST. As the pipette tip moves downwards within the well, the signal intensity in the event of the tip crossing the air-liquid border decreases relative to the tip position and volume of liquid remaining in the well. To account for this, the threshold value required to trigger a signal is also reduced as the residual volume decreases.
Protection against overflow (tip diving)
A missed liquid level detection can lead to severe outcomes, making the tip of the pipette to become immersed and possibly leading to well overflowing to create a contamination risk. Such a scenario might take place if the change in capacitance fails to reach the fast signal threshold as the tip moves across the air-liquid boundary. If this happens, the slow signal trigger will promote a ‘safety stop’ of the pipette tip under the liquid surface.
Detection of the true liquid level through bursting bubbles
If the tip touches a conductive bursting bubble, the fast signal may activate even if the tip is yet to cross the air-liquid boundary. Conversely, the slow signal will not activate, because the slow signal slope will not pursue the predicted profile. The liquid level detection cannot be validated without confirming a slow signal.
Prevention of non-bursting bubble and foam aspiration
If the tip touches a non-bursting and conductive foam or bubble, both slow and fast signal triggers may occur, which may lead to false liquid level detection and promote mid-air aspiration. The Aspiration Supervision can be used to detect this issue, thus making the tip movement and aspiration to stop instantly, including error handling as predefined.
Identification of incorrect labware
If a smaller diameter source tube defined in the method is wrongly mounted onto the worktable, monitoring the tip during aspiration will not be appropriate. This will cause the tip to exit the liquid and aspirate air.
The Aspiration Supervision function can be used to detect this problem. If the tip moves across the liquid-air boundary during aspiration, the resulting change in capacitance instantly stops the tip movement and aspiration, including error handling as predefined.
Detection of tip occlusion
During aspiration, if the tip is blocked by a clot, the volume remaining in the tip will be lower than predicted. Also, a filament may be hanging from the tip after aspiration. This will pose a contamination risk as the tip moves to empty the liquid into the destination container. The Retract Supervision can be used to detect this issue, provided the predicted liquid level difference before and after aspiration is 4 mm.
How does AST enhance pipetting performance?
Minimized dead volumes
AST’s improved sensitivity enables the detection of very small volumes: 2 μl of tap water and 10 μl of ethanol. This unique capability, in combination with the accuracy of the tip movements and precise well geometry definition in the application software, reduces the remaining dead volume in individual wells (Figures 4 and 5).
Figure 4. The FluentControl™ application software allows the user to define exception handling operations for specific aspirations in a protocol.
Figure 5. FluentControl enables precise definition of labware well geometries.
Improved pipetting precision
AST also ensures improved consistency and precision for liquid handling by reducing tip submergence during aspiration. Software features like Retract Supervision and Aspiration Supervision further enhance process security and reliability and help track all the aspiration, liquid level detection, and tip retraction steps.
Greater flexibility for automated error handling
During complex liquid level detection, the system causes an instant stop and retries automatically; however, it is not exactly known what will happen if the issue continues. For this purpose, an exception is created which can then be handled either automatically as preset by the user, or manually via a user prompt.
For each and every type of exception, default error handling settings on the basis of the liquid class are given. However, AST enables users to tune these settings for any particular liquid or each aspiration command, thus making it possible to treat errors in different ways for different pipetting steps.
More options to handle ‘not enough liquid’ exceptions
A ‘not enough liquid’ exception will be created if the quantified available volume in the well is lower than expected for the liquid transfer. In the case of traditional cLLD technologies, the only available option would be to aspirate to the base of the well, but again this would lead to partial air aspiration and would necessitate a visual check towards the conclusion of the run to guarantee that adequate amount of material is there for downstream processes.
The available volume can be more precisely calculated with AST by integrating the excellent mechanical precision with improved sensitivity. As a result, more options are available to handle ‘not enough liquid’ exceptions.
Whenever an exception takes place, users can program the system to aspirate as normal or modify the aspiration volume to correspond with the detected volume. The exact pipetted volume is subsequently logged and flagged in the output file.
Does AST require user intervention?
AST is set to run and eliminates the need for calibration or manual adjustments. The application software contains a database of liquids, with each allocated to one of the three sensitivity groups.
Based on the conductivity of the carrier and labware, the sensitivity profile is automatically changed, and the flexibility is further increased by providing an automatic sensitivity group determination technique for liquids that are not part of the database.
When a new technique is being developed, the aspiration process can be configured with the source labware, the aspiration volume, and the liquid type. Subsequently, AST will carry out automated high resolution liquid sensing as well as precise liquid level detection. It needs only user intervention when an error or exception cannot be resolved with predefined exception handling or automatic retries (Figure 6).
Figure 6. AST offers predefined sensitivities for numerous liquids.
The AST platform provided by Tecan enables liquid handling operations for Fluent™ laboratory automation workstations in a reliable and accurate way. This new technology can be used for those applications where high reliability and reproducibility are required, such as when precious samples and expensive reagents are used. As a result, the risk of errors is reduced considerably during aspiration and tip retraction.
The improved process security rendered by the adaptive threshold settings and refined split signal evaluation of the AST allow for fail-safe, multi-step liquid level detection and exception handling and ensure compliance with the rigorous regulatory directives encountered by various laboratories.
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