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European Biopharmaceutical Review

Fluid Thinking

The number of chemical compounds for testing and molecular targets for lead finding is increasing. This, entwined with the steadily escalating cost of drug discovery, means that companies will need to increase both process- and cost-efficiency.

Minimising the cost of screening can be achieved in several ways, including higher throughputs,miniaturising assay formats and streamlining the assay process.Assay miniaturisation in particular is recognised to produce a whole range of cost savings; by enabling assays to be performed in smaller volumes, reagent and compound use is optimised so costs are reduced significantly.With the right automation, throughput can be increased and assay quality and reproducibility improved.

Automation is fast, and also brings accuracy and repeatability by reducing human error.This is now so well-accepted that it is the accuracy and repeatability of the instruments themselves that is under the spotlight, especially with the focus on further reducing assay costs by miniaturising assays.As a result, automated liquid handling is now more critical than ever to increasing a laboratory’s overall efficiency. In response to these drivers, manufacturers are making constant improvements to the performance of liquid handlers based on a range of technologies. Each liquid handling technology has strengths and weaknesses for different applications, and should be assessed with the needs of your application(s) in mind.

Assessing Liquid Handling Technologies

Assay miniaturisation requires liquid handlers that are able to reproducibly deliver low volumes, accurately and without sample contamination, yet be flexible enough to handle a range of assays and liquid types without constant setup changes. High quality laboratory results require both good accuracy and precision.As volumes are miniaturised, even small inaccuracies are magnified proportionally, especially in protocols dependent on serial dilution.To save costs, assay miniaturisation requires that waste is also minimised, which includes dead volumes left in wells or tips,washing solvents and consumables.Other important criteria, though often overlooked, are the ease of use and reliability of the instrumentation. If a liquid handler is the key instrument in your process (which it often is), then you need it to have as little downtime as possible, whether that is re-programming, setup changes, recalibrating or repairing. Ease of use also ensures that knowledge is not limited to single, highly-trained operators and, combined with flexibility, offers a form of future-proofing if your processes change.

Air Displacement Pipetting
This is a contact technology which lacks accuracy at lower volumes and is chiefly used for high volume applications (microlitre volumes). Its accuracy is affected by pipette geometry and liquid properties (such as viscosity and surface tension) and also by ambient conditions. In general, fixed tips offer a more uniform geometry with a smaller bore, which gives better pipetting accuracy and positioning, but requires washing. Plastic disposable tips eliminate crosscontamination issues, but can give more variable accuracy and are typically an expensive and bulky consumable. Different heads can address different plate formats (96, 384) but the physical size of the microlitre volume tips means they are often unable to access high density plates such as 1536 and leave relatively large dead volumes in the well. Storage space for new and waste tips can also become an issue. Process speeds are good as the pipetting head often addresses a whole plate or quadrant of a plate at a time – which is excellent for plate replications – although time still needs to be allowed for wash steps or tip changing.To address serial dilution protocols and allow greater process flexibility,many instruments now offer alternative heads or tip loading methods where tips are arranged in columns or rows.Assay miniaturisation is pushing the limits of this technology in terms of accuracy and repeatability at low volumes.

Bulk Reagent Dispensing
Strictly speaking, this is liquid handling but not pipetting.These instruments often use peristaltic pumps or pressurised systems with valves to offer very fast, noncontact dispensing of microlitre to nanolitre volumes of common solutions from a reservoir or reservoirs.The more complex of these systems have the flexibility for each tip on each channel to dispense any volume into any well of the plate, but the dead volumes in the reservoirs are typically higher than conventional pipetting systems leave in the well. Bulk reagent dispensers are the workhorses of most labs and are easy to programme and use.However, they are often unable to aspirate and, when they can, generally need to over-aspirate.This can leave significant dead volumes in the tip which requires regular washing. Bulk reagent dispensers are not suited to applications with a variable component in every well, or for aspirate/dispense pipetting modes due to the washing requirements.Tip clogging, reliability and robustness are often major concerns with low volume devices.



Positive Displacement Pipetting
This is an accurate and precise technology for low to medium volumes (nanolitres to low microlitres). A piston draws up and dispenses the liquid, which also allows rapid in-well mixing.This piston is in direct contact with the liquid, rather than using an air gap or system liquid, which means that the technology is accurate over a very wide range of liquid viscosities and surface tensions and can even pipette highly viscous liquids, such as glycerol and PEG.This is a contact technology, but cross-contamination issues have been overcome by using low-cost disposable pipettes which incorporate disposable pistons to eliminate time-consuming tip washing.The pipettes themselves have been miniaturised: smaller tips mean that dead volumes are minimised because the smaller tips can access right to the bottom of plate wells to leave negligible waste in the wells and none in the tip.They also take up very little space either in storage or on disposal.These smaller tips also have precise positioning within wells, and facilitate access to high density plates such as 1536.Traditional serial dilution assays are easily miniaturised as pipettes can aspirate, dispense and mix directly across the plate. Speed is good; tip changing is extremely fast and the pipettes address a column of a plate at a time.The columnar arrangement of pipettes means that they can easily reformat between different plate types in the same protocol.The accuracy and precision of this technology, combined with its flexibility, means it can be applied to many different assays without the need for system setup changes.

Acoustic Ejection
This is a very accurate, non-contact technology which is optimal for extremely small volumes (picolitres to nanolitres).A destination plate is placed over the source plate and acoustic energy is used to rapidly fire a drop (of typically 1-5 nL) into the destination plate from one well at a time.This one-to-one transfer means multiple replications of a single source plate cannot be prepared efficiently by ‘stamping out’, as they can with the other technologies described here.Acoustic ejection is very quick to deliver very low volumes, but takes longer to deliver larger volumes to an entire plate as multiple drops from each source well are needed to make up the total volume.Using such small droplets means there are no problems with accessing high density plates, such as 1536+, as either source or destination plates. However, the technology does not eject from 96-well source plates. A minimum volume of liquid is required in the source wells in order to form the droplet for acoustic ejection so there are relatively high dead volumes in the source plate, especially compared to the very low volumes transferred.The technology can be calibrated for a range of viscosities, but is fundamentally limited to lower viscosity liquids and will need to be optimised for each fluid type used.The technology behind the drop formation and ejection is sensitive to plate variations, so it is most accurately used with a restricted range of plates with specific well geometry and materials.The direct, non-contact droplet transfers do not permit intra-well mixing, so the generation of concentration-effect curves are only possible by a direct volumetric approach using this technology.However, the dynamic range is not wide enough to do this in one step and so the preparation of intermediate stock solutions is required, as is subsequent back filling to equilibrate DMSO concentrations.

Pin Tool Technology
This contact technology dips an array of pins into the liquid to be transferred, relying on surface tension to retain small amounts of liquid in order to rapidly deliver low volumes (nanolitres). It is optimised for delivering repeatable volumes of the same liquid type into high density plates, particularly 384 and 1536 formats. However, because the technology relies on surface tension, transfer volumes are affected dramatically by both the liquid properties and the volumes in the source plate wells: variations will give inaccuracies. Pin tools are a contact technology and so require washing steps. Such instruments are less commonly used due to their inflexibility and the contamination issues.

Conclusion

Fundamentally, each of these technologies has its own blend of particular benefits (speed, accuracy, reliability, low or high volume dispenses, mixing, non-contact, flexibility and so on), drawbacks (including crosscontamination, slow, wash steps, set-up time and variability), and costs (such as reagents,waste, wash solvents, pipettes and plate types).The best choice for your application depends on your priorities and how flexible you need the process to be. Primarily, the push to assay miniaturisation has come from the need to save costs, but the calculation of cost savings should not rely solely on the reduction in sample volumes used in the assay.Accuracy and reliability of results are huge concerns for the screening scientist and difficult to attach a true cost to; but operator time, system downtime, processing time,waste and consumables (including plates, tips and wash solvents) are all factors to be weighed as well.

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Joby Jenkins is the Product Manager for Liquid Handling at TTP LabTech. Joby graduated with a first class honours degree in product design and manufacture from Loughborough University and joined TTP LabTech shortly after. Initially, Joby was a key engineer and project leader for the development of the mosquito® range of products before progressing to Product Manager. He has 11 years of experience in industrial R&D, focusing most recently on product management and sales. Email: joby.jenkins@ttplabtech.com
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