Automated Liquid Handling Robots: Bridging the Gap Between Nanoliter Liquid Handling and Larger Volume Dispensing

The introduction of automated nanoliter volume liquid handling instrumentation has revolutionized work flows, reducing the tedium of manual pipetting and the potential for manual error. In addition, assay miniaturization provides a number of cost and time-saving benefits for the research and drug discovery industry. Such benefits include a reduction in the amount of valuable compounds and reagents required per assay, which enables an increase in the assay range and size to be achieved, providing a greater amount of information per screen.

Until recently, it was difficult to reliably dispense volumes below 500 nL, a volume range often required by biologists and chemists to achieve successful final volume assay conditions. Inaccuracies that occur when dispensing low volumes can become proportionally magnified; therefore in order to ensure high-quality results, it is paramount to achieve consistency, accuracy, and precision at this low volume range.

The mosquito® HTS (TTP Labtech, Melbourn, Herts., U.K.) nanoliter liquid handling instrument is capable of rapid and accurate sample volume dispensing from 1200 nL down to 25 nL, regardless of viscosity. With its positive displacement pipetting technology and disposable tips, zero cross-contamination is ensured and users can quickly swap between applications, without labware or system setup changes. The instrument also allows users to employ a range of high-density plates (from 96 to 1536 wells) and to transfer samples between plate types for further process efficiency.

Nanoliter liquid handling applications

Nanoliter liquid handlers such as the mosquito HTS were designed primarily for plate replication and serial dilutions in compound screening laboratories. However, since their introduction, the number of nanoliter liquid handling applications has expanded rapidly, and the mosquito HTS is regularly employed in a wide range of applications, including the preparation of stock solutions, direct dispensing into plates, plate-to-plate transfer for compound screening and hit-picking, biological assay setup, nucleic acid extraction, rtPCR and qPCR preparation, as well as cell culture seeding and handling.

Despite the successful introduction of nanoliter liquid handling technology, it has become apparent that nanoliter volume ranges are not necessarily suitable for all assay types or formats. In particular, certain biological applications such as qPCR setup and protein crystallization screening and optimization regularly use volume ranges around and above 100 µL. In addition, although serial dilutions, plate replications, and reformatting requiring nanoliter liquid volume dispensing are routinely employed in a number of academic screening centers and pharmaceutical companies, these low volumes may not be beneficial for research establishments in which existing instrumentation is set up to use 96- or 384-well plates. Liquid handlers with an accurate and flexible volume range are therefore important, because they suit a wider range of applications and circumstances.

Microliter pipetting

  Figure 1 – mosquito HV.

Also included in TTP Labtech’s liquid handling portfolio is the mosquito HV, which offers the versatility to perform highly accurate microliter pipetting in the 0.5 to 5 µL volume range, while still retaining all the features of the mosquito HTS such as high precision, high speed, and accurate automatic dispensing (Figure 1).

With slight modifications to the head and tip clamping mechanism, it was possible to achieve pipetting and positioning accuracy across all plate types from 96- to 1536-well microplates using larger tips (internal diameter 0.7 mm, external 1.2 mm). In addition, these larger disposable tips are combined with mosquito’s positive displacement technology, to ensure zero cross-contamination, while retaining pipetting accuracy with liquids of even differing viscosities and surface tensions. The tips, although larger, are still able to reach to the bottom of wells, minimizing dead volume to under 50 nL per well.

Studies on the accuracy and precision performance carried out with mosquito HV using both DMSO- and aqueous-based sample solutions show comparable precision to the mosquito HTS nanoliter dispenser (Figure 2), with the pipetting accuracy of mosquito HV within 2%, and the coefficient of variation (relative standard deviation) less than 3% across the volume range (Table 1).

Figure 2 – Dispensing accuracy of mosquito HV and HTS with liquids of varying viscosity.
Table 1 – Volume transfer accuracy and repeatability

Intermediate volume dispensing

mosquito HV offers additional flexibility in the low volume range of 0.5 to 5 µL, where pipetting accuracy is difficult to achieve manually or using traditional, higher-volume pipetting instruments. This instrument, designed specifically for accurate and reproducible sample dispensing within this volume range, is of value for a wide range of chemical and biological-based applications. For example, dispensing within this volume range enables easy integration into preexisting screening protocols in high-content screening and HTS cell-based assays.

mosquito HV can be successfully employed for near assay serial dilutions using standard plates, where 2–8 µL of serially diluted compound are required. By dispensing larger initial volumes, it is possible to perform a wider serial dilution range. For example, starting with 5 µL, it is possible to remove two separate 2-µL aliquots and apply differing dilution ratios, i.e., 1-in-2 or 1-in-5. Furthermore, the use of larger starting volumes means that it is possible to achieve extended serial dilutions, e.g., up to 10×, while still retaining a high level of accuracy.

Using the TTP LabTech serial dilution wizard, it is possible to define and set up a range of serial dilutions simply by entering the required dilution series into the wizard template (with the final volume required), thereby eliminating time-consuming and error-prone manual programming effort (Figure 3).

Figure 3 – Serial dilution wizard layout.

Benefits of automated dispensing

In order to compare the advantages of using mosquito HV versus manual dispensing, a study was conducted to compare the dose response curves (DRC) of a group of 16 inhibitors in an enzyme-based assay. In this experiment, a 10-point (1:1) dilution series was set up in 384-well plates, starting with 3 µL of compound into 3 µL DMSO. Following this serial dilution step, 500 nL of each compound concentration was then transferred into 384-well plates and incubated with 25 µL of enzyme (2× concentrated). Enzymic activity was initiated by the addition of 25 µL substrate (2× concentrated), making the final volume to 50 µL.

While Figure 4 demonstrates that there was no significant difference in the dose response curves obtained where the dilution series and assay setup were performed either manually or with the mosquito HV, the study identified a significant reduction in the time and manual effort required using the automated mosquito HV. Specifically, the use of mosquito HV for conducting the serial dilution step took between 2 and 3 min per plate, with the total assay setup taking under 5 min per plate. This is compared to 20 min/plate for manual dispensing, where the accurate manual pipetting of such low volumes, especially into 384-well plates and over a large dilution series, required considerable effort and concentration. In addition to time savings, the risk of manual error and effort required to set up the 384-well plates was eliminated using mosquito HV, enabling the scientist to concentrate on science instead of tedious manual procedures.

Figure 4 – Comparison of dose response curves of compounds over a dilution series, set up using a) manual dispensing vs b) mosquito HV.

Similar to that observed with mosquito HTS, the mosquito HV is capable of plate replications and reformatting between different plate types, ranging from 96 to 1536 wells, addressing SBS format plates from any manufacturer. With five plate positions, the mosquito deck allows up to four daughter plates to be stamped from a mother plate directly to assay plates as part of the same protocol, saving valuable laboratory time and tip usage. This deck configuration also enables four 96-well source plates to be rapidly reformatted into a 384-well assay plate or even four 384-well source plates into a 1536-assay plate.

In addition to serial dilutions and plate reformatting, mosquito HV can be employed for low-volume liquid handling in the setup of a wide range of chemical and biological-based applications, including compound and small-molecule screening, cell or bead-based assays, enzyme kinetic assays, and ELISAs, to name a few. Indeed, the volume range dispensed by mosquito HV makes it more suitable than nanoliter dispensers for molecular biology applications and rtPCR or qPCR for the detection and quantification of RNA and DNA expression, RNAi screening, and SNP genotyping.

Conclusion

mosquito HV offers highly precise and accurate nanoliter and microliter liquid handling with robust, small-footprint instruments. Its ease of setup and use enables simple integration into a multiuser environment. The reliability, accuracy, and volume range over which the mosquito product range can dispense ensures effortless integration into any liquid handling procedure, speeding up laborious manual pipetting stages, reducing tedium and the risk of error in a high-throughput screening environment, and decreasing the cost and effort involved in the drug discovery process.

Wendy Gaisford, Ph.D., is the Science Writer at TTP Labtech, Melbourn Science Park, Melbourn, Hertfordshire SG8 6EE, U.K.; tel.: +44 1763 262626; fax: +44 1763 261964; e-mail: wendy.gaisford@ttplabtech.com.

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