Using Pipetting Robots for Flexible Repetitive Dispensing

Pipetting is one of the most common daily activities in life-science laboratories and takes up a large part of a technician or scientist’s working day. In a typical workflow, reagents, solutions and samples are pipetted in various volumes.

Additionally, there are procedures where the same volume needs to be repeatedly transferred from one source vessel to numerous destination vessels. Some common examples include aliquotting bodily fluid samples for biobanks and filling 96-well microplates with reagents or master mixes to prepare ELISA, qPCR, PCR or other clinical diagnostic and forensic tests.

These processes require the user to repeatedly refill pipette tips from the source and dispense the liquid to different destinations. Each cycle of repetitive pipetting requires the user to depress the pipette plunger twice and release it once with their thumb. This must be done with enough force and care to make sure that the liquid is transferred accurately and precisely.

Along with other pipetting activities, this repetitive process increases the risk of Repetitive Stress Disorders (RSI) of the users’ thumb, shoulder and hand, which are a well-known and costly medical problem [1].

As well as ergonomic issues, executing lengthy protocols that involve repetitive pipetting results in reduced efficiency and potential pipetting errors. Differences between the pipetting techniques of different uses can result in large deviations in the quantity pipetted and this causes significant downstream errors with regards to data analysis and interpretation.

Many companies have focused on improving this process because of the current issues with regards to user health/safety, poor downstream data and the time wasted carrying out this menial yet essential task. Until now, beneficial results have been limited.

The time needed to fill multi-well plates can be reduced using multi-channel pipettes. These allow 8- or 12- well positions to be filled in parallel. However, multi-channel pipettes are less precise and accurate and are also limited in that suitable consumables must have an exact distance of 9 mm between the wells.

Stepper pipettes, also known as repeater pipettes, provide a different solution for transferring the same volume to multiple destinations. They allow for a greater choice in consumables but require significant hands-on time and can only dispense a narrow range of volumes. Traditional robotic liquid handlers increase the speed but are expensive, limit the choice of consumables and can only be used with high-level expertise and a specific IT skill set.

Andrew Robot: A Hands-Off and Robust Solution to Repetitive Pipetting

Andrew Alliance has developed one of the most effective solutions to the challenge of repetitive liquid handling. It combines the positive aspects and eliminates the negative features of the repeater pipette and traditional automated liquid handling workstations. This solution is easy to implement and affordable and is made up of the pipetting robot, Andrew, combined with a graphical, user-friendly software for designing pipetting protocols. Andrew can achieve the same functionality of a repeater pipette using conventional mechanical pipettes (Figure 1). This is done automatically and with high levels of precision and accuracy. Therefore, Andrew provides a very competitive system for aliquotting activities and liquid distribution.

Repetitive pipetting by Andrew robot to dispense equal volume into multiple destinations from a single aspiration

Figure 1: Repetitive pipetting by Andrew robot to dispense equal volume into multiple destinations from a single aspiration

Andrew is the most flexible pipetting system and can change a pipette volume, eject and insert tips, mix, aspirate, reverse pipette, dispense liquid, deal with viscous solutions, forward pipette, adapt to the consumables on deck with artificial intelligence, create air gaps for eliminating dripping or tip touch for accuracy at low volumes, all from any source vessel to any destination vessel pre-determined by the user.

Andrew Accurately and Precisely Dispenses Equal Volumes Repetitively with Standard Manual Pipettes

The “repetitive dispensing” performance of Andrew was tested using a photometric method, measuring the aliquot volumes of Ponceau S dye solution in water as previously described [2]. An Andrew model 1000G was used to repetitively dispense varying dye volumes into clear, flat-bottom 96-well microplates. A spectrophotometer was used to measure the absorbance of the dye at 520 nm.

Andrew’s repetitive dispensing mode was tested using four Gilson pipetman P20, P100, P200 and P1000 (Table 1). The maximum volume tested was 100 mL and the minimum was 2 mL. For every tip, a pre-wet step was carried out by mixing the liquid at the source 10 times. This was to equilibrate the vapor pressure inside the tip to ensure more accurate results.

For the P1000 pipette, Eppendorf epTIP tips were used, while Gilson Diamond tips were used for the other three pipettes. Each volume tested in Table 1 was dispensed repeatedly as a series of eight aliquots in the “precise” mode (Figure 2), or 18 aliquots in the “fast” mode (Figure 3), with 10 - 12 series per tip and 4 - 5 tips per aliquotted volume. All experiments were carried out at a relative humidity of 34 - 40%, and at 21 – 24 oC.

Repetitive pipetting 8 aliquots of 10 µL with the “precise” mode using the P100 pipette

Figure 2: Repetitive pipetting 8 aliquots of 10 µL with the “precise” mode using the P100 pipette

Repetitive pipetting 18 aliquots of 10 µL with the “fast” mode using the P100 pipette

Figure 3: Repetitive pipetting 18 aliquots of 10 µL with the “fast” mode using the P100 pipette

By multiplying the series standard deviation by the expected aliquotted volume, the random error of the desired aliquotted volume that was dispensed respectively in each series was calculated. For systematic error calculation, the performance test results showing the superior accuracy of Andrew pipetting different volumes using single dispensing has been previously illustrated [2].

As a result, the single-dispensed volumes by the pipette which is best fit for the volume (for example P2 for 2 mL; P20 for 5, 10 and 20 mL; P100 for 50 and 100 mL), were also obtained. These were used as the reference standard.

Table 1: Andrew performance in “repetitive dispensing” mode

Table of Contents

Pipette (operation mode) Aliquoted Volume (µL) Number of Aliquots Andrew Systematic Error (µL) ISO8655 Systematic Error (µL) Andrew Random Error (µL) ISO8566 Random Error (µL) Time to fill 96-well microplate (min/sec)
Single-channel Multi-channel Single-channel Multi-channel min max
P1000 100 8 ±0.75 ±8 ±16 <4.8 <3 <12 10m23s 11m58s
(fast) 50 8 ±0.63 ±8 ±16 <4.5 <3 <12 7m2s 9m40s
P200 20 8 ±0.69 ±1.6 ±3.2 <1 <0.6 <1.2 9m51s 10m21s
(fast) 10 18 ±0.26 ±1.6 ±3.2 <0.7 <0.6 <1.2 7m24s 10m10s
P100 10 8 ±0.42 ±0.8 ±1.6 <0.6 <0.3 <0.6 9m30s 10m35s
(precise) 5 18 ±0.5 ±0.8 ±1.6 <0.6 <0.3 <0.6 7m3s 8m42s
P20 (precise) 2 8 ±0.29 ±0.2 ±0.4 <0.39 <0.1 <0.2 9m9s 10m14s

 

Andrew’s random and systematic errors of the tested aliquotted volumes for each pipette are shown in Table 1. These are compared with the reference maximal permissible random and systematic errors recommended by the ISO 8655 standards for single- and multi-channel pipettes [3]. In this test, Andrew dispensed liquid “on-the-fly” at the ambient humidity and temperature of a normal lab in daily operation.

However, the ISO 8655 standards are for dispensing the liquid into liquid at a very high humidity and constant temperature to prevent evaporation. Therefore, the data collected were obtained in strict daily laboratory conditions rather than reference ISO 8655 data which is not normally seen in real-life laboratory settings. Andrew’s systemic errors for the volume range 5 – 100 mL are all below the ISO 8655 standards of the single channel pipettes, despite the more demanding conditions. Andrew was still able to achieve an outstanding accuracy when dispensing volumes that are only 5% of nominal volumes of the pipettes.

Generally, Andrew’s precision of repetitive dispensing is as good as or better than the precision of the ISO 8655 recommended multi-channel pipette. Using the P1000 pipette, the maximum random errors were 4.8 and 4.5 mL in  8- and 18- aliquot series of 100 and 50 mL, respectively. This highlights that Andrew can maintain an excellent repetitive dispensing performance that is within the ISO standard of 6 mL random error for single dispense of the single channel pipette, and is much lower than the 12 mL of random error of the multi-channel pipette.

The random errors fall at the maximal permissible error of the ISO 8655 standards for the multi-channel pipette at lower volumes with the P100 (8 aliquots of 10 mL, 18 aliquots of 5 mL) and P200 (8 aliquots of 20 mL, 18 aliquots of 10 mL). The observed difference in maximum random errors of multi-dispensing 10 mL from the P200 and P100 pipette was 0.1 mL.

The time to completely fill one 96-well microplate with the P200 pipette in “fast” mode for 18 aliquots was approximately 1.75. This is 2.1 minute faster than the P100 in “precise” mode. Therefore, users can choose either of these pipetting mode options in Andrew Lab, according to the importance of processing time and the tolerable level for random errors.

It takes less than a minute to design the repetitive dispensing experiment for a 96-well plate in the Andrew Lab software. A further minute is required to arrange the labware (tip boxes, liquid source, microplate) for Andrew. As a result, with small variable processing times for Andrew to completely fill out a 96-well microplate, the hands-on time actually required from the human operator is only two minutes. This is a significant time saving when compared with using electronic repeater pipettes or multi-channel pipettes.

Andrew’s “Repetitive Pipetting” Mode – How Does it Work?

When in the “repetitive pipetting” mode, Andrew aspirates the liquid samples from the source at the maximum volume allowed by the pipettes (i.e. “nominal volume”). Andrew then moves the pipette to the correct destination and partially depresses the pipette plunger down to the appropriate level for dispensing the correct, smaller volume of liquid. Then, the plunger is released and Andrew moves to the next destination to dispense the same volume in the same way.

The process repeats until the remaining volume in the tip is no longer enough for the next disperse. The left over is dispensed back into the source, which, when applicable, saves valuable reagents. The tip end does not touch the bottom of the destination vessel nor does it dip into the liquid inside the destination, meaning the liquid is dispensed “on the fly”. This prevents contamination. The entire repetitive dispensing process is repeated until all the destinations are filled.

With repetitive pipetting, the first and last aliquots are known to be inaccurately dispensed when handling liquids different from water or in difficult environmental conditions (for example in quickly changing humidity and temperature). Andrew is programmed to discard the first and last aliquots back to the source in order to avoid this potential issue. As a result, Andrew calculates the number of aliquots per refill using the following formula:

The next smallest integer number is the final result. Pipette manufacturers generally recommend 10% of the nominal volume as the minimum dispensed volume for single dispensing. This is because pipettes perform better at volumes closer to their nominal volume (best at 35 – 100% nominal volume). Andrew, however, is programmed to repeatedly dispense the minimum volume at 5% of the nominal volume.

In some cases therefore, a desired aliquotted volume can be repetitively dispensed by two different pipettes. For instance, 15 mL is 15% of the P100 nominal volume and 7.5% of the P200 nominal volume. In this scenario, users designing the pipetting protocol for Andrew can choose between the two pipettes when selecting a repetitive dispensing mode: the “precise” mode is designed to achieve higher accuracy with fewer aliquots (Figure 2), whilst the “fast” mode is designed for producing more aliquots with a lower precision (Figure 3).

For the 15 mL example, Andrew can use a P200 pipette in “fast” mode or a P100 pipette in “precise” mode. In “precise” mode Andrew will dispense (100/15 -2) = 4.67 à 4 aliquots/refill. Using a P200 pipette in “fast” mode, it will dispense (200/15 -2) 11.33 à 11 aliquots/refill. Users can therefore select the best option according to their specific criteria and the cost/benefit analysis of the experiment.

Summary

The possibility and performance of repetitive dispensing standard mechanical single-pipettes has been demonstrated for the first time. The results are excellent and show that Andrew is an effective and concrete solution that can replace electronic pipettes and traditional liquid handlers for repetitive pipetting applications.

The Andrew robot provides effective automation and frees up user time whilst providing results in an efficient and timely fashion. It takes two minutes to design a repetitive dispensing protocol to fill a microplate, and a 96-well plate can be filled, completely unattended, in as little as seven minutes. With Andrew, laboratories can achieve a rapid return on their investment whilst also improving data reproducibility and preserving the well-being of the user, without needing to alter existing workflows or change consumables.

References

  1. McGlothlin et al. Health Hazard Evaluation, pp. 13. United States NIH for Occupational Safety and Health, Frederick, Maryland. 1995.
  2. I. Semac, G. Horak, A. Jordan, P. Zucchelli. Pipetting performances by means of the Andrew robot. www.AndrewAlliance. com [Online] 2014.
  3. ISO8655. www.iso.ch. 2002.

About Andrew Alliance S.A.

Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.

Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.

Start with meeting customer needs, end with customer feedback.

Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jul 22, 2019 at 7:57 AM

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