June 15, 2012 (Vol. 32, No. 12)

Michael D. O’Neil


Steady advances in automated liquid handling at the µL to nL level are enabling more scientists around the world to reap the benefits of increased throughput, decreased costs, and more efficient use of reagents. Many of the latest techniques were discussed at the recent “European Lab Automation” conference. For example, Hugues Ryckelynck, scientific associate II, oncology disease area, biochemistry unit, NIBR (Novartis Institute of Biomedical Research), described how to quickly implement automatic liquid handlers to dispense accurately and rapidly in the nL and μL ranges.

He noted that “automated liquid-handling systems represent a great opportunity to increase experimental throughput and reduce reagents costs through assay miniaturization.”

However, he emphasized that setting up an automatic liquid dispenser to operate optimally in the nL to low μL ranges requires careful observations at the bench, or the results are likely to be highly variable and of low quality. Ryckelynck went on to describe simple operations to be done by hand with air and positive-displacement pipettes in order to anticipate problems such as high liquid viscosity, surface bonding, foaming, and surface tension that will be faced during the development of assays on nL and μL automatic liquid dispensers.

He further stressed that “when pipetting nanovolumes, an important source of variation is also the carryover from the source and from well to well.”

Ryckelynck gave practical examples of procedures developed by his group to optimize liquid handling on air/positive displacement (pipettes) and injector (pressure-based) systems that can be applied when working at submicromolar and subnanomolar concentrations of target proteins or reagents. He explained that the choice of robotic dispenser, i.e., dispensing technique (injectors vs. pipette) and speed of dispensing, and the experimental setup in general, should be made based on the biophysical properties of compounds and solutions.

The usual programming for complex solutions would be to pipette in a single pipetting mode (take once, dispense once, and change the tips). A faster and more efficient technique is serial dispensing (take once, dispense many), which allows the user to save on reagents and consumables and reduces experimental variability when optimally set (changing tips is like getting a new tool with its own new variability).

By performing sequential dummy runs, Ryckelynck showed high variability at the beginning and at the end of serial dispenses with all the dispensing systems used. This variability can be compound/reagent-related, a carryover effect, a result of forward pipetting, or inherent to the dispensers.

Ryckelynck developed simple dispensing procedures in order to avoid these problems. These procedures involve: (i) working in the optimal dispensing series of the instruments and getting rid of carryover by performing prime and post-dispenses, and (ii) saving time by minimizing well-to-well contamination flaws by performing dispenses in logical series.

He gave practical examples of how compound dilution protocols that avoid contamination and time loss in the nL range can be carried out quickly, and be easily implemented on a positive-displacement system. He explained his “reverse-half-Log” dilution technique, which provides the double advantage of an internal control on all dilutions and data best suited to Log presentations.

In conclusion, Ryckelynck presented a flexible laboratory setup for an optimal use of nL and µL liquid handlers that is used in his unit.

Key features of this setup include the use of standalone instruments working on modules of experiments running in parallel: (i) bulk dispense of microvolumes for limited experimental conditions is performed with pressure-based dispensers, (ii) filling from complex sources is performed using an air-displacement liquid handler for microvolumes and nanoliter positive dispenser for nanovolumes, (iii) source and plate (re)organization is done using an air-displacement liquid handler while (iv) compound dilutions and nL transfer to limit solvent impact on the experiments is performed with a nanoliter positive dispenser.

Cycloolefin Microplates

Rainer Heller, Ph.D., is a leader in Greiner Bio-One’s high-throughput screening (HTS) group, where he is responsible for launching many new products dealing with HTS.

Greiner Bio-One boasts newly designed microplates made from cycloolefins. Due to their excellent optical, chemical resistance, and physical properties, cycloolefin microplates have become increasingly popular in research and high-throughput applications, Dr. Heller said.

A variety of different cycloolefin microplates are now, or will soon be, available from Greiner Bio-One for different purposes, including cell-based assays, compound storage, liquid handling, and biochemical assays.

The new 1,536-well SCREENSTAR Microplate, for example, is a cycloolefin microplate designed for microscopic applications, high-content screening, and high-throughput screening. The SCREENSTAR Microplate was co-developed by Greiner Bio-One and GNF Systems and features a black pigmented frame with a 190 µm ultra-clear film bottom for ideal compatibility with instrument optics.

Well bottoms display excellent optical properties for the highest optical transparency, with reduced autofluorescence in the lower UV range, low birefringence, and a refractive index of 1.53, the same as glass. Recessed microplate wells enable complete periphery access for high-magnification objectives. Cell culture treatment and sterility assure exceptional performance, he said, for high-content screening, especially with fluorescence microscopy in the lower UV range.

A smooth microplate top, absent of alphanumeric coding, facilitates flush lid mounting for use with ultra-high-throughput screening systems. A similarly constructed 96-well cycloolefin microplate will soon be available from Greiner Bio-One, and the company is currently developing a similar 384-well cycloolefin microplate.

Another application of cycloolefin microplates is for compound storage, as cycloolefins exhibit low water absorption, low impurities, high transparency, and resistance to polar solvents, particularly DMSO, which is commonly used to preserve biological samples.

In order to make the latest technical and design innovations available for HTS, Greiner Bio-One will soon be introducing a new 1,536-well cycloolefin microplate for compound storage, liquid handling (including acoustic systems and pin tools), and transmission measurements in biochemical assays.

This new 1,536-well microplate will follow the most relevant ANSI recommendations and feature a smooth microplate top, also absent of alphanumeric coding to facilitate flush lid mounting for use with the GNF ultra-high-throughput screening system. The wells are more tapered than in classic 1,536-well microplates, reducing the dead volume in different liquid-handling applications.

Human mesenchymal stem cells differentiated into adipocytes on a cell-culture-treated cycloolefin microplate surface. (Objective: 20 x; Device: LSM 710, Zeiss; Orange=DAPI-stained nuclei; Blue=calceine-stained cytoskeleton; Green=Lipid vesicles.) [Rainer Heller/Greiner Bio-One]

Regulated Bioanalysis

Joseph A. Tweed, a bioanalytical scientist working in the pharmacokinetics, dynamics, and metabolism department at Pfizer, described the development of an internal, regulated, automated sample-preparation and extraction platform for use on the Hamilton MICROLAB® STAR liquid-handling workstation. Tweed noted that the sample-preparation and extraction techniques used for regulated preclinical and clinical bioanalysis of serum, plasma, urine, and cerebrospinal fluid are often very repetitive and tedious tasks that can greatly benefit from automation.

His group chose the Hamilton STAR because of its air-displacement pipetting technology and its ability to pipet microliter (µL) volumes with reliable precision and accuracy. In addition, the Hamilton STAR offers flexible and customizable deck platforms with robotic manipulation arm(s) and integrated one-dimensional (1-D) and two-dimensional (2-D) bar code scanners, he said.

Tweed and his group developed a graphical user interface to couple with the Hamilton STAR liquid-handling method. This approach allows the bioanalytical scientist increased flexibility and customization of study- and assay-specific parameters for any given bioanalytical sample-preparation technique selected.

Tweed said the platform incorporates the ability to batch process study samples among five automated extraction techniques: protein precipitation, solid-phase extraction, liquid-liquid extraction, plate-based protein precipitation, and supported liquid extraction. Additional features include the ability to prosecute routine sample batches via an ordered laboratory information management system sequence or randomized 1-D or 2-D barcodes.

Tweed said that the software package and the modular method design provide a flexible and versatile approach for routine bioanalytical sample preparation. The advantages provided by this technology are that it offers increased throughput, improved chain-of-custody for study sample analysis, and a streamlined approach for routine bioanalytical sample preparation.

Tweed noted that after sample preparation has been accomplished, specimens are analyzed via liquid chromatographic tandem mass spectrometric analysis.

Direct Dilution Strategies

Toby Winchester, an application consultant for Titian Software, discussed his firm’s Mosaic Sample Management Software suite, which can be integrated both with standalone instruments and with integrated systems such as the Labcyte® POD™ (an automated plate handler), according to Winchester, who discussed the pros and cons of these various integration approaches (such as time to develop the software versus expertise necessary to run the process).

In particular, he noted that Mosaic offers the options of offline and online full integration—depending on the variability of the processes being controlled.

Offline integration is ideal for processes that are subject to frequent change (such as in an assay development environment), whereas online full integration is ideal for more developed processes not needing as high an expertise to run (as in support of safety studies). Online integration is also appropriate for processes that support more complex platforms such as Agilent’s BioCels.

With Mosaic, a workflow is requested and machine control scripts and commands are automatically generated, so the setup of each run takes just moments. Mosaic interfaces with the liquid handlers to obtain feedback about run results, and automatically records information about inventory changes and workflow progress.

Winchester went on to describe recent progress that has been made in addressing the complexities of modelling the dose-response fulfillment of the Echo® dispensers from Labcyte. These acoustic dispensers allow the dispensing of small volumes, down to 2.5 nL, that permit direct dilution, rather than serial dilution, to be used to develop dose-response curves. This permits error rates to be reduced to as low as 1.0% to 1.5% compared to the multiples of 10% per dispense step typically obtained for serial dilution approaches.

However, the Echo instruments do not have the dynamic range (typically 6 logs) required by researchers using dose response to determine IC50 (half maximal inhibitory concentration) in drug discovery. Consequently, the Echo liquid handlers require the use of an intermediate plate in order to achieve the required dynamic range. The handling of this intermediate plate has been problematic in optimizing a Mosaic solution for these instruments.

Winchester said that Titian has developed multiple solutions to manage the workflow and inventory, including the tracking of the intermediate plates, in order to drive improvements and workflow efficiencies in its customers’ substance management groups. He noted that the Mosaic Software notices and records when the Echo instrument cannot dispense a physical sample issue, such as precipitates. (See sidebar on previous page for information on Labcyte’s own new software for Echo liquid handlers.)

High-Precision Flow Control

Anne Le Nel, Ph.D., is R&D director of Fluigent, whose solutions are based on the company’s pressure-based flow technology (FASTAB™, for fast stabilization) and consist of different tools including Microfluidic Flow Control Systems (MFCS™) that are designed to independently control up to eight channels of a customer’s microfluidic system.

According to Dr. Le Nel, Fluigent’s FASTAB technology allows fast response and settling times (less than 200 milliseconds in specific conditions), pulseless delivery, and excellent flow stability (0.1% CV) compared to what is possible with syringe and peristaltic pumps. These characteristics are distinct advantages for the Fluigent technology and also allow it to be used successfully in long-period experiments.

In addition, she explained, the Fluigent technology provides precise pressure (within 0.1% full scale) in different pressure ranges, positive and negative (0–25 millibars, 0–69 millibars, 0–345 millibars, 0–1 bar, and 0–7 bars for the positive pressures), to meet different pressure demands and to control flow rates ranging from sub-nL/min to thousands of mL/min.

“Our flow-control and fluid-handling solutions are suited to many different applications: droplet manipulations, cell and particle applications, and chemical applications, among others,” Dr. Le Nel noted.

Droplets are a key tool in many microfluidic applications, and the Fluigent MFCS are especially suited to cell manipulation, Dr. Le Nel pointed out. The need of a highly stable and pulseless flow is very important when working with cells in order to avoid shear stress, cell damage, and to be able to fully control the flow of the cells (including stopping their flow). For all these reasons, the MFCS are especially adapted to cell manipulation, offering: a pulseless flow, an instantaneous control of the flow, and independent channels to stop the flow of cells anytime that is desired.

Dr. Le Nel described Fluigent’s MAESFLO software, which works with the company’s FASTAB technology and products to enable the control and monitoring of flow with a high level of precision and short response time. This software allows complex experiments with different pressure requirements to be completely automated using the Fluigent technology. Dr. Le Nel pointed out that the MAESFLO software has recently been upgraded to provide a feedback loop between external flow sensors and the pressure pump in order to monitor and control all the flow-relevant parameters: flow rate and pressure.

Fluigent’s MFCS tool was designed to control up to eight channels of a customer’s microfluidic system. (1) Users enter a pressure value for each MFCS-FLEX channel with MAESFLO software. (2) MFCS-FLEX will immediately and automatically provide the requested pressures. FASTAB technology uses a pneumatic path combined with a fast regulation algorithm. MFCS-FLEX regulates the pressure from a pneumatic pressure supply. (3) By connecting the MFCS-FLEX channels to the FLUIWELL, the pneumatic pressure allows users to precisely and smoothly control the liquid flow. Each FLUIWELL reservoir includes one pneumatic pressure connection and one microfluidic output. (4) By directly connecting the FLUIWELL to an application, samples flow from the tanks to the application.

Software Allows Auto-Adjustment of Acoustic Liquid Handling

Joe Barco, Ph.D., product manager of the Echo liquid handlers produced by Labcyte, recently talked about the company’s new software advance that reportedly enhances the capabilities of these devices.

Echo liquid handlers transfer volumes as small as 2.5 nL by the use of acoustic energy, thus avoiding the cost and contamination risk commonly associated with other liquid-handling systems requiring the use of tips.

The software feature is called Dynamic Fluid Analysis™ and it permits the Echo liquid-handling systems to adapt to fluid property changes such as surface tension and viscosity on a well-by-well basis, and to adjust the necessary sound energy in real time with no operator intervention required. This eliminates the operator calibration required by other liquid-handling devices.

“Developing calibrations for liquid handlers becomes more complicated at lower volumes. Echo instruments remove this hassle,” notes Dr. Barco.

He sees the new feature set as having particular application in sample transfers where reagents are highly variable, such as in crystallography, and in cell lysate, serum, and plasma transfers.

The Echo liquid-handling platform uses acoustic energy to transfer liquids. Sound waves eject precisely sized droplets from the source liquid into a microplate, microscope slide, or other surface suspended above the source.

























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