February 15, 2009 (Vol. 29, No. 4)

Gail Dutton

Novel Iteration Reduces Amount of Sample and Reagent Used and Speeds Processes

In the unending quest to do more with less, the biotech industry has embraced low-volume liquid handling, and is continuing to advance the tools and techniques that make it a laboratory mainstay. With microtiter well volumes often in the nanoliter range, the benefits are obvious. Researchers use much less sample and reagent and thereby cut expenses. They also get results faster by allowing more assays to be run with a given quantity of fluid.

Scienion recently launched the sciSWIFT sterile washfree identifiable fluid technology. It integrates storage and dispensing, which allows tasks that traditionally require two tools to be performed with one, according to Holger Eickhoff, Ph.D., CEO. These integrated liquid storage and dispensing cartridges are based upon disposable-pen technology, and use a 30 microliter reservoir that “can be filled on-site, or prefilled with a sample, reagent, or compound,” he says. They can be filled sterile, and work without tubing, thus removing one avenue for possible contamination. A sonicator resolves precipitates.  The design eliminates heat seals and helps ensure sample integrity.

“The sciSWIFT dispenses solution in volumes ranging from 100 picoliters to 1 microliter,” Dr. Eickhoff says. It boasts a closed environment and minimal dead volumes. Mounted into a sciSWIFTER, 16 different substances can be dispensed in parallel into many multiwall plate formats, with online drop control and optical volume determination built-in for each nozzle. 

The sciSWIFTER, marketed by Scienion and Matrical Bioscience, operates as a stand-alone device, but can be integrated into automatic robotics platforms. Because the devices function as both storage and dispensing platforms, primary and secondary assays, as well as dose-response tests can be made from the identical sample or dilution, with CVs of 2%.

This device includes RFID technology to identify and document content throughout the liquid-handling process. “RFID tagging isn’t widespread in the industry,” Dr. Eickhoff says, although its use is gaining ground. When integrated into sciSWIFT, it allows detailed information—for example, the compounds, when and where it was filled, and each time it was used—to be documented, in a way that remains with the compound as long as it is in that device. Consequently, “you can track usage for better process control,” Dr. Eickhoff points out. 

At a workshop held last year, Promega teamed with Labcyte and BMG Labtech to showcase the equipment needed to run Promega’s GloSensor cAMP (cyclic adenosine monophosphate) assay for automated GPCR signaling studies in 1,536-well format. “A 1,536-well plate is a good format. It’s relevant to a lot of screens, although the cAMP assay can be used in any format plate, from 96 wells to 3,456 wells,” according to Sarah Shultz, senior automated solutions scientist.

Higher density formats can be particularly challenging for users because the small size of the wells necessitates extraordinary accuracy in liquid volume handling and analysis. Promega’s goal in arranging the workshop was to simplify clients’ instrumentation decisions by assembling and demonstrating an optimal system and then put it through its paces for potential users. The workshop assayed against the LOPAC 1280™ library of pharmacologically active compounds. “Results show that it’s very fast and easy to use. We saw expected results,” she says, and used known controls. The entire process, from adding the cells to screening, takes less than 30 minutes.

In that workshop, Promega demonstrated that its GloSensor cAMP assay is as effective in 1,536-well plates as in 96-well plates, explains Shultz. The GloSensor cAMP Assay is a nonlytic, live-cell, bioluminescent assay that directly monitors the intracellular kinetics of cAMP, a key signaling molecule for many G-protein coupled receptors, Shultz explains. The assay has “a very high signal to noise ratio,” she adds.

For Labcyte, the project helped show that the acoustic droplet ejection technology used in its Echo series of dispensing equipment was as effective for aqueous solutions as it is for DMSO, even in 1,536-well or higher density plates. Acoustic droplet ejection has the advantage of taking samples without touching the sample material, which reduces the risk of contamination and also eliminates waste.

Using the Echo 555 with the Promega GloSensor cAMP Assay lets researchers easily run assays that otherwise would take a lot more work, says Howard Lee, applications specialist, Labcyte. “Promega needed a system to reliably dispense volumes into 1,536-well plates,” Lee explains. That level of density requires very precise instruments. “Accuracy is within eight percent of target volume and CV—the variation between droplets—is less than eight percent, too. We don’t use mixing with our assays,” Lee explains, “but there’s no reason users couldn’t take the plate out for that.”

BMG Labtech provided the reader for that project, the PHERAstar Plus. This simultaneous dual-emission high-throughput screening microplate reader includes fluorescence intensity, FRET, fluorescence polarization, time-resolved fluorescence, TR-FRET, laser-based AlphpaScreen®, advanced luminescence, BRET, UV/Vis absorbance, and advanced fluorescence polarization reading modes.

The PHERAster Plus combines separate mode-specific measurement electronics and a unique optical lens assembly that accurately reads the very small—0.1 mm wide—bioluminescent signals from samples in 1,536-well plates, according to E.J. Dell, Ph.D., business and applications development scientist. An automatic Z-height adjustment with a 100 micron resolution allows researchers to easily find the optimal height of their signal in solution in all plate formats up to 3,456.

The reader can collect kinetic data as quickly as 50 reading points per second, or as slowly as one measurement every 2.5 hours. It also offers TR-FRET decay monitoring and can collect TR-FRET curves with two microsecond resolution in real time. Output options include real time, raw data, graphs, and color pictorials. “The PHERAster Plus can read a 1,536-well plate in luminescence mode in less than 1.5 minutes. Sensitivity, determined by its limit of detection in luminescence mode, is less than 10 attamoles per well of ATP,” Dr. Dell says.

The point-of-care diagnostic market has grown from a few applications 10 to 15 years ago to more than 200 today, according to Tom Tisone, Ph.D., president, BioDot. BioDot, which initially concentrated on medical diagnostics, now sees potential clients in agriculture, industrial applications, and even biowarefare.

The continuing challenge, Dr. Tisone says, is trying to reduce the volume of sample and reagents used, which means smaller and smaller analytes and, therefore, smaller electrode patterns that can use smaller volumes of fluid.

BioDot takes a noncontact, ink-jet approach to dispensing solution on virtually any substrate, including membranes, microtitre plates, and glass slides.  Dispensing uses a microsolenoid coupled to a positive displacement system—a high-resolution syringe pump—for exact drop volumes. Three different configurations are available: Front Lines, AirJet Quanti, and BioJet Quanti. “The unique aspect is that these are fully quantitative systems. They have low coefficients of variation, in the one percent range for microliter volumes and in the four to five percent range for nanoliter volumes,” Dr. Tisone elaborates.

BMG Labtech’s PHERAstar Plus is a multidetection HTS microplate reader that features simultaneous dual- emission detection in all modes.

Labcyte’s acoustic droplet ejection technology focuses ultrasonic acoustic energy into a fluid sample to eject small droplets of liquid from open wells.

Assay Miniaturization

Thermo Scientific recently launched a bulk reagent dispensing device called the Multidrop Combi. Researchers at Merck, led by Dongyu Sun, Ph.D., tested it for assay miniaturization and optimization applications and found that it demonstrated acceptable accuracy and precision at the lowest specified volume—50 nanoliters to 40 microliters—for aqueous solutions dispensing. The device is capable of dispensing liquid ranging from 0.5 to 2,500 microliters onto plates ranging from 6 to 1,536 wells with heights from 5 to 50 mm.

Dr. Sun adds, “this platform is suitable for assay development and screening of miniaturized plate formats.”

The Multidrop Combi is designed to save the reagent by featuring minimal dead volume and backflushing that minimizes the need for re-runs. The device can be added to a lab-automation platform for high-throughput 24/7 operations.The cassettes are autoclavable and the equipment is designed for easy cleaning For traceability, its own SMART features counts the usage of the dispensing cassette, according to Thermo Scientific.

Scienion’s sciSWIFT sterile wash-free identifiable fluid technology is available in storage volumes between 30 µL (left) and 1 mL (right).

BioDot’s BioJet technology is based on the combination of a solenoid ink jet valve coupled to a positive displacement syringe.

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