January 15, 2012 (Vol. 32, No. 2)

Academic and industrial laboratories are under pressure to be more productive with limited resources. Laboratory automation is a critical and effective solution to the needs of modern science.

It is difficult to imagine a modern laboratory without at least one piece of automated equipment. Increased throughput and decreased volume dictate the need for efficient robotics and precise connectivity between analysis steps. The former “Lab Automation” conference, now known as SLAS, takes place in San Diego next month and will spotlight the breakthroughs that push the boundaries of laboratory development processes.

Last year, the Association for Laboratory Automation merged with the Society for Biomolecular Screening (SBS), launching the Society for Laboratory Automation and Screening (SLAS). SLAS is dedicated to advancing scientific research and discovery through laboratory automation and screening technology. Technologies profiled in this article represent just a few highlights from this exciting field.

“Most early microfluidic applications focused on separation and analysis of biochemical molecules,” comments Cristian Ionescu-Zanetti, chief technology officer at Fluxion Biosciences. “However, many cell-based assays also benefit from the advantages of the microfluidic approach, for example applying the shear flow to maintain physiologically relevant conditions or addressing single cells.

“Our development focused on cell-based assays to achieve the throughput and cost of biochemical assays with optimal biological relevance.” Fluxion Biosciences designed several proprietary microplates to enable cell-based assays in the microfluidic format.

“Biomolecular microfluidics trends toward lab-on-the-chip format, where all the valves and processors are integrated on the disposable chip. While this approach provides maximum automation, it also leads to high costs of disposables,” continues Dr. Ionescu-Zanetti.

“We pursued a radically different philosophy of product development: lab-off-the-chip.” Fluxion plates contain a network of passive microfluidic channels, while the controls reside within the external hardware station.

The movement of fluids is driven by air pressure applied to the sample loading chambers. The pressure pushes the sample out of the chamber and through the microchannel. If the cells are bound to the magnetic beads, magnetic capture occurs within the microchannel. The same microchannel may be connected to various other chambers, pneumatically actuated as needed for a particular application.

In addition to cell separation, Fluxion’s applications include cell imaging, automated patch clamping, cell migration assays, drug activity studies, and others.

“Isolation and concentration of circulating tumor cells (CTCs) hold tremendous potential in cancer companion diagnostics,” says Dr. Ionescu-Zanetti. “It is also technologically challenging. Microfluidics enables passing a few cells at a time through the separating region while maintaining the individual cell microenvironment.”

The company’s IsoFlux™ Rare Cell Access System captures the labeled cells on a small polymeric disc that forms the roof of the microfluidic separation channel. When the disc is decoupled from the channel, the cells remain in a small droplet ready for downstream assays.

This combination of microfluidic enrichment and delivery in a small volume opens the doors for continuous cancer monitoring via “liquid biopsy”. While frequent tissue biopsies to monitor cancer recurrence are often not practical, identification of tumor cells via simple blood draw may become routine in clinical patient management.

CellSpot technology isolates cells magnetically on a small disk that forms the roof of Fluxion Biosciences’ IsoFlux microfluidic flow channel. Once a run is complete, the disk is removed from the flow channel, and the intact target cells are transferred into a microcentrifuge tube in less than 20 µL, ready for downstream assays such as PCR, FISH, immunoassays, and sequencing.

Point-of-Care Diagnostics

“I tend to agree with Dr. Ionescu-Zanetti about the importance of the cost of disposables, but also want to emphasize that there is a series of additional key requirements for point-of-care diagnostics,” says John McDevitt, Ph.D., Brown-Wiess professor of bioengineering and chemistry, Rice University, and scientific founder of Force Diagnostics.

“We are now targeting the development of transformative medical microdevice technologies capable of producing lab quality results anywhere. We estimate that the cost of point-of-care applications, especially for resource-poor areas, should be just a few cents. Ability to mass produce the reagents, simplify chip design, and use plastics instead of silicone is how we can bring the cost down to this target range.”

Dr. McDevitt’s lab develops programmable bio-nanochips that combine novel microsensors with artificial intelligence. These sensors, which have a capacity to learn, allow for rapid analysis of just about any soluble chemistry, including biological molecules and chemical compounds, and can cover major cell-based assays.

For soluble chemistries, in the product’s reaction core are porous agarose beads woven from nanometer-size fibers. For cells, supported membranes are employed. The capture of analytes and cells is optimized by manipulating the pore and bead sizes. Every 30 nanometers of each fiber is coded with a capture reagent, such as an antibody.

“The bead is essentially a microsponge with a nanonet, with binding points distributed throughout. This structure enables 10,000-fold signal amplification in comparison with a standard ELISA plate,” says Dr. McDevitt. A single bead is loaded in each well of a plastic chip; each bead may have different derivatization, so that multiple analytes may be analyzed simultaneously.

“Our plug-and-play design can be utilized in place of almost any analytical instrument used for discovery, validation, or clinical applications. If the same tool is used throughout the development pipeline, the cost and interfacing complexities are eliminated, and the process can be significantly accelerated,” says Dr. McDevitt.

“The simplicity of this platform—which is now in six major clinical trials—will enable many more biomarkers to be developed for clinical applications.”

Simple sampling interface is another characteristic feature of Dr. McDevitt’s bio-nanochip, he asserts. To run the HIV diagnostic chip, a drop of blood is obtained by a needle stick and transferred into the key-hole port by a simple capillary tube. Analysis is performed by a portable “biotometer” with minimal user involvement.

With the aid of Force Diagnostics, the HIV diagnostic system is now in a clinical trial in Africa. Another advanced bio-nanochip application is POC diagnosis of heart attack based on gum swab. Multiple other salivary diagnostic products are in development.

A programmable bio-nano-chip system customized for measurement of cardiac biomarkers using oral fluid samples by researchers at Rice University.

Finding Perfect Protein Crystals

Protein crystallography is an important tool for investigation of biological molecules. Protein molecules can crystallize under certain conditions, forming regular lattices composed of multiple copies of the same molecule. When such a crystal is irradiated by a photon beam (x-ray), the photons scatter from the atoms and concentrate in sharp intense spots.

The molecular structure can be determined by analysis of the intensities and positions of the diffraction spots. Co-crystallization of interacting biomolecules or a biological molecule and a chemical drug helps elucidate the protein’s detailed function or find the inhibitor of this function.

Because of the complexity of such experiments, many structural studies require screening hundreds of samples until the crystal with the best resolution is identified.

Traditionally, the rate-limiting step of crystallography studies was a manual sample-loading step. Precise manipulations are required to extract the protein crystal from a liquid nitrogen storage container with special forceps and to place it in the exact position where the crystal can interact with the x-ray beam.

“Automated mounting was a key step in developing fully automated sample screening,” says Clyde Smith, Ph.D., senior staff scientist, Stanford Radiation Laboratory. “Without this manual step, remote data collection also became possible. Now our collaborators all over the world are able to visualize and collect the data stream via a computer interface.”

Stanford Automated Mounter is a robotic arm capable of locating the samples under liquid nitrogen, extracting the sample from storage cassette, and precisely transferring the crystal to the goniometer, a platform that rotates the crystal exposing it to the x-ray beam from various angles.

The robot uses a set of coordinates to find the sample location within the liquid nitrogen dewar. It is also equipped with fast response sensors, preventing it from bumping into the hardware. Periodic calibration ensures precision operation with no sample loss.

“Structure determination of RNA polymerase, a large and complex enzyme, was made possible only by automation of the crystallography process,” says Dr. Smith.

“The team had to screen several hundred samples to find a single crystal with required resolution. Manual screening on this scale is simply not practical.” To date, over 300,000 samples have been screened using Stanford Radiation Laboratory’s automated process.


Faster Stem Cell Therapy Development

Discovery of compounds affecting growth and differentiation of pluripotent stem cell is an active area of research. But the process of adaptation of stem cell cultures to the high-throughput format required for drug discovery has been slow.

Stem cells are difficult to grow, and the cultures have a tendency to spontaneously differentiate, or they do not differentiate consistently. To induce differentiation, embryonic stem cells are typically grown in hanging drops suspended from the lids of Petri dishes. In these drops, ES cells form embryoid bodies that need to be carefully collected and transferred into tissue culture plates for adherence.

“We started with a clear unmet need: to translate this manual process into an HTS format,” comments Michael Kowalski, Ph.D., senior applications scientist at Beckman Coulter.

“Moreover, we wanted to automate the optimization of the differentiation process itself. This means testing numerous combinations of differentiation factors at different concentrations.”

The BioRAPTR FRD noncontact dispenser became the key component of the solution. The device is able to dispense specific volumes of reagents in each well of a 384-well plate individually. Polypropylene plates were chosen because of low cell adherence, resulting in over 99% of wells forming a single embryoid body.

A Biomek Laboratory Automation Workstation transferred the embryoid bodies into the gelatin-coated plates. Again using the BioRAPTR, the team was able to test growth factors in hundreds of conditions on the same plate with the goal of identifying the set optimal for cardiomyocyte differentiation.

The “procardiomyocytic” compounds were selected from the literature. The statistical software created combinations of these factors resulting in an easily visualizable matrix. Automated Assay Optimization for BioRAPTR software converted the matrix into the dispense volumes. The BioRAPTR dispenser completed 914 individual pipetting steps in 7 minutes, Dr. Kowalski reports.

“We started with 6 percent differentiated cardiomyocytes,” he continues. “After completing the optimization schema, we found a set of conditions that can reproducibly generate over 40 percent cardiomyocytes on the plate. Automation provided a remarkable consistency for this highly complex biological process.” The team is currently working on optimizing differentiation of pluripotent human cells.

Post-translational modifications of chromatin facilitate or suppress gene expression. Changes in gene transcription due to dysregulations of these modifications have been linked to certain disease pathologies, primarily to various types of cancers. Consequently, enzymes that promote post-translational modifications have become important targets of drug discovery efforts.

According to Beckman Coulter, its BioRAPTR FRD noncontact dispenser is able to dispense specific volumes of reagents in each well of a 384-well plate individually.

Post-Translational Modifications

The protein methyltransferases (PMTs) methylate specific locations on histones using a universal methyl donating co-factor, S-adenosyl methionine (SAM). At least for one disease, MLL rearranged leukemia, a change of function of a PMT enzyme is recognized as a causative element of this malignancy.

“In MLL, recruitment of a specific PMT called DOT1L results in overexpression of leukemogenic genes,” says Margaret Porter Scott, director of biochemistry and molecular pharmacology, Epizyme.

“Finding small molecule inhibitors of this and other PMTs may lead to development of a new class of targeted cancer therapies as personalized therapeutics for genetically defined cancers.”

Epizyme has established an integrated discovery platform for PMT inhibitors. The new molecules from each PMT project are continuously aggregated and subjected to highly parallel pipeline analysis, including inhibition assays in a panel of many disease-relevant PMTs. As a result, Epizyme has now created a large and highly enriched proprietary library of novel PMT inhibitors.

“Different PMTs require particular substrates for maximum activity,” continues Dr. Porter Scott. “Some of them only work on full-length nucleosomes. When we started designing our screening schema, there were no published methods for assaying inhibitor libraries against a full-length nucleosome. We had to develop proprietary methods of PMT screening.”

Epizyme uses a battery of substrates ranging from native and recombinant histones to synthetic peptides in which methylation sites are systematically modified. Once the correct substrate is identified, screening moves into a high-throughput format.

Two advanced therapeutic leads generated by this process target DOT1L and EZH2, an enzyme implicated in non-Hodgkin lymphoma and several solid tumors; both are in preclinical development.

“As we learn more about the family of PMT enzymes, we start seeing analogies with the family of protein kinases. Just from one kinase co-factor, ATP, many different kinase inhibitors were discovered and are in clinical use today,” says Dr. Porter Scott.

“Similarly, our strategy is to target the PMTs as a family of SAM-utilizing enzymes, making full use of lessons learned from kinases and exploiting technological platforms that allow parallel processing of multiple enzymes of similar mechanism.

“Using this strategy, we have discovered and are developing small molecules with multiple modes of inhibition including competitive, noncompetitive, and uncompetitive antagonists of PMTs,” concludes Dr. Porter Scott.

One of the key factors impacting the development and evolution of automated laboratory equipment is the trend toward miniaturization. [Caleb Foster/fotolia.com]

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