The operational space of sample preparation is huge, with tens of thousands of possible starting materials and hundreds of relevant methods. The desired readout adds a third dimension to the decision matrix. In all cases, the objective is to “clean up” the sample, so the analyte of interest is preserved to the greatest degree possible, while removing potential interferences. Within that context, automation seeks to reduce human contact at critical points, thereby gaining speed, throughput, and reproducibility while eliminating human error.
Preparing samples for chemical analysis involves routine methods (or a combination thereof), for example extraction, digestion, grinding, preparative chromatography, filtration, dilution, reagent addition, crystallization, distillation, and other processes. Many of these operations are fully manual, while some occur nearly completely within robotic platforms. In either case, sample prep is straightforward and familiar.
Molecular analytic workflows present a different set of challenges, notes Mark Dupal, global portfolio manager for microfluidics and automation at PerkinElmer (Hopkinton, MA). Although many of these methods are already automated, sample collection and preparation are labor- and resourceintensive due to sample complexity.
Sample prep leading to next-generation sequencing, for example, usually involves the collection of human tissues or body fluids. That’s all well and good when the objective is quantitation of the obvious, for example plain vanilla genomic DNA. “There is plenty of genomic DNA in whole blood,” Dupal says.
Sequencing gets interesting when the object is detecting or quantifying circulating tumor cells or tumor DNA from plasma, or fetal DNA in a mother’s blood. These applications stand sample prep on its head, as “normal” patient DNA becomes not the target but an unwanted obfuscator to detecting rogue genes from cancer cells.
Moreover, the capture and analysis of such diagnostic DNA is confounded by the presence of circulating nontumor DNA arising from a patient’s healthy cells, and the fact that their concentration varies significantly with different cancers and stages of disease.
For pulling such low-abundance analytes from plasma, PerkinElmer offers the chemagic™ purification instrument and accessories. Like many gene preparation techniques, chemagic uses magnetic beads for cell-free DNA. Incubation proceeds normally, but instead of applying a magnet to the outside of the sample vial to collect the beads, the chemagic separation technology inserts magnetized rods into the sample, withdraws them, and then re-suspends the beads in a wash flask by demagnetizing and rotating the rods.
PerkinElmer claims high yields and purities, which is a requirement for medical diagnostics based on sequencing. “Finding these DNA fragments is like looking for a needle in a haystack,” Dupal says. “Plus with all the other cell-free DNA present, you have to extract as much target DNA as possible.”
Sample prep for molecular analytical workflows carries the same conundrum as for chemical-analysis workflows, namely whether to purchase a complete analytical system, including sample preparation, from one vendor or to cobble something together with components from several manufacturers.
Dupal makes a good case for the former:
“The choice typically depends on the end market. Basic researchers are more adept at tinkering, so they often don’t mind having components from several vendors. But in clinical settings, time is money. Relying on a single vendor for troubleshooting—a vendor that can support the entire system—is critical. There’s also comfort in knowing that a manufacturer that designed an analytical workflow has tested it extensively, including taking samples, extracting DNA, quantifying it, and delivering it to the sequencer. Many users appreciate that they’re not working with untested methods.”
What cost barrier?
Small companies and academic labs cite high costs as justification for sticking with manual sample preparation. With its line of liquid-handling robots, Opentrons (Brooklyn, NY) has broken the price barrier. “Our basic system is one-tenth to one-hundredth the cost of premium robots from well-known vendors,” says Opentrons cofounder Will Canine.
The secret is in the sourcing of technology and components. Opentrons has tapped into the extensive and growing hardware supply chain in Shenzhen, China, and embraces open-source technology. The global growth of desktop 3-D printers has made things like precise stepper motors and 3-axis controllers more affordable and higher quality than ever before.
Those same components are available to top-tier robotics companies; and given that, why don’t those companies use them? Canine suggests that companies with high-margin products cannot afford to cannibalize their own products; this is an idea posited by the highly successful book by Clayton Christensen, The Innovator’s Dilemma (https://goo.gl/s8uANp).
“But even this argument is an apples and oranges proposition. The big automation vendors serve laboratories conducting highly complex assays in high throughput. Our customers are the 90 percent that currently do everything by hand.”
In other words, these are small companies and academic groups with modest budgets that could benefit from the time savings and reproducibility of automated sample preparation.
Emerging protein therapeutics present the usual challenges of sequencing and higher-order structures, but often incorporate nongeneric chemical modifications. Agilent Technologies (Santa Clara, CA) introduced its AssayMAP peptide sampleprep system in 2013, perhaps in anticipation of the growing interest in next-gen protein drugs, whose sequencing and mapping is required for full characterization.
The system provides automated protein affinity purification, protein digestion, peptide cleanup through micro reversephase chromatography, and fractionation in preparation for mass-spectrometric (MS) analysis. AssayMAP also prepares samples for discovery and analysis of protein and peptide biomarkers. The platform includes Agilent’s Bravo liquid handler and single-use, ready-to-use cartridges for peptide cleanup and fractionation.
AssayMAP achieves the principal goal of sample prep: eliminating workflow bottlenecks, creating walkaway time, and providing reproducibility. The relevance in today’s proteomics environment is that sample preparation has become a rate-limiting step.
“AssayMAP facilitates peptide affinity purification and fractionation,” says Maryann Shen, PhD, LCMS global marketing program manager. “When a specific antibody is available, operators can use a streptavidin cartridge to immobilize the antibody, followed by affinity capture of the peptide of interest. Multiplexing the capture step is also possible. Once captured peptides elute, they fractionate on the AssayMAP before LC-MS analysis. AssayMAP is appropriate whenever there is a need for affinity purification, fractionation, and desalting.”
For addtional resources on automated sample preparation, including useful articles and a list of manufacturers, visit www.labmanager.com/sample-prep