Microarrayers on the Spot

Photo: Courtesy of Gene Machines SEEING SPOTS: Microarraying devices automate the task of arraying the hundreds or thousands of samples typically found on biochips. Shown here is Gene Machine's OmniGrid Accent, a contact-based printer with 48 pins that is capable of placing over 100,000 75-µm features on a standard microscope. Today's "big science" is all about high throughput, a concept elegantly epitomized by the DNA microarray. Biochips let researchers analyze the expression of thousands of genes simultaneously for applications ranging from genomic research to drug development to diagnostics. But the sheer power of this technology comes at a price, in terms of both expense and experimental complexity. Fundamentally, the quality of the microarray itself directly affects the reliability of the resultant data. With commercial arrays, researchers generally have little control over quality. But when they make their own arrays, quality control becomes a central issue. GEARING UP Scientists should first decide on the support and surface chemistry. The support must be amenable to any subsequent surface chemistry modifications, able to withstand the printing process, and stable under hybridization conditions. As researchers commonly probe arrays with fluorescently labeled samples, the support should exhibit minimal background fluorescence. The standard glass microscopic slide meets all these basic criteria. When attaching unmodified nucleic acids via ionic interactions, scientists often use amine- or lysine-coated slides, turning to aldehyde-functionalized slides to attach amino-modified probes. Alternatively, slides can be coated with thin layers of a polymeric reagent such as acrylamide. Regardless of the chemistry, the surface treatment should be homogeneous to avoid variations in probe immobilization, and must maintain the substrate's low background fluorescence. Sample preparation is also important. The target nucleic acid should be homogeneous, free of debris, and properly buffered. Numerous factors affect spot size and quality, including the printing mechanism, substrate characteristics, environmental factors such as humidity, and sample-solution properties such as salt concentration and viscosity. Researchers should also consider any potential variations introduced by the target nucleic acids themselves. For example, were consistent tissue-handling and target-preparation procedures employed for each sample being compared? Investigators must also give thought to array layout. If a single pin is used to array every sample, then each sample in the array can be positioned to correspond exactly to its position in the source plate. This approach simplifies analysis but is time- consuming. Using print heads with multiple pins is much faster, but the array's features will not correspond to sample positions in this case, so more forethought regarding layout is required. Many investigators employ tracking software to follow samples throughout the microarraying process. Photo: Courtesy of Brooks-API Automation HELPING HAND: A high-throughput microarrayer from Brooks-PRI Automation's Intelligent Automation Systems division GETTING SPOTTY Researchers can choose among a variety of printing technologies with different characteristic print speeds, positional resolutions, and deposition volumes. Most broadly, microarrayers fall into two categories: contact-based or noncontact-based instruments. Contact-based systems employ microspotting devices that transfer samples from reagent trays (usually microplate wells) onto solid surfaces. Whereas solid pins are designed to print one microarray per sample load, mechanisms that enable more than one printing per load are becoming increasingly popular, and include split pins, capillaries, tweezers, quills and other devices that feature a reservoir for sample uptake. Noncontact microarrayers depend on inkjet-printing technologies; this approach eliminates the microarray irregularities associated with mismatched or damaged pins. Most currently available inkjet-based microarrayers employ piezoelectric materials that, upon application of appropriate current, effect droplet formation by expanding and contracting. When comparing microarraying technologies on the market, expect to encounter different viewpoints along the way. Companies that use spring-loaded pins tout the speed and consistency of this approach, whereas those using other mechanisms emphasize the potential of tapping-induced substrate damage. These differences are not just academic. Contemporary inkjet microarrayers are generally more expensive than contact-based instruments but can load larger volumes of samples, feature faster print speeds, and can precisely deposit a wide range of user-specified volumes. In contrast, contact-based technologies are less expensive, offer greater spotting densities, and are readily scaled-up for high-throughput needs. THE SPIN ON PINS Contact-based microarrayers use pins to spot samples on the arrays. Some companies manufacture their own pins, whereas others purchase them from outside suppliers. Each pin type has an intrinsic variablility; researchers must ensure this will not hinder any intended applications. Some microarrayers are compatible with a variety of spotting technologies, giving researchers the option of purchasing pins from their manufacturer of choice. In some cases, users can alter the numbers and/or types of pins or other printing devices employed in a microarrayer. A number of microarrayer manufacturers use Sunnyvale, Calif.-based TeleChem International's Stealth Micro Spotting Device. These stainless steel pins contain chambers that collect a user-specified, set volume of 0.1-1.0 µL when dipped into a liquid sample. Stealth pins have flat tips with customizable widths to deliver spots with diameters of 50-300 µm. Direct contact between the liquid and a solid surface results in drop deposition. This technology enables multiple prints per load and is flexible, in that both the number of pins as well as the spot sizes can be varied. Also popular are the stainless steel MicroQuill® array pins from Tempe, Ariz.-based Majer Precision Engineering, which contain sample reservoirs enabling more than 400 repeat-spotting events. These pins uptake roughly 0.25-0.5 µL and deliver less than 1.0 nL per spot, producing spot diameters as low as 75 µm. The company's recently introduced MicroQuill HTG (Harder Than Glass™) features a high strength alloy coating for increased durability. Other companies also offer solid and repeat-spotting pins, including Point Technologies of Boulder, Colo., and V&P Scientific of San Diego, both of which specialize in customized production. Photo: Courtesy of Genomic Solutions Genomic Solutions' GeneTAC Library Management System Other distinguishing options include microplate capacity of slide and source, bundled control software, source-plate handling, and barcode reading. User-definable parameters include those for array production, tip washing and drying, spot densities, and total number of spots per slide. Many companies design their instruments to minimize losses due to evaporation and contamination from airborne contaminants. Such environmental control measures include enclosed sample chambers controlled for humidity and/or temperature, as well as high-efficiency particulate air (HEPA) filters. Additionally, some systems feature automated microplate lid removal and replacement to further bridle evaporation and contamination. MICROSPOTTER'S MARKETPLACE Piscataway, NJ-based Amersham Biosciences' Lucidea™ Array Spotter employs a stainless steel print head containing 24 matched capillary tube pens. Multiple prints can be carried out with each load, and the pens can handle a variety of sample viscosities. Apogent Discoveries of Portsmouth, NH, offers two microarrayers through its Cambridge, UK-based BioRobotics division. The Microgrid 610 is for high-density, high-throughput applications, whereas the Microgrid Compact is a lower-throughput instrument for investigators using smaller volumes. GeneMachines of San Carlos, Calif., offers two microarrayers. The OmniGrid Accent™ is a benchtop unit, and the high-throughput OmniGrid uses an optional automated plate handler offering random access delivery and retrieval of 96- or 384-well format plates. Genetix of Hampshire, UK, offers the QArray, QArraylite, and QArraymini. All three instruments are compatible with a variety of commercially available spotting devices. The QArraymini is a benchtop unit with a flexible 48-pin print head usable in a variety of pin modes. The QArraylite is suitable for high-throughput applications, and employs a print head with 16 or 24 solid or repeat-spotting pins. The high-end QArray employs a 24-pin print head that is also operable in a variety of modes. The GeneTAC™ G3 Library Management System from Genomic Solutions of Ann Arbor, Mich., employs solid titanium pins that use less than one nanoliter of sample per "dip and print" operation. This benchtop instrument also includes accessories for picking colonies, selective re-arraying, and other library management functions. Genomic Solutions recently acquired Irvine, Calif.-based Cartesian Technologies, and offers Cartesian printers. The PinArray™ (PA) family of contact arrayers utilizes TeleChem's Stealth pins. Researchers can choose from three PA platforms: the MicroSys 5100, a benchtop instrument for individual laboratory applications, and the PixSys PA and ProSys PA workstations for medium- and high-throughput microarraying, respectively. The SQ series of dispensing workstations employs Cartesian's synchronized nanoliter-quantitative aspirate-dispense (synQUAD™) technology for liquid handling, which scientists can use for high-throughput generation of relatively low-density microarrays. SynQUAD-based instruments coordinate the actions of a microsolenoid valve, syringe pump, and XY-positioning system. The syringe pump first displaces a user-specified, quantitative volume of fluid, which creates a positive pressure that is then released in a short-burst actuation of the normally closed microsolenoid valve. The resultant pressure wave causes a droplet to be ejected from the tip. Grosserkmannsdorf, Germany-based GeSIM offers the Nano-Plotter, a modular, piezoelectric-based microdispensing system featuring two interchangeable dispenser sets: one with smaller tips suitable for DNA microarraying, and the other with larger tips suitable for higher-viscosity solutions. Scientists can exchange the piezoelectric tips with commercially available plastic tips for pipetting applications. Yokohama, Japan-based Hitachi Software Engineering offers the SPBIO II Spotter through its MiraiBio division in Alameda, Calif. The SPBIO II uses one of its two microrobotic arms for transferring microplates to a station for lid removal and barcode reading, then to the sample loading area. The other arm loads and unloads microarray slides. The system has four zones separately controlled for humidity, and three that are independently regulated for temperature. Based in Cambridge, Mass., Intelligent Automation Systems, a division of Brooks-PRI Automation, offers a high-throughput microarrayer designed for mass production. The microarrayer is supplied with 32 or 48 solid-pin print heads, which can be configured for different grid patterns. Ebersberg, Germany-based MWG-Biotech offers the Affymetrix 427™ Arrayer, which employs Pin-and-Ring™ spot-deposition technology. The print head has eight user-replaceable pin/ring pairs; the pin and ring of each pair can be moved up and down separately but are kept in constant relation to one another with respect to the XY plane. A ring is immersed in a sample well while an aliquot is withdrawn and held in the ring's center via surface tension. The spring-loaded pin is driven through its associated ring, thus effecting spot deposition. The system can spot fluids with significantly different viscosities on a variety of flat-surface substrates. Packard Bioscience of Meriden, Conn., now part of Boston-based PerkinElmer Life Sciences, has developed both contact- and inkjet-based microarrayers. The contact-based SpotArray™ 24/72 microarrayers accommodate up to 48 TeleChem Stealth pins. An optional three-part print head creates more compact arrays by selectively lifting pin subsets during printing. The SpotArray Enterprise is a high-throughput piezoelectric-based system that can deliver a wide variety of sample types, including proteins, to virtually any substrate. The SpotArray Enterprise features a durable stage designed for a mass-production environment, with optional custom decks. A "quick-change" substrate platen allows one substrate set to be prepared off-line while the system processes another. PerkinElmer also offers the BioChip Arrayer™, a benchtop piezoelectric-based microdispenser for lower-throughput applications. Carlsbad, Calif.-based RoboDesign International's RoboArrayer™ system for high-density microarraying offers several customizable features, from the multilevel graphic user interface to environmental control modules. An optional robotic work cell automates lid removal as well as plate loading and unloading from sample plate "hotels" arranged in dual carousel-like formations. The robotic five-axis arm can randomly access up to 60 plates. TeleChem International's SpotBot, is a "personal microarrayer" for lower-throughput applications. According to the company, it can print virtually any biomolecule, including DNA, proteins, lipids or carbohydrates, to a wide variety of solid surfaces. A platen cooling system for unique protein applications is in development, and should be released before the end of the year, according to the company. Virtek Vision International of Ontario touts its ChipWriter™ Professional Arrayer (Pro) as an "all-in-one robotic system" for genomic and proteomic applications. An optional four-channel digital pipette module enables rapid re-arraying. The ChipWriter Compact is a benchtop system for those needing an economical microarrayer. V&P Scientific of San Diego offers a hand-held, manual arrayer for smaller laboratories with low-throughput needs. V&P also offers various accessories, such as a humidity chamber and wash station. The same basic microarraying unit is also available in different packaging formats from XENOPORE of Hawthorne, NJ, and Schleicher & Schuell of Dassel, Germany. ARRAYS AND BEYOND Even lower-throughput laboratories can now economically produce microarrays in-house, but do-it-yourself microarray production isn't for everyone. After all, printing arrays with multitudes of spots requires as many probes. Just maintaining the required clone sets, performing needed PCR reactions and/or synthesizing oligonucleotides can be time-consuming and laborious. Many companies, including some of those highlighted in this article, provide an alternative option by offering custom-ordered and/or premade stock microarrays. All of the instruments overviewed in this article serve to microarray pregenerated probes. Oligonucleotide probes can also be synthesized directly onto microarray supports, but microarrayers capable of in situ deposition are not commercially available at this time. Researchers can purchase arrays generated in situ from Santa Clara, Calif.-based Affymetrix, which uses a photolithography process, or Agilent Technologies of Palo Alto, Calif., which employs an inkjet-based method. Some researchers are turning to alternative, "nonprinting" technologies for array generation. For example, San Diego-based Nanogen's NanoChip® system uses microelectronics to direct biotinylated DNA probes to the desired destinations on microchips, where they are immobilized via interaction with chip-bound streptavidin. Deborah A. Fitzgerald (biotechnology@consultant.com) is a freelance writer in Birmingham, Ala. References 1. D. Rose, "Microfluidic technologies and instrumentation for printing DNA microarrays," in: Microarray Printing Technologies, M. Schena, ed., Natick, Mass.:Eaton Publishing Co., 2000. 2. M. Sánchez-Carbayo et al., "DNA microchips: Technical and practical considerations," www.bentham.org/coc-sample/carbayo/carbayo.htm. 3. Y.F. Leung et al., "Guide to microarray hardware--A researcher perspective," www.lab-on-a-chip.com/suppliers/comptab.html. 4. E. Zubritsky, "Spotting a microarray system," Analytical Chemistry, 72:761A-7A, 2000. 5. D.D.L. Bowtell, "Options available--from start to finish--for obtaining expression data by microarray," Nature Genetics, 21(suppl):25-32, 1999.

Microarrayers on the Spot

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Photo: Courtesy of Gene Machines
SEEING SPOTS: Microarraying devices automate the task of arraying the hundreds or thousands of samples typically found on biochips. Shown here is Gene Machine's OmniGrid Accent, a contact-based printer with 48 pins that is capable of placing over 100,000 75-µm features on a standard microscope.