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Lab on a chip: Poised on the brink Lab-on-a-chip instruments are being manufactured with moderate success, but widespread use of these systems is still in the future. Michael J. Felton
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ab on a chip (LOC) is a very difficult term to pin a meaning on. Not one of the systems currently on the market is an entire laboratory on a chip. All have bench-top sample delivery and detectors. “It’s a flashy word,” says Andreas Manz of Imperial College (U.K.), “ill-defined in terms of scientific content.” Although the term may be useful mainly “for venture capitalists,” Manz says, there is a dearth of functional alternatives. “Microfluidics” merely indicates microstructures carrying fluid, and “micro-TAS” should only apply to analytical monitors, according to Manz. “I would say that [LOC] would be an instrument platform that contains many functions encompassed within it,” says Steven Soper of Louisiana State University. With this as a basis for defining LOCs, why are they of interest, and why are more commercial models available every year? The simplest answer is that these systems have clear advantages over conventional bench-top systems, suggests Soper. This article will discuss systems that perform multiple functions on each chip, a capability that separates these devices from multiwell plates. Microarray techniques for the hybridization and detection of specific DNA, RNA, or proteins will not be included because they represent a field of their own. Chips to prepare for MALDI and other MS techniques are also a well-defined group and are excluded. Although not comprehen© 2003 AMERICAN CHEMICAL SOCIETY
sive, a list of companies with commercial LOC instruments that fit this definition is shown in Table 1. These instruments offer benefits such as interfacing
between functions, reduction in sample preparation, the ability to multiplex, and a decrease in the total time needed for an analysis.
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Table 1. Lab-on-a-chip products
Interfacing
Company
Product
Product description
Agilent Technologies, Inc. 395 Page Mill Rd. P.O. Box #10395 Palo Alto, CA 94303 877-424-4536 650-752-5000 www.agilent.com
2100
The 2100 uses Caliper’s LabChip LOC to analyze DNA, RNA, proteins, and cells.
Caliper Technologies Corp. 605 Fairchild Dr. Mountain View, CA 94043-2234 877-LabChip 650-623-0700 www.calipertech.com
LabChip Agilent 2100 250 drug discovery system AMS 90 SE
The AMS 90 SE is a high-throughput version of the Agilent 2100. The 250 runs enzyme and cell assays for smallmolecule analysis.
Eksigent Technologies, LLC 2021 Las Positas Ct., Ste. 161 Livermore, CA 94551 925-960-8869 www.eksigent.com
NanoLC
The system incorporates low-volume injectors, pumps with microflow sensors, and integrated UV absorbance detection. The system relies on 300 µm i.d. columns for separations. A multiplexed version is available supporting 8 LC separations at once.
Fluidigm Topaz crystallizer 7100 Shoreline Ct. South San Francisco, CA 94080 650-266-6000 www.fluidigm.com
The Topaz protein crystallization system uses a chip based on Stephen Quake’s work to mix small quantities of protein with different ratios of reagents to create samples for protein structure determination by X-ray crystallography.
Gyros US, Inc. Gyrolab workstation 11 Deer Park Dr., Ste. 100 Gyrolab Bioaffy Monmouth Junction, NJ 08852 877-433-9400 732-438-9400 www.gyros.com
The Gyrolab Bioaffy LOC for the Gyrolab workstation can measure reagents and samples and then perform single or multiplex assays and immunoassays.
Institute of Microchemical Integrated chemistry lab Technology KSP East 207, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan +81-44-811-6521 www1.odn.ne.jp/imt
The system uses a thermal lens microscope and glass chips, both developed by the University of Tokyo and the Kanagawa Academy of Science and Technology. Various channel patterns and custom fabrication are available.
Micronics, Inc. 8463 154th Ave., NE, Bldg. F Redmond, WA 98052 425-895-9197 www.micronics.net
The microFlow system is an ultra-lowpulse bench-top pump system complete with manifold and programmable software for precise fluid control and analysis. Active T and Active H lab cards may be used with the microFlow system; custom cards also available.
microFlow system Active H lab cards Active T lab cards Access cards
Nanostream, Inc. 580 Sierra Madre Villa Pasadena, CA 91107 626-351-8200 www.nanostream.com
Veloce microparallel LC system The Veloce system is used in conjuncBrio µPLC cartridge tion with Brio cartridges by life science researchers who need to analyze more samples in less time. Each reusable Brio cartridge incorporates 24 columns to enable parallel, reversed-phase LC separations.
Tecan Boston 200 Boston Ave. Ste. 3000 Medford, MA 02155 781-306-0827 www.tecan.com
LabCD
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The Tecan LabCD is shaped like a CD, performs fluidic manipulations by spinning the disc at different speeds, and can be used in Tecan workstations.
“Without a doubt, right now bench-top instruments are proven technologies,” says Soper. However, he suggests that the interface between individual bench-top systems is poor, except for well-known examples like LC/MS. Researchers are turning to robotic systems to carry samples and reagents from one device to another. However, Soper and Manz indicate that this solution is not the most elegant or logical. In part, the reason is sample size: Robots handle wells with volumes of tens to hundreds of microliters, but the microLC systems that they serve may handle between microliters to nanoliters. This dichotomy, Soper says, results in wasted material and sample preparation effort. Some instruments are being designed as a hybrid of robotics and LOCs. The Caliper 250 drug discovery system is robotic in that it handles well plates and brings them to the chips, where “sippers” draw sample from the wells and onto the chips. According to the company, the system performs enzymatic and cell-based assays. “One big, big advantage of the microfluidic chip for cellbased assays is that you can use fewer cells,” says Michele Boudreau of Caliper. Tecan’s LabCD is a compact discshaped LOC that can be handled and filled by robotic systems. Once loaded, it is spun to mix reactants and samples, and the products are then detected. Gyros AB produces LOCs in CD format for use with its lab workstation, which transfers samples from well plates to CDs. The Gyrolab Bioaffy CD is designed for protein quantification and needs as little as 500 nL of sample for each assay on the CD (104 per CD).
Sample prep Although related to integration, sample preparation is one of the most important areas that separates LOCs from the bench-top standard bearers. “You can’t just take blood and shoot it into your LC and expect to get reasonable results,” says Soper. Using LOCs, companies are taking a crack at preparing samples without preprocessing. Micronics says that its microFlow system, which uses its Active Card LOCs, can extract antibodies from an 80-µL sample of blood in 4 min. The
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system is commercially available only for researchers, but Micronics and others are working on methods to tackle sample prep for commercial LOC systems. Because LOCs can work with smaller sample volumes, they conserve precious unknowns, such as proteins, and reduce waste. For example, Fluidigm’s Topaz protein crystallization system uses very little protein (about 100 times less than alternative methods, the company says) so that a protein’s structure can be determined by X-ray crystallography. “There haven’t been any products introduced to crystallographers that solve the issue of obtaining structural data from a limited supply of protein sample,” says Kristin Spataro of Fluidigm. Each chip meters and mixes the sample protein with reagents to assist crystallization. In addition, the chip automatically sets three different ratios of crystallization reactions for each sample, tripling the number of conditions tested. Small sample volumes are also an advantage of Caliper’s LabChip for the Agilent 2100 Bioanalyzer. Boudreau indicates that the RNA application is especially popular because it needs as little as 1 µL of precious RNA sample per analysis.
Faster and easier Not every benefit is related to sample preparation. The small sample and reagent volumes used in these systems allow some reactions to proceed more quickly or with less effort than would non-LOC devices, say company representatives and researchers. “There are proven examples of how electrophoresis [on a chip] can really get [the] result you need to get in a significantly less amount of time than conventional capillary electrophoresis instruments,” says Soper. He adds that from a throughput standpoint, “These things are really the way to go.” Caliper’s AMS 90 SE electrophoresis instrument, which is meant to replace slab gel electrophoresis in high-volume laboratories, separates DNA segments by size. Two LabChips are available for the system; one separates DNA in 55 s, and the other will do it in 30 s with some loss in resolution. But Boudreau adds that time isn’t the only consideration for high-throughput analyses. “You could set up many gels to run simultaneously
In some cases, better data may be obtained with an LOC system than with traditional methods.
and beat the [LOC’s] time, but it would be much too labor-intensive,” she says. The LOC systems “are much less laborintensive than gels.” In Fluidigm’s protein crystallization LOC, the crystallization is often faster than traditional methods. According to Spataro, the kinetics on the chip reduces the crystallization time from possibly many months or even years to several weeks. This technique also saves more time and effort by reducing the number of times that the samples need to be examined for crystallization. A vastly different application, micro- or nano-LC, uses LOCs to allow faster gradient changes and separations. Eksigent’s NanoLC system incorporates a microfluidic flow sensor, two pumps, low-volume injection valves, and an integrated detector to perform HPLC using 10–100-nL injections. The chromatography is conducted by a 300 µm i.d. column. The flow sensor combined with the pump allows precise control of the gradient, and because of the small volume of the system, gradients can be changed extremely rapidly. “Rapid meaning 10 to 30 seconds for many of the gradients,” says Eksigent’s Dave Rakestraw. He says that through the use of microfabricated components and fast gradient times, analysis times can be four times faster compared to conventional HPLC.
Multiplexing and scaling Some instruments, such as LCs, are rarely designed to handle more than one sample at a time. “The pervasive issue is now how [to] run 1000 samples instead of running 1 at a time,” says Soper. The advantage of LOCs is that chips that can do one thing can often be scaled up to hundreds of analyses, although current
systems do not fully exploit this feature. Eksigent is multiplexing its NanoLC system, grouping eight systems together in one package. Another LC company, Nanostream, is taking a different approach by replacing columns with a chip that contains 24 columns. “Parallel obviously translates to a savings in time. There are also some other advantages you can exploit from the ability to run things in parallel,” says Surekha Vajjhala of Nanostream. One advantage is that reference standards and samples can be run simultaneously, using the same instrument conditions. Another possible advantage is analyzing multiple time-delayed samples from various reactions at once.
Better In some cases, better data may be obtained with an LOC system than with traditional methods. Boudreau says that for the kinase assay on the Caliper 250, the data are much more reproducible and have far fewer false results than homogeneous assays. By physically separating and measuring the phosphorylated and unphosphorylated peptides, screeners are more likely to achieve reliable, meaningful results. Eksigent also suggests that in some cases, its systems provide better data than those of traditional systems. “Taking the sample off [an] autosampler tray and injecting it into our system and running the same exact methods, [we] get almost identical results, except better resolution,” says Rakestraw.
Difficult disposables Although these systems may have advantages over conventional systems, they are newly commercialized instruments, and their success is not guaranteed. Several
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companies, such as ACLARA Bioscience, have already exited the LOC market. Soper suggests two reasons for the lack of widespread adoption: the novelty of the technology and economic considerations. “With any new technology, it takes about a 5- to 10-year lag period before it is actually accepted,” he says. Both the equipment and the chips can be costly; however, the equipment is a one-time expense and can be upgraded. The more complicated issue is the cost of the chips. “The chips don’t last forever,” says Soper. “They eventually break down, like an LC column.” Some are disposable and can be used only one time. Prices can range from $18 to $650 per chip depending on the system, making the cost of analysis potentially very high. The reasons for using disposable chips—and their high prices—are complex and are based on the application, the chip materials, and fabrication. One advantage is that chips offer flexibility, and “the more versatile you make an instrument, within reason, the more customers will want to buy one,” says Boudreau. If an LOC system is purchased to perform one function and then a new analysis is needed, a different chip can allow the new process. For instance, Caliper offers several chips for its 250 drug discovery system, including a calcium flux agonist, a calcium flux antagonist, and a 3-reagent chip. Disposable chips are also good for business. “Consumable products like chips are always good for a business since they continue producing revenue,” says Boudreau. Each chip maker produces chips for its own equipment; because the relevant technology is closely guarded, there is little chance that other manufacturers will produce chips for a given integrated system. “There are no thirdparty LabChip vendors or producers for these instruments—we have a strong position from ‘know-how’ and patent positions,” says Boudreau. One exception is Micronics, which enables other uses of its microfluidics and micropumping system, called the microFlow system. “Users of the microFlow system typically include researchers who are assessing fluidic properties, devising assays, and developing new products . . . whether on cards [chips] we provide or 508 A
their own,” says Micronics’ Karen Hedine. The microFlow system is composed of 3 ultra-low-pulse pumps, a manifold, and several different types of cards that are currently available only for research purposes. The company aims to customize lab card design services and produce cards from rapid prototype to commercial supply on behalf of clients and partners. Custom manufacture is also an important area in this field. The systems discussed and listed in the table are all non-custom products. Custom fabricators, such as Amic AB, Gesim, Mezzo Systems, Microalyne, Micronit, Scandinavian Micro Biodesign, Thales Nanotechnology, and ThinXXS, will take designs (or help design) and fabricate structures on the basis of an individual researcher’s needs. Micro Chemical Systems Ltd. (U.K.), also a fabricator, provides an interesting microreactor development kit that contains many of the necessary tools to begin experimenting with microreactors and microfluidics.
Conclusion This “coming of age” LOC technology offers clear advantages, but the field faces some economic hurdles. “As this technology evolves, things [will] get more accessible and less costly, so you [can] bet that is going to change dramatically,” says Soper. Spataro indicated that an advantage of the chips is that their density can increase, which makes them worth more to researchers but not more costly. Several established and new companies alike, such as Cepheid, Network Biosystems (with Shimadzu), and Surface Logix, are readying products for 2004, which will further broaden the LOC market. New materials are also being introduced, such as Agilent’s polyimide-based Chip-LC device, which was presented at the 2003 MicroTAS meeting. The chip also includes a nanoelectrospray tip for direct injection into an MS, eliminating dispersion after separation. Soper says, “There are definitely some high advantages, and people are just starting to use and . . . understand these advantages.” Michael J. Felton is an associate editor of Analytical Chemistry.
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