January 15, 2005 (Vol. 25, No. 2)
Biotech & Pharma Firm Appreciate Standardized Assay Formats
It has taken little more than a decade for microfluidics-based systems to become accepted and integrated into drug discovery by Big Pharma companies such as Merck, J&J, Pfizer, Lilly, Tashio, Amgen, Novartis, Serono, and Aventis.
Microfluidics emerged in the 1990s as a branch of physics and biotechnology that studied the behavior of fluids at the microscale level. The field quickly branched into the design of devices and systems in which nanoliter volumes of fluids could be controlled and used in analytical applications.
Microfluidic systems are of interest to Big Pharma because they provide intra-laboratory standardized assay formats. Kurt Stoeckli, Ph.D., vp and global head of lead discovery technologies at Aventis (www.aventis.com), states that the “use of microfluidics platforms has improved assay reproducibility leading to the generation of high-quality data across the discovery enterprise.”
Aventis reports that the use of a Caliper (www.calipertech.com) standardized microfluidic platform system has reduced the 1020% CVs observed using microtiter plate assays to a maximum CV of 5% with a much lower CV average.
Microfluidics-based systems now integrate the transport, delivery, and handling of nanoliter volumes of fluids with mechanical elements such as sensors, actuators, and electronics on a common substrate.
The functions performed by microfluidic networks include operations contributing to bioanalytical processes, such as sample dilution, sample injection, sample and reagent metering, mixing, incubation, components separation and detection.
Microfluidics, therefore, miniaturizes and integrates processes that were previously done at larger scale in separate operations and offer many advantages.
Microfluidic Origins
The microfluidics-based devices have intellectual roots in at least two diverse fields: microelectronics and bioanalytical chemistry. The computer chip paradigm inspired chemists and engineers to apply some of these same technologies to generate networks of channels through which fluids can be moved.
Not coincidentally, commercial microfluidics has its origins in California’s Silicon Valley. The advent of microprocessors in the early 1970s and the subsequent development of highly integrated microelectronic devices later in the decade paved the way for microfluidics.
The application of microfluidics to the bioanalytical realm derives from work done in the late 1980’s by Andreas Manz, then an analytical chemist at Ciba Geigy (now Novartis), who originated the TAS , an early lab-on-a-chip.
Manz envisioned the application of photolithography and chemical etching techniques for micromachining a complete microanalytical system on a chip to integrate sample preparation, chemical derivatization, electrophoretic separation, and detection using only nanoliters or picoliters of the test analyte.
In the course of investigating the TAS concept, Manz and his co-workers discovered that the phenomenon of electro-osmosis could be used to control the injection, flow and mixing of solutions.
Manz and coworkers joined forces with Jed Harrison’s group (University of Alberta, Canada) to demonstrate the practicality of such a device. Their studies showed that channels 10m deep by 30m wide etched in glass could be used for capillary electrophoresis, offering high-resolution separations in as little as 15 seconds.
More importantly, they showed that creating electric potential differences across chip elements permitted the manipulation of flow and mixing of fluids. The combination of exceptionally fast separation times and miniscule sample sizes demonstrated that such micro-systems were not simply elegant curiosities, but had practical advantages as well.
The integration of nucleic acid sample preparation, amplification, and hybridization, became another technological goal for the microfluidics community during the 1990s.
The first major commercial thrust toward micro-scale nucleic acid target amplification originated from work done by Allen Northrup and co-workers (Lawrence Livermore Laboratory).2 They demonstrated that PCR reactions could run up to ten times faster in their micro-scale device than by conventional means.
Kricka and Wilding (University of Pennsylvania School of Medicine) independently devised a thermocycler capable of amplifying DNA contained in less than five microliters of solution.
In 1994, J. Michael Ramsey and co-workers (U.S. Department of Energy’s Oak Ridge National Laboratory) began publishing a series of papers that extended the findings of Manz and Harrison and provided the fledgling microfluidics enterprise with some of its key intellectual property. The Ramsey group described a prototypical microfluidic device consisting of two perpendicular etched channels in the form of a cross with four reservoirs, one at each channel terminus.
The Future of Microfluidics
Although early microfluidic devices were usually made from glass, quartz, or silicon, which are all amenable to structuring via photolithography and wet-etching, several limitations of these materials are driving a shift of interest toward polymeric devices.
Methods for micromachining polymeric chips can be divided into direct fabrication and replication methods such as laser ablation, soft X-ray lithography, injection molding, soft lithography, and hot-embossing.
Microfluidics has a bright future moving well beyond the current crop of products and concepts in development. Technologies for rapid prototyping are making it possible to try out a variety of new concepts and approaches without incurring major expense or time expenditure.
As the sophistication of rapid prototyping increases, it will become increasingly possible to put usable devices into the hands of potential customers, obtain feedback, and redesign models almost in real time. A less likely scenario for the microfluidics sector relates to the once-prevalent computer chip vision, which calls for fabrication foundries mass-producing large lots of chips and benefiting from economies of scale.
It will be much more likely in the near-to-medium term to find companies producing modest lots of thousands, or even hundreds, of premium-priced, special purpose chips.
Microfluidics in Biodefense
Biowarfare defense is one area in which microfluidics might generate unit volumes approaching mass production levels. Government and industry sources see a personalized, hand-held device that will suck in air, process captured microbes, amplify their nucleic acids, and detect the products with a small microarray or some other form of multiplex analysis.
The Cepheid (www.cepheid.com) GeneXpert system is a slightly larger version of this vision. Micronics-Honeywell(www.micronics.net; www.honeywell.com) pictures a wristwatch-sized blood cell counting device to be worn by field military to monitor exposure to biowarfare agents.
Mass Market Diagnostics
Most diagnostic assays that have been envisioned for microfluidics-based devices fall short of the mass market scale. Large diagnostic markets are typically characterized by assays used to screen asymptomatic individuals or those with vague, common symptoms for disease.
Thyroid testing and prostate cancer screens are two examples of mass market diagnostics. A rare example of a mass market for already-diagnosed patients is glucose monitoring for insulin-dependent diabetics.
Microbiotechnology could find a significant market opportunity in the use of microarrays with microfluidic sample-processing front ends to test rapidly for blood infections, identify the organism, and determine the antibiotic susceptibility all in one step.
Perhaps the greatest opportunities for microfluidics lie in the realm of radically new concepts and devices that are not simply extrapolations of classical concepts and are possible only at the micro- or nano-scale.
By analogy to microelectronics, early devices tended to emulate larger-scale systems. Transistors replaced vacuum tubes, enabling construction of small portable radios whose components were longer lived than classical equivalents.
It took more than two decades for engineers to start to take advantage of large-scale integration, which permitted the construction of devices with levels of complexity not possible with macroscale components. We are still operating in a pre-large-scale integration environment, as pointed out by Stephen Quake.
Current approaches to addressing true biological integration now take the form of massive robotic fluidics workstations reminiscent of early vacuum tube-based computers. Quake’s group has made a good start at demonstrating that large scale microfluidic integration can have functional utility. They constructed devices that emulate random access computer memory and an electronic comparator circuit using dyes and biochemical reactions in place of manipulating electrons.
Conclusion
Microfluidics has a bright fu-ture. The field is increasingly characterized by microfluidic modules that contribute discrete functionality to overall systems.
Sample processing units for microarrays or mass spectrometry, patch clamping chips for screening drug candidates with ion channel targets, cell processing units for high-content screening systems, and target-amplification units for nucleic acid assay systems all exemplify value-adding contributions of microfluidics to overall systems.
Caliper, with its acquisition of Zymark’s fluid-handling expertise, is working with other companies such as Agilent (www.agilent. com), Bio-Rad (www.biorad. com), Qiagen (www.qiagen.com), Affymetrix (www. affymetrix. com), and others to extend microfluidics-enabled solutions.
The Caliper-Affymetrix partnership, for example, is focused initially on automating the GeneChip microarray target preparation steps of cDNA synthesis, purification, normalization, hybridization, washing, and staining for expression analysis.
