PROFILE

Successes and challenges of lab-on-a-chip

Successes and challenges of lab-on-a-chip

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The formation of Caliper Technologies

Company founding

An internet newsgroup and $3000 dollars were Caliper Technologies Corp.’s humble beginnings. The founder, Mike Knapp, Ph.D., paid Oak Ridge National Labs $3000 in 1994 for exclusive rights in a diagnostics field to the chip valving technology developed by Michael Ramsey, Ph.D., a Corporate Fellow at Oak Ridge. This original company was called Caliper Microanalytic Systems. Those rights were enough to convince the founding venture capitalist, Larry Bock, to team up with Knapp and establish a company with the lab-on-a-chip concept. They met in a biotech internet newsgroup when Knapp responded to a question Bock had posed regarding the academic leaders of the lab-on-a-chip movement. Coincidentally, Wally Parce, Ph.D., who also responded to that same internet newsgroup question, subsequently became Caliper’s Vice President of Research.

Larry Bock was with Avalon Ventures at the time. Unlike many other venture capital firms that waited for interesting business plans to come across their desks, Avalon partners actively identified areas that could benefit from new technology and started companies in those fields. They used their seed money to acquire intellectual property, exclusively sign up marquee scientific advisors, and attract early employees. This package was then used to draw other top venture capitalists in the next round of financing. One of the first things that the Bock and Knapp team did was go back to Oak Ridge and license all the remaining fields for the valving patents for $35,000. Afterwards they traveled the world for most of 1995 talking to microfluidic thought leaders and acquiring intellectual property from Harvard, Stanford, and Princeton Universities.

Key people

From its earliest days, Caliper was focused on product development. An important and novel feature of Caliper’s founding is that it was established on the idea of using microfluidics to create a “lab-on-a-chip.” Focusing on the idea of a lab-on-a-chip rather than a specific technology, idea, or single product concept has given Caliper the opportunity to pursue a portfolio of technologies and the development of a wide range of products.

By 1996, Caliper had raised $6 million in venture funds and assembled a key team of people. The two founding Vice Presidents after Knapp were Calvin Chow and Wally Parce, both from Molecular Devices and both with extensive product development experience. The next six employees were skilled in microfluidics, microfabrication, electrical and optical engineering, and chemistry. In addition, they all came from product development backgrounds, and each had worked in industry for many years. In December 1999, Caliper successfully completed an initial public offering (IPO). The money raised from the IPO, as well as from a subsequent offering in 2000, provided the funds that enabled Caliper to establish a substantial presence in the commercialization and research of microfluidics. The company now has 280 employees and a number of commercially available products utilizing the lab-on-a-chip strategy.

Acquiring intellectual property

We have always, at Caliper, had the conviction that we were at a seminal point in a transforming technology powerful enough to impact many fields. Thus, we knew it was critical to establish a comprehensive patent portfolio, especially in microfluidics. Although most young companies rely on outside legal firms for intellectual property (IP) counsel, Caliper decided at an early stage to hire an experienced patent attorney to protect and expand this important corporate asset. As a result, Caliper now owns, or has exclusive licenses to, over 100 issued US patents and more than 600 patents and patent applications worldwide. The IP estate is not merely numbers, however, MIT’s Technology Review Journal recently recognized not only the quantity of the Caliper patents, but the extremely high quality of the patents. (E. Jonietz, Technology Review, 2002, May, 71–77) Using their metric of “technological strength” they ranked Caliper as seventh in the Biotechnology/Pharmaceuticals field. In addition, their “current impact” metric, which takes into account how many other patents cite a company’s patents as prior art, gave Caliper the second highest score among all companies in all fields in this year’s survey.

Approach to microfluidic technology

The founding vision at Caliper was to develop microfluidic systems that consume low quantities of reagents, integrate multiple assay steps, and use computer control to govern the reaction conditions. Creating such systems presented a number of technical challenges for the team. Not only did they have to develop chip fabrication technology and fluid actuation systems, but they also had to build an effective, easy-to-use interface that would be compatible with current laboratory equipment.

Basic chip design

Caliper’s simplest chip is essentially a serial device that behaves very much like an assembly-line reactor. Samples, either chemicals or cells, move through the chip down a central channel where reagents and/or cells are added. The samples are then incubated, heated (if required), and separated. Finally, the reactants and/or products are measured by fluorescence detectors in proximity to the channel. The chips are made of two pieces of glass or quartz bonded together. One piece of the substrate has a network of fluid channels etched into it. The channel design is produced by the same photolithographic methods used in semiconductor manufacturing (that is why they are called “chips”).

World-to-chip interfaces

One of the most important issues in the development of microfluidic devices, and one that received significant attention at Caliper, is the world-to-chip interface. How do samples get into the chip? How does the user interact with the chip?

Sipper chips

The ability to get thousands of samples into the chip is the gating factor in making microfluidic chips useful in high-throughput applications. Caliper’s solution to this problem was to add a third dimension of flow to the chips in the form of a capillary, or sipper, attached into a drilled hole that connects to the fluid channels. The tip of the sipper accesses fluid samples held in a microplate, and the samples are brought onto the chip through the application of a vacuum (Fig. 1).

Fig. 1 Caliper sipper chip. The capillary addresses samples in a microtiter plate to make the chips usable for high throughput applications. Thousands of samples from a microplate are transported through the sipper and onto the chip for processing.

Chip holders

All of Caliper’s chips are held in a plastic caddy, giving the user a convenient hand-size frame with which to handle the chips. The caddy also supplies fluid reservoirs, in which to load samples or reagents, and a connection point for the instrument to make a pressure or a vacuum seal to the chip. Lastly, it prevents fingerprints from creating alternate paths for electrical current across the chip surfaces. For chips that test a limited number of samples (e.g., 6 to 12), a pipettor is used to load the samples into the fluid reservoirs in the plastic caddy that mate with the chip reservoirs. For chips that are being used in a high-throughput mode, the reservoirs are on a 384-well spacing (4.5 mm) suitable for multi-channel pipettors or automated liquid handling equipment (Fig. 2).

Fig. 2 Protein sizing chip for use on Agilent 2100 Bioanalyzer. The 16 fluid reservoirs in the plastic caddy mate with the glass chip underneath. The microfluidic chip itself occupies just the area under the reservoirs.

Multi-sipper chips

Caliper’s more complex chips utilize both a serial component and a parallel one. Adding a parallel dimension provides the high-throughput required for certain of Caliper’s applications. These multi-sipper chips have several parallel flow paths (i.e., 4 or 12 sippers) each with independent fluid channels allowing thousands or tens of thousands of samples to be processed. The spacing and pattern of multiple sippers are designed to mate with wells from a 384-well microplate. Our current sipper layout is 6 × 2 where the 6 sippers are spaced 4.5 mm apart and the 2-sipper direction is 9 mm apart (Fig. 3).

This is one solution that satisfies the 384-well mapping requirement of addressing each well once and only once. When each of these 4 or 12 independent flow paths is doing the same assay, samples may be tested approximately 4 to 12 times faster than would be possible with a single flow path.

Fig. 3 Caliper 12-sipper chip. Twelve sippers address 12 independent fluid paths allowing all samples from a microplate to be tested 12 at a time. The sipper pattern allows wells A1–A6 and C1–C6 to be sampled at one time. All wells can be addressed with this 2 × 6 pattern of sippers.

Sip-and-split™ chips

An alternative format that combines modest parallelization and extreme serialization is “sip-and-split™” In this case, one sipper brings in a sample that is then divided into many parallel channels. Each of the parallel channels has either different reagents added to the reaction or different reaction conditions (e.g., electric field or elevated temperatures). “Sip and split” chips are designed for experiments in which each sample is going to be tested against multiple reagents or conditions. The device tests the samples at the same rate as a single-sipper device but now provides as many as 8 to16 assays per sample. Caliper is using this concept to develop a genotyping product in which the microfluidic technology is used to test genetic variations known as single nucleotide polymorphisms (SNPs). For this application, either the patient samples are accessed through the sipper and tested against 12 different genetic loci, or thousands of locus-specific-reagents are accessed through the sipper and tested against 12 patient DNA samples.

Solving the challenge of moving fluid-electroomosis versus pressure

All of Caliper’s early research and technology acquisition, focused on the use of electroosmosis to move fluids through the chip. However, using electroosmosis to test sample compounds that could be either positively or negatively charged results in inconsistent sampling. To solve this challenge, we developed a scheme to put the unknown compounds in a high-salt buffer and space them with a low-salt buffer. The electric field was dropped almost entirely across the low-salt spacer buffer and prevented the samples from being affected by the electric field. This method successfully sampled all compounds at a consistent sampling and flow velocity and kept them uniformly spaced. However, this mode of flow also introduced new problems. The flow profile and the chemical composition of the fluid plugs produced a “fully-coupled” problem; that is, the characteristics of the flow profile determined the rate at which high- and low-salt buffers would disperse into each other, and in turn, the chemical composition of each of the plugs influenced and dictated the fluid flow. This made it relatively difficult to optimize new assays in the chip because when one variable changed, everything else changed too.

Importance of the Taylor–Aris dispersion theory

At the time Caliper was developing microfluidic systems that used electroosmotic flow, it was generally believed that pressure-driven flow would be unsuitable as a general flow actuation mechanism because motion-dependent dispersion would unacceptably spread a sample plug along the length of flow. However, Andrea Chow, Ph.D., the current leader of the microfluidic group, and I, the previous leader, realized that at the length scales relevant for microfluidics (tens of microns), and the velocity of interest (order of 0.1 cm s⁻¹), the dispersion is modeled by the specialized Taylor–Aris description, which accounts for a much slower rate of dispersion than normal (G. Taylor, Proc. R. Soc. (London), 1953, 210A, 186–203; US Patent no. 6150119). In flow at larger length scales, there are molecules that move along the center of the tube very rapidly, twice the average velocity, to be precise, and ones near the walls that move slowly, if at all. This differential velocity results in dispersion increasing linearly with time. In micron-scale formats, molecules rapidly diffuse across the flow streams; there are no molecules primarily trapped in fast streamlines and other ones primarily moving in slow streamlines. All molecules experience both fast and slow streamlines, and dispersion increases only as the square-root of time. Flow systems dominated by the Taylor–Aris form of dispersion have the very counterintuitive characteristic in which plugs of small molecules disperse less than plugs of large molecules. Small molecules, with their faster diffusion coefficients, move more rapidly from fast (centerline) to slow (near the wall) streamlines, thus keeping all molecules moving at nearly the same speed.

The interplay of dispersion and reactions is fascinating. If a plug of small molecules has a chance to bind (in the course of a reaction) with a large molecule (say an enzyme) or a cell, that plug of molecules will disperse far more than if it had not had a chance to bind with the large entities. The study and modeling of Taylor–Aris dispersion in lab-on-a-chip systems has become one of Caliper’s most important areas of expertise. The throughput of samples, and therefore the commercial value, of each chip is limited by the time interval between samples. That interval in turn, is limited by the rate of dispersion of one sample into its predecessor or successor. Accordingly, it makes a great deal of sense to be experts in every nuance of dispersion.

Shift in fluid control

Now, all of Caliper’s commercial products and products in development that have bulk fluid flow utilize vacuum (or pressure), not electroosmosis, to drive fluid flow. We continue to use electric fields to perform separations on the chips, and an understanding of electroosmotic flow (gleaned from the early days) is valuable to controlling this flow source. We use both pressure and electrokinetic forces to find the best solution for a given application and frequently combine both on a single chip. For example, both vacuum and electrophoresis are used in chips to screen inhibitors for a variety of different kinase enzymes (Fig. 4 and 5).

Fig. 4 Kinase chip using vacuum and electorphoresis, top view. Compounds to be tested for kinase inhibition are pulled into the chip, through the sipper, by vacuum applied at one fluid reservoir of the chip. Enzyme and substrate are pulled into the central channel by vacuum. The reaction proceeds in the absence of an electric field and later enters the separation channel in which an electric field is always present. The substrate and products have differential mobilities so characteristic signatures in the fluorescence trace are developed.

Fig. 5 Data trace from a high-throughput kinase assay. The magnitude of the peaks and valleys in the fluorescence vs. time is correlated to the magnitude of the inhibition of the compound being tested.

The substrates are fluorescent but not fluorogenic, meaning the fluorescence does not increase or decrease as the substrate is converted to product by the enzyme. The substrate and product do have different electrophoretic mobilities however, so placing the reaction in an electric field causes a characteristic signature in fluorescence generated by the extent of reaction. High-throughput DNA sizing is another example of an application involving vacuum and electrophoresis together on the same chip (Fig. 6 and 7). The DNA samples are brought onto the chip by vacuum, the markers are mixed with the samples by vacuum, and the injection and separation are driven by electrokinetics.

Fig. 6 High throughput DNA-sizing chip using both vacuum and electrokinetics. DNA samples are loaded into the chip through the sipper, mixed with markers and pulled towards the well by vacuum. Once completely loaded, the instrument applies an electric field between the “cross-injection voltage” wells to load the sample into the central intersection. Then an electric field applied across the “separation voltage wells” forces the DNA into the separation channel where it is separated through the sizing gel. These chips run up to 2000 samples, all processed down the same assembly-line.

Fig. 7 Instrument for running high throughput DNA sizing, AMS 90SE.

Sample accession interface

Although Caliper’s chips dramatically reduce the use of reagents placed on the chip, sample volumes in the microplates used in a typical laboratory are still relatively large. To reduce the amount of sample required, we are developing the “LibraryCard™ Reagent Array,” a novel microfluidic sample source in which nanoliter quantities of reagents are dried as spots on a glass substrate. The sipper adds a drop of liquid to the reagent spot, the reagent dissolves, and is then sipped up into the chip for testing (Fig. 8).

Fig. 8 LibraryCard™ Reagent Array: microfluidic reagent source. Thousands of nanoliter-size drops of compound or reagent are dried on a glass substrate. At run time, the sipper delivers a 10 nL drop of buffer to reconstitute the reagent spot and sips it into the chip for analysis.

The reproducibility of spotting and reconstitution on this system is remarkable (Fig. 9).

Fig. 9 Reproducibility of LibraryCard™ Reagent Array as compound source. Multiple copies of three different inhibitors, each at five concentrations were spotted on a card, reconstituted and run in a fluorogenic enzyme assay on a Caliper chip. The data trace shows a high level of fluorescence when no inhibitor is present. The magnitude of the dip below the baseline correlates with the inhibitor level.

This system not only dramatically reduces reagent consumption in microfluidic systems but also improves the data reproducibility. Reagents or compounds do not degrade as they would in aqueous form, and the reagents need only be diluted from the stock solutions once, eliminating another source of error. The system also has the capability to reduce the physical size of screening operations because the robotic infrastructure needed to transport a few LibraryCard array plates is dramatically less than the infrastructure required to store and handle thousands of microplates.

Development of double-depth chips

Early on we developed the ability to manufacture chips with two distinct depths of channels. It has been an essential element in combining pressure/vacuum with electrokinetics. Electrical resistance and hydrodynamic (fluid flow) resistance have fundamentally different dependencies on the channel cross-sectional area. These scalings have added an extra degree of flexibility in chip design, enabling us to nearly decouple the two mechanisms in which a pressure gradient can move fluid in one part of a chip, and separations can be performed in the absence of a pressure driven flow in another part of a chip. The decoupling is robust and reliable since it is passive, and only depends on the ratio of the channel depths that can be precisely controlled.

A two-pronged approach to business development

Caliper employs a dual business strategy to commercialize our microfluidic technology. Both of these strategies are based on the principle that the LabChip™ devices can be valuable, and even enabling, for many applications across many industries. However, it seems impossible to encompass in one company, all the expertise and resources required to develop, manufacture and sell all potential microfluidics products and still maintain competitiveness in core microfluidic innovation. One element of our strategy is for Caliper to develop and produce microfluidic chips and associated modules for other vendor companies to integrate into their application products. The second element is to develop and sell integrated system solutions directly to customers in certain markets. The experience of developing and selling our own systems—the instrument, chip, reagents, and software—for microfluidic assays has enhanced the building of our chip manufacturing process and infrastructure, and has focused the advancement of our microfluidic technology in a way that is more relevant to the needs of the market.

Partnering with others

Caliper’s original equipment manufacturer (OEM) business seeks to capitalize upon our focus of microfluidics as our core business. To effectively address the many different applications that benefit from microfluidics, Caliper has pursued co-development relationships with other companies that want to develop new microfluidic products for their markets.

The Agilent partnership

Almost five years ago, we embarked on the first of this type of collaborative effort with Agilent Technologies. The result has been a very successful relationship that has produced a benchtop system for macromolecular sizing and quantitation for research scientists (the Bioanalyzer), as well as a steady and expanding menu of assays. Each of these assays comes with its own chip, including the newest flow cytometer cell-based assay that can all be run on the same instrument system. Customers value the quantitative, reproducible data and the electronic data capture. Sales of Bioanalyzer chips and instruments have grown significantly since the launch of this system only four years ago.

Caliper’s goal is to establish a number of such OEM business relationships for which Caliper provides the microfluidic technology expertise, design, and manufacture of chips, and, the partner generally provides the application expertise, instrumentation, reagents, software, and commercialization.

Promoting new applications

To facilitate the emergence of new microfluidic applications, we have developed a program called the Application Developer Program (ADP) that puts microfluidic research and assay development capabilities into the hands of scientists and engineers in other companies. The ADP can serve as the first phase of an OEM business relationship where much of the activity is research oriented, or it can be a way for companies to develop solutions for their own internal use in order to address unresolved bottlenecks or unique experiment needs. The ADP often makes use of the Caliper 42 instrument (a workstation with wide-ranging microfluidic capabilities), and includes a training course, a standard chip set with model assays demonstrating basic microfluidic principles, and custom chip-design services. Caliper’s five ADP-based collaborations to date fulfill our goal of expanding microfluidics into new areas for which the expertise at Caliper is limited or non-existent: space (NASA), petrochemicals, agriculture, drug discovery (Millennium Pharmaceuticals), and chemistry synthesis (GlaxoSmithKline) applications.

Direct development of Caliper system solutions

For our own systems we have chosen to pursue high-throughput screening for pharmaceutical applications, high-throughput DNA and protein sizing for the research market, and SNP genotyping. Some systems are currently available commercially, and others are in development. All of these products use sipper chips that allow hundreds to tens of thousands of samples to be tested per chip. These products offer reduced reagent consumption, integrated assay capabilities, and high quality data. We have two ways of selling high-throughput chips. In one, the chip is part of a kit that includes conveniently packaged reagents, for example sizing gel and dyes. For applications in which the customer supplies the reagents, the chip is provided at a nominal price, and the user pays an incremental fee per data point generated from the chip.

Creation of Amphora

During the development of Caliper systems for high-throughput compound screening; it became apparent that the technology has the potential to change the process whereby pharmaceutical compounds are discovered and developed. The exquisite data quality provided by the assembly-line microfluidic chips could enable the building of large databases to compare the potency of many compounds against many targets. Pharmaceutical screening is currently done one target at a time against a large compound library. The database version would automatically give specificity of hits to targets. In addition, information about the relative potency of compounds against targets could help reveal the role of the target. Caliper’s superior data quality also allows one to find weak hits amidst noise—valuable information for understanding the structure-function relationship of chemicals. To maximally benefit from this capability, compounds to be tested must first be purified and characterized to exploit the high reproducibility in the Caliper screening systems. In addition, building such a database would require hundreds of screens per year rather than dozens that the largest pharmaceutical companies are currently doing.

Because of our intimate knowledge and experience with the technology, we decided to form a company dedicated to creating a database of compound potency against large numbers of targets. In September 2001, Amphora Discovery Corp. was created as an independently funded and managed company. The company is enabled by Caliper’s microfluidic chip capabilities and is aggressively pushing the limits of this technology. The relationship is symbiotic; Caliper technology enables Amphora’s business goals by providing technology, and Amphora’s demand for robust, broadly applicable, high-throughput screening products provides valuable input for Caliper’s product development, which ultimately benefits all of Caliper’s customers.

Caliper’s commercial microfluidic chip systems

Applying our approach to microfluidics and our business strategies, we have developed a number of commercial products encompassing our lab-on-a-chip concept. They include collaboration products in which we develop and supply the chips, as well as our own products where we sell chips, reagents, instruments, and software.

Agilent 2100 Bioanalyzer

Our first product, developed and commercialized in collaboration with Agilent Technologies, was the 2100 Bioanalyzer.

AMS 90 SE

The AMS 90 SE is Caliper’s product that performs high-throughput DNA sizing. It has one central channel down which all samples are sized and detected. Each chip runs between 1000 and 2000 samples.

Caliper 42

Our most flexible product is the Caliper 42. It uses planar chips and has pressure, vacuum or electrokinetic capability at each of the 8 chip fluid reservoirs.

Caliper 250 assay development and high-throughput screening system

The Caliper 250 that we sell for high-throughput screening applications uses the multiple sipper chips, containing 4 or 12 sippers.

Caliper’s commercial products in development

Single nucleotide polymorphism analysis

Caliper is developing a new product for the genetic analysis market that leverages our high-throughput screening systems to perform analysis of single nucleotide polymorphisms, or SNPs. This system uses the sip-and-split technology, i.e., it has one sipper but 12–16 tests per sample. This product has heating capability so it can run PCR-based experiments. Just as with the high-throughput screening systems, extremely high quality data from these reactions is anticipated.

LibraryCard™ Reagent Array

Caliper’s microfluidic reagent source, the LibraryCard array, which can supply nanoliter quantities of reagents to be tested on sipper chips, is being developed initially as an adjunct product to our high-throughput screening (HTS) systems. Customers will spot their compound libraries on the cards, making many copies at one time. These dried compound libraries will later be used as the compound source for all screening assays. Alternatively, Caliper or others will sell formatted compound libraries. In this way, microfluidic HTS can be a complete solution for customers to identify agonists and antagonists to their targets of interest. In addition, the LibraryCard reagent array is a platform that we plan to develop with other reagents for other products.

Conclusions

Caliper has generated a fantastic portfolio of technology and products over the previous seven years. Our commercial chip production capability, the patent estate, the microfluidics and product development expertise are the primary value-creating elements. Our unwavering vision towards, and confidence in the benefits that microfluidics can bring to many diverse applications, combined with our flexibility in technology and product development, are the traits that have driven Caliper in these early years.

Acknowledgements

Thank you to Margaret Hirst for valuable editorial advice and expert technical assistance in preparing this manuscript, to Paula Antonelli and Michelle Chan for research assistance, and to Lena Wu, Will Kruka, Mike Spaid, Andrea Chow, Don Morrissey, and Julie Wood for many good suggestions.

Anne R. Kopf-Sill
Caliper Technologies Corp., Mountain View, California, USA

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