An MD&DI November 1997 Column
R&D HORIZONSResearch in the biosensing industry has been fueled almost exclusively by the glucose-sensing market. New techniques, however, when fully optimized, could expand the field significantly.
The biosensor industry has come a long way since the mid-1970s, when the Yellow Springs Instrument Co. (Yellow Springs, OH) commercialized the first glucose monitor. The next major breakthrough didn't come until 1987, when MediSense (Cambridge, MA) introduced a pen-sized meter for home glucose monitoring. By the end of 1995, the industry was worth half a billion dollars.
Living sensors on a standard integrated circuit are magnified on the computer screen. Photo by Tom Cerniglio
The market's expansion has attracted the attention of some big guns in the biotech field. Hewlett-Packard, for example, recently purchased a stake in i-Stat (Princeton, NJ), and Abbott bought MediSense last year for about $867 million. Boehringer Mannheim and Bayer AG have competing biosensors on the market. New biosensing techniques are showing exceptional promise, and the future of the small start-ups that are commercializing the technology is not hard to guess. Still, upwards of 90% of the biosensor industry is focused solely on glucose sensing, even though the technology could potentially serve a wide range of niche markets.
A biosensor is "a sensing device with a biological or biologically derived sensing element, which is integrated within or intimately associated with a physical transducer," explains Anthony Turner, head of Cranfield University's Institute of BioScience and Technology (Bedfordshire, UK). Biosensors produce discrete or continuous digital electronic signals in proportion to the concentration of an analyte or group of analytes. The biological component of the sensor can be an enzyme, an antibodyany biological element that can detect a species, explains Mark Vreeke, a product development scientist at TheraSense, Inc. (Alameda, CA). Moreover, biosensors are not restricted to detecting biological species but can be used, for example, to detect neurotoxins. The biological sensing element is immobilized on an electrode using a membrane, or it can be incorporated in the membrane itself.
Biosensors can be divided roughly into two groups. Catalytic sensors use enzymes, microorganisms, or whole cells to catalyze a reaction with the target substance. Affinity systems use antibodies, receptors, and nucleic acids to bind with the target substance. Reactions are quantified using transducers based on electrochemical, optical, evanescent-wave, and other techniques, the method employed depending less on the target molecule than on the manufacturer's expertise.
Naturally, different sensor types have advantages and disadvantages, Vreeke notes. Electrochemical sensors, for example, can be used in turbid solutions, which can be troublesome for optical systems. On the other hand, optical sensors can be quite sensitive, particularly in systems based on fluorescence or chemiluminescence. Within the range of electrochemical sensors there are further subdivisions, such as potentiometric, amperometric, and conductimetric. A common potentiometric glucose sensor would operate by affixing glucose oxidase on top of a pH electrode and quantifying the change in potential as a function of the change in pH within the analyte solution. An amperometric system might entail oxidizing glucose and measuring the production of H2O2 as a change in current.
IMPLANTABLE GLUCOSE MONITORS
The feasibility of biosensing was first demonstrated by Leland Clark in the mid-1960s, when he measured glucose concentration in solution using what has since become known as the Clark oxygen electrode. Since 1991, Clark has headed the R&D branch of Synthetic Blood International (SBI) in Kettering, OH, focusing on the development of artificial blood and the commercialization of an implantable glucose monitorthe Holy Grail of the sensor industry. The primary obstacles to such a monitor involve calibration, biocompatibility, and stability.
The implantable glucose monitor under development at Synthetic Blood International (Kettering, OH) draws samples from tissue fluid rather than from blood.
Elmo Blubaugh, biosensor research associate with SBI, claims that his company has overcome these obstacles. SBI's design team has filed for two separate patents, and management is considering filing for a treatment investigational device exemption to obtain pilot data before beginning full-scale clinical trials. Pilot trials may also take place in Europe to speed the process.
Ease of Calibration. According to Blubaugh, SBI's glucose monitor avoids the problem of prolonged blood contact by drawing samples from tissue fluids, which have been shown to be a reliable indicator of blood-glucose levels. The difficulty with this approach is that tissues consume oxygen, and most enzyme-based glucose sensors are in fact oxygen sensors. Because the partial pressure of oxygen in tissue can dip quite low, an effective device would have to be able to achieve a linear response under tough conditions.
"We're still using an enzyme-based catalyzed oxidation of the glucose," Blubaugh explains, "but we're not using an artificial mediator." The structure of the inner enzyme layer attenuates the amount of glucose that gets through while providing facile transport and storage of oxygen. The two functions permit the monitor to draw what it needs from the surrounding tissue. "It's sort of a semiclosed system in which the oxygen is recycled in the enzyme layer itself," Blubaugh says. As a result, the enzyme is not subject to the partial pressure of oxygen in the surrounding tissue, improving the signal-to-noise ratio.
Such high sensitivity at lower partial pressure of oxygen distinguishes SBI's technology from other commercial techniques. "We can work at 2% oxygen, which would be a low body-tissue oxygen level, and we're linear up into the glucose range of 400500 mg/dl." In contrast, Blubaugh points to a clinical analyzer made by Yellow Springs Instrument Co. Its device, he says, "uses an outer membrane and inner enzyme layer and detects [the ratio of] hydrogen peroxide to oxygensimilar to what we're doing." The difference, he says, is that "their sensor works very well at 20% partial pressure of oxygenit's very linear through 800900 mg of glucose per deciliter. But reduce the oxygen content by one order of magnitude, and it has a drastic effect on kinetics."
Biocompatibility. To increase biocompatibility, SBI took a tip from the pacemaker industry and minimized the use of plastics. "We kept material use to a minimum," Blubaugh says. "We used titanium, platinum, silver, and some ceramics." The sensor and case are made from titanium, with the exception of the membrane that covers the sensor face, which must contact the solution. The device is currently about as large as a pacemaker and, like the pacemaker, uses an external telemetry system. Blubaugh would like to see the device get even smallera goal that goes hand in hand with decreasing its energy consumption.
Stability. Blubaugh contends that stability is not as big a problem as many would suggest. "In my experiments, I've had an enzyme sensor going for as long as five or six months, running at 400 to 600 mg/dlat room temperature. I don't see any stability issue." SBI expects its final device to have an implant life of two years.
Nonetheless, stability remains a chief focus of biosensor research across the globe. Although conceding that MediSense's glucose monitor has a shelf life of six months and could conceivably remain stable for a year or two, Cranfield University's Turner notes that "that's a very optimized case. A commercial sensor typically lasts around three monthsand that's a very good one."
The stability problem is being looked at from widely diverse perspectives. "People are trying to engineer greater stability into the protein molecule itself using genetic engineering or trying to find more stable sources," Turner says. Some work has focused on molecular imprinting. This involves making a nonbiological imprint of a molecule that will remain stable yet exhibit some of the same shape and binding characteristics of the original. Other research works with biomimicry, a specialty of Turner's. The idea, he says, is to identify the active part of the molecule and then organically synthesize it and cut out the redundant or unstable material.
"In biomimicry," Turner explains, "we're looking for active compounds that have similar structures to enzymes but greater stability, and we synthesize variants of those structures." The method makes extensive use of combinatory chemistry, which has now become standard in the drug discovery field. "With combinatory chemistry," Turner says, "the philosophy has been to make large quantities of different varieties of chemicals." To build a polymer, a chemist might start with 10 monomers, add a second monomer to each to make 10 polymers, mix them together, split them up, add another monomer, and repeat the process to generate an expanding number of variations.
"In about 10 hours you can generate a million different compounds," Turner says. "You can screen vast numbers of compounds, and if you don't get what you want, you throw them outbut it's only taken you a few hours." So far, the results have been extremely encouraging, says Turner, adding that "we're about to publish some of our first discoveries."
On this side of the Atlantic, researchers have made a breakthrough of a different sort. Mike Simpson, a scientist at the Department of Energy's Oak Ridge National Laboratory (Oak Ridge, TN), and Gary Sayler, who heads the Center for Environmental Biotechnology at the University of Tennessee (Knoxville), have developed what they affectionately term "critters on a chip." The device is deceptively simple, consisting of a coating of bioluminescent bacteria on top of a light-sensitive integrated circuit. The bacteria emit light in direct correlation to the analyte concentration. There are other whole-cell biosensors that are similar to the critter chip. Microphysiometers, Simpson notes, use a similar light-addressable potentiometric technique; based on micromachined silicon wafers, they detect the pH change that occurs when organisms secrete organic acid during metabolic processes. "But as far as we know," Simpson says, "the use of bioluminescent bacteria is unique."
The chips are the result of two separate lines of inquiry. "I had been working on what I call OASICsoptical application-specific integrated circuits," says Simpson. "Basically they're just normal computer chips that have been designed to be optically sensitive." At the same time, Sayler was working on engineering the biological degradation process using bacteria. Sayler's bacteria, a genetically engineered strain known as Pseudomonas fluorescens HS44, was created by fusing genes associated with the degradation of chemicals by microorganisms into the genes of the original bacteria. In the presence of the target chemicals, the mechanism for luminescence is tripped at the same time as the mechanism for degradation. As a result, Sayler explains, "the organism functions as a direct and immediate biosensor."
"So basically," Simpson says, "he's making this thing that glows, and I'm making this thing to detect light." It soon occurred to Simpson and Sayler that they could combine their ideas with signal processors, wireless transmitters, and related technology to create a useful device.
Their initial research was decidedly low tech. "Sayler's team made up a batch, and we placed it right on top of the chip and put it in a box," Simpson recalls. The bacteria were triggered to bioluminesce in the presence of a metabolite of naphthalene, which, in addition to being a common environmental pollutant, is the main constituent of mothballswhich is what the researchers used in the initial tests.
Sayler says that the team has done relatively little work specifically on the issue of stability but notes that the bacteria are extremely robust. In the current design, they are held in a polymer matrix that allows fluid and gas to get to them while preventing them from escaping. One elegantly simple innovation being considered is putting powdered milk in the matrix to feed the colony of bacteria. On the other end of the technological spectrum, Sayler says the team is investigating whether nanotechnology could create a feeding platform to provide nutrition for the organisms on the surface of the chip.
Manufacturing the critter chip is not significantly different from conventional chip making, although Sayler and Simpson had to deal with the possibility that the bacteria could interfere with the electronics. The team is working on several encapsulation techniques and has developed a way to attach bacteria to the chip that doesn't hurt them but does protect the chip from acidic secretions.
Any assay that could be made bioluminescent could potentially benefit from the technology. Tuberculosis screening, for example, has drawn Simpson's interest. Currently, deciding which antibiotic to use to treat TB requires several days to grow a culture and perform specificity testing. Simpson has been researching a recently developed method for TB screening that uses a chemical compound, firefly luciferase, to generate a luminescent response. The substance is catalyzed by adenosine triphosphate. The idea, he says, is to inject an antibody into the sample and monitor the luminescence: "If the luminescence starts to decay quickly after the antibiotic is injected, they know they've got a hit." Simpson hopes to create biosensing devices simple enough to use in an emergency vehicle and inexpensive enough to discard after use.
SURFACE PLASMON RESONANCE SENSORS
The point-of-care market is also the target of a different type of optical biosensor that has generated considerable interest since its commercial introduction a few years ago. The technology takes advantage of a quantum property of light known as surface plasmon resonance (SPR).
Quantech's (St Paul, MN) device for disposable testing.
"If you shine light at a metal surface," explains Robert McKiel, vice president of R&D at Quantech (St. Paul, MN), "at most wavelengths, light will be reflected off the metal; however, at a particular wavelength, the photons in the light will interact with the electrons in the metal surface, and you'll have a diminished reflection and absorption band." The condition of resonance depends on the medium that is in contact with the metal surface; any change in the refractive index of the medium will change the wavelength in a quantitative manner.
There are two ways to implement this phenomenon, McKiel says. "We illuminate the metal with broad-spectrum lightwhite lightand we look for the wavelength of light that is absorbed."
In contrast, the SPR technology developed by Biacore (Piscataway, NJ), a spin-off of the Swedish pharmaceutical firm Pharmacia, uses a single wavelength to induce SPR. "It's essentially a laser illumination," McKiel explains, "and what they look for is a change of angle in incident light. In order to use our approach, you can't have an absolutely smooth metalit's got to be rough in some sense." A rough surface permits interaction between the photons and electrons, which must exactly match each other in momentum. Quantech's device uses gold on a grating, very similar to the dispersion grating in a spectrophotometer. The gold can be coated with antibodies to specific target substances. The presence of the antibodies changes the absorption wavelength of the gold. Then, as the antigen binds to the antibody, another wavelength shift occurs, which can then be measured.
Quantech's focus is on point-of-care medical diagnostics. According to McKiel, there are currently 20 to 30 diagnostic tests commonly done in hospital labs that would be far more valuable to ER personnel if they could be performed at the patient's bedside. "What we're developing is an instrument to measure this SPR phenomenon and a disposable component to go with it," he explains.
The company's first product will measure CKMB, a valuable marker for myocardial infarct. After that, Quantech plans to introduce a product for measuring troponin, which, McKiel states, is gaining broad acceptance as a marker that can perform as well as or better than CKMB. Although the technology could be useful in a central lab, "that's not what we're going after," McKiel says. "We're going after those things that someone wants in a very fast fashion. The technique lends itself to speed. There's no need for a label, like there would be in an immunoassay, or for a fluorescent or radio marker. We can actually see the binding event."
Speed is a major characteristic of SPR systems. Biacore, for example, has a constant-flow system for developing immunoassays, wherein analytes are made to flow across the biosensor chip; those that adhere to ligands can be detected in real time without using detection reagents. The process can monitor biological interactions in complex mixtures. The instrument is versatile, capable of providing kinetic information, detecting association rates and dissociation rates, and determining affinity constants in a relatively quick expert platform.
In addition, the Biacore device consumes very small amounts of concentrations. The instrument requires only microgram or submicrogram concentrations and can perform kinetic analyses in about three to five minutes. The Biacore device, according to the company, could perform a typical ELISA, which now takes at least seven hours, in minutes. Biacore's research instrument might someday supplant calorimeters, microdialysis systems, and related devices that take a long time to generate results.
Although TheraSense's Vreeke suggests that SPR devices will carry a high price tag, McKiel claims that SPR is simple and inexpensive. "The fundamental component of the disposable part is the grating, which is just molded plastic. It has on top of it a very thin layer of gold, on the order of 50 to 70 nm in depth. So an ounce of gold will make hundreds of thousands of thesethe gold's less expensive than the plastic or the packaging." As for the antibodies, the cost will be typical of any immunoassayor even less, because SPR doesn't require a second antibody, chemiluminescent, or other marker.
"As biosensors go, SPR's a pretty good one," McKiel concludes. "It will be possible to manufacture in volume, and it will be cost-effective." The goal of point-of-care testing, he adds, is to move the patient out of the most expensive part of the hospitalthe ER. "Anything that does that can be priced a little higher."
"Biosensors offer potentially enormous diversity, but they've been dominated by one application," Turner laments, adding that a major challenge for the industry "will be to find the right niches." Despite research into new techniques, it may be some time before cross-disciplinary devices such as the critter chip become widely used. As Simpson points out, "engineers are trained in physics, not biology. Somewhere along the line, we'll need engineers who are trained in both."