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Emerald Group Publishing Limited
Copyright © 1999, MCB UP Limited
In theory, biosensors can fish out molecules of target compounds, or analytes, from nanomolar or smaller concentrations. Indeed, last year Bruce Cornell and a team from the Co-operative Research Centre for Molecular Engineering and Technology (Chatswood, Australia) reported they had built a nanoscale-sized biosensor with subpicomolar sensitivity the equivalent, as Cornell has described it, of detecting the increase in sugar content of Sydney Harbour if a single sugar cube were thrown from a ferry.
Applications for such exquisite devices the technological equivalents of a canary in a coal mine abound in medicine, industry, military and environmental monitoring. While some devices have been commercialised, many more are in various stages of development.
Globally, sales of biosensors totalled $440 million in 1997, reports David Kilmetz, a market research analyst for Frost & Sullivan. Medical applications account for more than 90 per cent of the market and half of all biosensor sales are hand-held glucose meters used by diabetics. By comparison, says Kilmetz, "the environmental and industrial segments have been slow to take off", as scientists hammer out problems with the stability of biomolecules and the way they interface with electronic circuitry.
Stability is a prominent factor in the huge success of glucose monitors. Unlike most biomolecular compounds, which evolved to work in living organisms, glucose oxidase is inherently stable outside the body. In addition, maintaining a healthy blood-sugar level requires sustaining a stable amount of, and hence analysing for, a single substance only. Finally, the market estimated at 120 million diabetics worldwide is enormous.
"Blood glucose was predestined as the perfect application of biosensors", says biochemist David Moss of the Karlsruhe Research Centre (Karlsruhe, Germany). "The biosensor community has spent the last 20-30 years looking for a second application as ideal as the glucose question", he says, but so far, "there isn't one".
Over the next five to ten years, however, Kilmetz expects biosensors to make their mark in the food and beverage industry, where they will track fermentation, measure sugar content and detect harmful bacteria, such as E. coli and Salmonella. Tougher environmental regulations are likely to drive development and use of biosensors in the environmental field.
In recent years, several small companies have emerged, aiming to commercialise new or improved versions of biosensors, often to fill niche markets. Among them is BioTul Bio Instruments GmbH (Munich, Germany), which recently sent its first prototype biosensor to a research laboratory in Rio de Janeiro. The German company makes biosensors that use an optical detection method called surface plasmon resonance (SPR) to quantify ligands and other molecular complexes in target compounds.
BioTul's version uses laser diodes, like those found in CD players, to read the slight shifts in light reflected off the surface where analytes are binding. The sophisticated, but inexpensive, laser diodes will increase resolution and improve measurement. It is a design that will start an "avalanche for an already mighty tool in bioscience", says BioTul's president.
Researchers at Nitrate Elimination Company, Inc. (NECi; Lake Linden, Michigan) are working on a biosensor to measure nitrate in water and wastewater. Toxic to infants and harmful to adults, nitrate is a common water contaminant. "Right now there isn't any good way to monitor it", says NECi's vice-president Ellen Campbell. The sensor being developed is based on the nitrate reductase enzyme, derived from corn seedlings. The hand-held probe will measure the flow of electrons as the enzyme reduces nitrate to nitrite.
In the medical field, Quantech Ltd (St Paul, Minnesota), received pre-market clearance from FDA for a biosensor that measures myoglobin a protein released by damaged muscles. It is the first of three "markers" to appear in the bloodstream after a heart attack. Scheduled for testing in emergency rooms by next year, the biosensor uses the antibody, antimyoglobin, to capture myoglobin from blood.
Quantech is using digital technology based on SPE to develop biosensors for two other cardiac markers, CK-MB and troponin I, and the pregnancy hormone, hCG. The goal is to have the instruments in emergency rooms or ambulances to analyse critical diagnostic factors faster than and just as accurately as a hospital's central lab.
Some small companies are developing biosensor technology that they hope will find many different niche markets. For example, engineers at F&S, Inc. (Blacksburg, Virginia) are building biosensor prototypes that use fibre optics, called long period gratings (LPG), as the transducing element.
These fibres give light travelling down their core a characteristic spectral fingerprint. Similar to the way the fibres signal changes of pressure, stress, temperature and acceleration in other applications, in a biosensor they detect how light changes speed as it passes through a thin film that coats one end of the fibre.
As analytes from the environment latch onto biomolecules anchored in the coating, its refractive index changes. So, light escaping from the end of the fibre shifts speed, altering its signature. A spectrometer detects the difference, which is then correlated to the concentration of target molecule.
Prototypes of a fibre optic biosensor that had polymyxcin B, a ligand, attached to the film substrate, detected endotoxin, a potential biological warfare agent, at 500 parts per trillion, in seconds. Bill Velander, a biochemical engineer at Virginia Polytechnic Institute (Blacksburg, Virginia) and one of the sensor's inventors, calls LPG fibre optic detection a "platform technology" that can be applied to many other analytes.
Scientists at Ikonos Corp. (Portland, Oregon) are using another platform technology to tailor inexpensive, custom-made biosensors to meet the needs of clients. Called molecular imprinting, it bypasses the fragility of natural biocomponents by making a "plastic antibody" of the target molecule.
To create a plastic template, a polymer and a cross-linking agent representing the molecule target, are mixed together and spin-coated onto a surface, such as a wafer. The film is exposed to UV light to cross link the polymer. The imprinting molecule is then removed in organic chemicals.
What is left behind is a "Swiss cheese" with holes the exact size, shape and functionality of the target molecule. "In some cases the film has better recognition or binding affinity than the natural antibody", says Christopher Sevrain, Ikonos's chief executive officer.
Bulky complex molecules like proteins are difficult to imprint, but molecules with molecular weights in the order of 20-400 work well. "Therapeutic drugs and drugs of abuse, such as cocaine and PCP, are an excellent fit for this technology", says Sevrain. Ikonos has been making molecular imprints for clients ranging from petrochemical processors to drugmakers. Some prototypes are now being field-tested.
The cost of Ikonos's services starts with a payment to determine the feasibility and details of imprinting the proposed molecular target. If the project gets the green light, Ikonos shares in the expense of developing the first "proof-of-concept" instrument, which typically costs $50,000-$125,000 and takes nine to 12 months to complete. The client gets exclusive rights to the particular molecule for a specific industrial application, and Ikonos gets a share of the royalties.
Not all biosensors under development employ complicated transducers and engineering. At least one harks back to classic mine canaries.
Microbial ecologist Carl Fliermans of Westinghouse Savannah River Technology Centre (SRTC; Aiken, South Carolina) is sifting through SRTC's cache of subsurface microbes to find organisms to detect land mines. The proper candidates will not only glow in the dark, but dine on trinitrotoluene (TNT). "Organisms from 100-1,000 feet below the earth's surface bioluminesce", Fliermans says. "And they've adapted so as to use whatever compounds come their way".
After initial screenings of more than 10,000 microbes, Fliermans's team has rounded up about 60 organisms that either light up or have a predilection for TNT. The next step will be adapting some to perform both tasks. When that is accomplished, the customised organisms will be sprayed over minefields and roadsides in the afternoon. By nightfall, microbes that landed atop buried land mines will bioluminesce as they digest the TNT in offgases. Observers flying over the terrain will map the firefly-like glow marking the weapons, so that they can be safely removed later on.