Biosensors

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By Amarjeet Bassi, PhD

Overview

Biosensors offer an innovative, but under-utilized technology that can provide significant potential in the early warning and detection of chemical and biological pollutants, hazardous agents and pathogens. Established at the University of Western Ontario (UWO) (London, ON), the Molecular and Nanobiosensor Laboratory has been funded by the Canada Foundation for Innovation, Ontario Innovation Trust, the JP Bickell Foundation, and Photonics Research Ontario Centre of Excellence. The research group at UWO includes Amarjeet Bassi, PhD (Chemical and Biochemical Engineering) and George Knopf, PhD (Mechanical and Materials Engineering) and graduate students Wei Wei Wang, Dennis Nancoo, Yutian Yin, Parya Sainepoor and research assistant Shikha Singh.

The team’s goal is to design, develop and produce micro- and nanoscale biosensor devices for use in environmental monitoring. Such biosensors could be interfaced directly to mobile, autonomous or semi-autonomous robotic devices for monitoring of hazardous zones, or they can be applied as part of integrated “biochips” for field analysis applications. One focus of research is the development and application of biological signal transducers and biophotonic transistor devices. The initial research projects underway involve the protein bacterio-rhodopsin. This article presents a brief introduction to biosensors and biological transducers with reference to the research currently in progress in our laboratory.

What is a Biosensor?

A biosensor consists of two components: a sensor or sensing material (usually biological in origin) and a signal transducer (usually physical, chemical or electrical). The two components are combined or integrated into one compact unit. The biosensor functions as an analytical device and produces a measurement signal (either discrete or continuous) in response to the concentration or level of either a single analyte or a group of analytes1 (Fig. 1).

The biological sensing material may be a protein such as an enzyme or antibody, a nucleic acid (DNA, RNA or PNA), antibody fragment, a whole microbial cell, or even a plant or animal tissue. The physico-chemical transducer can be electrochemical (e.g., pH, polarographic, potentiometric or conductometric probes), thermal (e.g., thermistors), optical (fibreoptic) or piezoelectric crystals.2 Some examples of commercially available biosensor devices include blood glucose analysers, home glucose meters and the general-purpose surface plasmon resonance BIACoreTM biosensor.3

Advantages, Limitations and Applications of Biosensors

Speed of response (typically less than a minute) and ease of use are the main advantages offered by biosensors. Typically the smaller the device, the faster and more sensitive the response. Biosensors can easily detect analytes in the micromolar to nanomolar range. In today’s world, where rapid information is needed, biosensors can serve exceptionally well in emergency situations or for on-site field applications. Micro-machining (e.g., of glass or silicon chips) can allow biosensor production with a high degree of miniaturization.

Extensive current research activity is focused on designing smaller and smaller integrated systems containing several hundred or more biosensors and chemical sensors for on-site analysis of toxins and threats to the environment. These integrated chips include microfluidic separation technology and associated electronics. Clinical applications and in vivo monitoring of blood components such as glucose, lactate, urea and creatinine in patients are other areas in which biosensor research is being targeted. A biosensor cassette for determining levels of glucose, lactate and urea in micro-samples of undiluted whole blood or plasma has been reported.4 This biosensor cassette required no maintenance and could be used for one week or 500 samples after insertion into an AVL OMNI® blood gas and electrolyte instrument.

Today, it is possible to incorporate several hundred electrodes on one square centimetre of silicon.5 Other usable techniques include ink-jet printing, photolithography and microcontact printing. Monolayers of biomolecules can be deposited at high densities using self-assembly techniques. Such micro- and nanosensors can achieve very high sensitivities of detection (parts per trillion).

In spite of these advantages, biosensor design suffers from two main limitations. The first is the instability of the biological sensing component (enzyme, antibody, tissue, etc.), which can lose its activity in hours or days depending on the nature of the molecule and exposure to environmental stresses, such as pH, temperature or ionic strength. The second limitation is on the size of the physico-chemical transducers being used in biosensors. Currently, the smallest signal transducers reported — such as the light-addressable potentiometric transducers — are in the millimetre to micrometre range.6 Further miniaturization could be achieved in principle by replacing the physico-chemical transduction devices with biological signal transducers.

Biological Signal Transducers

A biological signal transducer is a molecule, such as a protein, that can be used for converting the measured signal to electric signals such as a potential or current change. By replacing physico-chemical transducers in biosensors with biological systems, the construction of nanobiosensors becomes possible. These biosensors are only limited by the minimal optical fibre dia-meter available.

Several proteins can be exploited as biological transducers. Of interest are the optically active proteins such as bacteriorhodopsin (bR), the green fluorescent protein, the visual pigment protein (rhodopsin), photosynthetic reaction centres, cytochrome c, photosystems I and II, phycobiliproteins and phytochrome. In addition, nucleic acids, peptide nucleic acids (PNAs) and ribozymes can also be applied as molecular switches.7

Bacteriorhodopsin (bR)

Bacteriorhodopsin is a 26 kD, photoprotein found in the purple membrane of the archae microbe, Halobacterium salinarium.8 This extremophile lives in high-salt-containing marshes (25% NaCl). Under the oxygen starvation conditions found in these marshes, the microbe uses bR to capture light and generate a membrane potential. The membrane potential is used to drive ATP synthesis.9 Figure 2 shows the purple membranes (obtained in the Molecular and Nanobiosensor Laboratory) from H. salinarium (wild type). The purple membrane fragments contain mostly bR and can be used directly for biosensor applications making further purification of the protein unnecessary.

The bR-containing purple membranes are being produced in-house in a 10-litre New Brunswick Scientific Co. Inc. (Edison, NJ) stirred bioreactor. The membrane fragments are extracted using standard biochemical protocols and stored until use. The membranes are then immobilized in polyvinyl alcohol-chitosan gels or in silica sol-gel matrices. (Fig. 3) Strategies for optimizing the production, extraction and immobilization are being investigated.

The bR protein offers several advantages as a biological signal transducer: (i) it is mechanically robust; (ii) chemically and functionally stable even at high temperatures; and (iii) possesses both photonic and photovoltaic properties. When the protein absorbs light it undergoes a complex photo-cycle that generates intermediates with light absorption maximas spanning the entire visible region of the spectrum. This property can be exploited in developing all optical phototransducers.

Optical sensors, transducers and actuators are activated by photons instead of currents and voltages and are therefore free from electrical current losses, resistive heat dissipation, and friction forces that greatly diminish the performance and efficiency of conventional electronic systems. Exploiting biomolecular electronics can further reduce feature size by several orders of magnitude and decrease gate propagation delays because devices can be fabricated atom by atom. Efforts are underway in the UWO lab to develop phototransistors based on bR (Fig. 4). The light-suppression properties of the bR film are investigated and exploited to create an all-optical photonic transistor (PT). The simple PT is used for optical switching and small signal amplification tasks. Currently, a bench-scale platform has been set up and theoretical modelling is under way.

Environmental Toxicity Monitoring

Bioluminescence refers to the visible light emission in living organisms that accompanies the oxidation of organic compounds (luciferins) mediated by an enzyme catalyst (luciferase). Luminescent organisms, which include bacteria, fungi, fish, insects, algae and squid, have been found in marine, freshwater and terrestrial habitats, with bacteria being the most widespread and abundant luminescent organisms in nature. The enzymes involved in the luminescent (lux) system, including luciferase, as well as the corresponding lux genes, have been most extensively studied from the marine bacteria in the Vibrio and Photobacterium genera. The bacterial luminescence reaction, which is catalyzed by luciferase, involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (FMNH2) with the liberation of excess free energy in the form of a blue-green light at 490 nm.

The bacterial light emission is quite sensitive to the presence of toxicants in the environment, thus assays such as the Microtox assay have been developed for toxicity monitoring. The bR protein responds to coloured light and undergoes changes in tertiary structure. It can be made to exist in two states: the yellow absorbing (bR) state and the blue absorbing (M) state. As small changes in incident light can cause large state changes in the bR protein, this property is being exploited to monitor changes in bioluminescence emission from Vibrio fischeri. Both the photoelectric and the optical transmission properties of bR can be used for this purpose.

The interaction of the bacterial signal with the protein response will be investigated on prefabricated “biochips.” In a second project underway, the goal is to use bR protein directly as a sensor/transducer. Although the experiments are currently conducted on an optical bench (Fig. 5), the key sensing and light-transducing properties of bR are retained when reduced to the microscale. Thus, this rather amazing protein is serving as the workhorse in the Molecular and Nanobiosensor Laboratory’s search for better and cheaper environmental sensors.

References

1) Turner, A.P.F., I. Karube and G.S. Wilson. Biosensors: Fundamentals and Applications. 1987, Oxford University Press, Oxford.

2) White, S.F. and A.P.F Turner. In: Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. M.C. Flickinger and S.W. Drew, eds. 1997,Wiley, New York.

3) Kress-Rogers, E. Handbook of Biosensors and Electronic Noses: Medicine, Food and the Environment. 1996, CRC Press, Boca Raton.

4) Schaffar, B.P.H; H. Kontschieder, C. Ritter and H. Berger. Clin. Chem. 1999, 45:1678-79.

5) Turner, A.P.F., J.D Newman and A. Rickman. Optics for Environmental and Public Safety, Munich, Germany, June 19-23, 1995 Europto Series, Berlin, Germany.

6) Nicolini, C., M. Sartore, M. Zunino and M. Adami. Rev. Sci, Instrum. 1995, 6:4341-46

7) Soukup, G.A. and R.R. Breaker. Trends Biotechnol. 1999, 17(12):469-476.

8) Birge, R.R. Ann. Rev. Phys. Chem. 1990, 41:683-733.

9) Hampp, N.A. Chem Rev. 2000, 100: 1755-76.

Amarjeet Bassi, PhD is an associate professor in the department of Chemical and Biochemical Engineering, Faculty of Engineering, at the University of Western Ontario (London, ON). E-mail: abassi@uwo.ca; Tel.: 519-661-2111 x88324