Biological-Based Optical Sensors

Biosensors are devices that utilize a biochemical reaction in order to detect a specific chemical compound.

These sensor systems involve a biological recognition component such as receptors, nucleic acids, anti-bodies, or enzymes that are in direct contact with an electrochemical, electronic, or opto-electronic transducer. A variety of signal parameters such as changes in pH, oxygen consumption, ion concentra-tions, potential difference, current, resistance, or optical properties can be measured by an appropriate transducer.

Biosensors are divided into categories based on the method of signal transduction such as mass, electrochemical, thermal, or optical [Ivnitski et al., 1999]. Furthermore, biosensors can be classified as either direct-detection or indirect-detection systems. Direct-detection biosensors are designed such that the specific biochemical reaction, or target analyte, is measured directly by the transducer. In contrast, indirect-detection biosensors are those in which a preliminary biochemical reaction takes place and the products of this reaction are detected by the transducer.

Optical biosensors are an attractive solution for directly detecting infectious diseases, pathogens, and toxins. Some of these optical sensors are able to detect minute changes in the refractive index or material thickness that occur when cells bind to the immobilized receptors on the transducer surface. Several optical techniques have been reported for the detection of bacterial pathogens including monomode dielectric waveguides, surface plasmon resonance, ellipsometry, the resonant mirror, and the interferom-eter [Ivnitski et al., 1999]. Kelly et al. [1999] describe a simple, optical waveguide sensor for the detection of biological toxins. The biosensor works by optically tagging toxin receptors within a fluid phospholipid bilayer membrane that is formed on the surface of a planar optical waveguide. The process of toxin detection involves measuring the ratio of emission intensity from the donor–acceptor pair of fluorophores that are tagged onto the receptors. The ratio of fluorescent emission intensity depends on the concen-tration of toxin. The biosensor appears to be very sensitive with a high degree of specificity.

Recent advances in bio-analytical sensors have exploited the ability of certain enzymes to emit photons as a by-product of their reactions. This phenomenon, known as bioluminescence, can be used to detect the presence and physiological condition of cells. The concept of bioluminescence and the utilization of the light-emitting bacterium Vibrio fisheri for monitoring airborne toxins are presented below.

6.2.1 Bioluminescent Light Sources

The use of light for optical sensing, actuating, or communication requires a source of light radiation, a medium through which the light travels, and a detector to convert the light energy to another measurable form such as current or voltage. The transmission of information embedded in the light signal is accomplished by controlling any combination of the parts that comprise the system. Because both the source and the transmission medium determine the amount of light received by the detector, it is possible to convey information about both the source and the medium to the detector. If the source provides illumination while the medium is modulated or interrupted, then the detector can be designed to capture the modulated light while suppressing the effects of the ambient light conditions.

Any mechanism that causes an electron to vibrate will emit a stream of electromagnetic waves. If the electron is vibrated fast enough so that the wavelengths are in the 330- to 770-nm range of the electro-magnetic spectrum (see Figure 6.1), then visible light is emitted. These electroelectro-magnetic waves radiate in every direction away from the point of origin. The observed light has both a wavelength and intensity.

The frequency, f, of the electromagnetic wave is related to the vacuum wavelength, λ0, and is given by

f _λ0 =c (6.1)

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where c is the speed of light in a vacuum (~3 × 10⁸ m/s). Common artificial light sources include incandescent lamps, flourescent lamps, tungsten lamps, light-emitting diodes (LEDs), and lasers.

Some mechanisms can produce visible light through chemiluminescence, bioluminescence, and cathodoluminescence. Chemiluminescence occurs when the electron excitation energy necessary for photon emission is supplied by a chemical reaction. Phosphorous glow through oxidation in the air is one example. Bioluminescence is a subdivision of chemiluminescence and occurs in living organisms such as fireflies and glow-worms. Cathodoluminescence occurs when the excitation energy is supplied by an accelerated electron colliding with atoms. This causes an electron in the atomic structure to move from one orbit to another, which produces light. An example is the cathode ray tube used in television receivers, video terminals, and oscilloscopes.

6.2.2 Vibrio fisheri Bacteria

A variety of natural organisms emit visible light. These include squids, fish, insects, algae, and bacteria.

These organisms can be found in a wide range of environments, from marine and freshwater areas to terrestrial habitats. Of the various light-emitting organisms, bacteria are the most abundant and wide-spread throughout the world. All luminescent bacteria are known to be Gram-negative mobile rods.

These bacteria are mainly found in oceans living freely, symbiotically, saprophytically, or in a parasitic relationship with other higher-order organisms.

The Vibrio fisheri bacterium is one marine species found in the Pacific Ocean around Hawaii and the coastal areas of California. The most common function of Vibrio fisheri is to be a light source for other organisms. The squid Euprymma scolopes exploits Vibrio fisheri in a symbiotic relationship by allowing the bacteria to grow uninhibited in its light organ. Scientists have discovered that without the presence of high concentrations of Vibrio fisheri bacteria in immature squids, the light organ does not fully develop.

The squid is very specific in the type of Vibrio fisheri it allows to inoculate its light organ by using sophisticated epithelial structure to lure the bacteria. In return for infecting the light organ, the squid provides nutrients and protection to the Vibrio fisheri.

The luminescence of the bacteria appears as a faint glow and can only be observed in a dark environ-ment. The level of bioluminescence exhibited by the bacteria is highly dependent on cell density. This dependency is linked to the production of a chemical compound named the luxautoinducer, which provides communication between cells and allows individual bacteria to sense the response of the entire population. Both cellular and environmental factors control the bioluminescence reaction. These factors include the nutrient or growth medium, environmental toxicity, exposure to oxygen, and cell concen-trations.

In Vibrio fisheri, the autoinducer is termed N-(3-oxohexanoyl homoserine lactone). When Vibrio fisheri bacteria live freely in the ocean, there are approximately 10² cells/ml. The autoinducer is diffused out of the cell because of the naturally small concentration of bacteria. When the bacteria are present in high concen-trations, such as on the light organ of the squid (10¹⁰ to 10¹¹ cells/ml), the autoinducer accumulates until it reaches a critical concentration of about 5 to 10 nM—the amount required to activate the luminescence gene transcription, which triggers the specific luminescence enzymes. There are numerous enzymes involved FIGURE 6.1 Optical portion of the electromagnetic spectrum.

Visible

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in the light-emitting process of Vibrio fisheri. The luminescent reaction is catalyzed by the luciferase, which involves the oxidation of long-chain aldehyde and reduced flavin mononucleotide (FMNH2) and releases light:

FMNH₂ + RCHO + O₂→ FMN + RCOOH + H₂O + light (490 nm) (6.2) 6.2.3 Toxin Biosensor—An Illustrative Example

Many environmental monitoring systems employ a biological sensing element to detect the presence of toxins [Sandstrom and Turner, 1999]. Biosensor mechanisms that have been used for environmental applications include enzymes, antibodies, and microorganisms. These biological recognition elements can be interfaced to electrochemical, optical, or acoustic signal transducers. Currently, a laboratory-based MICROTOX^TM assay that utilizes bioluminescent bacteria is being used for a large number of applications.

However, a self-contained biosensor that can be easily transported to the investigated site is preferred for field applications that are inaccessible for the technician to gather samples or that have a high degree of risk due to the level of toxicity present in the environment.

The biological sensing elements are immobilized luminescent bacteria whose response to the toxins in the environment can be quantitatively measured. The level of bioluminescence exhibited by the bacteria is highly dependent on cell density. The bacterial luminescence reaction involves the oxidation of the 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. Both cellular and environmental factors control the bioluminescence reaction. These factors include the nutrient or growth medium, environmental toxicity, exposure to oxygen, and cell concentrations. Figure 6.2 is a plot of the normalized luminescence of the bacteria before and after the introduction of the toxin acetone. As soon as acetone was added, the luminescence of the bacteria decreased sharply but started to increase after absorbing the shock of being exposed to the toxin. It should also be noted that at the end of 10 min the bacteria were never able to fully recover from the toxin.

A simple opto-mechatronic device has been proposed by Knopf et al. [2000] that exploits the light-emission characteristics of Vibrio fisheri bacteria to measure the degree of toxicity in the surrounding air.

The bacteria are immobilized on polyvinyl alcohol gel capsules and placed inside a specially constructed, miniature light-sealed chamber. Enclosed along with the inoculated gel is an opto-electronic transducer that produces a train of pulses with a frequency proportional to the amount of light being emitted by

FIGURE 6.2 Normalized luminescence of immobilized Vibrio fisheri bacteria subjected to acetone at various times.

(Adapted from Knopf, G. K. et al., in OptoMechatronic Systems, Cho, H. S. and Knopf, G. K., Eds., Proc. Soc. Photo-Opt. Instrum. Eng., Vol. 4190, pp. 9–19, 2000.)

Time (min) Acetone 1.0

0.5

0.0 Normalized Luminescence

0 5 10 15 20 25 30 35

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the bacteria. The transducer response is amplified and broadcast to a distant site by a wireless radio-frequency (RF) transmitter. The RF receiver can be placed at a remote location for data reception, storage, and display. The simple design will enable the biosensor to be implemented in the field with very little preparation time and minimal operator training. Figure 6.3 is a block diagram showing the information flow through the basic components of the biosensor telemetry device. The level of bioluminescence exhibited by the bacteria population is proportional to the degree of toxicity, cell density, and airflow in the chamber. The intensity of bioluminescent light is focused onto a photodetector by means of a convex lens. The opto-electronic transducer converts the light intensity into a voltage signal that undergoes amplification and signal conditioning. The conditioned signal is converted to a pulse frequency signal and broadcast as a wireless signal via an RF transmitter. At a remote site the RF signal is picked up by a receiver and reconstructed for further processing, analysis, or data storage.