Whole-Cell Environmental Monitoring Devices: Bioluminescent

Chapter 14

Whole-Cell Environmental Monitoring Devices: Bioluminescent Bioreporter Integrated Circuits 1

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Steven Ripp , Bruce M. Applegate , David E. Nivens , Michael J. Paulus , George E. Jellison , Michael L. Simpson , and Gary S. Sayler 2

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Downloaded by STANFORD UNIV GREEN LIBR on September 22, 2012 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0762.ch014

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Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 Oak Ridge National Laboratory, P . O . Box 2008, M S 6006, Oak Ridge, TN 37831 2

Bioluminescent bioreporter integrated circuits (BBICs) are novel biosensor devices that utilize light emitting microorganisms as biological sensors for chemical contaminants. The microorganisms are coupled to a full-custom integrated circuit containing photodetector units and low-noise signal processing circuitry for measuring and communicating chemically induced cellular light responses. Two prototype BBICs have been designed and tested using naphthalene and toluene sensitive bioluminescent bioreporters. Measured sensitivities have approached the low partsper-billion range.

W h o l e - C e l l Biosensors Chemical sensing methods involving microorganisms have been widely applied for the detection and measurement o f industrial pollutants and toxicants. These 'whole-cell' biosensors consist of living microbial cells that respond in some manner to a target chemical, coupled to an instrument capable of detecting this response. The use o f intact microbes as chemical sensors allows for detection and monitoring o f chemical bioavailability rather than just chemical presence. This is in stark contrast to analytical techniques that simply determine whether a chemical is present and at what concentration, without providing requisite information as to the biological effect of the chemical. Corresponding author. © 2000 American Chemical Society

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In Chemical and Biological Sensors for Environmental Monitoring; Mulchandani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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In their earliest forms, whole-cell biosensors consisted o f electrochemical transducers that monitored basic changes in cell growth activity (i.e., oxygen utilization or carbon dioxide production) (I). The microbial cells and sensing elements have since evolved into more complex systems. Genetic engineering techniques have produced microorganisms that exhibit enhanced responses to specific biological or physical agents in their environment, and technological advances have produced highly sensitive transducers capable of detecting electrochemical, acoustic, or optical signals. Consequently, whole-cell biosensors are now being developed for the detection of a wide range of chemicals for environmental as well as medical, agricultural, defense, and food applications (2).

Bioluminescent Bioreporter Integrated Circuits A n innovative whole-cell biosensor currently undergoing testing is the bioluminescent bioreporter integrated circuit (BBIC) (3). The biological component of the B B I C consists of bacterial cells that emit light in response to a specific chemical, suite of chemicals, or physical agent within the cell's environment. The sensor is a photodetector unit capable of detecting the bacterial light signal. The bioluminescent bacteria, photodetectors, and all circuitry necessary for processing the light signals and communicating the results are situated on a 4 mm integrated circuit (Fig. 1). 2

Bioluminescent Bioreporters The bioluminescent bacteria adhered to the integrated circuit are genetically engineered cells capable of generating light in response to chemical exposure. Light production is dependent on a genetic operon designated lux, derived from luminescent



199 marine bacteria like Vibrio flscheri, Vibrio harveyi, or Photobacterium leiognathi (4). The lux operon consists of five genes, luxC, D, A, B , and E. luxA and B together encode for the enzyme luciferase which is responsible for generating the light response. Luciferase converts long-chain aldehydes into fatty acids with a concomitant production of photons, visible as blue-green light at 490 ran. The fatty acids are recycled back into the aldehyde substrate by a multienzyme fatty acid complex consisting of three proteins, a reductase, transferase, and synthetase encoded by the luxC, D, and E genes, respectively. Using genetic engineering techniques, the lux operon can be placed under the control o f a promoter element that is induced in the presence o f a specific chemical. Activation of the promoter results in transcription of the lux genes and light production, thereby forming a direct correlation between chemical presence and bioluminescence. lux fusions can consist of just the luxAB complex or the complete luxCDABE cassette. If only the luxAB genes are used, the cells must be supplied with an aldehyde substrate, usually decanal, before light production is possible. Utilization of the entire luxCDABE cassette, however, allows for a completely self-contained bioluminescent response, negating the requirement for any exogenous substrate additions. Consequently, these bioreporters can be used for continuous on-line, near real-time measurements. Bioluminescent bioreporters can also be engineered using a eukaryotic genetic system designated luc (5, 6). luc genes are derived from the firefly (Photinus pyralis or Luciola mingrelica) or click beetle (Pyrophorus plagiothalamus) and have been incorporated into Escherichia coli cells for biosensing applications. However, similar to the luxAB constructs described above, /wc-based bioreporters must be supplied with an exogenous substrate, luciferin, before bioluminescence can occur. Cells must also typically be destructively lysed. Thus, as currently designed, luc bioreporters cannot be continuous fully independent biosensors.

Pseudomonas fluorescens HK44 A well-studied example of a to-based bioluminescent bioreporter is the microorganism Pseudomonas fluorescens HK44, which is capable of sensing naphthalene, a common constituent of polyaromatic hydrocarbons (PAHs). P. fluorescens HK44 contains the complete luxCDABE cassette, and is therefore capable of intrinsic bioluminescence (7). The original parental P. fluorescens strain from which HK44 was derived was isolated from a manufactured gas plant facility heavily contaminated with PAHs. The strain was specifically isolated due to its ability to degrade naphthalene, which was experimentally determined to occur via a two pathway catabolic process situated on a plasmid designated p K A l (Fig. 2). The upper pathway converts naphthalene to salicylate, which is then further converted to catechol through the lower pathway. In strain HK44, genes within the lower pathway were replaced with the luxCDABE genes, producing a cell that continued to degrade naphthalene to salicylate, but rather than further converting salicylate to catechol, salicylate now served as the inducer for the artificial lux operon. Therefore, P.


200 fluorescens HK44 became a bioluminescent bioreporter for naphthalene (or salicylate), and has been applied in several experimental assessments to measure naphthalene bioavailability under environmental conditions (8-11). Similar bioluminescent bioreporters incorporating the complete luxCDABE cassette have since been engineered to detect a number of biological and physical agents (Table I).


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Figure 2. A) Genetic organization of the pKAl catabolic plasmid. Genes of the upper naphthalene regulatory system encode for proteins that mediate the conversion of naphthalene to salicylate. Salicylate is then further degraded to TCA cycle intermediates. B) In Pseudomonas fluorescens HK44, genes within the lower pathway were replaced with genes of the lux cassette to produce a bioluminescent bioreporter sensitive to naphthalene and salicylate.

Integrated Circuit The light response generated by the bioluminescent bioreporters is typically measured with optical transducers such as photomultiplier tubes, photodiodes, microchannel plates, charge-coupled devices, or photographic films. Additionally required is usually some means of transferring the bioluminescent signal to the transducer, which necessitates the need for fiber optic cables, lenses, or liquid light guides. What results is a large, bulky instrument anchored to power and optic cables. In contrast, integrated circuit technology positions all the required instrumentation onto a 2 mm by 2 mm silicon chip, and, by adhering the bioluminescent bioreporters directly to the chip, the need for external signal transfer cables can be bypassed (Fig. 3). In essence, the integrated circuit consists of only two main components;



201 Table I. Chemical and physical agents capable of detection by luxCDABE bioluminescent bioreporters Xylene 2,4-D Phenol Dinitrotoluene Benzene Ethylbenzene Zinc Lead Cadmium Cobalt Chromium Thallium Copper Polychlorinated Biphenyls (PCBs) Mercury Nitrate Nickel Naphthalene Toluene Arsenic Trichloroethylene D N A damaging agents (ultraviolet light) 4-chlorobenzoic acid Isopropylbenzene Oxidative stressors (peroxides, Environmental stressors (heat shock, alginate superoxide radicals) production)

photodetectors for capturing the on-chip bioluminescent bioreporter signals and signal processors for managing and storing information derived from bioluminescence. If required, remote frequency (RF) transmitters can also be incorporated into the overall integrated circuit design for wireless data relay, which can approach levels of satellite transmission coupled to global position sensing to create intelligent distributed biosensing networks. These networks will ultimately allow BBICs to communicate with each other, referencing time and location along with other physical parameters, to provide vector or transport information to pinpoint locations of potential biohazardous concern. The integrated circuit design is based on an industry standard complementarymetal-oxide-semiconductor (CMOS) process (12). C M O S possesses several characteristics desirable for B B I C applications, including low power drain, high noise immunity, the ability to perform digital, analog, and electro-optical signal processing, and highly reliable performance. Furthermore, the nature of C M O S design allows for the incorporation of accessory devices such as RF communication and positionsensing circuitry. C M O S is also amenable to optical application-specific integrated circuit (OASIC) fabrication. These 'personalized' integrated circuits can be produced quickly and at relatively low cost to custom fit specific applications. Therefore, BBICs can be crafted to fit a large number of user-defined functions.

Adhering bioreporters to integrated circuits BBICs will require methods for encapsulating and immobilizing bioreporter cells directly to integrated circuits. A large number of natural and synthetic polymers have been developed for cell encapsulation, all of which are potentially applicable in the BBIC format (13, 14). It must be demonstrated, however, that the matrix itself does



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Figure 3. Size of prototype integrated circuit compared to a U.S. penny. The magnified view displays the photodetector units and signal processing circuitry required for detecting and monitoring bioluminescence.

not induce bioluminescence nor optically interfere with bioluminescent signals. To best fulfill B B I C requirements, it is desirable to have a liquid matrix that can be combined with the cells, directly applied to the integrated circuit surface as a thin film, and allowed to polymerize. Alternatively, bioreporter cells can be encapsulated within blocks or sheets of polymer, from which thin slices can be dissected and attached to the chip. The encapsulation matrix additionally needs to provide an environment in which cell survivability can be maintained for extended periods. Thus, nutrients and co-factors, as well as a means of sustaining proper hydration, must be incorporated within the final matrix. A n encapsulation matrix functional in both aqueous and vapor phases is also desirable. Overcoming all of these obstacles within a single matrix is a difficult task, but experiments utilizing the family of hydrogel and xerogel polymers are showing great promise. These polymers, hydrophilic in nature and optically clear, are amenable to bioreporter encapsulation and have been shown in our own studies to not interfere with bioluminescent signals. Cell survivability, however, remains problematic, with bioreporters remaining fully functional for periods approaching one to two weeks. Current attempts are aimed at incorporating lyophilized bioreporter cells within the encapsulation matrix for long-term storage intervals. Bioreporters could then be resuscitated directly on the integrated circuit for use when needed.

Prototype integrated circuits We have developed two prototype integrated circuits for initial B B I C testing purposes. The first prototype was fabricated in a 1.2 ym C M O S process employing a



203 photodiode that has a strong response to the 490 nm bioluminescent signal (15). Leakage and noise of the photodiode was shown to be minimal (3). The second prototype was fabricated in a newer 0.5 um C M O S process to allow the inclusion of an on-chip remote frequency transmitter. Again, this photodiode generated very low noise (-120 eVsec with an integration time of 13 minutes). The front-end signal conditioning circuit for both chips was a current-tofrequency converter (CFC). When compared to conventional electrometer circuits, this signal conditioning circuit provided lower noise, faster recovery from overloads, and larger dynamic range. A digital signal proportional to the sum of the leakage and photo-current was generated by counting pulses from the C F C for a specified time (i.e., the integration time). To determine background sensitivity, multiple measurements were taken in the absence of bioluminescence with the integration time set to one minute. Leakage currents consistently produced a signal of approximately 6 ± 0.22 counts/minute. Since the integrated circuits will be in constant contact with encapsulated bioreporter cells, it was necessary that they be overlaid with a protective coating. Amorphous silicon dioxide is commonly used for this purpose, but is unsuitable in B B I C applications due to its susceptibility to attack by various biological and chemical agents. Rather, a silicon nitride film, which has been shown to be much more resistant to chemical and biological degradation, will be deposited on the integrated circuits (16). Encapsulated bioreporter cells can then safely be placed on the integrated circuit without harm to the circuit or the bioreporters. Conveniently, silicon nitride also possesses optical properties that will actually improve the response of the photodetectors to the 490 nm bioluminescent signal (17).

Prototype BBICs The integrated circuits described above were initially tested in the B B I C format using the bioluminescent bioreporters Pseudomonas fluorescens 5 R L and Pseudomonas putida T V A 8 . P. fluorescens 5 R L is a naphthalene bioreporter containing the same nah-lux fusion as described previously for the P. fluorescens HK44 bioreporter, but is incapable of metabolizing salicylate (18). P. putida T V A 8 is a bioreporter for B T E X (benzene, toluene, ethylbenzene, and xylene), representing water soluble components of petroleum fuels (19). Toluene was chosen as the representative chemical inducer for strain T V A 8 . Since only two integrated circuits were available for initial testing, bioreporter cells could not be directly affixed to the circuits. Rather, a flow cell was developed to confine the bioreporters directly above the integrated circuit within a light-tight environment. As more integrated circuits become available, actual adhesion of bioreporters to the circuits will be performed to produce a fully functional B B I C as shown in Figure 1. Bioluminescence was induced in the bioreporter cells and a control sample of cells by exposure to naphthalene or toluene vapor. Bioluminescent data was transferred from the B B I C directly to a computer interface. The minimum detectable concentrations achieved by P. fluorescens 5RL and P. putida T V A 8 on the B B I C both approached approximately 10 ppb (Fig. 4).


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Integration time (minutes) Figure 4. Minimum detectable concentrations of toluene and naphthalene as a function of integration time for the prototype BBIC containing either the Pseudomonas putida TVA8 (toluene) or Pseudomonas fluorescens 5RL (naphthalene) bioluminescent bioreporters.

Towards better BBICs Improving B B I C technology relies on refining integrated circuit design as well as enhancing bioreporter attributes. Integrated circuit complexity has approximately doubled every year since its inception, with corresponding increases in reliability and decreases in costs. Therefore, improvements in chip design are likely unmitigated. New chips with more sensitive photodetectors and faster processing units are currently undergoing testing, and will undoubtedly lead to B B I C chemical detection limits below the current 10 ppb threshold. These integrated circuits can also carry sensors to monitor environmental parameters such as temperature, p H , or oxygen concentration, thereby producing a more comprehensive 'laboratory-on-a-chip' device. Bioluminescent bioreporters are also evolving into organisms suitable for a wide range of B B I C applications. Genetic engineering modifications are now underway to create mammalian /ax-based bioreporters that will produce bioluminescence at 37°C without cell lysis or exogenous reagent requirements. Such bioreporters, when placed on integrated circuits, may have huge potential in the continuous, on-line monitoring of medically important diagnostic agents or in the rapid assessment of novel chemicals for combinatorial chemistry purposes.

Acknowledgments Funding support provided by The Perkin-Elmer Corporation, N A S A , Department of Energy, and the National Institutes of Health.

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11. 12. 13. 14. 15. 16. 17. 18. 19.

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Whole-Cell Environmental Monitoring Devices: Bioluminescent

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