Issue



Biosensor monitoring of the cleanroom environment


04/01/2006







Integrated circuit sensors: Combining the living bacterial cell with microchips

By Steven Ripp, Ph.D., research assistant professor at The University of Tennessee’s Center for Environmental Biotechnology

Keeping a cleanroom free from contamination is an essential yet often difficult process, especially when it comes to microorganisms. Besides their main route of entry via personnel working in the cleanroom, microbes maintain their presence through introduced equipment or raw materials, in the air supply, and anywhere water is present, such as in compressed air, condensates from inefficiently designed or malfunctioning equipment, or the moist skin-to-garment interface. Any breakdown in regular cleaning and disinfection procedures can also drastically magnify biocontamination levels. And, once microorganisms gain hold, they can persist and survive under fairly harsh environmental conditions for lengthy periods using their well-honed repertoire of stress-response mechanisms.

Indeed, biocontamination events can, and will, occur in the cleanroom environment; thus, microbial surveillance becomes paramount. Doing so requires the application of various detection methodologies, the most conventional of which is identification based on morphological evaluation and the ability to grow the microbes on certain media under certain conditions. The fastest of these tests can take several hours, but the vast majority requires overnight incubation, at minimum. And since not all microbes are cultivable, a certain underestimation of microbial occurrence is always inherent in plate-count assays. As a result, quantitative verification of cleanliness cannot be discerned.

The polymerase chain reaction (PCR) provides a much higher degree of sensitivity using genetic material as its basis for identification. However, it can be a fastidious procedure requiring sample preprocessing, trained technical expertise, and specialized instrumentation. Immunoassays, like the Enzyme-Linked Immunosorbent Assay (ELISA), take advantage of antibody specificity to identify a microorganism. The procedure, though, requires multiple steps, a typically large and expensive spectrophotometer, and trained personnel. But, if a person wants to simply determine if biocontaminants are present without regard to their specific identity, an adenosine triphosphate (ATP) assay can be performed.

Because ATP is used by all living organisms, its presence in the cleanroom facility indicates biological infiltration. The ability to distinguish extracellular ATP from intracellular ATP in these assays provides a means for differentiating between viable and nonviable biological contaminants. The assay is very simple, rapid, and cost-effective; yet, again, ATP cannot identify a specific microorganism, nor can it quantitatively confirm the number of microorganisms present.

The current selection of common microbial detection methodologies clearly doesn’t meet the needs of the cleanroom practitioner. Consequently, significant R&D effort continues to be directed toward the creation of the ideal detection system-one that would encompass all the following traits:

  • Target specificity - Ability to precisely identify the biocontaminant down to species and strain within complex sample matrices, and distinguish viable targets from nonviable targets.
  • Sensitivity - Target must be detectable at single-cell concentrations within minimal sample volumes.
  • Multiplexed - Ability to detect several targets simultaneously.
  • Detection time - Occurs within minutes.
  • Effortless sampling - No multi-step preprocessing, no cleanup, no extraneous reagent additions, and no pre-enrichment.
  • Ease of use - A kit-like format containing non-hazardous disposables, requiring minimal to no user training.
  • Automated - Highly automated and operable in either high-throughput benchtop instruments or field-portable single-sample detectors.
  • Cost-effective - Minimal cost per sample.

No detection method supporting all these features has yet to be devised. However, many new approaches are just now beginning to embrace at least some of the goals listed above. These methods include detectors based on chromatography, flow cytometry, infrared or fluorescence spectroscopy, mass spectrometry, nucleic acid microarrays, quantum dots, and other technologies too numerous to mention. Also included within this group are biosensors.

Biosensor and bacteriophage

Biosensors are described as an interconnection of a biological entity with an analytical transducer. The biological portion is responsible for sensing a target analyte and generating a signal that is then detected, quantified, and processed electronically, optically, or mechanically by the interfaced transducer. The biological component can take on a variety of forms ranging from biomolecular (antibodies, DNA, enzymes, receptors) to living cells (bacterial, yeast, mammalian) or viruses (bacteriophage), and can be designed for specific or nonspecific detection of numerous chemical, biological, or physical targets.

For the most part, biosensors are fairly simple devices capable of maintaining high specificity and sensitivity within complex sample matrices, with little requisite sample pretreatment. In combination with small size, portability, rapidity, and real-time to near real-time output, biosensors are increasingly demonstrating utility toward microbiological recognition.1, 2

Some of the most unique and interesting biosensor elements currently being used for identifying bacteria are the bacteriophage. Bacteriophage, or phage for short, are viruses that infect viable bacterial cells. A phage’s life begins when it randomly bumps into a compatible host bacterium, after which it injects its nucleic acid into the cell. The phage’s nucleic acid hijacks the bacterial cell and diverts all host resources toward the production of scores of new phage, which are then released en mass when the host cell ruptures. The cycle continues as these new phage find, infect, and propagate within additional hosts.


Figure 1: Bacteriophage is a virus that looks akin to an alien-landing pod. With its six legs, the bacteriophage attaches to the surface of the much-larger bacteria.
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Prior to the discovery of antibiotics, the selectively destructive capacity of phage was often used to treat bacterial infections. Pioneered in Soviet Georgia, these so-called phage therapies successfully treated cases of cholera and bubonic plague, and prevented infections in war-wounded soldiers.3 Bacteriophage did their job so well because they were highly specific, destroying only the “bad” bacteria while leaving the “good” bacteria alone.

Unfortunately, phage therapy was given the cold shoulder when the miracle of antibiotics was realized. But with the escalation of antibiotic-resistant microorganisms, the exploitation of phage for targeted annihilation of bacteria has gained renewed interest.

Phage typing

Rather than using bacteriophage to simply destroy (or lyse) bacteria, the exquisite specificity of phage infection can be utilized to detect and identify particular bacterial species in what is called phage typing.4 Phage-typing schemes are widely available for all the major bacterial pathogens, and can be used to uniquely detect the specific presence of a targeted viable bacterium within a mixed-population sample. By applying the fundamentals of phage typing, a person can create a variety of biosensors for extremely selective bacterial recognition.

T. Neufeld and others developed an amperometric biosensor based on the premise that phage added to a sample will destroy target bacteria, resulting in the release of intracellular enzymes.5 Thus, the detection of an electrical current from selectively activated enzymes suggests that cell lysis occurred and, therefore, that the target bacterium was present. The resulting microamperage produced also yields an estimate of target-cell concentration that, at its lower limit, approached one colony-forming unit (cfu) per 100 ml within a six- to eight-hour detection period.

Using the same concept, DJ Squirrell and others conveyed intracellular enzyme release into a light reaction moderated by the firefly luciferase (luc) genes.6 Light signals could be easily detected by a luminometer and then quantified, establishing a lower detection limit of 100 cells per ml in approximately one hour.

The multiplication of phage within the host cell, and the subsequent release of large quantities of phage upon host-cell lysis, can also be used as a sign that a target cell is present. These tests, referred to as phage amplification assays, link escalating phage titers to biosensor-compatible endpoints such as changes in absorbency due to increased rates of bacterial death or by the addition of fluorescent probes that bind to viable cells only. Approximately 104 cfu per ml can be detected within five hours in these assay formats.7


Figure 2: Pictured here are lux-based bioluminescent bacterial colonies growing on a Petri plate. Image courtesy of University of Tennessee.
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If we move toward the beginning of the phage-infection cycle, where nucleic acid is injected into the host cell, we can begin to see that the phage itself is nothing more than a vessel that carries and transfers genetic material. However, it is the transfer of DNA or RNA to a specific host cell that makes the phage much more interesting in terms of biosensing applications. For instance, what if we could insert a marker into the phage genome? That marker, or reporter gene, would then be transmitted to the host cell upon phage infection, resulting in an easily identified, tagged target cell.

One of the first commercially available kits to take advantage of phage-incorporated reporter genes was the bacterial ice nucleation diagnostic (BIND) assay.8 A reporter gene (inaW) responsible for the initiation of ice-crystal formation was inserted into a Salmonella-specific phage. Transfer of the inaW gene from the reporter phage to the Salmonella host induced freezing, which was monitored through the introduction of an orange-colored indicator dye.

Although sensitive and capable of detecting less than 10 Salmonella per ml, the assay never gained widespread use due, somewhat, to its complexity. Nowadays, simpler phage-reporter assays have been developed using reporter genes like green fluorescent protein (GFP), luc, and bacterial luciferase (lux), which yield easily measurable fluorescent or bioluminescent signals when inserted and expressed in the host cell.

For example, T. Funatsu and others constructed a GFP reporter phage capable of detecting individual Escherichia coli cells using charge-coupled device (CCD) imaging.9 Meanwhile, S. Bardarov and others used luc-based reporter phage, supplementary luciferin substrate, and a luminometer to identify Mycobacterium tuberculosis at concentrations approaching 1000 cfu per ml.10 And, J. Chen and M.W. Griffiths were able to visualize Salmonella contamination directly inside an eggshell via infusion of lux-based reporter phage and CCD imaging.11

Bioluminescent signals

Although GFP, luc, and lux each has advantages and disadvantages, the laboratory at the University of Tennessee’s The Center for Environmental Biotechnology has been advocating the bacterial lux genes for several years simply because lux functions autonomously; it doesn’t require the addition of substrate or specialized excitation in order to see its bioluminescent light signal (see Figure 2). The lux gene cassette used-which comprises five genes designated luxA, luxB, luxC, luxD, and luxE-originates from naturally luminescent bacteria that live, for example, within glowing appendages seen in marine organisms such as the bobtail squid.12

Two other genes associated with the lux cassette are luxR and luxI, which act somewhat like a switch that turns the light on and off in a process called quorum sensing. Quorum sensing discourages light production when only a few cells are present-why waste valuable cellular energy producing light when the cumulative light signal created from only a few cells is negligible? Using cell-derived chemical mediators (autoinducer molecules), quorum-sensing bacteria can communicate with one another to establish population densities and, therefore, understand when a sufficient number of cells are present (a quorum) for collectively generating a concentrated light response.


Figure 3: The reporter phage carrying the luxI reporter gene specifically attaches to and infects the target bacterium. Subsequent transfer of the luxI gene to the host cell obligates host cell synthesis of autoinducer molecules that diffuse out of the cell and trigger bioluminescence emission in neighboring bioreporter cells. Thus, based on simple light signatures, one can identify the original bacterial target.
Click here to enlarge image

By combining lux-based bioluminescence, quorum sensing, and phage reporters, the university lab has recently created a novel phage diagnostic for E. coli that can be applied toward other bacterial targets as well, simply by substituting other host-specific phage.13 The reporter phage was genetically manipulated to contain the luxI gene responsible for the production of the quorum-sensing autoinducer molecules.

Thus, after infection and commandeering by the reporter phage, the E. coli cell becomes a biological factory churning out autoinducer molecules. The diffusion of these autoinducer molecules out of the cell and into the surrounding medium serves as the signal that phage infection occurred and, therefore, that E. coli is present in the sample. So, now all that’s remaining is the detection of the autoinducer molecules. This can be achieved using mass spectrometry, but the cost and complexity involved in such an analysis is prohibitive.

Alternatively, the lab genetically engineered a bacterial cell-or, more specifically, a bioluminescent bioreporter cell-using the luxA, luxB, luxC, luxD, and luxE genes to autonomously sense autoinducers and communicate their presence via simple bioluminescent light emission. Thus, the synthesis of autoinducer molecules by the target bacterial cell, initiated by phage-specific infection, causes the bioreporter cell to emit light at 490 nm, and the intensity of this light response can be correlated back to the autoinducer concentration and the corresponding target bacterial concentration.

The steps involved in the assay-which requires merely the addition of reporter phage and bioreporter bacteria to the sample-are shown in Figure 3. Although only a prototype at this stage, the binary phage-reporter sensing system was able to detect down to approximately 100 E. coli cfu per ml within less than 24 hours in a high-throughput 96-well microtiter plate assay.

Light-measuring equipment

While the measurement of light is a fairly straightforward process, it is often tied to bulky, expensive laboratory-based instruments designed to accommodate microtiter plates and little else. On-site monitoring-where bioreporter microorganisms excel due to their propensity to subsist, remain stable, sense, and adapt to widely fluctuating environmental conditions-remained difficult under this constraint. Thus, there has always been great impetus toward miniaturization of the analytical instrumentation associated with light measurement.

With this in mind, the lab developed an integrated circuit microluminometer that can directly interface with bioluminescent bioreporters to detect and measure bioluminescence emission. The microluminometer, which is referred to as a bioluminescent bioreporter integrated circuit (BBIC), uses cost-effective industry-standard complementary metal oxide semiconductor (CMOS) technology. Using CMOS, a single 1.5 x 1.5 mm integrated circuit can be inlaid with millions of task-specific transistors, allowing the BBIC to not only monitor light, but to also house ancillary functions such as radio frequency (RF) wireless telemetry, global positioning, temperature sensing, or time stamping.


Figure 4: The biosensor consists of a handheld wand to which attaches disposable target-specific bioluminescent bioreporter cartridges that can be interchanged depending on monitoring needs. Data derived from bioluminescense emission is downloaded wirelessly to provide near real-time monitoring of biocontamination events. Image courtesy of University of Tennessee.
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The current BBIC design confines the microluminometer chip in a handheld wand-type biosensor consisting of a disposable reaction cartridge containing the bioreporters (see Figure 4). A cartridge, chosen based on the particular bacterial target for the assay, is plugged into the biosensor wand, and the sample is added. If the target bacteria are present, a light response is registered by the microluminometer and relayed in real-time back to the user.


Figure 5: The size of the microchip is extremely small, as shown on this penny. Image courtesy of University of Tennessee.
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At present, the biosensor is capable of detecting only a single target at a time, but with modifications, the microluminometer can be subdivided into individualized cells for isolated patterning of multiple bioreporters on a single chip platform, thereby allowing for multiplexed detection. The emerging technology platform provided by BBIC-based microluminometer sensing, in conjunction with target-specific phage reporter systems, presents a unique on-the-spot monitoring tool for application toward the maintenance and verification of cleanroom sterility.

References

  1. Deisingh AK, Thompson M. Biosensors for the detection of bacteria. Can J Microbiol 2004; 50 (2):69-77.
  2. Ivnitski D, Abdel-Hamid I, Atanasov P, Wilkins E. Biosensors for detection of pathogenic bacteria. Biosens Bioelectron 1999; 14:599-624.
  3. Stone R. Stalin’s forgotten cure. Science 2002; 298:728-31.
  4. Marks T, Sharp R. Bacteriophages and biotechnology: a review. J Chem Technol Biotechnol 2000; 75:6-17.
  5. Neufeld T, Schwartz-Mittelmann A, Biran D, Ron EZ, Rishpon J. Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria. Anal Chem 2003; 75 (3):580-5.
  6. Squirrell DJ, Price RL, Murphy MJ. Rapid and specific detection of bacteria using bioluminescence. Anal Chim Acta 2002; 457 (1):109-14.
  7. Favrin SJ, Jassim SA, Griffiths MW. Development and optimization of a novel immunomagnetic separation-bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth. Appl Environ Microbiol 2001; 67 (1):217-24.
  8. Irwin P, Gehring A, Tu SI et al. Minimum detectable level of Salmonellae using a binomial-based bacterial ice nucleation detection assay (BIND). J AOAC Int 2000; 83 (5):1087-95.
  9. Funatsu T, Taniyama T, Tajima T, Tadakuma H, Namiki H. Rapid and sensitive detection method of a bacterium by using a GFP reporter phage. Microbiol Immunol 2002; 46 (6):365-9.
  10. Bardarov S, Dou H, Eisenach K et al. Detection and drug-susceptibility testing of M. tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system. Diagn Microbiol Infect Dis 2003; 45 (1):53-61.
  11. Chen J, Griffiths MW. Salmonella detection in eggs using lux+ bacteriophages. J Food Protect 1996; 59:908-14.
  12. Meighen EA. Molecular biology of bacterial bioluminescence. Microbiol Rev 1991; 55:123-42.
  13. Ripp S, Jegier P, Birmele M et al. Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents. J Appl Microbiol 2006; 100 (3):488-99.

Steven Ripp is a research assistant professor at the University of Tennessee’s Center for Environmental Biotechnology in Knoxville. Previously he received a Ph.D. in Microbiology and Molecular Genetics from Oklahoma State University. He has done extensive research in the construction of recombinant bacterial and bacteriophage bioreporter strains for the detection and monitoring of food-borne pathogens, biological toxicants, environmental pollutants, environmental stressor mechanisms, and components of bio-microelectronic computer processing systems, and has a number of patents pending in these areas. Ripp also has written numerous industry-related articles and publications. He can be reached at saripp@utk.edu.