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Volume 16 | Number 3 | Fall 2004

Safety Issue


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Innovative Biosensors Are Opening New Frontiers

Georgia Tech Interferometric Biosensor
Georgia Tech’s novel interferometric biosensor is one of many university-based sensors under development.

For more than a decade, the poultry industry has actively pursued better methods for controlling foodborne pathogens on fresh product. Starting with the introduction of systematic microbial screening of products and processes and progressing to the many new rinse and anti-microbial treatment steps that have been added to most processing operations, strong progress is being made. Yet, the long time delay between sample collection and obtaining microbial screening results continues to hamper the efficiency of these programs. In the absence of simple, inexpensive, and rapid microbial detection techniques, little feedback is available to help plants recognize changing microbial conditions as they are occurring. This, in turn, prevents them from better managing the intervention resources they are using to control microbial contaminants.

Fortunately, university research labs and commercial instrument manufacturers have been hard at work trying to find new ways of closing the time gap between taking a sample and getting results. The majority of this work has been driven by the needs of the medical diagnostics sector. But food safety concerns and terrorism threats have managed to focus some of this research on the unique performance needs of industries like poultry. Some promising developments are starting to emerge, particularly in the area of biosensors.

What Are Biosensors?
Biosensors are electronic devices capable of measuring the presence, identity, and/or quantity of an organism in a rapid, usually unattended manner. Most do so through integrated detection mechanisms that generate a signal that corresponds to the concentration of the organism, a particular protein component, or nucleic acid that is present. Techniques for recognizing the presence of the target vary from the use of selectively attached labels (colorometric agents, florescent dyes, etc.), which the sensor in turn can detect and measure, to direct detection of organism binding to the sensor surface that causes subtle, but measurable, changes in the sensor’s optical or electrical properties. What distinguishes a biosensor from other types of sensors is its use of biological materials as part of the recognition element. The most common biological materials used in biosensors are antibodies and nucleic acids (DNA or RNA).

Antibody usage has its root in conventional immunoassay analysis techniques, in which antibodies for a specific organism are used to selectively capture that organism (or elements of it) present in the sample. The unique ability of antibodies to bind only one specific molecule or microbial species within a sample containing high background microbial flora makes this one of the most popular rapid screening techniques in use today. Antibodies can be used to attract whole cells and cell proteins to the sensor, making them extremely versatile and ideally suited for both laboratory and field screening applications.

Like antibodies, the affinity of DNA and RNA molecules for their complementary targets can be used to capture an organism’s genetic material within a sample. Unlike antibodies, this technique can be used to determine very precise details about the organism that could prove critical in defining its origin or risk status. For instance, is the organism part of a unique and dangerous strain originating from a known contamination source? Because amounts of nucleic acid typically extracted from a sample are small, amplification techniques are usually needed to allow their subsequent measurement. One of the most powerful and widely used amplification techniques is polymerase chain reaction (PCR); however, it adds additional levels of sample handling, processing sophistication, cost, and time delays, making it more suitable for detailed laboratory analysis activities.

Once the biological element of the sensor captures the whole organism or a selected molecular component, a wide variety of signal transduction methods can be used to detect and measure its presence. These methods can generally be classified as optical, electrical, or mass-based measurements. Optical approaches include interferometry, surface plasmon resonance, fluorescence, and chemiluminescence. The first two of these measure index of refraction changes when a captured object binds to a surface-attached receptor without the use of any sort of labeling. Fluorescence and chemiluminescence measurements require the detection of light generated by a label attached to a secondary or reporter antibody or nucleic acid. Electrical transducers measure changes in current (amperometry), resistance, or conductance when target binding occurs. Finally, mass-based sensors use piezoelectric (quartz crystal microbalance) or magnetoelastic (cantilever) transducers as very sensitive balances to detect changes in weight when a molecule or microbe binds to the surface. While the specifics for any one biosensor device will vary, typical sensitivities of from 100 to 10,000 colony forming units (cfu)/milliliter (ml) are achievable in less than an hour with antibody-based detection systems.

Georgia Tech Interferometric BiosensorPhoto courtesy of Georgia Tech
Georgia Tech Interferometric Biosensor
Photo courtesy of Innovative Biosensors
Georgia Tech Interferometric Biosensor
Photo courtesy of Michigan State University
Georgia Tech Interferometric Biosensor
Photo courtesy of Penn State University
Pictured top to bottom: Georgia Tech Interferometric Biosensor, CANARY™ Biosensor System, Michigan State Electrochemical Biosensor, and Penn State Magnetoelastic Sensor Reader.

Emerging Designs
While biosensors have been around since the 1970s, they have only recently begun to appear commercially for food processing and safety applications. The majority of commercial offerings are based on traditional signal transduction methods, such as surface plasmon resonance and fluorescence, and are targeted at laboratory-based testing operations. However, a number of promising new designs are emerging from university research laboratories with the potential to broaden their value to operational support.

Georgia Tech Interferometric Biosensor: Researchers are in the later stages of developing a biosensor whose key elements are a laser diode, a waveguide, and an image detector. As molecules or microbes bind to capture antibodies on the waveguide surface, they alter the propagation speed of light within the waveguide. When that beam is optically combined with a reference beam, an interference pattern is created that is observed by the image detector. Changes in that interference pattern allow the sensor to accurately measure the amount of binding taking place on the waveguide surface. Laboratory tests with Salmonella and Campylobacter have shown the current design to have detection sensitivities of 5,000 cfu/ml and 500 cfu/ml, respectively, in less than 30 minutes. Additional optimization of the fluidics and assay protocol is expected to yield detectability as low as 100 cfu/ml. More importantly, the sensor has been designed not only to be inexpensive and portable, but also to be used within a processing plant environment to provide feedback to help control operating performance.

CANARY™: Based on a novel sensor designed by researchers at the Massachusetts Institute of Technology (MIT), the CANARY™ (Cellular Analysis and Notification of Antigen Risks and Yields) system, offered by Innovative Biosensors, uses a genetically engineered biosensor composed of a human cell line that expresses a selective antibody. When a target is bound, the cell responds with a cascade of events that triggers bioluminescence that is detected using a simple luminometer. Tests have shown the current design can detect 500 cfu/g of E. coli O157:H7 on lettuce within 5 minutes using a simple protocol.

Michigan State Electrochemical Biosensor: A disposable, reagentless biosensor that uses conductive polymer molecules to convert binding events between an antibody and target into electrical signals on a membrane surface has been designed by Michigan State University scientists. The membrane surface can be removed for subsequent confirmation of the target using conventional microbiological methods. The device is small and rugged for field use. Both E. coli O157:H7 and Salmonella in fresh produce and meat products have been detected within 10 minutes.

Penn State Magnetoelastic Sensor: Analogous to the concept of an acoustic bell, magnetoelastic transducers resonate in response to an externally applied magnetic field impulse. Changes in environmental parameters, including an increase in mass due to binding of target pathogen to immobilized antibodies, result in the modulation of the characteristic resonant frequency which can be monitored using a remote pickup device. In recent work from this lab, as little as 100 cells/ml of E. coli O157:H7 were detected in buffer solution.

Opening New Frontiers
What may ultimately separate these sensors, however, down the road could be their ability to be used in remote settings, such as on-line in a processing plant, and to be operated automatically as part of a feedback mechanism for process control. Here simplicity of operation and ruggedness will play increasingly important roles as will system and operating costs. But with the emergence of such systems, pathogen control methods and water reuse have the potential to be monitored and adjusted more dynamically through the generation of more timely and cost-effective pathogen load tracking and statistical control methodologies.

 

Major portions of this article were extracted from “Biosensors: Speeding Up Detection of Pathogens” by J. Craig Wyvill and David Gottfried, WATT PoultryUSA, September 2004, with the permission of Watt Publishing. Any reprint of this article without the expressed permission of Watt Publishing is prohibited.