For the food industry, microbial contamination results in spoilage of food products and in food that has to be disposed of. There is a need to control and avoid the presence of pathogens from the food production line by constant monitoring and inspection of manufacturing processes.
The detection of eventually contaminated batches of food products translates into actions such as recalling through tracking systems from the distribution lines and their final destinations at supermarket stores.
The costs of contamination for the industry are twofold, caused by a missed income from their marketing and the costs of disposal of deteriorated foods. Therefore, it is of the utmost importance to reduce to a minimum, the potential contamination routes.
In addition, the food components and intermediate products need to be adequately treated, using food-grade bacteriostatic chemicals such as lactic acid and acetic acid. Meat, poultry, processed egg products, milk, seeds and vegetables are the main sources of bacteria that are transferred through cross-contamination during food preparation.
Major foodborne illnesses are caused by salmonella, listeria monocytogenes, staphylococcus aureus, and e coli O157:H7 in raw and cooked foods. The threshold for acceptance of a pathogen depends on its ability to grow at refrigerated conditions and on the type of food and how it will be consumed.
The traditional microbiological culturing methods require longer times due to the time that bacteria need to recover and grow in the enrichment medium and on selective agar plates. Molecular methods can be applied for species identification after bacteria have reached the exponential phase in culture broth.
The food matrix makes difficult the process of elution of the bacteria and frees polymerase chain reaction (PCR) inhibitors that may affect the subsequent analyses, while the number of bacteria may be too low for their isolation. Therefore, enrichment by culturing in selective broth assures improved purification of bacterial DNA, devoid of food matrix compounds.
A pre-enrichment broth is run overnight or for a period of 16 hours, resulting in rapid detection that leads to quick interventions such as change in the destination of the contaminated foods.
There is a need to provide alternative methods in rapid bacteria identification. A biosensor is an analytical device, used for the detection of an analyte, consisting of a bio-recognition component, biotransducer component, physicochemical detector and electronic system which includes a signal amplifier, processor and display.
The transducer or the detector element (works in a physicochemical way) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal that can be more easily measured and quantified. A biosensor reader device and electronics can be combined with a CMOS-image acquisition system.
The sensitivity of the biosensor is the key to microbe detection. The ideal characteristics of a biosensor for implementation in food analysis are: fast detection, portability and low cost.
The sensitive biological recognition component uses biomolecules from organisms or receptors modelled after biological systems to interact with the bacteria of interest, such as aptamers (nucleic acid sequences with high affinity for a bacterial surface protein) or by recognition of bacterial antigens (such as bacteriophage tailspike protein). Immunoassays are based on species-specific antibodies that recognise bacteria with high specificity.
Jack Letourneau, New York, US
An ideal biosensor should detect target molecules easily without multiple steps. A number of methods are used for the rapid detection of biomolecules in solution. The interaction is measured by the biotransducer which outputs a measurable signal proportional to the presence of the target analyte in the sample.
Biosensors can be classified by their biotransducer type. The most common types of biotransducers used in biosensors are: electrochemical, optical, electronic, piezoelectric and gravimetric biosensors.
Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) have moved towards an integrated platform based on low-cost, miniaturised and sensitive detection of bacteria coupled with a smartphone platform, composed of interdigitated electrodes on a micro-hole silicon substrate and a microfluidic chamber based on a nano-porous filter paper, to preconcentrate the bacteria.
Other physico-chemical methods exploited in biosensors are: surface acoustic wave biosensors, quartz crystal microbalances (QCM), optical ellipsometry (OE), oblique-incidence reflectivity difference (OI-RD), resonant cavity and resonant waveguide grating (RWG) biosensors.
Optical biosensors have also been studied with an integrated Mach-Zehnder interferometer for the detection of l monocytogenes; reflectometric interference spectroscopy (RIfS) applied in monitoring several different target molecules at the same time.
On-chip surface-enhanced Raman spectroscopy (SERS), Fourier transformation-infrared spectrometry (FT-IR) and field-effect transistor technologies have been applied in biosensor detection.
Thin film transistors (TFTs) have been applied either as potentiometric sensors for the detection of different biomolecular interactions or as circuit elements for the conditioning and read-out of signals. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene-based electrical devices have been widely developed for high performance label-free chemical/biological sensors.
Magnetic nanoparticle detection systems are based on an alternate current (AC) susceptometer measuring a wide frequency range, from one Hz up to 500 kHz. The magnetic flux change is measured by a detection-coil system connected to the AC source, delivering the current to the excitation coil that gives the excitation field. Signals are fed into a low-noise lock-in amplifier.
This biosensor can detect the concentration of different molecules in a liquid. The software elaboration of the magnetic field measurements in Tesla units results in a graphical resolution of plots with the frequency on the x-axis and magnetic susceptibility on the y-axis.
Recently, this method was applied to measuring the presence of mycobacterium tuberculosis in sputum samples, with a sensitivity in the range of 100–1000 colony forming units/ml.
Some biosensors can be automated and many methods also have multiplex capability and high-throughput performance. Cytometric bead arrays (CBAs) make use of a fluidic method in which microspheres, individually coded using different ratios of red and infrared fluorophores, serve as the solid support for a sandwich-type immunoassay.
Captured molecules are attached to the microspheres and bound to the target, while a fluorescent molecule is used to generate the signal, read by a flow cytometer, which determines the identity of each individual bead and the target associated signal on each bead.
Methods such as lateral flow-based dipsticks with species-specific antibodies are mostly used for species identification, especially after bacteria enrichment by incubation in culture broth at 24 to 48 hours.
Lateral flow immuno-separation is a method exploited in many commercial kits for bacteria identification. Lateral flow imuno-assays are based on a solid support, such as nitrocellulose porous membranes suitable for chromatographic separation of high molecular weight complexes. Dipsticks with printed microfluidic channels on paper (µPADs) are used, where the capture antibody is conjugated with a detection molecule, depending on whether the detection is based on colourimetry, chemiluminescence or gold nanoparticles.
Several types of substrates, membranes and detection systems are available, such as colorimetric or fluorescent quantification. In lateral flow immuno-assays for identification of salmonella spp, the detection antibody is labelled with gold nanoparticles and detected by densitometry using UV wavelength.
In a different setting, mastitis bacteria are detected using biotinylated antibodies and streptavidin coupled with carbon nanotube labels that were visualised by flatbed scanning of the pixel gray intensity. CdSe/ZnS quantum dots (QDs) have also been used to visualise salmonella spp, which show high stability, but are excited on average at 50 percent of the molecules on the sensor.
Biosensor devices for pathogen detection generally consist of several elements, including a biological capture molecule (affinity probes or antibodies), a labelled antibody interacting with the bacteria captured from the solution (in case of beads, a double label is already present on each different bead type), and a signal detection system.
Fluorescent methods make use of a detection system that read the fluorescence signal of excited molecules. Antibodies can be directly labelled, or recognised by labelled protein A or anti-IgG antibodies, by means of covalent binding to cyanines and alexa flour dyes, whit excitation/emission wavelength in the green/blue spectrum, and the bright, intense and photo-stable HiLyte dyes, which are insensitive to pH changes.
Universal Pops, Virginia, US
Steven Depolo, Miami, US
Planar protein arrays in microplates, antibodies bound to microarray glass slides, and middle-throughput wells in microplates have been combined with magnetic separation, and fluorescent silica nanoparticles in solution, and applied to the detection of pathogens and their toxins in immunoassay format.
Protein chips methods are based on the binding to a glass surface of an antibody or an aptamer recognising the bacterial species, for the capture of the bacteria to the surface.
In previous experiments, captured aptamers bound to streptavidin coated surface and captured antibodies directly bound to the epoxy-modified glass slides were tested. Since the bacterial wall harbours several protein antigens in different locations in the membrane, it is possible to use the same antibody either as capture antibody and also as detection probe.
In the protein chip experiment, an activated surface such as clean glass treated with a suitable chemistry, such as epoxy-groups, for the binding of amino residues (by a Schiff reaction), is spotted with capture antibodies, streptavidin or proteins with high affinity for a bacterial species.
The sensitivity (limit of detection) of protein chips method applied to bacteria detection is approximately 100 cfu/ml and immunomagnetic separation can be added in order to increase this value.
The detection method makes use of laser scanners that are highly performant, due to the need to obtain a high sensibility even at a low concentration of the targets. Improvements have been made to increase the sensitivity of fluorescence detection using a low cost CMOS webcam and dedicated software readers.
It was reported that by applying a multi-wavelength LED illuminator and suitable filters, the photocamera can acquire a video processed for signal-to-noise reduction by a software, achieving a Limit-of-Detection of 30 µM fluorescein in independent spots.
In a different system, a portable microarray slide reader measuring 19 cm in length has been developed that performs as well as commercial laser scanners but is much cheaper.
The scientific and technology advancements have provided biosensor tools that may be used by industry workers in their laboratory to check the quality and safety of their food manufacturing lines with results much faster than previous years. Therefore, it is possible to implement HACCP procedures and have a tighter control of food productions.