A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.
It consists of 3 parts:
A common example of a commercial biosensor is the blood glucose biosensor, which uses the enzyme glucose oxidase to break blood glucose down. In doing so it first oxidizes glucose and uses two electrons to reduce the FAD (a component of the enzyme) to FADH2. This in turn is oxidized by the electrode (accepting two electrons from the electrode) in a number of steps. The resulting current is a measure of the concentration of glucose. In this case, the electrode is the transducer and the enzyme is the biologically active component.
Recently, arrays of many different detector molecules have been applied in so called electronic nose devices, where the pattern of response from the detectors is used to fingerprint a substance. . In the Wasp Hound odor-detector, the mechanical element is a video camera and the biological element is five parasitic wasps who have been conditioned to swarm in response to the presence of a specific chemical.[2] Current commercial electronic noses, however, do not use biological elements.
A canary in a cage, as used by miners to warn of gas, could be considered a biosensor. Many of today's biosensor applications are similar, in that they use organisms which respond to toxic substances at a much lower concentrations than humans can detect to warn of the presence of the toxin. Such devices can be used in environmental monitoring, trace gas detection and in water treatment facilities.
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Many optical biosensors based on the phenomenon of surface plasmon resonance (SPR) are evanescent wave techniques. This utilises a property of gold and other materials; specifically that a thin layer of gold on a high refractive index glass surface can absorb laser light, producing electron waves (surface plasmons) on the gold surface. This occurs only at a specific angle and wavelength of incident light and is highly dependent on the surface of the gold, such that binding of a target analyte to a receptor on the gold surface produces a measurable signal.
Surface plasmon resonance sensors operate using a sensor chip consisting of a plastic cassette supporting a glass plate, one side of which is coated with a microscopic layer of gold. This side contacts the optical detection apparatus of the instrument. The opposite side is then contacted with a microfluidic flow system. The contact with the flow system creates channels across which reagents can be passed in solution. This side of the glass sensor chip can be modified in a number of ways, to allow easy attachment of molecules of interest. Normally it is coated in carboxymethyl dextran or similar compound.
Light of a fixed wavelength is reflected off the gold side of the chip at the angle of total internal reflection, and detected inside the instrument. This induces the evanescent wave to penetrate through the glass plate and some distance into the liquid flowing over the surface.
The refractive index at the flow side of the chip surface has a direct influence on the behaviour of the light reflected off the gold side. Binding to the flow side of the chip has an effect on the refractive index and in this way biological interactions can be measured to a high degree of sensitivity with some sort of energy. The refractive index of the medium near the surface changes when biomolecules attach to the surface, and the SPR angle varies as a function of this change.
Other evanescent wave biosensors have been commercialised using waveguides where the propagation constant through the waveguide is changed by the absorption of molecules to the waveguide surface. One such example, Dual Polarisation Interferometry uses a buried waveguide as a reference against which the change in propagation constant is measured. Other configurations such as the Mach-Zehnder have reference arms lithographically defined on a substrate. Higher levels of integration can be achieved using resonator geometries where the resonant frequency of a ring resonator changes when molecules are absorbed.
Other optical biosensors are mainly based on changes in absorbance or fluorescence of an appropriate indicator compound and do not need a total internal reflection geometry. For example, a fully operational prototype device detecting casein in milk has been fabricated. The device is based on detecting changes in absorption of a gold layer.[3] A widely used research tool, the micro-array, can also be considered a biosensor.
Nanobiosensors use a immobilized bioreceptor probe that is selective for target analyte molecules. Nanomaterials are exquisitely sensitive chemical and biological sensors. Nanoscale materials demonstrate unique properties. Their large surface area to volume ratio can achieve rapid and low cost reactions, using a variety of designs.[4]
Biological biosensors often incorporate a genetically modified form of a native protein or enzyme. The protein is configured to detect a specific analyte and the ensuing signal is read by a detection instrument such as a fluorometer or luminometer. An example of a recently developed biosensor is one for detecting cytosolic concentration of the analyte cAMP (cyclic adenosine monophosphate), a second messenger involved in cellular signaling triggered by ligands interacting with receptors on the cell membrane.[5] Similar systems have been created to study cellular responses to native ligands or xenobiotics (toxins or small molecule inhibitors). Such "assays" are commonly used in drug discovery development by pharmaceutical and biotechnology companies. Most cAMP assays in current use require lysis of the cells prior to measurement of cAMP. A live-cell biosensor for cAMP can be used in non-lysed cells with the additional advantage of multiple reads to study the kinetics of receptor response.
Electrochemical biosensors are normally based on enzymatic catalysis of a reaction that produces or consumes electrons (such enzymes are rightly called redox enzymes). The sensor substrate usually contains three electrodes; a reference electrode, a working electrode and a counter electrode. The target analyte is involved in the reaction that takes place on the active electrode surface, and the reaction may cause either electron transfer across the double layer (producing a current) or can contribute to the double layer potential (producing a voltage). We can either measure the current (rate of flow of electrons is now proportional to the analyte concentration) at a fixed potential or the potential can be measured at zero current (this gives a logarithmic response). Note that potential of the working or active electrode is space charge sensitive and this is often used. Further, the label-free and direct electrical detection of small peptides and proteins is possible by their intrinsic charges using biofunctionalized ion-sensitive field-effect transistors.[6]
Another example, the potentiometric biosensor, (potential produced at zero current) gives a logarithmic response with a high dynamic range. Such biosensors are usually often made by screen printing the electrode patterns on a plastic substrate, coated with a conducting polymer and then some protein (enzyme or antibody) is attached. They have only two electrodes and are extremely sensitive and robust. They enable the detection of analytes at levels previously only achievable by HPLC and LC/MS and without rigorous sample preparation. All biosensors usually involve minimal sample preparation as the biological sensing component is highly selective for the analyte concerned. The signal is produced by electrochemical and physical changes in the conducting polymer layer due to changes occurring at the surface of the sensor. Such changes can be attributed to ionic strength, pH, hydration and redox reactions, the latter due to the enzyme label turning over a substrate ([2]). Field effect transistors, in which the gate region has been modified with an enzyme or antibody, can also detect very low concentrations of various analytes as the binding of the analyte to the gate region of the FET cause a change in the drain-source current.
The use of ion channels has been shown to offer highly sensitive detection of target biological molecules.[7] By imbedding the ion channels in supported or tethered bilayer membranes (t-BLM) attached to a gold electrode, an electrical circuit is created. Capture molecules such as antibodies can be bound to the ion channel so that the binding of the target molecule controls the ion flow through the channel. This results in a measurable change in the electrical conduction which is proportional to the concentration of the target.
An Ion Channel Switch (ICS) biosensor can be created using gramicidin, a dimeric peptide channel, in a tethered bilayer membrane.[8] One peptide of gramicidin, with attached antibody, is mobile and one is fixed. Breaking the dimer stops the ionic current through the membrane. The magnitude of the change in electrical signal is greatly increased by separating the membrane from the metal surface using a hydrophilic spacer.
Quantitative detection of an extensive class of target species, including proteins, bacteria, drug and toxins has been demonstrated using different membrane and capture configurations.[9][10]
Piezoelectric sensors utilise crystals which undergo an elastic deformation when an electrical potential is applied to them. An alternating potential (A.C.) produces a standing wave in the crystal at a characteristic frequency. This frequency is highly dependent on the elastic properties of the crystal, such that if a crystal is coated with a biological recognition element the binding of a (large) target analyte to a receptor will produce a change in the resonance frequency, which gives a binding signal. In a mode that uses surface acoustic waves (SAW), the sensitivity is greatly increased. This is a specialised application of the Quartz crystal microbalance as a biosensor.
Thermometric and magnetic based biosensors are rare.
There are many potential applications of biosensors of various types. The main requirements for a biosensor approach to be valuable in terms of research and commercial applications are the identification of a target molecule, availability of a suitable biological recognition element, and the potential for disposable portable detection systems to be preferred to sensitive laboratory-based techniques in some situations. Some examples are given below:
Commercially available gluocose monitors rely on amperometric sensing of glucose by means of glucose oxidase, which oxidises glucose producing hydrogen peroxide which is detected by the electrode. To overcome the limitation of ameperometric sensors, a flurry of research is present into novel sensing methods, such as fluorescent glucose biosensors.
The Interferometric Reflectance Imaging Sensor (IRIS) was developed by the Unlu research group at Boston University for the purpose of label-free biosensing. Using simple lenses and low-powered, coherent LED’s, the device offers exquisite sensitivity and reproducibility and is able to image with remarkable resolution beyond the classical diffraction limit. This relatively cheap solution also presents minimal hazards when compared to a laser illumination source.
One specific form of photometric biosensing technique developed by researchers at Boston University is interferometric reflectance imaging. Using optical interference techniques, imaging of antibodies were successfully performed. This was achieved without altering the antibody structure or using bio-markers such as fluorescent proteins. The basis of this technique stems solely from optical interference. By using a reflective substrate such as silicon, light reflected from proteins will interfere with light reflected from the substrate. In result, interference patterns are generated that alter the intensity of the reflected light. This phenomena is measurable by a camera.
Proteins have indices of refraction based on their concentration. When light is shined on the proteins, a portion of the light is transmitted through the molecules and reflected off the silicon's surface. The interference of the light initially reflected off the proteins and the light reflected off the surface of the silicon will have a relative phase difference (after being transmitted back through the protein) contributing to a wavelength-dependent sinusoidal variation in the total amount of reflected light (captured by the imaging device).
The Interferometric Reflectance Imaging Sensor (IRIS) was developed by the Unlu research group at Boston University for the purpose of label-free biosensing. Using simple lenses and low-powered, coherent LED’s, the device offers exquisite sensitivity and reproducibility and is able to image with remarkable resolution beyond the classical diffraction limit. This relatively cheap solution also presents minimal hazards when compared to a laser illumination source.
The IRIS operates solely on optical reflection. The ability for it to image with extremely high spatial resolution stems from the integration of a diffuser into the design of the microscope. The diffuser randomizes the directionality of the light from a single LED source (called Köhler illumination) which allows for sharp focusing of incident light without back-imaging the source in the image projection.
Practical uses of this device include the detection of bacterial and viral infections in underdeveloped countries. When pathogen specific growth factors are introduced into a microarray, only spots with the targeted pathogens will grow and increase in concentration. In turn, this dictates a change in the reflected intensity compared to pre-growth. Thus, by measuring how reflectance changes over time, unknown pathogens and their growth rates can be easily characterized and identified.
There are several applications of biosensors in food analysis. In food industry optic coated with antibodies are commonly used to detect pathogens and food toxins. The light system in these biosensors has been fluorescence, since this type of optical measurement can greatly amplify the signal.
A range of immuno- and ligand-binding assays for the detection and measurement of small molecules such as water-soluble vitamins and chemical contaminants (drug residues) such as sulfonamides and Beta-agonists have been developed for use on SPR based sensor systems, often adapted from existing ELISA or other immunological assay. These are in widespread use across the food industry.
An important part in a biosensor is to attach the biological elements (small molecules/protein/cells) to the surface of the sensor (be it metal, polymer or glass). The simplest way is to functionalize the surface in order to coat it with the biological elements. This can be done by polylysine, aminosilane, epoxysilane or nitrocellulose in the case of silicon chips/silica glass. Subsequently the bound biological agent may be for example fixed by Layer by layer depositation of alternatively charged polymer coatings[14]
Alternatively three dimensional lattices (hydrogel/xerogel) can be used to chemically or physically entrap these (where by chemically entraped it is meant that the biological element is kept in place by a strong bond, while physically they are kept in place being unable to pass through the pores of the gel matrix). The most commonly used hydrogel is sol-gel, a glassy silica generated by polymerization of silicate monomers (added as tetra alkyl orthosilicates, such as TMOS or TEOS) in the presence of the biological elements (along with other stabilizing polymers, such as PEG) in the case of physical entrapment.[15]
Another group of hydrogels, which set under conditions suitable for cells or protein, are acrylate hydrogel, which polymerize upon radical initiation. One type of radical initiator is a peroxide radical, typically generated by combining a persulfate with TEMED (Polyacrylamide gel are also commonly commonly used for protein electrophoresis),[16] alternatively light can be used in combination with a photoinitiator, such as DMPA (2,2-dimethoxy-2-phenylacetophenone).[17] Smart materials that mimic the biological components of a sensor can also be classified as biosensors using only the active or catalytic site or analogous configurations of a biomolecule.[18]