biosensor

A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component.^[1]

It consists of 3 parts:

• the sensitive biological element (biological material (eg. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc), a biologically derived material or biomimic) The sensitive elements can be created by biological engineering.
• the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified;
• associated electronics or signal processors that is primarily responsible for the display of the results in a user-friendly way.^[2]

The most widespread 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. 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 level than us to warn us of their presence. Such devices can be used in environmental monitoring, trace gas detection and in water treatment facilities.

Principles of Detection

Photometric

Many optical biosensors based on the phenomenon of surface plasmon resonance are evanescent wave techniques. This utilises a property shown 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.

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.

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. ^[4] 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

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, an active electrode and a sink electrode. An auxiliary electrode (also known as a counter electrode) may also be present as an ion source. The target analyte is involved in the reaction that takes place on the active electrode surface, and the ions produced create a potential which is subtracted from that of the reference electrode to give a signal. 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.

Another example, the potentiometric biosensor, works contrary to the current understanding of its ability. Such biosensors are screenprinted, conducting polymer coated, open circuit potential biosensors based on conjugated polymers immunoassays. 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. 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([1]).

Others

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 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.

Applications

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:

• Glucose monitoring in diabetes patients <-- historical market driver
• Other medical health related targets
• Environmental applications e.g. the detection of pesticides and river water contaminants
• Remote sensing of airborne bacteria e.g. in counter-bioterrorist activities
• Detection of pathogens^[5]
• Determining levels of toxic substances before and after bioremediation
• Detection and determining of organophosphate
• Routine analytical measurement of folic acid, biotin, vitamin B12 and pantothenic acid as an alternative to microbiological assay
• Determination of drug residues in food, such as antibiotics and growth promoters, particularly meat and honey.
• Drug discovery and evaluation of biological activity of new compounds.
• Protein engineering in biosensors http://www.springerlink.com/content/672p4l4l45xk02j2/
• Detection of toxic metabolites such as mycotoxins ^[6]

Biosensors in food analysis

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.

Surface Attachment of the biological elements

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 biologicla agent may be for example fixed by Layer by layer depositation of alternatively charged polymer coatings^[7]
Alternatively three dimentional 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. ^[8]
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)^[9], alternatively light can be used in combination with a photoinitiator, such as DMPA (2,2-dimethoxy-2-phenylacetophenone)^[10].

See also

References

External links

• Biosensors and Bioelectronics journal (elsevier.com)
• What are biosensors
• Biosensor Applications - Drug and explosives detection products from Sweden
• Scratching at the surface of biosensors - an *Instant Insight discussing how surface chemistry lets porous silicon biosensors fulfil their promise from the Royal Society of Chemistry
• Biosensor Forum - Social network for researchers and organizations