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As our modern world evolves in discovering new and faster technologies, medicine and treatments. The aim of these industries is to lower costs, while increasing efficiency and ability of their products. In order for the products to be as they intended, an integral process is to achieve this is the analysis of the subject. If we take a few examples such as the food industry, in order to carry out quality control of the food products, the company employed methods such as chromatograph, spectroscopy and titration. These methods are time consuming, required trained analylist, not allow continuous monitoring of the sample and also require additional steps in extraction 1. These are problems faced by industries, costing them time and money. Too combat this, industries and other disciplines such as environmental researchers, medical fields and many more needed analytical technologies which were fast, sensitive and cost effective. An early answer lied in an obvious field of medical diagnostics. As treatments where prolonged by sampling taking, then sending them to laboratories and waiting for the tests to be sent back. This was a very long process especially for patients with potentially fatal illnesses, as they had to endure pain and wait for answers from the doctors. Inevitably this was the driving force for the research and development of biosensors 2.
The birth of biosensors had an impact on vast areas of the science field, ranging from Biochemistry and Molecular Biology to Mechanics and Engineering. Their success comes from, the potential to be miniaturized, due to technologies are becoming increasingly intelligent and reliant as the size of component become smaller thanks to such things as micro chips. Biosensors can be automated, minimizing human effort and allows for scientists to carry out other important work, making the process of analyses more efficient. The simple construction and storage costs of biosensors are low. Also they are portable, to allow for greater mobility of the sensors. This is a great advantage as they can be taken everywhere such as analysis needed for the environment. However the brilliance of biosensors and biosensor design comes from the specificity and selectivity from the analytical interface which is in connected to a chemical or physical transducer. The brilliant feature of biosensors is, as simply annalist's use natures own techniques of specificity and selectivity to detect biochemical processes 1. Such as to detect a specific antigen in a sample with many substrates, the analytical interface could contain the antibodies for that antigen therefore only that specific antigen which corresponds and bind to the antibody will be detected using suitable transducers. This is illustrated by scheme 1.
Figure 1. Basic mechanism of biosensors
The biosensors are made up of an analytical interface (the chemical sensor or recognition element) attached to a chemical or physical transducer. The analyte is the sample being analysed, which can range from antigen to microbial cells and many more. The analytical interface is the surface which contains the recognition element according to the analyte and can be cells, receptors, antibodies and many more. The transducer is the component that detects the interaction between the anlyate and the analytical interface. Transducers have the ability to continuously monitor the reaction and can detect a range of interactions, such as oxygen consumption, change in pH , conductivity and many more 1.
This report will on the different varieties of analysis that biosensors have provided. Also to review the development of biosensors and their design to accommodate the anylate, to produce fast and accurate results.
Biosensors in Biochemistry
The potential for success of biosensors in biochemistry is high. In this section we will be exploring a variety of fields within biochemistry, which have potential to revolutionaries in our modern world. Blood glucose biosensors are part of a multibillion dollar industry and we will see the change in design over time which makes them more accommodating towards home use and make lives easier for patients with diabetes. We will be looking at an interesting breed of biosensors, stochastic biosensors, have the potential to sense variety of analytes using membrane bound receptors.
Stochastic Biosensors in Biochemistry
Stochastic biosensors have the ability to detect multiple target analytes. This is achieved by the unique current signatures detected by the transducer for each analylate; we will see how these signatures are derived, later on. Firstly we need to explore our analytical interface, using pores based on staphylococcal -haemolysin (HL) in lipid bilayer spread across a polymer film e.g. Teflon3. The unique ability of this pore allows analysts to use as the analytical interface of the biosensor. Due to recent discoveries on engineering proteins to our advantage, has allowed researches to use this technique on the αHL to sense particular analytes. The wild typeHL is engineered at a specific position in its channel, to allow binding of specific analytes. Now with the modified channel of the HL pore, it will give allow analyse a broader scope of analyte. Another unique feature of HL pores, the ability to maintain a unique current signature when reproduced; therefore allowing analysts to communicate their results from their own labs.8 As mentioned before the ability of these sensors to detect multiple analytes, makes these biosensors potentially very good. However there needs to be a way to derive the signal produced thus the identification of each analyte. Organic molecules such as 2,4,6-Trinitrotoulene better known as TNT, among other molecules that can be detected using stochastic sensing. TNT can be detected by engineering the channel of the HL, so that the nitroaromatic molecule bind to it. This is done by engineering the pore to contain aromatic side chain amino acids ( Tye, Phe, Trp). These are introduced this at a particular position in the wild type HL. TNT can be identified and quantified in an aqueous environment. This can done because TNT has a unique resident time and amplitude.8
2.1.1 Transduction in Stochastic Sensing
In experiments, the lipid bilayer spread across a polymer film, causes compartmentalisation where there are two chambers. Next we need a current, this is achieved by using a salt buffer solution (1M of NaCl or KCl), the salt will dissociate into their respected positive (Na+ or K+) and negative (Cl-) ions. The flow of these ions will cause a current. At high concentration of the salt solution, keeps the HL channel open. Thus allowing for a constant current to be monitored. If an analyte is added and there is a successful binding event between the channel and analyte, the flow of ions is blocked hence modulations in ionic current can be monitored (blockage in pore causes decrease in current through the pore). When the analyte is released from the binding site, the channel is re-opened and a current is increased. This simple concept of blocking and unblocking depending on analyte is information on concentration of analyte and also the identity of the analyte.8
Figure 2: Diagram showing the modified HL pore place on lipid bilayer. Analyte (represented as green circle) bound in the channel and also the direction of current flow.3
By keeping fixed potential and monitoring modulations of ionic current caused by analyte blocking we can record the frequency of blocking events ( ) , which gives us the concentration of analyte. Measuring the mean residence time ( ) in conjunction with amplitude (extent of current blockage), we can get the identity of analyte (scheme 2).
Figure 3: showing the analyte binding and releasing events with the binding site in the channel. Where residence time ( ) in conjunction with amplitude ( Ib , extent of current block), gives identity of analyte. The frequency of blocking events ( ), which gives us the concentration of analyte. Io is the current of the open channel.8
Glucose Biosensors in Biochemistry
1ST Generation of Glucose Biosensors
This generation of biosensors uses a platinum (Pt) cathode, allowing for the reduction of oxygen. A reference electrode Ag/AgCl, this allows to measure the concentration of oxygen as it proportional to the current. We can do this when a potential of -0.6V vs Ag/AgCl electrode is applied to a Pt electrode. However for the oxygen to be reduced at the Pt cathode, oxygen has to diffuse from the bulk solution. Hence the rate of diffusion from the bulk solution will have an effect on the rate of electrochemical reduction of oxygen. However this was thought to be not such a good strategy to detect glucose concentrations. The solution came from an electron acceptor replacing an electron donor. The electron acceptors is artificially incorporated in the biosensor system.4
2nd Generation of Glucose Biosensors
This generation of biosensors work, like a chain of builders passing a brick from one side to another, where the brick represents an electron for the 2nd generation of glucose biosensors. The redox reaction between analyte and enzyme, causing electron transfer, reducing the enzyme. The reduced enzyme goes though another electron transfer causing the artificial electron acceptor (mediator) to be reduced. Finally the mediator will now be reduced at the electrode, which causes a current thus allowing us to detect this current. However some techniques does not involve external mediator, rather the transfer of electron from the enzyme directly done.4
3rd Generation of Glucose Biosensors
The 3rd generation of biosensors requires an electric conductor such as NMP+ TCNQ. If we had GOD/FADH2 , we can use the electrode to oxidize, GOD/FADH2 directly.4 NMP -TCNQ have been used of electron transfer with redox enzyme. These are mediators which are also electrodes making the need for the external mediator mentioned in section 2.2.2. These methods and trails lead to a massive market in home blood glucose testing.
Transduction in Glucose Biosensors
For industrial, environmental and clinical field, these biosensors are highly sensitive so we don't not need to use large amount of analyte for testing. They are reliable and cheap which make these biosensors ideal for these fields.
Glucose biosensors use electrochemical transducers (i.e.amperometric transducers). They detect the oxidation of the biochemical reaction and thus measuring the fluctuation in current on the electrode. This technique relies on the concentration of analyte, if there is an error in measurement current there will be a response and also give a normal dynamic range. These are based on 2 systems an indirect and direct system. Direct system has engineered electrodes where in the natural electron donor is replaced by an electron acceptor. So that the biological reaction that takes place are joined by the biology and electrochemistry, thus allowing us to detect a current by the transfer of electron.4
Home Blood Glucose Biosensors
The 1st successful blood glucose biosensor was developed by Genetic International with the support of University of Cranfield and Oxford. The sensor was named Exac Tech device.5 This device requires 10-50 drop of blood and the test results took 30 seconds. This device was a whole blood calibrated meter and did not have a data port. As ergonomics goes, the display screen was user friendly and so was the manner in which the device is used.
MediSense Sof-TechTM was another biosensor developed which offered multiple information with much effort. With a press of a button, glucose testing, blood collecting and lancing took into effect. In comparison with Exac Tech devices, MediSense Sof-TechTM just needed 2-3 of blood and time for the results took 20 seconds. To add blood is taken from sensitive areas such as forearms and upper arm, causing less pain to the patient.
Free StyleTM blood glucose monitoring system is another evolutionary glucose biosensor. Compared to the above biosensors , this biosensors need just 0.3 of blood, reducing the pain, encouraging patients to use the device more often. In turn the disposal of blood is more efficient and controlling hygiene. Also this device allows for multiple sites from where blood can be extracted including, thighs, fingertip, calf's. This analyer uses Pyroloquinoline quinone glucose dehydrogenase (PQQ-GDH).5
In recent reviews there is growing interest in PQQ-GDH based glucose biosensors. This enzyme is dominating the home blood glucose biosensor market. However researchers believe that the reliability is not the best. To overcome this, an approach similar to section 2.1 where protein was modified. Engineered PQQ-GDH increased the activity and selectivity to glucose, in turn it was stable under physiological pH and temperature.6
Quality control in our modern day is essential as it could pose a serious health risks to millions of people. Waterborne disease cost $20 billion in the US alone, due to citizens falling ill because of untreated water. European Union directives such as the Water Framework Directive and the Marine Strategy Framework Directives have implemented the use of environmental biosensors. As these biosensors produce results rapidly, are sensitive and accurate, they are cost effective and allow for continuous monitoring for the analytes. In this section we will discuss the different recognition elements such as enzymes, antibodies and DNA which are coupled to a suitable transducer, for the detection of pesticides / herbicides, chemical toxins and pathogens. 10, 13
3.1.1 Enzyme Sensors for Environmental Biosensors
The use of enzymes as the recognition elements, allows for high selectivity due to the specific bind of substrate on the active site of the enzyme. One way for detecting analytes using enzymes is by an inhibitory interaction between the enzyme and analyte. Enzymes such as hydrolase (choline esterase), can be used to detect potential dangerous chemicals. Hydrolase normal function is to use water to hydrolyse the substrate.10
Enzyme biosensors can be used to detect Arsenic and organophosphates (pesticides) using a hydrolase such as acetyl choline esterase (AChE). Detection of pesticides is done in the presence of acetyl choline chloride (AChCl), along with AChE. The normal function of AChE would be to hydrolyse AChCl, however when there is pesticide present, it will inhibit AChE. Thus deactivating AChE and a decrease in signal will be detected. AChE can also be used to detect Arsenic (AsO33- or As(III) ) as along with pesticide , they both are water contaminants. Similar to the detection of pesticides, acetyl choline iodine (AChI) is used as the substrate that is hydrolysed by AChE. As(III) inhibits AChE therefore the hydrolyses of AChI will be stopped allowing for detection of the metal.
3.1.2 Transduction for Enzyme Biosensors
An appropriate transducer used is electrochemical transducers (i.e. amperometric). For the example of AChE enzyme used to detect pesticide and As(III), the enzyme are immobilized on to a screen printing electrodes (SPE). Sanllorente-Mendez et al. used screen printing carbon electrode (SPCE) which have the immobilized AChE enzyme by covalent linkage. SPE and SPCE were used because are they eliminate memory effects in the analysis at trace levels. The method of detection of both pesticide and As(III), we will be looking at the way Sanllorente-Mendez et al. detected As(III). In order to determine As(III) in a sample, a potential must be applied and a steady current must be defined. Then the addition of AChI causes an oxidative signal, as the immobilized AChE hydrolyses AChI to make thiocholine iodide and acetic acid. If there is As(III) present in the sample then the hydrolysis will be stopped due to the inhibition of AChE by As(III). Thus will cause a decreased in the oxidative signal. Thus the oxidation signal reported to be proportional to the concentration of As(III). Amperometric biosensor were used because they deliver, low costs, are reproducible and have disposable electrodes (no need to re-clean the electrode once used).10, 15
3.2.1 Immunosensors in Environmental Biosensors
The use of antibodies in environmental biosensors, allows analysts to detect viruses, bacteria and spores. Immunoassays are put into 3 categories of antibodies which are obtained by different methods. Monoclonal Antibodies (mAbs) solutions, where antibodies are produced in a laboratory (in vitro) using B cells which produce antibodies for our target analyte and fused with myeloma cells (hybridoma cell lines). This results in binding to a single part of the antigen that the antibody recognises. Another category of antibodies is Polyclonal Antibodies (pAbs), the antibodies can bind to several different recognition sties of the antigen, compared to mAbs. pAbs are obtained in vivo (within a living organism). Finally, the methods employed by engineering proteins in section 2.1.1, are taking effect in a similar way for immuonsensors. Antibody fragments can be engineered to the specifically bind to antigen analyte. Some examples of analylates which can be detected by immuonsensors are, Hormones, Bisphenol A, Surfactants.10, 13
3.2.2 Transduction for Immunosensors
There are several transducers used for immuonsensors. Electrochemical transduction such amperometric, potentiometric can be coupled with antibody recognition element. Oxidation and reduction current can be detected at the electrode surface, when a electrical potential is added. Farre et al. reported the detection of 2,4 - dicholrophenoxacetic acid (2,4-D), a artificially plant hormone used as a pesticide, using amperometric immuonsensors. Another method of detecting analyte using immunosensors, is by change in mass. Acoustic transducers, have a oscillating piezoelectric crystal with a resonant frequency. When there is an antigen-Abs interaction, there is change in the frequency of the surface bound crystal, due to change in mass at the surface.10
3.3.1 DNA and Nucleic acid in Environmental Biosensors
DNA and nucleic acid can be used as recognition element, which can detect specific gene according to the single stranded DNA (ssDNA) on the recognition element. One method employed to obtain ssDNA or RNA for our target analyte through the use of aptamer technology.14
The method using aptamer technology is as follows: a library of oligonucleotides (approximately 105) is used to select an ssDNA or RNA for our analyte. As the library of oligonucleotides is random, some nucleic acid will have a low binding affinity too others. By using series of washing up steps to remove non-specific and low affinity binding nucleic acid, leaving only oligonucleotides which have a high binding affinity and are specific to our target analyte. 13, 14, 12 Next the specific and selective sequence goes through a process of polymerase chain reaction (PCR), where copies of this sequences can be copied.11 The detection of analyte by the ssDNA or RNA, does not happen through the sequence of the strand but the ssDNA or RNA are short, it can assemble itself into a 3-D structure. The analyte is recognised by the ssDNA or RNA by the structure.
3.3.2 Transduction for DNA and Nucleic acid sensors
We can combine several traducers with optical transducer such as electrochemical and mass-based detection. However the best used transduction techniques to detect pathogens and other environmental pollutants are fluorescence and colorimetry.14 Fluorescence, a fluoropore gets excited by light. When the fluoropore returns back down to ground state energy, it emits a wavelength which then can be detected.13 Using fluorescence in conjunction with real-time PCR give a qualitative and quantitative detection of organisms such as Salmonella enterica. Colorimetry is another method detecting using DNA and nucleic acid as recognition element. This detection technique is based on UV-Vis luminescence spectroscopy. Due to binding of the analyte and the nucleic acid (recognition element) causes conformational change, thus a change in optical signal will be detected.14