Saturday, April 7, 2012

Biosensor Technology

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BIOSENSOR TECHNOLOGY


The thesis here is done to develop an understanding of biosensors in general and then appreciate the construction of antibody sensors which are a boon to the medical industry to detect pathogens in the body much earlier than any known conventional pathology � lab test. This device can ultimately save lives because of the quick diagnosis that medical practitioners can provide to their patients. Moreover an antibody sensor can detect even traces of an antibody in a sample and thereby provide very accurate analytical data.


A Brief Introduction Biosensors were originally developed to meet the requirements of the medical industry. For example, doctors wanted to know the blood � sugar levels of their patients in the shortest possible time with sufficient accuracy to enable a wise diagnosis. Biosensors are basically measuring instruments that can detect the presence of certain biomolecules and measure the quantity of that substance with sufficient accuracy using a unique property of that substance in its interaction with some other base (reference) material. But now they are universally used to monitor bioprocesses in bioreactors- the most complex of which is the human body itself- the marvel of creation, the sceptre of divine power. Our aim is to study the fundamental theory of working of a general biosensor and then discover the flexibility and the accuracy of an Antibody Sensor. An antibody sensor is just a special biosensor that uses the unique properties of antibody � antigen reaction to its advantage, to detect antibodies.


Now we shall consider the basic construction and working of a biosensor. What we need to keep


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in mind is that at ground zero we are going to use some unique property of the substance to be detected,


when it reacts with a reference molecule [which could be part of the biosensor equipment]. This unique


property can be a particular change in colour ( optical phenomenon ) during the course of the reaction, or a


change in the sound during the reaction ( acoustic phenomenon ) , or change in electrode potential of a


particular substrate or change in the pH of the reaction medium . The only necessity here is that the change


being measured should be unique to that substance and also be of significance in the greater perspective � that the change is actually a measure of the presence of that particular molecule that we want to observe. Once the chemical or physical change [or a combination of both] has been picked up by the sensing part of the biosensor this is fed to a transducer. A transducer is the component of the biosensor that converts the observed chemical or physical change to an electrical signal which can in turn be inter converted between other electromagnetic fields. The output of the transducer is generally obtained as the mechanical deflection of a needle across a properly marked dial or as a digital display.


Though different situations require different types of biosensors the basic principles underlying their construction and working is the same. A good biosensor is one that can endure the market � it should be produced economically, that is, using existing factory techniques. It should serve the purpose for which it was initially required. For example, if it were built for the purpose of research then it should be very accurate, with a reasonable compromise of cost.


Theory Of The Detector Analyser (Biomaterial Properties)


The detector does the work of biomolecular recognition. Biomolecular recognition provides the basis for bioreactor control processes in the biochemical industry. The detector is the core of the biosensor. It does the real analysis that can give us the required data. The detector is basically a setup that allows a reagent to react with the substrate in stoichiometric quantities and then by monitoring the change in concentration of the reagent or the substrate � produce a noticeable physical (optical, acoustic), or chemical (pH, electrode potential) change that can be picked up by the transducer to be converted to an appropriate electrical signal.


Proteins by their virtue of their structural and chemical properties are the best candidates for detector (biomolecular recognition) molecules. It is because of the unique presence of an amino group and a carboxylic group in the same molecule and the varied chiralty that the alpha � carbon can exhibit that makes proteins so stereo specific in their reactions. There are some very special proteins called enzymes that are of special interest because of the factors listed below


They are one of those classes of organic compounds about which sufficient research has already been done and hence their chemistry is pretty well understood.


Some are readily available.


They are very specific in their reactions.


They generally yield products that can be easily recognised and measured by already existent stoichiometric methods.


Generally purified enzymes are used for the process of analysis (albeit mainly for the analysis of chromosomes). Purified doesn’t necessarily mean absence of all other proteins but just that the absence of proteins that could react in that particular situation under those conditions to mess up the analysis. Non reactive proteins could remain in the analyser matrix. Highly purified enzymes (with zero impurities) are used in the analysis of protein structure and modification, or for previously uncharted tests.


The requisites of analytic enzymes are


Specificity - in reaction.


Purity - absence of reactive proteins that may interact with the detection and analytical systems.


Stability � capable of long term storage and stability even during reaction.


Kinetic Properties � the sample compound should not contain some entity that inhibits enzyme action.


pH � the enzyme pH should be optimum under the required conditions.


Solubility � the enzyme should be such that adsorption or aggregation should not affect its activity.


Cost Effectiveness.


Once all these requisites are satisfied we have found the enzyme that will help us in our analysis.


Now comes in the factor of transforming the chemical signals to electrical signals. We do this because among all fields we have understood very well, we have understood the electrical field the best. We have learnt to manipulate electric signals to meet our needs whenever required.


Whenever enzymes are used as anylate molecules, they are first combined with electrodes to check for phenolic compounds, because phenols are enzyme inhibitors (this is why smokers are in a greater health risk because the carcinogens in tobacco smoke are mainly phenolic in nature and hence inhibit the activity of the enzymes in the smoker’s body).





Antibodies are another class of proteins that have properties favourable for use in biosensors. They are not catalytic. They are very specific and bind only to a type specific compounds called antigens, that are specific to each antibody. The advantages of using antibodies are


They are very selective (too selective even between strains).


They are ultra-sensitive (for example, a TNT biosensor developed in Germany can detect down to one atto-mole (10^(-18)) of TNT).


They bind very powerfully to the respective antigens, thus making the analysis more accurate.


Their major disadvantage is that they are non-catalytic.


Nucleic acids are proteins whose specificity arises from the fact that they form base pairs that are very specific. Adenine pairs only with Guanine and Thymine, or Uracil pairs only with Cytosine. They are used mainly in the identification of DNA or RNA sequences.


Nucleic acids are used to engineer new enzymes, whose activity is less dependent on the reaction environment conditions like pH, temperature, ionic concentration, etc.


Receptors are proteins that are found on the inside surface neighbourhood of the plasma membranes that cover all cells. They are principally involved in transport processes at the cellular level. They have specificity equal to that of antibodies. Their unique property that distinguishes them from antibodies is that they can bind not just with specific molecules but all molecules with a specific structure. This is a great tool in the detection of previously undetected, that is, analytical science.


Their only major disadvantage is that they are not readily available and their synthesis is complicated.


Receptors have a great advantage in that they can detect Action Potential in nerve synapses. Thus they are frequently used to test the effect of mind-altering drugs.


Neurotransmitters and hormonal receptors are the body’s own biosensors.


Tissue Materials are sources of enzymes in crude form. They are composed of mashed tissue and are mainly employed when the required biomaterial is in excess in a certain tissue and that the other proteins present will not affect the analysis considerably.


The advantages of using tissue materials


The enzymes are present in their natural state. So they are more enhanced in their behaviour, and the enzyme activity will be stabilised.


They work when purified enzymes fail under certain conditions.


They are definitely much cheaper than purified enzymes.


The only major disadvantage is that the lack of purity leads to loss in selectivity of the reaction to some extent.


The classical example for this case is the use of banana pulp mixed with graphite (to enhance conductivity) and coupled with a glass electrode, which serves as a biosensor for dopamine (a chemical found in the brain).


Microorganisms can also be used as a source of non-purified enzymes. The advantages of using them are


They are very cheap sources of enzymes.


They are less sensitive to inhibition by the contents in the anylate.


They are more tolerant to change in reaction conditions than any other form of enzymes.


They have long working lifetime.


The disadvantages of microorganisms for biomaterials are


Sometimes they take a long response time.


They characteristically have a long recovery time.


Since microorganisms contain many enzymes they become less selective in the analysis.


The best example is that of yeast being used in the brewing industry.


Once a suitable biomaterial has been selected for the biosensor it is now required that the biomaterial be immobilised so that the biomaterial is in constant contact with the anylate and the transducer simultaneously. There are various ways of achieving this


Adsorption on a surface (for example, on paper) is the oldest method.


Microencapsulation involves the trapping of a biomaterial between two semipermeable layers. It is a very robust (lesser reaction condition dependency) method and is almost universally applied.


Entrapment involves extracting the biomaterial with a monomer solution and then polymerising the solution thus obtained into a gel thereby trapping the biomaterial. This method slows down the reaction considerably.


Cross Linkage involves bonding the biomaterial to a rigid support but this method is not preferred because it is not practical in many occasions.


Covalent Bonding of the biomaterial with a nucleophilic group, which in turn is linked to a rigid support is a very good idea.


As a side � effect of immobilisation of the biomaterial, the working lifetime of the material is greatly increased.








Principles Of Transduction





Let us first look at a few definitions that will help in proceeding into the material with more confidence.


A Transducer is a device that transforms a chemical signal to an electric signal.


A Principle of Transduction is the use of a well-defined technique to capture a unique property of the event being detected.


In a chemical event the main changes immediately observable changes are optical, acoustic and most generally electrical. Optical or acoustic changes can also be detected electrically, for example by using a photoelectric device or a microphone respectively, which ultimately convert the input optical and acoustic signals to electric signals.


Considering the potential change or the current change during the course of a reaction we can transduce an electrical output. Accordingly we have Potentiometric or Amperometric principles of transduction.


Potentiometry belongs to the class of Voltammetric measurements where the potential difference between an electrode and a reference for a zero current is measured in real time for the purpose of analysis and transduction. The most commonly used instruments for this purpose are standard electrodes. The enzyme that needs to be used should be such that it liberates a product that will react with the electrode component and change the electrode potential so that it can be recorded. For example, the use of a fluoride sensitive electrode is recommended to be used with a Hydrogen peroxide liberating enzyme. The liberated peroxide reacts with an organic fluoride to liberate fluorine. This fluorine reacts with the fluorine sensitive electrode to and changes the electrode potential. Ammonium ion sensitive electrodes are useful in biosensors (because of ammonia liberating enzymes).


Gas sensitive electrodes are also used because some enzyme reactions evolve a good amount of identifiable gas. They basically consist of a glass plate covered by a thin liquid electrolyte film. This setup is immersed into the analysing mixture. Any reactant gas that is liberated reaches the electrolyte and changes the glass electrode potential, which is recorded. Solid electrolytes are also used to detect gases. This is because solids can adsorb a lot of gases on their surfaces and react with the same. A classical example is PtrOPt for oxygen detection. When oxygen gas passes through the porous Zirconium dioxide it gets adsorbed and reacts with the electrode setup changing the electrode potential and hence creating an electrical signal.


The next method of transduction is Amperometry. It belongs to the class of voltammetric measurements recording an electrode current as a function of applied potential.


In general voltametry involves the application of an increasing (decreasing) potential to a cell until oxidation (reduction) of anylate occurs. At this point there is a sharp rise in cell current to reach a maximum. The height of this peak bears a direct relation with the concentration of the anylate. To measure this current one can also directly apply the oxidation (reduction) potential of the anylate compound if it were already known.


Conductimetry


Based on the fact that ionic solutions have conductivity that can be altered by a chemical or biochemical reaction. The relation between conductimetry and anylate concentration depends upon the nature of the reaction.


FET Based Sensors


All the above-discussed types, especially potentiometric, of transduction can be constructed on a FET Chip using embedded software technology. This leads to miniaturisation of the circuitry so a vast repertoire of sensors can be put in one package.


Optical Sensors


With the advent of optical fibres and their subsequent development it is now possible to build transducers based purely on optical signals. This gives the designer more flexibility and greater order of miniaturisation of the sensor. Here the transducers wok exclusively on the physical aspect of the reaction, like fluorescence spectroscopy, surface plasmon resonance, absorption spectrum, light scattering, internal reflection spectroscopy, and luminescence spectroscopy.


Piezoelectric devices


These devices are based on the electric field generated by the vibrations of a special crystal, whose frequency is affected by the mass of substance adsorbed on the crystal’s surface. This can be controlled by an active biochemical reaction.


Surface acoustic waves A very non-conventional method.


Thermal involving thermistors.








Electronics Of The Transducer


The transducer is a very important part of the biosensor because it controls the accuracy of the


output that can be obtained out of the instrument. No matter how accurate the sensing part of the biosensor


is but the real accuracy of any instrument lies in the final output read by the user. If the conversion of a


chemical or physical change to an electric signal and the reconversion of the electric signal to an easily


readable form is not faithful then the error that could occur in the final reading would be too lousy an


excuse on the part of a competitive technologist, and it would lead to the loss of the importance of that


particular biosensor in the market irrespective of how innovative a technique it used to detect even the


faintest of the substrate. So it should be the duty of a designer to make sure the electronics is alright so that


he doesn’t lose out in the market. Back to professional literature � transducers are of mainly types


Resistive Transducers


Inductive Transducers


Capacitive Transducers


A Resistive Transducer, as the name implies, is based on the change in magnitude of the current flowing through the detector with change in resistance of a conducting component in the instrument. Change in potential drop is only a measure of the current through the conductor, so its measurement need not be done. A wide variety of resistors are being used for this purpose.


Many reactions proceed with either evolution or absorption of heat, that is, a change that can be observed as a rise or fall in temperature of the reaction environment. Resistors in general have temperature dependant resistances, but their temperature coefficients are not constant themselves and are complex functions of temperature itself. This makes the use of normal resistors inaccurate and more difficult to handle because of the unnecessary mathematics involved. Thermistors are resistors whose resistance at a certain temperature is dependent on that temperature and their resistance decreases with increase in temperature, and vice � versa. In more technical terms we can say that thermistors have a negative temperature coefficient. A thermistor will be used in a biosensor in such a way that it is exposed to the reaction and as the temperature changes its resistance changes correspondingly and affects a constant current already flowing through it. The designer should calibrate an ammeter in such a way that it directly measures the concentration of the substrate being detected. This is possible because the heat change during a reaction is directly proportional to the concentration of the reactants. By making the concentration of the testing reactant large we can make the proceedings of the reaction dependant only on the concentration of the substrate. Many such biosensors are already in the market and are doing well. The biochemical industry requires constant monitoring of processes that are very temperature sensitive, and thermistors serve the purpose very well here. Enzymes are extremely sensitive proteins that react only under very specific temperature and chemical conditions. A biosensor that could monitor enzyme activity in a patient can be of critical help in the cases of emergency, as in the case of people who suffer from diabetes mellitus who need to constantly monitor the concentration of insulin in their blood.


A Capacitive Transducer, makes use of a capacitive element in a circuit to measure a change in a system. Its working is based on the fact that the impedance offered by a capacitive circuit element varies inversely as the frequency of the electrical signal input to it. Here the charge on a capacitor is measured as a function of applied voltage.





An Inductive Transducer uses an inductive element in transduction. It is based on the principle of an induced magnetic field in a coil when a variable potential is applied across another coil in the neighbourhood. Many coils can be coupled together in a fashion that will enable generous control of the accuracy of the transduced signal.


Performance Factors


Selectivity


This is the most important factor by which the biological component of a biosensor (detector) discriminates between different substrates (anylates). Though it is the main role played by the biological component; the transducer, to a certain extent, can effect selectivity.


Sensitivity


This usually needs to be sub millimolar but can go down to femtomolar ranges.


Accuracy


This is usually around 5% around the true value, that is, (+/-) 5%.


Nature of the Solution


The pH, temperature, and ionic strength are important factors in determining the activity of the biomaterial.


Times


The Response Time is around 0 seconds for a typical biosensor (but this is longer than a typical chemical sensor would take).It is the time between the beginning of the test and the output reading.


The Recovery Time should not exceed a few minutes. This is the time that the biosensor needs between two consecutive readings.


The Working Lifetime is greatly dependent on the stability of the biomaterial. It can vary from a day upto a year. This is the maximum time upto which a biosensor can be used economically.


Antibody Sensors





An antibody sensor is a very sensitive biosensor that is based on an antibody � antigen reaction. Any pathogen (disease causing entity) in the blood immediately initiates a chemical reaction that produces antibodies that can neutralize the pathogen. This process is called immunity. Immunity is the method by which the body recognises and neutralises any pathogen (or in general foreign entity) so that it doesn’t cause any disease. Accordingly it is classified into natural, acquired and artificial immunities. Immunity is something that an individual acquires when he is exposed to a pathogen either active or deadweakened. One can artificially introduce dead or weakened pathogens into the system of an organism so that the body develops immunity to the pathogen without getting the disorder. This way, the host is better prepared when virulent forms (active � disease causing) of the pathogen enter the system because the body can already recognise and neutralise the pathogen immediately. Thus the chances that the pathogen can infect the host seriously is greatly reduced. This process is called vaccination. Edward Jenner, in London, pioneered this technique for small � pox but without the forethought of its universal applicability. Louis Pasteur, of France, discovered the general application of vaccination (with the proper logic behind his approach) by chance, while experimenting on chicken cholera.


Today, we have the required medicines for the treatment of most diseases, but the death blow strikes when it comes to the proper diagnosis of the disease. To this day we do not have ample proper pathogen � lab tests that are quick and accurate. A compromise has to be made between time and accuracy. There are many cases where an early diagnosis could have saved lives. Antibodies are one of the first biomolecules created when a new pathogen enters the system, as a response to the antigens of the pathogen. So if there is an effective method of determining the antibodies in the blood of an individual, then we would know the individual’s ailment immediately and this would make quick and accurate diagnoses possible. An antibody sensor can immediately detect the presence of an antibody in a blood sample.


Antigens are compounds that cause a response in the host when they are injected. This property is called Immunogenecity. Antigenecity is the property of antigens by which they react with the antibodies. The antibody � antigen reaction is a stereospecific reaction and is dependent on the structure of the antibody (protein) that will interact with the antigen. A popularly encountered situation is when the Antibody for antigen ‘X’ reacts with antigen ‘Y’. This is not a deviation from normal antibody � behaviour. We must remember that a specific antibody reacts with a particular antigen because the stereochemistry of the reaction is such that only a particular arrangement of molecules in space can enable the proceeds of the reaction well. Infact, in antibodies this factor is so large that the mechanism actually seems absolute and pre �determined by some power. But this mechanism is actually just the special case of a general mechanism that we know all too well. It’s stereochemistry. Actually an antibody will react with an antigen (protein) that will resemble the actual antigen at the antigen � antibody-binding site. A very good example at this conjuncture is that some looped proteins (converted to antigens by suitable carrier molecules) are attacked by antibodies that were developed against the whole protein with the closed loop, or that developed against the closed loop itself, but were unaffected by the antibodies against the opened loop or other configurations of the loop. At the same time we must remember that the reaction still does exhibit strict stereochemistry. Likewise protein denaturation is enough to stop the antibody � antigen reaction for an antibody that was developed for the original denatured protein. For example, the antibody against Ribonuclease did not react against a ribonuclease whose four disulphide bonds were oxidised and hence whose shape was changed.


‘Antigens’ can be designed in the form of haptens. Haptens are smaller molecules that have the structural properties to induce the production of antibodies in the host when adequately transported using carrier molecules. Thus the concentration of haptens can be monitored using an adequate transducer. The concentration of antibodies in the sample is directly proportional to the decrease in concentration of the haptens. This is because haptens that have reacted with the antibodies are bound with the antibody in an antigen � antibody complex. These ‘captured’ haptens are not picked up by the biomaterial (detector) because the complex, on the whole, has a structure different from the structure of the hapten.


For example, if the structure of a virus is determined in the lab, or at least a virus whose structure closely resembles the structure of the virus is found, a suitable hapten can be designed. Now this hapten is introduced into a sample of blood of the patient. A transducer can be set up such that it measures the concentration of unbound hapten in the sample, that is, hapten that is not part of the antibody � antigen complex. The hapten is actually introduced bonded to a carrier molecule, so that it can induce antibody production. The production of antibodies in the sample of blood is actually a measure of the preparedness of the patient’s immune system to attack by a pathogen, which the blood cells view as antigens. As antibodies are produced they react with the haptens (viewed as antigens by the blood cells) to form a complex that deactivates the hapten chemically. Thus this hapten molecule does not show up on the transducer reading as it is ‘bound’ to the antibody. Thus there is a fall in concentration of the hapten, which is directly proportional to the amount of antibodies generated. This in turn depends on the condition of the immunity of the individual, whose blood sample is under scrutiny. If a person already has had the disease then the blood sample will react immediately and produce a sufficient quantity of antibodies immediately. So the concentration of the hapten will drop sharply. This can be interpreted as a glow in an LED in the instrument (if it were calibrated as such). Thus a competitive medical practitioner can easily know if the individual was already exposed to this disease or is suffering from it right now. If antibodies already exist in the blood, then the concentration of the hapten will be low from the very beginning, and may just rise a little later. In this case it can be concluded that the individual already has the disease.


Antibody sensors are extremely accurate and ultra � sensitive. They can sense very small amounts of antibodies in a sample. Thus they check for the real first effect of a pathogen on the body, instead of waiting for the physical symptoms to show up.


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