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62 Cards in this Set
- Front
- Back
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Define a biosensor? |
A chemical sensing device in which a biologically derived recognition entity is coupled to a transducer, to allow quantitative analysis in a complex biochemical matrix. - biological component - physical component |
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Give an example of a biological component of a biosensor? |
- enzyme - antibody - nuleic acid - tissue - microbial - polysaccaride |
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Give an example of a physical component of a biosensor? |
- transducer - amplifier - Electric potential - Electric current - Electric conductance - Electric impedence - Intensity and phase of EM radiation - Mass - Temperature - Viscosity |
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Giver a brief overview of how a biosensor works? |
The analyte (a substrate) undergoes a biological reaction to form a product, facilitated by a catalyst. The biological change associated with the catalyst is transmitted to a linked transducer. The transducer senses the changes and converts them to an electronic signal. The signal is then amplified and processed to be displayed in a digital form. |
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What are the 6 important features of a biosensor? |
1) Specific for its purpose 2) The reaction must take place independently to any physical parameters (pH, temperature) 3) Sensitivity - accurate, precise, reproducible and linear range 4) If the biosensor is intended for invasive therapy it must by tiny and biocompatible - non-toxic and non-antigenic 5) The complete biosensor should be cheap, small, portable and easy to use for a semi-skilled operator. |
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What are the 4 ways in which the biorecognition element can be coupled to the biosensor? |
- Membrane entrapment - Physical adsorption - Porous entrapment - Covalent bonding |
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Describe the process of membrane entrapment? |
A semipermeable membrane separate the analyte and the bioelement - this confines the enzyme whilst allowing free passage for the reaction substrates and products |
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Describe the process of physical adsorption? |
Dependent on a combination of Van der Waal forces, hydrophobic forces, H bonds, and ionic forces to attach the biomaterial to the surface of the sensor. |
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Describe the process of porous entrapment? |
Based on forming a porous encapsulation matrix around the biological material that can help in binding it to the sensor. |
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Describe the process of covalent binding. |
The sensor surface is treated as a reactive group to which the biological materials can bind. |
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What are some medical applications of biosensors? |
- General healthcare monitoring - Screening for disease - Clinical analysis and diagnosis of disease |
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Which medical application provides the biggest market for biosensors? |
Testing blood glucose in diabetics |
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How do glucose biosensors work? |
The glucose monitor |
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Describe how glucose biosensors work? |
- They immobilise the enzyme glucose oxidase; this enzyme is very specific to glucose levels and can be measured in the presence of others. - Glucose oxidase catalyses the oxidation of glucose to H2O2 and gluconic acid. An electrode can detect a drop in the O2 concentration. - Another way to detect the glucose concentration is to use peroxidase to catalyse H2O2, the peroxidase enzymes are immobilised, the H2O2 will then oxidise another chemical and induce an electrical signal. |
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Describe the dipstick works? |
- Strips of plastic or card, with mobilised enzymes on the surface. - Can be used to detect the presence of certain molecules in the urine. 1) measuring glucose: peroxidase breaks down H2O2 and the colourless H donor, are all immobilised on a cellulose fibrous pad. - A chemical that changes colour is potassium iodide chromogen, which will go from green to brown. -The dipstick also gives a quantitative measurement when comparing to a standardised colour chart. |
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What is the underlying principle of electrochemical biosensors? |
Many chemical reactions produce or consume ions or electrons, causing soume change in the electrical properties of the solution that can be used as a measuring parameter. Electrochemical biosensors can be classified based on the measuring electrical parameters - Conductometric - Potentiometric - measures change in potential difference - Ampometric - measures current |
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List the types of biosensor. |
- Resonance based - Thermal detection - Ion-selective field effect transistors (ISFET) - Electrochemical - Conductometruc - Potentiometric - Amperometric |
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What are the 3 most common type of biosensors in point of care testing? (testing at or near the sight of care) |
- Amperometric - Optical - Potentiometric |
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Give an overview of an amperometric biosensor? |
The reduction and oxidation (redox) of certain chemical species when subjected by an electrical potential facilitates measurements of specific analytes. |
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What is the principle of an amperometric biosensor? |
- When provided with a constant driving potential, the analyte bound to the receptor will undergo a redox reaction that can be quantified by measuring the electron exchange from the working electrode (anode) to the counter electrode (cathode). The electron exchange is a current and is limited by the diffusion of the analyte through a selectively permeable membrane and various boundary layer phenomena, but in a well designed test strip will be linearly proportional to the concentration of target analyte. |
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What are some applications of an amperometric biosensor? |
In clinical science, redox reactions can be catalysed by enzymes. For example, in glucose testing, the enzyme glucose oxidase or glucose dehydrogenase are commonly used. |
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What type or biosensor does pulse oxymetry use, what are the governing principles and what are some advantages? |
Optical biosensor - measures the difference in the visible and near IR absorbance of the fully oxygenated and deoxygenated arterial blood. - it is continuous, non-invasive and instantaneous measurement of blood oxygenation. |
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What percentage of the current world market for biosensors account for blood glucose sensors and what is the cost? |
85% £2.5 billion |
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Why is the market for glucose biosensors so high? |
Diabetes is an epidemic with multiple complications; 270 million develop complications each year. |
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What are the complications of diabetes? |
percentage of disease caused by diabetes - 70% eye diseases - 40% nerve damage - 70% heart conditions - 30% kidney disease - >60% of all non-traumatic limb amputations |
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What is the redox reaction with glucose oxidase? |
glucose + O2 --> gluconic acid + H2O2 (glucose is oxidised to the acid, enzyme is reduced) |
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What is the redox reaction with glucose dehydrogenase? |
Glucose + NAD+ --> gluconic acid + NADH (glucose is oxidised to the acid, enzyme is reduced) |
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What are the advantages of glucose oxidase as an enzyme in glucose biosensors? |
Inexpensive x) requires O2 as a cosubstrate x) as oxygen is depleted in the sample, performance decreases |
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What are the advantages of glucose dehydrogenase as an enzyme on glucose biosensors? |
Not oxygen dependent x) the cofactor NAD+ is expensive and unstable |
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Describe the first generation of amperometric glucose biosensors? |
No connection between the enzyme and the electrode. Measures the end product of substrate conversion. Based on glucose oxidase immobilised close to an electrode. Uses the oxidation of glucose to transfer electrons. |
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Describe the second generation of amperometric glucose biosensors? |
Not dependent on local oxygen concentration but uses a mediator to link transfer the electrons produced by the reaction, at the active site, to the electrode surface. The mediator is usually ferrocene - (reduced form): ferrocyanide <--> ferricyanide (oxidised form). |
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What are the advantages of the second generation redox mediators? |
- They have a wide range of redox potentials - The redox potentials are independent of pH - Easy to manufacture |
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Describe how the Clear Blue pregnancy biosensor works? |
- the antibody is plotted on the nitrocellulose membrane - the antibody adsorbed latex is sprayed onto wick material - as a reservoir - the urine sample re suspends the latex from the wick and it is carried in solution through the nitrocellulose membrane. - hCG binds to the antibody in the latex - the antibody is plotted as the line captures the hormone-latex complex and a blue line forms. |
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What are the chemical reactions that take place in a cholesterol monitor? |
Cholesterol esterase: cholesterol esters + H2O --> cholesterol + FA Cholesterol oxidase: cholesterol + O2 --> cholest-4-en-3-one +H2O2 Peroxidase: H2O2 + colourless dye --> coloured dye + H2O measured at 642 nm |
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What is a potentiometric biosensor? |
Makes use of the ion-selective electrodes in order to transduce the biological reaction into an electrical one |
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Describe the mechanism of the potentiometric biosensor? |
an immobilised enzyme membrane surrounding the probe from a pH-meter, where the catalysed reaction generates or absorbs hydrogen ions. The reaction occurring next to the thin sensing glass membrane causes a change in pH which may be read directly from the pH-meter's display. |
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What are the 2 classes of redox enzymes? |
- Extrinsic - Intrinsic active sites |
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Give an example of an extrinsic redox enzyme? |
Cytochrome c peroxidase |
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Give an example of an intrinsic enzyme? |
Glucose oxidase |
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What are the disadvantages of intrinsic redox enzymes?
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electron transfer occurs within the confines of the enzyme. It is difficult to achieve electron transfer between the electrode surface and enzyme active site. the electron donating or accepting species required usually via a co-substrate which binds at a site remote to the active site centre. |
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What are the advantages of extrinsic redox enzymes? |
This is the best case scenario because the active site is accessible and there is no need for an electron transfer pathway between the active site and the protein surface. (electrons transfer easily from one exposed site to the next)
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Which redox enzymes are used in the majority of studies? |
Intrinsic |
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What is the most overriding problem of intrinsic redox enzymes? |
The 'buried' nature of the active site, means electron transfer rates are low. |
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Give 5 problems associated with protein electrochemistry? |
1) The redox active site is often shielded by the protein structure so electron transfer is either slow or not possible 2) The protein diffusion to the electrode can be slow. If the protein is not tethered to the electrode it is relying on simple diffusion that it will come close enough 3) however if it is tethered it could cause a change in protein structure, altering the active site. 4) The stability of many proteins is limited outside their natural environment 5) Many proteins undergo adsorption at bare metal electrode followed by degradation. |
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Give 2 major sources of free radicals in the body? |
- by-product of oxidative respiration in the mitochondria - macrophages |
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What diseases are linked with free radicals? |
- heart disease - atherosclerosis - ischaemia - neurodegenerative diseases |
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What is the main reason free radicals are so damaging? |
lipid peroxidation |
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Which diseases result from lipid peroxidation? |
80% of cholesterol is produced in the liver, there is increasing evidence that lipid peroxidation is involved in liver damage - Peroxidised lipids damage arteries leading to atherosclerosis. |
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Describe where lipid peroxidation has its greatest affect? |
Fats that have been chemically damaged by oxygen free radicals. Cell membranes consist mainly of layers of phospholipids. Free radicals attack the phospholipid cell membranes causing injury and eventual cell death due to DNA strand breakage. |
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State the stages of lipid peroxidation?
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Initiation Propagation Termination |
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Describe the initiation step of lipid peroxidation? |
Initiation unsaturated lipid (fatty acid) present in larger amounts in the lipid membrane is attacked by ROS (•O2-, •OH, superoxide anion, hydrogen peroxide, and hydroxyl radical) and this produces a fatty acid free radical. |
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Describe the propagation step of lipid peroxidation? |
The fatty acid free radical reacts with O2 to form a lipid peroxyle free radical. This radical reacts with another FFA producing a different fatty acid radical and a lipid peroxide. The cycle continues as the new fatty acid reacts in the same way - chain reaction. This can go on to cause widespread cell damage and ultimately DNA damage. |
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How can the propagation step of lipid peroxidation stop? |
A radical must react with another free radical species to form a non-radical species. |
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Describe the termination step of lipid peroxidation? |
In nature there are specific molecules that activate the termination of the chain reaction called antioxidants. - Vitamin A, C, E - Enzymes: superoxide dismutase, catalase, peroxidase |
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What are the final products of lipid peroxidation? |
Reactive aldehydes - malondialdehyde (MDA) (marker of oxidative stress) and 4-hydroxynonenal (product of lipid peroxidation) |
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What is the problem with measuring MDA as a marker of free radical detection? |
Melondialdehyde is an endpoint measurement, it is only produced at the end of a destruction process that the free radicals have formed in lipid peroxidation. |
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Describe the third generation of biosensors? |
Direct electron transport between enzyme active site and the electrode surface. Since the active site of GOx is 'shielded' the aim is to get the active site to communicate with the electrode. |
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How does the second generation biosensors communicate with the electrode? |
It involves soluble electron transfer mediators, which communicate by diffusion to and from the active site. |
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What is the difference between the 1st and 2nd generations compared to the third generation? |
The GOx is immobilised at the gold electrode |
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What is horse entrapment? |
An example of immobilising the biological element onto the electrode surface. Horseradish peroxidase (HRP) is combined with carbon paste which dries to form a porous substrate. It detects the electron transfer when H2O2 is reduced to H2O |
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What is different when horseradish peroxidase is used as the biological component of the biosensor? |
HRP is oxidised by the conversion of peroxide into water, it take electrons from the electrode and is reduced. So, the other way round - usually the biological element is reduced and the biological change is an oxidation reaction. |
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What are the applications of HRP? |
ELISA and Immunohistochemistry due to its monomeric nature and the ease with which it produces coloured products. |
