Biosensors and Microsystems L11-18 Flashcards
Define a biosensor.
A biosensor is a chemical sensing device in which a biologically derived recognition entity is coupled to a transducer, to allow quantitative or qualitative analysis in a complex biochemical matrix.
Biosensors incorporate a specific ____1____ element (that creates a recognition event) and a ____2____ element (that converts the recognition event into a recordable signal).
- Biological
- Physical
Basic Principle of a Biosensor
Coupling the biorecognition element to a transducer can be achieved in four ways. State each.
- Membrane Entrapment
- Physical Adsorption
- Porous Entrapment
- Covalent Bonding
Define analyte.
A substance or chemical constituent that is of interest in an analytical procedure.
What characteristics must biosensors exhibit? (4)
- Repeatability – Intra-assay variability
- Reproducibility – Inter-assay variability
- Specificity/selectivity (to analyte of interest)
- Sensitivity – Linear range, detection limit, response time
Resonance Based Biosensors
In resonant biosensors, a light-wave ____1____ is coupled with an antibody, or bioelement.
When the analyte molecule, or antigen, gets attached to the membrane, the ____2____ of the membrane changes (due to surface flexing).
The resulting change in the mass subsequently changes the resonant frequency of the ____1____ (light hits somewhere else).
This frequency change is then measured.
The degree of flexion = ____3____ of antigen.
- Transducer
- Mass
- Concentration
Thermal Detection Biosensors
Exploits the absorption or production of heat, that in turn changes the ____1____ of the medium in which the reaction takes place.
Predominantly enzyme based.
When the analyte comes in contact with the enzyme, the energy change of the enzyme reaction is measured and calibrated against the analyte ____2____.
The total heat produced or absorbed is ____3____ to the total number of molecules in the reaction.
Common applications of this type of biosensor include the detection of pesticides and pathogenic bacteria.
- Temperature
- Concentration
- Proportional
Ion-Selective Field Effect Transistors (ISFET)
Ion sensitive biosensors are semiconductor FETs having an ion-sensitive surface.
The surface electrical ____1____ changes when the ions and the semiconductor interact.
This change in the ____1____ can be subsequently measured.
The ISFET can be constructed by covering the sensor electrode with a polymer layer.
This polymer layer is ____2____ permeable to analyte ions.
The ions diffuse through the polymer layer, causing a change in the FET surface ____1____.
This type of biosensor is primarily used for ____3____ detection.
- Potential
- Selectively
- pH
Electrochemical Biosensors
The underlying principle for this class of biosensors is that many chemical reactions produce or consume ions or electrons, causing some change in the electrical properties of the solution that can be used as a measuring parameter.
Electrochemical biosensors can be classified into 3 types, based on measured electrical parameters.
What are they? (name + parameter)
- Conductometric - measures conductance
- Potentiometric - measures changes in potential difference
- Amperometric - measures current
Conductance = inverse of ______
Resistance
Ohm’s Law deals with the relationship between voltage and current. This relationship states what?
The potential difference (voltage) across an ideal conductor is directly proportional to the current through it.
State the equation for Ohm’s law.
Include key.
V = I R
where:
V is the potential difference between two points
I is the current flowing through the resistance.
R is the resistance to current flow
Conductometric Biosensors
Based on measuring changes in ____1____ of a selective material.
Since the inverse of resistivity is conductivity, these sensors are interchangeably called conductometric sensors or chemiresistors.
There are two types of these sensors:
- A selective material, which can change its conductivity upon interaction with chemical species is clamped between two contact electrodes and the ____1____ of the entire device is measured. Used in____2____ sensing.
- The chemically interactive layer is at the top of an electrode, which is immersed in the solution of ____3____. Used in biosensing
- Resistance
- Gas
- Electrolyte
Potentiometric biosensors make use of ion-selective electrodes in order to ____1____ the biological reaction into an electrical signal.
This consists of an immobilised enzyme membrane surrounding the probe from a pH-meter where the catalysed reaction
generates or absorbs ____2____ ions.
The reaction occurring next to the thin sensing glass membrane
causes a ____3____ in pH.
- Transduce
- Hydrogen
- Change
An amperometric biosensor is a high ____1____ biosensor that can detect electroactive species present in biological test samples.
Since the biological test samples may not be intrinsically
electroactive, enzymes are needed to ____2____ the
production of reactive species.
In this case, the measured parameter is current.
- Sensitivity
- Catalyze
Name the 2 major areas of commerical success for biosensors.
- Diabetes monitoring (blood glucose measuring)
- ClearBlue® (pregnancy test)
Blood glucose biosensors account for approximately 85% of the current world market for biosensors (~£2.5billion)
What is the reasons why the glucose market was particularly receptive to the introduction of biosensors?
The high (and increasing) prevalence of diabetes in developed nations.
Most enzyme biosensors (glucose included) rely on ______ enzymes.
Redox
The most commonly used enzymes in the design of glucose biosensors contain redox groups that change redox state during the biochemical reaction.
Enzymes of this type are glucose ____1____ (GOx) and glucose ____2____ (GDH).
In nature, ____1____ enzymes such as GOx act by oxidising their substrates, accepting electrons in the process and thereby changing to an inactivated ____3____ state.
- Oxidase
- Dehydrogenase
- Reduced
State the advantages and disadvantages of glucose oxidase redox enzyme biosensors.
Advantages
- Inexpensive
Disadvantages
- Requires oxygen as a cosubstrate. Consequently, as oxygen is depleted in the sample, performance decreases.
State the advantages and disadvantages of glucose dehydrogenase redox enzyme biosensors.
Advantages
- Oxygen independent
Disadvantages
- The cofactor (NAD+) is expensive and unstable
State the chemical equation that happens in a glucose oxidase redox biosensor.
Glucose + O2 → gluconolactone + H2O2
State the chemical equation that happens in a glucose dehydrogenase redox biosensor.
Glucose + NAD+ → gluconolactone + NADH
The earliest approaches to the construction of amperometric glucose biosensors were based on GOx immobilised close to an electrode. The depletion of oxygen was monitored, using a ______ oxygen electrode
Clark
What are the advantages of using a redox mediator in a biosensor? (3)
- They have a wide range of redox potentials
- The redox potentials are independent of pH
- Easy to manufacture
Name the analyte used in pregnancy testing.
Why is this used?
The hormone HCG.
HCG is present in pregnant women.
Redox enzymes can be divided into 2 classes depending on the mechanism of electron transfer.
What are they? + Examples?
- Intrinsic (e.g. Cytochrome c peroxidase)
- Extrinsic (e.g. Glucose oxidase)
Describe intrinsic redox enzymes. (3)
- Take part naturally in electron-transfer outside the confines of the enzyme.
- Electron transfer between prosthetic group and substrate in vicinity of active centre.
- No requirement for ET pathway from active site to protein surface.
Describe extrinsic redox enymes. (4)
- Electron transfer occurs within the confines of the enzyme.
- Difficult to achieve electron transfer between electrode surface and enzyme active site.
- Electron donating or accepting species required (co-substrate) which binds at site remote to active centre.
- Electron transport pathway presumed to exist between co-substrate binding site and active centre — possibly involves enzyme surface.
What is the difference between intrinsic and extrinsic redox enzymes?
The positioning of the active site within the structure.
Free radicals (O2ˉ and NO) are continuously generated during cell ____1____ and ____2____.
- Respiration
- Metabolism
Define lipid peroxidation.
Lipid peroxidation is the oxidative degradation of lipids. It is the process in which free radicals “steal” electrons from the lipids in cell membranes, resulting in cell damage.
Lipid Peroxidation
Initiation
- Reactive oxygen species (ROS), such as OH·, O2ˉ and NO, combine with a hydrogen atom to make ____1____ and a fatty acid radical.
Propagation
- The fatty acid radical reacts with molecular oxygen to create a lipid peroxyl radical.
- This radical reacts with another free fatty acid, producing a different fatty acid radical and a lipid peroxide, or a ____2____ peroxide if it reacted with itself.
- This cycle continues, as the new fatty acid radical reacts in the same way.
Termination
- When a radical reacts with a non-radical, it produces another radical, resulting in a “chain reaction mechanism”.
- This radical reaction can only be stopped by the interaction of two radicals to produce a non-radical species.
- In nature there are specific molecules that accelerate termination by capturing free radicals and preventing them from damaging the cell ____3____.
- Antioxidants include vitamins A, C and E. Other anti-oxidants are enzymes and include superoxide dismutase, catalase, and peroxidase.
Final products of lipid peroxidation
- The end products of lipid peroxidation are reactive ____4____, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE).
- Water
- Cyclic
- Membrane
- Aldehydes
Xanthine oxidase catalyses the conversion of xanthine to uric ____1____ and superoxide (O2- free radical).
This is our method of generating superoxide at will.
We can therefore design a biosensor for free radical/oxidative stress ex vivo.
Transfer of ____2____ is the same as cyto c peroxidase, just with superoxide instead.
- Acid
- Electrons
Define amperometry.
The detection of ions in a solution based on electric current or changes in electric current.
Amperometric Glucose Measurement
Amperometric Glucose Measurement
Amperometric Glucose Measurement
Read
In medical and biochemical research, when the domain of the sample is reduced to micrometer regimes, e.g. living cells or their subcompartments, the real-time measurement of chemical and physical parameters with high spatial resolution and negligible perturbation of the sample becomes extremely challenging.
The larger the sensor the more it can detect and therefore it can be less sensitive.
The smaller the sensor the less it can detect and therefore it must be more sensitive to retain functionality.
A traditional strength of chemical sensors (optical, electrochemical, etc.) is the minimization of chemical interference between sensor and sample, achieved with the use of inert, ‘biofriendly’ matrices or interfaces.
However, when it comes to penetrating individual live cells, even the introduction of a sub-micron sensor tip can cause biological damage and resultant biochemical consequences.
In contrast, individual molecular probes (free sensing dyes) are physically small enough but usually suffer from chemical interference between probe and cellular components.
What are PEBBLE sensors (Probes Encapsulated By Biologically Localized Embedding)?
Nano-scale spherical devices consisting of sensor molecules (fluorescent sensing dyes) entrapped in a chemically inert matrix.
PEBBLE sensors (Probes Encapsulated By Biologically Localized Embedding) are nano-scale spherical devices consisting of sensor molecules (____1____ sensing dyes) entrapped in a chemically inert matrix.
This protective coating eliminates interferences such as protein binding and/or membrane/organelle sequestration which alter dye response.
Conversely, the nanosensor matrix also provides ____2____ to the cellular contents, enabling dyes that would usually be ____3____ to cells to be used for intracellular sensing.
In addition, the inclusion of reference dyes allows quantitative, ratiometric fluorescence techniques to be used.
PEBBLEs have been used to measure ____4____ such as calcium, potassium, nitric oxide, oxygen, chloride, sodium and glucose.
- Fluorescent
- Protection
- Toxic
- Analytes
Define ratiometric.
Describing any system in which an output is directly proportional to an input.
One of the advantages of the nanosensors are that they are ______.
Ratiometric
Fibre-Optic Precursor Technology
Pulled (tapered) fibre optic probes as intracellular sensors have been used. The tips of these fibres were either coated with a layer of plasticized PVC (glue) containing sensor materials, or they had a small amount of sensor material on the end which was photo-polymerized. The sensor materials stuck down by the PVC polymer are effectively fluorophores. This fluorophore responds to pH.
The probe is pushed through the cell membrane into the intracellular space. Any changes in pH in this immediate area will be recognized by the probe causing the fluorophore to fluoresce. This light then travels down the fibre and can be measured. The level of fluorescence is dependent on the pH.
What are the major problems of this technology? (3)
- Made a big hole in the membrane (to get the probe into the cell). This causes damage and can lead to apoptosis.
- Can only measure pH changes in the small vicinity of the fibre tip. This is unlikely to give an accurate representation of the entire cell.
- Entire fibre is very big in relation to the cell meaning that a maximum of only 2 or 3 fibres could be used on a single cell. Therefore you cannot make up for the second problem. Multiple fibres in a single cell would likely kill the cell regardless.
Name the 2 major problems with free sensing dyes.
What nanoscale biosensor technology overcomes this?
- The fluorescence intensity will change from cell to cell due to the different concentrations of dye in each cell.
- Interference by things like non-specific protein binding (which happens a lot in a cellular environment).
PEBBLEs
What delivery method is used for inserting PEBBLEs into cell culture?
Give an advantage of this.
Give a disadvantage of this
Gene gun.
Advantage - This method forces the nanosensors through the cell membrane into the cytoplasm and are even forced into the nucleus (which makes the Gene Gun especially useful).
Disadvantage - Punctures the cells leaving holes in them. Like the fibre-optic method can cause apoptosis and cell death. Some of the fired nanosensors pass right through the cell.
What is the advantage of using a gene gun as a PEBBLE delivery method?
Good penetration. This method forces the nanosensors through the cell membrane into the cytoplasm and some are even forced into the nucleus.
The bioreactor is a network of tubes.
Cells enter via inoculation tube. The cells spread out and populate the entire reactor. The cells are kept healthy by the constant perfusion of air and oxygen and the removal of old medium and CO2 from the respective inlet/outlet tubes.
