Biosensors Flashcards
Bio sensor Definition
Chemical signalling device in which a biologically derived recognition entity is coupled to a transducer, allowing quantitative or qualitative analysis in complex biochemical matrix
Couple biorecognition element to transducer (4)
Membrane entrapment
Physial Adsorption
Porous entrapment
Covalent Bonding
Membrane Entrapment
semi-permeable membrane separates analyte and bioelement
Physical Adsorption
Dependent on combination of van der Waals forces, hydrophobic, hydrogen and ionic forces to attach biomaterial/analyte to the surface of the sensor
Porous Entrapment
Form porous encapsulation matrix around biological material - helping it bind to sensor, e.g. carbon paste
Covalent Binding (most common)
sensor surface treated as reactive group to which biological material binds
Biosensors must exhibit
repeatability - intra-assay
reproducibility - inter-assay
specificity/selectivity
sensitivity - linear range, detection limit, response time
Resonance Biosensors 2 steps included
light-wave transducer coupled with antibody/bioelement
- analyte binds - increases mass membrane - distort sensor
- changes resonant frequency and angle of deflection
Thermal Detection Biosensor 3 steps and example
- analyte comes into contact with enzyme
- energy change measured and calibrated against conc
- total heat produced or absorbed proportional no. molecules
detection pesticides/pathogenic bacteria
Ion-selective field effect transistor
semi-conductive FET
ion selective surface - gate between source (+ve) + drain (-ve)
- surface electrical potential changes when ions and semi-conductor interact
- measure change in potential
e.g. pH measurement
Conductometric/Chemisresistor definition
measure conductance (opposite resistance) how easy electrons flow
Potentiometric definition
measure change in potential difference (voltage)
charge +ve - hydrogen production
Amperometric definition
measure change in current
flow of electrons
Ohms Law
V=IR
Conductometric types (2)
- material clamped between 2 contact electrodes (gas sensing CO)
- active layer immersed in electrolyte solution
Potentiometric
ion-selective electrodes
immobilised enzyme membrane surrounding pH meter
difference in pH between two areas
Amperometric
high sensitivity and selective
detect electroactive species
may require redox enzymes to produce electroactive species - oxidative = active
electrode reduced rate proportional to concentration
Commercial (2)
Blood glucose - diabetes
Pregnancy test - clear blue
Technical Issues (3)
Fragile
many different materials needed
issues - bonding, connection, reading
Blood Glucose biosensor required
diabetes --> increased heart disease 2-4x lead to blindness 40% increase serious kidney 60% increase ampuations
Blood glucose chemistry
redox enzymes
glucose oxidase - oxidise substrate and become reduced
transfer electron to oxygen –> hydrogen peroxide
glucose dehydrogenase - “
reduces NAD+ to NADH in order to regen
Glucose Oxidase pros and cons
+ cheap
-require oxygen and performance reduces throughout
Glucose Hydrogenase
+ independent of oxygen
-NAD+ expensive and unstable
Redox mediators (electron shuttles)
independent of local oxygen conc
allowing operation at lower potentials
–> minimise detection inteferents
Ferrocene states and features (2) -
oxidated - ferricyanide
reduced - ferrocyanide
range of redox potentials - independent of pH
easy manufacture
Clear Blue Pregnancy Test
Antibody plotted on nitrocellulase (result strip)
Antibody adsorbed latex sprayed onto wick material (reservoir/test strip)
Pregnancy test substrate/hormone and mechansim
human chorionic gonadotrophin (hcG)
both antibodies complimentary to hcG
urine resuspends latex and carried through nitrocellulase
hormone bound by capture and form blue latex line
Redox enzyme - intrinsic example and description
e.g. cytochrome c oxidase
electron-transfer outside confines of enzyme
electron-transfer between prosthetic group and substrate in vicinity of active site
not require ET path from active site to protein (directly)
Extrinsic enzyme - most biosensors
glucose oxidase
ET within confines of enzyme
difficult to achieve ET between active site and electrode
electron donating/receiving species required - bind at separate site
ET pathway with co-substrate, bind site and active centre
expensive
Difficult to make direct communication between extrinsic and electrode
deeply buried active centre
ET rates low due to distance
denaturation at electrode (Hg electrode)
Extrinsic solution
electrode mimic intermolecular recognition
cytochrome c peroxidase
water soluble, haeme-containing
reduced by cytochrome c
reduces water to Hydrogen peroxide
Bind cytochrome c to electrode to
achieve direct interaction with electrode
using DTSSP - dimer - binds 2 cytochrome c
Inappropriate production of radicals creates (4)
cardiovascular dysfunction
arteriosclerosis
ischameia
neurodegenerative diseases
Free radicals features (2)
hard to detect as unstable and high interactivity
try to detect end point/products
mopped up by antioxidants
Possible detect radicals details (3)
by cytochrome c on electrode
using xanthine oxidase or cells
superoxide reduces cytochrome c
Human glioblastoma cells (3)
destroy pathogen with O2- by NAPDH oxidase
leak some - collateral damage
first produce NO. then O2-, NO. react with O2-
Third gen glucose sensor
direct electronn transport between enzyme active site and electrode surface
Glucose Oxidase structure
dimer - active site buried
co-factor FAD/FADH2 1 (per subunit)
electrostatic site at entrance active site -> +ve
Lysine residues in Glucose oxidase react
with DTSSP
so bind active site of GoX to electrode surface
Horse radish peroxide use with electrode (3)
re-reduced by carbon electrodes
electrodes fabricated using screen printing tech
HRP mixed into carbon ink - printed directly on electrode
HRP reduced by
by electrode
oxidised by hydrogen peroxide
immunosensor using antigen bound to GOD
PEBBLE sensors meaning and definiton (4)
Probes encapsulated by Biologically Localised Embedding
(polyacrylamide porous shell with fluorophore inside)
nano-scale spherical devices with sensor molecules in chemically inert matrix
PEBBLE structure (2)
protective coating eliminates interferences - protein binding/membrane or organelle sequestration
–> which alter dye response
matrix protects cell contents - dye would be toxic otherwise
PEBBLEs able to measure
calcium, potassium, nitric oxide, oxygen, chloride, sodium and glucose
Fibre-optic probe
fluorophore on the tip - measure pH, glucose, oxygen
Fibre-optic negatives
punch hole in cell
measure very small area
PEBBLE extras
reference dye
enzyme create product that reacts with dye
Characterisitics of PEBBLE (3)
non-invasive in cell - not seen as threat
cell viability 97%
Response time <1ms
Sol-gel also used as matrix
not as rigid
not change shape of enzymes within
Ratiometric Device
e.g. oxygen sensor
silica glass
ruthenium dye - orange, decreases with higher oxygen
oregon dye - green, not sensitive to oxygen
Free dye problem
Bind other proteins such as Bovine Serum Albumin
Nanosensor not able to enter
Nuclear membrane - very selective
Gene Gun
deliver DNA and PEBBLEs
helium shoots dried PEBBLEs off a disk
embed in cells
able to penetrate nuclear membrane
Gene Gun -ve
shoot right through cell
multiple ruptures
Phagocytic Nanosensor Internalisation
Sensors ingested
remain encapsulated
However not free in membrane
Lipid Transfection
Nanosensors internalised into liposome
free in cytoplasm
PEBBLEs +ve
and synthesis
cheap,easy, fast
water in oil microemulsions - then concentratedm washed nad vacuum filtered
PEBBLE modification
attach molecules to outside of matrix - specific attachment to specific cellular membrane
Amine groups on surface of nanosensor
bind ester groups on biotin
then bind avidin and biotin
then bind antibody
SeLCA (future)
Sensing living cell arrays
combine optical nanoprobes delivered to cell interior
and electrochem sensing arrays to measure extracell
Microtitre-plate cell retainers
Insert Micro-ring electrodes of gold and platinum into wells (electrodes)
monitor different analytes
sharp borders stop cells sticking together
Myalgic Encephalomyelitis/Chronic Fatigue
Decreased function of pyruvatedehydrogenase complex
- -> increased conversion pyruvate to lactate via lactate dehydrogenase pathway
- -> intracellular acidosis
ME sensors
pH nanosensors
Dichloroacetate
up-regulates function of pyruvate dehyrdogenase
–> inhibition of inhibitory kinase
neurological SE
