2. Immobilization of Biomelecule
(a) Activates sensing of analyte in flow systems
e.g. body fluids, bioreactor fluids, or water.
(b) Sufficient addition of biomolecule to the
sample.
(c) Biosensor can be reused or re-generated.
Stability of immobilized biomolecules is however a
problem.
3. Types of Optical Biosensors
1- Depends upon changes in the intrinsic optical property
(absorbance, emission, polarization, or luminescence
decay time) of the biomolecule produced by its interaction
with the analyte. (problems: lower sensitivity, difficult in
complex study, separation of Fluorescence or Absorbance
signal.
2- Using optical labels/ probes by covalent bonding with Biomolecule.
(Shift of analytical wavelength- Adjustable decay time/ anisotropy)
Example: Luminescent nanoparticles/ dyes
4. Enzymatic Biosensors
(Catalytic- Highly specific)
Cross section of a fiber-optic enzymatic biosensor. The
analyte (substrate) enters the enzyme layer where it is
converted into products. The indicator (sensing) layer
consists of an indicator dye in a polymer layer and
registers the formation of reaction products.
5. Product of enzyme analyte reaction
1. Directly detected (if colored or luminiscent)
2. Using optical transducer
In both cases the signal depends upon the concentration
of Analyte/ substrate.
Some enzymes require coenzymes (NAD or FAD) for activation
regarding a specific reaction.
Enzyme is either
1. Chemically immobilized on glass, cellulose, nylon, etc.
2. Physically entrapped in sol-gel, hydrogels, etc.
6. Simpler optical sensor (chemiluminescent/ Bioluminescent
-No indicator layer
The reaction of chemical analyte with enzyme- Produces light
Glucose Biosensors
D-glucose + O2 D-gluconolactone + H2O2
GOx
D-gluconolactone + H2O D-gluconate + H+
The concentration of glucose thus can be related to
-O2 consumpsion,
-The H2O2 production
-Decrease in pH
7. Typical signal shapes can be obtained if a glucose
sensor based on immobilized glucose oxidase and using
an oxygen sensor as the transducer (in contact with air-
saturated buffer) is exposed to flowing samples
containing various levels of oxygen and glucose,
respectively, and then again to air-saturated buffer.
8. Determining glucose concentration by the
amount of H2O2 formed is the most realistic
Strategy because it works at zero background.
However it is also interrupted by the initial pO2
.
Other example
- Diphenylanthracene (DPA) in a sol-gel based glucose
biosensor as probe for Oxygen
- Polycondensation of Tetraethoxysilane (TEOS) for
inorganic glassy matrix whose porosity and size of
pore network
DPA + GOx entrapment into the sol-gel network.
Good for preparation of optical sensor membrane as
no signal in UV-Vis. region
9. Other optical biosensors:
- Use polyaromatic hydrocarbons (pyrene,
decacyclene and their derivatives)
However
- Ruthenium(II) complexes with ligands such as bipyridyl
(Rubipy),1,10-phenanthroline (Ru-phen) and 4,7-
diphenyl- 1,10-phenanthroline (Ru-dpp) replaced the
polycyclic aromatic hydrocarbons.
Have visible absorption, - longer decay times
(0.6-6 µs) - good Photostability
So widely used as oxygen probes
10. -Platinum(II) and palladium(II) porphyrins – are used
luminescent oxygen indicators.
Advantages; Stable chemically/ photochemicaly, Better
brightness, long luminescence lifetimes.
Papkovsky used a phosphorescent platinum(II)
complex with octaethylporphyrin (PtOEP) dissolved in
polystyrene as the oxygen transducer for glucose
sensing.
The sensor was applied to the determination of 0.05-1.2
mM of glucose.
- limit of detection was 0.05 mM
11. Dual Glucose sensors based on
- Europium probe acting as a reporter for hydrogen.
peroxide.
- Iridium-trisbipyridine complex as a reporter for oxygen.
Glucose oxidase was entrapped in a hydrogel membrane.
Other approach:
The concentration of glucose may also be related to the
amount of protons (pH approach). However very few
glucose sensor are made.
Example: The fluorescence of the pH probe 8-
hydroxypyrene-1,3,6- trisulfonate in a protonpermeable
hydrogel was used to monitor pH changes during
enzymatic reaction. LOD was 0.1mM.
12. According to Freeman and Seitz
The H2O2 transducer was used a biosensor as it used the
oxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-
dione) catalyzed by (POx).
luminol + 2H2O2 + OH- 3-aminophthalate + N2+3H2O + hv
The intensity of chemiluminescence at 430 nm is
proportional to the concentration of H2O2 and in case of the
glucose biosensor equal to concentration of glucose.
H2O2 was measurable at concentrations up to 1 µM.
Pox
