optical biosensor.pptx by taimoor khan awkum
- 2. Optical biosensor Optical biosensors utilizes optical techniques to detect and quantify any chemical, biochemical or biological components in a sample. selective, rapid and extremely sensitive measurements
- 3. Components Fig. 2 shows the key components of a biosensor
- 4. Two main components • Bio recognition element for interaction with biological or chemical species. • Transduction a transduction principle which converts physical or chemical response of biological element into optical signal.
- 5. • Label based A fluorescent or a colorimetric label is used and the intensity of the fluorescence or the change in color respectively is observed to detect an analyte • Label free These biosensors detect the interaction between biological element and analyte based on changes in optical properties i,e refractive index or light scattering
- 7. Optical sensors • Absorbance in chemical reaction • Reflectance / transmittance • Refractive index • Surface plasmon resonance
- 8. Transducer mechanism Absorption • The product or altered substrate absorb light at specific wavelength. This change correlate with conc. of analyte. Fluorescence Some reaction lead to the production of fluorescent products or alteration of fluorescence properties of an initial fluorescent molecule. Luminescence • Certain reactions produce light as a product of the enzymatic reaction, which is directly measured
- 9. Surface Plasmon Resonance Biosensors An Optical technique used to study and measure molecular interaction in real time It detects the shift in refractive index caused by molecular interaction at a metal surface via surface plasmon wave. Provide a non invasive, label free detection in real time.
- 10. Surface plasmon resonance Fig. 1 A light beam moves from denser medium into a lighter medium such as from glass to air. Within the medium it undergoes total internal reflection
- 11. a b The free electron on biochip surface absorb the incident light photons and convert it to surface plasmon waves. It create electron density waves that propagates along the surface of the metal. As result of absorption dark zone appears in refracted light. The absorbed light force electrons in metal to oscillate and this oscillating is called plasmon. Fig. 2 Fig. 3
- 12. What is surface plasmon? • Surface plasmon occurs when photons from incident wave interact with electrons in the metal surface • The coupling results in the creation of plasmon, a group of excited electrons on surface of metal thereby forming a surface plasmon wave.
- 13. Principle of SPR • SPR is an optical phenomenon that provide a non invasive, label free or observing binding interaction between an injected analyte and immobilized biomolecule in real time. • SPR angle: depend upon the refractive index of a medium near metal surface. • Any change in refractive index will cause change in SPR angle.
- 14. Total Internal Reflection Fig. 4: TIR strikes in electrically conducting gold layer at the surface b/w the media of different refractive index.
- 15. Fig. 5 When sample in a buffer passed over the gold plate the two surfaces; 1. Metal surface of high refractive index 2. Buffer having sample of low refractive index, So when the light pass from it from high index to low index and form a total internal reflection
- 16. • Index of the refraction of surface bound layer is proportional to concentration of the bound molecule. Fig. 6 functionalized metal surface having ligands attaches through specific binding
- 17. Applications • Studying interaction between two molecules • Biomolecule interaction • DNA RNA interaction • To quantify proteins or other biomolecules at surface • Hormone – protein interaction
- 18. SPR Biosensor applications • Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance (Yano et al., 2022) • Used Au nanoparticles (AuNPs) functionalized with complementary DNA https://doi.org/10.1038/s41598-022-05036-x
- 19. Metal detection • Mercury (Hg) – the gold surface sensors functionalized with thiol containing agent (marcaptopropanic acid) form self assembling monolayer. • Sample containing Hg (having affinity for MPA) adheres to functionalized surface. • SPR measurement is taken, by the resulting change in refractive index near sensor surface
- 20. Food monitoring • Detection of Foodborne Pathogens – for example bacteria, viruses, Salmonella, E. coli • Detect the presence of histamine, tyramine etc by immobilization of various antibodies specific to agents. • Mycotoxin detection – secondary metabolites produced by fungal species.
- 21. • Detect specific biomarkers associated with cancer (PSA, HER2 in breast cancer) • drug-target interactions (i,e. kinase inhibitor in cancer) • Target validation using SPR
- 22. Example
- 23. Optical Biosensor Based on Redox Enzyme An analytical device that combines biological components with physiochemical transducer to monitor and quantify biological reactions by converting substrate into product through enzymatic reactions.
- 24. Enzyme-based optical biosensors for organophosphate • class of pesticide pollutant • Bio recognition element acetylcholinesterase (AChE) organophosphorus hydrolase (OPH)
- 25. Biosensing transducers • Photoluminescence (PL) • Chemiluminescence (CL) • Colorimetric (CM) biosensing • Surface Plasmon Resonance Transducer
- 26. Photoluminescence Based on the Beer-Lambert law Log (Io/I) = A = cl • where Io is the intensity of incident light; • I is the intensity of transmitted light; • A is absorbance (molar absorbance of the analyte at a specific wavelength) • C is the concentration of analyte; • l is the path length of light through solution.
- 27. Luminescence Transducers • Involve generation of light as result of reaction between a luminophore and substrate molecule. • Luminophore – molecule capable of emitting light. • Intensity of light is proportional to conc. Of target analyte in sample.
- 28. Chemiluminescence Transducers • Emission of light as result of chemiluminescence reaction between reagent and analyte. • Enzyme-based optical biosensors • H2O2 biosensor -- based on the peroxidase- mediated oxidation of luminol by H2O2. Disadvantage • The main disadvantages of this type of optical transducer are finite lifetime due to reagent consumption and steady-state mass transfer required to get a constant signal
- 39. Paper based fluorescence biosensor
- 41. Optical biosensor: Fluorophore • Molecular Beacons (attached with fluorophore) – oligonucleotide with stem and loop structure labeled with fluorophore at one end and quencher at another end.
- 42. Optical DNA probes Transduction approaches: Fluorescent quenching(F and Q) Fluorescent enhancement Fluorescent resonance energy transfer (FRET) ⁻ Based on energy transfer b/w donor fluorophore in its exited state and acceptor chromophore ⁻ Donor = fluorescent molecule Sekar R.B. & Periasamy A. (2003). Fluorescence resonance energy transfer (FRET)
- 43. Conditions: 1. Donor and acceptor to be in close proximity If they are too far apart then no energy transfer may occur. If both are closer then the donor transfer their energy to a acceptor molecule. That acceptor molecule is going to fluoresce and you are going to see a change in wavelength of acceptor molecule 2. The absorption spectrum of the donor molecule must overlap the emission spectrum of the acceptor molecule.