Fluorescence Based Sensors

Prabhat Kumar, Ph.D. Definition

Fluorescent sensors: An analyte responsive fluorescent molecule/super molcule/supramolecule which changes its characteristics in the presence of an analyte. A fluorescent sensor is the complete optical sensing device: • the light source • the analyte-responsive (supra)molecular moiety properly immobilized • the optical system • the light detector (photomultiplier or photodiode) connected to • appropriate electronics for displaying the signal.

Fluorescent sensor (in literatures): • Analyte-responsive Molecular/Supermolecular/Supramolecular fluorescent moiety • Fluorescent molecular/supramolecular sensor is used, to avoid confusion

Fluorescence Based Sensors ǁ Session 1 Types, Importance and Applications

Types of Sensors: Based on /signal transducer converting the information into an optical signal) • Chemical sensors (chemosensors): Analyte-responsive moiety is of abiotic origin • : Biological macromolecule (e.g. protein) as fluorescent molecular sensors Importance of Fluorescent sensors: High demand in • • Clinical biochemistry • Medicine and • Environment Application: Detection of chemical and biochemical analytes Cations: H+, Li+, Na+, K+, Ca2+, Mg2+, Zn2+, Pb2+, Al3+, Cd2+, etc. Anions: halide , citrates, carboxylates, phosphates, ATP, etc. Neutral molecules: sugars, e.g. glucose, etc.)

Gases: O2, CO2, NO, etc. Fluorescence Based Sensors ǁ Session 1 Discovering New Sensors?

Necessity of new fluorescent sensors: New fluorescent molecular sensors for particular applications should show • Improved selectivity • Higher selectivity • Response time • Local observation • Minimum perturbation of the microenvironment and • Biochemical analytes: amino acids, coenzymes, carbohydrates, nucleic acids etc. Success of new fluorescent sensors: • Remote sensing using optical fibers • Great improvement in the sensitivity • The spatial or • Temporal resolution of instruments

Fluorescence Based Sensors ǁ Session 1 Classes of Sensors

Classes of fluorescent sensor:

Class 1: that undergo quenching upon collision with an analyte (e.g. O2, Cl) Class 2: fluorophores that can reversibly bind an analyte. If the analyte is • a proton: fluorescent pH-indicator • an : fluorescent chelating agent Fluorescence can be either • CEQ type: Enhancement of Quenching • CEF type: Chelation Enhancement of Fluorescence: fluorogenic: 8-hydroxyquinoline Class 3: fluorophores linked to a receptor via a spacer The design of such sensors, fulfil the criteria of • Selectivity and • affinity in the field of

Fluorescence Based Sensors ǁ Session 1 Classes of Sensors

Class 3: The changes in photophysical properties of the fluorophore upon • Electron transfer, • Charge transfer • Energy transfer • Formation or disappearance of Exciplex and/or absorption and fluorescence dynamic quenching resonance energy transfer In the case of ion recognition: Formation or disappearance • the receptor is called an ionophore, and of Exciplex and/or Excimer • the whole molecular sensor is called a fluoroionophore.

Again, Fluorescence can be either • CEQ type: Chelation Enhancement of Quenching • CEF type: Chelation Enhancement of Fluorescence: fluorogenic

Fluorescence Based Sensors ǁ Session 1 Classes of Sensors

CEQ type: Chelation Enhancement of Quenching

CEF type: Chelation Enhancement of Fluorescence: Fluorogenic

Reference: Lakowicz, Principles of fluorescence Fluorescence Based Sensors ǁ Session 1 Why Fluorescence, why not Absorbance?

Why is fluorescence rather than absorption for high-sensitivity detection? Fluorescence is more sensitive. In Absorbance: The difference in intensity between light passing through the reference and the sample. In fluorescence: Intensity is measured directly, without comparison with a reference beam. Advantage: The measurement of the fluorescence relative to a dark background, as compared to the bright reference beam in an absorbance measurement. For Example, Concentration of sample: 10–10 M; molar extinction coefficient: 105 M–1 cm–1. Absorbance: 10–5 per cm, (percentage transmission of 99.9977%; Absorption: 0.0023%) In contrast, Fluorescence detection at 10–10 M is readily accomplished with .

Fluorescence Based Sensors ǁ Session 1 Conclusions

• An analyte responsive fluorescent molecule/supramolecule which changes its fluorescence is called Fluorescent sensors. • Types of Sensors: Chemical sensors (chemosensors) & Biosensors. • Fluorescent sensors are used to in Analytical, Clinical, Biochemistry, Medicine & Environment to detect Chemical and biochemical analytes: Cations, Anions, Neutral molecules, Gases. • Need of new fluorescent sensors with improved selectivity, higher sensitivity, better Response time whose success lies in remote sensing using optical fibers, better sensitivity, higher resolution of instruments. • Three Classes of fluorescent sensor: Class 1: quenching upon collision with an analyte Class 2: fluorophores that can reversibly bind an analyte. Class 3: fluorophores linked to a receptor via a spacer In case of class 2 and class 3 the fluorescence may be CEQ or CEF type • Fluorescence is more sensitive than Absorbance.

Fluorescence Based Sensors ǁ Session 1 Assignment

1. Why fluorescence is used for sensing application? 2. Define fluorescent sensor. 3. Why do we need new sensors? 4. How many types of sensors are there? 5. Describe different class of sensors? 6. Define fluorogenicity. 7. Define fluoroionophores. 8. Rationalize the following

References: B. Valeur; Molecular Fluorescence J. R. Lakowicz, Principles of fluorescence Non-fluorescent Fluorescent

Fluorescence Based Sensors ǁ Session 1