Part 2: L2, Photophysics and Responses / Detection

Question 1

6 common organic based fluorophores

Answer 1

• DNS-Cl
• 5-IAF
• FITC
• TRITC
• Acrylodan
• NBD-Cl
• Planar, rigid, aromatic heterocycles
• Rigid is important to limit vibrational quenching
• n to pi* or pi to pi* charge transfer

Question 2

Basic Pi to Pi* transitions

Answer 2

• Simplest process
• 1 electron moved up to pi*
• Double bond to diradical
• High energy excitation for simple alkene

Question 3

Conjugate pi to pi* transition

Answer 3

• Delocalised around aromatic ring -> lower energy
• Better tissue penetration due to longer wavelength (redshift)

Question 4

N to pi* transitions

Answer 4

• N to pi* from high energy lone pair (non-bonded)
• Large redshift

Question 5

Charge transfer transitions

Answer 5

• Electrons physically moved in space
• Analogous to resonance
• Single electron to LUMO
• EWGs stabilise resonance form -> lower E -> redshift

Question 6

pH responsive Fluorophores

Answer 6

• Reports by intensity only (hard to use if concentration is not known)
• Difficult to differentiate between an organelle that doesn’t uptake the molecule vs when the molecule is in a non-fluorescent state

Question 7

Potential responsive fluorophore

Answer 7

• Application of charge transfer transition
• Useful in investigating hyperpolarisation of membranes
• Polarisation in the membrane interacts with the charge transfer -> hyperpolarisation makes excitation easier -> longer wavelengths, lower energy (redshift)
• Measuring intensity change

Question 8

Peroxide sensing

Answer 8

• Alkene hydroborated, treated with peroxide to form alcohol (1st year chemistry)
• Peroxide can be generated under stress conditions
• Peroxide-generating organelles turning on fluorescence
• Same issue with both ‘turned off’ agent and lack of agent producing dark signal

Question 9

Enzyme sensing

Answer 9

• e.g. nitroreductase (NTRII only active in hypoxic cells)
• Originally used in gene modification and splicing to show successful transfection
• Can also detect hypoxia
• Wavelength distribution of emission shows cell pH
• Bioreduction produces fluorescent pH indicator
• N lone pair used in excitation process

Question 10

Ratiometric sensing

Answer 10

• Looking at two different wavelengths and comparing the intensity of the two
• e.g. modified peroxide sensor emitting at 450nm vs 500nm depending on peroxide exposure
• Colour observed shows concentration (based on ratio)
• Gets rid off ‘turn off’ issue in other types of agents