CM4027 – Advanced Analytical Chemistry Part 2 Fluorescence Sensing Mechanisms I

Section Overview

CM4027 - Advanced Analytical Chemistry Part 2 Fluorescent Sensors for Bioanalysis - 6 Core Lectures This section broadly covers some key aspects of fluorescence-based sensing:

• CB-CM4027 - Fluorophores and Sensing Responses
• 1. Fluorescence, Fluorophores, and Sensing Responses
• 2. Fluorescence Sensing Mechanisms I
• 3. Fluorescence Sensing Mechanisms II
• 4. Fluorescence Sensing Mechanisms III
• 5. Introduction to Fluorescence Bioimaging
• 6. Strategies to Overcome Core Challenges in Bioimaging

Assessment: Part of Final Exam Lecture slides will be made available on Canvas over the next few weeks.

Brief Theoretical Overview

Instrumentation Options

Chemical / Biological Spectroscopic Probes

(Bio)analytical Applications

Research and Industry Contexts

Fluorescence Sensing Principles

Sensing requires a fluorescence response to analyte.

• A fluorescence sensor can be designed where fluorescence output selectively responds to the presence of the target analyte.
• For quantitative sensing, the response should be proportional to analyte concentration.
• The fluorescence response can originate due to many factors, such as
• Chemical reaction (Breaking/making new bonds)
• Physical changes (solvent polarity, viscosity, temperature, etc)
• Electron/energy transfer (within / with the fluorophore)
• Combinations of the above.

Fluorescent Dye (+ co-reagents) + Analyte = Distinct Fluorescence Response

N N N(CH3)2 8 Rotation controls emission viscosity increase F A R F A R × off on DNA binding controls emission FRET process switched off after reaction no FRET protease cleavage site A QD A Quantum Dot fluorescent protein organic dye quencher nanoparticle FRET QD CB-CM4027 - Fluorescence Sensing Mechanisms I

Fluorescence Sensing Mechanisms

Across these lectures we study six mechanisms of fluorescence sensing that utilise well understood photophysical phenomena;

• Collisional quenching (Stern-Volmer quenching)
• Förster resonance energy transfer (FRET)
• Aggregation induced emission (AIE)
• Two-state fluorescent sensors
• Photoinduced electron transfer (PET)
• Supramolecular recognition fluorescent sensors

Note that in some cases, more than one mechanism may be operative. CB-CM4027 - Fluorescence Sensing Mechanisms |

FRET no FRET protease cleavage site QD A QD A Quantum Dot fluorescent protein organic dye quencher nanoparticle

Fluorescent Dye (+ co-reagents) + Analyte = Distinct Fluorescence Response

Fo = 1 + K[Q] =1 + kato[e] F

fluorescent probe in hydrophobic core (BODIPY) oxygen probe (Ru (II) complex) BODIPY Ru(II) complex Non-Fluorescent Aggregates

Dynamic and Static Quenching

Spectroscopy Online. Special Issues-08-02-2019, Volume 34, Issue 8. Pages: 12-14

• Quenching is often described as being static or dynamic.
• Dynamic quenching involves a quenching event by some mechanism that occurs once the luminophore is in the excited state, which leads to a decrease in the emission intensity (quenching).
• Dynamic quenching is characterised by a decrease in luminescence lifetime.
• Static quenching is a process that inhibits the initial formation of the emissive excited state. There is no apparent change in emission lifetime, just a decrease in the emission intensity.
• There are several mechanisms that make static or dynamic quenching possible, and a combination of mechanisms can be operative.

CB-CM4027 - Fluorophores and Sensing Responses

Collisional Quenching

Static Quenching

F*+ Q =(F.Q)* f(t) Y=To" -Q ką[Q] -Q hv F + Q Mediation of Quenching Q F + Q . F Q F F + Q F Collisions Direct Association or Indirect Association Concentration V ISC Fo/F -Slope=K Slope-kq To"Kp 1 1 To/2 O O T [Q] [Q] Mechanism of Quenching Dexter ET FRET Ground-State Non-Fluorescent Complex F F Inner-Filter Effects Exciplex PET + Sphere-of-Action Dynamic Quenching Static Quenching Trivial Quenching 1 No emission F Q K F.Q Fo /F and To/T Higher temperature Higher temperature F

Collisional Quenching and Stern-Volmer

Microchim Acta 187, 71 (2020)

• Collisional quenching is a dynamic quenching process.
• A solution based, excited state fluorophore is de-activated by contact with a diffusing quencher and is returned to the ground state. The respective molecules, fluorophore and quencher, are not chemically altered in the process.
• Examples of collisional quenchers include molecular oxygen, halogen ions, halogen/amine-containing molecules, electron-deficient molecules (e.g. nitroaromatics), heavy atom-containing molecules, heavy metal ions.
• For collisional quenching, the decrease in intensity is described by the Stern-Volmer equation.
• The equation can be used in linear form as a calibration curve for analyte determinations. However, the quenching mechanism should be well understood using other photophysical techniques to verify. And, given the acute sensitivity of fluorescence to fluorophore environment, careful considerations of interferences is needed.

CB-CM4027 - Fluorescence Sensing Mechanisms I

Tryptophan Measurement with Quantum Dots

Measurement of tryptophan (quencher) using fluorescent Quantum Dots

Stern-Volmer equation Fo F = 1 + K|Q] = 1 + kgto[@] Fo - Initial intensity F - Measured intensity K - Stern-Volmer constant [Q] - Quencher concentration kg - Quenching rate constant To- Initial luminescence lifetime

a 1000 8 Stern-Volmer calibration curve 800 Intensity (a.u.) 600 CO2H 400 Fo/F 1.1 NH2 200 ·NÍH 0.9 0 0.2 0.4 [L-Trp] (UM) 0 0 0 4 8 12 16 20 470 500 530 560 590 620 650 Wavelength (nm) Calibration Plot y =0.4301x+1.0296 R2 = 0.9991 6 F0/F 4 2 [L-Trp] (UM)

Factors Affecting Collisional Quenching

• Collisional (dynamic) quenching requires the transport of quencher to the fluorophore where a quenching event can occur by some mechanism.
• In solution, the interaction between fluorophore and quencher is diffusion controlled and so will depend on the diffusion rate.
• In many systems, the diffusion rate can be described by the Stokes-Einstein equation and indicates some of the key factors affecting diffusion. It follows that the local viscosity, temperature and molecular size impact bimolecular quenching.
• Knowing these key parameters in a Stern-Volmer analysis can permit the calculation of the quenching rate constant, kg, which is related to the quenching efficiency.
• Deviations from linearity in Stern-Volmer analyses can be indicative of a combination of static or 'static-like' processes occurring in addition to dynamic quenching (e.g. 'sphere of action').

Stern-Volmer equation Stokes-Einstein equation Fo = 1 + K|Q]= 1 + kgto[@] F Fo - Initial intensity F - Measured intensity K - Stern-Volmer constant [Q] - Quencher concentration kg - Quenching rate constant To- Initial luminescence lifetime D = kT/6πηR D - Diffusion coefficient k - Boltzmann constant T - Temperature n - Solvent viscosity R - Molecular radius

Stern-Volmer Plots: Linearity Deviations

Stern-Volmer Plots: Deviations from linearity due to 'static-like' and dynamic quenching F (F· Q*) I Non fluorescent -hv Ks. F + Q F . Q FO/F 1 Slope = KDKs To 1 1 Kapp - KD +Ks 1 0 [Q] [0]/ (1-05) [Q] A combined 'Kapp' analysis CB-CM4027 - Fluorescence Sensing Mechanisms I kg [Q]

Dynamic Oxygen Sensing using Stern-Volmer

RSC Chem. Biol., 2021, 2, 1520-1533. Analyst, 2017, 142, 3400-3406.

• Oxygen concentration measurement is suited to dynamic (collisional) quenching responses.

Ratiometric fluorescence responsive nanoparticles for oxygen sensing in live cells. Fo = 1 + K|Q]= 1 + kgto[@] F 1.3 1.7 y =- 0.001x+1.6225 R2 = 0.9525 1.6 1.25 1.5 1.2 1.4 5.1.15 y = 0.0009x + 19 1.3 R2 = 0.9743 1.1 y =0.0008x + 1 R2 = 0.9867 1.2 1.05 1.1 1 1 0 100 200 300 Concentration O2 (umol/L) Top: Stern-Volmer calibration curve fitting to lifetime analysis and ratiometric readout. Left: BODIPY emission remains unchanged but Ru(II) emission is quenched with increasing [O2]. CB-CM4027 - Fluorescence Sensing Mechanisms | Ru(II) sensor at surface and insensitive BODIPY reference at core. Dual-emission and ratiometric sensing. ---- 8 umol 400 600 A -23 umol -43 umol 350 500 84 umol -120 umol 300 -133 umol 400 250 221 umol -266 umol -291 umol 200 300 150 200 100 100 50 0 - 0 450 550 650 750 Wavelength (nm) Excitation Intensity (a.u.) fluorescent probe in hydrophobic core (BODIPY) oxygen probe (Ru (II) complex) BODIPY Ru(II) complex Emission Intensity (a.u.) -70 umol -177 umol 1601 1547

Dynamic Oxygen Sensing with Ruthenium(II) Complexes

J. Am. Chem. Soc. 2014, 136, 43, 15300-15309. Ruthenium(II) complexes for dynamic oxygen concentration measurement within the mitochondria using fluorescence lifetime.

2+ [Ru(bpy)3]2+ · Long-lived luminescence N · Triplet excited states 1000 500 0 0 200 400 600 800 1000 Time (ns) 250 Frequency (kCounts) 200 150 100 50 0 0 200 400 600 800 1000 B Time (ns) As [O2] increases within the mitochondria, the probe lifetime decreases due to increased quenching. Frequency (kCounts) 300 200 100 1.1 K 0.9 0 50 100 150 200 250 Concentration O2 / uMol Stern-Volmer calibration curves for [O2] using Ru(II) complex. 0 hr + stress agent (Antimycin A) Io To = = 1 + k __ [2] I 2.3 B T 2.1 y =0.0044x +1 R2 = 0.9725 (ii) 37 °℃ 1.9 T T ~ 1.7 T T 1.5 (i) 1.3 K y = 0.0031x+ 1 R2= 0.952 0 C 0 200 400 600 800 1000 2 hr Time (ns) A mitochondria targeted oxygen probe that responds dynamically to changing oxygen concentration within live Hela cells. CB-CM4027 - Fluorescence Sensing Mechanisms | N Ru · Large Stokes-shift · Visible A Abs/Em N N A Frequency (kCounts) 1500 400 TO/T 18 ℃

Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET)

• FRET occurs when the emission spectrum of a donor fluorophore overlaps with an acceptor absorption spectrum. Energy is transferred from the excited state donor to the ground state acceptor due to long-range dipole-dipole interactions.
• It is important to realise that the acceptor does not have to be fluorescent. Also, the FRET process itself does not involve the emission of a photon.
• Although the term fluorescence resonance energy transfer is in common use, it may be used erroneously, and care must be taken not to assume the acceptor is fluorescent in a FRET process.
• As the equation suggests, the rate (and efficiency) of FRET depends inversely on the sixth power of the distance between the donor and acceptor.
• Usefully, FRET distances are on the order of about 30 - 60 Å (up to about 10 nm) which correlates well with the scale of biomolecules enabling the use of donor- acceptor pairs for bioanalysis.

D A KT = - (R) FRET is sensitive to small changes in distance FLUORESCENCE INT. or ABSORPTION Donor Emission Acceptor Absorption 2 / WAVELENGTH Donor, D and Acceptor, A Ro - Forster distance (the distance at which the energy transfer efficiency is 50%) Tp - Donor fluorescence lifetime r - Distance between D and A KT - Rate of energy transfer CB-CM4027 - Fluorescence Sensing Mechanisms I