Fluorescence Sensing Mechanisms III in Advanced Analytical Chemistry

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.

Fluorescence Sensing

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 FRET no FRET protease go cleavage site A QD A QD Quantum Dot fluorescent protein organic dye quencher nanoparticle CB-CM4027 - Fluorescence Sensing Mechanisms III

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 III 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[@] F fluorescent probe in hydrophobic core (BODIPY) oxygen probe (Ru (II) complex) BODIPY Ru(II) complex Non-Fluorescent Aggregates

Photoinduced Electron Transfer (PET)

Photoinduced electron transfer (PET) sensors are characterized by a fluorophore linked to a receptor that controls its fluorescence based on capability of quenching by electron transfer from receptor to fluorophore.

• In a PET system, the fluorophore is tethered via linker to a receptor. The receptor possesses lone pairs of electrons, usually localized on N atoms.

. The fluorescence arising from the excited fluorophore can be quenched by electron transfer from the receptor. . This is usually because HOMO of the receptor (non-bonding orbital) is higher than the vacated orbital following electron excitation at the fluorophore. . If the receptor lone pairs are not available for PET due to bonding an analyte, then a fluorescence response can be observed. This is exploited for sensing. hv PET - on or off ‘linker' or Fluorophore Receptor 'spacer' ⇋ hvʼ Analyte binding at receptor - OFF/ON response E LUMO 1 - PET HOMO - HOMO- 1 HOMO Excited Fluorophore Free Receptor OFF +F = 0.003 ( in methanol) E LUMO hvF HOMO N ON +F = 0.14 (in methanol) A +F = 0. 38 (in 0.01M HCI ) CB-CM4027 - Fluorescence Sensing Mechanisms III

PET Sensing of Mn(II) in Live Cells

Chem. Commun., 2015, 51, 2605-2608.

• A 'light-switch' fluorescence turn-on sensor for Mn(II) based on a PET mechanism.
• In the Mn(II) free state; the nitrogenous macrocycle receptor efficiently quenches BODIPY fluorescence via PET.
• In the Mn(II) bound state; PET is inhibited due to Mn(II) hosting at the macrocycle.

CB-CM4027 - Fluorescence Sensing Mechanisms III OFF ON 150- Relative Emission Intensity 100- Mn2+ 50- 0- T T T T 500 520 540 560 580 600 2(nm) Turn-on sensing of endogenous Mn2+ using a BODIPY- macrocycle conjugate via PET mechanism. F F F B + N Z PET 2+ PET Mn N N O O N N N 2+ _ N O . -N N- -N N R R M1: R= Me M2: R= CH2COOMe R' R Ctrl + Mn a b M1 B N O Mn

Protonation Interference in PET Sensing

Acc. Chem. Res. 2019, 52, 10, 2818-2831 and refs within . A problem in PET sensing is protonation of the basic groups that usually inhibits their participation in PET (i.e. the HOMO of the receptor must be higher in energy than the HOMO of the fluorophore for PET). This results in a non-specific fluorescence response at the fluorophore. . Of course, this interference is also what makes PET sensors attractive as pH probes. . In the examples here, Zn(II) sensing is achieved over a wide pH range by altering the molecular structure to lower the pKa of the tertiary amine at the DPA receptor to about 2.1 (i.e. case A to case B; switching from napthalimide to a BODIPY fluorophore). This allows Zn(II) sensing across the range pH 3 - 10. · DPA is di(2-picoyl)amine. A CH3 CH3 C 8 - Fluorescence Intensity 7 - 6 - 5- 4- N N Ň 3- 2 4 6 8 10 12 PH D B PET EN N: N Zn2+ N F/Fmax E 0.4 0.2 0.4- 0.0 2 3 4 5 6 7 8 9 10 11 PH 0.2- 0.0 + 1 2 3 4 5 6 7 8 pH CB-CM4027 - Fluorescence Sensing Mechanisms III 1.0 0.8- 0.8 × 0.6 0.6 F/F max Zn2 N N. N .- N= B + F F F F BDA N .O .N O PET Zn2 HN HŇ N N `Zn2+-N. NIDPA

Overcoming Protonation Interference in PET Sensing

Acc. Chem. Res. 2019, 52, 10, 2818-2831 and refs within

• An alternative approach to overcoming protonation interference is to lower the energy of the HOMO of the fluorophore so that PET occurs even if the receptor is protonated (e.g. using electron withdrawing groups).

CB-CM4027 - Fluorescence Sensing Mechanisms III 0 O CI N4 N5 B LUMO 1 HOMO HOMO -5.85 eV -N DPA R' ₡-Z O e- N HOMO e- HOMO HOMO 1 -6.23 eV -N -- H+ DPA-H+ E R e- HOMO N4 R=H -6.48 eV HOMO N5 R=CI e- e- HOMO 11 N HOMO -7.82 eV -N-Zn2+ DPA-Zn2+ 11 N non-classical PET case - protonation does not inhibit PET R' = DPA, DPA-H+, and DPA-Zn2+ DPA N N. 0 N OCH, NMO e- -6.10 eV R=OCH3 NMO N classic PET case - protonation inhibits PET -6.55 eV LUMO O Fluorophore R

Exploiting Protonation for pH-Controlled Theranostics using PET

Acc. Chem. Res. 2019, 52, 10, 2818-2831 and refs within

• Protonation of amine receptors is a useful mechanism for pH sensing. The fluorophore and receptor can be tuned to the desired pKa to suit the pH range of application.

. In healthy tissue, bloodstream pH is about 7.4. However, in cancerous solid tumours, the microenvironment pH is more acidic, about 6.2 - 6.8. . Here, pH sensitivity was exploited for theranostic effect by designing a PET probe with pKa close to that of tumour environments. · Cancer labelling is achieved via pH response based on the PET mechanism. . However, in this case, the fluorophore PET acceptor is also very good singlet oxygen photo-sensitizer and so therapy can be triggered with selectivity for cancer cells once PET is turned off. 1 A .N N -2+ N 102 PET NH HN NH O HN H+ 302 O O CI3 CI3-H+ B Control Light CI3 CI3+Light 20um 20um 20um 20um (A) Chemical structure and sensing mechanism of chrysophanol-amine to H+. (B) Fluorescence images of singlet oxygen generation in MCF-7 cancer cells (stained with DCFH-DA, a 1O2 indicator). CB-CM4027 - Fluorescence Sensing Mechanisms III +

Supramolecular Recognition Fluorescent Sensors

A fluorescent supramolecular sensor exploits non-covalent recognition or 'host-guest' binding to elicit a fluorescence response. . There are many examples of supramolecular recognition, including; bioderived systems (e.g. protein receptors), macrocyclic receptors, metal-organic frameworks, etc. . The key challenges of supramolecular sensing are to incorporate fluorophores without disrupting the recognition, and to design a total system that is responsive upon recognition (turn on, off, ratiometric, lifetime, etc). (a) NH2 * H2N=C ZI NH2 NH2 DAPI O COO- NH 43 N N N N N N CB7-CF (b) FRET + Î + DNA (b) fluorophore C receptor Reversible CB-CM4027 - Fluorescence Sensing Mechanisms III Binding-Based Sensing (BBS) (a) fluorophore receptor spacer Reversible + C HO

Hypoxia Sensing using Dye-Captured Calixarenes

Angew. Chem. Int. Ed. 2019, 58, 2377. · Calixarenes are attractive host molecules in supramolecular biosensing due to their low cytotoxicity, water solubility, and wide availability with varying cavity sizes. · Here, an azo-calix[4]arene as host and rhodamine dye as guest system is devised. In normal media, the host-guest system is stable, and the rhodamine fluorescence is quenched. . However, in hypoxic conditions, the azo bridge is selecltivty reduced to amines and cleaves to release the rhodamine guest, switching on its fluorescence. . The authors suggested three intrinsic merits associated with this supramolecular sensing system: 1) no elaborate synthesis; 2) highly reliable sensing system selective for hypoxia; 3) easy adaptability in that this specific design strategy could be generalized into a universal sensing platform. 'Indicator displacement assay' Normoxia Hypoxia CO2H CO2H CO2H HO2C + H2N NH2 N. N N=N 'N N 1-1 CO2CH3 OH HO HO 63 Rho123 Hypoxia + 63.Rho123 "OFF" 63 "ON" CB-CM4027 - Fluorescence Sensing Mechanisms III

Anion Recognition by Supramolecular Capture

Org. Lett. 2019, 21, 21, 8746-8750 . This study is a good example of modifying sensor designs to tune selectivity to various analytes. · Anthracene as fluorophores are built into cages as receptors for anion detection. · Structural and intermolecular bonding interactions control selectivity. CB-CM4027 - Fluorescence Sensing Mechanisms III NH HIN HN N N H HN H HN N NH NH HN HN 2 3 Selective Fluorescence Response to F-or NO3" Sensor 2 1,5 1 1-10/10 0,5 0 F- CI- Brī H2PO4" SO42- C2042- NO3 CIO4" BF4" PF6 SCNT SO22- c) 3 2,5 Sensor 3 2 1,5 I-lo/lo 1 0,5 0 L 6 Brī 1 -0,5 H2PO4 SO42- C2042- NO3 CIO4 BF4" PF6 SCN- SO32- -1 - -0,5 NH N N N