Squaraine dyes: fluorescence turn-on for protein detection and quantification

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27 July 2021

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Squaraine dyes: fluorescence turn-on for protein detection and quantification

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SQUARAINE DYES

Sonja Visentin,

(a)

_{Cosmin Butnarasu,}

(a)

_{Nadia Barbero,}

(b)

_{Claudia Barolo,}

(b)

(a)

_{Department of Molecular Biotechnology and Health Sciences, University of Torino, via Gioacchino Quarello 15A, 10135 Torino}

(b)

_{Department of Chemistry, NIS Interdepartmental and INSTM Reference Centre, University of Torino, via Pietro Giuria 7, 10125 Torino}

In the last decades great attention was dedicated to the development of new dyes as markers in biological applications. Polymethine dyes (i.e. squaraines and cyanines) deserve to be counted among innovative potential photosensitizers (PSs) because they offer numerous advantages such as their easiness in designing NIR molecules simply by elongating the central bridge and/or tuning the lateral functional groups [1], providing NIR compounds with absorption that perfectly match the phototherapeutic window (650-850 nm). However, in physiological conditions, their chemical instability and self-aggregation properties limit their widely applications [2]. Several squaraines exhibit a fluorescence “turn-on” when bound to proteins which translate into an increase in fluorescence quantum yield and lifetimes due to the changes in the environment (Figure 1) [3].

I

NTRODUCTION

UK-IT JOINT MEETING ON PHOTOCHEMISTRY

24-26 June 2019, Lipari

FLUORESCENCE “TURN-ON” FOR PROTEIN

DETECTION AND QUANTIFICATION

SQ in organic

media SQ in aqueousmedia SQ + BSA in aqueous

media

E

XPERIMENTAL

P

ART

There is an urgent need and challenge for the development of novel fluorescent probes to serve as a platform for a variety of biological applications.

A

IMS

• Spectroscopic characterization: UV/Vis absorption and steady-state fluorescence spectra of a constant concentration of the squaraine dye were recorded upon increasing the concentration of the protein.

• Time-domain lifetime measurements of the Protein-Squaraine adducts.

• Kinetic measurements of the formation of Protein-Squaraine complexes.

• Quantum yield measurements.

• Transmission electron microscopy (TEM) characterization of the adducts. R = C2H5 VG1-C2 R = C8H17 VG1-C8 R = C2H5 VG10-C2 R = C8H17 VG10-C8 INDOLENINE series BENZOINDOLENINE series

Squaraine dyes have a structure-relationship influence on the kinetic interaction with PGM (and BSA [3]).

Squaraine showed a significant increase of fluorescence

intensity when proteins (especially BSA and PGM) were added

probably due to the interactions established with the

hydrophobic domains of the protein.

Protein-dyes adducts could be employed as potential probes or

photosensitizers for different applications (bioimaging,

photodynamic therapy, etc).

R

ESULTS AND

D

ISCUSSION

UV/Vis absorption spectroscopy

Upon addition of protein the UV/Vis

absorption spectra of squaraine

changes due to the interactions

established between the two species. Addition of increasing concentrations of PGM to a constant concentration of squaraine results in a disaggregation

effect with a greater amount of

solubilized squaraine (Figure 3).

Steady-state fluorescence spectroscopy

Squaraine molecules are almost non

non emissive when they are

suspended in water, however a

gradual addition of protein (such as BSA or PGM) gave an enhancement in fluorescence intensity (turn-on), sometimes with a bathochromic shift of about 11-12 nm for all the studied squaraines (Figure 4).

T

AKE HOME MESSAGE

Kinetics of interaction

The dye backbone structure as well as the length of the alky chain play a crucial role in the kinetics of the interaction with PGM. The bulkier the dye’s molecular structure the slower the interaction (Figure 6).

VG1-C2 formed an immediately stable complex, while VG10-C2 and VG1-C8

showed similar behaviour with a

reaction time within 20 minutes. VG10-C8 requested the longer time to form a steady complex.

Lifetime measurements

The complex formation between

VG1-C2 and VG1-C8 with PGM was further confirmed by time-correlated single photon counting (TCSPC).

For instance, time-resolved

fluorescence analysis indicated that

VG1-C8 alone exhibits a

mono-exponential decay, whereas

bi-exponential decay with significantly increased lifetimes were observed in the presence of PGM (Figure 7).

Fluorescence “turn-on”

The addition of the proteins to a water solution of squaraine generally yielded

a significant increase of the

fluorescence intensity (Figure 5).

Particularly, the greater enhancement of fluorescence intensity was observed especially in presence of bovine serum albumin (BSA) and porcine gastric mucin (PGM).

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Figure 1. Squaraines are freely soluble in organic solvents while in water form self aggregates. Even though, when PGM is added to an aqueous solution of squaraine a fluorescence turn-on is observed.

Figure 3. UV/Vis absorption spectra of the four squaraines upon increasing concentrations of PGM.

Figure 4. Steady-state fluorescence spectra of VG1-C8 upon increasing concentrations of PGM.

Figure 7. Time-domain lifetime of VG1-C8 alone and in presence of increasing concentrations of PGM.

Figure 6. Kinetic behavior of the squaraine-PGM complexes formation. Figure 5. Maximum of the fluorescence intensity of the four squaraines alone

and in presence of different proteins.

REFERENCES

[1] G. M. Paternò et al., Near-infrared emitting single squaraine dye aggregates with large Stokes shift, J. Mater. Chem. C, 2017, 5, 7732-7738

[2] I. A. Karpenko et al., Squaraine as a bright, stable and environment- sensitive far-red label for receptor-specific cellular imaging, Chem. Commun., 2015, 51, 2960-2963.

[3] N. Barbero et al., Squaraine Dyes: Interaction with Bovine Serum Albumin to Investigate Supramolecular Adducts with Aggregation-Induced Emission (AIE) Properties, Chem. Asian J., 2019, 14, 896-903.

Water _{Pepsin ctDNA}

Protease Trypsin TransferrinFibrinogen PGM BSA 0 10 20 30 50 75 100 125

Fluorescence intesity (a.u.)

VG1-C2 VG10-C2 VG1-C8 VG10-C8 Up to

65x

increase 7.5 10.0 12.5 15.0 17.5 20.0 0 2000 4000 6000 8000 10000 12000 Time (ns) Counts VG1-C8 VG1-C8 + PGM 500 550 600 650 700 750 0.00 0.15 0.30 0.45 Wavelength (nm) Abs VG1-C2 2 µM VG1-C2 2 µM + PGM 20 µg/mL VG1-C2 2 µM + PGM 40 µg/mL VG1-C2 2 µM + PGM 80 µg/mL VG1-C2 2 µM + PGM 120 µg/mL VG1-C2 2 µM + PGM 200 µg/mL VG1-C2 2 µM + PGM 300 µg/mL 500 550 600 650 700 750 0.0 0.1 0.2 0.3 Wavelength (nm) Abs VG10-C2 2 µM VG10-C2 2 µM + PGM 20 µg/mL VG10-C2 2 µM + PGM 40 µg/mL VG10-C2 2 µM + PGM 80 µg/mL VG10-C2 2 µM + PGM 120 µg/mL VG10-C2 2 µM + PGM 200 µg/mL VG10-C2 2 µM + PGM 300 µg/mL 500 550 600 650 700 750 0.0 0.1 0.2 Wavelength (nm) Abs VG10-C8 2 µM VG10-C8 2 µM + PGM 20 µg/mL VG10-C8 2 µM + PGM 40 µg/mL VG10-C8 2 µM + PGM 80 µg/mL VG10-C8 2 µM + PGM 120 µg/mL VG10-C8 2 µM + PGM 20 µg/mL VG10-C8 2 µM + PGM 300 µg/mL 500 550 600 650 700 750 0.0 0.1 0.2 Wavelength (nm) Abs VG1-C8 2 µM VG1-C8 2 µM + PGM 20 µg/mL VG1-C8 2 µM + PGM 40 µg/mL VG1-C8 2 µM + PGM 80 µg/mL VG1-C2 2 µM + PGM 120 µg/mL VG1-C8 2 µM + PGM 200 µg/mL VG1-C8 2 µM + PGM 300 µg/mL VG1-C2 VG10-C2 VG10-C8 VG1-C8

Herein we investigate from a spectroscopic point of view the interaction between several proteins and a series of squaraine dyes with different substitutions (Figure 2).

Squaraine dyes that exhibit a fluorescence

turn-on when bound to proteins could be potentially

employed as fluorescent markers for biological applications [3]. 630 665 700 735 0 25 50 75 100 Wavelength (nm)

VG1-C2 1 µM VG1-C2 1 µM + PGM 1 µg/mL VG1-C2 1 µM + PGM 3 µg/mL VG1-C2 1 µM + PGM 6 µg/mL VG1-C2 1 µM + PGM 9 µg/mL VG1-C2 1 µM + PGM 12 µg/mL VG1-C2 1 µM + PGM 20 µg/mL VG1-C2 1 µM + PGM 40 µg/mL VG1-C2 1 µM + PGM 60 µg/mL VG1-C2 1 µM + PGM 100 µg/mL VG1-C2 1 µM + PGM 150 µg/mL 630 665 700 735 770 0 25 50 75 100 Wavelength (nm)

VG1-C8 0.1 µM VG1-C8 0.1 µM + PGM 10 µg/mL VG1-C8 0.1 µM + PGM 30 µg/mL VG1-C8 0.1 µM + PGM 60 µg/mL VG1-C8 0.1 µM + PGM 100 µg/mL VG1-C8 0.1 µM + PGM 150 µg/mL VG1-C8 0.1 µM + PGM 200 µg/mL VG1-C8 0.1 µM + PGM 300 µg/mL VG1-C8 0.1 µM + PGM 500 µg/mL VG1-C8 0.1 µM + PGM 900 µg/mL 665 700 735 770 0 25 50 75 100 Wavelength (nm)

VG10-C2 1 µM VG10-C2 1 µM + PGM 3 µg/mL VG10-C2 1 µM + PGM 6 µg/mL VG10-C2 1 µM + PGM 10 µg/mL VG10-C2 1 µM + PGM 20 µg/mL VG10-C2 1 µM + PGM 60 µg/mL VG10-C2 1 µM + PGM 100 µg/mL VG10-C2 1 µM + PGM 150 µg/mL VG10-C2 1 µM + PGM 200 µg/mL VG10-C2 1 µM + PGM 300 µg/mL VG10-C2 1 µM + PGM 500 µg/mL 665 700 735 770 0 25 50 75 100 Wavelength (nm)

VG10-C8 0.1 µM VG10-C8 0.1 µM + PGM 10 µg/mL VG10-C8 0.1 µM + PGM 20 µg/mL VG10-C8 0.1 µM + PGM 4 µg/mL VG10-C8 0.1 µM + PGM 60 µg/mL VG10-C8 0.1 µM + PGM 100 µg/mL VG10-C8 0.1 µM + PGM 150 µg/mL VG10-C8 0.1 µM + PGM 300 µg/mL VG10-C8 0.1 µM + PGM 600 µg/mL VG10-C8 0.1 µM + PGM 900 µg/mL VG1-C2 VG10-C2 VG10-C8 VG1-C8 𝑭 𝑭_𝟎 = 𝟑. 𝟓 𝑭 𝑭_𝟎 = 𝟒. 𝟐 𝑭 𝑭_𝟎 = 𝟔𝟏 𝑭 𝑭_𝟎 = 𝟒𝟎 0 ₂₀ ₄₀ 150 300 450 VG10-C8 VG10-C2 VG1-C8 VG1-C2 Time (min) PGM Squaraine