A turn-on fluorescent probe for mucin glycoproteins detection

(1)

27 July 2021

AperTO - Archivio Istituzionale Open Access dell'Università di Torino

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A turn-on fluorescent probe for mucin glycoproteins detection

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(2)

A

TURN

-

ON FLUORESCENT PROBE FOR MUCIN GLYCOPROTEINS DETECTION

Cosmin Butnarasu,

(a)

_{Nadia Barbero,}

(b)

_{Claudia Barolo,}

(b)

_{Daniela Pacheco,}

(c)

_{Paola Petrini,}

(c)

_{and Sonja Visentin,}

(a)

(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}

(c) _{Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, Milan, 20133, Italy}

cosminstefan.butnarasu@unito.it

Mucins are a family of long polymeric glycoconjugates having high molecular weight, produced by the gastrointestinal, respiratory, reproductive, pancreatic, hepatic and renal epithelium (Figure 1). Alterations or overexpression of mucins are associated with diseases like chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis and several types of cancer [1]. Particularly, in the last years, great attention was addressed to expression of mucins in various cancers such as pancreatic adenocarcinomas, colon and rectal cancer, breast cancer, ovarian cancer and gastric carcinoma.

The early diagnosis is a key factor for outcome, treatments, and healthcare. Thus, the identification and detection of specific and sensitive biomarkers has become extremely important in the last decades [2].

I

NTRODUCTION

15

TH

_I

_{NTERNATIONAL WORKSHOP ON CARCINOMA}

-ASSOCIATED MUCINS

29th _{July – 2}nd _{August 2019 – Robinson College, Cambridge, UK.}

E

XPERIMENTAL

P

ART

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 PGM.

• Time-domain lifetime measurements of the

PGM-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

R

ESULTS AND

D

ISCUSSION

UV/Vis absorption spectroscopy 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 4).

Steady-state fluorescence spectroscopy

Squaraine molecules are

almost 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), (Figure 5).

Kinetics of interaction

The bulkier the dye’s molecular structure the slower the

interaction (Figure 6).

Lifetime measurements

An increase in fluorescence

lifetime was observed in 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 8).

Figure 4. UV/Vis absorption spectra of the four squaraines upon increasing concentrations of PGM.

Figure 5. 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 PGM.

Figure 6. Kinetic behavior of the squaraine-PGM complexes formation.

Figure 8. Maximum of the fluorescence intensity of the four squaraines alone and in presence of different proteins.

REFERENCES

[1] S. K. Behera, et al., Exploring the role and diversity of mucins in health and disease with special insight into non-communicable diseases. Glycoconj. J., 2015, 32, 575–613. [2] K. Chen, et al., Mucins as biomarkers in cancer in Mucins and Cancer, Future Medicina, 2013, 34–49

[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 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 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 𝑭𝟎= 𝟒. 𝟐 𝑭 𝑭_𝟎= 𝟔𝟏 _𝑭𝑭_𝟎= 𝟒𝟎

Up until now, fluorometric assays have received remarkable attention due to their convenience,

unparalleled sensitivity, simplicity, rapid

implementation, noninvasive monitoring capability and usability in biological samples

Herein we investigate from a spectroscopic point of view the interaction between porcine gastric mucin (PGM) and a series of squaraine dyes with different substitutions (Figure 3).

Squaraine dyes exhibit a

fluorescence turn-on when bound

to proteins (Figure 2) and

potentially could be employed as fluorescent markers for biological applications [3]. Eyes Mouth Lungs Stomach Pancreas Intestines _Reproductive tract

Figure 1. Mucins glycoproteins can be found in several tissues and organs in the human body.

SQ in organic

media SQ in aqueousmedia SQ + BSA in aqueous

media

Figure 2. 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.

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Squaraine dyes have a structure-relationship influence on the kinetic interaction with PGM.

Squaraine showed a significant increase of fluorescence intensity when PGM was 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). PGM Squaraine 0 ₁₀ 20 150 300 450 VG10-C8 VG10-C2 VG1-C8 VG1-C2 Time (min) 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