Spectroscopic study of squaraines as protein-sensitive fluorescent dyes

Abstract

A series of new symmetrical and asymmetrical squaraines were synthesised and efficiency of their use as fluorescent probes for the specific detection of proteins was studied. Spectral-luminescent properties of the squaraines were measured in presence of bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, avidin from hen egg white (AVI), lysozyme, and trypsin. All investigated squaraines show considerable (in 24–190 times) emission increase in the presence of BSA. At the same time the fluorescent response of the studied dyes in the presence of other albumins is significantly lower – emission enhances up to 24 times. The 3-oxo-substituted indolenine dye 9(74) demonstrates sufficient fluorescence increasing value and emission intensity level in the presence of BSA as well as of HSA and ovalbumin. Dyes containing N-carboxyalkyl group demonstrate sufficient emission enhancement (up to 16 times) and noticeable fluorescent signal in the presence of avidin from hen egg white. Squaraines slightly increase or even decrease their emission intensity in the presence of hydrolases lysozyme or trypsin.

Introduction

Fluorescence detection of proteins at long-wavelength excitation is widely used for biomedical application due to such benefits of near-infrared-based methods as possibility to use of non-expensive diode lasers operating at 635–650 nm [1] and decreased autofluorescence from biomolecules beyond 600 nm [2]. Squarylium dyes are suitable for these purposes owing to their unique physico-chemical properties namely effective light absorption in the visible and near-infrared (NIR) regions [3], sharp and intensive fluorescence [4] and photoconductivity [5]. Since the squaraines contributed greatly to various areas of optoelectronic fields [6], these dyes are also successfully used as long-wavelength fluorescent probes and labels in biological assay techniques [7], [8].

Squaraines derived from different heterocyclic methylene bases can be used as labels and probes for selective biomolecules detection because of easiness of their structure functionality. One approach to structural modification consists in introduction of substituents into the aromatic ring or the N-atom of the heterocyclic moiety. We have already realised other approach which lied in an introduction of different groups in the squaric ring. This leads to red-shift in absorption and fluorescence spectra, but the influence of such substituents on the spectral properties of the dye in various solvents and in presence of proteins was not investigated [9].

Welder et al. [3] described spectral properties of 3-oxo-substituted squaraines in presence of various proteins and concluded that both the symmetrical and unsymmetrical squarylium dyes showed enhancement of fluorescence intensity upon noncovalent interactions with proteins, however, symmetrical dye interacted strongly with HSA, β-lactoglobulin A and trypsinogen whereas unsymmetrical one showed markedly greater binding affinity to HSA and BSA. Furthermore, Terpetschnig et al. [10] studied spectral characteristics of 10 squarylium dyes in free form and when bound to BSA and reached a conclusion that the most suitable protein probes for use in biomedical applications were the symmetrical indolenine-based squaraines which displayed the highest (28-fold) fluorescence intensity increase upon proteins binding.

In this paper we synthesise a series of symmetrical and unsymmetrical squaraine dyes 1, 3–10 (Scheme 1, Scheme 2, Scheme 3, Scheme 4) and evaluate them for detection of a variety of proteins such as bovine serum albumin (BSA), human serum albumin (HSA) ovalbumin, avidin from hen egg white (AVI), and hydrolases such as trypsine and lysozyme. The influence of the dye molecules' structures on selectivity towards certain protein is studied.