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An Introduction to Small Molecule Fluorescent Sensors

Liam D. Adair1,2,3, Kylie Yang1,3, and Elizabeth J. New1,2,3

1 School of Chemistry, The University of Sydney, NSW, Australia

2 Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, NSW, Australia

3 The University of Sydney Nano Institute (Sydney Nano), The University of Sydney, NSW, Australia

Vision has underpinned biological discovery throughout human history, from astronomical observations since prehistoric times, to Mendel's discovery of genetics by observing the colours of sweet pea plants. However, as our understanding of biological systems develops, so does our need to see smaller and smaller structures, and to look deeper and deeper into cells. Today, biology is increasingly investigated at the molecular scale, and there are numerous chemical tools and assays that can be used to observe relevant biomolecules.

Fluorescent sensors are an important tool for biological research as they are able to sense analytes or chemical reactions of interest, and report on their presence through a fluorescence output. Recent advances in technology - from confocal microscopes to flow cytometers to imaging plate-readers - have been accompanied by the development of more selective and sensitive fluorescent sensors, which have enabled the discovery of new biological processes involved in health and disease. While many fluorescent sensors are commercially available, there are many more that have been reported in the literature and are yet to be used widely.

While many imaging technologies have been designed for the simple application of common commercial fluorophores and fluorescent sensors, it is nonetheless important for the end-user to have an understanding of the principles of fluorescence and the mechanisms by which fluorescent sensors operate in order to ensure that sensors are being used appropriately and optimally. This is particularly important for bespoke fluorescent sensors, which may not fit the standard profile of more common commercially available systems. In this chapter, we outline the basic principles of fluorescence and fluorescent sensing. We then explain common classes of fluorophores and the mechanisms by which fluorescent sensors operate.

1.1 What is Fluorescence?

Fluorescence is the emission of light from a substance in an electronically excited state. Specifically, it describes the rapid emission of a photon from a singlet excited state as it returns to the ground state. An electron is promoted from an orbital in the ground state to a higher energy empty orbital by absorption of a photon; the ground state S0 is excited to an excited state Sn. This excited state can then relax to the ground state in several ways, but fluorescence is the process when the molecule returns to the ground state by emission of a photon. A substance that undergoes fluorescence decay is known as a fluorophore.

The orbitals involved in fluorescence are the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) in the ground state of the molecule. A photon with energy equal to the difference between the HOMO p to LUMO p* energies can promote an electron, generating an excited state. A Jablonski diagram (Figure 1.1) is a simple schematic often used for illustration of the electronic states of a molecule and the transitions between them.

Figure 1.1 Jablonski diagram showing: 1. Absorption of a photon to excite electrons from the S0 state to the S1 excited state; 2. non-radiative decay or relaxation from higher vibrational excited states to S1; and 3. emission of a photon as fluorescence.

Vibrational relaxation, or non-radiative decay, is the process in which the excited state relaxes to its lowest vibrational level. This is rapid and takes place on 10-12-10-10?second time scale [1]. This rapid relaxation means that emission occurs from the lowest vibrational level.

Internal conversion (IC) is a non-radiative transition between two electronic states with the same spin state, or multiplicity [2]. Kasha's rule states that appreciable luminescence will only be observed from the lowest energy excited state, as IC is orders of magnitude faster than fluorescence (10-12?second compared to ~10-8?second) [3].

Excited singlet states (denoted Sn) have their excited electron spin paired with the ground state electron. In excited triplet states (Tn), the excited electrons have a parallel spin to the ground state electron and are no longer spin-paired (Figure 1.2).

Another complex excited state process where energy can be dissipated, that competes with fluorescence, is intersystem crossing (ISC). ISC is a non-radiative transition between two electronic states with different spin states, or multiplicities. ISC therefore describes the transition from a singlet state (Sn) to a triplet excited state (T1). Relaxation from T1 to S0 leads to phosphorescence, which is a form of luminescence characterised by having longer-lived emission than fluorescence [4]. This is because emission from the first excited triplet state (i.e. phosphorescence) is spin-forbidden, whereas emission from the first excited singlet state (i.e. fluorescence) is spin-allowed. Phosphorescence is several orders of magnitude slower than fluorescence emission (~10-4?second) [4] (Figure 1.3).

Figure 1.2 Illustration of electron spin states in a singlet ground state S0, singlet excited state Sn, and triplet excited state Tn.

Figure 1.3 Jablonski diagram illustrating absorption, vibrational relaxation, internal conversion, intersystem crossing, fluorescence, and phosphorescence.

Fluorescence spectra are generally presented as a plot of the fluorescence intensity (arbitrary unit) against the wavelength, given in nm (or wavenumber, cm-1). The emission spectra of fluorophores are dependent on the structure of the molecule, and therefore the mechanism by which it fluoresces, and the chemical environment. Fluorescent molecules tend to be aromatic, with extended conjugation and a large system of delocalised p-electrons. These p-electrons are involved in excitation and in general, the more extended the conjugation, the longer the wavelength of photon absorbed, the longer the wavelength emitted.

1.2 Why Is Fluorescence Useful?

There are many characteristics of fluorescence that are advantageous for application in imaging and spectroscopy. Fluorescence is highly sensitive, making it suitable for detection of biological analytes that can be present at extremely low concentrations. This is because fluorophores can be detected in solution at nanomolar to micromolar concentrations, and fluorophores can be identified with high specificity among non-fluorescent material. Furthermore, fluorescence is a fast process, with excitation and emission occurring on the nanosecond time scale, and this allows fluorophores to be imaged with high temporal resolution, enabling the study of detailed dynamic processes in real time. In addition, fluorescence is a convenient method for studying the interactions between different molecular components in a chemically complex system as multiple fluorophores can be used concurrently, which allows for several analytes or molecules and their interactions to be simultaneously tracked.

Fluorescence imaging is amongst the most popular and widely used techniques for molecular imaging. Fluorescence imaging techniques have become indispensable tools allowing the study of the production, localisation, movement, and biological activity of biomolecules in vitro and in vivo. Fluorescence techniques have found use in biochemistry, biophysics, flow cytometry, diagnostics, DNA sequencing, etc. [5-10]. Fluorescence microscopy has become more powerful due to developments such as fluorescence lifetime imaging microscopy (FLIM) and super resolution microscopy [11-15]. The resolution for a standard optical microscope in the visible light spectrum is about 200?nm laterally and 500?nm axially [12]. This has been improved with the use of super resolution microscopy, where the molecular blinking of fluorophores is detected and used to build images that can achieve resolutions of around 20?nm, much below the diffraction limit of visible light [16-21].

As a technique, fluorescence imaging offers many advantages, but its power depends on the availability of fluorescent labels and sensors. Because there are few naturally occurring fluorophores within cells, fluorescence microscopy frequently requires the addition of a stain or probe, which functions to label a structure of interest, certain cell types, organelles, proteins, or chemical species. Microscopy can be performed on fixed specimens, as well as live cells or in vivo, as it is minimally invasive, sensitive, and selective. Furthermore, fluorescence imaging provides good spatial and temporal resolution, which allows dynamic biological processes to be monitored in real time.

Recent decades have seen great advances in the field of fluorescence sensing. Brighter fluorophores with improved biological...