Chapter 1
Fluorescence and Molecular Recognition

Weijie Zhang and Laraib

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, China

During the past decade, there has been a remarkable growth in the use of fluorescence for molecular recognition. A general concept of this method was called "fluorescent sensor or probe," which could identify molecules with a concomitant fluorescence signal. Nowadays, such fluorescent sensors have been widely used in the fields of in vivo imaging, surgical navigation, immunochemistry, clinical diagnosis, environmental analysis, criminal investigation, food safety, and other fields because of their simplicity, high selectivity, and sensitivity in fluorescent assays. Recognition at the molecular level is a fundamental characteristic to glean insights into the processes of biology and chemistry. In this chapter, we present the principles of fluorescence and molecular recognition, highlight recent work that uses these methods, and discuss the applications and future directions as they apply to molecular recognition.

1.1 Advancing Fluorescence Theory

1.1.1 The Discovery of Fluorescence

For many years, fluorescence has been an intriguing scientific technique that enables researchers to examine minute aspects of live organisms by revealing hidden components through brilliant hues. Understanding life beneath the microscope has been made easier by the discovery of fluorescence. In the Florentine Codex, Franciscan missionary Bernardino de Sahagún (1499-1590) recorded the use of a wood called "coatli" that had medicinal qualities and could alter the color of water. Because the water looked bluish in the sun, they utilized this wood to make drinking cups that assisted individuals with urinary issues [1]. In 1565, Spanish scientist Nicolás Monardes wrote in "Historia medicinal de las cosas que se traen de nuestras Indias Occidentales" of the blue-tinted, sparkling look of water when mixed with the Mexican plant Lignum nephriticum [2]. Monardes' paper, which highlighted the special optical qualities of kidney wood, Lignum Nephriticum, was translated into Latin in 1574 by the Flemish botanist Charles de L'Écluse (Figure 1.1). When Vincenzo Casciarolo from Bologna burned a stone he named "lapis solaris" in 1603, it turned out to be barium sulfate, which gave off purple-blue light [3]. The Bolognian stone's ability to emit light was identified by Galileo Galilei in 1612 and was subsequently given the name phosphorescence [4, 5].

Figure 1.1 The blue fluorescence of the Lignum nephriticum in water.

Source: Mark Muyskens et al. (2006)/American Chemical Society.

In 1646, German Jesuit priest Athanasius Kircher published "Ars Magna Luciset Umbrae" (translation: The Grand Mastery of Brightness and Gloom), which explains that light passing through a wood infusion appears yellow, but when reflected, it appears blue. Robert Boyle extended Monarde's research in 1670 and found that some salts caused wood to lose its capacity to alter the color of water after repeated applications. Additionally, he found that adding vinegar eliminated the tint, while potassium carbonate restored it. Boyle was the first to employ fluorescence as a measure of pH [6].

Scottish scientist David Brewster made the first observation of fluorescence in concentrated solutions in 1833, when he found chlorophyll fluorescence, which happens when sunlight is absorbed by a combination of alcohol and leaves, turning the light orange, yellow, and greenish [7].

In 1842, Edmond Becquerel made the remarkable discovery that calcium sulfate emitted ultraviolet light with a wavelength greater than the light it absorbed [8]. In 1858, he developed the first phosphoroscope, which measured the duration of phosphorescence [9].

This phenomenon was understood through research on quinine. Once thought to be a powerful medication for treating infectious disorders like malaria, quinine is an alkaloid that is extracted from Cinchona plants in South America. In 1845, Sir John Frederick William Herschel discovered the luminescence in a quinine solution and coined this effect as "epipolic dispersion" [10].

He reported a distinct blue hue in two articles that were published in the Journal of the Royal Society of London. At that time, scientific understanding was insufficient to accurately identify this color. Though fully transparent and colorless when placed between the eye and a light source, the blue color was seen under specific lighting circumstances and angles.

In his 1852 work "On the Change of Refrangibility of Light," George Gabriel Stokes first proposed the idea of "dispersive reflection" in the sunlight spectra [11]. He proved that fluorescence only happens when UV light is reflected onto a quinine solution, causing a Stokes shift - longer wavelengths of light being emitted than absorbed.

After proposing the use of fluorescence for investigation in 1864, Gabriel Stokes was known as the "Father of Fluorescence" [12]. In 1875, Eugen Lommel proposed the notion that fluorescence can only happen when a body absorbs radiation [13]. The words "fluorophore" and "chromophore" were later coined by R. Meyer in 1897 [14] and O.N. Witt in 1876 [15], respectively. E. Merritt and E. Nichols investigated in 1905 how dyes absorb light at various wavelengths to get excited and then release fluorescent light [16].

Heinrich Lehmann and Otto Heimstaedt invented the first fluorescence microscope between 1911 and 1913 (Figure 1.2), which was used to investigate autofluorescence in a variety of biological materials [17]. Using a microscope, Stanislav von Prowazek investigated dye binding in live cells in 1914 [18]. Theoretical explanations of how certain mechanisms might reduce fluorescence brightness were provided by Stern and Volmer in 1919 [19]. This understanding is important for applications like sensing small changes in concentration. S.J. Vavilov and W.L. Levshin's 1923 study on the interaction of polarized light with fluorescent materials yielded significant details for polarization microscopy methods [11]. Energy efficiency in transforming absorbed energy into fluorescent light output was measured by S.J. Vavilov in 1924, and it is essential for photonic applications [20]. The foundation for the study of dynamic systems incorporating fluorescent materials was established by F. Perrin's in his 1925 study on polarized light emission from excited dye molecules [21]. For theoretical models and experimental designs in a variety of domains, E. Gaviola's 1926 measurement of the nanosecond range of electronic excitations' duration following energy absorption is essential [22].

Figure 1.2 The first fluorescence microscope by Otto Heimstaedt and Heinrich Lehmann.

Source: Carl Zeiss AG/Wikimedia Commons/Public Domain.

Fluorescent dyes employed in biological staining to cause secondary fluorescence in tissues are referred to as fluorochromes, a name Haitinger first used in 1934 [13]. In 1935, the Jablonski diagram, which showed electronic states and their transitions, was created by Alexander Jablonski, who also established fluorescence lifetimes [23]. In order to better understand fluorescence intensity, Francis Perrin extended Jablonski's work by adding the ideas of fluorescence polarization and quantum yield [7]. The efficiency of energy transmission between donor-acceptor pairs spanning nanometers, as shown in resonance energy transfer, is the main emphasis of T. Förster's 1948 quantum mechanical concepts, which also explain dipole interaction and energy transfer in biochemistry research. Knowledge of photosynthesis was greatly improved by pioneering research on delayed fluorescence in photosynthetic plants by Robert Emerson and William Arnold [24, 25].

Biological imaging was transformed after 1970 by the advancement of fluorescence probe technology, which included the revelation and manufacture of green fluorescent protein (GFP). Modern techniques like live-cell imaging and super-resolution microscopy were developed, and these are now essential resources for life sciences research.

1.1.2 The Mechanism of Fluorescence

The term "fluorescence" has a long history, going all the way back to its first discovery, which was detailed in the historical research mentioned above. Fluorescence is the result of a fluorescent molecule, known as a fluorochrome, absorbing light at one wavelength and releasing energy as radiation at another. The crucial relationship between energy and wavelength is that each fluorochrome interacts with particular wavelengths to produce fluorescence.

Photon-excited molecules undergo relaxation processes, which fall into two categories: radiative and nonradiative. Fluorescence and phosphorescence emission are examples of radiative processes, but infrared emission between vibrational levels is frequently regarded as a nonradiative activity. Whereas phosphorescence moves between states of differing multiplicity, fluorescence moves between states of the same multiplicity. Intramolecular nonradiative reactions, which include internal conversion,...