**3.1 Organic analysis**

In the period reviewed so many papers using fluorescence spectrophoometry for analysis, characterization, and as a tool for identification of several compounds are appeared in the literature. In this section, few recent methods will be summarized.

For example, the design and development of artificial molecular systems for sensing anions in biologically relevant conditions is a challenging task in supramolecular chemistry. In particular, sensing fluoride anion has attracted increasing interest in the molecular recognition community because of its pivotal importance in many areas of biological and chemical sciences. In recent years high levels of fluoride in drinking water have caused numerous human diseases, creating a crucial need for artificial sensors to detect fluoride anions in an aqueous environment. Recently, highly sensitive fluorescence "Turn-On"

Current Achievement and Future Potential of Fluorescence Spectroscopy 227

fluorescence-based sensors (Chandrasekhar et al., 2009). This methodology consists of assembly of phosphorus-supported coordinating platforms whose fluorescence properties are modulated by binding with Cu(II) as well as by the number of coordinating arms that the ligand possesses. We believe that this design is quite general and can be applied for

In recent decades, colorimetric and fluorometric sensors have been used in various scientific fields. In biology, for instance, such sensors are useful reagents for living cell imaging. It is important to design novel sensors because they have the potential to overcome many technical limitations in experiments. Imidazo[1,2-a]pyrazin- 3(7H)-one (*imidazopyrazinone*) often is used as a bioluminescent substrate, and it is an attractive core structure for useful sensors. In this regard, a new series of imidazopyrazinones [7-benzylimidazo[1,2 a]pyrazin-3(7H)-one derivatives] have been prepared and their fluorescent properties in the presence

Copper amine oxidases (CAOs) are a large family of copper-containing quinone-dependent amine oxidases that can be found in all living organisms including bacteria, yeast, plants, and mammals. Human CAOs have been implicated in a number of diseases, including atherosclerosis, cardiovascular diseases, diabetes, Alzheimer's disease, and cancer. To study the kinetic behavior of highly potent inhibitors of the CAO bovine plasma amine oxidase (BPAO), it has been sought a sensitive and real time assay to monitor low levels of enzyme activity. The most sensitive assay for CAOs is the fluorometric coupled assay, which monitors generation of hydrogenperoxide during substrate turnover using horseradish peroxidase (HRP) as a secondary detecting enzyme. In this regard, a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6- (aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA) has been reported, which displays extremely tight binding to BPAO (*K*m 183 ( 14 nM) and yet is metabolized fairly quickly (*k*cat 0.690 ( 0.010 s-1), with the aldehyde turnover product (2-(6 formylnaphthalen-2 yloxy)ethyl)trimethyl-ammonium serving as a real time reporting fluorophore of the enzyme activity (Ling et al., 2009). This allowed for the development of a fluorometric noncoupled assay that is two orders of magnitude more sensitive than the spectrophotometric benzylamine assay. ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to

Organic molecules containing a fluorophoric unit combined with site(s) for guest binding purposes have found application in building up florescent signaling systems for biomedical research and chemical logics. Metal ions can act effectively as guests for these molecules because of their ability to enhance, shift or quench luminescent emissions of these organic ligands by coordination. The changes brought about by metal binding are mechanistically of four types, photoinduced electron transfer (PET), photoinduced charge transfer (PCT), formation of monomer/excimer, energy transfer and proton transfer. Transition metal ions with partially filled d-orbitals are known to induce fluorescence quenching by oxidative or reductive PET and energy transfer processes. In this regard, dioxomolybdenum(VI) complexes [MoO2(B2)H2O], [MoO2(B2)EtOH], [MoO2(B3)EtOH] and [MoO2(B4)EtOH] were synthesized using several Schiff base ligands (B1, B2, B3, and B4) (Gupta et al., 2009). These ligands (B1, B2, B3, and B4) were prepared by condensation of 1-(2-pyridyl)-5-methyl-3 pyrazole carbohydrazide with salicylaldehyde, o-hydroxy acetophenone, 5-bromo

selective detection of other type of metal ions also.

respond *directly* to the enzyme activity *in real time*.

of various metal ions (Mn+) have been studied (Hirano, et al., 2010).

indicators, triisopropylsilyl-protected coumarin derivatives, for fluoride anion with remarkable selectivity in organic and aqueous media have been developed (Sokkalingam & Lee C-H., 2011). This developed method exhibited new fluorescent sensors systems for fluoride anion detection that proved to be simple, inexpensive, and highly selective and, achieve accurate determination with a low detection limit. In addition, the results of this study showed that the system can detect inorganic fluorides as quickly as organic ones by simply introducing chelating agents such as crown ethers. The easily prepared indicator system synthesized here could be an ideal chemodosimeter for detecting and determining fluoride anion in both organic and aqueous solutions and could lead to development of a convenient and reliable detection method for fluoride anion in practical and commercial applications.

Fluorescence is a powerful tool for structural and functional studies of a diversity of molecules. Among the various fluorophores, pyrene derivatives are attractive fluorescent probes. Therefore, pyrene "click" conjugates of 7-deazapurine and 8-aza-7-deazapurine nucleosides and a basic pyrene compound has been synthesized (Ingale et al., 2011). The influence of the nucleobase on fluorescence quenching was studied on nucleoside and oligonucleotide level. This study showed that the favorable photophysical properties of 8 aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching. This improves the utility of pyrene fluorescence reporters for detection of oligonucleotides.

Selective recognition of Ag+ ions and amino acids is an important area of research due to their involvement in chemical, biological, and environmental applications. Silver compounds are used as antimicrobial agents, and the activity is closely related with the interaction of Ag+ with sulfurdryl (−SH) groups. There are several chemosensors reported in the literature for the recognition of Ag+ ion in nonaqueous and aqueous systems. However, the molecular receptors which can recognize Ag+ followed by amino acids are indeed limited in the literature. Recently, a new 1,1′-thiobis(2-naphthoxy)-based receptor molecule (L) containing a benzimidazole moiety has been synthesized (Dessingou et al., 2011). The selectivity of L has been explored in aqueous methanol, resulting in selective (7.5 ± 0.5)-fold *switch-on* fluorescence response toward Ag+ among 14 different transition, alkali, and alkaline earth metal ions studied.

There is a growing interest in the development of molecular sensors that can detect selectively metal ions even in low concentrations. Among the various techniques used for this purpose fluorescence-based methods have gained in importance because of their sensitivity. These methods depend upon the change of fluorescence intensity and/or a shift in the fluorescence band of the sensor upon interaction with the metal ion. Although such a methodology has been successful for diamagnetic metal ions, its application to paramagnetic metal ions is fraught with difficulties in view of the fact that the latter quench fluorescence either via energy or electron transfer and only in some instances a fluorescence enhancement has been observed. In this regard, development of fluorescence-based sensors for Cu(II) has assumed importance in view of the fact that it is an essential trace element and yet at slightly increased concentrations, it is toxic, being implicated in gastrointestinal, liver, and kidney diseases as well as in neurological diseases such as Alzheimer's or Parkinson's. Therefore, Chandrasekhar et al. have demonstrated a simple approach for the design of

indicators, triisopropylsilyl-protected coumarin derivatives, for fluoride anion with remarkable selectivity in organic and aqueous media have been developed (Sokkalingam & Lee C-H., 2011). This developed method exhibited new fluorescent sensors systems for fluoride anion detection that proved to be simple, inexpensive, and highly selective and, achieve accurate determination with a low detection limit. In addition, the results of this study showed that the system can detect inorganic fluorides as quickly as organic ones by simply introducing chelating agents such as crown ethers. The easily prepared indicator system synthesized here could be an ideal chemodosimeter for detecting and determining fluoride anion in both organic and aqueous solutions and could lead to development of a convenient and reliable detection method for fluoride anion in practical and commercial

Fluorescence is a powerful tool for structural and functional studies of a diversity of molecules. Among the various fluorophores, pyrene derivatives are attractive fluorescent probes. Therefore, pyrene "click" conjugates of 7-deazapurine and 8-aza-7-deazapurine nucleosides and a basic pyrene compound has been synthesized (Ingale et al., 2011). The influence of the nucleobase on fluorescence quenching was studied on nucleoside and oligonucleotide level. This study showed that the favorable photophysical properties of 8 aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching. This improves the utility of pyrene fluorescence reporters for detection

Selective recognition of Ag+ ions and amino acids is an important area of research due to their involvement in chemical, biological, and environmental applications. Silver compounds are used as antimicrobial agents, and the activity is closely related with the interaction of Ag+ with sulfurdryl (−SH) groups. There are several chemosensors reported in the literature for the recognition of Ag+ ion in nonaqueous and aqueous systems. However, the molecular receptors which can recognize Ag+ followed by amino acids are indeed limited in the literature. Recently, a new 1,1′-thiobis(2-naphthoxy)-based receptor molecule (L) containing a benzimidazole moiety has been synthesized (Dessingou et al., 2011). The selectivity of L has been explored in aqueous methanol, resulting in selective (7.5 ± 0.5)-fold *switch-on* fluorescence response toward Ag+ among 14 different transition, alkali, and

There is a growing interest in the development of molecular sensors that can detect selectively metal ions even in low concentrations. Among the various techniques used for this purpose fluorescence-based methods have gained in importance because of their sensitivity. These methods depend upon the change of fluorescence intensity and/or a shift in the fluorescence band of the sensor upon interaction with the metal ion. Although such a methodology has been successful for diamagnetic metal ions, its application to paramagnetic metal ions is fraught with difficulties in view of the fact that the latter quench fluorescence either via energy or electron transfer and only in some instances a fluorescence enhancement has been observed. In this regard, development of fluorescence-based sensors for Cu(II) has assumed importance in view of the fact that it is an essential trace element and yet at slightly increased concentrations, it is toxic, being implicated in gastrointestinal, liver, and kidney diseases as well as in neurological diseases such as Alzheimer's or Parkinson's. Therefore, Chandrasekhar et al. have demonstrated a simple approach for the design of

applications.

of oligonucleotides.

alkaline earth metal ions studied.

fluorescence-based sensors (Chandrasekhar et al., 2009). This methodology consists of assembly of phosphorus-supported coordinating platforms whose fluorescence properties are modulated by binding with Cu(II) as well as by the number of coordinating arms that the ligand possesses. We believe that this design is quite general and can be applied for selective detection of other type of metal ions also.

In recent decades, colorimetric and fluorometric sensors have been used in various scientific fields. In biology, for instance, such sensors are useful reagents for living cell imaging. It is important to design novel sensors because they have the potential to overcome many technical limitations in experiments. Imidazo[1,2-a]pyrazin- 3(7H)-one (*imidazopyrazinone*) often is used as a bioluminescent substrate, and it is an attractive core structure for useful sensors. In this regard, a new series of imidazopyrazinones [7-benzylimidazo[1,2 a]pyrazin-3(7H)-one derivatives] have been prepared and their fluorescent properties in the presence of various metal ions (Mn+) have been studied (Hirano, et al., 2010).

Copper amine oxidases (CAOs) are a large family of copper-containing quinone-dependent amine oxidases that can be found in all living organisms including bacteria, yeast, plants, and mammals. Human CAOs have been implicated in a number of diseases, including atherosclerosis, cardiovascular diseases, diabetes, Alzheimer's disease, and cancer. To study the kinetic behavior of highly potent inhibitors of the CAO bovine plasma amine oxidase (BPAO), it has been sought a sensitive and real time assay to monitor low levels of enzyme activity. The most sensitive assay for CAOs is the fluorometric coupled assay, which monitors generation of hydrogenperoxide during substrate turnover using horseradish peroxidase (HRP) as a secondary detecting enzyme. In this regard, a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6- (aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA) has been reported, which displays extremely tight binding to BPAO (*K*m 183 ( 14 nM) and yet is metabolized fairly quickly (*k*cat 0.690 ( 0.010 s-1), with the aldehyde turnover product (2-(6 formylnaphthalen-2 yloxy)ethyl)trimethyl-ammonium serving as a real time reporting fluorophore of the enzyme activity (Ling et al., 2009). This allowed for the development of a fluorometric noncoupled assay that is two orders of magnitude more sensitive than the spectrophotometric benzylamine assay. ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond *directly* to the enzyme activity *in real time*.

Organic molecules containing a fluorophoric unit combined with site(s) for guest binding purposes have found application in building up florescent signaling systems for biomedical research and chemical logics. Metal ions can act effectively as guests for these molecules because of their ability to enhance, shift or quench luminescent emissions of these organic ligands by coordination. The changes brought about by metal binding are mechanistically of four types, photoinduced electron transfer (PET), photoinduced charge transfer (PCT), formation of monomer/excimer, energy transfer and proton transfer. Transition metal ions with partially filled d-orbitals are known to induce fluorescence quenching by oxidative or reductive PET and energy transfer processes. In this regard, dioxomolybdenum(VI) complexes [MoO2(B2)H2O], [MoO2(B2)EtOH], [MoO2(B3)EtOH] and [MoO2(B4)EtOH] were synthesized using several Schiff base ligands (B1, B2, B3, and B4) (Gupta et al., 2009). These ligands (B1, B2, B3, and B4) were prepared by condensation of 1-(2-pyridyl)-5-methyl-3 pyrazole carbohydrazide with salicylaldehyde, o-hydroxy acetophenone, 5-bromo

Current Achievement and Future Potential of Fluorescence Spectroscopy 229

containing ligands, but in most cases these complexes with imidazole rings coordinated to the metal center are nonemissive or weakly emissive and only display deprotonating process [Ayers, 2002, as cited in Cheng, 2010). In this regards, tripodal ligands 1,3,5-tris{4- ((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}-2,4,6-trimethylbenzene (L1), 1,1,1 tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}propane (L2), 2,2′,2′′ tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)ethyl}amine (L3), and corresponding Ru(II) complexes [(bpy)6L(Ru(II))3](PF6)6, have been synthesized (Cheng et al., 2010) . This study showed that the fluorescence spectra of these complexes are strongly dependent on the pH of the buffer solution. These complexes act as pH-induced off–on–off fluorescence switch through protonation and deprotonation of the imidazole-containing ligands. For the same purpose, two novel tetrapodal ligands tetrakis{4-((1,10 phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L1), tetrakis {3-((1,10 phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L2), and corresponding Ru(II) complexes [(bpy)8L(Ru(II))4](PF6)8 have been synthesized (Cheng et al., 2011). The two complexes act as off–on–off fluorescence pH switches with a maximum on–off ratio of 5. This on–off ratio is moderate compared with those reported for imidazole-containing Ru(II) complexes (Cheng et al., 2010). They have potential utility to detect pH variations of external

environment due to their interesting photon-dependent photophysical properties.

metals.

Crystal engineering of coordination polymers and supramolecules have attracted lot of attention due to their potential applications as functional materials, as well as their fascinating architectures and topologies (Moulton & Zaworotko, 2010, as cited in Leong, 2009). A successful strategy in the construction of such networks is to utilize appropriate multidentate ligands with flexible backbone that are capable of binding metal ions in different modes. In this regards, a series of metal (Cu(II), Ni(II), Mn(II), Zn(II), Mg(II), Ca(II), and Al(III)) complexes containing the 4-methylumbelliferone-8-methyleneiminodiacetic acid (H3muia, also named as Calcein Blue) has been synthesized (Leong et al., 2009). In this study, the fluorescence spectroscopy has been used to investigate the hydrogen bonding interactions along with π–π stacking in the synthesized complexes solid-state structures. Solid-state fluorescence studies indicate that complexes of muia have the similar emission properties as in the solution state. Transition metal ions quench the fluorescence of muia while alkali earth and post-transition metal complexes of muia exhibit strong blue emission. A considerable number of papers have focused on the use of anthracene containing compounds as protein photo cleavers (Hasewage et al., 2006, as cited in Oliveria, 2007), organic light-emitting diodes and materials (Jou, et al., 2006, as cited in Oliveria, 2007), crystal engineering , molecular imprinted polymers, sensors and chemosensors (Magri, et al., 2005, as cited in Oliveria, 2007). Anthracene is one of the most employed chromophores due to its ability to induce PET (photoinduced electron transfer) processes. In this regard, a new scorpionate system (L) containing an emissive anthracene pendant arm, derived of O1,O7-bis(2-formylphenyl)-1,4,7-trioxaheptane and tren, has been synthesized (Oliveira et al., 2007). The fluorescence spectroscopy studies conducted suggest that this ligand is an effective complexation molecular device for several divalent metal ions of biological importance as well as for Al(III) and Cr(III), both metals of great relevance in medicine and environmental chemistry. The results of this study could be used as a starting point to develop a more efficiently fluorescence chemosensor based on macrocyclic ligands for these

salicylaldehyde and 5-nitro salicylaldehyde, respectively. Due to the presence of a substituted 1-(2-pyridyl) pyrazole unit, these ligands exhibit fluorescent emissions. As the ligands are capable of using different binding modes, according to the demands of the guest metal ions, their emission properties also change accordingly.

#### **3.2 Inorganic analysis**

The development of sensors for metal ions in solution has always been of particular importance for cations with biological and environmental interest. The molecular devices converting metal ions recognition in physical recordable signal are continuously growing. In the last few years great attention has been paid to fluorescent chemosensors and many new systems were synthesized. In particular, an effective fluorescent sensor for metal ions is a system able to interact with the metal ion in solution signaling its presence by changing fluorescence properties, as the wavelength or emission intensity, as well as by the appearance of a new fluorescence band different from those of the free sensor. Recently, paper reviews ligand molecules containing fluorophores synthesized and employed in metal ions sensing in solution in the last few years has been published (Formica et al. 2012). The large number of references reported in the review highlights the synthesis of new fluorescent chemosensors able to sense and signal metal ions in solution. This is a prosperous and still emerging field. In this review the authors concluded that the discovery of newer and more efficient emitting units is desirable. In this case, research is now focused on the synthesis of fluorescent units able to shift the emission in an optical range where the biological noise is reduced in the near infrared region (NIR) thus to increase the sensitivity of the chemosensor for in vivo analysis.

Metal–polypyridine complexes are extensively used in photochemical applications, such as solar energy conversion (Akasheh & AI-Rawashdeh, N.A.F, 1990), due to their peculiar excited-state dynamics. These complexes, of which ruthenium tris-bipyridine ([Ru(bpy)3]2+) is considered the prototype, exhibit a visible absorption band due to the singlet metal-toligand charge transfer state. The principle of the dye sensitized solar cells is based on the use of such metal-based molecular systems, of which the RuN3 ([Ru(dcbpyH2)2(NCS)2]) dye is the most popular, adsorbed onto a semiconductor substrate (usually TiO2). In this regards, femtosecond-resolved broadband fluorescence studies are recently reported for [M(bpy)3]2+ (M=Fe, Ru), RuN3 and RuN719 ((Bu4N)2[Ru(dcbpyH)2(NCS)2]2-) complexes in solution (Bram et al. 2011). In this study, the pump wavelength dependence of the fluorescence of aqueous [Fe(bpy)3]2+ and the solvent and ligand dependence of the fluorescence of Rucomplexes excited at 400 nm have been investigated. The RuN3 and RuN719 are asymmetric complexes contrary to [Ru(bpy)3]2+, which allows us to explore the effects of molecular geometry on the ultrafast relaxation dynamics of this class of molecules.

Ru(II) polypyridyl complexes have been widely used as DNA, cation, and anion sensors, because their outstanding photophysical and electrochemical properties are quite sensitive to external stimuli (Schmittel, 2007, as cited in Cheng, 2010). Transfer of protons can be regarded as one of the simplest external stimuli and can induce the switching of properties such as fluorescence and UV–Vis absorption for pH sensors, so some Ru(II) polypyridyl complexes containing imidazole fragment have been synthesized. Imidazole-containing ligands are poor π-acceptors and good π-donors and have the appreciable ability to control orbital energies by proton transfer compared with pyridine-, pyrazine-, and pyrimidine-

salicylaldehyde and 5-nitro salicylaldehyde, respectively. Due to the presence of a substituted 1-(2-pyridyl) pyrazole unit, these ligands exhibit fluorescent emissions. As the ligands are capable of using different binding modes, according to the demands of the guest

The development of sensors for metal ions in solution has always been of particular importance for cations with biological and environmental interest. The molecular devices converting metal ions recognition in physical recordable signal are continuously growing. In the last few years great attention has been paid to fluorescent chemosensors and many new systems were synthesized. In particular, an effective fluorescent sensor for metal ions is a system able to interact with the metal ion in solution signaling its presence by changing fluorescence properties, as the wavelength or emission intensity, as well as by the appearance of a new fluorescence band different from those of the free sensor. Recently, paper reviews ligand molecules containing fluorophores synthesized and employed in metal ions sensing in solution in the last few years has been published (Formica et al. 2012). The large number of references reported in the review highlights the synthesis of new fluorescent chemosensors able to sense and signal metal ions in solution. This is a prosperous and still emerging field. In this review the authors concluded that the discovery of newer and more efficient emitting units is desirable. In this case, research is now focused on the synthesis of fluorescent units able to shift the emission in an optical range where the biological noise is reduced in the near infrared region (NIR) thus to increase the sensitivity

Metal–polypyridine complexes are extensively used in photochemical applications, such as solar energy conversion (Akasheh & AI-Rawashdeh, N.A.F, 1990), due to their peculiar excited-state dynamics. These complexes, of which ruthenium tris-bipyridine ([Ru(bpy)3]2+) is considered the prototype, exhibit a visible absorption band due to the singlet metal-toligand charge transfer state. The principle of the dye sensitized solar cells is based on the use of such metal-based molecular systems, of which the RuN3 ([Ru(dcbpyH2)2(NCS)2]) dye is the most popular, adsorbed onto a semiconductor substrate (usually TiO2). In this regards, femtosecond-resolved broadband fluorescence studies are recently reported for [M(bpy)3]2+ (M=Fe, Ru), RuN3 and RuN719 ((Bu4N)2[Ru(dcbpyH)2(NCS)2]2-) complexes in solution (Bram et al. 2011). In this study, the pump wavelength dependence of the fluorescence of aqueous [Fe(bpy)3]2+ and the solvent and ligand dependence of the fluorescence of Rucomplexes excited at 400 nm have been investigated. The RuN3 and RuN719 are asymmetric complexes contrary to [Ru(bpy)3]2+, which allows us to explore the effects of molecular

Ru(II) polypyridyl complexes have been widely used as DNA, cation, and anion sensors, because their outstanding photophysical and electrochemical properties are quite sensitive to external stimuli (Schmittel, 2007, as cited in Cheng, 2010). Transfer of protons can be regarded as one of the simplest external stimuli and can induce the switching of properties such as fluorescence and UV–Vis absorption for pH sensors, so some Ru(II) polypyridyl complexes containing imidazole fragment have been synthesized. Imidazole-containing ligands are poor π-acceptors and good π-donors and have the appreciable ability to control orbital energies by proton transfer compared with pyridine-, pyrazine-, and pyrimidine-

geometry on the ultrafast relaxation dynamics of this class of molecules.

metal ions, their emission properties also change accordingly.

**3.2 Inorganic analysis** 

of the chemosensor for in vivo analysis.

containing ligands, but in most cases these complexes with imidazole rings coordinated to the metal center are nonemissive or weakly emissive and only display deprotonating process [Ayers, 2002, as cited in Cheng, 2010). In this regards, tripodal ligands 1,3,5-tris{4- ((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}-2,4,6-trimethylbenzene (L1), 1,1,1 tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}propane (L2), 2,2′,2′′ tris{4-((1,10-phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)ethyl}amine (L3), and corresponding Ru(II) complexes [(bpy)6L(Ru(II))3](PF6)6, have been synthesized (Cheng et al., 2010) . This study showed that the fluorescence spectra of these complexes are strongly dependent on the pH of the buffer solution. These complexes act as pH-induced off–on–off fluorescence switch through protonation and deprotonation of the imidazole-containing ligands. For the same purpose, two novel tetrapodal ligands tetrakis{4-((1,10 phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L1), tetrakis {3-((1,10 phenanthroline-[5,6-d]imidazol-2-yl)phenoxy)methyl}methane (L2), and corresponding Ru(II) complexes [(bpy)8L(Ru(II))4](PF6)8 have been synthesized (Cheng et al., 2011). The two complexes act as off–on–off fluorescence pH switches with a maximum on–off ratio of 5. This on–off ratio is moderate compared with those reported for imidazole-containing Ru(II) complexes (Cheng et al., 2010). They have potential utility to detect pH variations of external environment due to their interesting photon-dependent photophysical properties.

Crystal engineering of coordination polymers and supramolecules have attracted lot of attention due to their potential applications as functional materials, as well as their fascinating architectures and topologies (Moulton & Zaworotko, 2010, as cited in Leong, 2009). A successful strategy in the construction of such networks is to utilize appropriate multidentate ligands with flexible backbone that are capable of binding metal ions in different modes. In this regards, a series of metal (Cu(II), Ni(II), Mn(II), Zn(II), Mg(II), Ca(II), and Al(III)) complexes containing the 4-methylumbelliferone-8-methyleneiminodiacetic acid (H3muia, also named as Calcein Blue) has been synthesized (Leong et al., 2009). In this study, the fluorescence spectroscopy has been used to investigate the hydrogen bonding interactions along with π–π stacking in the synthesized complexes solid-state structures. Solid-state fluorescence studies indicate that complexes of muia have the similar emission properties as in the solution state. Transition metal ions quench the fluorescence of muia while alkali earth and post-transition metal complexes of muia exhibit strong blue emission.

A considerable number of papers have focused on the use of anthracene containing compounds as protein photo cleavers (Hasewage et al., 2006, as cited in Oliveria, 2007), organic light-emitting diodes and materials (Jou, et al., 2006, as cited in Oliveria, 2007), crystal engineering , molecular imprinted polymers, sensors and chemosensors (Magri, et al., 2005, as cited in Oliveria, 2007). Anthracene is one of the most employed chromophores due to its ability to induce PET (photoinduced electron transfer) processes. In this regard, a new scorpionate system (L) containing an emissive anthracene pendant arm, derived of O1,O7-bis(2-formylphenyl)-1,4,7-trioxaheptane and tren, has been synthesized (Oliveira et al., 2007). The fluorescence spectroscopy studies conducted suggest that this ligand is an effective complexation molecular device for several divalent metal ions of biological importance as well as for Al(III) and Cr(III), both metals of great relevance in medicine and environmental chemistry. The results of this study could be used as a starting point to develop a more efficiently fluorescence chemosensor based on macrocyclic ligands for these metals.

Current Achievement and Future Potential of Fluorescence Spectroscopy 231

fluorescence spectrum, of NAB reveals a maximum whose intensity increases with the different γ-CD's growing concentrations. It is noteworthy mentioning that significant alterations in the physicochemical properties of the included molecule (NAB) are observed upon forming the inclusion complex with γ-cyclodextrin, such as stability and solubility in

Imidazoline-derived drugs are a family of drugs that is structurally distinguished by the existence of the heterocyclic ring of imidazoline that enables these drugs to interact with adrenergic receptors via stimulating presynaptic and postsynaptic a-adrenoceptors (Parini et al., 1996). The majority of the imidazoline-derived drugs are frequently used for their agonist activity, whereas others are used for either antihypertensive, antihistaminic, or agonistic activity (Kaliszan et al., 2006). However, the principle pharmaceutical applicability of these drugs is due to their vascoconstrictive effects. Three typical imidazoline-derived drugs were selected for this study; Naphazoline (NP) (4,5-Dihydro-2-(1 naphthalenylmethyl-1H-imidazole), Antazoline (AN) (4,5-Dihydro-Nphenyl-N- (phenylmethyl)-1H-imidazole-2-methanamine, 2-[(N-benzyl anilino) methyl]-2-imidazole, and Xylometazoline (XM) (2-(4-tert-Butyl-2,6-dimethylbenzyl)-2-imidazoline). While CDs have a wide range of applications, using CDs as additives in various separation and pharmaceutical sciences is still the foremost application of them. Hence, adding the CDs to the separation media can significantly enhance the separation process, whereas employing CDs as additives to drugs formulation can promote the bioavailability through enhancing the stability and solubility of selected drugs. In this regard, the inclusion complexes of selected imidazoline-derived drugs, namely Antazoline (AN), Naphazoline (NP) and Xylometazoline (XM) with β-cyclodextrin (β-CD) were investigated using steady-state fluorescence (Dawoud & Al-Rawashdeh, N. 2008)). Their results confirmed the formation of the inclusion complexes between the three studied drugs and β-cyclodextrin using steadystate fluorescence spectroscopy. Importantly, the results of their study showed that the geometrical size and polarity of various substituents, such as phenyl and naphthyl groups, have dramatically altered the stability and geometrical configuration of the inclusion complexes. These studies present the state-of-the-art of macromolecular binders and provide detailed illustrative examples of recent developments bearing much promise for

Propranolol is a beta-adrenergic blocking drug widely prescribed for the treatment of cardiac arrhythmia, sinus tachycardia, angina pectoris and hypertension (Parfitt, 1990). It has also been suggested for use in a number of other conditions including dysfunctional labour and anxiety. When administered over a long period of time it reduces mortality caused by hypertension and lengthens survival in patients with coronary heart disease. It is also used in low activity sports, reducing cardiac frequency, contraction force and coronary flow. Therefore, it has been included in the list of forbidden substances by the International Olympic Committee. The Spanish Olympic Committee has decided that only a qualitative determination of propranolol in urine is necessary. In this regards, a sensitive fluorescence optosensor for the drug propranolol to use in the analysis of pharmaceutical preparations and as a doping test for the qualitative analysis of propranolol in human urine without lengthy preliminary procedures have been developed (Fernandez-Sanchez et al. 2003).The effect of proteins presents in urine samples were evaluated using the developed flowthrough fluorescence optosensor. The results of this study showed that the proposed methods for analysis were satisfactorily applied to commercial formulations and urine

aqueous media.

future pharmaceutical applications.

Since some of lanthanide ions, especially Eu3+ and Tb3+, posses good luminescence characteristics (high color purity) based on the 4 f electronic transitions, a variety of rare earth compounds activated by Eu3+ and Tb3+ have been studied for practical applications (Zhang, et al., 1999, as cited in Xu, et al., 2004). Polydimethylsiloxane (PDMS) materials have broad application in a variety of industrial area so their well-established surface modifying properties. In this regard, Eu(III)-containing polymer complex was synthesized, in which polydimethylsiloxane was used as a polymer ligand (Xu, et al., 2004). The result of this study showed that the fluorescence intensity change of Eu(III)-PDMS complex displays typical fluorescent concentration quenching behavior, in which the emission intensity of the complex was enhanced with increasing content of Eu (III) ion and reaches a maximum at 2 wt.%, and then decreased with further increasing content of Eu(III) ion. These results indicate that the complex contains ionic aggregates in which Eu(III) ions are located close together. Eu(III)-PDMS could transform harmful ultraviolet radiation to blue luminescence that is needed by plants effectively. So Eu(III)-PDMS has a great application foreground.

Schiff base ligands have been extensively studied in coordination chemistry mainly due to their facile syntheses, easily tunable steric, electronic properties and good solubility in common solvents. Transition metal complexes with oxygen and nitrogen donor Schiff bases are of particular interest (You et al., 2004) because of their ability to possess unusual configurations, be structurally labile and their sensitivity to molecular environments. In this regard, the preparation and structures of nickel(II) zinc(II) and cadmium(II) complexes with the related Schiff base ligand N-2-pyridylmethylidene-2- hydroxy-phenylamine have been investigated (Majumder et al. 2006). The results of this study showed that the complexes with zinc(II) and cadmium(II) metals can serve as potential photoactive materials as indicated from their characteristic fluorescence properties. Since the complexes with zinc(II) and cadmium(II) metals possess an intense fluorescence property at room temperature, which is not observed for complexes with Ni(II). It is suggested that complexes with Zn(II) and Cd(II) exhibit potential applications as photoactive materials.

#### **3.3 Pharmaceutical analysis**

Over the last three decades, wide ranges of thermodynamic data concerning the guest host complexation have been reported. Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucopyranose units linked together via oxygen bridges at the 1 and 4 positions (a-(l,4)-glycoside bonds) (Bender& Komiyama, 1978). This class of organized media possesses a hydrophilic upper and lower rims lined with hydroxyl groups and a hydrophobic cavity due to C3H, C5H, and C6H hydrogen's and O4 ether oxygen. This structure gives CDs the ability to extract a variety of organic guest molecules of appropriate size and hydrophobicity from the bulk aqueous solution (Szejtli, 1988). Complexation of various guest compounds with CDs generally results in the improvement of some physical properties of the guest molecules, such as stability, bioavailability, membrane permeability, and solubility. In luminescence studies, CDs have been employed to enhance fluorescence emission of different luminophors (Bender & Komiyama, 1978; Szejtli, 1988) and to induce room temperature phosphorescence under appropriate conditions. In this regards, there is a lot of literature reported in several journals of interest, as an example, the inclusion of the anti-inflammatory drug, Nabumetone (NAB), in γ-cyclodextrin (γ-CD) was studied by fluorescence measurements (Al-Rawashdeh. N.A.F., 2005). Nabumetone is poorly soluble in water and exhibits intrinsic fluorescence. The results of this study showed that the emission

Since some of lanthanide ions, especially Eu3+ and Tb3+, posses good luminescence characteristics (high color purity) based on the 4 f electronic transitions, a variety of rare earth compounds activated by Eu3+ and Tb3+ have been studied for practical applications (Zhang, et al., 1999, as cited in Xu, et al., 2004). Polydimethylsiloxane (PDMS) materials have broad application in a variety of industrial area so their well-established surface modifying properties. In this regard, Eu(III)-containing polymer complex was synthesized, in which polydimethylsiloxane was used as a polymer ligand (Xu, et al., 2004). The result of this study showed that the fluorescence intensity change of Eu(III)-PDMS complex displays typical fluorescent concentration quenching behavior, in which the emission intensity of the complex was enhanced with increasing content of Eu (III) ion and reaches a maximum at 2 wt.%, and then decreased with further increasing content of Eu(III) ion. These results indicate that the complex contains ionic aggregates in which Eu(III) ions are located close together. Eu(III)-PDMS could transform harmful ultraviolet radiation to blue luminescence that is needed by plants effectively. So Eu(III)-PDMS has a great application foreground.

Schiff base ligands have been extensively studied in coordination chemistry mainly due to their facile syntheses, easily tunable steric, electronic properties and good solubility in common solvents. Transition metal complexes with oxygen and nitrogen donor Schiff bases are of particular interest (You et al., 2004) because of their ability to possess unusual configurations, be structurally labile and their sensitivity to molecular environments. In this regard, the preparation and structures of nickel(II) zinc(II) and cadmium(II) complexes with the related Schiff base ligand N-2-pyridylmethylidene-2- hydroxy-phenylamine have been investigated (Majumder et al. 2006). The results of this study showed that the complexes with zinc(II) and cadmium(II) metals can serve as potential photoactive materials as indicated from their characteristic fluorescence properties. Since the complexes with zinc(II) and cadmium(II) metals possess an intense fluorescence property at room temperature, which is not observed for complexes with Ni(II). It is suggested that complexes with Zn(II)

Over the last three decades, wide ranges of thermodynamic data concerning the guest host complexation have been reported. Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucopyranose units linked together via oxygen bridges at the 1 and 4 positions (a-(l,4)-glycoside bonds) (Bender& Komiyama, 1978). This class of organized media possesses a hydrophilic upper and lower rims lined with hydroxyl groups and a hydrophobic cavity due to C3H, C5H, and C6H hydrogen's and O4 ether oxygen. This structure gives CDs the ability to extract a variety of organic guest molecules of appropriate size and hydrophobicity from the bulk aqueous solution (Szejtli, 1988). Complexation of various guest compounds with CDs generally results in the improvement of some physical properties of the guest molecules, such as stability, bioavailability, membrane permeability, and solubility. In luminescence studies, CDs have been employed to enhance fluorescence emission of different luminophors (Bender & Komiyama, 1978; Szejtli, 1988) and to induce room temperature phosphorescence under appropriate conditions. In this regards, there is a lot of literature reported in several journals of interest, as an example, the inclusion of the anti-inflammatory drug, Nabumetone (NAB), in γ-cyclodextrin (γ-CD) was studied by fluorescence measurements (Al-Rawashdeh. N.A.F., 2005). Nabumetone is poorly soluble in water and exhibits intrinsic fluorescence. The results of this study showed that the emission

and Cd(II) exhibit potential applications as photoactive materials.

**3.3 Pharmaceutical analysis** 

fluorescence spectrum, of NAB reveals a maximum whose intensity increases with the different γ-CD's growing concentrations. It is noteworthy mentioning that significant alterations in the physicochemical properties of the included molecule (NAB) are observed upon forming the inclusion complex with γ-cyclodextrin, such as stability and solubility in aqueous media.

Imidazoline-derived drugs are a family of drugs that is structurally distinguished by the existence of the heterocyclic ring of imidazoline that enables these drugs to interact with adrenergic receptors via stimulating presynaptic and postsynaptic a-adrenoceptors (Parini et al., 1996). The majority of the imidazoline-derived drugs are frequently used for their agonist activity, whereas others are used for either antihypertensive, antihistaminic, or agonistic activity (Kaliszan et al., 2006). However, the principle pharmaceutical applicability of these drugs is due to their vascoconstrictive effects. Three typical imidazoline-derived drugs were selected for this study; Naphazoline (NP) (4,5-Dihydro-2-(1 naphthalenylmethyl-1H-imidazole), Antazoline (AN) (4,5-Dihydro-Nphenyl-N- (phenylmethyl)-1H-imidazole-2-methanamine, 2-[(N-benzyl anilino) methyl]-2-imidazole, and Xylometazoline (XM) (2-(4-tert-Butyl-2,6-dimethylbenzyl)-2-imidazoline). While CDs have a wide range of applications, using CDs as additives in various separation and pharmaceutical sciences is still the foremost application of them. Hence, adding the CDs to the separation media can significantly enhance the separation process, whereas employing CDs as additives to drugs formulation can promote the bioavailability through enhancing the stability and solubility of selected drugs. In this regard, the inclusion complexes of selected imidazoline-derived drugs, namely Antazoline (AN), Naphazoline (NP) and Xylometazoline (XM) with β-cyclodextrin (β-CD) were investigated using steady-state fluorescence (Dawoud & Al-Rawashdeh, N. 2008)). Their results confirmed the formation of the inclusion complexes between the three studied drugs and β-cyclodextrin using steadystate fluorescence spectroscopy. Importantly, the results of their study showed that the geometrical size and polarity of various substituents, such as phenyl and naphthyl groups, have dramatically altered the stability and geometrical configuration of the inclusion complexes. These studies present the state-of-the-art of macromolecular binders and provide detailed illustrative examples of recent developments bearing much promise for future pharmaceutical applications.

Propranolol is a beta-adrenergic blocking drug widely prescribed for the treatment of cardiac arrhythmia, sinus tachycardia, angina pectoris and hypertension (Parfitt, 1990). It has also been suggested for use in a number of other conditions including dysfunctional labour and anxiety. When administered over a long period of time it reduces mortality caused by hypertension and lengthens survival in patients with coronary heart disease. It is also used in low activity sports, reducing cardiac frequency, contraction force and coronary flow. Therefore, it has been included in the list of forbidden substances by the International Olympic Committee. The Spanish Olympic Committee has decided that only a qualitative determination of propranolol in urine is necessary. In this regards, a sensitive fluorescence optosensor for the drug propranolol to use in the analysis of pharmaceutical preparations and as a doping test for the qualitative analysis of propranolol in human urine without lengthy preliminary procedures have been developed (Fernandez-Sanchez et al. 2003).The effect of proteins presents in urine samples were evaluated using the developed flowthrough fluorescence optosensor. The results of this study showed that the proposed methods for analysis were satisfactorily applied to commercial formulations and urine

Current Achievement and Future Potential of Fluorescence Spectroscopy 233

complexes with low toxicity and improved therapeutic properties. In this regards, 3- Carbaldehyde chromone thiosemicarbazone and its transition metal (Cu(II), Zn(II) and Ni(II)) complexes were synthesized and characterized systematically (Li et al., 2010). The results of this study showed that the Zn(II) complex can emit blue fluorescence under UV light in solid state and may be used as an advanced material for blue light emitting dioxide devices. However, almost the solid fluorescence of Cu(II) and Ni(II) complexes could not be observed. Thus, the fluorescence property of these complexes was used as a helpful tool to understand the interaction mechanism of small molecule compounds binding to DNA. It was believed that the information obtained from the present work would be useful to

The quantitative determination of micro-amounts of nucleic acid has attracted a great deal of attention in the fields of medicine and molecular biology. Many methods have been developed, such as direct determination, including ultraviolet absorption and determination of ribose or deoxyribose in nucleic acid, spectrophotometry, chemiluminescence, electrochemical chromatography, including high-performance liquid chromatography and paper chromatography, capillary electrophoresis, and resonance light scattering. However, low sensitivity and easy interruption by protein and other biomolecules existed in these methods in common. However, the fluorometric methods make predominant concern because of their high sensitivity and selectivity. Generally, the fluorescence intensity of DNA must be enhanced by fluorescent probes because it emits weak fluorescence itself. In this regard, method for the determination of DNA based on the fluorescence intensity of the gatifloxacin-europium(III) (GFLX-Eu3+) complex that could be enhanced by DNA was developed (Wang et al. 2011). The GFLX-Eu3+ complex showed an up to 6-fold enhancement of luminescence intensity after adding DNA. On the basis of the above findings, the fluorescence enhancement effect of the GFLX-Eu3+ complex by DNA was investigated in this

The green fluorescent proteins (GFPs) originated from the bioluminescent jellyfish Aequorea victoria, were discovered by Shimomura in the early 1960s (Shimomura et al., 1962). In the last few years, green fluorescent protein (GFP) has become one of the most widely used tools in molecular and cell biology. As a noninvasive fluorescent marker in living cells, GFP allows for numerous applications where it functions as a probe of gene expression, intercellular tracer or as a measure of protein-protein interactions. The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein–protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. Recently, the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and the decrease in decay time has been observed by Fu et al. (Fu et al., 2008). Furthermore, single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, longer duration time and suppressed blinking were observed. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the

Detection of DNA in solution is an important problem in a large variety of biochemical assays. The most popular agents for DNA quantitation are fluorescent dyes that strongly

develop new potential antioxidants and therapeutic agents for some diseases.

study in detail.

structure.

samples, which offers excellent analytical parameters, such as sensitivity, selectivity, versatility, and ease of use. The development of optosensing techniques has led to a shorter turnaround analysis time and reduced costs for doping controls. As a large part of the samples prove to be non-doped, rapid analytical methods such as doping tests that provide reliable 'yes/no' responses are of increasing interest. These tests can usually be described as systems that 'filter' samples to select those with analyte content levels 'similar to' or 'higher than' a previously established threshold. These 'probably doped' samples must then be examined with more exact instrumental methods. Doping tests can significantly cut costs and save time.

Anthraquinones are known to be present in many different families such as Polygonaceae, Leguminosae, Rubiaceae, Liliaceae and Rhamnaceae. Recently, a number of pharmacological tests revealed that the anthraquinone derivatives present various biological activities including antifungal (S K. Agarwal, 2000, as cited in He, 2009), antimicrobial (Y. W. Wu, as cited in He, 2009), anticancer (J. Koyamma, as cited in He, 2009) , antioxidant (G. C. Yen, as cited in He, 2009), and antihuman cytomegalovirus activity (D. I. Barnard. As cited in He, 2009). In general, fluorescence detection is sensitive and selective. The five anthraquinones are known to possess natural fluorescence, but it is difficult to analyse and determine their contents by conventional fluorimetry due to their similar molecule structures. Therefore, recently a simple, rapid and sensitive reversed-phase highperformance liquid chromatography (RP-HPLC) method, using fluorescence detection, to simultaneously quantify aloe-emodin, emodin, rhein, chrysophanol and physcion in medicinal plants and their pharmaceutical preparations was developed by He et al. (He et al., 2009). Their method was suitable for use as a tool for routine quality assurance and standardization of the anthraquinone from the raw material and commercially available pharmaceutical preparations containing rhubarb.

Fluorimetry from the early fifties has been among the most frequently used techniques for determining both therapeutically and abuse drugs; probably due to its excellent selectivity and the low detection limits. Also, the fluorimetric technique is the recommended choice for quantifying the purity of active principles. The research on analytical fluorescence applications is in continuous expansion to new automated or semi-automate processes like classic or emergent methodologies on the continuous-flow field. In this way, fluorescencebased methods have found a wide range of analytical applications (Calatayud & Zamora, 1995)). In this regard, a new strategic tool to predict the native fluorescence of organic molecules has been proposed (Albert-Garcia et al. 2009). For this purpose, the molecular connectivity indices of different organic substances (pharmaceuticals and pesticides) were calculated. The work presented in this paper was focused to present a new tool for enhancing the research yield on new analytical applications of fluorescence.

#### **3.4 Biological and biomedical analysis**

Cancer has overtaken heart disease as the world's top killer by 2011, part of a trend that should be more than double global cancer cases and deaths by 2030. Cisplatin (cis diamminedichloroplatinum(II)) is one of the most effective anticancer drugs in the treatment of a variety of tumors and it has been clinically used widely. However, its limited usefulness in the development of resistance in tumor cells and the significant side effects have generated new areas of research, which mainly focused on searching for new metal-based

samples, which offers excellent analytical parameters, such as sensitivity, selectivity, versatility, and ease of use. The development of optosensing techniques has led to a shorter turnaround analysis time and reduced costs for doping controls. As a large part of the samples prove to be non-doped, rapid analytical methods such as doping tests that provide reliable 'yes/no' responses are of increasing interest. These tests can usually be described as systems that 'filter' samples to select those with analyte content levels 'similar to' or 'higher than' a previously established threshold. These 'probably doped' samples must then be examined with more exact instrumental methods. Doping tests can significantly cut costs

Anthraquinones are known to be present in many different families such as Polygonaceae, Leguminosae, Rubiaceae, Liliaceae and Rhamnaceae. Recently, a number of pharmacological tests revealed that the anthraquinone derivatives present various biological activities including antifungal (S K. Agarwal, 2000, as cited in He, 2009), antimicrobial (Y. W. Wu, as cited in He, 2009), anticancer (J. Koyamma, as cited in He, 2009) , antioxidant (G. C. Yen, as cited in He, 2009), and antihuman cytomegalovirus activity (D. I. Barnard. As cited in He, 2009). In general, fluorescence detection is sensitive and selective. The five anthraquinones are known to possess natural fluorescence, but it is difficult to analyse and determine their contents by conventional fluorimetry due to their similar molecule structures. Therefore, recently a simple, rapid and sensitive reversed-phase highperformance liquid chromatography (RP-HPLC) method, using fluorescence detection, to simultaneously quantify aloe-emodin, emodin, rhein, chrysophanol and physcion in medicinal plants and their pharmaceutical preparations was developed by He et al. (He et al., 2009). Their method was suitable for use as a tool for routine quality assurance and standardization of the anthraquinone from the raw material and commercially available

Fluorimetry from the early fifties has been among the most frequently used techniques for determining both therapeutically and abuse drugs; probably due to its excellent selectivity and the low detection limits. Also, the fluorimetric technique is the recommended choice for quantifying the purity of active principles. The research on analytical fluorescence applications is in continuous expansion to new automated or semi-automate processes like classic or emergent methodologies on the continuous-flow field. In this way, fluorescencebased methods have found a wide range of analytical applications (Calatayud & Zamora, 1995)). In this regard, a new strategic tool to predict the native fluorescence of organic molecules has been proposed (Albert-Garcia et al. 2009). For this purpose, the molecular connectivity indices of different organic substances (pharmaceuticals and pesticides) were calculated. The work presented in this paper was focused to present a new tool for

Cancer has overtaken heart disease as the world's top killer by 2011, part of a trend that should be more than double global cancer cases and deaths by 2030. Cisplatin (cis diamminedichloroplatinum(II)) is one of the most effective anticancer drugs in the treatment of a variety of tumors and it has been clinically used widely. However, its limited usefulness in the development of resistance in tumor cells and the significant side effects have generated new areas of research, which mainly focused on searching for new metal-based

enhancing the research yield on new analytical applications of fluorescence.

and save time.

pharmaceutical preparations containing rhubarb.

**3.4 Biological and biomedical analysis** 

complexes with low toxicity and improved therapeutic properties. In this regards, 3- Carbaldehyde chromone thiosemicarbazone and its transition metal (Cu(II), Zn(II) and Ni(II)) complexes were synthesized and characterized systematically (Li et al., 2010). The results of this study showed that the Zn(II) complex can emit blue fluorescence under UV light in solid state and may be used as an advanced material for blue light emitting dioxide devices. However, almost the solid fluorescence of Cu(II) and Ni(II) complexes could not be observed. Thus, the fluorescence property of these complexes was used as a helpful tool to understand the interaction mechanism of small molecule compounds binding to DNA. It was believed that the information obtained from the present work would be useful to develop new potential antioxidants and therapeutic agents for some diseases.

The quantitative determination of micro-amounts of nucleic acid has attracted a great deal of attention in the fields of medicine and molecular biology. Many methods have been developed, such as direct determination, including ultraviolet absorption and determination of ribose or deoxyribose in nucleic acid, spectrophotometry, chemiluminescence, electrochemical chromatography, including high-performance liquid chromatography and paper chromatography, capillary electrophoresis, and resonance light scattering. However, low sensitivity and easy interruption by protein and other biomolecules existed in these methods in common. However, the fluorometric methods make predominant concern because of their high sensitivity and selectivity. Generally, the fluorescence intensity of DNA must be enhanced by fluorescent probes because it emits weak fluorescence itself. In this regard, method for the determination of DNA based on the fluorescence intensity of the gatifloxacin-europium(III) (GFLX-Eu3+) complex that could be enhanced by DNA was developed (Wang et al. 2011). The GFLX-Eu3+ complex showed an up to 6-fold enhancement of luminescence intensity after adding DNA. On the basis of the above findings, the fluorescence enhancement effect of the GFLX-Eu3+ complex by DNA was investigated in this study in detail.

The green fluorescent proteins (GFPs) originated from the bioluminescent jellyfish Aequorea victoria, were discovered by Shimomura in the early 1960s (Shimomura et al., 1962). In the last few years, green fluorescent protein (GFP) has become one of the most widely used tools in molecular and cell biology. As a noninvasive fluorescent marker in living cells, GFP allows for numerous applications where it functions as a probe of gene expression, intercellular tracer or as a measure of protein-protein interactions. The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein–protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. Recently, the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and the decrease in decay time has been observed by Fu et al. (Fu et al., 2008). Furthermore, single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, longer duration time and suppressed blinking were observed. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.

Detection of DNA in solution is an important problem in a large variety of biochemical assays. The most popular agents for DNA quantitation are fluorescent dyes that strongly

Current Achievement and Future Potential of Fluorescence Spectroscopy 235

agrochemicals (pesticides, fungicides, insecticides, etc), which have attracted attention worldwide due to their usage in agriculture. Therefore, the potential increase in fluorescence of a benzimidazole-type fungicide (carbendazim) due to complexation with cucurbit[6]uril was reported (Saleh & Al-Rawashdeh, N.A.F., 2006). The fluorescence enhancement of the fungicide carbendazim by cucurbit[6]uril has been observed in solution due to formation of a host–guest inclusion complex. The enhancement of the carbendazim fluorescence (maximum factor of 10) is accompanied by a significant blue shift in the spectrum (11 nm). In general, Saleh and Al-Rawashdeh work (Saleh & Al-Rawashdeh, N.A.F., 2006) demonstrates the potential usefulness of cucurbit[6]uril in fluorometric analysis of fungicide

Recycling in agriculture of organic wastes produced by various animal breeding, such as pig slurry (PS), is a common practice throughout the world, which has recently raised serious environmental concerns. In particular, Cu(II) and Zn(II) ions, which are used abundantly as pig feed additives, may be introduced in relatively large amounts into PS-amended soils, thus representing an actual risk of phytotoxicity and/or leaching downward the soil with potential endangering groundwater quality (L'Herroux et al., 1997, Saviozzi et al., 1997, Giusquiani et al., 1998, Nicholson et al., 1999, Aldrich et al., 2002, Taboada-Castro et al.,

Bioavailability, mobility and transport of metal ions in soils are strongly influenced by binding reactions with soil organic matter, and especially its humified fractions, i.e., humic substances (HS), of which humic acid (HA) is the major component (Aldrich et al., 2002, as cited in Hernandez, at al. 2006). For these reasons, the effects of PS application on the compositional, structural and functional properties of native soil HA, and especially on their Cu(II) and Zn(II) binding behavior, need to be accurately evaluated. Therefore, The effect of the consecutive annual additions of pig slurry on the Cu(II) and Zn(II) binding behavior of soil HAs was investigated in a field experiment by a fluorescence titration method (Hernandez et al. 2006). In Hernandez et al. study (Hernandez et al. 2006), a fluorescence titration method was used for determining metal ion complexing capacities and stability constants of metal ion complexes of humic acid isolated from pig slurry and unamended and amended soils. The results of this study is expected to have a large impact on bioavailability, mobilization, and transport of Cu(II) and Zn(II) ions in pig slurry-amended

Moreover, a luminescent sensor utilizing the substrate 2,6pyridine-dicarbox-aldehydebis-(*o*hydroxyphenylimine) has been developed for low concentration detection of the environmental mercuric ion pollutants (Kanan et. al 2009). The sensor selectively detects mercury in the presence of several ions that are commonly found in aquatic environments. The sensor was found to be highly selective with strongest binding was observed between the mercuric ions and the substrate at a pH range of 6.5-7.5 which makes the substrate a distinctive fluorescent sensor for detecting mercury under normal environmental conditions. No effect was demonstrated with the addition of any other metal ions commonly

Dissolved organic carbon (DOC) refers to the hundreds of dissolved compounds found in water that derive from organic materials, and is composed of 'organic acids', 'organic bases', and 'neutral groups'. The amount of DOC in the hydrosphere (700 gigatons) is almost the

found in water, making this compound selective to mercuric ions.

for agricultural and environmental applications.

2002, as cited in Hernandez, at al. 2006 ).

soils (Hernandez et al. 2006).

interact with nucleic acids and significantly increase their emission intensity in the DNA complex. Fluorescent dyes are used in real-time PCR, DNA-based cell quantitation, gel staining, chromatin and other DNA-based approaches (Glazer and Rye, 1992, Jing et al., 2003, Lakowicz, 2006, Le Pecq and Paoletti, 1967, Lim et al., 1997, Szpechcinski et al., 2008, as cited in Dragan, 2010). In this regard, both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which noncovalently binds dsDNA were investigated (Dragan et al. 2010). It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. In this study an experimental data obtained for a PicoGreen® (PG)/DNA quantitation assay was presented, which is in complete agreement with the results of our theoretical analysis. Furthermore, it has been shown, both theoretically and experimentally, that DNA assays based on the MEF of PG demonstrate sensitivity to DNA concentration of ≈1 pg/ml, which is several orders of magnitude more sensitive than without the silver nanoparticles, suggesting the broader practical use of this approach (metal-enhanced PicoGreen fluorescence) for the ultra-sensitive detection of double stranded nucleic acids.

Identification of living organisms (eukaryotes, bacteria, viruses etc.) by means of quantitative analysis of their specific DNA sequences, which represent different genomes, is a challenging aim, which faces many scientists today. It also concerns the search and detection of different microorganism mutations and strains of pathogenic bacteria and causes of severe diseases in humans. In recent short communication, further development of the "Catch and Signal" technology—principles of a 2-color DNA assay for the simultaneous detection/quantification of two genome-specific DNAs in one well was presented (Dragan et al., 2011) In this method a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization has been utilized. Furthermore, it is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver nanoparticles, which is ideal for MEF. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Their results clearly showed that the 2-color DNA assay can effectively be employed as a new "Rapid Catch and Signal" technological platform in the creation of an ultra-sensitive, sequence-specific approach for the fast analysis of genetic material from different organisms, for potential analysis of bacteria and virus pathogens, and search for possible mutations and sequence variations. This technology being fast, ultrasensitive and inexpensive can effectively compete with the PCR technique, especially for the routine and rapid analysis in Point-of-Care settings and bio-medical laboratories.

#### **3.5 Environmental analysis**

For the past 20 years there has been continued growth in the applications of fluorescence spectroscopy in physical and biological sciences. Because of the sensitivity of fluorescence detection, the fluorometeric method has been one of the selected techniques to determine compounds at low concentrations (Lakowicz, 2006). Examples of such compounds are

interact with nucleic acids and significantly increase their emission intensity in the DNA complex. Fluorescent dyes are used in real-time PCR, DNA-based cell quantitation, gel staining, chromatin and other DNA-based approaches (Glazer and Rye, 1992, Jing et al., 2003, Lakowicz, 2006, Le Pecq and Paoletti, 1967, Lim et al., 1997, Szpechcinski et al., 2008, as cited in Dragan, 2010). In this regard, both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which noncovalently binds dsDNA were investigated (Dragan et al. 2010). It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. In this study an experimental data obtained for a PicoGreen® (PG)/DNA quantitation assay was presented, which is in complete agreement with the results of our theoretical analysis. Furthermore, it has been shown, both theoretically and experimentally, that DNA assays based on the MEF of PG demonstrate sensitivity to DNA concentration of ≈1 pg/ml, which is several orders of magnitude more sensitive than without the silver nanoparticles, suggesting the broader practical use of this approach (metal-enhanced PicoGreen fluorescence) for the ultra-sensitive detection of

Identification of living organisms (eukaryotes, bacteria, viruses etc.) by means of quantitative analysis of their specific DNA sequences, which represent different genomes, is a challenging aim, which faces many scientists today. It also concerns the search and detection of different microorganism mutations and strains of pathogenic bacteria and causes of severe diseases in humans. In recent short communication, further development of the "Catch and Signal" technology—principles of a 2-color DNA assay for the simultaneous detection/quantification of two genome-specific DNAs in one well was presented (Dragan et al., 2011) In this method a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization has been utilized. Furthermore, it is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver nanoparticles, which is ideal for MEF. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Their results clearly showed that the 2-color DNA assay can effectively be employed as a new "Rapid Catch and Signal" technological platform in the creation of an ultra-sensitive, sequence-specific approach for the fast analysis of genetic material from different organisms, for potential analysis of bacteria and virus pathogens, and search for possible mutations and sequence variations. This technology being fast, ultrasensitive and inexpensive can effectively compete with the PCR technique, especially for the

routine and rapid analysis in Point-of-Care settings and bio-medical laboratories.

For the past 20 years there has been continued growth in the applications of fluorescence spectroscopy in physical and biological sciences. Because of the sensitivity of fluorescence detection, the fluorometeric method has been one of the selected techniques to determine compounds at low concentrations (Lakowicz, 2006). Examples of such compounds are

double stranded nucleic acids.

**3.5 Environmental analysis** 

agrochemicals (pesticides, fungicides, insecticides, etc), which have attracted attention worldwide due to their usage in agriculture. Therefore, the potential increase in fluorescence of a benzimidazole-type fungicide (carbendazim) due to complexation with cucurbit[6]uril was reported (Saleh & Al-Rawashdeh, N.A.F., 2006). The fluorescence enhancement of the fungicide carbendazim by cucurbit[6]uril has been observed in solution due to formation of a host–guest inclusion complex. The enhancement of the carbendazim fluorescence (maximum factor of 10) is accompanied by a significant blue shift in the spectrum (11 nm). In general, Saleh and Al-Rawashdeh work (Saleh & Al-Rawashdeh, N.A.F., 2006) demonstrates the potential usefulness of cucurbit[6]uril in fluorometric analysis of fungicide for agricultural and environmental applications.

Recycling in agriculture of organic wastes produced by various animal breeding, such as pig slurry (PS), is a common practice throughout the world, which has recently raised serious environmental concerns. In particular, Cu(II) and Zn(II) ions, which are used abundantly as pig feed additives, may be introduced in relatively large amounts into PS-amended soils, thus representing an actual risk of phytotoxicity and/or leaching downward the soil with potential endangering groundwater quality (L'Herroux et al., 1997, Saviozzi et al., 1997, Giusquiani et al., 1998, Nicholson et al., 1999, Aldrich et al., 2002, Taboada-Castro et al., 2002, as cited in Hernandez, at al. 2006 ).

Bioavailability, mobility and transport of metal ions in soils are strongly influenced by binding reactions with soil organic matter, and especially its humified fractions, i.e., humic substances (HS), of which humic acid (HA) is the major component (Aldrich et al., 2002, as cited in Hernandez, at al. 2006). For these reasons, the effects of PS application on the compositional, structural and functional properties of native soil HA, and especially on their Cu(II) and Zn(II) binding behavior, need to be accurately evaluated. Therefore, The effect of the consecutive annual additions of pig slurry on the Cu(II) and Zn(II) binding behavior of soil HAs was investigated in a field experiment by a fluorescence titration method (Hernandez et al. 2006). In Hernandez et al. study (Hernandez et al. 2006), a fluorescence titration method was used for determining metal ion complexing capacities and stability constants of metal ion complexes of humic acid isolated from pig slurry and unamended and amended soils. The results of this study is expected to have a large impact on bioavailability, mobilization, and transport of Cu(II) and Zn(II) ions in pig slurry-amended soils (Hernandez et al. 2006).

Moreover, a luminescent sensor utilizing the substrate 2,6pyridine-dicarbox-aldehydebis-(*o*hydroxyphenylimine) has been developed for low concentration detection of the environmental mercuric ion pollutants (Kanan et. al 2009). The sensor selectively detects mercury in the presence of several ions that are commonly found in aquatic environments. The sensor was found to be highly selective with strongest binding was observed between the mercuric ions and the substrate at a pH range of 6.5-7.5 which makes the substrate a distinctive fluorescent sensor for detecting mercury under normal environmental conditions. No effect was demonstrated with the addition of any other metal ions commonly found in water, making this compound selective to mercuric ions.

Dissolved organic carbon (DOC) refers to the hundreds of dissolved compounds found in water that derive from organic materials, and is composed of 'organic acids', 'organic bases', and 'neutral groups'. The amount of DOC in the hydrosphere (700 gigatons) is almost the

Current Achievement and Future Potential of Fluorescence Spectroscopy 237

Ueda (Soh & Ueda 2011). Furthermore, the analytical methods presented in this study are

Fluorescence spectroscopy is an instrumental technique whose theory and methodology has been widely exploited for studies of molecular structure and function. It is therefore applicable to address molecular problems in food. Since many foods exhibit auto fluorescence it is considered highly relevant for characterization purposes and highthroughput screening. The main advantages of molecular fluorescence spectroscopy are its sensitivity and selectivity, in addition to its ease of use, instrumental versatility, speed of

Recently, it has been shown that excitation-emission matrix (EEM) fluorescence spectroscopy and three-way statistical methods, concretely Parallel Factor Analysis (PARAFAC) can be used for distinguishing between wine samples of different appellations, or between wine samples obtained with different ageing procedure (Airado-Rodrguez et al., 2011). At first, excitation-emission matrices (EEMs) were obtained and examined with the aim of identifying the main families of fluorescent compounds. Then PARAFAC was applied for exploratory analysis and the scores of the four selected components were obtained. The set of sample score values obtained in the PARAFAC decomposition were plotted against each other, to visualize clustering trends of samples belonging to different appellations. The potential of the autofluorescence of wine, through its Excitation-emission matrices (EEMs), combined with the three-way method PARAFAC for the purpose of discrimination of wine according to the appellation of origin, is shown in this paper. Wine samples were monitored with rapid and non-destructive front face fluorescence spectroscopy, yielding information about the wine throughout its appellation and ageing conditions. PARAFAC revealed four groups of fluorophores that were responsible for the main fluorescence of Spanish wines. Two of them were assigned to benzoic-like phenolic acids and phenolic aldehydes, and to monomeric catequins and polymeric proanthocyanidin dimers, respectively. The exploration of the matrix of score values obtained by PARAFAC reveals

Rapid measurements of milk properties and discrimination of milk origin are necessary techniques for quality control of milk products. A study was undertaken to evaluate the potential of using front face fluorescence spectroscopy (FFFS) and synchronous fluorescence spectroscopy (SFS) for monitoring the quality of forty-five ewe's milk samples originating from different feeding systems by Hammamia et al. (Hammamia et al., 2010). Whereas, the physicochemical analyses and fluorescence spectra were conducted on samples during lactation periods (the first 11 weeks). Results obtained showed a good discrimination among milk samples with regard to feeding systems given to the ewes throughout the lactation periods. In addition, a better discrimination was observed with front face fluorescence

Fumonisins (FBs) are worldwide distributed and produced by Fusarium verticillioides and Fusarium proliferatum, mainly in corn and corn-based products (Soriano & Dragacci, 2007, as cited in Silva et al., 2009). Although several other fumonisin analogues have been characterized, fumonisin B1 (FB1) remains the most abundant in naturally contaminated

some distribution trends of the samples according with their appellation.

spectroscopy than with synchronous fluorescence spectroscopy.

classified based on the detection mechanisms.

analysis and its non-destructive character.

**3.6 Food analysis** 

same as the amount of carbon in the atmosphere (750 gigatons). However, a large proportion of rainwater DOC is still uncharacterized. A novel method for qualitatively characterizing DOC compounds in rainfall is to use fluorescence. A large proportion of DOC is fluorescent and this fraction can be used to characterize DOC compounds (Baker & Spencer, 2004, as cited in Muller, 2008), yet its full potential for analyzing rainwater DOC compounds has yet to be achieved. Fluorescence can be used to fingerprint fluorophores, identify DOC compounds, and detect small concentration levels which may have otherwise gone undetected, with just 0.04 ml samples. Given the importance of better characterization of rainwater DOC, and the presence of fluorescent DOC compounds, using fluorescence spectrophotometry, primarily sought to examine the fluorescent DOC compounds present in precipitation in Birmingham, UK has been investigated (Catherine et al., 2008). Furthermore, how the fluorescence of the identified DOC compounds varies with meteorological parameters, by assessing the variations with stratiform/convective storm types; examining the variations with air mass type and source area using back-trajectory analysis; and investigating variations with wind speed and wind direction have been investigated (). The results of this study demonstrate the utility of fluorescence analysis for identifying and characterizing rainwater DOC compounds. This research has revealed information regarding fluorescent rainwater DOC compounds in Birmingham, UK and provides evidence in support of using fluorescence spectrophotometry as a means of qualitatively characterizing rainfall DOC.

Organic pollution originating from industrial, agricultural and municipal wastewater discharge has become a common environmental problem in many lakes, rivers, estuaries and coastal waters. The commonly-used methods to study the composition of dissolved organic matter (DOM) (nuclear magnetic resonance, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, size exclusion chromatography, etc.) need complicated sample pretreatment procedures and are not suitable for on-line or real-time determination. Excitation-emission matrix (EEM) fluorescence spectroscopy has been suggested as a powerful tool to characterize aquatic DOM which could detect the specific fluorescent fractions in organic matter from different sources with much higher sensitivity (Coble et al., 1996, as cited in Guo, 2010). Therefore, wastewater dissolved organic matter (DOM) from different processing stages of a sewage treatment plant in Xiamen was characterized using fluorescence spectroscopy by Gue at al. (Guo et al., 2010). The results of this study showed that excitation emission matrix fluorescence of wastewater DOM from a sewage treatment plant revealed high concentration of degradable protein-like fluorescence and the occurrence of xenobiotic-like component. This technique provides a fast and sensitive way to monitor the qualitative and quantitative variation of DOM during the whole sewage treatment process.

Perylene-3,4:9,10-tetracarboxylic bisimide, so-called perylene bisimide (PBI) is known as red vat dyes (*fluorescent dyes*), and has been applied to pigments such as automotive finishes due to its light-fastness, thermal stability, and chemical inertness . Perylene bisimide (PBI) is a fluorescent dye which has strong emission and high photostability. Although PBI has been widely used for industrial materials, the application of PBI in analytical fields was limited mainly due to its high hydrophobicity. In recent years, however, unique and useful analytical methods based on PBI platform are being successfully developed by utilizing the characteristic features of this compound including its high hydrophobicity. In this regard, the recent trend of environmental and biological analysis using PBI was reviewed by Soh & Ueda (Soh & Ueda 2011). Furthermore, the analytical methods presented in this study are classified based on the detection mechanisms.
