**3.8 Nanomaterials**

Noble metal nanocrystals exhibit extraordinary plasmonic properties. The excitation of their localized surface Plasmon resonance modes results in the confinement of electromagnetic waves in regions below the diffraction limit near the metal surface. On the other hand, the localized plasmon modes of elongated metal nanocrystals are inherently anisotropic. The optical signal amplification is therefore expected to be strongly dependent on the excitation polarization direction. Recently, the strong polarization dependence of the plasmonenhanced fluorescence on single gold nanorods has been reported (Ming et al., 2009). In this study, it has been observed that the fluorescence from the organic fluorophores that are embedded in a mesostructured silica shell around individual gold nanorods was enhanced by the longitudinal Plasmon resonance of the nanorods. The polarization dependence is ascribed to the dependence of the averaged electric field intensity enhancement around the individual gold nanorods on the excitation polarization direction. The maximum fluorescence enhancement occurs when the longitudinal Plasmon wavelength of the Au nanorods is nearly equal to the excitation laser wavelength.

Organic dyes, such as fluorescein, rhodamine, and cyanine, and fluorescent proteins, such as green fluorescent protein (GFP) and its variants, are popular probes for cellular organelles and lipid and protein dynamics, due to their small sizes (<5 nm), high specificity, and aqueous solubility. However, because these fluorescent markers rapidly photobleach, have low quantum yield, and exhibit blinking, it is difficult to obtain quantitative spatial and temporal data on the cellular structures they probe (Yao et al., 2005, as cited in Muddana et al., 2009). Recently, the principle photophysical properties of calcium phosphate nanoparticles (CPNPs) using steady-state and time resolved fluorescence spectroscopy, to demonstrate the potential of these particles for biological imaging and drug delivery and to understand the underlying photophysical mechanisms of encapsulation-mediated fluorescence enhancement have been characterized (Muddana et al., 2009). The enhanced

in tap water and tea samples. This study showed the application of a PCT dye for preparation of a new Cu2+ sensitive optical chemical sensor for the first time. The sensor

Heavy metal pollution is a global problem and it causes threat to the environment and human beings. Among the different heavy metal ions, mercury has received considerable attention due to its highly toxic and bioaccumulative properties. It is released from coal burning power plants, oceanic and volcanic emissions, gold mining, and solid waste incineration. Mercury vapor lamps, fluorescent lamps, electrical switches, batteries, thermometers and electrodes are the second largest sources of mercury discharge to the environment. In this regard, an ultrasensitive and selective spectrofluorimetric determination of Hg(II) using 2,5-dimercaptothiadiazole (DMT) as a fluorophore was developed (Vasimalai & John, 2011). In this study, the practical application of the present method was demonstrated by determining Hg(II) in tap water, river water and industrial waste water samples. The obtained results have a good agreement with inductively coupled plasma atomic emission spectrometric (ICP-AES) and atomic absorption spectrometric (AAS) methods. According to the literature, this is the first report for the lowest detection

shows a high selectivity and quick response for Cu2+ over other common metal ions.

with the highest selectivity for Hg(II) in a water medium by the fluorimetric method.

nanorods is nearly equal to the excitation laser wavelength.

Noble metal nanocrystals exhibit extraordinary plasmonic properties. The excitation of their localized surface Plasmon resonance modes results in the confinement of electromagnetic waves in regions below the diffraction limit near the metal surface. On the other hand, the localized plasmon modes of elongated metal nanocrystals are inherently anisotropic. The optical signal amplification is therefore expected to be strongly dependent on the excitation polarization direction. Recently, the strong polarization dependence of the plasmonenhanced fluorescence on single gold nanorods has been reported (Ming et al., 2009). In this study, it has been observed that the fluorescence from the organic fluorophores that are embedded in a mesostructured silica shell around individual gold nanorods was enhanced by the longitudinal Plasmon resonance of the nanorods. The polarization dependence is ascribed to the dependence of the averaged electric field intensity enhancement around the individual gold nanorods on the excitation polarization direction. The maximum fluorescence enhancement occurs when the longitudinal Plasmon wavelength of the Au

Organic dyes, such as fluorescein, rhodamine, and cyanine, and fluorescent proteins, such as green fluorescent protein (GFP) and its variants, are popular probes for cellular organelles and lipid and protein dynamics, due to their small sizes (<5 nm), high specificity, and aqueous solubility. However, because these fluorescent markers rapidly photobleach, have low quantum yield, and exhibit blinking, it is difficult to obtain quantitative spatial and temporal data on the cellular structures they probe (Yao et al., 2005, as cited in Muddana et al., 2009). Recently, the principle photophysical properties of calcium phosphate nanoparticles (CPNPs) using steady-state and time resolved fluorescence spectroscopy, to demonstrate the potential of these particles for biological imaging and drug delivery and to understand the underlying photophysical mechanisms of encapsulation-mediated fluorescence enhancement have been characterized (Muddana et al., 2009). The enhanced

**3.8 Nanomaterials** 

photophysical properties together with excellent biocompatibility make CPNPs ideal for bioimaging applications ranging from single-molecule tracking to in vivo tumor detection while pH-dependent dissolvability of calcium phosphate offers the possibility of timed codelivery of drugs to control cell function.

Fluorescent nanoparticles have attracted increasing research attention due to their promising applications covering electro-optics to bio-nanotechnology. In this regard, monodispersed water-soluble fluorescent carbon nanoparticles (CNPs) were synthesized directly from glucose by a one-step alkali or acid assisted ultrasonic treatment (Li et al., 2011). The results showed that the particle surfaces were rich in hydroxyl groups, giving them high hydrophilicity. The CNPs could emit bright and colorful photoluminescence covering the entire visible-to-near infrared (NIR) spectral range. In this study they conclude that combining free dispersion in water (without any surface modifications) and attractive photoluminescent properties, CNPs should serve as a promising candidate for a new type fluorescence marker, bio-sensors, biomedical imaging, and drug delivery for applications in bioscience and nanobiotechnology.

Recent advances in ultrasensitive protein biosensors have brought significant impacts to proteomics, biomedical diagnostics, and drug discovery (Zhu et al. 2001, as cited in Huang & Chen, 2008). Advanced nanoscale biosensors based on nanoparticles, nanowires, and other nanomaterials have been developed to detect various proteins with improved sensitivity, specificity, and reliability (Fu et al., 2007, as cited in Huang, 2008). Ultrasensitive fluorescence nanosensors can detect the fluorescence signal from a fluorescence tag bound specifically with a single target molecule, but the plain fluorescence intensity measurement can hardly discriminate against a nonspecifically bound tag. Therefore, an electrically modulated fluorescence protein assay that can detect specific fluorescence from a single molecule assembled on an Au nanowire by manipulating the molecule with an electrical potential applied on the nanowire have been developed (Suxian et al., 2008). In their study, they conclude that the simple electrically modulated fluorescence detection method can be generally applied to various bioassays. The essential requirement of the method is to selectively modulate the specific fluorescence from the target molecules by an external reference field, which can be achieved by electrical, optical, magnetic, mechanical, or biochemical interactions etc.

Inorganic nanomaterials have been widely used in biological and environmental fields such as bio-labeling, imaging, drug delivery, separation processes and optical sensing. In these applications, the size and the shape of nanomaterials have very important effects on their properties. Recently, much attention has been given to one- dimensional nanomaterials for building various sensors, due to its high activity, high surface-to-volume ratio, easy assembly in an array for the device and especial suitability for intracellular detection by inserting it into cell (Zhang et al., 2004; Park et al., 2007, as cited in Xu et al., 2011). Therefore, a fluorescence sensor for selective detection of Cu(II) realized by covalently immobilizing derivatives of rhodamine6G (R6G) on the surface of silicon nanowires (SiNWs) has been designed and fabricated (Xu et al., 2011) The fabricated SiNWs-based chemosensor can be electively used for detection of Cu(II) with Cu(II)-special fluorescence enhancement over other metal ions. The Cu(II) sensor exhibits a good selectivity and sensitivity.

Current Achievement and Future Potential of Fluorescence Spectroscopy 243

quenching induced by gold nanoparticles has been observed (Falco et al., 2011). The results showed that laser-induced fluorescence spectroscopy can be used to investigate the alterations in the physiological response of plants induced by gold nanoparticles, and that both excitation wavelengths, 405 nm and 532 nm, were able to detect the presence of the gold nanoparticles inside the plants. Even though, further investigations must be conducted to clarify the

With the increasing nutrient pollution of freshwater ecosystems, lots of reservoirs, including those used as drinking water resources, suffer from extensive cyanobacterial blooms in the summer. Most of cyanobacteria produce a broad range of compounds with various chemical and toxicological properties. Therefore, their occurrence in recreational or drinking water reservoirs represents high health risk for humans and other organisms. In vivo fluorescence methods have been accepted as a quick, simple, and useful tool for quantification of phytoplankton organisms. In this regard, a case study in which fluorescence methods were employed for the selective detection of potentially toxic cyanobacteria in raw water at the drinking water treatment plant has been presented (Gregor et al., 2007). In this study the author demonstrated that presence of cyanobacteria in raw water can be easily monitored by phycocyanin fluorescence. Measured values were in good correlation with the other parameters of cyanobacterial biomass (chlorophyll a, cell counts). However, the blue lightexcited fluorescence of eukaryotic algae should be also monitored to avoid false positive

Photodynamic therapy (PDT) is a treatment modality that may in some cases replace invasive, more harmful or more expensive therapies for certain disorders such as cancer (Dougherty et al., 1998; Ochsner et al., 1997, as cited in Fischer at al., 2002). The PDT treatment process uses photosensitizing compounds that are selectively retained in abnormal tissue some hours to days after administration. At an optimal time after the administration, the photosensitizer is activated with visible light, causing the formation of reactive oxygen species that can initiate a destruction process of the tissue in which they are located. In addition to this photosensitization process, however, most photosensitizers used in PDT show characteristic fluorescence properties (Wagnieres at al., 1998, as cited in Fischer at al., 2002). In this regard, in vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement has been described (Fischer et al., 2002). In their study, in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data were described. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the

Currently, the use of 5-aminolevulinic acid (5-ALA) (Zaak, et al., 2001, as cited in Bulgakova et al., 2009) offers the most promising outlook for a fluorescence diagnosis method to reveal superficial bladder cancer. In this regard, a methodological approach combining fluorescence imaging with in vivo local fluorescence spectroscopy (LFS) was clinically tested in order to improve the specificity of photodynamic diagnosis of superficial bladder cancer after intravesical instillation of Alasense (Bulgakova et al., 2009). This preliminary study, which included 62 patients, suggests that in vivo LFS in the course of fluorescence cystoscopy examinations could possibly minimize false positive fluorescence cases and reduce the required number of biopsies from 5-aminolevulinic acid (5-ALA)-based agent

processes of penetration, translocation, and accumulation of nanoparticles in plants.

signals.

image processing procedure.

induced red fluorescence zones.

Drug delivery systems (DDS) are intensively investigated in the recent years for their great potential to improve the therapeutic index of small molecular drugs. In this regard, recently, new types of fluorescent nanoparticles (FNPs) were prepared through ionic self-assembly of anthracene derivative and chitosan for applications as drug delivery carriers with real-time monitoring of the process of drug release (Wei et al., 2011). In this study, the potential practical applications as drug delivery carriers for real-time detection of the drug release process were demonstrated using Nicardipine as a model drug. Upon loading the drug, the strong blue fluorescence of FNPs was quenched due to electron transfer and fluorescence resonance energy transfer (FRET). With release of drug in vitro, the fluorescence was recovered again. The relationship between the accumulative drug release of FNPs and the recovered fluorescence intensity has been established. Therefore, they conclude that such FNPs may open up new perspectives for designing a new class of detection system for monitoring drug release.

Finally, the current state-of-the-art of gold nanoparticles in biomedical applications targeting cancer has been revised (Cai et al., 2008). In which, gold nanospheres, nanorods, nanoshells, nanocages, and surface enhanced Raman scattering nanoparticles have been discussed in detail regarding their uses in *in vitro* assays, *ex vivo* and *in vivo* imaging, cancer therapy, and drug delivery.

#### **3.9 In vivo fluorescence spectroscopy**

Monitoring phytoplankton classes and their abundance is a routine task in marine scientific research. With the frequent occurrence of red tide (a colloquial term used to refer to one of a variety of natural phenomena known as a harmful algal blooms), there is an urgent need for rapid analytical methods that can provide qualitative and quantitative information. Therefore, in vivo synchronous fluorescence spectra (SFS) of phytoplankton samples for determining the relative abundance of specific classes of phytoplankton (plant-like organisms that can form dense, visible patches near the water's surface) was investigated (Li et al., 2008). This study demonstrates the potential for determining phytoplankton class abundance by in vivo SFS. The database could be expanded to include more phytoplankton species grown under different nutrient, temperature, and photon flux densities. This work brings an in situ method for determining major phytoplankton class abundance by fluorescence measurement of seawater and deserves further studies.

Magnetic nanoparticles have been widely used in biomedical research such as magnetic carriers for bioseparation (Dolye et al., 2002, as cited in Yoo at al., 2007), enzyme and protein immobilization (Cao et al., 2003, as cited in Yoo at al., 2007 ), and contrast-enhancing media (Wu et al., as cited in Yoo at al., 2007). In this regard, in vivo fluorescence imaging method of novel threadlike tissues (Bonghan ducts) inside the lymphatic vessels of rats with fluorescent magnetic nanoparticles has been investigated (Yoo et al., 2007). The results of this study showed new applications of nanoparticles for in vivo imaging of hardly detectable tissues using fluorescence reflectance imaging and magnetophoretic control.

Chlorophyll fluorescence has been used as an accurate and nondestructive probe of photosynthetic efficiency, which can directly or indirectly reflect the impacts of environmental factors and changes in the physiological state of the plants (Baker, 2008, as cited in Falco et al., 2011). Recently, the first in vivo observation of chlorophyll fluorescence

Drug delivery systems (DDS) are intensively investigated in the recent years for their great potential to improve the therapeutic index of small molecular drugs. In this regard, recently, new types of fluorescent nanoparticles (FNPs) were prepared through ionic self-assembly of anthracene derivative and chitosan for applications as drug delivery carriers with real-time monitoring of the process of drug release (Wei et al., 2011). In this study, the potential practical applications as drug delivery carriers for real-time detection of the drug release process were demonstrated using Nicardipine as a model drug. Upon loading the drug, the strong blue fluorescence of FNPs was quenched due to electron transfer and fluorescence resonance energy transfer (FRET). With release of drug in vitro, the fluorescence was recovered again. The relationship between the accumulative drug release of FNPs and the recovered fluorescence intensity has been established. Therefore, they conclude that such FNPs may open up new perspectives for designing a new class of detection system for

Finally, the current state-of-the-art of gold nanoparticles in biomedical applications targeting cancer has been revised (Cai et al., 2008). In which, gold nanospheres, nanorods, nanoshells, nanocages, and surface enhanced Raman scattering nanoparticles have been discussed in detail regarding their uses in *in vitro* assays, *ex vivo* and *in vivo* imaging, cancer

Monitoring phytoplankton classes and their abundance is a routine task in marine scientific research. With the frequent occurrence of red tide (a colloquial term used to refer to one of a variety of natural phenomena known as a harmful algal blooms), there is an urgent need for rapid analytical methods that can provide qualitative and quantitative information. Therefore, in vivo synchronous fluorescence spectra (SFS) of phytoplankton samples for determining the relative abundance of specific classes of phytoplankton (plant-like organisms that can form dense, visible patches near the water's surface) was investigated (Li et al., 2008). This study demonstrates the potential for determining phytoplankton class abundance by in vivo SFS. The database could be expanded to include more phytoplankton species grown under different nutrient, temperature, and photon flux densities. This work brings an in situ method for determining major phytoplankton class abundance by

Magnetic nanoparticles have been widely used in biomedical research such as magnetic carriers for bioseparation (Dolye et al., 2002, as cited in Yoo at al., 2007), enzyme and protein immobilization (Cao et al., 2003, as cited in Yoo at al., 2007 ), and contrast-enhancing media (Wu et al., as cited in Yoo at al., 2007). In this regard, in vivo fluorescence imaging method of novel threadlike tissues (Bonghan ducts) inside the lymphatic vessels of rats with fluorescent magnetic nanoparticles has been investigated (Yoo et al., 2007). The results of this study showed new applications of nanoparticles for in vivo imaging of hardly detectable tissues using fluorescence reflectance imaging and magnetophoretic control.

Chlorophyll fluorescence has been used as an accurate and nondestructive probe of photosynthetic efficiency, which can directly or indirectly reflect the impacts of environmental factors and changes in the physiological state of the plants (Baker, 2008, as cited in Falco et al., 2011). Recently, the first in vivo observation of chlorophyll fluorescence

fluorescence measurement of seawater and deserves further studies.

monitoring drug release.

therapy, and drug delivery.

**3.9 In vivo fluorescence spectroscopy** 

quenching induced by gold nanoparticles has been observed (Falco et al., 2011). The results showed that laser-induced fluorescence spectroscopy can be used to investigate the alterations in the physiological response of plants induced by gold nanoparticles, and that both excitation wavelengths, 405 nm and 532 nm, were able to detect the presence of the gold nanoparticles inside the plants. Even though, further investigations must be conducted to clarify the processes of penetration, translocation, and accumulation of nanoparticles in plants.

With the increasing nutrient pollution of freshwater ecosystems, lots of reservoirs, including those used as drinking water resources, suffer from extensive cyanobacterial blooms in the summer. Most of cyanobacteria produce a broad range of compounds with various chemical and toxicological properties. Therefore, their occurrence in recreational or drinking water reservoirs represents high health risk for humans and other organisms. In vivo fluorescence methods have been accepted as a quick, simple, and useful tool for quantification of phytoplankton organisms. In this regard, a case study in which fluorescence methods were employed for the selective detection of potentially toxic cyanobacteria in raw water at the drinking water treatment plant has been presented (Gregor et al., 2007). In this study the author demonstrated that presence of cyanobacteria in raw water can be easily monitored by phycocyanin fluorescence. Measured values were in good correlation with the other parameters of cyanobacterial biomass (chlorophyll a, cell counts). However, the blue lightexcited fluorescence of eukaryotic algae should be also monitored to avoid false positive signals.

Photodynamic therapy (PDT) is a treatment modality that may in some cases replace invasive, more harmful or more expensive therapies for certain disorders such as cancer (Dougherty et al., 1998; Ochsner et al., 1997, as cited in Fischer at al., 2002). The PDT treatment process uses photosensitizing compounds that are selectively retained in abnormal tissue some hours to days after administration. At an optimal time after the administration, the photosensitizer is activated with visible light, causing the formation of reactive oxygen species that can initiate a destruction process of the tissue in which they are located. In addition to this photosensitization process, however, most photosensitizers used in PDT show characteristic fluorescence properties (Wagnieres at al., 1998, as cited in Fischer at al., 2002). In this regard, in vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement has been described (Fischer et al., 2002). In their study, in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data were described. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the image processing procedure.

Currently, the use of 5-aminolevulinic acid (5-ALA) (Zaak, et al., 2001, as cited in Bulgakova et al., 2009) offers the most promising outlook for a fluorescence diagnosis method to reveal superficial bladder cancer. In this regard, a methodological approach combining fluorescence imaging with in vivo local fluorescence spectroscopy (LFS) was clinically tested in order to improve the specificity of photodynamic diagnosis of superficial bladder cancer after intravesical instillation of Alasense (Bulgakova et al., 2009). This preliminary study, which included 62 patients, suggests that in vivo LFS in the course of fluorescence cystoscopy examinations could possibly minimize false positive fluorescence cases and reduce the required number of biopsies from 5-aminolevulinic acid (5-ALA)-based agent induced red fluorescence zones.

Current Achievement and Future Potential of Fluorescence Spectroscopy 245

excitation and emission, no Raman from the solvent, deep penetration in tissues, single excitation wavelength for many dyes, avoid chromatic aberrations, and no expensive UV optics (for UV excited fluorophores) needed. Even though it has some disadvantages such as only is suitable for fluorescence images (reflected light images is not currently available), the technique is not suitable for imaging highly pigmented cells and tissues which absorb near infrared light, and laser source is expensive. It is worth mentioning that all analysis that can be done using fluorescence spectroscopy can be done using a multi-photon excitation fluorescence microscopy. However, multi-photon excitation fluorescence microscopy has a unique analysis applications over the fluorescence spectroscopy such as: analysis of deep tissue imaging (Brain, skin, etc), can prime photochemical reaction within subfemtoliter volumes inside solutions, cells and tissues (photolabile "caged" compounds), can be used for imaging of living specimen for longer period of time, and for live animal imaging

It could be concluded at the end of this chapter, that the fluorescence spectroscopy is intensely employed as a powerful spectroscopic tool for quantitative analysis, characterization, and quality control in different fields such as inorganic, organic, pharmaceutical, biological and biomedical, food, and environmental analysis. Furthermore, fluorescence spectroscopy has so many applications in optical sensors, and nanamaterials. Compare with other analytical techniques, it must suffice here to add that fluorescence measurements are rapid, accurate and require only very small quantities of sample (nanomole or less) with high selectivity. The principal advantage of fluorescence over radioactivity and absorption spectroscopy is the ability to separate compounds on the basis of either their excitation or emission spectra, as opposed to a single spectra. This advantage is further enhanced by commercial fluorescent dyes that have narrow and distinctly

Airado-Rodrguez, D., Duran-Meras, I., Galeano-Daz, T., Wold, J. P. (2011). Front-face

Akasheh, T.S., Al-Rawashdeh, N.A. F. (1990). Sodium Lauryl Sulfate-Ruthenium(II)

Albert-Garcia, J.R., Anto◌َ n-Fos, G.M., Duartb, M.J., Zamoraa, L. L., Calatayuda, J. M.

*Composition Analysis*, 24, pp.257-264, ISSN: 0889-1575.

*and Pigments*. 83, pp. 211-217, ISSN: 0143-7208.

79, 2, (July 2009), pp. 412-418, ISSN: 0039-9140. Al-Rawashdeh. N.A.F. (2005). Interactions of Nabumetone with

fluorescence spectroscopy: A new tool for control in the wine industry. *J. Food* 

Interactions: Photogalvanic and Photophysical Behavior of Ru(II)-Mimine Complexes. *J. Phys. Chem.,* 94, 23, (Nov. 1990), pp. 8594-8598, ISSN: 0022-3654. Aksuner, N., Henden, E., Yilmaz, I., Cukurovali, A. (2009). A highly sensitive and selective

fluorescent sensor for the determination of copper(II) based on a schiff base. *Dyes* 

(2009). Theoretical prediction of the native fluorescence of pharmaceuticals. *Talanta*,

Fluorescence Measurements. *J. Incl. Phenom. Macrocyclic Chem.,* 51, 1-2, (Feb. 2005),

γ


(intrinsic fluorophores).

separated excitation and emission spectra.

pp. 27-32, ISSN: 0923-0750.

**5. Conclusion** 

**6. References** 
