**2.1 9,10-Anthracenedipropionic acid (ADPA)**

Ground state of molecular oxygen exists as triplet state with a lowest lying excited state being a singlet state. The singlet state of oxygen can be generated in solution via energy transfer from excited photosensitizers (S, e.g., humic substance or Rose Bengal; Eq. (1)) [17].

*Photophysical Detection of Singlet Oxygen DOI: http://dx.doi.org/10.5772/intechopen.99902*

$$\mathbf{S} \xrightarrow{\mathbf{S}} \mathbf{S}"\xrightarrow{\mathbf{O}\_2} \mathbf{S} + ^1\mathbf{O}\_2 \tag{1}$$

The idea to detect environmentally relevant concentration prompted development of molecular probe that can trap SO and can also be used in studying kinetics. Lindig et.al was the first to introduced 9,10-anthracenedipropionic acid (ADPA) as a quantitative efficient <sup>1</sup> O2 detection probe [18]. The benefit of using ADPA is that it is water soluble and reacts rapidly and irreversible with <sup>1</sup> O2 to form an endoperoxide with the result of photobleaching of absorbance peak approximately at 378 nm (**Figure 2**) [20]. Craig et.al was the pioneer to demonstrate the use of ADPA for the quantitative measure of <sup>1</sup> O2 from the surface of a series of porphyrin-incorporated hydrogels in comparison to the pervious study where ADPA used to ingress into the materials under investigation [19].

#### **2.2 9,10-dimethyl anthracene (DMA)**

Another derivative of anthracene i.e. 9,10-dimethyl anthracene (DMA) reacts almost irreversibly with <sup>1</sup> O2 in various organic and aqueous medium with a significantly high rate constant (6.8 × 107 –5.7 × 1010 M−1S−1) with the result of producing non fluorescent 9,10-endoperoxide [21–24]. Elim Albiter et.al reported a facile photosensitized oxidation of 9,10 demethylanthracene with <sup>1</sup> O2 in presence of safranin O/ silica composite as a heterogeneous photosensitizer [21] in which they reported that oxidation rate does not depend on surface of the composite rather depend only the initial concentration of DMA, light intensity and the amount of composite formed. Their result correlates with the result if the same reaction performed in homogeneous medium. In a similar type experiment Eitan Gross et al. explored DMA inside liposome to study the kinetics of DMA with <sup>1</sup> O2 in presence of photosensitizer [22].

#### **2.3 9,10-diphenyl anthracene (DPA)**

Addition of two phenyl group in 9 and 10 position of anthacene generates a stable and specific <sup>1</sup> O2 trap, 9,10-diphenyl anthracene (DPA) with higher stability of endoperoxide by reaction with <sup>1</sup> O2. However, DPA is not a very suitable candidate as the detection method is based on decrease in absorbance at 355 nm band [25]. V. Nardello et al. enhanced the water solubility of 9,10-diphenyl anthracene (DPA) derivative

**Figure 2.** *The formation of endoperoxide upon reaction of ADPA with 1 O2 (adapted from [19]).*

#### **Figure 3.**

*(a) Interaction of bis-9,10-anthracene-(4-trimethylphenylammonium) dichloride (BPAA) with singlet oxygen resulting into the formation of water soluble endoperoxide (BPAAO2) and (b) UV–Vis absorption spectra of a: BPAA 10−4 M in water and b: the corresponding endoperoxide of BPAA, 10−4 M in water [adapted from [14]].*

by adding two quaternary ammonium functionality with the phenyl ring that do not interfere with <sup>1</sup> O2 and also resulting compound [bis-9,10-anthracene-(4-trimethylphenylamonium) dichloride] BPAA is very much stable with common oxidising agent (**Figure 3**) [14].

UV–Vis absorption band of BPAA is ranging from 320 to 420 nm which is originated from anthracene core structure and once it binds with <sup>1</sup> O2 (**Figure 3**) the absorbance band is quenched totally confirming the formation of endoperoxide.

### **3. Fluorescent probe for the detection of singlet oxygen species**

Among the various available techniques to detect ROS the fluorescence based methodology is an excellent one because of its high sensitivity, high spatial resolution in imaging techniques and also simplicity during data collection [26, 27]. Fluorescent probes are generally non fluorescent before being oxidised by some oxygen species and they are very much specific to some oxidant. Combination of 1 O2 trap and a fluorophores like fluorescein [27, 28] reactive dienes [29] including 9,10-disubstituted anthracene [30–33] etc. are the usual method to develop a fluorescent <sup>1</sup> O2 probe. In fact <sup>1</sup> O2 has huge affinity for biomolecules having cisoid-diene structure and easily undergo [2 + 4] cycloaddition reaction [1, 6, 27]. Thus for most of the probes the fluorescent signals were obtained after [2 + 4] cycloaddition reaction between the probe and 1 O2 (**Figure 4**).

#### **3.1 1,3 Diphenylisobenzofuran (DPBF)**

D. Song et.al [13] synthesized a series of compounds of 1,3 Diphenylisobenzofuran (DPBF) which can acts as ratiometric fluorescence detection probe having singlet oxygen binding rate constant of 9.6 × 108 M−1S−1 in water [13]. Once DPBF reacts with 1 O2 forms nonfluorescent endoperoxide or 1,2-dibenzoylbenzene thus fluorescence signal becomes off. To overcome this practical difficulties they synthesised three more derivatives of DPBF namely phenanthrene substituted phenylisobenzofuran

*Photophysical Detection of Singlet Oxygen DOI: http://dx.doi.org/10.5772/intechopen.99902*

#### **Figure 4.**

*Chemical structure of fluorescence detection probes for 1 O2, (a) DPBF, (b) PPBF, (c) PyPBF and (d) StPBF (adapted from [13]).*

(PPBF), pyrene substituted phenylisobenzofuran (PyPBF) and 4-(diphenylamino) stilbene substituted derivative (StPBF) by substituting one phenyl group of DPBF (**Figure 5**) [13].

These <sup>1</sup> O2 probes exhibit significant red shift in their emission spectrum as the conjugation increases from DBPF to StPBF.

Upon interaction with <sup>1</sup> O2 species the fluorescence signal of DPBF is getting turn off while PPBF, PyPBF and StPBF demonstrate a blue shift of the emission signal with significant ratiometric enhancement of fluorescence as shown in **Table 1**.

#### **Figure 5.**

*(a) Fluorescence spectra of StPBF in absence (red in colour) and with gradually increasing concentration of <sup>1</sup> O2. The spectra gets blue shifted from 505 nm to 435 nm and fluorescence intensity increases with concentration of 1 O2 as directed by arrow in spectra and inset shows the observable colour change from green (in absence) and in presence of 1 O2 (blue in colour). (b) Graph of observed rate constant (Kobs) vs. concentration of various 1 O2 probe (adapted from [13]).*


#### **Table 1.**

*Photophysical data of <sup>1</sup> O2 probes [13]*
