2.1.1 Types of Probing Agents for ROS
Hydrogen Peroxide Oxidized luminol, which is 5-amino-2, 3-diaza-1,
4-naphthoquinone, can react with H 2 O 2 to generate the excited state of
3-aminophthalate ion, AP* [2]. It is also an active fluoregen to
•
O 2
À
. For the
exclusive detection of H 2 O 2 , the luminol solution was added to the reaction mixture,
which was then kept in the dark for ca. 30 min to eliminate
•
O 2
À . Then hemoglobin
(Hem) as an oxidant is added into the mixture to form fluorescence. A detection limit
of about 1 nM can be achieved through this probe. Besides HEM, K 3 Fe(CN) 6 could
also be used as an oxidant to trigger the fluorescence [3]. The application of luminol
is mainly restricted in basic system since it could be used only at higher pH
(pH > 10).
Lucigenin (bis-N-methylacridinium nitrate) is another typical fluorescent probe for
both H 2 O 2 and
•
O 2
À [4], which could be operated at moderate pH (pH ¼ 9). A
dioxetane can be produced after the oxidation, which decomposes to the excited state
of N-methyl acridone. Using the detection of H 2 O 2 as an example, lucigenin solution
was injected into the suspension of TiO 2 after stopping UV irradiation on TiO 2
powder. On the injection, bluish-green fluorescence appeared and decayed in several
seconds. The time integral of the fluorescence intensity is proportional to the amount
of H 2 O 2 , which is used to estimate the H 2 O 2 concentration with calibrations.
Superoxide Oxygen Anion As mentioned above, luminol is also widely used for
probing
•
O 2
À . The detection limit of
•
O 2
À with luminol chemiluminescence is
estimated to be 0.1 nM. As a reference, MCLA (2-methyl-6-(4-methoxyphenyl)-3,
7-dihydroimidazo (1, 2-a) pyrazin-3-one, hydrochloride), a probe for both
•
O 2
À and
1
O 2 , can be used to confirm the exclusive detection of
•
O 2
À together with luminol
[5]. The priority of MCLA is its applicability to neutral pH system.
Singlet Oxygen Sensor A singlet oxygen sensor green (SOSG) was employed to
detect
1 O 2 , which produces an endoperoxide that fluoresces at 528 nm under the
excitation at 488 nm [6]. SOSG shows no appreciable response to
• OH or
• O 2
À .
However, when it was used under light illumination, significant problems in
photosensitized
1 O 2 generation and photodecomposition were clearly demonstrated.
1 O 2 generated in air can also be detected with the terrylene diimide (TDI) derivative,
which produces fluorescent diepoxide [7].
Hydroxyl Radical Fluorescein derivatives are commonly used for the probing of
•
OH. For example, 3
0 -( p-hydroxyphenyl) fluorescein (HPF) selectively reacts with
•
OH to form a strongly emissive fluorescein molecule but does not react with the
other ROS [8]. Nonfluorescent Amplex Red is also a commonly used probe for
•
OH
and
• O 2
À , forming the fluorescent product resorufin [9]. Other probes including
terephthalic acid and coumarin derivatives were also active to generate the fluorescent product for detecting
• OH in solution, which have no absorption at the excitation wavelength for photocatalysts. Different from the detection methods for
• O 2
À
and H 2 O 2 , the probe molecules were present during the photocatalytic reaction, and
18
2 In Situ Characterization of Photocatalytic Activity
Hydrogen Peroxide Oxidized luminol, which is 5-amino-2, 3-diaza-1,
4-naphthoquinone, can react with H 2 O 2 to generate the excited state of
3-aminophthalate ion, AP* [2]. It is also an active fluoregen to
•
O 2
À
. For the
exclusive detection of H 2 O 2 , the luminol solution was added to the reaction mixture,
which was then kept in the dark for ca. 30 min to eliminate
•
O 2
À . Then hemoglobin
(Hem) as an oxidant is added into the mixture to form fluorescence. A detection limit
of about 1 nM can be achieved through this probe. Besides HEM, K 3 Fe(CN) 6 could
also be used as an oxidant to trigger the fluorescence [3]. The application of luminol
is mainly restricted in basic system since it could be used only at higher pH
(pH > 10).
Lucigenin (bis-N-methylacridinium nitrate) is another typical fluorescent probe for
both H 2 O 2 and
•
O 2
À [4], which could be operated at moderate pH (pH ¼ 9). A
dioxetane can be produced after the oxidation, which decomposes to the excited state
of N-methyl acridone. Using the detection of H 2 O 2 as an example, lucigenin solution
was injected into the suspension of TiO 2 after stopping UV irradiation on TiO 2
powder. On the injection, bluish-green fluorescence appeared and decayed in several
seconds. The time integral of the fluorescence intensity is proportional to the amount
of H 2 O 2 , which is used to estimate the H 2 O 2 concentration with calibrations.
Superoxide Oxygen Anion As mentioned above, luminol is also widely used for
probing
•
O 2
À . The detection limit of
•
O 2
À with luminol chemiluminescence is
estimated to be 0.1 nM. As a reference, MCLA (2-methyl-6-(4-methoxyphenyl)-3,
7-dihydroimidazo (1, 2-a) pyrazin-3-one, hydrochloride), a probe for both
•
O 2
À and
1
O 2 , can be used to confirm the exclusive detection of
•
O 2
À together with luminol
[5]. The priority of MCLA is its applicability to neutral pH system.
Singlet Oxygen Sensor A singlet oxygen sensor green (SOSG) was employed to
detect
1 O 2 , which produces an endoperoxide that fluoresces at 528 nm under the
excitation at 488 nm [6]. SOSG shows no appreciable response to
• OH or
• O 2
À .
However, when it was used under light illumination, significant problems in
photosensitized
1 O 2 generation and photodecomposition were clearly demonstrated.
1 O 2 generated in air can also be detected with the terrylene diimide (TDI) derivative,
which produces fluorescent diepoxide [7].
Hydroxyl Radical Fluorescein derivatives are commonly used for the probing of
•
OH. For example, 3
0 -( p-hydroxyphenyl) fluorescein (HPF) selectively reacts with
•
OH to form a strongly emissive fluorescein molecule but does not react with the
other ROS [8]. Nonfluorescent Amplex Red is also a commonly used probe for
•
OH
and
• O 2
À , forming the fluorescent product resorufin [9]. Other probes including
terephthalic acid and coumarin derivatives were also active to generate the fluorescent product for detecting
• OH in solution, which have no absorption at the excitation wavelength for photocatalysts. Different from the detection methods for
• O 2
À
and H 2 O 2 , the probe molecules were present during the photocatalytic reaction, and
18
2 In Situ Characterization of Photocatalytic Activity