Angewandte
Chemie
DOI: 10.1002/anie.201109052
Fluorescent Probes
Measurement of pH Values in Human Tissues by Two-Photon
Microscopy**
Hee Jung Park, Chang Su Lim, Eun Sun Kim, Ji Hee Han, Tae Hee Lee, Hoon Jai Chun, and
Bong Rae Cho*
Gastroesophageal reflux disease (GERD) is a common
gastrointestinal disorder that is associated with the pH value
of the esophageal lumen. It is a chronic condition in which
mucosal damage is caused by the reflux of acidic gastric
the fluorophore and pyridine as the protonation site, with the
expectation that protonation at the pyridyl nitrogen atom
would result in a red-shifted fluorescence, thereby detecting
the pH by ratiometry (Scheme 1). We adopted a naphthalene
[
1]
contents into the esophagus. Nearly two thirds of the
patients that have GERD-related symptoms are endoscopy-
negative, which is called nonerosive reflux disease
[
2,3]
(
NERD).
Currently available methods for the diagnosis
of NERD include 24 h esophageal pH monitoring, endoscopic
[
4,5]
exam, and a proton-pump inhibitor (PPI) test.
However,
Scheme 1. Proposed mechanism of the equilibrium between NP1 and
NP1H .
none of these methods has yet been accepted as the gold
standard. An interesting hypothesis on the pathophysiology
of GERD proposes that as the dilated intercellular spaces
+
(
DIS) are widened and the tight junctions between the cells
derivative as the fluorophore reporter because two-photon
(TP) probes derived from similar compounds have been
successfully utilized in live-tissue imaging. Pyridine was
are weakend, the refluxed acid infiltrates into the esophagus,
[
6]
[9]
thereby stimulating the nociceptor. To test this hypothesis
and possibly diagnose NERD, it is crucial to measure the pH
deep inside the esophageal tissue. However, except for a one-
photon (OP), near-infrared (NIR), fluorescence-lifetime, pH-
sensitive probe that has been applied to the mouse model,
there is no reliable method of measuring pH values deep
inside human tissue.
chosen as the protonation site because the pK of pyridine is
a
approximately 5.0, which is suitable to detect a slightly acidic
environment. Herein, we report that NP1 is an efficient TP
probe that can estimate pH values in live cells and human
tissues through the use of TPM.
The synthesis of NP1 is summarized in the Supporting
Information. Its solubility as determined by a fluorescence
method is 5 mm in universal buffer solution (pH 7.0), which is
sufficient to stain the cells (Figure S1 in the Supporting
Information). Under this simulated physiological condition,
[7]
An attractive approach to the determination of pH values
deep inside living tissue is the use of two-photon microscopy
(
TPM). TPM, which employs two NIR photons as the
excitation source, offers a number of advantages over one-
photon microscopy (OPM), which include increased depth of
penetration (more than 500 mm), localized excitation, and
prolonged observation time as a result of the use of less-
damaging, lower-energy light. However, there has been no
report on the targeted design and evaluation of a ratiometric
pH probe that is applicable for measuring pH values by TPM.
In this context, we have developed a ratiometric pH probe
that is derived from 2-methoxy-6-(5-oxazolyl)naphthalene as
NP1 has an absorption maximum at l = 342 nm (e =
abs
À1
À1
17000m cm ) and a fluorescence maximum at l = 500 nm
fl
(F = 0.084), which is almost 160 nm red-shifted from the labs.
The unusually large Stokesꢀ shift that is observed for NP1 is
likely to be a result of a great stabilization of charge-transfer
excited state.
[
8]
The absorption and emission spectra of NP1 showed
À
Á
N
T
[10]
gradual bathochromic shifts with the solvent polarity E
in the order, 1,4-dioxane < dimethylformamide (DMF) <
EtOH < H O (Figure S2 and Table S1). The large bathochro-
2
[
+]
[+]
[
*] Dr. H. J. Park, C. S. Lim, J. H. Han, Prof. Dr. B. R. Cho
mic shift for the emission spectra (ca. 70 nm) indicates the
utility of NP1 as a polarity probe. Furthermore, the value of lfl
for NP1 in DMF is similar to that measured in HeLa cells (see
Figure 2b below, see also Table S1), which suggests that this
solvent can adequately represent the polarity of the intra-
cellular environment.
Department of Chemistry, Korea University
145, Anam-ro, Sungbuk-gu, Seoul, 136-713 (Korea)
E-mail: chobr@korea.ac.kr
Prof. Dr. E. S. Kim, Prof. Dr. H. J. Chun
School of Medicine, Korea University (Korea)
T. H. Lee
The UVabsorption of NP1 gradually decreased at 350 nm
and concomitantly increased at 410 nm with a decrease in the
pH value (Figure S3a), presumably as a result of protonation
at the pyridyl nitrogen atom, thereby enhancing the intra-
molecular charge transfer (ICT) and labs (Scheme 1). The
behavior of the emission spectra was similar, except that the
longer wavelength band decreased slightly. The relative
Department of Chemistry, Chungnam National University (Korea)
+
[
] These authors contributed equally to this work.
[
**] This work was supported by grants from the Korea Healthcare
Technology R&D Project, the Ministry of Health & Welfare, the
Republic of Korea (A111182), and the NRF (No. 2011-0020477).
Angew. Chem. Int. Ed. 2012, 51, 2673 –2676
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2673