1. ATu4O.3.pdf CLEO:2016 © OSA 2016
Highly Stable Two-photon Oxygen Imaging Probe
Based on a Ruthenium-Complex Encapsulated in a
Silica-coated Nanomicelle
Aamir A. Khan,1∗ Susan K. Fullerton-Shirey,2 Genevieve D. Vigil,1 Yide Zhang,1 and
Scott S. Howard1
1Department of Electrical Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
2Department of Chemical & Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA 15261, USA.
*E-mail: akhan3@nd.edu
Abstract: A nanomicelle-based ruthenium-complex oxygen imaging probe is coated with a
silica shell. The biostability of the silica-coated probe is improved by a factor of 4, while the
oxygen-sensitivity is reduced by a factor of 3.
© 2016 Optical Society of America
OCIS codes: (180.4315) Nonlinear microscopy, (170.3650) Lifetime-based sensing.
1. Introduction
Optical probes for measuring and imaging oxygen are essential for the research in many areas of engineering and
medicine [1]. Optical approaches for quantitative oxygen imaging involve measuring the emission intensity (I) or
emission lifetime (τ) of a phosphorescence dye, both of which are correlated to the oxygen concentration in the
microenvironment of the dye molecules. Ruthenium(II) metal complexes are commercially available oxygen-sensitive
dyes for a wide range of applications. Previous work by this research team demonstrated a scheme to encapsulate a
hydrophobic oxygen dye ([Ru(dpp)3]2+
) in Pluronic® nanomicelles [2] for oxygen imaging in vitro and in vivo [3–5].
It is shown in ref. [2] that, while the [Ru(dpp)3]2+
/Pluronic® nanomicelle probe is stable in DI water for several
months, it phase separates within a few hours in biological media which is filled with ions, e.g., phosphate buffered
saline (PBS; 7.4 pH) and cell culture growth medium. To address this shortcoming, this paper offers a modification over
the standard [Ru(dpp)3]2+
nanomicelle probe in order to improve its biostability. We demonstrate that a silica coating
can be used to improve the stability of the micelles in biological media by a factor of 4. Quantitative characterization
is also performed to show that the silica shell also reduces the probe’s oxygen sensitivity by a factor of 3, which still
remains comparable to several other oxygen sensing probes. Coupled with easy preparation and a large two-photon
cross-section [2], the silica-coated [Ru(dpp)3]2+
nanomicelle probe can be a viable choice for deep, 3D, imaging of
dissolved oxygen in vivo. Potential applications of this improved probe include blood oxygen monitoring, ischemia
and bone angiogenesis studies, and functional brain imaging in vivo [6–8].
2. Preparation method
A scheme for coating surfactant micelles with silica is described in [9] and is used here with some modifications. A
1% w/w solution of Pluronic® F127 surfactant is prepared in deionized water. The solution is buffered at a pH of ∼ 3
by adding 3.4% w/w of citric acid monohydrate and 2.4% w/w of trisodium citrate dihydrate. The buffered surfactant
solution is used as a precursor for preparing [Ru(dpp)3]2+
nanomicelles. The micelles are prepared according to the
protocol described in the literature [2]. Sodium silicate solution (27.5 wt. % SiO2) is diluted in water at 34% w/w.
For silica coating of the prepared nanomicelles, 50µL of diluted sodium silicate solution is added to 1 mL of micelle
solution while stirring vigorously. In the buffered acidic medium, silica penetrates the hydrophilic periphery of the
micelles and deposits on the outside of the hydrophobic core, forming a shell [9] as depicted in Fig. 1a.
3. Stability observations
The standard [Ru(dpp)3]2+
/F127 nanomicelles (without silica coating) phase separate in biological media within a
few hours due to the presence of electrolytes. In ref. [2], phosphate buffered saline (PBS; 7.4 pH) is found to be the
harshest of all the media tested, yielding a half-life of only 5 hours. Therefore, the silica-coated [Ru(dpp)3]2+
micelles

2. ATu4O.3.pdf CLEO:2016 © OSA 2016
pO
2
(hPa)
0 50 100 150 200
1
1.1
1.2
1.3
1.4
1.5
1.6
Least-squares fit
Measured data
τ0 5.02 µs
Ksv 2.85x10-3
hPa-1
τ0
τ = 1 + Ksv
· pO2
τ0
τ
(c)
Hydrophilic tail
Hydrophobic head
Water
[Ru(dpp)3
]2+
Silica shell
(a)
t = 0 t = 0 t = 12 ht = 0 t = 0 t = 12 h
Dye
precipitate
t = 12 h
uncoated micelles silica-coated micelles
(b)
Figure 1. (a) Structure of a silica-coated Pluronic® nanomicelle with a hydrophobic core encap-
sulating [Ru(dpp)3]2+
. (b) Dye precipitation occurs during the 12 hours period when the uncoated
nanomicelles are kept in PBS. In contrast, the silica-coated micelles remain stable and show no vi-
sual signs of phase separation. (c) Stern-Volmer plot of the silica-coated [Ru(dpp)3]2+
nanomicelles
showing oxygen-sensitivity.
are tested for stability in PBS. Fig. 1b shows that the most of the dye have been precipitated from the solution after
12 hours for uncoated micelles while the coated micelles show no visual sign of degradation during the same period
of time. Even after 20 hours, no visual indication of phase separation is found in the silica-coated micelle solution in
PBS. These results show that the silica shell acts like a steric stabilizer for the [Ru(dpp)3]2+
/F127 nanomicelles.
4. Stern-Volmer characterization
While coating the micelles with a silica shell improves stability, it could also potentially limit oxygen diffusion to
the core, where the dye molecules reside, thus reducing the probe’s oxygen-sensitivity. Therefore it is important to
show that the modified micelles still retain comparable oxygen sensitivity. The Stern-Volmer relationship describes
the oxygen-sensitivity of a probe based on the principle of collisional quenching. The relationship is defined as
τ0/τ = 1+KSV pO2, where τ0 is the emission lifetime in the absence of oxygen, τ at a particular partial pressure
of oxygen (pO2), and KSV is the Stern-Volmer constant. KSV also serves as a measure of oxygen sensitivity as it
defines the dynamic range of the probe’s lifetime.
A gas divider (Horiba Scientific) is used to produce mixtures of air and pure nitrogen in different ratios. Each
mixture, corresponding to a different partial pressure of oxygen, is dissolved in the probe solution and the emission
lifetime is measured with the techniques described in the literature [2]. The measured lifetime data is least-square
fitted to the Stern-Volmer equation to find the values of τ0 and KSV as shown in Fig. 1c. It can be seen that the silica
coating reduces the oxygen-sensitivity of [Ru(dpp)3]2+
nanomicelle probe by a factor of 3 (from 8.47 · 10−3 hPa to
2.85·10−3 hPa), albeit the sensitivity remains comparable to that of some of the other oxygen-sensing probes [2].
References
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