Development of a hydroxyl radical ratiometric nanoprobe
Matt King*, Raoul KopelmanDepartment of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
Accepted 6 December 2002
Abstract
A hydroxyl radical nanoprobe has been developed by covalently attaching coumarin-3-carboxylic acid (CCA) to amine-functionalized
polyacrylamide (AFPA) nanoparticles (40–100 nm in size). CCA is a non-fluorescent aromatic compound that reacts with hydroxyl radical
(�OH) to produce a fluorescent product, 7-hydroxy-CCA (7-OH-CCA). Texas Red-Dextran (a reference dye) is encapsulated within the
nanoprobe matrix to allow for ratiometric measurements to be made that correct for variations in source intensity, sample and instrument
geometry and dilution.
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Nanoprobe; PEBBLE; Hydroxyl radical
1. Introduction
The hydroxyl radical (�OH) is one of the most reactive
and shortest lived of the reactive oxygen species (ROS) [1].
The ROS include superoxide, singlet oxygen, nitric oxide,
hydrogen peroxide and peroxynitrite. The lifetime of �OH in
biological systems is believed to be about 1 ns [2]. Because
of this, methods used to detect �OH have often been indirect,
using markers such as lipid peroxidation, protein oxidation
and the hydroxylation of DNA bases [1]. Other methods
include ESR [5] (using a spin trap such as DMSO), HPLC
[6] and fluorescence [3,4,7]. Two different methods can be
used for the detection of �OH. One is the direct reaction of a
probe molecule with �OH. The other method is to use a
scavenger that creates a radical species with a longer life-
time. The probe molecule then reacts with this radical
species [7,8].
One of the main challenges of producing a functional
OH probe is to have a reproducible method of generating�OH for the purposes of calibration and testing of the
probe. One method is the photolysis of hydrogen peroxide
by ultraviolet light [9]. This method is simple and clean but
depends on the concentration of hydrogen peroxide and the
intensity of light absorbed. In an another method, a redox
active metal (typically an iron or copper chelate) catalyzes
the dismutation of superoxide [8]. One example of this is
the xanthine oxidase–[Fe(III)EDTA]� system shown in
Eqs. (1)–(5).
XanthineðXÞ þ 2O2 ! 2O2� þ uric acid
ðin the presence of xanthine oxidaseÞ (1)
2O2� þ Hþ ! H2O2 þ O2 (2)
½FeðIIIÞEDTA�� þ O2� $ ½FeðIIÞEDTA�2� þ O2 (3)
½FeðIIÞEDTA� þ O2� þ 2Hþ ! ½FeðIIIÞEDTA�� þ H2O
(4)
½FeðIIÞEDTA�2� þH2O2 ! ½FeðIIIÞEDTA�� þOH� þ �OH
(5)
The third method is the generation of �OH by the Fenton or
Haber–Weiss system with a molecule such as ascorbic acid
that can be oxidized to regenerate the metal catalyst [10].
Coumarin-3-carboxylic acid (CCA) reacts directly with�OH to produce the highly fluorescent compound, 7-hydroxy-
coumarin-3-carboxylic acid (7-OH-CCA) (Fig. 1) [3], and
this reaction has been shown to be specific for detection of�OH [3,4]. 7-OH-CCA has excitation maxima around 325
and 385 nm and emits at 450 nm.
Sensors and Actuators B 90 (2003) 76–81
Fig. 1. Reaction of CCA with OH to produce 7-OH-CCA.
* Corresponding author.
E-mail address: [email protected] (M. King).
0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0925-4005(03)00100-X
In previous work, CCA has been conjugated to biomo-
lecules by reaction of the succinimidyl ester of CCA
(SECCA) with the amine groups on the biomolecules [4].
This method has been used to study �OH effects caused by
localized generation of �OH [4].
In this study, �OH PEBBLE probes have been prepared
using a similar reaction (Fig. 2). A comprehensive review of
PEBBLE technology is contained in [11]. SECCA reacts
with amine groups on an amine-functionalized polyacryla-
mide (AFPA) PEBBLE. The number of reactive amine
groups on a PEBBLE increases with the square of the
diameter of the PEBBLE.
The large surface to volume ratio of these PEBBLE
nanoprobes compared to that of a single larger probe
enhances their performance as a radical sensor. Hydroxyl
radicals can only be sensed on the surface of the probe
because of their high reactivity. Many PEBBLE nanoprobes
can be used inside a cell without taking up a significant
portion of the cell’s volume.
Ratiometric measurement uses the ratio of two fluores-
cence peaks instead of the absolute intensity of one peak.
This form of measurement eliminates effects due to fluctua-
tions in intensity of the light source, the concentration of
dyes or PEBBLEs, the alignment of optics or the placement
of the sample cell [11]. To make these probes ratiometric, a
reference dye, Texas Red-Dextran (emission peak, 595 nm)
is added during the PEBBLE synthesis. This reference dye is
encapsulated in the matrix to protect it from the outside
environment and from reaction with �OH. The ratio of the
two fluorescence peaks is measured instead of the absolute
intensity (Fig. 3).
2. Experimental methods
2.1. Chemicals
All chemicals except the following listed were ordered
from Aldrich (Milwaukee, WI). N-(3-Aminopropyl)metha-
crylamide hydrochloride (APMA) from Polysciences (War-
rington, PA), 7-OH-SECCA, SECCA, Texas Red-Dextran
from Molecular Probes (Eugene, OR), xanthine oxidase was
purchased from Sigma.
2.2. Preparation of AFPAA PEBBLEs
A solution of 600 mg APMA, 600 mg acrylamide,
360 mg bisacrylamide and 50 ml (25 mg/ml) Texas Red-
Dextran, dissolved in 4 ml phosphate buffer was prepared
by sonication. During this time, 1590 mg dioctyl sulfosuc-
cinate (AOT), 3.2 ml Brij 30 and 43 ml of hexane were
added to a 100 ml round-bottomed flask. Argon was then
bubbled through the solution for 15 min. Two milliliter of
acrylamide monomer solution was then added. After 15
min, 80 ml 10% ammonium persulfate and 60 ml N,N,N0,N0-tetramethylethylenediamine (TEMED) was added to initi-
ate the polymerization. The solution was allowed to stir
overnight and then the hexane was removed by vacuum
evaporation. The residue left in the flask was re-suspended
in ethanol and washed three times with a solution of
50% ethanol/50% phosphate buffered saline (PBS, (v/v))
Fig. 2. Conjugation of SECCA to AFPA PEBBLEs (R is AFPA PEBBLE).
Fig. 3. �OH PEBBLE probe. The reference dye (Texas Red, shown in
gray) is encapsulated in the matrix during the synthesis. The AFPA
PEBBLEs are washed and re-suspended after the synthesis is complete,
then SECCA is reacted with the amine groups on the AFPA PEBBLE to
form a covalent bond between CCA (�OH probe dye, shown in black) and
the AFPA PEBBLE.
Fig. 4. Drawing of experimental cell used for the production of OH by
hydrogen peroxide photolysis with illumination area shown in center.
M. King, R. Kopelman / Sensors and Actuators B 90 (2003) 76–81 77
three times. The volume of the solution was reduced
to 20 ml by filtration and moved to a vacuum filter for
drying.
2.3. Preparation of �OH PEBBLEs
The 570 mg of Texas Red containing amine functiona-
lized polyacrylamide (AFPA) PEBBLEs were dissolved in
30 ml 0.1 M sodium bicarbonate by sonication. Three
milliliter of a SECCA solution (1.66 mg/ml in dimethyl-
formamide (DMF)) was then added to the PEBBLE–
bicarbonate solution. The reaction was allowed to con-
tinue overnight, transferred to an Amicon fliter, and
washed three times with 50% ethanol/50% PBS (v/v)
(Fig. 4).
2.4. PEBBLE fluorescence measurements
2.4.1. Experimental set-up 1
This set-up was used for the production of OH by
hydrogen peroxide photolysis shown by Eq. (6).
H2O2 þ hn ! 2�OH (6)
The excitation source was a mercury arc lamp with a UV-
filter cube (excitation 300–400 nm). Measurements were
obtained with an inverted microscope with a spectrograph
and liquid nitrogen cooled CCD (Princeton Instruments,
Trenton, NJ). This experimental set-up was used for mea-
surements using hydrogen peroxide photolysis for �OH
production. The sample cell was made from quartz with a
diameter of 30 mm. The radical was only produced in the
irradiated area. A drawing of the sample cell is shown in
Fig. 5.
2.4.2. Experimental set-up 2
A Fluoromax-2 (Jvon-Harba Spex) fluorimeter was
also used for the experiments with the xanthine oxi-
dase–[Fe(III)EDTA]� method of generating hydroxyl
radicals and also for test for response from hydrogen
peroxide.
2.5. Radical generation
Hydroxyl radical production was accomplished using two
different methods, hydrogen peroxide photolysis and the
xanthine oxidase–[Fe(III)EDTA]� system described in [8].
2.6. Tests for interference from hydrogen peroxide
Hydrogen peroxide. A stock solution of 2 mg/ml CCA
PEBBLEs in 7.2 mM phosphate buffer was prepared by
sonication. One milliliter of this stock solution was pipetted
into a quartz cuvette and placed in a Fluoromax-2 (Jvon-
Harba Spex) fluorimeter. The experiment was repeated
twice without any ethanol to check reproducibility and then
was repeated four more times, each time using an increasing
amount of ethanol. The 10 ml of 30% (w/w) hydrogen
peroxide was then pipetted into the cuvette and the solution
was constantly stirred during the experiment. Excitation
was at 366 nm (6 nm bandpass slit width) and the emission
wavelength was 450 nm (3 nm bandpass slit width).
3. Results and discussion
A radical scavenging test using ethanol (a �OH scaven-
ger) was done to see if the response from the PEBBLE was
due to �OH or hydrogen peroxide. The results (Fig. 5) show
that the fluorescence response is due to �OH. The two lines
marked 0% are from two separate runs without any ethanol
present. Each run was conducted separately and only the
amounts of ethanol and buffer were changed for each run.
The number written beside the line corresponds to the
concentration of (vol.%/v) ethanol. The concentration of
CCA and hydrogen peroxide were kept the same. This test
was done using PEBBLEs that did not contain a reference
dye and the intensity listed is the absolute intensity in counts
per second (CPS).
Ethanol is an �OH scavenger. The response from CCA
decreases with the addition of a small amount of ethanol and
this indicates that CCA is responding to �OH and not
Fig. 5. �OH scavenging experiment using ethanol.
78 M. King, R. Kopelman / Sensors and Actuators B 90 (2003) 76–81
hydrogen peroxide. Then PEBBLEs used in this experiment
do not contain a reference dye and the difference between
the two measurements at 0% shows that absolute intensity
measurements can be unreliable, even if the conditions are
exactly the same.
A reference dye was then added to the next batch of
PEBBLEs and the response of the PEBBLEs is shown in
Fig. 6(a-c). In the experiment shown in Fig. 6(a-c), 250 ml of
2 mg/ml PEBBLEs, 25 ml of 30% (w/w) hydrogen peroxide
and 3.75 ml 10 mM pH 7.2 phosphate buffer were added to
the sample cell. An amount of 250 ml more of PEBBLE
solution was added to the reaction mixture after 51 and 112 s
(at sharp drop in graph of Fig. 6(a)).
The Texas Red signal increases immediately (Fig. 6(b))
after each addition of PEBBLEs and then remains fairly
constant. The CCA signal (Fig. 6(c)) actually decreases at
the time of each addition and then increases slowly to an
approximately steady value and then declines near the end of
the experiment—see following page. The signal then
increases until most of the PEBBLEs in the reaction zone
are depleted and an equilibrium is established. PEBBLE
response is shown by the ratio of the two peaks in Fig. 6(a)
and is relatively independent of the concentration of PEB-
BLEs in the solution.
Hydrogen peroxide photolysis is in principle simple and
clean. However, calibration requires knowing the exact
concentration of hydrogen peroxide and the intensity of
the light absorbed. Another method (see Eqs. (1)–(5)) is
to used xanthine oxidase to produce superoxide and then
use a metal catalyst to produce �OH. Theoretically, one �OH
is produced for every three superoxide molecules. The
reduction of cytochrome c absorbance at 550 nm (usually
Fig. 6. Ratiometric response of �OH PEBBLE. (a) Ratio of CCA/Texas Red peaks. (b) Texas Red (reference dye) response during experiment. (c) CCA (�OH
probe dye) response.
M. King, R. Kopelman / Sensors and Actuators B 90 (2003) 76–81 79
plotted as change in absorbance 550 versus time) is used to
measure the amount of superoxide produced by xanthine
oxidase (Fig. 7).
The enzyme can degrade over time and lose activity, so
the superoxide production must be checked before the
experiment is run. At a high enough concentration of
Fe(III)–EDTA, the production of superoxide to OH is 3:1.
In the OH production experiments, the amount of xanthine/
xanthine oxidase was kept constant, and a solution of OH
PEBBLEs and Fe(III)EDTA was substituted for the cyto-
chrome c solution. The total volume was kept constant and
the results of the two runs are shown in Fig. 8a and b.
The production of superoxide leveled off after approxi-
mately 300 s but remained relatively constant between the
two runs. For the Texas Red Reference dye this peak over-
laps both the Texas Red and CCA peaks and makes it
difficult to get quantitative data using this method—see
following page.
4. Concluding remarks
A ratiometric nanoprobe has been prepared for the detec-
tion of hydroxyl radical. The inclusion of a reference dye
eliminates effects due to fluctuations in source intensity,
PEBBLE concentration, alignment of optics, and the place-
ment of cells fluctuations and placement of the sample cell.
This nanoprobe also has a large surface to volume ratio,
Fig. 7. Production of superoxide by xanthine oxidase.
Fig. 8. (a–b) Production of �OH by xanthine oxidase–Fe(III)EDTA system measured with �OH PEBBLEs.
80 M. King, R. Kopelman / Sensors and Actuators B 90 (2003) 76–81
which allows for a much greater sensing area than is possible
with a single large probe.
The high reactivity and short lifetime of �OH present a
unique challenge in the production of a reliable and calibra-
table probe. But it is also a necessary one to be met in order to
determine the precise role that �OH plays in human diseases.
Acknowledgements
The authors acknowledge support from the National
Institutes of Health Grant number 8R01-EB00250 and the
National Cancer Institute Grant number N01-CO-07013.
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Biographies
Matt King acquired his BS degree in chemistry at Bemidji State University
in Bemidji, MN, in 1998. He is currently a graduate student at the
University of Michigan (Ann Arbor, MI) and his main fields of interest are
the production and detection of free radicals and reactive oxygen species.
Raoul Kopelman is currently Kasimir Fajans Collegiate Professor of
chemistry, physics and applied physics and a member of the Biophysics
Program and the Center for Biological Technology at the University of
Michigan, Ann Arbor. He has a BS and Dipl Eng in chemical engineering
from the Technion, Israel, Institute of Technology, as well as an MS in
physical chemistry. After having received a PhD at Columbia University,
he spent 2 years at Harvard University, 2 years as an instructor at the
Technion, and 2 years at the California Institute of Technology before
coming to Michigan. He is a fellow of the American Physical Society and
the American Association for the Advancement of Science, and received
the Edward Morley Award from the American Chemical Society.
Kopelman is the author of over 400 scientific papers. Current research
interests are in non-classical reaction kinetics, scanning photon and exciton
tunneling microspectroscopy, super-molecular antenna, and ultra-small
opto-chemical sensors and actuators. Kopelmans research contributions
include the discovery of pseudolocalized phonons; the measurement of
exciton exchange and super-exchange energies in molecular crystals; the
demonstration of quantum percolation via exciton trapping and annihila-
tion; the initiation of non-classical reaction kinetics experiments and
Monte Carlo simulations; the concept and practice of active sub-
wavelength light sources and their application to near-field scanning
microscopy; and the construction and application of optical nanosensors
and nanoprobes for chemical and biomedical applications.
M. King, R. Kopelman / Sensors and Actuators B 90 (2003) 76–81 81