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: kopelman@umich.edu (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