Sensors and Materials, Vol. 14, No. 6 (2002) 309-338

MYUTokyo
S &M0492

Production, Characteristics and Applications of
Fluorescent PEBBLE Nanosensors:
Potassium, Oxygen, Calcium and pH Imaging
inside Live Cells
Murphy BrasueP, Raoul Kopelman 1 *, Jonathan W. Aylott 1 , Heather Clark1 ,Hao Xu 1, Marion Hoyer2, Terry J. Miller2, Ron Tjalkens2 and Martin A. Philbert2
Department of Chemistry' and Department of Environmental Health Sciences2,
The University of Michigan, Ann Arbor, Michigan48109, U.S.A.
(Received October 5, 2001; accepted February 23, 2002)
Key words: optodes, PEBBLEs, nanoparticles, nanospheres, acrylamide, cross-linked decyl meth­acrylate, sol-gel, pH, calcium, potassium, oxygen

A novel platform for the intracellular monitoring of key biological components has
been developed, using three different nanoparticle fabrication technologies. These nano­
optodes are termed PEBBLEs (Probes Encapsulated By Biologically Localized Embed­ding). The sensors, based on polyacrylamide, cross-linked decyl methacrylate, and silica­
based sol-gel, have been characterized in aqueous solution and also tested in intracellularsurroundings. Each matrix can be combined with specific "free dyes", ionophores, or
enzymes to produce sensors selective for the biological component of interest. · Sphericalsensors less than 600 nm in diameter (and reducible to below 100 nm) have been madefrom all three matrices. Acrylamide-based sensors have been used to monitor intracellular
pH and calcium (with proven selectivity over Mg 2+). Decyl methacrylate has beensuccessfully applied to intracellular potassium monitoring with probes 1,000 times more
selective for potassium than sodium. Sol-gel has proven useful for monitoring intracellularoxygen at physiologically interesting concentrations. PEBBLEs, with a wide range of both
simple and complex sensing schemes, provide a unique tool for minimally invasive
intracellular monitoring, with many significant advantages over free dyes as well as overfiber-optic sensors.

*Corresponding author, e-mail address: kopelman@umich.edu

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1.

Sensors and Materials, Vol. 14, No. 6 (2002)

Introduction

A traditional strength of chemical sensors (optical, electrochemical, etc.) is the minimi­
zation of the chemical interference between the sensor and sample, achieved with the useof inert, biocompatible matrices or interfaces. However, with respect to penetrating
individual live cells, the size of the sensor results in physical inte,ference, accompanied byserious biological damage and resultant biochemical consequences.
Optical methods for analyzing intracellular ion concentrations have become widelyused biological tools.0-4) Injection of fluorescent indicator dyes into a cell, combined withratiometric imaging, confocal microscopy, two-photon fluorescence, or fluorescence life­
time imaging has provided insights into concentration and spatial location of ions through­out a single cell. One challenge of using these optical methods is that each dye for use in
intracellular measurements must be chosen carefully and assessed for its ability to enterand then provide accurate and reliable information from within a cell. Problems such as
toxicity to the cell, intracellular sequestration, non-ratiometric properties, protein binding
and intracellular buffering must be evaluated.<5-3l While some indicator dyes work wellwithin a cell, many fluorescent probes suffer from the above problems, thus limiting theindicator dyes available for reliable intracellular measurements. For example, common
calcium probes can buffer a cell when used in high concentrations,<9l and pH indicatorssuch as carboxyfluorescein and BCECF can be affected by self-quenching and by binding
to proteins.
By entrapping the indicator dyes within a polymer matrix, optodes provide a method forminimizing many of the undesired interactions that occur between fluorescent probes andthe cell. The matrix allows ions or neutral analyte species to diffuse through and bind with
the indicator, but prevents mobility of the indicator dyes through the cell, preventing
sequestration and self-quenching. In short, the matrix protects the cell from the dyes andthe dyes from cell components. Another advantage of using an optode rather than a free
dye is the ability to use complex (tandem) sensing schemes to tailor selectivity, dynamic
range, and the co-localization of reference dyes (to correct for light source or sensor
concentration fluctuations). However, optodes have traditionally been bulky and imprac­
tical for single cell measurements, mainly due to the connecting fibers, which take up asignificant amount of space inside the cell. Also, the thickness of the polymer film has
prevented rapid measurements, as diffusion through the matrix can be slow. To overcome
this problem, the last decade saw significant progress in miniaturization of electrodes andfiber-optic optodesY 0- l However, even reductions to tips with 100 nm radius or below are
not satisfactory.O l l In contrast, individual molecular probes (free sensing dyes) arephysically sufficiently small, but usually suffer from chemical interference between theprobe and cellular components. Furthermore, their use in complex, multi-dye sensing
schemes is impractical.
A recent development in sensor design attempts to combine the advantages of sensor
tips and molecular probes, i.e., to simultaneously avoid both physical and chemicalinterference between sensor (probe) and sample (cell or organelle). These sphericalnanosensors have been termed PEBBLEs and, due to their chemically inert matrices andtheir small physical sizes, provide an almost non-perturbing, real time measurement inside
intact biological systems. The small size, while important in minimizing cell perturbation,
11

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311

also allows rapid measurement, because even with slow diffusion, the nanosensors reachequilibrium quickly. It also allows improved cell delivery.
The use of fluorescent indicator molecules in encapsulated form (polyacrylamide
PEBBLEs) has proven valuable in the study of a number of intracellular analytesc12-15l (H+,Ca2+, Mg2+, 02). However, there are many ions for which no fluorescent indicator dye issufficiently selective or even available. An alternate class of optical nanosensors was thus
required.
Decyl methacrylate (DMA) based sensors, with much higher selectivity for most ions,relies on the chemical equilibrium (or steady state) among different sensor components.
These sensors act as "bulk optodes" or ion selective optodes (ISO) where the matrix(hydrophobic liquid polymer) contains a selective lipophilic ionophore ("optically silent"),
a fluoroionophore and an ionic additive.crn. l6-l9l The operation of the entire system is basedon having a thermodynamic equilibrium that controls ion exchange (for sensing cations) orion co-extraction (for sensing anions), i.e., an equilibrium based correlation between
different ion species. To achieve sensor miniaturization, fluorescence (rather than absor­
bance), has been utilized. Fabrication of PEBBLEs based on the described scheme, in thesubmicron size range, has been demonstrated for K+, and extension to existing ionophores
for other cations as well as anion analytes involves a simple optimization of the matrix forthe required sensing elements.<20J Alternative liquid polymer sensing particles have also
been recently developed by Tsagkatakis et al. <21 l and Peper et al. <22l utilizing a dispersion
method to cast PVC particles and plasticizer free dodecyl acrylate microspheres for ion
sensing.
Sol-gel "glass" has also been used as the matrix for the fabrication of PEBBLE
nanosensors, because of its superior properties over organic polymers. Sol-gel glass is aporous, high purity, optically transparent and homogeneous material,c23l thus making it an
ideal choice as a sensor matrix for quantitative spectrophotometric measurements. Also, it
is chemically inert, photostable and thermally stable, compared with polymer matrices.
The preparation of sol-gel "glasses" is technically simple and tailoring the physico­chemical properties (i.e. pore size or inner-surface hydrophobicity) of sensor materials canbe achieved easily by varying the processing conditions and the amount or type of reactants
used. Pore size can be optimized such that the analyte is able to diffuse easily and interact
with the sensing molecules while the latter are prevented from leaking out of the matrix(also true for polyacrylarnide based sensors). Furthermore, this "glass" is produced underso-called soft chemical conditions, allowing the inclusion of biomolecules.
A range of sol-gel sensor configurations has been described in the literature, including
monoliths, thin films, miniaturized probe-tips and powders.<23l Immobilization of thesensing reagent in a supportive matrix is a critical step in the fabrication of optical sensors.
It can be achieved by either chemical or physical entrapment ( of the fluorescent dye
molecules) in the pore structures of the sol-gel network. An important advantage ofphysical entrapment is the minimal alteration in the spectral and binding properties of thesensing molecules, due to the weak interactions with the supporting matrix. A key
advantage of sol-gel sensors is that the "soft" chemical techniques used are ideal for theentrapment of enzymes and proteins. For instance, one can incorporate oxidase enzymes
into proven ratiometric oxygen sensing sol-gel PEBBLEs<24l for the sensing of analytes
such as glucose (using the glucose oxidase enzyme).

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The nanoemulsion process for preparing PEBBLEs is subtle and there is no universal
method for making hydrophilic, hydrophobic, and ampiphilic nanospheres that contain thecorrect matrix and chemical components, in their proper proportions. Thus, switchingfrom single dye containing hydrophilic polyacrylamide nanospheres to multicomponent,
hydrophobic, liquid polymer sensors or to inert glass sol-gel sensors is not yet a routine
procedure. Specific methods for producing sensors from all these matrices are described inthis report.

2.

Materials and Methods

2.1 Materials for PEBBLE production

Reagents for PEBBLEs Acrylamide, N,N-methylenebis[acrylamide], N,N,N',N'­tetraethylmethy lenediamine (TEMED), tetraethyl orthosilicate (TEOS),
hexanedioldimethacrylate (HDDMA) and kainic acid were purchased from Aldrich (Mil­waukee, WI). Ammonium persulfate, Bis (2-ethylhexyl) sebacate (DOS), Brij 30, dioctyl
sulfosuccinate, sodium salt (AOT), chromoionophore III or 9-(Diethylamino)-5-[(2octoyldecyl)imino]benzo[a]phenoxazine (ETH 5350), potassium ionophore III or 2Dodecyl-2-methyl-1,3-propanediylbis[N-[5 'nitro(benzo-15-crown-5)-4,-yl]carbamate](BME-44), potassium tetralds-[3,5-bis(trifluoromethyl)phenyl borate (KTFPB), polyeth­
ylene glycol (PEG) 5'000 monomethyl ether, polyethylene glycol (PEG) 6'000, polyethyl­ene glycol (PEG) 20'000, tetrahydrofuran (THF) potassium peroxodisulfate, potassium
chloride (KCl), and sodium chloride (NaCl) were obtained from Fluka (Ronkonkoma, NY)
and used without further purification. Decyl methacrylate (DMA) was obtained from
Pfaltz & Bauer (Waterbury, CT). 5 (and 6)-carboxynaphthofluorescein (CNF), 5-(and -6) carboxy-4,,5,-dimethylfluorescein (CDMF), 2,,7,-bis-(2-carboxypropyl)-5-(and-6)­
carboxyfluorescein (BCPCF), fluorescein-5-(and -6)-sulfonic acid (FSA), 5-(and-6)-carboxy
SNAFL®-1 (SNAFL), sulforhodamine 101 (SR), Calcium Crimson, Calcium Green,
Calcium Orange, Calcium Green 5N and Oregon Green 488-dextran were purchased from
Molecular Probes, Inc. (Eugene, OR). Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline) chlo­ride ([Ru(dpp)3]Clz), was purchased from GFS Chemicals, Inc.(Columbus, OH). Ethanol(200 proof) was obtained from Pharmco Products Inc. (Brookfield, CT).
Gases 02 (99.99%, extra dry grade) and N2 (99.99%, extra dry grade) were obtained
from Cryogenic Gases (Detroit, Ml).

2.2 PEBBLE fabrication

Preparation of Polyacrylamide PEBBLEs The polymerization solution consistedof 0.4 mM fluorescent ionophore, 27% acrylamide and 3% N,N-methylenebis (acrylamide)
in 0.1 M phosphate buffer, pH 6.5. One mL of this solution was then added to a solution
containing 20 mL hexane, 1.8 mmol AOT and 4.24 mmol Brij 30. The solution was stirred
under nitrogen for 20 min, while cooling in an ice bath. The polymerization was initiatedwith 24 µL of a 10% ammonium persulfate solution and 12 µL TEMED, then the solution
was stirred at room temperature for 2 h. Hexane was removed by rotary evaporation, thenthe probes were rinsed of surfactant with ethanol, to yield a product consisting of 20 and
200 nm probes.<1 2.13l

Sensors and Materials, Vol. 14, No. 6 (2002)

313

Decyl methacrylate (DMA) PEBBLE Preparation A batch of PEBBLE sensors
typically consisted of 210 mg of DMA, 180 mg of HDDMA, 300 mg of dioctyl sebacate,with 10-30 mmol/kg each of ionophore, chromoionophore, and ionic additives added after
spherical particle synthesis. The spherical particles were prepared by dissolving decyl
methacrylate, HDDMA, and DOS in 2 mL of hexane. To a 100 mL round bottom flask, ina water bath on a hot plate stirrer, 75 mL of pH 2 HCl was added along with 1.8 g of PEG
5'000 monomethyl ether, or PEG 6'000, or PEG 20'000 and stirred and degassed. The
hexane dissolved monomer cocktail was then added to the reaction flask under nitrogenand stirred at full speed, and the water bath temperature was raised to 80°C over 30--40 min.
6 mg of potassium peroxodisulfate was then added to the reaction and stirring was reduced
to medium speed. The temperature was kept at 80°C for two more hours, and then thereaction was allowed to return to room temperature and stirred for 8-12 h. The resulting
polymer was suction filtered through a Fisherbrand glass microanalysis vacuum filter
holder with a Whatman anodise filter (0.2 µm pore diameter). The polymer was rinsed
three times with water and three times with ethanol to remove excess PEG and unreacted
polymer. THF was then used to leach out the DOS and then the PEBBLEs were again
filtered and rinsed. They were allowed to dry in a 70°C oven overnight. Dry polymer wasthen weighed out, and DOS, BME-44, ETH 5350 and KTFPB were added to this dry
polymer, so that the resulting polymer would have 40% DOS, 20 mrnole/kg BME-44, 10
mmole/kg ETH 5350, and 10 mrnole/kg KTFPB. Sufficient THF was added to this mixture
to just wet the PEBBLEs. The PEBBLEs were allowed to swell for eight hours and then the
THF was removed by roto-evaporation. The resulting PEBBLE sensors were rinsed withdouble distilled water and allowed to air dry. c20l
Preparation of Sol-Gel PEBBLEs The reaction solution consisted of PEG MW
5'000 monomethyl ether (3 g), ethanol (6 mL), Oregon Green-dextran MW 10,000 (0.1mM), [Ru(dpp)3]2+ (0.4 mM), and 30% wt. ammonia water (3.9 mL) with ammonia serving
as a catalyst and water being one of the reactants. Upon mixing, the solution became
transparent and tetraethyl orthosilicate (TEOS) (0.5 mL) was added dropwise to initiate thehydrolysis of TROS. The solution was then stirred at room temperature for 1 hour to allow
the sol-gel reaction to reach completion.
A liberal amount of ethanol was then added to the reaction solution and the mixture wastransferred to an Amicon ultrafiltration cell (Millipore Corp., Bedford, MA). A 100 kDa
membrane was used to separate the reacted sol-gel particles (PEBBLEs) from the unreacted
monomers, PEG, ammonia and dye molecules, under a pressure of 10 psi. The PEBBLEs
were further rinsed with 500 mL ethanol to ensure that all unreacted chemicals had been
removed from the PEBBLEs. The PEBBLE solution was then passed through a suction
filtration system (Fisher, Pittsburgh, PA) with a 2 µm Whatman anodise filter to separate
the larger size particles from the smaller ones. The filtrate (containing the smaller particles)
was filtered again, this time with a 0.02 µm Whatman anodise filter, to collect the particles
which were then dried to yield a final product consisting of sol-gel PEBBLEs in the size
range of 100-600 nm in diameter.<24l314

Sensors and Materials, Vol. 14, No. 6 (2002)

2.3 Imaging and optics
Previous publications give the specific methods for calibration and imaging used foreach specific matrix; acrylamide,U2-15J decyl methacrylate,c20i and sol-gel.c24l
Sensor calibrations were accomplished using either a FluoroMax-2 spectrofluorometer(ISA Jobin Yvon-Spex, Edison, NJ), slits set to 2 nm for both the emission and excitation,or an Olympus inverted fluorescence microscope, IMT-II (Lake Success, NY), using
Nikon 50 mm f/1.8 camera lenses to project the image into an Acton 150 mm spectrograph(Acton, MA) with spectra read on a Princeton Instruments, liquid nitrogen-cooled, 1024 x
256 CCD array (Trenton, NJ), using a mercury arc lamp and an appropriate dichroic mirror
module for wavelength selection.
Images of PEBBLE loaded cells were obtained using an Olympus Fluo View 300
scanning confocal microscope system equipped with an Ar-Kr and He-Ne laser or anequivalent system. Data were acquired using the Olympus FluoView package.
2.4 Cell culture
Alveolar macrophages were recovered from rat lung lavage using Krebs-Henseleit
buffer. Macrophages were maintained in a 5% CO2 , 37° C incubator in Dulbecco's
Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 0.3% penicillin,
streptomycin and neomycin. PEBBLE suspensions ranging from 0.3-1.0 mg/mL wereprepared in DMEM and incubated with alveolar macrophage overnight.
Human neuroblastoma cells (cell line SH-SY5Y) were maintained in DMEM contain­
ing 10% fetal bovine serum and 0.3% penicillin, streptomycin and neomycin and subcul­
tured into 60 mm petri dishes and incubated at 37°C in a 5% CO2 environment. The cells
were used when they reached a confluency of about 80%.
Rat C6-glioma cells were cultured in DMEM containing 400 mg/L D-glucose, 2 mM L­
glutamine, 10% fetal bovine serum, 0.3% penicillin, streptomycin and neomycin andincubated at 37°C in a 5% CO2 environment. Cells were released from culture dishes bytrypsin treatment one day prior to the experiments and plated at a density of 150,000 cells
per plate on uncoated 22 mm glass coverslips in 35 mm culture dishes.
3.

Results and Discussion

3.1 PEBBLE size distribution

Each method for PEBBLE production has been optimized to produce uniform distribu­
tions of small particles, based on the specific matrix. A key requirement is the ability tocollect these particles (dry) and then re-suspend them in aqueous solution. For acrylamide
PEBBLEs, the described fabrication method gives a bi-modal distribution of 20-nm- and· 200-nm-diameter particles.02. l The size of particles in this bi-modal distribution can be
adjusted by changing the concentrations of the surfactants. These variously sized particles
can be separated by filtration. Decyl methacrylate PEBBLEs range from 300-700 nm indiameter.<20J Sol-gel PEBBLEs are typically 100-600 nm in diameter.<24l Figure 1 shows
SEM and TEM micrographs of acrylamide, decyl methacrylate, and sol-gel PEBBLEs.
Recently all three have been made with dimensions below 100 nm in-house.
J3

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Sensors and Materials, Vol. 14, No. 6 (2002)

(a)

(b)

(c)

(d)

Fig. 1. (a-b) Transmission electron micrographs of PEBBLE sensors embedded biolistically (viagene gun), at 900 psi, into the cytoplasm of neuroblastoma cells: (a) Two 200 nm PEBBLEs, near orinside the cell nucleus. (b) One 20 nm PEBBLE next to a 1° lysosome in the cell cytoplasm. Original
magnification is indicated in the figure and the inset. (c) SEM of gold coated decyl methacrylate
PEBBLEs produced in emulsion polymerization with PEG 5'000 monoethyl ether as surfactant. Size
distributions of 500 nm±40 nm =48% and 600 nm±40 nm =37% are found for the PEBBLEs
produced. (d) Scanning electron micrograph showing the size distribution of sol-gel-PEG particles
produced by the modified Stober method (50% particles<200 nm).

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3.2 Polyacrylamide PEBBLE performance

The major benefits of using a dye containing polyacrylamide matrix (compared to free
dye alone) are the protection of the cell from dye cytotoxicity and of the dye from protein
binding and other cellular processes, as well as the ability to include reference dyes in thesensing scheme. One example of the effect of protein binding is demonstrated with CNF
pH dye. CNF is a highly photostable, ratiometric dye for pH, which is not used for
intracellular applications because of the error induced by macromolecule binding. How­ever, protected by the acrylarnide matrix, CNF becomes a viable tool for intracellular pH
study. Figure 2 illustrates the benefit of entrapping CNF in an acrylamide matrix.
Incubation of the free dye with as little as 0:01 % albumin induces alterations in emission
ratios of almost 90% (pH was maintained constant as measured with a standard pH
electrode) which is an error equivalent to 1 pH unit. The same dye, protected in the
PEBBLE, shows minimal perturbation by albumin, with the resulting error equivalent toabout one hundredth of a pH unit.
There is a wide range of available fluorescent dyes with good selectivity for pH andcalcium. Thus calcium and pH sensing presented a good opportunity to compare PEBBLE
response to that of a free dye in similar calibration environments. Complete details onthese comparisons can be found in previously published work.< 13 . I 4l Tables 1 and 2
summarize the results for pH sensitive dyes and calcium dyes, respectively. Both dye
solutions and PEBBLEs utilized the inert sulforbodamine 101 (SR) dye as an internal
standard. It can be seen that, with few exceptions, in PEBBLES the slope of the calibration
decreases, resulting in reduced sensitivity of the measurements, but the linear ranges are
not significantly affected by incorporating the dye in the PEBBLE. The acrylamide matrix
does not adversely affect the reversibility of the indicator dye and can be tailored to entrap
the dye for periods Jong enough to perform sensing in live cells. Figure 3 illustrates thereversibility of pH PEBBLEs containing the fluorescent pH probe CDMF and the reference

0

Cl)

0

•

160

Cl)

free CNF

140

0

120

...0 .::a:cu 100
Cl)

-= ·e
:::,

u:::

C:

0

80

Ill

60

as w

40

Cl)
C)
C:

.J::.

0

'if.

"iii

•
CNF in PEBBLES

20
0
0.00

0.01

0.02

0.03

%AlbuminFig. 2. Effect of protein on sensors.

0.04

0.05

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Sensors and Materials, Vol. 14, No. 6 (2002)

Table 1Results of calibrations of five pH sensitive dyes and the corresponding PEBBLE sensors. An internal
standard, sulforhodamine 101, was added to each dye solution and was contained within thepolymeric matrix of each PEBBLE sensor.
CNF: 5-(and 6-) carboxynaphthofluorescein
CDMF: 5-(and -6-) carboxy-4',5'-dimethylfluorescein
BCPCF: 2,,7,-bis-(2-carboxypropy1)-5-(and-6)-carboxyfluorescein
FSA: fluorescein-5-(and -6)-sulfonic acid
SNAFL: 5-(anq-6)-carboxy SNAFL®-1
SR: sulforhodamine 101
PH Indicatorslope"±SD
Linear Range
H units
CNF (dye)7.8±0.4
7.0-7.7
CDMF+SR (dye)3.3±0.5
6.2-7.4
BCPCF+SR (dye)6.5-7.6
1.3±0.07
5.8-7.2
FSA+SR (dye)3.1±0.3
SNAFL (dye)--0.75±0.01
7.2-7.7
CNF (PEBBLEs)
7.0-7.7
0.24±0.01
CDMF+SR(PEBBLEs)
6.2-7.4
0.67±0.05
BCPCF+SR(PEBBLEs)
0.43±0.07
6.2-7.2
FSA+SR (PEBBLEs)
5.8-7.0
3.4±0.4
SNAFL (PEBBLEs)
7.2-8.0--0.49±0.02"Ratio of normalized fluorescence intensity vs pH.

intercept

r2

n

-53
-19
-7.3
-17
6.8
-0.66
-3.0
-1.6
-17
4.6

0.98
0.96
0.99
0.99
0.99
0.96
0.99
0.90
0.99
0.99

6
12
6
7
4
5
6
6
5
7

Table 2
Results of calibrations of three calcium selective dyes and the corresponding PEBBLE sensors. Aninternal standard, sulforhodamine 101, was added to each dye solution and was contained within thepolymeric matrix of each PEBBLE sensor.
Calcium Indicator

Linear RangeµM calcium
Calcium Green+SR(dye)0-0.15
Calcium Orange+SR(dye)0--0.15
Calcium Green+ 5N SR(dye)3-30
Calcium Green+SR(PEBBLEs)
0--0.15
Calcium Orange+SR(PEBBLEs)
0 -0.1
Calcium Green+ 5N SR(PEBBLEs) 0-5

slope±SD

interceptr2n30±0.71.5±0.70.010±0.05
7.3±0.05
1.3±0.05
0.022±0.007

1.7
1.0
0.99
0.97
1.0
1.0

0.94
0.95
0.95
0.99
0.79
0.99

4
6
7
6
5
4

dye SR. The pH of the solution was varied between pH 6.4 and pH 7.0 for several cycles.
The CDMF emission maximum was ratioed against the SR emission maximum andplBtted, clearly demonstrating the full reversibility of the sensors. Assays of the leachingof CDMF, and calcium green PEBBLEs (each containing SR reference dye) show that lessthan 50% of each dye, leaches from the PEBBLEs in a 48 h time period. The leaching ratesof all dyes were comparable. On the time scale of the current single cell experiments,
usually a few hours, the dye loss is acceptable. With comparable leaching rates for318

Sensors and Materials, Vol. 14, No. 6 (2002)
1.4

cc

1.2
1.0
0.8

0 0.6
cc

0.4
0.2
0.0

0

2

3

4

5

6

Acquisition Number
Fig. 3. The pH ofa solution ofCDMF/SR PEBBLEs was varied back and forth from pH 6.4 to pH
7.0, for several measurements. The ratio ofthe two peaks in the spectrum attained from each ofthemeasurements was then plotted.

indicator and reference dyes, the problem is minimized due to the ratiometric sensing
scheme used with these PEBBLEs.

3.3 Decyl methacrylate (DMA) PEBBLE peiformance
While the protection given by the acrylamide matrix prevents protein and macro­molecule interference with sensing dyes, it does not prevent iori interference. In spite ofadvances in fluorescent indicator molecule development, there are still a good number ofions for which the most selective ionophores are nonfluorescent ("optically silent"). These
ionophores stem from the century-long development of ion-selective electrodes (TSEs).
The fluorescence response scheme we used to follow analyte binding of nonfluorescentionophores in OMA-based PEBBLE sensors closely follows much previous work on PVC-based fiber potassium-selective ion sensors. 00-25·26l The hydrogen-ion selective
chromoionophore competes with the optically silent ionophore as cations enter the liquid
polymer matrix. A lipophilic additive maintains ionic strength in the matrix and aids inpreventing the co-extraction of anions. It allows for charge neutrality in the membranewithout negative counter-ions being brought from the solution into the membrane.0°l
The work described here takes advantage of an indicator with two fluorescenceemission maxima (A1 ,Ai), giving a relative intensity that changes with the degree ofprotonation (IT). IT* can be evaluated in terms of the fluorescence intensity ratio, FJ..i!FJ..1,
given by the protonated chromoionophore intensity FJ..2 and the deprotonatedchromoionophore intensity FJ..1 (see Fig. 4 for Spectra).<25l The experimentally obtainedSpectra are normalized to the isoemmisive point cif the dye and eq. (1) is used to determine
the degree of protonation of the dye at any given analyte concentration at a known pH(superscripts P and D denote the completely protonated state and completely deprotonatedstate of the chromoionophore, respectively, lack of superscript denotes intermediate
points)<20l:* II, the degree ofprotonation is equivalent to (1-a). <25i319

Sensors and Materials, Vol. 14, No. 6 (2002).'!l 1000
·2
::,
.ci

�

·;;;

.,
.,
::,

800

600
400
200
0
550

600

700

650

750

800

Wavelength (nm)Fig. 4. Normalized emission spectra from suspended K + PEBBLE sensors using the pH
chromoionophore ETH5350 for ion-correlation spectroscopy in tande.rp with BME-44 for themonitoring ofK + activity. [A] 10 mM KCl, [BJ50 mM KC!, [CJ 200 mM KCl, [DJ 500 mM KCl, and[E] 2.0 M KCl, all buffered at pH 7.2 with 10 mM Tris buffer.

(1)

The degree of protonation of the indicator spectra obtained from the PEBBLE calibra­tion is related to the analyte concentration by using the theoretical treatment of ion­exchange sensors developed by Buhlmann et aZ.,C10l Bakker and Simon,C1 6l Morf et at.,( 17l
and Shortreed et al. cz5J They defined IT as the relative portion of the protonatedchromoionophore,c25l 11=[CH]/[C,ot]. IT is a function of the metal ion activity a{+ insolution, the hydrogen ion activity aH+ in solution, the interfering cations ar (where K0P\ isthe selectivity coefficient for the interfering ion) and the constants [L,01], [C,01], [R,0,-J,
which are total ionophore (ligand) concentration, total chromoionophore concentration,and total lipophilic charge site concentration, in the membrane. Note that [CH] is the
protonated chromoionophore concentration and [ CJ is the free base concentration. The
following equation has been shown to apply for a K+ PEBBLE sensor when accounting formonovalent interfering cations.

[[L,01]
a v +K°.Ptaz+=_I_(IT-1-l)a
I
I}
J
+
Kexch
H
Rr�t
ctot]

[ ]- II[

ll-l

(2)

Sensors and Materials, Vol. 14, No. 6 (2002)

320

Calibration of a K+ sensor based on these principles is shown in Fig. 5: For potassium
sensing the chromoionophore is ETH5350, the ionophore is BME-44, and the lipophilic
additive is KTFPB.<20- ) The data points for potassium and sodium responses are plottedalong with corresponding theoretical curves based on eq. (2). The constant Kexch isdetermined from a line fit to the experimental data. Then the theoretical curve is plottedusing the experimentally determined Kexch and the constants R,0,, C,01 and L,01 to find the
26

0.8

t::

0.6

0.4
0.2

0
2

4

6

8

10

Log(axJaH+)

B

0.8
0.6

c::

0.4

•

0.2
0
2

4

6

Log(aKJaH+)

8

10

Fig. 5. (A) Response of BME-44-based decyl methacryiate PEBBLEs to potassium (0), andsodium (L':s.), along with theoretical curves. Theoretical curves are constructed by solving eq. (2) for
a1.., W being K+ or Na+ in this case), for a given value of P. The solid lines delimit values for log (adaH+) typically found in intracellular media and the dashed lines delimit the typical extracellularratios.(27l (B) Response of K+ PEBBLEs to standard additions of KC! in tris buffer (0) compared to
The theoretical lines are drawn
a similar experiment run in a constantbackground of 0.5 M Na+
33
using eq. (2) with log K0P'ij = - . .

ce).

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Sensors and Materials, Vol. 14, No. 6 (2002)

expected a1+ for a given value of P. Dashed lines delimit typical extracellular activity ratios
find the solid lines delimit the intracellular levels (log (aKjaH+)).<27>
We find that the response matches well with the theory, which is gratifying considering
the small size of the systems. The dynamic range at pH 7.2 extends from 0.63 mM to 0.63
M aK+ · The log of the selectivity for potassium vs. sodium, detemlined by measuring thehorizontal separation of the response curves at II= 0.5, is -3.3. This value, when used toplot the expected response in a sample with a constant 0.5 M Na+ interference, matches theexperimental data obtained (see Fig. 5). This value indicates a selectivity similar to or
better than that obtained for other and larger matrices incorporating BME-44, e.g., -3.1 in
PVC-based fiber optic work, and -3.0 in PVC-based microelectrodes.(26> It also exactly
matches the value given in the review by Buhlmann et al.,C10> for a thin PVC film sensor.
This selectivity should be more than sufficient for measurements in intracellular media
where the potassium concentration(27) is about 100 mM and sodium is about 10 mM.
DMA based potassium sensors have a lifetime of 30 min due to component leachingfrom the liquid polymer membrane. This is consistent with the lifetime of PVC-based
optodes of the same composition.<26> After 30 min the sensor response can deviate up to 7%
from the initial calibration data at lower K+ concentration. After 90 min the deviation is up
to 13% at lower K+ concentrations. The deviations are smaller at larger K+ concentrations.
However, as the PEBBLEs are single use sensors made for quick measurements inside
cells that only survive for a short period of time, this is acceptable.
The sensors also prove to be fully reversible. Figure 6 shows the PEBBLEs, starting in
an initial solution of 45 mM KCl which is diluted to 12 mM, then brought back to 45 mM
by standard addition buffer and 2 M KCl solution.

8
7
N

6

u.. 5
0

3
2

0

50

100

Time (Sec)

150

200

Fig. 6. Reversibility of K+ sensitive PEBBLEs. The PEBBLES started in 45 mM K+, buffered at pH
7.2 with TRIS. The solution was then diluted with buffer to a concentration of 12 mM K+ ; sufficient
standard KC! solution was then added to bring the concentration of the solution back to 45 m.M K•.

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Sensors and Materials, Vol. 14, No. 6 (2002)

3.4 Sol-gel PEBBLE peiformance

Oxygen is a key parameter in biological systems and is either produced by autotrophic
photosynthesis in the presence of light, or consumed by different metabolic processes. For
example, oxygen plays a crucial role in the cellular aerobic energy metabolism since itis
used as an electron acceptor at the end of the aerobic pathway of glucose oxidation. Thus,
knowledge of oxygen gradients in complex biological samples is of paramount importancefor the understanding and quantification of these processes. <28•29l The numerous biologicalroles of molecular oxygen create the need for noninvasive, sensitive, and selective detec­tion methods that are capable of real-time oxygen measurements.
The oxygen sensing sol-gel pebbles are based on the quenching of luminescence byoxygen. The oxygen quenching process is ideally described by the linear Stern-Volmer
equation:where [0 and I are the luminescence intensities in the absence and presence of oxygen at apartial pressure of p[02], respectively, and Ksv is the Stern-Volmer constant, which
depends directly upon the diffusion constant of oxygen, the solubility of oxygen, and thequenching efficiency and lifetime of the excited-state of the fluorophore.<30l
The fluorescence emission spectra of the sol-gel PEBBLE sensors, in different gaseous
oxygen concentrations, are shown in Fig. 7. The sensor response was determined from theratio (R) of the fluorescence intensities of [Ru(dpp)3]2+ to Oregon Green 488-dextran.
These plots illustrate a number of features that are important to the optimization of thesensor design. The overall gas phase quenching response, QG, is given by:

30000
� 25000·2

N2.ci 20000
� 15000·.;£ 10000

Air
02

5000
0
450

500

550

600

650

Wavelength (nm)700

Fig. 7. Gaseous phase emission spectra of sol-gel oxygen PEBBLE sensors excited at 488 nm; topline: purged with N2 ; middle line: in air; bottom line: purged with 02 . The peak on the left is thefluorescence emission of the reference dye (oregon green) and the peak on the right is the fluorescenceemission of the indicator dye (ruthenium dye).

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Sensors and Materials, Vol. 14, No. 6 (2002)

where /N2 and /02 denote intensities in 100% N2 and 100% 02, respectively. The measured
value of Q0 for the sol-gel PEBBLEs is -92%. The calibration curve for the ratiometric sol­gel PEBBLEs showing the response to gaseous oxygen is displayed in Fig. 8. The quasi­
linearity (r2 = 0.995) of the Stern-Volmer plot implies a single :fluorophore class accessible
to molecular oxygen.<31J This data is in agreement with thin film sol-gel oxygen sensors<32J
and has the advantage of being a ratiometric measurement.
The reversibility of the previously reported Ru-based sol-gel sensors in the gas phasehas also been retained in these nanosensors.<24l Non-optimized measurements indicate that
response times for a 90% oxygen concentration change should be well below 1 s, which isa direct result of the small size of the PEBBLES. The sensors showed at least 98 % recovery
each time after the sensing environments were varied among air, oxygen and nitrogensaturated conditions.
Figure 9 shows the response of the TEOS based sol-gel PEBBLEs to dissolved oxygen(DO). The Q 00 for the sol-gel oxygen PEBBLEs is -80%. (Quenching response todissolved oxygen, Q00, is defined in a similar way to Q0 , where /N2 and /02 are replaced by/ in fully deoxygenated water and I in fully oxygenated water, respectively). This value
represents a slight reduction in performance vis-a-vis gaseous oxygen; however, it is agreat improvement with regards to TEOS-based sol-gel films.<33J These sol-gel films, madeusing TEOS as the precursor, had an excellent response to oxygen in the gas phase, i.e., Q0= 90%, but a poor quenching response to dissolved oxygen, Q 00, of only about 20%.
MacCraith et al. <33J have subsequently reported significant improvements to the Q00 ratio
by preparing organically modified sol-gel (Ormosil) films using methyltriethoxysilane(MTEOS) and ethyltiiethoxysilane (ETEOS) as the precursors. The success of the Ormosil
films in raising the Q00 ratio to 70-80% was largely attributed to the increased hydropho­
bicity of the film which reduced the water solubility in the film and caused the partitioning

5.5
5.0
4.5

...

4.0
3.5
3.0

d: 2.5

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� 2.0
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-

1.5

1.0
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0.0
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ll:

m

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w

w

100

% Oxygen Concentration

Fig. 8. Stern-Volmer plot of relative fluorescence intensity ratios for ratiometric sol-gel oxygen
PEBBLEs in gaseous phase.

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Sensors and Materials, Vol. 14, No. 6 (2002)
450000� 400000"i::::,

350000N2
Air300000i!' 250000·.;
C
20000d., 150000
0C

0100000
50000
0

500

550

600

650

700

Wavelength (nm)Fig. 9. Aqueous phase emission spectra of sol-gel oxygen PEBBLEs excited at 488 nm: top line:
PEBBLE solution purged with N2 ; middle line: PEBBLE solution purged with air; bottom line:
PEBBLE solution purged with 02•

of oxygen out of solution and into the film.<32l This would greatly increase the accessibilityof oxygen molecules to the indicator dye molecules. It is thought that the high Q00 responseof the TEOS sol-gel PEBBLEs might be caused by the PEG content of the sensing matrix,
PEG playing a role analogous to the Ormosil precursors and thus partitioning the oxygen
preferentially into the sol-gel PEBBLEs. It is well known that oxygen has a higher
solubility in organic liquids than in water,(34) so it should dissolve better in an organic phasecompared to an aqueous phase. In summary, doping the sol-gel PEBBLEs with PEG adds
organic components to the sensing matrix, thus encouraging the partitioning of oxygen into
the matrix, and increases the accessibility of oxygen to the entrapped indicator dye
molecules. This is in addition to the role of PEG in preventing particle aggregation in the
PEBBLE sensor fabrication.
Figures 10 and 11 show the reversibility of the sol-gel PEBBLES to changing dissolved
oxygen concentrations and the Stem-Volmer plot of fluorescence intensity ratios to oxygen
concentrations, respectively. This data shows that although the performance of the sol-gel
PEBBLEs is slightly reduced in the aqueous phase, as opposed to the gas phase, the sensors
still demonstrate good reversibility and reproducibility, as evidenced by the error bars in
the calibration curve. The dashed line in Fig. 11 shows the extent of the biologically
relevant regime of oxygen concentrations. We note that in this regime (from Oto -30 ppm
oxygen), the Stern-Volmer plot is quasi-linear (r2 = 0.988). The sensors showed at least
95% recovery each time the sensing environments were varied among air, 02, and N2
saturated sensor solutions.
Leaching of dye molecules out of the sol-gel matrix of the PEBBLE sensors is a major
concern and is highly dependent on the dye. Factors such as the molecular size of the dye(small dyes can more readily diffuse through the pores and leak out of the matrix) and thesolubility of the dye in the matrix and in water play a significant role.c24l Generally, sol-gel

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Sensors and Materials, Vol. 14, No. 6 (2002)

....

0.8
0.7
C:

0.6

.Q

0.5

C:

0.4

'lo

r

1.....

0.3

20

0

40 60
80
Time (minutes)

100

120

Fig. 10. Reversibility of PEBBLE sensor response to dissolved oxygen. Alternating measurements
were taken in air-saturated, Orsaturated and Ni-saturated PEBBLE sensor solutions.

3.0
2.5

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/

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.(II

I I

.,_,,

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so

Oxygen Concentration (ppm)Fig. 11. Stem-Volmer plot of relative fluorescence intensity ratios for ratiometric sol-gel oxygen
PEBBLEs in aqueous phase.
supports provide excellent stability with respect to dye leaching.c35l In particular, ruthe­
nium complexes often have excellent stability inside the sol-gel matrix and in agreementwith previous reports,<28•32•35-37l the indicator dye [Ru(dpp)3] 2+ shows no signs of leaching.
Fot the reference dye, the large size of the dextran molecular backbone to which the
Oregon Green dye molecules are bound should greatly reduce leaching. Indeed, an
investigation into the stability of the sol-gel PEBBLEs showed excellent stability of thesol-gel PEBBLE sensors with respect to dye 1eaching.c24l According to the dilution factor,a rough estimate provides an upper limit of 1 % for the amount of dye molecules leached outof the sensing matrix over a three day period. In addition, we note again that the typicalbiological application is performed over a time scale of only a few hours.

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Sensors and Materials, Vol. 14, No. 6 (2002)

We have found that the sol-gel matrix of PEBBLE sensors prevents macromolecules,
such as proteins, from diffusing through the matrix (as expected). The matrix thus protects
the entrapped dyes from the intracellular environment, preventing interference with thefluorescent properties of the dyes.C5l Without this shielding of a dye, its fluorescence would
behave unpredictably inside a given cell, making calibration of even ratiometric dyes
difficult or impossible. The effects of nonspecific protein binding were investigated by theaddition of bovine-serum albumin (BSA, 5% solution), and the results are shown in Fig. 12(a). Adding as little as 0.14% BSA to a solution containing [Ru(dpp)3] and Oregon Green488-dextran dye (at the same molar ratio in solution as inside the PEBBLEs) changed thefluorescence intensity ratio of the two dyes by a factor of more than 2.3 (i.e., an increase of
2+

�

140

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Free dye solution

80
60

a:

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0.00

0.10

0.05

0.15

0.20

0.25

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E° 300000.§ 280000-f! 260000·!

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-

Oregon Green- -- - - ·- ,,__ � - .; � --=,-;::::::::;=- ._ c,c ===� � �..- � � - �+-�-1
0.00
0.04
0.06
0.08
0.10
0.02
0.12
0.14
0.16
·--·- - ccc%Albumin(b)
Fig. 12. (a) Effect of protein on sol-gel PEBBLEs and free dye solution. (b) Effect of protein on freedye molecules in solution. It is apparent that the fluorescence intensity of [Ru(dpp)3] 2+ changed
drastically upon interaction with BSA, while the intensity of Oregon Green remained largely
unchanged.

Sensors and Materials, Vol. 14, No. 6 (2002)

327

over 130%). This change was mostly due to the change in the fluorescence intensity of[Ru(dpp)3] 2+ after the addition of BSA, while the intensity of Oregon Green remained
basically unchanged (Fig. 12(b)). However, under the same conditions, the PEBBLE
sensors containing these two dyes were not affected by the addition of BSA and a change
in fluorescence intensity ratio of at most 4% was observed even when an increased
concentration of BSA (0.23%) was added, demonstrating the same protection as given todyes in the acrylamide matrix.<13J The susceptibility of the sol-gel PEBBLEs to heavy
metal ions (Hg2+ and Ag +) and to one of the notorious collisional quenchers (I-) was also
examined.C24l Hg(NO 3)2, Ag(NO 3) and KI were added, respectively, to a PEBBLE solution
and to a free dye solution of [Ru(dpp)3] 2+ (same concentrations as in PEBBLEs) up to aconcentration of about 200 ppm. There was a 5-10% decrease in the fluorescence intensityof the [Ru(dpp)3] 2+ free dye each time, while for the PEBBLEs no measurable effect could
be observed.

3.5 Response time of PEBBLE nanosensors

In order to follow biological pertmbations in real time, a fast response is required fromthe PEBBLE sensors. Most PEBBLE sensors depend on bulk equilibrium between sensor
and solution phase (the oxygen sensor depends on steady-state). The diffusion of analyte
inside acrylamide and sol-gel should be similar to that in aqueous solution phase, while thediffusion of analyte in the hydrophobic DMA matrix is much slower. In all cases the small
size of the PEBBLE sensors gives a rapid response time, despite the need for bulk
equilibrium.
A reliable method for determining the response time of acrylamide sensors, to Ca2+,
used an Olympus IX50 inverted microscope equipped with a mercury arc lamp and a PMT.
Calcium selective probes were premixed with a caged calcium ion, and this solution wasinserted into a quartz capillary. The calcium was uncaged with a pulse of UV light from a
Quanta-Ray 10 ns Nd/Yag laser (Quanta-Ray, Mountainview, CA) equipped with afrequency tripler and coupled into an optical fiber positioned over the capillary. In Fig.
13(a), the response time of the PEBBLE sensors was compared to that of the free dye (nopolymer matrix), so as to separate the diffusion time through the solution from the diffusion
time through the matrix. As can be seen, the 90% response time of the PEBBLE sensor to
the increase in free calcium is on the order of one millisecond or less. Theoretically, with
an approximate diffusion constant of 10---u cm2/s, the average diffusion time should be about
ten microseconds for a 100-nm-radius sensor, and 100 nanoseconds for a IO-nm-radiussensor.
The measured transition times for sol-gel are on the order of20-30 s, but these times aremuch longer than the intrinsic response time of the PEBBLEs, due to the significant
contribution of the time used to saturate the solution with 02 or N2 (see Fig. 10). It isdifficult to measure the exact response time, because changing between oxygenated anddeoxygenated PEBBLE solutions itself takes time. The measured transition times (includ­ing the time of saturating the solution) are only an upper limit of the response time. We
believe that the PEBBLE sensors should intrinsically have shorter response times thanpreviously reported thin film and fiber optic sol-gel sensors (on the order of seconds or
minutes) simply because of the smaller sizes of these sensors. According to the Einstein328

Sensors and Materials, Vol. 14, No. 6 (2002)

0.6

0.5

Ql
u
C
Ql
u
(J)

0.4

0.3

0
::,

Response witi1
cage present

0.2

0.1
-0.006-0.004-0.002

0.000

0.002

0.004

0.006

Time (sec)
(a)
50

40

u.
u.

30

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.

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2

0 20

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1
15

10

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0

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5

10

15

Time (sec)

20

25

(b)

Fig. 13. (a) Response time of ac1ylamide PEBBLE sensors. Calcium was released using a single 10
ns UV pulse from a Nd/Yag laser which photolysed the cage, releasing free calcium into a solution of
PEBBLEs. The observed response time, less than l ms, was indiscernible from that of thecorresponding dye not entrapped in a polymer matrix. (h) Response time of K+ sensitive PEBBLEs
to added KCl and added buffer solutions. It can be seen that response in the forward cLirection is about
0.5 s (0-40 mM KCl, A) and the reverse (see inset) slightly longer (40-20 mM KCl, B), 0.8 s.

diffusion equation, where X2=2Dr, a shorter diffusion length X (which is directly related tothe size of the sensor) results in a much shorter time for oxygen molecules to diffuse
through the sensing matrix (which is basically the response time). A lower limit can thus beestimated, using D""2x10-9 m2/s (diffusion constant of oxygen in water) and X""3x1Q- 7 m,

Sensors and Materials, Vol. 14, No. 6 (2002)

329

giving r""20xl0-6 s, i.e. a response time in the microsecond range. An upper limit can be
estimated considering that the PEBBLE sensor dimensions are 10-100 times smaller thanthose of thin film sensors, and have a spherical shape. This should give the PEBBLE
sensors a response time in the millisecond range.
For DMA-based K+ sensors, using BME-44 as the ionophore, ETH5350 as thechromoionophore and KTFPB as the ionic additive,<20J the ratio of the protonatedchromoionophore to free base was analyzed vs. time in response time measurements. It
was found (see Fig. 13(b)) that in going from log aK+faH+ = 3.6 to 5.7 the response time
(10%-90% signal change) was about 0.5 s (for a concentration change of over 2 decades).
In the reverse direction, the response was about 0.8 s. This fast, sub-second response timeof the PEBBLEs is a direct result of their small size. Diffusion in decyl methacrylate is in
the range of 10-s cm2/s, with small variations that depend on cross linker content.08• )Thus, for a PEBBLE radius of about 300 nm, we expect a diffusion time of about 10- 3 s.
This is consistent with the experimental values, which are again upper limit values due tothe solution mixing times.
To summarize, using acrylamide or sol-gel PEBBLES, one can monitor changes withresponse times on the microsecond timescale or, using the decyl methacrylate PEBBLEs,
one can monitor changes on the 0.5 s time scale. Below are examples of some actual
biological measurements taken with PEBBLE nanosensors.
39

3.6 Biological applications of PEBBLE nanosensors

An acrylamide PEBBLE selective for calcium (containing Calcium Crimson in theacrylamide matrix<40l) was produced in order to test for response to calcium flux in an
intracellular environment. The PEBBLE sensors were also used to monitor calcium inphagosomes within rat alveolar macrophage, because of the ease with which macrophage
phagocytosed the particles. This method for delivering the PEBBLEs into cells provided asimple, yet important, test of the PEBBLE sensors in a challenging (acidic) intracellularenvironment. Macrophage that had phagocytosed 20 nm calcium-selective PEBBLE
sensors (Fig. 14) were challenged with a mitogen, Concanavalin A (Con A), inducing aslow increase in intracellular calcium, which was monitored over a period of 20 min (Fig.
14). PEBBLE clusters confined to the phagosome enabled the con-elation of ionic fluxes
with stimulation of this organelle.
Acrylamide PEBBLEs have also been utilized to observe the toxic effects of theindustrial chemical DNB (m-dinitrobenzene).< 14J Exposure to m-dinitrobenzene (DNB), a
compound widely used as an industrial intermediate in the manufacture of dyes, plastics,
and explosives, induces brain stem glio-vascular lesions in nuclei with high energy
requirements, particularly those in the auditory pathway.<41J Neurotoxicity associated with
DNB exposure is thought to be mediated by loss of ATP production secondary to thedisruption of cellular redox potentiaJ.<42l Considering the potential of m-DNB to undergo
redox cycling in the reducing environment of the mitochondrion, we have investigatedwhether disruption of mitochondrial function, subsequent to m-DNB exposure, resulted
from an uncontrolled release of calcium associated with the onset of the mitochondrial
permeability transition (MPT). Utilizing calcium selective PEBBLE sensors, it was
discovered that the half-maximal rate of calcium release (EC50) occuned at a 10-fold lower

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Sensors and Materials, Vol. 14, No. 6 (2002)

2000-r-----------------------,

1800
1600
1400
2
_e. 1 00

"j 1000

!

800
600
400
200

---==--_,r

0 +--""T"""----r----,---..---�--,---,---,----,----1

�

�

�

�

� � � �Wavelength (nm)

�

m

m

Fig. 14. Top image. Rat alveolar macrophage with calcium PEBBLE sensors (60x). Left: Nomarski
illumination and Right: fluorescence illumination. Bottom. The increasing intracellular calcium,
monitored by calcium PEBBLEs in alveolar macrophage following stimulation with 30 µg/mL
Concanavalin A.
concentration of m-DNB in human SY5Y neuroblastoma cells than in human C6 glioma
cells. This finding supports the hypothesis that sensitivity to m-DNB-induced calcium
release is associated with differential regulation of the MPT in the cell lines evaluated. To
further illustrate that the observed increases in cytosolic calcium are caused by release fromintracellular stores, rather than disruption of the plasma membrane by m-DNB, cells
containing calcium selective PEBBLE sensors were imaged using laser scanning confocal
microscopy (Fig. 15). As in fluorimetry studies, increased cytosolic calcium was observed
upon m-DNB exposure, as indicated by an increase in the fluorescence intensity of331

Sensors and Materials, Vol. 14, No. 6 (2002)

Fig. 15. Laser scanning confocal microscopy images of m-DNB-stimulated calcium release in
SYSY neuroblastoma cells lipofected with Calcium Green/SR PEBBLEs. Calcium Green andrhodamine fluorescence were monitored at time zero, prior to addition of m-DNB (A) and 5 min
following addition (B).

Calcium Green-containing PEBBLE sensors.c 14l The fluorescence intensity of the SR
internal standard dye did not change following treatment with m-DNB, since it is inert withrespect to ion concentration, thus demonstrating the utility of ratiometric PEBBLE sensors
for measuring intracellular calcium fluxes.
In order to demonstrate how DMA PEBBLEs (400-600 nm) can be used for intracellu­
lar analysis, it is first necessary to introduce these sensors into the cells. The larger size ofthese liquid polymer PEBBLEs is prohibitive to liposomal delivery. Using the group's
experience in introducing acrylamide PEBBLE sensors into cellsC14•1 l gene gun insertion5

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Sensors and Materials, Vol. 14, No. 6 (2002)

was chosen to insert the DMA PEBBLEs into cultured cells. DMA PEBBLEs were.
delivered into rat C6-glioma cells using a BioRad (Hercules, CA) Biolistic PDS-1000/He
system at a firing pressure of 650 psi with a vacuum of 15 Torr on the system. A thin filmof PEBBLEs suspended in water was dried on the target membrane. Following PEBBLE
delivery, the cells were rinsed three times with Hanks lx buffered salt solution andincubated in the same solution during analysis.c20>
Immediately following PEBBLE delivery, cells were placed on the INT-II Olympus
inverted fluorescent microscope. The gating software for the CCD was set to take continu­
ous spectra at 1.3 s intervals. After 20 s, and again after 60 s, 50 µL of 0.4 mg/mL kainicacid was injected into the microscope cell. Kainic acid is known to stimulate cells by
causing the opening of ion channels. Confocal microscopy was used to determine thelocalization of the PEBBLE sensors after gene gun delivery. Figure 16(a) shows theconfocal fluorescent image of the PEBBLEs overlaid with a Nomarski differential interfer­
ence contrast image of the cells. The image indicates that the PEBBLE sensors are
localized in the cytoplasm of the glioma cells. Figure 16(b) shows the PEBBLE sensors
inside the cells responding to kainic acid addition to the cell medium, at 20 and 60 s. One
can see that log (aK+faH+) increases, indicating either an increase in K+ concentration or adecrease in H+ concentration (increase in pH). The amount of kainic acid added is notknown to affect the pH of cells in culture. Thus the change is likely due to increasing
intracellular concentration of K+ ; this is the expected trend. The membrane of C6-glioma
cells can initiate an inward rectifying K+ current indicated by specific K+ channels, adocumented role in the control of extracellular potassium_C43l Thus when stimulated with a

7. 8 ,------------------,
7.7,_ 7.6::l:: 7 -5.!!! 7.4

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.3 7

6.9
6.8
6.7 --�-- --,--�----,--�-�
0

20

40

60

80

100

120

Time (sec)

(a)

(b)

Fig. 16. (a) Confocal image of decyl methacrylate K+ PEBBLE fluorescence, overlaid with Nornarsld
image of rat C6-glioma cells. 488 excitation, 580 long pass filter. (b) Ratio data of decyl
methacrylate K+ PEBBLEs in C6-glioma cells during the addition of kainic acid (50 µL of 0.4 mg/mL) at 20 s and at 60 s. Ratios were converted to log (aKjaH+) using solution calibration of the
PEBBLEs.

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Sensors and Materials, Vol. 14, No. 6,,(2002)

channel opening agonist, the K+ concentration within the glioma cells is indeed expected toincrease.
Sol-gel PEBBLEs were also delivered into rat C6-glioma cells using the BioRad genegun system with the same parameters as described above. A thin film of PEBBLEs
suspended in ethanol was dried on the target membrane. Following PEBBLE delivery thecells were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and incubated withDPBS during measurements .CZ4l
Figure 17 (a) shows the confocal images of C6-glioma cells containing sol-gel PEBBLEs
under Nomarski illumination. It can be seen that the cells still maintained their morphol­ogy after the gene gun injection of PEBBLEs and showed no indications of cell death. The
fluorescence confocal images of these cells are presented in Fig. 17 (b) and 17 (c). The

(a)

(b)

(c)

Fig. 17. Confocal images of rat C6-glioma cells loaded with sol-gel PEBBLEs by gene gun injection.
(a) Nomarski illumination. (b) Oregon Green fluorescence of PEBBLEs inside cell overlaid with
Nomarski illumination. (c) [Ru(dpp)3]2+ fluorescence of PEBBLEs inside cells overlaid with Nomarski
illumination.

Sensors and Materials, Vol. 14, No. 6 (2002)

334

green fluorescence of Oregon Green 488-dextran and the red fluorescence of [Ru(dpp) 3]2+
were excited, respectively, by reflecting the 488-nm (Ar-Kr) and 543-nm (He-Ne) laserlines onto the specimen, using a double dichroic mirror. The Oregon Green fluorescencefrom the PEBBLEs inside the cells (Fig. 17 (b)) was detected by passage through a 510 nm
long-pass and a 530 nm short-pass filter, and the fluorescence of [Ru(dpp)3] 2+ (Fig. 10 (c))
through a 605 nm (45 nm band-pass) barrier filter. A 40X, 1.4 NA oil immersion objective
was used to image the Oregon Green and [Ru( dpp)3] 2+ fluorescence. The distribution of
PEBBLEs shown in the overlaid images (Fig. 17 (b) and Fig. 17 (c)) shows that the green
and red fluorescence were truly from PEBBLEs inside cells. It should be noted that mostof the PEBBLEs were loaded into the cytoplasm, but there were also some in the nucleus.
Figure 18 shows the response of oxygen sensitive sol-gel PEBBLEs, inserted inside rat C6
glioma cells, to changing intracellular oxygen concentrations. After gene gun injection, thecells were immersed in DPBS and a spectrum was taken of these cells, using 480±10 nm
excitation light. The air-saturated DPBS was then replaced by nitrogen-saturated DPBS, tocause a decrease in the intracellular oxygen concentration, and the response of the oxygen
PEBBLE sensors inside the cells was monitored during a time period of 2 min. As can beseen, the fluorescence intensity of [Ru(dpp)3]2+ increased gradually as the oxygen level
inside the cells decreased. Average intracellular oxygen concentrations were determinedon the basis of the Stern-Volmer calibration curve, obtained using the fluorescencemicroscope-Acton spectrometer system, and are summarized in Table 3. The compara­tively large errors are due to the low resolution of the spectrometer. We note that ourmeasured intracellular oxygen value (when cells were in air-saturated DPBS) is compa­
rable with the value of -7.1 ppm measured inside the much larger islets of Langerhans. C44l
These results show that the PEBBLE sensors are responsive when loaded into cells and thatthey retain their spectral characteristics, enabling a ratiometric measurement to be made. c24l

150�---------�- - - �

!!
·2
:,

N2 2min.

-f 100�

N2 25 s"iii.!:

Air Sat.
DPBS

50

(I)
(I)

0
:,

450

500

550

600

650

700

750

Wavelength (nm)Fig. 18. Fluorescence spectra of a typical ratiometric sensor measurement of molecular oxygen
inside rat C6-glioma cells; bottom line: cells (loaded with sol-gel PEBBLEs) in air-saturated DPBS;
middle line: cells in Nrsaturated DPBS, 25 s after replacing the air-saturated DPBS; top line: cells in
Nrsaturated DPBS, after 2 min.

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Sensors and Materials, Vol. 14, No. 6 (2002)
Table 3
Real time measurements of intracellular oxygen.

Cells in air-saturated buffer
Cells in Ni-saturated buffer (after 25 s)
Cells in Ni-saturated buffer (after 120 s)
Air-saturated buffer solution

4.

Average intracellular oxygen concentrations (ppm)
7.9 ± 2.1
6.5 ± 1.7:::; 1.5
8.8 ± 0.8

Concluding remarks

We have established polyacrylamide, polydecyl methacrylate and silica sol-gel hybrids
as viable platforms for the construction of nanooptical fluorescent probes (PEBBLEs).
The use of established microemulsion protocols for acrylamide and the addition of PEG
stabilization for decyl methacrylate and sol-gel particles has allowed us to reach submicron
diameter sizes. A PEBBLE with a 20 nm radius only occupies I ppb of a standard
mammalian cell (assumes 20 µm radius). This allows for insertion of a host of PEBBLEs
into a single viable cell, with little mechanical or chemical perturbation of the cellularenvironment. All three classes of PEBBLE preserve the basic bio-compatibility principle:
No interference between cell and PEBBLE. This is achieved by encapsulating theindicator dye inside the matrix, in contrast to other strategies, where the active groups areon the outer surface of the nanoparticle.
The three matrices allow one to use a variety of sensing schemes so as to tailor
PEBBLEs for the specific monitoring of any analyte for which selective probes areavailable. Fluorescent free dyes, optically silent ionophores, proteins, and enzymes are all
viable options for the construction of PEBBLEs by choosing the correct matrix platform.
So far PEBBLEs h.ave been developed for H+, Ca2+, K+, Na+,Mg2+, Zn2+, c1-,No2-, 02, NO,
and glucose. The utility of ratiometric measurements, along with the tandem use of dyes inorder to create ratiometric sensors, has been demonstrated. Also demonstrated was theability to use highly selective but nonfluorescent ("optically silent") ionophores innanooptical PEBBLEs. The theory describing ion exchange in micron-sized polymer films
also works for nanometer sized decyl methacrylate PEBBLEs. The flexibility in thecomposition of the matrix and sensor components permits this design to be used for anumber of different analytes and analyte concentrations. The small size allows for novel
intracellular studies.
Initial studies have also shown that the new PEBBLEs can be delivered to viable cells
and these cells can be observed in a time resolved manner, responding to changes in theirenvironment. The small size of the PEBBLE sensors allows for the insertion of more than
one type of sensor. The second sensor could be spectrally resolved from the primarysensors in either emission or excitation, or the PEBBLE concentration could be adjusted sothat signals from single PEBBLEs can be resolved spatially. In this manner one could
study complex biological responses, where a host of changes may occur in response to asingle agonist; this could be done in a single viable cell, and in real time. PEBBLEs are an
excellent tool for gaining further understanding of biological processes.

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Sensors and Materials, Vol. 14, No. 6 (2002)

Acknowledgment
The authors thahk Steve Parus for instrumentation expertise, Susan Barker for technicalassistance, Professor Mark E. Meyerhoff and Theresa M. Ambrose for help in the initialstages of utilizing DMA and Maria J. Moreno for assistance with the data normalization
formalism for DMA PEBBLEs, Rhonda Lightle and Chris Edwards for transmission
electron microscopy, the University of Michigan Transgenic Core for technical assistance,
and the University of Michigan Electron Microbeam Analysis Laboratory (funded in partby NSF grant EAR-5)628196) for use of the SEM. We also gratefully acknowledge NIH
Grants 2R01-GM50300-04A l (Kopelman) and R01-ES08846 (Philbert) for funding, as
well as NCI Contract N0 l-CO-07013.

Reference

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

J. Slavik: Fluorescent Probes in Cellular and Molecular Biology (CRC Press: Boca Raton, FL,1994).
W.T.Mason: Biological Techniques (Academic Press: San Diego, CA, 1993).
R. Nuccitelli: Methods in Cell Biology (Academic Press: San Diego, CA, 1994).
B. Herman: Fluorescence Microscopy, 2 ed. (Springer: New York, NY, 1998).
M. L. Graber, D. C.DiLillo, B.L.Friedman and E.Pastoriza-Munoz: Analytical Biochemistry
156 (1986) 202.
M. CohenKashi, M. Deutsch, R. Tirosh, H. Rachmani and A.Weinreb: Spectrochimica Acta
Part A-Molecular and Biomolecular Spectroscopy 53 (1997) 1655.
C. C. Overly, K D. Lee, E. Berthiaume and P. J. Hollenbeck: Proceedings of the National
Academy of Sciences of the United States of America 92 (1995) 3156.
B.Morelle, J.M.Salmon, J. Vigo and P.Viallet: Cell Biology and Toxicology 10 (1994) 339.
W. N. Ross: Biophysical Journal 64 (1993) 1655.
P. Buhlmann, E. Pretsch and E. Bakker: Chemical. Reviews 98 (1998) 1593.
W. Tan, R. Kopelman, S. L.R. Barker and M.T.Miller: Analytical Chemistry 71 (1999) 606A.
H. A.Clark, S.L.R.Barker, R.Kopelman, M.Hoyer and M.A. Philbert: Sensors and Actuators
B 51 (1998) 12.
H. A. Clark, M.�foyer, M.A. Philbert and R. Kopelman: Analytical Chemistry 71 (1999) 4831.
H. A. Clark, R. Kopelman, R. Tjalkens and M. A. Philbert: Analytical Chemistry 71 (1999)
4837.
H.A.Clark, M. Hoyer, S. Parus, M.Philbert and R. Kopelman: Mikrochimica Acta 131 (1999)
121.
E. Bakker and W..Simon: Analytical Chemistry 64 (1992) 1805.
W. E.Morf, K. Seiler, B. Lehmann, C. Behringer, K. Hartman and W. Simon: Pure & Applied
Chemistry 61 (1989) 1613.
K. Kurihara, M. Ohtsu, T. Yoshida, T. Abe, H. Hisamoto and K. Suzuki: Analytical Chemistry71 (1999) 3558.
K. Suzuki, H. Ohzora, K. Tohda, K. Miyazaki, K. Watanabe, H.Inoue and T. Shirai: Analytica
Chimica Acta 237 (1990) 155.
M. Brasuel, R. Kopelman, T. J. Miller, R. Tjalkens and M.A.Philbert: Analytical Chemistry 73(2001) 2221.
I. Tsagkatakis, S. Peper and E.Bakker: Analytical Chemistry 73 (2001) 315.
S. Peper, I.Tsagkatakis and E. Bakker: Analytica Chimica Acta 442 (2001) 25.

Sensors and Materials, Vol. 14, No. 6 (2002)

337

23 D.R. Uhlmann, G. Teowee and J.Boulton: Journal of Sol-Gel Science and Technology 8(1997) 1083.
24 H. Xu, J. W. Aylott, R. Kopelman, T. J. Miller and M.A. Philbert: Analytical Chemistry 73(2001) 4124.
25 M.Shortreed, E.Bakker and R. Kopelman: Analytical Chemistry 68 (1996) 2656.
26 M. R. Shortreed, S. Dourado and R. Kopelman: Sensors and ActuatorsB 38-39 (1997) 8.
27 D.Ammann: Ion-Selective Microelectrodes (Springer: Berlin, 1986).
28 I. Klimant, F.Ruchruh, G.Liebsch, A. Stangelmayer and 0.S. Wolfbeis: Mikrochimika Acta
131 (1999) 35.
29 I. Lahdesmaki, L.D. Scampavia, C.Beeson and J. Ruzicka: Analytical Chemistry 71 (1999) 5248.
30 J.N.Demas andB.A.DeGraff: Analytical Chemistry 63 (1991) 829A.
31 W. Xu, R. Scmidt, M.Whaley, J.N.Demas,B. A.DeGraff, E.K. Karikari andB. A.Farner:
Analytical Chemistry 67 (1995) 3172.
32 C. M. McDonagh,B.D.MacCraith and A.K. McEvoy: Analytical Chemistry 70 (1998) 45.
33 A.K. McEvoy, C.M. McDonagh andB.D. MacCraith: Analyst 121 (1996) 785.
34 The Merck Index 12th edition; Merck&Co: NJ, 1996.
35 C. M.Ingersoll and F. V. Bright: Chemtech 27 (1997) 26.
36 M. L.Bossi, M. E. Daraio and P. F. Aramendia-: Journal of Photochemistry Photobiology A 120(1999) 15.
37 M. T.Murtagh, M. R.Shahriari and M. Krihak: Chemical Materials 10 (1998) 3862.
38 T. M. Ambrose and M. E.Meyerhoff: Electroanalysis 8 (1996) 1095.
39 T. M.Ambrose and M. E. Meyerhoff: Analytical Chemistry 69 (1997) 4092.
40 H. A. Clark, Ph.D Dissertation, Ph.D Thesis,University of Michigan, Ann Arbor, 1999.
41 J.L.Holton, C. C. Nolan, S. A.Burr, D. E. Ray and J.B.Cavanagh: Acta Neuropathologica 93(1997) 159.
42 M. A. Philbert, C. C. Nolan, J.E. Cremer, D.Tucker and A. W.Brnwn: Neuropathology &Applied Neurobiology 13 (1987) 371.
43 A. Emmi, H. J. Wenzel and P. A. Schwartzkroin: Journal of Neuroscience 20 (2000) 3915.
44 S. -K. Jung, W. Gorski, C.A.Aspinwall and L. M.Kauri: Analytical Chemistry 71 (1999) 3642.

About the Authors
Dr. Raoul Kopelman and Dr. Martin A. Philbert have a continuing collaboration for thedevelopment, characterization, and application of nanodevices for sensing, imaging, andtherapeutic applications on the subcellular level. PEBBLEs, the sensing element of theseendeavors, are serving as the developmental platform for both imaging and therapeutic
applications.

Dr. Raoul Kopelman is the Kasimir Fajans Collegiate Professor of Chemistry, Physics andApplied Physics. A member of the Biophysics Program, he is also formally associatedwith the University of Michigan Centers for Ultrafast Optics and for Biologic
Nanotechnology. Dr. Kopelman is an expert in laser chemistry, optical materials scienceand nanofabrication of optochemical sensors and actuators. He is also an expert on
spectroscopy, molecular excitonics, reaction kinetics in confined domains, and Monte
Carlo simulations on phase transitions and surface chemistry. Dr. Kopelman received his
Ph.D. from Columbia University in 1960 and served as a postdoc in chemical physics at338

Sensors and Materials, Vol. 14, No. 6 (2002)

Harvard, 1960-62, and after that as Senior Research Fellow at Caltech. He has served as a
Professor of Chemistry at the University of Michigan since 1966. In 1991 he was made a
Professor of Chemistry & Physics and a member of the Biophysics Program at the
University of Michigan. In 1994 he was made The Kasimir Fajans Collegiate Professor ofChemistry, Physics & Applied Physics at the University of Michigan. In 1998 he was afounding member of the University of Michigan Biologic Nanotechnology Center at the
Medical School. Dr. Kopelman's broad range of research interests in analytical andphysical chemistry has led to more than 400 peer-reviewed publications.

Dr. Martin A. Philbert is a Professor of Toxicology and is a neurotoxicologist andexperimental neuropathologist with expertise in the biochemical and pathologic outcomesof exposure to toxic chemicals. In 1988 Dr. Philbert received his Ph.D. in neurochemistry/
biochemistry from the London Univ., Royal Postgrad. Medical School and served as a
postdoc. in neurotoxicology at Rutgers University from 1988-1990. From 1990-1995 heserved as a research Assistant Professor in Neurotoxicology at Rutgers University and has
been a member of the Michigan Toxicology faculty since 1995. The expertise of Dr.
Martin A. Philbert is essential to the study of PEBBLE sensor delivery, function, andeffectiveness as applied to living biological systems.

Graduate students and postdoc researchers who play and have played a key role in
PEBBLE development in Dr. Kopelman's lab include the following:
Dr. Murphy Brasuel (Ph.D., 2002) played a role in liquid polymer PEBBLE production(decyl methacrylate PEBBLEs for K+, Na+, and CI-), (1996-present).
Dr. Jonathan W. Aylott (postdoc 1998-2001) worked on sol-gel and acrylarnide PEBBLE
development (oxygen and glucose sensing). He is currently a Lecturer of Analytical
Chemistry at the University of Hull, UK.
Dr. Heather Clark (Ph.D., 1999) was a key player in initial PEBBLE studies, including
work on 02, Ca2+, H+, and Mg2+ acrylarnide PEBBLEs as well as initial studies on liquid
polymer PEBBLE development as well cell delivery method exploration. She is currently
a Postdoctoral Fellow at the Center for Biomedical Imaging Technology and Departmentof Physiology, University of Connecticut Health Center.
Hao Xu played a role in sol-gel and acrylamide PEBBLE development for oxygen andglucose sensing (1996-present).
Graduate students and postdoc researchers who play and have played a key role in
PEBBLE biological application in Dr. Philbert's lab include:
Dr. Marion Hoyer (postdoc 1997-1999) assisted in the initial stages of PEBBLE delivery
to cells and in designing biologically relevant experiments for PEBBLE application. She iscurrently working on automobile regulations for air quality with the EPA.
Terry J. Miller (1997-present) is currently involved in culturing of cells and running theconfocal microscope experiments involving PEBBLEs in single cells.
Dr. Ron Tjalkens (postdoc 1999-2001) was instrumental in setting up confocal experi­ments for PEBBLE application in the study of DNB toxicity. He is currently an AssistantProfessor of Toxicology and Neuroscience in the Department oflnterdisciplinary Toxicol­
ogy at Texas A&M University.