Fluorescence-based zinc ion sensor for zinc ion release from pancreatic cells

Article abstract of DOI:10.1021/ac060764i

In this paper, we describe the synthesis and characterization of analytical properties of fluorescence-based zinc ion-sensing glass slides and their application in monitoring zinc ion release from beta pancreatic cells in cell cultures. To fabricate the s

Full text of DOI:10.1021/ac060764i

Anal. Chem. 2006, 78, 5799-5804
Fluorescence-Based Zinc Ion Sensor for Zinc Ion
Release from Pancreatic Cells
Georgeta Crivat,† Kazuya Kikuchi,‡ Tetsuo Nagano,§ Tsvia Priel, Michal Hershfinkel, Israel Sekler,
Nitsa Rosenzweig,† and Zeev Rosenzweig*,†
Department of Chemistry and the Advanced Materials Research Institute, University of New Orleans,
New Orleans, Louisiana 70148, Department of Material and Life Science, Graduate School of Engineering,
2-1 Yamada-oka, Suita City, Osaka, Japan, Graduate School of Pharmaceutical Sciences, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, and Department of Cell Physiology, Medical School, Ben Gurion University,
Beer Sheva, Israel
large amount of zinc compared to other tissues, zinc is involved
in insulin synthesis, storage, and secretion.8 Insulin is costored
as a hexamer complexed with two zinc ions. It is found in a
crystalline state and stored within vesicles.9 When pancreatic cells
are stimulated by elevated glucose concentration, insulin is
coreleased with zinc through exocytosis. The dissociation of the
insulin-zinc complex occurs as a result of exposure to the
extracellular pH.8,10 The dissociation results in the formation of
insulin monomers, the biologically active form of insulin.8,11,12 The
role of zinc ions in insulin secretion and in the pathology of
diabetes is not entirely understood; however, numerous reports
have suggested that diabetes affects zinc homeostasis.13 Abnor-
mally low levels of zinc, which is typically associated with a poor
renal zinc ion reuptake, have been found in many diabetes
patients.13 Zinc-sensing glass slides could find application in studies
aiming to understand the role of zinc in the pathogenesis and
pharmacology of diabetes.
A number of analytical techniques have been used to detect
and quantify zinc in biological samples, including inductively
coupled plasma atomic emission spectroscopy,14,15 atomic absorp-
tion spectrophotometry,16,17 X-ray fluorescence18 and radioisotope
detection.19 However, none of these techniques can be used for
real time zinc ion detection in cellular systems. Fluorescence
sensors that were developed in the last two decades have provided
a new analytical tool for the detection of intracellular ion levels.
Most studies have concentrated on the fabrication of sensors for
pH and calcium ion measurements in cells.20,21 This is due to the
In this paper, we describe the synthesis and characteriza-
tion of analytical properties of fluorescence-based zinc ion-
sensing glass slides and their application in monitoring
zinc ion release from beta pancreatic cells in cell cultures.
To fabricate the sensors, the zinc ion indicator ZnAF-2
{6-[N-[N′,N′-bis(2-pyridinylmethyl)-2-aminoethyl]amino-
3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]xan-
thene]-3-one} was modified to include a sufficiently long
linking aliphatic chain with a terminal carboxyl functional
group. The recently synthesized ZnAF-2 zinc ion indicator
provided high zinc ion selectivity in physiological solutions
containing millimolar levels of calcium and other possible
interfering cations. The carboxyl-modified ZnAF-2 was
conjugated to the activated surface of glass slides, which
then served as zinc ion sensors. It was possible to grow
pancreatic cells directly on the zinc-sensing glass slide or
on a membrane placed on these glass slides. The sensors
were used to monitor zinc ion release events from glucose-
stimulated pancreatic cells. The study showed that the
zinc ion sensors responded effectively to the release of
zinc ions from pancreatic cells at the nanomolar level with
high selectivity and rapid subsecond response time.
Zinc is one of the most abundant transition metals in the body.
It is an essential element required by all cells, with various
functions; for example, the control of gene transcription and
metalloenzyme function.1-7 In pancreatic islets, which contain
* Corresponding author. E-mail: zrosenzw@uno.edu.
† University of New Orleans.
(8) Chausmer, A. B. J. Am. Coll. Nutr. 1998, 17, 109-115.
(9) Kennedy, R.; Huang, L.; Aspinwall, C. J. Am. Chem. Soc. 1996, 118, 1795-
1796.
‡ Graduate School of Engineering.
§ The University of Tokyo.
Ben Gurion University.
(1) Hirano, T.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Nagano, T. J. Am. Chem.
(10) Dodson, G.; Steiner, D. Curr. Opin. Struct. Biol. 1998, 8, 189-194.
(11) Berson, S. A.; Yalow, R. S. Am. J. Med. 1966, 40, 67-69.
(12) Helmerhorst, E.; Stokes, G. B. Arch. Biochem. Biophys. 1986, 245, 238-
242.
Soc. 2000, 122, 12399-12400.
(2) Dudek, E. Epilepsy Curr. 2001, 1, 66.
(3) Cohen-Kfir, E.; Lee, W.; Eskandari, S.; Nelson, N. Proc. Natl. Acad. Sci.
2005, 102, 6154-6159.
(13) Rabinovitch, A.; Suarez-Pinzon, W.; Strynadka, K.; Lakey, J.; Rajotte, R. J.
Clin. Endocrinol. Metab. 1996, 81, 3197-3202.
(4) Ruiz, A.; Walker, M. C.; Fabian-Fine, R.; Kullmann, D. M. J. Neurophysiol.
(14) Zhefeng, F.; Liming, D.; Xiaotao, J. CJI 2001, 3, 53.
(15) Wilharlitz, P.; Dreew, S.; Krismer, R.; et al. Mikrochim. Acta 1997, 125,
45.
(16) El-Yazigi, A.; Al-Saleh, I.; Al-Mefty, O. Clin. Chem. 1984, 30, 1358-1366.
(17) El-Yazigi, A.; Al-Saleh, I.; Al-Mefty, O. Clin. Chem. 1986, 32, 2187-2190.
(18) Cesareo, R. Eur. J. Nucl. Med. 1976, 1, 49-56.
2004, 91, 1091-109.
(5) Minami, A.; Sakurada, N.; Fuke, S.; Kikuchi, K.; Nagano, T.; Oku, N.; Takeda,
A. J. Neurosci. Res. 2006, 83, 167-176.
(6) Takeda, A.; Minami, A.; Seki, Y.; Oku, N. J. Neurosci. Res. 2004, 75, 225-
229.
(7) Lin, D. L.; Cohen, A. S.; Coulter, D. A. J. Neurophysiol. 2001, 85, 1185-
1196.
(19) Mazen, A.; Gradwohl, G.; de Murcia, G. Anal. Biochem. 1998, 172, 39-42.
(20) Nguyen, T.; Rosenzweig, Z. Anal. Bioanal. Chem. 2002, 374, 69-74.
10.1021/ac060764i CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/18/2006
Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 5799

Figure 1. Schematic diagram describing the synthesis of carboxyl-functionalized ZnAF-2 and its activation with EDC/NHS.
availability of fluorescent probes for pH and calcium ion level
determination. Zinc ion fluorescent indicators that were used for
the analysis of zinc ion in cells include ultraviolet excitable
quinoline-based dyes, such as 6-methoxy-8-quinolyl-para-toluene-
sulfonamide (TSQ),22 Zinquin,23 and visible light-excitable fluoro-
phores, such as Fluozin-3.24 A common limitation of these dyes is
their limited zinc ion selectivity, particularly in the presence of
calcium. In a recent study, Chang et al. developed a new zinc ion-
sensitive dye named Zin-naphthopyr 1 (ZNP1).25 It is based on a
hybrid seminaphthofluorescein platform and affords single-excita-
tion, dual-emission ratiometric imaging of intracellular zinc ions
through controllable zinc ion-induced switching between a fluo-
rescein and naphthofluorescein tautomeric forms. The tautomeric
chemosensor features excitation and emission maxima in the
visible range; excellent selectivity for zinc ions over ubiquitous
intracellular metal ions such as sodium, potassium, calcium, and
magnesium; and a dissociation constant (Kd) for zinc ions of <1
nM. Recently, Kikuchi and co-workers synthesized a new zinc-
sensitive fluorophore, {6-[N-[N′,N′-bis(2-pyridinylmethyl)-2-ami-
noethyl]amino-3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]-
xanthene]-3-one} (ZnAF-2).1 This fluorophore is structurally and
spectroscopically similar to fluorescein. The excitation and emis-
sion wavelengths of ZnAF-2 are 492 and 514 nm, respectively. The
fluorescence intensity of ZnAF-2 increases with increasing zinc
ion concentration. This increase is attributed to photoinduced
electron transfer (PET) when ZnAF-2 chelates zinc ions.1 Whereas
similar in zinc-sensing properties to ZNP1, ZnAF-2 exhibits higher
long-term stability. The cellular permeability of ZNP1, ZnAF-2, and
other zinc ion-sensitive dyes remained a problem, since it is
difficult to measure zinc ion release from cells if the zinc ion-
sensing dye is cell-permeable. Our current study describes a new
(21) Nguyen, T.; Dumitrascu, G.; McNamara, K. P.; Rosenzweig, N.; Rosenzweig,
Z. Anal. Chem. 2001, 73, 240-3246.
(22) Jindal, R. M.; Taylor, R. P.; Gray, D. W.; Esmeraldo, R.; Morris, P. J. Diabetes
1992, 41, 1056-1062.
(23) Qian, W. J.; Aspinwall, C. A.; Battiste, M. A.; Kennedy, R. T. Anal. Chem.
2000, 72, 711-717.
(24) Qian, W. J.; Gee, K. R.; Kennedy, R. T. Anal. Chem. 2003, 75, 3468-3475.
(25) Chang, C. J.; Jaworski, J.; Nolan, E. M.; Sheng, M.; Lippard, S. J. Proc. Natl.
Acad. Sci. 2004, 101, 1129-1134.
5800 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

Figure 2. Schematic diagram describing the fabrication of zinc-sensing glass slides, including chemical attachment of carboxyl-functionalized
ZnAF-2 to amino-modified silica substrate and the selective deprotection of the nosyl sulfonamide group from the zinc ion binding site.
approach to overcome the cell permeability problem of zinc ion-
sensitive dyes in order to facilitate their use in measurement of
zinc ion release from cells. It makes use of a modified ZnAF-2 in
the fabrication of zinc ion-sensing glass slides. The paper describes
the analytical properties of the zinc-sensitive glass slides and their
application in the measurement of glucose-stimulated zinc ion
release from beta pancreatic cells.
magnesium chloride, Dulbecco’s modified Eagle’s medium, anti-
biotic-antimycotic and fetal bovine serum qualified for cell
cultures were obtained from Invitrogen Corporation. HEPES
buffer, N-hydroxysulfosuccinimide sodium salt, N-(3-dimethylami-
nopropyl)-N′-ethylcarbodiimide, benzenethiol 99%, and zinc sulfate
were obtained from Sigma-Aldrich. Trimethoxysilylpropyldieth-
ylenetriamine was purchased from United Chemical Technologies,
Inc. Nitrilotriacetic acid was purchased from Spectrum Chemical
Mfg. Corp. Cover glasses (22 mm) were purchased from VWR
Scientific. All reagents were used as received.
EXPERIMENTAL SECTION
Materials and Reagents. The zinc ion indicator {6-[N-[N′,-bis-
(2-pyridinylmethyl)-2-aminoethyl]amino-3′,6′-dihydroxyspiro[iso-
benzofuran-1(3H),9′-[9H]xanthene]-3-one} (ZnAF-2) was modified
to include a carboxyl-terminated aliphatic chain. TPEN (N,N′,N′-
tetrakis(2-pyridylmethyl)ethylenediamine was obtained from Dojin-
do Laboratories. Trypsin-EDTA was purchased from Gibco.
Magnesium chloride, sodium chloride, potassium carbonate,
calcium chloride, magnesium sulfate, and potassium phosphate
were obtained from EM Industries, Inc. Aqueous solutions were
prepared using 18-MΩ deionized water and were conditioned
using the water purification system Barnstead Nanopure Diamond.
Phosphate buffer (PBS, pH ) 7.2) without calcium chloride and
Fluorescence Spectroscopy and Microscopy. Emission
spectra were obtained using a PTI international (model QM1)
fluorometer equipped with a 75-W continuous Xe arc lamp as a
light source. Fluorescence microscopy measurements were car-
ried out using an inverted fluorescence microscope (Olympus IX-
70). A 100-W mercury lamp was used as the light source for
excitation and a 20× microscope objective was used to collect
the fluorescence. The fluorescence microscope filter cube con-
sisted of a 480/30-nm bandpass excitation filter, a 500-nm dichroic
mirror, and a 535/40-nm bandpass emission filter. A 250-mm
Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 5801

spectrograph (Roper Scientific, Acton SpectraPro 2300) was
attached to the side port of the microscope and used to collect
emission spectra of the observed samples. A high-performance
charge-coupled device (CCD) camera (Roper Scientific,
Photon Max 512B) was attached to the exit port of the 250-mm
spectrograph and used for digital fluorescence imaging and
spectroscopy of the samples. The Roper Scientific software
WinSpec 32 was used for data acquisition and spectral analysis.
A typical exposure time was 0.1 s. Each data point was the average
of 10 randomly selected fields of view in the obtained images.
Silanization of Glass Slides. Prior to silanization, glass slides
underwent acidic treatment to remove particulate matter and
organic impurities. The acidic treatment also ensured the release
of hydroxyl groups of the silica glass slides.26,27 Preparation of
glass slides for silanization consisted of an immersion in 1:1
concentrated HCl/MeOH for 30 min, rinsing with deionized water,
treatment with H2SO4 for 30 min, and boiling in deionized water
for 5 min. A solution of 1% silane trimethoxysilylpropyl diethyl-
enetriamine (DETA) was hydrolyzed in 1 mM acetic acid for 5
min prior to silanization. The silanization reaction was carried out
by immersing the treated glass slides in the DETA solution for
30 min at room temperature. The silanized slides were thoroughly
rinsed with deionized water, dried under N2, and cured at 120 °C
for 5 min.
Synthesis of ZnAF-2 Modified with a Carboxyl-Terminated
Aliphatic Chain. The synthesis of ZnAF-2 modified with a
carboxyl-terminated aliphatic chain is described in Figure 1.
Compound 1 was synthesized following Kikuchi’s earlier work.1
Compound 1 (70 mg, 92 µmol) was dissolved in 400 µL of DMF
with 50 µL of diisopropylethylamine and 50 µL of diisopropylcar-
bodiimide. A 50-mg portion of N-hydroxysuccinamide was then
added to the solution. The reaction mixture was stirred at room
temperature for 24 h. It was then evaporated to dryness and
resuspended in 0.1% TFA. The product was separated by HPLC
with an ODS column. Eluent: initial, 20% acetonitril, 0.1% TFA;
final, 80% acetonitril, 0.1% TFA. The collected fractions were
lyophilized, and compound 2 was obtained (19 µmol, 21% yield).
Compound 2 (16 mg, 19 µmol) was dissolved in 200 µL of DMF
with 15 mg of 4-piperidinebutanoic acid. A 8.5-µL portion of
triethylamine was then added to the solution. The reaction mixture
was stirred at room temperature for 24 h, then the solution was
neutralized with 80 µL of 2 N HCl. The solution was separated by
HPLC with an ODS column. Eluent: initial, 20% acetonitril, 0.1%
TFA; final, 80% acetonitril, 0.1% TFA. The collected fractions were
lyophilized, and compound 3 was obtained (13 µmol, 68% yield).
Compound 4 was obtained by deprotection of 3, according to the
literature.1
Figure 3. Emission spectra of 5 µM carboxyl-modified ZnAF-2 at
increasing zinc ion concentrations: (a) 0, (b) 1, (c) 3, and (d) 5 µM.
The excitation wavelength was 498 nm.
the stability of the intermediate by forming longer-lived sulfo-NHS
ester intermediates. To activate the modified ZnAF-2, we prepared
a 500-µL HEPES buffer solution at pH 7.5 that contained 1 µM of
the dye, 20 µM EDC, and 50 µM sulfo-NHS. The preparation of
ZnAF-2-modified glass slides is shown in Figure 2. The glass slides
were incubated in the activated ZnAF-2 solution for 6 h at room
temperature. After the coupling reaction, the glass slides were
thoroughly rinsed with deionized water and sonicated to remove
the adsorbed reagents. It should be noted that the secondary
amine of ZnAF-2, which is a part of its zinc binding site, was
protected with a nossyl group to preclude the possibility of
modifications that would negatively affect the zinc-sensing proper-
ties of ZnAF-2 during the fabrication of the zinc-sensing glass
slides. The protecting group was removed from the zinc binding
site once ZnAF-2 was covalently bound to the glass surface. The
selective deprotection of the nosyl sulfonamide group was carried
out in aprotic solvent (DMF), with 3 molar equiv of K2CO3 salt
and 1.5 equiv of thiolate under argon conditions for 6 h at room
temperature.
Cell Culture and Maintenance. Min-6 cells were kindly
provided by Bryan Wolf of Childrens Hospital of Philadelphia. The
cells were cultured at 37 °C under 5% CO2 on polycarbonate
membranes of cell culture inserts, which allows media access from
both sides of the cell. They were grown in Dullbeco’s modified
Eagle’s medium supplemented with 15% fetal bovine serum, 1%
Binding of ZnAF-2 to Silanized Glass Slides. Figure 2
describes the activation of the carboxyl-modified ZnAF-2 with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The activa-
tion resulted in the formation of a highly reactive O-acylisourea
intermediate that could easily react with the primary amines of
the silanized glass slides to form stable amide bonds. The active
ester intermediate (O-acylisourea) is quite unstable in aqueous
solution28 and could easily hydrolyze to regenerate the carboxyl
group. Using sulfo-NHS (N-hydroxysulfosuccinimide) increased
antibiotic-antimycotic, 4% L-glutamine, and 0.06 mM â-mercap-
toethanol.
RESULTS AND DISCUSSION
Zinc Ion Fluorescence-Sensing Properties of Modified
ZnAF-2. Figure 3 shows the zinc ion concentration dependence
of the fluorescence intensity of a 5 µM solution of the modified
ZnAF-2 dye. The excitation and emission wavelengths were 498
and 518 nm, respectively. It can be seen that the fluorescence
(26) Halliwell, C. M.; Cass, A. E. G. Anal. Chem 2001, 73, 2476-2483.
(27) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24,
3040-3047.
(28) Hermanson, G. T. Bioconjugate Techniques: Carbodiimides; Academic
Press: Rockford, IL, 1996, pp 169-176.
5802 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

in the dye distribution on the glass slides, which resulted from
the heterogeneous distribution of amine groups on the silanized
glass surface. To reduce the variation in the fluorescence intensity
measurements, each data point was composed of 10 spectral
measurements of randomly selected spots on the glass slide. This
decreased the variation in the average reading to ∼10%. Photo-
stability measurements were carried out to optimize the experi-
mental conditions in order to avoid photobleaching during
fluorescence microscopy measurements. The zinc ion-sensing
glass slides exhibited photostability properties similar to glass
slides coated with fluorescein. To minimize photobleaching, we
employed a 10X microscope objective with a numerical aperture
of 0.25, limited the exposure of the glass slides to excitation light
by employing a normally closed electronic shutter that opens only
during the exposure time of the digital camera, and limited the
number of exposures during any given experiment. Under these
conditions, we could carry out repeated measurements during 30-
min zinc release experiments from cells without significant
photodecomposition of the zinc ion-sensing indicator. Selectivity
studies were carried out to determine whether the covalent
immobilization of ZnAF-2 to the glass slides affected the zinc
selectivity of ZnAF-2. The selectivity studies showed that the
response factor of the glass slide-based zinc sensors were 1.30 (
0.21 for calcium, 1.10 ( 0.2 for magnesium, 1.16 ( 0.1 for sodium,
and 1.3 ( 0.3 for iron. The affinity of these cations reached
saturation at micromolar concentrations. Reversibility measure-
ments were performed to demonstrate the capability of the zinc-
sensing glass slides to detect multiple exocytotic events. The glass
slide-based sensors were first treated with 1 µM zinc solution,
and a 2-fold fluorescence increase was recorded instantly. A
fluorescence decrease to the basal level was observed when 1
mM TPEN solution was added to the glass slides. TPEN is an
effective zinc ion chelator. At millimolar concentration, TPEN
displaced the zinc ions from the glass slides, which resulted in a
fluorescence decrease of the sensor. The sample was thoroughly
rinsed with a 100 mM HEPES buffer to remove the TPEN and
zinc ions from the sensors, then the glass slide-based sensors were
exposed again to a zinc-containing solution. As expected, a 2-fold
fluorescence increase of the sensor was observed. The response
factor decreased gradually with the number of repeated zinc ion
exposures, indicating that some of the binding sites were
permanently blocked or that the sensing dyes were pho-
tobleached. It was possible, however, to run at least five repeated
exposures without a significant loss of zinc ion sensitivity. Time-
dependent fluorescence measurements of the sensors at 520 nm
(λex ) 498 nm) were carried out to determine the response time
of the zinc ion-sensing glass slides. The fluorescence intensity
was measured 10 s prior to adding 80 µL of 1 µM zinc ion to the
sensor while recording the fluorescence intensity for another 10
s. The fluorescence intensity of the sensor increased due to the
addition of zinc in less than 1 s. Figure 4 shows the fluorescence
spectra of a glass slide-based zinc sensor at increasing zinc
concentration. It can be seen that the fluorescence intensity
increased with increasing zinc ion concentration between 0 and
80 nM. The dynamic range of the glass slide-based zinc sensors
was between 1.25 and 20 nM. This concentration range is lower
than the one observed for free ZnAF-2 solution1 due to the limited
number of ZnAF-2 molecules that are covalently attached to the
Figure 4. Emission spectra of a zinc ion-sensing glass slide in
solutions of increasing zinc ion concentrations: (a) 0, (b) 5, (c) 20,
and (d) 80 nM. The excitation wavelength was 498 nm.
Figure 5. Emission spectra of a zinc ion-sensing glass slide prior
to and following glucose-stimulated zinc release from Min-6 cells. The
spectra were taken at 1-s time intervals.
intensity increased by ∼3-fold when the concentration of zinc in
the solution increased from 0 to 5 µM. The excitation and emission
wavelengths were very similar to the excitation and emission
wavelengths of unmodified ZnAF-2 and other fluorescein deriva-
tives.
Analytical Properties of ZnAF-2-Modified Glass Slides.
Zinc ion-sensitive glass slides were fabricated according to the
protocol described in the Experimental Section. The concentration
of ZnAF-2 in the preparation solution was optimized to realize the
maximum zinc ion response. It was found that a solution
concentration of 1 µM ZnAF-2 resulted in a maximum response
factor of 2.1 ( 0.2. The response factor was defined as Fmax/F0
where Fmax was the maximum fluorescence intensity of ZnAF-2-
modified glass slides (obtained at 1 µM zinc ion concentration)
and F0 was the fluorescence intensity in the absence of zinc.
Fluorescence intensity measurements of the zinc-sensing glass
slides indicated a large variation of up to 25% among different spots
on the glass surface. This was attributed to significant variations
Analytical Chemistry, Vol. 78, No. 16, August 15, 2006 5803

glass slide. However, this dynamic range still enables the detection
of release events in pancreatic cells.
Application of the Zinc Sensors for the Measurement of
Zinc Release from Pancreatic Cells. Min-6 cells were chosen
for these experiments since they retain the characteristics of the
glucose transport system and glucose metabolism of normal
pancreatic beta cells and secrete insulin in response to chemical
stimulation by glucose or potassium.29 We used 500 K cells/mL
in our cell experiments. A polycarbonate membrane in which
Min-6 cells were grown was placed over the zinc-sensing glass
slides. The membrane and glass slides were washed with a
solution of Krebs-Ringer buffer at pH 7.4 that contained 500 nM
TPEN to complex and remove free zinc ions from the solution.
The glass slides and membrane were then washed and placed in
a Krebs-Ringer buffer free of TPEN. Fluorescence measurements
of the zinc-sensing glass slide under zinc-free conditions were
obtained. The cells were then treated with 20 mM glucose to
induce zinc release. Figure 5 shows the fluorescence spectra of a
zinc-sensing glass slide prior to and following stimulation of the
Min-6 cells with 20 mM glucose. An instant increase in the
fluorescence of the zinc-sensing glass slide by ∼90% is clearly
seen. We also employ fluorescence microscopy measurements to
monitor the release of zinc ions from Min-6 cells. In these
experiments, suspended Min-6 cells were placed directly on a zinc
ion-sensing glass slide. Figure 6a shows a transmission image of
the cells when placed on the slide. Figure 6b shows the
fluorescence image of the cells prior to stimulation with glucose.
Only a residual fluorescence is seen. Figure 6c shows the
fluorescence image of the glass slide that was taken immediately
following stimulation of the cells with 20 mM glucose. A large
increase in the fluorescence intensity of the glass slide is observed,
which was attributed to zinc release. The fluorescence increase
varied largely between cells. Some cells did not register a
fluorescence increase when stimulated, whereas others showed
a 2-fold fluorescence intensity increase in response to stimulation.
This is indicative of a large cell-to-cell variation in the response
of pancreatic cells to stimulation.
SUMMARY
Zinc ion sensors were fabricated by the covalent attachment
of ZnAF-2, a newly synthesized zinc ion fluorescence indicator,
to amino-modified glass slides. Detailed characterization studies
indicated that the glass slide-based zinc sensors have the high
sensitivity and selectivity and fast response time needed to
measure zinc ion release events from pancreatic cells. The sensor
was used to measure the release of zinc from Min-6 cells when
stimulated by elevated glucose levels. The study revealed large
variations in zinc ion release between individual cells. It should
be noted that quantitative analysis of released zinc is complicated,
since binding of released zinc to free insulin competes to some
extent with the binding of released zinc ions to the surface-bound
ZnAF-2 molecules. The binding of zinc ions to insulin is pH-
dependent. This could result in a pH-dependent negative bias of
the measured levels of released zinc ion from the cells. The
analytical properties of the newly developed sensors could be
further improved by decreasing the heterogeneity of dye distribu-
Figure 6. (a) A 10X transmission image of Min-6 cells in suspen-
sion, (b) fluorescence image of a zinc ion-sensing glass slide prior to
stimulation with glucose, and (c) fluorescence image of the same zinc
ion-sensing glass slide following the stimulation and zinc ion release
from beta cells deposited on the glass slide.
tion on the silanized glass slides. In the future, we will explore
alternative approaches to silanization to fabricate more homoge-
neous sensors.
ACKNOWLEDGMENT
This work was supported by NSF Grant CHE-0314027. The
authors acknowledge the support of Laurie Locascio and Michael
Gaitan of the National Institute of Standards and Technology
(NIST) and the NIST administration for enabling the completion
of this research project at NIST in the aftermath of Hurricane
Katrina.
Received for review April 24, 2006. Accepted June 7,
2006.
AC060764I
(29) Ishihara, H.; Asano, T.; Tsukuda, K.; Katagiri, H.; Inukai, K.; Anai, M.;
Kikuchi, M.; Yazaki, Y.; Miyazaki, J.-I.; Oka, Y. Diabetologia 1993, 36, 1139-
1145.
5804 Analytical Chemistry, Vol. 78, No. 16, August 15, 2006