communications
Fluorescence
Optical Detection of Glucose by Means of Metal
Nanoparticles or Semiconductor Quantum Dots**
Lily Bahshi, Ronit Freeman, Ron Gill, and Itamar Willner
The use of metal nanoparticles (NPs) or semiconductor
quantum dots (QDs) for sensing and biosensing has attracted
growing research efforts.[1,2] The unique optical features of
metallic NPs have been extensively implemented to develop
biosensing procedures.[3] For example, the color changes of
aggregated AuNPs as a result of interparticle plasmon
coupling were used for the development of DNA sensors,[4]
aptamer-based biosensors,[5] nucleic acid-based Hg2þ or Agþ
sensors,[6] and DNAzyme-based Pbþ2 sensors.[7] AuNPs have
also been used for the amplified surface plasmon resonance
(SPR) sensing of biorecognition events through the coupling
of the local plasmon of the NPs with surface plasmon.[8]
Similarly, the biocatalytic growth of metal NPs has been used
to probe enzyme activity[9] and to track enzyme inhibitors.[10]
For example, the biocatalyzed oxidation of glucose was
followed optically by the H2O2-stimulated reduction of
AuCl4
and the catalytic growth of AuNPs.[9a] Similarly,
the 1,4-dihydronicotinamide adenine dinucleotide (NADH)
cofactor reduced AuCl4–, resulting in the growth of AuNPs.[11]
This process was implemented for the analysis of NADþ-
dependent enzymes. Semiconductor QDs have been applied
to follow biocatalytic transformations.[12] For example, the
activity of tyrosinase was probed by following the quenching
of the luminescence of QDs by the reaction product of
the biocatalytic process.[12d] QDs have also been used for
the fluorescent detection of glucose.[13] For example glucose
was monitored by analyzing the quenching of QDs by the
enzyme-generated H2O2.[14] In the present report we use
the NADþ-dependent glucose dehydrogenase (GDH) and
semiconductor CdSe/ZnS QDs or metallic AuNPs to develop
integrated hybrid systems for the optical detection of glucose.
CdSe/ZnS QDs were functionalized with methylene blue
(MBþ)-modified avidin (average loading of avidin and MBþ
were 3 units per QD and 6 units per QD, respectively) that was
covalently linked to the glutathione (GSH) protecting capping
layer associated with the QDs (Figure 1). Since MBþ is
reduced by NADH[15] and the fluorescence of the QDs is
quenched by MBþ via a fluorescence resonance energy
[] Prof. I. Willner, L. Bahshi, R. Freeman, R. Gill
Institute of Chemistry
The Center for Nanoscience and Nanotechnology
The Hebrew University of Jerusalem
Jerusalem 91904 (Israel)
E-mail: willnea@vms.huji.ac.il
[] This research is supported by the Converging Technology
Program administered by the Israel Science Foundation.
DOI: 10.1002/smll.200801403
676 ß 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
transfer (FRET) process, we anticipated that the modified
QDs would act as fluorescent labels for the detection of
NADH. That is, the reduction of MBþ by NADH to colorless
MBH is anticipated to eliminate the FRET process and
activate the fluorescence of the QDs. Indeed, Figure 2A
depicts the time-dependent luminescence changes upon
reaction of the MBþ-modified QDs with 1  10
3 M NADH.
As the reaction time is prolonged, the fluorescence of the QDs
intensifies. This is consistent with the fact that MBH is
colorless and hence does not quench the QDs. As the
reduction of the capping layer is controlled by the bulk con-
centration of NADH, the extent of regenerated fluorescence
of the QDs provides a quantitative measure for the
concentration of NADH. Figure 2B shows the calibration
curve corresponding to the luminescence intensities of the
QDs upon interaction with variable concentrations of NADH
for a fixed interval of 24 min.
The success in analyzing NADH by means of the
MBþ-functionalized QDs suggested that coupling of
the NADþ-dependent GDH to the system could enable the
quantitative analysis of glucose through the biocatalyzed
oxidation of glucose and the concomitant formation of
NADH. Accordingly, GDH was functionalized with biotin,
and the biotinylated GDH (B-GDH) was linked to the avidin
layer (ca. 6 enzyme units per particle) (Figure 3A). The
resulting integrated enzyme–QD conjugates were then used
for the optical detection of glucose. Figure 3B shows the time-
dependent changes in the luminescence of the QDs upon
their interaction with 5  10
4 M glucose and 1  10
3 M
NADþ. As the time interval of the reaction is prolonged,
the luminescence of the QDs intensifies. This is consistent with
Figure 1. Schematic sensing of NADH by the MBþ-functionalized CdSe/
ZnS QDs.
small 2009 5, No. 6, 676–680
 the fact that as the biocatalytic oxidation of glucose is
prolonged, the concentration of NADH increases, and hence
the fluorescence of the QDs is higher. Thus, glucose could be
quantitatively monitored by analyzing the biocatalytically
generated NADH at a fixed time interval of the enzymatic
process. Figure 3C shows the calibration curves upon
analyzing variable concentrations of glucose while allowing
the biocatalytic process to proceed for two fixed intervals that
corresponded to 30 and 60 min, respectively. Control experi-
ments revealed that all of the components in the integrated
system are essential to alter the fluorescence of the QDs.
Exclusion of either glucose or NADþ or B-GDH from the
system did not affect the luminescence of the QDs. These
results imply that the biocatalyzed oxidation of glucose and the
formation of NADH are essential to stimulate the lumines-
cence of the QDs.
The GDH-mediated oxidation of glucose with the
concomitant generation of NADH was also used for the
optical detection of glucose by means of AuNPs. Towards this
goal, GDH was modified with mono-N-hydroxysuccinimide
(NHS)-functionalized AuNPs (1.4-nm diameter) (Figure 4A).
Transmission electron microscopy (TEM) measurements
Figure 2. A) Time-dependent fluorescence changes upon the interaction indicated that each of the proteins is modified with ca. 12–
of the MBþ-functionalized QDs with 1 mM NADH. Spectra were recorded at
15 AuNPs (Figure 4B). As the AuNPs catalyze the NADH-
3 min intervals. B) Calibration curve corresponding to the optical analysis
of different concentrations of NADH by the functionalized QDs. Each mediated reduction of AuCl4
with the concomitant enlarge-
þ
sample was analyzed after reacting the MB -functionalized QDs with ment of the particle, one may apply this process to analyze
variable concentrations of NADH for a fixed 24 min interval. NADH or glucose (Figure 4A). The AuNPs (1.4-nm diameter)
linked to GDH do not exhibit a plasmon absorbance in the
visible absorption regions. The enlargement of the AuNPs by
the biocatalytically generated NADH yields, however,
particles with a distinct absorbance in the
visible spectral region. Figure 4C shows the
time-dependent absorbance changes of the
AuNP-GDH buffer solution upon interac-
tion with 1.6 mM glucose and 2.5 mM
NADþ. As the time of the biocatalytic
process is prolonged, the plasmon absor-
bance of the particles intensifies. These
results are consistent with the fact that
higher concentrations of NADH, generated
upon prolonging the biocatalytic transfor-
mation, lead to bigger NPs with higher
absorbance. One may note that the
NADH-mediated enlargement of the NPs
does not only increase the absorbance, but
the absorbance peak of the plasmon is also
red-shifted. For example, the plasmon
absorbance of the NPs generated after
50 min of growth is shifted by 8 nm as
compared to the plasmon absorbance of the
NPs formed after 10 min (from 527 nm to
535 nm). The red-shift in the absorbance of
the NPs is consistent with their biocatalytic
Figure 3. A) Sensing of glucose by the MBþ-functionalized CdSe/ZnS QDs. B) Time- growth. Control experiments revealed that
dependent fluorescence changes upon the interaction of 0.5 mM glucose with the in the absence of glucose or NADþ or
þ
MB -functionalized QDs. Spectra were recorded at 6 min intervals. C) Calibration curve
AuNP-GDH, no absorbance changes are
corresponding to the optical analysis of different concentrations of glucose by the
MBþ-functionalized QDs. Each sample was analyzed by reacting the functionalized QDs with observed. Also, the application of GDH
different concentrations of glucose for two fixed time intervals of 30 min and 60 min, instead of AuNP-GDH did not lead to the
respectively. formation of AuNPs. These results indicate

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 communications
that the AuNP seeds associated with GDH
act as catalysts for the biocatalytic growth
of the NPs.
The catalytic growth of the AuNPs was
then used for the optical detection of
glucose. A mixture of AuNP-GDH and
NADH (generated biocatalytically) was
reacted with AuCl4– in the presence of
variable concentrations of glucose for a
fixed time of 50 min, and the growth of the
AuNPs was followed spectroscopically.
Figure 4D depicts the absorbance changes
upon analyzing different concentrations of
glucose. As the concentration of glucose
increases, the absorbance of the resulting
NPs intensifies. These results are consistent
with the growth of the AuNPs.
In conclusion, the present study has
demonstrated the use of semiconductor
QDs or AuNPs for probing the biocata-
lyzed oxidation of glucose by NADþ-
dependent GDH. The detection limit for
analyzing glucose by the QDs is 1  10
5 M,
whereas the limit of detection for analyzing
glucose by the biocatalytic growth of the
AuNPs is 1  10
4 M. The practical utility of
the system is, however, questionable due to
the long analysis times. Table 1 summarizes
the limits of detection of glucose by
different QD- or NP-based sensor systems.
While the electrochemical sensors yield an
almost instantaneous amperometric read-
out signal in a glucose concentration range
of 5–30 mM, relevant for diabetes, the
optical biosensors reveal enhanced sensi-
tivities, yet longer analysis times. This
suggests that the optical biosensor may be
applied for analysis of low volume body
fluids that contain limited amount of
glucose, for example subcutaneous fluids.

Figure 4. A) Colorimetric detection of glucose by the NADH-mediated growth of 1.4-nm

Experimental Section
diameter AuNPs. B) TEM image of a GDH enzyme modified with AuNPs. C) Time-dependent
Materials: Ultrapure water from NANOpure
absorbance changes of the AuNP-GDH solution upon interaction with 1.6 mM glucose in the
presence of 0.1 mg AuCl4
. D) Calibration curve corresponding to the optical analysis of Diamond (Barnstead International) source
different concentrations of glucose by the biocatalytic growth of the AuNPs for a fixed interval was used throughout the experiments. Hops
of 50 min. Absorbance was measured at l ¼ 530 nm. Yellow Core/Shell EviDots (CdSe/ZnS QDs) in
toluene were purchased from Evident Tech-

Table 1. Detection limits of glucose by different QD or NP-based sensor systems.

Method System Detection Limit [M] Reference

3
Electrochemical (Direct Wiring) AuNPs/Glucose Oxidase 1  10 [16a]
Electrochemical (Direct Wiring) AuNPs/GDH 1  10
3 [16b]
Electrochemical (Analysis of H2O2) PtNPs/Glucose Oxidase 0.1  10
3 [16c]
Optical (Displacement of AuNPs by glucose) CdTe QDs/AuNPs hybrid 1  10
8 [13a]
Optical (Quenching of QDs) CdTe/Glucose Oxidase/Peroxidase/Hydroquinone 5  10
8 [13b]
Optical (Generation of enhanced fluorescence) CdSe/ZnS QDs/GDH-NADþ 1  10
5 Present Study
Optical (Biocatalytic growth of AuNPs) AuNPs/GDH-NADþ 1  10
4 Present Study

678 www.small-journal.com ß 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim small 2009 5, No. 6, 676–680
nologies. Bis(sulfosuccinimidyl) suberate (BS3) was purchased
from Pierce Biotechnologies. NHS-AuNPs (1.4-nm diameter) were
purchased from NanoProbes Inc. Monocarboxy-MBþ-NHS ester
was purchased from EMP Biotech GmbH. All other chemicals and
enzymes were purchased from Sigma.
Preparation of GSH-capped QDs: QDs were precipitated from
toluene solution by addition of 2 mL methanol to 0.5 mL QDs in
toluene, followed by centrifugation for 5 min at 3000 rpm. The
resulting precipitate was dissolved in 1 mL chloroform, to which
was added 200 mL of a GSH solution (containing 0.142 g GSH and
40 mg KOH in 2 mL methanol) and the resulting mixture was
shaken. After the addition of 1.5 mL of 1 mM NaOH solution in
water, all particles were transferred to the water phase. The QDs
solution was separated from the chloroform by centrifugation for
1 min. The excess GSH was removed by two successive QD
precipitation steps using NaCl and methanol followed by
centrifugation. The resulting QDs were dissolved in 400 mL of a
10 m M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer (pH 7.4).
Preparation of avidin-capped QDs: To 1 nmol GSH-capped QDs
were added 100 mL of a BS3 stock solution (1 mg mL
1) and the
mixture was shaken for 15 min. The QDs were purified by
precipitation, dissolved in 10 mM HEPES buffer (pH 7.4) to which
was added 1 mg of avidin, and then the solution was shaken for
1.5 h. Finally, the excess of avidin was removed by two successive
precipitation steps, and the purified particles were dissolved in
400 mL of 10 mM HEPES buffer.
Preparation of MBþ-capped QDs: To the buffer solution of
1 nmol avidin-QD was added 0.5 mmol of monocarboxy-MBþ-NHS
ester dissolved in 150 mL methanol. After 1 h of shaking, excess of
MBþ-NHS was removed by two successive precipitation steps of
QDs, which were then dissolved in HEPES buffer.
Preparation of B-GDH: 1 mg GDH and 10 mL of a B-NHS stock
solution (1 mg in 1 mL DMSO) was shaken for 1.5 h, then GDH was
purified from excess of biotin using a centricon filtration device
(50 000 cutoff, Millipore Inc.).
Preparation of MBþ and GDH-MBþ-capped QDs: To the
solution of MBþ-QDs were added 10 nmol of B-GDH, the mixture
was shaken for 30 min, and then the GDH-MBþ-QDs were purified
by three precipitation steps.
Preparation of AuNP-GDH: A solution of 0.01 mg GDH was
reacted with 6 nmol of NHS-AuNPs in 10 mM HEPES buffer (pH 7.4)
for 1.5 h at room temperature. The AuNP-modified enzymes were
then purified and separated from the free NPs using a centricon
filtration device (50 000 cutoff, Millipore Inc.).
Biocatalytic enlargement assay: The GDH-mediated oxidation of
glucose in the presence of NADþ, monitored by the enlargement of
AuNPs (as optical labels) was performed in two steps: 1) A solution
of HEPES buffer (5 mM, pH 7.4) that contained 2.5 mM NADþ, 0.27 U
of AuNP-GDH hybrid and different concentrations of glucose was
allowed to react for 30 min at room temperature, and 2) a 200 mL
aliquot of this mixture was added to 800 mL aqueous growth
solution consisting of 27 mg cetyl trimethylammonium bromide
(CTAB) and 0.1 mg HAuCl4
. The solution was allowed to react for
50 min at 38 8C, and the absorbance spectra were recorded.
Determination of Avidin/MBþ/B-GDH residue loading on the
particles: The loading of the different residues on the QDs was
determined spectroscopically. The spectra of the QDs were
small 2009 5, No. 6, 676–680 ß 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
recorded before and after modification with the different
modifiers. From the difference spectrum, and knowing the
extinction coefficient of the residues (avidin: 105 400 M
1 cm
1
at l ¼ 282 nm, MBþ: 71 547 M
1 cm
1 at l ¼ 660 nm, B-GDH:
39 190 M
1 cm
1 at l ¼ 282 nm), the concentrations of the
different units were determined. Knowing the concentration and
extinction coefficient of the QDs (100 000 M
1 cm
1 at
l ¼ 540 nm), the average loading of units per particle was
calculated.
Instruments: Real-time fluorescence measurements were
carried out using a photon-counting spectrometer (Edinburgh
Instruments, FLS 920) equipped with a cooled photomultiplier
detection system connected to a computer (F900 v.6.3 software,
Edinburgh Instruments). UV–Vis spectroscopy measurements were
carried out using a Shimdzu UV-2401PC. NP sizes were measured
using a Tecnai F20 G2 TEM using 300 mesh grids covered with
carbon/formvar.
Keywords:
glucose . gold . metal . nanoparticles . quantum dots
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Received: September 24, 2008
Revised: November 9, 2008
Published online: February 18, 2009
small 2009 5, No. 6, 676–680