Probing biocatalytic transformations with CdSe-ZnS QDs

Article abstract of DOI:10.1021/ja066636t

CdSe/ZnS QDs enable the optical probing of the biocatalytic oxidation of tyrosine derivatives and of the scission of peptides by thrombin. CdSe/ZnS QDs were modified with tyrosine methyl ester or with a tyrosine-containing peptide. The tyrosine units were reacted with tyrosinase/O₂ to yield the respective l-DOPA and quinone derivatives. The luminescence of QDs modified by the enzyme-generated quinone units is quenched. The quinone-functionalized peptide associated with the QDs was cleaved by thrombin, a process that restored the luminescence of the QDs. Copyright

Full text of DOI:10.1021/ja066636t

Published on Web 11/10/2006
Probing Biocatalytic Transformations with CdSe-ZnS QDs
Ron Gill, Ronit Freeman, Jian-Ping Xu, Itamar Willner,* Shira Winograd, Itzik Shweky, and Uri Banin*
Institute of Chemistry and the Center for Nanoscience and Nanotechnology, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel
Received September 14, 2006; E-mail: willnea@vms.huji.ac.il
Scheme 1a
The unique size-controlled photonic properties of semiconductor
quantum dots (QDs) find increasing interest for sensing and
biosensing. Numerous studies have employed QDs as fluorescent
1
labels for biorecognition events, such as the formation of immuno-
2
3
complexes or nucleic acid-DNA duplexes. The use of semicon-
ductor QDs as reporter units for biocatalytic processes is, however,
scarce, and only few examples demonstrated the application of the
photophysical properties of semiconductor QDs to probe biocatalytic
reactions. CdS QDs were coupled with biocatalysts, and the
resulting photocurrent was employed to follow the enzyme activity.4
Fluorescence resonance energy transfer (FRET) from CdSe-ZnS
QDs to dye units incorporated into replicated DNA or into telomers
5
was used to follow polymerase or telomerase functions. Similarly,
the DNase-catalyzed scission of a duplex DNA, consisting of CdSe-
and dye-tethered hybridized nucleic acids, was probed by following
the FRET quenching of the particle in the duplex structure and the
regeneration of the QDs’ fluorescence upon the cleavage of the
DNA.6 Recently, the activities of proteolytic enzymes were
monitored by fluorescence resonance energy transfer within QD-
peptide conjugates.7 Here we wish to report on the monitoring of
tyrosinase activity and of thrombin hydrolytic activities by following
the fluorescence quenching within CdSe-ZnS QD-peptide con-
jugates. In contrast to previous studies, we generate by means of
tyrosinase the quencher units in the peptide, and we restore the
QD fluorescence by the cleavage of the quencher units.
,8
a
(A) Tyrosinase-induced oxidation of tyrosine by O2. (B) Probing
tyrosinase activity through the quenching of the luminescence of the QDs
by quinone units. (C) Probing tyrosinase and thrombin activities through
the quenching of the luminescence of the QDs by the generation of the
quinone-containing peptide and the regeneration of the luminescence of
the QDs by the thrombin-induced scission of the peptide.
Tyrosinase catalyzes the oxidation of tyrosine to L-DOPA that
is subsequently oxidized to the respective o-quinone (Scheme 1A).9
The enzyme oxidizes phenol derivatives; for example, tyramine is
oxidized to the catechol derivative. Elevated amounts of tyrosinase
were found in melanoma cells, and it is considered as a marker for
these cells.10 Previous studies have demonstrated the optical analysis
of tyrosinase through the growth of Au NPs by the L-DOPA
product.11 Scheme 1B depicts the biocatalyzed reactions that were
analyzed by probing the fluorescence properties of the CdSe-CdS-
ZnS multishell QDs.
2
QDs with tyrosinase/O resulted in the formation of L-DOPA units
that are further oxidized to the o-quinone residues. The latter
products are expected to act as quenchers of the fluorescence of
the QDs and thus provide a path for the optical detection of
tyrosinase. Figure 1A shows the fluorescence intensity of the QDs
prior to the interaction with tyrosinase, curve a, and at time intervals
of reaction with the biocatalyst. The fluorescence of the QDs is
quenched in the presence of tyrosinase, and as the reaction time
intervals are prolonged, the quenching degree is intensified (after
10 min, the fluorescence intensity decreased by 45%). Control
experiments revealed that the fluorescence of the QDs was almost
CdSe-core CdS(2MLs)/Zn0.5Cd0.5S(3MLs)/ZnS(2MLs) multishell
QDs, with a diameter of 7.3 ( 1.0 nm (core diameter ) 2.6 nm),
were prepared according to the literature procedure.6 These QDs
were further transformed into water-soluble QDs by ligand exchange
,12
unaffected (<10%) by excluding tyrosinase or O from the system.
2
Thus, the biocatalyzed oxidation of the tyrosine units led to the
6
with 3-mercaptopropionic acid (MPA). QDs were mixed with either
fluorescence quenching of the QDs. Complementary FTIR experi-
ments revealed that the tyrosinase-induced reaction, indeed, gener-
l000-fold excess of tyrosine methyl ester or 100-fold excess of
peptide 1, in a 10 mM HEPES buffer, pH ) 7.4, containing 10
mM 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
-1
ated the o-quinone product (characteristic band at 1660 cm , Figure
S2 in Supporting Information). Also, the monitoring of the lifetime
of the CdSe-ZnS QDs revealed that it was shortened as the
biocatalyzed oxidation of tyrosine proceeded (Figure S3 in Sup-
porting Information). For example, the lifetime (major component)
of the tyrosine-functionalized CdSe-ZnS QDs was 9.4 ns, and after
treatment with tyrosinase for 10 min, it was shortened to 6.5 ns
(see Supporting Information).
(EDC). The mixtures were placed on a shaker for 2 h and then
purified by precipitation of the particles by addition of 2-fold
volume acetone, followed by centrifugation at 1500 rpm for 1 min
(
see details of synthesis and solubilization in Supporting Informa-
tion).
The loading of the QDs with the tyrosine units was determined
spectroscopically, and it corresponded to ca. 60 tyrosine units per
particle (Figure S1 in Supporting Information). Interaction of the
The successful quenching of the fluorescence of the QDs by the
biocatalyzed oxidation of tyrosine allowed us to develop an optical
15376
9
J. AM. CHEM. SOC. 2006, 128, 15376-15377
10.1021/ja066636t CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
33%) is, however, substantially lower than that with the tyrosine-
functionalized QDs since the peptide introduced a longer distance
that separates the QD/quencher pairs.
The QDs modified with the o-quinone residues were then reacted
with thrombin. Figure 2A, curve c, shows the fluorescence spectrum
of the QDs after reaction with thrombin, 10 U, for 6 min. The
original fluorescence intensity of the QDs in the absence of the
quencher was recovered, implying that the quencher units were
cleaved off of the particles. Figure 2B depicts the time-dependent
decrease of the fluorescence of the QDs in the presence of tyrosinase
and the subsequent recovery of the fluorescence upon the thrombin-
induced removal of the quencher units. It should be noted that the
tyrosinase-induced oxidation of the peptide 1 is stopped after 10
min of reaction (ca. 30% quenching of the QDs) and subsequently
subjected to the scission of the peptide by thrombin, due to the
limited stability of the QD-peptide conjugation. Longer time
intervals for the oxidation of 1 result in partial precipitation of the
QD-peptide conjugates, eliminating the recovery of the QDs’
fluorescence upon treatment with thrombin.
Figure 1. (A) Luminescence spectra of the tyrosine methyl ester-
functionalized QDs upon the reaction with tyrosinase, 2 U, for (a) 0 min;
(b) 0.5 min; (c) 2 min; (d) 5 min; (e) 10 min. (B) Decrease in the
luminescence spectra of the tyrosine methyl ester-functionalized QDs upon
the reaction with (a) different concentrations of tyrosinase for a fixed time
interval of 6 min; (b) control experiment where 2 U of tyrosinase was
employed in the absence of O2. All data were recorded in 10 mM phosphate
buffer solution, pH 6.3, under air (unless otherwise stated) at 25 °C.
In conclusion, we described the successful use of semiconductor
QDs as optical probes for biocatalytic transformations. Besides the
fundamental significance of the results, the method might be of
value for the development of QD-based sensors for following
tyrosinase activity.
Acknowledgment. This research was supported by the Ger-
man-Israeli Program (DIP). We thank Prof. Chaim N. Sukenik,
Chemistry Department, Bar-Ilan University, for the FTIR-ATR
measurements on our samples.
Figure 2. (A) Luminescence spectra of the 1-functionalized QDs (a) before
treatment with tyrosinase; (b) after treatment with tyrosinase, 4 U, for 10
min; (c) after reaction with thrombin, 10 U, for 6 min. (B) Time-dependent
luminescence intensities upon reacting the 1-functionalized QDs with
tyrosinase (point a) and thrombin (point b). All data were recorded in 10
mM phosphate buffer solution, pH 6.3, under air (unless otherwise stated)
at 25 °C.
Supporting Information Available: Synthesis and functionalization
of the particles, FTIR data, and transients of the lifetimes of the tyrosine
methyl ester-functionalized QDs before and after reaction with tyro-
sinase. This material is available free of charge via the Internet at http://
pubs.acs.org.
assay for tyrosinase activity. Figure 1B shows the fluorescence
intensity of the tyrosine-functionalized QDs upon reaction with
different concentrations of tyrosinase for a fixed time interval of 6
min. As the concentration of the biocatalyst increased, the quenching
efficiency was enhanced. Tyrosinase could be assayed with a
sensitivity limit corresponding to 0.2 U of tyrosinase.
References
(
(
(
1) (a) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater.
2005, 4, 435-446. (b) Costa-Fernandez, J. M.; Pereiro, R.; Sanz-Medel,
A. Trends Anal. Chem. 2006, 25, 207-218.
2) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.; Clapp, A.
R.; Uyeda, H. T.; Deschamps, J. R.; Lassman, M. E.; Mattoussi, H. J.
Am. Chem. Soc. 2005, 127, 6744-6751.
In order to generalize the application of semiconductor QDs as
optical reporters for biocatalytic transformations, the peptide 1 was
covalently coupled to the MPA-modified QDs in the presence of
EDC (Scheme 1C). The peptide includes a tyrosine residue that is
oxidizable to the respective L-DOPA and o-quinone units and, thus,
is anticipated to yield the quencher units upon reaction with
tyrosinase. The peptide includes, however, the appropriate amino
acid sequence that is cleavable by the proteolytic enzyme thrombin.
Thus, the hydrolytic cleavage of the o-quinone units by thrombin
is anticipated to restore the fluorescence properties of the QDs.
The loading of 1 on the QDs was determined spectroscopically,
and it corresponded to 15. Figure 2A shows the fluorescence
properties of the peptide 1-functionalized CdS-ZnS QDs prior to
the reaction with tyrosinase, curve a, and after the tyrosinase-
induced oxidation of the tyrosine residue, curve b. The fluorescence
intensity is quenched due to the biocatalyzed formation of the
o-quinone derivative. The fluorescence quenching efficiency (ca.
3) (a) Mitchell, G. P.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc.
1999, 121, 8122-8123. (b) Parak, W. J.; Gerion, D.; Zanchet, D.; Woerz,
A. S.; Pellegrino, T.; Micheel, C.; Williams, S. C.; Seitz, M.; Bruehl, R.
E.; Bryant, Z.; Bustamante, C.; Bertozzi, C. R.; Alivisatos, A. P. Chem.
Mater. 2002, 14, 2113-2119.
(
(
(
4) Pardo-Yissar, V.; Katz, E.; Wasserman, J.; Willner, I. J. Am. Chem. Soc.
2003, 125, 622-623.
5) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I.
J. Am. Chem. Soc. 2003, 125, 13918-13919.
6) Gill, R.; Willner, I.; Shweky, I.; Banin, U. J. Phys. Chem. B 2005, 109,
23715-23719.
(7) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H.
T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat.
Mater. 2006, 5, 581-589.
(8) Shi, L.; De Paoli, V.; Rosenzweig, N.; Rosenzweig, Z. J. Am. Chem. Soc.
2006, 128, 10378-10379.
(
9) Korner, A. M.; Pawelek, J. M. Science 1982, 217, 1163-1165.
(10) Angeletti, C.; Khomitch, V.; Halaban, R.; Rimm, D. L. Diagn. Cytopathol.
004, 31, 33-37.
11) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566-1571.
(12) Xie, R.; Kolb, U.; Basche, T.; Mews, A. J. Am. Chem. Soc. 2005, 127,
480-7488.
2
(
7
JA066636T
J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006 15377