Bioluminescent nanosensors for protease detection based upon gold nanoparticle-luciferase conjugates

Article abstract of DOI:10.1039/b915612g

This communication reports the use of click chemistry to site-specifically conjugate bioluminescent Renilla luciferase proteins to gold nanoparticles (Au NPs) for sensing protease activity. The bioluminescent emission from luciferase was efficiently quenched by Au NPs, but significantly recovered after the proteolytic cleavage.

Full text of DOI:10.1039/b915612g

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Bioluminescent nanosensors for protease detection based upon gold
nanoparticle–luciferase conjugatesw
Young-Pil Kim,a Weston L. Daniel,b Zuyong Xia,a Hexin Xie,a Chad A. Mirkinb and
Jianghong Rao*a
Received (in Cambridge, UK) 30th July 2009, Accepted 28th September 2009
First published as an Advance Article on the web 12th October 2009
DOI: 10.1039/b915612g
This communication reports the use of click chemistry to
site-specifically conjugate bioluminescent Renilla luciferase
proteins to gold nanoparticles (Au NPs) for sensing protease
activity. The bioluminescent emission from luciferase was
efficiently quenched by Au NPs, but significantly recovered after
the proteolytic cleavage.
eight mutations and shows higher stability and improved
catalytic efficiency than the wild-type luciferase.22
A
site-specific bioconjugation strategy was devised to conjugate
the Luc8 to Au NPs with click chemistry and an intein-
mediated protein splicing reaction (Scheme 1). The cyclo-
addition between azides and alkynes, known as the ‘‘click’’
reaction, is highly efficient and specific, can take place in
water, and has been successfully applied to in vitro and
in vivo applications, including the coupling of biomolecules
to nanoparticles.23–26 An intein-mediated ligation method27,28
was implemented to site-specifically introduce the azide moiety
to Luc8 to facilitate the conjugation. MMP-2 was chosen as
the model protease because of its important role in promoting
tumor progression and invasion.29,30 A single peptide substrate
of MMP-2 (IPVSLRSG) was employed as the sensing domain
in the nanoconjugate.
Nanoparticles functionalized with biomolecules hold promise
for many potential applications in nanotechnology and
nanomedicine.1–7 Among the many types of nanoparticles,
gold nanoparticles (Au NPs) have been of special interest
due to their biocompatibility and outstanding biophysical
properties and have already been utilized in many medical
applications.8–10 It has been demonstrated that Au NPs have
superior quenching efficiency compared to molecular quenchers
for emission from small organic dyes,11 fluorescent proteins,12
and even semiconductor quantum dots.13 This quenching
ability enables the design of fluorescent nanosensors based
on Au NPs for biosensing applications.14–17 However, these
fluorescent probes are often amenable to fast photobleaching
and excitation-induced cytotoxicity.18 Bioluminescence (BL)
offers an alternative to circumvent this problem and has been
drawing much attention for in vitro monitoring19 and
noninvasive imaging of specific targets in vivo.20 While small
organic dyes have been reported to quench bioluminescence
emission through bioluminescence resonance energy transfer,21
the utility of Au NPs as luminescence quenchers has not been
explored in the literature. Herein, we show that Au NPs can
efficiently quench the emission from the bioluminescent
protein luciferase and that this Au NP quenching effect can
be applied to design nanosensors for the detection of matrix
metalloproteinase-2 (MMP-2) activity.
As depicted in Scheme 1, oligo(ethylene glycol)-modified Au
NPs 1 (B5 nm Au core diameter) with carboxyl groups at their
termini were modified with an alkyne derivative 2 via a
carbodiimide-mediated reaction, resulting in alkynated Au
NP (3, Au–R). A recombinant Luc8 protein (4, Luc8-pep-
GyrA) was expressed with the substrate peptide (IPVSLRSG)
and the Mex GyrA intein protein fused at its C-terminus.27
The thioester intermediate, formed via the catalysis of GyrA,
was reacted with cysteine-azide (5, Cys-N3) to produce the
Luc8-pep fusion with an azide-modified C-terminus (6,
Luc8-pep-N3). The final product (7, Luc8-pep-Au NP) was
generated from 3 and 6 by copper(I)-catalyzed azide–alkyne
[3 + 2] cycloaddition.
The bioluminescent protein used in this work is
a
Renilla reniformis luciferase mutant dubbed Luc8, which has
a Molecular Imaging Program at Stanford (MIPS), Departments of
Radiology and Chemistry, Stanford University, Stanford,
CA 94305-5484, USA. E-mail: jrao@stanford.edu;
Fax: +1 650 736 7925; Tel: +1 650 736 8563
b Department of Chemistry and International Institute for
Nanotechnology, Northwestern University, 2145 Sheridan Road,
Evanston, IL 60208-3113, USA.
E-mail: chadnano@northwestern.edu;
Fax: +1 847 467 5123, +1 847 467 2907
w Electronic supplementary information (ESI) available: Experimental
section, the determination of the number of Luc8 per Au NP, signal
recovery of Luc8 after the click reaction, the quenching efficiency of
the conjugate, and time-dependent monitoring of MMP-2 activity. See
DOI: 10.1039/b915612g
Scheme
1 Conjugation of gold nanoparticles (Au NPs) with
recombinant luciferase proteins via click chemistry and an intein-based
ligation method.
ꢀc
This journal is The Royal Society of Chemistry 2010
76 | Chem. Commun., 2010, 46, 76–78

View Article Online
We examined the relative migration of the Luc8-pep-GyrA
protein by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) to confirm its intein-based ligand
modification by 5 (Cys-N3). Luc8-pep-GyrA showed two
strong bands under non-reducing conditions (lane 1 in
Fig. 1) corresponding to the monomeric form (59 kDa) and
the oxidized dimer form (118 kDa) of the protein. Addition of
only the Cys-N3 group to the fusion protein resulted in few
cleavage products (lane 2). However, efficient cleavage by
Cys-N3 took place in the presence of 2-mercaptoethanesulfonic
acid (MESA) as evidenced by the reduced intensity of fusion
protein band, and the appearance of two bands corresponding
to the azide-modified Luc8 fusion protein and the free GyrA
intein (lane 4). In comparison, MESA treatment alone also
produced two cleavage bands (lane 3) corresponding to the
Luc8-pep (37 kDa) and the GyrA intein (21 kDa), but the
unmodified Luc8-pep appeared to have a slightly faster
mobility than the azide-modified Luc8-pep. Purification with
a Ni-nitrilotriacetic acid (NTA) affinity column removed the
His6 tagged GyrA intein and any uncleaved fusion protein
from the reaction and gave the pure azide-modified fusion
protein (Luc8-pep-N3) (lane 5). This result confirmed the
successful site-specific introduction of the azide group to the
Luc8 fusion protein.
Fig. 2 Click chemistry-based conjugation of Au–R and Luc8-pep-N3:
agarose (1.2%) gel electrophoresis (a) and UV-visible spectroscopy
analysis (b). In agarose gel electrophoresis, Au–R was subjected to
different reaction conditions, where one of the components was
removed in the reaction solution: without Luc8-pep-N3 (lane 1),
without CuSO4 (lane 3), or without ascorbate (lane 4). The extinction
graphs were displayed for Au–R (black line) and for Luc8-pep-Au
NP (gray line).
confirmed this conjugation using UV-Vis spectroscopy after
purifying the mixture with 100 kDa cut-off microfiltration.
Compared to the unconjugated Au NPs (Au–R), which had a
strong absorption at 516 nm, the Luc8-conjugated Au NPs
displayed a slightly red-shifted spectrum (520 nm) because of
their surface modification (Fig. 2(b)). Finally, we determined
that the conjugation afforded 4.7 Æ 1.2 proteins per Au NP
by displacing its monolayer with 2-mercaptoethanol and
quantifying the luciferase concentration with a Bradford assay
(ESI,w Fig. S1).
Before using the Luc8-pep-Au NP conjugates in the MMP-2
detection assay, we examined the effect of Cu(I) on the activity
of the Luc8 protein, because it has been reported to deactivate
the function of the protein.32 When the Luc8 was treated with
Cu(II) and ascorbate the activity of the Luc8 decreased when
compared to the untreated protein. However, this activity loss
could be partially recovered by treatment of the protein with
L-cysteine. Up to 40% of the initial BL signal of Luc8 was
regained (ESI,w Fig. S2). Among the other Cu(I)–chelating
agents that were tested, dithiothreitol (DTT) was also effective
at restoring the Luc8 activity. However, DTT displaced the
oligo(ethylene glycol) groups from the Au NP surface33 and
destabilized the Au NPs. To further verify the quenching
efficiency of Au NP, the carboxyl Au NP was directly
conjugated with Luc8-pep-N3via 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC) reaction with different ratios of
Luc8 to Au NP, and the maximum quenching efficiency
was observed at the ratios of 2 : 1 and 1 : 1 (Luc8 : Au NP),
which corresponds to 85%, as compared to the initial BL
(ESI,w Fig. S3).
Au–R was treated with Luc8-pep-N3 in the presence of
CuSO4 and ascorbate to couple the Au NPs with azide-
modified luciferase by click chemistry. The conjugation of
the protein to the Au NP surface was confirmed with electro-
phoresis, UV-Vis spectroscopy, and a Bradford assay. The
Luc8 conjugated Au NPs displayed a strong band shift in
agarose gel electrophoresis (lane 2 in Fig. 2(a)) when compared
to the negative controls lacking either Luc8-pep-N3 (lane 1),
CuSO4 (lane 3), or ascorbate (lane 4). This result clearly
indicated that the Cu(I)-mediated click reaction enabled
the conjugation of Au–R to Luc8-pep-N3.31 We further
Fig. 1 NuPAGE analysis of cysteine-N3 ligation by intein-mediated
cleavage. Luc8-pep-GyrA protein (lane 1) was incubated with different
thiol groups for 16 h at 4 1C: 10 mM Cys-N3 (lane 2), 20 mM MESA
(lane 3), and 20 mM MESA/10 mM Cys-N3 (lane 4). The purified
sample from lane 4 is displayed in lane 5. The reaction mixture was
loaded into each well after mixing with lithium dodecyl sulfate (LDS)
loading buffer in the absence of reducing agent. The arrows indicate
the position of Luc8 protein fused with the Cys-N3. The molecular
weight standard is displayed in the far left lane. Electrophoresis was
carried out on 12% gel in bis (2-hydroxyethyl)amino-tris(hydroxy-
methyl)methane (Bis-Tris) buffer containing LDS for 45 min.
Finally, we evaluated whether the quenched bioluminescent
nanosensor could measure the activity of MMP-2, which
specifically cleaves the amide bond between Ser-Lys34 in the
peptide sequence linking the Luc8 to the Au NP. The BL
emission of the Luc8-pep-Au NP conjugate solution was
enhanced about 15-fold over the background signal 1 h after
adding MMP-2 (Fig. 3(a)). Sufficient enzymatic cleavage was
observed within 1 h under the given conditions (ESI,w Fig. S4),
and the increase in BL signal was linearly dependent on the
logarithmic concentration of MMP-2, with a dynamic range
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 76–78 | 77

View Article Online
(NRF-2009-352-D00098). C. A. M. is also grateful for a
National Security Science and Engineering Faculty Fellows
Program award.
Notes and references
1 C. A. Mirkin, R. L. Letsinger, R. C. Mucic and J. J. Storhoff,
Nature, 1996, 382, 607.
2 N. L. Rosi and C. A. Mirkin, Chem. Rev., 2005, 105, 1547.
3 M. Bruchez, Jr, M. Moronne, P. Gin, S. Weiss and
A. P. Alivisatos, Science, 1998, 281, 2013.
4 E. Katz and I. Willner, Angew. Chem., Int. Ed., 2004, 43, 6042.
5 M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161.
6 C. M. Niemeyer, B. Ceyhan and P. Hazarika, Angew. Chem., Int.
Ed., 2003, 42, 5766.
7 R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger and
C. A. Mirkin, Science, 1997, 277, 1078.
8 N. L. Rosi, D. A. Giljohann, C. S. Thaxton, A. K. Lytton-Jean,
M. S. Han and C. A. Mirkin, Science, 2006, 312, 1027.
9 J. M. Nam, C. S. Thaxton and C. A. Mirkin, Science, 2003, 301,
1884.
10 R. A. Sperling, P. Rivera Gil, F. Zhang, M. Zanella and
W. J. Parak, Chem. Soc. Rev., 2008, 37, 1896.
11 B. Dubertret, M. Calame and A. J. Libchaber, Nat. Biotechnol.,
2001, 19, 365.
12 P. Hazarika, F. Kukolka and C. M. Niemeyer, Angew. Chem., Int.
Ed., 2006, 45, 6827.
Fig. 3 Detection of protease activity by using different Luc8-pep-Au
NP conjugates: click and intein-based conjugate (a and b) and
randomly coupled conjugate (c and d). Left graphs represent
bioluminescence signals with different MMP-2 enzyme concentrations
(0, 50, 100, 200, 500, and 1000 ng mLÀ1, from bottom to top), and the
correlation between peak intensity and enzyme concentration is
displayed in right graphs.
13 Y. P. Kim, Y. H. Oh, E. Oh, S. Ko, M. K. Han and H. S. Kim,
Anal. Chem., 2008, 80, 4634.
14 A. Verma, H. Nakade, J. M. Simard and V. M. Rotello, J. Am.
Chem. Soc., 2004, 126, 10806.
15 D. J. Maxwell, J. R. Taylor and S. Nie, J. Am. Chem. Soc., 2002,
124, 9606.
16 E. Oh, M. Y. Hong, D. Lee, S. H. Nam, H. C. Yoon and
H. S. Kim, J. Am. Chem. Soc., 2005, 127, 3270.
17 D. S. Seferos, D. A. Giljohann, H. D. Hill, A. E. Prigodich and
C. A. Mirkin, J. Am. Chem. Soc., 2007, 129, 15477.
18 R. A. Hoebe, C. H. Van Oven, T. W. Gadella, Jr,
P. B. Dhonukshe, C. J. Van Noorden and E. M. Manders,
Nat. Biotechnol., 2007, 25, 249.
from 50 ng mLÀ1 to 1 mg mLÀ1 (Fig. 3(b)). As a control, a
Luc8-pep-Au NP nanoconjugate was prepared via an
EDC-mediated coupling reaction between the lysine residues
of the Luc8-pep-N3 and the carboxyl groups of the Au NP. As
expected, this conjugate did not show a MMP-2 dependent
increase in bioluminescence over the background intensity
(Fig. 3(c) and (d)). This result indicates that the site-specific
orientation of Luc8 on the Au NP is critical for detecting
protease activity.
19 Y. Zhang, M. K. So, A. M. Loening, H. Yao, S. S. Gambhir and
J. Rao, Angew. Chem., Int. Ed., 2006, 45, 4936.
In conclusion, we demonstrated the conjugation of luciferase
to Au NPs via click chemistry and an intein-mediated ligation
and the subsequent detection of protease activity based on the
Au NP-quenched BL. The Cu(I)-catalyzed click reaction
allowed for a rapid and efficient conjugation between the
nanoparticle and the luciferase protein, and the intein-
mediated ligation enabled this protein to be site-specifically
immobilized on the Au NP, both of which are critical for the
function of the nanosensor. This conjugation method should
be generally applicable to other proteins and nanoparticles.
Moreover, the Au NP–luciferase system can avoid the
disadvantages in fluorescence-based approaches such as
photoexcitation-derived photobleaching and phototoxicity.
We anticipate that our method will help expand the utility of
functionalized nanoparticles in biology and nanotechnology
research.
20 P. R. Contag, I. N. Olomu, D. K. Stevenson and C. H. Contag,
Nat. Med., 1998, 4, 245.
21 Z. Xia and J. Rao, Curr. Opin. Biotechnol., 2009, 20, 37.
22 A. M. Loening, T. D. Fenn, A. M. Wu and S. S. Gambhir,
Protein Eng., Des. Sel., 2006, 19, 391.
23 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8,
1128.
24 C. R. Becer, R. Hoogenboom and U. S. Schubert, Angew. Chem.,
Int. Ed., 2009, 48, 4900.
25 J. L. Brennan, N. S. Hatzakis, T. R. Tshikhudo, N. Dirvianskyte,
V. Razumas, S. Patkar, J. Vind, A. Svendsen, R. J. Nolte,
A. E. Rowan and M. Brust, Bioconjugate Chem., 2006, 17, 1373.
26 G. von Maltzahn, Y. Ren, J. H. Park, D. H. Min,
V. R. Kotamraju, J. Jayakumar, V. Fogal, M. J. Sailor,
E. Ruoslahti and S. N. Bhatia, Bioconjugate Chem., 2008, 19, 1570.
27 Z. Xia, Y. Xing, M. K. So, A. L. Koh, R. Sinclair and J. Rao,
Anal. Chem., 2008, 80, 8649.
28 P. C. Lin, S. H. Ueng, M. C. Tseng, J. L. Ko, K. T. Huang,
S. C. Yu, A. K. Adak, Y. J. Chen and C. C. Lin, Angew. Chem.,
Int. Ed., 2006, 45, 4286.
29 A. John and G. Tuszynski, Pathol. Oncol. Res., 2001, 7, 14.
30 M. Egeblad and Z. Werb, Nat. Rev. Cancer, 2002, 2, 163.
31 The combination of Au–R and Luc8-pep-N3 had a much greater
yield compared to when the azide was on the Au NP and alkyne
was on the Luc8 under the same reaction conditions.
32 A. J. Link and D. A. Tirrell, J. Am. Chem. Soc., 2003, 125, 11164.
33 C. S. Thaxton, H. D. Hill, D. G. Georganopoulou, S. I. Stoeva and
C. A. Mirkin, Anal. Chem., 2005, 77, 8174.
This work was supported in part by the National
Cancer Institute (Grant 1R01CA135294-01), the Burroughs
Wellcome Fund, the Stanford University National Cancer
Institute Centers of Cancer Nanotechnology Excellence
(Grant 1U54CA119367-01), the Northwestern University
National Cancer Institute Centers of Cancer Nanotechnology
Excellence, and the National Research Foundation of
Korea Grant funded by the Korean Government
34 B. E. Turk, L. L. Huang, E. T. Piro and L. C. Cantley,
Nat. Biotechnol., 2001, 19, 661.
ꢀc
This journal is The Royal Society of Chemistry 2010
78 | Chem. Commun., 2010, 46, 76–78