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Article history:
Received 25 October 2010
wide and varied including drug delivery, immobilization of
biomolecules, cell targeting vectors, sensors, enhancement of
electron transfer between electrode surfaces and proteins,
labeling of biomolecules and colorimetric detection of biomole-
cules [3,4,8e13]. Protein-AuNP hybrids have been identified as
derivative of the naturally occurring green fluorescent protein from
theNorth Pacific jellyfishAequorea Victoria- at AuNP hybridswithout
compromising their biological activity. This research demonstrated
that biological fluorophores can be incorporated into nanoparticle-
based devices, leading to optically coupled hybrid architecture
building blocks for the construction of biomimetic antennae and
light-harvesting devices [20,21]. Iosin et al. [22] investigated the
fluorescence quenching of tryptophan residues of bovine serum
albumin (BSA) by AuNPs to monitor the direct interaction between
* Corresponding authors. Tel.: þ61 8 83023546; fax: þ61 8 83023683.
E-mail addresses: naba.dutta@unisa.edu.au (N.K. Dutta), namita.choudhury@
Contents lists availab
Biomat
ev
Biomaterials 32 (2011) 2786e2796unisa.edu.au (N.R. Choudhury).
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Nanosized noble metal particles and their conjugates have
attracted significant attention owing to their unique chemical,
physical, electronic and surface properties; and their many
potential applications in the construction of new and improved
devices in the fields of photochemistry, electrochemistry, optics,
magnetics, electronics, catalysts and sensors [1e7]. The reported
usage of gold nanoparticles (AuNPs) and their bioconjugates is
versatile probes in immunohistochemistry and related techniques
[3,4,14]. The application of protein-functionalized AuNPs in the
detection of antigens, which are specific biomarkers in disease
diagnostics, has also been advanced [3,15,16].
Fluorescent-based detection schemes that use quenched emis-
sion of fluorescence by ligand functionalized AuNPs have generated
significant recent interest in biotechnology for fluorescence immu-
noassay and biosensors [16e19]. Hazarika et al. [16] reported
reversible binding of yellow fluorescent protein (EYFP)-a mutantAccepted 21 December 2010
Available online 3 February 2011
Keywords:
Biomimetic protein
Self-assembly
Gold-protein bioconjugates
Gold nanoparticles
Photophysical properties of bioconjugates0142-9612/$ e see front matter
2011 Elsevier Ltd.
doi:10.1016/j.biomaterials.2010.12.030a b s t r a c t
In this investigation we report the synthesis of optically coupled hybrid architectures based on a new
biomimetic fluorescent protein rec1-resilin and nanometer-scale gold nanoparticles (AuNPs) in a one-step
method using a non-covalentmode of binding protocol. The presence of uniformly distributed fluorophore
sequences, eSer(Thr)-Tyr-Gly- along the molecular structure of rec1-resilin provides signnificant oppor-
tunity to synthesizefluorophore-modifiedAuNPs bioconjugateswithuniquephotophysical properties. The
detailed analyses of the AuNP-bioconjugates, synthesized under different experimental conditions using
spectroscopic, microscopic and scattering techniques demonstrate the organizational pathways and the
electronic and photophysical properties of the developed AuNP-rec1-resilin bioconjugates. The calculation
of the bimolecular quenching constant using the SterneVolmer equation confirms that the dominant
mechanism involved in quenching of fluorescence of rec1-resilin in the presence of AuNP is static. Photo-
acoustic infrared spectroscopy was employed to understand the nature of the interfacial interaction
between the AuNP and rec1-resilin and its evolutionwith pH. In such bioconjugates the quenched emission
of fluorescence byAuNP on thefluorophoremoiety of rec1-resilin in the immediate vicinity of the AuNP has
significant potential for fluorescence-based detection schemes, sensors and also can be incorporated into
nanoparticle-based devices.cCSIRO Materials Science and Engineering, Clay toria, AustraliaIan Wark Research Institute, ARC Special Research Centre, Mawson Lakes Campus, University of South Australia, Mawson Lakes, South Australia 5095, Australia
bCSIRO Livestock Industries, Level 6, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Queensland 4067, AustraliaSelf-organization, interfacial interaction
of gold nanoparticle complexes derived
protein rec1-resilin
Sundar Mayavan a, Naba K. Dutta a,*, Namita R. Cho
Christopher M. Elvin b, Anita J. Hill c
journal homepage: www.elsAll rights reserved.nd photophysical properties
om resilin-mimetic fluorescent
hury a,*, Misook Kim b,
le at ScienceDirect
erials
ier .com/locate/biomateria ls

fragment, genomic DNA from adult D. melanogaster was used as
template for PCR with primers designed to amplify a 946-base-pair
fragment (including codons for an N-terminal His6 affinity tag) that
was inserted into a T7-promoter expression vector. Recombinant
rec1-resilin production was induced with IPTG in E. coli BL21(DE3)/
pLysS and the protein was purified from the cell-free extract by
a combination of anion exchange and NieNTA affinity chromatog-
raphy. Full details of experimental procedures are given elsewhere
[25]. The soluble protein thus prepared has concentration range
from 200 to 300 mg ml1. Rec1-resilin solutions of required
concentration were prepared in phosphate buffered saline (PBS)
unless otherwise indicated.
terials 32 (2011) 2786e2796 2787BSA protein and the surface of gold AuNPs. Sanpui et al. [23]
employed recombinant green fluorescent protein (GFP) for single-
step synthesis of gold nanoparticles (AuNPs) and associated
quenched fluorencence characteristics in an aqueous medium. The
final properties of the protein-AuNP hybrids are very sensitive not
only to the chemical composition of the protein and the size of
AuNPs; but also on their topological organization including the
distance of the fluorophores from AuNP surfaces. Herein, we report
the synthesis of AuNPs/rec1-resilin bioconjugates of size specificity,
aggregation and unique photophysical properties using pre-orga-
nized engineered biomimetic protein rec1-resilin [24] as a
nanoreactor.
Native resilin is a member of the family of natural elastic
proteins that includes elastin, gluten, gliadin, abductin and spider
silks [24e27]. It occurs as a highly elastic extracellular skeletal
component in insects and is purported to be the most resilient
material known. The elasticity of resilin is best known for its roles
in insect flight and the remarkable jumping ability of fleas, and
spittle bugs. Recently, we have reported the synthesis of a resilin-
mimetic polypeptide rec1-resilin from the repeat sequences of the
first exon of the Drosophila melanogaster CG15920 gene using
recombinant DNA technology [24,25]. Its physical, physiochemical
and thin film characteristics have also been discussed in detail
[27e29]. Structurally rec1-resilin consists of 310 amino acid resi-
dues, (molecular weight: 28.4 kD) containing repeat sequences of
the resilin gene CG15920 (19e321 residues in the N-terminal
region of a 620 amino acid sequence). The composition of this
protein dominated by 18 copies of a 15-residue repeat sequence:
GlyGlyArgProSerAspSerTyrGlyAlaProGlyGlyGlyAsn with a molecular
weight of 28.4 KD. The amino acid composition, alignment of
repeated amino acid sequences, and structural consensus of rec1-
resilin are given in supplementary information S_1 (see Figure S_1
and Table S_1 for details). The structure of this protein is unique
due to the abundance of fluorophore amino acid residues Tyr-
conserved in all the 18 copies of the repeat units-within the
polypeptide chain. Tyr is present in two major distinctly different
tripeptide environments in rec1-resilin, respectively Ser-Tyr-Gly
and Thr-Tyr-Gly. Tyr is a natural fluorophore and the sequence -Ser
(or Thr)-Tyr-Gly- has also been identified as the sequence respon-
sible for the formation of the chromophore and the unique fluo-
rescence characteristics of A. victoria green fluorescent protein
(GFP) [30,31]. This structural feature of the protein provides
unique opportunity to synthesize fluorophore-modified AuNP and
also to exploit it for in-depth understanding of the protein’s
conformational dynamics and interaction with AuNP due to
change in the environment and conjugation. In this study, the
organizational pathways and unique electronic and photophysical
properties of rec1-resilin and the AuNP-rec1-resilin bioconjugates
have been demonstrated using spectroscopic, microscopic and
scattering techniques. This one step facile protocol for the
synthesis and surface engineering of AuNPs has the potential to be
used as sensors and create a convenient reporter molecule,
because of the quenched fluorescence of the rec1-resilin upon
binding to the gold nanoparticles [16,30e32].
2. Experimental
2.1. General synthetic procedure of rec1-resilin
We have reported the synthesis of resilin-mimetic polypeptide
rec1-resilin from the repeat sequences of the first exon of the D.
melanogaster CG15920 gene using recombinant DNA technology
[25,26]. Summarily the exon-1 of the D. melanogaster gene
CG15920 was cloned and expressed in Escherichia coli and was
S. Mayavan et al. / Biomapurified to make rec1-resilin. To express the CG15920 gene2.2. Procedure of synthesis of gold-rec1-resilin bioconjugates
The primary reaction for the formation of AuNP involved tuning
of the interaction and organization of Au (III) cations (auric chlo-
ride) within pre-organized rec1-resilin followed by the reduction to
zero-valent AuNP using an excess of sodium borohydride (NaBH4)
at room temperature. The solutions of rec1-resilinwere prepared in
phosphate buffered saline (PBS). A series of the bioconjugates with
different molar ratios of rec1-resilin/AuNP were prepared at room
temperature by keeping the concentration of the protein constant
while varying the concentration of Au-precursor (Table 1). To
investigate the effect of pH on the characteristic structure, func-
tions and the conformational changes of the protein rec1-resilin/
AuNP bio-conjugate, solutions at fixed concentration but different
pH were also prepared. In the seeded growth method, first small
seeds were prepared by the reduction of Au-precursor (auric
chloride) with sodium borohydride, NaBH4 in the presence of rec1-
resilin at room temperature. Additional Au-precursor was then
introduced and successively reduced to obtain larger nanoparticles.
All of the measurements in the following discussion were per-
formed at room temperature (293 K).
2.3. Instrumentation
UVeVis absorption spectra, fluorescence emission measure-
ments and dynamic light scattering (DLS) of the prepared solu-
tions were measured using a CARY 1E Scan UVeVis spectrometer,
Cary fluorescence spectrophotometer and Malvern Zetasizer Nano
ZS, ZEN3600, Malvern Instruments Ltd respectively. Photo-
acoustic-Fourier transform infrared spectroscopy (PA-FTIR) was
performed using a Nicolet Magna Spectrometer (Model 750)
equipped with an MTEC (Model 300) photoacoustic cell. Carbon
black was used as reference. Helium was used due to its high
thermal conductivity. A gas flow rate of 10 cm3 s1 was used. TEM
images were acquired with a Philips 200 EX electron microscope.
The samples for TEM studies were prepared by placing a drop of as
prepared colloidal solutions on carbon-coated copper grids fol-
lowed by drying.
Table 1
Sample designation and physical characteristics of AuNPs.
S C1 mM C2 mM C3 mM Cr NAu
Au_2 2.015 6.59 4.28 2.12 62
Au_21 2.015 65.9 42.8 21.24 427
Au_106 2.015 329.6 214.0 106.20 376
Au_212 2.015 659.2 428.0 212.40 e
Au_425 2.015 1318.6 856.1 424.86 e
S_Au_425 2.015 1318.6 856.1 424.86 835
S ¼ Sample designation, C1 ¼ Concentration of r1-R, C2 ¼ Concentration of AuCl3
C3 ¼ Concentration of Au(III), Cr ¼ molar ratio of Au(III) to r1-R, NAu ¼ Average
number of Au particles per nano clusters. S_Au is seeded growth; all others are
normal growth.