Multifunctional linker for orthogonal decoration of gold nanoparticles with DNA and protein

Article abstract of DOI:10.1039/c4ra01773k

A novel trifunctional linker was prepared to allow multiple orthogonal functionalization of gold nanoparticles (AuNPs) with biomolecules. Thiooctic groups contained within the linker act as AuNP surface anchors, while two additional orthogonal functional groups are used for attachment of heme and DNA using amide and copper catalyzed Huisgen cycloaddition, respectively. We demonstrate that heme can act as a platform for reconstitution of a functional heme protein such as myoglobin. Using multifunctional linker, fully functional Au-DNA-Mb hybrids were obtained and such multifunctional constructs expand the synthetic toolbox for nanomaterial tailoring and design of biosensors and novel catalytic materials.

Full text of DOI:10.1039/c4ra01773k

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Multifunctional linker for orthogonal decoration of
gold nanoparticles with DNA and protein†
Cite this: RSC Adv., 2014, 4, 17980
Dania M. Kendziora, Ishtiaq Ahmed and Ljiljana Fruk*
Received 28th February 2014
Accepted 31st March 2014
DOI: 10.1039/c4ra01773k
www.rsc.org/advances
13
A novel trifunctional linker was prepared to allow multiple orthogonal loss of inherent properties such as strong surface plasmon or
14
functionalization of gold nanoparticles (AuNPs) with biomolecules. hyperthermia effect. Despite the advances achieved in
Thiooctic groups contained within the linker act as AuNP surface attaching different classes of proteins (antibodies, STV, perox-
anchors, while two additional orthogonal functional groups are used idases, uorescent proteins) and DNA to the surface of metallic
for attachment of heme and DNA using amide and copper catalyzed NPs such as Ag, Au and quantum dots, protein attachment still
Huisgen cycloaddition, respectively. We demonstrate that heme can suffers both from little control over the stereochemistry and
act as a platform for reconstitution of a functional heme protein such stoichiometry of the attachment and in some cases, protein
12,15
as myoglobin. Using multifunctional linker, fully functional Au–DNA– deactivation.
Mb hybrids were obtained and such multifunctional constructs expand For development of sensing platforms or drug delivery
the synthetic toolbox for nanomaterial tailoring and design of systems, where i.e. cell binding species need to be attached
biosensors and novel catalytic materials.
together with therapeutic agents, multiple modications of NPs
are required. Up till now this has been achieved through the
addition of two or more ligands containing different functional
groups in various ratios to which desired molecular species can
Introduction
16,17
be attached.
However, this oen complicates the surface
Due to their unique chemical and physical properties, metallic
nanoparticles have found numerous applications in bionano-
technology, in particular in design of biosensing platforms and
coverage analysis, in particular the determination of the quan-
18
tity of different species on the surface. To achieve multi-
functionality, Beltram et al. prepared a linker containing a thiol
group for AuNP binding and carboxy- and amino groups for
biomolecule attachment, which might lead to cross-linking
between amino and carboxy groups of different AuNPs and
therefore aggregation of NPs if the conditions are not carefully
1–4
drug delivery systems. Most recent developments include the
use of AuNPs for gene regulation due to the control of RNAse
5
6
protein or design of vaccines against tumor cells. However, all
of these applications require bio functionalization of AuNPs
with various biomolecules such as DNA, peptides or anti-
19
7
controlled. In general, use of multifunctional linkers for NPs
modication is scarce and one example was demonstrated by
Graham and coworkers while designing surface resonance
bodies. One of the most commonly used method to enable the
8
attachment of biomolecules is a thiol–gold surface interaction,
which has successfully been employed for immobilization of
20
9
10
Raman scattering (SERRS) probes. A SERRS active dye con-
taining AgNP binding group benzotriazole was introduced to
the side chains of commercially available polymers featuring an
additional carboxyl group for further modication with e.g.
amino-modied oligonucleotides. Although this method facili-
tated the direct labeling of AgNP with SERRS active, NP binding
dye and a biomolecule, it is limited to one particular SERRS
label and introduction of the new optical labels requires the
synthesis of new ligands limiting the practicality of the
thiolated DNA or cystein containing peptides and proteins.
However, when trying to bind biomolecules onto an inorganic
nano core, oen the rst challenge is a choice of a mild and
biocompatible chemical strategy that enables the attachment
without compromising the biological activity of the biomolecule
7,11,12
or the stability of NPs.
Namely, NPs aggregation can occur
under biological conditions, which subsequently leads to the
Centre for Functional Nanostructures, Karlsruhe Institute of Technology (KIT),
Wolfgang Gaede Street 1a, 76131 Karlsruhe, Germany. E-mail: Ljiljana.Fruk@kit.
edu; Fax: +49 721 608 48495; Tel: +49-721-608 45800
20
approach.
To enable the addition of multiple and structurally different
†
Electronic supplementary information (ESI) available: Synthesis of trifunctional species onto Au NPs using single linker, we designed tri-func-
linker and data for the optimization of nanoparticle synthesis. See DOI:
tional linker containing AuNP anchoring group and two
1
0.1039/c4ra01773k
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additional functional groups to enable binding of a range of
different species (biomolecules, drugs, tags) in various desired
combinations. To demonstrate the applicability of such linker,
we present proof of concept study where amine and alkyl/azide
functional groups of the multifunctional linker are used for
Huisgen cycloaddition with DNA and amide coupling of
cofactor heme, which can subsequently be used for the immo-
21
bilization of protein through cofactor reconstitution.
Results and discussion
Synthesis of orthogonal linker and Au NPs
A one-pot synthesis of AuNPs was previously reported using
lipoic acid derivatives containing thioctic moieties for Au
binding, which resulted in spherical NPs of varying sizes
Fig. 1 (a) Synthesis of AuNP using trifunctional linker L1. (b) UV-vis
depending on the ratio between a ligand and a gold spectrum of AuNP–L1 after synthesis using a linker to Au ratio of 1 : 20.
22,25
precursor.
Therefore, we designed trifunctional linkers in (b) TEM image of AuNP–L1. Scale bar represents 20 nm.
such way to contain thioctic moiety able to bind and stabilize
AuNP surface.
In addition, such trifunctional linker contains an amine to containing linker was used only dark precipitate was formed
which different functional moieties can be added through indicating the precipitation of AuNP aggregates. This is prob-
amide coupling and bioorthogonal alkyne or azide groups ably due to the reduction of azide to amine during the one pot
capable of undergoing the copper-catalyzed Huisgen 1,3-dipolar synthesis of AuNPs in the presence of NaBH making it inca-
4
26,27
31
cycloaddition (Scheme 1).
Namely, Huisgen 1,3-dipolar pable of undergoing subsequent click reactions. Therefore
cycloaddition has already successfully been employed to enable only multifunctional linker containing alkyne was used in the
28
mild and specic attachment of DNA to proteins or for cova- subsequent experiments. The most stable and monodisperse
29
lent crosslinking of Au NPs.
7.4 nm AuNP–L1 were prepared using 1 :20 ratio of the linker to
Two different trifunctional linkers L1 and L2 shown in precursor as seen from UV-vis and TEM images (Fig. 1b and c)
Scheme 1 were synthesized from commercially available Fmoc- and were further employed for attachment of DNA and heme.
Lys(Boc)-OH 1 and were used for preparation of Au NPs. Reac-
tions conditions were rst optimized using different ratios of
linker to HAuCl precursor followed by reduction with NaBH
4 4
Orthogonal attachment of DNA and heme cofactor
(Fig. 1a). Besides alkyne and azide containing linker, unmodi- Huisgen dipolar cycloaddition was used to attach azide modi-

ed lipoic acid (LA) was used to prepare lipoic acid coated Au ed DNA (azDNA) using sodium ascorbate and copper sulfate
NPs to be employed as a control in the subsequent experiments. together with the stabilizing ligand tris(benzyltriazolylmethyl)
NP formation was directly assessed by appearance of the plas- amine (TBTA) resulting in highly water dispersible DNA-modi-
mon absorption maximum around 520 nm in UV-vis spectra ed AuNPs (AuNP–L1–D). Successful formation of AuNP–L1–D
30
characteristic for smaller AuNPs. However, when azide conjugate was conrmed by agarose gel. As shown in Fig. 2,
AuNP–L1 precipitated in the gel pocket while AuNP–L1–D
migrated within the gel indicating the presence of DNA strands
attached to the surface of Au. Negative control in which AuNP–
L1 was mixed with azDNA without Cu catalyst at rst showed an
electrophoretic shi probably due the non specic binding of
azDNA, however such NP–DNA conjugates were not stable as the
black precipitates were formed aer removal of azDNA by
centrifugation and washing.
To conrm the multifunctionality of the attached L1, AuNP–
L1–D was further modied with heme using HOBt/HBTU
mediated coupling of the free amine on the AuNP surface and
carboxylic groups of the heme molecule (Fig. 3). We have chosen
heme for several reasons. First, heme is an important cofactor
for a range of enzymes such as metabolically important P450
32,33
proteins or peroxidases.
As such, it has already successfully
Scheme 1 Synthesis of trifunctional linkers L1 and L2. Shown is the
schematic representation of the organic synthesis of alkyne and azide-
modified linkers. (a) EDC–HCl, HOBt, NMM, THF, 12 h, r.t., (b) piperidin,
MeCN, 2 h, r.t., (c) lipoic acid, EDC–HCl, HOBt, NMM, 18 h, r.t., (d) TFA,
been used to alter existing and introduce new functionalities to
these proteins using modied heme reconstitution.
Second, heme itself has shown potential to be used as an
21,34,35
CH Cl
2 2
, 2 h, r.t.
effective catalyst for a range of reactions and has been utilized
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the specic absorption of heme (405 nm) and AuNP (520 nm)
showed that approx. 82 heme molecules per NPs are present.
Heme reconstitution and Au–DNA-protein functionality
Once heme attachment was conrmed, the Au hybrid was
further used for the attachment of myoglobin (Mb) protein
using apoMb (Mb without the cofactor) reconstitution. ApoMb
was prepared by extraction of native heme using Teale's
24,38
method
and 8-fold excess of apoMb was added to the heme
modied AuNP–L1–D, incubated for 4 hours (Fig. 3) and
successful reconstitution was conrmed by UV-vis spectros-
copy. As shown in Fig. 3, reconstitution resulted in a shi of
heme Soret band absorbance from 390 to Mb specic 406 nm
and a change of peak intensity and 520 (AuNP)/Soret band ratio.
Using the same methodology as described before, an amount of
Fig. 2 1% agarose gel of the modification of AuNP–L1 towards AuNP–
D–Mb conjugates. Shown is the electrophoretic separation of different
AuNP conjugates after 20 min at 100 V. AuNPs appear as red bands in
the gel without additional staining.
53 immobilized Mb was calculated, showing a reconstitution
yield of 65%.
Successful Mb reconstitution was further conrmed by
agarose gel electrophoresis as shown in Fig. 2. As mentioned
Aer heme earlier, agarose gel electrophoresis not only provided an addi-
36,37
for design of articial enzymatic systems.
coupling, analysis of AuNP–L1–D–heme with UV-vis spectros- tional proof for the attachment of DNA molecules onto the
copy and comparison of molar concentrations calculated from AuNP surface (high mobility band observed in lane 2, Fig. 2),
Fig. 3 Preparation of AuNP–DNA–Mb conjugates using trifunctional linker. Modification of the amine containing DNA–AuNP with heme and
subsequent reconstitution with apoMb to yield functional AuNP–L1–D–Mb conjugates. (i) 1000 eq. heme, 1500 eq. HBTU, 2000 eq. HOBt, 3600
eq. DIPEA, 10 min, r.t., (ii) add to 1 eq. AuNP–L1–D, 48 h, r.t., (iii) 1 eq. apoMb, 16 h, r.t. Also shown is the UV-vis spectra of the heme-modified
AuNP-DNA before and after reconstitution with apoMb indicating a shift in the Soret band of the heme cofactor upon reconstitution.
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L1–D with cDNA at room temperature for 4 h, the successful
hybridization was demonstrated by a change in electrophoretic
mobility and ethidium bromide staining (Fig. 4a). Subse-
quently, the peroxidase activity of attached Mb was investigated
using oxidation of non uorescent substrate Ampliu Red into
39

uorescent resorun (Fig. 4b) in the presence of H
2 2
O . In such
way, enzymatic activity of Mb can directly be assessed using

uorescence measurements.
Measured peroxidase activity of AuNP–L1–D–heme aer
incubation with apoMb clearly demonstrates that a functional
enzyme is present on the surface of the NP. In addition, the
activity of the Mb reconstituted on the surface of AuNPs is
comparable to that of the control, covalently bound AuNP–Mb
conjugates. Furthermore, AuNP–L1, AuNP–L1–D and AuNP–L1–
D–heme have negligible or low activity in comparison to AuNP–
L1–D–Mb. Native Mb appears to be twice as active as both
AuNP–Mb and AuNP–L1–D–Mb hybrids, which might be a
consequence of the reduced measured uorescence due to the
quenching effects of the AuNP.
Conclusions
Herewith we describe the synthesis of multifunctional linkers
for one-pot synthesis of stable AuNPs, which can further be
modied with different molecular species. AuNP containing
orthogonal functional groups were used to prepare AuNP
conjugates containing both DNA and heme cofactor. Heme can
be employed as an anchor for number of proteins through
reconstitution as demonstrated by reconstitution of apo
myoglobin. Using multifunctional linker, fully functional Au–
DNA–Mb hybrids were obtained. Such multifunctional
constructs can expand the NP toolbox for biosensing and
assembly of novel catalytical materials as well as aide the
Fig. 4 (a) 1% agarose gel of hybridization of AuNP–L1–D with cDNA
before (left) and after ethidium bromide staining (EtBr, right). (b) preparation of novel multifunctional “target and deliver” ther-
Enzymatic activity by AmpliFlu Red assay. Shown are the measured
apeutic agents, which is the subject of our ongoing studies.
fluorescence signals for different precursors towards the assembly of
AuNP–DNA–Mb conjugates after a reaction time of 15 min.
Experimental
Materials and method
but also for the coupling of heme (small decrease in mobility in All chemicals were purchased from Sigma-Aldrich and used
lane 3 as compared to lane 2, Fig. 2). In addition, as seen from without further purication. Azido–DNA was obtained from
lane 4, electrophoretic mobility further decreases upon the Atdbio (Southampton, UK).
reconstitution of apoMb on the NP surface due to the change in
the size of the hybrid construct. Apo Mb was employed as a
negative control, which is not visible without staining (lane 5).
Furthermore, additional control sample containing AuNP–Mb
conjugates prepared by direct amide coupling of native Mb to
lipoic acid AuNP-0 was added to the gel. As it can be seen in lane
Synthesis of linker
Multifunctional linkers were prepared starting from commer-
cially available Fmoc-Lys(Boc)-OH (Scheme 1). Details on the
synthesis of each component are presented in ESI.†
6
, such conjugates show electrophoretic mobility comparable to
Preparation of Au NPs
that of Au–DNA–Mb constructs (lane 2). Finally, additional
controls were preformed to rule out non specic interaction of For the synthesis of AuNPs modied protocol of previously
22
native Mb with AuNP–L1, AuNP–L1–DNA and AuNP–L1–D– reported strategy was employed. Briey, all glassware was
heme and no change in electrophoretic mobility was observed cleaned thoroughly with aqua regia, washed with water and
ꢀ
upon addition to the protein.
dried in an oven at 70 C. In a typical synthesis 312 mL
To conrm that fully functional multi-AuNP hybrid is HAuCl $H O (50.8 mM, 15.8 mmol, 20 eq.) were stirred in 50 mL
4
2
obtained using trifunctional linker, rst, the hybridization double distilled water with 39.6 mL linker (20 mM, 0.79 mmol, 1
ability of attached DNA was explored. Aer incubation of AuNP– eq.) for 1 h at room temperature. 144 mL NaBH (880 mM, 126.4
4
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0
mmol, 160 eq.) were added in 4 steps over the course of 30 min. complementary ssDNA cDNA (5 -CTTCACGATTGCCACTTTC-
0
The solution was stirred over night and analyzed with UV-vis CAC-3 ) added in a 100 fold excess in water and le to hybridize
(Cary 300 Scan (Varian) scanned from 200–800 nm with water as for 4 h. Gel electrophoresis showed a shi in electrophoretic
reference).
mobility and the staining with ethidium bromide (EtBr) addi-
tionally proved the formation of dsDNA.
TEM study
The TEM samples were prepared by ultrasonic dispersion of the Covalent coupling of Mb to AuNP
particle solution on a carbon support lm (3 nm, TED Pella Inc.
To activate the carboxyl groups 0.165 mL of EDC and NHS (each,
California). TEM experiments were performed using a Philips
0.1 M, 16.5 nmol, 500 eq.) was added to 1 mL AuNP-01 : 10 (33
CM200FEG/ST microscope. Diameters were calculated from N ¼
nM, 33 pmol, 1 eq.) for 15 min. Followed by coupling of 116 mL
of Mb (285 mM, 33 nmol, 100 eq.). AuNP–Mb was puried by
centrifugation (15 min, 13 200 rpm) and re-suspended in
phosphate buffer.
100 with the help of ImageJ. Concentrations of AuNP were
calculated based on the NP size using reported extinctions
22
coefficients at the absorption maximum of 520 nm and were
usually in the range of 100 nM.
Mb activity assay
Coupling of DNA to AuNPs
To test for the peroxidase-like activity of Mb, 50 mL of a solution
containing 10 mM AmpliFluRed™ and 100 mM H O in sodium
2 2
phosphate buffer (pH 6) were added to 50 mL of each sample (0.3
mM, 15 pmol). Formation of resorun was followed by
measuring uorescence emission at 590 nm aer excitation at
The Cu-catalyzed Huisgen cycloaddition was performed using
previously published approach. In a typical reaction 5 mL
AuNP–L1 (100 mM, 0.5 nmol, 1 eq.) were incubated with 50 mL
23
azDNA (100 mM, 5 nmol, 10 eq., sequence: (T)
ACC), 2.5 mL sodium ascorbate (40 mM, 100 mol, 200 eq.) and a
premixed solution of 300 mL TBTA (333 mM in H O–DMSO–
8
GAT CTC TTC
530 nm.
2
tBuOH 4 : 3 : 1, 100 nmol, 200 eq.) and 2 mL copper sulfate
pentahydrate (25 mM, 50 nmol, 100 eq.). The reaction mixture
was incubated over night at 4 C. To stabilize the DNA on the
Acknowledgements
ꢀ
surface 2 mL TETBS300 (20 mM Tris–HCl, 300 mM NaCl, 5 mM Presented work was supported by FET grant Single Molecule
EDTA, 0.05% Tween-20, pH 7.5) was added to the mixture and Activation and Computing (FOCUS), grant agreement no 270483
le to incubate for 2 h at room temperature before purication as well as DFG-CFN project A5.7 We would like to thank Pascal
by several centrifugation and wash steps (20 min, 13 200 rpm). Bockstaller for his help with TEM measurements.
The residue was suspended in TETBS300 to yield a red solution.
Notes and references
Heme coupling
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