Biosensors and Bioelectronics 43 (2013) 148–154
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Dendrimer functionalization of gold surface improvesthe measurement of protein–DNA interactions by surface plasmonresonance imaging
Flavien Pillet a,b,c, Aurore Sanchez d,e, Cecile Formosa a,f, Marjorie Severac g,Emmanuelle Trevisiol a,b,c, Jean-Yves Bouet d,e,n,1, Veronique Anton Leberre a,b,c,nn,1
a Universite de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, Franceb INRA, UMR792, Ingenierie des Syst�emes Biologiques et des Procedes, F-31400 Toulouse, Francec CNRS, UMR5504, F-31400 Toulouse, Franced Universite de Toulouse, Universite Paul Sabatier, Laboratoire de Microbiologie et Genetique Moleculaires, F-31000 Toulouse, Francee Centre National de Recherche Scientifique, LMGM, F-31000 Toulouse, Francef Centre National de la Recherche Scientifique, Laboratoire d’Analyse et d’Architecture des syst�emes (LAAS), Toulouse, Franceg CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, F-31077 Toulouse Cedex 4, France
a r t i c l e i n f o
Article history:
Received 25 October 2012
Received in revised form
3 December 2012
Accepted 4 December 2012Available online 20 December 2012
Keywords:
Surface plasmon resonance imaging
Phosphorus-dendrimer
Protein–DNA interaction
High throughput detection
Label-free detection
63/$ - see front matter & 2012 Elsevier B.V. A
x.doi.org/10.1016/j.bios.2012.12.023
esponding author at: Universite de Toulou
oire de Microbiologie et Genetique Mole
Tel.: þ33 561 33 59 06; fax: þ33 561 33 58 8
responding author at: Universite de Toulouse
de Rangueil, F-31077 Toulouse,
Tel.: þ33 561 55 94 71;
3 561 5594 00.
ail addresses: [email protected] (J.-Y. Bou
[email protected] (V.A. Leberre).
th authors contribute equally to this work.
a b s t r a c t
Surface Plasmon Resonance imaging (SPRi) is a label free technique typically used to follow
biomolecular interactions in real time. SPRi offers the possibility to simultaneously investigate
numerous interactions and is dedicated to high throughput analysis. However, precise determination
of binding constants between partners is not highly reliable. We report here a dendrimer functionaliza-
tion of gold surface that significantly improves selectivity of the detection of protein–DNA interactions.
We showed that amino–gold surface functionalization with phosphorus dendrimers of fourth genera-
tion (G4) allowed complete coverage of the gold surface and the increase of the surface roughness. We
optimized the conditions for DNA probe deposition to allow accurate detection of a well-known
protein–DNA interaction involved in bacterial chromosome segregation. Using this G4-functionalized
surface, the specificity of the SPRi response was significantly improved allowing discrimination
between protein and DNA interactions of different strengths. Kinetic constants similar to those
obtained with other techniques currently used in molecular biology were only obtained with the G4
dendrimer functionalized surface. This study demonstrated the benefit of using dendrimeric surfaces
for sensitive high throughput SPRi analysis.
& 2012 Elsevier B.V. All rights reserved.
1. Introduction
Association of DNA with proteins is a phenomenon of utmostbiological importance. Indeed, almost all aspects of cellularfunction, such as transcriptional regulation, chromosome main-tenance, replication and DNA repair depend on the interaction ofproteins with DNA. Understanding these biochemical mechan-isms requires efficient systems to characterize interactions
ll rights reserved.
se, Universite Paul Sabatier,
culaires, F-31000 Toulouse,
6.
; INSA,UPS,INP; LISBP, 135
et).
between the different partners and in view of such an importantrole played by DNA–protein interactions, various techniques haveevolved over the years to elucidate such interactions (Dey et al.,2012). Label based methods are the most commonly used tofollow interaction between molecular probes deposited on asurface and targets molecules injected. Labeling occurs eitherdirectly on the target molecules prior to the interaction or in astep after binding of the target molecules on the sensing sites.However, the labeling is time and cost consuming and maydisturb interactions, particularly with proteins (Tsouti et al.,2011). In addition, in some label based techniques, the interactioncannot be monitored in real time (end point detection).
Biosensors with label free technologies are a potential solutionto these problems. Surface Plasmon Resonance (SPR) is an opticaltechnique that can measure changes in refractive index near aplanar surface in real time. It is based on the evanescent waveformation at the interface between a free electron rich metal(gold, silver, etc.) and a dielectric medium, classically either liquid
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154 149
or air (Huang et al., 2007). During an SPR experiment, biomole-cular interactions observed between probes or ligands covalentlybound to the metal surface (sensor surface) and target or analyteinjected, induce perturbations of the evanescent wave due to localvariation of refractive index. The result is a decrease of resonanceangle defined as the angle of minimum reflectance (Cooper,2003). This method has been mostly developed by BIAcore(Amano et al., 1999; Maesawa, 2003). The use of surface plasmonresonance (SPR) based instrumentation for investigating biomo-lecule interactions has grown substantially over the past decade(Guo, 2012), but the principal limitation in conventional SPRis low multiplexing. Indeed a maximum of 36 interactions arefollowed per run (Rich and Myszka, 2007).
The Surface Plasmon Resonance imaging technique (SPRi)combines the SPR principle with an imaging system that allowshigh throughput measurement of interaction. The metal surface isvisualized with a CCD camera via an imaging lens (Scarano et al.,2010; Kodoyianni, 2011). During the experiment, the reflectedlight is monitored by a CCD camera at a fixed angle defined justabove the resonance angle and more than a thousand interactionswere visualized in real time (Pillet et al., 2010). Probe grafting ongold surface is currently based on thiol chemistry (Wang et al.,2004; Herne and Tarlov, 1997; Pillet et al., 2011), the Au–Sinteraction is considered as one of the strongest between goldand non-metal biomolecule ligands (Biener et al., 2005).
We previously applied this chemistry to the SPRi study of aprotein–DNA complex formed during active segregation of theEscherichia coli F plasmid (Bouet et al., 2007; Bouet and Lane,2009). The interaction between the DNA–binding protein SopBand the centromeric site sopC was fully characterized for the DNAbinding requirements, allowing the complete description of theSopB binding site (Pillet et al., 2011). This study has revealed thatthe 16-bp of the sopC site is involved, at different levels, in thebinding efficiency of SopB. However, several limitations wereobserved in quantifying protein–DNA interactions. The curveshapes did not allow the determination of kinetic constantsbecause the dissociation of the complex was not significant inthe conditions used. As previous results obtained with BIAcoresystem showed correct dissociation between sopC and SopB(Ah-Seng et al., 2009), we proposed that the impossibility toobtain acceptable curves in SPRi could be due to several limitingfactors such as probe attachment, probe accessibility or reactivity
Fig. 1. Schematic illustration of surface functionalization and DNA grafting used in t
directly on gold surface (a). (B) Surface dendrimer functionalization: after cysteamine
were bound to the amine surface (b) then amino-modified oligonucleotides were depos
amine by immersion in a NaBH4 solution (d).
of the gold surface. For example, (i) high probe concentration mayinduce steric hindrance or mass transport problems on the sur-face and thus disturb interactions; (ii) interaction between probesand targets could be limited by the relative planar gold surface;and (iii) Adsorption of target proteins on gold could increase thesurface background.
To circumscribe the surface background and improve theprobe accessibility, we built a pseudo-three-dimensional surfaceon the gold prism by functionalizing the biosensor with dendri-mers. In the literature, few cases of dendrimeric surfaces weredescribed in SPR and SPRi but the dendrimer grafting and probeattachments are complicated and time-consuming (Chen et al.,2002; Altintas et al., 2011). To reduce the chemical steps requiredfor surface functionalization, we used phosphorus dendrimers offourth generation (G4) with aldehyde function at the periphery(Launay et al., 1994). These dendrimers have previously beenused successfully for microarrays applications on glass surface(Trevisiol et al., 2003; Le Berre et al., 2003) but not on gold surfacefor SPRi studies.
Here we showed that a dendrimeric surface used in SPRiallows a better discrimination between protein and DNA interac-tions, compared to untreated-gold surface. Kinetic constantsdetermined with G4 dendrimer-coated gold surface are compar-able to the ones obtained with other techniques commonly used.
2. Materials and methods
2.1. Preparation of nanostructured surfaces
SPRi-BiochipsTM were designed exclusively by Horiba Scientific-
Genoptics (France) with the Kretschmann configuration (Kretschmannand Raether, 1968). The sensing surface, coated with a 50 nm thingold layer, was used either untreated or functionalized with G4dendrimers (Fig. 1). The G4 dendrimers with an aldehyde endgroup were produced by the LCC Lab as previously described(Launay et al., 1994). To generate dendrimer surfaces, goldsensorchip surfaces were first cleaned by immersion for 20 minin a solution containing H2O2 (30%)/–NH3 (30%) and deionizedwater in a 1:1:5 ratio (v/v/v) (Manera et al., 2008). Surfaces werethen washed for 5 min in water and absolute ethanol, and dried.The cystamine coating was applied by immersion of precleaned
his study. (A) Direct grafting: thiol-modified oligonucleotides were immobilized
coating on gold surface (a), phosphorus G4-dendrimer with aldehyde end groups
ited and covalently fixed on the dendriprism (c) and Imine bonds were reduced in
Table 1SopB binding efficiencies determined by different techniques and immobilization
procedures. Dissociation constants (KD(nM)) for the indicated probes were
determined by electromobility shift assays (EMSA), surface plasmon resonance
(SPR) or SPRi assays. For the SPRi assays, gold surfaces were either grafted with G4
dendrimers or directly functionalized. For the SPR assay, the probe used was a
136-bp duplex DNA containing a single sopC binding site corresponding to C2
sequence.
C2 C19 C39 C40 Refs.
EMSA 3 17 37 250 Pillet et al. (2011)
SPRiG4 dendrimers 4 9 19 NA This work
Functionalized Gold 13 18 14 15 This work
SPR 2.3 ND ND ND Ah-Seng et al. (2009)
ND: not determined; NA: not available.
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154150
gold sensorchip surfaces for 2 h at room temperature in 90%ethanol solution containing 20 mM of cystamine dihydrochloride(Sigma-Aldrich). After washing for 5 min in 90% ethanol anddrying, amino–gold surfaces were incubated for 5 h in thedendrimer solution (50 mM in tetrahydrofurane, THF). The func-tionalized gold surfaces with G4 dendrimers were washedsequentially for 5 min in THF and absolute ethanol, then driedand kept at room temperature until spotting.
2.2. Atomic force microscopy
Images were recorded in contact mode in air using MLCTAUHW cantilevers (Bruker) with a nominal spring constantof 0.5 N/m on a Nanowizard 2 (JPK Instruments, Germany).The applied force was kept at 5 nN during the experiment. Thecantilever spring constant was measured at 0.528 N/m bythe thermal noise method (Hutter and Bechhoefer, 1993). Forroughness analysis, high resolution height images of the samecenter offset (0.5, 1 and 5 mm) of the surface were processed andanalyzed with the power spectral density method (JPK dataprocessing software ), consisting of average roughness (Ra)measurements on five boxes of five different sizes for each image(Formosa et al., 2012).
2.3. Probes and targets used
Synthetic 65-mer oligonucleotide probes, modified at their50 ends with a thiol (SH) or an amine (NH2) group were obtainedfrom Sigma-Aldrich (France) (Table S1) and spotted on SPRi-sensorchip surfaces functionalized or not with dendrimers. 30-bpdouble strand DNA probes were obtained by spontaneous foldingof each 65-mers oligonucleotide (Fig. 2(A) and Pillet et al., 2011).Expression and purification of SopB protein were carried out aspreviously described (Bouet et al., 2007; Ah-Seng et al., 2009).Propidium iodide (PI) was obtained from Invitrogen (France).
2.4. Spotting conditions
Probe deposition on different surfaces was performed withChip Writer Pro contact spotter (Biorad, France) and a 260 mmsolid pin 947NS7 (Array it Corporation, USA). Two spotting bufferswere employed for these experiments: (i) a 3X SSC buffer (Sigma-
Aldrich, France) containing 450 mM NaCl and 45 mM sodiumcitrate Ph 7.4 and (ii) a 0.1 M phosphate buffer pH 9. Oligonucleo-tide probes were prepared at different concentrations from0.25 mM to 20 mM by dilution in the spotting buffer. Iminefunctions are formed by interactions between amino oligonucleo-tides and aldehyde end groups of dendrimers. The reduction inamine functions was performed during 2 h in the presence of3.5 mg mL�1 of NaBH4 (Sigma-Aldrich, France) at room tempera-ture. Finally, surfaces were washed for 5 min in ultra-pure water,dried and kept at 4 1C in the dark until use.
2.5. SPRi assays
A SPRi–Plex system from Horiba-Scientific Genoptics (France)was employed for SPRi experiments. The best angle of incidencewas chosen before each kinetic measurement as previouslydescribed (Pillet et al., 2010). Biomolecular interactions weremeasured at a flow rate of 50 mL min�1 in the running buffer R(20 mM HEPES pH 7.4, 100 mM KCl, 0.1% of bovine serumalbumin (BSA) and 50 mg ml�1 sonicated salmon sperm DNA).SopB protein and propidium iodide (PI) were diluted in R bufferprior to injection with a 200 mL loop. Regeneration of surface wasperformed with NaOH (50 mM) at 200 mL min�1, followed by awash in running buffer (200 ml). Reflectivity values were obtained
with the Genoptics software SPRiAnalysis 1.2.1 and kinetic curveswere calculated with ScrubberGen based on 1:1 Langmuir bindingmodel (O’Shannessy et al., 1993). Measurements were performedon three replicates per prism on at least three prisms. Theinteractions between probes and targets were determined aftersubtraction of the background signal from the surface. Quantifi-cation of DNA deposition was evaluated with PI, according to themeasurement of reflectivity values at the end of the injection step(240 s). Kinetic curves of SopB–sopC interactions were comparedto the signal obtained between SopB and the non-specific DNAprobe (C1). KD for different sopC were calculated from the ratio ofkinetic constants Koff(s�1)/Kon(M�1s�1) obtained from the fittedcurves.
3. Results and discussion
The main objective of this study was to increase the sensitivityand specificity of SPRi to allow more accurate calculation ofkinetic constants during protein–DNA interactions (with the bestsensitivity and discrimination possible). For this, we used a well-characterized protein–DNA interaction model involved in thesegregation machinery of the E. coli plasmid F (Ah-Seng et al.,2009; Pillet et al., 2011). SopB protein binds to the centromerecomposed of 10 highly conserved binding sites (sopC). Here, wedesigned five DNA probes consisting of variants of the sopC
sequence that have different affinities with SopB (Table 1;Fig. 2). We tested different surface chemistries to determine thebest DNA grafting conditions for each (Fig. 1), and kineticconstants were then estimated for each surface to quantify thesensitivity and specificity of protein–DNA interaction using SPRi.
3.1. Grafting of DNA probes on different surfaces
DNA probes were grafted on untreated-or G4 dendrimerfunctionalized-gold surface. Unlike conventional SPR, the surfacefunctionalization and the probe grafting steps are not controlledduring experiments because they are performed outside thefluidic system (Guedon et al., 2000). Thus, in SPRi the efficiencyof probe grafting on the surface is an important step to bemonitored. To evaluate the quantity of DNA bound to thebiosensor surface, we tested different molecules currentlyemployed to quantitatively assess DNA content, including propi-dium iodide (PI), SYBR Safe, Picogreen and Hoechst 33258(Lapinsky et al., 1991; Rengarajan et al., 2002). The best resultsfor DNA quantification on the surfaces were obtained with PI(data not shown), an intercalating agent with no sequence
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154 151
preference and with a stoichiometry of one dye per 4–5 base pairsof DNA (Nocker et al., 2006)).
First, we defined the optimal concentration of PI for DNAdetection and found that linear detection responses wereobtained between 5 and 20 mg mL�1 of PI for DNA deposited onG4 dendrimer and gold surfaces (Fig. S1). In all our subsequentexperiments we used PI at 10 mg mL�1. To evaluate the appro-priate spotting conditions on gold and G4 dendrimer surfaces,thioland amino-modified probes were deposited at 10 mM onboth surfaces using SSC buffer (pH 7.4) or phosphate buffer (pH 9)(Fig. 3A and B). On gold surface, the SPRi signal was divided byfive with amino-modified DNA deposition compared with thiol-modified probes and we observed a decrease of �20% in DNA-probe grafting in SSC buffer at pH 9 in comparison with pH 7.4(Fig. 3A). Hence, we confirmed the grafting efficiency of thiol-modified DNA at pH 7.4 on gold surface as previously described(Pillet et al., 2010). On G4 dendrimer surface, DNA-grafting wasonly observed with amino-modified probes deposited at pH 9 asexpected (Fig. 3B, Le Berre et al., 2003). This pH was required toform imine bonds between amino function of oligonucleotides
Fig. 2. Description of DNA probes used. (A) Sequence of a typical 65-mer
oligonucleotide and its folding in a double strand DNA duplex. A 50 thiol-modified
oligonucleotide is shown. (B) SopB binding site and variant sequences tested in
SPRi. Only the 16 pb corresponding to the SopB binding site tested is shown; the
remaining sequence being invariable (see (A)). Variations relative to the SopB
binding site were indicated as black boxes.
gold G4
1.6
1.2
0.8
0.4
0
1.8
1.4
1.0
0.6
0.2
gold
thiol-DNA amino-DNA
Ref
lect
ivity
var
iatio
ns (%
)
Fig. 3. Confirmation of G4 dendrimer grafting. (A, B) Comparison of probe deposition on
Hatched bars indicate amino-modified DNA and full bars the thiol-modified DNA. Oligo
pH 9 (gray). Propidium iodide was injected at 10 mg mL�1. Reflectivity values were ca
functionalized prism surfaces. Vertical deflection images of gold (left panel) and G4 de
(5�5 mm) and (D) Surface roughness measured from AFM height images of untreated-
are the average of three measurements for each of the indicated square surface.
and aldehyde functions of G4 dendrimers. Importantly, no signalwas detected when thiol-modified probes were used on the G4dendrimer functionalized surface, indicating that the gold surfacewas fully covered by G4 dendrimers.
In order to confirm the homogeneous G4 dendrimer functio-nalization, we performed Atomic Force Microscopy (AFM) surfacecharacterization. Fig. 3(C) presents AFM images of untreated- orG4 functionalized-gold surfaces. The roughness was calculated(see Section 2) from different AFM images of 0.5, 1 and 5 mm(Fig. 3D). For both surfaces, roughness curves rapidly reach aplateau indicating that they were homogeneous throughout thesurface. On the G4 dendrimer grafted-gold surface, the roughnessappeared two-fold higher than on untreated-gold surface. Thisincrease in roughness is consistent with the shape and structureof the G4 dendrimers (Fig. 1B). Furthermore, a decrease of thestandard deviations was observed on G4 dendrimer surfacecompare to the untreated one, suggesting that the G4 dendrimergrafting layer was more homogeneous than that of the goldsurface. These results, along with the prevention of thiol probesgrafting on this surface (Fig. 3B), thus confirm the full andhomogeneous functionalization of the gold surface by G4dendrimers.
3.2. Optimal conditions for DNA grafting
To estimate the ideal probe concentration, we took advantageof the high throughput capability of the SPRi technique bydepositing each probe at seven different concentrations. TheDNA deposition was then quantified after injection of 10 mg mL�1
of PI (Fig. 4A). The signal increased linearly from 0.25 mM up to5 mM and between 1 mM and 10 mM on untreated- and G4dendrimer grafted-gold surfaces, respectively. At higher DNAconcentrations, the signals continue to increase but with a lowerslope. We deduced that the best conditions for DNA depositionranged from 0.25 mM to 5 mM on untreated-gold surface and from1 mM to 10 mM on G4 dendrimer surface.
0.2
0.16
0.12
0.08
0.04
0
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0.10
0.06
0.02
G4
thiol-DNA amino-DNA
0 50 100 150 200 250
0.80.70.60.50.4
0.2
0
0.3
0.1
gold
G4
Rou
ghne
ss (n
m)
Square surface (x103 nm2)
Ref
lect
ivity
var
iatio
ns (%
)
gold and G4 dendrimer surfaces. Probes were deposited at 10 mM on both surfaces.
nucleotides were spotted with SSC buffer at pH 7.4 (black) or phosphate buffer at
lculated at the end of injection after background subtraction. (C) AFM imaging of
ndrimer functionalized gold (right panel) surfaces were shown at the microscale
(circle) and dendrimer G4functionalized- (square) gold surface. Roughness values
0.4
0.3
0.2
0.1
00 5 10 15 20
Probe concentrations (µM)
0.5Probe concentrations (µM)
1 2.5 100.25 5
1.2
1.0
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0.6
0
0.4
0.2
Ref
lect
ivity
var
iatio
n (%
)R
efle
ctiv
ity v
aria
tion
(%) gold
G4
gold
G4
C1 C2
2.551020
C1 C2 C19 C39 C40
0.5
Fig. 4. Determination of optimal DNA probe concentrations for surface deposition.
(A) Analysis of oligonucleotide probe deposition on gold and dendrimer surfaces.
Reflectivity values obtained after injection of 10 mg ml�1 of PI and corrected for
surface background, represent average responses obtained with five different
probes at the indicated probe concentrations deposited on dendrimer surface
(filled square) or gold surface (filled circle). Continuous lines indicated the
responses proportional to probe concentration. A typical SPRi image of the G4
dendrimer-grafted gold surface captured at the end of the injection step was
shown in the inset. Five different probes (C1, C2, C19, C39 and C40) were spotted
at four concentrations (mM) as indicated on the right. (B) SPRi analysis of SopB
binding to sopC spotted on gold and dendrimer surfaces. C2 DNA-probes
(corresponding to the consensus binding site) were spotted at the indicated
concentrations. SopB protein was injected at 200 nM on gold (black bars) or
dendrimer (grey bars) surfaces. Reflectivity values show average responses
obtained after subtraction of C1 probe (randomized binding site). Examples of
SPRi images after SopB protein injection on gold and G4 surfaces grafted with
C1 and C2 probes are presented in the inset.
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154152
At higher probe concentrations we assume that steric hindranceand/or mass transport limitation disturbs interactions, which couldexplain why the signals increased at lower slopes. The lowerresponses obtained on G4 dendrimer-grafted surface compared tountreated-gold surface may be explained either by a lower efficiencyof DNA deposition or by an exponential decay of the evanescentwave from the surface with G4 dendrimer (7 nm of diameter)coating (Frutos and Corn, 1998). However the consequence of thisdistance could decrease the steric hindrance and the mass transporton the G4 dendrimer surface.
3.3. Optimal conditions allowing protein–DNA interactions
We then investigated the best conditions to analyze protein–DNA interaction on both surfaces. For this we used the SopB–sopC
model; C2 (SopB consensus binding site) and C1 (randomizedsite) probes were deposited at different concentrations and SopBprotein was injected at 200 nM (Fig. 4B). On untreated-gold
surface, the SopB–sopC interaction was readily visualized at thelowest probe concentration tested (0.25 mM). Reflectivity valuesincreased until 2.5 mM then decreased up to 10 mM. On the G4dendrimer surface, reflectivity variations were detected from1 mM and increased until 5 mM. Similar profiles were obtainedat lower SopB concentrations on both surfaces (Fig. S2), showingthat the most appropriated probe concentration must be evalu-ated first to avoid signal saturation. Globally, these resultsdemonstrate that the best concentrations for probe spotting arebelow 2.5 and 5 mM on untreated- and G4 dendrimer grafted-goldsurfaces, respectively.
Although the responses with PI were four-fold lower than ongold surface (Fig. 4A), the response with SopB at 2.5 mM of probesis only �40% less than on untreated-gold surface and evenidentical at higher concentration due to protein saturation ongold surface (Fig. 4B). The G4 dendrimer surface thus appears tobe highly sensitive for detecting protein–DNA interactions.
3.4. Dendrimer grafting increases the detection
of specific interaction
SopB binding was studied with several sopC sequences ofdifferent interaction strengths (Fig. 2B) deposited on the studiedsurfaces. At 2.5 mM of probes spotted on the G4 dendrimer surface(Fig. 5, left panels) the best response was obtained as expectedwith the SopB binding site C2 displaying an estimated KD of 4 nM(Table 1) comparable to 3 nM obtained in electrophoretic mobi-lity shift assay (EMSA) (Pillet et al., 2011) and 2.3 nM found inBIAcore SPR analysis (Ah-Seng et al., 2009). For lower affinitybinding sites, with C19 and C39, KD were estimated at 9 nM and19 nM, respectively. A non-significant response was observedwith C40 because reflectivity variations were identical to thenegative control. By EMSA, C40 probe was found to bind SopBwith a 100-fold lower affinity than C2 (Pillet et al., 2011). Bycontrast, at 5 mM deposited probes on dendrimer surface aresponse was observed with C40 (Fig. 5, central panels). In thiscondition, the reflectivity variations for C40 were equivalent tothose of C39 indicating that, at 5 mM probe deposition, thediscrimination between these probes were not possible. Thisresult confirms that the optimal probe concentration describedabove must be below 5 mM on G4 dendrimer grafted surface toprevent mass transport limitation and/or steric hindrance effect.
At 2.5 mM spotted probes on untreated-gold surface, kineticcurves of SopB–sopC interactions were similar for the SopBbinding site (C2) and lower affinity binding sites (C19, C39 andC40) with dissociation constants (KD) estimated around 15 nM(Fig. 5 right panels; Table 1). The discrimination between SopBbinding site and lower affinity binding sites was thus impossible.This can be explained by the fact that the probe concentration of2.5 mM was close to saturation. However, below 2.5 mM, we didnot observe better discrimination between SopB and the differentprobes (data not shown), indicating that steric surface hindrancerestrained protein–DNA interaction. By contrast, on gold surfacefunctionalized with G4 dendrimer, the probe concentration of2.5 mM was non-saturating. Thus, we demonstrated that it waspossible with SPRi technology to discriminate the SopB affinitiesof the consensus binding site and different lower affinity sites, bymodifying the biosensor surface. This can be explained by a betteraccessibility of the DNA probes to the proteins, obtained throughthe pseudo-three-dimensional (3D) structure of the G4 dendri-mer, probably reducing steric hindrance and/or mass transporteffects. To verify this hypothesis we investigated the functiona-lization of gold by Gold Nano Particles (GNP) of 10 nm with a sizesimilar to the G4 dendrimer. Compared to the untreated-goldsurface, GNP surface allows a better discrimination between C2and C40 but differences were lower than that of G4 dendrimer
G4 dendrimer 2.5 µM G4 dendrimer 5 µM Gold 2.5 µM
C19 C19C19
C39 C39C39
C40 C40C40
C1 C1C1
C2 C2C2
Fig. 5. SPRi analysis of SopB binding on sopC probes deposited on G4 dendrimer surface and gold surface. Probes C1, C2, C19, C39 and C40 were deposited at 2.5 mM and
5 mM on dendrimer surface and at 2.5 mM on gold surface. Probe C1 represents randomized binding site, C2 the SopB binding site and remaining probes are considered as
lower-affinity binding sites. SopB was injected at various concentrations (50, 100 and 200 nM). Fitted curves were represented by orange lines. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154 153
surface (Fig. S3). We deduced that the presence of a pseudo-3Dstructure was not sufficient by itself to explain our results andthose already reported on glass slide microarrays (Le Berre et al.,2003). Alternatively or complementary to this pseudo-3D struc-ture, it is possible to consider that the probes spotted on thedendrimeric surface are better organized than on gold- or GNP-surfaces, and provide better accessibility of the probes to theproteins. Moreover, a gold surface fully covered by G4 dendrimeralso probably prevents direct interactions between target pro-teins and the gold surface. By reducing background signal, G4dendrimers increase the sensitivity of the SPRi response and thusthe possibility to discriminate between protein–DNA interactionsof slightly different affinities.
4. Conclusion
In this work we investigated the functionalization of a goldsurface sensorchip to improve sensitivity and specificity in SPRifor calculation of kinetic constants. We compared different
surfaces for protein–DNA interactions. Although we optimizedprobe deposition conditions, we were not able to significantlyimprove the sensitivity and discrimination of the SPRi responseon untreated-gold surface compared to previous analysis (Pilletet al., 2011). This was probably due to several limitations such asgold planar surface, steric hindrance and mass transport occurringon the biosensor surface. By contrast, kinetic constants obtainedwith G4 dendrimer-grafted gold surface allowed discrimination ofinteractions between SopB and different sopC sequences. More-over, we found SPRi values comparable to those obtained withother techniques such as EMSA and SPR (with BIAcore system).Here, we describe for the first time a functionalization procedurebased on G4 dendrimer with aldehyde end groups, grafted onamino–gold surface, that allows a reliable discrimination ofinteractions between different DNA probes sequences and SopBprotein. The G4 dendrimers induce a pseudo-3D structure on thesurface that optimizes protein–DNA interactions. The completecoverage of the surface by G4 dendrimers reduces the contact andadsorption of target protein on gold and consequently decreasesbackground and potentially protein denaturation.
F. Pillet et al. / Biosensors and Bioelectronics 43 (2013) 148–154154
This new grafting chemistry on amino–gold surface is easy tocarry out and we demonstrate that the G4 dendrimer functiona-lization is particularly useful in SPRi to analyze protein and DNAinteractions and could be developed for high throughput SPRscreening.
Funding
This work was supported by l’Agence National pour la Recherche(2010 BLAN 1316 01).
Aknowledgement
We thank the centre Pierre Potier UMS 3039 for the use ofSPRi-Plexand AFM equipments. We also thank our colleagues inToulouse at the Biopuces-Bionanotechnologie team (LISBP) andthe LMGM for fruitful discussions and critical reading of themanuscript. We are grateful to Calum Johnston for Englishimprovement of the manuscript. We thank Anne-Marie Caminadeand Jean-Pierre Majoral from LCC for helpful collaboration indendrimer chemistry.
Appendix A. Supporting information
Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2012.12.023.
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