Analytica Chimica Acta 805 (2013) 95 100

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Analytica Chimica Acta

j ourna l ho mepage: www.elsev ier .co

Bimeta hlysubstra sse

Liyan Bi, WeState Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, PR China

h i g h l i g h t s

The bimetaarrays werver nanopgold nanop

The SERS the silver to a large nanopartic

The SERS testing aginteractionbiotin.

g r a p h i c a l a b s t r a c t

a r t i c l

Article history:Received 8 JunReceived in reAccepted 26 OAvailable onlin

Keywords:Bimetallic goldLocalized surfaSurface enhanStreptavidin/b

1. Introdu

Much efnanoparticlalloys and chave intere

CorresponE-mail add

0003-2670/$ http://dx.doi.ollic goldsilver nanoplatee fabricated by coating sil-articles uniformly on thelate arrays.intensity increased withnanoparticle coating, duenumber of hot spots andle interfaces.substrate was used for

ainst the supramolecular between streptavidin and

e i n f o

e 2013vised form 24 October 2013ctober 2013e 4 November 2013

silver nanoplate arrayce plasmon resonance (LSPR)ced Raman scattering (SERS)iotin assemblies

a b s t r a c t

The silver-modied gold nanoplate arrays as bimetallic surface-enhanced Raman scattering (SERS) sub-strates were optimized for the surface-enhanced Raman detection of streptavidin/biotin monolayerassemblies. The bimetallic goldsilver nanoplate arrays were fabricated by coating silver nanoparticlesuniformly on the gold nanoplate arrays. Depending on silver nanoparticle coating, the localized surfaceplasmon resonance (LSPR) peak of the bimetallic goldsilver nanoplate arrays blue-shifted and broadenedsignicantly. The common probe molecule, Niel Blue A sulfate (NBA) was used for testing the SERS activityof the bimetallic goldsilver nanoplate arrays. The SERS intensity increased with the silver nanoparticlecoating, due to a large number of hot spots and nanoparticle interfaces. The platforms were tested againsta monolayer of streptavidin functionalized over the bimetallic goldsilver nanoplate arrays showing thatgood quality spectra could be acquired with a short acquisition time. The supramolecular interactionbetween streptavidin (strep) and biotin showed subsequent modication of Raman spectra that implieda change of the secondary structure of the host biomolecule. And the detection concentration for biotinby this method was as low as 1.0 nM. The enhanced SERS performance of such bimetallic goldsilvernanoplate arrays could spur further interest in the integration of highly sensitive biosensors for rapid,nondestructive, and quantitative bioanalysis, particularly in microuidics.

2013 Published by Elsevier B.V.

ction

fort has been devoted to the synthesis of bimetallices such as goldsilver mixed systems, through bothoreshell structures [18]. The bimetallic nanoparticlessting applications in electronic technologies, catalysis,

ding author. Tel.: +86 25 83795719; fax: +86 25 83795719.ress: wqian@seu.edu.cn (W. Qian).

chemical sensing and as substrates in SERS. The interest of bimetal-lic nanoparticles resides on the fact that these systems displaydifferent optical and surface chemical properties with respect tothe pure monometallic systems. For technological applications itwould be interesting to synthesize coreshell bimetallic nanopar-ticles. The coreshell bimetallic nanoparticles not only preservethe plasmonic properties of the core metal, but also have secondmetal as the external shell, which is able to interact with a widerrange of adsorbates. Although silver nanoparticle shows a higherenhancement factor in comparison to that of gold nanoparticle,

see front matter 2013 Published by Elsevier B.V.rg/10.1016/j.aca.2013.10.045llic goldsilver nanoplate array as a higte for detection of streptavidin/biotin a

Jian Dong, Wei Xie, Wenbo Lu, Wei Tong, Lin Tao,m/locate /aca

active SERSmblies

iping Qian

96 L. Bi et al. / Analytica Chimica Acta 805 (2013) 95 100

gold nanoparticle have the advantage of easier preparation witha high degree of homogeneity. For this reason, many attempts toobtain goldsilver composite nanoparticles by depositing silvernanoparticle on preformed gold nanoparticles have been carriedout in orderticle [2,9].

The prepchange in tThe optical tics of the effect [10].which requused for biooverlapping[11]. It has yield molectering ampnoble metathe SERS tedetection onucleic acidnative to the vibratiotroscopy is groups withof both molactions betwchain of theare based oAll of thesesystem an site identi

In this wtion of the SERS activinanoplate acoating, Furexhibited hoptical proparrays wereSERS substrstreptavidinlevel.

2. Experim

2.1. Chemic

Hydrogenitrate salmide (CTABpotassium (NH3H2O, chloride (NChemical R40) and phSangon BioAminopropobtained fr(18 M).

2.2. Instrumentation

Absorption spectra of gold nanoplate colloid, gold nanoplatearray and bimetallic goldsilver nanoplate arrays were recorded

a Shtructilvercannmentuipp

ors a lase

densing ostrepas dil

10

in dreme

oced

Modieet

ater, a strenol nsed

[25]

Synthhe f goltic m

werntain

wasr immsoluhe seing tds (1by ad

6 m smauri

Fabriicalllate ced ITs forbled anopprepM A

mL tirrin. Th

by al timons a

Prepa bim

imm to induce a higher homogeneity in the silver nanopar-

aration of bimetallic nanoparticles normally implies ahe plasmonic properties of the constituent metals [7].properties are important to determine the characteris-electromagnetic mechanism associated with the SERS

SERS is a nondestructive and noninvasive techniqueires minimal sample preparation and has been widelylogical sensing as it can minimize photobleaching, peak, and background signal in complex biological systemsshown great promise as a powerful analytical tool thatular ngerprints and good sensitivity based on scat-lication in the near vicinity of nanoscale roughenedl substrates. With its high sensitivity and selectivity,chnique has broad biological applications including thef small bioactive molecules [1214], proteins [15,16],

[1719], and cells [20,21], making it a viable alter-uorescence detection methods [22]. Beyond providingnal ngerprints of a molecular material, Raman spec-also sensitive to the interaction between biotinylated

the streptavidin host protein [23]. The strong afnityecules is derived from intra- and intermolecular inter-een tryptophan (Trp) residues and the nonpolar side

protein with the nonpolar moieties of the biotin, whichn hydrogen bonding and hydrophobic interactions [24].

characteristics make the strept/biotin supramolecularideal model for biomolecular recognition and bindingcation.ork, we presented the fabrication and characteriza-

bimetallic goldsilver nanoplate arrays with superiorties. We also demonstrate that LSPR of goldsilverrrays were highly dependent on the silver nanoparticlethermore, the bimetallic goldsilver nanoplate arraysigh SERS activity. Both formation mechanism and theerties of the different bimetallic goldsilver nanoplate

explored in detail. In addition, we used such bimetallicates to probe the supramolecular interaction between-derivatized surfaces and biotin at a monolayer

ental

als

n tetrachloroauric acid (HAuCl44H2O, 99.9%), silvert (AgNO3, 99.97%), cetyltrimethylammounium bro-, 99%), sodium citrate, sodium borohydride (NaBH4),iodide (KI), l-ascorbic acid (AA), ammonia water2528%), formaldehyde (HCHO, 3740%), and sodiumaCl) were all purchased from Shanghai Sinopharmeagent Co. Ltd (China). Polyvinylpyrrolidone (PVP-osphate buffer solution (PBS) was purchased fromtech Co. Ltd (China). Niel Blue A sulfate (NBA), -yltriethoxysilane (APTES), biotin and streptavidin wereom SigmaAldrich. Indium tin oxide (ITO) glasses2) were purchased from Xiamen ITO Photoelectricity. Ltd. (China). All reagents were used as received with-purication. Aqueous solution used in the experimentsed by deionized water from Milli-Q system (resistivity

using microsgoldsplus sexperitem eqdetectHeNepowerscatterbiotin/tion wto 1.0 soakedmeasu

2.3. Pr

2.3.1. A sh

ized wunder an ethathen rifor 3 h

2.3.2. In t

sions osynthe34 nmtion cocitratein coloNaBH4ring. Tdegradthe seepared AA, andcases awere p

2.3.3. Typ

nanopmodicolloidassemgold n

As-of 60 mand 0.6with sformedtrolledseveraganic i

2.3.4. The

ed byimadzu UV3600 UV-vis-NIR spectrophotometer. Theures of the gold nanoplate array and bimetallic

nanoplate arrays were carried out on Zeiss ULTRA-ing electron microscope (SEM) at 15 kV. The Ramans were collected with a Renishaw Invia Reex sys-ed with Peltier-cooled charge-coupled device (CCD)nd a Leica microscope. Samples were excited with ar (785 nm) with a spot size of approximately 2 m andity of around 3 104 W cm2. Subsequently, the Ramanf the analytes were tested, including NBA, strep and

complex. Before SERS spectra collection, NBA solu-uted to various concentrations ranging from 1.0 10610 M. The bimetallic goldsilver nanoplate arrays wereifferent analytes for 2 h, and dried naturally for SERSnts.

ures

cation of glass substrateof ITO glass was sonicated in detergent solution, deion-acetone and ethanol for 15 min, respectively. After driedam of high purity nitrogen, the substrate was soaked in

solution of 1% (v/v) APTES for 12 h at room temperature, 5 times in ethanol with sonication and dried at 120 C.

esis of gold nanoplaterst step, we synthesized size-selected colloidal disper-d nanoplate according to previously reported chemicalethods [26]. The gold seeds of a size in the range of ca.e produced as follows: a 10 mL volume of aqueous solu-ing 2.43 104 M HAuCl4 and 2.50 104 M trisodium

prepared in a conical ask. The solution became pinkediately after the addition of 300 L of ice-cold 0.5 M

tion which was sustained for 12 min with vigorous stir-eds were kept at 25 C for 25 h which was essential forhe residual NaBH4. The nanoplates were formed when00 L) were added to a growth solution, which was pre-ding 400 L of 2.43 104 M HAuCl4, 800 L of 0.1 ML of 5 104 M KI into 60 mL of 5 102 M CTAB in somell quantity of NaOH or HCl. After 3 h, the gold nanoplatesed by salt-induced aggregation [27].

cation bimetallic goldsilver nanoplate arrayy, 1.0 mL of 3.0 mM PVP was added in 10.0 mL of goldolloid under strong stirring for more than 30 min. TheO glass described previously was immersed into the

24 h. The monodisperse gold nanoplates then self-uniformly onto the surface of the glass substrates, andlate arrays formed.ared gold nanoplate arrays were immersed into 60 mLgNO3 aqueous solution. Then 0.6 mL NH3H2O solutionHCHO solution were quickly added to the above solutiong for 8 min. The bimetallic goldsilver nanoplate arraye location of silver nanoparticle deposition was con-djusting the reaction time. These products were washedes by deionized water and pure ethanol to remove inor-nd other impurities.

ration of functional interfacesetallic goldsilver nanoplate arrays were amino modi-ersing the array in a solution of 104 M strep solution

L. Bi et al. / Analytica Chimica Acta 805 (2013) 95 100 97

array

in PBS for 1of the biotibimetallic gof biotin rantimes with dry with air

3. Result a

3.1. Bimeta

We synnanoplate aviously [28amine-termcontrolled cof the gold narrays treatt = 3, 5 and nanoplate igold nanopatively smoa reducing small silverarrays, whinanoshell (nanoparticldensity (Figof silver nananoparticllic goldsilvperiod, the

3.2. Compo

As showgoldsilvercarbon, as wnitrogen siglayer of silithe existenof 24 keV,characteristcorrespond3.1 keV corr[29]. The stthe bimetallic goldsilv

A)(D) presents the top view SEM images of the gold nanoplate array andic gold-silver nanoplate arrays treated by gold nanoplate arrays for different

time: t = 3, 5 and 8 min.

/1 to 1.2/1. The observed ratio of gold/silver could be tailoredtrolling the conditions of the reaction time.

asmonic properties of bimetallic goldsilver nanoplate

surface plasmon resonance band of metal nanoparticlesly depends on the size, shape, composition, and dielectricty of the nanoparticles and the local environment [3033]. Indy, LSPR peak of the bimetallic goldsilver nanoplate arraysnitored during the growth of silver nanoshell.

UVvis absorbance spectra of the gold nanoplate arraysFig. 1. Schematic illustration for fabrication of the bimetallic gold-silver nanoplate

2 h and rinsed thoroughly with water. The absorptionn was accomplished by immersing the functionalizedoldsilver nanoplate arrays in different concentrationging from 105 M to 109 M in PBS and washing severalin PBS solution. Finally, the samples were softly blown.

nd discussion

llic goldsilver nanoplate array

thesize size-selected colloidal dispersions of goldccording to chemical synthetic methods reported pre-]. Gold nanoplate array can be obtained by immersinginated ITO glass into the gold nanoplate colloids underonditions. Fig. 2AD presents the top view SEM imagesanoplate array and the bimetallic goldsilver nanoplateed by gold nanoplate array for different reaction time:8 min. As shown in Fig. 2A, the edge length of the golds estimated to be 111 5 nm, with a density of 422 20lates per spot. Almost all the gold nanoplates have a rel-oth surface. Reduction reaction of Ag+ with HCHO asagent is the key to the growth of silver nanoshell. And

nanoparticles can be attached to the gold nanoplatech contribute to the formation of continuous silveras shown in Fig. 1). After reaction for t = 3 min, silveres are attached to the gold nanoplate arrays with lower. 2B). With increasing time, the size and the densitynoparticle grow larger (Fig. 2C). When t = 8 min, silveres coat the gold nanoplate arrays uniformly, bimetal-er nanoplate arrays forming (Fig. 2D). Over this timepale-yellow solution gradually darkens in color.

sition characterization

Fig. 2. (bimetallreaction

from 0by con

3.3. Plarray

Thestrongproperthis stuare mo

Then in Fig. 3, the SEMEDS result of the bimetallic nanoplate arrays indicates the existence of silver, gold,

ell as silicon and nitrogen elements. The silicon andnals are attributed to the silicon substrate with a thincon dioxide. The low-intensity peak of carbon provesce of cetyltrimethylammounium bromide. In the range

there are several peaks which are attributed to theic M and L lines of gold and silver. The peak at 2.1 keVs to the M peak of gold; while the peaks at 2.9 andespond to the L and L peaks of silver, respectivelyrong peaks demonstrate that the main components oflic goldsilver arrays are gold and silver. The bimetal-er arrays with the corresponding ratios of Ag/Au range

display a scorresponddemonstratthe plasmogold nanopbimetallic gformed. Asto silver na1100 nm. spectra of sincrease froto 930 nmlength peakThe peak pos, which is use for detection of strep/biotin assemblies.trong absorbance peak at 1150 nm (Fig. 4A). Thiss to the LSPR band of the gold nanoplate arrays ased by our previous work [28]. To further understandn interaction between the silver nanoparticles andlate arrays, UVvis measurement of randomly orientedoldsilver nanoplate arrays on a glass substrate is per-

shown in Fig. 4B, the characteristic peaks belongingnoparticle and gold nanoplate occur at 400 nm andIt showed that the representative UVvis absorbanceamples for t = 3, 5 and 8 min. When the reaction timem t = 0 to t = 8 min, the LSPR peaks shift from 1150

and became increasingly broader. The larger wave- demonstrates a continuous shift as shown in Fig. 4B.sition versus reaction time is also plotted in Fig. 4C, and

98 L. Bi et al. / Analytica Chimica Acta 805 (2013) 95 100

Fig. 3. (A) SEM micrograph of bimetallic gold-silver nanoplate arrays. Inset shows the corresponding EDS spectrum; (B) SEM micrograph of gold nanoplate array. Inset showsthe corresponding EDS spectrum.

it follows the same trends as blue-shift with increasing reactiontime. The changes of the LSPR signal are the result of interaction ofbetween gold nanoplates and silver nanoparticles due to aggrega-tion and differences in dielectric environment. The plasmon peakat 400 nm suggested nanoparticle-to-nanoparticle plasmon cou-pling. During the reaction, the UVvis absorbance spectra of thesilver nanoparticle colloid do not change and its intensity weak-ens which is correlated with the decrease of the concentration ofsilver nanoparticles in solution (Fig. 4D) [34]. It dedicates that sil-ver nanoparticle steadily grows on the surface of gold nanoplatearrays. The SEM images conrm the silver nanoparticle coating withincreasing the reaction time and eventually forms a rough silvernanoshell.

3.4. SERS p

One of thactive SERSon them [3nanoplate aeffect owinnanoparticlbecause ofa self-asse

Fig. 4. (A) TheThe optical aband 8 min, resoptical absorbtime t = 3, 5 an

nanoparticles without any further chemical modication. Fig. 5Ashows high specic SERS spectra of 1 107 M NBA on the differ-ent goldsilver nanoplate array, which revealed the characteristicpeak of NBA at 592 cm1 and 1638 cm1, formed by the positivelycharged nitrogen [24]. The SERS signals increased dramatically withincreasing reaction time, and reached its maximum as the bimetal-lic goldsilver nanoplate array forming.

Typical SERS spectra from the different concentrations of NBAon the bimetallic goldsilver nanoplate arrays [36,37] are shownin Fig. 5B. Well-resolved peaks are obtained at concentrations aslow as 1010 M. The SERS capability of the bimetallic goldsilvernanoplate arrays are evaluated by estimating the enhancementfactor (EF) by the approach developed by Le Ru et al. [38].

ERS/CSERS(IRS/CRS)

ISERSa cerRamae conroperties of the bimetallic goldsilver nanoplate array

e major goals of the present study is to prepare highly substrates for investigating the molecules adsorbed5]. As mentioned above, the bimetallic goldsilverrrays are expected to exhibit efcient enhancementg to the presence of a large number of hot spots ande interfaces. NBA was selected as the probe molecule

its distinct Raman features and its ability to formmbled monolayer or sub-monolayer on gold/silver

EF = IS

whereunder is the analyt optical absorbance spectra of as-deposited gold nanoplate array. (B)sorbance spectra of bimetallic gold-silver nanoplate arrays for t = 3, 5pectively. (C) The plot of LSPR peak versus the reaction time. (D) Theance spectra of silver nanoparticle colloids with different reactiond 8 min, respectively.

Fig. 5. (A) Thnanoplate arraband of NBA frof various conreaction 8 mintration of NBAgold-silver nais the Raman intensity obtained for the SERS substratetain concentration CSERS of Raman label molecule. IRSn intensity obtained under non-SERS conditions at ancentration of CRS. We choose the 592 cm1 peak ofe SERS spectra of NBA by using different bimetallic gold-silverys for t = 3, 5 and 8 min shown in Fig. 2AD. Inset spectra are the 592om different bimetallic gold-silver nanoplate arrays. (B) SERS spectracentrations of NBA on the bimetallic gold-silver nanoplate arrays for

shown in Fig. 2D. Inset shows the SERS intensity of different concen- (592 cm1). (C) SERS enhancement factor of the different bimetallicnoplate arrays mentioned above.

L. Bi et al. / Analytica Chimica Acta 805 (2013) 95 100 99

Fig. 6. (A) Rananoplate arrasilver nanopla(104, 103, 102, nanoplate arra

the NBA moof EF = 7.4 (CSERS = 1.0 shown in Fiarray is abo

3.5. Detecti

The Ramgoldsilverence, and thVibrationalassociated wof the protea majority obetween 11residues [3are susceptto biotin anshown in Fcontributiois possible tonly to strepphan residu

Table 1Assignment of the Raman bands in the spectra of streptavidin and the strepta-vidin/biotin complex shown in Fig.6.

Experimentalcm1)

Literature Assignment

For etenseand T

126presonfoed bvidin

in Fi dowband (

759 870 965 993

1075 1142 1201 1261 1325 1349 1375 1468 1487

biotin.less inTrp17 nals atwhich helix cof dilutstreptashowntion isman spectra of streptavidin functionalized bimetallic gold-silverys (a), and biotin on the streptavidin functionalized bimetallic gold-te arrays (b). (B) Raman spectra of biotin with different concentrations10 and 1 nM) on the streptavidin functionalized bimetallic gold-silverys. Inset shows the SERS intensity of different concentration of biotin.

lecule as reference peak in the study. We get a value 107 for the bimetallic goldsilver nanoplate arrays

1010 M, IRS = 2627 and CRS = 2.0 102 M) [28]. Asg. 5C, the SERS EF of the bimetallic goldsilver nanoplateut 20 times stronger than that of gold nanoplate arrays.

on of streptavidin/biotin assemblies

an spectra of streptavidin-derivatized bimetallic nanoplate arrays (Fig. 6A) are rst obtained as a refer-e signals have been identied and assigned in Table 1.

bands are observed at 960, 990, 1139, and 1468 cm1

ith the Trp/Val, Trp, CN/Trp, and -CH2/-CH3 modesin, respectively [3943]. Tryptophan residues comprisef the observed Raman spectra of strep, while features60 and 1280 cm1 are associated with other amino acid9]. In the present sample, some of these Raman bandsible to changes in conformation once strep is linkedd make it possible to study their interaction [23]. Asig. 6B, there are complex Raman spectra where bothn of strep and biotin are present with some overlap. Ito detect specic regions on the spectra that correspond/biotin binding. The bands assigned to different trypto-es on strep get slightly modied when interacting with

Raman banlic goldsilvsubstrate fo

4. Conclus

In this pnanoplate abimetallic goptical propthe bimetaThe LSPR basignicant bticle coatinarrays was arrays. Wedue to a laWith improbimetallic gdetect goldanalysis of from the trybly of this receptor fu

Acknowled

We gratProject (Gradation of CTechnology

References

[1] I. Srnov-[2] L. Rivas, S

9722.[3] S. Link, Z.

3529.band (cm1)

761 Streptavidin (Trp18)879 Streptavidin (Trp17)960 Streptavidin (Trp, Val)

1006 Streptavidin (Trp16)1080 Streptavidin, Biotin (Glu, Thr)1132 Streptavidin, Biotin (Trp13, -C-N)1212 Streptavidin, Biotin (Tyr7a)1270 Streptavidin, Biotin (amide III helix)1320 Streptavidin (Ser (-CH2))1348 Streptavidin, Biotin (Trp7)1382 Streptavidin, Biotin (Trp7)1462 Streptavidin (-CH2 ring)1449 Streptavidin (-CH2, -CH3)

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Bimetallic goldsilver nanoplate array as a highly active SERS substrate for detection of streptavidin/biotin assemblies1 Introduction2 Experimental2.1 Chemicals2.2 Instrumentation2.3 Procedures2.3.1 Modification of glass substrate2.3.2 Synthesis of gold nanoplate2.3.3 Fabrication bimetallic goldsilver nanoplate array2.3.4 Preparation of functional interfaces

3 Result and discussion3.1 Bimetallic goldsilver nanoplate array3.2 Composition characterization3.3 Plasmonic properties of bimetallic goldsilver nanoplate array3.4 SERS properties of the bimetallic goldsilver nanoplate array3.5 Detection of streptavidin/biotin assemblies

4 ConclusionAcknowledgementsReferences