Sensors and Actuators B 149 (2010) 212220

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Sensors and Actuators B: Chemical

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Surface plasmon resonance instrument as a refraultrathin lms

Huamin sz WBochu Wa Bioengineerinb Division of Bic BioNavis Ltd,d KSV, Biolin Sc

a r t i c l e i n f o

Article history:Received 22 November 2009Received in reAccepted 20 MAvailable onlin

Keywords:Surface plasmRefractometerLB lmRefractive indLB monolayerEthylene glycoSucroseStearic acid

a b s t r a c t

A surface plasmon resonance (SPR) setup in Kretschmann conguration is being utilized as a refractome-ter for both liquids as well as ultrathin lms. The SPR signal detection technology used is based on a

1. Introdu

Surface pthat may ocstants of opand a dielecis a powerfu

Correspon CorresponUniversity ofHelsinki, Finla

E-mail add(M. Yliperttula

0925-4005/$ doi:10.1016/j.vised form 29 April 2010ay 2010e 16 June 2010

on resonance

exthicknessl

goniometer approach providing a wide angular scan range which facilitates highly accurate liquid andgas phase measurements. Attention was paid to improve sample handling and preparation. In order toavoid cross-contamination betweenmeasurements an easily removable and exchangeablemolded PDMSow cell was used during the measurements. By careful choice of components for liquid handling thedead volume of the system could be reduced down to some microliters.

The angular change and thus the refractive index for sucrose, ethylene glycol (EG) and ethanol solu-tions with different concentrations, the thickness and refractive index of deposited Langmuir-Blodgett(LB) lms, and the interaction kinetics between a biotin containing self-assembled monolayer (SAM) andstreptavidin were determined. The measured refractive indices of sucrose, EG and ethanol solutions cor-responded well with literature values. LB lms were characterized by measuring the complete SPR curvein an angular scan range from 40 to 78. A two-color SPR approach combined with two-media measure-ments was successfully employed for simultaneous and unambiguous determination of both refractiveindex and thickness of stearic acidmonolayers. The thickness obtained for the stearic acidmonolayerwas2.66nm, and the refractive indices at 635 and 670nm were 1.5800 and 1.4138, respectively. The devel-oped sensor-plate holder enabled functionalization of the SPR gold chip outside the instrument, thereforemaking the sample handling more exible. The afnity constant obtained for the streptavidinbiotininteraction was 1.01108 M. The total angle SPR method used in this study clearly shows its potentialto be used as a refractometer for both liquids and ultrathin lms, as well as for traditional liquid phasebiomolecular kinetic studies.

2010 Elsevier B.V. All rights reserved.

ction

lasmon resonance (SPR) is a charge-density oscillationcur at the interface of two media with dielectric con-posite signs such as a metal (typically gold and silver)tric medium [1]. The analytical technique based on SPRl method used to detect changes in the refractive index

ding author.ding author at: Division of Biopharmaceutics and Pharmacokinetics,Helsinki, Faculty of Pharmacy, P.O. Box 56, Viikinkaari 5 E, 00014nd.resses: wangbc@cqu.edu.cn (B. Wang), marjo.yliperttula@helsinki.).

of the adjacent medium next to a metal [2]. Due to its many advan-tages, for example high sensitivity, real-timemonitoring, label-freedetection etc., SPR has merely established itself as a powerful tech-nique for a variety of liquid phase chemical and biological sensorapplications such as bimolecular interactions [36], quantitativemeasurements [7,8], and membrane studies [912].

Liquid phase SPR applications often rely on measuring relativechanges in angular position or reection intensities. The reason forthis being theuse of detection technologies providing limited infor-mationnot enabling full utilizationof theSPRphenomenon. Inmostcases found in the literature, the sensitivity of the surface plasmonresonance signal is measured as the interaction kinetics either asa binding (association) or a dissociation between the moleculesand the proteins on the surface, with an angular range limitedto a few degrees only. Although, the SPR phenomenon provides

see front matter 2010 Elsevier B.V. All rights reserved.snb.2010.05.048Lianga,b, Heini Mirantob,c, Niko Granqvistb,d, Januanga,, Marjo Yliperttulab,

g College, Chongqing University, 400044 Chongqing, Chinaopharmaceutics and Pharmacokinetics, University of Helsinki, FinlandTampere, Finlandientic Oy, Helsinki, Finlandm/locate /snb

ctometer for liquids and

. Sadowskic, Tapani Viitalab,

H. Liang et al. / Sensors and Actuators B 149 (2010) 212220 213

the means for real-time interaction studies, it also allows a morethorough optical characterization of liquids and (ultra)thin lmsprovided that the SPR detection technology is properly designed. Acomplete SPR curve holds all the necessary information to extractthe refractivsurface, golas the SPRimaginary pto assume oorder to extdeposited oplasmon reties it is poproblem. Foof a liquid iindex of the

The optiLangmuirBmon due tofull SPR cuare widely uthinlmthifrom time cination of stime-resolvalso an attrization, espangular infdifferent mdeterminatculty in pehas been thprepare thesample coato be prepauser speciside the insspecic suracterizationa completel

Here weSPR detectiof liquids adetection teand linkedboth air anThe goniomSPR curvewenabling thlms. A custtechnologyprovidedusrefractive in

In orderand make idevelopedoutside thecic sampleso commonfore, our aand customand improvexamples incentrationsglycol (EG)of ethanol

The Kr

ono-cicingviding ag balor as equ

erim

strum

cipleodiuipmalysisw celass sn on

coateuousrized by a polarizer before entering the prism, i.e. its electricctor (E-vector) is parallel to the plane of incidence, denedincident beam and a vector perpendicular the reecting sur-fter propagating the inside of the prism, light is reected aterface of glass and gold (moreprecisely at the glass/dielectricinterface covered by a thin gold lm), due to a bigger inci-gle than the critical angle of the total internal reection. The

ity changes of the reected beam can bemonitored as a func-the angle of incidencewith the detector and analyzed by theanalyzing software.During themeasurementsbuffer is ownh the ow cell in the uidic system equipped with a peri-pump. Theowcell is composed of a owchannel, and liquidg in the channel is in direct contact with the gold surface ofss slide. The sample that includes biomolecules, for instance,ted by means of an injector valve into the buffer ow andver the gold surface of the glass slide. When molecules are

ed to the gold surface, the refractive index (RI) of themediumnt to gold surface changes from that of a pure buffer (back-RI) and is observed as the surface plasmon resonance signal.e index and the thickness of adsorbed layers on ametald being the most used due to its inertness. However,curve is a product of thickness d and the real and theart of the refractive index n (d*n), it is often necessaryr know either the refractive index or the thickness inract one of these parameters for the layer adsorbed orn the gold chip. By simultaneously measuring surfacesonance curves in different media with known proper-ssible to gain additional information for resolving thisr plain liquids the situation is simpler as the thicknesss innite and therefore it is possible to t the refractivesolutions by using known concentrations of the liquid.

cal characterization of ultrathin lms (e.g. phospholipidlodgettmono- andmultilayers) by SPR is not very com-a lack of commercial instruments capable of measuringrves in a wide angular range. Although ellipsometerssed and have established themselves as techniques forckness andoptical constantsdeterminations they sufferonsuming measurements, which often leads to exam-teady-state systems [13]. The SPR technology allowsed monitoring of dynamic processes and is thereforeactive alternative method for ultrathin lm character-ecially if the detection technology provides absoluteormation, a wide angular scan range and the use ofedia or two laser wavelengths enabling unambiguousion of layer thickness and refractive index. Another dif-rformingmeasurementswith SPRdetection technologye sample preparation, more specically how to actuallysample surface coatings of interest. Often, the substratetings are xed and provided by a supplier or supposedred inside the instrument. This is not as exible as ifc surfaces could be functionalized and prepared out-trument. Moreover, the possibility to functionalize userfaces outside the instrument allows a step by step char-of each functionalization step from a clean surface to

y functionalized surface.utilize an SPR instrument having a goniometer based

on technology for a thorough optical characterizations well as ultrathin lms. In the goniometer based SPRchnology both the laser and photodetector are movedtogether enabling a wide angular scan range coveringd liquid phase ranges in the same mechanical setup.eter approach also allows us to collect the completeith high accuracy providing absolute angle informationorough optical characterization of liquids and ultrathinommodicationof the goniometer basedSPRdetectionto include two lasers with two different wavelengthswith a solution for unambiguous determination of bothdex and thickness of ultrathin layers.to simplify sample preparation for SPR measurementst more exible, traceable chips and chip holders wereand utilized to coat the substrates both inside andinstrument. Wide angular scan ranges and user spe-coatings prepared outside the instrument are not yetly used when it comes to SPR measurements. There-im is to demonstrate the feasibility of the presentmodied goniometer based SPR detection technology,ed sample handling with a number of measurementcluding the following: (1) sucrose solutions with con-ranging from 5 to 300mM, (2) a series of ethylenesolutions with concentrations up to 2wt%, (3) a seriessolutions with concentrations up to 5wt%, (4) stearic

Fig. 1.

acid mnonspecontainstreptautilizinenablintwo-conel wa

2. Exp

2.1. In

Printom mThe eqnal anthe o

A gof Cr) oprismcontinp-polaeld veby theface. Athe intmediadent anintenstion ofsignalthrougstalticowinthe glais injecows oattachadjacegroundchangeetschmann type conguration for surface plasmon signal detection.

and multilayered LangmuirBlodgett (LB) lms, (5)interaction of 0.5mg/ml BSA with SAM and (6) biotinSAM interacting with an increasing concentration of(1.2520nM). All the measurements were performeddual channel ow cell with one laser wavelengthckground noise subtraction, except in the case of thepproach for LB lm characterization where each chan-ipped with different wavelength lasers.

ental

entation

of the signal detection: We used a standard and cus-ed KSV SPR 200 instrument [14] for signal detection.ent includes the light source, prism, detector and sig-software as well as the pump for liquid handling and

ll with the Kretschmann prism conguration (Fig. 1).lide with a sputtered gold layer (50nm of Au on 2nme side together with the ow cell is pressed against thed with an index matching elastomer in order to ensureproceeding of the light. The light beam of the laser is

214 H. Liang et al. / Sensors and Actuators B 149 (2010) 212220

Optical system: The KretschmannATR conguration comprises apermanent triangle prism made of BK7 optical glass, and a remov-able sensor slide which can easily be inserted into the instrumentwithout affecting the optical geometry of the system. The lightsource of thwavelengthmium stearof 635 andThe laser anscanning sytion of lightis no wandeinterface du

Flow sysdevice, whitwo separatpumpwithnected to aanalysis resvalveworkithus givingis loaded in

The buffgrated perisyringe to ttion ports finjected intby turning t

The owber. PDMS iheight andthe materiaan insulatoenable the mreaction-limPEEK, and tume from tchamber).

Gold senwas utilizedcell, chip hoically and tthen replaccuted in thechip is appgold substrin Fig. 2b. Twith 50nmsensor chipmaking it poutside the

Immobilpatible withused for inoutside theavoided andtionalized w

2.2. Data ha

Data acqsoftware insists of twomeasuremelasers was ebe sampled

chemrtion mer, (3)

outs neeely low baseline noise is important, the integration time ofnal up to 1min or more can be utilized.user interface has two main modes of measurement, the

r scanning (AS) mode and the reected light in xed angleode. In the FA mode the laser angle is set to a falling slopeanges on the surface are seen as reectance changes. Thede measures reectance versus angle with a user-denedr range and resolution and the dip position can be moni-n-line in the sensorgram view of the user interface. This

and post-processing of the curves by tting Fresnel equa-llow calculation of the surface RI or the layer thickness as an of time.al tting with Fresnell equations: In order to obtain refrac-dex or thickness of LB lms, the experimental SA curvestted with the Winspall 3.01 computer program, which is

on the Fresnel equations and the recursion formalism and isvailable fromtheMax-Planck Institute for PolymerResearch, Germany) [16]. The AS curve for pure gold was simulatedorder to obtain the effective parameters of the thickness

electric constant of pure gold before tting the LB lms AS. The curves for sucrose solution were performed using theimulation with Winspall 3.01 to obtain the refractive indexose solution.ddition, the change in SPR angle minimum or the changection intensity at constant angle for EG and ethanol solu-ere used for calibration curve purposes. The calibration

data were tted in Origin 7.0 software with the smallestminimum-linear regression method. The calibration curvealuated based on the correlation coefcient (R) and standardon (SD). The repeatability was tested by ve injections of ae system is composed of a laser diode with an emissionof 670nm. For two-color SPR measurements of cad-

ate (CdSA) two laser diodes with emission wavelengths670nm were used for the two channels, respectively.d detector are xed at the end of two bars of the anglestemdrivenbya steppermotor inorder to assure collec-for all angles of incidence. It should be noted that therering of the light spot reected from the measurementring the full wide angular range scan.tem: The used conguration is a two-channel systemch means that the two optical measuring spots and thee uidic channels are operated by the same peristalticow rates from 5 to 200l/min. Fluid channels are con-6-port injector valve commonly used for ow injectionearch. The instrument is equipped with one 12-portng as two6-port systems connected to the same switch,simultaneous injections for both channels. The sampleto a sample loop determining the sample volume.er container is connected through tubing to the inte-staltic pump. The sample is injected manually with ahe corresponding sample loop through the two injec-or channel 1 and channel 2, respectively. The sampleo the loop is loaded into the main stream of buffer owhe handle switch of the injector.cell is molded from poly-dimethylsiloxane (PDMS) rub-s used because it is easy to prepare owcells of differentvolume, based on the experimental set up. Moreover,l is relatively inert and the PDMS ow cell also acts asr. The depth of the ow cell used was about 80m, toinimizing of the sample amount and obtaining correctited kinetic values [15]. The tubing used was 125m

he dead volume of the system was about 10l (the vol-he output of the 12-port valve to the inlet of the ow

sor chips and chip holder: The removable chip holderwhile changing the slides into the ow cell. The ow

lder and prism are separated from each other automat-he chip holder can be pulled out. A new chip can beed into the holder and the Dock closing can be exe-software to drive the pieces together again. Thus the

lied into the holder either before or after coating theate. A schematic picture of the chip holder is presentedhe glass slide (Schott D 263, 20mm12mm0.5mm)gold layer sputtered on 2nm of Cr was used as the

. The gold slide can easily be replaced into the holderossible to prepare the substrate coating both inside andequipment.izer: An equipment called immobilizer (Fig. 2a), com-

sensor slides and slide holder of the instrument wassitu coating or functionalization of the sensor platesinstrument. In this way possible contamination can bethe two channels can be individually coated or func-ith different molecules, if needed.

ndling

uisition: The instrument is controlled by user interfacestalled on a PC. A standard conguration which con-670nm lasers (one laser per channel) was used for allnts, except for the two-color approachwhere one of thexchanged for a 635nm laser. The response signal canwith user-dened time base. Our measurements were

Fig. 2. Sple inseelastom

carriedpling iextremthe sig

Theangula(FA) mand chAS moangulatored omodetions afunctio

Signtive inwere basedfreely a(Mainzrst inand dicurvessame sof sucr

In ain reetions wcurvesquarewas evdeviatiatic pictures of (a) the immobilizer accessory and (b) the SPR sam-echanism. The numbered parts are: (1) prism, (2) index matching

SPR sensor slide, (4) PDMS ow cell and (5) closing mechanism.

with a standard 1Hz sample rate. If high speed sam-ded, the sampling can be set to a 200Hz sample rate. If

H. Liang et al. / Sensors and Actuators B 149 (2010) 212220 215

sample close to the limit of quantitation (LOQ) in order to estimatethe maximum error for repeatability from the SD of the injections.Error rate for the experiments was estimated by RMS equation (1)averaging the SDs of the linear t and repeatability experiment. (2)LOD was debaseline du

RMSerror (

Reference dwas takenthe analytewavelengthneglected in

2.3. Sample

Chemica84099), CdCserum albuSigma, Inc.Pierce. HS-02) and HS-TH012-02)Ethanol (gr

Gold subammonia/hammonia (water (1:1:gold slidesrinsedwithfor use.

Sucrose,10, 25, 50with SPR incol (EG) con5wt% downdiluted intoand ethanoscanmodesfor creatingand for me(LOQ). The

Stearic aLtd, Helsinklayers on thsitionswere(330mmface pressuafter spreadstant barriemonolayerthe pre-detstabilizingcedure. Thestearic acidfor SA, thebefore cons

Monolaysurface preswith pH valbymeans otted with

BSA andSAMprepar

xperimater.

withanneG3-bimm(EG3ucedas kehe thand

eaklyG3 Sents.red teptathe ients) asptavates [eaned EGM inm, anld suted fandogen. The prepared EG3/EG3-biotin SAMs were used foreasurements. After BSA solution of 0.5mg/ml in 20mM/150mM NaCl (pH 7.4) was injected to prevent nonspeciction [19], streptavidin solution of 1.25, 2.5, 10 and 20nMjected to measure the specic interaction between strepta-nd biotin-SAM. The measurements were performed at thete of 10L/min. The buffer used was 20mM HEPES/150mMH 7.). The specic interaction kinetic between streptavidintin was calculated with the software of Scrubber-2 packageby Myszka and collaborators [20,21].

ults and discussion

gular scan of pure gold slides in air and in water

pure gold slide was measured in both air and water. Therves of pure gold are shown in Fig. 3. The sharp difference ofonance angles between the SPR signals measured in air andned as three times and LOQ as ve times the SD ofring the experiments performed:

%) = SQR((

SDSlopeSlope

)2+(

SDRepeatAVGRepeat

)2) 100 (1)

ata for refractive index (RI) concentration dependencyfrom CRC [17], and it is assumed that the dn/dc ofis linear below 5wt%. Differences in temperature andbetween reference and experimental conditions arethe analysis as we measure changes in RI.

preparation and measurements

ls: Stearic acid (CatalogNo. 85680), sucrose (CatalogNo.l2 (Catalog No. 20912), ethylene glycol (EG) and bovinemin (BSA) (Catalog No. A4503) were purchased fromStreptavidin (Catalog No. 21125) was purchased from(CH2)11-(OCH2OCH2)3-OH (EG3) (Catalog No. TH002-(CH2)11-(OCH2OCH2)3-biotin (EG3-biotin) (Catalog No.were purchased from ProChimia Surfaces Sp. z.o.o.

ade A) was purchased from Altia group.strate cleaning: The gold substrates were cleaned withydrogen peroxide solution. The solution consists of 30%NH4OH), 30% hydrogen peroxide (H2O2) and Milli-Q5). The solution was heated until boiling (8090 C) andwere put in it for 10min. The slides were removed andMilli-Qwater, and then dried by blowingwith nitrogen

EG and ethanol solutions: The sucrose solutions of 5,and 300mM in water were prepared and measuredthe mode of angular scan. A series of ethylene gly-centrations and ethanol concentrations ranging fromwards for ethanol and 2wt% downwards for EG werewater. EGexperimentsweredone in angular scanmodel experimentswere done in both angular andxed angle(ASandFA, respectively). Themeasurementswereusedcalibration curves, testing for repeatability of injectionsasuring the levels of detection (LOD) and quantitationmeasurements were done in ambient conditions.cid (SA) LB lms: A KSV Minitrough (KSV Instrumentsi, Finland) was used for the deposition of stearic acide cleaned gold slides for SPR measurements. The depo-carried out at 23 Cusing a thermostated Teon trough

75mm). The compression, to the pre-determined sur-re for deposition, of the monolayer was started 10mining the monolayer substance on the subphase. A con-r speed of 10mm/min was used for compressing theto the pre-determined surface pressure. After reachingermined surface pressure, the monolayer was left forat least for 10min before starting the deposition pro-speed for the deposition of mono- and multilayers ofwas 5mm/min. In the case of multilayer depositions

deposited layers were allowed to dry in air for 15minecutive layers were deposited.ers and multilayers of SA were deposited at a constantsure of 30mN/m froma subphase (104 CdCl2 inwater)ue of 6. The SA mono- and multilayers were performedf SPRmeasurement in air. The obtained SPR curveswereWinspall 3.01 to obtain the thickness of the layers.streptavidin with thiol self-assembled monolayers (SAM):ation on SPR gold substrates [18]. Thiol SAMswere pre-

Fig. 3. Eand in w

paredtwo chnel 2, Ein thebiotinintrodtion wfrom tfor 5 sand wThen Esuremmeasuthis strdetectsurem(pH 7.4

StresubstrThe clEG3 an0.24mparalThe gosonicaexcessof nitrthe mHEPESinteracwas invidin aow raNaCl (pand biowritten

3. Res

3.1. An

TheSPR cuthe resental (dots) and tted (solid) SPR curves of pure gold substrate in air

an immobilizer outside the SPR equipment to enablels to be coated individually (channel 1, EG3 and chan-iotin). After the cleaned gold substrates were mountedobilizer, EG3 of 0.2mM or the mixture of EG3 and EG3-/EG3-bitoin, 4:1) of total concentration 0.2mM wereto two different channels and the self-assembled reac-pt overnight. The gold substrates were then removediol solution, and the gold substrates were sonicatedrinsed with ethanol three times to remove excessbound thiols and dried under a stream of nitrogen.

AM and EG3/EG3-biotin SAM were used for SPR mea-Nonspecic interaction between BSA and SAM washrough injecting 0.5mg/ml BSA in PBS (pH 7.4). Aftervidin solutionof30g/ml inPBS (pH7.4)was injected tonteraction between streptavidin and biotin-SAM. Mea-were performed at the ow rate of 10l/min with PBSbuffer.idinbiotin kinetics: The SAM was rst prepared on gold18] by starting with the thiol solutions in pure ethanol.d gold substrates were immersed in the mixture of3-biotin (EG3/EG3-biotin, 5:1) of total concentrationpure ethanol ushed with N2 and then sealed withd it was kept as a self-assembled reaction overnight.bstrates were then removed from the thiol solution,

or 5 s and rinsed with ethanol three times to removeweakly bound thiols, and nally dried under a stream

216 H. Liang et al. / Sensors and Actuators B 149 (2010) 212220

Fig. 4. ExperimcdSA LB layers

in water iswide angulble to measwithout takshows thatand with drobtained afWinspall. Ttive gold lagold layer th(), obtaineand 3.8152pure gold iobtained inbe explaineboth cases,et al. [22].

3.2. Stearicangle scan, t

Monolayangular scadots signifycurves. Thewithdiffereproperties oindex. The afunction ofin which aan organicresonance arelationsprtal condition is the diand the meditions so kthe layer thshifts and th

The meacurves in Figthe ones obWinspall arcan be seen

The SA LB layers are deposited at pH 6 as cadmium stearate andtransferred onto gold surface under a surface pressure of 30mN/m.The tting was performed, assuming that the refractive index ofSA is 1.45 [23]. The thicknesses from tting as function of number

rs presented in Fig. 5 indicate a linear relation, as expected.ckness as a function of the number of the layers in the plot is/layer, which is in close agreementwith the layer thicknesst 2.5nm presented in literature [24].approach above for determining thin lm thicknesses can

e used if the refractive index (n) is known or assumed, andrsa the refractive index can only be determined if the thinickness is known or assumed. This originates from the factis impossible to obtain both n and d from a single SPR

ment [25]. This is an obvious limitation for standard SPRentation when there is a need for characterizing proper-

thin lms, especially when it comes to monolayers with aess of just a few nanometers. However, by custom modify-SPR instrumentation tohold twodifferent laser light sourceso different wavelengths it is largely possible to overcomeitation mentioned above, thus allowing an unambiguousination of refractive index, n, and thickness, d, for ultra-

lms like LB layers. This approach is based on the fact thatnd [12], and by using two different wavelengths there willsets of different refractive indices which eventually can

d to extract a unique refractive index for the used wave-and thickness of the thin layer [25,26]. However, also thelor method has one limitation. In order to nd n and ded tofferesioninedwerlatioonolater)l 2:

ffereedialatioor th

motherental (dots) and tted (solid) SPR curves for pure gold, 1, 3, and 5.

clear implying the capacity of the detection. Due to thear scan range (4078) of the instrument it was possi-ure SPR curves in both, air and water simultaneously,ing the slide out of the instrument. In addition, this alsoit is possible to work in both gas and in liquid phases,y and wet samples. The parameters of pure gold wereter the measured curves for pure gold were tted withhe gold parameters, which in our case mean an effec-yer representing also the Cr adhesion under the gold,ickness, refractive index (n) and absorption coefcientd from tting of pure gold in air are 53.21nm, 0.2034, respectively. The parameters obtained from tting ofn water are 54.37nm, 0.1972 and 3.8107. The resultsair and in water are very close and the discrepancy cand by an intermediate layer of Au/air and Au/water forrespectively, in a similar way as described by Sadowski

acid (SA) mono- and multilayer LB lms with totalwo-media and two-color SPR approach

ers and multilayers of SA were measured in air withn mode. The obtained curves are shown in Fig. 4. The

the measured curves, and the lines are the ttedobvious shifts between the pure gold and the lms

nt layernumberareobserved,which imply thedifferent

of layeThe thi2.68nmof abou

Theonly bvice velm ththat itexperiinstrumties ofthickning thewith twthe limdetermthin spr = kbe twobe uselengthtwo-cowe netwo didisperdetermand posion reacid mand wchannetwo ditwo-msion reused flayer. Ain anof the three LB lms, e.g. different thickness or refractivengular shifts of SA layers with respect to pure gold as anumber of layers are shown in the lower line of Fig. 5,good linear relation is clearly seen. In the presence oflayer at metal/dielectric interface, the position of thengle (spr) shifts to higher values. As to spr, there is the= knd,wherek is a constant reecting theexperimen-ns, d is the geometrical thickness of organic layer, andfference between the real refractive indices of the layerdium [12]. The SA layers are deposited in the same con-and n should be unchanged. The spr just depends onickness d. Therefore the linear relation between angulare number of layers is reasonably based on spr = knd.sured curves were tted to the theoretical model (solid. 4)withWinspall 3.01. The ttedmeasured curves andtained by Winspall are accepted if the curves tted bye deeper than the experimentally measured signals, asin Fig. 4.

Fig. 5. The andots representillustrate lineaand 0.9927, reknow the dispersion relation of the material for thent wavelengths used for the measurements. Often thisrelation cannot be found in literature and it has to beby other methods. Here, we shortly present an easy

ful approach for simultaneously obtaining the disper-n of layers, refractive index and thickness. The stearicayer was measured in two different media (both in airwith the two-color SPR approach (channel 1: 635nm;670nm), see Fig. 6. The unique refractive index (usingnt wavelengths) and thickness were obtained from thismeasurement. This also enabled extraction of disper-n between the two wavelengths, which could then bee two-color SPR approach for the stearic acid mono-re detailed description of the procedure will be givenmanuscript [27]. The results for the two-media mea-

gle and thickness as a function of the number of CdSA LB layers. Themeasurement and simulation to SPR data respectively. The solid linesr regression t to the data (R2 for angle and thickness were 0.99905spectively). The slope for thickness is 2.68nm/layer.

H. Liang et al. / Sensors and Actuators B 149 (2010) 212220 217

Fig. 6. Resultwavelengths o(intersection i

surementsin Fig. 6 givboth wavelwavelength2.585, respe

Fig. 7. The reswith two-colo2.7nm.

Fig. 8. (a) Angin water. (b) Adots are fromdata (R2 = 0.99

d 67edianm tsultsess fo635 antwo-mto 670The rethickns for SA monolayers determined by two-media measurements forf 635nm (a) and 670nm. (b). The RI and thickness for 635 and 670nmn (a) and (b)) are 1.58, 2.66nm and 1.4138, 2.585nm, respectively.

are shown in Fig. 6. The intersection of the two curveses the unique refractive index (RI) and thickness for

engths. The RI and thickness for the SA monolayer fors of 635nmand670nmare1.5800, 2.66nmand1.4138,ctively. The dispersion for the SA monolayer between

ults of simultaneous determination of refractive index and thicknessr SPR measurements. The RI and thickness (intersection) is 1.41 and

surement imonolayerwas 1.4138color measutwo-mediaalso very cmentwith tdeterminedvalue for thThey havedard deviatresults furthor custom mis a suitable

3.3. Sucros

The sucsured in anangular scaas a functiovides us wishows theconcentratidifferent cotion of conclinear relatof sucrose sof sucrose ibe conrmetration theTherefore,angle chang

The meashown in Fular change as a function of time for 5, 10, 25, 50 and 300mM sucrosengular change as function of concentration of sucrose solution. Themeasurement, and the solid line is a linear regression tting to the999).

0 nm, n (635nm, 670nm)=0.1662, obtained from themeasurements is then used to shift the RI of 635nmo get the real intersection at a wavelength of 670nm.are shown in Fig. 7. The unique RI at 670nm and ther the SA monolayer obtained from the two-color mea-

n water are 1.41 and 2.7nm, respectively. The RI of SAfor 670nm obtained from the two-media measurement, which is in good agreement with the RI from the two-rements. Additionally, the three thickness values fromand two-color measurements, 2.66, 2.585 and 2.7 arelose to each other and these are also in good agree-he single average thickness (2.68nm) for SAmultilayersabove by assuming the refractive index. The average

ese four thicknesses is: (2.66+2.585+2.7 +2.68) =2.656.very small standard deviation (SD) and relative stan-ion (RSD), which are 0.05 and 1.89%, respectively. Theseer support the fact that the goniometer based standardodied SPR instrument shows high performance and

to be used as a refractometer for ultrathin lms.

e solutions

rose solutions of different concentrations were mea-gular scan mode at the ow rate of 50l/min. In thenmode, themeasured curves anddynamic angle changen of time can be simultaneously acquired, which pro-th more information for the measured samples. Fig. 8angle change as a function of time for ve differentons, 5, 10, 25, 50 and 300mM. Based on the angle forncentrations from Fig. 8a, the angle change as a func-entrations is achieved (Fig. 8b), which indicates a goodion. This angle change is a result of the refractive indexolution, the medium close to gold. The refractive indexncreases linearly at the lower concentration, which cand by the literature (Fig. 10) while at the higher concen-refractive index versus concentration is nonlinear [17].the linear refractive index change results in the lineare.sured SPR curves as a function of concentration are

ig. 9. These curves are tted to theoretical model (solid

218 H. Liang et al. / Sensors and Actuators B 149 (2010) 212220

Fig. 9. Measu300mM sucro

Fig. 10. Refraliterature [18]sion tting (R2

tting are asY=1.3331+4.5

curves in Fifor tting isRefractomesucrose solFig. 10. Theones in liteSPR systemthe refractiter at the wmeasured rear relationfrom SPR arHowever, thin literaturesuring condand 25 C fo

hanol and EG solutions using angular and xed angle scans

anol was measured both with angular scan and xed angleG was measured only with AS mode. For the ethanol experi-, the laseline glt%.

expendtplayees t aientsdataR vaseents ta shiFA wticalred (dots) and tted (solid) SPR curves for water, 5, 10, 25, 50 and

3.4. Et

Ethscan, Ementsfrom bethyle0.139w

Alltions aare disanalyscoefc

Theas thecan belinearminimand inTheorese in water.

ctive index of sucrose solution from SPR, Abbe Refractometer andas a function of concentration. The solid lines are the linear regres-respectively 0.99994, 0.99986 and 0.99959). The equation from the

follows: Y=1.33107+4.91485E5X, Y=1.33116+4.89234E5X,7272E5X.

g. 9) with Winspall 3.01. The refractive index of waterassumed to be 1.33096, from determination of Abbe

ter (Fig. 10). The obtained refractive indices of theution as a function of the concentration are shown inobtained refractive indices are in accordance with therature (squares in Fig. 10). In order to compare theto be used as a refractometer, we also determined

ve indices of sucrose solution with Abbe Refractome-avelength of 633nm at 25 C (triangles in Fig. 10). Theefractive indices from Abbe Refractometer have a lin-with concentrations. Moreover, the refractive indicese equal to the ones measured by Abbe Refractometer.ey are somewhat different from the refractive indices[17], which is possibly a result from the different mea-itions, at 670nmand roomtemperature for SPR, 633nmr Abbe and 589.3nm and 20 C for literature.

change in Rmethod, a cpass througdeviation oused in erro

The SD oimum errorone of the slargest relatdone, and tment. The eaveraging, aterms of thbaseline ofconversionalso in Tabl

The LODwhich indiingful chanlower leveltive RMS ermeasuringthe AS moddynamic rato test thewith PDMS

3.5. Nonspethiol self-as

The dynwith the xnonspecicintensitiesSAMs are dis more thaalmost closreturns to bstreptavidincic bindinthe sensogrclearly lowbinding sigBSA adsorpinteractionowest concentrations that were possible to be detectedne were 0.096wt% for AS and 0.0095wt% for FA. Forycol AS experiment the lowest concentration used was

riments were plotted as signal versus mass concentra-tedwith RMS linear t (Fig. 11), and the linear t resultsd in Table 1. For reference the theoretical dn/dc of there also displayed, as well as the signal to RIU conversion, in Table 2.for all experiments correspond well with the linear t,

lues in each case are close to one (perfect linear t). Itfrom the R values, that the AS experiments give better

han the FA experiments. This is reasonable as the SPRfts linearly with the change of RI of the sample solution,e measure the near-linear area of the SPR-band slope.ly, all the intercepts should be zero in all cases, as zeroI should result in zero signal change. In an analyticalalibration curve would be corrected with this value toh zero and thus eliminate any offset error. The standardf a t is the RMS error for the line t, and it is furtherr calculations.f repetition injections was used to estimate the max-of the experiment (as the repetition experiments are

mallest ones in the experiments and therefore have theive error of the samples). For the EGno repetitionswerehe error for this was estimated from the EtOH experi-xperimental analytical error was calculated using RMSnd the results are displayed in Table 2. LOD and LOQ ine RI of the analyte were calculated from the SD of theeach experiment and converted to RI units by using thecoefcient of the linear ts. The results are displayede 2.

and LOQ values for the experiments are excellent,cates that we are able to measure analytically mean-ges in RI in the range of 106 at best. The FA mode has aof detection, but it has poorer linearity and larger rela-ror than the AS mode. The FA mode is clearly better forreally small changes in RI because of its sensitivity, ande is respectively better for experiments where a largernge isneeded.Other solventanalytepairs couldbeuseddynamic range further, as long as they are compatible-rubber.

cic and specic binding of BSA and streptavidin tosembled monolayers (SAM)

amic change of adsorption of BSA on the SAM obtaineded angle scan are shown in Fig. 12b. Relatively clearadsorption of BSA can be observed. Moreover, the

of nonspecic adsorption for EG3 and EG3/EG3-biotinifferent. The adsorption of BSA to EG3/EG3-biotin SAMn for EG3 SAM. Amount of BSA binding to EG3 SAM ise to zero because the signal for BSA binding to EG3 SAMaseline after washing with buffer. After that, 30g/mlin PBS is injected to both channels to investigate spe-

g of streptavidin on EG3/EG3-biotin SAM. Fig. 12a showsam of streptavidin binding. The signal for EG3 SAM iser than for EG3/EG3-biotin. Moreover, the streptavidinnal for EG3/EG3-biotin is also greatly more than that oftion (Fig. 12a), which further implies a strong specicbetween streptavidin and biotin.

H. Liang et al. / Sensors and Actuators B 149 (2010) 212220 219

Fig. 11. SPR angle minimum change as a function of ethanol (crosses) and ethylene glycol (squares) concentration in angular scan mode. Inset. Fixed angle intensity as afunction of ethanol concentration in xed angle experiment.

Table 1Linear t data for the experiments. Slope and SD in units are deg/wt% for AS and RU/wt% for FA. Y-intercept unit is deg for AS and RU for FA. Unit for dn/dc is RI/wt%. Thecalibration coefcient is (ds/dc)/(dn/dc) = (ds/dn) in 1/deg for AS and 1/RU for FA.

Slope (ds/dc) Y-intercept R SD (t) Theor (dn/dc) Conv. coef.

EG-AS 0.109748 1.55E04 0.999647 0.002618 9.49E04 115.62EtOH-AS 0.06817 0.0014 0.99977 0.0019 6.00E04 113.62EtOH-FA 0.01914 5.60E05 0.9972 1.04E04 6.00E04 31.90

Table 2Relative errors in arbitrary units. RMS error in percentages. Background SD, in deg for AS and in RU for FA. LOD and LOQ for change in RI for the experiments.

Err (slope) Err (rep) RMS err % SD (bg, signal) LOD (RIU) LOQ (RIU)

EG-AS 0.0239 0.0500 5.54 7.03E04 1.82E05 3.04E05EtOH-AS 0.0279 0.0462 5.40 1.35E03 3.55E05 5.92E05EtOH-FA 0.00542 0.0681 6.83 6.27E05 5.90E06 9.83E06

Fig. 12. SPR sensorgram for binding of BSA and streptavidin to the functionalizedsurface, thiol self-assembled monolayer (SAM). (a) Specic binding of streptavidinsolution of 30g/ml to biotinylated thiol SAM. (b) Nonspecic binding of BSA solu-tion of 0.5mg/ml to thiol self-assembled monolayer (SAM). The SPR measurementswere performed at the ow rate of 10l/min. EG3: HS-(CH2)11-(OCH2OCH2)3-OH,EG3-biotin: HS-(CH2)11-(OCH2OCH2)3-biotin. EG3 SAM and EG3/EG3-biotin (molarratio 4:1) were prepared with the immobilizer outside the equipment.

Fig. 13. SPR sensorgram for the interaction of streptavidin with the functional-ized surface, EG3-biotin SAMs. Streptavidin solutions of 1.25, 2.5, 10 and 20nMwere respectively injected. The measurements were performed at the ow rate of10l/min. EG3 SAM and EG3/EG3-biotin (molar ratio 5:1) were prepared outsideSPR equipment.

220 H. Liang et al. / Sensors and Actuators B 149 (2010) 212220

3.6. Determination of kinetics between streptavidin and biotin

The measured SPR sensograms are shown in Fig. 13. The senso-grams are processedwith the Scrubber-2 software to obtain afnityof streptavidin with biotin-SAMs [20]. The calculation based onFig. 13 gives a value of KA of 1.01108 M1, which is the afn-ity constant for single sub-unit of streptavidin to the immobilizedbiotin. This value of KA is comparablewith the solution-based valueof 2107 M1outside the

4. Conclus

Based onSPR instrumwell as forvided by ththe two-meneously detthin lms. M40 to 78 esensor-platinstrumentslide can bepractically echannels enmeasureme

When mcorrection,angular moincreases, blem. This chigh degreethe other isinjection.

Acknowled

The authfunding theTuppuraineconcerningvaluable op

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s

received his MS degree in food science at Henan University of Tech-4. He worked a short time in food industry, where after he in 2005D student at the Division of Biopharmaceutics and Pharmacokinetics,rmacy of University of Helsinki, Finland and Bioengineering CollegeUniversity, PR China. His current research interests focus on targetedand ultrathin lms.

(earlier Heikkil) received herMS (Technology) degree fromHelsinkiechnology in 2009. Currently she is nalizing her studies in aMastersm in biotechnology at the University of Helsinki.

st received his MS degree in polymer chemistry from University2010. Currently he is working as an application scientist at KSVe is also a PhD student at the Division of Biopharmaceutics and

tics, Faculty of Pharmacy of University of Helsinki.

ski received his MS degree from the Warsaw University of Tech-5 and his PhD degree in physics from the Department of TechnicalHelsinki University of Technology in 1978. He has been working atrsity of Technology (19791982), University of Joensuu (19821988),l Research Centre of Finland (19892006), and currently he is thector of BioNavis Ltd. His areas of expertise are in the elds of biosen-phasis on optical detection methods, mainly the surface plasmonenomenon. He is an author of several scientic publications and

received his PhD degree in physical chemistry from bo Akademi999. From 2000 to 2009 he worked for KSV Instruments as a scien-R&D group and as a technical sales manager. In 2010 he joined theand Nanotechnology Group in the Division of the Biopharmaceuticskinetics at the University of Helsinki. His current research is focusedemical characterization of interfaces and nanoscale layers in closebiophysics, nanotechnology and drug delivery.

eceivedhisMEdegree in engineeringmechanics fromChongqingUni-ina in 1988. After receiving his PhD degree in biomechanics in 1996g University he worked as a senior visiting researcher at the Gunma

Japan (19971999). He has been a professor of Bioengineering Col-ing University since 1999. His current interests include isolation andnatural products, screening of bioactive compounds and controlled.

tula received herMS and PhDdegrees in physical chemistry fromUni-inki in 1987 and 1993, respectively. During her studies she workedtronics with multilayered thin lm technology and at KSV Instru-g biophysical properties of LangmuirBlodgett lms. After that shesity of Helsinki to study bio-optoelectronical properties of the bac-

in thin lms. After working at CEA in Saclay, France with ultraopy of molecular wires she worked 10 years in drug discovery atFinland. She has been a professor of Biopharmaceutics and Pharma-he University of Helsinki since 2007. Her current interests includeparticle drug and gene delivery research, and development of theals based drug discovery tools for drug and gene testing.

Surface plasmon resonance instrument as a refractometer for liquids and ultrathin filmsIntroductionExperimentalInstrumentationData handlingSample preparation and measurements

Results and discussionAngular scan of pure gold slides in air and in waterStearic acid (SA) mono- and multilayer LB films with total angle scan, two-media and two-color SPR approachSucrose solutionsEthanol and EG solutions using angular and fixed angle scansNonspecific and specific binding of BSA and streptavidin to thiol self-assembled monolayers (SAM)Determination of kinetics between streptavidin and biotin

ConclusionsAcknowledgementsReferencesBiographies