Analytica Chimica Acta 551 (2005) 98–104

Acousto-optic tunable filter-surface plasmon resonanceimmunosensor for fibronectin

Yuan Tian, Yanhua Chen, Daqian Song, Xia Liu, Shuyun Bi,Xin Zhou, Yanbo Cao, Hanqi Zhang∗

College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, China

Received 8 March 2005; received in revised form 28 June 2005; accepted 13 July 2005Available online 24 August 2005

Abstract

An acousto-optic tunable filter-surface plasmon resonance (AOTF-SPR) immunosensor based on wavelength-modulation was appliedto detect fibronectin by direct, sandwich and colloidal Au-enhanced immunoassay. The design of the wavelength-modulation AOTF-SPRimmunosensor is based on fixing the incident angle of light and measuring the reflected intensity of light in the wavelength range spanning4 u-e immunoassayo©

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40–790 nm. Fibronectin was determined in the concentration range 2.5–30, 0.5–30, and 0.25–30�g/mL for direct, sandwich and colloidal Anhanced immunoassay, respectively. The results demonstrate that AOTF-SPR biosensor can be applied to direct and enhancedf biomolecule.2005 Elsevier B.V. All rights reserved.

eywords: AOTF; SPR; Immunosensor; Enhanced immunoassay; Fibronectin

. Introduction

Surface plasmon resonance (SPR) spectroscopy is a pow-rful tool for in situ real-time characterization of solid/liquid

nterfaces[1]. Assembly of monolayers and chemical reac-ions occurring on thin film of metal surface can be monitoredy SPR spectrum. The chemical modification of the metal sur-

ace alters the refractive index and thickness of the thin film,esulting in changes in the resonant angle or wavelength ofPR spectrum. Because of the advantages such as real-timeonitoring, label-free detection, crude sample analysis, etc.

2], SPR technique has been extensively applied to the studiesf antibody–antigen interaction[3], DNA-protein interac-

ion [4], DNA-nucleic acid hybridization[5], drug–serumlbumin interaction[6]. Other papers have reported the com-ination of SPR and mass spectrometry[7]. Moreover, this

echnique has also been expanded to imaging and holographyormats[8].

∗ Corresponding author. Tel.: +86 431 5168352; fax: +86 431 5112355.E-mail address: analchem@jlu.edu.cn (H. Zhang).

When the SPR technique for biosensors is appto measure the small changes in the refractive inthat accompany protein binding processes, the sysdisplay limited sensitivities. The detection limit for tbiosensors where, for example, a protein is adsoonto a gold surface from an aqueous buffer solutioin the order of 10 pg mm−2 adsorbed protein[9]. Butnow, biacore AB have developed new machines ware designed to detect very small changes in respunits (RU) and are used with low molecular weight copounds typically <1000 Da[10]. Moreover, some methods for enhanced sensitivity in SPR biosensors baseusing sandwich assay[11], liposome[12], latex particle[13], colloidal Au [14–16] have been reported. But inthese methods the traditional SPR devices were apWe reported a sensitivity-enhancement of wavelenmodulation SPR biosensor for determining human[17].

Fibronectin is a dimeric glycoprotein consisting of tmonomers of approximately 220–250 kDa held togethedisulfide bonds[18]. The concentration of fibronectin

003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2005.07.017

Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104 99

blood plasma is 200–400�g/mL. It exists in two forms,an insoluble, polymerized form, which is a major com-ponent of extracellular matrices and basement membrane,and a soluble form present in most body fluids. Fibronectinplays a major role in embryogenesis, blood clotting, inflam-mation, tissue repair, tumorigenesis, and cellular migration[19,20]. Some established immunoassay techniques such asenzyme-linked immunosorbent assay (ELISA)[21], SDS-PAGE [22] are widely used for detecting fibronectin butsuffer from time-consuming procedures and complicatedoperation.

An acousto-optic tunable filter (AOTF) is electronicallytunable spectral bandpass filter, which is based on the princi-ple of acousto-optic interaction in an anisotropic medium.The filter can diffract incident white light into a specificwavelength light when a specific radio frequency (RF) isapplied to it. The wavelength of the diffracted light canbe turned over large optical regions by simple chang-ing the frequency of the applied RF[23–25]. The scan-ning speed of the AOTF, which is controlled by the tran-sit time of an acoustic wave across an optic beam, canbe as fast as a few microseconds. Not only monochro-matic light but also polychromatic light can be diffractedfrom the AOTF when more than one RF signals are simul-taneously applied to the AOTF. So, the AOTF can beused as a polychromator, for example, multidimensionalfl olids ningr ty ofi

sys-t n-b frac-t g acd -o n filmo opti-c oret con-v ntly, aS usinga ourl

tionA TFw d ont thec van-t bility( theA irecti Au-e mon-s minet

2. Materials and methods

2.1. Materials and equipment

Fibronectin, fibronectin antiserum (titer, 1:120) andbovine serum albumin (BSA) were purchased from Shang-hai Biology Product Research Institute. 3-Mercaptopropionicacid (MPA) and colloidal Au were obtained fromSigma (St. Louis, MO, USA). 1-Ethyl-3-(3-dimethylamino-propyl)carbodimide (EDC) andN-hydroxysuccinimide(NHS) were purchased from Shanghai Lizhu DongfengBiotechnology Co. All the biological reagents were kept at4◦C. All other chemicals were of analytical reagent grate.All solutions were prepared with doubly distilled water. The0.01 mol/L phosphate-buffered saline (PBS; pH 7.4) and0.1 mol/LNaOH were prepared.

The halogen tungsten lamp and adjustable optical devicewere purchased from Changchun (P.R. China) Fifth OpticsPrecision Instrument. The AOTF is obtained from ShanghaiSilicate Research Institute.

2.2. Apparatus

Traditional angle-modulation SPR biosensing deviceavailable from BIAcore is based on fixing a discrete excitationwavelength and measuring the angle of incident light, there-f ectedl er,t asedo ngthi thei rmso ctedl att

OTFi TF-S s thatr ew ngths urce.T r iss el om-i heni anu k isu lightb amp oldfi eent thepc llc cellb

uorimeter [26,27]. Because of the advantages (all state, non-moving parts, fast scanning ability, wide tuange) the AOTF has been used to develop a varienstruments.

To date, there are a few reports about AOTF-SPRem. In 1995, Jory et al.[28] developed a surface plasmoased optical sensor using an AOTF. A gold-coated dif

ion grating was used as optical coupler. By addinhemically active overlay to the system NO2 in N2 wasetected. In 1998, Carusoa et al.[29] reported acoustoptic surface-plasmon-resonance measurements of thin gold. An equilateral sapphire prism was used asal coupler. The AOTF SPR system was found to be mhan two orders of magnitude more sensitive thanentional angle-dependent SPR measurements. RecePR biosensor setup based on wavelength-modulationn AOTF as the wavelength selector was installed in

aboratory.In this paper, we reported a wavelength-modula

OTF-SPR biosensor for detecting fibronectin. The AOas used as the wavelength selector and is designe

he basis of fixing angle of incidence and measuringhange of resonant wavelength. The AOTF offers such adage as being all-solid state, having rapid scanning a�s), so it can monitor reaction in real-time. ApplyingOTF-SPR biosensor we determined fibronectin by d

mmunoassay, sandwich immunoassay and colloidalnhanced immunoassay. The experimental results detrate that AOTF-SPR biosensor can be applied to deterhe biomolecules.

ore, the SPR-reflected spectra are shown in terms of reflight intensity versus angle of the incident light. Howevhe wavelength-modulation SPR biosensing device is bn fixing incident angle and measuring resonant wavele

nstead of employing a fixing wavelength and modulatingncident angle of light. The SPR spectra are shown in tef reflected light intensity versus wavelength of the refle

ight. The intensity of the reflected light is the minimumhe resonant wavelength.

In this study, a SPR immunosensor based on As applied. The device of wavelength-modulation AOPR immunosensor used in this paper is the same a

eported previously[30]. In this device, the AOTF, whosavelength range is 440–790 nm, is used as waveleelector, a halogen tungsten lamp is used as light sohe schematic diagram of the AOTF-SPR biosensohow in Fig. 1. The polychromatic light emitted from thamp passing through a parallel optical tube and becng a parallel light beam, travels through AOTF and ts diffracted into TM- and TE-polarized light beam andndiffracted zero-order light beam. The dark beam blocsed to shut out TE-polarized light beam and zero-ordeream. The parallel TM-polarized monochromatic light beasses through an optical prism with a 50 nm thick glm and excites surface plasmon at the interface betwhe gold film and the analytes. The output light fromrism is detected by a photomultiplier tube. A 180�L flowell (18 mm× 5 mm× 2 mm) was used for the reaction. Ahemicals and biochemicals were injected into the flowy a syringe.

100 Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104

Fig. 1. Schematic diagram of the wavelength-modulation AOTF-SPR biosensor.

The AOTF used consists of an anisotropic TeO2 crys-tal and a LiNbO3 piezoelectric transducer. A direct volt-age signal from 0 to 5 V is supplied to the voltage-controlled oscillator (VCO), which is in conjunction withpiezoelectric transducer of AOTF, by digital to analog con-verter controlled by computer. When the voltage signal ischanged, the center wavelength of diffracted TM-polarizedlight beam is changed. That is to say, one voltage corre-sponds to one wavelength. The relationship between trans-mitted wavelength (λ) and supplied voltage (v) of AOTFis described by the polynomial with the equation:λ =817.6094 − 130.96174v − 18.17996v2 + 26.9456v3 −8.37204v4 + 0.93962v5 + 0.00177v6 − 0.00474v7 (R =0.9998). For the AOTF used in this paper, the low volt-age corresponds to long wavelength, while the high volt-age corresponds to short wavelength. In the AOTF-SPRimmunosensor, SPR spectrum is shown in terms of reflectedlight intensity versus applied voltage of AOTF. When theplasmon resonance phenomenon occurs, the voltage at whichreflected light intensity is the minimum is the resonantvoltage. The resonant voltage is corresponding to the res-onant wavelength of conventional wavelength-modulationSPR biosensor. A smaller change in refractive index orlayer thickness at the sensor surface will cause a shift ofthe resonant voltage in SPR-reflected spectrum. The rela-tionship between supplied voltage of AOTF used in thisp lana-t usly[

theA teris-t TFua elec-t lighti nma n bem f theA ti COa ltager

Though the AOTF-SPR biosensor setup has so many obvi-ous advantages, its sensitivity is limited by the resolution ofAOTF used in this work. If a high resolution AOTF was used,the sensitivity of AOTF-SPR should be improved. The perfor-mance of AOTF used in this work is studied by a 1m gratingmonochromator, whose wavelength range is 200–800 nm andwavelength resolution is 0.01 nm. The resolution [full widthat half-maximum (FWHW)] of AOTF used in this work wasdetermined to be 4.8 nm at 628 nm.Fig. 2shows the typicalAOTF spectral output. When the applied voltage is 1.500 and1.505 V, the two beams of light for AOTF can be resolved bymonochromator.

2.3. Surface modification and antibody immobilization

Molecular self-assembling is applied to form sensingmembrane on the gold substrate. The immunosensor sub-strates were rinsed with doubly distilled water and PBS bufferto keep the resonant voltage constant. Then, the surface wasexposed to solution containing 10 mmol/L MPA for 1 h; it wasactivated by injecting a solution containing NHS (40 mg/mL)and EDC (40 mg/mL). The fibronectin antiserum at 1:10 (v/v)was injected into the flow cell to monitor its assembling onthe activated surface. The assembling of the antibody was

F 0 V,B

aper and transmitted wavelength, and the more expion about the AOTF-SPR setup were reported previo30].

Compared with other traditional SPR biosensors,OTF-SPR biosensor setup has many obvious charac

ics. The instrument is miniaturization, because the AOsed is very small, only 60 mm× 40 mm× 30 mm. Therere no moving parts in the biosensor, because AOTF is

ronically tunable spectral bandpass filter. The reflectedntensities at all wavelengths in the range of 440–790lmost simultaneously are measured. The reaction caonitored in real-time, because the scanning speed oOTF is on the order of microseconds (�s). The instrumen

s cheap and its operation is simple. Inputting AOTF Vvoltage range, the SPR spectrum of corresponding vo

ange can be obtained.

ig. 2. The typical AOTF spectral output: A: the applied voltage is 1.50: the applied voltage is 1.505 V.

Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104 101

Fig. 3. Scheme of the evaluated immunoassay procedures: (a) direct immunoassay; (b) sandwich immunoassay; (c) colloidal Au-enhanced immunoassay.Fibronectin antibody ( ); Fibronectin antigen (); Colloidal Au-fibronectin antigen ( ).

carried out for 16 h to organize the processing antibody onthe MPA surface and thus the immunosensor membrane wasstable. Then, PBS buffer was injected into the flow cell towash off non-covalently bound antibody. Prior to the analy-sis, BSA solution was applied to block the unoccupied siteson the gold film.

2.4. Immunoassay procedure

In this paper, fibronectin was determined by the meth-ods of direct immunoassay, sandwich immunoassay andcolloidal Au-enhanced immunoassay using the wavelength-modulation AOTF-SPR biosensor. The assay procedures areshow inFig. 3.

After the fibronectin antibody was immobilized on thebiosensor surface, fibronectin was diluted to different con-centrations with PBS, then different concentration fibronectinwas injected into the flow cell and the response due toantibody–antigen reaction was monitored. The fibronectinwas injected into the flow cell at 18◦C and reacted withfibronectin antibody for 120 min; in the method of sand-wich immunoassay, after the antibody was immobilized onthe biosensor surface, the solution containing fibronectinwas injected into the flow cell. After the resonance volt-age remains almost constant, fibronectin antiserum at 1:10( aket denti-c rentc ll at1 edi thei tedi ctedi tiono

sys-t tingm g lows rans-p nto

the flow cell by a syringe not a pump, so the diffusion rate ofreagent is low. To reduce the mass transport limitation, thereaction times were increased.

2.5. Antigen and colloidal gold conjugates

Colloidal gold was coated with the various proteins bycharge adsorption according to published procedures[14,33].In a typical process, the antigen is diluted with water ata concentration of 50�g/mL and 10 nm diameter colloidalAu solutions is adjusted to pH 8.0 with 0.1 mol/L NaOH.While stirring the colloid vigorously, the 50�g/mL of anti-gen solution is rapidly added so as to cover entirely theparticles present in the solution and thereby stabilize themagainst coagulation by salts. The mixture is then incubatedfor 10 min at room temperature and the solution is furtherbuffered with 0.01 mol/L PBS. The antigen and colloidal goldconjugates can be stored between 2 and 8◦C for several dayswithout loss of activity. Prior to the determination, colloidalAu–fibronectin was diluted to different concentrations withPBS and then was injected into the flow cell.

3. Results and discussion

3

PAs ges ofr ve ofMo shiftw thent imen-t sheda so-nc olt-a withP was

v/v) as secondary antibody was injected for 16 h to mhe immunosensor membrane organized and stable. Ially, after the secondary antibody was immobilized, diffeoncentration fibronectin was injected into the flow ce8◦C for 120 min; in the method of colloidal Au-enhanc

mmunoassay, after the antibody was immobilized onmmunosensor surface, colloidal Au–fibronectin was dilunto different concentrations with PBS and then was injento the flow cell for 60 min. The response due to the reacf antibody with colloidal Au–antigen was monitored.

The mass transport limitation is lies in SPR biosensorems[31]. Some methods have been found for eliminaass transport in SPR biosensor system, include usin

urface densities and fast flow rates to minimize mass tort effects[32]. In this study, all reagents were injected i

.1. Modification of the SPR biosensor surface

By fast voltage scanning of AOTF monitoring the Melf-assembly process on the gold substrate, the chanesonant voltage were measured. The adsorption curPA at the surface of gold film is shown inFig. 4. The shiftf the resonant voltage reached about 95% of its totalithin 7 min. Further increasing the self-assembly time,

he resonant voltage keeps almost constant. This experal result implied that the self-assembly has been finind the monolayer is formed. The maximum shift of reant voltage for the formation of this monolayer is−0.024 V,orresponding to 2.12 nm. The virtual shift of resonant vge is−0.023 V after the biosensor surface was washedBS buffer. Almost no change in the resonant voltage

102 Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104

Fig. 4. The kinetic adsorption curve of MPA on the gold surface. The errorbars represent the standard deviation of the values determined from the threesame assays.

observed when the biosensor surface was washed with PBSbuffer, which suggested that sulfide bond of MPA is easy toform S Au binding and the binding is tight.

The kinetic curve of the reaction between MPA and NHSis shown inFig. 5. The maximum shift of resonant volt-age is−0.160 V. In fact, the shift of the resonance voltageis −0.027 V, corresponding to 2.42 nm, after the biosensorsurface was washed with PBS buffer until the resonant volt-age kept constant. It can be seen that almost 85% of boundNHS molecules were removed after washing the surface withbuffer. The result is due to the non-specific binding from theNHS to the sensor surface and partly due to the refractiveindex of NHS being different from that of buffer.

3.2. Antibody protein immobilization

Fibronectin antiserum was diluted to 1:10 (v/v) with thePBS buffer at pH 7.4 in this assay. Then antiserum solutionwas injected into the flow cell to monitor its assembling on

.

Fig. 6. The kinetic adsorption curve of fibronectin antibody on modifiedbiosensor surface.

the activated immunosensor surface. By fast voltage scanningof AOTF, SPR spectrum of antibody assembling at differenttime can be obtained. The adsorption curve of fibronectinantibody immobilization is shown inFig. 6. As expected,when biosensor surface prepared with MPA is exposed tofibronectin antibody, the response is obvious. The maxi-mum shift of resonance voltage is−0.097 V, correspondingto 8.94 nm. After 16 h, there are only a few changes in theresonant voltage when the biosensor surface was washedwith PBS buffer. This suggested that the fibronectin anti-body had bound tightly on the biosensor surface. Prior to theimmunoassay, non-specific binding sites were blocked withBSA.

3.3. Direct and enhanced immunoassay

The sensitivity of direct immunoassay for fibronectin islow. In order to improve sensitivity a sandwich immunoassayand a colloidal Au-enhanced immunoassay for the detectionof fibronectin were developed.Fig. 7 shows the SPR spec-trum of 5�g/mL fibronectin in three methods. The curveA in Fig. 7 is the SPR spectrum when the biosensor sur-face was washed with PBS buffer until the resonant voltagekept constant after the fibronectin antibody immobilization.The curves B, C and D are the SPR spectrum when the5 ella oas-s ltagei d-w vely.T canb Au-e nb eableb nantv ave-l

Fig. 5. The kinetic curve of the interaction between NHS and MPA

�g/mL fibronectin solution was injected into the flow cnd direct, sandwich and colloidal Au-enhanced immunay was applied, respectively. The shift of resonant vos −0.008,−0.016,−0.031 V in the method of direct, sanich and colloidal Au-enhanced immunoassay, respectihe results show that the sensitivity of the immunoassaye improved by the methods of sandwich and colloidalnhanced assays. It can be seen fromFig. 7that the interactioetween fibronectin antibody and antigen causes a noticroadening of the SPR spectrum and a shift of the resooltage towards lower voltage, that is, the resonant wength moves towards longer wavelength.

Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104 103

Fig. 7. The SPR spectrum of 5�g/mL fibronectin in three methods: (a)direct immunoassay; (b) sandwich immunoassay; (c) colloidal Au-enhancedimmunoassay.

In the method of sandwich immunoassay, after the firstantibody was immobilized on the biosensor surface, the solu-tion containing 50�g/mL fibronectin as sandwich antigenwas injected into the flow cell. The shift of resonant volt-age is−0.038 V in 120 min. Then, secondary antibody offibronectin was injected for 16 h. After the secondary anti-body was immobilized, different concentrations fibronectinwas injected into the flow cell. In the method of colloidal Au-enhanced immunoassay immunoassay, 2.5�g/mL colloidalAu–fibronectin conjugate leads to a−0.023 V shift in reso-

Fig. 8. The relationships between the analyte concentration and the shift ofresonant voltage for three immunoassay methods.

nance voltage. Furthermore, the blank experiment was done.The solution only containing colloidal Au but no antigen,whose colloidal Au concentration is corresponding with thecolloidal Au of 2.5�g/mL colloidal Au–antigen conjugate,was injected into the flow cell and the shift of SPR spectrumresonant voltage was monitored. The resonant voltage is nochange for 60 min. This suggests that the colloidal Au do notbind with antibody.

The relationships between the analyte concentration andthe shift of resonant voltage for three immunoassay methodsare shown inFig. 8. The AOTF-SPR biosensor has a goodresponse to fibronectin in the concentration range 2.5–30,0.5–30 and 0.25–30�g/mL for the method of direct, sand-wich and colloidal Au-enhanced immunoassay, respectively.A significant increase in sensitivity is provided through theuse of protein–Au colloidal conjugates in this AOTF-SPRbiosensing scheme. The results suggest that colloidal Auenhancement immunoassay may be well useful for the detec-tion of biomolecules in the wavelength-modulation AOTF-SPR immunosensor.

Fig. 9. The elution curve of the immmnosensor surface.

104 Y. Tian et al. / Analytica Chimica Acta 551 (2005) 98–104

3.4. Regeneration of biosensor surface

The same immobilized surface can be used for a seriesof determinations. Once a series of determinations are per-formed, the immmnosensor surface can be regenerated bydissociation of the immunocomplex with 0.1 mol/L NaOHfor 5–10 min. The rinsed time must change according to thethick of self-assembly monolayers sensing membrane. Thethicker is the sensing membrane, the longer is the rinsed time.Fig. 9 shows the antibody–antigen complex desorbed fromMPA monolayer. By this treatment, the immmnosensor sur-face can be used repeatedly.

4. Conclusion

In recent years, SPR biosensors, which include angularmodulation, wavelength-modulation and so on, have beenextensively applied to the analysis of biomolecular interac-tions in real-time. In this paper, a wavelength-modulationSPR immunosensor based on AOTF was described. TheAOTF, whose wavelength range is 440–790 nm, was usedas the wavelength selector. The wavelength is modulated byinputting AOTF 0–5 V voltage and one voltage correspondsto one wavelength. Because of the characteristic of rapidscanning of AOTF, the AOTF-SPR system can realize deter-m ver,t sori therS thisp ncedi ands tionA ationrf oas-s emenm ivityo ens-i menti o bea

R

nal.

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[ tt,

[ io-

[[ .R.

Mar-

[ rog.

[[[ Tech-

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ining the biomolecular interactions in real-time. Howehe analytical sensitivity of the AOTF-SPR immunosens limited by the molecular mass of the analyte as oPR devices, as well as the resolution of used AOTF. Inaper, the methods of sandwich and colloidal Au-enha

mmunoassay for detecting fibronectin were reportedhown enhanced sensitivity for the wavelength-modulaOTF-SPR immunosensor. The determined concentr

ange of fibronectin is 2.5–30, 0.5–30 and 0.25–30�g/mLor direct, sandwich and colloidal Au-enhanced immunay, respectively. These results show that these enhancethods are simple and effective for improving the sensitf the immunoassay. The current AOTF-SPR immunos

ng device and the methods of direct and enhancemmunoassay for the detection of fibronectin could alspplied to construct other immnosensors.

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