BioSystems 98 (2009) 5155

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BioSystems

journa l homepage: www.e lsev ier .com/ l

Variabl ce

Nyeon-S o KChang-Sua Department o t, Wesb School of Elec 01, Soc Department o th Kor

a r t i c l

Article history:Received 12 MReceived in reAccepted 17 M

Keywords:Variable waveSurface plasmSalmonellaPenicillin

riabla ma

e was. Oururedcells

penicillinexpean o

As a result, the resonant wavelength shifted about 0.26 nm/min owing to the salmonellaantibodyantigen reaction. Thus, we could detect the change in wavelength (0.8 nm/min) through theinteraction of penicillin and penicillinase for 15 min using variable wavelength SPR sensor.

2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduc

The SPRbiochemistrand to mon2004). Thelight weighapplied in c

Considerrecent yearsincreasing.is actively pas the methods of detecattenuatedhigh-refracKopelman,methods haadvantagesdifculties

CorresponE-mail add

0303-2647/$ doi:10.1016/j.btion

has become widely used in the elds of chemistry andy to characterize a biological surface (Hoa et al., 2007)itor binding events (Mao and Brody, 2007; Rella et al.,SPR sensor has many merits such as high sensitivity,t, low cost, and linear characteristics. Thus, it has beenhemical sensors and biosensors.ing the research and development in bioengineering in, interest in the measurement of various biomaterials is

In addition, research on overcoming the detecting limitrogressing and improving measurement systems as wellods for detecting measurement technologies. The meth-ting biomaterials using optics with high sensitivity aretotal reection (ATR) (Soldatkin et al., 2003) using a

tion index prism, a ber-optic optode (Rosenzweig and1996) and thin-lm optical waveguide devices. Theseve a relatively high accuracy, but they also have dis-

, like as requiring pretreatment, long analysis time, andin manufacturing processes and applications.

ding author. Tel.: +82 53 950 6829; fax: +82 53 950 7932.ress: swkang@knu.ac.kr (S.-W. Kang).

Currently, sensors using the SPR are often used inantigenantibody reactions (Tang et al., 2007; Ayela et al., 2007),protein and DNA detection (Yang et al., 2007). Most of the sensorsusing the SPR phenomenon are Kretschmann-type (Ritchie, 1957;Eum et al., 2003) SPR sensors, and in some cases, a few researchersuse an SPR sensor with optical waveguides (Dostalek et al., 2001)and optical bers.

In this study, we fabricated a variable wavelength SPR sensorusing a white light source (4001800 nm) and multimode opticalber, and observed the movement of resonant wavelength using anoptical spectrum analyzer (OSA). The fabricated SPR sensor, whichhas a variable wavelength, has a wider sensing area than SPR sen-sors using monochromatic light source. It could measure the shiftin resonant wavelength for a smaller change in refractive index ofsample than that of a Kretschmann-type SPR which has been nor-mally used until now. In addition, we conrmed the feasibility of abiosensor by measuring the movement of resonant wavelength onthe basis of the antibodyantigen reaction of salmonella and theinteraction between penicillinase and penicillin.

2. Theory

As has been known for several decades, the SPR phenomenonis a surface electromagnetic wave that propagates along the inter-face between a metal and a dielectric material. A model predicting

see front matter 2009 Elsevier Ireland Ltd. All rights reserved.iosystems.2009.05.008e wavelength surface plasmon resonan

ik Eum a, Do-Eok Kim b, Se-Hyuk Yeom b, Byoung-Hb Park c, Shin-Won Kang b,

f Agriculture and Biological Engineering, Purdue University, 225 South University Streetrical Engineering and Computer Science, Kyungpook National University, Daegu 702-7f Sensor and Display Engineering, Kyungpook National University, Daegu 702-701, Sou

e i n f o

arch 2009vised form 8 May 2009ay 2009

lengthon resonance

a b s t r a c t

In this study, we fabricated a novel vadetects resonance conditions such asscopic refractive index. Such a changand a penicillinasepenicillin reactionantibody on the sensor chip. We measreaction for 30 min by injecting 5 107ple chamber. Also, after immobilizingwavelength during the reaction. Penicshift of resonant wavelength for eachoptical ber, a part of sensor chip andocate /b iosystems

(SPR) in biosensing

ang b, Kyu-Jin Kim b,

t Lafayette, IN 47907, USAuth Koreaea

e wavelength surface plasmon resonance (SPR) sensor, whichximum attenuation wavelength, measuring change of micro-measured to detect a salmonella antigenantibody reaction

experiments were performed after immobilizing a salmonellathe shift in resonant wavelength during the antigenantibody/ml concentration of salmonella antigen solution into the sam-illinase on the sensor chip, we measured the shift in resonant

solution at 10 mM was injected into the sample chamber. Theriment was measured using a white light source, multimodeptical spectrum analyzer.

52 N.-S. Eum et al. / BioSystems 98 (2009) 5155

resonance conditions can be obtained using the plane-wave Fres-nel reection equations for a structure containing multiple planarlayers. Surface plasmons (SPs) are excited by incident laser beams,causing resonance with evanescent waves at a certain resonanceangle or rescent waveswith a peneNelson, 200(ATR) methof surface p

kSPW = k0

where k0 isspace (k0 =direct const

The basrefraction in

Thus, reconstant, inin the case oat a xed incthat of a surphase-matcunder this rwave is exp

kx = k0npsiand the refrelation to t

np = n() =Also, in

resonant phthe same cothe wave vexpressed a

kSPW k0

Here, the pindicated in

= () = If kSPW a

of the incideFinally, a

produces adecrease (Jospecic mativity of theThus, resontor. This shi

3. Experimen

3.1. Congura

The systemconditions sucIn this systemlength band. Tn = 1.515), throthe TM mode.(n = 1.5151.51prism (BK7, n =

The samplprism was tot

Fig. 1. Schematic diagram of variable wavelength SPR sensor.

s into the OSA (AQ-4303B, Ando) through the lens and optical ber (3 M Co.).e above-mentioned process, the shift in resonant wavelength was measured., we are preparing a new potable wavelength variable SPR system, capa-time measurement and analysis, using small Halogen lamp and spectrumbased on above system.

sor Ch

used arismmon otor.etermthe abs. The2O5 wgth baness oactionr of 10ttering.

nufact

immosolustirr

hip inmmobp inylamin

Fig. 2. Photograph of fabricated SPR sensor.onance wavelength. The SPs are resonated with evanes-and decay exponentially away from the phase boundarytration depth on the order of 200 nm (Brockman and0). This is the so-called attenuated total reectance

od proposed by Kretschmann. The propagation constantlasmon waves (SPWs) can be expressed as

mn2sm + n2s

(1)

the propagation constant of incident light in the free2/0, 0 being the free space wavelength), m is theant of gold, and ns is the refractive index of the solution.ic principle of the SPR sensor is due to the complex

dex of a thin metal lm.sonance occurs according to the changes in dielectriccidence angle, and incident wavelength. In particular,f the variable wavelength SPR sensor, resonance occursidence angle when the wave vector of incident light andface plasmon become equal, that is, while satisfying thehing condition kev =ksp. After xing the incidence angleesonance condition, the wave vector kx of an incidentressed

n (2)

raction index of the prism np is expressed as Eq. (3) inhe wavelength component

(n() jn()) (3)the case of the variable wavelength SPR sensor, theenomenon occurs only in TM-polarized light. Undernditions using an incidence variable angle SPR sensor,

ector kSPW on the surfaces of the metal and sample iss

metalsamplemetal + sample

(4)

ermittivity , a wavelength-dependent component, isEq. (5) in relation to the refraction index

() j() = n()2 (5)nd kx are under the same conditions, the optical energynt wave is converted to that of a surface plasmon wave.specic wavelength meeting the condition kSPW =kx

resonance, and the intensity of incident light rapidlyhnston and Mar, 1999). Under this condition, when a

terial is injected into the chamber, the complex permit-thin metal lm is changed by the injected material.

ance wavelength was shifted by the change in SPW vec-ft in resonant wavelength is analyzed using the OSA.

tal

tion of Sensor System

of this variable wavelength SPR sensor, which detects resonanceh as a maximum attenuation wavelength, is shown in Figs. 1 and 2., we used a white light source (AQ-6315A, Ando) with a wide wave-he light coming out from the light source focused into the prism (BK7,ugh a lens and a polarizer (Suruga Seiki Co.), and was polarized inThe sensor chip was inserted under the prism. We used matching oil7, Merck) to reduce the difference in the refractive index between the

1.51) and the sensor chip.e chamber was manufactured using Teon. The light coming into theally reected at the thin metal lm of the sensor chip. The reected

light goeUsing th

Alsoble realanalyzer

3.2. Sen

Weto the pthe plasevapora

To dtured byapparatuWhen Tawavelenthe thickhigh refrRF poweand spuin Fig. 3

3.3. Ma

ForantibodyThen wesensor c

To ithe chi(dimethip

cover glass (n = 1.522, Matsunami Co.) with a similar refractive indexas a substrate. A thin gold layer, which is easy to use for excitingn the substrate and chemically safe, was evaporated using a vacuum

ine the change of the detecting band on the gold sensor chip manufac-ove-mentioned method, we evaporated Ta2O5 using an RF sputteringthickness of the evaporated Ta2O5 was estimated to be about 300 .

as evaporated on the sensor chip, it was possible to choose a resonantnd in the variable wavelength SPR sensor system. Also, by adjustingf the Ta2O5 sensing layer, it was possible to measure the sample withindex. The following evaporation conditions of Ta2O5 were used: an

0 W, a working pressure of 70 mTorr, a substrate temperature of 100 Ctime of 30 min. The shape of the manufactured sensor chip is shown

ure of Sample and Sensing Layer

bilizing the salmonella antibody, we prepared 100 ml of salmonellation at 50g/ml using 1 mg/ml goat anti-Salmonella solution (KPL).ed it at room temperature for 12 h while soaking the manufacturedthis solution.ilize penicillinase on the manufactured sensor chip, we immersed

0.3 M GAmethanol solution for 3 h including 0.4 M 1-[3-o)propyl]-3-ethylcarbodiimide hydrochloride (EDC) and 0.1 M N-

N.-S. Eum et al. / BioSystems 98 (2009) 5155 53

Fig. 3. Schematic diagram of Au/Ta2O5 sensor chip.

hydroxysuccinBuffered Salining layer by puCleaning waslized penicillinfrom Bacillus c

The ow c2001) is show

Fig

6. Resonance characteristics of salmonella antibodyantigen reaction.

asurement and Results

sonadyA

valupresella,Fig.

4. Me

4.1. ReAntibo

To esystemsalmonFig. 4. Flow chat of penicillinase immobilization.

imide (NHS). After cleaning the reacted sensor chip with Phosphatee (PBS) solution, we immobilized penicillinase on the Au/Ta2O5 sens-tting the device in 0.4 M EDC + 0.1 M NHS + PBS (20) solution for 2 h.

performed more than three times with PBS, and we nally immobi-ase on the chip through a reaction in the penicillinase (-lactamaseereus, 10000 units/mg, Aldrich) solution.

hart of penicillinase immobilization (Kim et al., 2005; Barie and Rapp,n in Fig. 4, and its schematic diagram is the same as that in Fig. 5.

. 5. Schematic diagrams of penicillinase immobilization.

the salmon50g/ml, wduring theantigen sol30 min. Figwhich is mto the samp

As in Fig10 min withing the antiand the absalmonella

From thebody immochamber prfound thatantigenan

Fig. 7. Resonce Characteristics Produced by Salmonellantigen Reaction

ate the possible application as a biosensor to the sensorented in this paper, we measured SPR phenomena usingwhich are food-poisoning bacteria. After immobilizing

ella antibody on the sensor chip using concentration ofe measured the amount of shift in resonant wavelength

antigenantibody reaction by injecting the salmonellaution at 5 107 cells/ml to the sample chamber for. 6 shows the amount of resonant wavelength shift,easured for 30 min after injecting the antigen solutionle chamber.. 7, the resonant wavelength moved linearly for aboutthe salmonella antibodyantigen reaction after inject-

gen solution. We observed weak binding for 1020 minsence of binding 20 min after the injection of theantigen solution.se results, we found that the reaction between the anti-

bilized on the sensor chip and the antigen injected to theogresses actively for 10 min with the injection. We alsothe shift in resonant wavelength with the salmonellatibody reaction is about 0.26 nm/min.nance wavelength shift by salmonella antibodyantigen reaction.

54 N.-S. Eum et al. / BioSystems 98 (2009) 5155

4.2. Resonance Characteristics Produced by Interaction BetweenPenicillinase and Penicillin

After immobilizing penicillinase on the Au/Ta2O5 sensor chip,we measured the movement of resonant wavelength by injecting10 mM penicillin solution to the sample chamber. Such measure-ment was performed similarly to that in the case of salmonella.

As a result, we observed that the resonant wavelength increaseswith time, as shown in Figs. 8 and 9. We also observed a changein the concentration of solution due to the reaction between thepenicillinase immobilized on the sensor chip and the injected peni-cillin.

We could make an analogy since there was a rapid reactionbetween the penicillinase and the penicillin for 15 min and therewere no other reactions 15 min after the sample injection.

Also, the shift in resonant wavelength due to the penicillinaseand penicillin reaction was considered to occur at about 0.8 min1

until the reaction was terminated.Due to the evaporation of Ta2O5 on the sensor chip, the reso-

nant phenomenon shifted by about 100 nm, compared with thatof a non-evaporated Ta2O5 thin lm: thus, such a phenomenonoccurred at 796 nm. On the basis of this phenomenon, it isexpected that the sensitivity of sensor systems can be increasedand that the samples with high refractive index can be ana-lyzed.

Fig. 9. R

5. Conclusion

Most incident light of variable angle SPR sensors using amonochromatic light source have a narrow measurement range anddifculties in measuring samples at low concentrations.

In this sand evaluattages of varthe shift inOSA.

From thpenicillinp0.26 min1

ment of resshowed a 0

These reis sufcientindex, detematerials in

For portbased on thlight sourceber havinglength varianeeding con

Acknowled

This woGrant fund2007-357-D

This woFoundation(No. R11-20

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gments

rk was supported by the Korea Research Foundationed by the Korean Government (MOEHRD) (No. KRF-00093).

rk was supported by Korea Science and Engineering(KOSEF) grant funded by the Korea government (MEST)08-105-03004-0).

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Nyeon-Sik Eum received his Ph.D. in Sensor Engineering from Kyungpook NationalUniversity, South Korea in 2004. He is currently Postdoctoral at the Bindleybioscuence center, Purdue University, U.S.A. Current research interests include nano-SPR, bio-sensor and micro uidics.

Shin-Won Kang received his Ph.D. in Biomedical Science from University of Keio,Japan in 1993. He is currently Professor at the School of Electrical Engineering andComputer Science, Kyungpook National University, South Korea. His research inter-ests are opto-electronic functional device, OLED, bio-sensor and nano device.

Variable wavelength surface plasmon resonance (SPR) in biosensingIntroductionTheoryExperimentalConfiguration of Sensor SystemSensor ChipManufacture of Sample and Sensing Layer

Measurement and ResultsResonance Characteristics Produced by Salmonella Antibody-Antigen ReactionResonance Characteristics Produced by Interaction Between Penicillinase and Penicillin

ConclusionAcknowledgmentsReferences