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B i o s e n s o r s & B i o e l e c t r o n i c s 10 (1995) i-ix [ BIOANALYTICAL HISTORY REPORT

B i o s e n s i n g w i t h s u r f a c e p l a s m o nr e s o n a n c e - - - h o w it a ll s t a r te dBo Liedberg, C laes Ny lander* & Ingemar Lundst r6m

L ab o ra t o ry o f A p p l i ed Ph ys ic s, D ep a r t me n t o f Ph ys ic s an d Meas u rem en t T ech n o l o g y , L i n k 6 p i n g U n i v e r si t y ,S-581 83 Link6ping , Sweden

Abs t r a c t : A sub jec t ive descr ip t ion i s g iven o f how the develo pm ent o f su r facep l a s mo n r e s o n an ce fo r i mmu n o s en s i ng b eg an . T h e m a i n d i f fe r en ces b e t w eenthe in i t ia l exper im ents and a com mercia l ly avai l ab le ins t rumen ta t ion arepo in ted ou t . For the p rac t i ca l use o f su r face o r i en ted methods fo r b iosens ingi t i s no ted tha t the ar rangements a round the op t i ca l sys tem i t se l f , such as thesens ing ch ip o r sample ce l l , a re mos t impor tan t . I t i s concluded tha t thei n s t ru men t a t i o n d ev e l o p ed can b e u s ed n o t o n l y fo r i mmu n o s en s i n g b u t a l sofor " rea l t ime b iospeci f i c in terac t ion analys i s" in genera l . I t i s po in ted ou ttha t the use o f su r face p lasmon resonance fo r de tec t ion i s on ly one poss ib i l i tyand th a t m any new (op t i ca l) meth ods fo r rea l t ime b iospecif i c in terac t ionanalys i s have been and wi l l be developed .

I N T R O D U C T I O NT o o u r k n o w l e d g e t h e u s e o f s u rf a c e p la s m o nr e s o n a n c e f o r b i o s e n s i n g p u r p o s e s w a s f i r s t d e m -o n s t r a t e d i n 1 9 8 3 b y L i e d b e r g e t a l . , a l t h o u g h a tt h is t i m e i t h a d a l r e a d y b e e n u s e d f o r s e v e r a ly e a r s t o s t u d y o r g a n i z e d o r g a n i c m o n o - a n dm u l t i l a y e r s o n m e t a l s u r f a c e s ( P o c k a r d e t a l . ,1 9 7 8 ; S w a l e n e t a l . , 1 9 8 0 ) . A p r a c t i c a l a n dc o m m o n l y u s e d m e t h o d b y w h i c h t o e x c it et h e s u r f a c e p l a s m o n w a s i n i ti a ll y s u g g e s t e d b yK r e t s c h m a n n ( 1 9 71 ) . I n t h is m e t h o d l i g h t f a ll st h r o u g h a g l a s s ( p r i s m ) u n d e r t o t a l r e f l e c t i o nc o n d i ti o n s a n d o n t o a m e t a l f i lm e v a p o r a t e d o n t ot h e g l as s. A t t h e b e g i n n i n g o f t h e 1 9 8 0s o u r g r o u pd e m o n s t r a t e d t h a t s u r f a c e p l a s m o n r e s o n a n c e i nt h e K r e t s c h m a n n c o n f i g u r a t i o n i s w e l l s u i t e d f o r

* Sens i to r AB, Box 76 , S- -581 02 Link6ping , S weden .

b o t h g a s a n d b i o m o l e c u l a r s e n s i n g p u r p o s e s( N y l a n d e r e t a l . , 1 9 8 2 / 8 3 ; L i e d b e r g e t a l . , 1 9 8 3 ) .A s a r e s u lt o f t h e s e i n it ia l o b s e r v a t i o n s P h a r m a c i ao f S w e d e n b e c a m e i n t e r e s t e d i n u si n g s u r f a c ep l a s m o n r e s o n a n c e a s a n i n s t r u m e n t a t i o n t o b ed e v e l o p e d f o r t h e s t u d y o f i n te r a c ti o n s b e t w e e nb i o m o l e c u l e s . A p r o j e c t w a s i n i t i a t e d a t P h a r m a -c i a i n 1 9 8 4 a n d i n 1 9 8 6 a s e p a r a t e c o m p a n y ,P h a r m a c i a B i o s e n s o r , w a s f o r m e d f o r t h e d e v e l -o p m e n t o f n e w b i o s e n s o r te c h n o l o g y . T h e s ed e v e l o p m e n t s r e s u lt e d i n t h e l a u n c h o f B I A c o r ei n 1 99 0 a n d B I A l i t e i n 1 9 9 4. S u c h i n s t r u m e n t a t i o nc a n , f o r e x a m p l e , b e u s e d f o r r e a l t im e b i o s p e c if i ci n t e r a c t i o n a n a l y s is w i t h o u t t h e u s e o f l a b e l l e dm o l e c u l e s . I n t h e p r e s e n t c o m m u n i c a t i o n w ew o u l d l i k e to d e s c r i b e t h e d e v e l o p m e n t o f su r f a c ep l a s m o n r e s o n a n c e f o r b i o s e n s i n g p u r p o s e s b yd i s c u s s i n g w o r k c a r r i e d o u t i n S w e d e n o v e r t h ep a s t d e c a d e o r s o .

0956-5663/95/$09.50 i


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B . L i e d b e r g et al. B i o s e n s o r s & B i o e l e c t r o n i c sHOW IT ALL BEGANAt the physics department in Link6ping Univer-sity we were asked to develop laboratory exercisesfor the undergraduate students. One idea wasto build a simple set-up to demonstrate thephenomenon of surface plasmon resonance. Wehad at that time developed a quartz crystalmicrobalance sensor for anaesthetic gases(halogenated hydrocarbons) based on a siliconeoil as the sensing layer (Kindlund and Lundstr6m,1982/83). As part of this research we sought todiscover whether the refractive index changesthat must occur in the silicone oil on theadsorption of the anaesthetic molecules couldgive rise to appreciable shifts of the surfaceplasmon resonance angle. The study showed thatas a measurable parameter surface plasmonresonance performed quite well compared witha commercial instrumentation based on the quartzcrystal microbalance (Nylander e t a l . 1982/83).At the Labora tory of Applied Physics we usedellipsometry to study the adsorption of organicmolecules on solid surfaces. Furthermore we hadalready used such techniques both for biosensingpurposes (Arwin and Lunds tr6m, 1987; 1988)and for detailed studies of macromolecular inter-actions at surfaces (J6nsson e t a l . , 1982; Elwinge t a l . , 1987). With this background knowledgewe decided to try immunosensing with surfaceplasmon resonance; this resulted in a paper whichwas published in 1983 (Liedberg e t a l . ) . Beforewe describe this initial experiment, a very briefdescription of the physics of surface plasmonresonance will be given.

SURFACE PLASMON RESONANCEA surface plasmon is a charge density waveoccurring at the interface between a metal anda dielectricum. A surface plasmon can be excitedby light as demonstrated in Fig. la. At thesurface plasmon resonance angle the energy andmomentum (along the interface) coincide for theincident photon and the charge density wave.The photon energy is then transferred to thecharge density wave. This phenomenon can beobserved as a sharp dip in the reflected lightintensity (Fig. lb). The dielectric function of themetal should have a (large) negative real part atthe chosen wavelength of the light. Surfaceplasmon resonance (SPR) occurs therefore in the

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A N G LE O F IN C I D E N C E ( deg )F i g . I . ( a ) S c h e m a t i c d r a w i n g o f t h e e x p e r i m e n t a l s e t -u p u s e d i n o u r i n i ti a l e x p e r i m e n t s . T h e l i g h t s o u r c ew a s a H e - N e l a s e r (6 3 2 . 8 n m ) a n d t h e d e t e c t o r ap h o t o d i o d e . ( b ) C a l c u la t e d r e f le c t a n c e c u r v e s f o r t h r e em e t a l s i n a ir . M e t a l t h i c k n e s s e s 5 6 n m ( A g ) ; 4 8 . 5 n m

( A u ) , a n d 8 .5 n m ( A l ) .visible region in so-called free electron-like metalssuch as silver and gold. Furthermore, the thick-ness of the metal film should be a fraction ofthe wavelength.

Outside the metal there exists an evanescentelectric field, a field that decays exponentiallywith distance from the meta l surface with a decaylength of the order of 0.2 to 0.3 of the wavelengthof light. This evanescent field interacts with theclose vicinity of the metal. Changes in the opticalproperties of this region will therefore influencethe (SPR) angle, which is the basis of the use ofSPR for (bio-)sensing purposes.

THE INITIAL IMMUNOSENSINGEXPERIMENTA silver film evaporated onto a microscope slidewas used as the sensing surface, and a goniometer


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Biosensors & B ioelec t ron ics B iosens ing wi th sur face p lasm on resonancearrangement with a photodiode as a light detectorwas used to measure the position of the resonanceangle. An antigen (an immunoglobulin in thiscase) was spontaneously adsorbed on the silversurface. The subsequent binding of an antibody(a-IgG) was detected as shown in Fig. 2. In orderto monitor the SPR-angle shift more directly thechange in photocurrent at a given resonance anglewas detec ted upon injection of the antibodies (Fig.3a). From the (maximum) slope of such curvesa calibration curve was constructed (Fig. 3b).The sample cell used consisted of a channel(approx. 2 ram) formed between the sensingsurface and a second glass slide. With this setupit was possible to determine a-IgG concentrationsdown to about 0.2/xg/ml.

The simplicity of the experimental set-up andthe reasonable sensitivities obtained using ournon-optimized equipment made SPR an interest-ing candidate for the basis of a practical immuno-sensor.

PHARMACIA BIOSENSOR, BIAcore ANDBIAliteAt the beginning of the 1980s Pharmacia becameinterested in the possibilities of biosensor tech-nology and a decision was made to investigatethe possibility of developing methods for thedirect detection (without labels) of biomolecularinteractions. In 1984 a project investigating theuse of surface plasmon resonance for biosensing

purposes was initiated. Researchers wereemployed from both the Laboratory of AppliedPhysics and the Swedish Defense Research Insti-tute in Umeh, where studies of biomolecularinteractions were also undertaken (J6nsson e ta l . , 1985). In 1986 a company called PharmaciaBiosensor was formed to develop, produce andmarke t a product for real time biospecific interac-tion analysis. A large effort was made to providean instrumentation that would be easy to use--a regenerable sensing chip to which biomoleculescould be coupled using known coupling chemistr-ies. Furthermore an efficient liquid handling anda small sample cell were developed to makekinetic analysis possible. The first products werelaunched in 1990; namely the instrument BIAcoreand sensing chips that had a dextran layer ontop of the metal surface as a sensing matrix. Thisdevelopment has been described by J6nsson e ta l . (1991) and J6nsson and Malmqvist (1992).Recently a new instrument, BIAlite, has beenintroduced, which has the same analytical per-formance as BIAcore, but where the samplehandling is manual and not computer-controlledas in a BIAcore.

The performance of the commercial instrumen-tation (for immunosensing) is summarized in Fig.4. The comparison between Fig. 3 and 4 speaksfor itself. The commercial development hasincreased the sensitivity and accuracy of SPRfor biosensing purposes by several orders ofmagnitude. However, as described later, the mostinteresting application may not be to determine

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A i r, /v 4 1 2 4 3

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i I / i6 8 6 9 7 0 71A N G L E O F I N C I D E N C E ( d e g re e s )

Fig. 2 . Expe r imen ta l resu lt s showing the adsorp tion o f a pro te in m olecu le , an an t igen ( IgG) and the subsequen tb ind ing o f i ts an t ibody (an t i - lgG) . The exper imen ts were per fo rm ed on a s i l ver f i lm, 60 nm th ick ( f rom Liedberget al., 1983).

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B. L iedberg et al.

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Fig. 3 . (a) Kinetics of the antibody binding to a layero f spon taneous ly adsorbed an tigens a t d i ff eren t an t ibodyconcentrations me asur ed as the change in reflected l ightat a given angle o f incidence close to the resona nce angle.(b) Ca l ibrat ion curve cons tructed fo r the m ax im umder iva t ive o f the curves in (a ) ( f rom Liedberg et al.,1983).

concentrations but to follow biospecific interac-tions in real time.There are of course many differences betweenthe commercial instrumentation and our initialexperimental set-up. A few of the most importantones are briefly described below (see also Fig.5 ) .iv

Biosensors & BioelectronicsINITIAL EXPERIMENT VERSUSDEVELOPED INSTRUMENTATIONSensing surfacePharmacia Biosensor uses a hydrogel, a carboxy-methylated dextran layer, on the surface ofa gold film as the matrix for biomolecularinteractions. The dilute dextran layer, about100 nm thick in swollen form, utilizes the evan-escent field in an efficient way and provides thesensing surface with carboxyl groups to whichbiomolecules can be coupled using known tech-niques (JOnsson e t a l . , 1991). Since the dextranlayer contains 97 to 98% water, interactionbetween the covalently bound molecule and theother molecule in an interaction pair will takeplace in a more native surrounding than interac-tions that would otherwise take place directly onthe metal surface. Furthermore, it is possible toprovide an extended matrix, even if it is dilute,with more coupling sites per unit area than abare surface and thereby increase the sensitivityof the method (L6f~s e t a l . , 1991; Liedberg e ta l . , 1993). The matrix is also regenerab le, forexample, through the use of a buffer of low pHwhich desorbs all but the covalently boundbiomolecules from the matrix. The dextran mol-ecules are attached to the gold surface by alinker layer consisting of alkane thiols. This useof self assembled monolayers appears to be oneof the first commercial applications of suchlayers spontaneously formed on gold (LOf~s andJohansson, 1990).Sample cell and microfluidic systemA very important development concerns theliquid handling and sample delivery to the sensingsurface. By using micromachining methods anintegrated microfluidic cartridge was fabricated,which contains sample and buffer loops, andwhich together with the sensing chip automatical lyforms a sample cell 50 ~m high and 0.5 mm widewith a volume of 6 0 n l . This enables very efficientand accurate delivery of the analyte to the sensingsurface making (non-diffusion limi ted) kineticstudies possible (Sj61ander and Urbaniczky,1991). The microfluidic cartridge is made ofsilicone rubber. The valves controlling the liquidflow are operated by a small air pressure.


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Biosensors & Bioelectronics Biosensing with surface plasmon resonance~ U l I I II I I II I I I II I I II I I U I I l l lU I I I I l U l I l U l I I I I I I I I I I II I I I l U l I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I l U l I l U l I ~__-=- ~ , -- oN _=_= ~ .. ~ --= \ r - . ~ r ~ = 7--= X I : ~ O / a -=-- " ~ t~ ~ _ \ _ ~ - 0 0 / , -

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