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Physica B 394 (2007) 297–300

www.elsevier.com/locate/physb

Coupled plasmon effects for the enhancementof fluorescent immunoassays

Anne Barnetta,c,�, Evgenia G. Matveevaa, Ignacy Gryczynskia,b,Zygmunt Gryczynskia, Ewa M. Goldysc

aDepartment of Molecular Biology and Immunology, Health Science Center, University of North Texas, Fort Worth, TX 76107, USAbDepartment of Cell Biology and Genetics, Health Science Center, University of North Texas, Fort Worth, TX 76107, USA

cDepartment of Physics, Division of Information and Communication Sciences, Macquarie University, North Ryde, NSW 2109, Australia

Abstract

We present a study of fluoroimmunoassays using Rhodamine-RedX labelled antibodies enhanced by nanoparticle silver island films

(SIFs) in the presence of thin metallic layers. Our results show enhancement of the immunoassay signal by up to 10-fold for SIFs on glass

and up to 50-fold enhancement for SIFs supported by a metal surface. An explanation for these effects is proposed by considering

plasmonic coupling within the system and modification of the fluorophore spontaneous emission rate.

r 2007 Elsevier B.V. All rights reserved.

PACS: 73.20.Mf; 87.64.Ni

Keywords: Plasmonic coupling; Silver island films; Myoglobin immunoassay

1. Introduction

It is well known that the presence of a metal surfacemodifies the properties of an oscillating dipole (such as afluorophore) present in the vicinity [1–4]. A fluorophorewithin distances around 50–200 A from the metal surface isable to produce an increased radiative output over that of afluorophore in free space. This effect has been attributed tothe presence of a locally enhanced electric field fromplasmonic oscillations in the nearby metal [5,7] as well as amodification in the fluorescence lifetime caused by dis-tance-dependent changes in the radiative decay rates (orequivalently the fluorescence lifetime) caused by modifica-tion of the photonic mode density due to the presence ofthe metal [1].

This work aims to investigate a system which utilises theincrease in fluorescence intensity for a fluorophore in thepresence of a metallic nanostructure as well as the greatly

e front matter r 2007 Elsevier B.V. All rights reserved.

ysb.2006.12.085

ng author. Department of Physics, Division of Informa-

unication Sciences, Macquarie University, North Ryde,

stralia. Tel.: +61 2 9850 7748; fax: +61 2 9850 8115.

ss: anneb@ics.mq.edu.au (A. Barnett).

enhanced radiative scattering efficiency provided by anunderlying metal layer. A novel bioassay platform forenhanced fluoroimmunoassays has been developed basedon these concepts and applied to an immunoassay for animportant cardiac disease marker, myoglobin.

2. Immunoassay description and methods

An immunoassay is a method by which highly specificchemical binding events between two types of proteinmolecules (an antigen and an antibody) are used to detectthe presence and/or quantify the concentration of a proteinpresent in the examined sample. In addition to the antigenand antibody proteins that undergo specific binding, animmunoassay requires a label that reports a positivebinding event. In this work fluorescent molecules act asreporters.The two immunoassays carried out in this study are a

model immunoassay based on the binding reaction betweena rabbit IgG antigen and antibody and an immunoassayfor myoglobin. A model immunoassay uses a well under-stood antibody–antigen combination so that the effect of

ARTICLE IN PRESSA. Barnett et al. / Physica B 394 (2007) 297–300298

other experimental steps in the assay can be studiedseparately from the well controlled model immunoreaction.In this work the aim was to compare the fluorescenceoutput from surface-immobilised myoglobin immunoas-says performed on various supporting substrates. Plainglass substrates were compared to substrates supportingthin metal layers and/or silver nanoparticles. The modelimmunoassay was performed first to study the validityand potential response of an immunoassay performed ondifferent substrates.

A detailed procedure for the model immunoassay hasbeen outlined previously [8]. To summarise, rabbit IgGantigen was non-covalently immobilised on the substrate ofinterest. Rhodamine-RedX fluorescently labelled anti-rabbit IgG conjugate was added to the sample and controlslides. Following the final incubation period, excesslabelled antibody was removed and the surface boundantigen–antibody level measured by excitation with a532 nm laser diode. The occurrence of a successful bindingevent was reported by fluorescence emission from thefluorescent label attached to the antibody. The fluorescencesignal from the surface is measured via a fibre optic fromabove (see Fig. 1).

The more complex ‘sandwich’ immunoassay was used tostudy the specific binding between the myoglobin antigenand antibody. An extra antibody binding step was includedin this approach (see Fig. 1). This produced a highlysensitive assay suitable for a low concentration of antigen.The main steps of this immunoassay format are describedin a previous publication [9]. In the present work, themyoglobin antigen was bound between two myoglobinantibodies, a surface-immobilised (non-covalently bound)‘capture’ antimyoglobin antibody and a Rhodamine-RedXfluorescently labelled ‘reporter’ myoglobin antibody asillustrated in Fig. 1. Fluorescence measurements of thelevel of bound labelled antibody were carried out at thecompletion of the immunoassay in a similar manner to themodel immunoassay.

For both the model and myoglobin immunoassays,control immunoassays were performed to account forsignal due to binding events not directly between theantigen and antibody of interest [8,9]. For the model

antigen

labelled antibody

antibody

fibre

excitation

support

sl ide

MODEL IMMUNOASSAY

MYOGLOBIN IMMUNOASSAY

Fig. 1. Experimental setup for immunoassay measurement. Schematic

representation of model immunoassay and myoglobin immunoassay

labelled binding event.

immunoassay, a control was achieved by replacing therabbit IgG antigen with an IgG antigen from a differentsource (goat), and for the myoglobin immunoassay zeroconcentration of myoglobin antigen was employed toachieve a control. From these control immunoassays,non-specific binding events between unrelated moleculesresulted in labelled antibody remaining bound to thesurface or another molecule rather than bound to theantigen of interest. This signal was measured and removedfrom the sample results, so that only signal from the strongspecific antigen–antibody binding reaction was retained.Immunoassays were carried out on four different

substrates (see Fig. 2). A plain glass microscope slide wasused as a reference substrate and compared to substratesconsisting of a thin metal layer (Au or Ag), silver islandfilms (SIFs) formed on plain glass or on thin metal layers.The thin metal layer (48 nm thick Au or 50 nm thick Ag)was vapour deposited onto glass microscope slides by theEMF Corporation [10]. A thin silica layer (10 nm on gold,5 nm on silver) was added to protect the metal and act as aspacer between the metal and SIFs. Thin metal layers of Auor Ag are capable of supporting surface plasmon reso-nances at visible wavelengths, with thicknesses around50 nm producing optimal coupling under total internalreflection conditions [6]. Although coupling through theprism in this manner is not performed in this case, layerthicknesses appropriate for this were employed in order toprovide a consistent experimental setup for comparisonwith further work in this area. An experimental approachsimilar to that employed here has been demonstrated usinga 200 nm Ag film combined with SIFs [4]. The SIF surfacewas formed by chemical reduction of silver nitrate [8,9,11].One half of the surface of each slide was modified bydepositing SIF by chemical reduction of silver nitrate usingDðþÞglucose, while the other half was left unmodified andused as a reference. Excitation of the labelled Rhodamine-RedX fluorophore was via a 532 nm solid state laser,illuminating the surface bound immunoassay from aboveat an angle around 45� to the vertical (see Fig. 1). Thepeak in the emission spectrum was around 590 nm forthe Rhodamine-RedX fluorophores employed. Multiplemeasurements of each immunoassay were made for all sub-strates considered in order to build up a statistically signifi-cant result level. The myoglobin antigen concentration

SIF formed by

chemical reduction

Silica layer,

5-10nm

not to scale

Thin metal layer

(48nm Au or 50nm Ag)

Glass microscope slide

Fig. 2. Summary of four immunoassay supporting substrates considered.

Further details provided in main text.

ARTICLE IN PRESS

0

60

70

SIF on glass

En

han

cem

en

t (r

el to

gla

ss)

Model Immunoassay Myoglobin Immunoassay

≈ 10x enhancement

for SIF on glass

≈ 50x enhancement

for SIF on metal50

40

30

20

10

Glass Silver Gold SIF on silver SIF on gold

Supporting substrate

Fig. 3. Peak fluorescence output enhancement relative to glass from myoglobin and model immunoassays performed on various nanometallic substrates.

Table 1

Rhodamine-RedX fluorescence output signal summary

Substrate Model immunoassay Myoglobin immunoassay

Peak signal SD (%) Peak signal SD (%)

Glass 1.0 18.8 1.0 7.6

Silver 1.7 11.2 2.0 21.8

Gold 1.7 4.4 1.8 11.7

SIF on glass 9.6 9.6 7.8 12.1

SIF on silver 49.7 11.2 50.5 4.3

SIF on gold 55.7 9.3 46.6 15.7

Excitation at 532 nm, peak signal at 590 nm.

A. Barnett et al. / Physica B 394 (2007) 297–300 299

used was 1000 ng/mL, corresponding to around 10 timesgreater than an accepted clinically relevant cut-off level ataround 100 ng/mL [9].

For both immunoassays a known concentration andvolume of labelled protein solution was used. As a result,measurements made of the fluorescence signal from theexcess labelled supernatant removed from the completedimmunoassay allowed for scaling between different sam-ples to take into account varying surface binding levels. Inthis manner immunoassays performed on different sub-strates with varying available surface binding areas may bedirectly compared.

3. Experimental results

Fluorescence emission spectrum measurements weretaken from different spots in each immunoassay reactionwell, with a minimum of six measurements taken from twowells for each immunoassay-substrate combination. Thepeak fluorescence emission at 590 nm was determined foreach measurement and averaged to give a final fluorescencesignal. This approach takes into account natural variationin surface binding levels across the reaction well. Thestandard deviation of the measurements from the calcu-lated mean value was determined to give a measure of thelevel of spread (uncertainty) in the results. Error bars inFig. 3 correspond to one standard deviation calculatedfrom the raw data measurements of peak fluorescence at590 nm. A summary of the experimental results is presentedin Table 1. The peak fluorescence emission signal for animmunoassay performed on a given substrate was com-pared to that measured from the same immunoassayperformed on glass. By setting the immunoassay signal onglass to unity, the signal from other substrates may benormalised to the glass signal level, providing a measure ofthe signal change from these substrates compared to thatfrom bare glass. The results for both, model and myoglobin

immunoassays, are represented graphically in Fig. 3. Thisshows clear enhancement of the fluorescence signal in allcases over that detected from the immunoassay performedon glass. When the supporting substrate consisted of SIFsformed on glass, the fluorescence signal from the immu-noassay was enhanced by around 10 times over the signalproduced from the same immunoassay performed on aplain glass substrate. For a substrate of silver islandsformed on a thin metal layer of gold or silver, thefluorescence emission was enhanced by a further factor offive resulting in around 50 times enhancement compared tothat from plain glass. As demonstrated in Fig. 3 theseenhancement results are within experimental error, repre-sented as one standard deviation from the calculated meanfluorescence emission level derived from measurementsfrom different spots of a number of reaction wells.

4. Discussion

The immunoassay results have shown modification ofthe fluorescence output signal from fluoroimmunoassaysperformed on different substrates. We interpret these

ARTICLE IN PRESSA. Barnett et al. / Physica B 394 (2007) 297–300300

observations by considering enhanced radiative propertiesof the system in combination with quantum yield increasesof plasmon mediated fluorescence.

It is well known that atoms surrounded by media otherthan free space, such as mirrors, [12] will experience achange in their radiative properties [13,14]. The presence ofthe media will modify the decay rates of the atoms withboth an increase or decrease in the spontaneous emissionrate possible depending upon the geometry [12,15,16].A fluorophore present within the enhanced field regionassociated with a plasmon supporting structure mayundergo an increase in spontaneous emission rate andassociated quantum efficiency as well as experiencing ashortening of fluorescence lifetime [1,7].

Two of the supporting substrates investigated containsmall metallic nanostructures in the form of SIFs, capableof supporting localised surface plasmon dipole oscillations[7]. When deposited on a glass substrate, the ensemble ofoscillating dipoles (localised surface plasmons) on the silverislands will experience a weak dipole–dipole couplingmediated by the distance between the islands. The additionof a thin metal layer to the supporting substrate provides asurface plasmon polariton mediated coupling mechanismbetween the localised surface plasmon dipoles. Thisstrengthens the dipole–dipole interaction and provides adramatic enhancement of the radiative scattering efficiencyof the system [4,17,18]. Similarly enhanced coupling hasbeen achieved through waveguide modes for metallicnanostructures deposited on appropriate waveguide sup-porting structures [4].

The combination of these two effects, an increasedfluorescence output due to high localised electric fieldintensities as well as a highly efficient radiative system, hasproduced the enhancement of fluorescence intensity fromthe fluoroimmunoassays performed. Fluoroimmunoassaysperformed on a weakly coupled SIF on glass systemproduced an enhanced output of around 10 times that of aplain glass surface, while immunoassays performed on thestrongly coupled system of silver islands on a thin metallayer produced a further fivefold increase in signal outputdue to the increase in radiative scattering from the system.

The results presented here suggest that fluorescence-enhancing substrates can be effectively optimised byimproving the electromagnetic coupling between the nanos-tructures on the substrate surface, for example by the use ofa continuous underlying metal layer. In general, enhance-ment of the local electric field may be achieved through theuse of small nanostructures (with small radiative scatteringefficiency), whilst large scattering efficiency is achievedthrough use of large nanostructures (which do not amplifythe field as well as small structures). We have presented away of combining two key contributions to fluorescenceenhancement, requiring mutually exclusive conditions.

5. Conclusion

In this work we presented results that show the highpotential of a metal mirror surface modified with silverisland films (SIF) for developing a universal platform forany surface assays by detection of the enhanced fluores-cence signal. The initial results for myoglobin immunoas-say and for model immunoassay show that using a thinmetal layer in combination with SIF gives approximately50-fold enhancement. For the protein concentrations usedthis is equivalent to a myoglobin concentration 5 timeslower than a generally accepted clinical cut-off level [9].The unique combination of locally enhanced electric fieldsfrom localised surface plasmons supporting nanostructuresand a highly efficient radiative system resulting fromsurface plasmon mediated enhanced dipole–dipole cou-pling has produced the highly enhanced fluorescenceoutput.

Acknowledgements

This work was supported by NIH (NCI CA 114460,NIBIB EB-1690), BITC, and Philip Morris USA, Inc.Results were partially reported at ACS meeting (Atlanta,GA, USA, March 2006).

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