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R E V IE W

Tanja Steinkamp Uwe Karst

Detection strategies for bioassays based on luminescent

lanthanide complexes and signal amplification

Received: 19 April 2004 / Accepted: 12 May 2004 / Published online: 5 August 2004

Springer-Verlag 2004

Abstract Two attractive detection strategies for bioas-says are reviewed in this article. Both approaches use thehighly sensitive time-resolved luminescence detection oflanthanide complexes in combination with a signal

amplification scheme. While enzyme-amplified lantha-nide luminescence (EALL) has been an establishedtechnique for more than a decade, nanoparticles dopedwith luminescent lanthanide complexes have beenintroduced very recently. In this paper, the basic prop-erties and major applications of both techniques arepresented, and their future perspectives are discussedcritically.

Keywords Lanthanides Luminescence Bioassays Amplification

Introduction

Enzyme assays and immunoassays are commonly usedtools in clinical chemistry and are applied to the analysisof a large number of important analytes [1, 2]. They arecharacterized by high selectivity and sensitivity.Improvements in the field of bioassays can be achievedeither by optimizing the biochemical processes involvedor by developing or improving detection schemes. Time-resolved lanthanide luminescence detection has beenestablished as a powerful approach in recent years, andhas been summarized in several reviews [3, 4]. This

review focuses on a particular aspect in the field of time-resolved lanthanide luminescence detection: the combi-nation with an amplification step yielding a gain inselectivity and sensitivity.

The four lanthanide ions Tb(III), Eu(III), Sm(III)and Dy(III) exhibit characteristic emission spectra withnarrow line-shaped emission bands in the visible rangeof the electromagnetic spectrum. However, it is hard to

evoke this emission because of the low molar absorp-tivity of the naked metal ions [5]. To circumvent thisproblem, the lanthanide ions are complexed to organicligands possessing energy levels close to those of themetal ions. The principle of the energy transfer is de-picted in Fig. 1. In these complexes, it is possible toexcite the ligand by irradiation of light at its character-istic excitation wavelength. The absorbed energy is thentransferred from the ligand to the central lanthanide ion,which subsequently emits light at its characteristicemission wavelengths [5]. Figure 2 shows the emissionspectra of Tb(III) and Eu(III) complexes, respectively.The energy transfer, which has a low probability by the

laws of quantum mechanics, accounts for the two greatadvantages of lanthanide luminescence: first, the ex-tremely large Stokes shift (often exceeding 200 nm) andsecond, the long-lived luminescence. Typical decay timesof the lanthanide complexes are in the high microsecondor the low millisecond range, allowing time-resolvedmeasurements with comparably simple and inexpensiveinstrumentation. The principle of time-resolved mea-surements is explained in Fig. 3. After excitation with alight pulse, mostly from a xenon lamp or a laser, a delaytime is used to allow the (almost) complete decay ofscattered excitation light and of background fluores-cence of organic molecules, typically exhibiting lifetimes

in the nanosecond range. Afterwards, the long-livedfluorescence of the lanthanide complexes is recordedselectively. For these reasons, time-resolved fluorescencemeasurements of lanthanide chelates are frequentlymore sensitive and more selective than conventionalfluorescence detection methods.

Based on these properties, lanthanide luminescencehas evolved to become a highly sensitive and selectivedetection method. Lanthanide chelates have becomepopular labels for immunoassays [6, 7], and attractivedetection schemes have been developed and commer-

T. Steinkamp U. Karst (&)Department of Chemical Analysis and MESA Institutefor Nanotechnology, University of Twente,P.O. Box 217, 7500 AE Enschede, The NetherlandsE-mail: u.karst@utwente.nlFax: +31-53-4894645

Anal Bioanal Chem (2004) 380: 2430DOI 10.1007/s00216-004-2682-2

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cialized, including the DELFIA [8] and the FIAgen [9]techniques. However, a further improvement in thesensitivities of these methods would allow the detectionof even lower analyte concentrations. Therefore, thecombination of amplification strategies with lanthanideluminescence is a highly promising approach.

In recent years, two attractive techniques for thecombination of lanthanide luminescence measurementswith a coupled amplification step have become available.The first comprises the enzyme-catalyzed formation of aligand, which is, in contrast to the substrate, able totransfer energy to a lanthanide cation. This strategy

combines the enzymatic amplification of establishedenzyme-linked immunosorbent assays (ELISA) with thesensitivity of lanthanide luminescence techniques, and

is known as enzyme-amplified lanthanide luminescence(EALL). The second uses luminescent lanthanide com-plexes that are entrapped in nanoparticles. If the nano-particles are used as labels in immunoassays, every singleparticle comprises the luminescent properties of a largenumber of individual lanthanide complexes. Althoughboth strategies were developed to fulfill similar require-ments, they differ significantly with respect to possibleapplications. The EALL technique is not only capable ofdetecting affinity reactions with bound enzyme labels,such as antibodyantigen or biotinstreptavidin binding,but also enzymatic conversions themselves. Although

one reaction step more (addition of the lanthanidesolution) is required compared with assays based onconventional fluorophores, the total effort is only

transition involving lightabsorption or emissiontransition involving lightabsorption or emission

transition reducingTb(III)-fluorescence

radiationless transition

S1

ground state of the ligand

absorption

ligandfluorescence

ISC

T1

phosphorescence

energytransfer

Ln3+

luminescence

S0

Fig. 1 Mechanism of theenergy transfer from the ligandto the lanthanide(III) ion

460 480 500 520 540 560 580 600 620 640 660 680 700

0

1

2

3

4

5

6PDC/Tb(III)PDC/Eu(III)

Intensity[-]

[nm]

Fig. 2 Emission spectra of Tb(III) and Eu(III), complexed to 2,6-pyridinedicarboxylic acid (PDC)

0 10 200 400 600 800 1,0000.0

0.2

0.4

0.6

0.8

1.0

I [-]

t [s]

backgroundlanthanide luminescence

time window for detection

pulsed excitation

Fig. 3 Principle of time-resolved luminescence measurements

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slightly higher for an EALL system. It should also benoted that this additional step is not significant in anautomated analyzer system. Luminescent nanoparticles,however, cannot change their fluorescent propertiesupon an enzymatic reaction, and are therefore limited tothe detection of affinity assays. On the other hand, thedetection scheme for affinity assays using nanoparticlesis simpler and can be easily transferred to relatedapplications.

Within this review, the most prominent features andapplications of both techniques are introduced andcritically discussed with particular emphasis on possiblefuture trends. Related techniques utilizing other types ofnanoparticles, including those based on semiconductormaterials (quantum dots) [10, 11, 12], lanthanide-doped solid-state materials [13, 14], or polymers con-taining ruthenium complexes with long-lived fluores-cence [15] are characterized by attractive properties aswell, but are not the subject of this review.

Enzyme-amplified lanthanide luminescence

The term EALL was introduced by Evangelista et al [16,17] in 1991. In a pioneering article, they introduced anew detection scheme for highly sensitive bioanalyticalassays. Here, enzymes are either used as a label or as theanalyte of interest. A method for the determination ofalkaline phosphatase (aP) with the use of 5-fluorosali-cylic phosphate (FSAP) and Tb(III) has been developed(see Fig. 4) and a model immunoassay for Rat IgG hasbeen performed with alkaline phosphatase as marker.The effectiveness of the EALL principle was furtherdemonstrated by applying the detection scheme to thedetermination of other hydrolytic and redox enzymes,

namely xanthine oxidase, b-galactosidase and glucoseoxidase. For xanthine oxidase and b-galactosidase, twoother salicylic acid (SA) derivatives, salicylaldehyde andsalicyl-b-D-galactoside, were employed as substrates forthe enzymatic reaction. Both were, after conversion toSA, complexed to Tb(III). In contrast, for the determi-nation of glucose oxidase, 1,10-phenanthroline-2,9-dicarboxylic acid dihydrazide has been used as substrate.In this case, the resulting 1,10-phenanthroline-2,9-dicarboxylic acid forms a luminescent complex withEu(III). This article indicated that EALL had the

potential to be used as an alternative to ELISA andradioimmunoassays (RIAs).

The same authors used the EALL principle fordetection in nucleic acid hybridization assays in micro-well, dot-blot and Southern blot formats [18]. For theassay, which was performed in microwells, the dena-tured target DNA sequence (pBR322 linearized withHind III enzyme) was hybridized with a biotinylatedprobe (pBR322 BioProbe), and after washing andblocking the unspecific binding sites, aP-labeled avidinwas added. For the detection of the aP label, FSAPsolution in buffer was given into the well. After incu-bation, in which the FSAP was cleaved in an aP-cata-lyzed reaction to FSA (see Fig. 4), a solution containingTb(III) and EDTA was added and the resulting lan-thanide luminescence was recorded. In the dot-blot andSouthern blot assays, which were performed on nylonmembranes, 5-tert-octylsalicyl phosphate was used asreagent instead of the FSAP. The authors report ahigher sensitivity of the non-isotopic EALL method incomparison to radioisotopic detection. Various bio- andimmunoassays were developed, which routinely use the

EALL principle as the detection method. Those assaysemploy aP as marker and use the cleavage of a salicylicacid phosphate derivative to the respective SA derivativewith subsequent formation of a luminescent complexwith Tb(III) and EDTA. Figure 5 illustrates the basicprinciple of most of those assays.

These include the determination of thyrotropin(thyroid-stimulating hormone, TSH) in serum [19]. Thesamples are pipetted into the wells of a microtiter strip,which was coated previously with monoclonal anti-TSHantibody. Biotinylated detection antibody is added,which subsequently reacts with the aP-labeled strepta-vidin. Detection is performed as described above with

FSAP as substrate.The same assay, and a related one for thyroxine, was

used to prove the applicability of 4-methylumbelliferylphosphate, which was selected from 33 candidate fluor-ogenic chelators for Eu(III) and Tb(III) as substrate forEALL with Eu(III) [20]. In this case, the fluorescence isquenched with the ongoing enzymatic reaction becauseonly the phosphate forms luminescent complexes withEu(III), whereas the generated 4-methylumbelliferone isnot a sensitizer for lanthanide luminescence.

Later, it was found that phosphotyrosine but nottyrosine itself forms luminescent complexes with Tb(III).Based on this, fluorometric and time-resolved immuno-

fluorometric assays for acidic phosphatase and protein-tyrosine phosphatase activity have been developed [21].

COO-

OPO3H-

F

COO-

OHFaP

pH 12-13

Tb(III), EDTA

COO

O TbF

N

OOC

OOC

N

OOC

OOC

3-

Fig. 4 Reaction sequence of the aP-catalyzed cleavage of FSAP toFSA with subsequent complexation to Tb(III)/EDTA

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Some assays for the determination of a-fetoproteinhave also been performed using the EALL technique [22,23, 24]. These assays are based, as in the case of thethyrotropin assays, on the biotin-streptavidin interac-tions, and aP was again used as label. However, themeasurement principles and the employed enzyme sub-strates were evolved in order to enhance the sensitivity.Lianidou and coworkers for example, used secondderivative synchronous scanning fluorescence spectrom-etry for the screening of 14 different bidentate ligands,some of which are compounds of pharmaceutical inter-est. The most promising, diflunisal (2,4-difluoro-4-hydroxy-3-biphenylcarboxylic acid), has been convertedto its phosphate ester, which has been found useful in theEALL-type immunoassay for a-fetoprotein. The samegroup introduced a sandwich-type immunoassay forinterleukin 6, a cytokine that has been implicated in the

pathology of several diseases, using diflunisal phosphateas enzyme substrate for aP [25]. In another paper, theydescribed the determination of tumor necrosis factor-a[26], a potent pre-inflammatory and immunoregulatorycytokine, which is produced by a large number of dif-ferent cell types in response to bacterial toxins andinflammatory and other invasive stimuli. Therefore, it isa critical mediator for the diagnosis of a wide variety ofdiseases.

Another approach has been chosen for the determi-nation of chronic myelogenous leukemia-specific mRNAsequences, also named the Philadelphia translocation.This DNA abnormality can be directly associated withmalignancy. Here, the analyte is additionally amplifiedby a polymerase chain reaction (PCR), after labeling ofthe PCR primers with biotin or a hapten [27, 28, 29, 30],The immunoassay technique is analogous to the previ-ously mentioned assays, and detection is performed withthe FSA/Tb(III)/EDTA-system too. Similar attemptshave been made to determine prostate-specific antigenmRNA [31, 32]. Improved quantification of the PCRproducts was achieved using an internal standard pro-cedure [33]. As template, a 308 bp DNA fragment wasused, and an internal standard of identical size andidentical primers, but with a different centrally locatedsequence of 26 bp, was synthesized. As the target ana-lyte is co-amplified with a known amount of the internalstandard, the latter compensates for the varying effi-ciency of the amplification. Again, the FSA/Tb(III)/EDTA system is used for the final detection afterhybridization with digoxigenin-modified probes and

attachment of an anti-digoxigenin-aP conjugate. Thesame group later published a similar approach forsandwich-type DNA hybridization assays [34]. A relatedtwo-cycle amplification scheme led to a 30-foldimprovement of the signal and a tenfold improvement ofthe signal-to-noise ratio compared to a single cycle ofamplification [35]. However, the repeatability for thetwo-round amplification scheme is only moderate, withrelative standard deviations of over 10%.

Barrios and Craik [36] recently used the FSA/Tb(III)/EDTA system to assay the substrate specifity of prote-ases. In a library of tetrapeptides, FSA was bound to theN-terminus of each peptide. Upon cleavage of the

respective amide bond by a protease (compare to Fig. 6),FSA is released and detected as described above. Inprinciple, this approach should also allow a rapidquantitative analysis of other proteases based on dedi-cated FSA-modified substrates.

Other groups made attempts to further increase thenumber of applications for EALL by using differentenzymes and substrates. Xie et al [37] described anEALL method that uses p-hydroxybenzoic acid as

Y

Y YA

nalyte

A

nalyte

detection

antibody

capture

antibody

B AB A aP

FSAP

FSA

Tb(III)/EDTA

pH 13

fluorescent complex

Fig. 5A, B Principle of an immunoassay with detection by meansof EALL. The detection antibody is marked with biotin (B), whichinteracts with aP labeled avidin (A)

NN

NH2N N

O

OR2

OR3

OR4

O R1

F

OHH

H

H

H

Protease HO

O

F

OH

Tb/EDTA

COO

O TbF

N

OOC

OOC

N

OOC

OOC

3-

Fig. 6 Reaction sequenceemploying the FSA/Tb(III)/EDTA system to evaluate the

substrate specifity of proteases(R1R4 are the rest of therespective amino acids)

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substrate for horseradish peroxidase (HRP) with sub-sequent detection by means of Tb(III) luminescence.The method has been used for the determination oftuberculosis antibodies. The same group used p-hy-droxybenzoic acid to study hemin catalysis [38]. Heminis often used as substitute for HRP and in this case, itcatalyzes the dimerization of p-hydroxybenzoic acid,p-hydroxyphenylacetic acid and p-hydroxyphenylprop-ionic acid in the presence of H2O2. The reaction schemeis depicted in Fig. 7. It was found out that only thedimer ofp-hydroxybenzoic acid, di-p,p-hydroxybenzoicacid, forms a luminescent complex with Tb(III),

whereas all educts and dimers of the two other sub-strates do not sensitize Tb(III). This implies that inrespect to Tb(III) the complexing site was the twocarboxylic acid groups of the dimer of p-hydroxyben-zoic acid. Additionally, a simple label assay for ahemin-BSA-conjugate was performed which exhibited adetection limit for the conjugate of 210)10 mol/l. Incontrast to these results, it was later found that thedimers of a variety of substrates for the above de-scribed reaction, in this case catalyzed by HRP, doform luminescent complexes with Tb(III) in the pres-ence of CsCl [39, 40]. Hence, the authors of this articlesuggest that the complexation sites for the lanthanide

ion are not the carboxylic acid groups but the twohydroxyl groups of the dimer. The limit of detectionfor HRP with p-hydroxyphenylpropionic acid asenzyme substrate was in this case 210)12 mol/l.This reaction was further applied to the detection oftwo different ELISAs, one for the determination ofgoat anti-rabbit IgG, the other for human anti-gliadinIgG in serum. It turned out that detection by means

of EALL was applicable to both assays with good re-sults.

Until now, most EALL schemes have been performedat alkaline pH, where the luminescence intensities yieldthe highest values. However, addition of EDTA at highpH is required in order to avoid precipitation of lan-thanide hydroxide. This is very unfavorable in terms ofthe limits of detection of the EALL methods, becauseEDTA itself is able to sensitize the lanthanide ions to aminor extent. One of the first EALL methods that can beperformed at neutral pH was developed by Steinkampet al [41] after screening a small library of tripod ligands.The ligand bis(2-pyridylmethyl)-(2-hydroxybenzyl)-amine (HL1) has been converted to its acetic acid ester,which was subsequently cleaved in an esterase-catalyzed

reaction to the acid. Detection was performed by meansof Tb(III)-sensitized luminescence. Figure 8 shows theester hydrolysis with subsequent complexation. Owing tothe fact that the tetradentate tripod ligand forms highlystable complexes with Tb(III) and that the luminescencemeasurements are performed at neutral pH, the additionof co-complexing agents like EDTA can be avoided andbackground fluorescence is further decreased. With thisdetection principle, a limit of detection of 10)9 mol/l foresterase from porcine liver could be achieved [41]. Basedon this reaction, a method for the direct monitoring of anenzymatic conversion has been developed, which wascompared to reaction monitoring by means of electro-

spray ionization mass spectrometry (ESI-MS) [42].Very recently, another EALL system that operates at

nearly neutral pH without the addition of EDTA hasbeen developed for the determination of esterases or ofxanthine oxidase (XOD) [43]. It employs PDC, which isable to sensitize Tb(III) as well as Eu(III) in a veryefficient way. PDC has been converted to a variety ofalkyl esters, which are then cleaved in an esterase-cata-lyzed reaction to the diacid again. The reaction scheme isshown in Fig. 9. 2,6-pyridinedicarboxaldehyde has beenused for the determination of XOD, which also yieldsPDC when oxidized in the presence of XOD.

HO COOH2

HO COOH

HO COOH

Tb(III)/EDTA

pH 13

-O COO-

-O COO-

Tb

N

-OOC

-OOC

N

-OOC

-OOC

H2O2,

Hemin

Fig. 7 Hemin-catalyzed dimerization of p-hydroxybenzoic acid inthe presence of H2O2; the dimer is subsequently complexed to

Tb(III)/EDTA

Fluorescence at 545 nm

Esterase

- HAc

+ Tb(III)

Acetic acid esterof HL1

NN

N

O

O

NN

N

OH

HL1

Fig. 8 Esterase-catalyzed ester cleavage of the acetic acid ester ofHL1; the resulting HL1 is complexed to Tb(III) and theluminescence intensity is read out

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Luminescent lanthanide complexes in nanoparticles

An attractive approach to enhancing the signal intensityof lanthanide luminescence is to enclose a large amountof luminescent lanthanide chelates in one nanoparticle,which is then used as a label in an affinity assay. Themajority of the commercially available nanoparticleshave until now contained Eu(III)/b-diketonate chelatecomplexes with a polystyrene cover, exhibiting similarspectroscopic properties to the free chelate complexes in

solution.Harma et al [44, 45, 46, 47, 48, 49, 50] applied these

nanoparticles to different approaches to the determina-tion of prostrate specific antigen (PSA). A commerciallyavailable nanoparticle with an average diameter of107 nm and tris(naphthyltrifluorobutanedione) as ligandfor Eu(III) was employed. The assay was performed inanti-PSA microplates. Biotinylated PSA was given ontothe plate and the streptavidin-coated nanoparticle wasadded after incubation and washing. Evaluationwas performed by means of TRF imaging after the platewas dried [44]. The principle of the described assay isexplained in Fig. 10. The assay was further investigated

with respect to solid-phase association and dissociation,non-specific binding, and affinity constants of the vari-ous binding site density nanoparticle-antibody biocon-jugates [47]. Later, the assay was applied to thedetermination of real samples, namely PSA in serum[49]. Recently, a paper from these authors described thereplacement of the microtiter plate by microparticles ofsize of 60920 lm in diameter [50].

Eu(III) chelate-dyed nanoparticles with a size of92 nm have recently been used as donors in a homoge-neous proximity-based immunoassay for estradiol [51].Regarding this competitive assay, the nanoparticles wereinitially coated with 17b-estradiol (E2) specific re-

combinant antibody Fab S16 fragments. The coatedparticles were placed into the BSA-blocked wells of amicrotitration plate. 17b-estradiol was then added whichbinds to the nanoparticles, and after incubation of theplate an E2-Alexa680-conjugate was pipetted into thewells. AlexaFluor 680 is a near-infrared fluorescent labelwhose excitation spectrum overlaps with the emissionspectrum of Eu(III). Hence, after the E2-Alexa680-conjugate is bound to the remaining free binding sites onthe nanoparticle, it is possible to excite the Alexa labelwith the emission light of the nanoparticle due to the

Forster energy transfer taking place between the twoluminescent substances [52]. The principle of this assay isdepicted in Fig. 11. Detection limits of 70 pmol/l for E2could be achieved.

R = Me, Et, Pr, iPr

NOO

OR OR

NOO

OH OHTb

3+

NOO

O-

O-

Esterase Tb(III)

Fig. 9 Reaction sequence of the esterase-catalyzed hydrolysis of

PDC esters with subsequent complexation to Tb(III)

Y

Y YAnal

yte

Anal

yte

detection

antibody

capture

antibody

YYY Y

nanoparticle

containing

Eu(III) chelate complexes

Fig. 10 Principle of an immunoassay employing nanoparticlescontaining Eu(III) chelate complexes

Y

Analyte

Analyte

detection

antibody

YYY Y

E2

E2

E2

Alexa680

Alexa680

nanoparticle

containing

Eu(III) chelate complexes

Analyte

Analyte

Fig. 11 Principle of the competitive proximity-based nanoparticleassay for the determination of 17b-estradiol

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Conclusions and future perspectives

Despite the attractive properties of both approachespresented in this review, they are still only used by a lim-ited number of scientists. With respect to the EALLtechnique, this is mainly due to the need for laboriousdevelopment steps in order to apply the principle to otherenzymes beyond those described in the literature. It is not

trivial to develop a new EALL system, as the potentialsubstrate must, aside from the required interaction withthe enzyme, be characterized by a change from no ob-served energy transfer to a strongly sensitizing effect forone of the four lanthanide cations upon conversion. Thiscertainly hampers the application of EALL to a broaderrange of enzymes. Furthermore, the EALL technique re-quires a little more experimental effort when compared toclassical ELISA detection, and it provides significantadvantages only in those cases where the sensitivity of thedetection scheme is the bottleneck for the total sensitivityof a bioassay. On the other hand, many situations exist inwhich EALL could solve important analytical problems

and would certainly be worth the experimental work mainly the cases of enzyme determination where ultimatesensitivity is required.

A large expansion in the field of luminescent nano-particles based on lanthanide chelates may be expectedwithin the next few years. The reason for this is the pos-sibility of applying this method in a more general way,making it easier for industry to provide commercialsolutions. Furthermore, the complexity of the detectionscheme is lower compared to ELISAs and it shouldfacilitate an at least slight increase in the speed of analysis.Furthermore, multiplexing with at least four differentcolors should in principle be possible and will add new

tools for multianalyte measurements in the future.For both methods, however, their potential applica-

tion to the field of bioassays is far from being fullyexploited, and new highly selective and sensitive detec-tion schemes based on these approaches can certainly beexpected soon.

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