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Thin solid europium(III) dye layers as donors in time-resolved fluorescence resonance energy transfer assays
Applied Surface Science 255 (2009) 6529–6534
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Thin solid europium(III) dye layers as donors in time-resolved fluorescence resonance energy transfer assays Harri Ha¨rma¨ a,b,*, Riikka Suhonen c, Terho Kololuoma c, Ari Ka¨rkka¨inen d, Mika Hara a, Pekka Ha¨nninen a a
Laboratory of Biophysics, University of Turku, Tykisto¨katu 6A, FIN-20520, Turku, Finland Department of Biotechnology, University of Turku, Tykisto¨katu 6A, FIN-20520, Turku, Finland c VTT, Kaitova¨yla¨ 1, FIN-90570, Oulu, Finland d BraggOne Oy, Kaitova¨yla¨ 1, FIN-90570, Oulu, Finland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2008 Received in revised form 11 February 2009 Accepted 11 February 2009 Available online 25 February 2009
Lanthanide chelates and lanthanide nanoparticle labels are attractive donors for separation-free timeresolved fluorescence resonance energy transfer (TR-FRET) assays. In fully dyed nanoparticles, the inner volume of nanoparticle labels in TR-FRET assays are incapable of participating to energy transfer due to large distances to acceptors on the surface. Our interest was to study surface-based TR-FRET and, therefore, various europium(III) (Eu) chelate layers were investigated for TR-FRET efficiency. Eu(III) chelates incorporated in a siloxane layer, Eu(III) chelate covalently coupled on silanized surface and Eu(III) labeled protein surface were prepared and compared to nanoparticle-based TR-FRET. Energy transfer between the solid-phase donors and Cy5-labeled protein were obtained with signal-tobackground ratios ranging from 1.2 to 9.9. In this study, a thin layer prepared using Eu(III)-labeled protein gave the most efficient TR-FRET. This thin donor layer was tested in a competitive separationfree immunoassay of human albumin (hAlb). hAlb was measured in a clinically relevant concentrations from 0.05 to 10 mg l1 with the coefficient of variation ranging from 1.0% to 12.4%. ß 2009 Elsevier B.V. All rights reserved.
Keywords: TR-FRET Europium Thin solid surface Immunoassay
1. Introduction Luminescence resonance energy transfer (TR-FRET) techniques are based on non-radiative transfer of energy between donors and acceptors via the Fo¨rster mechanism. Lanthanide chelates of europium (Eu) and terbium (Tb) are attractive donors as they have exceptionally long emissive lifetimes, large Stokes’ shifts, and narrow band emission lines [1–3]. These properties make sensitized acceptor emission detectable with temporal and spectral separation from interfering background emission [2,4,5]. Large Fo¨rster distances have been measured for lanthanide chelates in comparison to short-lived donors allowing larger intermolecular distances and more efficient energy transfer [1]. TR-FRET has been widely applied in homogeneous bioaffinity assay systems to measure molecular interaction in real time and in high throughput screening [6–11]. Soluble label molecules have been main interest area in TRFRET studies as rapid kinetics and simple coupling chemistry have been achieved. However, labeled solid surfaces can result in an
* Corresponding author at: Laboratory of Biophysics, University of Turku, Tykisto¨katu 6A, FIN-20520 Turku, Finland. Tel.: +358 2 3337065; fax: +358 2 3337060. E-mail address: harri.harma@utu.fi (H. Ha¨rma¨). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.02.047
advantage: A high number of labels in close proximity to donors or acceptors in the solid surface ensure that donors or acceptors are available at appropriate distances for high signal generation and efficient energy transfer. Surface layer thickness and, therefore, signal within the Fo¨rster distance for TR-FRET is also readily adjustable. Radiative energy transfer may also be reduced as intermolecular distances of the TR-FRET pairs, donor solid-phase and soluble acceptor, become large. This holds true especially in particle-based assay systems where a relatively low number of particles are used as compared to soluble labels. Separation-free TR-FRET assay formats using nanoparticles as donors have been reported recently [12–14]. In model assay systems biotin and estradiol have been detected with reasonable sensitivity and reproducibility. Problems related to nanoparticle TR-FRET assays are related to donors that are incapable of participating to an energy transfer process [12]. These donors are mainly located in an inner part of a particle having distances clearly larger than the Fo¨rster radius. Donors located solely in the outer surface layer would obviously reduce the number of donors which are not participating in the energy transfer process. Therefore, we focused our study on thin donor layers. Over the years, solid surface based FRET studies have been described. Most assay formats on surfaces rely on fluorescence and quenching mechanisms of organic fluorophores [15,16]. Pe´rez-Luna et al. exploited TR-FRET between fluorophore and a
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gold film to make a fluorescence-based reversible biosensor [15]. Seidel et al. established a competitive immunoassay on goldcoated nano-titer plates [17]. We have prepared various thin donor layers using different Eu(III) chelates and coupling strategies for assay purposes. Highly fluorescent Eu(III) surface were prepared and successful TR-FRET was measured for all prepared surfaces. Protein-based dye surface led to a functional assay setup and hAlb was measured in a separation-free assay system in a clinically relevant concentration range. This study is a step forward in our aims to develop separation-free methods for receptor-ligand studies using lipid layers.
Sweden) and tris(dibenzoylmethane)mono(phenantroline)europium(III) (ADS051RE) dye from American Dye Source. EuCl3 (99.9%), Naphthoyltrifluoroacetone (NTA) and trioctylphosphine oxide (TOPO) for NTE (NTA/TOPO/Eu(III)) were obtained from AlfaAesar, Acros Organics and Sigma–Aldrich, respectively. Eu(III)nanoparticles were from Seradyn (Indianapolis, IN, USA). Monoclonal antibody against human albumin (hAlb-mAb) was from Medix Biochemica and highly purified human albumin (hAlb) from Sigma–Aldrich. Polypropylene substrates were purchased from Plastone (Nurmija¨rvi, Finland) and low fluorescent microtiter plates were from Nunc (Denmark).
2. Materials and methods
2.2. Conjugates
2.1. Reagents
Streptavidin (SA) was labeled with Eu(III) chelate (1a) as described earlier [20]. 480 mg of SA was reacted with 20-fold molar excess of Eu-chelate and unbound dye was separated using size exclusion column. Streptavidin was quantified using absorbance at 280 nm after subtracting the absorbance of the Eu-chelate at 280 nm. The number of Eu(III) chelates per streptavidin was determining by comparing the fluorescence of the streptavidinlabeled Eu(III) chelate in DELFIA enhancement solution to fluorescence of a known Eu(III) standard. Labeling of streptavidin with Cy5-NHS was carried out according to the instructions provided by the manufacturer: 100 mg of streptavidin was dissolved in 100 mL of 100 mM carbonate buffer, pH 9.3. Cy5-NHS ester was added in 100 mL volume and the reaction was allowed to proceed for 30 min. Unbound dye was separated with NAP-5 and NAP-10 columns 100 mM phosphate buffer, pH 7.4, as eluent. The labeling degree of the SA-Cy5 was determinated ratiometrically using absorbance at 280 and 650 nm. Human albumin specific monoclonal antibody (hAlb-mAb) was labeled with Eu(III) chelate 2, human albumin (hAlb) was labeled
Fluorescent 9-dentate Eu(III) chelates {2,20 200 ,2000 -{[2-(4-isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis (mehylenenitrilo)}tetrakis(acetato)} (chelate 1a) and {2,20 200 ,2000 -{[2-(4isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(aglucopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis (mehylenenitrilo)}tetrakis(acetato)} (chelate 1b) (Fig. 1) [18], Innotrac assay buffer (50 mM TRIS-HCl, pH 7.8, containing 0.9 g/l NaCl, 0.05 g/l NaN3, 0.5 g/l BSA, 0.1 g/l Tween 40, 0.05 g/l bovine g-globulin, and 2 mM DTPA), wash solution (5 mM TRIS-HCl, pH 7.8, containing 9 g/l NaCl, 0.05 g/l Tween 20, and 1 g/l Germall II) and microtiter plates were purchased from Innotrac Diagnostics (Turku, Finland). Fluorescent Eu(III) chelate {2,20 ,200 ,2000 -{[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]-bis(methylenenitrilo)}tetrakis(acetato)} [19] (chelate 2, Fig. 1) was obtained from Wallac, PerkinElmer Life and Analytical Sciences (Turku, Finland). Cy5NHS ester was purchased from Amersham Biosciences (Uppsala,
Fig. 1. Structures of 9-dentate Eu-chelates (1a) and (1b) having different sugar moieties and 7-dentate chelate (2).
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with Cy5 and the labeling degrees of the purified antibody and albumin conjugates were determined as previously described [22]. BSA was biotinylated with 35-fold molar excess of NHS-LCbiotin as described previously [21]. Biotinylation was confirmed with an assay based on Eu(III)-labeled SA and SA-coated microtiter wells. 2.3. Preparation of three thin solid dye layers 2.3.1. Surface 1: spin coated surfaces Two different fluorescent Eu(III) dyes, NTE and ADS051, were used in preparation of thin solid dye layers on a polypropylene substrate. Eu(III) chelates were mixed with photopatternable Braggone siloxane (Braggone, Oulu, Finland). O2 plasma etched (200 W, 5 min) polypropylene substrates were spin coated with siloxane-dye solution at speed of 3000 rpm for 30 s, and then dried at 100 8C for 1 h. NTE dye concentration in siloxane was optimized between 10 and 50 mM, for ADS051 concentration was optimized using concentrations from 0.05 to10 mM. Thicknesses of spin coated siloxane dye films were evaluated by using Veeco 3300 optical white light interferometer (Veeco Instruments, Plainview, NY). 2.3.2. Surface 2: covalently coated surfaces Polypropylene substrate was silanized and, thereafter, Eu(III)ITC chelate 1b was covalently coupled to the amino functionalized silane via reactive isothiocyanate group in the chelate. The substrates were placed in toluene, heated to 60 8C and 1% of 3aminopropyl-trimethoxysilane (APTMS) was added. Temperature was kept in 60 1 8C for 4 min. The substrates were rinsed with toluene and dried with air. Silanized substrates were immersed in 1.5 and 0.3 mM solution of the Eu(III)-ITC chelate 1b in 100 mM carbonate buffer, pH 9.8, at room temperature overnight. After incubation the substrates were rinsed with water and dried with air. 2.3.3. Surface 3: adsorbed protein surfaces Eu(III)-labeled streptavidin was adsorbed on the polystyrene microtiter plate by placing a 5 ml drop of 10 mg l1 Eu(III)-labeled streptavidin in 100 mM phosphate buffer, pH 7.4, to the center of a microtiter well. The droplets were incubated in dark at room temperature for 30 min and washed twice with the wash solution.
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2.4. TR-FRET assays Properties of prepared solid Eu(III) surfaces (surfaces 1–3) and Eu(III) nanoparticles as TR-FRET donors were evaluated by adsorbing streptavidin labeled with Cy5 acceptor on the substrates containing Eu(III) layers and on the nanoparticles (Fig. 2). This was carried out in a 100 mM phosphate buffer pH 7.4 In a negative control test, SA-Cy5 conjugate was applied in the commercial assay buffer containing high concentration of proteins and detergents to block the binding of SA-Cy5 to the surfaces efficiently. In the protein-based assays biotinylated BSA was required to conjugate two streptavidin species containing donor and acceptor dyes. After immobilization of SA-Eu and a washing step, 50 ng of biotinylated BSA was allowed to react with the SA-Eu substrate. Final washing step was employed and SA-Cy5 was added to the substrate containing SA-Eu and biotinylated BSA. In a control assay, 1 mM of free biotin was added together with biotinylated BSA. Each nanoparticle assay contained 2 108 or 2 107 Eu(III) nanoparticles and 50 ng SA-Cy5 conjugate in 20 ml of phosphate buffer or the commercial assay buffer. The assays were incubated for 20 min at room temperature in the dark. Five micro liters of the assay mixture was transferred onto a polypropylene substrate, and time-resolved fluorescence intensity signals were measured for both Eu donor and Cy5 acceptor. In all assays, the binding surface area was kept constant, the assay volume was 5 ml and 50 ng of SA-Cy5 was added to each test. This held true in studies conducted with the Eu(III) layers as well as with the nanoparticle assay having 2 108 Eu(III) particles. We estimated that 5 ml drop spread over a 3 mm diameter spot with a surface area of 7.1 mm2. Surface area of 2 108 nanoparticles of 107 nm diameter was 7.2 mm2. Such a high number of particles yielded significant Eu(III) signal and background signal in the TRFRET measurement. Therefore, a 10-fold lower number of particles was tested in the TR-FRET assay for more optimal performance. 2.5. Competitive hAlb immunoassay Eu(III)-labeled human albumin monoclonal antibody hAlbmAb-Eu was immobilized on a microtiter plate in a total volume of 5 mL using 100 mM phosphate buffer, pH 7.4, for 45 min. The
Fig. 2. Schematic representations of the prepared europium surfaces and FRET assays on the surfaces.
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droplet containing 100 mg antibody was carefully placed in the center of a microtiter well and the well was covered to prevent evaporation. No mixing was carried out in order to avoid any movement of the droplet. The well was washed twice with the wash solution using a Delfia Platewash (PerkinElmer). Albumin calibrators were added to the wells in 40 mL assay buffer and mixed in the plate shaker for 15 min. Thereafter, 20 mL of Cy5labeled human albumin (300 ng) in assay buffer was added to the well for additional 20 min incubation. The sensitized acceptor fluorescence signal was measured using the plate reader.
A simple protein adsorption strategy was applied to study the efficiency of fluorescence resonance energy transfer between solid Eu(III) surfaces and labeled streptavidin. The close proximity achieved by attaching acceptor-labeled protein (SA-Cy5) directly onto the donor surface resulted in many cases a relatively high signal-to-background ratio. Sensitized Cy5 emission could be readily measured with spectral resolution at 665 nm where interference of Eu(III) donor emission was minimal. The excitation maximum of SA-Cy5 was 650 nm. Thus sensitized SA-Cy5 emission could be resolved temporally from the non-sensitized Cy5 emission using UV excitation. The labeling degree of Cy5-labeled streptavidin was 2.3. The adsorption of SA-Cy5 was prevented on the surfaces by adding large excess of proteins and detergent in solution. The ratio of sensitized Cy5 signal between bound and non-bound SA-Cy5 in a separation-free experiment was used to estimate TR-FRET efficiency.
2.6. Fluorescence intensity and spectral measurements Time-resolved fluorescence intensity measurements were carried out with VictorTM 1420 multilabel counter (Wallac) with 340 nm excitation and 615 nm emission wavelengths for Euchelates. Sensitized emission of Cy5-acceptor was measured at 665 nm. Eu(III) emission was measured with 400 ms delay, 400 ms integration window and 1000 ms cycle time and acceptor emission with 75 ms delay, 100 ms integration window and 2000 ms cycle time. Emission spectra and decay profiles of the ADS051 and NTE Eu(III) labels in water, and SA-Eu, SA-Cy5 and mixture of SAEu:bioBSA:SA-Cy5 in 100 mM phosphate buffer, pH 7.4, were measured with a Varian Cary Eclipse spectrofluorometer (Varian, Palo Alto, CA). The excitation wavelength for Eu(III) labels and conjugates was 325 nm measured using 100 ms delay, 900 ms window and 1000 ms cycle time and 615 nm emission wavelength. The excitation maximum for SA-Cy5 conjugate was 635 nm. Lifetimes of Eu(III) labels were measured with 325 nm excitation wavelength and 615 nm emission wavelength 100 ms delay, 25 ms gate and 2000 ms cycle time. Photometric measurements were carried out with Shimadzu Biospec 1601 E (Kyoto, Japan).
3.1. Eu(III) nanoparticles The fluorescence properties of the commercial nanoparticles, 107 nm in diameter, containing NTE chelating structure were studied. The fluorescence lifetime of the nanoparticles was 720 ms measured at the excitation and emission maxima of 340 and 614 nm, respectively [24]. The fluorescence resonance energy was transferred efficiently between Eu(III)-nanoparticles and Cy5-labeled streptavidin (Table 1). A specific TR-FRET-based bioaffinity immunoassay has been reported on nanoparticles previously [12]. The inner volume of the particles containing Eu(III) chelate without participating to energy transfer process has been associated with inefficient overall energy transfer [12]. Particle core-related chelates contributed solely to background signal at the measured acceptor wavelength. By decreasing the number of Eu(III) nanoparticles the background signal could be reduced enhancing the signal-to-background ratio. However, reduction of particle concentration did not remove the fact that background signal was still obtained from the inner volume. Therefore thin layer of Eu(III) was prepared and studied for TRFRET efficiency.
3. Results and discussion Various thin solid Eu(III) layers were investigated for optimal surface-based donors and TR-FRET efficiency: commercial polystyrene nanoparticles embedded with Eu(III) chelate, surface 1 – spin-coated siloxane surfaces incorporated with two different Eu(III) chelates, surface 2 – a silane surface with covalently coupled Eu(III) chelate and surface 3 – immobilized protein labeled with Eu(III) chelate. The prepared layers were characterized for their energy transfer efficiency and spectral properties. FRET efficiency is known to be dependent on donor lifetime. Therefore, we measured the fluorescence emission lifetimes and spectral properties of the different chelating structures. The fluorescence property data were measured in aqueous solution because the signal from surfaces was too weak to obtain reliable spectroscopic data. All Eu(III) chelating structures used gave reasonably long lifetimes (see below). This led to small variation between the different surfaces in fluorescence energy transfer experiments.
3.2. Surface 1: siloxane:Eu(III) NTE and ADS051 (spin coated) The fluorescence excitation maxima of ADS051RE and NTE were 359 nm and 337 nm, respectively. These Eu(III) complexes had the emission maximum at 614 nm, characteristic for the studied Eu(III) chelates. Emission lifetimes of ADS051 and NTE were 470 and 533 ms, respectively. A minor short lifetime component was also detected for ADS051. The layer thickness of the spin-coated siloxane layers was 10 nm. This was measured for siloxane without incorporated dyes. The dye solutions were not expected to affect the layer thickness because the relative volume of the dye solution having a low viscosity was 100-fold smaller compared to the siloxane solution. The optimal concentrations of ADS051 and NTE in the siloxane
Table 1 Europium dye layer properties and measurement data. Surface
Eu particles 2 108 Eu particles 2 107 Eu-siloxane (NTE) Eu-siloxane (ADS) Eu-silane SA-Eu *
Dye surface thickness (nm)
Surface area per assay (mm2)
FRET signal at 665 (nm) No BSA
BSA
1 107 1 107 10 10 1 <10
7.2 0.72 7.1 7.1 7.1 7.1
943 76 2 7 2 1
173 7 2 4 3 1
FRET ratio between bioBSA and buffer containing no bioBSA.
470 949 504 989 1411 058
bioBSA 143 811 111 661 268 017
6 404
FRET ratio no BSA/BSA
Relative Eu signal without acceptor
5.4 9.9 1.2 1.7 6.6 6.3*
70 7.0 1.0 7.5 2.7 1.0
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layer were optimized for 5 mM and 20 mM, respectively. The ADS051 concentration was approximately two orders of magnitude higher due to the weaker luminescent properties compared to NTE chelate structure [25]. On spin-coated thin layers with NTE and ADS051 dyes the energy transfer was inefficient (Table 1). A major reason for this could have been a low adsorption of SA-Cy5 on these layers. However, we studied the adsorption properties of SA labeled with Eu(III) on solid siloxane and found that protein adsorption was comparable to highly adsorbing commercial polystyrene microtiter plate surfaces (data not shown). This indicated that the low protein adsorption was not a reason for inefficient TR-FRET. Eu(III) chelate incorporated into a thin layer (10 nm) was thought be an efficient donor in the TR-FRET studies due to distance-related properties. However, no such effect was found. The reason for low efficacy remains unclear. We obtained high fluorescence intensities at 615 nm indicating that the chelating structure was intact within siloxane matrix. One could speculate that the dyes may have distributed into the layers unfavorably for fluorescence energy transfer.
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Fig. 3. Fluorescence emission spectra of SA-Eu, streptavidin-Cy5 conjugate and SAEu:bioBSA:SA-Cy5 in phosphate buffer. Excitation wavelength for SA-Eu and FRET samples was 325 nm (time-resolved fluorescence) and 635 nm (fluorescence) for SA-Cy5 conjugate.
3.3. Surface 2: silane:Eu(III) chelate 1b (covalently coated) 3.5. Comparison of the methods Monomolecular layer of APTMS and covalently attached Eu(III) chelate 1b was estimated to yield Eu(III) layer with thickness of approximately 1 nm. Covalent coupling of Eu(III) chelate directly on the prepared silane surface further reduced the layer thickness form nanoparticle and siloxane surfaces leading to efficient energy transfer. The excitation maximum of the chelate was 325 nm and the emission maximum was 613 nm and the lifetime was 925 ms. We measured the highest signal-to-background ratio of 6.6 for the sensitized Cy5 signal between bound and non-bound SA-Cy5 having the equal surface area in comparison to other surfaces under study. 3.4. Surface 3: protein:Eu(III) chelate 1a (adsorpted) Streptavidin (protein) was labeled with Eu(III) chelate 1a giving a labeling degree of 7.8. The spectral studies indicated that the SAEu(III) excitation maximum was 325 nm and the emission maximum was 613 nm. The emission lifetime of the europium chelate 1a was 995 ms, which was somewhat longer than the lifetime of the free chelate, 920 ms. The lifetimes corresponded well to the previously measured values [19]. The labeled streptavidin was immobilized to polystyrene microtiter well surface for FRET studies. The surface layer thickness was approximately 5 nm as estimated from the diameter of streptavidin [23]. Initially attempts were made to demonstrate TRFRET on prepared Eu(III) surfaces without success due to low signal levels and sensitivity constrains using the spectrofluorometer. Thus, complex of SA-Eu:bioBSA:SA-Cy5 was used to demonstrate and characterize TR-FRET between Eu(III) donor and SA-Cy5 acceptor in solution. Increase of Cy5 emission at 665 nm and simultaneous decrease in Eu(III) emission at 615 nm compared to non-complexed SA-Eu and SA-Cy5 was monitored in the timeresolved fluorescence emission spectrum of SA-Eu:bioBSA:SA-Cy5 (Fig. 3). The sensitized SA-Cy5 emission decayed in 123 ms being significantly longer than the non-sensitized decay time of Cy5, 1 ns [26]. Protein–protein interactions have been long studied using FRET approach. We applied the same strategy to prepare functional TRFRET surface. As shown above high energy transfer signal was detected in solution and this was also observed on SA-Eu(III) surfaces (Table 1). The signal-to-background ratio of 6.3 was calculated between the complex of SA-Eu:bioBSA:SA-Cy5 and soluble free SA-Cy5 without bioBSA.
Varying Eu(III) signal level was observed on the studied surfaces. This variation largely depended on the used Eu(III) chelate, fluorescence lifetime, layer thickness and applied chelate concentration. For the FRET experiments, the optimal concentrations of the dyes at surfaces were determined by titration. Typically low concentration of Eu(III) chelate was preferred in order to achieve low background signal at the emission wavelength of 665 nm for Cy5. The Eu(III) siloxane surfaces (surface 1) were impractical for FRET studies due to low performance in the FRET tests even under optimized conditions. Eu(III)-silane layer (surface 2), Eu(III) protein layer (surface 3) and Eu(III) nanoparticles gave significantly higher signal-to-background ratios in the experiments (Table 1). The surface 2 and nanoparticle surfaces were further tested by applying additional bioBSA protein layer prior to applying SA-Cy5. The additional protein layer significantly reduced the signal-to-background ratio of these surfaces (data not shown). Obviously, the distance between the FRET pairs was too long for efficient energy transfer. The experiment with labeled proteins gave a signal-to-background ratio of 6.3. This test was constructed initially to contain bioBSA between the labeled SA molecules making protein-based energy transfer assays possible. These results suggested that the protein-based surface was the most practical means of preparing a donor surface for TR-FRET assay because similar signal-to-background ratio was achieved using a functional assay i.e. additional protein in between the TR-FRET pairs compared to non-functional assays on solid-phase Eu(III) layers. Our primary target is to prepare functional dye surfaces for receptor-ligand studies in a homogeneous TR-FRET concept using lipid membranes as a receptor host. The data prepared using the model system suggests that the fluorescence energy transfer expands over a distance of bioBSA (4 nm) corresponding to distances found for lipid bilayers [27,28]. 3.6. Competitive hAlb immunoassay The successful fluorescence energy transfer measured on a protein surface encouraged us to investigate human albumin in a clinically relevant assay range. The competitive immunoassay of hAlb was carried out using immobilized Eu(III)-labeled anti-hAlb monoclonal antibody on polypropylene substrate and hAlb labeled
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functional TR-FRET-based receptor-ligand assay development on planar lipid bilayers. Acknowledgements The work was supported by the Finnish Funding Agency for Technology and Innovation, Tekes. References [1] [2] [3] [4] [5] [6] [7] [8] Fig. 4. Calibration curve and within assay imprecision of the competitive nonseparation human albumin immunoassay. Each data point represents average of triplicate measurements.
with Cy5 (Fig. 4). The labeling degrees of hAlb-mAb-Eu and hAlbCy5 were 5.0 and 1.6, respectively. The dynamic range of the assay was 0.05–10 mg/l with the coefficient of variation from 1.0 to 12.4%. The assay sensitivity was 0.05 mg/l as measured using two SD at the zero concentration. 4. Conclusions Fluorescence resonance energy transfer on various solidphases was studied. All prepared Eu(III) surfaces exhibited fluorescence resonance energy transfer as probed by adsorbing or reacting Cy5-labeled streptavidin on the surfaces. We found that the most promising surface for TR-FRET studies was prepared using proteins labeled with fluorochromes and adsorbing one of the label pair onto a substrate. Using this approach a functional immunoassay was successfully developed for hAlb. The study showed the usefulness of surface-based TRFRET in bioaffinity assays encouraging us further towards
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Thin solid europium(III) dye layers as donors in time-resolved fluorescence resonance energy transfer assays Harri Ha¨rma¨ a,b,*, Riikka Suhonen c, Terho Kololuoma c, Ari Ka¨rkka¨inen d, Mika Hara a, Pekka Ha¨nninen a a
Laboratory of Biophysics, University of Turku, Tykisto¨katu 6A, FIN-20520, Turku, Finland Department of Biotechnology, University of Turku, Tykisto¨katu 6A, FIN-20520, Turku, Finland c VTT, Kaitova¨yla¨ 1, FIN-90570, Oulu, Finland d BraggOne Oy, Kaitova¨yla¨ 1, FIN-90570, Oulu, Finland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 November 2008 Received in revised form 11 February 2009 Accepted 11 February 2009 Available online 25 February 2009
Lanthanide chelates and lanthanide nanoparticle labels are attractive donors for separation-free timeresolved fluorescence resonance energy transfer (TR-FRET) assays. In fully dyed nanoparticles, the inner volume of nanoparticle labels in TR-FRET assays are incapable of participating to energy transfer due to large distances to acceptors on the surface. Our interest was to study surface-based TR-FRET and, therefore, various europium(III) (Eu) chelate layers were investigated for TR-FRET efficiency. Eu(III) chelates incorporated in a siloxane layer, Eu(III) chelate covalently coupled on silanized surface and Eu(III) labeled protein surface were prepared and compared to nanoparticle-based TR-FRET. Energy transfer between the solid-phase donors and Cy5-labeled protein were obtained with signal-tobackground ratios ranging from 1.2 to 9.9. In this study, a thin layer prepared using Eu(III)-labeled protein gave the most efficient TR-FRET. This thin donor layer was tested in a competitive separationfree immunoassay of human albumin (hAlb). hAlb was measured in a clinically relevant concentrations from 0.05 to 10 mg l1 with the coefficient of variation ranging from 1.0% to 12.4%. ß 2009 Elsevier B.V. All rights reserved.
Keywords: TR-FRET Europium Thin solid surface Immunoassay
1. Introduction Luminescence resonance energy transfer (TR-FRET) techniques are based on non-radiative transfer of energy between donors and acceptors via the Fo¨rster mechanism. Lanthanide chelates of europium (Eu) and terbium (Tb) are attractive donors as they have exceptionally long emissive lifetimes, large Stokes’ shifts, and narrow band emission lines [1–3]. These properties make sensitized acceptor emission detectable with temporal and spectral separation from interfering background emission [2,4,5]. Large Fo¨rster distances have been measured for lanthanide chelates in comparison to short-lived donors allowing larger intermolecular distances and more efficient energy transfer [1]. TR-FRET has been widely applied in homogeneous bioaffinity assay systems to measure molecular interaction in real time and in high throughput screening [6–11]. Soluble label molecules have been main interest area in TRFRET studies as rapid kinetics and simple coupling chemistry have been achieved. However, labeled solid surfaces can result in an
* Corresponding author at: Laboratory of Biophysics, University of Turku, Tykisto¨katu 6A, FIN-20520 Turku, Finland. Tel.: +358 2 3337065; fax: +358 2 3337060. E-mail address: harri.harma@utu.fi (H. Ha¨rma¨). 0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2009.02.047
advantage: A high number of labels in close proximity to donors or acceptors in the solid surface ensure that donors or acceptors are available at appropriate distances for high signal generation and efficient energy transfer. Surface layer thickness and, therefore, signal within the Fo¨rster distance for TR-FRET is also readily adjustable. Radiative energy transfer may also be reduced as intermolecular distances of the TR-FRET pairs, donor solid-phase and soluble acceptor, become large. This holds true especially in particle-based assay systems where a relatively low number of particles are used as compared to soluble labels. Separation-free TR-FRET assay formats using nanoparticles as donors have been reported recently [12–14]. In model assay systems biotin and estradiol have been detected with reasonable sensitivity and reproducibility. Problems related to nanoparticle TR-FRET assays are related to donors that are incapable of participating to an energy transfer process [12]. These donors are mainly located in an inner part of a particle having distances clearly larger than the Fo¨rster radius. Donors located solely in the outer surface layer would obviously reduce the number of donors which are not participating in the energy transfer process. Therefore, we focused our study on thin donor layers. Over the years, solid surface based FRET studies have been described. Most assay formats on surfaces rely on fluorescence and quenching mechanisms of organic fluorophores [15,16]. Pe´rez-Luna et al. exploited TR-FRET between fluorophore and a
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gold film to make a fluorescence-based reversible biosensor [15]. Seidel et al. established a competitive immunoassay on goldcoated nano-titer plates [17]. We have prepared various thin donor layers using different Eu(III) chelates and coupling strategies for assay purposes. Highly fluorescent Eu(III) surface were prepared and successful TR-FRET was measured for all prepared surfaces. Protein-based dye surface led to a functional assay setup and hAlb was measured in a separation-free assay system in a clinically relevant concentration range. This study is a step forward in our aims to develop separation-free methods for receptor-ligand studies using lipid layers.
Sweden) and tris(dibenzoylmethane)mono(phenantroline)europium(III) (ADS051RE) dye from American Dye Source. EuCl3 (99.9%), Naphthoyltrifluoroacetone (NTA) and trioctylphosphine oxide (TOPO) for NTE (NTA/TOPO/Eu(III)) were obtained from AlfaAesar, Acros Organics and Sigma–Aldrich, respectively. Eu(III)nanoparticles were from Seradyn (Indianapolis, IN, USA). Monoclonal antibody against human albumin (hAlb-mAb) was from Medix Biochemica and highly purified human albumin (hAlb) from Sigma–Aldrich. Polypropylene substrates were purchased from Plastone (Nurmija¨rvi, Finland) and low fluorescent microtiter plates were from Nunc (Denmark).
2. Materials and methods
2.2. Conjugates
2.1. Reagents
Streptavidin (SA) was labeled with Eu(III) chelate (1a) as described earlier [20]. 480 mg of SA was reacted with 20-fold molar excess of Eu-chelate and unbound dye was separated using size exclusion column. Streptavidin was quantified using absorbance at 280 nm after subtracting the absorbance of the Eu-chelate at 280 nm. The number of Eu(III) chelates per streptavidin was determining by comparing the fluorescence of the streptavidinlabeled Eu(III) chelate in DELFIA enhancement solution to fluorescence of a known Eu(III) standard. Labeling of streptavidin with Cy5-NHS was carried out according to the instructions provided by the manufacturer: 100 mg of streptavidin was dissolved in 100 mL of 100 mM carbonate buffer, pH 9.3. Cy5-NHS ester was added in 100 mL volume and the reaction was allowed to proceed for 30 min. Unbound dye was separated with NAP-5 and NAP-10 columns 100 mM phosphate buffer, pH 7.4, as eluent. The labeling degree of the SA-Cy5 was determinated ratiometrically using absorbance at 280 and 650 nm. Human albumin specific monoclonal antibody (hAlb-mAb) was labeled with Eu(III) chelate 2, human albumin (hAlb) was labeled
Fluorescent 9-dentate Eu(III) chelates {2,20 200 ,2000 -{[2-(4-isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(a-galactopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis (mehylenenitrilo)}tetrakis(acetato)} (chelate 1a) and {2,20 200 ,2000 -{[2-(4isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(aglucopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis (mehylenenitrilo)}tetrakis(acetato)} (chelate 1b) (Fig. 1) [18], Innotrac assay buffer (50 mM TRIS-HCl, pH 7.8, containing 0.9 g/l NaCl, 0.05 g/l NaN3, 0.5 g/l BSA, 0.1 g/l Tween 40, 0.05 g/l bovine g-globulin, and 2 mM DTPA), wash solution (5 mM TRIS-HCl, pH 7.8, containing 9 g/l NaCl, 0.05 g/l Tween 20, and 1 g/l Germall II) and microtiter plates were purchased from Innotrac Diagnostics (Turku, Finland). Fluorescent Eu(III) chelate {2,20 ,200 ,2000 -{[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]-bis(methylenenitrilo)}tetrakis(acetato)} [19] (chelate 2, Fig. 1) was obtained from Wallac, PerkinElmer Life and Analytical Sciences (Turku, Finland). Cy5NHS ester was purchased from Amersham Biosciences (Uppsala,
Fig. 1. Structures of 9-dentate Eu-chelates (1a) and (1b) having different sugar moieties and 7-dentate chelate (2).
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with Cy5 and the labeling degrees of the purified antibody and albumin conjugates were determined as previously described [22]. BSA was biotinylated with 35-fold molar excess of NHS-LCbiotin as described previously [21]. Biotinylation was confirmed with an assay based on Eu(III)-labeled SA and SA-coated microtiter wells. 2.3. Preparation of three thin solid dye layers 2.3.1. Surface 1: spin coated surfaces Two different fluorescent Eu(III) dyes, NTE and ADS051, were used in preparation of thin solid dye layers on a polypropylene substrate. Eu(III) chelates were mixed with photopatternable Braggone siloxane (Braggone, Oulu, Finland). O2 plasma etched (200 W, 5 min) polypropylene substrates were spin coated with siloxane-dye solution at speed of 3000 rpm for 30 s, and then dried at 100 8C for 1 h. NTE dye concentration in siloxane was optimized between 10 and 50 mM, for ADS051 concentration was optimized using concentrations from 0.05 to10 mM. Thicknesses of spin coated siloxane dye films were evaluated by using Veeco 3300 optical white light interferometer (Veeco Instruments, Plainview, NY). 2.3.2. Surface 2: covalently coated surfaces Polypropylene substrate was silanized and, thereafter, Eu(III)ITC chelate 1b was covalently coupled to the amino functionalized silane via reactive isothiocyanate group in the chelate. The substrates were placed in toluene, heated to 60 8C and 1% of 3aminopropyl-trimethoxysilane (APTMS) was added. Temperature was kept in 60 1 8C for 4 min. The substrates were rinsed with toluene and dried with air. Silanized substrates were immersed in 1.5 and 0.3 mM solution of the Eu(III)-ITC chelate 1b in 100 mM carbonate buffer, pH 9.8, at room temperature overnight. After incubation the substrates were rinsed with water and dried with air. 2.3.3. Surface 3: adsorbed protein surfaces Eu(III)-labeled streptavidin was adsorbed on the polystyrene microtiter plate by placing a 5 ml drop of 10 mg l1 Eu(III)-labeled streptavidin in 100 mM phosphate buffer, pH 7.4, to the center of a microtiter well. The droplets were incubated in dark at room temperature for 30 min and washed twice with the wash solution.
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2.4. TR-FRET assays Properties of prepared solid Eu(III) surfaces (surfaces 1–3) and Eu(III) nanoparticles as TR-FRET donors were evaluated by adsorbing streptavidin labeled with Cy5 acceptor on the substrates containing Eu(III) layers and on the nanoparticles (Fig. 2). This was carried out in a 100 mM phosphate buffer pH 7.4 In a negative control test, SA-Cy5 conjugate was applied in the commercial assay buffer containing high concentration of proteins and detergents to block the binding of SA-Cy5 to the surfaces efficiently. In the protein-based assays biotinylated BSA was required to conjugate two streptavidin species containing donor and acceptor dyes. After immobilization of SA-Eu and a washing step, 50 ng of biotinylated BSA was allowed to react with the SA-Eu substrate. Final washing step was employed and SA-Cy5 was added to the substrate containing SA-Eu and biotinylated BSA. In a control assay, 1 mM of free biotin was added together with biotinylated BSA. Each nanoparticle assay contained 2 108 or 2 107 Eu(III) nanoparticles and 50 ng SA-Cy5 conjugate in 20 ml of phosphate buffer or the commercial assay buffer. The assays were incubated for 20 min at room temperature in the dark. Five micro liters of the assay mixture was transferred onto a polypropylene substrate, and time-resolved fluorescence intensity signals were measured for both Eu donor and Cy5 acceptor. In all assays, the binding surface area was kept constant, the assay volume was 5 ml and 50 ng of SA-Cy5 was added to each test. This held true in studies conducted with the Eu(III) layers as well as with the nanoparticle assay having 2 108 Eu(III) particles. We estimated that 5 ml drop spread over a 3 mm diameter spot with a surface area of 7.1 mm2. Surface area of 2 108 nanoparticles of 107 nm diameter was 7.2 mm2. Such a high number of particles yielded significant Eu(III) signal and background signal in the TRFRET measurement. Therefore, a 10-fold lower number of particles was tested in the TR-FRET assay for more optimal performance. 2.5. Competitive hAlb immunoassay Eu(III)-labeled human albumin monoclonal antibody hAlbmAb-Eu was immobilized on a microtiter plate in a total volume of 5 mL using 100 mM phosphate buffer, pH 7.4, for 45 min. The
Fig. 2. Schematic representations of the prepared europium surfaces and FRET assays on the surfaces.
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droplet containing 100 mg antibody was carefully placed in the center of a microtiter well and the well was covered to prevent evaporation. No mixing was carried out in order to avoid any movement of the droplet. The well was washed twice with the wash solution using a Delfia Platewash (PerkinElmer). Albumin calibrators were added to the wells in 40 mL assay buffer and mixed in the plate shaker for 15 min. Thereafter, 20 mL of Cy5labeled human albumin (300 ng) in assay buffer was added to the well for additional 20 min incubation. The sensitized acceptor fluorescence signal was measured using the plate reader.
A simple protein adsorption strategy was applied to study the efficiency of fluorescence resonance energy transfer between solid Eu(III) surfaces and labeled streptavidin. The close proximity achieved by attaching acceptor-labeled protein (SA-Cy5) directly onto the donor surface resulted in many cases a relatively high signal-to-background ratio. Sensitized Cy5 emission could be readily measured with spectral resolution at 665 nm where interference of Eu(III) donor emission was minimal. The excitation maximum of SA-Cy5 was 650 nm. Thus sensitized SA-Cy5 emission could be resolved temporally from the non-sensitized Cy5 emission using UV excitation. The labeling degree of Cy5-labeled streptavidin was 2.3. The adsorption of SA-Cy5 was prevented on the surfaces by adding large excess of proteins and detergent in solution. The ratio of sensitized Cy5 signal between bound and non-bound SA-Cy5 in a separation-free experiment was used to estimate TR-FRET efficiency.
2.6. Fluorescence intensity and spectral measurements Time-resolved fluorescence intensity measurements were carried out with VictorTM 1420 multilabel counter (Wallac) with 340 nm excitation and 615 nm emission wavelengths for Euchelates. Sensitized emission of Cy5-acceptor was measured at 665 nm. Eu(III) emission was measured with 400 ms delay, 400 ms integration window and 1000 ms cycle time and acceptor emission with 75 ms delay, 100 ms integration window and 2000 ms cycle time. Emission spectra and decay profiles of the ADS051 and NTE Eu(III) labels in water, and SA-Eu, SA-Cy5 and mixture of SAEu:bioBSA:SA-Cy5 in 100 mM phosphate buffer, pH 7.4, were measured with a Varian Cary Eclipse spectrofluorometer (Varian, Palo Alto, CA). The excitation wavelength for Eu(III) labels and conjugates was 325 nm measured using 100 ms delay, 900 ms window and 1000 ms cycle time and 615 nm emission wavelength. The excitation maximum for SA-Cy5 conjugate was 635 nm. Lifetimes of Eu(III) labels were measured with 325 nm excitation wavelength and 615 nm emission wavelength 100 ms delay, 25 ms gate and 2000 ms cycle time. Photometric measurements were carried out with Shimadzu Biospec 1601 E (Kyoto, Japan).
3.1. Eu(III) nanoparticles The fluorescence properties of the commercial nanoparticles, 107 nm in diameter, containing NTE chelating structure were studied. The fluorescence lifetime of the nanoparticles was 720 ms measured at the excitation and emission maxima of 340 and 614 nm, respectively [24]. The fluorescence resonance energy was transferred efficiently between Eu(III)-nanoparticles and Cy5-labeled streptavidin (Table 1). A specific TR-FRET-based bioaffinity immunoassay has been reported on nanoparticles previously [12]. The inner volume of the particles containing Eu(III) chelate without participating to energy transfer process has been associated with inefficient overall energy transfer [12]. Particle core-related chelates contributed solely to background signal at the measured acceptor wavelength. By decreasing the number of Eu(III) nanoparticles the background signal could be reduced enhancing the signal-to-background ratio. However, reduction of particle concentration did not remove the fact that background signal was still obtained from the inner volume. Therefore thin layer of Eu(III) was prepared and studied for TRFRET efficiency.
3. Results and discussion Various thin solid Eu(III) layers were investigated for optimal surface-based donors and TR-FRET efficiency: commercial polystyrene nanoparticles embedded with Eu(III) chelate, surface 1 – spin-coated siloxane surfaces incorporated with two different Eu(III) chelates, surface 2 – a silane surface with covalently coupled Eu(III) chelate and surface 3 – immobilized protein labeled with Eu(III) chelate. The prepared layers were characterized for their energy transfer efficiency and spectral properties. FRET efficiency is known to be dependent on donor lifetime. Therefore, we measured the fluorescence emission lifetimes and spectral properties of the different chelating structures. The fluorescence property data were measured in aqueous solution because the signal from surfaces was too weak to obtain reliable spectroscopic data. All Eu(III) chelating structures used gave reasonably long lifetimes (see below). This led to small variation between the different surfaces in fluorescence energy transfer experiments.
3.2. Surface 1: siloxane:Eu(III) NTE and ADS051 (spin coated) The fluorescence excitation maxima of ADS051RE and NTE were 359 nm and 337 nm, respectively. These Eu(III) complexes had the emission maximum at 614 nm, characteristic for the studied Eu(III) chelates. Emission lifetimes of ADS051 and NTE were 470 and 533 ms, respectively. A minor short lifetime component was also detected for ADS051. The layer thickness of the spin-coated siloxane layers was 10 nm. This was measured for siloxane without incorporated dyes. The dye solutions were not expected to affect the layer thickness because the relative volume of the dye solution having a low viscosity was 100-fold smaller compared to the siloxane solution. The optimal concentrations of ADS051 and NTE in the siloxane
Table 1 Europium dye layer properties and measurement data. Surface
Eu particles 2 108 Eu particles 2 107 Eu-siloxane (NTE) Eu-siloxane (ADS) Eu-silane SA-Eu *
Dye surface thickness (nm)
Surface area per assay (mm2)
FRET signal at 665 (nm) No BSA
BSA
1 107 1 107 10 10 1 <10
7.2 0.72 7.1 7.1 7.1 7.1
943 76 2 7 2 1
173 7 2 4 3 1
FRET ratio between bioBSA and buffer containing no bioBSA.
470 949 504 989 1411 058
bioBSA 143 811 111 661 268 017
6 404
FRET ratio no BSA/BSA
Relative Eu signal without acceptor
5.4 9.9 1.2 1.7 6.6 6.3*
70 7.0 1.0 7.5 2.7 1.0
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layer were optimized for 5 mM and 20 mM, respectively. The ADS051 concentration was approximately two orders of magnitude higher due to the weaker luminescent properties compared to NTE chelate structure [25]. On spin-coated thin layers with NTE and ADS051 dyes the energy transfer was inefficient (Table 1). A major reason for this could have been a low adsorption of SA-Cy5 on these layers. However, we studied the adsorption properties of SA labeled with Eu(III) on solid siloxane and found that protein adsorption was comparable to highly adsorbing commercial polystyrene microtiter plate surfaces (data not shown). This indicated that the low protein adsorption was not a reason for inefficient TR-FRET. Eu(III) chelate incorporated into a thin layer (10 nm) was thought be an efficient donor in the TR-FRET studies due to distance-related properties. However, no such effect was found. The reason for low efficacy remains unclear. We obtained high fluorescence intensities at 615 nm indicating that the chelating structure was intact within siloxane matrix. One could speculate that the dyes may have distributed into the layers unfavorably for fluorescence energy transfer.
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Fig. 3. Fluorescence emission spectra of SA-Eu, streptavidin-Cy5 conjugate and SAEu:bioBSA:SA-Cy5 in phosphate buffer. Excitation wavelength for SA-Eu and FRET samples was 325 nm (time-resolved fluorescence) and 635 nm (fluorescence) for SA-Cy5 conjugate.
3.3. Surface 2: silane:Eu(III) chelate 1b (covalently coated) 3.5. Comparison of the methods Monomolecular layer of APTMS and covalently attached Eu(III) chelate 1b was estimated to yield Eu(III) layer with thickness of approximately 1 nm. Covalent coupling of Eu(III) chelate directly on the prepared silane surface further reduced the layer thickness form nanoparticle and siloxane surfaces leading to efficient energy transfer. The excitation maximum of the chelate was 325 nm and the emission maximum was 613 nm and the lifetime was 925 ms. We measured the highest signal-to-background ratio of 6.6 for the sensitized Cy5 signal between bound and non-bound SA-Cy5 having the equal surface area in comparison to other surfaces under study. 3.4. Surface 3: protein:Eu(III) chelate 1a (adsorpted) Streptavidin (protein) was labeled with Eu(III) chelate 1a giving a labeling degree of 7.8. The spectral studies indicated that the SAEu(III) excitation maximum was 325 nm and the emission maximum was 613 nm. The emission lifetime of the europium chelate 1a was 995 ms, which was somewhat longer than the lifetime of the free chelate, 920 ms. The lifetimes corresponded well to the previously measured values [19]. The labeled streptavidin was immobilized to polystyrene microtiter well surface for FRET studies. The surface layer thickness was approximately 5 nm as estimated from the diameter of streptavidin [23]. Initially attempts were made to demonstrate TRFRET on prepared Eu(III) surfaces without success due to low signal levels and sensitivity constrains using the spectrofluorometer. Thus, complex of SA-Eu:bioBSA:SA-Cy5 was used to demonstrate and characterize TR-FRET between Eu(III) donor and SA-Cy5 acceptor in solution. Increase of Cy5 emission at 665 nm and simultaneous decrease in Eu(III) emission at 615 nm compared to non-complexed SA-Eu and SA-Cy5 was monitored in the timeresolved fluorescence emission spectrum of SA-Eu:bioBSA:SA-Cy5 (Fig. 3). The sensitized SA-Cy5 emission decayed in 123 ms being significantly longer than the non-sensitized decay time of Cy5, 1 ns [26]. Protein–protein interactions have been long studied using FRET approach. We applied the same strategy to prepare functional TRFRET surface. As shown above high energy transfer signal was detected in solution and this was also observed on SA-Eu(III) surfaces (Table 1). The signal-to-background ratio of 6.3 was calculated between the complex of SA-Eu:bioBSA:SA-Cy5 and soluble free SA-Cy5 without bioBSA.
Varying Eu(III) signal level was observed on the studied surfaces. This variation largely depended on the used Eu(III) chelate, fluorescence lifetime, layer thickness and applied chelate concentration. For the FRET experiments, the optimal concentrations of the dyes at surfaces were determined by titration. Typically low concentration of Eu(III) chelate was preferred in order to achieve low background signal at the emission wavelength of 665 nm for Cy5. The Eu(III) siloxane surfaces (surface 1) were impractical for FRET studies due to low performance in the FRET tests even under optimized conditions. Eu(III)-silane layer (surface 2), Eu(III) protein layer (surface 3) and Eu(III) nanoparticles gave significantly higher signal-to-background ratios in the experiments (Table 1). The surface 2 and nanoparticle surfaces were further tested by applying additional bioBSA protein layer prior to applying SA-Cy5. The additional protein layer significantly reduced the signal-to-background ratio of these surfaces (data not shown). Obviously, the distance between the FRET pairs was too long for efficient energy transfer. The experiment with labeled proteins gave a signal-to-background ratio of 6.3. This test was constructed initially to contain bioBSA between the labeled SA molecules making protein-based energy transfer assays possible. These results suggested that the protein-based surface was the most practical means of preparing a donor surface for TR-FRET assay because similar signal-to-background ratio was achieved using a functional assay i.e. additional protein in between the TR-FRET pairs compared to non-functional assays on solid-phase Eu(III) layers. Our primary target is to prepare functional dye surfaces for receptor-ligand studies in a homogeneous TR-FRET concept using lipid membranes as a receptor host. The data prepared using the model system suggests that the fluorescence energy transfer expands over a distance of bioBSA (4 nm) corresponding to distances found for lipid bilayers [27,28]. 3.6. Competitive hAlb immunoassay The successful fluorescence energy transfer measured on a protein surface encouraged us to investigate human albumin in a clinically relevant assay range. The competitive immunoassay of hAlb was carried out using immobilized Eu(III)-labeled anti-hAlb monoclonal antibody on polypropylene substrate and hAlb labeled
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functional TR-FRET-based receptor-ligand assay development on planar lipid bilayers. Acknowledgements The work was supported by the Finnish Funding Agency for Technology and Innovation, Tekes. References [1] [2] [3] [4] [5] [6] [7] [8] Fig. 4. Calibration curve and within assay imprecision of the competitive nonseparation human albumin immunoassay. Each data point represents average of triplicate measurements.
with Cy5 (Fig. 4). The labeling degrees of hAlb-mAb-Eu and hAlbCy5 were 5.0 and 1.6, respectively. The dynamic range of the assay was 0.05–10 mg/l with the coefficient of variation from 1.0 to 12.4%. The assay sensitivity was 0.05 mg/l as measured using two SD at the zero concentration. 4. Conclusions Fluorescence resonance energy transfer on various solidphases was studied. All prepared Eu(III) surfaces exhibited fluorescence resonance energy transfer as probed by adsorbing or reacting Cy5-labeled streptavidin on the surfaces. We found that the most promising surface for TR-FRET studies was prepared using proteins labeled with fluorochromes and adsorbing one of the label pair onto a substrate. Using this approach a functional immunoassay was successfully developed for hAlb. The study showed the usefulness of surface-based TRFRET in bioaffinity assays encouraging us further towards
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