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Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer
Analytica Chimica Acta 585 (2007) 120–125
Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer Tiina Kokko ∗ , Leena Kokko, Tero Soukka, Timo L¨ovgren Department of Biotechnology, University of Turku, Tykist¨okatu 6A, 6th Floor, FIN-20520 Turku, Finland Received 16 October 2006; received in revised form 12 December 2006; accepted 13 December 2006 Available online 19 December 2006
Abstract A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin–quencher is bound to Eu–streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher–biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2–2 nmol L−1 . In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay. © 2006 Elsevier B.V. All rights reserved. Keywords: Fluorescence resonance energy transfer; Homogeneous; Non-competitive; Hapten
1. Introduction Haptens are low molecular weight analytes, e.g. many drugs, steroids, metabolites and pollutants, which are too small to be recognized simultaneously by two binders (for example, antibodies). Many of the traditional non-competitve assay techniques, however, require the analyte to be bound to two different binders in order to be detected. First the analyte is bound to the capture antibody and then detected with another analytic spesific labeled antibody. Therefore, the measured signal is obtained from those labeled recognizing agents, which are bound to the analyte. Thus, the signal increases with the concentration of the analyte. Since haptens are not suitable for two-site assays, most of the assays designed to measure haptens are competitive, where the analyte to be measured is competing with a labeled analyte analogue for the binding sites of a single recognizing agent. Thus, in competitive assay the signal is measured from those labeled analyte analogues that are bound to the recognizing agent. Increasing the amount of analyte decreases the amount
∗
Corresponding author. Tel.: +358 2 333 8067; fax: +358 2 333 8050. E-mail address: [email protected] (T. Kokko).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.12.021
of bound labeled analyte analogue, which means that increasing the amount of analyte decreases the obtained signal. Sensitivity can be defined as the smallest amount of analyte, which generates a distinguishable difference in the signal from the background (zero amount of analyte) [1,2]. In competitive assays the signal at zero concentration is high whereas in noncompetitive assays the signal at zero amount of analyte is small. When the concentration is slightly elevated from zero only a small change in the signal occurs. It is typically easier to detect a small change from small signals than in high signals. Hence, the non-competitive assays are capable of detecting smaller changes in analyte concentrations and are, therefore, considered to have better sensitivity than competitive assays [3]. Since non-competitive assays have better sensitivity than competitive assays, quite a few different non-competitive assay techniques for haptens have been designed. For example, in immunometric assays antibodies bound to analyte are separated from those antibodies that are not bound to analyte. After the separation the analyte-bound antibodies are measured. A non-competitive digoxin assay uses digitoxigenin-coupled polyacrylamide beads to separate the unbound antibodies [4]. These techniques rely on the laborious physical separation step, so they are time consuming to do.
T. Kokko et al. / Analytica Chimica Acta 585 (2007) 120–125
Homogeneous assays are separation free, and, therefore, less tedious to perform than heterogeneous assay. Fluorescence resonance energy transfer (FRET) is a commonly used technique in homogeneous assays. When the donor and the acceptor are in close proximity with each other, the non-radiative energy transfer occurs from the excited donor molecule to the acceptor [5]. In addition to small distance, an overlapping of the emission spectrum of the donor with the excitation spectrum of the acceptor is also required [6]. During measurement either the emission of the acceptor or the fluorescence of the donor, in which case the acceptor acts as a quencher, is measured. For example, Arai et al. has demonstrated a FRET based noncompetitive open sandwich assay where separate VH and VL fragments are labeled with donor and acceptor. FRET can occur only if the antigen has induced heterodimerization between VH and VL fragments thus bringing the donor and acceptor in close proximity. However, this assay requires a suitable antibody fragment that has a weak VH –VL interaction when the antigene is not bound to it [7]. Another FRET method was described by Pulli et al. which utilizes anti-immuno complex (anti-IC) Fab fragments [8]. In that assay europium labeled antimorfine and Cy5-labeled anti-IC Fab are in close enough proximity for FRET only when the antimorfine is bound to the antigen. However, anti-IC antibodies are rather difficult to develop. This article presents a homogeneous non-competitive assay for biotin. Moreover, the objective is to demonstrate a principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer for a small analyte, which can only be recognized by one binder at a time. Thus, biotin–streptavidin complex was used as a model system. Since the interaction of biotin and streptavidin is strong (Kd ∼ 10−15 mol L−1 ), it is ideal for testing this type of protocol. The assay requires only one binder/recognizing agent, which is labeled with europium chelate, and an analyte analogue, which is conjugated to quencher dye. The assay utilizes FRET to quench the fluorescence of those europium(III) labeled streptavidins that are not occupied by biotin of the sample. 2. Experimental 2.1. Measurement buffer and instrument Buffer for all assays and dilutions contained 0.05 mol L−1 Tris–HCl, pH 7.75, 0.9% (w/v) NaCl, 0.05% (w/v) NaN3 , 0.01% (v/v) Tween 40, 0.05% (w/v) bovine-␥-globulin, 20 mol L−1 diethylenetriaminepentaacetate (DTPA) and 0.5% (w/v) bovine serum albumin (BSA) (purchased from Innotrac Diagnostics, Turku, Finland). Fluorescence was measured using time-resolved fluorescence mode at Victor 1420 Multilabel counter from Wallac, Perkin-Elmer Life and Analytical Sciences (Turku, Finland). Excitation and measuring wavelengths were 340 and 615 nm, respectively. Delay time and measurement time were both 400 s. All the washings and aspirations of the wells were done using 1296-026 Delfia Platewash. For the shakings of the Plates
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1296-003 Delfia Plateshake was used. Both of these instruments were acquired from Wallac, Perkin-Elmer Life and Analytical Sciences. 2.2. BSA-treated microtitration plates All assays were made in Low Fluor Maxisorp 96-well microtitration plates purchased from Nunc (Roskilde, Denmark). Wells were treated with BSA prior to the assays to prevent non-specific binding by adding 250 L of solution containing 0.1% (w/v) BSA (Bioreba, Switzerland), 0.1% (w/v) Germall II (ISP, Wayne, NJ, USA) and 3% (w/v) trehalose (Sigma–Aldrich, St. Louis, MO, USA) in 0.05 mol L−1 Tris–HCl buffer, pH 7.2. Plates were incubated for 1 h, in room temperature with low shaking. After incubation wells were aspirated and dried. 2.3. Streptavidin coated low capacity wells 96-well Low Fluor Maxisorp microtitration plates were coated with streptavidin in order to make low capacity biotin binding wells suitable for competitive biotin assay. Into the wells 100 L of 0.05 mol L−1 citric acid and 0.1 mol L−1 sodium phosphate, pH 5.0, containing 0.2 ng L−1 streptavidin was added. Plates were incubated over night at +35 ◦ C in moist conditions. After incubation wells were washed twice and 250 L of saturation solution (50 mmol L−1 Tris, 0.15 mol L−1 NaCl, 0.05%, w/v, NaN3 , 6%, w/v, sorbitol, 0.2%, w/v, bovine serum albumin, pH 7.0) was added. Plates were again incubated over night at room temperature in moist conditions. After incubation wells were aspirated and stored +4 ◦ C. 2.4. Europium(III)-chelate labeled streptavidin Streptavidin, purchased from Societa Prodotti Antibiotici (Milano, Italy), was labeled with a long lifetime fluorescent 9-dentate europium(III)-chelate (2,2 ,2 ,2 -{[2(4-isothiocyanatophenyl)ethylimino]-bis(methylene)bis{4{[4-(␣-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2diyl}-bis(methylenenitrilo)}tetrakis(acetato)europium(III)) [9]. Streptavidin (480 g) was dissolved in water and 5, 10 or 15fold molar excess of europium(III)-chelate (dissolved in water) was added. pH was adjusted by adding 30 L of 1 mol L−1 carbonate buffer, pH 9.8, and the total volume of the reaction was adjusted with water to 600 L. Reactions were incubated over night at room temperature. After incubation reactions were purified with gel filtration using Superdex 200 Matrix (Pharmacia Biotech, GE Healthcare, Fairfield, CT, USA). Buffer in the purifications was TSA (50 mmol L−1 Tris, 0.9%, w/v, NaCl and 0.5%, w/v, NaN3 , all purchased from Sigma–Aldrich), pH 7.75. After purification quantity of streptavidin was calculated from absorbance at 280 nm after subtracting the absorbance caused by the europium(III)-chelate. The amount of europium was measured by comparing the fluorescence of the labeling reaction to the fluorescence of known europium standard.
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Purified product was diluted in Delfia Enhancement Solution (purchased from Wallac, Perkin-Elmer Life and Analytical Sciences) and incubated for 30 min before measurement. Labeling degrees for streptavidins labeled with europium(III)-chelate were calculated as europium chelates per streptavidin. 2.5. Biotin–quencher conjugates N-(2-Aminoethyl)biotinamide, hydrobromide was purchased from Molecular Probes Inc. (Eugene, OR, USA). Cy5-NHS and Cy7-NHS esters were purchased from Amersham Biosciences, GE Healthcare. Alexa Fluor 680 carboxylic acid succinimidyl ester, dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester), QSY7 and QSY9 carboxylic acids, succinimidyl esters were acquired from Molecular Probes Inc. and ATTO612Q-NHS-ester from Atto-Tec (Siegen, Germany). Approximately 0.5 mg of quencher dye was dissolved in 50 mmol L−1 carbonate buffer, pH 9.0 and 20% (v/v) of DMF (N,N-dimethylformamide, Sigma–Aldrich) was added. Approximately 10-fold molar excess of N-(2-aminoethyl)biotinamide, hydrobromide was dissolved in 50 mmol L−1 carbonate buffer, pH 9.0. Quencher dye and biotin solutions were mixed. Reactions were protected from light and incubated at +35 ◦ C over night. Reactions were frozen until purification. Biotin–quencher conjugates were purified with RP-HPLC using equipment from Pharmacia, GE Healthcare together with Genesis C18 column from Jones Chromatography (Grace Vydac, Hesperia, CA, USA). Gradient purification was done using 50 mmol L−1 triethylammonium acetate buffer (TEAA; Fluka Biochemica, Steinheim, Switzerland) in water and 50 mmol L−1 TEAA in acetonitrile (from J.T. Baker, Phillipsburg, NJ, USA) as buffers. The amount of acetonitrile was increased from 30 to 100% in 30 min. After purification the concentrations of biotin–quencher conjugates were measured with absorbance. The molar absorptivities and the measuring wavelengths (absorbance maximum of the quencher dyes) were provided by the manufacturer. 2.6. Measurement of quenching efficiency Quenching efficiencies of different quenchers were tested by finding the maximum and minimum signal for each quencher. Volume of 50 L of either 4 nmol L−1 or 2 nmol L−1 Eu–SA (labeling degree 4.2) in assay buffer was added to BSAtreated microtitration wells. Thereafter, 50 L of 5 mol L−1 d-biotin (from Sigma–Aldrich) in assay buffer or assay buffer without biotin (background) was added to obtain maximum and minimum signals, respectively. Wells were incubated in room temperature for 30 min with low shaking. After incubation 50 L of 50 nmol L−1 biotin–quencher conjugate was added and incubation was continued in room temperature for 15 min with low shaking. Fluorescence of the europium(III)chelate was measured using Victor 1420 Multilabel Counter. To optimize the needed biotin–quencher concentration, titrations were done using biotin–quencher conjugate concentrations from 0 to 50 nmol L−1 . Protocol was the same as described above.
2.7. Heterogeneous competitive assay The heterogeneous competitive assay was used to characterize biotin–quencher conjugations and as a comparison for developed homogeneous non-competitive assay. 2-(((N-(Biotinoyl)amino)hexanoyl)amino)ethylamine was purchased from Molecular Probes and conjugated with europium chelate of 4-[2-(4-aminophenyl)ethynyl]-2,6-bis {[N,N-bis(carboxymethyl)-amino]methyl}pyridine [10,11] as described by Kuningas et al. [12]. Streptavidin low capacity wells were prewashed once and 150 L of d-biotin standards from 0.05 nmol L−1 to 0.5 mol L−1 or appropriately diluted samples were added into the wells. Wells were then incubated in room temperature, with low shaking for 25 min. Thereafter, 50 L of europium–biotin conjugate of 85 nmol L−1 was added and incubation was continued for 45 min in room temperature and with low shaking. After incubation wells were washed four times. Delfia Enhancement Solution (200 L) (purchased from Wallac, Perkin-Elmer Life and Analytical Sciences) was added and wells were incubated for 20 min in room temperature with low shaking. Fluorescence of the europium(III)-chelate was measured with Victor 1420 Multilabel Counter. 2.8. Homogeneous non-competitive biotin assay The biotin assay was done using QSY9, Cy7 and ATTO612Q quenchers conjugated with biotin. First 50 L of 3 nmol L−1 Eu–SA and 50 L of biotin standards from 0 to 2 nmol L−1 with two replicas were added to BSA-treated wells. After 30 min of incubation 50 L of 20 nmol L−1 biotin–QSY9 and biotin–Cy7 or 25 nmol L−1 biotin–ATTO612Q was added. Fluorescence of the europium was measured after 5 min of incubation with Victor 1420 Multilabel Counter. 3. Results and discussion The different situations depending on the amount of biotin during measurement are presented in Fig. 1 Streptavidin labeled with europium(III)-chelate (Eu–SA) has emission at 615 nm when excited with 340 nm. Maximum signal of the assay is obtained when four biotins are bound to Eu–SA, hence no quenching occurs. If biotin has bound to some of the binding sites of Eu–SA and the rest are occupied by the biotin–quencher conjugates the emission of europium is decreased. This is due to fluorescence resonance energy transfer, which can occur when the donor (europium(III)-chelate) and the acceptor (quencher dye molecule) are in close proximity, resulting in quenching the emission of europium. If all the binding sites of Eu–SA are occupied by the biotin–quencher conjugate, maximum quenching is observed. This also represents the background of the assay. Interaction between biotin and streptavidin is strong (Kd ∼ 10−15 mol L−1 ) and therefore biotin does not easily dissociate from streptavidin. During the homogeneous assay the biotin to be measured was allowed to interact with the Eu(III)-chelate labeled streptavidin before the biotin–quencher conjugate was added. Since the biotin to be measured did not significantly dissociate from the streptavidin, there was no com-
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Fig. 1. Different situations depending on the amount of biotin bound to streptavidin during measurement. (A) Streptavidin labeled with europium(III)-chelate emits at 615 nm when excitated at 340 nm. (B) When streptavidin is bound to four biotin maximum europium signal is obtained. (C) When some of the binding sites are not bound to biotin, biotin–quencher complex will bind to free biotin binding sites. Hence, portion of the fluorescence of europium(III)-chelate is quenched. (D) All the binding sites are occupied by biotin–quencher conjugate, hence maximum amount of the fluorescence is quenched.
petition over streptavidin binding sites between the biotin and the biotin–quencher conjugate. Thus, the biotin–quencher could only bind to those binding sites that were not already occupied by biotin. 3.1. Reagents Streptavidin was labeled with intrinsically fluorescent 9dentate europium(III)-chelate. The obtained labeling degrees were 3.2, 4.2 and 6.3 for 5, 10 and 15-fold excess of europium(III)-chelate in labeling reactions, respectively. One streptavidin is capable of binding four biotins and each biotin is conjugated with one quencher molecule. Thus, the maximum number of quenchers per streptavidin is four. Therefore, Eu–SA with labeling degree of 4.2 was chosen for the assay to attain in average one quencher molecule per one europium(III)-chelate. The fractions of purification of conjugation reactions of biotin to quencher dyes were characterized with absorbance measurements and heterogeneous competitive biotin assay. Absorbance measurements indicated the amount of quencher and heterogeneous competitive biotin assay was used to measure the amount of biotin in the fractions. Fractions that contained both biotin and quencher dye were selected. 3.2. Quenching efficiencies Quenching efficiencies for different quencher conjugates were tested and results are presented in Table 1. Quenching efficiencies were calculated using Eq. (1): Quenching efficiency =
maximum signal − signal of zero dose × 100% maximum signal
(1)
The maximum signal of the assay was obtained when there was a high excess of free biotin. Thus, no biotin–quencher conjugate was able to bind to the europium(III)–streptavidin and
the emission of europium(III)-chelate was not quenched (situation B in Fig. 1). The background signal (=signal of zero dose) was measured when there was no free biotin in the well. Thus, all of the binding sites of streptavidin were occupied by biotin–quenchers (situation D in Fig. 1). As can be seen from Table 1, different quenchers were able to quench slightly different amounts of the fluorescence of europium(III)-chelate. The best quenchers, QSY9, QSY7, Cy7 and ATTO612Q, all quenched nearly 100% of the signal when biotin–quencher concentration was 50 nmol L−1 . All of these dyes, except Cy7, are non-fluorescent, hence designed to quench fluorescence of other fluorophores. In addition, Cy7 is the only quencher, whose excitation spectrum does not overlap with the main emission peak (615 nm) of europium(III)-chelate. However, the excitation spectrum of the Cy7 does overlap with other parts of the emission spectrum of europium(III)-chelate, e.g. local emission maximum at 700 nm, which enables appropriate quenching [13]. QSY9 and QSY7 have similar chromophore structures differing only in their solubility. Therefore, QSY9, Cy7 and ATTO612Q were chosen for more detailed study in order to find possible differences among them. Different amounts of biotin–quenchers were used to detect the amount needed for maximum quenching efficiency. Using 4 nmol L−1 Table 1 Quenching efficiencies of different quenchers (biotin-quencher concentration 50 nmol L−1 ) Quencher
Bio-dabcyl Bio-ATTO612Q Bio-QSY9 Bio-QSY7 Bio-Cy5 Bio-Cy7 Bio-Alexa Fluor 680
Eu–streptavidin concentrations 2 nmol L−1
4 nmol L−1
90 98 97 97 67 96 78
90 97 97 97 72 97 80
All the values are given in percentage.
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Fig. 2. Titration of the quenching efficiencies with different amounts of quencher–biotin conjugate with standard error bars. biotin–QSY9 (), biotin–Cy7 (䊉) and biotin–ATTO612Q (). Maximum quenching efficiency was achieved with 20 nmol L−1 of biotin–Cy7 and biotin–QSY9 and 25 nmol L−1 of biotin–ATTO612Q.
Eu–SA, with labeling degree of 4.2, the quenching was titrated with quencher concentrations from 0 to 50 nmol L−1 . Titration curves are presented in Fig. 2. Maximum quenching was obtained for Cy7 and QSY9 at 20 nmol L−1 and for ATTO612Q at 25 nmol L−1 . Thus, in order to obtain maximum quenching, with presented incubation times, 1.25-fold excess of Cy7 or QSY9 and 1.6-fold excess of ATTO612Q quenchers per binding site of streptavidin was needed. Since the concentration of Eu–SA was 4 nmol L−1 and the labeling degree was 4.2, the europium concentration was approximately 16.8 nmol L−1 . In the case of one quencher being capable of completely quenching the emission energy of one europium(III)-chelate, the theoretical quenching for the quencher concentration of 5 nmol L−1 would be 30%. However, the observed quenching efficiencies with quencher concentration of 5 nmol L−1 for ATTO612Q, QSY9 and Cy7 were 66, 68 and 74%, respectively. Since the quenching efficiency is relative to the distance between the quencher dye and the europium(III)-chelate, the fluorescence lifetime of those europium(III)-chelates that are close to quencher is diminished completely. The fluorescence lifetime of those chelates that are further from the quencher dye are only somewhat decreased and some fluorescence can still be measured. In conclusion, according to these results one quencher dye can affect the fluorescence of more than one europium(III)-chelate.
Fig. 3. Standard curves with standard error bars of homogeneous noncompetitive biotin assays done with different quenchers, biotin–QSY9 (), biotin–Cy7 (䊉) and biotin–ATTO612Q (). The dotted lines indicate the detections limits of the assays.
in order to get an increase in the signal. Hence, the results below 0.2 nmol L−1 of biotin concentration are at background level (results not shown). The high quenching efficiency of the quenchers also necessitates the proportion of the response to concentration changing when the biotin concentration is altered and, therefore, the standard curves are not linear. In the case of monovalent binders this problem would not exist. The lowest limits of detection were calculated as concentrations that equate 3% of the background signal. Hence, the practical ranges for the assays were: 0.2–2, 0.25–2 and 0.5–2 nmol L−1 for Cy7, ATTO612Q and QSY9, respectively. The most suitable quencher dye appears to be Cy7. It has the best quenching efficiency profile (Fig. 2) and the highest specific signals compared to other quencher dyes (Fig. 3). Standard curve for traditional heterogeneous competitive assay is presented in Fig. 4. Dynamic range for the competitive assay is approximately 0.2–1 nmol L−1 . In theory non-competitive assay should be able to distinguish smaller analyte amounts from the background than a competitive assay. However, in the case of biotin assay the quadrovalent
3.3. Non-competitive biotin assay Standard curve for biotin assay was obtained by measuring signals for different biotin concentrations while the amounts of biotin–QSY9 and biotin–Cy7 conjugates were 20 nmol L−1 and biotin–ATTO612Q was 25 nmol L−1 , and Eu–SA was 3 nmol L−1 . Obtained standard curves for quenchers QSY9, Cy7 and ATTO612Q are presented in Fig. 3 As explained previously, one quencher can quench the emission of more than one europium(III)-chelate. This deteriorates the detection limit because multiple biotins per streptavidin are needed
Fig. 4. Standard curve with standard error bars for heterogeneous competitive assay for biotin.
T. Kokko et al. / Analytica Chimica Acta 585 (2007) 120–125
nature of streptavidin and the ability of one quencher dye to quench the emission of more than one europium(III)-chelate increases the smallest amount of biotin needed to create a distinguishable difference in the signal. However, the homogenous non-competitive assay does have somewhat larger practical range than the heterogeneous competitive assay. Also the actual concentration of biotin in non-competitive assay well is currently three-fold smaller since the sample volume is one third of the total volume (50 L of total 150 L), whereas in competitive assay 150 L of sample is used. If the sample volume is increased, lower biotin concentration could be measured with the non-competitive assay. Furthermore, the homogeneous assay requires only 35 min and no washing steps. The traditional heterogeneous assay requires 90 min and two washing steps. 4. Conclusions We have demonstrated a novel principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer between europium(III)-chelates and quencher dyes. Since the principle requires strong interaction between the analyte and the recognizing agent, the method was tested using biotin–streptavidin complex. The practical range was 0.2–2 nmol L−1 , when using the most suitable quencher, Cy7. Due to the high quenching efficiencies of the quenchers and the quadrovalent nature of streptavidin, the amount of biotin needed to observe an increase in the fluorescence was higher than expected. Therefore, with the non-competitive homogeneous
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concept, similar detection sensitivity was obtained than with the heterogeneous competitive assay with significantly faster assay time. However, the excellent quenching efficiencies of the quencher dyes improve the potentials of future studies where monovalent binders are used. Acknowledgement This study was supported by TEKES, the Finnish Funding Agency for Technology and Innovation. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
R.P. Ekins, Clin. Chem. 44 (1998) 2015. R.P. Ekins, F.W. Chu, Clin. Chem. 47 (1991) 1955. P. Saviranta, U.S. Patent 6,037,185 (2000). D. Brustolin, M. Sirtoli, G. Tarenghi, Ther. Drug Monit. 14 (1992) 72. P.R. Selvin, T.M. Rana, J.E. Hearst, J. Am. Chem. Soc. 116 (1994) 6029. T. F¨orster, Ann. Physik. 6 (1948) 55. R. Arai, H. Ueda, K. Tsumoto, W.C. Mahoney, I. Kumagai, T. Nagamune, Protein Eng. 13 (2000) 369. T. Pulli, M. H¨oyhty¨a, H. S¨oderlund, K. Takkinen, Anal. Chem. 77 (2005) 2637. P. von Lode, J. Rosenberg, K. Pettersson, H. Takalo, Anal. Chem. 75 (2003) 3193. H. Takalo, E. H¨anninen, J. Kankare, Helv. Chim. Acta 76 (1993) 877. H. Takalo, V.-M. Mukkala, H. Mikola, P. Liitti, I. Hemmil¨a, Bioconjugate Chem. 5 (1994) 278. K. Kuningas, T. Rantanen, U. Karhunen, T. L¨ovgren, T. Soukka, Anal. Chem. 77 (2005) 2826. M. Latva, I. Hemmil¨a, K. Blomberg, P. Hurskainen, U.S. Patent 5,998,146 (1999).
Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer Tiina Kokko ∗ , Leena Kokko, Tero Soukka, Timo L¨ovgren Department of Biotechnology, University of Turku, Tykist¨okatu 6A, 6th Floor, FIN-20520 Turku, Finland Received 16 October 2006; received in revised form 12 December 2006; accepted 13 December 2006 Available online 19 December 2006
Abstract A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin–quencher is bound to Eu–streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher–biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2–2 nmol L−1 . In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay. © 2006 Elsevier B.V. All rights reserved. Keywords: Fluorescence resonance energy transfer; Homogeneous; Non-competitive; Hapten
1. Introduction Haptens are low molecular weight analytes, e.g. many drugs, steroids, metabolites and pollutants, which are too small to be recognized simultaneously by two binders (for example, antibodies). Many of the traditional non-competitve assay techniques, however, require the analyte to be bound to two different binders in order to be detected. First the analyte is bound to the capture antibody and then detected with another analytic spesific labeled antibody. Therefore, the measured signal is obtained from those labeled recognizing agents, which are bound to the analyte. Thus, the signal increases with the concentration of the analyte. Since haptens are not suitable for two-site assays, most of the assays designed to measure haptens are competitive, where the analyte to be measured is competing with a labeled analyte analogue for the binding sites of a single recognizing agent. Thus, in competitive assay the signal is measured from those labeled analyte analogues that are bound to the recognizing agent. Increasing the amount of analyte decreases the amount
∗
Corresponding author. Tel.: +358 2 333 8067; fax: +358 2 333 8050. E-mail address: [email protected] (T. Kokko).
0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.12.021
of bound labeled analyte analogue, which means that increasing the amount of analyte decreases the obtained signal. Sensitivity can be defined as the smallest amount of analyte, which generates a distinguishable difference in the signal from the background (zero amount of analyte) [1,2]. In competitive assays the signal at zero concentration is high whereas in noncompetitive assays the signal at zero amount of analyte is small. When the concentration is slightly elevated from zero only a small change in the signal occurs. It is typically easier to detect a small change from small signals than in high signals. Hence, the non-competitive assays are capable of detecting smaller changes in analyte concentrations and are, therefore, considered to have better sensitivity than competitive assays [3]. Since non-competitive assays have better sensitivity than competitive assays, quite a few different non-competitive assay techniques for haptens have been designed. For example, in immunometric assays antibodies bound to analyte are separated from those antibodies that are not bound to analyte. After the separation the analyte-bound antibodies are measured. A non-competitive digoxin assay uses digitoxigenin-coupled polyacrylamide beads to separate the unbound antibodies [4]. These techniques rely on the laborious physical separation step, so they are time consuming to do.
T. Kokko et al. / Analytica Chimica Acta 585 (2007) 120–125
Homogeneous assays are separation free, and, therefore, less tedious to perform than heterogeneous assay. Fluorescence resonance energy transfer (FRET) is a commonly used technique in homogeneous assays. When the donor and the acceptor are in close proximity with each other, the non-radiative energy transfer occurs from the excited donor molecule to the acceptor [5]. In addition to small distance, an overlapping of the emission spectrum of the donor with the excitation spectrum of the acceptor is also required [6]. During measurement either the emission of the acceptor or the fluorescence of the donor, in which case the acceptor acts as a quencher, is measured. For example, Arai et al. has demonstrated a FRET based noncompetitive open sandwich assay where separate VH and VL fragments are labeled with donor and acceptor. FRET can occur only if the antigen has induced heterodimerization between VH and VL fragments thus bringing the donor and acceptor in close proximity. However, this assay requires a suitable antibody fragment that has a weak VH –VL interaction when the antigene is not bound to it [7]. Another FRET method was described by Pulli et al. which utilizes anti-immuno complex (anti-IC) Fab fragments [8]. In that assay europium labeled antimorfine and Cy5-labeled anti-IC Fab are in close enough proximity for FRET only when the antimorfine is bound to the antigen. However, anti-IC antibodies are rather difficult to develop. This article presents a homogeneous non-competitive assay for biotin. Moreover, the objective is to demonstrate a principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer for a small analyte, which can only be recognized by one binder at a time. Thus, biotin–streptavidin complex was used as a model system. Since the interaction of biotin and streptavidin is strong (Kd ∼ 10−15 mol L−1 ), it is ideal for testing this type of protocol. The assay requires only one binder/recognizing agent, which is labeled with europium chelate, and an analyte analogue, which is conjugated to quencher dye. The assay utilizes FRET to quench the fluorescence of those europium(III) labeled streptavidins that are not occupied by biotin of the sample. 2. Experimental 2.1. Measurement buffer and instrument Buffer for all assays and dilutions contained 0.05 mol L−1 Tris–HCl, pH 7.75, 0.9% (w/v) NaCl, 0.05% (w/v) NaN3 , 0.01% (v/v) Tween 40, 0.05% (w/v) bovine-␥-globulin, 20 mol L−1 diethylenetriaminepentaacetate (DTPA) and 0.5% (w/v) bovine serum albumin (BSA) (purchased from Innotrac Diagnostics, Turku, Finland). Fluorescence was measured using time-resolved fluorescence mode at Victor 1420 Multilabel counter from Wallac, Perkin-Elmer Life and Analytical Sciences (Turku, Finland). Excitation and measuring wavelengths were 340 and 615 nm, respectively. Delay time and measurement time were both 400 s. All the washings and aspirations of the wells were done using 1296-026 Delfia Platewash. For the shakings of the Plates
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1296-003 Delfia Plateshake was used. Both of these instruments were acquired from Wallac, Perkin-Elmer Life and Analytical Sciences. 2.2. BSA-treated microtitration plates All assays were made in Low Fluor Maxisorp 96-well microtitration plates purchased from Nunc (Roskilde, Denmark). Wells were treated with BSA prior to the assays to prevent non-specific binding by adding 250 L of solution containing 0.1% (w/v) BSA (Bioreba, Switzerland), 0.1% (w/v) Germall II (ISP, Wayne, NJ, USA) and 3% (w/v) trehalose (Sigma–Aldrich, St. Louis, MO, USA) in 0.05 mol L−1 Tris–HCl buffer, pH 7.2. Plates were incubated for 1 h, in room temperature with low shaking. After incubation wells were aspirated and dried. 2.3. Streptavidin coated low capacity wells 96-well Low Fluor Maxisorp microtitration plates were coated with streptavidin in order to make low capacity biotin binding wells suitable for competitive biotin assay. Into the wells 100 L of 0.05 mol L−1 citric acid and 0.1 mol L−1 sodium phosphate, pH 5.0, containing 0.2 ng L−1 streptavidin was added. Plates were incubated over night at +35 ◦ C in moist conditions. After incubation wells were washed twice and 250 L of saturation solution (50 mmol L−1 Tris, 0.15 mol L−1 NaCl, 0.05%, w/v, NaN3 , 6%, w/v, sorbitol, 0.2%, w/v, bovine serum albumin, pH 7.0) was added. Plates were again incubated over night at room temperature in moist conditions. After incubation wells were aspirated and stored +4 ◦ C. 2.4. Europium(III)-chelate labeled streptavidin Streptavidin, purchased from Societa Prodotti Antibiotici (Milano, Italy), was labeled with a long lifetime fluorescent 9-dentate europium(III)-chelate (2,2 ,2 ,2 -{[2(4-isothiocyanatophenyl)ethylimino]-bis(methylene)bis{4{[4-(␣-galactopyranoxy)phenyl]ethynyl}-pyridine-6,2diyl}-bis(methylenenitrilo)}tetrakis(acetato)europium(III)) [9]. Streptavidin (480 g) was dissolved in water and 5, 10 or 15fold molar excess of europium(III)-chelate (dissolved in water) was added. pH was adjusted by adding 30 L of 1 mol L−1 carbonate buffer, pH 9.8, and the total volume of the reaction was adjusted with water to 600 L. Reactions were incubated over night at room temperature. After incubation reactions were purified with gel filtration using Superdex 200 Matrix (Pharmacia Biotech, GE Healthcare, Fairfield, CT, USA). Buffer in the purifications was TSA (50 mmol L−1 Tris, 0.9%, w/v, NaCl and 0.5%, w/v, NaN3 , all purchased from Sigma–Aldrich), pH 7.75. After purification quantity of streptavidin was calculated from absorbance at 280 nm after subtracting the absorbance caused by the europium(III)-chelate. The amount of europium was measured by comparing the fluorescence of the labeling reaction to the fluorescence of known europium standard.
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Purified product was diluted in Delfia Enhancement Solution (purchased from Wallac, Perkin-Elmer Life and Analytical Sciences) and incubated for 30 min before measurement. Labeling degrees for streptavidins labeled with europium(III)-chelate were calculated as europium chelates per streptavidin. 2.5. Biotin–quencher conjugates N-(2-Aminoethyl)biotinamide, hydrobromide was purchased from Molecular Probes Inc. (Eugene, OR, USA). Cy5-NHS and Cy7-NHS esters were purchased from Amersham Biosciences, GE Healthcare. Alexa Fluor 680 carboxylic acid succinimidyl ester, dabcyl (4-((4-(dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester), QSY7 and QSY9 carboxylic acids, succinimidyl esters were acquired from Molecular Probes Inc. and ATTO612Q-NHS-ester from Atto-Tec (Siegen, Germany). Approximately 0.5 mg of quencher dye was dissolved in 50 mmol L−1 carbonate buffer, pH 9.0 and 20% (v/v) of DMF (N,N-dimethylformamide, Sigma–Aldrich) was added. Approximately 10-fold molar excess of N-(2-aminoethyl)biotinamide, hydrobromide was dissolved in 50 mmol L−1 carbonate buffer, pH 9.0. Quencher dye and biotin solutions were mixed. Reactions were protected from light and incubated at +35 ◦ C over night. Reactions were frozen until purification. Biotin–quencher conjugates were purified with RP-HPLC using equipment from Pharmacia, GE Healthcare together with Genesis C18 column from Jones Chromatography (Grace Vydac, Hesperia, CA, USA). Gradient purification was done using 50 mmol L−1 triethylammonium acetate buffer (TEAA; Fluka Biochemica, Steinheim, Switzerland) in water and 50 mmol L−1 TEAA in acetonitrile (from J.T. Baker, Phillipsburg, NJ, USA) as buffers. The amount of acetonitrile was increased from 30 to 100% in 30 min. After purification the concentrations of biotin–quencher conjugates were measured with absorbance. The molar absorptivities and the measuring wavelengths (absorbance maximum of the quencher dyes) were provided by the manufacturer. 2.6. Measurement of quenching efficiency Quenching efficiencies of different quenchers were tested by finding the maximum and minimum signal for each quencher. Volume of 50 L of either 4 nmol L−1 or 2 nmol L−1 Eu–SA (labeling degree 4.2) in assay buffer was added to BSAtreated microtitration wells. Thereafter, 50 L of 5 mol L−1 d-biotin (from Sigma–Aldrich) in assay buffer or assay buffer without biotin (background) was added to obtain maximum and minimum signals, respectively. Wells were incubated in room temperature for 30 min with low shaking. After incubation 50 L of 50 nmol L−1 biotin–quencher conjugate was added and incubation was continued in room temperature for 15 min with low shaking. Fluorescence of the europium(III)chelate was measured using Victor 1420 Multilabel Counter. To optimize the needed biotin–quencher concentration, titrations were done using biotin–quencher conjugate concentrations from 0 to 50 nmol L−1 . Protocol was the same as described above.
2.7. Heterogeneous competitive assay The heterogeneous competitive assay was used to characterize biotin–quencher conjugations and as a comparison for developed homogeneous non-competitive assay. 2-(((N-(Biotinoyl)amino)hexanoyl)amino)ethylamine was purchased from Molecular Probes and conjugated with europium chelate of 4-[2-(4-aminophenyl)ethynyl]-2,6-bis {[N,N-bis(carboxymethyl)-amino]methyl}pyridine [10,11] as described by Kuningas et al. [12]. Streptavidin low capacity wells were prewashed once and 150 L of d-biotin standards from 0.05 nmol L−1 to 0.5 mol L−1 or appropriately diluted samples were added into the wells. Wells were then incubated in room temperature, with low shaking for 25 min. Thereafter, 50 L of europium–biotin conjugate of 85 nmol L−1 was added and incubation was continued for 45 min in room temperature and with low shaking. After incubation wells were washed four times. Delfia Enhancement Solution (200 L) (purchased from Wallac, Perkin-Elmer Life and Analytical Sciences) was added and wells were incubated for 20 min in room temperature with low shaking. Fluorescence of the europium(III)-chelate was measured with Victor 1420 Multilabel Counter. 2.8. Homogeneous non-competitive biotin assay The biotin assay was done using QSY9, Cy7 and ATTO612Q quenchers conjugated with biotin. First 50 L of 3 nmol L−1 Eu–SA and 50 L of biotin standards from 0 to 2 nmol L−1 with two replicas were added to BSA-treated wells. After 30 min of incubation 50 L of 20 nmol L−1 biotin–QSY9 and biotin–Cy7 or 25 nmol L−1 biotin–ATTO612Q was added. Fluorescence of the europium was measured after 5 min of incubation with Victor 1420 Multilabel Counter. 3. Results and discussion The different situations depending on the amount of biotin during measurement are presented in Fig. 1 Streptavidin labeled with europium(III)-chelate (Eu–SA) has emission at 615 nm when excited with 340 nm. Maximum signal of the assay is obtained when four biotins are bound to Eu–SA, hence no quenching occurs. If biotin has bound to some of the binding sites of Eu–SA and the rest are occupied by the biotin–quencher conjugates the emission of europium is decreased. This is due to fluorescence resonance energy transfer, which can occur when the donor (europium(III)-chelate) and the acceptor (quencher dye molecule) are in close proximity, resulting in quenching the emission of europium. If all the binding sites of Eu–SA are occupied by the biotin–quencher conjugate, maximum quenching is observed. This also represents the background of the assay. Interaction between biotin and streptavidin is strong (Kd ∼ 10−15 mol L−1 ) and therefore biotin does not easily dissociate from streptavidin. During the homogeneous assay the biotin to be measured was allowed to interact with the Eu(III)-chelate labeled streptavidin before the biotin–quencher conjugate was added. Since the biotin to be measured did not significantly dissociate from the streptavidin, there was no com-
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Fig. 1. Different situations depending on the amount of biotin bound to streptavidin during measurement. (A) Streptavidin labeled with europium(III)-chelate emits at 615 nm when excitated at 340 nm. (B) When streptavidin is bound to four biotin maximum europium signal is obtained. (C) When some of the binding sites are not bound to biotin, biotin–quencher complex will bind to free biotin binding sites. Hence, portion of the fluorescence of europium(III)-chelate is quenched. (D) All the binding sites are occupied by biotin–quencher conjugate, hence maximum amount of the fluorescence is quenched.
petition over streptavidin binding sites between the biotin and the biotin–quencher conjugate. Thus, the biotin–quencher could only bind to those binding sites that were not already occupied by biotin. 3.1. Reagents Streptavidin was labeled with intrinsically fluorescent 9dentate europium(III)-chelate. The obtained labeling degrees were 3.2, 4.2 and 6.3 for 5, 10 and 15-fold excess of europium(III)-chelate in labeling reactions, respectively. One streptavidin is capable of binding four biotins and each biotin is conjugated with one quencher molecule. Thus, the maximum number of quenchers per streptavidin is four. Therefore, Eu–SA with labeling degree of 4.2 was chosen for the assay to attain in average one quencher molecule per one europium(III)-chelate. The fractions of purification of conjugation reactions of biotin to quencher dyes were characterized with absorbance measurements and heterogeneous competitive biotin assay. Absorbance measurements indicated the amount of quencher and heterogeneous competitive biotin assay was used to measure the amount of biotin in the fractions. Fractions that contained both biotin and quencher dye were selected. 3.2. Quenching efficiencies Quenching efficiencies for different quencher conjugates were tested and results are presented in Table 1. Quenching efficiencies were calculated using Eq. (1): Quenching efficiency =
maximum signal − signal of zero dose × 100% maximum signal
(1)
The maximum signal of the assay was obtained when there was a high excess of free biotin. Thus, no biotin–quencher conjugate was able to bind to the europium(III)–streptavidin and
the emission of europium(III)-chelate was not quenched (situation B in Fig. 1). The background signal (=signal of zero dose) was measured when there was no free biotin in the well. Thus, all of the binding sites of streptavidin were occupied by biotin–quenchers (situation D in Fig. 1). As can be seen from Table 1, different quenchers were able to quench slightly different amounts of the fluorescence of europium(III)-chelate. The best quenchers, QSY9, QSY7, Cy7 and ATTO612Q, all quenched nearly 100% of the signal when biotin–quencher concentration was 50 nmol L−1 . All of these dyes, except Cy7, are non-fluorescent, hence designed to quench fluorescence of other fluorophores. In addition, Cy7 is the only quencher, whose excitation spectrum does not overlap with the main emission peak (615 nm) of europium(III)-chelate. However, the excitation spectrum of the Cy7 does overlap with other parts of the emission spectrum of europium(III)-chelate, e.g. local emission maximum at 700 nm, which enables appropriate quenching [13]. QSY9 and QSY7 have similar chromophore structures differing only in their solubility. Therefore, QSY9, Cy7 and ATTO612Q were chosen for more detailed study in order to find possible differences among them. Different amounts of biotin–quenchers were used to detect the amount needed for maximum quenching efficiency. Using 4 nmol L−1 Table 1 Quenching efficiencies of different quenchers (biotin-quencher concentration 50 nmol L−1 ) Quencher
Bio-dabcyl Bio-ATTO612Q Bio-QSY9 Bio-QSY7 Bio-Cy5 Bio-Cy7 Bio-Alexa Fluor 680
Eu–streptavidin concentrations 2 nmol L−1
4 nmol L−1
90 98 97 97 67 96 78
90 97 97 97 72 97 80
All the values are given in percentage.
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Fig. 2. Titration of the quenching efficiencies with different amounts of quencher–biotin conjugate with standard error bars. biotin–QSY9 (), biotin–Cy7 (䊉) and biotin–ATTO612Q (). Maximum quenching efficiency was achieved with 20 nmol L−1 of biotin–Cy7 and biotin–QSY9 and 25 nmol L−1 of biotin–ATTO612Q.
Eu–SA, with labeling degree of 4.2, the quenching was titrated with quencher concentrations from 0 to 50 nmol L−1 . Titration curves are presented in Fig. 2. Maximum quenching was obtained for Cy7 and QSY9 at 20 nmol L−1 and for ATTO612Q at 25 nmol L−1 . Thus, in order to obtain maximum quenching, with presented incubation times, 1.25-fold excess of Cy7 or QSY9 and 1.6-fold excess of ATTO612Q quenchers per binding site of streptavidin was needed. Since the concentration of Eu–SA was 4 nmol L−1 and the labeling degree was 4.2, the europium concentration was approximately 16.8 nmol L−1 . In the case of one quencher being capable of completely quenching the emission energy of one europium(III)-chelate, the theoretical quenching for the quencher concentration of 5 nmol L−1 would be 30%. However, the observed quenching efficiencies with quencher concentration of 5 nmol L−1 for ATTO612Q, QSY9 and Cy7 were 66, 68 and 74%, respectively. Since the quenching efficiency is relative to the distance between the quencher dye and the europium(III)-chelate, the fluorescence lifetime of those europium(III)-chelates that are close to quencher is diminished completely. The fluorescence lifetime of those chelates that are further from the quencher dye are only somewhat decreased and some fluorescence can still be measured. In conclusion, according to these results one quencher dye can affect the fluorescence of more than one europium(III)-chelate.
Fig. 3. Standard curves with standard error bars of homogeneous noncompetitive biotin assays done with different quenchers, biotin–QSY9 (), biotin–Cy7 (䊉) and biotin–ATTO612Q (). The dotted lines indicate the detections limits of the assays.
in order to get an increase in the signal. Hence, the results below 0.2 nmol L−1 of biotin concentration are at background level (results not shown). The high quenching efficiency of the quenchers also necessitates the proportion of the response to concentration changing when the biotin concentration is altered and, therefore, the standard curves are not linear. In the case of monovalent binders this problem would not exist. The lowest limits of detection were calculated as concentrations that equate 3% of the background signal. Hence, the practical ranges for the assays were: 0.2–2, 0.25–2 and 0.5–2 nmol L−1 for Cy7, ATTO612Q and QSY9, respectively. The most suitable quencher dye appears to be Cy7. It has the best quenching efficiency profile (Fig. 2) and the highest specific signals compared to other quencher dyes (Fig. 3). Standard curve for traditional heterogeneous competitive assay is presented in Fig. 4. Dynamic range for the competitive assay is approximately 0.2–1 nmol L−1 . In theory non-competitive assay should be able to distinguish smaller analyte amounts from the background than a competitive assay. However, in the case of biotin assay the quadrovalent
3.3. Non-competitive biotin assay Standard curve for biotin assay was obtained by measuring signals for different biotin concentrations while the amounts of biotin–QSY9 and biotin–Cy7 conjugates were 20 nmol L−1 and biotin–ATTO612Q was 25 nmol L−1 , and Eu–SA was 3 nmol L−1 . Obtained standard curves for quenchers QSY9, Cy7 and ATTO612Q are presented in Fig. 3 As explained previously, one quencher can quench the emission of more than one europium(III)-chelate. This deteriorates the detection limit because multiple biotins per streptavidin are needed
Fig. 4. Standard curve with standard error bars for heterogeneous competitive assay for biotin.
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nature of streptavidin and the ability of one quencher dye to quench the emission of more than one europium(III)-chelate increases the smallest amount of biotin needed to create a distinguishable difference in the signal. However, the homogenous non-competitive assay does have somewhat larger practical range than the heterogeneous competitive assay. Also the actual concentration of biotin in non-competitive assay well is currently three-fold smaller since the sample volume is one third of the total volume (50 L of total 150 L), whereas in competitive assay 150 L of sample is used. If the sample volume is increased, lower biotin concentration could be measured with the non-competitive assay. Furthermore, the homogeneous assay requires only 35 min and no washing steps. The traditional heterogeneous assay requires 90 min and two washing steps. 4. Conclusions We have demonstrated a novel principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer between europium(III)-chelates and quencher dyes. Since the principle requires strong interaction between the analyte and the recognizing agent, the method was tested using biotin–streptavidin complex. The practical range was 0.2–2 nmol L−1 , when using the most suitable quencher, Cy7. Due to the high quenching efficiencies of the quenchers and the quadrovalent nature of streptavidin, the amount of biotin needed to observe an increase in the fluorescence was higher than expected. Therefore, with the non-competitive homogeneous
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concept, similar detection sensitivity was obtained than with the heterogeneous competitive assay with significantly faster assay time. However, the excellent quenching efficiencies of the quencher dyes improve the potentials of future studies where monovalent binders are used. Acknowledgement This study was supported by TEKES, the Finnish Funding Agency for Technology and Innovation. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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