Miniaturization of a Homogeneous Fluorescence Immunoassay Based

The miniaturization of a homogeneous competitive im- munoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plasti...
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Anal. Chem. 2001, 73, 5172-5179

Miniaturization of a Homogeneous Fluorescence Immunoassay Based on Energy Transfer Using Nanotiter Plates as High-Density Sample Carriers Uwe Schobel,*,†,‡ Ingrid Coille,† Andreas Brecht,§,| Michael Steinwand,⊥ and Gu 1 nter Gauglitz†

Institute of Physical and Theoretical Chemistry, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany, DC-LCPPM, EPFL, CH-1014 Lausanne-Ecublens, Switzerland, Applied Biosystems PE Deutschland GmbH, ¨ berlingen, Germany Askaniaweg 6, D-88662 U

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 × 2 cm2 containing 25 × 25 wells, corresponding to ∼15 000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelengthexcitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 µg/L, or 1.11 × 10-16 mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 µg/L, or 1.87 × 10-13 mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 µg/L. High-throughput screening (HTS) of analytical processes has been one of the main research subjects in clinical diagnostics,1 genome and proteome research,2 and pharmaceutical screening3 during the past decade. Among them, pharmaceutical screening * To whom correspondence should be addressed. Phone: [49] (0)931 30 986 30. Fax: [49] (0)931 52650. E-mail: [email protected]. † Institute of Physical and Theoretical Chemistry. § DC-LCPPM. ⊥ Applied Biosystems PE Deutschland GmbH. ‡ Current address: Institut Virion\Serion GmbH, Konradstraβe 1, D-97072 Wu ¨ rzburg, Germany. | Current address: Cytion SA, Chemin de Croisettes 22, 1022 Epalinges, Switzerland.

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has currently the most pressing need for improvement in HTS technology. The explosion of the number of available compounds fueled by combinatorial chemistry and the accelerated identification of new pharmaceutical targets catalyzed by genomics has led to a strong demand for increased throughput in HTS programs. In 1998, for example, HTS laboratories read an average of 55 000 wells per week. In 2003, the expected average reading will increase by more than 500% to 350 000 wells per week, with peak throughput rates of 100 000 wells per day (ultra HTS).4 The necessity to simultaneously conserve or reduce consumable costs and samples has inevitably induced the miniaturization of HTS.5,6 The traditional 96-well microtiter plate, with assay volumes from 50 to 250 µL per well, is currently in the process of being replaced by 384- and 1536-well plates with 50-1 µL per well. Assays performed on plates with larger numbers of smaller wells, including 3456, 6144 and 9600 wells with volumes from 2 µL to 0.2 µL are under investigation.7-9 This reduction in scale has led to the development of new equipment. In response, several manufacturing techniques for the high-density microvolume microplate devices, such as injection molding, polymer casting and drilling, silicon etching and laser ablation have been developed,10-12 and are readily available. A range of new micropipetting systems based on ink-jet principles (thermal, solenoid, or piezoelectric-actuated) has been introduced for delivery of pico- to nanoliter volumes of samples or reagents.13-16 A diverse range of bioanalytical techniques has been adapted to the new high-density, low-volume microwell format. There has (1) Robinson, B. W. S.; Erle, D. J.; Jones, D. A.; Shapiro, S.; Metzger, W. J.; Albelda, S. M.; Parks, W. C.; Boylan, A. Thorax 2000, 55, 329-339. (2) Emili, A. Q.; Cagney, G. Nature Biotechnol. 2000, 18, 393-397. (3) Beggs, M.; Block, H.; Diels, A. J. Biomol. Screening 1999, 4, 143-149. (4) Fox, S.; Farr-Jones, S.; Yund, M. A. J. Biomol. Screening 1999, 4, 183-186. (5) Stahl, W. J. Biomol. Screening 1999, 4, 117-118. (6) Burbaum, J. J. J. Biomol Screening 2000, 5, 5-8. (7) Mere, L.; Bennett, T.; Coassin, P.; England, P.; Hamman, B.; Rink, T.; Zimmerman, S.; Negulescu, P. Drug Discovery Today 1999, 4, 363-369. (8) Schullek, J. R.; Butler, J. H.; Ni, Z.-J.; Chen, D.; Yuan, Z. Anal. Biochem. 1997, 246, 20-29. (9) Oldenburg, K. R.; Zhang, J.-H.; Chen, T.; Maffia, A., III.; Blom, K. F.; Combs, A. P.; Chung, T. D. Y. J. Biomol. Screening 1998, 3, 55-62. (10) Pitts, W. K.; Martin, M. D.; Belolipetskiy, S.; Crain, M.; Hutchins, J. B.; Matos, S.; Walsh, K. M.; Solberg, K. Nucl. Instrum. Methods Phys. Res., Sect. A 1999, 438, 277-281. (11) Prins, M. W.; Weekamp, J. M.; Giesberg, J. B. J. Microchem. Microeng. 1999, 9, 362-363. (12) Duffy, D. C.; Gillis, H. L.; Lin, J.; Sheppard, N. F., Jr.; Kellogg, J. Anal. Chem. 1999, 71, 4669-4678. 10.1021/ac010456e CCC: $20.00

© 2001 American Chemical Society Published on Web 09/28/2001

been relatively little progress in formatting multistep separation assays, such as enzyme immunoassays17 or immunofluorimetric assays,18 to a high-density microwell format. Most of the reported miniaturized assays are so-called simple “mix-and-measure”-type homogeneous assays, such as scintillation proximity assays,19 fluorescence correlation spectroscopy based assays,20 fluorescence polarization assays,21 and fluorescence resonant energy transfer assays.7 Assays relying on fluorescence detection are known to be highly sensitive, but the achievable limit of detection is often restricted by background signals, such as light scattering and sample autofluorescence. Because of reduced light scattering at longer wavelengths and minimal sample autofluorescence, particularly above 600 nm, background signals can be minimized by using far-red excitable fluorescent dyes.22 Several classes of polymethine cyanine dyes that are excitable in the far-red (635-1100 nm), including heptamethine cyanine dyes,23 squarine cyanine dyes,24 and sulfoindocyanine dyes,25,26 have been developed. In a previous study, we demonstrated the suitability of the long-wavelength excitable sulfoindocyanine dyes Cy5 and Cy5.5, as a donoracceptor pair with an exceptionally high Fo¨rster distance R0 of 7.6 nm, for energy-transfer immunoassay (ETIA) application.27 This article describes the miniaturization of this homogeneous, competitive ETIA to a final assay volume of 70 nL (nano-ETIA). Disposable plastic nanotiter plates with dimensions of 2 × 2 cm2 (see Figure 1) containing 25 × 25 truncated pyramid shaped wells with a volume of 70 nL/well, corresponding to ∼15 000 wells on a traditional 96-well microtiter plate footprint, were used as the sample carrier and were manufactured by polymer casting. Sample handling was accomplished by using a piezoelectrically actuated micropipet. As a model system, an antibody-based assay for the detection of the pesticide simazine in drinking water was chosen. The assay characteristics of the optimized nano-ETIA are compared with the performance obtained with the ETIA in 96-well microtiter plates (micro-ETIA). Advantages and limitations of the nano-ETIA are presented and discussed. (13) Laurell, T.; Wallman, L.; Nilsson, J. J. Micromech. Microeng. 1999, 9, 369376. (14) Harris, T. M.; Massimi, A.; Childs, G. Nature Biotechnol. 2000, 18, 384385. (15) Niwa, O.; Kurita, R.; Liu, Z.; Horiuchi, T.; Torimitsu, K. Anal. Chem. 2000, 72, 949-955. (16) Roda, A.; Guardigli, M.; Russo, C.; Pasini, P.; Baraldini, M. BioTechniques 2000, 28, 492-496. (17) Dzgoev, A.; Mecklenburg, M.; Larsson, P.-O.; Danielsson, B. Anal. Chem. 1996, 68, 3364-3369. (18) Ha¨rma¨, H.; Lehtinen, P.; Takalo, H.; Lo ¨vgren, T. Anal. Chim. Acta 1999, 387, 11-19. (19) Beveridge, M.; Park, Y.-W.; Hermes, J.; Marenghi, A.; Brophy, G.; Santos, A. J. Biomol. Screening 2000, 5, 205-211. (20) Moore, K. J.; Turconi, S.; Ashman, S.; Ruediger, M.; Haupts, U.; Emerick, V.; Pope, A. J. J. Biomol. Screening 1999, 4, 335-353. (21) Kowski, T. J.; Wu, J. J. Comb. Chem. High Throughput Screening 2000, 3, 437-444. (22) Cullander, C. J. Microsc. 1994, 176, 281-286. (23) Mason, J. C.; Patonay, G. J. Heterocyclic Chem. 1996, 33, 1685-1688. (24) Oswald, B.; Lehmann, F.; Simon, L.; Terpetschnig, E.; Wolfbeis, O. Anal. Biochem. 2000, 280, 272-277. (25) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis C. J.; Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105-111. (26) Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A. S. Bioconjugate Chem. 1996, 7, 356-362. (27) Schobel, U.; Egelhaaf, H.-J.; Brecht, A.; Oelkrug, D.; Gauglitz, G. Bioconjugate Chem. 1999, 10, 1107-1114.

Figure 1. NTP with 25 × 25 cavities and a volume of 70 nL/well in comparison to a 96-well microtiter plate. The scale at the lower edge of the photograph is in centimeters.

EXPERIMENTAL SECTION Materials. N-Hydroxysuccinimidyl (NHS) esters of Cy5 (monofunctional) and Cy5.5 (bisfunctional) were purchased from Amersham Life Science (Braunschweig, Germany). Solvents of analytical grade, Sephadex G-25 columns, polyethyleneimine (50% v/v aqueous solution), and all other common chemicals and biochemicals were purchased from Sigma Chemical Co. (Deisenhofen, Germany) and Fluka (Neu-Ulm, Germany). The triazine herbicide simazine (2-chloro-4,6-bis(ethylamino)-1,3,5-triazin) was obtained from Riedel de Hae¨n AG (Seelze, Germany). Triazine derivatives 2-chloro-4-isopropylamino-6-carboxypentylamino-1,3,5triazine (atrazine caproic acid, ACA), 2-chloro-4-amino-6-carboxypentylamino-1,3,5-triazine (deisopropylatrazine caporoic acid, DeACA) and 2-chloro-4-isopropylamino-6-carboxyphenyl-p-amino1,3,5-triazine (atrazine benzoic acid, ABA) and the polyclonal antisimazine antibody (IgG) were received as a gift from Dr. Ram Abuknesha, GEC London. Drinking water samples were kindly provided by Institut Dr. Ja¨ger (Tu¨bingen, Germany). Buffers and standards were prepared using deionized water. [4-(2-Hyroxyethyl)-piperazino]-ethanesulfonic acid (HEPES) buffer, pH 7.4, containing 100 mM HEPES and 750 mM NaCl was used for performing the immunoassay. Sodium carbonate-bicarbonate buffer used for the synthesis of the conjugates contained 100 mM Na2CO3 and was adjusted to pH 9.7 using 100 mM NaHCO3. The simazine standard solutions were diluted in deionized water from a stock solution of c(simazine) ) 1 mg/mL in DMF in order to obtain a serial dilution ranging from 0.01 to 3000 µg/L over 12 dilution steps. Drinking water samples were fortified at levels ranging from 0.1 to 6.0 µg/L from the simazine stock solution. Nanotiter plates (NTP) with dimensions of 2 × 2 cm2 (see Figure 1) containing 25 × 25 truncated pyramid-shaped wells with a footprint of 0.6 × 0.6 mm2 and a volume of 70 nL/well were manufactured by polymer casting using acrylnitrile-butadienestyrene copolymer (Terulan 877M-71300) and were obtained from BIAS (Bremen, Germany). Transparent adhesive tape Arcare 7759 for sealing the filled NTP was a gift from Adhesive Research Inc. (Glen Rock, PA). Polystyrene 96-well microtiter plates were from Perkin-Elmer (U ¨ berlingen, Germany). Apparatus. Apparatus Used for Performing the Nano-ETIA. Sample handling was accomplished using a microdispensing Analytical Chemistry, Vol. 73, No. 21, November 1, 2001

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device developed in cooperation with BIAS, Bremen/Germany. Briefly, on a three-axis positioning system consisting of three linear stages of the M-500 series (Physik Instrumente, Berlin, Germany), a piezoelectrically actuated micropipet type TMP (GeSiM mbH, Dresden, Germany) was mounted by means of an aluminum holder. To control the generation of droplets by applying voltage pulses to the piezoceramic body of the pipet (amplitude, 70 V; pulse duration,50 µs; frequency, 200 Hz), a DOS software controlled module, “control unit multi-dos” (GeSiM mbH, Dresden, Germany), was used. Available drop volumes for single droplets were between 100 and 500 pl. For visualization of droplet shape, a stroboscope plug-in module (GeSiM mbH, Dresden, Germany) and a video camera were integrated into the dispensing device. The camera was mounted on an aluminum holder, enabling a continuous monitoring of the pipet. To avoid evaporation of the dispensed liquids, the NTP was handled in a closed humid chamber and cooled to the point of condensation by means of a peltier element. Fluorescence readout was performed using the scanning laserinduced fluorescence detector “BioScan” built as a functional module by Applied Biosystems (U ¨ berlingen, Germany). As excitation lightsource, a 20 mW He-Ne laser was used. The laser beam was guided over the NTP using a 2D scanner (ScanLab GmbH, Mu¨nchen, Germany) that was based on beam deflection using analogue galvano-driven mirrors and a dichroitic mirror (beam splitter) Q650LP (AHF Analysentechnik, Tu¨bingen, Germany). The emitted light was detected in the epifluorescent mode through imaging optics by a photomultiplier tube R5929 (Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany). Two filters, a long-pass filter EFLP 665 (Omega Optical Inc./Photomed GmbH, Seefeld, Germany) and a band-pass filter HQ 670/20 (AHF Analysentechnik, Tu¨bingen, Germany), were introduced in the emission beam path to block the laser. The device takes full images of the NTP. For recording a fluorescence image, the following system parameters were used: PMT voltage, 1000V; scan speed, 150 m/s; data interval (spacial resolution), 20 µm; and integration time, 100 µs. The obtained NTP image consisted of 1000 × 1000 pixels. The time required for scanning one NTP was 2.8 min. Data evaluation was performed by integrating the intensity values of the 30 × 30 pixel of each well using software from LLG (Go¨ttingen, Germany). All images were corrected for the inhomogeneous distribution of the fluorescence intensity over the area of a NTP. Apparatus Used for Performing the Micro-ETIA. Absorption spectra were recorded on a Zeiss Specord M500 spectrophotometer (Jena, Germany). Fluorescence data were obtained with a Perkin-Elmer LS-50 B luminescence spectrometer (U ¨ berlingen, Germany) equipped with a red-sensitive photomultiplier R928. Drinking water samples were analyzed in 96-well microtiter plates using the Perkin-Elmer luminescence plate reader LSR 200 (U ¨ berlingen, Germany). Procedure. Labeling of IgG and BSA with Cy5 and Cy5.5, Respectively. A 20-µL portion of reactive dye (for molar ratios, see Table 1) in absolute DMF was added to a solution of 500 µg of protein (3.3 nmol IgG or 7.2 nmol BSA) in 250 µL of sodium carbonate-bicarbonate buffer and mixed thoroughly by gentle vortexing. The reaction was incubated at room temperature for 5174 Analytical Chemistry, Vol. 73, No. 21, November 1, 2001

Table 1. Molar Ratio of Reactive Dye (TD)/Protein Used in the Labeling Procedure in Comparison with the Obtained Dye (TD)/Protein Ratio of the Conjugates

protein

reactive dye/TD

[reactive dye/TD]/ [protein]

[attached dye/TD]/ [protein]

IgG BSA BSA BSA-Cy5.5 (1:1.6) BSA-Cy5.5 (1:1.6) BSA-Cy5.5 (1:1.6) BSA-Cy5.5 (1:1.6) BSA-Cy5.5 (1:2.1) BSA-Cy5.5 (1:2.1)

Cy5-NHS Cy5.5-NHS Cy5.5-NHS ACA-NHS ACA-NHS ACA-NHS ACA-NHS ABA-NHS DeACA-NHS

20 10 18 7 15 20 40 35 40

3.7 1.6 2.1 0.4 1.3 1.8 3.6 3.8 3.5

30 min. Unconjugated dye was separated from protein-dye conjugate by gel permeation chromatography over Sephadex G-25 using HEPES buffer as the eluent. The dye-to-protein ratios were determined spectrophotometrically as described in ref 27 and are presented in Table 1. Labeling of the BSA-Cy5.5 Conjugates with Triazine Derivatives. A 3.76-mg (18 µmol) portion of dicyclohexylcarbodiimide (DCC) was added to a solution of 16.5 µmol of triazine derivative (TD) and 2.1 mg (18 µmol) of N-hydroxysuccinimide (NHS) in 200 µL of absolute N,N-dimethylformamide (DMF) kept on ice. The mixture was stirred for 6 h at room temperature and filtered to remove the precipitated dicyclohexylurea. From this solution, various amounts of reactive TD (for molar ratios see Table 1) were added to a solution of 400 µg (5.8 nmol) of BSA-Cy5.5 conjugate in 250 µL of sodium carbonate-bicarbonate buffer and mixed thoroughly by gentle vortexing. The reaction condition and duration, as well as the purification of the conjugate, were the same as described above. The TD concentrations of the conjugates were determined by means of reflectometric interference spectroscopy.28 The obtained TD/protein ratios are given in Table 1. Cross-Reactivity Determination. Cross-reactivity of the polyclonal IgG toward different structurally related triazine pesticide derivatives was determined using a label-free optical detection system based on reflectometric interference spectroscopy, as described in ref 29. Nano-ETIA Procedure. Surface Modification. NTP were coated with a 3% aqueous solution of polyethyleneimine for 12 h at 25 °C. After washing, the plate was incubated with 3 mg/mL BSA in HEPES buffer for 12 h at 25 °C, washed, and dried. Titration of Antibodies: To 450 µL of a serial dilution of the ACA-BSA-Cy5.5 conjugate (3.6:1:1.6), covering a concentration range between 0 and 30 µg/L, 50 µL of 200 nM IgG-Cy5 solution (final concentration, 20 nM) containing 100 µg/mL chicken egg albumin (OVA) as background protein was added. After 10 min incubation at 25 °C, 70 nL of the preincubated solutions was transferred into the cavities of the NTP, the plate was sealed, and the fluorescence intensities were read out. The ACA concentration yielding the maximum quenching was used for calibration. For comparison, these titration experiments were also performed in the micro-ETIA. (28) Piehler, J.; Brecht, A.; Giersch, T.; Hock, B.; Gauglitz, G. J. Immunol. Methods 1997, 201, 189-206. (29) Piehler, J.; Brecht, A.; Giersch, T.; Kramer, K.; Hock, B.; Gauglitz, G. Sensors Actuators B 1997, 38-39, 432-437.

Figure 2. Schematic representation of the nano-ETIA procedure: (I) after droplet shape optimization using stroboscopic illumination, sample, and labeled compounds are sequentially dispensed into the cavities of the NTP; (II) sealing of the NTP and incubation of the mixture for 10 min; and (III) readout of the fluorescence intensity (λexc ) 633 nm, λem ) 665-680 nm), which is modulated by ET from Cy5 to Cy5.5 (arrow).

Calibration Curves. A schematic representation of the immunoassay detection procedure is given in Figure 2. After optimization of the electrical parameters for the micropump by visualization of the droplet shape using a stroboscopic illumination, 56 nL of the simazine standard solution (0-3000 µg/L), 7 nL of the IgGCy5 conjugate, and 7 nL of the TD-BSA-Cy5.5 conjugate were sequentially dispensed into the wells (25 wells/simazine concentration, step I). For avoiding nonspecific binding, all of the conjugates were dissolved in HEPES buffer containing 100 µg/ mL OVA as background protein. The plate was sealed, incubated for 10 min at 25 °C (step II), and the fluorescence was recorded using the NTP reader BioScan (step III). The obtained image was analyzed as described above. As a reference signal, at each simazine standard concentration, the IgG-Cy5 fluorescence in the absence of TD-BSA-Cy5.5 (IR(standard)) was recorded. For data presentation, the fluorescence intensities (I) were divided by the maximum fluorescent value (I0). The I/I0 values were plotted against the simazine concentration and were fitted to a four-parameter logistic function. Precision profiles were calculated from the standard errors of the mean fluorescence responses for n replicates, as described in ref 30. Drinking Water Samples. Eight drinking water samples collected from six cities in the southern part of Germany were fortified to a concentration range from 2.0 to 6.0 µg/L and analyzed using the nano-ETIA. No sample pretreatment was required other than thorough degassing. Each water sample was aliquoted into 50 wells (56 nL/well), and 7 nL of IgG-Cy5 was added to each well. To 25 of these wells, 7 nL of ACA-BSA-Cy5.5 conjugate was added, but the remaining 25 wells received only 7 nL of the HEPES buffered OVA (100 µg/mL) solution (control signal, IC(matrix)) to determine the influence of the sample matrixes on the IgG-Cy5 fluorescence. To correct for matrix effects, the measured intensities (I(raw)) were multiplied by the quotient of the fluorescence values of the control, IC(matrix), and reference, IR(standard), solutions.

I ) I(raw) ×

IC(matrix) IR(standard)

(1)

GC/MS measurements of the fortified drinking water samples were kindly performed by Institut Dr. Ja¨ger, (Tu¨bingen, Germany).

Micro-ETIA Procedure. Titration of Antibodies. The same protocol as described for the nano-ETIA was employed to perform the titration. An IgG-Cy5 concentration of 10 nM (final concentration, 1 nM) and TD-BSA-Cy5.5 concentrations between 0 and 30 µg/L were used. After a 10-min incubation at 25 °C, emission spectra were recorded using the Perkin-Elmer LS-50 B, whereby the excitation monochromator was set at 647 nm, and the emission was detected from 662 to 800 nm. The TD concentration yielding the maximum quenching was used for calibration. Calibration Curves. Standard curves were obtained by adding first 50 µL of 1.5 µg/mL IgG-Cy5 conjugate and then 50 µL of TD-BSA-Cy5.5 conjugate to 400 µL of the simazine standard solution (from 0.01 to 3000 µg/L). To avoid nonspecific binding, all of the conjugates were dissolved in HEPES buffer containing 100 µg/mL OVA as background protein. After incubation for 20 min at 25 °C, the Cy5 fluorescence intensity was measured using the settings λexc ) 647 nm and λem ) 667 nm. As a reference signal at each simazine standard concentration, the IgG-Cy5 fluorescence in the absence of TD-BSA-Cy5.5 (IR(standard)) was recorded. Drinking Water Samples. Ten drinking water samples, collected from eight cities in the southern part of Germany, were fortified covering a concentration range from 0.1 to 2.2 µg/L and were analyzed as described above. To reduce nonspecific binding of the proteins to the well surface, the microtiter plates were coated by adding 300 µL of 3 mg/mL BSA dissolved in HEPES buffer to each well and incubating for 12 h at 25 °C. The plates were emptied, washed three times with HEPES buffer, and dried. Each water sample was aliquoted into six wells (200 µL/ well) and 25 µL of 10 nM IgG-Cy5 was added. Following a 10 min incubation at 25 °C, to three of these wells, 25 µL of ABABSA-Cy5.5 conjugate was added, but the remaining three wells received only 25 µL of the HEPES buffered OVA (100 µg/mL) solution (control signal, IC(matrix)) in order to determine the influence of the sample matrixes on the IgG-Cy5 fluorescence. The mixtures were incubated for 10 min at 25 °C. Fluorescence intensities were measured at λexc ) 640 nm and λem ) 667 nm using an integration time of 30 s. The evaluation of the fluorescence signals was performed as described above. (30) Dudley, R. A.; Edwards, P.; Ekins, R. P.; Finney, D. J.; McKenzie, I. G. M.; Raab, G. M.; Rodbard, D.; Rodgers, R. P. C. Clin. Chem. 1985, 31, 12641271.

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Figure 3. Titration of IgG-Cy5 (3 µg/mL) with increasing concentrations of ACA-BSA-Cy5.5 (0-30 µg/L) in NTP (volume of 70 nL). Left, picture of the filled NTP; right, evaluated fluorescence signals (n ) 12).

RESULTS AND DISCUSSION Optimization of the Nano-ETIA. Antibody Concentration. In a first experiment, the absolute limit of detection (LOD) of the NTP reader BioScan for the detection of the IgG-Cy5 conjugate was determined. The labeling ratio of the IgG-Cy5 conjugate was adjusted to ∼4, because in previous investigations, this value was found to yield the maximum fluorescence intensity/antibody.27,31 A serial dilution of this IgG-Cy5 conjugate, ranging from 1 to 100 nM, was dispensed into an NTP and analyzed. From the graph, showing a linear relation between the IgG-Cy5 concentration and the fluorescence intensity (data not shown), an LOD of 1.3 nM was determined. To obtain a sufficiently high signal-tonoise ratio the nano-ETIA was established by employing an antibody concentration of c(IgG) ) 20 nM. Signal Dynamic and Cy5.5/ACA Labeling Ratio. In the homogeneous ETIA, a triazine derivative (TD) attached to the BSA-Cy5.5 conjugate competes with the free analyte for a limited number of antibody binding sites. At low analyte concentrations, the TD-BSA-Cy5.5 conjugate is preferentially bound to the antibody binding sites. Because of the close proximity of Cy5.5 and Cy5 in the [Cy5-IgG]-[TD-BSA-Cy5.5] complex, the IgG-Cy5 fluorescence is efficiently quenched by energy transfer. A higher analyte concentration leads to a decreased occupation of the antibody binding sites by the TD conjugate. This results in an increased IgG-Cy5 fluorescence. As described previously,27 approximately 50% of the IgG-Cy5 quenching is due to Fo¨rstertype resonant energy transfer to the TD-BSA-Cy5.5 conjugate and results in sensitized fluorescence of the Cy5.5. The sensitized and the directly excited Cy5.5 fluorescence overlap the quenched IgG-Cy5 signal and, thus, limit the signal dynamic of the assay. Consequently, by employing the lowest possible Cy5.5 concentration, the highest ETIA signal dynamic is obtained. This can be achieved by using high-affinity TD and an optimized Cy5.5/TD labeling ratio. In the nano-ETIA, the immunizing hapten ACA was used as a high-affinity competitive TD. To assess the effect of the Cy5.5/ ACA ratio on the signal dynamic of the assay, four ACA-BSACy5.5 conjugates were synthesized with Cy5.5/ACA ratios ranging from 4 to 0.4 (see Table 1). With these conjugates, the maximum quenching of the IgG-Cy5 fluorescence was determined by means of titration experiments using the micro-ETIA set up (see the Experimental Section). ACA-BSA-Cy5.5 solutions covering (31) Schobel, U.; Egelhaaf, H.-J.; Fro ¨hlich D.; Brecht, A.; Oelkrug, D.; Gauglitz, G. J. Fluoresc. 2000, 10, 147-154.

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an ACA concentration range from 0 to 30 µg/L were prepared and preincubated with 20 nM IgG-Cy5. At Cy5.5/ACA ratios > 1, significant interference of the sensitized and directly excited Cy5.5 fluorescence with the quenched Cy5 signal was observed (data not shown). This limited the signal dynamic at the Cy5.5/ ACA ratios of 4 and 1.2 to 53% and 59%, respectively. The maximal signal dynamic of 64% could be obtained with Cy5.5/ACA ratios < 1 (at c(ACA) ) 30 µg/L), where only minor interference of the Cy5.5 fluorescence with the Cy5 signal occurred. Hence, the ACA-BSA-Cy5.5 conjugate with the lowest Cy5.5/ACA ratio (labeling ratio 3.6:1:1.6) was used to establish the nano-ETIA. ACA Concentration. The appropriate ACA concentration for the nano-ETIA was determined by performing a titration using the preincubated ACA-BSA-Cy5.5 and IgG-Cy5 solutions from above. A 70-nL portion of each solution was dispensed into 12 cavities of the NTP (in each second well). Figure 3 shows the obtained picture of the NTP (left) and the evaluated fluorescence signals (right). No contamination of the free wells could be observed, confirming the high positioning accuracy of the dispensed droplets. At an ACA concentration of 10 µg/L, a maximum fluorescence quenching of 47% was achieved. This decrease of signal dynamic by 17%, when compared to the results from the micro-ETIA (64% at c(ACA) ) 30 µg/L; see above), can be explained by a higher surface/volume ratio of the cavities of the NTP (76.3 l/cm), as compared to that of the 96-well microtiter plate (8.8 l/cm), leading to increased nonspecific adsorption of the proteins. This effect can be reduced by either choosing a different kind of plastic or by further improving the surface modification of the NTP. Nano-ETIA Calibration Curve. Using the optimized conditions, a calibration curve was recorded. An overview of the nanoETIA procedure is shown in Figure 2. Briefly, after optimization of the electrical parameters for the pipet by visualization of the droplet shape using a stroboscopic illumination, 56 nL of the simazine standard solution (0-3000 µg/L), followed by 7 nL of the IgG-Cy5 conjugate and 7 nL of the ACA-BSA-Cy5.5 conjugate are dispensed sequentially into the wells of a NTP. The time required for dispensing one solution in all 625 cavities of the NTP was ∼2 min. The liquid handling, that is, rinsing the pipet, aspirating the solution from the reservoir, controlling the droplet shape at the stroboscopic unit and positioning the pipet above the first well took another 50 s. Therefore, the total time required to dispense all solutions of a calibration curve, including the reference signals, was 17 min. Because of the closed humid

Figure 5. Precision profiles of the nano- and micro-ETIA calibration curves from Figure 4. Table 2. Analytical Parameters for the Nano-ETIA and Micro-ETIA Derived from the Calibration Curves in Figure 4B assay

IC50a [µg/L]

micro-ETIAc 4.80 ( 0.20 nano-ETIAd 4.83 ( 0.65

Figure 4. Calibration of simazine with 20 nM IgG-Cy5 and 10 µg/L ACA-BSA-Cy5.5 using the nano-ETIA: A, image of the filled NTP; B, evaluated fluorescence intensities plotted versus the simazine concentration. For comparison, the calibration curve of the microETIA obtained using the same assay conditions is also presented.

chamber, which was cooled to the point of condensation, the dispensed liquids were stable against evaporation for at least 30 min. After finishing the liquid dispensing, the plate was sealed with a transparent adhesive tape and incubated for 10 min, and the fluorescence was recorded using the NTP reader BioScan. The obtained image was analyzed as described in the Experimental Section. In Figure 4A, the obtained picture of the NTP is presented. At low simazine concentrations, an efficient quenching of the IgGCy5 fluorescence can be observed. Higher simazine concentrations led to a progressive binding of the analyte to the antibody binding sites, which results in an increased Cy5 fluorescence. The evaluated fluorescence intensities are plotted in Figure 4B. In the same figure, a micro-ETIA calibration curve recorded at the same assay conditions is displayed. Both calibration curves show an identical IC50 value of 4.8 µg/L. From the precision profiles in Figure 5, it can be seen that the precision of the nano-ETIA is approximately 2-fold lower than the precision of the micro-ETIA. This is the consequence of the lower signal dynamic and, therefore, sensitivity of the nano-ETIA (slopes of the nano- and microETIA at the IC50 are equal to 0.73 and 0.81, respectively; see Table 2), and the higher standard deviations of the NTP fluorescence responses. Despite the higher LOD and limit of quantification (LOQ) that are obtained for the nano-ETIA (see Table 2), the actual amount of simazine that could be measured per sample became smaller by a factor of approximately 1700. With the miniaturized assay, it was possible to detect as little as 111 amol, corresponding to approximately 6.7 × 107 molecules of simazine. The maximum residue level of 0.1 µg/L for each pesticide in drinking water, which was established by the European Economic

slope at IC50a LODb [L/µg] [µg/L] 0.81 ( 0.01 0.73 ( 0.06

0.15 0.32

LOD [mol]

LOQb [µg/L]

187 fmol 111 amol

0.85 3.30

a IC and slope are determined from the curve fit. b LOD and LOQ 50 are determined from the calibration curves according to the IUPAC35 definition as 3 × sd0 and 10 × sd0, respectively, where sd0 is the standard deviation of the matrix blank signal. c n ) 3. d n ) 25.

Communities (EEC) in the directive 80/778,32 however, could not be achieved with either the nano- or micro-ETIA at the assay conditions used above. The optimization of the two experimental variables, that is, the antibody concentration and the affinity constant of the competitive TD,33 would certainly lead to an improved ETIA performance; however, the improvement of the nano-ETIA performance is currently limited as a result of experimental restrictions set by the hardware available. As discussed, these restrictions are the present LOD of the NTP reader BioScan, the precision of the microdispensing device, and the nonspecific binding properties of the NTP. Because the limits of the assay itself could, therefore, not be shown with the nanoETIA, the effect of the further assay optimization on the achievable LOD and LOQ is demonstrated using the micro-ETIA. Further Optimization of the Micro-ETIA. In the direct competitive ETIA, in which two equilibrium reactions take place simultaneously, leading to the [Cy5-IgG]-[TD-BSA-Cy5.5] (KTD) and [Cy5-IgG]-[simazine] (Ksimazine) immunocomplexes, the ratio between the affinity constants, KTD/Ksimazine, is the key to obtaining an immunoassay with low LOD. Because affinity constants of polyclonal antibodies cannot be adequately assessed as a result of the heterogeneous multiple specificities,34 the KTD/ Ksimazine ratio is expressed as the cross-reactivity value (CR) of the antibody toward the investigated TD. CR values were calculated as CR ) IC50(TD)/IC50(simazine). IC50 values were obtained from dose-response curves recorded using a label-free (32) EEC Drinking Water Guideline; European Economic Community: Brussels, 30 August, 1980; 80/778/EEC, no. L229, EEC. (33) Ballesteros, B.; Barcelo´, D.; Sanchez-Baeza, F.; Camps, F.; Marco, M.-P. Anal. Chem. 1998, 70, 4004-4014. (34) Steward, M. W.; Steensgard, J. Antibody Affinity: Thermodynamic Aspects and Biological Significance; CRC Press. Inc.: Boca Raton, FL, 1983; p 79.

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Table 3. Cross Reactivities of the Polyclonal Anti-Simazine Antibody toward Selected Triazine Derivatives

Table 4. Analytical Parameters Derived from the Calibration Curves in Figure 6A triazine signal derivative dynamic, [%] DeACA ABA ACA

41 50 55

IC50a, [µg/L] 0.86 ( 0.06 1.18 ( 0.19 2.96 ( 0.28

slope at LODb, LOQb, IC50a, [L/µg] [µg/L] [µg/L] 0.52 ( 0.04 0.55 ( 0.09 0.72 ( 0.04

0.03 0.01 0.10

0.19 0.08 0.38

a IC and slope are determined from the curve fit. b LOD and LOQ 50 are determined from the calibration curves according to the IUPAC35 definition as 3 × sd0 and 10 × sd0, respectively, where sd0 is the standard deviation of the matrix blank signal.

Figure 6. A, simazine calibration curves using DeACA (cDeACA ) 6 µg/L), ABA (cABA ) 4 µg/L) and ACA (cACA ) 2 µg/L) as competitive TD. Means of triplicates ( sd are plotted. B, precision profiles of the three calibration curves.

optical detection system based on reflectometric interference spectroscopy, as described in ref 29. The structures and the determined CR values of the three structurally related simazine derivatives, ACA, ABA, and DeACA, used to further optimize the micro-ETIA are summarized in Table 3. As expected, the highest CR value of 135% was obtained for ACA. For ABA and DeACA, CR values of 81.8 and 0.36%, respectively, were obtained. For each of these TDs, a TD-BSACy5.5 conjugate with a Cy5.5/TD ratio lower than 1 (see above, Table 1) was synthesized, and a competitive micro-ETIA was set up. The titration experiments, performed using an antibody concentration as low as 1 nM, revealed optimum TD concentrations of 2, 4, and 6 µg/L for ACA, ABA, and DeACA, respectively. Using these TD and antibody concentrations, the micro-ETIA calibration curves shown in Figure 6A were recorded. The analytical parameters derived from the calibration curves are given in Table 4. The signal dynamic and, hence, the assay sensitivity decreases as the CR of the employed TD is reduced (see above). 5178 Analytical Chemistry, Vol. 73, No. 21, November 1, 2001

Simultaneously, the IC50 values are reduced from 2.96 (ACA) to 1.18 (ABA) and to 0.86 µg/L (DeACA). As can be seen from the precision profiles in Figure 6B, the highest precision at simazine concentrations lower than 1 µg/L is obtained for the ABA calibration curve. From this calibration curve, the lowest LOD and LOQ of 0.01 and 0.08 µg/L, respectively, were obtained. These values are well below the limit established by the EEC (see above). Nano- and Micro-ETIA Validation. Validation of the optimized nano- and micro-ETIA, using ACA and ABA as competitive TD and antibody concentrations of 20 nM and 1 nM, respectively, was performed by determining the standard calibration curves, precision, and accuracy using standard and fortified drinking water samples that were from different geographical areas in the southern part of Germany. To minimize the random error in the nano-ETIA (1/xn dependence), for each sample, n ) 25 replicates were measured. This allowed accurate determination of the contribution of systematic errors to the analysis error. For economic reasons, only three replicates were performed for the micro-ETIA validation. The standard calibration curve of the nano-ETIA, as displayed in Figure 4, was linear from 1 to 30 µg/L. The intra-assay precision of this range varied between 7 and 12% (n ) 25), as expressed in terms of % CV of dose. The standard calibration curve of the microETIA (see Figure 6A) was linear from 0.08 to 15 µg/L, with intraassay precision values between 6 and 12% (n ) 3; see Figure 6B). The recorded reference signals, that is, IgG-Cy5 fluorescence at each standard concentration in the absence of tracer used to correct for interferences caused by matrix effects of drinking water samples (see eq 1 in the Experimental Section), was constant over an analyte concentration range from 0 to 300 µg/L (y ) 0.96 10-4x). The accuracies of the nano- and micro-ETIA were studied by recovery experiments in which fortified drinking water samples with simazine levels from 0.1 to 6.0 µg/L were analyzed (see Table 5). No sample pretreatment was required other than thorough degassing. The representative recoveries of these experiments were between 88 and 110% (96.8 ( 6.9%; n ) 8) and 77% to 125% (101.4 ( 15.1%; n ) 10) for the nano- and micro-ETIA, respectively. In general, recoveries of 70-120% are considered acceptable at the micrograms-per-liter level.36 The relative standard deviation (RSD) of these replicate fortifications was between 26.1 and 62.3% (nano-ETIA) and 9.8 and 31.3% (micro-ETIA). According to the recommendations of the AOAC, the within-laboratory RSD should fall within the range of 43 (0.1 µg/L)-21% (10 µg/L).37 This (35) Currie, L. A. Pure Appl. Chem. 1995, 67, 1699-1723.

Table 5. Accuracy Assessment of the Nano- (n ) 25) and Micro-ETIA (n ) 3) Using Fortified Drinking Water Samples Collected from Different Cities in the Southern Part of Germany assay

sample matrix

fortified level meas concn recovery [µg/L] [µg/L] [%]

Konstanz Konstanz Konstanz Heiligenberg Aschaffenburg Aschaffenburg Bad Du ¨ rrheim Nagold

2.00 2.20 2.50 3.00 3.50 4.00 5.00 6.00

2.20 ( 1.37 2.00 ( 0.90 2.20 ( 0.77 3.00 ( 1.43 3.50 ( 1.40 3.90 ( 1.04 4.60 ( 1.20 5.70 ( 1.46

110 91 88 100 100 98 92 95

micro-ETIA Konstanz Konstanz Nagold Konstanz Zwiefalten Hettingen Engstingen Gottmadingen Aschaffenburg Kirchberg

0.10 0.15 0.35 0.43 0.55 0.65 0.80 1.10 1.50 2.20

0.10 ( 0.03 0.16 ( 0.05 0.27 ( 0.04 0.50 ( 0.05 0.55 ( 0.10 0.57 ( 0.06 1.00 ( 0.15 0.93 ( 0.17 1.50 ( 0.21 2.55 ( 0.25

100 107 77 116 100 88 125 85 100 116

nano-ETIA

Figure 7. Accuracy studies to assess the correlation between ETIA and GC/MS measurements using fortified drinking water samples: A, correlation nano-ETIA and GC/MS; B, correlation micro-ETIA and GC/MS.

criterion is fulfilled for the micro-ETIA but not for the nano-ETIA, indicating that the precision of the nano-ETIA has to be further improved. In addition to improving the precision of the microdispensing device (see above), higher precision can also be achieved by a reconfigured assay protocol. Either the number of pipetting steps needs to be reduced or the dispensed volumes of the (36) Conacher, H. B. S. J. Assoc. Off. Anal. Chem. 1990, 73, 332-334. (37) Horwitz, W.; Kamps, L. R.; Boyer, K. W. J. Assoc. Off. Anal. Chem. 1980, 63, 1344-1353.

conjugates needs to be increased while the total assay volume is kept constant. Similarly, the optimized assays were compared with GC/MS by analyzing seven and six fortified drinking water samples using the nano- and micro-ETIA, respectively (see Figure 7). When analyzing the goodness of the correlation between the nano-ETIA and the GC/MS results, the regression coefficient (r2) was 0.996. The correlation obtained between micro-ETIA and GC/MS was r2 ) 0.987. A t-test revealed for both assays no concentrationlevel-independent (constant) and no concentration-level-related (proportional) bias from the equation y ) x (R ) 0.05). This result demonstrates that by using an appropriate matrix effect correction procedure, the homogeneous ETIA can be applied as a routine screening method to assess drinking water quality, even at volumes as low as 70 nL. CONCLUSIONS The homogeneous competitive energy-transfer immunoassay (ETIA) has been successfully performed in disposable plastic nanotiter plates at a total assay volume of 70 nL. The optimized nano-ETIA showed an IC50 value identical to that of the reference micro-ETIA performed in traditional 96-well microtiter plates, even though the assay volume was reduced by a factor of more than 3500. Validation of the nano-ETIA revealed excellent accuracy, demonstrating the effective correction for interferences caused by matrix effects. However, the precision and the limit of detection of the nano-ETIA needs to be further improved. The characteristics of the finally optimized micro-ETIA, where an LOD far below the EEC limit and precision values within the range recommended by the AOAC were obtained, showed that by further improving the performance of the hardware available (the LOD of the scanning laser-induced fluorescence detector, the precision of the piezoelectrically actuated micropipet, and the nonspecific binding properties of the NTP), a miniaturized assay can be obtained that is useful as a cost-effective, high-throughput screening routine method to assess drinking water quality. ACKNOWLEDGMENT The authors are grateful to Dr. Ram Abuknesha (GEC London, U.K.) for his valuable gift of the polyclonal anti-simazine antibody (project RIANA/ENV4-CT95-0066, supported by the European Commission under the Environment and Climate Program) and to Prof. Dr. W. Ja¨ger of Institut Dr. Ja¨ger (Tu¨bingen, Germany) for performing the GC/MS measurements of the drinking water samples. The close and productive cooperation with the project partners Bremer Institut fu¨r angewandte Strahltechnik-BIAS (Bremen, Germany) and Laser Laboratorium Go¨ttingen-LLG (Go¨ttingen, Germany) is gratefully acknowledged. This work was supported by the German Ministry of Education, Science, Research and Technology under the “Mikrosystemtechnik 19941998” program (project LINDAU/16SV541/VDI-VDE IT) and by the DFG (Graduiertenkolleg “Quantitative Analyse und Charakterisierung pharmazeutisch und biochemisch relevanter Substanzen” at the University of Tu¨bingen). Received for review April 23, 2001. Accepted July 30, 2001. AC010456E Analytical Chemistry, Vol. 73, No. 21, November 1, 2001

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