ENSAM: Europium Nanoparticles for Signal Enhancement of Antibody Microarrays on Nanoporous Silicon Kerstin Järås,† Asilah Ahmad Tajudin,‡ Anton Ressine,‡ Tero Soukka,§ György Marko-Varga,4 Anders Bjartell,⊥ Johan Malm,† Thomas Laurell,*,‡ and Hans Lilja†,# Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, Malmö University Hospital, Malmö, Sweden, Department of Electrical Measurement, Lund University, Lund, Sweden, Department of Biotechnology, University of Turku, Turku, Finland, Department of Analytical Chemistry, Lund University and AstraZeneca R&D Lund, Lund, Sweden, Department of Clinical Sciences, Division of Urological Cancers, Lund University, Malmö University Hospital, Malmö, Sweden, Departments of Clinical Laboratories, Surgery (Urology), and Medicine (GU-Oncology), Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Received September 10, 2007
Abstract: To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (β-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection e 0.14 ng/mL, dynamic range > 105) compared to the streptavidin-europium chelate assay (limit of detection e 0.7 ng/mL, dynamic range > 104). Analysis of a titration series showed strong linearity of ENSAM (R2 ) 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100–200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for * Corresponding author: Thomas Laurell, Dept. Electrical Measurements, Div. Nanobiotechnology, Lund University, Box 118, 22100 Lund, Sweden. Phone: +46 46 222 7540. Fax: +46 46 222 4527. E-mail: thomas.laurell@ elmat.lth.se. † Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University. ‡ Department of Electrical Measurement, Lund University. § Department of Biotechnology, University of Turku. 4 Department of Analytical Chemistry, Lund University and AstraZeneca R&D Lund. ⊥ Department of Clinical Sciences, Division of Urological Cancers, Lund University. # Memorial Sloan-Kettering Cancer Center.
1308 Journal of Proteome Research 2008, 7, 1308–1314 Published on Web 02/08/2008
analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing. Keywords: prostate-specific antigen • prostate cancer • lanthanides • europium • antibody microarrays • timeresolved fluorescence • nanoparticles • nanobeads • porous silicon • protein chip
Introduction Signal enhancement is an important part of assay development. In immunoassays, signal can be enhanced in a variety of ways, for example, by detection with rolling-circle amplification,1–5 catalyzed signal amplification with colorimetric readout,6–8 or indirect detection with biotinylation of the antigen or detection antibody followed by incubation with multilabeled streptavidin.9 Another approach could be use of multilabeled nanoparticles coated with detection antibody or streptavidin. Although other signal amplification techniques might reach as low as single-molecule detection sensitivities, multilabeled nanoparticles give signals, directly proportional to the number of binding events. This could be regarded as a benefit for accurate quantification. Signal enhancement is particularly important for miniaturized techniques like protein microarrays. Protein chip technology is becoming increasingly established within the fields of diagnostics and protein characterization.10–17 However, as compared to 96-well assays, the planar, multiplexed format of microarrays imposes special requirements. Catalyzed signal amplification techniques like enzyme-linked immunosorbent assay (ELISA) require elaborate protocols and advanced detection principles in miniaturized formats. Miniaturized techniques have, however, great potential benefit, since both sample volume and analytes can be reduced. The biological focus of this paper is prostate cancer and the detection of the biomarker prostate-specific antigen (PSA). In routine clinical practice today, PSA in serum is measured by immunoassays as an indicator of prostate cancer. However, an elevated PSA value can be caused by benign prostate hyperplasia as well as by prostate cancer. To improve prostate cancer diagnostics, more biomarkers of cancer are sought. In anticipation of future diagnostic requirements, possibly entailing hundreds of antibodies, protein microarrays are under devel10.1021/pr700591j CCC: $40.75
2008 American Chemical Society
technical notes
ENSAM on Nanoporous Silicon
Figure 1. Complementary nanotechniques for improved limits of detection. (A) Porous silicon matrix used for protein microarrays. The scanning electron micrographs show a sequential zoom into a typical surface (left column, top view; right column, side view). The macropores of micrometer size are clearly seen, combined with a nanomorphology (50–100 nm) as seen in the inset zoom, lower right. (B, upper) Schematic of the nanoparticles used in the ENSAM assay. Many fluorophores are incorporated into each particle. (B, lower) The nine-dentate europium chelate used in the microarray assay that serves as a reference for the ENSAM protocol.
opment. Assays using protein microarrays will require high sensitivity for tumor markers, both because the levels of some biomarkers may be very low, and because less than 1 µL of sample may be available to determine levels of a single biomarker. For prostate cancer research, signal enhancement is important for developing protein microarray assays with detection limits as low as the serum concentrations of human kallikreins, for which the normal values range from around 0.03 ng/mL for hK2 to 0.6 ng/mL for PSA.18,19 Earlier studies by our group have shown proof of principle for PSA analysis with antibody microarrays using FITC-labeled detection antibodies20 or FITC-labeled PSA.21 Today, one of the standard assays for PSA uses lanthanide chelates and time-resolved fluorescence (TRF). TRF was first discussed in 197822 and applied to immunoassays in the early 1980s.23 In TRF, a short delay between excitation and detection of emitted light depresses any autofluorescence of substrate or background, improving the signal-to-noise ratio due to exceptionally slow decay of lanthanide fluorescence.23,24 On the basis of TRF detection, PerkinElmer Life Science developed the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). DELFIA, which is based on the dissociation of lanthanide from the chelate in order to form complexes with β-diketones, detergents, or synergistic agents,25 is known as one of the most sensitive and reliable immunoassay platforms.26 The translation of these assays to a miniaturized format is an attractive approach to reduce the consumption of sample and analyte. Lövgren et al.27 showed proof of principle for TRF detection of intrinsically fluorescent lanthanide chelates on microparticles, and subsequently, signal amplification using polystyrene nanoparticles that incorporate europium chelate (β-diketone) was developed.28–34 Dosev et al.35 and Nichkova et al.36,37 have detected protein microstripe patterns by lan-
thanide oxide particles with conjugated antibodies or streptavidin. However, for the first time, europium chelate nanoparticles have been successfully applied to spotted protein microarrays using TRF detection. To reach high sensitivity in protein microarrays, the substrate or chip is also of utmost importance. The substrates used in this work are three-dimensional nanoporous silicon surfaces developed in-house (Figure 1A), obtained by electrochemical dissolution of silicon wafers. They have proven to be highly compatible with protein chip technology, and they outperform most of the commercial array surfaces with respect to spot quality,38,49 spot density,21 linear range, and sensitivity.39 Interestingly, these surfaces were compatible with matrixassisted laser desorption/ionization (MALDI) mass spectrometry, and they can be simultaneously analyzed by fluorescence, enabling a dual-readout mode of detection.40 In this paper, we describe how TRF can be used for antibody microarray detection on our high-performance nanoporous silicon surfaces. Two antibody microarray assays were developed as model systems for detection of biotinylated PSA in serum. The first assay uses europium chelate-incorporated nanoparticles coated by streptavidin (Figure 1B, upper), whereas the second assay uses streptavidin labeled with a nine-dentate europium chelate (Figure 1B, lower). In addition proof of principle was shown for analyzing PSA in plasma, obtained from patients undergoing clinical PSA-testing, using the ENSAM assay in a sandwich approach.
Experimental Section Proteins and Reagents. The monoclonal antibody against PSA (2E9) was produced as previously described.41,42 Recombinant proPSA was expressed in insect cells as previously Journal of Proteome Research • Vol. 7, No. 3, 2008 1309
technical notes 43
demonstrated and subsequently biotinylated. Streptavidin purchased from Societa Prodotti Antibiotici - BioSpa (Milan, Italy) was labeled as described earlier44 (6 europium chelates per streptavidin) with intrinsically fluorescent 9-dentate europium chelate ({2,2′,2″,2′′′-{[2-(4-isothiocyanatophenyl)ethylimino]-bis(methylene)bis{4-{[4-(R-D- galactopyranoxy)phenyl]ethynyl}pyridine-6,2 diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III)),45 kindly provided by Jaana Rosenberg (Department of Biotechnology, University of Turku, Turku, Finland). Labeled and purified streptavidin was diluted in Delfia enhancement solution and the fluorescence compared to europium(III) ion calibrator, obtained from Wallac, to determine the average number of europium(III) ions attached to streptavidin. The streptavidin concentration was determined by the Bradford assay as well as UV absorption at 280 nm (correcting for the absorption due to the europium(III) chelate). Europium chelate (β-diketone)-incorporated polystyrene nanoparticles, 47 nm in diameter (Seradyn, Inc., Indianapolis, IN), coated with streptavidin, were prepared as described by Härmä et al.30 The nanoparticle concentration is measured based on mass/volume ratio, confirmed by filtering particles on membrane followed by particle counting from scanning electron microscope images. The number of chelated europium ions in the particle solution was determined from a standard curve of europium(III) ion calibrator in DELFIA enhancement solution producing equivalent fluorescence. Signals of nanoparticles and europium-labeled streptavidin, as well as standard curve of calibrator, were obtained by the Victor 1420 multilabel counter (Perkin-Elmer Life Sciences/Wallac Oy, Wellesley, MA). Analytic Samples. Human female serum, which normally lacks any detectable PSA, was obtained from a healthy volunteer. These serum samples were spiked with biotinylated proPSA in a titration series ranging from 0.07 to 7000 ng/mL. EDTA-anticoagulated patient plasma collected for routinely performed clinical PSA-testing was used to assess analytical performance of the “sandwich-type” ENSAM assay. In additon, we also used the Prostatus PSA Free/Total DELFIA, Dissociation Enhanced Lanthanide FluoroImmunoAssay (PerkinElmer, Wallac Oy, Turku, Finland), which is a commercial 96-well immunoassay, as reference method to determine the total PSA concentrations. Fabrication of Nanoporous Silicon. The fabrication of nanoporous silicon by anodic dissolution of p-type, borondoped monocrystalline silicon wafer has been described in detail earlier.38 Briefly, the silicon used was of -orientation, 6–8 Ω cm resistivity (P-type, boron) and purchased from Addison Engineering (San Jose, CA). The 3 in. silicon wafer was placed in an electrolytic cell for electrochemical etching(2 mA/ cm2 wafer). The electrolyte solution contained 3.6% hydrofluoric acid and 90.7% dimethyl formamide (Merck, Darmstad, Germany). Anodization was performed for 70 min. During the procedure, the positive charge carriers in the silicon wafer migrate toward the anodic side of the wafer. At the anodic side of the wafer, silicon is solubilized, and the micro- and nanopores are formed. Antibody Microarray with Europium-Labeled Streptavidin. Monoclonal anti-PSA capture antibody (0.5 mg/ml ), 2E9, was arrayed onto the nanoporous silicon chips using a piezoelectric microdispenser developed in-house,46 followed by three washes in PBS-Tween (0.05% Tween 20 in 10 mM PBS) to remove loosely bound antibodies. Arrayed chips were then blocked with 24 µL of 5% (w/v) nonfat dry milk (Bio-Rad) in PBS-Tween for 30 min to prevent nonspecific binding. The three washes were 1310
Journal of Proteome Research • Vol. 7, No. 3, 2008
Järås et al. repeated before exposing each chip to 24 µL of human female serum spiked with biotinylated PSA. Following a 2-h incubation, the chips were again washed three times. The chips were then incubated for 2 h in the dark with 24 µL of europium-labeled streptavidin diluted to 0.73 µg/mL in PBS. Washing was performed again before dipping the chips quickly in MilliQ water. The chips were dried with high-pressure air before signal detection. All steps were performed at room temperature. ENSAM: Antibody Microarray with Streptavidin-Coated Europium Nanoparticles. The ENSAM procedure was similar to that described above, but instead of incubating with europium-labeled streptavidin, the chips were placed in an Eppendorf tube with nanoparticle solution. Streptavidin-coated europium chelate-incorporated nanoparticles were diluted to 4 × 107 particles/µL in 560 µL of Prostatus Assay buffer (PerkinElmer). Incubation was carried out by end-over-end rotation in the dark for 2 h. Subsequently, the chips were washed once in PAB-Tween (0.05% Tween 20 in Prostatus Assay buffer) for 5 min in the dark. The chips were allowed to dry before signal detection. All steps were performed at room temperature. ENSAM: Sandwich Approach. The analysis was performed as the above ENSAM assay with exceptions as follows. The chips were incubated with undiluted patient plasma for 1 h, washed three times in PBS-Tween as described above, and incubated with 24 µL of biotinylated monoclonal anti-PSA detector antibody H117 (4.2 µg/mL in PBS). The chips were subsequently washed three times in PBS-Tween, incubated 1 h with the nanoparticles as described above, and washed twice in PAB-Tween for 5 min in the dark. Time-Resolved Fluorescence Detection and Analysis. A standard epifluorescence microscope (Nikon Eclipse E600, Nikon, Tokyo, Japan) was equipped with a rotating shutter and a xenon flash-lamp to enable TRF imaging (Perkin-Elmer Life Sciences, Turku, Finland) as previously described.31 The chips were focused in bright-field mode using a halogen light source, and the xenon flash lamp was then used for time-resolved imaging with pulsed excitation at 340 nm wavelength. The rotating shutter was used to close the light path to the camera during the excitation pulses and the delay time (between the excitation and the light collection). The chips were analyzed under the following conditions: excitation pulse length < 10 µs, emission wavelength 615 nm, delay time after excitation 300 µs, signal detection period 600 µs, repetition frequency of excitation and detection 50 Hz, and a total measurement time of 60 s for each image. Fluorescent images were obtained by a cooled CCD-camera (Astrocam model TE3/A/S, Perkin-Elmer Life Sciences, Cambridge, U.K.). Image analysis was then performed using Image-Pro Plus Version 4.5.1.29 (Media Cybernetics, Inc., Silver Spring, MD). Mean Spot Intensity and Limit of Detection. The intensity of each spot was quantified using Image-Pro Plus and the circle method. Mean intensity was calculated by dividing integrated optical density by the circle area. Local background was quantified using the same method and subtracted from the mean intensity of each spot to generate the mean spot intensities presented in the graphs and tables. The limit of detection (LOD) was defined as the lowest PSA concentration for which the mean spot intensity was at least two standard deviations above the mean intensity of the background, calculated from 6 to 7 background locations.
ENSAM on Nanoporous Silicon
technical notes
Figure 2. Schematic of the microarray protocols. (A) The assay based on europium-labeled streptavidin entails dispensing of the capture antibody (1), subsequent washing, blocking, and washing again (not shown), followed by incubation with biotinylated PSA spiked into female serum (2). Part 3 of the figure is a close-up illustration of step 2. The chip is finally incubated with 9-dentate europium-labeled streptavidin and washed before TRF microscope readout (4). (B) The ENSAM procedure is the same, except the final incubation is with streptavidin-coated europium nanoparticles (2 and 3).
Results and Discussion Comparison of Europium Chelates and Europium Nanoparticles. Figure 2 outlines the two antibody microarray assays, ENSAM and the assay based on europium-labeled streptavidin. The two assays were used to analyze a titration series of biotinylated PSA spiked into human female serum. In Figure 3A, the signal enhancement provided by the nanoparticle is clearly visible. ENSAM compared to europium-labeled streptavidin yielded around 10-fold signal enhancement, calculated as the ratio between the mean spot intensities at each PSA concentration. The limits of detection (LODs) were obtained from repeated runs of the two microarrays at the lowest PSA concentrations for each assay. As control, female serum without PSA was used in the two assays. No signals were detectable (data not shown). The LODs were 0.48–0.70 ng/mL for the streptavidin-europium assay and 0.07–0.14 ng/mL for the nanoparticle assay. In comparison to earlier studies by our group using FITC-labeled PSA,21 the europium-labeled streptavidin reached a slightly lower limit of detection (0.48–0.7 ng/mL as compared to 0.7 ng/mL for FITC-labeled PSA). With the chelate-incorporated nanoparticles, the LOD was improved by another order of magnitude, down to 0.07–0.14 ng/mL. The LOD of the ENSAM assay corresponds to approximately 100–200 zmol PSA per spot. The dynamic ranges were found to be 0.7–7000 (4 orders of magnitude dynamic range) versus 0.07–7000 (5 orders of
magnitude dynamic range) ng/mL for the europium-labeled streptavidin and ENSAM assays, respectively. Linearity of ENSAM and Aspects of TRF Detection. To further validate the ENSAM assay, the linearity was analyzed using seven serum samples spiked with biotinylated PSA in a range from 0.98 to 69.2 ng/mL (Figure 3B). Prostatus PSA Free/ Total DELFIA was used as the reference method. The linear regression of the mean spot intensity versus PSA concentration corresponded to a coefficient of determination (R2) equal to 0.99, indicating good linearity within the working range. The inset table of Figure 3B lists the standard deviations of the corresponding mean spot intensities at each PSA concentration. It should be noted that the current assays, based on TRF detection, have the advantage of high tolerance toward complex biofluids such as blood serum. Commonly, protein microarray assays require a 4- to 10-fold dilution to suppress background interference. In this work, crude plasma samples obtained from patients undergoing clinical PSA-testing and female serum samples spiked with biotinylated PSA were successfully analyzed without dilution. Any autofluorescence of the surface itself as well as signal from any nonspecifically bound serum proteins would be depressed by the time-resolved detection. PSA Analysis in Patient Plasma Subject to Clinical PSATesting. Our comparison of the two assays described above provided evidence for a gain in signal enhancement contributed by the nanoparticles. In these assays, we spiked biotin-labeled Journal of Proteome Research • Vol. 7, No. 3, 2008 1311
technical notes
Järås et al.
Figure 3. (A) Titration of biotinylated proPSA spiked into female serum. Streptavidin labeled with europium (lower curve) and europium chelate-incorporated nanoparticles coated with streptavidin (upper curve) were used for detection. Mean spot intensities and standard deviations were calculated from the spots detected by a 20× lens. The images shown were obtained from the nanoparticle microarray through a 10× lens, and the size of each spot is around 50 µm in diameter. (B) The linearity of the nanoparticle assay was studied by analyzing human female serum samples spiked with biotinylated PSA in a range from 0.98 to 69.2 ng/mL. The PSA concentrations were determined using DELFIA as reference method (x-axis). Each mean spot intensity (y-axis) was calculated from 6 microarray spots (20× lens). The inset table shows the PSA concentrations as measured by DELFIA, mean spot intensities, and the corresponding standard deviations of the mean spot intensities.
Figure 4. (A) Illustration of the ENSAM sandwich approach used to show proof of principle for detection of PSA in plasma from three different patients in disease. The plasma samples were analyzed using the ENSAM sandwich antibody microarray as well as the Prostatus DELFIA reference assay. The microarray results are presented as images (obtained through a 10× lens), the corresponding mean spot intensities, and CVs. Mean spot intensities and CVs were calculated from the 12 microarray spots displayed in the images. The total PSA concentration is the one measured by DELFIA. (B) Mean spot intensities of the three patient samples plotted against total PSA concentration as measured by the DELFIA.
PSA into human female serum. However, in order to avoid a laborious, time-consuming assay design where each and every sample needs to be labeled prior to analysis (and therefore also subjected to increased risk for ex vivo/in vitro decay), we developed a modified “sandwich-type” version of the ENSAM assay (Figure 4A). These analyses provided a proof of principle for the detection of the PSA in patient plasma samples collected for the routinely performed clinical PSA-testing using the Prostatus DELFIA assay to determine the PSA levels. Figure 4B shows the mean spot intensities of the microarrays plotted against PSA concentration measured by the Prostatus DELFIA assay. 1312
Journal of Proteome Research • Vol. 7, No. 3, 2008
Improved Signal Readout via Nanotechnology. In this work, two nanomaterials were utilized: nanoporous silicon and europium chelate-incorporated nanoparticles. The nanoporous silicon protein chip surface is composed of deep macro pores in which the walls have a nanomorphology (Figure 1A).38,47 Not only does the fractal pore morphology provide a high surface area, but most importantly, the macroporous structure is instrumental for making the silicon dioxide surface behave hydrophobically to the antibody fluid.38 This occurs as a result of the low ratio of solid versus air contact area for the spotted antibody droplet on the surface. The resulting high contact angle for the spotted antibody droplet
technical notes
ENSAM on Nanoporous Silicon yields a reduced spot size compared to that of flat and nonporous surfaces. Yet, the porous silicon is hydrophilic at the molecular level, which in turn minimizes denaturation of the deposited antibody. The nanoparticles consist of polystyrene beads with a diameter of 47 nm incorporated with europium chelate (Figure 1B upper). Onto the carboxyl-groups of the particle surface, streptavidin is covalently coupled. Since each bead contains a large number of europium chelates, signal is enhanced compared to that from streptavidin labeled with 9-dentate europium chelate (Figure 1B lower). ENSAM Compared to Prostatus DELFIA Assay. The current gold standard of immunoassays is the 96-well format. One such assay is Prostatus PSA Free/Total DELFIA, which is used to determine the total and free PSA concentration of human serum samples. Since both DELFIA and ENSAM utilize TRF and have similar LODs, an interesting objective is to compare the sample consumption of the two assays. The sample volume used is 25 µL per microtiter plate well in the DELFIA assay and 24 µL per chip in the ENSAM assay. However, in the ENSAM assay, this volume covers the 600 assay points arrayed on the chip surface, each of which could be used for analysis of a different antigen. This clearly demonstrates the potential for high-level multiplex analysis using the same volume needed for a single measured well in the 96-well format. From the same assay volume at least 600 spots could be detected simultaneously in the microarray format, which therefore could offer an important advantage compared to the 96-well assay format used for the Prostatus DELFIA assay. It should also be noted that the DELFIA technology is not suitable for work on solidphase microarrays as the signal generation requires a separate (dissociative) enhancement step, where the spatial information of bound labels is lost.
Conclusions In the future, we anticipate that individual clinical samples, retrieved from large size bio repositories linked to long-term following up, will be expected to cover a larger number of assays, such as biomarker screens in clinical studies. The discovery of novel biomarkers is envisioned to be a cornerstone in modern healthcare, in which selection of drug treatments will heavily rely on the analysis of biomarkers in various biological fluids. For this reason, investigators are already developing multiplex assays, including analyses of pathway biology, protease and kinase activities, phosphorylation, and glycosylation, as well as transcript profiling. With protein chip technology, signal enhancement is important to enable monitoring of low-abundance biomarkers. This work describes how time-resolved fluorescence can be used to reach high sensitivity for antibody microarray detection on a nanoporous silicon substrate. The large number of europium molecules per nanoparticle in the ENSAM assay provided a 10-fold signal amplification, decreasing the LOD 1 order of magnitude compared to the LOD of microarrays with europium-labeled streptavidin. Low picogram per milliliter biotinylated PSA could be detected in undiluted human serum. At this early stage of ENSAM development, the operational stability of the assay fulfils the requirements for a protein microchip array. The sensitivity of the microarray assay for PSA is more than adequate to cover relevant diagnostic cut-points (ranging from 0.6 to 4 ng/mL) that are currently considered for identifying and counselling men with elevated risk of malignant or benign
prostate disease. The microarray format will allow multiplex detection of prostate cancer biomarkers. These biomarkers might include PSA and several other human kallikreins that have shown strong correlation with malignant prostatic disease.18,48 Future work will focus on multiplex detection of these kallikreins on a single chip.
Acknowledgment. The authors thank Ingrid Wigheden, Gun-Britt Eriksson, Kerstin Håkansson, and Birgitta Frohm for providing critical reagents (antibodies and PSA) and expert technical assistance. Also thanks to Ms Christina Möller and Ulla Fält for technical assistance with time-resolved fluorescence imaging and to Dr. Janet Novak for help with preparation of this manuscript. This study was supported by grants from the European Union 6th Framework contract LSHC-CT-2004-503011 (P-Mark), the Foundation for Strategic Research - Biomedical Engineering for Better Health Programme, Swedish Research Council, SWEGENE, the National Cancer Institute contract P50-CA92629 - SPORE Pilot Project 7, the Swedish Cancer Society project no. 3555 and 4294, Wallenberg Foundation, Crafoord Foundation, Carl Trygger Foundation, Fundacion Federico, Thulefjord Foundation, and Royal Physiographical Society in Lund. References (1) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Inaugural article: immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (18), 10113–10119. (2) Zhou, H.; Bouwman, K.; Schotanus, M.; Verweij, C.; Marrero, J. A.; Dillon, D.; Costa, J.; Lizardi, P.; Haab, B. B. Two-color, rollingcircle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. GenomeBiology 2004, 5 (4), R28. (3) Lizardi, P. M.; Huang, X.; Zhu, Z.; Bray-Ward, P.; Thomas, D. C.; Ward, D. C. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 1998, 19 (3), 225–32. (4) Jarvius, M.; Paulsson, J.; Weibrecht, I.; Leuchowius, K. J.; Andersson, A. C.; Wahlby, C.; Gullberg, M.; Botling, J.; Sjoblom, T.; Markova, B.; Ostman, A.; Landegren, U.; Soderberg, O. In situ detection of phosphorylated PDGF receptor beta using a generalized proximity ligation method. Mol. Cell. Proteomics 2007, 6, 1500–1509. (5) Wiltshire, S.; O’Malley, S.; Lambert, J.; Kukanskis, K.; Edgar, D.; Kingsmore, S. F.; Schweitzer, B. Detection of multiple allergenspecific IgEs on microarrays by immunoassay with rolling circle amplification. Clin. Chem. 2000, 46 (12), 1990–1993. (6) Rapkiewicz, A.; Espina, V.; Zujewski, J. A.; Lebowitz, P. F.; Filie, A.; Wulfkuhle, J.; Camphausen, K.; Petricoin, E. F., 3; Liotta, L. A.; Abati, A. The needle in the haystack: application of breast fineneedle aspirate samples to quantitative protein microarray technology. Cancer 2007, 111 (3), 173–184. (7) Calvert, V. S.; Collantes, R.; Elariny, H.; Afendy, A.; Baranova, A.; Mendoza, M.; Goodman, Z.; Liotta, L. A.; Petricoin, E. F.; Younossi, Z. M. A systems biology approach to the pathogenesis of obesityrelated nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis. Hepatology 2007, 46 (1), 166–72. (8) Nishizuka, S.; Charboneau, L.; Young, L.; Major, S.; Reinhold, W. C.; Waltham, M.; Kouros-Mehr, H.; Bussey, K. J.; Lee, J. K.; Espina, V.; Munson, P. J.; Petricoin, E., 3; Liotta, L. A.; Weinstein, J. N. Proteomic profiling of the NCI-60 cancer cell lines using new highdensity reverse-phase lysate microarrays. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (24), 14229–14234. (9) Ingvarsson, J.; Larsson, A.; Sjoholm, A. G.; Truedsson, L.; Jansson, B.; Borrebaeck, C. A.; Wingren, C. Design of recombinant antibody microarrays for serum protein profiling: targeting of complement proteins. J. Proteome. Res. 2007, 6, 3527–3536. (10) Ekins, R. P.; Chu, F. W. Multianalyte microspot immunoassay-microanalytical “compact disk” of the future. Clin. Chem. 1991, 37 (11), 1955–1967. (11) Joos, T. O.; Stoll, D.; Templin, M. F. Miniaturised multiplexed immunoassays. Curr. Opin. Chem. Biol. 2002, 6 (1), 76–80.
Journal of Proteome Research • Vol. 7, No. 3, 2008 1313
technical notes (12) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Global analysis of protein activities using proteome chips. Science 2001, 293 (5537), 2101–2105. (13) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Protein microarray technology. Trends Biotechnol. 2002, 20 (4), 160–166. (14) MacBeath, G.; Schreiber, S. L. Printing proteins as microarrays for high-throughput function determination. Science 2000, 289 (5485), 1760–1763. (15) Miller, J. C.; Zhou, H.; Kwekel, J.; Cavallo, R.; Burke, J.; Butler, E. B.; Teh, B. S.; Haab, B. B. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 2003, 3 (1), 56–63. (16) Zhu, H.; Hu, S.; Jona, G.; Zhu, X.; Kreiswirth, N.; Willey, B. M.; Mazzulli, T.; Liu, G.; Song, Q.; Chen, P.; Cameron, M.; Tyler, A.; Wang, J.; Wen, J.; Chen, W.; Compton, S.; Snyder, M. Severe acute respiratory syndrome diagnostics using a coronavirus protein microarray. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (11), 4011–4016. (17) Ho, S. W.; Jona, G.; Chen, C. T.; Johnston, M.; Snyder, M. Linking DNA-binding proteins to their recognition sequences by using protein microarrays. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (26), 9940–9945. (18) Lilja, H.; Ulmert, D.; Bjork, T.; Becker, C.; Serio, A. M.; Nilsson, J. A.; Abrahamsson, P. A.; Vickers, A. J.; Berglund, G. Long-term prediction of prostate cancer up to 25 years before diagnosis of prostate cancer using prostate kallikreins measured at age 44 to 50 years. J. Clin. Oncol. 2007, 25 (4), 431–436. (19) Shaw, J. L.; Diamandis, E. P. Distribution of 15 human kallikreins in tissues and biological fluids. Clin. Chem. 2007, 53 (8), 1423– 1432. (20) Jaras, K.; Ressine, A.; Nilsson, E.; Malm, J.; Marko-Varga, G.; Lilja, H.; Laurell, T. Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective. Anal. Chem. 2007, 79 (15), 5817–5825. (21) Ressine, A.; Finnskog, D.; Malm, J.; Becker, C.; Lilja, H.; MarkoVarga, G.; Laurell, T. Macro/nano-structured silicon as solid support for antibody arrays. Surface design, reproducibility and assay characteristics enabling discovery of kallikrein gene products for prostate disease diagnostics. NanoBiotechnology 2005, 1 (1), 93–104. (22) Wieder, I. Background rejection in fluorescent immunoassay. In Immunofluorescence and Related Staining Techniques; Knapp, W., Holubar, K. Wick, G., Eds.; Elsevier: Amsterdam, 1978; pp 67–80. (23) Siitari, H.; Hemmila, I.; Soini, E.; Lovgren, T.; Koistinen, V. Detection of hepatitis B surface antigen using time-resolved fluoroimmunoassay. Nature 1983, 301 (5897), 258–260. (24) Ekins, R. P.; Dakubu, S. The development of high-sensitivity pulsed-light, time-resolved fluoroimmunoassays. Pure Appl. Chem. 1985, 57 (3), 473–482. (25) Soini, E.; Lovgren, T. Time-resolved fluorescence of lanthanide probes and applications in biotechnology. CRC Crit. Rev. Anal. Chem. 1987, 18 (2), 105–154. (26) Dickson, E. F.; Pollak, A.; Diamandis, E. P. Ultrasensitive bioanalytical assays using time-resolved fluorescence detection. Pharmacol. Ther. 1995, 66 (2), 207–235. (27) Lovgren, T.; Heinonen, P.; Lehtinen, P.; Hakala, H.; Heinola, J.; Harju, R.; Takalo, H.; Mukkala, V. M.; Schmid, R.; Lonnberg, H.; Pettersson, K.; Iitia, A. Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry. Clin. Chem. 1997, 43 (10), 1937–1943. (28) Soukka, T.; Antonen, K.; Harma, H.; Pelkkikangas, A. M.; Huhtinen, P.; Lovgren, T. Highly sensitive immunoassay of free prostatespecific antigen in serum using europium(III) nanoparticle label technology. Clin. Chim. Acta 2003, 328 (1–2), 45–58. (29) Soukka, T.; Paukkunen, J.; Harma, H.; Lonnberg, S.; Lindroos, H.; Lovgren, T. Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology. Clin. Chem. 2001, 47 (7), 1269–1278. (30) Harma, H.; Soukka, T.; Lovgren, T. Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostatespecific antigen. Clin. Chem. 2001, 47 (3), 561–568. (31) Vaisanen, V.; Harma, H.; Lilja, H.; Bjartell, A. Time-resolved fluorescence imaging for quantitative histochemistry using lan-
1314
Journal of Proteome Research • Vol. 7, No. 3, 2008
Järås et al.
(32)
(33)
(34)
(35)
(36)
(37)
(38) (39)
(40) (41)
(42)
(43)
(44)
(45) (46) (47) (48)
(49)
thanide chelates in nanoparticles and conjugated to monoclonal antibodies. Luminescence 2000, 15 (6), 389–397. Valanne, A.; Huopalahti, S.; Soukka, T.; Vainionpaa, R.; Lovgren, T.; Harma, H. A sensitive adenovirus immunoassay as a model for using nanoparticle label technology in virus diagnostics. J. Clin. Virol. 2005, 33 (3), 217–223. Valanne, A.; Huopalahti, S.; Vainionpaa, R.; Lovgren, T.; Harma, H. Rapid and sensitive HBsAg immunoassay based on fluorescent nanoparticle labels and time-resolved detection. J. Virol. Methods 2005, 129 (1), 83–90. Jaakohuhta, S.; Harma, H.; Tuomola, M.; Lovgren, T. Sensitive Listeria spp. immunoassay based on europium(III) nanoparticulate labels using time-resolved fluorescence. Int. J. Food Microbiol. 2007, 114 (3), 288–294. Dosev, D.; Nichkova, M.; Liu, M.; Guo, B.; Liu, G. Y.; Hammock, B. D.; Kennedy, I. M. Application of luminescent Eu:Gd2O3 nanoparticles to the visualization of protein micropatterns. J. Biomed. Opt. 2005, 10 (6), 064006. Nichkova, M.; Dosev, D.; Gee, S. J.; Hammock, B. D.; Kennedy, I. M. Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated Eu:Gd2O3 nanoparticles as fluorescent labels. Anal. Chem. 2005, 77 (21), 6864–6873. Nichkova, M.; Dosev, D.; Perron, R.; Gee, S. J.; Hammock, B. D.; Kennedy, I. M. Eu3+-doped Gd2O3 nanoparticles as reporters for optical detection and visualization of antibodies patterned by microcontact printing. Anal. Bioanal. Chem. 2006, 384 (3), 631– 637. Ressine, A.; Ekstrom, S.; Marko-Varga, G.; Laurell, T. Macro-/ nanoporous silicon as a support for high-performance protein microarrays. Anal. Chem. 2003, 75 (24), 6968–6974. Steinhauer, C.; Ressine, A.; Marko-Varga, G.; Laurell, T.; Borrebaeck, C. A.; Wingren, C. Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon substrates. Anal. Biochem. 2005, 341 (2), 204–213. Finnskog, D.; Ressine, A.; Laurell, T.; Marko-Varga, G. Integrated protein microchip assay with dual fluorescent- and MALDI readout. J. Proteome Res. 2004, 3 (5), 988–994. Lilja, H.; Christensson, A.; Dahlen, U.; Matikainen, M. T.; Nilsson, O.; Pettersson, K.; Lovgren, T. Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin. Clin. Chem. 1991, 37 (9), 1618–1625. Petterson, K.; Vehnianen, M.; Viloma, S.; Makinen, M. L.; Manstala, P.; Lovgren, T. Comparison of recombinant fab fragment to Mab on immunoassay of human alpha-fetoprotein. Clin. Chem. 1995, 41 (6), S81–S81. Lovgren, J.; Rajakoski, K.; Karp, M.; Lundwall, A.; Lilja, H. Activation of the zymogen form of prostate-specific antigen by human glandular kallikrein 2. Biochem. Biophys. Res. Commun. 1997, 238 (2), 549–555. Kokko, T.; Kokko, L.; Lovgren, T.; Soukka, T. Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer. Anal. Chem. 2007, 79 (15), 5935–5940. von Lode, P.; Rosenberg, J.; Pettersson, K.; Takalo, H. A europium chelate for quantitative point-of-care immunoassays using direct surface measurement. Anal. Chem. 2003, 75 (13), 3193–3201. Laurell, T.; Wallman, L.; Nilsson, J. Design and development of a silicon microfabricated flow-through dispenser for on-line picolitre sample handling. J. Micromech. Microeng. 1999, 9 (4), 369–376. Bardyshev, N. I.; Mokrushin, A. D.; Puryaeva, T. P.; Serebryakova, N. V.; Starkov, V. V. Nanopores in macroporous silicon. Inorg. Mater. 2004, 40 (11), 1127–1132. Steuber, T.; Vickers, A. J.; Haese, A.; Becker, C.; Pettersson, K.; Chun, F. K.; Kattan, M. W.; Eastham, J. A.; Scardino, P. T.; Huland, H.; Lilja, H. Risk assessment for biochemical recurrence prior to radical prostatectomy: significant enhancement contributed by human glandular kallikrein 2 (hK2) and free prostate specific antigen (PSA) in men with moderate PSA-elevation in serum. Int. J. Cancer 2006, 118 (5), 1234–1240. Ressine, A.; Marko-Varga, G.; Laurell, T. Porous silicon protein microarray technology and ultra-/superhydrophobic states for improved bioanalytical readout. Biotechnol. Annu. Rev. 2007, 13, 149–200.
PR700591J
