Sensitive Quantitative Protein Concentration Method Using

Anal. Chem. 2008, 80, 9781–9786

Sensitive Quantitative Protein Concentration Method Using Luminescent Resonance Energy Transfer on a Layer-by-Layer Europium(III) Chelate Particle Sensor Harri Ha¨rma¨,*,† Lars Da¨hne,‡ Sari Pihlasalo,† Janne Suojanen,† Jouko Peltonen,§ and Pekka Ha¨nninen† Laboratory of Biophysics and Medicity, University of Turku, Tykisto¨katu 6A, FIN-20520 Turku, Finland, Capsulution Nanoscience AG, Volmerstrasse 7b, D-12489 Berlin, Germany, and Laboratory of Paper Coating and Converting, Åbo Akademi University, Porthaninkatu 3, FIN-20500 Turku, Finland A particle-based protein quantification method was developed. The method relies on adsorption of proteins on particles and time-resolved fluorescence resonance energy transfer (TR-FRET). Layer-by-layer (LbL) particles containing europium(III) chelate donor were prepared. A protein labeled with an acceptor was adsorbed onto the particles and near-infrared energy transfer signal was detected in time-gated detection mode. Sample proteins efficiently occupied the particle surface preventing binding of the acceptor-labeled protein leading to a particle sensor with a significant signal change. We detected subnanomolar protein concentration using the rapid and simple mix-and-measure method with a coefficient of variation below 10%. Compared to known protein concentration methods, the developed method required no hazardous substances or elevated temperature to reach the highsensitivity level. Protein quantification is a key element in biochemical studies. Over the years, there has been a great interest to develop high sensitivity methods for protein quantification. Today one of the driving forces is the expanding need for sensitive protein quantification in proteomics. Total protein concentration measurements have typically relied on photon absorption in methods such as UV280, biureat,1 bicinchoninic acid,2 Bradford,3 and Lowry.4 Fluorescence is known to be an orders of magnitude more sensitive detection method than absorbance. Therefore, several fluorescence-based methods have been developed for protein quantification such as fluorescamine,5 3-(4-carboxybenzoyl) quino* Corresponding author. E-mail: [email protected]. Fax: + 358 2 3337060. † University of Turku. ‡ Capsulution Nanoscience AG. § Åbo Akademi University. (1) Gormall, A. G.; Bardawill, C. J.; David, M. M. J. Biol. Chem. 1949, 177, 751–766. (2) Smith, P. K.; Krohn, R. I.; Hermansen, G. T.; Malia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76–85. (3) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (4) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265–275. (5) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Science 1972, 178, 871–872. 10.1021/ac801960c CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

line-2-carboxaldehyde (CBQCA),6,7 o-phthaldialdehyde,8 and NanoOrange9,10 methods. CBQCA and NanoOrange offer the highest sensitivity of the methods at the low nanomolar range. Protein adsorption is involved in a wide range of phenomena and applications within biosciences. Proteins readily adsorb on various surfaces, which is being exploited in, e.g., diagnostics and chromatographic techniques. Often particles are used as adsorbents due to reproducible size and variable surface properties. The layer-by-layer (LbL) technology offers an elegant opportunity to vary the surface properties widely by deposition of functionalized layers with a thickness in the nanometer range on planar surfaces and colloidal particles.11-13 These polyelectrolyte layers may carry desired properties such as dyes which can be deposited onto particles leading to increased functionality for drug delivery or sensor purposes. Time-resolved fluorescence is a highly sensitive detection technology yielding a sensitivity orders of magnitude higher than conventional fluorescence.14 A number of methods utilizing particles and time-resolved fluorescence have been studied, and high-sensitivity assays have been reported.15-18 Recently, chelated lanthanide ion particles have been synthesized, and low detection limits have been achieved in immunoassays.19 Such nanosized dye particles have been also applied for resonance energy transfer (6) You, W. W.; Haugland, R. P.; Ryan, D. K.; Haugland, R. P. Anal. Biochem. 1997, 244, 277–282. (7) CBQCA, http://probes.invitrogen.com/media/pis/mp06667.pdf. (8) Benson, J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 619–622. (9) Jones, L. J.; Haugland, R. P.; Singer, V. L. Biotechniques 2003, 34, 850– 861. (10) NanoOrange, http://probes.invitrogen.com/media/pis/mp06666.pdf. (11) Decher, G.; Schlenoff, J. B. Multilayer Thin Films; Wiley: Weinheim, Germany, 2003. (12) Peyratout, C. S.; Da¨hne, L. Angew. Chem., Int. Ed. 2004, 43, 3762–3783. (13) Leporatti, S.; Voigt, A.; Mitlo ¨hner, R.; Sukhorukov, G.; Donath, E.; Mo ¨hwald, H. Langmuir 2000, 16, 4059–4063. (14) Hemmila¨, I. Applications of Fluorescence in Immunoassays; Wiley: New York, 1991. (15) Ha¨rma¨, H.; Soukka, T.; Lo ¨vgren, T. Clin. Chem. 2001, 47, 561–568. (16) Soukka, T.; Ha¨rma¨, H.; Paukkunen, J.; Lo ¨vgren, T. Anal. Chem. 2001, 73, 2254–2260. (17) Zhao, X.; Tapec-Dytioco, R.; Tan, T. J. Am. Chem. Soc. 2003, 125, 11474– 11475. (18) Ye, Z.; Tan, M.; Wang, G.; Yuan, J. Anal. Chem. 2004, 76, 513–518. (19) Huhtinen, P.; Kivela¨, M.; Kuronen, O.; Hagren, V.; Takalo, H.; Tenhu, H.; Lo ¨vgren, T.; Ha¨rma¨, H. Anal. Chem. 2005, 15, 2643–2648.

Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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EXPERIMENTAL SECTION Materials. Monodisperse melamine-formaldehyde particles of 600 and 1000 nm in diameter were purchased from Microparticles GmbH (Berlin, Germany). Bovine serum albumin (BSA), γ globulin, thyroglobulin, Bradford reagent, poly(styrenesulfonate) sodium salt (PSS, MW 70 000), and poly(allylamine) hydrochloride (PAH, MW 70 000) were from Sigma-Aldrich (Buchs, Switzerland). Polyacrylic acid (PAA, MW 100 000) was obtained from Polyscience (Warrington, PA). The polyelectrolytes were dialyzed with molecular weight cutoff (MWCO) 30 000 membranes before use. The chelate {2,2′,2′′,2′′′-{[4-[(4-isothiocyanatophenyl)ethynyl]pyridine2,6-diyl]-bis(methylenenitrilo)}tetrakis(acetato)}europium(III) was

synthesized at the University of Turku.21 Alexa Fluor 680 NHS (Alexa680) was purchased from Invitrogen (Carlsbad, CA). Delfia enhancement solution was obtained from Innotrac Diagnostics (Turku, Finland). The NAP5 column was from GE Healthcare (Uppsala, Sweden). Instrumentation. Fluorescence emission spectra were measured with Varian Cary Eclipse (Varian, Palo Alto, CA). Timeresolved fluorescence emission spectra were monitored using a 340 nm excitation wavelength with 20 nm excitation and 2.5 nm emission slits and 100 µs decay and 900 µs delay times at 615 and 730 nm. The lifetimes were detected with the same parameters. Fluorescence of resonance energy transfer was measured at 730 nm using 340 nm excitation wavelength, 75 µs decay and 50 µs delay times with a Victor2 Multilabel counter from PerkinElmer Life Sciences. In detection of europium(III) chelate signal, 400 µs decay and 400 µs delay times were applied. The electrophoretic mobility measurements were carried out with Zetasizer 3000 HAS from Malvern Instruments (Worcestershire, U.K.). Preparation of Layer-by-Layer Particles. The europium(III) chelate was coupled to PAH by incubating 0.14 mM of PAH with 6 mM of the chelate in 500 µL of 200 mM carbonate buffer, pH 10, overnight. The reaction mixture was applied to the NAP5 column and eluted with water. This did not necessarily result in a product without starting materials. The aim was rather to change the buffer used in the coupling procedure. The melamine-formaldehyde particles offered a positive surface charge of +20 mV determined by measuring the electrophoretic mobility. The deposition of the polyelectrolyte layers were performed by incubating the particles in a solution of 1 g L-1 polyelectrolyte, 0.2 M NaCl, and 50 mM acetate buffer at pH 5.6 for 20 min. The excess of the polyelectrolyte solution after each deposition was removed by centrifugation and three washing cycles with water. Five layers were deposited in a sequence of PSS/PAH-Eu/PSS/PAH-Eu/PAA. The ζ-potential alternates between -45 mV for PSS as the outermost layer, -28 mV for PAA as the outermost layer, and +32 mV for PAH-Eu as the outermost layer. The fluorescence spectral studies were carried out with 600 nm particle using 1.6 × 109 particles and 32 nM concentration of BSA-Alexa680 in a total volume of 300 µL in 5 mM sodium citrate buffer, pH 3. Labeling of BSA with Alexa680. A total of 500 µg of BSA was dissolved in 100 µL of 50 mM phosphate buffer pH 7.4 and 1.0 mg of Alexa Fluor 680 NHS was dissolved in 20 µL of DMF. Thereafter, 3 µL of Alexa Fluor 680 NHS was mixed with the BSA solution. The reaction was allowed to proceed for 30 min, and unreacted dye was purified using a NAP5 column and PBS as an eluent. Protein Concentration Measurement. Incubations for protein concentration measurement were carried out in a microcentrifuge tube in a total volume of 175 µL. First, protein was diluted to 5 mM sodium citrate buffer, pH 3, in a total volume of 150 µL. Thereafter, 600 or 1000 nm LbL particles were added into the protein solution using 1.6 × 107 or 4.6 × 106 particles, respectively. Subsequently, BSA-Alexa680 was mixed with the suspension to the final concentration of 130 pM. The reaction mixture was

(20) Kokko, L.; Sandberg, K.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2004, 503, 155–162.

(21) Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.; Hemmila¨, I. Bioconjugate Chem. 1994, 5, 278–282.

Scheme 1. Schematic Illustration of the Principle of the Particle Sensor for the Total Concentration Measurement of Proteinsa

a

Acceptor (A)-labeled protein was adsorbed on a particle carrying surface-bound Eu(III) chelates in a surface layer composition of PSS/PAH-Eu/PSS/PAH-Eu/PAA. Adsorption of acceptor-labeled BSA on the Eu(III)-particle resulted in detectable TR-FRET signal. Sample protein efficiently occupied the surface preventing the adsorption of acceptor-labeled protein, and low TR-FRET signal was detected.

(RET) studies successfully.20 Although dyed nanoparticles have been useful for measurement of analytes in a homogeneous bioaffinity assay format, the particles are have not yielded optimal results for transferring energy in RET studies. This has been suggested to be due to the dyeing process which results in incorporation of the dye within the entire volume of a particle. In a RET approach, solely dyes incorporated to the outer surface layer can efficiently participate in energy transfer.20 Therefore, we synthesized particles with europium(III) chelates coupled to a surface layer of a particle for efficient energy transfer. This could be realized by the coating of monodisperse spherical particles with a specific sequence of europium(III)-doped polyelectrolyte films by LbL technology. Colloidal particles with nanostructured LbL layers served also as an efficient protein adsorption surface. The prepared particles were applied for protein concentration measurement using RET as the detection principle (Scheme 1). The developed method was simple: mixing of sample, particles, and label molecules followed by the detection of TR-FRET signal in a microtiter well. The detection limit of the technique was in the subnanomolar range for different proteins.

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Figure 1. Time-resolved fluorescence emission spectra for 600 nm particle containing europium(III) chelate (solid) and for sensitized signal (dashed) of acceptor-labeled protein adsorbed onto donor particles.

incubated for 10 min at room temperature, and 70 µL of the solution was transferred to a microtiter plate well for the fluorescence measurement. The pH test was performed at the BSA-Alexa680 concentration of 2.6 nM using universal buffer containing 100 mM of sodium tetraborate, citric acid, tris(hydroxymethyl)aminomethane, potassiumdihydrogen phosphate, and potassium chloride. Universal buffer was selected to obtain the least possible difference in the measurement conditions. The fluorescence spectra were measured under the assay condition having approximately 200-fold more particles and labeled protein in a total volume of 300 µL. RESULTS AND DISCUSSION Preparation and Characterization of Europium(III) LbL Particles. In this study, two europium(III) chelate particles were fabricated for total concentration measurement of proteins (Scheme 1). Positively charged core particles of 600 and 1000 nm in diameter were coated with PSS. A negatively charged surface was required for assembly of positively charged PAH carrying primary amine groups. Prior to assembly, amino-reactive isothiocyanato europium(III) chelates were covalently bound to the amino groups. Thereafter, additional layers of PSS and PAH-Eu were deposited. Finally, a layer of PAA was introduced to protect the europium(III) ion from dissociation at low pH in the protein concentration measurement and protecting the chelate from local environment changes and, therefore, signal changes due to protein adsorption. PAA was applied as the last layer due to reduce stabilization problems, when PSS was deposited. Layer thicknesses were estimated to be 1.5-2.0 nm per layer yielding still an adequate Fo¨rster distance from PAH-EU to the acceptor dye for RET studies.12,13 The europium(III) content of the prepared single particles was measured. This was carried out by dissociating europium(III) ions to the commercial signal enhancement solution and comparing the signal level to a known europium(III) calibrator. A single 600 or 1000 nm particle contained 4.6 × 104 or 7.5 × 104 europium(III) chelates, respectively. Typical excitation and emission spectra were measured for the soluble europium(III) chelate and the 600 nm LbL particle (Figure 1). An additional fluorescence excitation band was found at

the low UV region possibly due to particle-chelate interactions. The fluorescence lifetime of the 600 nm LbL particle was approximately 400 µs being equal to the soluble chelate. The sensitized energy transfer signal was monitored at the emission wavelength of Alexa680 after BSA-Alexa680 adsorption onto the particle. The lifetime of the sensitized signal at 730 nm was reduced to approximately 200 µs from that of the particle. This indicated a successful energy transfer from donor particle to acceptor-labeled protein. The FRET efficiency appeared to be in the range of 30% as estimated at the donor emission channel (Figure 1). The particle and BSA-Alexa680 concentrations were more than 100-fold higher than in the actual protein concentration measurement due to insensitivity problems related to the spectrofluorometer. Therefore, we measured the reduction in fluorescence signal at the donor emission wavelength, 614 nm, using the plate fluorometer when BSA-Alexa680 was adsorbed onto the particle at BSA-Alexa680 concentrations of 0 (0 ng), 0.13 (1.5 ng), and 3.9 (50 ng) nM. The BSA-Alexa680 concentration of 3.9 nM was used to saturate the particle surface with acceptor-labeled protein. The fluorescence measured at the donor emission wavelength decreased by approximately 70% at surface saturation (data not shown). One can assume that in a particle donor system not all the donors can participate in the FRET process. This is due to an unfavorable distance of Eu(III) chelates with respect to the acceptor molecules because the acceptor dye positioning is laterally random with respect to the donor labels; one can also argue that the orientation of the donor molecules is random. However, the millisecond long lifetime of the donor allows orientation changes within the relatively soft LBL environment of the donor and the protein coupled acceptor to take place with respect to each other within the time-window of the excited-state of the donor. In a previous study using the dual particle TR-FRET system and commercial polystyrene Eu(III) particles as donors, no reduction at the Eu(III) emission channel was observed.22 Although Valanne et al. had applied the specific binding component in the assay system leading to a larger donor-acceptor distance compared to that in the current study, our data suggests that the particle-based FRET efficiency was relatively high. This can be attributed at least partially to the LbL shell structure of Eu(III) donors positioning donor and acceptor molecules in close proximity to one another and reducing the effect of core related background fluorescence compared to Eu(III) chelate incorporated polystyrene particles.20,22 The efficiency was approximately 10% at BSA-Alexa680 concentration used in the protein quantification as estimated from the fluorescence signal decrease of the donor. No TR-FRET signal was found when no acceptor was present in solution. Protein Concentration Measurement. Once the particles were prepared and characterized, they were used to quantitatively measure the concentration of proteins. The sensor element was optimized for measuring bovine serum albumin and, thereafter, applied for other proteins under the BSA-optimized conditions. The studied sensor concept relied on the adsorption of labeled and sample protein on the LbL particles. Therefore, the adsorption process was expected to be strongly dependent on the pH of the solution. We investigated the pH dependence of protein adsorption (22) Valanne, A.; Lindroos, H.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2005, 539, 251–256.


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Figure 2. Time-resolved fluorescence signal was monitored as a function of pH for soluble europium(III) chelate (2), 600 nm particle containing europium(III) chelate (b), and sensitized signal of acceptorlabeled protein adsorbed onto donor particles (9).

on the prepared LbL particles with the labeled protein and followed the sensitized RET signal. The highest specific signal, i.e., highest adsorption tendency was found between pH 4 and 5 (Figure 2). This could be explained by the changes in surface charge of the protein and particles. BSA and PAA possessed relatively low pI values of 4.6 and 4.8, respectively. As pH approached the pI values, improved adsorption was found due to protonation of the surface groups and reduction in repulsive electrostatic forces. This led to a scheme, where proteins could be measured with an equal surface charge property between the proteins and the particles. The time-resolved fluorescence signal of the soluble europium(III) chelate was lost below pH 6 while high signal was measured at pH 8 (Figure 2). Again high instability was observed at high pH. However, europium(III) chelate-labeled LbL particles showed only minor pH dependence over the studied range. At low and high pH no more than one-third of the signal was lost. Obviously PAH-coupled chelate, additional PAA layer on the particle, and generally, the layer formation protected europium(III) chelate from dissolution leading to a stable structure for the particle sensor. A surface area of 1.1 × 106 nm2 was calculated for the 600 nm particle. Typically BSA occupies an area of 55 nm2 on a flat surface.23,24 Therefore 20 000 BSA or labeled BSA molecules could be maximally adsorbed on a single particle. This corresponded to approximately 18 ng of Alexa680-labeled BSA molecules in an assay. We measured the sensitized signal saturation of the assay by monitoring the signal change as a function of the BSA-Alexa680 concentration (Figure 3). The signal saturation and, therefore, the surface saturation of the 600 nm particle were reached at the BSAAlexa680 amount of 15 ng. This was well in accordance with the predicted surface-bound mass of BSA. The same signal saturation level for BSA-Alexa680 was achieved with the 1000 nm particle, when the surface area was taken into an account (data not shown). Rapid signal generation is a desired property in a protein concentration measurement. We investigated the adsorption of (23) Tencer, M.; Charbonneau, R.; Lahoud, N.; Berini, P. Appl. Surf. Sci. 2007, 253, 9209–9214. (24) Maste, M. C. L.; Pap, E. H. W.; van Hoek, A.; Norde, W.; Visser, A. J. W. G. J. Colloid Interface Sci. 1996, 180, 632–633.

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Figure 3. Saturation curve for BSA-Alexa680 adsorbed onto the 600 nm particle without competing sample protein.

Figure 4. Kinetic study on adsorption of BSA-Alexa680 to 600 (9) and 1000 (b) nm europium(III) particles at the concentration of 0 (filled) and 1 (open) µM of competing BSA.

BSA-Alexa680 on the particles as a function of time (Figure 4). Under the experimental conditions, the equilibrium was reached in 5 min. In comparison to commercial total protein concentration measurement methods, the developed method was rapid. The Bradford test is also rapid, but it is not competitive in assay sensitivity (see below). The commercial NanoOrange10 and CBQCA7 methods having a high and equal sensitivity compared to the developed method and require more than 30 and 60 min assay time, respectively. Therefore, the developed particle sensor can potentially speed up the overall laboratory work when high sensitivity is required. We estimated that a relatively low concentration of the large particle size used could be critical for assay kinetics. However, no change in signal was detected after the equilibrium was reached indicating that the particle size had insignificant effect on the measurement. We did not follow the kinetics further than 30 min because our aim was to develop a rapid protein total concentration assay. At longer times, large particle size may eventually affect the test but this was out of the scope of this study. To show the potential of the developed method for measurement of protein total concentration, we chose three differently sized proteins for testing: BSA (MW 66 000 g mol-1) γ globulin (treated as immunoglobulin, MW 150 000 g mol-1), and thyro-

Figure 5. Calibration curves for BSA (9), γ globulin (b), and thyroglobulin (2). The curves have been presented on the nanomolar (A) or microgram per liter (B) scale. Figure A contains a calibration curve for BSA measured with the Bradford method (1).

globulin (MW 670 000 g mol-1). Each protein was diluted and measured at the concentration range from 0.010 to 1000 nM (Figure 5A). The lowest detection limit was detected for thyroglobulin being the largest molecule among the measured molecules. Consistently the highest detection limit was monitored for the smallest molecule. This was due to size-dependent adsorption and displacement properties. With the same adsorption properties, a large protein occupied a larger area on the particles than smaller proteins. Therefore, a higher concentration of proteins was required for smaller proteins to efficiently displace the labeled competitor. However, on a gram/liter scale, the calibration curves of the proteins overlapped each other (Figure 5B). This suggested that the adsorption properties of different proteins were equal. We considered that low pH reduced all variations between the different proteins in the assay and had a major role in adsorption. The protein concentration measurement was competitive in nature, and the dynamic range of the assay could be varied by changing the concentration of the assay components. Competitive assays are typically limited to a concentration dynamic range of 2 orders of magnitude, which we have also consistently found for the developed method. The signal-to-background ratio of the measurement shown here was more than an order of magnitude. Over two-logs signal-to-background ratio was measured at the cost of reduced sensitivity (data not shown). High dynamics in signal

was achieved due to close proximity between donor and acceptor. Even closer proximity could have been achieved by omitting the PAA layer on the europium(III)-labeled PAH. However, protection of the chelate on the particle with PAA was seen as important and valuable against signal variations and was applied in this study. We also measured a calibration curve for BSA using the Bradford method according to the manufacturer’s protocol. The dynamic range of the Bradford method at the concentration scale was narrower than that of the developed method. As expected, the sensitivity of the Bradford method was poor as compared to the particle sensor (Figure 5A,B). The particle sensor measured protein at approximately a 100-fold lower concentration range than the widely used Bradford method with a coefficient of variation below 10%. NanoOrange and CBQCA are the most sensitive commercial protein concentration methods available. These commercial tests and the developed method have a detection limit of approximately 100 µg L-1.7,10 The commercial methods have, however, drawbacks as the assay protocols include heating steps (90 °C, NanoOrange) or toxic compounds must be applied (QBQCA). In addition, CBQCA measurement relies on the use of aliphatic primary amines of proteins. Therefore, the method is not suitable for the concentration measurement of proteins conjugated to, e.g., biotin or fluorochromes through amines. Also proteins, which have gone through post-translational modifications, may not be readily measured with methods using the protein sequence for signal generation.25 The developed particle sensor was based entirely on adsorption of proteins onto the LbL particle and subsequent detection of luminescent resonance energy transfer with common plate fluorometers without any need for hazardous or lengthy assay steps. Therefore, the particle sensor is more suitable for determining the concentration of labeled and modified proteins. Rotello’s group has developed particle-based sensors for multiprotein analysis in proteomics.26 The quenching mechanism used in their study reached a detection limit of more than 100 nM for BSA with photometric detection. Although the principle is appealing, the sensitivity does not meet the demand for a detection technique in proteomics. Having 1 × 108 cells and a protein expression copy number of 1000, a sensitivity of low nanomolar range is required.27 Our sensor could potentially lead to more powerful detection schemes providing subnanomolar sensitivity in a simple, rapid, and user-friendly format. We have chosen the LbL chemistry on the sensor which gives versatile opportunities for varying the coupling chemistry. Different polyelectrolytes with different functional groups can be assembled to provide surfaces with a desired chemistry. CONCLUSIONS Europium(III) chelate nanoparticles have been used for RET studies in combination with near-infrared emitting dyes for specific detection of bioaffinity binding partners in the past. Unfavorable background signal was estimated to limit the use of these dyed (25) Noble, J. E.; Knight, A. E.; Reason, A. J.; Di Matola, A.; Bailey, M. J. Mol. Biotechnol. 2007, 37, 99–111. (26) You, C.-C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I.; Erdogan, B.; Krovi, S. A.; Bunz, E. H. F.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318– 323. (27) Corthals, G.; Wasinger, V. C.; Hochstrasser, D. F.; Sanchez, J.-C. Electrophoresis 2000, 21, 1104–1115.


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nanoparticles due to the dyes incapability of participating in RET signal generation.20 Therefore, LbL technology provided an excellent platform for a particle-based RET sensor concept as labeling of surface layers was readily performed with desired surface properties, opening possibilities toward more selective quantification of various proteins. A new and simple method for quantification of proteins was developed. The technique was based on the combination of two well-known phenomena: surface adsorption and TR-FRET. The technique was powerful in detecting low concentrations of proteins, and no lengthy assay steps or hazardous substances were required. We achieved a detection limit comparable to most sensitive commercial methods. The rapid and user-friendly method is potentially useful in speeding up overall laboratory work when

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high sensitivity is needed. As the principle of the method relies on an entirely different concept from the known methods, the particle sensor can be applied to measure the concentration of target proteins, e.g., labeled and modified proteins, which may be outside the reach of other methods. Further development of the technique toward higher sensitivity is ongoing in our laboratory. ACKNOWLEDGMENT The work was supported by the Finnish Funding Agency for Technology and Innovation, Tekes. Received for review September 16, 2008. Accepted October 14, 2008. AC801960C

Sensitive Quantitative Protein Concentration Method Using

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