Ultrasensitive Protein Concentration Measurement Based on Particle

May 19, 2009 - Sari Pihlasalo,* Jonna Kirjavainen, Pekka Ha¨ nninen, and Harri Ha¨ rma¨. Laboratory of Biophysics and Medicity, University of Turku...
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Anal. Chem. 2009, 81, 4995–5000

Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching Sari Pihlasalo,* Jonna Kirjavainen, Pekka Ha¨nninen, and Harri Ha¨rma¨ Laboratory of Biophysics and Medicity, University of Turku, Tykisto¨katu 6A, 20520 Turku, Finland A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition of the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The protein concentration is determined by observing the magnitude of fluorescence altered by quenching the fluorescence on the gold particles in a homogeneous assay format. Under optimal low pH conditions, the assay allowed the determination of picogram quantities (7.0 µg/L) of proteins with an average variation of 4.5% in a 10 min assay. The assay sensitivity was more than 10fold improved from those of the commonly used most sensitive commercial methods. In addition, the particle sensor provides a simple and rapid assay format without requirements for hazardous test compounds and elevated temperature. Eleven different proteins were tested with the constructed sensor exhibiting a protein-to-protein variability less than 15% allowing protein concentration measurements without the need for recalibration of different proteins. Sensitive analytical methods for quantification of protein concentration in solution are important in biological laboratories.1 Bradford,2 Lowry,3 and BCA4 methods are the most commonly used techniques to determine protein concentrations. However, these methods have limitations in sensitivity, protein-to-protein variability, and dynamic range. Sample treatment and measurement procedures can also in some cases be impractical and time-consuming. The methods may require exact or lengthy incubation time with multiple steps at elevated temperatures and hazardous reagents. For instance, the most sensitive commercial NanoOrange5 and CBQCA1 methods require sample heating and cooling steps in order to reach adequate reproducibility and sensitivity. The methods that rely on the interaction * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +358 2 333 7060. (1) You, W. W.; Haugland, R. P.; Ryan, D. K.; Haugland, R. P. Anal. Biochem. 1997, 244, 277–282. (2) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (3) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265–275. (4) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, 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. (5) Jones, L. J.; Haugland, R. P.; Singer, V. L. BioTechniques 2003, 34, 850– 861. 10.1021/ac9001657 CCC: $40.75  2009 American Chemical Society Published on Web 05/19/2009

of a dye with proteins,2-4,6 detergent-coated proteins,5,7 or a specific protein functional group1,8 are dependent on protein surface characteristics and the number of the functional groups. Thus these methods are susceptible to protein-to-protein variability and need to be calibrated for each target separately. The adsorption of colloidal gold particles to biological molecules have been exploited in immunocytochemical and histochemical applications as probes for electron microscopy purposes and in cell receptor and function studies.9-11 The adsorption and binding characteristics of citrate stabilized gold particles have been utilized for different analytical purposes.12-20 Rohringer and Holden have applied the nonspecific adsorption of proteins to gold particles for qualitative assaying of proteins in a separation assay.12 The method for determining protein concentration in solution using nonspecific adsorption of sample protein onto gold surface has been previously reported.13,14 The ability of proteins to aggregate gold particles under acidic conditions was the basis of the method and broadening of the light absorption maximum typical to gold particles was detected. The assay was sensitive to minor changes in pH, gold particle size, and different proteins used and limited in dynamic range and sensitivity. The same principle has also been applied in a sol particle immunoassay introduced by Leuvering et al.15 The sensitivity of light absorption spectroscopy is, however, far less than that of fluorescence and other luminescence methods. Recently a method based on the covalent binding of thiols onto the gold particle and fluorescence resonance energy transfer has been developed to determine a (6) Peterson, G. L. Anal. Biochem. 1977, 83, 346–356. (7) Lee, S. H.; Suh, J. K.; Li, M. Bull. Korean Chem. Soc. 2003, 24, 45–48. (8) Liu, J.; Hsieh, Y.-Z.; Wiesler, D.; Novotny, M. Anal. Chem. 1991, 63, 408– 412. (9) Faulk, W. P.; Taylor, G. M. Immunochemistry 1971, 8, 1081–1083. (10) Hayat, M. A. Colloidal Gold: Principles, Methods and Applications, Vols. 13; Academic Press: San Diego, CA, 1989-1991. (11) Horisberger, M. Scanning Electron Microscopy 1981, 2, 9–31. (12) Rohringer, R.; Holden, D. W. Anal. Biochem. 1985, 144, 118–127. (13) Stoscheck, C. M. Anal. Biochem. 1987, 160, 301–305. (14) Ciesiolka, T.; Gabius, H.-J. Anal. Biochem. 1988, 168, 280–283. (15) Leuvering, J. H. W.; Thal, P. J. H. M.; Van der Waart, M.; Schuurs, A. H. W. M. J. Immunoassay 1980, 1, 77–91. (16) Chen, S.-J.; Chang, H.-T. Anal. Chem. 2004, 76, 3727–3734. (17) Phillips, R. L.; Miranda, O. R.; You, C.-C.; Rotello, V. M.; Bunz, U. H. F. Angew. Chem., Int. Ed. 2008, 47, 1–5. (18) Miranda, O. R.; You, C.-C.; Phillips, R.; Kim, I.-B.; Ghosh, P. S.; Bunz, U. H. F.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 9856–9857. (19) Krovi, S. A.; Erdogan, B.; Han, G.; Kim, I.-B.; Bunz, U. H. F.; Rotello, V. M. Polym. Prepr. 2006, 47, 599–600. (20) You, C.-C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I.-B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H. F.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318–323.

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concentration of different small molecule thiols. In this assay, thiols displaced an organic dye molecule, nile red, adsorbed onto the gold surface thus enhancing fluorescence intensity of the displaced dye.16 The method was specific only for thiols and was not applied to measure the concentration of other molecules such as proteins. Rotello et al. have utilized the ability of differently coated gold nanoparticles to quench the fluorescence of conjugated polyelectrolytes to identify different bacteria, DNA, and proteins.17-19 However, the sensitivity of the method was limited in protein quantification and varied significantly between different proteins.20 Previously, we have reported a FRET-based protein quantification assay using adsorption competition of sample protein and acceptor dye labeled protein onto europium(III) labeled donor particles.21 In the FRET-based assay, the sensitivity was limited to 100 µg/L being equal to the sensitivity of the most sensitive commercial methods. Here, we developed a highly sensitive and easy-to-use method for total protein concentration measurement using protein adsorption onto gold particles and fluorescence quenching. In the developed assay, colloidal gold quenches fluorescence of dye-labeled protein at close proximity to the gold surface. The method is based on the competitive assay principle with noncovalent adsorption of sample protein and dipyrrylmethene-BF2 530 (BF530) labeled bovine serum albumin (BSA) on nanosized gold particles in a homogeneous assay format. The assay condition was optimized such that the dye-labeled BSA was adsorbed on cold particles and the luminescence was quenched, when no competing sample protein was present. Addition of sample protein efficiently occupies the particle surface preventing adsorption of the dye-labeled BSA onto the gold particles and thus labeled BSA remained fluorescent in solution (Figure 1). The analytical conditions were optimized for pH, buffer components, incubation time, concentration of BSABF530 and gold particles, and gold particle size. Eventually the method was applied to investigate different proteins with varying size and isoelectric properties.

facturer) were obtained from British Biocell International (Cardiff, U.K.). Streptavidin (>97%) was purchased from BioSPA (Milan, Italy), and all other proteins (albumin from bovine serum, >99%; ferritin saline solution from equine spleen, type I (85 g/L), purity not provided by the manufacturer; histone from calf thymus, type II-A, high purity stated by the manufacturer; pepsin from porcine stomach mucosa, 32%; catalase from bovine liver, 65%; albumin from chicken egg white, 97%; albumin from sheep serum, >98%; albumin from porcine serum, 99%; γ-globulins from bovine blood, 98%; thyroglobulin from bovine thyroid, 99%) were from SigmaAldrich (Munich, Germany). Dipyrrylmethene-BF2 530 (BF530) dye was synthesized as described earlier.22 All the reagents used to prepare buffer solutions were obtained from SigmaAldrich (Munich, Germany), except citric acid and sodium acetate were from FF-Chemicals (Yli-Ii, Finland) and 2-aminoisobutyric acid from TCI Europe (Zwijndrecht, Belgium). The purity of the chemicals used was >98%. High purity Milli-Q water was used to prepare all aqueous solutions. Methods. Labeling of Bovine Serum Albumin with BF530. Dipyrrylmethene-BF2 530 (BF530) dye was conjugated to bovine serum albumin as described previously for mouse monoclonal IgG anti-hAFP.23 The labeling degree of BSA-BF530 was 4.8. Competitive Protein Assay. The competitive protein assay was optimized for gold particle and BSA-BF530 concentrations, gold particle size, assay buffer conditions, and microtiter well incubation time (4 min-5.6 h) using BSA as a model protein. Universal buffer containing 5.0 mM sodium tetraborate, citric acid, tris(hydroxymethyl)aminomethane, potassiumdihydrogen phosphate, and potassium chloride was used to study the effect of pH, at a pH range from 1.5 to 11.5. The concentration of the universal assay buffer was varied from 5.0 µM to 100 mM. In addition, the following buffer components (5.0 mM concentration) were tested: citrate pH 1.5, 2.0, 2.5, 3.0, and 4.5, citrate/phosphate pH 3.0 and 4.5, mandelic acid pH 3.0 and 4.5, acetate pH 4.5, propionic acid pH 4.5, 3-chloro-4-hydroxy-phenylacetic acid pH 4.5, phthalate pH 4.5, acrylic acid pH 4.5, isonicotinic acid pH 4.5, glycin pH 2.5 and 3.0, 2-aminoisobutyric acid pH 2.5, and aminocaproic acid pH 4.5. Eleven proteins with different isoelectric point and size were tested for the assay. In a typical microtiter well assay, 70 µL of the sample protein solution in 5.0 mM glycin buffer pH 3.0 and 20 µL of the gold particles 20 nm in diameter in water were mixed. BSA-BF530 was added in 20 µL of 1.0 mM phosphate buffer, pH 7.4, containing 0.10 mM potassium chloride. The final concentrations of gold particles and BSA-BF530 were 64 pM and 0.89 nM, respectively. Fluorescence emission intensities were measured after 10 min of incubation with the Victor2 multilabel counter (Wallac, PerkinElmer Life and Analytical Sciences, Turku, Finland) using 530 nm excitation and 572 nm emission wavelengths. Stability of diluted BSA-BF530 was examined by measuring the fluorescence signal of the diluted BSA-BF530 stored in different buffers. These buffer solutions were stored at +4 °C and diluted in the assay buffer (5.0 mM glycin buffer pH 3.0) before the stability measurement.

MATERIALS AND METHODS Materials. Colloidal gold particles (20, 40, 80, and 100 nm in diameter having ∼100% monodispersity according to the manu-

RESULTS AND DISCUSSION We have developed a sensitive protein quantification method based on protein adsorption on nanosized gold particles and

(21) Ha¨rma¨, H.; Dähne, L.; Pihlasalo, S.; Suojanen, J.; Peltonen, J.; Hänninen, P. Anal. Chem. 2008, 80, 9781–9786.

(22) Meltola, N. J.; Wahlroos, R.; Soini, A. E. J. Fluoresc. 2004, 14, 635–647. (23) Meltola, N. J.; Kettunen, M. J.; Soini, A. E. J. Fluoresc. 2005, 15, 221–232.

Figure 1. Schematic illustration of quenching assay using gold particles and BF530 labeled BSA. Quenching of fluorescence signal was detected by adsorption of BSA-BF530 onto gold particles in the absence of sample protein (a). As sample protein occupied the particle surface, BSA-BF530 remained in solution and high fluorescence signal was detected (b).

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Figure 2. The effect of pH on the signal-to-background ratio (ratio between signal at 700 and 0 mg/L BSA) of the developed assay. BSABF530 was adsorbed to the 20 nm gold particles in a universal buffer in order to reduce the effect of assay buffer components to the adsorption process.

fluorescence quenching of BF530 conjugated to BSA. Different concentrations of proteins were competing with BSA-BF530 from adsorption onto the gold particles in a homogeneous quenching assay format (Figure 1). The fluorescence of the dye was efficiently quenched, when bound onto the particle, and an increasing, high signal level was measured with increasing sample protein concentration. We studied the effect of pH ranging from 1.5 to 11.5 for the assay performance (Figure 2). A universal buffer was used to measure the influence of pH in order to minimize the effect of the different buffer components to the adsorption process. The most efficient adsorption of BSA-BF530 onto gold and, therefore, the highest signal-to-background ratio (ratio between signal at 700 and 0 mg/L BSA) in the assay was reached at pH 3-4. We extended the investigation to the pH range of 1.5-4.5 using 12 different buffer compositions (see Materials and Methods) to find the optimal signal-to-background ratio. Independent of the buffer used, nearly identical signals were measured. These measurements implied that the buffer components had an insignificant effect on protein adsorption. Mainly pH appears to determine the functionality of the sensor suggesting that the method is widely applicable as sample buffer composition needs not to be strictly defined. We chose pH 3.0 glycin buffer for further studies and optimized the concentration of the glycin buffer between 5.0 µM and 100 mM. The highest signal-to-background ratio was obtained using 5.0 mM glycin buffer (data not shown). The pH effect on adsorption is explained by the properties of a protein and the particle surface. Mainly the surface charge characteristics determined protein adsorption to gold particles used. At pH close to the protein isoelectric point, protein exhibits an zwitterionic form and its solubility to an aqueous solution reaches the minimum.24,25 The gold particle surface carried a negative charge introduced by citrate ions giving necessary (24) Weiser, H. B. A Textbook of Colloid Chemistry; Wiley & Sons: New York, 1949. (25) Yin, F.; Zhang, Y.; Wu, Y.; Cai, Y.; Xie, Q.; Yao, S. Anal. Chim. Acta 2001, 444, 271–277.

colloidal stability to the gold particles and preventing aggregation.26 Protein adsorption to the negatively charged particles increases with pH lower than the isoelectric point of the protein. This stems from the fact that proteins and particles possess opposite charges.27,28 Citric acid is a triprotic acid having pKa values of 3.15, 4.77, and 6.40. At pH below 3, the particle surface charge approaches zero evidently lowering the electrostatic interaction and reducing protein adsorption. Our results are in accordance to the reported properties of these binding partners (Figure 2). BSA adsorbs efficiently to the gold particle surface at pH below its isoelectric point 4.7,29,30 exhibiting positive charge. As expected, the adsorption decreases at pH below 3 and above 4. This dynamic pH range gives an adequate window to quantify proteins reducing the variation due to minor pH changes of the buffer. In addition, entropic effects may have influenced on protein adsorption to nanoparticles.31,32 At low pH, denaturation of proteins was likely to expose protein hydrophobic moieties leading to hydrophobic interactions at the particle surface and, therefore, to entropic changes. The protein assay sensitivity was optimized for the concentration of gold particles and BSA-BF530. The highest signal-tobackground ratio was reached at a concentration of 64 pM for particles and 890 pM for BSA-BF530. The assay sensitivity was 500 pg in a microtiter well giving the signal-to-background ratio of 1.2. This corresponded to a concentration of 7.0 µg/L or 100 pM (Figure 3a). The achieved sensitivity was considerably improved from our layer-by-layer particle sensor reported earlier and exceeds that of the most sensitive commercially available tests.21 Sensitivity levels of 20 and 10 ng have been achieved for BSA with the NanoOrange5 and CBQCA1 protein quantification methods, respectively. We have earlier measured an assay sensitivity of 1000 ng for the widely used Bradford method.21 Our homogeneous assay format also led to a low assay variation, 4.5%. Commercial sensitive protein quantitation assays are timeconsuming. Incubation times more than 30 and 60 min are required in the NanoOrange5 and CBQCA1 methods, respectively. The developed assay reached equilibrium in 10 min suggesting that the overall laboratory work can be speeded up, when high sensitivity protein quantification is required. After reaching the equilibrium in 10 min, the signals were essentially unchanged in all concentrations tested (Figure 3b). This indicates that BSA adsorbed strongly to the nanoparticles in an irreversible manner inhibiting the exchange of BSA with BSA-BF530 over time. The fluorescence could also be read directly after the addition of BSABF530 (before 10 min) in case a very rapid detection is desired. Still high sensitivity can be achieved, but this may result in an increase of assay variation. The stability of the reagents used in the protein quantification can be limited by storage time and conditions.1,3,5 Protein adsorption and its loss on test tubes under the storage can be a severe limitation. Therefore, the fluorescence signal of the diluted (26) Frens, G. Nat.-Phys. Sci. 1973, 241, 20–22. (27) Hemmersam, A. G.; Rechendorff, K.; Besenbacher, F.; Kasemo, B.; Sutherland, D. S. J. Phys. Chem. C 2008, 112, 4180–4186. (28) Shen, D. Z.; Xue, Y. H.; Kang, Q.; Xie, Q. J.; Chen, L. X. Microchem. J. 1998, 60, 1–7. (29) Lee, W.-K.; Ko, J.-S.; Kim, H. M. J. Colloid Interface Sci. 2002, 246, 70–77. (30) Chun, K. Y.; Stroeve, P. Langmuir 2002, 18, 4653–4658. (31) Ferna´ndez, A.; Ramsden, J. J. J. Biol. Phys. Chem. 2001, 1, 81–84. (32) Lee, S. H.; Ruckenstein, E. J. Colloid Interface Sci. 1988, 125, 365–379.

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Figure 4. The fluorescence signal stability of labeled BSA at the 4.9 nM storage condition. The stability of the labeled protein was investigated with the developed method. The curves are the fits of the data to the equation signal ) a ln(t/h) + b, where t is time and a and b are constants.

Figure 3. The calibration curve (a) and kinetic measurements (b) of the BSA protein assay. Gold particles of 20 nm in diameter and various concentrations of BSA were mixed, and the kinetic data was monitored after addition of BSA-BF530.

BSA-BF530 was studied over time stored at a concentration of 4.9 nM at different buffer solutions corresponding to the end-user storage conditions (Figure 4). The signal loss at high salt concentration was due to decreased electrostatic repulsions between the test tubes and protein enhancing adsorption.33,34 This was significantly reduced at low salt concentrations and pH 7.4 leading to high signal stability. Although a minor decrease of fluorescence signal was observed, the assay sensitivity or performance was unaffected compared to the assay performed with freshly prepared BSA-BF530 dilution. The gold colloid suspension is stable at least 1 year according to the manufacturer, and its stability was not further investigated. We assume that the sensitivity of the method is still limited by the adsorption of the diluted sample and labeled protein onto test tubes. Although our results show that a stable storage condition can be found, adsorption of proteins may occur to plastic tube walls rapidly even under optimized buffer conditions significantly reducing the assay sensitivity. Elucidation of the problem would be a target of a (33) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233– 244. (34) Chang, Y.-K.; Chou, S. Y.; Liu, J.-L.; Tasi, J.-C. Biochem. Eng. J. 2007, 35, 56–65.

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separate investigation where various nonsticky surfaces are compared to the currently used test tubes, pipet tips, and microtiter wells. The effect of particle size ranging from 20 to 100 nm was studied for optimal assay performance. The absorbance of the diluted particle solutions was measured with a spectrophotometer, and the absorption coefficient was calculated for each particle size using the gold chloride concentration provided by the manufacturer. The linear relationship between the logarithms of absorption coefficient and the particle diameter was found suggesting that the particle concentrations were correctly provided.35 According to Jones et al., the total surface area of the differently sized particles should be kept constant to yield the same surface coverage and quenching.36 However, we observed different quenching efficacies for the particles. Thus, we selected a number of particles for each particle size giving equal background signal (no BSA sample in the assay) to the comparative study. With the optimized number of particles, the sensitivity and signal response did not vary with the particle size (Figure 5). The least amount of gold (mass) was required in the assay with 20 nm particles, which were used throughout this study. In protein quantification, it is desirable that different proteins can be determined with high accuracy in order to measure proteins of unknown structures. We studied the response of the assay to a proteins with different properties (Figure 6): the molecular weight varied from 14 to 670 kDa, the isoelectric points ranged from 1.0 to 10, the proteins contained a varied number of subunits having physical or covalent intersubunit interactions, and the proteins possessed various shapes. As discussed above, protein adsorption onto gold particle favors test conditions at the isoelectric point of the protein or below,37,38 which is supported also by (35) Liu, X.; Atwater, M.; Wang, J.; Huo, Q. Colloids Surf., B 2007, 58, 3–7. (36) Jones, G. M.; Wofsy, C.; Aurell, C.; Sklar, L. A. Biophys. J. 1999, 76, 517– 527. (37) Geoghegan, W. D.; Ackerman, G. A. J. Histochem. Cytochem. 1977, 25, 1187–1200. (38) Goodman, S. L.; Hodges, G. M.; Trejdosiewicz, L. K.; Livingston, D. C. Scan. Electron Microsc. 1979, 3, 619–628.

Figure 5. The response of the BSA assay using four different gold particle sizes. The number of particles was optimized individually to each assay due to differences in quenching efficiency of differently sized particles.

Figure 6. The response curves of different proteins measured with the particle sensor. The average coefficient of variation (CV) for signal is shown in the insert for assays of individual proteins, all proteins, and all proteins excluding ferritin at each measured concentration.

our results for BSA (Figure 1). In assaying 11 different proteins, the protein-to-protein signal variability was low. This is a consequence of the correctly chosen pH. At low pH, most of the proteins carry a net positive charge and may be entirely or partially denaturated increasing adsorption on surfaces. This apparently applies to a large number of different proteins improving universality of the developed method. Although pepsin is an acidic protein (pI 1.0),39 it was assayed with equal efficacy compared to (39) Chern, C.-S.; Lee, C.-K.; Chang, C.-J. Colloid Polym. Sci. 2004, 283, 257– 264.

the other tested proteins. Only ferritin (pI 6.1)40 showed a significantly lower response compared to other proteins. It is a cytosolic protein and exhibits an unusually high thermal and chemical stability due to its metal binding properties compared to other proteins.41-43 The assay sensitivity for ferritin was nearly equal to other proteins. However, a lower signal was detected at concentrations above 20 µg/L, which may be due to the high structural stability and, therefore, low adsorption behavior of the protein.33 This suggested that the protein intrinsic structural properties may have an effect on adsorption and, therefore, on sensoring efficacy. For example, proteins may possess open and closed conformations,44 hard and soft proteins may adsorb to surfaces with varying efficiencies,33 and proteins may take a pHdependent extended or globular conformation.32 Although varying properties can be identified, still proteins of different structural features could be measured with nearly equal efficiencies under the optimized conditions with the current sensor concept. The developed method was relatively robust and detected signal values within 15% coefficient of variation for different proteins. The corresponding coefficient of variation was 20% for the NanoOrange5 and 30% for the CBQCA1 and BCA4 methods as deduced from the literature. The protein-to-protein variability of the developed method was comparable to the NanoOrange method, when the molecular weight of the protein varied from 18 to 150 kDa. However, the variation in the signal increased significantly for small proteins in the NanoOrange method. The 15% coefficient of variation for the particle sensor included ferritin having different assay signal levels from those of the other proteins. When excluding ferritin data, we obtained an average coefficient of variation of 13%. Within each protein assay, the coefficient of variation was on average 5.3% (Figure 6). In a competitive assay, the sensitivity is determined by the affinity of the binding partners and the number of assay components. The smaller the number of assay components the more sensitive assay is gained. In the developed method, proteins competed from the surface sites on the nanoparticles and both the protein assayed and the labeled protein must adsorb. High protein adsorption efficacy, i.e., high affinity was achieved under optimized assay conditions. This ensured an effective quenching of the label and, therefore, low background signal. The comparison of fluorescence signals at high and zero BSA concentration gave 97% quenching efficacy when background fluorescence of microtiter wells containing assay buffer was subtracted. In comparison to our previous FRET-based particle sensor, the sensitivity improvement was significant.21 This is partially due to the fact that in the current sensor, signal was increased as sample was added and small changes in fluorescence signal could be observed. In the FRET-based assay, high fluorescence signal was detected at zero sample concentration making the differentiation of low signal changes and, therefore, small sample concentrations more difficult. The observed assay sensitivity of the current gold particle (40) Passaniti, A.; Roth, T. F. Biochem. J. 1989, 258, 413–419. (41) De Domenico, I.; Vaughn, M. B.; Li, L.; Bagley, D.; Musci, G.; Ward, D. M.; Kaplan, J. EMBO J. 2006, 25, 5396–5404. (42) Santambrogio, P.; Levi, S.; Arosio, P.; Palagi, L.; Vecchioq, G.; Lawson, D. M.; Yewdall, S. J.; Artymiuk, P. J.; Harrison, P. M.; Jappelli, R.; Cesareni, G. J. Biol. Chem. 1992, 287, 14077–14083. (43) Martsev, S. P.; Vlasov, A. P.; Arosio, P. Protein Eng. 1998, 11, 377–381. (44) De´r, A.; Kelemen, L.; Fa´bia´n, L.; Taneva, S. G.; Fodor, E.; Pa´li, T.; Cupane, A.; Cacace, M. G.; Ramsden, J. J. J. Phys. Chem. B 2007, 111, 5344–5350.

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sensor was 500 pg (7 µg/L) which corresponded to the surface coverage of approximately 4%. This was calculated assuming that BSA occupies a 50 nm2 surface area45,46 on the particle in a suspension containing 4.2 × 109 gold particles (64 pM). This calculation suggests that small changes in the surface coverage were detected, and the adsorption of the protein to the nanoparticle must be strong preventing the loss of the sample protein and an exchange of the sample protein with the labeled protein. CONCLUSIONS A new and highly sensitive method to detect protein total concentrations was developed. The method relies on protein adsorption on gold colloids and quenching of fluorescently labeled protein. The BSA assay sensitivity of 500 pg was more than 10fold higher than that reported for most sensitive commercial protein quantification methods. We have previously shown that a sensitive particle sensor can be constructed using europiumlabeled particles and fluorescence resonance energy transfer from the europium chelates on the particle to acceptor labeled protein.21 Our current data shows that sensitive time-gated fluorescence detection is not required to achieve low detection limits. In fact, (45) Zsom, R. L. J. J. Colloid Interface Sci. 1986, 111, 434–445. (46) Teichroeb, J. H.; McVeigh, P. Z.; Forrest, J. A. Eur. Phys. J., E 2009, 28, in press.

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conventional fluorescence is more amenable to end-users as plate fluorometers without the time-gating mode are more commonly in use. The particle sensor is based on nonspecific adsorption of proteins to the solid surface, which is in contrast to methods based on, e.g., specific identification of protein chemical groups,1,8 or the interaction of the protein surface with dye2-4,6 or detergent molecules.5,7 This assay principle led to a small variation between parallel measurements and exhibited less than 15% protein-toprotein variability. The low protein-to-protein variability was achieved by selecting low pH conditions reducing differences in the electrostatic interaction between different proteins and the gold particle. This potentially results in more reliable quantification of proteins without a need for calibration of each protein. In addition, the method is based on the simple mix-and-measure principle, and no time-consuming heating steps or hazardous chemicals are used making the method more user-friendly to endusers. ACKNOWLEDGMENT This work was supported by the Finnish Funding Agency for Technology and Innovation (Tekes). Received for review January 22, 2009. Accepted April 16, 2009. AC9001657