Sensitive and Colorimetric Detection of the Structural Evolution of

Jan 26, 2009 - Structural Evolution of Superoxide Dismutase with. Gold Nanoparticles. Surin Hong,† Inhee Choi,† Suseung Lee,† Young In Yang,† ...
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Anal. Chem. 2009, 81, 1378–1382

Sensitive and Colorimetric Detection of the Structural Evolution of Superoxide Dismutase with Gold Nanoparticles Surin Hong,† Inhee Choi,† Suseung Lee,† Young In Yang,† Taewook Kang,‡ and Jongheop Yi*,† School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 151-744, Korea, and Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 121-742, Korea The detection and characterization of protein aggregates are critical in terms of advanced diagnostic applications and investigations of protein stability. A variety of analytical methods (e.g., circular dichroism, size exclusion chromatography, and fluorescence microscopy) have been used in this regard, but they are limited in the trace detection of the structural evolution of protein aggregation. Here we report the gold nanoparticle (AuNP)-based highly sensitive and colorimetric detection of the temporal evolution of superoxide dismutase (SOD1) aggregates implicated in the pathology of amyotrophic lateral sclerosis (ALS). For the temporal discrimination of SOD1 aggregation, AuNPs were conjugated with SOD1 monomers (SOD1-AuNPs). Upon exposure of the probes (SOD1AuNPs) with SOD1 aggregates, significant changes in both surface plasmon resonance spectra and concomitant colors were observed which are attributed to the formation of probe aggregates of variable sizes onto the SOD1 aggregates. Protein aggregation is one of the key elements of the biophysical stability of a protein, as well as a pathological feature of many disorders in the human body. In certain cases, misfolding and aggregation, which appear to be linked to cytotoxicity, are mediated by a variety of factors (e.g., native state stability and structural propensity) and are thought to be involved in neurodegenerative diseases such as Huntington’s, Alzheimer’s, and Parkinson’s diseases.1 Understanding the detailed process (including the biophysical characteristics) by which protein aggregation occurs holds the potential for novel diagnosis of these diseases; thus, appropriate analytical methods are required for the characterization of protein aggregates in a sample. In this context, several methods have been utilized to detect protein aggregates, including size exclusion chromatography,2-4 circular dichroism,5,6 and fluorescence * To whom correspondence should be addressed. Phone: +82-2-880-7438. Fax: +82-2-885-6670. E-mail: [email protected]. † Seoul National University. ‡ Sogang University. (1) Soto, C. FEBS Lett. 2001, 498, 204–207. (2) Ye, H. Anal. Biochem. 2006, 356, 76–85. (3) Oliva, A.; Llabres, M.; Farina, J. B. J. Pharm. Biomed. Anal. 2001, 25, 833–841. (4) Wen, J.; Arakawa, T.; Philo, J. S. Anal. Biochem. 1996, 240, 155–166. (5) Wang, W. Int. J. Pharm. 2005, 289, 1–30.

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microscopy.7,8 These methods have been frequently employed to characterize protein aggregates and show high accuracy in terms of the measured properties; however, they possess limited sensitivity in detecting the stepwise structural evolution of protein aggregation. Therefore, a highly sensitive and simple analytical method is required to overcome this problem. In this study, we propose a newly designed gold nanoparticle (AuNP)-based colorimetric detection system for the sensitive visualization and characterization of the structural evolution of protein aggregates using their own monomer-conjugated AuNP. Noble metal nanoparticles have been exploited in sensing applications such as biological labels, markers, and stains for various microscopies,9-12 due to the sensitivity of the environmental changes in the localized surface plasmon band. In particular, AuNPs have been employed as molecular-recognition elements or amplifiers13 in biosensing systems and as components of nanoscale optical devices in extinction-based methods.14-17 Sensing is based on the sensitive change in the refractive index arising from the formation of AuNP probe aggregates according to the specific interaction between protein aggregates and probes. Thus, the own monomer of protein aggregates is an essential element in developing a sensing probe as an intermediate recognizing seed in the evolution of protein aggregates. As a proof-of-concept test, superoxide dismutase (SOD1) protein was chosen and conjugated on a AuNP surface (SOD1AuNP) for detection of the structural evolution of SOD1 aggregates. SOD1 is well-known as a highly stable dimeric metalloenzyme that catalyzes the dismutation of superoxide radical to (6) Rakhit, R.; Crow, J. P.; Lepock, J. R.; Kondejewski, L. H.; Cashman, N. R.; Chakrabartty, A. J. Biol. Chem. 2004, 279, 15499–15504. (7) Demeule, B.; Gurny, R.; Arvinte, T. Int. J. Pharm. 2007, 329, 37–45. (8) Giorgadze, T. A.; Shiina, N.; Baloch, Z. W.; Tomaszewski, J. E.; Gupta, P. K. Diagn. Cytopathol. 2004, 31, 300–306. (9) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. (10) Penn, S. G.; He, L.; Natan, M. J. Curr. Opin. Chem. Biol. 2003, 7, 609– 615. (11) Hutter, E.; Fendler, J. H. Adv. Mater. 2004, 16, 1685–1706. (12) Endo, T.; Yamamura, S.; Nagatani, N.; Morita, Y.; Takamura, Y.; Tamiya, E. Sci. Technol. Adv. Mater. 2005, 6, 491–500. (13) Kang, T.; Hong, S.; Choi, I.; Sung, J. J.; Kim, Y.; Hahn, J.-S.; Yi, J. J. Am. Chem. Soc. 2006, 128, 12870–12878. (14) Lee, S.; Perez-Luna, V. H. Anal. Chem. 2005, 77, 7204–7211. (15) Kneipp, J.; Kneipp, H.; Rice, W. L.; Kneipp, K. Anal. Chem. 2005, 77, 2381– 2385. (16) Simonian, A. L.; Good, T. A.; Wang, S. S.; Wild, J. R. Anal. Chim. Acta 2005, 534, 69–77. (17) Schofield, C. L.; Haines, A. H.; Field, R. A.; Russell, D. A. Langmuir 2006, 22, 6707–6711. 10.1021/ac802099c CCC: $40.75  2009 American Chemical Society Published on Web 01/26/2009

hydrogen peroxide and molecular oxygen; however, its aggregated structure in motor neurons is a key feature of the pathology of both sporadic and familial amyotrophic lateral sclerosis (ALS).1,18 The major advantage of the proposed sensing system is that it enables us to discriminate the evolutionary state of SOD1 aggregation based on sensitive changes in the local dielectric environment of SOD1-AuNP associated with the formation of probe aggregates of different sizes, which occurs when an extremely small amount of SOD1 aggregate is exposed to the probe solution. A distinct change in the dielectric properties of the probe that occurs with different SOD1 aggregate samples induces a significant change in the surface plasmon resonance spectra, accompanied by a concomitant color change of the SOD1-AuNP solution. This property makes the method more sensitive than existing methods and much simpler in terms of visualizing by the naked eye, in addition to both UV-vis spectroscopy and scanometric readout. Finally, the proposed sensor system provides quantitative information of use in tracking the structural evolution of SOD1 aggregates and provides the potential for definitive diagnosis; it may also be possible to apply this method in characterizing other target proteins with a tendency to aggregate. EXPERIMENTAL SECTION Preparation of SOD1 Protein. The wild-type human SOD1 gene was cloned into the pET23b (+) (Novagen) vector, and the protein was expressed in E. coli. Cultures were induced by the addition of 0.5 mM isopropyl β-D-thiogalactopyranoside for 3-6 h at 30 °C; cells were then lysed by sonication in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Proteins were eluted with a linear gradient of ammonium sulfate (0.75-0 M) in 50 mM sodium phosphate (pH 7.0), 150 mM NaCl, 0.1 mM EDTA, and 0.25 mM DTT. Wild-type SOD1 was released with high specificity from the column between 1.3 and 0.8 M ammonium sulfate. To produce apotype SOD1, demetallation of Zn and Cu metals was performed. The purified wild-type apo-SOD1 molecules were diluted with an acidic phosphate-buffered saline (PBS) solution (pH ) 5.4) to a concentration of 0.1 mg/mL. SOD1 aggregates were prepared by treatment with a solution containing 20% (v/v) trifluoroethanol (TFE). Preparation of Gold Nanoparticle Probes. AuNP solution was prepared as described previously.19 Briefly, the particles were prepared by boiling 200 mL of aqueous 0.005% (w/w) hydrogen tetrachloaurate(III) trihydrate (HAuCl4) stirred vigorously in a flask connected to a water-cooling column and, subsequently, adding 15.3 mg of sodium citrate (Na3C6H5O7 · 2H2O). Once the color of the solution began to change, the heat was turned off and the solution was allowed to cool overnight to room temperature. The diameter of the nanoparticles was 19 ± 3 nm by transmission electron microscopy. To prepare the SOD1conjugated AuNP (SOD1-AuNP) solution, 100 µL of SOD1 (0.1 mg/mL solution) was added to 1 mL of the nanoparticle solution, and the SOD1 was covalently assembled on the (18) Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Chem. Rev. 2006, 106, 1995–2044. (19) Chah, S.; Hammond, M. R.; Zare, R. N. Chem. Biol. 2005, 12, 323–328.

Scheme 1. Schematic Diagram Showing the Colorimetric Detection System Applied to the Structural Evolution of SOD1 Aggregatesa

a The samples of SOD1 aggregate were induced by TFE in weak acidic conditions (pH ) 5.4) with an incubation time of ∼4 weeks. After addition of the SOD1 aggregate samples into the prepared SOD1-AuNP solution, color changes in the probe solution (by shift of the surface plasmon peak) can be observed by the naked eye and measured by UV-vis spectroscopy.

nanoparticle for 4 h.20-22 Unreacted excess SOD1 was removed by twice-repeated centrifugation for 10 min at 13 000 rpm. Circular Dichroism (CD) Spectroscopy. CD spectra were recorded for 10 mg/mL SOD1 monomer and for several states of TFE-induced aggregates using a Jasco J-715 spectropolarimeter equipped with a Naslab temperature controller. The cell path length was 1 mm for the 190-270 nm regions. Optical Absorption Spectroscopy. Optical absorption spectroscopy measurements were performed in a spectrophotometer (HP 8453) using 1 cm path length quartz cuvettes. Spectra were collected over the 400-800 nm wavelength range. Scanometric Detection for Nanoprobe Quantification. After adding SOD1 aggregate samples to the SOD1-AuNP probe, the solution was spotted and dried on a C18 thin-layer chromatography (TLC) plate. The plate was scanned using a flatbed scanner, and the scanned image was adjusted using Adobe Photoshop software.23,24 RESULTS AND DISCUSSION Nanoparticle Functionalization for the Recognition of SOD1 Aggregates. We examined the feasibility of developing a nanoparticle-based sensor for detecting the structural evolution of SOD1 aggregates using AuNP modified with SOD1 monomers (SOD1-AuNP). The principle behind the sensor operation is shown in Scheme 1. The SOD1-AuNP probes were designed using small AuNPs and self-assembled SOD1 monomers. A total of 100 µL of 0.1 mg/mL SOD1 solution was added to 1 mL nanoparticle solution to fabricate the SOD1-AuNP sensing probe; the self-assembled SOD1 protein was then covalently bound to the nanoparticle (sulfur group of cysteine to the gold surface) in 4 h. The SOD1-AuNP solution maintained its stability for several months, meaning that in the future it may be used as a diagnostic (20) Hyun, J.; Kim, J.; Craig, S. L.; Chilkoti, A. J. Am. Chem. Soc. 2004, 126, 4770–4771. (21) Cse, M. A.; McLendon, G. L.; Hu, Y.; Vanderlick, T. K.; Scoles, G. Nano Lett. 2003, 3, 425–429. (22) Mateo-Marti, E.; Briones, C.; Roman, E.; Briand, E.; Pradier, C. M.; MartinGago, J. A. Langmuir 2005, 21, 9510–9517. (23) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (24) Nam, J. M.; Wise, A. R.; Groves, J. T. Anal. Chem. 2005, 77, 6985–6988.

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probe with a long availability period. Samples of SOD1 aggregate induced by TFE under acidic conditions (pH ) 5.4) were incubated for between 0 and 4 weeks; this treatment has also been used extensively to study the aggregation of other proteins.25 The various SOD1 aggregate samples were introduced to the prepared SOD1-AuNP solution in order to examine morphological growth during SOD1 aggregation. A total of 20 µL of SOD1 aggregates was injected, as predetermined by optimization via comparison of the maximum color change in each sample within a rapid response time (The amount of injected volume meant an extremely low concentration of target SOD1 aggregates when added to the SOD1-AuNP solution; therefore, this sensor system is useful for sensitive screening of the temporal evolution of SOD1 aggregates using a small sample amount.). We assume that the individual monomer on the probes may act as an intermediate seed for further aggregation of SOD1 protein:26,27 it promotes the formation of probe aggregates, resulted in binding of the probes to the SOD1 aggregate. Greater numbers of SOD1-AuNP aggregates formed on the SOD1 aggregates with their progressive structural evolution. This formation of SOD1-AuNP aggregates of varying sizes onto the SOD1 aggregate causes distinct shifts in the surface plasmon resonance peak associated with the color change of the SOD1-AuNP solution, as detected by UV-vis absorption spectroscopy. In the case of unmodified AuNPs, we observed no interparticle aggregation induced by further association between AuNP and SOD1 aggregates (i.e., the color of AuNP solution did not change; data not shown). Simple Optical Detection for the Structural Evolution of SOD1 Aggregates. The interaction between SOD1-AuNP and SOD1 aggregates at different incubation times was assessed by optical absorbance spectroscopy (see Figure 1). The SOD1-AuNP solution shows a narrow absorbance peak at 526 nm, which indicates that the SOD1-AuNPs did not aggregate with each other, instead remaining well dispersed as individual particles. The addition of SOD1 aggregates at different growth stages to the SOD1-AuNP solution induced probe aggregates of varying sizes following association between the probes and SOD1 aggregates. This formation of probe aggregates of varying sizes is reflected in a significant red-shift and broadening upon interaction with SOD1 aggregates. A significant change in absorbance intensity is clearly observed at 526 and 660 nm for the AuNP solutions. Figure 1A shows that the addition of a 2 day aggregate sample to the SOD1-AuNP solution did not significantly induce competitive probe aggregates within a detection time of 50 min, although a weak longitudinal surface plasmon peak arose from minor SOD1-AuNP aggregation on the SOD1 aggregates. When 2 and 3 week aggregate samples were added to the probe solution, the extinction spectra of the surface plasmon peak were shifted and broadened, and an intensive longitudinal surface plasmon peak appeared. Finally, for a 4 week aggregate sample, the surface plasmon peak at 526 nm had almost disappeared and the plasmon (25) Stathopulos, P. B.; Rumfeldt, J. A. O.; Scholz, G. A.; Irani, R. A.; Frey, H. E.; Hallewell, R. A.; Lepock, J. R.; Meiering, E. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7021–7026. (26) GhoshMoulick, R.; Bhattacharya, J.; Mitra, C. K.; Basak, S.; Dasgupta, A. K. Nanomedicine 2007, 3, 208–214. (27) Frieden, C. Protein Sci. 2007, 16, 2334–2344.

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Figure 1. Optical absorbance spectra of SOD1-AuNP solution due to association after the addition of different SOD1 aggregates with incubation times between 0 days and 4 weeks (A). The final color of SOD1-AuNP solution with SOD1 aggregates after 50 min of detection time (B) changed from red (a, 2 day aggregate) to purple (b, 2 week aggregate), then bluish-purple (c, 3 week aggregate), and finally blue (d, 4 week aggregate).

resonance red-shift induced a concomitant longitudinal surface plasmon peak. The shift of the surface plasmon absorbance band was found to be proportional to the growth state of the SOD1 aggregate. This result indicates that, with continued aggregate growth of SOD1, there occurs greater formation of probe aggregates onto SOD1 aggregates. This finding is thought to reflect specific interactions of the SOD1 monomer on the AuNP with the aggregates, exclusively mediated via the recognition seed in SOD1 aggregation. Figure 1B shows the final color of the SOD1-AuNP solutions after a detection time of 50 min for the structural evolution of SOD1 aggregates. In each case, the red-shift of the surface plasmon absorption band follows the color change of the SOD1-AuNP solution, from red (a in Figure 1B, 2 day aggregate) to purple (b, 2 week aggregate), then bluish-purple (c, 3 week aggregate), and blue (d, 4 week aggregate). Both the rate and extent of absorbance change are dependent on the temporal evolution of the SOD1 aggregates, with the maximum absorbance in each state of SOD1 aggregate observed for the sample incubated for 4 weeks. On the basis of the red-to-blue color change provoked by shifts in the plasmon resonance according to the formation of the probe aggregates onto the SOD1 aggregates, it is possible to monitor the growth state-dependent SOD1 aggregates by the naked eye, without the need for complex analytical tools. Detection Sensitivity for the Structural Evolution of SOD1 Aggregates. Time-dependent sensitive changes in absorbance intensity of the SOD1-AuNP solution (useful for detection of the

Figure 2. Sensitivity of the proposed colorimetric detection system compared with a circular dichroism (CD) spectrum. (A) The extinction spectra of the plasmon resonance peak were shifted and broadened, and the longitudinal surface plasmon peak appeared after the addition of SOD1 aggregates: (a) 2 weeks, (b) 3 weeks, and (c) 4 weeks every 10 min. (B) CD spectra in the far-UV region were observed between SOD1 incubated with TFE and freshly native SOD1 to ensure the physiological relevance of the in vitro aggregation system of SOD1; however, the CD spectra of the samples of SOD1 aggregate incubated for 2-4 weeks are not distinguishable.

structural evolution of SOD1 aggregates) are observed by UV-vis spectroscopy. In Figure 2A, the addition of a 2 week aggregate sample (a in Figure 2A) to the SOD1-AuNP solution every 10 min steadily induced the longitudinal surface plasmon peak within a detection time of 50 min. When 3 week aggregate sample (b in Figure 2A) was exposed to the SOD1-AuNP solution, the extinction spectra of the transverse surface plasmon peak was shifted and broadened. Concomitantly, the longitudinal surface plasmon peak intensified due to changes in the refractive index surrounding the particles. Finally, for 4 week aggregate sample (c in Figure 2A), the surface plasmon peak at 526 nm had almost disappeared and the dynamic red-shift from 526 to 700 nm of the surface plasmon band induced a concomitant longitudinal surface plasmon peak. To compare sensitivity in terms of the evolution of SOD1 aggregates, the far-UV spectra of SOD1 after various periods of incubation with TFE was measured by CD spectroscopy, as commonly used in the identification of protein structures. As shown in Figure 2B, differences in CD spectra were also observed between TFE-induced SOD1 aggregates and freshly prepared wildtype SOD1. This indicates that the secondary structure of native SOD1 was mainly composed of β-sheets (60%) and random coils (30%), with the remainder being R-helixes; this result is consistent with the findings of X-ray crystal structure analysis.28-30 Changes in the secondary structure of SOD1 (i.e., a decrease in the β-sheet and R-helix contents of SOD1, together with an increase in the random coil content) were observed after 2 days of incubation with TFE under the same acidic conditions. These (28) Leinweber, B.; Barofsky, E.; Barofsky, D. F.; Ermilov, V.; Nylin, K.; Beckman, J. S. Free Radical Biol. Med. 2004, 36, 911–918.

structural changes of SOD1 may be accompanied by conformational damage; however, we observed little signal change in CD spectra as SOD1 aggregates grew over different incubation periods ranging from 2 to 4 weeks (see red dashed rectangle in Figure 2B). This result indicates little difference in the secondary structure that contributes to the CD signal with greater SOD1 aggregation and that this analytical method cannot sensitively discriminate the temporal growth state of SOD1 aggregates. This method is also inefficient because it requires a high concentration of SOD1 aggregates (about 100 times more concentrated than that required by our proposed optical method) to obtain useful signals in residue ellipticity. Therefore, the proposed SOD1-AuNP sensing system has great advantages as a highly sensitive and visual detection system for screening the structural evolution of extremely small amounts of SOD1 aggregates. Time-Dependent Absorbance Change of SOD1-AuNP Solution with SOD1 Aggregates. After the absorbance data are fitted as a function of detection time, the position of the surface plasmon peak can be averaged for the different SOD1 aggregate samples. By measuring the absorbance at a fixed 526 nm wavelength with each SOD1 aggregate sample (Figures 1A and 2A), it is possible to determine the detection-time-dependent change in extinction of the SOD1-AuNP solution with SOD1 aggregates at different incubation times (Figure 3). The intensity of each SOD1-AuNP solution decreases as a function of detection time, indicating that the red-shift and broadening of the spectrum results in decreased absorbance at a fixed wavelength. In other (29) Tiwari, A.; Hayward, L. J. J. Biol. Chem. 2003, 278, 5984–5992. (30) Nagami, H.; Yoshimoto, N.; Umakoshi, H.; Shimanouchi, T.; Kuboi, R. J. Biosci. Bioeng. 2005, 99, 423–428.

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Figure 3. Change in absorbance at 526 nm of SOD1-AuNP solution with SOD1 aggregate samples as a function of detection time. By measuring the absorbance at a fixed 526 nm wavelength with each SOD1 aggregate sample from Figures 1A and 2A, we observed a detection-time-dependent change in extinction of the SOD1-AuNP solution with SOD1 aggregate samples.

Figure 4. Quantification method for SOD1-AuNP probes spotted on a TLC plate. The spot intensity values are proportional to the density of SOD1-AuNP associations (with more SOD1 aggregates, less red color is evident), and the density of the AuNPs is proportional to the amount of target SOD1 aggregates present.

words, this SOD1-AuNP-based detection system has a response with various decrements; it enables the discrimination of the state of structural evolution of SOD1 aggregates in regard to detection time. Importantly, the noticeable changes in absorbance intensity occurred within 40 min, suggesting that the time required for detection must be rapid to enable significant and sensitive determination of the structural evolution of SOD1 aggregates and providing an appropriate analytical time for the distinct intensity of the absorbance signals. Colorimetric Assays. To develop a simpler way to monitor the structural evolution of SOD1 aggregates for target verification and quantification, colorimetric assays were made by spotting 3 µL of the solution containing SOD1-AuNP/SOD1 aggregates on a TLC plate every 10 min for each sample. As shown in Figure 4, the spotted dots show different colors as a function of incubation (i.e., aggregate growth) and detection time. Initially, each spot retains the color of the 2 day SOD1 aggregate solution mixture,

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which remains pink during the entire 50 min detection time; however, in the case of 2 and 3 week SOD1 aggregates, the spot color ranges from pink at a detection time of 0 min to reddishpurple and finally purple upon aggregation of the SOD1-AuNPs, depending on the detection time. The spot color of the 4 week SOD1 aggregate changes from pink to purple, then bluish-purple, and finally blue. This phenomenon is attributable to increased aggregation of the organized SOD1-AuNPs onto the SOD1 aggregate upon drying the solution on the plate. Repetitions of the spot test with another set of independently prepared probes afforded the same narrow range. In each simple test using the SOD1-AuNP probe, the color change of the SOD1-AuNP solution enabled us to simply discriminate the structural evolution of SOD1 aggregates within a fast response time. Therefore, this sensitive colorimetric detection system based on protein-conjugated AuNP is feasible as a sensing marker for the definitive diagnosis of SOD1 aggregates by the naked eye and may also be applied to other proteins with a tendency to aggregate or fibrillate. CONCLUSION We developed a simple and sensitive colorimetric detection system for the structural evolution of SOD1 aggregates. AuNP, which has special optical properties and the potential to be used as an ideal colorimetric sensor, was functionalized with SOD1 monomer to readily interact with SOD1 aggregates and was used to detect structural evolution via optical spectroscopy. The SOD1 monomer immobilized on the AuNP surface played an important role in the growth process of the protein aggregation as an intermediate binding seed. In each instance, the SOD1 aggregates induced by TFE for different incubation times in an acidic condition were used as target samples to analyze the performance of the proposed sensor system. An obvious advantage of this approach is that optical measurement based on the change in absorption intensity of the nanoparticle appears as a color change in the probe solution, which makes analysis more sensitive and easier compared with conventional methods. A final colorimetric assay, which is sensitive to aggregate growth and detection time, may be conveniently used to sense the temporal growth state of SOD1 aggregates, thereby enabling a simple diagnosis. The proposed method and founding principles may be possible to apply the method to other proteins for the recognition and detection of their structural evolution. ACKNOWLEDGMENT This work was supported by Grant No. R01-2006-000-10239-0 from the Basic Research Program of the Korea Science & Engineering Foundation and a Seoul National University Grant from the Engineering-Medical Multidisciplinary R&D Project. It was also supported by Grant No. 101-082-032 from the Ministry of Environment as “The Eco-technopia 21 project” and the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-331D00134). Received for review October 6, 2008. Accepted December 30, 2008. AC802099C