Highly Sensitive Protein Concentration Assay over a Wide Range via

May 12, 2010 - In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilli...
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Anal. Chem. 2010, 82, 4325–4328

Letters to Analytical Chemistry Highly Sensitive Protein Concentration Assay over a Wide Range via Surface-Enhanced Raman Scattering of Coomassie Brilliant Blue Xiao Xia Han,† Yunfei Xie,‡ Bing Zhao,‡ and Yukihiro Ozaki*,† Department of Chemistry and Research Center for Single Molecule Vibrational Spectroscopy, School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan, and State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, P. R. China In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilliant blue G-250 (CBBG) to proteins. In a protein-CBBG liquid mixture, surfaceenhanced Raman scattering (SERS) is sensitive to the amount of unbound CBBG molecules adsorbed on silver surfaces, and the bound CBBG amount is directly related to the target protein concentration. Accordingly, a novel method for detecting total protein concentration in a solution has been developed based on SERS of unbound CBBG with an internal standard of silicon. Two obvious advantages of the proposed protein assay over conventional Bradford protein assay are its much wider linear concentration range (10-5-10-9 g/mL) and 200 times lower limit of detection (1 ng/mL), which demonstrates its great potential in rapid, highly sensitive concentration determination of high and low-abundance proteins. Protein concentration determination, known as a protein assay, is a basic biochemical method and often necessary before processing protein samples. Currently, the measurement used most commonly in protein assays is absorbance of light, and there are several colorimetric assays for proteins including Lowry, Biuret, Bradford, and bicinchoninic acid (BCA) assays.1 The Bradford assay with its advantages of rapidness, convenience, and relative sensitivity has been used extensively. This protein assay is based on an absorbance shift from 465 to 595 nm of a band arising from Coomassie brilliant blue G-250 (CBBG) when binding to proteins occurs in an acidic solution. The disadvantage of the Bradford assay is, however, its narrower linear concentration range (0.2-20 µg/mL), which presents a severe problem when the concentration of a target protein is outside the range.2-4 * To whom correspondence should be addressed. E-mail: [email protected]. † Kwansei Gakuin University. ‡ Jilin University. (1) olson, B. J. S. C.; Markwell, J. Current Protocols in Protein Science; John Wiley & Sons: Somerset, NJ, 2007; Chapter 3.4. (2) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (3) Zor, T.; Seliger, Z. Anal. Biochem. 1996, 236, 302–308. (4) Stoscheck, C. M. Methods Enzymol. 1990, 182, 50. 10.1021/ac100596u  2010 American Chemical Society Published on Web 05/12/2010

As an ultrasensitive and promising analytical tool,5-8 the surface-enhanced Raman scattering (SERS) technique is widely applied to various qualitative analyses, for instance, investigations of adsorption, orientation, and electron transfer of molecules on a metal surface.9-14 Moreover, SERS is sensitive to the amount of molecules adsorbed on a metal surface, based on which highly sensitive and quantitative detection methods have been accomplished under optimized conditions (e.g., usage of internal standards for SERS measurement).15-18 In the present study, we have developed a novel SERS-based method to probe protein concentrations in a solution. In a protein-CBBG liquid mixture, we have found that the amount of unbound CBBG molecules remarkably decreases with the increase of the protein amount due to high affinity of CBBG to proteins, which results in the decrease of SERS intensity of unbound CBBG after the addition of silver nanoparticles. We found that there was a direct relationship between the SERS intensity of unbound CBBG molecules and the target protein concentration, and accordingly, the target protein concentrations can be determined by the SERS intensity of unbound CBBG molecules in a protein-CBBG mixture. Silicon, with a Raman peak at 520 cm-1, was used as an internal standard to eliminate instrumental variables (e.g., laser power) and differences in focus.16 Because (5) Aroca, R. Surface-Enhanced Vibrational Spectroscopy; John Wiley & Sons, Ltd.: Chichester, U.K., 2006. (6) Kneipp, K.; Moskovits, M.; Kneipp, H. Surface-Enhanced Raman Scattering: Physics and Applications, Springer: Berlin, Germany, 2006. (7) Nie, S.; Emory, S. R. Science 1997, 275, 1102–1106. (8) Natan, M. J. Faraday Discuss. 2006, 132, 321–328. (9) Persson, B. N.; Zhao, K.; Zhang, Z. Phys. Rev. Lett. 2006, 96, 207401. (10) Quagliano, L. G. J. Am. Chem. Soc. 2004, 126, 7393–7398. (11) Yu, Q. M.; Golden, G. Langmuir 2007, 23, 8659–8662. (12) Wang, Y. F.; Zhang, J. H.; Jia, H. Y.; Li, M. J.; Zeng, J. B.; Yang, B.; Zhao, B.; Xu, W. Q.; Lombardi, J. R. J. Phys. Chem. C 2008, 112, 996–1000. (13) Zhou, Q.; Li, X. W.; Fan, Q.; Zhang, X. X.; Zheng, J. W. Angew. Chem., Int. Ed. 2006, 45, 3970–3973. (14) Murgida, D. H.; Hildebrandt, P. Acc. Chem. Res. 2004, 37, 854–861. (15) Bell, S. E. J.; Sirimuthu, N. M. S. J. Am. Chem. Soc. 2006, 128, 15580– 15581. (16) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012–1024. (17) Xu, S. P.; Ji, X. H.; Xu, W. Q.; Li, X. L.; Wang, L. Y.; Bai, Y. B.; Zhao, B.; Ozaki, Y. Analyst 2004, 129, 63–68. (18) Stevenson, R.; Ingram, A.; Leung, H.; McMillan, D. C.; Graham, D. Analyst 2009, 134, 842–844.

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of high sensitivity and selectivity of SERS, this new SERS-based method allows one to detect proteins over a much wider concentration range with a lower limit of detection than the Bradford assay and other protein assays commonly used in biochemical laboratories.1 EXPERIMENTAL SECTION Materials. Brilliant blue G-250, bovine serum albumin (BSA), human serum albumin (HSA), human IgG, human serum (H4522), and silver nitrate were purchased from Sigma-Aldrich Co., Ltd., and they were used without further purification. All other chemicals were from Wako Co., Ltd., and ultrapure water (18 MΩ cm-1) was used throughout the present study. Phosphate buffered saline (PBS, 0.01 M, pH 7.2) contained 0.8% NaCl, 0.02% KH2PO4, 0.02% KCl, and 0.12% Na2HPO4 · 12H2O. The protein assay reagent contained 0.01% (w/v) BBG, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid. The negatively charged colloidal silver was prepared by aqueous reduction of silver nitrate (10-3 M, 200 mL) with trisodium citrate (1%, 4 mL).19 The plasmon absorption maximum of the silver colloid is located at 433 nm, and the average size of silver nanoparticles is about 50 nm (Supporting Information, SI-Figure 1). Pretreatment of Protein Samples. The protein assay reagent was diluted 100 times with PBS, and then the protein samples were added, respectively, to it. After 2 min, silver colloid was added and the volume ratio of the diluted protein assay reagent, protein sample, and silver colloid was 1:1:1 and the concentration of Ag colloid used in SERS measurements is 3 × 10-4 M. SERS Measurement. One minute after mixing the proteinCBBG mixture with the silver colloid, an amount of 10 µL of each sample was dripped onto a silicon chip for a SERS measurement. SERS spectra were measured with a HoloSpec f/1.8i spectrograph (Kaiser Optical Systems Inc.), and the 785 nm of a NIR diode laser (Invictus) was used as an excitation source. The laser power at the sample was about 15 mW, and the exposure time for each SERS measurement was 15 s. All SERS spectra shown here were collected with baseline correction. RESULTS AND DISCUSSION Figure 1 shows concentration-dependent SERS spectra of CBBG molecules in silver colloid. It can be seen that SERS intensity of CBBG is sensitive to the amount of CBBG molecules added to the silver colloid. It is noted that no new Raman band can be found in the SERS spectrum of CBBG compared to its normal Raman spectrum (SI-Figure 2 in the Supporting Information). Therefore, the CBBG molecules probably adsorb on the silver surfaces nonspecifically,20 which induces the SERS effect of CBBG. Considering the unsaturated level of CBBG molecules adsorbed onto the silver surfaces and the appropriate SERS intensities, 0.5 ppm CBBG (after being mixed with proteins) was used for each protein sample. After addition of a protein-CBBG mixture, the absorbance maximum of silver colloid shows a red shift from 433 to 437 nm, which is probably due to the adsorption of the unbound CBBG molecules on silver surfaces.21 An ag(19) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391–3395. (20) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 5935–5944. (21) Graham, D.; Thompson, D. G.; Smith, W. E.; Faulds, K. Nat. Nanotechnol. 2008, 3, 548–551.

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Figure 1. Structure of CBBG (the inset) and concentration-dependent SERS spectra of CBBG in Ag colloid (the volume ratio of BBG solution to Ag colloid is 2:1).

Figure 2. SERS spectra observed from the BSA-CBBG mixtures with the BSA concentrations of (a1) 10-3, (a2) 10-4, (b1) 10-10, and (b2) 10-11 g/mL.

gregation state of silver nanoparticles was monitored by UV-vis spectra of the silver colloid after adding a CBBG-BSA mixture, from which one can see that changes due to the aggregation state of nanoparticles are negligible during the first several minutes (SI-Figure 3 in the Supporting Information). To determine SERS spectra of bound and unbound (to proteins) CBBG in protein-CBBG mixtures, we measured BSA concentration-dependent SERS spectra of CBBG (Figure 2). We found that for the higher BSA concentrations, [BSA] > 10-5 g/mL, only the Raman peak due to silicon can be observed (spectra a1 and a2 in Figure 2), indicating that BSA molecules are at the saturation level in the mixtures and these bound CBBG molecules cannot be detected by SERS. During the formation of the protein-CBBG complex, both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change.1 After the complex formation, bound CBBG molecules block most of hydrophobic and positive sites of BSA, which makes it difficult for most of CBBG-bound BSA molecules to adsorb onto silver surfaces.22-24 In the case of the lower BSA concentrations,

Figure 3. Concentration-dependent SERS spectra of CBBG with the BSA concentrations of (0) 0, (1) 10-5, (2) 10-6, (3) 10-7, (4) 10-8, and (5) 10-9 g/mL.

[BSA] < 10-9 g/mL, the SERS intensity of CBBG was similar to that without BSA, indicating that the influence of a much smaller amount of BSA molecules on the adsorption of unbound CBBG is undetectable. Figure 3 shows BSA concentration-dependent SERS spectra of unbound CBBG molecules over a concentration range from 10-5 to 10-9 g/mL. It is noted that the SERS intensity of CBBG distinctly increases with the decrease in the concentration of BSA. The limit of detection of the proposed method for BSA was determined by comparing I1176/I520 of BSA at 10-9 g/mL (0.356) and 0 g/mL(0.458) and is as low as 1 ng/mL, which is 200 times lower than that of the conventional Bradford assay.2,3 Another important finding in the present study is the linear concentration range of the proposed protein assay as seen in Figure 4, which is wider than not only the conventional Bradford method but also other currently used assays for protein concentration determination.1,4 We consider that the large surface area of the silver nanoparticles in a solution enables the unbound CBBG molecules in a CBBG-BSA mixture to adsorb on the silver surfaces over a large concentration range, resulting in the wider linear concentration range for proteins. For quantitative analysis of target proteins, the intensity ratio of a strong peak at 1176 cm-1 due to the C-H in-plane bending mode20 of CBBG and a peak at 520 cm-1 due to silicon17 (I1176/I520) was measured. The (I1176/I520) versus the BSA concentration is plotted over the concentration range from 10-5 to 10-9 g/mL. It is noted that it is linear (I1176/I520 ) -0.384-0.079 log[protein], R2 ) 0.974). Therefore, the limit of detection and linear concentration range of the conventional Bradford assay have significantly been improved by using of SERS. To investigate whether the proposed method is universal for other proteins or protein mixtures, HSA and human IgG solutions with selected concentrations were examined by using the same (22) Han, X. X.; Jia, H. Y.; Wang, Y. F.; Lu, Z. C.; Wang, C. X.; Xu, W. Q.; Zhao, B.; Ozaki, Y. Anal. Chem. 2008, 80, 2799–2804. (23) Han, X. X.; Kitahama, Y.; Itoh, T.; Wang, C. X.; Zhao, B.; Ozaki, Y. Anal. Chem. 2009, 81, 3350–3355. (24) Han, X. X.; Zhao, B.; Ozaki, Y. Anal. Bioanal. Chem. 2009, 394, 1719– 1727.

Figure 4. SERS intensity ratio of 1176 to 520 cm-1 (I1176/I520) versus the BSA (O) concentration, and (I1176/I520) of HSA (9) and human IgG (1) versus their concentrations. The calibration curve is obtained by BSA.

method. Figure 4 also shows SERS responses of CBBG after the addition of individual proteins with different concentrations. One can see that the degree of scatter around the calibration curve obtained by BSA is very low especially for HSA, which is consistent with the similarity between BSA and HSA in their primary structures. For a protein sample with unknown concentration, there will be three possible situations when using this SERS-based protein assay. If a SERS spectrum of unbound CBBG is clear and the (I1176/I520) is lower than that without BSA (Figure 3 (spectra 0)), the target protein concentration can be directly determined by the calibration curve obtained by BSA; if a SERS spectrum of unbound CBBG is clear with a similar I1176/I520 as CBBG without BSA, the target protein concentration is lower than 10-9 g/mL; if a SERS spectrum of unbound CBBG is undetectable, the target protein concentration is higher than 10-5 g/mL and should be diluted before further SERS measurement. On the basis of the above equation (I1176/I520 ) -0.384-0.079 log[protein]) and the intensity ratio (I1176/I520) obtained from 100 timesdiluted human serum in the proposed assay, the protein

Figure 5. Time-dependent (I1176/I520) of CBBG with the BSA concentration of 10-8 g/mL. The laser was off during each time interval. Analytical Chemistry, Vol. 82, No. 11, June 1, 2010

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concentration of the human serum used in the present study was determined as 70 mg/mL, which is exactly within the total protein range of 40-90 mg/mL reported by Sigma Co., Ltd. Figure 5 shows a time-dependent change in the SERS intensity ratio (I1176/I520). One can see that only a slight variation in the SERS intensity of CBBG occurs over a time range of 30 min, which is very important when a large number of protein samples are required to be measured one after another by the SERS-based method. Thus, the proposed method is compatible to high-throughput protein assays.

ACKNOWLEDGMENT This work was supported by Support Project to Assist Private Universities in Developing Bases for Research (Research Center for Single Molecule Vibrational Spectroscopy) by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and KAKENHI (Grant-in-Aid for Scientific Research) on Priority Area “Strong Photon-Molecule Coupling Fields (Grant No. 470, 20043032)” from MEXT. This work was also supported by the 111 project (Grant B06009), China. X.X.H. acknowledges the support from the Japanese Society for Promotion of Science (JSPS, Grant P09248).

CONCLUSIONS By using SERS rather than the absorbance measurement in the Bradford protein assay, we have developed a SERS-based method for protein concentration determination. The proposed method enables one to determine protein concentrations over a much wider linear concentration range with a lower limit of detection than currently used protein assays. Although some ions contained in sample solutions may interfere with the adsorption of CBBG to silver surfaces, this method may be applied to routine high-throughput assays for proteins by use of proper buffer controls. Extension of the work including its application to a broad range of proteins is now in progress.

SUPPORTING INFORMATION AVAILABLE UV-vis spectrum and SEM image of silver colloid used in the study; normal Raman spectrum of CBBG and a SERS spectrum of CBBG in a BSA-CBBG mixture; time-dependent UV-vis spectra of the silver colloid after mixed with a BSA-CBBG mixture. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review March 5, 2010. Accepted May 9, 2010. AC100596U