Label-Free Highly Sensitive Detection of Proteins in Aqueous

Mar 27, 2009 - Label-Free Highly Sensitive Detection of Proteins in Aqueous Solutions Using Surface-Enhanced Raman Scattering ... avidin, catalase, an...
0 downloads 0 Views 1MB Size
Download PDF
Anal. Chem. 2009, 81, 3329–3333

Label-Free Highly Sensitive Detection of Proteins in Aqueous Solutions Using Surface-Enhanced Raman Scattering Xiao X. Han,†,‡ Genin Gary Huang,‡ Bing Zhao,*,† and Yukihiro Ozaki*,‡ State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, People’s Republic of China, and Department of Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan We detected concentration-dependent surface-enhanced Raman scattering (SERS) spectra of several label-free proteins (lysozyme, ribonuclease B, avidin, catalase, and hemoglobin) for the first time in aqueous solutions. Acidified sulfate was used as an aggregation agent to induce high electromagnetic enhancement in SERS. Strong SERS spectra of simple and conjugated protein samples could easily be accessed after the pretreatment with the aggregation agent. The detection limits of the proposed method for lysozyme and catalase were as low as 5 µg/ mL and 50 ng/mL, respectively. This detection protocol for label-free proteins has combined simplicity, sensitivity, and reproducibility and allows routine qualitative and relatively quantitative detections. Thus, it has great potential in practical high-throughput protein detections. Surface-enhanced Raman scattering (SERS), with its great ability to detect single molecules, has been proved to have great potential in ultrasensitive biomolecule detection.1-7 Label-free direct8-11 and Raman dye-labeled indirect methods12-15 are two * To whom correspondence should be addressed. E-mail: [email protected] (B.Z.); [email protected] (Y.O.). † Jilin University. ‡ Kwansei Gakuin University. (1) Moskovits, M. Rev. Mod. Phys. 1985, 57, 783–825. (2) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev. 1999, 99, 2957–2975. (3) Nie, S; Emory, S. R. Science 1997, 275, 1102–1106. (4) Tian, Z. Q.; Ren, B. Annu. Rev. Phys. Chem. 2004, 55, 197–229. (5) Aroca, R. Surface-Enhanced Vibrational Spectroscopy; John Wiley & Sons Ltd.: Chichester, U.K., 2006; pp 141-176. (6) Kneipp, K., Moskovits, M., Kneipp, H., Eds. Surface-Enhanced Raman ScatteringsPhysics and Applications; Springer: Heidelberg and Berlin, 2006. (7) Graham, D.; Goodacre, R. Chem. Soc. Rev. 2008, 37, 873–1076. (8) Habuchi, S.; Cotlet, M.; Gronheid, R.; Dirix, G.; Michiels, J.; Vanderleyden, J.; De Schryver, F. C.; Hofkens, J. J. Am. Chem. Soc. 2003, 125, 8446– 8447. (9) Drachev, V. P.; Thoreson, M. D.; Khaliullin, E. N.; Davisson, V. J.; Shalaev, V. M. J. Phys. Chem. B 2004, 108, 18046–18052. (10) Pavan Kumar, G. V.; Ashok Reddy, B. A.; Arif, M.; Kundu, T. K.; Narayana, C. J. Phys. Chem. B 2006, 110, 16787–16792. (11) 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. (12) Cao, Y. C.; Jin, R.; Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676–14677. (13) Driskell, J. D.; Uhlenkamp, J. M.; Lipert, R. J.; Porter, M. D. Anal. Chem. 2007, 79, 4141–4148. (14) Han, X. X.; Cai, L. J.; Guo, J.; Wang, C. X.; Ruan, W. D.; Han, W. Y.; Xu, W. Q.; Zhao, B.; Ozaki, Y. Anal. Chem. 2008, 80, 3020–3024. 10.1021/ac900395x CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009

major SERS-based detection protocols for DNA, proteins, and other biological molecules. The direct method is based on vibration information of target proteins themselves, whereas the indirect one detects target proteins by SERS signals of Raman dyes linked to the target proteins. In general, the label-free strategy is a better way for reliable and convenient detection for any analyte compared to the Raman dye-labeled one, although the low Raman cross section from biomolecules themselves makes it more difficult to carry out highly sensitive measurements by the former method. Hemoproteins, one kind of conjugated protein, are wellcharacterized by surface-enhanced resonance Raman scattering (SERRS).16-19 However, SERS-based label-free identification of proteins that have no conjugated chromophore remains a great challenge. Roughened metal surfaces and dried colloids cannot yield reproducible SERS spectra because of irreproducible SERS substrates or different orientation of proteins on a metal surface. No previous SERS-based study for proteins allows routine detection of label-free proteins with high sensitivity in aqueous solutions because halide ions, which are commonly used aggregation agents, can form a strongly bonded surface layer that repels the adsorption of analytes.20,21 Weak aggregation induced by alkaline metals is another barrier that makes it difficult to obtain strong SERS signals from label-free proteins. So far, few SERS-based studies have reported the detection limits of their methods for label-free proteins. In the present study, SERS was used, for the first time, as a detection method for qualitative and relatively quantitative detection of proteins in aqueous solutions over a wide range of concentrations. On the basis of strong aggregation of silver nanoparticles in aqueous solutions we have developed a rapid detection method for labelfree proteins. In acidic conditions, we use sulfate instead of halide as an aggregation agent, according to the weak binding ability of (15) Han, X. X.; Kitahama, Y.; Tanaka, Y.; Guo, J.; Xu, W. Q.; Zhao, B.; Ozaki, Y. Anal. Chem. 2008, 80, 6567–6572. (16) Eng, L. H.; Schlegel, V.; Wang, D.; Neujahr, H. Y.; Stankovich, M. T.; Cotton, T. Langmuir 1996, 12, 3055–3059. (17) Lecomte, S.; Wackerbarth, H.; Soulimane, T.; Buse, G.; Hildebrandt, P. J. Am. Chem. Soc. 1998, 120, 7381–7382. (18) Xu, H.; Bjerneld, E. J.; Ka¨ll, M.; Bo ¨rjesson, L. Phys. Rev. Lett. 1999, 83, 4357–4360. (19) Bizzarri, A. R.; Cannistraro, S. Appl. Spectrosc. 2002, 56, 1531–1537. (20) Bell, S. E. J.; Sirimuthu, N. M. S. J. Am. Chem. Soc. 2006, 128, 15580– 15581. (21) Bell, S. E. J.; Sirimuthu, N. M. S. J. Phys. Chem. A 2005, 109, 7405–7410.

Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

3329

SO42- to silver surfaces.20 In this way, we could obtain SERS spectra of three proteins with no chromophore (lysozyme, ribonuclease B, and avidin) and three hemoproteins (catalase, hemoglobin, and cytochrome c). Moreover, these target proteins could all be distinguished clearly from each other with high reproducibility. There are three key features in our proposed method. First, for the first time we obtained strong SERS signals of both simple and conjugated proteins by using acidified sulfate as an aggregation agent. The proteins were involved in the aggregation agent for inducing H+ and proteins mediated silver aggregation. Second, the present method has salient advantages of rapidness and high sensitivity in protein detections. After being mixed with the target protein involved aggregation agent, silver colloid aggregated instantaneously and then could be used for SERS measurement immediately. Moreover, we first observed concentration-dependent SERS spectra of target proteins in solutions, and the detection limits of the proposed method for lysozyme and catalase were 5 µg/mL and 50 ng/mL, respectively. Third, protein samples were detected in aqueous solutions, which may effectively avoid the irreproducibility from roughed and dried metal substrates. Therefore, this detection protocol for label-free proteins has combined simplicity, sensitivity, and reproducibility and has great potential in both practical quantitative and qualitative protein detections. EXPERIMENTAL SECTION Biochemicals and Chemicals. Ribonuclease B, avidin, and cytochrome c were purchased from Sigma-Aldrich Co., Ltd. Lysozyme was obtained from ICN Biomedicals, Inc. Catalase, hemoglobin, silver nitrate, trisodium citrate, and all other chemicals were purchased from Wako Co., Ltd. Triply distilled water was used throughout the present study. A phosphate buffer (PB) solution (0.1 M, pH 7.0) containing NaH2PO4 and Na2HPO4 was used to dissolve protein samples in this study. The aggregation agent was prepared by using 0.1 M Na2SO4 (adjust pH to 3 with H2SO4). Preparation of Silver Colloid. Colloidal silver was prepared by an aqueous reduction of silver nitrate (10-3 M, 200 mL) with trisodium citrate (1%, 4 mL) using a method of Lee and Meisel.22 The plasmon absorption maximum of the silver colloid we prepared was located at 431 nm. Field emission scanning electron microscope (FE-SEM) images of the colloidal silver were measured with a JEOL JSM-6700F FE-SEM with an accelerating voltage of 3 kV (Supporting Information SI-Figure 3). SERS Measurements. Before each SERS measurement, the concentrations of protein samples were diluted 1/10 with the proposed aggregation agent, and then we mixed the diluted proteins with silver colloids (1:5, v/v). An amount of 20 µL of each sample was added to an open differential scanning calorimetry (DSC) pan for carrying out SERS measurements. All SERS spectra were measured with a HoloSpec f/1.8i spectrograph (Kaiser Optical Systems Inc.), and the 785 nm line of a NIR diode laser (Invictus) was used as an excitation source. Laser power at the sample was set at ∼15 mW. The typical exposure time for each Raman/SERS measurement in this study (22) Lee, P. V.; Meisel, D. J. Phys. Chem. 1982, 86, 3391–3395.

3330

Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

Figure 1. Aggregation protocol of silver colloid for label-free proteins.

Figure 2. Raman spectrum of 5 mg/mL catalase in a PB solution (a), (b) a SERS spectrum of catalase (mixed 5 mg/mL catalase in a PB solution with silver colloid (1:5, v/v)), (c) a SERS spectrum of catalase diluted by 0.1 M Na2SO4 (mixed the diluted proteins with silver colloid (1:5, v/v)), and (d) a SERS spectrum of 50 ng/mL catalase obtained by the present method.

was 20 s. After SERS spectra collection, we did not treat them except for baseline correction. RESULTS AND DISCUSSION As illustrated in Figure 1, each target protein is first diluted with the proposed aggregation agent and then mixed with the silver colloid. All SERS spectra were measured in aqueous solutions. In this way, we detected, respectively, three target proteins with no chromophore (lysozyme, ribonuclease B, and avidin) and three hemoproteins (catalase, hemoglobin, and cytochrome c) over a wide range of concentrations. Several control experiments were carried out to confirm the reliability of the proposed method (Figure 2). We cannot observe a Raman spectrum of catalase from catalase solutions (PB solution, pH 7.0) except for two typical Raman bands of phosphate (Figure 2a). In contrast, strong SERS spectrum of catalase can be obtained by using the present method at even lower concentration (Figure 2d). The protocol of using sulfate as an aggregation agent to induce strong SERS has been used to detect some small molecules quantitatively and qualitatively.20,21,23 In this proposed detection system, we modified the sulfate aggregation agent and observed strong SERS of six target label-free proteins. Three important factors contribute to the strong SERS enhancement together. First, weak binding of SO42- can induce much stronger SERS, which lies in the higher solubility product of silver sulfate (Ksp ) 1.20 × 10-5 mol3 dm-9) compared to that of other common salts used for aggregation (e.g., AgCl, Ksp ) 1.77 × 10-10 mol2 (23) Bell, S. E. J.; Mackle, J. N.; Sirimuthu, N. M. S. Analyst 2005, 130, 545– 549.

Figure 3. UV-vis spectra of a silver colloid (a) and (b) silver aggregates induced by the aggregation agent with 0.5 µg/mL catalase).

dm-6).23 Therefore, the presence of SO42- results in weakly bound anions on silver surfaces and then makes it easier for some groups of target proteins (e.g., COO-, tyrosine, and tryptophan residues) to access the Ag surfaces by physical and hydrophobic interactions.24,25 The second factor is the protocol of involving proteins in the aggregation agent before adding silver colloid. We could not observe SERS signals of the target proteins when adding protein samples after silver nanoparticles being aggregated by the proposed aggregation agent, which demonstrated that this protocol also greatly contributed to strong SERS of the target proteins. At low pH condition (lower than the pI of most proteins), all the target proteins carry net positive charges, so these proteins can adsorb on silver surfaces by electrostatic interactions and also other interactions that we have demonstrated above. Accordingly, multiple interaction sites of an individual protein may bridge two or more silver nanoparticles and induce the subsequent SERS. Third, excess H+ cations are indispensable because over the range of our concerned concentrations, no SERS spectra could be obtained from the target proteins treated with neutral salt solutions (Figure 2, curves b and c). It is very likely that more H+ can induce stronger silver aggregation due to electrostatic interactions between negatively charged silver nanoparticles and the cations20 in this system. Thus, in the proposed method, strong silver aggregation is mediated by both the target proteins and H+, owing to which the proteins that adsorbed on the silver nanoparticle junctions show strong SERS effect. Broad bands were observed at around 800 nm in UV-vis spectra of the aggregated silver solutions at different catalase concentrations (Supporting Information SI-Figure 1). One can find only slight shifts of the broad bands, which indicated that the acidified sulfate plays a major role in the aggregation of silver nanoparticles. Figure 3 shows UV-vis spectra of a silver colloid before and after the aggregation by the present method. According to the surface plasmon resonance (SPR) of silver aggregates, we (24) Stewart, S.; Fredericks, P. M. Spectrochim. Acta, Part A 1999, 55, 1615– 1640. (25) Carney, J.; Braven, H.; Seal, J.; Whitworth, E. IVD Technol. 2006, 11, 41– 51.

Figure 4. Raman spectrum of solid lysozyme and concentrationdependent SERS spectra of lysozyme measured by the proposed method.

chose 785 nm as an excitation wavelength for electromagnetic enhancement.26,27 Figure 4 shows concentration-dependent SERS spectra of lysozyme measured by using the present method. One can see amide I (1661 cm-1) and amide III (1235 cm-1) bands due to the R-helix structure and bands assigned to aromatic residues of Phe (1005 cm-1), Trp (1557, 1342, 508 cm-1), and Tyr (856 cm-1).28-33 Note that the SERS spectra of lysozyme have little deviation from a Raman spectrum of lysozyme (bottom spectrum in Figure 4). In contrast, there were significant differences between SERS and Raman spectra of some label-free proteins according to our previous SERS studies.6 It is probably that under the present experimental conditions, lysozyme adsorbs on the silver surface mainly by physical interactions and the variation in the lysozyme concentration has little effect on the adsorbing state between lysozyme and silver nanoparticles. Moreover, the features of simple structure and high stability in low pH conditions of lysozyme are also helpful for explaining the similarity between its Raman and SERS spectra shown in Figure 4. These results also demonstrate the possibility of the proposed method for the detection of native proteins. We also obtained SERS spectra of two glycoproteins (i.e., ribonuclease B and avidin) by use of the proposed method (Figure 5). As in the case of the SERS spectra of lysozyme depicted above, SERS spectra of the two proteins mainly show vibrations of amide bands (1672, 1241, and 1252 cm-1) and aromatic residues such as Phe (1004, 1032, 1005, 1207, and 1617 cm-1), Tyr (643 and 852 cm-1), and Trp (539, 758, and 1369 cm-1).30-33 We can find clear differences in peak positions and relative intensities among these SERS spectra and those from lysozyme, which (26) Schatz, G. C.; Van Duyne, P. R. Handbook of Vibrational Spectroscopy Vol. 1: Electromagnetic Mechanism of Surface-Enhanced Spectroscopy; Chalmers, J. M.,; Griffiths, P. R., Eds. John Wiley & Sons Ltd.: Chichester, U.K., 2002; pp 759-774. (27) Wang, H.; Levin, C. S.; Halas, N. J. J. Am. Chem. Soc. 2005, 127, 14992– 1499. (28) Grabbe, E. S.; Buck, R. P. J. Am. Chem. Soc. 1989, 111, 8362–6366. (29) Podstawka, E.; Ozaki, Y.; Lroniewicz, L. M. Appl. Spectrosc. 2004, 58, 570– 580. (30) Lord, R. C.; Yu, N. T. J. Mol. Biol. 1970, 50, 509–524. (31) Ahern, A. M.; Garrell, R. L. Langmuir 1991, 7, 254–261. (32) Herne, T. M.; Ahern, A. M.; Carrell, R. L. J. Am. Chem. Soc. 1991, 113, 846–854. (33) Kumar, G. V. P.; Reddy, B. A. A.; Arif, M.; Kundu, T. K.; Narayana, C. J. Phys. Chem. B 2006, 110, 16787–16792.

Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

3331

Figure 5. SERS spectra of ribonuclease B and avidin with concentrations of 500 µg/mL (upper) and 50 µg/mL (lower), respectively.

Figure 6. Concentration-dependent SERS spectra of catalase.

demonstrate the difference in selective enhancements due to preferential adsorption of certain groups for the different proteins. Furthermore, the adsorption is reproducible in the solutions under the same aggregation agent for individual protein, which is a crucially important issue for an analytical method. Thus, we can easily identify proteins by the differences in SERS spectra of the target proteins. For hemoproteins, the proposed method is much more sensitive than for lysozyme and glycoprotein because of much larger Raman cross section from heme. Figure 6 shows concentration-dependent SERS spectra of catalase with typical vibrations of heme, e.g., ν10B1g (1612 cm-1), ν42Eu (1264 cm-1), and ν7A1g (677 cm-1).34-37 The strong SERS signals imply that we can detect low concentrations of hemoproteins in solutions even without the resonance SERS effect. Accordingly, the present method may be used in relatively quantitative detection of catalase in organism with the advantages of simplicity and sensitivity. The same effects were found to be useful for detecting other two hemoproteins, hemoglobin and cytochrome c (Figure 7). Vibration modes of ν10B1g (1615 cm-1), ν4A1g (1373/1372 cm-1), and ν14B1g (1127/1123 cm-1) are also involved in these SERS spectra. Apparent differences in both peak positions and relative (34) Niaura, G.; Gaigalas, A. K.; Vilker, V. L. J. Electroanal. Chem. 1996, 416, 167–178. (35) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1993, 115, 12446–12458. (36) Spiro, T. G., Ed. Biological Applications of Raman Spectroscopy; John Wiley & Sons Ltd.: Chichester, U.K., 1988; Vol 3. (37) Feng, M.; Tachikawa, H. J. Am. Chem. Soc. 2008, 130, 7443–7448.

3332

Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

Figure 7. SERS spectra of hemoglobin and cytochrome c with concentrations of 50 µg/mL (upper) and 5 µg/mL (lower), respectively.

intensities were observed by comparing the SERS spectra of these three hemoproteins, which are probably owing to different adsorbing states of heme on silver surface after interactions with silver nanoparticles. Therefore, hemoproteins can be easily distinguished by the proposed method. According to distance-dependent enhancement of SERS, a very close distance (below 2-3 nm) between analytes and the metal surface is required for SERS to occur.38 As for proteins, a typical kind of biomacromolecule, most of them fold into unique threedimensional structures and have larger diameters (1 to 20 nm), which often lead to different orientation and partial enhancement28 of the target proteins due to random adsorption and to probable irreproducibility in SERS spectra of individual proteins from dried silver films (Supporting Information SI-Figure 2A). When silver nanoparticles are aggregated by the proposed protein involved aggregation agent, target proteins with net positive charges are sandwiched among silver nanoparticles in aqueous solutions, and then vibration information of whole proteins would be probably displayed in their SERS spectra with remarkable enhanced reproducibility (Supporting Information SI-Figure 2B). Concentration-dependent SERS intensities of catalase and lysozyme are plotted in Figure 8. Strong SERS bands at 677 and 1005 cm-1 were chosen, respectively, for Gaussian curve fit based on the relative intensities. Error bars addressed the sample-to-sample variability in SERS intensities. One can see the remarkable increase in the SERS intensities of the two proteins over a large range of concentrations, which demonstrates the ability of the proposed method in relatively quantitative detection under the saturation level of these label-free proteins that adsorbed on the silver surfaces. The detection limits of the proposed method for lysozyme and catalase were as low as 5 µg/mL and 50 ng/mL, respectively. It implies that the proposed SERS-based method with its advantages of rapidness and appropriately high sensitivity is compatible with high-throughput methods. Furthermore, due to the high selectivity of SERS, the SERS intensity of catalase is at least 30-fold stronger than that of lysozyme at the same protein concentrations, which indicates the possibility of selectively (38) Lakowicz, J. R.; Geddes, C. D.; Gryczynski, I.; Malicka, J.; Gryczynski, Z.; Aslan, K.; Lukomska, J.; Matveeva, E.; Zhang, J.; Badugu, R.; Huang, J. J. Fluoresc. 2004, 14, 425–441.

Figure 8. Concentration-dependent SERS intensities of catalase (677 cm-1) and lysozyme (1005 cm-1) measured by the present method. Each datum indicates an average of 5-7 measurements, and each error bar indicates the standard deviation.

detecting one protein in a certain protein mixture by the present SERS-based method. CONCLUSION We have greatly improved SERS-based detection methods for label-free proteins in aqueous solutions. In comparison to conventional mass spectroscopy based methods, our protocol can be used for intact protein detection without digestion and with high sensitivity and high selectivity due to SERS. Moreover, by using acidified sulfate as an aggregation agent, proteins can be identified rapidly in solutions. Both simple and conjugated proteins can be well-distinguished in aqueous solutions by the proposed protocol. All the observed SERS spectra of target proteins imply that it can be used as a practical method to directly detect label-free proteins with the advantages of rapidness, reproducibility, and appropriately high sensitivity. Extension of this work to more proteins and protein mixtures is now in progress. ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation (Grant Nos. 20573041, 20773044, 20873050) of P. R.

China; by the Program for Changjiang Scholars and Innovative Research Team in University (IRT0422), Program for New Century Excellent Talents in University; the 111 project (B06009), the Development Program of the Science and Technology of Jilin Province (20060902-02). This work was also supported by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Area “Strong Photon-Molecule Coupling Fields (No. 470, 20043032)” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We also thank China Scholarship Council for their fund support. SUPPORTING INFORMATION AVAILABLE UV-vis spectra of aggregated silver solutions at different catalase concentrations, SERS spectra of lysozyme on a silver island film and by the proposed system, respectively, and SEM images of silver nanoparticles used in this study. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 1, 2008. Accepted March 7, 2009. AC900395X

Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

3333