Separation of Acidic and Basic Proteins by Nanoparticle-Filled

Anal. Chem. 2006, 78, 8004-8010

Separation of Acidic and Basic Proteins by Nanoparticle-Filled Capillary Electrophoresis Cheng-Ju Yu, Chih-Lin Su, and Wei-Lung Tseng*

Department of Chemistry, National Sun Yat-sen University and Center for Nanoscience & Nanotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan

We present the first example of the analysis of acidic and basic proteins by nanoparticle-filled capillary electrophoresis. Compared to the didodecyldimethylammonium bromide (DDAB)-coated capillary, the DDAB-capped gold nanoparticles (AuNPs) as pseudostationary phase were found to form more stable coating on the capillary wall, thus leading to greater separation efficiency and high reproducibility. In addition to their advantages for protein separation, DDAB-capped AuNPs can generate high reversed electroosmotic flow, which is 75% greater than DDAB at pH 3.5. To allow strong interactions with proteins, the AuNPs were modified with poly(ethylene oxide) via noncovalent bonding to form gold nanoparticles/polymer composites (AuNPPs). Using a capillary dynamically coated with DDAB-capped AuNPs and filled with AuNPPs under acidic conditions (10 mM phosphate, pH 3.5), we have demonstrated the separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations of migration time less than 0.6%. Additionally, the proposed method has been applied to the analyses of biological samples, including saliva, red blood cells, and plasma. With simplicity, high resolving power, and high reproducibility, the proposed method has shown great potential for proteomics applications and clinical diagnosis. Nanoparticles and nanoparticle-based materials have a great impact on many scientific fields, resulting in the development of a variety of important technologies.1 Much research has been carried out in relation to the use of metal nanoparticles in nanotechnology.2 Gold nanoparticles (AuNPs) are one of the most popular materials and offer many advantages, including sizedependent optical, electric, and magnetic properties, long-term stability, high surface area-to-volume ratio, as well as ease of chemical modification.3 Besides, biomolecules containing thiol (SH) or amino (NH2) groups can be adsorbed spontaneously onto gold surfaces to generate well-organized, self-assembled mono* To whom correspondence should be addressed. E-mail: tsengwl@ mail.nsysu.edu.tw. Fax: 011-886-7-3684046. (1) (a) West, J. L.; Halas, N. J. Annu. Rev. Biomed. Eng. 2003, 5, 285-292. (b) Portney, N. G.; Ozkan, M. Anal. Bioanal. Chem. 2006, 384, 620-630. (2) (a) Eustic, S.; EI-Sayed, M. A. Chem. Soc. Rev. 2006, 35, 209-217. (b) Welch, C. M.; Compton, R. G. Anal. Bioanal. Chem. 2006, 384, 601-619. (3) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346.

8004 Analytical Chemistry, Vol. 78, No. 23, December 1, 2006

layers.4-6 A number of differently sized and shaped AuNPs may be functionalized with a variety of molecules, such as polymers,4 proteins,5 and DNA.6 For example, AuNPs conjugated with proteins have been used as novel labels for detection of cancer cells7 and as building blocks for the formation of nanostructured materials.8 Because AuNPs inherently exhibit many desirable characteristics, they are extremely attractive candidates for use in biosensor,9 drug carrier,10 nanochemical devices,11 and cell imaging.12 However, although AuNPs provide a large surface area to interact with a column surface, analyte, or both,13 very little research has been devoted to understanding their impact on separation science. A thin film of dodecanethiol-protected AuNPs, as an efficient stationary phase for gas chromatography, has been demonstrated in the separation of four kinds of organic compounds.14 The selfassembled monolayer and multilayer of AuNPs on the modified capillary column have been shown useful for capillary electrochromatography.15 Capillaries and microchannels coated with AuNPs have been performed in capillary electrophoresis (CE) and microchip CE to improve selectivity and control the electroosmotic (4) (a)Bhattacharjee, R. R.; Chakraborty, M.; Mandal, T. K. J. Phys. Chem. B 2006, 110, 6768-6775. (b) Schneider, G.; Decher, G.; Nerambourg, N.; Praho, R.; Werts, M. H.; Blanchard-Desce, M. Nano Lett. 2006, 6, 530536. (5) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (6) (a) Thaxton, C. S.; Georganopoulou, D. G.; Mirkin, C. A. Clin. Chim. Acta 2006 363, 120-126. (b) You, C.-C.; De. M.; Rotello, V. M. Curr. Opin. Chem. Biol. 2005, 9, 639-646. (c) Fritzsche, W.; Taton, T. A. Nanotechnology 2003, 14, R63-R73. (7) (a) Pissuwan, D.; Valenzuela, S. M.; Cortie, M. B. Trends Biotechnol. 2006, 24, 62-67. (b) Huang, X.; EI-Sayed, I. H.; Qian, W.; EI-Sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2115-2120. (8) Huang, Y.; Chiang, C.-Y.; Lee, S. K.; Gao, Y.; Hu, E. L.; Yoreo, J. D.; Belcher, A. M. Nano Lett. 2005, 5, 1429-1434. (9) Willner, I.; Baron, R.; Willner, B. Adv. Mater. 2006, 18, 1109-1120. (10) Yang, P.-H.; Sun, X.; Chiu, J.-F.; Sun, H.; He, Q.-Y. Bioconjugate Chem. 2005, 16, 494-496. (11) Tseng, R. J.; Huang, J.; Ouyang, J.; Kaner, R. B.; Yang, Y. Nano Lett. 2005, 5, 1077-1080. (12) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.; Anderson, M. F.; Franzen, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003, 125, 47004701. (13) Nilsson, C.; Nilsson, S. Electrophoresis 2006, 27, 76-83. (14) (a) Gross, G. M.; Grate, J. W.; Synovec, R. E. J. Chromatogr., A 2004, 1029, 185-192. (b) Gross, G. M.; Nelson, D. A.; Grate, J. W.; Synovec, R. E. Anal. Chem. 2003, 75, 4558-4564. (15) (a) Yang, L.; Guihen, E.; Holmes, J. D.; Loughran, M. O’Sullivan, G. P.; Glennon, J. D. Anal. Chem. 2005, 77, 1840-1846. (b) O’Mahony, T.; Owens, V. P.; Murrihy, J. P. Guihen, E.; Holmes, J. D.; Glennon, J. D. J. Chromatogr., A 2003, 1004, 181-193. 10.1021/ac061059c CCC: $33.50

© 2006 American Chemical Society Published on Web 10/31/2006

flow (EOF).16 Also, chemical modification of nanoparticle surfaces plays an important role in enhancing separation efficiency in CE. Recently, Chang’s group has developed nanoparticle-filled capillary electrophoresis (NFCE), allowing separation of long doublestranded DNA with peak efficiency greater than 106 plate/m.17 In NFCE, AuNPs were modified with poly(ethylene oxide) (PEO) through noncovalent bonding to form AuNPs/polymer composites (AuNPPs). To further investigate the separation mechanism, the migration of λ-DNA was monitored in real time using a chargecoupled device imaging system when the capillary was filled with AuNPPs.18 Based on these results, it appears that AuNPs can be used to separate proteins by CE. Through the interaction of gold surface with thiol groups of cysteine residues and amino groups of lysine residues of the proteins,19 we can reasonably suppose that the mobility of proteins will be significantly altered by the AuNPs. To date, a number of chemical approaches have been actively explored to prepare the AuNPs; however, chemical reduction of ionic gold using sodium citrate as reducing agent is still a general route for biological application.20 The citrate-capped AuNPs tend to be fairly unstable in solutions of high ionic strength or low pH, resulting in the limitation of providing the possibility to optimize the pH for the best separation.21 Surfactant-protected AuNPs are a promising alternative to separation of proteins in CE since they not only have the potential to prevent protein adsorption on the capillary wall but also have been successfully used for the creation of nanorods and nanowires.22 In this work, we introduced a simple approach for highly efficient separation of acidic and basic proteins using didodecyldimethylammonium bromide (DDAB) bilayerprotected AuNPs (Scheme 1A) as dynamic coating additives. Capillaries modified with the AuNPs were shown to generate a stable, fast, and reversed EOF in the pH range of 3.0-5.0. In order to improve the efficiency of separation, the DDAB-protected AuNPs were further modified with PEO molecules (MW 8 000 000) (Scheme 1B). The separation of acidic and basic proteins with peak efficiencies ranging from 71 000 to 1 007 000 plates/m and relative standard deviations (RSD) of migration time less than 0.6% has been accomplished. Using the preceding method for analyses of saliva, red blood cells (RBCs), and plasma has also been demonstrated. EXPERIMENTAL SECTION Chemicals. Hydrogen tetrachloroaurate(III) dehydrate, sodium borohydride, PEO (MW 8 000 000), DDAB, sodium hydroxide, H3PO4, NaH2PO4, and Na2HPO4 were purchased from Aldrich (Milwaukee, WI). Buffer solutions were 10 mM NaH2PO4 (either 10 mM Na2HPO4 or H3PO4 was used to adjust the pH range from (16) (a) Pumera, M.; Wang, J.; Grushka, E.; Polsky, R. Anal. Chem. 2001, 73, 5625-5628. (b) Neiman, B.; Grushka, E.; Lev, O. Anal. Chem. 2001, 73, 5220-5227. (17) (a) Huang, M. -F.; Kuo, Y.-C.; Huang, C.-C.; Chang, H.-T. Anal. Chem. 2004, 76, 192-196. (b) Lin, Y.-W.; Huang, M. -F.; Chang, H.-T. Electrophoresis 2005, 26, 320-330. (18) Tseng, W.-L.; Huang, M.-F.; Huang, Y.-F.; Chang, H.-T. Electrophoresis 2005, 26, 3069-3075. (19) (a) Aubin-Tam, M.-E.; Hamad-Schifferli, K. Langmuir 2005, 21, 1208012084. (b) De, Roe, C.; Courtoy, P. J.; Baudhuin, P. J. Histochem. Cytochem. 1987, 35, 1191-1198. (20) Frens, G. Nature 1973, 241, 20-22. (21) Zhu, T.; Vasilev, K.; Kreiter, M.; Mittler, S.; Knoll, W. Langmuir 2003, 19, 9518-9525. (22) Gole, A.; Murphy, C. J. Chem. Mater. 2004, 16, 3633-3640.

Scheme 1. Cartons for (A) AuNPs (B) AuNPPs. Schematic Illustration of a Capped AuNP Showing the Relative Size of the Particle Core, Bilayer, Protein, and PEO

3 to 6). All proteins and silver enhancement solution were obtained from Sigma (St. Louis, MO). Apparatus. A double-beam UV-visible spectrophotometer (Cintra 10e, GBC Scientific Equipment Pty Ltd., Dandenong, Victoria, Australia) was used to measure the absorbance of the AuNPs and the AuNPPs. A H7100 transmission electron microscopy (TEM) (Hitachi High-Technologies Corp., Tokyo, Japan) operating at 75 keV was used to collect TEM images of asprepared AuNPs. A commercial UV absorbance detector (ECOM) was use at 220 and 280 nm for proteins and methanol (neutral marker), respectively. A high-voltage power supply from Bertan (Hicksville, NY) was used to drive electrophoresis. The entire detection system was enclosed in a black box with a high-voltage interlock, and the high-voltage end of the separation system was put in a laboratory-made plexiglass box for safety. Data acquisition (10 Hz) and control were performed by DataApex Software (DataApex, Prague, Czech Republic). Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) with 75-µm i.d. and 365µm o.d. were used for protein separations without any coating process in advance. Synthesis of AuNPs and AuNPPs. The DDAB-capped AuNPs were prepared according to reported methods.23 Briefly, 375 µL of 0.048 M HAuCl4 solution was mixed with 60 mL of 1.4 mM DDAB solution under vigorous stirring. Then, 200 µL of freshly prepared 0.4 M NaBH4 solution was added all at once, followed by rapid inversion mixing for 2 min. The solution changed color from pale yellow to wine, indicating the formation of the 6-nm AuNPs (density 19.3 g/cm3). The concentration of the original 6-nm AuNPs is ∼45 nM (2.71 × 1013 particles/mL), which we denote in this study as 1×. The TEM images (not shown) confirmed that the size of the AuNPs is 6 ((0.9) nm. The preparation of the AuNPPs was conducted by mixing the AuNPs directly with PEO. The PEO samples, molecular weights (Mw) of 8 × 106, were dissolved in 10 mM phosphate buffer (pH 3.5). Aliquots of 0.5% PEO solutions (1-10 mL) were added separately to different concentrations of the AuNP solutions (0.11×) such that the final volume of the mixture was 50 mL and the final concentrations of PEO ranged from 0.01 to 0.3%. The solutions were equilibrated at ambient temperature and pressure (23) Zhang, L.; Sun, X.; Song, Y.; Jiang, X.; Dong, S.; Wang, E. Langmuir 2006, 22, 2838-2843.

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overnight. Using this approach, these composite particles remained stable for more than 1 month at room temperature. Capillary Coating. Before coating, the untreated capillary was flushed with 0.5 M NaOH at 1 kV for 10 min, followed by water for 1 min. Subsequently, the capillary was coated by rinsing for 60 min with 0.1 mM DDAB or 10 min with 0.1× AuNPs, which were prepared in 10 mM phosphate buffer at pH 3.0. Before conducting separations, the capillaries were filled with the 0.1 mM DDAB, AuNPs, and AuNPPs by applying a low pressure (syringe pushing), respectively. Standard protein solutions were injected into capillaries at 20-cm height for 10 s, and the separations were conducted at -8 kV. Peak efficiency was calculated using the peak width at half-height method. Protein recovery was obtained by calculating peak area ratios of 80- (60 cm to detector) to 60-cm (40 cm to detetor) capillary. Within-day migration time and peak height reproducibilities were determined for 12 successive injections without 0.1× AuNPs. The day-to-day migration time reproducibility was determined over five consecutive days. The capillary was stored in 0.1 mM DDAB and 0.1× AuNP solutions overnight, respectively. Dark-Field Scattering. The basic design of the dark-field scattering system has been previously described.24 Briefly, the dark-field scattering system is made of an Olympus IX70 inverted microscope (Tokyo, Japan), a DP70 digital camera (Olympus), and high-numerical-aperture dark-field condenser (NA ) 1.2-1.4; U-DCW, Olympus). The white light from a 100-W halogen lamp and a focusing lens within a condenser are angled with respect to the objective (40×; NA ) 0.55) so that the illuminating light does not directly enter the objective; this arrangement results in a low background. A DP70 camera provides images at a resolution of up to 4080 × 3072 pixels, which represents a 755 × 545 µm detection area when using a 40× objective. The AuNP-modified capillary was treated with silver enhancement solution for 2 min25 and then subsequently washed with deionized water. After being silver stained, scattering images for AuNP-modified capillary were observed using dark-field scattering microscopy. We further used the ImageJ program (http://rsb.info.nih.gov/ij/) to analyze the images. Sample Preparation. Saliva samples (500 µL) collected from a healthy adult male were diluted to 2-fold with deionized water. Subsequently, the diluted sample was centrifuged at 3000 rpm for 10 min; the supernatant fraction was analyzed without further pretreatment. Fresh blood (10.0 µL) from a healthy adult male was diluted to 1.0 mL using phosphate-buffered saline, which consisted of 37.0 mM phosphate (pH 7.4) and 38.0 mM sodium chloride, followed by centrifugation and removal of the supernatant to leave only the intact cells without the plasma. The cell pellet was then diluted and lysed by adding 2.0 mL of deionized water. To obtain plasma samples, the collected whole blood samples were immediately centrifuged at 2500 rpm for 10 min at room temperature. The plasma samples were stored at -20 °C. RESULTS AND DISCUSSION Characterization of DDAB-Capped AuNPs and AuNPModified Capillary. The resonance wavelength of the surface plasmon in metallic nanoparticles is highly dependent on the (24) Tseng, W.-L.; Lee, K.-H.; Chang, H. -T. Langmuir 2005, 21, 10676-10683. (25) Wang, J.; Polsky, R.; Xu, D. Langmuir 2001, 17, 5739-5741.


Figure 1. UV-visible absorption spectra of DDAB-capped AuNPs as a function of (A) solution pH and (B) concentration of phosphate solution (pH 3.5). The (A) pH and (B) ionic strength of AuNPs solution are indicated next to the respective curves.

surrounding medium.26 A change in the refractive index of the surrounding medium exhibits a red shift in the resonance wavelength. Also, the aggregation of AuNPs results in the longitudinal plasmon resonance.27 To characterize the stability of DDAB-capped AuNPs as a function of electrolyte concentration and pH, spectral shifts in the surface plasmon resonance were monitored by a commercial UV-visible absorption spectrometer. The peak of the plasmon resonance band is located at 521 nm, indicating the formation of gold nanoparticles. Observed absorption spectra show no shifts in the wavelength corresponding to the plasmon adsorption of the AuNPs from pH 3.0 to 9.0 (Figure 1A). We suggest that DDAB-capped AuNPs are very stable as a function of solution pH. Also, the stability of DDAB-capped AuNPs was characterized at different electrolyte concentrations (Figure 1B). Similarly, the UV-visible spectra after addition of different concentrations of phosphate (pH 3.5) are almost identical, indicating that even the large concentration of phosphate does not destabilize the colloidal solution. Swami et al. have reported the same phenomena mentioned above.28 In contrast to citrate-capped AuNPs, these properties provide the possibility to optimize the pH and electrolyte concentration for the best separation of proteins but no aggregation of AuNPs. In previous studies, we demonstrated that dark-field microscopy is a very versatile imaging technique that has no limitations with respect to the characterization of the composition or size of the sample surface.24 Although various techniques are commonly used to characterize the adsorption of AuNPs on solid surfaces, such as TEM, scanning electron microscopy, and atomic force microscopy, the capillary should be cut to provide detailed information on such features as the degree of aggregation of AuNPs or the roughness and uniformity of the surface.29 However, dark-field microscopy based on the intense Rayleigh scattering of submicrometer particles does not apply to very small metal particles, because Rayleigh scattering decreases as the sixth power of diameter.30 The sensitivity of DDAB-capped AuNPs absorbed on the capillary was improved by treatment of the silver enhance(26) McFarland, A. D.; Van Duyne, R. P. Nano Lett. 2003, 3, 1057-1062. (27) Roll, D.; Malicka, J.; Gryczynski, I.; Gryczynski, Z.; Lakowicz, J. R. Anal. Chem. 2003, 75, 3440-3445. (28) Swami, A.; Kumar, A.; Sastry, M. Langmuir 2003, 19, 1168-1172. (29) Kaupp, S.; Wa¨tzig, H. J. Chromatogr., A 1997, 781, 55-65.

Figure 2. Dark-field images of (A) untreated capillary and (B) AuNPmodified capillary after the treatment of silver enhancement for 2 min. Exposure times: 50 ms; scattering detection area using a 40× objective: 755 µm × 545 µm, which corresponds to 2592 (horizontal) × 1944 (vertical) pixels.

ment solution, which resulted in silver plating where the nanoparticles were bound to the substrate. Without destroying the capillary, Figure 2B shows that adsorption of DDAB-capped AuNPs on the capillary was observed by dark-field microscopy after silver enhancement for 2 min. The silver-enhanced AuNPs with green color (∼70 nm) were 10-fold enlarged compared to 6-nm colloidal gold nucleating cores.31 Some spots display a yellow color and a strong scattering intensity, which points out that a small number of the aggregation of AuNPs. In comparison to an untreated capillary (Figure 2A), dark-field microscopy indeed demonstrated that DDAB-capped AuNPs adsorbed on the capillary wall, reversing the surface charge and thus the EOF. Separation in AuNP-Modified Capillaries. The bilayer structures of double-chained surfactants are attractive for wall coating in CE due to their greater surface coverage and longterm coating stability. In contrast to single-chained surfactants, they provide excellent separation performances for basic proteins, such as high peak efficiencies, excellent run-to-run reproducibility, and high bulk EOF.32 Recently, Lucy’s group has demonstrated that basic proteins were separated efficiently using a DDAB-coated capillary.33 The stability of DDAB-coated capillary increased with (30) Boyer, D.; Tamarat, P.; Maali, A.; Lounis, B.; Orrit, M. Science 2002, 297, 1160-1163. (31) Schultz, S.; Smith, D. R.; Mock, J. J.; Schultz, D. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 996-1001. (32) (a) Wang, C.; Lucy, C. A. Anal. Chem. 2005, 77, 2015-2021. (b) Yassine, M. M.; Lucy, C. A. Anal. Chem. 2005, 77, 620-625.

Figure 3. Comparison of the separations of acidic and basic proteins under three different conditions at pH 3.5 using (A) DDABmodified and (B, C) AuNP-modified capillaries. The separation buffer was (A) 25 mM phosphate buffer containing 0.1 mM DDAB, (B) 10 mM phosphate buffer containing 0.1× AuNPs, and (C) 25 mM phosphate buffer containing 0.1× AuNPs. Electrophoresis conditions: 80-cm capillary (60 cm to detector); applied voltage, -8 kV; hydrodynamic injection at 20-cm height for 10 s; and direct UV detection at 220 nm. Peak identities: 1, R-chymotrypsinogen (5 µM); 2, ribonuclease A (5 µM); 3, trypsinogen (5 µM); 4, cytochrome c (5 µM); 5, lysozyme (2.5 µM); 6, BSA (0.5 µM); 7, carbonic anhydrase (1 µM); 8, ovalbumin (10 µM); 9, myoglobin (5 µM); 10, R-lactalbumin (5 µM).

increasing ionic strength of the running buffer, resulting in improving the peak efficiency and reproducibility of migration time. However, the adsorption of low-pI proteins on the capillary wall was inevitable due to DDAB’s cationic headgroup. Under acidic condition (pH 3.5, 10 mM phosphate), Figure 3A shows that poor separation efficiency and tailing were observed for acidic proteins in the presence of 0.1 mM DDAB. To overcome this problem, we present, to the best of our knowledge, the first attempt to use DDAB-capped AuNPs as the pseudostationary phase for protein separation in CE. We suggest that AuNPs have exceptionally high affinity for proteins due to hydrophobic interaction and covalent conjugation, which is believed to occur mainly by direct attachment of cysteine and lysine residues of protein to gold surface (Table 1).19 To understand the impact of pH on peak efficiency, we tested the separations of proteins by NFCE at pH values ranging from 3.0 to 6.0 when the capillary was filled with (33) (a) Melanson, J. E.; Baryla, N. E.; Lucy, C. A. Anal. Chem. 2000, 72, 41104114. (b) Yassine, M. M.; Lucy, C. A. Anal. Chem. 2004, 76, 2983-2990.


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Table 1. Properties of Proteins Investigated in This Study. proteins

source

MW

pI

radius (nm)a

hydrophobic parameterb

cysteine lysinec

R-chymotrypsinogen A ribonuclease A trypsinogen cytochrome c lysozyme bovine serum albumin carbonic anhydrase ovalbumin myoglobin R-lactalbumin

bovine pancreas bovine pancreas bovine pancreas bovine heart chicken egg white bovine plasma bovine erythrocytes chicken white horse heart bovine milk

25 600 13 700 25 000 13 000 14 000 66 000 29 000 45 000 17 000 14 200

8.7 9.6 9.3 9.5 11.0 4.7 6.3 4.7 7.4 4.7

1.94 1.58 1.88 1.55 1.59 2.66 2.03 2.34 1.70 1.59

950 780 910 1020 890 1000 1000 980 1000 1050

10/19 8/2 12/14 1/18 8/8 35/60 2/26 6/32 0/19 8/12

a Spherical radius, calculated by assuming that all the proteins are spherically shaped. The radii of these proteins were calculated from their volumes using r ) (3V/4π)1/3. b Reference 47. c The data were obtained from the Protein Data Bank.

0.1× AuNPs. At pH 6.0, the peak efficiencies for lysozyme and bovine serum albumin (BSA) are 81 000 and 23 000 plates/m, respectively. Adjustment of the pH to 3.0 resulted in peak efficiency improvement relative to higher pH conditions (450 000 and 62 000 plates/m for lysozyme and BSA, respectively), but at the expense of resolving power. Figure 3B indicates that the separation of proteins (Table 1) by NFCE was successful at pH 3.5 using 10 mM phosphate buffer containing 0.1× AuNPs (10fold dilution of the concentration of the original AuNPs). Peak efficiencies for the proteins ranged from 36 000 to 624 000 plates/ m. We suggested that the resolution was significantly improved due to the different degree of interaction between AuNPs and proteins. It is important to point out that the separation was unsuccessful when using 25 mM phosphate buffer containing 0.1× AuNPs (Figure 3C). Additionally, the AuNP-coated capillary generated a stable EOF after 10 min of coating in the presence of 0.1× AuNPs. The reversed EOF was 8.3 × 10-4 cm2 V-1 s-1 with RSD of less than 0.7% (n ) 60). In comparison, the capillary coated with 0.1 mM DDAB (pH 3.0, 25 mM phosphate buffer) required 60 min of electrokinetic rinsing to achieve a reproducible EOF. At pH 3.5, DDAB-capped AuNPs produced a 75% faster reversed EOF than DDAB, indicating a relatively high surface coverage on the capillary wall. The long-term reproducibility of AuNPmodified capillary was then investigated. The peak efficiency remains unchanged for over 60 consecutive runs for a 5-day period without ever regenerating or refreshing the capillary. The RSD values of migration time and peak height for all protein were 83%), indicating less protein adsorption on the capillary wall (Supporting Information Table S-1). The limits of detection, at a signal-to-noise ratio of 3 for 10 proteins, were in the range from 955.1 to 79.2 nM. However, the separation efficiency became insufficient at concentrations above the entanglement threshold of PEO, 0.07% (Figure 4C,D, respectively). To clarify the deleterious effect of excess PEO, we also collected UV-visible absorption spectra of (36) Cocke, D. L.; Wang, H.; Chen, J. Chem. Commun. 1997, 23, 2331-2332. (37) Azegami, S.; Tsuboy, A.; Izumi, T.; Hirata, M.; Dubin, P. L.; Wang, B.; Kokufuta, E. Langmuir 1999, 15, 940-947. (38) Crothers, D. M.; Gartenberg, M. R.; Schrader, T. E. Methods Enzymol. 1991, 208, 118-146. (39) Yu, C. -J.; Tseng, W. -L. Electrophoresis. In press.

Table 2. Comparison of Electrophoretic Mobility (µep), RSD of Migration Time, and Efficiency (N) for Proteins Under Three Different Conditions 0.1 mM DDABa

R-chymotrypsinogen A ribonuclease A trypsinogen cytochrome c lysozyme BSA carbonic anhydrase ovalbumin myoglobin R-lactalbumin

0.1× AuNPPs (0.05% PEO)c

0.1× AuNPsb

µep × 10-5 (cm2 V-1 s-1)

RSD (%) (n ) 3)d

efficiency (plate/m)

µep × 10-5 (cm2 V-1 s-1)

RSD (%) (n ) 3)d


µep × 10-5 (cm2 V-1 s-1)

RSD (%) (n ) 3)d


14.8 16.0 16.7 22.8 22.8 nd 24.7 nd 27.6 28.2

0.93 0.98 1.12 nde nd nd 2.04 nd 2.23 2.20

50 000 35 000 73 000 nd nd nd 32 000 nd 74 000 26 000

18.3 19.6 20.3 26.0 26.7 28.8 29.9 30.7 31.8 32.6

0.47 0.48 0.50 1.22 0.57 0.58 0.66 0.56 0.60 0.61

294 000 283 000 624 000 329 000 387 000 50 000 36 000 57 000 188 000 239 000

19.3 20.5 21.3 27.2 28.1 29.9 31.3 32.0 33.4 34.2

0.40 0.40 0.41 0.46 0.49 0.50 0.52 0.45 0.53 0.56

744 000 619 000 794 000 638 000 1 007 000 397 000 78 000 71 000 310 000 302 000

a Prepared in 10 mM phosphate buffer at pH 3.5. b Conditions are the same as those in Figure 3C. c Conditions are the same as those in Figure 4B.dMigration time. e nd, not determined because the peak was not detected or resolved.

Figure 5. Effect of PEO concentration on UV-visible adsorption spectra of 0.1× AuNPs in 10 mM phosphate buffer at pH 3.5. The concentration of PEO is (a) 0.01, (b) 0.05, (c) 0.07, (d) 0.10, and (e) 0.30%, respectively.

Figure 4. Separation of acidic and basic proteins using 0.1× AuNPs modified with (A) 0.01, (B) 0.05, (C) 0.07, and (D) 0.10% PEO. Other conditions are the same as those in Figure 3C.

the 0.1× AuNPs in the presence of different amounts of PEO (Figure 5). In the absorption spectrum recorded above 0.07%, particle aggregation is indicated by spectral shifts in the surface plasmon resonance. A change in the absorbance is due to the aggregation or agglomeration of the AuNPs in PEO solutions. A similar phenomenon has been reported in the study of the interaction between AuNPs and polymer solutions.40

Analysis of Biological Samples. With high separation efficiency and reproducibility, our proposed methods have been further considered to test a complex sample. To demonstrate this potential, we tested a salivary sample from a normal male by NFCE. Figure 6A indicates that sharp peaks were detected in the saliva sample by NFCE using 0.1× AuNPs modified with 0.05% PEO. The peak for lysozyme (chicken egg white) was observed after 2.5 µM lysozyme was spiked into the saliva sample (Figure 6B). It demonstrates that this method allows analysis of proteins in a high-salt matrix, which mainly consisted of 15 mM NaCl and 6 mM KCl.41 Interestingly, the peak efficiency for lysozyme is superior to that observed on the above results. The improvement may be attributed to the effect of saliva matrix. Contrastively, the analyses of RBCs and plasma have been considered more contributive to clinical diagnosis. For example, the concentration of RBC hemoglobin is an indicator used to diagnose and monitor many diseases, such as diabetes, leukemia, (40) Huang, C. -C.; Huang, Y. -F.; Chang, H. -T. J. Nanosci. Nanotechnol. 2004, 4, 622-627. (41) van Ruth, S. M.; Grossmann, I.; Geary, M.; Delahunty, C. M. J. Agric. Food Chem. 2001, 49, 2409-2413.


8009

Figure 6. Analysis of saliva samples by NFCE using 0.1× AuNPs modified with 0.05% PEO. Saliva samples were diluted to 2-fold using deionized water. Samples (A) without and (B) with spiking of 2.5 µM lysozyme were injected by raising the capillary inlet 20-cm height for 10 s. The other conditions are the same as Figure 4B.

renal tubular acidosis, and osteoposis.42 Figure 7A shows that three major peaks corresponding to methemoglobin, carbonic anhydrase, and hemoglobin were detected by our proposed methods. By applying a standard addition method, we estimated that the amounts of hemoglobin per cell were ∼430 ( 20 amol (n ) 5), which is very close to the literature value of 450 amol.43 Under the same conditions, Figure 7B indicates that there are many peaks detected in the plasma samples. The big peak at ca. 17.8 min corresponds to human serum albumin (HSA), which is present at less than 50 g/L in a normal plasma sample.44 The RSD values for the migration times of HSA in plasma samples was 0.52%. Although we did not identify the peaks corresponding to transferrin, immunoglobulins, and other plasma proteins, our results demonstrated that this method is a straightforward one for diagnostic purposes. CONCLUSION In this paper, we have demonstrated a new method for the separation of acidic and basic proteins by NFCE in the presence of reversed EOF. The DDAB-capped AuNPs form a dynamic wall coating that is effective at preventing protein adsorption and providing fast reversed EOF. The separation of proteins with pI (42) (a) Chiang, W.-L.; Chu, S.-C.; Lai, J.-C.; Yang, S.-F.; Chiou, H.-L.; Hsieh, Y.-S. Clin. Chim. Acta 2001, 314, 195-201. (b) Yoshida, K. Tohoku J. Exp. Med. 1996, 178, 345-356. (c) Nagai, R.; Kooh, S. W.; Balfe, J. W.; Fenton, T.; Halperin, M. L. Pediatr. Nephrol. 1997, 11, 633-636. (43) Chen, S.; Lillard, S. J. Anal. Chem. 2001, 73, 111-118. (44) Choi, S.; Choi, E. Y.; Kim, D. J.; Kim, J. H.; Kim, T. S.; Oh, S. W. Chin. Chim. Acta 2004, 339, 147-156. (45) Shin, Y. K.; Lee, H. J.; Lee, J. S.; Paik, Y. K. Proteomics 2006, 6, 11431150. (46) Reiseter, B. S.; Miller, G. T.; Happ, M. P.; Kasaian, M. T. J. Neuroimmunol. 1998, 91, 156-70. (47) Handbook of Biochemistry and Molecular Biology; Fasman, G. D., Ed.; CRC Press: Boca Raton, FL, 1967.


Figure 7. Analyses of (A) lysed RBC and (B) plasma samples by NFCE using 0.1× AuNPs modified with 0.05% PEO. Lysed RBCs and plasma were diluted to 200- and 130-fold, respectively. The other conditions are the same as Figure 4B.

ranging from 4.7 to 11.0 was successful by using 0.1× AuNPs modified with 0.05% PEO, with the advantages of high efficiency, excellent reproducibility, and stable EOF. Without any pretreatment, this approach can be applied for analyses of biological samples, including saliva, red blood cells, and plasma. This study reveals that the resolving power of separation depends on interaction between proteins and AuNPPs, which could significantly alter the electrophoretic mobility of proteins. The high peak efficiency observed in basic proteins suggests the suitability of this method for the proteomic analysis of basic peptide and proteins. For example, we should be able to use the proposed method for determination of highly basic, macrophage proteins in mammalian cells45 and myelin basic protein in cerebral spinal fluid.46 Interestingly, we found that the peak efficiencies for acidic proteins and separation time could be further improved by increasing AuNP concentration (Supporting information Figure S-1). This is a subject of further investigations. Therefore, we believe strongly that separation performed by NFCE could be further optimized by using AuNPs with different size, shape, and concentration. ACKNOWLEDGMENT We thank the National Science Council (NSC 94-2119-M-110016-) of Taiwan and National Sun Yat-sen University for financial support of this work. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 10, 2006. Accepted September 14, 2006. AC061059C

Separation of Acidic and Basic Proteins by Nanoparticle-Filled

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