Quantification of Proteins by Functionalized Gold Nanoparticles Using

Apr 27, 2012 - Prosopis farcta beans increase HDL cholesterol and decrease LDL cholesterol in ostriches (Struthio camelus). Arash Omidi , Hossein Ansa...
1 downloads 0 Views 1MB Size
Download PDF
Letter pubs.acs.org/ac

Quantification of Proteins by Functionalized Gold Nanoparticles Using Click Chemistry Kui Zhu,†,‡ Yi Zhang,†,§ Sha He,† Wenwen Chen,† Jianzhong Shen,‡ Zhuo Wang,*,† and Xingyu Jiang*,† †

CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China ‡ Department of Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China § Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China S Supporting Information *

ABSTRACT: This letter presents a click-chemistry-based assay for proteins (CAP) that allows quantitative determination of the concentration of proteins, using azide- and alkyne-functionalized gold nanoparticles (AuNPs). Compared with conventional methods, CAP has a broader linear range for detection of proteins with good selectivity. CAP enables the analysis of total proteins in various sera and milk samples.

A

require the aid of advanced instruments or be poorly selective in complex samples. The Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction (click chemistry) in homogeneous aqueous solution at room temperature has opened up new approaches for biochemical assays.11−14 Our previous work demonstrates that click chemistry allows high sensitivity and selectivity for the detection of Cu(I).14,15 In the presence of Cu(II) with sodium ascorbate as the reductant, AuNPs functionalized with azide and alkyne undergo aggregation as the result of the Cu(I)catalyzed azide/alkyne cycloaddition (CuAAC) reaction. The azide, alkyne, and their conjugation process are generally tolerant to a wider range of the solvents, temperature, and pH than other techniques.16−18 Referring to the mechanism of the BCA assay where Cu(I) is reduced by the protein in a homogeneous alkaline solutions containing Cu(II), we hypothesized that a click-chemistry-based assay for proteins (CAP) could also quantify the concentrations of proteins (Scheme 1). Because of the high sensitivty and selectivity associated with click chemistry-based assays14,15 and the colorchange-based visual readout enable by gold nanoparticles, we believe that CAP can potentially overcome some disadvantages of existing methods for quantifying proteins. To the best of our knowledge, this is the first example in which a functionalized AuNP-based colorimetric assay has been used to quantify total protein concentration. In this strategy, Cu(II) is reduced to Cu(I) by proteins in the alkaline solution, where Cu(I) subsequently acts as a catalyst to promote the tethering reaction between azide- and alkyne-

ccurate quantification of proteins are critical for various applications, ranging from the diagnosis of diseases, determination of enzyme activity, to the evaluation of the safety and quality of food. For example, sensitive analytical methods for quantification of the concentration of total proteins are significant clinical markers for many diseases, the most common of which includes the markers of hepatopathy.1 Moreover, to ensure the safety and quality of food, the concentration of protein is a necessary standard, e.g., milk and dairy products. Exisiting methods to determine the protein concentrations have several limitations. For example, the Kjeldahl assay allows indirect determination of proteins by the quantity of nitrogen. Illegal dopants, such as melamine, can increase the proportion of nitrogen and thus a false increase in the concentration of proteins.2 Researchers are working toward protein assays that are highly specific, rapid, sensitive, simple, and convenient to operate. The Lowry assay,3 UV spectroscopy,4 the Bradford assay,5 and the bicinchoninic acid (BCA) assay are generally applied to determine protein concentration in laboratories.6 There is no single method that yields precise results, as each method has its own advantages and limitations. Combining the traditional method with new sensors or readout technologies, such as gold nanoparticles (AuNPs), may improve the efficiency of the quantitative analysis of proteins. AuNP-based protein detection has been developed with many variations, such as colorimetric detection,7 fluorescence quenching,8 time-resolved fluorescence resonance energy transfer (TR-FRET),9 and surface plasma resonance (SPR).10 Although bringing some new insight, these methods, however, are suffering from several inevitable shortcomings. The sensitivity of these methods, however, is moderate and varied significantly between different proteins; moreover they may © 2012 American Chemical Society

Received: April 21, 2012 Accepted: April 25, 2012 Published: April 27, 2012 4267

dx.doi.org/10.1021/ac3010567 | Anal. Chem. 2012, 84, 4267−4270

Analytical Chemistry

Letter

Scheme 1. Click-Chemistry-Based Assay for Proteins (CAP) by Two Types of AuNPs, Each Modified with Thiols Terminated with an Azide or an Alkyne Functionalized Group

Figure 1. (A) Typical photographs of parts a−g show the essential compounds of CAP at room temperature, and the various compounds are listed in the table below; (B) corresponding UV−vis spectra of the solutions from parts a−g. (Alkaline solution is the biuret reagent.)

purple after incubation at room temperature. This phenomenon verified that the buiret assay has worse sensitivity than CAP for detection of proteins. In order to show that copper was necessary as a catalyst, we carried out a pair of tests, one with biuret reagent and protein (Figure 1A,g) and the other one without copper (with everything else) in the solution (Figure 1A,d). The color of the copper-catalyzed click reaction of the functionalized AuNPs can be easily distinguished in Figure 1A,g. To show that the reduction of Cu(II) by protein is critical to our experiments, we detected proteins in the presence of bathocuproine disulfonic acid (BCDSA), a Cu (I) chelator and inhibitor of the CuAAC reaction.20 When Cu(I) was sequestered by BCDSA, the mixed AuNPs solution still kept a red color even upon the addition of proteins and Cu(II). (Figure S2 in the Supporting Information) We optimized the sensitivity of CAP with different concentrations of AuNPs and biuret reagents to achieve the maximum absorbance for the reagents (Figure S3 in the Supporting Information). Moreover, we studied the effects of temperature, time, and pH values on the click reaction. CAP was completed in a short time (less than 10 min) and simplifies the procedure, while commercial protein quantification assays were time-consuming, for example, the BCA assay.6 At room temperature, the reaction could proceed smoothly, which made the assay easy to operate (Figure S3 in the Supporting Information). We investigated the standard curve of the protein with a series of gradient concentrations of bovine serum albumin (BSA), under optimal conditions. The absence or presence of BSA with a concentration down to 30 μg/mL could be determined by the naked eye without the aid of any advanced instruments. For a lower limit of detection (LOD), 0.2 μg/mL (3 S/N, singal-noise-ratio) could be performed with the help of a UV−vis spectrophotometer. The linear range of BSA is from 30 to 2500 μg/mL, with the regression equation y = 0.113x + 0.0096 (R2 = 0.9906). The dynamic range of CAP is broader than both the Bradford and BCA method (Figure 2). This method is therefore less time-consuming and more

modified AuNPs, resulting in the nanoparticles to aggregate with a color change of the solution. We prepared AuNPs functionalized with azide and alkyne functional groups by ligand exchange reactions using the previously reported method where the lowest detectable concentration of Cu(II) is 1 μM.14 The AuNPs are modified by thiol-PEG (thiol terminated in polyethylene glycol) and the azide/alkyne ligands for click chemistry at a molar ratio of 5:1 thiol-PEG−azide/alkyne, respectively. PEG is effective in preventing nonspecific adsorption of proteins on the surface of AuNPs and keeping the AuNPs stably dispersed.19 The homogenously dispersed solution of the two kinds of AuNPs exhibit characteristic SPR absorption band at 526 nm (Figure S1 in the Supporting Information), and the average diameter is approximately 14 nm.14 Proteins can reduce Cu(II) to Cu(I) in situ in alkaline solution, then cross-link the azide- and alkyne-functionalized AuNPs resulting in the aggregation of the nanoparticles and the color change (Figure 1A). The absorbance spectra corresponding to each bottle in Figure 1A also validated the aggregation of AuNPs. (Figure 1B). We tested the specificity of the assay, and it indicated that copper played a key role in the assay of proteins using click chemistry. The alkaline solution used in Figure 1 is the biuret reagent. The biuret assay is used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms a complex in an alkaline solution. The biuret reagent is made of potassium hydroxide and hydrated copper(II) sulfate, together with potassium sodium tartrate. So the biuret reagent was used here to provide the alkaline condition and the partial source of Cu(II) ion. When we added an equal amount of proteins into the functionalized AuNPs (Figure 1A,b) by weight, there was almost no difference compared to the control (Figure 1A,a). When we added biuret reagent with excess Cu(II) ion into the homogeneous solution, the strong alkaline solution would not result in aggregation of the functionalized AuNPs (Figure 1A,c). We also compared the traditional biuret assay for the assay of proteins (Figure 1A,f). The solution appeared light 4268

dx.doi.org/10.1021/ac3010567 | Anal. Chem. 2012, 84, 4267−4270

Analytical Chemistry

Letter

the variation of the affinity between different proteins and the dye, and some proteins have difficulty in dissolving in the acidic medium. Compared with the Bradford and BCA assays,5,6 CAP is more tolerant to most detergents. However, it is still suspectible to cationic surfactants such as lecithin and CTAB because the cationic surfactants might cause the aggregation of the AuNPs.21 We applied CAP to determine several kinds of proteins from various sources and compared the results with BCA and Bradford methods (Table S3 in the Supporting Information). Similar to the various purified proteins, there is also no significant difference between the crude proteins and sera. The three assays almost gave the same trend for one sample, but the Bradford assay showed a lower response compared to the other two methods. We next determined the amount of proteins in milk samples purchased from a local supermarket in Beijing. Prior to performing the assay, all samples were incubated for 30 min at room temperature. We applied all three methods to analyze the milk samples. The Bradford assay was easily interfered with by the complicated mixture, while the BCA and CAP methods worked well (Tables S4−S6 in the Supporting Information). The components of the sample could result in response suppression or an enhancement effect, which is from the components of the sample other than the target analyte.22,23 The total protein in skim milk can be assayed directly without any pretreatment, but fresh milk and yogurt samples need to be centrifuged to remove the fat before the assay. The supernatant fat must be removed to eliminate lipid interference in determining protein content.24 The standard BCA assay can overestimate the protein content of lipoproteins. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions.25 We confirmed that the presence of lipids gave excessively high absorbances with the BCA assay (Table S2 in the Supporting Information). To overcome the interference from lipids, we centrifuged fresh milk and yogurt samples before the assay. Skim milk samples can be directly used for CAP. Although BCA and CAP matched well in the determination of various proteins and sera, they showed differences in the milk detection and the determining results also did not fit the amounts labeled on the packages. There are several reasons accounting for this discrepancy. (1) The complicated components and unknown additive might still be the major factor. The milk samples which are in the markets contain lots of stabilizing agents for long storage. Moreover, different additives which make milk friendly to consumers are also used to adjust the taste. All of these additives influence the protein assay procedures and make the results difficult to match the labeled data on the packages. (2) All the tested milk samples contained sugars. The BCA assay is more sensitive to the interference from reducing sugars, possibly because the protocol makes it possible for the sugars to have the opportunity to reduce Cu(II) to Cu(I).6 However, the CAP assay can be completed without the side reactions in a short time (less than 10 min) and lower temperature (room temperature, see Figure S3 in the Supporting Information) than the BCA assay. So, this discrepancy of BCA and CAP on the detection of milk samples is due to the different reaction conditions of the two assays. (3) The residue of antimicrobial agents can be another reason. Peniclillamine easily interferes with BCA.26 In some cases, either illegal use of veterinary drugs or noncompliance of producers with existing animal-treatment protocols, residues of these substances may enter the food

Figure 2. Calibration curves of Bradford, BCA assay, and CAP. The linear ranges of Bradford, BCA, and CAP are from 20 to 200 μg/mL, 50 to 200 μg/mL, and 30 to 2500 μg/mL, respectively. The insert is the linear range of CAP by plotting the absorbance value vs log (protein).

accurate for quantifying a crude protein sample with less need for dilution compared to existing methods. To test the general applicability of our approach, we investigated the response of the assay to proteins with different properties. These proteins we selected have molecular weights that vary from 14.6 to 149.9 kDa, isoelectric points (pI) that range from 1.0 to 11.0, as well as various number of subunits and different shapes (Table S1 in the Supporting Information). OVA (ovalbumin), has (human serum albumin), BSA, Hb (hematoglobin), and IgG (immunoglobulin G) were widely used to investigate the discrepancy among the Bradford, BCA, and Lowry assays . Also, we added several commonly used proteins to extend the pI range from 1.0 to 11.0. These proteins also represent a wide range of species from which they originate: bacteria, birds, and mammals. Smith et al. used seven proteins to illustrate the protein−protein variation for the BCA method compared to the other method.6 The molecular weights of the proteins had little effect on the reproducibility of CAP. Proteins with diverse isoelectric points showed different responses to the functionalized AuNPs (Table S1 in the Supporting Information). Each protein was analyzed by the three assays (BCA, CAP, and Bradford), respectively, and the standard deviation (SD) was used to evaluate the results of the corresponding assays. SDs of BCA, CAP, and Bradford assays for various proteins are 0.302, 0.301, and 0.342, respectively. This suggested that CAP has a better stability than Bradford assay, eventhough the intrinsic structural properties of protein might still have a negative effect on CAP, especially the slightly acidic proteins showed much higher efficacy than most alkaline proteins. Only pepsin (pI 1.0) and alkaline proteins showed significantly different responses compared with BCA and CAP. Moreover, the Bradford assay is more easily influenced by various proteins compared with CAP. We tested the specificiy of this assay by assaying potential contaminants and buffer components most frequently used in laboratory. The interferences included various surfactants, amino acids, reductants, inorganic salts, and so on. CAP was generally more tolerant to the presence of compounds that interfere with the Bradford and BCA assays (Table S2 in the Supporting Information). Glucose could be detected with Fehling or Benedict’s reagents but showed low response to Cu(II) at room temperature. Thus glucose had little effect on the detection of proteins using CAP. In the Bradford assay, proteins and the Coomassie Brilliant Blue G-250 form a protein−dye noncovalent complex. This method suffers from 4269

dx.doi.org/10.1021/ac3010567 | Anal. Chem. 2012, 84, 4267−4270

Analytical Chemistry

Letter

(15) Zhou, Y.; Wang, S. X.; Zhang, K.; Jiang, X. Y. Angew. Chem., Int. Ed. 2008, 47, 7454−7456. (16) Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272− 1279. (17) Rangan, K. J.; Yang, Y. Y.; Charron, G.; Hang, H. C. J. Am. Chem. Soc. 2010, 132, 10628−10629. (18) Rijkers, D. T. S.; van Esse, G. W.; Merkx, R.; Brouwer, A. J.; Jacobs, H. J. F.; Pieters, R. J.; Liskamp, R. M. J. Chem. Commun. 2005, 41, 4581−4583. (19) Liu, D. B.; Xie, Y. Y.; Shao, H. W.; Jiang, X. Y. Angew. Chem., Int. Ed. 2009, 48, 4406−4408. (20) (a) Faizullah, A. T.; Townshend, A. Anal. Chim. Acta 1985, 172, 291−296. (b) Suzuki, T.; Ota, Y.; Kasuya, Y.; Mutsuga, M.; Kawamura, Y.; Tsumoto, H.; Nakagawa, H.; Finn, M. G.; Miyata, N. Angew. Chem., Int. Ed. 2010, 49, 6817−6820. (21) Kuong, C. L.; Chen, W. Y.; Chen, Y. C. Anal. Bioanal. Chem. 2007, 387, 2091−2099. (22) Niessen, W. M. A.; Manini, P.; Andreoli, R. Mass Spectrom. Rev. 2006, 25, 881−899. (23) Monaci, L.; Brohée, M.; Tregoat, V.; van Hengel, A. Food Chem. 2011, 127, 669−675. (24) Kessler, R. J.; Fanestil, D. D. Anal. Biochem. 1986, 159, 138− 142. (25) Morton, R. E.; Evans, T. A. Anal. Biochem. 1992, 204, 332−334. (26) Wiechelman, K. J.; Braun, R. D.; Fitzpatrick, J. D. Anal. Biochem. 1988, 175, 231−237.

chain. (4) The Kjeldahl assay allows indirect determination of proteins by the quantity of nitrogen and is recommended by the Chinese government. Nevertheless, the Kjeldahl method is easily influenced by contamination such as melamine. In conclusion, we presented a new system for the quantitative determination of proteins based on the CuAAC reaction and functionalized AuNPs. CAP for protein detection is more tolerant to most of detergents and has a broader linear range than the BCA and Bradford assays. The analysis of real samples shows the potential of CAP in practical applications.

■

ASSOCIATED CONTENT

S Supporting Information *

Description of the materials and method used and further experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Z.W.); [email protected] (X.J.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS K.Z. and Y.Z. contributed equally to this work. We gratefully acknowledge grants from the Ministry of Science and Technology (2009CB930001, 2011CB933201), the NSFC (Grant Nos. 30830082, 31072171, 21025520, 90813032, and 21105018), the Program for Changjiang Scholars and Innovative Research in University (Grant No. IRT0866), the Chinese Academy of Scienes (Grant No. KJCX2-YW-M15), and the support from the Youth Innovation Promotion Association, CAS.

■

REFERENCES

(1) Gornall, A. G.; Bardawill, C. J.; David, M. M. J. Biol. Chem. 1949, 177, 751−766. (2) Chu, P. W. S.; Chan, K. M.; Cheung, S. T. C.; Wong, Y. C. TrAC, Trends Anal. Chem. 2010, 29, 1014−1026. (3) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265−275. (4) Layne, E. Methods Enzymol. 1957, 3, 447−455. (5) Bradford, M. M. Anal. Biochem. 1976, 72, 248−254. (6) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76−85. (7) Wei, H.; Li, B. L.; Li, J.; Wang, E. K.; Dong, S. J. Chem. Commun. 2007, 43, 3735−3737. (8) Krovi, S. A.; Erdogan, B.; Han, G.; Kim, I. B.; Bunz, U. H. F.; Rotello, V. M. Polym. Prepr. 2006, 47, 599−600. (9) Härmä, H.; Dähne, L.; Pihlasalo, S.; Suojanen, J.; Peltonen, J.; Hänninen, P. Anal. Chem. 2008, 80, 9781−9786. (10) Mallon, C. T.; Jose, B.; Forster, R. J.; Keyes, T. E. Chem. Commun. 2010, 46, 106−108. (11) Clark, P. M.; Dweck, J. F.; Mason, D.; Hart, C.; Buck, S. B.; Peters, E. C.; Agnew, B. J.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2008, 130, 11576−11577. (12) Salic, A.; J Mitchison, T. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2415−2420. (13) Dieterich, D. C.; Link, A. J.; Graumann, J.; Tirrell, D. A.; Schuman, E. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9482−9487. (14) Qu, W. S.; Liu, Y. Y.; Liu, D. B.; Wang, Z.; Jiang, X. Y. Angew. Chem., Int. Ed. 2011, 50, 3442−3445. 4270

dx.doi.org/10.1021/ac3010567 | Anal. Chem. 2012, 84, 4267−4270