NANO LETTERS
Seeding-Induced Self-assembling Protein Nanowires Dramatically Increase the Sensitivity of Immunoassays
2009 Vol. 9, No. 6 2246-2250
Dong Men,†,‡,§ Yong-Chao Guo,†,§ Zhi-Ping Zhang,† Hong-ping Wei,† Ya-Feng Zhou,† Zong-Qiang Cui,† Xiao-Sheng Liang,†,‡ Ke Li,†,‡ Yan Leng,†,‡ Xiang-Yu You,†,‡ and Xian-En Zhang*,† State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 and Graduate School, Chinese Academy of Sciences, Beijing 100039, China Received February 3, 2009; Revised Manuscript Received March 31, 2009
ABSTRACT Aiming to build a supersensitive and easily operable immunoassay, bifunctional protein nanowires were generated by seeding-induced selfassembling of the yeast amyloid protein Sup35p that genetically fused with protein G and an enzyme (methyl-parathion hydrolase, MPH), respectively. The protein nanowires possessed a high ratio of enzyme molecules to protein G, allowing a dramatic increase of the enzymatic signal when protein G was bound to an antibody target. As a result, a 100-fold enhancement of the sensitivity was obtained when applied in the detection of the Yersinia pestis F1 antigen.
Molecular self-assembly (MSA) is the spontaneous association of molecules under equilibrium conditions into stable, structurally well-defined aggregates that are joined by noncovalent bonds. MSA is ubiquitous in biological systems and underlies the formation of a wide variety of complex biological structures.1 The self-assembling of misfolded proteins leads to the formation of well-ordered fibrillar aggregates that are called amyloid fibrils. These amyloid fibrils are observed in association with a range of disorders, debilitating human diseases, and physiological microbial processes.2 Many studies have shown that amyloid fibrils are non-native protein aggregates that contain numerous β-sheets and a highly ordered cross-β core structure.3 The property of self-organized prionlike proteins has been exploited in the fields of biomedicine, biotechnology, materials science, and nanotechnology. A prospective application of amyloid fibrils is functionalization, which is the expression of functional ligands on the fibrillar surface at the nanoscale. Successfully functionalized fibrils express various functional ligands, such as fluorophores,4 biotin,5 cytochrome,6 enzymes,7 and other functional tags. The potential applications of functionalized nanowires include scaffolds for cell adhesion,8 architecture with enzyme activity, nanowires for use in the electronics * To whom correspondence should be addressed. Tel: +86 10 5888 1508. Fax: +86 10 5888 1559. E-mail:
[email protected]. † State Key Laboratory of Virology, Wuhan Institute of Virology. ‡ Graduate School. § These authors contributed equally to this work. 10.1021/nl9003464 CCC: $40.75 Published on Web 04/29/2009
2009 American Chemical Society
industry,9 and diagnostic devices5 and templates for the assembly of inorganic materials.10 Sup35p, isolated from Saccharomyces cereVisiae, is one of a number of amyloid proteins that are currently in use. The self-assembly of Sup35p into amyloid fibrils is a convenient model for studying both amyloid formation and conformational transmission.11 Recombinant proteins in which the C-terminal domain of Sup35p is substituted with green fluorescent protein (GFP) have been used to study the mechanism of prion-mediated disease. It has been observed that Sup35p can adequately fuse with exogenous functional molecules without significantly impairing its own assembly. Furthermore, the fibrils formed by Sup35p have high chemical stability and their length can be controlled.9 Because of these advantages, Sup35p is considered a good candidate for developing functional nanosized building blocks. Previous studies have demonstrated that the growth of Sup35p fibrils is a self-nucleation process.12 The fragments of preformed fibrils act like seeds and can increase the rate of prion protein aggregation.13 Many researchers have reported that seeding events are also observed during the assembly of amyloid fibrils in vitro.14 Seeding events during mammalian amyloid protein aggregation have been used to detect prion diseases.15,16 By observing the assembly process of Sup35p in nature, we developed a method for the construction of bifunctional protein nanowires. Figure 1 illustrates the formation process of bifunctional protein nanowires. Sup35p prion domain-functional molecule A and
Figure 1. Schematic diagram of the construction of bifunctional protein nanowires by using seeding events and self-assembly. In the finalized bifunctional nanowires, the Sup35 acts as the skeleton carrying a few copy protein G molecules but a large number of enzyme molecules.
Sup35p prion domain-functional molecule B are constructed and expressed as monomers, respectively, through gene manipulation. Sup35p carrying the functional molecule A aggregate forms the protein nanowires by self-assembling. The protein nanowires are then broken into smaller fragments to be used as seeds. The seeds are then mixed with the Sup35p protein monomers carrying the functional molecule B at appropriate proportion. The resultant protein nanowires formed via self-assembly exhibit two different functions derived from A and B, respectively. On the basis of this design, an enzyme molecule and protein G were fused to the C-terminal of Sup35p. The prion domain of Sup35p acts as a protein nanowire skeleton for attaching many copies of the enzyme molecule to a few protein G molecules (Figure 3a). The resultant protein G-enzyme-nanowires have two independent functions. One is recognizing and binding to the Fc portion of immunoglobulin G (IgG) molecule, derived from the protein G moiety; the other function is offering measurable signals derived from the enzymatic catalysis. Since the bifunctional protein nanowires enable the integration of a large number of enzyme molecules into the small seed fragments of recombinant protein G, theoretically, the bifunctional protein nanowires as an ELISA reagent can produce much stronger signals compared to the conventional ELISA procedure, in which one capture antibody usually couples with a single copy or a few copies of enzyme molecules. The sensitivity of the immunoassay can thus be greatly enhanced. MPH is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. We have previously demonstrated that the active form of MPH in solution is monomeric with its C and N terminals exposed at its surface.17 The enzyme has been exploited to be an analytical enzyme for several reasons: easy to fuse to single-chain antibodies through gene fusion without obvious loss of activity, less background noise in an eukaryotic system because this enzyme is only found in some bacteria, and reasonable catalytic activity.18 To produce Nano Lett., Vol. 9, No. 6, 2009
Figure 2. Sup351-61-MPH catalytic nanowires and Sup351-61protein G seeds. (a) Electron micrograph of Sup351-61-MPH catalytic nanowires aggregates. (b) Enzyme activity of MPH under different states. (c) Electron micrograph of seeds formed by Sup351-61-protein G. (d) Distribution of seed lengths.
MPH catalytic nanowires, MPH was fused to the C-terminal of the first 61 amino acids of Sup35p by gene fusion.4,19 The fusion gene was then expressed in Escherichia coli (Supporting Information). The purified fusion protein Sup351-61MPH was incubated at 4 °C overnight to allow self-assembly (Supporting Information). A large number of filaments in the Sup351-61-MPH solution were observed by transmission electron microscope (TEM) (Figure 2a). The diameter of the filaments was approximately 14.2 ( 1.1 nm, and the length was up to 10 µm. This indicates that the fusion of MPH molecules to Sup35p did not impair the formation of the nanowires. The activity of these catalytic nanowires (Supporting Information) was measured subsequently. Before the assay, the soluble monomer Sup351-61-MPH was removed by repeated centrifugation and washing. Since the MPH molecules in the nanowires were not in a homogeneous state, only the apparent activity was given. The results of the assay showed that the Sup351-61-MPH wires restored about 150% of the activity of free MPH (Figure 2b). Normally, an aggregated enzyme shows a decreased activity because of an increased substrate diffusion limit.7 The reason of slightly increased enzyme activity in this experiment is not clear yet. Perhaps, aggregation in nanowires is correlated with the formation of a hydrophobic environment.20 For some enzymes, the main driving force of the enzyme-substrate binding is the hydrophobic interactions.21 Therefore, the hydrophobic microenvironment due to the protein aggregations attracts the comparatively hydrophobic substrate to the surface of the nanowire, which enhances the catalytic efficiency. Another possible reason may be that the formation of catalytic fibrillar structure results in the conformational change of MPH molecule toward higher catalytic kinetics. These should be clarified in the follow-up study. 2247
Figure 3. Characterization of bifunctional protein nanowires. (a) Schematic diagram of bifunctional protein nanowire-mediated signal amplification for immunoassays. The bifunctional protein nanowires were produced as described in Figure 1. (b) Functional evaluation of bifunctional protein nanowires. Indirect ELISA was performed to confirm the bifunctional profiles (antibody capture and signal production) of protein G catalytic nanowires for the detection of antigen F1.
Seeding events are crucial for introducing molecular capture units into the catalytic partners. Here, fusion proteins comprising protein G and Sup35p1-61 (Supporting Information) that had the ability to bind antibodies and undergo self-assembly were genetically constructed for seed production. The preformed Sup351-61-protein G wires were sonicated into extremely short fragments (Supporting Information), which were subsequently used as seeds (Figure 2c). Taking account of about 1000 seeds, the lengths of most seeds ranged from 20 to 40 nm with an average of 36.6 nm (Figure 2d). The seeds were added to the monomer Sup351-61-MPH solution in order to induce the formation of protein G-MPH bifunctional nanowires. Obviously, the signal amplification ability is determined by the ratio of MPH to protein G molecules in the nanowires. The longer the nanowires are, the more enzyme molecules are incorporated into the seed, resulting in a stronger signal. However, filaments that are too long may cause other problems such as liquid handling and nonspecific adsorption. Therefore, we attempted to control the length of the nanowires by simple manipulation of the assembly condition as previously described.9 Here, by changing the ratio of Sup351-61-protein G seeds to soluble Sup351-61-MPH molecules, it was possible to generate nanowires of varying average lengths. For example, at the ratio of 1:1 (w/w), the average length of the filaments was 100 ( 20 nm. Increasing the soluble Sup351-61-MPH concentration increased the length of the filaments (Supporting Information, Figure 1). At a seed to soluble Sup351-61-MPH ratio of 1:16, most filaments were approximately 1 µm in length. Longer filaments could be obtained by increasing the ratio further; however, the resultant filaments had more variable lengths. To evaluate the biological functions of the bifunctional protein nanowires, the assembled nanowires were subjected to a typical ELISA, instead of a typical enzyme-conjugated second antibody (Figure 3a). The detection procedure was similar to that of the conventional indirect ELISA (Supporting Information). Purified F1 protein from Y. pestis was immobilized on a microplate as a target antigen. F1-specific monoclonal antibodies were captured on the microplate through antigen-antibody reaction, followed by addition of the bifunctional protein nanowires. After washing, the color2248
development reaction was performed by adding methyl parathion solution. The signal intensity was directly correlated to the concentration of antigen F1 immobilized in the microplate wells (Figure 3b). The length of the bifunctional protein nanowires was optimized. Nanowires assembled at progressively increasing ratios of seeds to soluble monomers were examined by ELISA. The results indicated that about 500 nm (1:8, Supporting Information, Figure 1) yielded the lowest standard deviations and a higher signalto-noise ratio (data not shown). These results indicate that protein G and MPH molecules both retained their biological functions on the nanowires, and that the two molecules were responsible for immunological recognition and signal generation respectively. To compare our bifunctional protein nanowire-based detection with the conventional ELISA and the fusion protein G-MPH chimera-based immunoassays, parallel assays were performed on a pathogen F1 modified microplate. Compared to the recombinant protein G-MPH, which has a 1:1 signal molecule to capture molecule ratio, the bifunctional protein nanowires possessed much higher molar ratios of MPH to protein G. As predicted, the sensitivity of detection of F1 using the bifunctional protein nanowires was over 1000-fold higher than that of the protein G-MPH chimera and 100-fold higher than that of the conventional HRP-conjugated antibody-based method. The detection limit was found to be 2 ng/mL (70 pmol/ L) with an OD cutoff value of 0.2, which was 2-fold higher than that of the negative control (Figure 4). The extraordinary high sensitivity is due to the large copy number of the enzyme molecules on the bifunctional protein nanowires. Although there are a few protein G copies in one nanowire, there may be only one protein G copy that could actually bind to the target and the others would have very little chance to do so because of steric hindrance. This in fact increases the ratio of the MPH molecules to protein G bond to the target. As result, the detection sensitivity is significantly enhanced. The signal attributed to the nanowires for the negative control was slightly higher owing to the lower dispersibility of the nanowires (Figure 4). There are substantial interests in the detection of pathogens or biomarkers of cancers or other diseases at ultralow levels.22 Nano Lett., Vol. 9, No. 6, 2009
Figure 4. Comparison of the sensitivity of bifunctional protein nanowires, commercial ELISA reagent, and MPH-protein G chimera for F1 antigen detection. F1 antigen at 10-fold dilutions was immobilized on a microplate. The detection procedure was similar to conventional indirect ELISA. The color development was achieved by MPH and horseradish peroxidase (HRP) and measured at 405 nm (MPH) and 450 nm (HRP), respectively, by using a microplate reader (BIO-TEK).
To address the needs, some super- or ultrasensitive methods have been developed, such as immuno-PCR (IPCR),23,24 Biobarcode assay (BCA),25 and the assays employing gold nanoparticles, nanowires,26 or nanotubes.27 Compared with them, the proposed method in this study offers several merits. First, the seeds induced bifunctional protein nanowires resulting in high molar ratios of signal molecules to binding molecules, which generates significant signal amplification, thereby leading to higher immunoassay sensitivity. The sensitivity remains to be further increased if an enzyme with higher catalytic activity or a binding partner with higher affinity to the target were incorporated. For example, the MPH used in this study has a specific activity of about 50 U/mg, while the commercial ELISA kit used in this study for comparison experiment contained horseradish peroxidase (HRP) that possesses a higher specific activity (200-300 U/mg). If HRP could be fused, it would yield further several folds enhancement in signal. Second, the bifunctional protein nanowires are designed as a ready-to-use reagent, supposedly to replace the secondary antibody (with enzyme labeling) used in ELISA. This meets the requirement of the conventional ELISA process that is widely adopted in clinical or field tests today. Therefore, there is no particular instrumental demand. Third, the bifunctional protein nanowires were prepared by genetic fusion technology and MSA. This process is easy to perform and control and is thus fit for production in high yield and orientation control overcoming the function reduction caused by random chemical cross-linking in the conventional antibody labeling with enzymes, which is important for practical application. Orientation control of the enzymeantibody coupling by gene manipulation has been suggested in recent years.28 The seeding-induced self-assembling Nano Lett., Vol. 9, No. 6, 2009
method is a new approach for the orientation control of enzyme-antibody coupling. Furthermore, successful construction of the bifunctional protein nanowires shows the capability of building multifunctional nanowires for other delicate systems, such as nanosensors and targeting nanodelivery vehicles because it can integrate various functional ligands into one fibril by using seeding events. Although the bifunctional protein nanowires have obvious advantages, a few problems remain to be solved before practical application. The major one is nonspecific adsorption that may cause pseudo signals. Another problem encountered is the diversity in length of the fibril, which is a cause of a high standard deviation. These problems could be largely alleviated by optimizing the assay system, which may be achieved by optimizing the washing and blocking conditions. One alternative is to introduce molecules with higher binding affinity, for example, streptavidin (monomer) seeds and biotinylated antibody pair, to further improve the signal-to-noise level. In conclusion, the self-assembly of amyloid fibrils has been employed for the first time to construct bifunctional protein nanowires that dramatically increased sensitivity in the detection of the Y. pestis F1 antigen. The protocol should have broad interests in immunoassays and could be used as a model to develop multifunctional protein nanostructures for many other applications. Acknowledgment. This work was supported by the National Natural Science Foundation of China (30700745 and 90606028) and the Ministry of Health China (infectious disease control, Project No. 2008ZX10004-004). Z.P.Z. and Z.Q.C. were supported by the National Basic Research Program of China (2006CB933100) and the Nanoscience Project of China Academy of Sciences (No. kjcx2-sw-h12). We thank Professor R. F. Yang for providing Y. pestis F1 antigen and antibody, Z. Q. Li and A. Smallwood for valuable comments, and H. J. Wang for figure assistance. Supporting Information Available: Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Whitesides, G. M.; Mathias, J. P.; Seto, C. T. Science 1991, 254 (5036), 1312–1319. (2) Chien, P.; Weissman, J. S.; DePace, A. H. Annu. ReV. Biochem. 2004, 73, 617–656. (3) Kelly, J. W. Nat. Struct. Biol. 2000, 7 (10), 824–826. (4) King, C. Y.; Diaz-Avalos, R. Nature 2004, 428 (6980), 319–323. (5) Kodama, H.; Matsumura, S.; Yamashita, T.; Mihara, H. Chem. Commun. 2004, (24), 2876–2877. (6) Baldwin, A. J.; Bader, R.; Christodoulou, J.; MacPhee, C. E.; Dobson, C. M.; Barker, P. D. J. Am. Chem. Soc. 2006, 128 (7), 2162–2163. (7) Baxa, U.; Speransky, V.; Steven, A. C.; Wickner, R. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (8), 5253–5260. (8) Gras, S. L.; Tickler, A. K.; Squires, A. M.; Devlin, G. L.; Horton, M. A.; Dobson, C. M.; MacPhee, C. E. Biomaterials 2008, 29 (11), 1553–1562. (9) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger, H.; Lindquist, S. L. Pro. Natl. Acad. Sci. U.S.A. 2003, 100 (8), 4527– 4532. (10) Reches, M.; Gazit, E. Science 2003, 300 (5619), 625–627. (11) Uptain, S. M.; Lindquist, S. Annu. ReV. Microbiol. 2002, 56, 703– 741. (12) Serio, T. R.; Cashikar, A. G.; Kowal, A. S.; Sawicki, G. J.; Moslehi, J. J.; Serpell, L.; Arnsdorf, M. F.; Lindquist, S. L. Science 2000, 289 (5483), 1317–1321. 2249
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NL9003464
Nano Lett., Vol. 9, No. 6, 2009
