A highly sensitive and selective diagnostic assay based on ... - Nature

LETTERS PUBLISHED ONLINE: 8 MARCH 2009 | DOI: 10.1038/NNANO.2009.38

A highly sensitive and selective diagnostic assay based on virus nanoparticles Jin-Seung Park1†, Moon Kyu Cho2†, Eun Jung Lee1†, Keum-Young Ahn1, Kyung Eun Lee3, Jae Hun Jung4, Yunjung Cho5, Sung-Sik Han3, Young Keun Kim6 * and Jeewon Lee1 * Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction1–5 can reduce the risk of death from heart attacks6–10. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range11. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays11–16. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers. Despite considerable advances in protein (disease marker) detection, the current enzyme linked immunosorbent assay (ELISA) based detection methods have several drawbacks. First, antibodies are usually laid on various kinds of surface in a random fashion, which may make protein markers inaccessible to antibodies, except affinity attachment that requires the additional modification of both antibodies and the surface ( for example, biotinylation of antibodies and streptavidin conjugation to surface17). Second, a low ratio of antibodies to protein markers may limit sensitivity. Third, inefficient capture of protein markers by antibodies that are displayed on a two-dimensional surface may also decrease the sensitivity. By breaking through these barriers, a significant increase in detection sensitivity would be achieved if the following crucial improvements are made: (i) convenient and efficient control of antibody orientation upon the surface immobilization of antibodies, (ii) a substantial increase in density and the ratio of antibodies to protein markers, and (iii) the capture of protein markers by antibodies that are displayed on a three-dimensional surface, leading to more frequent antibody–marker binding. We synthesized hepatitis B virus (HBV) capsid-derived nanoscale particles (termed chimeric nanoparticles here), at the surface of which the B domains of staphylococcal protein A (SPAB) with a specific affinity for the Fc domain of immunoglobulin G (refs 18,19) are displayed with high density. The HBV core protein, consisting of four long a-helix bundles (Fig. 1a), when expressed in bacteria

assembles into core–shell particles that closely resemble the native capsid structure of the virus20,21. Through electron cryomicroscopy analysis, Bo¨ttcher and colleagues20 demonstrated that a single HBV core protein truncated after residue 149 forms core–shell particles that contain 240 subunits and have an overall diameter of 36 nm. The dimer clustering of the subunits produces spikes on the surface of the shell particle, and the immunogenic epitope is located at the tips of prominent surface spikes (Fig. 1a). The surface-exposed spike tip corresponds to the loop segment consisting of the residues from D78 to D83 of the single core protein. The P79A80 in the loop segment was replaced with the tandem repeated SPAB sequences, which were subsequently exposed on the surface of the synthesized chimeric nanoparticles with high density (Fig. 1a). In the chimeric nanoparticle synthesis, we also added the hexahistidine sequence to the N-terminus of the truncated HBV core protein so that the chimeric nanoparticles would have a strong affinity for nickel (Fig. 1a). Transmission electron microscopy (TEM) image analysis (Fig. 1a) revealed that the HBV capsid-derived chimeric nanoparticles that were expressed in E. coli assembled into spherical nanoparticles with a nearly native diameter. Consequently, the chimeric nanoparticles have a dual affinity for the Fc domain of antibodies (IgG) and nickel. Recently, genetically engineered viruses and virus-like particles have been increasingly used to make biodetection devices including diagnostic assays22–27. These viral particles can display on their surface various peptides and proteins that are used for detecting and/or quantifying biomolecules of interest. A three-dimensional assay system was developed by combining the chimeric nanoparticles with a nickel nanohair structure or porous membrane, then adding antibodies to specifically capture protein markers, as illustrated in Fig. 1b. (See Supplementary Fig. S1 for scanning electron microscopy (SEM) images of the nickel nanohairs and porous membrane used in this study.) The term ‘nanohair’ refers to an array of nanowires (see Methods) in which some wires are exposed to the air, the rest being embedded in the body of a supporting organic or inorganic template (Fig. 1b; see also Supplementary Fig. S1). In these structures, the air-exposed portion of the nanowires has a greatly increased surface-to-volume ratio. When antibodies are added to the chimeric nanoparticles that are already attached to the nickel nanohair surface as a result of the affinity interaction between the hexahistidine and nickel (Fig. 1a), the Fc domain of antibody (IgG) is specifically bound to the surface SPAB of the chimeric nanoparticles, and hence the antigen-specific variable domains of IgG are fully accessible to protein markers. Consequently, as illustrated in Fig. 1b, the efficient

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Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea, 2 Department of Micro/Nano Systems, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea, 3 School of Life Sciences and Biotechnology, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea, 4 Cardiovascular Division, Department of Internal Medicine, Kangnam Sacred Heart Hospital, Hallym University Medical Center, Seoul 150-950, Republic of Korea, 5 College of Medicine, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea, 6 Department of Materials Science and Engineering, Korea University, Anam-Dong 5-1, Seongbuk-Gu, Seoul 136-713, Republic of Korea; † These authors contributed equally to this work; * e-mail: [email protected]; [email protected] NATURE NANOTECHNOLOGY | VOL 4 | APRIL 2009 | www.nature.com/naturenanotechnology © 2009 Macmillan Publishers Limited. All rights reserved.

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NATURE NANOTECHNOLOGY Native HBV capsid particle

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Figure 1 | Three-dimensional diagnostic assay based on virus nanoparticles. a, Schematic and TEM images of native hepatitis B virus (HBV) capsid particles and chimeric nanoparticles synthesized in E. coli. The structures of HBV core protein and capsid particles are from Protein Data Bank (www.rcsb.org) (PDB ID: 1QGT). b, Schematic of the diagnostic system performed in a 96-well microplate and the assay principle in detail. Briefly, antibodies that recognize the disease marker (troponin I in this case) bind to the chimeric nanoparticle and are oriented in a specific way. Troponin I binds to the antibodies and detection is achieved with secondary antibodies conjugated with quantum dots.

three-dimensional assay system was developed with the following significant advantages: (i) maximum accessibility of protein markers to antibodies, enabled both by the controlled orientation of the antibodies and the three-dimensional manner of protein capture, and (ii) a dramatically increased density and ratio of antibodies to protein markers on the three-dimensional nanohair surface. The captured markers are detected by sensing photoluminescence emitted by quantum dots conjugated to the secondary antibodies (Fig. 1b). 260

From Fig. 2a, it is clear that the ELISA-based assay (see Methods) did not detect troponin I (in PBS buffer or healthy sera) at concentrations lower than 0.1 nM, but gave highly reproducible signals at each troponin I concentration. Using the chimeric nanoparticles and nickel nanohair system developed here, the sensitivity was surprisingly boosted to attomolar level (Fig. 2b), which represents 100,000-fold higher sensitivity than the highest level (0.25 pM) reported to date28 and also six to seven orders of magnitude greater sensitivity than current ELISA assays11–16. Furthermore, troponin I in acute

NATURE NANOTECHNOLOGY | VOL 4 | APRIL 2009 | www.nature.com/naturenanotechnology © 2009 Macmillan Publishers Limited. All rights reserved.

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PBS buffer and healthy sera spiked with troponin I. The attomolar detection limit of troponin I was also comparable to that of the nickel nanohair-based assay. Furthermore, the assay with antibodies directly immobilized on the PVDF surface showed significantly lower sensitivities than assays using the chimeric nanoparticles (Fig. 4b). This is probably due to the orientation of the antibodies; those immobilized directly on the PVDF surface may be random and have lower accessibility to troponin I. The orientation of antibodies immobilized on the chimeric nanoparticles seems crucial for the sensitivity of the assay. We also tested the PVDF-based assay system in the clinical diagnosis of 26 AMI patients (see Supplementary Table S1) who were confirmed to have experienced an AMI, and the assay results were compared with the ELISA-based assay (Fig. 5a,b). In the ELISAbased assay (Fig. 5b), three (nos 4, 9 and 18) AMI patient sera were not positively detected; that is, the absorbance signals were below the clinical cutoff value (indicated by the horizontal dotted line), and the signals from nine (nos 1, 2, 3, 5, 8, 11, 16, 17 and 23) AMI patient sera were positive but very close to the clinical cutoff. Meanwhile, the chimeric nanoparticles and PVDF-based assay gave clear positive signals for all 26 AMI patient sera, therefore indicating 100% clinical specificity (Fig. 5a). The ELISA assay results were not surprising because the clinical specificity of the ELISA kit is reported by the supplier to be 87.5%. Furthermore, antibodies directly immobilized on the PVDF surface failed to diagnose the 1,000-times diluted AMI patient sera, whereas the chimeric nanoparticles and PVDF-based assay could detect troponin I in the diluted patient sera (Fig. 5c), indicating that this three-dimensional assay can discriminate the onset of AMI even with an extremely small quantity of patient sera. Using the HBV capsid-derived chimeric nanoparticles and threedimensional nanostructures (nickel nanohair and PVDF membrane) we were able to develop a highly sensitive and specific assay system for the specific AMI marker, troponin I. Although HBV capsid was used in this study as a model viral scaffold for the surface display of SPAB , other viruses or virus-like particles could also be used for the production of chimeric nanoparticles displaying the surface SPAB. Owing to the controlled orientation of densely immobilized antibodies and the three-dimensional manner of protein capture, the assay sensitivity and clinical specificity were significantly enhanced as compared to the conventional ELISA assay. Although the AMI diagnosis system was used as proof-of-concept in this study, this approach should be generalized to the detection of almost all protein markers.

Methods myocardial infarction (AMI) patient sera was successfully detected using the same assay system and procedure, while the troponin Ifree PBS buffer and healthy sera gave only negligible signals (Fig. 2b). As shown in step B in Fig. 3, to prevent false signals arising from non-specific binding of quantum dot secondary antibodies to the bare nickel surface, a sufficient amount of chimeric nanoparticles were added to the washed surface of the nickel nanohair (see Fig. 2b and also Supplementary Fig. S2 for this experiment). One of the distinct advantages of this assay system is that one nickel nanohair structure can be repeatedly used for multiple samples. Through washing and rinsing (step A of Fig. 3a), the used nickel nanohair was refreshed and reused for another sample assay. All of the three separate nickel nanohair structures were successfully used for the consecutive assay of eight different samples. Each consecutive assay showed reproducible and consistent signals for all the samples tested (Fig. 3b). A polyvinylidene fluoride (PVDF) membrane with an average pore size of 450 nm (see Supplementary Fig. S1) was selected as a suitable nanostructure with a hydrophobic pore surface on which the chimeric nanoparticles were easily immobilized, and was used to construct another type of three-dimensional assay system. As seen in Fig. 4a, antibodies attached on the chimeric nanoparticles could reproducibly detect troponin I, at all concentrations, in both

Biosynthesis of HBV capsid-derived chimeric nanoparticles. Following assembly PCR using appropriate primers, we prepared the two gene clones derived from the HBV core protein (HBVcAg) gene and code for the synthesis of N-NdeI-hexahistidineHBVcAg(1–78)-G4SG4T-XhoI-C and N-BamHI-G4SG4-HBVcAg(81–149)-HindIIIC. To replace the P79A80 of the HBVcAg with the tandem repeat of SPAB (residues 209–271), the two different clones, N-XhoI-SPAB-EcoRI-C and N-EcoRI-SPABBamHI-C were prepared. Through the sequential ligation of the four above gene clones into plasmid pT7-7, we constructed the plasmid expression vector pT7-Chimera-HBV encoding the synthesis of N-His6-HBVcAg(1–78)-SPAB-SPAB-HBVcAg(81–149)-C. After the complete DNA sequencing of gel-purified plasmid expression vector, E. coli strain BL21(DE3)[F2ompThsdSB(rB2mB2)] was transformed with pT7-ChimeraHBV, and ampicillin-resistant transformants were selected. The experimental procedures for gene expression, purification and TEM image analysis of chimeric nanoparticles are well described in our previous report29. Manufacture of nickel nanohair structure. The nickel nanohair structure was fabricated by electrodeposition using a nanoporous anodized aluminium oxide (AAO) membrane (Anodisc 25, Whatman International, UK) as a template. The thickness and nominal pore diameter of the AAO template were 60 mm and 200 nm, respectively. The detailed procedure can be found in the Supplementary Methods. Construction of the three-dimensional diagnostic systems using virus nanoparticles. For the nickel nanohair-based system, the nickel nanohair structure was first placed in the Costar 96-well plate (cat. no. 3599, Corning). Before immobilizing the chimeric nanoparticles, the nickel nanohair in each well was washed four times for 15 min using 0.3 M sulphuric acid and then six times for 10 min using distilled water, then completely dried. Next, the background


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Figure 3 | A washable and reuseable assay system. a, Four-step protocol for washing and reusing the nickel nanohairs for detection. b, Consecutive assays of eight different troponin I (Tn) samples using three separate systems, showing good reproducibility. A, B, C, D, dotted and solid arrows, and PL1/PL2 correspond to those in a. Black areas represent net PL increase. photoluminescence from the nickel nanohair structure was measured using a microplate reader (GENios, Tecan) with excitation and emission at 420 and 650 nm, respectively. PBS buffer (50 ml) containing the 38-nM chimeric nanoparticles was added to the nickel nanohair structure, followed by slow agitation for 30 min, after which it was washed with 50 mM Tris buffer ( pH 7.4). Rabbit anti-troponin polyclonal antibody (200 ml, 5 mg ml21; cat. no. ab470003, Abcam) in PBS buffer was added to the chimeric nanoparticles that were already immobilized on the nickel nanohair, by slowly stirring the nickel nanohair in the antibody-containing PBS buffer for 2 h. 262

For the PVDF-based system, PVDF membrane (Immobilion-FL, IPFL 10100, Millipore) in a Costar 96-well plate was pre-wetted with methanol for 1 min and washed with a PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 , 2 mM KH2PO4 , pH 7.4) for 5–10 min. Before the PVDF membrane was completely dried, 10 ml of PBS buffer containing the purified chimeric nanoparticles (0.15 mg ml21) was dropped onto a designated spot on the membrane. The membrane was then slowly stirred for 1 h in the blocking solution (1% skimmed milk) and washed twice with the PBS buffer for 30 min. Goat anti-troponin I polyclonal antibodies (200 ml, 20 mg ml21 in PBS buffer; cat. no. 70-XG82,



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Figure 5 | Clinical specificity and sensitivity of the viral chimeric nanoparticle-based assay. a,b, Assays of sera from 16 healthy individuals and 26 AMI patients using the chimeric nanoparticles and PVDF membrane system (a) and an ELISA-based diagnosis (b). The chimeric nanoparticles and PVDF membrane system unambiguously detected troponin I in all patients, but the ELISA-based diagnosis failed to detect three patients (nos 4, 9, 18) and revealed nine ambiguous signals close to the clinical cutoff signal (horizontal dotted line). c, Assays with antibodies attached to chimeric nanoparticles, but not those immobilized directly on PVDF membranes, could detect troponin I in a sample diluted 1,000 times. 74/M and 75/F represent the age and gender of the AMI patients. NATURE NANOTECHNOLOGY | VOL 4 | APRIL 2009 | www.nature.com/naturenanotechnology © 2009 Macmillan Publishers Limited. All rights reserved.

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Fitzgerald) was added to the chimeric protein nanoparticles that were already immobilized onto the PVDF membrane, by slowly stirring the membrane in the antibody-containing PBS buffer for 2 h. Detection of troponin I and diagnosis of AMI patients. To the three-dimensional diagnostic system consisting of anti-troponin I antibodies, HBV capsid-derived chimeric nanoparticles, and nickel nanohair structure (or PVDF membrane), 200 ml troponin (human cardiac troponin I-T-C complex, cat. no. 8T62, HyTest) that had been properly diluted in PBS buffer, AMI patient or healthy serum was added, then stirred for 20 s, and incubated at room temperature for 1 h. After washing for 5 min using PBS buffer, mouse anti-troponin I monoclonal antibodies (200 ml, 3.2 mg ml21; cat. no. 4T21, HyTest) in PBS buffer was added, stirred for 20 s, incubated at room temperature for 1 h, and then washed twice for 5 min using PBS buffer. Q-dot(CdSe)-secondary Ab conjugate (200 ml, 1 nM, Qdotw 655-Goat F(ab0 )2 anti-mouse IgG conjugate, cat. no. Q11021MP, Invitrogen) was added, stirred for 20 s, incubated for 1 h, and finally washed twice for 10 min with PBS buffer. Photoluminescence was then measured using a microplate reader (GENios, Tecan) with excitation and emission at 420 and 650 nm, respectively. All the ELISA assay experiments in this study were conducted using the commercial ELISA troponin assay kit (Troponin I EIA, cat. no. 25-TR1HU-E01, 96 wells, ALPCO Diagnostics) that was developed for in vitro diagnostic use. According to the enclosed protocol, Troponin I EIA provides a reliable assay for the quantitative measurement of human cardiac-specific troponin I with clinical specificity of 87.5%. The procedure described in the Troponin I EIA protocol was strictly followed for the troponin I assay, and the summarized assay procedure can be found in the Supplementary Methods. The entire list of AMI patient and healthy sera can be found in Supplementary Table S1.

Received 11 September 2008; accepted 7 February 2009; published online 8 March 2009 References 1. Adams, J. E. et al. Cardiac troponin I. A marker with high specificity for cardiac injury. Circulation 88, 101–106 (1993). 2. Adams, J. E., Schechtman, K. B., Landt, Y., Ladenson, J. H. & Jaffe, A. S. Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I. Clin. Chem. 40, 1291–1295 (1994). 3. Thygesen, K., Alpert, J. S. & White, H. D. Universal definition of myocardial infarction. J. Am. Coll. Cardiol. 50, 2173–2195 (2007). 4. Morrow, D. A. et al. National Academy of Clinical Biochemistry Laboratory Medicine practice guidelines: clinical characteristics and utilization of biochemical markers in acute coronary syndromes. Clin. Chem. 53, 552–574 (2007). 5. Gibler, W. B. et al. Practical implementation of the guidelines for unstable angina/non-ST-segment elevation myocardial infarction in the emergency department. Ann. Emerg. Med. 46, 185–197 (2005). 6. Antman, E. M. et al. Cardiac-specific troponin I levels to predict the risk of mortality in patients with acute coronary syndromes. N. Engl. J. Med. 335, 1342–1349 (1996). 7. Wu, A. H. B. & Jaffe, A. S. The clinical need for high-sensitivity cardiac troponin assays for acute coronary syndromes and the role for serial testing. Am. Heart J. 155, 208–214 (2008). 8. Benamer, H. et al. Elevated cardiac troponin I predicts a high-risk angiographic anatomy of the culprit lesion in unstable angina. Am. Heart J. 137, 815–820 (1999). 9. Heeschen, C., van den Brand, M. J., Hamm, C. W. & Simoons, M. L. Angiographic findings in patients with refractory unstable angina according to troponin T status. Circulation 100, 1509–1514 (1999). 10. Wong, G. C. et al. Elevations in troponin T and I are associated with abnormal tissue level perfusion: A TACTICS-TIMI 18 substudy. Circulation 106, 202–207 (2002). 11. Rosi, N. L. & Mirkin, C. A. Nanostructures in biodiagnostics. Chem. Rev. 105, 1547–1562 (2005). 12. Hirsch, L. R., Jackson, J. B., Lee, A., Halas, N. J. & West, J. A whole blood immunoassay using gold nanoshells. Anal. Chem. 75, 2377–2381 (2003). 13. Nam, J. M., Park, S. J. & Mirkin, C. A. Bio-barcodes based on oligonucleotidemodified nanoparticles. J. Am. Chem. Soc. 124, 3820–3821 (2002).

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DOI: 10.1038/NNANO.2009.38

14. Niemeyer, C. M. & Ceyhan, B. DNA-directed functionalization of colloidal gold with proteins. Angew. Chem. Int. Ed. 40, 3685–3688 (2001). 15. Chien, R. J. et al. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Natl Acad. Sci. 100, 4984–4989 (2003). 16. Wang, J., Polsky, R., Merkoci, A. & Turner, K. L. DNA-based amplified bioelectronic detection and coding of proteins. Angew. Chem. Int. Ed. 43, 2158–2161 (2004). 17. Rowe, C. A. et al. Array biosensor for simultaneous identification of bacterial, viral and protein analytes. Anal. Chem. 71, 3846–3852 (1999). 18. Gouda, H. et al. NMR study of the interaction between the B domain of staphylococcal protein A and the Fc portion of immunoglobulin G. Biochemistry 37, 129–136 (1998). 19. Deisenhofer, J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus ˚ . resolution. Biochemistry 20, 2361–2370 (1981). aureus at 2.9- and 2.8-A 20. Bo¨ttcher, B., Wynne, S. A. & Crowther, R. A. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386, 88–91 (1997). 21. Crowther, R. A. et al. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 77, 943–950 (1994). 22. Fischlechner, M. & Donath, E. Viruses as building blocks for materials and devices. Angew Chem. Int. Ed. 46, 3184–3193 (2007). 23. Toellner, L., Fischlechner, M., Ferko, B., Grabherr, R. M. & Donath, E. Virus-coated layer-by-layer colloids as a multiplex suspension array for the detection and quantification of virus-specific antibodies. Clin. Chem. 52, 1575–1583 (2006). 24. Yang, L. M. C. et al. Virus electrodes for universal biodetection. Anal. Chem. 78, 3265–3270 (2006). 25. Willis, S. et al. Virus-like particles as quantitative probes of membrane protein interactions. Biochemistry 47, 6988–6990 (2008). 26. Datta, M. et al. High relaxivity gadolinium hydroxypyridonate-viral capsid conjugates: nanosized MRI contrast agents. J. Am. Chem. Soc. 130, 2546–2552 (2008). 27. Fischlechner, M. et al. Fusion of enveloped virus nanoparticles with polyelectrolyte-supported lipid membranes for the design of bio/nonbio interfaces. Nano Lett. 7, 3540–3546 (2007). 28. Apple, F. S., Smith, S. W., Pearce, L. A., Ler, R. & Murakami, M. M. Use of the centaur TnI-ultra assay for detection of myocardial infarction and adverse events in patients presenting with symptoms suggestive of acute coronary syndrome. Clin. Chem. 54, 723–728 (2008). 29. Ahn, J. Y. et al. Heterologous gene expression using self-assembled supramolecules with high affinity for HSP70 chaperone. Nucl. Acids Res. 33, 3751–3762 (2005).

Acknowledgements This study was supported by the National Research Laboratory Project (a main project that supported this work) of the Ministry of Education, Science and Technology (grant no. ROA-2007-000-20084-0), the Korea Health 21 R&D Project of the Ministry of Health, Welfare and Family Affairs of the Republic of Korea (grant no. A050750), the Pioneer Research Center Program (grant no. M10711160001-08M1116-00110), the Microbial Genomics and Applications Center at KRIBB, the Seoul R&BD program (no. 10920), and the Korea Research Foundation (grant no. KRF-2006-005-J03603).

Author contributions J.L. conceived the viral particle-based assay experiments, conducted the data analysis and wrote the paper. Y.K.K. conceived the nanohair concept, conducted the data analysis and co-wrote the paper. J.S.P. and E.J.L. performed the viral particle synthesis and assay experiments and collected the data. M.K.C. performed the nanohair synthesis and SEM analysis. J.S.P., E.J.L. and M.K.C. contributed equally to this work. K.Y.A. analysed assay data. K.E.L. and S.-S.H. contributed TEM analysis tools. J.H.J. and Y.C. contributed human sera for diagnostic assay.

Additional information Supplementary Information accompanies this paper at www.nature.com/ naturenanotechnology. Reprints and permission information is available online at http://npg. nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to J.L. and Y.K.K.
