Anal. Chem. 2008, 80, 583-587
Biologically Produced Bifunctional Recombinant Protein Nanoparticles for Immunoassays Anu Ja 1a 1 skela 1 inen,†,‡ Reija-Riitta Harinen,†,§ Tero Soukka,† Urpo Lamminma 1 ki,† †,| ,⊥ Teemu Korpima 1 ki, and Marko Virta*
Department of Biochemistry and Food Chemistry, University of Turku, 20014 Turku, Finland, and Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland
Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and selfassembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles. Nanoparticles have been developed for an increasing number of applications. Various luminescent/fluorescent/electrochemical micro- and nanosize particles are employed as labels in different detection technologies.1,2 In particular, quantum dots3,4 and lanthanide-containing particles utilizing time-resolved fluorometry * Corresponding author. Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland. Phone, +358919157586; fax, +35819159322; e-mail,
[email protected]. † University of Turku. ‡ Current address: Innotrac Diagnostics, Biolinja 12, FI-20750 Turku, Finland. § Current address: Thermo Fisher Scientific, Ratastie 2, FI-01621 Vantaa, Finland. | Current address: Raisio Diagnostics Oy, Joukahaisenkatu 1, FI-20520 Turku, Finland. ⊥ University of Helsinki. (1) Schultz, D. A. Curr. Opin. Biotechnol. 2003, 14, 13-22. (2) Chan, W. C.; Nie, S. Science 1998, 281, 2016-2018. (3) Marks, K. M.; Nolan, G. P. Nat. Methods 2006, 3, 591-596. (4) Riegler, J.; Nann, T. Anal. Bioanal. Chem. 2004, 379, 913-919. 10.1021/ac071382v CCC: $40.75 Published on Web 01/08/2008
© 2008 American Chemical Society
(TRF)5 have been explored in biological sample analysis.6,7 In general, nanoparticles offer superior specific activity over the conventional labels.8 Functionalization of nanoparticles for use in applications such as bioaffinity assays usually entails multiple distinct production and purification steps, each requiring quality control. Particles are usually synthesized using for example organic or inorganic procedures. When separate label molecules or ions are needed they are introduced either during particle synthesis or afterward. In the case of especially inorganic particles, surfaces must be modified to ensure biocompatibility and water solubility9,10 and usually to enable conjugation of biological binding molecules to the nanoparticles.11 Biocompatibility is an important issue for in vitro diagnostics because it improves the solubility of particulate reagents and minimizes both particle aggregation and nonspecific interactions between sample components and assay reagents. Binding molecules, such as antibodies, must also be separately produced, typically by means of biological processes such as cell culture (eukaryotic or prokaryotic) or animal immunization, followed by isolation and purification. Bioconjugation to nanoparticles is performed actively (i.e., by forming covalent bonds via chemical reactions) or passively (i.e., hydrophobic and/or electrostatic adsorption), after which the nonbound excess binding molecules are separated from the particles. Conjugation often occurs in a random orientation, resulting in considerable loss of binding capacity. Overall, identical and stably functionalized particles are often difficult and expensive to produce. Economy and straightforward production are important, particularly for applications that require large quantities of homogeneous functionalized particles. Consequently, further studies are required to identify novel nanomaterials for use as particles and alternative cost-effective ways of production. The utilization of protein-based particles as alternative particle shells solves some of the issues related to the manufacturing (5) Hemmila, I.; Mukkala, V. M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441-519. (6) Huhtinen, P.; Kivela¨, M.; Kuronen, O.; Hagren, V.; Takalo, H.; Tenhu, H.; Lo ¨vgren, T.; Ha¨rma¨, H. Anal. Chem. 2005, 77, 2643-2648. (7) Ha¨rma¨, H.; Soukka, T.; Lo ¨vgren, T. Clin. Chem. 2001, 47, 561-568. (8) Soukka, T.; Harma, H.; Paukkunen, J.; Lo¨vgren, T. Anal. Chem. 2001, 73, 2254-2260. (9) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (10) Zheng, M.; Huang, X. Y. J. Am. Chem. Soc. 2004, 12, 12047-12054. (11) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158.
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process and biocompatibility.12,13 A versatile, globular protein, apoferritin, has long been a focus of interest due to its unique ability to take up various ions and molecules.14,15 This property of apoferritin has also been applied to successfully produce particles that can be used as labels in bioaffinity assays for both nucleic acids16 and proteins.17,18 Recently, we presented an alternative, simple method of generating functional ferritin-based nanoparticles for use as label agents in a bioaffinity assay.19 The theory is to replace chemical bioconjugation of the particle and binding molecules with genetic fusion. The functionalized nanoparticles (comprising the entity of the particle shell and binding moieties) are synthesized biologically from generic raw materials by microbial cells. Production is combined with a simple purification method during which label activity is introduced. The process results in particles with inherent binding activity that are readyto-use label agents for bioaffinity assays. Apoferritin, referred to as “ferritin”, is a 24-meric protein, with the 24 amino terminal ends of the monomers located on the surface of the protein shell. Biological binding molecules can be attached stably (covalently) in the correct orientation to these ends by fusion of the genes encoding human ferritin and the binding molecule. Fusion polypeptides are overproduced in microbes as insoluble forms (inclusion bodies)20 that are easily isolated and purified from the rest of the biomass by generic nonchromatographic procedures. These inclusion bodies are solubilized at low pH, after which the unfolded polypeptide chains are allowed to self-assemble to form particles with binding molecules protruding from the surface. Eu3+ ions are passively introduced into the particles during self-assembly. Europium ions are used as the label moiety, since they can be detected sensitively by time-resolved fluorometry upon chelation with the appropriate agents.21 The method for preparing ferritin particles is directly compatible with the use of other marker molecules detected with different technologies (i.e., fluorescence, electrochemical, and magnetic resonance imaging), instead of europium.15,18,22,23 Apart from the straightforward production system, the biological particles described here have several properties that support their efficiency as label agents in bioaffinity assays: (1) binding molecules are favorably oriented on the surface (binding sites are exposed to the solution), (2) particles have inherent binding activity, water solubility, and likely biocompatibility, (3) conjugation of the binding molecule by genetic fusion is stable as the binding moiety is covalently connected to the particle, (4) ferritin (12) Ceyhan, B.; Alhorn, P.; Lang, C.; Schuler, D.; Niemeyer, C. M. Small 2006, 2, 1251-1255. (13) Soto, C. M.; Blum, A. S.; Vora, G. J.; Lebedev, N.; Meador, C. E.; Won, A. P.; Chatterji, A.; Johnson, J. E.; Ratna, B. R. J. Am. Chem. Soc. 2006, 128, 5184-5189. (14) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (15) Aime, S.; Frullano, L.; Geninatti, C. S. Angew. Chem., Int. Ed. 2002, 41, 1017-1021. (16) Liu, G.; Wang, J.; Lea, S. A.; Lin, Y. ChemBioChem. 2006, 7, 1315-1319. (17) Liu, G.; Wu, H.; Wang, J.; Lin, Y. Small 2006, 2, 1139-1143. (18) Liu, G.; Wang, J.; Wu, H.; Lin, Y. Anal. Chem. 2006, 78, 7417-7423. (19) Ja¨a¨skela¨inen, A.; Harinen, R.-R.; Soukka, T.; Lamminma¨ki, U.; Korpima¨ki, T.; Pelliniemi, L. J.; Virta, M. Small 2007, 3, 1362-1367. (20) Baneyx, F.; Mujacic, M. Nat. Biotechnol. 2004, 22, 1399-1408. (21) Hemmila, I.; Mukkala, V. M. Crit. Rev. Clin. Lab. Sci. 2001, 38, 441-519. (22) Liu, G.; Wu, H.; Dohnalkova, A.; Lin, Y. Anal. Chem. 2007, 79, 5614-5619. (23) Wong, K. K. W.; Douglas, T.; Gider, S.; Awschalom, D. D.; Mann, S. Chem. Mater. 1998, 10, 279-285.
584 Analytical Chemistry, Vol. 80, No. 3, February 1, 2008
Figure 1. Scheme of functionalized nanoparticles based on the globular protein shell of ferritin that contain Eu3+ ions as the label. RTSH-ferritin particles display single chain Fv (scFv) of an antibody against thyroid stimulating hormone, TSH, on the surface, enabling specific binding to the analyte (TSH).
is a stable protein particle body,16,19,24 and (5) particles are small (
