Nanoencapsulated Microcrystalline Particles for Superamplified Biochemical Assays Dieter Trau,* ,†,‡ Wenjun Yang, † Matthias Seydack, § Frank Caruso, | Nai-Teng Yu, † and Reinhard Renneberg † Department of Chemistry and Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, SAR Hong Kong, 8sens.biognosticAG, Robert Roessle-Strasse 10, D-13125 Berlin, Germany, and Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany We report on the preparation and utilization of a novel class of particulate labels based on nanoencapsulated organic microcrystals with the potential to create highly amplified biochemical assays. Labels were constructed by encapsulating microcrystalline fluorescein diacetate (FDA; average size of 5 0 0 nm) within ultrathin polyelectrolyte layers of poly(allylamine hydrochloride) and poly(sodium 4 -styrenesulfonate) via the layer-by-layer technique. Sub- sequently, the polyelectrolyte coating was used as an “interface” for the attachment of anti-mouse antibodies through adsorption. A high molar ratio of fluorescent molecules present in the microcrystal core to biomol- ecules on the particle surface was achieved. The ap- plicability of the microcrystal-based label system was demonstrated in a model sandwich immunoassay for mouse immunoglobulin G detection. Following the im- munoreaction, the FDA core was dissolved by exposure to organic solvent, leading to the release of the FDA molecules into the surrounding medium. Amplification rates of 7 0 -2 0 0 0 -fold (expressed as an increase in assay sensitivity) of the microcrystal label-based assay com- pared with the corresponding immunoassay performed with direct fluorescently labeled antibodies are reported. Our approach provides a general and facile means to prepare a novel class of biochemical assay labeling systems. The technology has the potential to compete with enzyme-based labels as it does not require long incubation times, thus speeding up bioaffinity tests. Biochemical assays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoagglutination assays (IAA), fluorescent immunoassays (FIA), and those based on DNA hybridization or receptor ligand interactions play an important role in medical diagnostics, food, and environmental analyses. 1,2 A common feature of most assay techniques is that they require a label to detect the interaction of a biocompound with an analyte. The basic requirements for a biolabel system are high sensitivity, low limit of detection, and preserved biomolecule functionality. In the present trend of applying nonradioactive labeling strategies, fluorescent molecules and enzymes are widely employed as labels. Both strategies offer good sensitivity and limits of detection. These parameters can be improved by increasing the ratio of fluorescent dyes to biomolecules (i.e., the F/ P ratio) for fluorescent-based label techniques and by increasing the substrate incubation time for enzyme-based label systems. The F/ P ratio is a key parameter that has an impact on sensitivity and is widely used for evaluation of a fluorescent label in biochemical assay technologiessa high number of labeling molecules per biomolecule is desired. For example, the F/ P value is typically 4-8 for a conventional covalently coupled fluorescent immunolabel, e.g., an immunoglobulin G (IgG) -FITC conjugate. Since conjugation of the fluorescent molecules to the biomolecules is typically achieved by covalent binding, the fluorescent probes used in FIAs should possess a reactive group for bioconjugation, high water solubility, low nonspecific adsorption, and good photostability. 3 Additionally, they should have a high molar extinction coefficient and fluorescence quantum yield. However, covalent binding of a large number of label molecules usually leads to a decrease of specific binding activity of the biomolecule and the quantum yield due to self-quenching effects. Antibodies labeled with more than four to six fluorophores per protein may also exhibit reduced specificity and binding affinity. 4 To prevent such complications, the optimal dye/ protein ratio is normally kept below ∼4, which highly limits the potential sensitivity improvement of FIAs. 5 These aforementioned demands placed on the performance of fluorescent probes vastly limit the number of suitable candi- dates. The sensitivity of fluorescence assays is determined by the number of light quanta emitted per analyte molecule. Increasing the dye/ biomolecule ratio while minimizing dye self-quenching to obtain signal amplification (and hence sensitivity) is highly * To whom correspondence should be addressed. E-mail: trau@ ust.hk. Fax: 852-3106-4857. † Department of Chemistry, The Hong Kong University of Science and Technology. ‡ Bioengineering Graduate Program, The Hong Kong University of Science and Technology. § 8sens.biognosticAG. | Max Planck Institute of Colloids and Interfaces. (1) Tijssen, P. Practice and theory of enzyme immunoassays, 8th ed.; Elsevier: New York, 1985. (2) Kessler, C. Nonradioactive Labeling and Detection of Biomolecules; Springer- Verlag: New York, 1992. (3) Hemmila, I. A. Applications of fluorescence in immunoassays; J. Wiley & Sons: New York, 1991. (4) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene OR, 1996. (5) Johnson, G. D. Antibodies. A Practical Approach; IRL: Oxford, U.K., 1989. Anal. Chem. 2002, 74, 5480-5486 5480 Analytical Chemistry, Vol. 74, No. 21, November 1, 2002 10.1021/ac0200522 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/25/2002