ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 334 (2004) 135–144 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.08.005 Ultrasensitive detection of biomolecules with Xuorescent dye-doped nanoparticles Wei Lian a , Sally A. Litherland b , Hassan Badrane a , Weihong Tan c , Donghai Wu d , Henry V. Baker a , Paul A. Gulig a , Daniel V. Lim e , Shouguang Jin a,¤ a Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA b Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA c Department of Chemistry, University of Florida, Gainesville, FL 32610, USA d Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China e Department of Biology and Center for Biological Defense, University of South Florida, Tampa, FL 33620, USA Received 11 May 2004 Abstract Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the Xuorescent dye tris(2'2-bipyridyl)dichlo- roruthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of Xuorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various Xuorescence labeling techniques, includ- ing Xuorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein micro- array. By combining the high-intensity luminescent nanoparticles with the speciWcity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic Xuorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed Xuorescent nanoparticles in various biodetection systems.  2004 Elsevier Inc. All rights reserved. Fashionable Xuorescence techniques have changed the face of today’s science and technology. Fluorescent labeling techniques have been used extensively in both biological research and clinical diagnosis. To achieve sensitive detections, there is an increasing demand for Xuorescent labeling probes that are more intense and stable. The traditional Xuorophores such as FITC 1 are not photostable in addition to the problem of relatively low Xuorescence intensity [1–3]. Although the new gener- ation of Xuorophores such as the Alexa series have dramatically increased the labeling eYciencies, the molecular nature of the dyes determines the limitations in Xuorescence intensity and photostability. Because of the advanced computational technologies and the diYcult * Corresponding author: Fax: +1 352 392 3133. E-mail address: sjin@mgm.uX.edu (S. Jin). 1 Abbreviations used: FITC, Xuorescein isothiocyanate; FLISA, Xuorescent-linked immunosorbent assay; TEOS, tetraethylorthosilicate; NP, nanoparticle; NHS, N-hydroxysuccinimide; DETA, trimethoxysilylpropyldiethylenetriamine; PE, phycoerythrin; SEM, scanning electron micro- scope; TEM, transmission electron microscope; BSA, bovine serum albumin; PBS, phosphate-buVered saline; PBMC, peripheral blood mononuclear cells; DAPI, 4',6-diamido-2-phenylindole dihydrochloride; ChAT, choline acetyltransferase; F.I., Xuorescence intensity, QD, quantom dots; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; Ag, antigen.