2627 Pure Appl. Chem., Vol. 80, No. 12, pp. 2627–2641, 2008. doi:10.1351/pac200880122627 © 2008 IUPAC Flow cytometer with mass spectrometer detection for massively multiplexed single-cell biomarker assay* Scott D. Tanner ‡ , Dmitry R. Bandura, Olga Ornatsky, Vladimir I. Baranov, Mark Nitz, and M. A. Winnik Department of Chemistry, University of Toronto, 80 St. George Street, Room 18, Toronto, Ontario M5S 3H6, Canada Abstract: This paper describes the development and application of new metal-tagging reagents and a novel mass spectrometer (MS) detector for a flow cytometer that enables highly multiplexed measurement of many biomarkers in individual cells. A new class of tag- ging reagents, based on an acrylic polymer backbone that incorporates a reproducible num- ber of lanthanide elements, has been developed. When linked to antibodies that specifically recognize target proteins of interest, determination of the tag elements is diagnostic for the presence and quantification of the antigen. The use of enriched stable isotope tags provides the opportunity for multiparametric assay. The new instrument uses inductively coupled plasma (ICP) to vaporize, atomize, and ionize individual cells that have been probed using the metal-labeled antibodies. The elemental composition, specifically of the metal tags, is recorded simultaneously using a time-of-flight (TOF)-MS that has been specifically designed for high-speed analysis during the short transient corresponding to the individual cell event. The detector provides for well-resolved atomic fingerprints of many elemental and isotopic tags, with little overlap of neighboring signals (high abundance sensitivity) and wide dy- namic range both for a single antigen and between antigens. Keywords: flow cytometer; ICP-MS; metal tags; leukemia cell lines; multiparametric analy- sis; element-tagged immunoassay. INTRODUCTION Astonishing advances in the understanding, and sometimes treatment, of diseases have been enabled with the application of biotechnologies that were developed to advance the Human Genome Project and its postgenome fall-out. It is becoming increasingly apparent that delineation of cell signaling pathways and the genesis of disease states require the simultaneous knowledge of the many genes and proteins that are the machinery of the cell. Flow cytometry (FC) is an analytical art that recognizes a limited number (typically 4, though as many as 17 have been reported [1] with heroic effort) of proteins in single cells at high throughput (typ- ically 600, but up to 30 000, cells per second). The method requires the availability of a suite of non- cross-reactive antibodies that are specific for each target antigen, and these antibodies are tagged with *Paper based on a presentation at the International Symposium on Metallomics 2007 (ISM 2007), 28 November–1 December 2007, Nagoya, Japan. Other presentations are published in this issue, pp. 2565–2750. ‡ Corresponding author: Tel.: 1-416-946-8850; E-mail: sd.tanner@utoronto.ca