Four bead sizes (1.75, 3, 4.5, and 6 m) and three different fluorescent colors (blue, green, and yellow) are utilized to encode 12 proteins (Determine 1A). markers. An enclosed 60 pL-sized microchamber defines a Rabbit Polyclonal to PIK3CG high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells. Circulating tumor cells (CTCs) are tumor cells that shed from tumor sites and enter into blood circulation. CTCs symbolize the molecular characteristics of the tumor and are generally believed a real-time liquid biopsy for malignancy patients. 1 CTCs are extraordinarily rare in a few milliliter LTX-315 of blood samples, down to a single cell, requiring single-cell LTX-315 resolution for molecular analysis. Genetic and transcriptional profiling of single CTCs has been reported.2C4 However, techniques for quantitatively profiling multiple proteins (e.g., organ-specific markers, proliferative markers, drug targets, and drug resistance-associated markers) with a single CTC resolution have not yet been achieved.1 The most common method is the immunofluorescence staining in which cells are fixed and stained with fluorescent antibodies, but is usually limited to 3C4 membrane and intracellular proteins.5 Fluorescent flow cytometry is the dominant workhorse in the field of single-cell protein quantification, featured with high throughput and high level of multiplexing, but is limited by inability to deal with a very low quantity of cells.6,7 Love and co-workers have developed the microengraving technique in which a large array of microwells isolate individual cells and quantify three secreted proteins by antibodies immobilized in the microwells.8C10 Therefore, it is greatly needed to develop a technology for single-cell, multiplexed detection of membrane and intracellular proteins in isolated CTC population that has a very small quantity of cells and also contains contaminating leukocytes. In the past five years, we have developed a microfluidics-based single cell barcode chip (SCBC) technology to simultaneously detect a dozen of functional proteins released from a single cell, including secreted, membrane, and intracellular proteins.11,12 The SCBC platform utilizes a large number of 1 nL sized microchambers to isolate single cells for protein measurement and a miniaturized, spatially encoded antibody microarray that enables multiplexed protein detection. A theoretical model was also developed to establish a new signaling network model that promoted understanding of the regulation mechanism based on single-cell proteomic data obtained from the SCBC platform.13 Further, Fan et al. altered the spatially encoded antibody microarray with multiplex fluorescence labeling and then successfully achieved codetection of 45 secreted proteins at the single cell level.14 However, for functional proteins with low copy figures, a 1 nL sized microchamber defines a low concentration of proteins that are released from single cells upon lysis, leading to difficulty in reliable protein detection. For this reason, a reduction of microchamber size from 1 nL to 60 pL prospects to an increase of protein concentration by 16 and a decrease of protein diffusion LTX-315 time that greatly enhance the limit of detection of low abundant functional proteins. However, a 60 pL sized microchamber cannot accommodate the spatially encoded antibody barcode that we developed before, requiring a new approach to create miniaturized antibody microarray with enhanced density. In this study, a beads-on-barcode antibody microarray (BOBarray) has been created to accomplish multiplexed protein detection by assigning two impartial identifiers (bead size and fluorescent color) to each protein. Four bead sizes (1.75, 3, 4.5, and 6 m) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL sized microchambers that isolate single cells for cell lysis and the subsequent detection of proteins, including organ-specific markers and drug targets. These protein markers help to characterize the organ-of-origin and drug targets of isolated CTCs from peripheral blood samples of malignancy patients. The number of 60 pL sized microwells around the microchip for protein.
Four bead sizes (1
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