Browsing by Author "Takayama, Shuichi"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA
    (Springer Nature, 2014-05-02) Frampton, John P.; White, Joshua B.; Simon, Arlyne B.; Tsuei, Michael; Paczesny, Sophie; Takayama, Shuichi; Pediatrics, School of Medicine
    Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.
  • Thumbnail Image
    Item
    Aqueous two-phase systems enable multiplexing of homogeneous immunoassays
    (World Scientific, 2014) Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi; Pediatrics, School of Medicine
    Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD).
  • Thumbnail Image
    Item
    Preprogrammed capillarity to passively control system-level sequential and parallel microfluidic flows
    (Royal Society of Chemistry, 2013) Kim, Sung-Jin; Paczesny, Sophie; Takayama, Shuichi; Kurabayash, Katsuo; Pediatrics, School of Medicine
    In microfluidics, capillarity-driven solution flow is often beneficial, owing to its inherently spontaneous motion. However, it is commonly perceived that, in an integrated microfluidic system, the passive capillarity control alone can hardly achieve well-controlled sequential and parallel flow of multiple solutions. Despite this common notion, we hereby demonstrate system-level sequential and parallel microfluidic flow processing by fully passive capillarity-driven control. After manual loading of solutions with a pipette, a network of microfluidic channels passively regulates the flow timing of the multiple solution menisci in a sequential and synchronous manner. Also, use of auxiliary channels and preprogramming of inlet-well meniscus pressure and channel fluidic conductance allow for controlling the flow direction of multiple solutions in our microfluidic system. With those components orchestrated in a single device chip, we show preprogrammed flow control of 10 solutions. The demonstrated system-level flow control proves capillarity as a useful means even for sophisticated microfluidic processing without any actively controlled valves and pumps.
  • Thumbnail Image
    Item
    Preprogrammed, Parallel On-Chip Immunoassay Using System-Level Capillarity Control
    (ACS, 2013) Kim, Sung-Jin; Paczesny, Sophie; Takayama, Shuichi; Kurabayashi, Katsuo; Pediatrics, School of Medicine
    Fully manual use of conventional multiwell plates makes enzyme-linked immunosorbent assay (ELISA)-based immunoassays highly time-consuming and labor-intensive. Here, we present a capillarity-driven on-chip immunoassay that greatly saves time and labor with an inexpensive setup. Our immunoassay process starts with pipetting multiple solutions into multiwells constructed on a microfluidic device chip. Subsequently, capillarity spontaneously transports multiple sample solutions and common reagent solutions into assigned detection channels on the chip in a purely passive and preprogrammed manner. Our device implements capillarity-driven immunoassays involving four sample and six reagent solutions within 30 min by orchestrating the functions of on-chip passive components. Notably, our immunoassay technique reduces the total number of pipetting processes by ~5 times, as compared to assays on multiwell plates (48 vs 10). This assay technique allows us to quantify the concentrations of C-reactive protein and suppressor of tumorigenicity 2 with a detection limit of 8 and 90 pM, respectively. This device should be useful for sophisticated, parallel biochemical microfluidic processing in point-of-care settings under limited resources.