-
[show abstract]
[hide abstract]
ABSTRACT: We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein-protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0-5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein-protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.
Proceedings of the National Academy of Sciences 12/2011; 109(2):419-24. · 9.68 Impact Factor
-
Chao Ma, Rong Fan,
Habib Ahmad,
Qihui Shi,
Begonya Comin-Anduix,
Thinle Chodon,
Richard C Koya,
Chao-Chao Liu,
Gabriel A Kwong,
Caius G Radu,
Antoni Ribas,
James R Heath
[show abstract]
[hide abstract]
ABSTRACT: Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (∼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.
Nature medicine 06/2011; 17(6):738-43. · 27.14 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network.
Biophysical Journal 05/2011; 100(10):2378-86. · 3.65 Impact Factor
-
ChemPhysChem 10/2010; 11(14):3063-9. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Electrolyte transport through an array of 20 nm wide, 20 microm long SiO(2) nanofluidic transistors is described. At sufficiently low ionic strength, the Debye screening length exceeds the channel width, and ion transport is limited by the negatively charged channel surfaces. At source-drain biases >5 V, the current exhibits a sharp, nonlinear increase, with a 20-50-fold conductance enhancement. This behavior is attributed to a breakdown of the zero-slip condition. Implications for energy conversion devices are discussed.
Nano Letters 04/2009; 9(4):1315-9. · 13.20 Impact Factor
-
Rong Fan,
Ophir Vermesh,
Alok Srivastava,
Brian K H Yen,
Lidong Qin,
Habib Ahmad,
Gabriel A Kwong,
Chao-Chao Liu,
Juliane Gould,
Leroy Hood,
James R Heath
[show abstract]
[hide abstract]
ABSTRACT: As the tissue that contains the largest representation of the human proteome, blood is the most important fluid for clinical diagnostics. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are routinely used in clinical tests. Reasons for this include the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases and the rapid degradation of proteins in sampled blood. We report an integrated microfluidic system, the integrated blood barcode chip that can sensitively sample a large panel of protein biomarkers over broad concentration ranges and within 10 min of sample collection. It enables on-chip blood separation and rapid measurement of a panel of plasma proteins from quantities of whole blood as small as those obtained by a finger prick. Our device holds potential for inexpensive, noninvasive and informative clinical diagnoses, particularly in point-of-care settings.
Nature Biotechnology 12/2008; 26(12):1373-8. · 29.50 Impact Factor
