Donut-shaped chambers for analysis of biochemical processes at the cellular and subcellular levels.

In order to study cell-cell variation with respect to enzymatic activity, individual live cell analysis should be complemented by measurement of single cell content in a biomimetic environment on a cellular scale arrangement. This is a challenging endeavor due to the small volume of a single cell, the low number of target molecules and cell motility. Micro-arrayed donut-shaped chambers (DSCs) of femtoliter (fL), picoliter (pL), and nanoliter (nL) volumes have been developed and produced for the analysis of biochemical reaction at the molecular, cellular and multicellular levels, respectively. DSCs are micro-arrayed, miniature vessels, in which each chamber acts as an individual isolated reaction compartment. Individual live cells can settle in the pL and nL DSCs, share the same space and be monitored under the microscope in a noninvasive, time-resolved manner. Following cell lysis and chamber sealing, invasive kinetic measurement based on cell content is achieved for the same individual cells. The fL chambers are used for the analysis of the same enzyme reaction at the molecular level. The various DSCs were used in this proof-of-principle work to analyze the reaction of intracellular esterase in both primary and cell line immune cell populations. These unique DSC arrays are easy to manufacture and offer an inexpensive and simple operating system for biochemical reaction measurement of numerous single cells used in various practical applications.

[1]  V. Koshkin,et al.  Single-cell-kinetics approach to discover functionally distinct subpopulations within phenotypically uniform populations of cells. , 2013, Analytical chemistry.

[2]  A. Schmid,et al.  Single-cell analysis in biotechnology, systems biology, and biocatalysis. , 2012, Annual review of chemical and biomolecular engineering.

[3]  P S Dittrich,et al.  Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells. , 2013, Analytical chemistry.

[4]  A. Woolley,et al.  Advances in microfluidic materials, functions, integration, and applications. , 2013, Chemical reviews.

[5]  Lloyd M Smith,et al.  Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. , 2010, Analytical chemistry.

[6]  R. Zare,et al.  Microfluidic platforms for single-cell analysis. , 2010, Annual review of biomedical engineering.

[7]  Q. Fang,et al.  Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay. , 2012, Analytical chemistry.

[8]  David R Walt,et al.  Analytical chemistry on the femtoliter scale. , 2010, Angewandte Chemie.

[9]  Luke P. Lee,et al.  Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays. , 2006, Analytical chemistry.

[10]  Tomoyuki Iwasawa,et al.  Single-cell chemical lysis method for analyses of intracellular molecules using an array of picoliter-scale microwells. , 2008, Analytical chemistry.

[11]  D. Marshall,et al.  Microfluidics for single cell analysis. , 2012, Current opinion in biotechnology.

[12]  N. Malys,et al.  What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase. , 2011, Biochemical and biophysical research communications.

[13]  Nancy L Allbritton,et al.  Continuous analysis of dye-loaded, single cells on a microfluidic chip. , 2011, Lab on a chip.

[14]  Andreas Schmid,et al.  Chemical and biological single cell analysis. , 2010, Current opinion in biotechnology.

[15]  Yusi Fu,et al.  Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors. , 2012, Analytical chemistry.

[16]  M. Magnani,et al.  Deciphering the single-cell omic: innovative application for translational medicine , 2012, Expert review of proteomics.

[17]  J Christopher Love,et al.  Massively parallel detection of gene expression in single cells using subnanolitre wells. , 2010, Lab on a chip.

[18]  N. Zurgil,et al.  Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device. , 2012, Analytical chemistry.

[19]  N. Zurgil,et al.  Polymer live-cell array for real-time kinetic imaging of immune cells. , 2010, Biomaterials.

[20]  Mehmet Toner,et al.  Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device. , 2004, Analytical chemistry.

[21]  N. Allbritton,et al.  Micro total analysis systems for cell biology and biochemical assays. , 2012, Analytical chemistry.

[22]  P. Masson,et al.  Structure, activities and biomedical applications of human butyrylcholinesterase. , 2009, Protein and peptide letters.

[23]  Helene Andersson-Svahn,et al.  Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays , 2010, PloS one.

[24]  D. Chiu,et al.  Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets. , 2005, Analytical chemistry.

[25]  Takehiko Kitamori,et al.  Microchip-based cellular biochemical systems for practical applications and fundamental research: from microfluidics to nanofluidics , 2011, Analytical and Bioanalytical Chemistry.

[26]  A. Minton,et al.  How can biochemical reactions within cells differ from those in test tubes? , 2006, Journal of Cell Science.

[27]  David A. Rand,et al.  Measurement of single-cell dynamics , 2010, Nature.

[28]  N. Friedman,et al.  Dynamic single-cell measurements of gene expression in primary lymphocytes: challenges, tools and prospects. , 2013, Briefings in functional genomics.

[29]  R. Voorman,et al.  Comparison of Skin Esterase Activities from Different Species , 2006, Pharmaceutical Research.

[30]  X. Zheng,et al.  Single cell analysis at the nanoscale. , 2012, Chemical Society reviews.

[31]  Subhajyoti De,et al.  Cellular crowding imposes global constraints on the chemistry and evolution of proteomes , 2012, Proceedings of the National Academy of Sciences.