Analytical chemistry on the femtoliter scale.

The compartmentalization of reactions in femtoliter (fL) containers and integration of fL containers into arrays not only enhances and accelerates chemical and biochemical analysis but also leads to new scientific methods and insights. This review introduces various fL container and array formats and explores their applications for the detection and characterization of biologically relevant analytes. By loading analytes, sensing elements, or cells into fL arrays, one can perform thousands of analytical measurements in parallel. Confining single enzyme molecules in fL arrays enables one to analyze large numbers of individual enzyme molecules simultaneously in solution. New nanofabrication techniques and progressively more sensitive detection methods drive the field of fL analytical chemistry. This review focuses on the progress and challenges in the field of fL analytical chemistry with examples of both basic and applied research.

[1]  D S Goodsell,et al.  Inside a living cell. , 1991, Trends in biochemical sciences.

[2]  D R Walt,et al.  Array-to-array transfer of an artificial nose classifier. , 2001, Analytical chemistry.

[3]  Helen Song,et al.  Reactions in droplets in microfluidic channels. , 2006, Angewandte Chemie.

[4]  David R Walt,et al.  Fiber-based single cell analysis of reporter gene expression in yeast two-hybrid systems. , 2007, Analytical biochemistry.

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

[6]  David G Spiller,et al.  Encoded microcarriers for high-throughput multiplexed detection. , 2006, Angewandte Chemie.

[7]  Christoph A. Merten,et al.  Drop-based microfluidic devices for encapsulation of single cells. , 2008, Lab on a chip.

[8]  P. Burke,et al.  Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin , 2009, Biotechnology progress.

[9]  Boris Rotman,et al.  MEASUREMENT OF ACTIVITY OF SINGLE MOLECULES OF β-D-GALACTOSIDASE , 1961 .

[10]  J.V. Sweedler,et al.  Nanofluidics: Systems and Applications , 2008, IEEE Sensors Journal.

[11]  Shinji Katsura,et al.  Single-molecule PCR using water-in-oil emulsion. , 2003, Journal of biotechnology.

[12]  David R Walt,et al.  Optical imaging fiber-based single live cell arrays: a high-density cell assay platform. , 2002, Analytical chemistry.

[13]  D. Chiu,et al.  Dynamic modulation of chemical concentration in an aqueous droplet. , 2007, Angewandte Chemie.

[14]  David R. Walt,et al.  Fiber-Optic Microarray for Simultaneous Detection of Multiple Harmful Algal Bloom Species , 2006, Applied and Environmental Microbiology.

[15]  S. Weiss,et al.  Single-molecule fluorescence studies of protein folding and conformational dynamics. , 2006, Chemical reviews.

[16]  D. Chiu,et al.  Droplets for ultrasmall-volume analysis. , 2009, Analytical chemistry.

[17]  E. Delamarche,et al.  Self-assembled microarrays of attoliter molecular vessels. , 2003, Angewandte Chemie.

[18]  Nico A J M Sommerdijk,et al.  A virus-based single-enzyme nanoreactor. , 2007, Nature nanotechnology.

[19]  C. G. Sunner.  Emulsions in theory and practice. , 1960 .

[20]  S. Turner,et al.  Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations , 2003, Science.

[21]  David R Walt,et al.  Detection of Salmonella spp. using microsphere-based, fiber-optic DNA microarrays. , 2005, Analytical chemistry.

[22]  C. Joo,et al.  Fueling protein–DNA interactions inside porous nanocontainers , 2007, Proceedings of the National Academy of Sciences.

[23]  Antoine M. van Oijen,et al.  Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited , 2006, Nature chemical biology.

[24]  Peng Chen,et al.  Single-Molecule Kinetic Theory of Heterogeneous and Enzyme Catalysis , 2009 .

[25]  Monpichar Srisa-Art,et al.  Microdroplets: a sea of applications? , 2008, Lab on a chip.

[26]  David R Walt,et al.  Simultaneously monitoring gene expression kinetics and genetic noise in single cells by optical well arrays. , 2004, Analytical chemistry.

[27]  E. Yeung,et al.  Single-molecule reactions in liposomes. , 2007, Angewandte Chemie.

[28]  Albert,et al.  High-speed fluorescence detection of explosives-like vapors , 2000, Analytical chemistry.

[29]  G. Haran,et al.  Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy , 2001 .

[30]  David R Walt,et al.  Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays. , 2006, Journal of the American Chemical Society.

[31]  D. Craig,et al.  Differences in the Average Single Molecule Activities of E. coli β-Galactosidase: Effect of Source, Enzyme Molecule Age and Temperature of Induction , 2003, Journal of protein chemistry.

[32]  Yonghao Zhang,et al.  Microfluidic DNA amplification--a review. , 2009, Analytica chimica acta.

[33]  J. Shendure,et al.  Materials and Methods Som Text Figs. S1 and S2 Tables S1 to S4 References Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome , 2022 .

[34]  Rithy K. Roth,et al.  Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays , 2000, Nature Biotechnology.

[35]  Lars Edman,et al.  The fluctuating enzyme: a single molecule approach , 1999 .

[36]  Andrew D Griffiths,et al.  Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis. , 2009, Analytical chemistry.

[37]  Zhaohui Li,et al.  Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels. , 2008, Journal of the American Chemical Society.

[38]  David R Walt,et al.  Design, implementation, and field testing of a portable fluorescence-based vapor sensor. , 2009, Analytical chemistry.

[39]  Daniel Bratton,et al.  An Integrated Device for Monitoring Time‐Dependent in vitro Expression From Single Genes in Picolitre Droplets , 2008, Chembiochem : a European journal of chemical biology.

[40]  David R Walt,et al.  Distinct and long-lived activity states of single enzyme molecules. , 2008, Journal of the American Chemical Society.

[41]  L. Hood,et al.  Systems medicine: the future of medical genomics and healthcare , 2009, Genome Medicine.

[42]  David R Walt,et al.  Multianalyte single-cell analysis with multiple cell lines using a fiber-optic array. , 2007, Analytical chemistry.

[43]  G. Whitesides,et al.  New approaches to nanofabrication: molding, printing, and other techniques. , 2005, Chemical reviews.

[44]  G. Whitesides,et al.  Formation of droplets and bubbles in a microfluidic T-junction-scaling and mechanism of break-up. , 2006, Lab on a chip.

[45]  David R Walt,et al.  Living bacterial cell array for genotoxin monitoring. , 2004, Analytical chemistry.

[46]  D. Weitz,et al.  Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. , 2009, Lab on a chip.

[47]  Horst Vogel,et al.  An integrated self-assembled nanofluidic system for controlled biological chemistries. , 2008, Angewandte Chemie.

[48]  D. S. Gill,et al.  Optical multibead arrays for simple and complex odor discrimination. , 2001, Analytical chemistry.

[49]  E. Yeung,et al.  Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes. , 1994, Analytical chemistry.

[50]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[51]  David R Walt,et al.  Optical‐fiber bundles , 2007, The FEBS journal.

[52]  Jérôme Bibette,et al.  Emulsions: basic principles , 1999 .

[53]  Thomas Hirsch,et al.  A simple strategy for preparation of sensor arrays: molecularly structured monolayers as recognition elements. , 2003, Chemical communications.

[54]  David R. Walt,et al.  Ordered nanowell arrays , 1996 .

[55]  Brian W Matthews,et al.  Crystallization of beta-galactosidase does not reduce the range of activity of individual molecules. , 2003, Biochemistry.

[56]  Edward S. Yeung,et al.  Differences in the chemical reactivity of individual molecules of an enzyme , 1995, Nature.

[57]  Norman J. Dovichi,et al.  STUDIES ON SINGLE ALKALINE PHOSPHATASE MOLECULES : REACTION RATE AND ACTIVATION ENERGY OF A REACTION CATALYZED BY A SINGLE MOLECULE AND THE EFFECT OF THERMAL DENATURATION : THE DEATH OF AN ENZYME , 1996 .

[58]  S. Turner,et al.  Real-Time DNA Sequencing from Single Polymerase Molecules , 2009, Science.

[59]  David R Walt,et al.  Fibre optic microarrays. , 2010, Chemical Society reviews.

[60]  Hans H. Gorris,et al.  Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies. , 2009, Journal of the American Chemical Society.

[61]  R. Ismagilov,et al.  Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability. , 2009, Angewandte Chemie.

[62]  R N Zare,et al.  Chemical transformations in individual ultrasmall biomimetic containers. , 1999, Science.

[63]  M. Márquez,et al.  Micro/Nano Encapsulation via Electrified Coaxial Liquid Jets , 2002, Science.

[64]  D R Walt,et al.  Application of high-density optical microwell arrays in a live-cell biosensing system. , 2000, Analytical biochemistry.

[65]  Seung-Yong Jung,et al.  Fast mixing and reaction initiation control of single-enzyme kinetics in confined volumes. , 2008, Langmuir : the ACS journal of surfaces and colloids.

[66]  David R. Walt,et al.  Bead-based Fiber-Optic Arrays , 2000, Science.

[67]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[68]  P. Luisi,et al.  Cell‐free Protein Synthesis through Solubilisate Exchange in Water/Oil Emulsion Compartments , 2004, Chembiochem : a European journal of chemical biology.

[69]  E. Cox,et al.  Real-Time Kinetics of Gene Activity in Individual Bacteria , 2005, Cell.

[70]  D. Chiu,et al.  Electro-generation of single femtoliter- and picoliter-volume aqueous droplets in microfluidic systems , 2005 .

[71]  Mircea Cotlet,et al.  Single-enzyme kinetics of CALB-catalyzed hydrolysis. , 2005, Angewandte Chemie.

[72]  Robert Wilson,et al.  Codierte Mikropartikel für Hochdurchsatz‐Mehrfachanalysen , 2006 .

[73]  Daniel T Chiu,et al.  Microfluidic and optical systems for the on-demand generation and manipulation of single femtoliter-volume aqueous droplets. , 2006, Analytical chemistry.

[74]  O. Wolfbeis,et al.  A spreader-bar approach to molecular architecture: formation of stable artificial chemoreceptors. , 1999, Angewandte Chemie.

[75]  David R Walt,et al.  Very high density sensing arrays. , 2008, Chemical reviews.

[76]  A. Grossman,et al.  Localization of bacterial DNA polymerase: evidence for a factory model of replication. , 1998, Science.

[77]  Trevor Douglas,et al.  Viruses: Making Friends with Old Foes , 2006, Science.

[78]  Hiroyuki Fujita,et al.  Highly coupled ATP synthesis by F1-ATPase single molecules , 2005, Nature.

[79]  D. Walt,et al.  Microsphere-based rolling circle amplification microarray for the detection of DNA and proteins in a single assay. , 2009, Analytical chemistry.

[80]  Nam Ki Lee,et al.  Single-molecule approach to molecular biology in living bacterial cells. , 2008, Annual review of biophysics.

[81]  Andrew D Griffiths,et al.  Amplification of complex gene libraries by emulsion PCR , 2006, Nature Methods.

[82]  Aldo Jesorka,et al.  Liposomes: technologies and analytical applications. , 2008, Annual review of analytical chemistry.

[83]  David R Walt,et al.  Duplexed sandwich immunoassays on a fiber-optic microarray. , 2006, Analytica chimica acta.

[84]  Charles C. Richardson,et al.  University of Groningen Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder , 2018 .

[85]  J. Kauer,et al.  Convergent, self-encoded bead sensor arrays in the design of an artificial nose. , 1999, Analytical chemistry.

[86]  Bill W Colston,et al.  High-throughput quantitative polymerase chain reaction in picoliter droplets. , 2008, Analytical chemistry.

[87]  Hiroyuki Fujita,et al.  Microfabricated arrays of femtoliter chambers allow single molecule enzymology , 2005, Nature Biotechnology.

[88]  X. Xie,et al.  Probing Gene Expression in Live Cells, One Protein Molecule at a Time , 2006, Science.

[89]  David R Walt,et al.  Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics. , 2006, Nano letters.

[90]  J. Elf,et al.  Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell , 2007, Science.

[91]  David M. Rissin,et al.  Stochastic inhibitor release and binding from single-enzyme molecules , 2007, Proceedings of the National Academy of Sciences.

[92]  A. Folch,et al.  Large-scale single-cell trapping and imaging using microwell arrays. , 2005, Analytical chemistry.

[93]  N. Dovichi,et al.  Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few beta-galactosidase molecules. , 1995, Analytical biochemistry.

[94]  Xiang-Dong Fu,et al.  Profiling alternative splicing on fiber-optic arrays , 2002, Nature Biotechnology.

[95]  O. Orwar,et al.  A nanofluidic switching device. , 2003, Journal of the American Chemical Society.

[96]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[97]  Monika Milewski,et al.  Decoding randomly ordered DNA arrays. , 2004, Genome research.

[98]  J. Brody,et al.  Single-molecule enzymology of chymotrypsin using water-in-oil emulsion. , 2005, Biophysical journal.

[99]  N. Dovichi,et al.  Escherichia coli β-galactosidase is heterogeneous with respect to the activity of individual molecules , 1998 .

[100]  Hiroyuki Kishi,et al.  Single-cell microarray for analyzing cellular response. , 2005, Analytical chemistry.

[101]  X. Xie,et al.  Single-molecule enzymatic dynamics. , 1998, Science.

[102]  Helen Song,et al.  Reaktionen in Mikrofluidiktröpfchen , 2006 .

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

[104]  X. Xie,et al.  Living Cells as Test Tubes , 2006, Science.

[105]  Manuel A Palacios,et al.  Polymer nanofibre junctions of attolitre volume serve as zeptomole-scale chemical reactors. , 2009, Nature chemistry.

[106]  E. Yeung,et al.  Monitoring the Reactions of Single Enzyme Molecules and Single Metal Ions , 1997 .

[107]  David R Walt,et al.  Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection. , 2006, Analytical chemistry.

[108]  Piotr Garstecki,et al.  Formation of bubbles and droplets in parallel, coupled flow-focusing geometries. , 2008, Small.

[109]  R. Nolte,et al.  Viruses and protein cages as nanocontainers and nanoreactors , 2009 .

[110]  Jörg Enderlein,et al.  Fluorescence spectroscopy of single molecules under ambient conditions: methodology and technology. , 2003, Chemphyschem : a European journal of chemical physics and physical chemistry.

[111]  R. Ismagilov,et al.  Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics. , 2008, Lab on a chip.

[112]  David R Walt,et al.  Multiplexed spectroscopic detections. , 2008, Annual review of analytical chemistry.

[113]  G. Whitesides.  The 'right' size in nanobiotechnology , 2003, Nature Biotechnology.

[114]  Johannes A A W Elemans,et al.  Self-assembled nanoreactors. , 2005, Chemical reviews.

[115]  D R Walt,et al.  High-density fiber-optic DNA random microsphere array. , 2000, Analytical chemistry.

[116]  E. Rhoades,et al.  Watching proteins fold one molecule at a time , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[117]  Petra Schwille,et al.  A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices , 2005, Chembiochem : a European journal of chemical biology.

[118]  H. Stone,et al.  Formation of dispersions using “flow focusing” in microchannels , 2003 .

[119]  J. S. Johnson,et al.  Biocompatible surfactants for water-in-fluorocarbon emulsions. , 2008, Lab on a chip.

[120]  Helene Andersson-Svahn,et al.  Detection and analysis of low-abundance cell-surface biomarkers using enzymatic amplification in microfluidic droplets. , 2009, Angewandte Chemie.

[121]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[122]  Vladimir M. Mirsky,et al.  Spreader‐bar‐Technik in der Molekülarchitektur: Bildung von künstlichen Rezeptoren , 1999 .