Abstract

Zero-Mode Waveguides were first introduced for Fluorescence Correlation Spectroscopy at micromolar dye concentrations. We show that combining zero-mode waveguides with fluorescence correlation spectroscopy in a continuous flow mixer avoids the compression of the FCS signal due to fluid transport at channel velocities up to ~17 mm/s. We derive an analytic scaling relationship δkONkON=δkOFFkOFF~kON+kOFFkON0.1DBDFDBeSNR converting this flow velocity insensitivity to improved kinetic rate certainty in time-resolved mixing experiments. Thus zero-mode waveguides make FCS suitable for direct kinetics measurements in rapid continuous flow.

© 2008 Optical Society of America

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  1. J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
    [CrossRef]
  2. E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
    [CrossRef] [PubMed]
  3. D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
    [CrossRef] [PubMed]
  4. D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
    [CrossRef] [PubMed]
  5. D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
    [CrossRef]
  6. E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
    [CrossRef]
  7. D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
    [CrossRef] [PubMed]
  8. M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
    [CrossRef] [PubMed]
  9. K. K. Kuricheti, V. Buschmann, and K. D. Weston, "Application of fluorescence correlation spectroscopy for velocity imaging in microfluidic devices," Appl. Spectrosc. 58, 1180-1186 (2004).
    [CrossRef] [PubMed]
  10. M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
    [CrossRef] [PubMed]
  11. M. Leutenegger, M. Gósch, A. Perentes, P. Hoffmann, O. J. Martin, and T. Lasser, "Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture," Opt. Express 14, 956-969 (2006).
    [CrossRef] [PubMed]
  12. J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
    [CrossRef]
  13. S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
    [CrossRef]
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  16. P. G. Gloersen, "Ion-beam etching," J. Vac. Sci. Technol. 12, 28-35 (1975).
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    [CrossRef]
  18. K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
    [CrossRef]
  19. T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
    [CrossRef]

2007 (1)

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

2006 (3)

M. Leutenegger, M. Gósch, A. Perentes, P. Hoffmann, O. J. Martin, and T. Lasser, "Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture," Opt. Express 14, 956-969 (2006).
[CrossRef] [PubMed]

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

2005 (1)

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

2004 (2)

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

K. K. Kuricheti, V. Buschmann, and K. D. Weston, "Application of fluorescence correlation spectroscopy for velocity imaging in microfluidic devices," Appl. Spectrosc. 58, 1180-1186 (2004).
[CrossRef] [PubMed]

2003 (2)

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

2000 (1)

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

1998 (1)

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

1997 (1)

S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
[CrossRef]

1979 (1)

R. Lee, "Microfabrication by ion-beam etching," J. Vac. Sci. Technol. 16, 164-170 (1979).
[CrossRef]

1975 (1)

P. G. Gloersen, "Ion-beam etching," J. Vac. Sci. Technol. 12, 28-35 (1975).

1974 (2)

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
[CrossRef] [PubMed]

1972 (1)

D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Alaverdyan, Y.

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

Austin, R. H.

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

Bakajin, O.

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

Blom, H.

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Boned, A.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Brody, J. P.

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

Buschmann, V.

Conchonaud, F.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Cox, E.

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

Craighead, H.

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Dintinger, J.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Eaton, W. A.

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

Ebbesen, T. W.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Elson, E.

D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Elson, E. L.

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
[CrossRef] [PubMed]

Foquet, M.

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Gérard, D.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Gloersen, P. G.

P. G. Gloersen, "Ion-beam etching," J. Vac. Sci. Technol. 12, 28-35 (1975).

Gósch, M.

Gösch, M.

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Guo, L.

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

Haupts, U.

S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
[CrossRef]

Heino, T.

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Hertzog, D. E.

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Hoffmann, P.

Holm, J.

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Ivorra, B.

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

Jäger, M.

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Käll, M.

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

Knight, J. B.

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

Kong, X.

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Korlach, J.

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Kuricheti, K. K.

Lasser, T.

Lee, R.

R. Lee, "Microfabrication by ion-beam etching," J. Vac. Sci. Technol. 16, 164-170 (1979).
[CrossRef]

Lenne, P.-F.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Leutenegger, M.

Levene, M.

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Lipman, E. A.

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

Magde, D.

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
[CrossRef] [PubMed]

D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Maiti, S.

S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
[CrossRef]

Marguet, D.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Martin, O. J.

Michalet, X.

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Mohammadi, B.

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

Pakzeh, T.

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

Perentes, A.

Rigler, R.

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Rigneault, H.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Rindzevicius, T.

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

Samiee, K.

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

Santiago, J. G.

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Schuler, B.

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

Sepulveda, B.

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

Turner, S.

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Vishwanath, A.

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

Webb, W.

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Webb, W. W.

S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
[CrossRef] [PubMed]

Weiss, S.

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

Wenger, J.

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Weston, K. D.

Anal. Chem. (3)

D. E. Hertzog, B. Ivorra, B. Mohammadi, O. Bakajin, and J. G. Santiago, "Optimization of a microfluidic mixer for studying protein folding kinetics," Anal. Chem. 78, 4299-4306 (2006).
[CrossRef] [PubMed]

D. E. Hertzog, X. Michalet, M. Jäger, X. Kong, J. G. Santiago, S. Weiss, and O. Bakajin, "Femtomole mixer for microsecond kinetic studies of protein folding," Anal. Chem. 76, 7169-7178 (2004).
[CrossRef] [PubMed]

M. Gösch, H. Blom, J. Holm, T. Heino, and R. Rigler, "Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy," Anal. Chem. 72, 3260-3265 (2000).
[CrossRef] [PubMed]

Appl. Spectrosc. (1)

Biophys. J. (1)

K. Samiee, M. Foquet, L. Guo, E. Cox, and H. Craighead, "λ -Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides," Biophys. J. 88, 2145-2153 (2005).
[CrossRef]

Biopolymers (2)

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Fluorescence correlation spectroscopy. II. An experimental realization," Biopolymers 13, 29-61 (1974).
[CrossRef] [PubMed]

J. Phys. Chem. C. (1)

T. Rindzevicius, Y. Alaverdyan, B. Sepulveda, T. Pakzeh, and M. Käll, "Nanohole plasmons in optically thin gold films," J. Phys. Chem. C. 111, 1207-1212 (2007).
[CrossRef]

J. Vac. Sci. Technol. (2)

P. G. Gloersen, "Ion-beam etching," J. Vac. Sci. Technol. 12, 28-35 (1975).

R. Lee, "Microfabrication by ion-beam etching," J. Vac. Sci. Technol. 16, 164-170 (1979).
[CrossRef]

Opt. Express (2)

M. Leutenegger, M. Gósch, A. Perentes, P. Hoffmann, O. J. Martin, and T. Lasser, "Confining the sampling volume for Fluorescence Correlation Spectroscopy using a sub-wavelength sized aperture," Opt. Express 14, 956-969 (2006).
[CrossRef] [PubMed]

J. Wenger, D. Gérard, P.-F. Lenne, H. Rigneault, J. Dintinger, T. W. Ebbesen, A. Boned, F. Conchonaud, and D. Marguet, "Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations," Opt. Express 14, 12,206-12,216 (2006).
[CrossRef]

Phys. Rev. Lett. (2)

D. Magde, E. Elson, and W. Webb, "Thermodynamic fluctuations in a reacting system-measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

J. B. Knight, A. Vishwanath, J. P. Brody, and R. H. Austin, "Hydrodynamic Focusing on a Silicon Chip: Mixing Nanoliters in Microseconds," Phys. Rev. Lett. 80, 3863-3866 (1998).
[CrossRef]

Proceedings of the National Academy of Sciences (1)

S. Maiti, U. Haupts, and W. W. Webb, "Fluorescence correlation spectroscopy: Diagnostics for sparse molecules," Proceedings of the National Academy of Sciences 94, 11,753-11,757 (1997).
[CrossRef]

Science (2)

E. A. Lipman, B. Schuler, O. Bakajin, and W. A. Eaton, "Single-molecule measurement of protein folding kinetics," Science 301, 1233-1235 (2003).
[CrossRef] [PubMed]

M. Levene, J. Korlach, S. Turner, M. Foquet, H. Craighead, and W. Webb, "Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations," Science 299, 682-686 (2003).
[CrossRef] [PubMed]

Other (2)

J. Widengren and U. Mets, Single Molecule Detection in Solution, chap. 3, pp. 69-120 (Wiley-VCH, 2002).
[CrossRef]

J. Enderlein and C. Zander, Single Molecule Detection in Solution, chap. 2, pp. 21-67 (Wiley-VCH, 2002).
[CrossRef]

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Figures (8)

Fig. 1.
Fig. 1.

Diffraction-limited FCS collects fluorescent emission from molecules within a confocal detection profile. The duration of the fluorescence signal from a single molecule is limited by the characteristic time to diffuse or flow advectively out of the observation profile. (a) Optical path in a custom instrument. The polychroic beam splitter DCRe, 500-nm long pass filter LP, and band-pass filter BP excluded light at the excitation wavelength 488 nm from the detection path. The second dichroic mirror DCRx gives the option to detect either red or green fluorescence. The pinhole PH is a fiber end. A beam splitter BS and camera CCD assist sample alignment using the 60× objective.

Fig. 2.
Fig. 2.

Normalized autocorrelation curves acquired from diffraction-limited FCS with local fluid flow. Data are identified through markers, and calculations are distinguished by line width (thick or thin) and line color (black or gray). The legend tabulates velocities at the center of the channel.

Fig. 3.
Fig. 3.

Diffraction-limited FCS’s ability to distinguish diffusion constants becomes buried in noise at high fluid velocity. (a) The signal function ΔG is the difference between the correlation function at diffusion constant D=5.1×10-12 m2/s and 1.1D for varied velocities. The signals at 5.5 mm/s, 11 mm/s, and 17 mm/s are multiplied by a factor of 10 for clarity. (b) Uncertainties in the correlation data from Fig. 2.

Fig. 4.
Fig. 4.

(a) and (b) A zero-mode waveguide method confines intensity fluctuations measurement to diffusers proximate to the metal substrate. (c) and (d) Repeated scanning electron micrographs including those shown indicate typical scales h=163.3±8.8 nm, R=209.3±4.4 nm, and r=26.1±1.3 nm.

Fig. 5.
Fig. 5.

ZMW correlation curves. (a) The averages of seven normalized correlation data series and the averages of their fits. (b) The fitted diffusion constants at channel-center velocities of 0 mm/s, 5.5 mm/s, 11 mm/s, and 17 mm/s. Markers indicate data. Lines indicate calculations.

Fig. 6.
Fig. 6.

ZMWFCS’s ability to distinguish diffusion constants persists at high channel-center velocity. (a) The signal function ΔG is the difference between the correlation function at diffusion constant D=5.1×10-12 m2/s and 1.1D. (b) Uncertainties in the correlation data from Fig. 5.

Fig. 7.
Fig. 7.

Applying Eq. 11 to signal and uncertainty functions in Fig. 3 and Fig. 6 shows that diffraction-limited FCS loses sensitivity as channel-center fluid velocity increases. In contrast, ZMW FCS retains sensitivity at high values of channel-center fluid velocity.

Fig. 8.
Fig. 8.

Substrates and fluorescent labels injected at opposite inlets of a T-mixer form bound complexes at the outlet. The present example is transcription factor binding. A fluorescently labeled transcription factor serves as the ligand, and DNA containing the binding site takes the role of the substrate. Flow velocity maps a spatial interval to a reaction time interval Δt.

Equations (30)

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G ( τ ) = 1 + < δ I ( t ) δ I ( t + τ ) > < I > 2
S ( r ) = S 0 exp ( 2 x 2 + y 2 ω xy 2 2 z 2 ω z 2 )
g ( τ ) = ( 1 + 4 D τ ω xy 2 ) 1 ( 1 + 4 D τ ω z 2 ) 1 2 exp ( ( v τ ω xy ) 2 ( 1 + 4 D τ ω xy 2 ) 1 )
Vol = ( S ( r ) d r 3 ) 2 ( S ( r ) ) 2 d r 3
Δ G ( τ ) = G D ( τ ) G 1.1 D ( τ )
Δ G ~ G D Δ D = 4 g τ ω xy 2 ( 1 + 4 D τ ω xy 2 ) 2 [ 1 + 4 D τ ω xy 2 ( v τ ω xy ) 2 ] 0.1 D
D τ x ω xy 2 = 2 + 4 + Pe g 2 Pe g 2
g ZMW ( τ ) π 4 ( 2 T π + ( 1 2 T ) exp ( T ) erfc ( T ) ) R ( 1 + R 2 ) 2 π 2 erf ( R T ) R T
N = 1 A ( I GOLD + SPHERES I GOLD I GOLD + SPHERES ) 2
r ( z ) R ( R r ) ( h z h ) 2
SNR = τ ( Δ G ( τ ) δ G ( τ ) ) 2
Δ D CRITICAL ~ 0.1 D SNR
d p B dt = k ON p F k OFF p B
p B ( ) = k ON k ON + k OFF
Δ p B ( t ) = p B ( ) p B ( t )
Δ p B ( t ) p B ( ) = exp [ ( k ON + k OFF ) t ]
k ON = p B ( ) k TOT
k OFF = [ 1 p B ( ) ] k TOT
δ x = v δ t = 2 D t
δ t t 2 D t v t = 2 Pe
Pe = ( vt ) v D
δ k ON k ON = δ k OFF k OFF = [ Δ p B p B ( ) ln ( p B ( ) Δ p B ) ] 1 δ p B p B ( )
t BEST = 1 k ON + k OFF
G ( τ ) = 1 + N F Q F 2 g F ( τ ) + N B Q B 2 g B ( τ ) ( N F Q F + N B Q B ) 2
G ( τ ) = 1 + 1 N [ g F ( τ ) p B ( g F ( τ ) g B ( τ ) ) ]
p B f ( τ ) = g F ( τ ) N [ G ( τ ) 1 ] D B D F 0.1 D B Δ G
p B , EST = τ f ( τ ) δ f ( τ ) 2 τ 1 δ f ( τ ) 2
δ p B , EST ~ 0.1 D B D F D B 1 SNR
δ k ON k ON BEST = δ k OFF k OFF BEST = k ON + k OFF k ON 0.1 D B D F D B e SNR
δ k ON k ON BEST = δ k OFF k OFF BEST = k ON + k OFF k ON e Δ D CRITICAL D F D B

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