Abstract

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7μm) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed.

© 2010 Optical Society of America

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  1. C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
    [CrossRef]
  2. H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
    [CrossRef]
  3. O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
    [CrossRef]
  4. O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
    [CrossRef] [PubMed]
  5. B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
    [CrossRef] [PubMed]
  6. S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
    [CrossRef] [PubMed]
  7. P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
    [CrossRef] [PubMed]
  8. X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
    [CrossRef] [PubMed]
  9. M. C. Rodriguez and G. A. Rivas, “Label-free electrochemical aptasensor for the detection of lysozyme,” Talanta 78, 212–216 (2009).
    [CrossRef] [PubMed]
  10. P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
    [CrossRef]
  11. M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
    [CrossRef] [PubMed]
  12. E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
    [CrossRef] [PubMed]
  13. T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
    [CrossRef]
  14. M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
    [CrossRef] [PubMed]
  15. M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
    [CrossRef] [PubMed]
  16. M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
    [CrossRef] [PubMed]
  17. J. W. Goodman, Introduction to Fourier Optics, 3rd ed.(Roberts, 2005), pp. 103–108.
  18. A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
    [CrossRef] [PubMed]

2010 (1)

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

2009 (4)

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. C. Rodriguez and G. A. Rivas, “Label-free electrochemical aptasensor for the detection of lysozyme,” Talanta 78, 212–216 (2009).
[CrossRef] [PubMed]

2008 (1)

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

2007 (1)

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

2006 (3)

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

2005 (1)

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

2004 (3)

C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
[CrossRef]

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

2003 (1)

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

2002 (1)

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

2000 (1)

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Angenendt, P.

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Auyeung, R. Y. C.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Bruce, R.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Buus, S.

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

Cahill, D. J.

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Chrisey, D. B.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Daub, M.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

de Heij, B.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Duroux, L.

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

Duroux, M.

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Elrod, S.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Fitch, J.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Glokler, J.

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Goodman, J. W.

J. W. Goodman, Introduction to Fourier Optics, 3rd ed.(Roberts, 2005), pp. 103–108.

Gurevich, L.

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

Gutmann, O.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Henderson, E.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Herron, J. N.

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Hsieh, H. B.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Johnson, J.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Jonkheijm, P.

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

Kim, H.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Klitgaard, S.

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Kold, A.

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

Kold di Gennaro, A.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

Konthur, Z.

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Kowalski, B.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Krivacic, B.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Krizman, D. B.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Kuehlewein, R.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Lehrach, H.

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Liu, X. H.

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Lövgren, T.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

Lynch, M.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Matusiak, R.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Mosher, C.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Nettikadan, S.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Neves-Petersen, M. T.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Niekrawietz, R.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Niemeyer, C. M.

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

Parracino, A.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

Pascher, T.

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

Petersen, S. B.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Pettersson, K.

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

Pique, A.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Polivka, T.

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

Prestwich, G. D.

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Radke, K.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Reinbold, S.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Ringeisen, B. R.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Rivas, G. A.

M. C. Rodriguez and G. A. Rivas, “Label-free electrochemical aptasensor for the detection of lysozyme,” Talanta 78, 212–216 (2009).
[CrossRef] [PubMed]

Roder, G. A.

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

Rodriguez, M. C.

M. C. Rodriguez and G. A. Rivas, “Label-free electrochemical aptasensor for the detection of lysozyme,” Talanta 78, 212–216 (2009).
[CrossRef] [PubMed]

Roy, J.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Schroder, H.

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

Skovsen, E.

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

Snabe, T.

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Steinert, C. P.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Strobl, C. J.

C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
[CrossRef]

Sundström, V.

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

Torres, F.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

von Guttenberg, Z.

C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
[CrossRef]

Waldmann, H.

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

Wang, H. K.

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Weinrich, D.

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

White, D.

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

Wixforth, A.

C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
[CrossRef]

Wu, P. K.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Xu, J. T.

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Yartsev, A.

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

Young, H. D.

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

Zengerle, R.

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Anal. Chem. (1)

P. Angenendt, J. Glokler, Z. Konthur, H. Lehrach, and D. J. Cahill, “3D protein microarrays: performing multiplex immunoassays on a single chip,” Anal. Chem. 75, 4368–4372 (2003).
[CrossRef] [PubMed]

Angew. Chem., Int. Ed. Engl. (1)

P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer, and H. Waldmann, “Chemical strategies for generating protein biochips,” Angew. Chem., Int. Ed. Engl. 47, 9618–9647 (2008).
[CrossRef]

Bioconjug. Chem. (1)

X. H. Liu, H. K. Wang, J. N. Herron, and G. D. Prestwich, “Photopatterning of antibodies on biosensors,” Bioconjug. Chem. 11, 755–761 (2000).
[CrossRef] [PubMed]

Biomed. Microdev. (1)

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “A highly parallel nanoliter dispenser for microarray fabrication,” Biomed. Microdev. 6, 131–137 (2004).
[CrossRef]

Biophys. J. (1)

M. T. Neves-Petersen, S. Klitgaard, T. Pascher, E. Skovsen, T. Polivka, A. Yartsev, V. Sundström, and S. B. Petersen, “Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues,” Biophys. J. 97, 211–226 (2009).
[CrossRef] [PubMed]

Biosens. Bioelectron. (1)

T. Snabe, G. A. Roder, M. T. Neves-Petersen, S. Buus, and S. B. Petersen, “Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology,” Biosens. Bioelectron. 21, 1553–1559(2006).
[CrossRef]

Biotechnol. Prog. (1)

B. R. Ringeisen, P. K. Wu, H. Kim, A. Pique, R. Y. C. Auyeung, H. D. Young, D. B. Chrisey, and D. B. Krizman, “Picoliter-scale protein microarrays by laser direct write,” Biotechnol. Prog. 18, 1126–1129 (2002).
[CrossRef] [PubMed]

IEEE Trans. Ultrason. Ferroelect. Freq. Contr. (1)

C. J. Strobl, Z. von Guttenberg, and A. Wixforth, “Nano- and pico-dispensing of fluids on planar substrates using SAW,” IEEE Trans. Ultrason. Ferroelect. Freq. Contr. 51, 1432–1436(2004).
[CrossRef]

J. Biomol. Sceening (1)

H. B. Hsieh, J. Fitch, D. White, F. Torres, J. Roy, R. Matusiak, B. Krivacic, B. Kowalski, R. Bruce, and S. Elrod, “Ultra-high-throughput microarray generation and liquid dispensing using multiple disposable piezoelectric ejectors,” J. Biomol. Sceening 9, 85–94 (2004).
[CrossRef]

J. Nanosci. Nanotechnol. (2)

E. Skovsen, M. T. Neves-Petersen, M. Duroux, A. Kold, L. Duroux, and S. B. Petersen, “Immobilizing biomolecules near the diffraction limit,” J. Nanosci. Nanotechnol. 9, 4333–4337(2009).
[CrossRef] [PubMed]

M. T. Neves-Petersen, M. Duroux, E. Skovsen, L. Duroux, and S. B. Petersen, “Printing novel molecular architectures with micrometer resolution using light,” J. Nanosci. Nanotechnol. 9, 3372–3381 (2009).
[CrossRef] [PubMed]

Lab Chip (1)

O. Gutmann, R. Kuehlewein, S. Reinbold, R. Niekrawietz, C. P. Steinert, B. de Heij, R. Zengerle, and M. Daub, “Fast and reliable protein microarray production by a new drop-in-drop technique,” Lab Chip 5, 675–681 (2005).
[CrossRef] [PubMed]

Mol. Cell. Proteomics (1)

S. Nettikadan, K. Radke, J. Johnson, J. T. Xu, M. Lynch, C. Mosher, and E. Henderson, “Detection and quantification of protein biomarkers from fewer than 10 cells,” Mol. Cell. Proteomics 5, 895–901 (2006).
[CrossRef] [PubMed]

Protein Sci. (2)

A. Parracino, M. T. Neves-Petersen, A. Kold di Gennaro, K. Pettersson, T. Lövgren, and S. B. Petersen, “Arraying prostate specific antigen PSA and Fab anti-PSA using light assisted molecular immobilization technology,” Protein Sci. 19, 1751–1759 (2010).
[CrossRef] [PubMed]

M. T. Neves-Petersen, T. Snabe, S. Klitgaard, M. Duroux, and S. B. Petersen, “Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces,” Protein Sci. 15, 343–351 (2006).
[CrossRef] [PubMed]

Proteomics (1)

M. Duroux, E. Skovsen, M. T. Neves-Petersen, L. Duroux, L. Gurevich, and S. B. Petersen, “Light-induced immobilisation of biomolecules as an attractive alternative to microdroplet dispensing-based arraying technologies,” Proteomics 7, 3491–3499 (2007).
[CrossRef] [PubMed]

Talanta (1)

M. C. Rodriguez and G. A. Rivas, “Label-free electrochemical aptasensor for the detection of lysozyme,” Talanta 78, 212–216 (2009).
[CrossRef] [PubMed]

Other (1)

J. W. Goodman, Introduction to Fourier Optics, 3rd ed.(Roberts, 2005), pp. 103–108.

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Figures (8)

Fig. 1
Fig. 1

Immobilization setup: illumination setup used for light-assisted molecular immobilization using a UV light diffraction pattern.

Fig. 2
Fig. 2

Single-aperture masks used (left), false color images of the expected diffraction patterns obtained after Fourier transforming each mask (middle), and the experimentally obtained fluorescence intensity images of immobilized PSA-AF555 molecules (right). (a) Mask with a small circular pinhole aperture. (b) Mask with a short slit. (c) Mask with a long slit. It is apparent from the images that the patterns of light-induced immobilized biomolecules correspond reasonably well with the predicted diffraction patterns used to expose the film of fluorescently labeled biomolecules.

Fig. 3
Fig. 3

Fluorescence emitted by PSA-AF555 after UV-light- assisted immobilization using a pinhole as the single-aperture mask, together with the integrated fluorescence intensity profile along the direction indicated by the arrow. The observed distance between the central intense peak and the first dip in the airy profile is 6 6.4 μm , in good agreement with theoretical calculations.

Fig. 4
Fig. 4

Fluorescence emitted by PSA-AF555 after UV-light- assisted immobilization using a small slit as the single-aperture mask, together with the integrated fluorescence intensity profile along the direction indicated by the arrow.

Fig. 5
Fig. 5

Fluorescence emitted by PSA-AF555 after UV-light- assisted immobilization using a large slit as the single-aperture mask together with the integrated fluorescence intensity profiles along the directions indicated by the arrows. The smallest visible resolved features are the fringes separated by 0.7 μm , which is close to the diffraction limit of 0.5 μm for the optics used for the immobilization.

Fig. 6
Fig. 6

Zoom onto a 2 × 2 array of spots each showing the immobilization patterns obtained when carrying out light-assisted immobilization using a small slit as mask. Notice the high degree of reproducibility, which is typical of this technique.

Fig. 7
Fig. 7

Dual-aperture (two pinholes) transmission mask used (left), false color image of the expected diffraction pattern obtained after Fourier transforming the mask (middle), and the experimentally obtained fluorescence intensity image of immobilized PSA-AF555 molecules (right).

Fig. 8
Fig. 8

Fluorescence image of the PSA-AF555 protein pattern immobilized using the dual-aperture mask, together with the integrated intensity profiles along the directions indicated by the arrows. The radial intensity has been integrated over all angles to show the fluorescence intensity profile as a function of the distance from the center of the diffraction pattern. The smallest visible resolved fringes are separated by 0.8 μm .

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