Abstract

Thin (<10-nm) adsorbed organic films are visible to the unaided eye, if the substrate is first covered with a dielectric film giving a strong interference color, a so-called sensitive color. It is found that a single low-index dielectric film on an absorbing substrate gives optimal sensitivity. A detection limit in the subnanometer range is predicted and confirmed by experiments. Two practical designs using silicon and glass substrates are discussed. These slides can be produced by industrial methods and have proved to give good visualization of monomolecular protein films, e.g., antigen–antibody layers. They have a detection limit of 0.7 nm or 100 ng of protein/cm2 surface.

© 1985 Optical Society of America

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References

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  1. I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
    [CrossRef] [PubMed]
  2. K. B. Blodgett, I. Langmuir, “Built-up Films of Barium Stearate and their Optical Properties,” Phys. Rev. 51, 964 (1937).
    [CrossRef]
  3. A. Rothen, “The Ellipsometer, an Apparatus to Measure Thickness of Thin Surface Films,” Rev. Sci. Instrum. 16, No. 2, 26 (1945).
    [CrossRef]
  4. M. F. Shaffer, J. H. Dingle, “A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir,” Proc. Soc. Exp. Biol. Med. 38, 528 (1938).
  5. A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
    [CrossRef] [PubMed]
  6. I. Giaever, R. J. Laffin, “Visual Detection of Hepatitis B Antigen,” Proc. Natl. Acad. Sci. USA 71, 4533 (1974).
    [CrossRef] [PubMed]
  7. I. Giaever, “Diagnostic Device for Visually Detecting Presence of Biological Particles,” U.S. Patent3,979,184.
  8. R. J. Laffin, “Visual Detection of Hepatitis B Surface Antigen and Antibody,” in Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2 (Plenum, New York, 1977), p. 147.
    [CrossRef]
  9. M. Born, E. Wolf, Principles of Optics (Pergamon, New York, 1980).
  10. H. Kubota, “Interference Color,” Prog. Opt. 1, 211 (1961).
    [CrossRef]
  11. D. B. Judd, G. Wyszecki, Color in Business, Science and Industry (Wiley, New York, 1975), pp. 17, 19, 20.
  12. R. M. A. Azzam, N. M. Bashara, Ellipsometry and Polarized Light (North-Holland, New York, 1977), p. 6.
  13. G. J. Chamberlin, D. G. Chamberlin, Colour (Heyden, London, 1980).
  14. H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
    [CrossRef] [PubMed]
  15. E. Ingelstam, L. Johansson, “Sur la sensibilité différentielle de l'oeil aux couleurs dans l'échelle de Newton notamment pour les teintes sensible,” Opt. Acta 2, 139 (1955).
    [CrossRef]
  16. M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
    [CrossRef]
  17. H. Elwing, L.-A. Nilsson, “Diffusion-in-Gel Thin Layer Immunoassay (DIG:TIA): Optimal Conditions for Quantitation of Antibodies,” J. Immunol. Methods 38, 257 (1980).
    [CrossRef]
  18. H. Nygren, M. Stenberg, “Electrophoresis of Ligands over a Surface Coated with a Binding Receptor—a Novel Methodological Principle for Electroimmunoassays,” FEBS Lett. 135, 73 (1981).
    [CrossRef] [PubMed]
  19. Slides of the two types described in the text are available under the tradename Sagax slides. Sagax is a registered trademark of SAGAX Instrument AB, Box 7003, S-172 07 Sundbyberg, Sweden.

1983 (1)

H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
[CrossRef] [PubMed]

1981 (1)

H. Nygren, M. Stenberg, “Electrophoresis of Ligands over a Surface Coated with a Binding Receptor—a Novel Methodological Principle for Electroimmunoassays,” FEBS Lett. 135, 73 (1981).
[CrossRef] [PubMed]

1980 (2)

M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
[CrossRef]

H. Elwing, L.-A. Nilsson, “Diffusion-in-Gel Thin Layer Immunoassay (DIG:TIA): Optimal Conditions for Quantitation of Antibodies,” J. Immunol. Methods 38, 257 (1980).
[CrossRef]

1974 (1)

I. Giaever, R. J. Laffin, “Visual Detection of Hepatitis B Antigen,” Proc. Natl. Acad. Sci. USA 71, 4533 (1974).
[CrossRef] [PubMed]

1973 (1)

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

1961 (1)

H. Kubota, “Interference Color,” Prog. Opt. 1, 211 (1961).
[CrossRef]

1955 (1)

E. Ingelstam, L. Johansson, “Sur la sensibilité différentielle de l'oeil aux couleurs dans l'échelle de Newton notamment pour les teintes sensible,” Opt. Acta 2, 139 (1955).
[CrossRef]

1945 (1)

A. Rothen, “The Ellipsometer, an Apparatus to Measure Thickness of Thin Surface Films,” Rev. Sci. Instrum. 16, No. 2, 26 (1945).
[CrossRef]

1938 (1)

M. F. Shaffer, J. H. Dingle, “A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir,” Proc. Soc. Exp. Biol. Med. 38, 528 (1938).

1937 (2)

I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
[CrossRef] [PubMed]

K. B. Blodgett, I. Langmuir, “Built-up Films of Barium Stearate and their Optical Properties,” Phys. Rev. 51, 964 (1937).
[CrossRef]

Adams, A. L.

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

Azzam, R. M. A.

R. M. A. Azzam, N. M. Bashara, Ellipsometry and Polarized Light (North-Holland, New York, 1977), p. 6.

Bashara, N. M.

R. M. A. Azzam, N. M. Bashara, Ellipsometry and Polarized Light (North-Holland, New York, 1977), p. 6.

Blodgett, K. B.

K. B. Blodgett, I. Langmuir, “Built-up Films of Barium Stearate and their Optical Properties,” Phys. Rev. 51, 964 (1937).
[CrossRef]

Born, M.

M. Born, E. Wolf, Principles of Optics (Pergamon, New York, 1980).

Chamberlin, D. G.

G. J. Chamberlin, D. G. Chamberlin, Colour (Heyden, London, 1980).

Chamberlin, G. J.

G. J. Chamberlin, D. G. Chamberlin, Colour (Heyden, London, 1980).

Dingle, J. H.

M. F. Shaffer, J. H. Dingle, “A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir,” Proc. Soc. Exp. Biol. Med. 38, 528 (1938).

Elwing, H.

H. Elwing, L.-A. Nilsson, “Diffusion-in-Gel Thin Layer Immunoassay (DIG:TIA): Optimal Conditions for Quantitation of Antibodies,” J. Immunol. Methods 38, 257 (1980).
[CrossRef]

Fischer, G. C.

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

Giaever, I.

I. Giaever, R. J. Laffin, “Visual Detection of Hepatitis B Antigen,” Proc. Natl. Acad. Sci. USA 71, 4533 (1974).
[CrossRef] [PubMed]

I. Giaever, “Diagnostic Device for Visually Detecting Presence of Biological Particles,” U.S. Patent3,979,184.

Ingelstam, E.

E. Ingelstam, L. Johansson, “Sur la sensibilité différentielle de l'oeil aux couleurs dans l'échelle de Newton notamment pour les teintes sensible,” Opt. Acta 2, 139 (1955).
[CrossRef]

Johansson, L.

E. Ingelstam, L. Johansson, “Sur la sensibilité différentielle de l'oeil aux couleurs dans l'échelle de Newton notamment pour les teintes sensible,” Opt. Acta 2, 139 (1955).
[CrossRef]

Judd, D. B.

D. B. Judd, G. Wyszecki, Color in Business, Science and Industry (Wiley, New York, 1975), pp. 17, 19, 20.

Klings, M.

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

Kubota, H.

H. Kubota, “Interference Color,” Prog. Opt. 1, 211 (1961).
[CrossRef]

Laffin, R. J.

I. Giaever, R. J. Laffin, “Visual Detection of Hepatitis B Antigen,” Proc. Natl. Acad. Sci. USA 71, 4533 (1974).
[CrossRef] [PubMed]

R. J. Laffin, “Visual Detection of Hepatitis B Surface Antigen and Antibody,” in Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2 (Plenum, New York, 1977), p. 147.
[CrossRef]

Langmuir, I.

I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
[CrossRef] [PubMed]

K. B. Blodgett, I. Langmuir, “Built-up Films of Barium Stearate and their Optical Properties,” Phys. Rev. 51, 964 (1937).
[CrossRef]

Nilsson, L.-A.

H. Elwing, L.-A. Nilsson, “Diffusion-in-Gel Thin Layer Immunoassay (DIG:TIA): Optimal Conditions for Quantitation of Antibodies,” J. Immunol. Methods 38, 257 (1980).
[CrossRef]

Nygren, H.

H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
[CrossRef] [PubMed]

H. Nygren, M. Stenberg, “Electrophoresis of Ligands over a Surface Coated with a Binding Receptor—a Novel Methodological Principle for Electroimmunoassays,” FEBS Lett. 135, 73 (1981).
[CrossRef] [PubMed]

Rothen, A.

A. Rothen, “The Ellipsometer, an Apparatus to Measure Thickness of Thin Surface Films,” Rev. Sci. Instrum. 16, No. 2, 26 (1945).
[CrossRef]

Sandström, T.

H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
[CrossRef] [PubMed]

M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
[CrossRef]

Schaefer, V.

I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
[CrossRef] [PubMed]

Shaffer, M. F.

M. F. Shaffer, J. H. Dingle, “A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir,” Proc. Soc. Exp. Biol. Med. 38, 528 (1938).

Stenberg, M.

H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
[CrossRef] [PubMed]

H. Nygren, M. Stenberg, “Electrophoresis of Ligands over a Surface Coated with a Binding Receptor—a Novel Methodological Principle for Electroimmunoassays,” FEBS Lett. 135, 73 (1981).
[CrossRef] [PubMed]

M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
[CrossRef]

Stiblert, L.

M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
[CrossRef]

Vroman, L.

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

Wolf, E.

M. Born, E. Wolf, Principles of Optics (Pergamon, New York, 1980).

Wrinch, D. M.

I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
[CrossRef] [PubMed]

Wyszecki, G.

D. B. Judd, G. Wyszecki, Color in Business, Science and Industry (Wiley, New York, 1975), pp. 17, 19, 20.

FEBS Lett. (1)

H. Nygren, M. Stenberg, “Electrophoresis of Ligands over a Surface Coated with a Binding Receptor—a Novel Methodological Principle for Electroimmunoassays,” FEBS Lett. 135, 73 (1981).
[CrossRef] [PubMed]

J. Immunol. Methods (3)

A. L. Adams, M. Klings, G. C. Fischer, L. Vroman, “Three Simple Ways to Detect Antibody-Antigen Complex on Flat Surfaces,” J. Immunol. Methods 3, 227 (1973).
[CrossRef] [PubMed]

H. Nygren, T. Sandström, M. Stenberg, “Direct Visual Detection of a Protein Antigen—Importance of Surface Concentration,” J. Immunol. Methods 59, 145 (1983).
[CrossRef] [PubMed]

H. Elwing, L.-A. Nilsson, “Diffusion-in-Gel Thin Layer Immunoassay (DIG:TIA): Optimal Conditions for Quantitation of Antibodies,” J. Immunol. Methods 38, 257 (1980).
[CrossRef]

Mater. Sci. Eng. (1)

M. Stenberg, T. Sandström, L. Stiblert, “A New Ellipsometric Method for Measurements on Surfaces and Surface Layers,” Mater. Sci. Eng. 42, 65 (1980).
[CrossRef]

Opt. Acta (1)

E. Ingelstam, L. Johansson, “Sur la sensibilité différentielle de l'oeil aux couleurs dans l'échelle de Newton notamment pour les teintes sensible,” Opt. Acta 2, 139 (1955).
[CrossRef]

Phys. Rev. (1)

K. B. Blodgett, I. Langmuir, “Built-up Films of Barium Stearate and their Optical Properties,” Phys. Rev. 51, 964 (1937).
[CrossRef]

Proc. Natl. Acad. Sci. USA (1)

I. Giaever, R. J. Laffin, “Visual Detection of Hepatitis B Antigen,” Proc. Natl. Acad. Sci. USA 71, 4533 (1974).
[CrossRef] [PubMed]

Proc. Soc. Exp. Biol. Med. (1)

M. F. Shaffer, J. H. Dingle, “A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir,” Proc. Soc. Exp. Biol. Med. 38, 528 (1938).

Prog. Opt. (1)

H. Kubota, “Interference Color,” Prog. Opt. 1, 211 (1961).
[CrossRef]

Rev. Sci. Instrum. (1)

A. Rothen, “The Ellipsometer, an Apparatus to Measure Thickness of Thin Surface Films,” Rev. Sci. Instrum. 16, No. 2, 26 (1945).
[CrossRef]

Science (1)

I. Langmuir, V. Schaefer, D. M. Wrinch, “Built-Up Films of Proteins and their Properties,” Science 85, No. 2194, 76 (1937).
[CrossRef] [PubMed]

Other (7)

I. Giaever, “Diagnostic Device for Visually Detecting Presence of Biological Particles,” U.S. Patent3,979,184.

R. J. Laffin, “Visual Detection of Hepatitis B Surface Antigen and Antibody,” in Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2 (Plenum, New York, 1977), p. 147.
[CrossRef]

M. Born, E. Wolf, Principles of Optics (Pergamon, New York, 1980).

D. B. Judd, G. Wyszecki, Color in Business, Science and Industry (Wiley, New York, 1975), pp. 17, 19, 20.

R. M. A. Azzam, N. M. Bashara, Ellipsometry and Polarized Light (North-Holland, New York, 1977), p. 6.

G. J. Chamberlin, D. G. Chamberlin, Colour (Heyden, London, 1980).

Slides of the two types described in the text are available under the tradename Sagax slides. Sagax is a registered trademark of SAGAX Instrument AB, Box 7003, S-172 07 Sundbyberg, Sweden.

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Figures (9)

Fig. 1
Fig. 1

Reflection from a surface with and without a dielectric surface layer. With the dielectric layer, the reflected beam is split into a number of components. When the optical path is different for the components, they can add up to a smaller amplitude than in the case without the dielectric layer.

Fig. 2
Fig. 2

Reflected monochromatic intensity I from an antireflection layer. The layer has a perfectly matching refractive index but a varying thickness. The reflectivity vanishes for a layer with an optical path length of π/4, curve a. The change of reflectivity dI/dh is largest at π/8 and vanishes at π/4, curve b. The contrast, dI/dh ·1/I, tends to infinity at π/4, curve c. Arbitrary units.

Fig. 3
Fig. 3

White light reflection and color contrast of a ns = 2.25 substrate with a nf = 1.50 dielectric layer: a, reflected intensity vs the thickness of the dielectric layer; b, c, and d, color contrast for a 1-nm thick organic layer calculated by the CIELUV, CIELAB, and FMC II models, respectively.

Fig. 4
Fig. 4

Chromaticity coordinates for interference colors of a nf = 1.50 dielectric layer on a ns = 2.25 substrate and thicknesses from 0 to 600 nm.

Fig. 5
Fig. 5

Same as Fig. 3 but showing only FMC II. The intensity, curve a, and the sensitivity, curve b, functions are shown for thicknesses up to 600 nm.

Fig. 6
Fig. 6

Maximum sensitivity, curve a, and minimum reflectivity, curve b, for perfect single-layer antireflection coatings with different film indices. In each case the substrate has the matching index n f 2.

Fig. 7
Fig. 7

Influence of deviations from the perfect index for antireflection, nf = 1.50, on a ns = 2.25 substrate. The upper graph, curve a, shows the value obtained after correction of the thickness, while the lower graph, curve b, gives the sensitivity at nominal thickness.

Fig. 8
Fig. 8

Silicon slide with an SiO layer with varying thickness from 0 to 150 nm that is covered with a 2–3-nm thick SiO2 layer. A strip in the middle of the slide is coated with a 7.5-nm thick biolayer.

Fig. 9
Fig. 9

Viewing arrangement to make specular reflection compensate for low reflectivity. The lamp is seen reflected in the slide, while the background scatters light diffusely. The intensity of the background can be varied at constant apparent slide lightness by changing the distance between the lamp and table.

Equations (5)

Equations on this page are rendered with MathJax. Learn more.

n f = n s .
I r = 2 I i ( n f 1 n f + 1 ) 2 [ 1 + cos ( α h ) ] ,
α = 4 [ ( n f ) / λ ] .
Δ I r = d I r d h Δ h = 2 I i ( n f 1 n f + 1 ) 2 α sin ( α h ) Δ h .
S = Δ I r I r = α sin ( α h ) 1 + cos ( α h ) Δ h .

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