Abstract

The most sensitive existing assays used to determine antibody levels in blood serum samples require a tracer material, e.g., radioisotope, fluorofore, or enzyme, to identify the specific analyte. Surface plasmon spectroscopy has been applied recently as a no-label technique for the assay of specific antibody solutions with the antigen proteins immobilized on a metal surface. It is found that the metal surface configuration originally proposed for the surface plasmon immunoassay (SPI) is unstable and unsuitable for the assay of specific antibodies in a large mixture of proteins such as in a blood serum. Nevertheless, by properly designing the metal surface structure, the SPI can be made an extremely practical device. Preliminary results for the assay of dinitrophenyl (DNP) and keyhole limpet hemocyanin (KLH) antibodies in blood serum samples, indicate that the SPI, in addition to providing a simple and fast measurement, is comparable with existing approaches, such as radioimmunoassy or enzyme-linked immunosorbent assay both in sensitivity and specificity.

© 1990 Optical Society of America

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References

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  1. R. Edwards, Immunoassay: An Introduction (William Heinemann Medical Books, London1985), p. 3.
  2. A. Voller, D. E. Bidwell, “Enzyme Immunoassays,” in Alternative Immunoassays, W. P. Collins, Ed. (Wiley, New York, 1985), Chap. 6.
  3. M. J. O’Sullivan, “Enzyme Immunoassay,” in Practical Immunoassay. The State of the Art, Wilfrid R. Butt, Ed. (Marcel Dekker, New York, 1984), Chap. 3.
  4. A. Otto, “Spectroscopy of Surface Polaritons by Attenuated Total Reflection,” in Optical Properties of Solids, New Developments, B. O. Seraphin, Ed. (North-Holland, Amsterdam, 1975), Chap. 13.
  5. B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
    [CrossRef]
  6. M. T. Flanagan, R. H. Pantell, “Surface Plasmon Resonance and Immunosensors,” Electron. Lett. 20, 968–970 (1984).
    [CrossRef]
  7. E. Kretschmann, “Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingungen,” Z. Phys. 241, 313–324 (1971).
    [CrossRef]
  8. H. Raether, “Surface Plasma Oscillations and their Applications,” Phys. Thin Films 9, 145–261 (1977).
  9. I. Pockrand, “Surface Plasma Oscillations at Silver Surfaces with Thin Transparent and Absorbing Coatings,” Surf. Sci. 72, 577–588 (1978).
    [CrossRef]
  10. H. Raether, “Surface Plasmons and Roughness,” in Surface Polaritons, V. M. Agranovich, D. L. Mills, Ed. (North-Holland, Amsterdam, 1982), Chap. 9.
  11. E. Fontana, R. H. Pantell, “Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy,” Phys. Rev. B 37, 3164–3182 (1988).
  12. D. S. Campbell, “Mechanical Properties of Thin Films,” in Handbook of Thin Film Technology, L. I. Maissel, R. Glang, Eds. (McGraw-Hill, New York, 1970), p. 12.30.
  13. D. S. Campbell, Ref. 12, p. 12.9.
  14. J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.
  15. D. Fasman, Ed., Handbook of Biochemistry and Molecular Biology: Proteins Vol III. (CRC Press, Cleveland1976), pp. 372–380.
  16. N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.
  17. C. Bonaventura, J. Bonaventura, “Respiratory Pigments: Structure and Function,” in The Mollusca, P. W. Hochachka, Ed. (Academic, New York, 1983), Vol. 2, pp. 1–50.
  18. B. Blomback, L. A. Hanson, Eds. Plasma Proteins, (Wiley, New York, 1976), p. 45.
  19. I. Lundstrom, “Models of Protein Adsorption on Solid Surfaces,” Prog. Colloid Polym. Sci. 82, 70–76 (1985).

1988 (1)

E. Fontana, R. H. Pantell, “Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy,” Phys. Rev. B 37, 3164–3182 (1988).

1985 (1)

I. Lundstrom, “Models of Protein Adsorption on Solid Surfaces,” Prog. Colloid Polym. Sci. 82, 70–76 (1985).

1984 (1)

M. T. Flanagan, R. H. Pantell, “Surface Plasmon Resonance and Immunosensors,” Electron. Lett. 20, 968–970 (1984).
[CrossRef]

1983 (1)

B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
[CrossRef]

1978 (1)

I. Pockrand, “Surface Plasma Oscillations at Silver Surfaces with Thin Transparent and Absorbing Coatings,” Surf. Sci. 72, 577–588 (1978).
[CrossRef]

1977 (1)

H. Raether, “Surface Plasma Oscillations and their Applications,” Phys. Thin Films 9, 145–261 (1977).

1971 (1)

E. Kretschmann, “Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingungen,” Z. Phys. 241, 313–324 (1971).
[CrossRef]

Bidwell, D. E.

A. Voller, D. E. Bidwell, “Enzyme Immunoassays,” in Alternative Immunoassays, W. P. Collins, Ed. (Wiley, New York, 1985), Chap. 6.

Bonaventura, C.

N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.

C. Bonaventura, J. Bonaventura, “Respiratory Pigments: Structure and Function,” in The Mollusca, P. W. Hochachka, Ed. (Academic, New York, 1983), Vol. 2, pp. 1–50.

Bonaventura, J.

C. Bonaventura, J. Bonaventura, “Respiratory Pigments: Structure and Function,” in The Mollusca, P. W. Hochachka, Ed. (Academic, New York, 1983), Vol. 2, pp. 1–50.

N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.

Campbell, D. S.

D. S. Campbell, “Mechanical Properties of Thin Films,” in Handbook of Thin Film Technology, L. I. Maissel, R. Glang, Eds. (McGraw-Hill, New York, 1970), p. 12.30.

D. S. Campbell, Ref. 12, p. 12.9.

Coch, E. E.

J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.

Edwards, R.

R. Edwards, Immunoassay: An Introduction (William Heinemann Medical Books, London1985), p. 3.

Flanagan, M. T.

M. T. Flanagan, R. H. Pantell, “Surface Plasmon Resonance and Immunosensors,” Electron. Lett. 20, 968–970 (1984).
[CrossRef]

Fontana, E.

E. Fontana, R. H. Pantell, “Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy,” Phys. Rev. B 37, 3164–3182 (1988).

Krafka, C.

J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.

Kretschmann, E.

E. Kretschmann, “Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingungen,” Z. Phys. 241, 313–324 (1971).
[CrossRef]

Landrum, J.

N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.

Liedberg, B.

B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
[CrossRef]

Lundstrom, I.

I. Lundstrom, “Models of Protein Adsorption on Solid Surfaces,” Prog. Colloid Polym. Sci. 82, 70–76 (1985).

B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
[CrossRef]

Lynch, D. W.

J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.

Nylander, C.

B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
[CrossRef]

O’Sullivan, M. J.

M. J. O’Sullivan, “Enzyme Immunoassay,” in Practical Immunoassay. The State of the Art, Wilfrid R. Butt, Ed. (Marcel Dekker, New York, 1984), Chap. 3.

Otto, A.

A. Otto, “Spectroscopy of Surface Polaritons by Attenuated Total Reflection,” in Optical Properties of Solids, New Developments, B. O. Seraphin, Ed. (North-Holland, Amsterdam, 1975), Chap. 13.

Pantell, R. H.

E. Fontana, R. H. Pantell, “Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy,” Phys. Rev. B 37, 3164–3182 (1988).

M. T. Flanagan, R. H. Pantell, “Surface Plasmon Resonance and Immunosensors,” Electron. Lett. 20, 968–970 (1984).
[CrossRef]

Pockrand, I.

I. Pockrand, “Surface Plasma Oscillations at Silver Surfaces with Thin Transparent and Absorbing Coatings,” Surf. Sci. 72, 577–588 (1978).
[CrossRef]

Raether, H.

H. Raether, “Surface Plasma Oscillations and their Applications,” Phys. Thin Films 9, 145–261 (1977).

H. Raether, “Surface Plasmons and Roughness,” in Surface Polaritons, V. M. Agranovich, D. L. Mills, Ed. (North-Holland, Amsterdam, 1982), Chap. 9.

Senozam, N. M.

N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.

Voller, A.

A. Voller, D. E. Bidwell, “Enzyme Immunoassays,” in Alternative Immunoassays, W. P. Collins, Ed. (Wiley, New York, 1985), Chap. 6.

Weaver, J. H.

J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.

Electron. Lett. (1)

M. T. Flanagan, R. H. Pantell, “Surface Plasmon Resonance and Immunosensors,” Electron. Lett. 20, 968–970 (1984).
[CrossRef]

Phys. Rev. B (1)

E. Fontana, R. H. Pantell, “Characterization of Multilayer Rough Surfaces by Use of Surface-Plasmon Spectroscopy,” Phys. Rev. B 37, 3164–3182 (1988).

Phys. Thin Films (1)

H. Raether, “Surface Plasma Oscillations and their Applications,” Phys. Thin Films 9, 145–261 (1977).

Prog. Colloid Polym. Sci. (1)

I. Lundstrom, “Models of Protein Adsorption on Solid Surfaces,” Prog. Colloid Polym. Sci. 82, 70–76 (1985).

Sens. Actuators (1)

B. Liedberg, C. Nylander, I. Lundstrom, “Surface Plasmon Resonance for Gas Detection and Biosensing,” Sens. Actuators 4, 299–304 (1983).
[CrossRef]

Surf. Sci. (1)

I. Pockrand, “Surface Plasma Oscillations at Silver Surfaces with Thin Transparent and Absorbing Coatings,” Surf. Sci. 72, 577–588 (1978).
[CrossRef]

Z. Phys. (1)

E. Kretschmann, “Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflachenplasmaschwingungen,” Z. Phys. 241, 313–324 (1971).
[CrossRef]

Other (12)

H. Raether, “Surface Plasmons and Roughness,” in Surface Polaritons, V. M. Agranovich, D. L. Mills, Ed. (North-Holland, Amsterdam, 1982), Chap. 9.

R. Edwards, Immunoassay: An Introduction (William Heinemann Medical Books, London1985), p. 3.

A. Voller, D. E. Bidwell, “Enzyme Immunoassays,” in Alternative Immunoassays, W. P. Collins, Ed. (Wiley, New York, 1985), Chap. 6.

M. J. O’Sullivan, “Enzyme Immunoassay,” in Practical Immunoassay. The State of the Art, Wilfrid R. Butt, Ed. (Marcel Dekker, New York, 1984), Chap. 3.

A. Otto, “Spectroscopy of Surface Polaritons by Attenuated Total Reflection,” in Optical Properties of Solids, New Developments, B. O. Seraphin, Ed. (North-Holland, Amsterdam, 1975), Chap. 13.

D. S. Campbell, “Mechanical Properties of Thin Films,” in Handbook of Thin Film Technology, L. I. Maissel, R. Glang, Eds. (McGraw-Hill, New York, 1970), p. 12.30.

D. S. Campbell, Ref. 12, p. 12.9.

J. H. Weaver, C. Krafka, D. W. Lynch, E. E. Coch, “Physics Data: Optical Properties of Metals Part. I (Fachinformationszentrum, FRG1981) pp. 62–65.

D. Fasman, Ed., Handbook of Biochemistry and Molecular Biology: Proteins Vol III. (CRC Press, Cleveland1976), pp. 372–380.

N. M. Senozam, J. Landrum, J. Bonaventura, C. Bonaventura, “Hemocyanin of the Giant Keyhold Limpet, Megathura crenulata,” in: Invertebrate Oxygen Binding Proteins, J. Lamy, J. Lamy, Eds. (Marcel Dekker, New York, 1981), pp. 703–718.

C. Bonaventura, J. Bonaventura, “Respiratory Pigments: Structure and Function,” in The Mollusca, P. W. Hochachka, Ed. (Academic, New York, 1983), Vol. 2, pp. 1–50.

B. Blomback, L. A. Hanson, Eds. Plasma Proteins, (Wiley, New York, 1976), p. 45.

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Figures (11)

Fig. 1
Fig. 1

Surface plasmon at an interface separating two semi-infinite regions, one of which is a metal. The maximum amplitude occurs at the interface and falls off exponentially within each region. The wave has an electric field normal to the boundary, and propagates along the x-direction with a phase velocity smaller than the velocity of light in the dielectric medium. At optical wavelengths the metal has a permittivity that is negative; whereas, the of the dielectric is positive. This condition is necessary to establish the surface plasma wave.

Fig. 2
Fig. 2

Prism method to couple a laser beam to a SP. The probing laser beam polarized in the x-z plane strikes the prism base coated with a metal film a few hundred angstroms thick. The prism provides the conditions for the existence of an evanescent wave within the dielectric medium if the inequality n1 > n2 is satisfied. Surface plasmon excitation is observed as a dip in the reflected intensity, and the minimum occurs when the phase velocity of the incident wave in the plane of the interface equals the SP phase velocity.

Fig. 3
Fig. 3

Surface plasmon immunoassay (SPI) principle. (a) The silver film is exposed to an aqueous solution containing antigen proteins. The resonance curve for a metal–solution configuration will be shifted to larger angles with respect to the one obtained in air because of a larger value for the solution refractive index, as illustrated in (b). Next, the metal surface with the antigen coating is placed in contact with a solution containng antibodies, which upon binding will further shift the resonance curve as indicated in (d). Measuring the reflected intensity vs time at the angle θ = θSP where θSP is the resonance angle before antibody attachment, yields the plot depicted in (e). The initial slope of the reflectance vs time plot is proportional to the antibody concentration in solution.

Fig. 4
Fig. 4

Experimental apparatus for measuring antibody concentration. A rotation stage provides the relative movements of the prism with respect to the incoming laser beam, around an axis normal to the incidence plane. The light source is a He–Ne laser tube operating at λ = 6328 Å and the glass substrate is coated with a Ag film ≈ 500 Å thick. The laser beams entering and exiting the prism are collected by a pair of photodetectors, and the ratio between the photocurrents is obtained by the ratiometer, thus eliminating fluctuations from the laser source. The ratio signal is read out and/or sent to the strip chart recorder where the reflectance vs time curves can be plotted.

Fig. 5
Fig. 5

Details of the contact between metal surface and cavity. The O-ring is sandwiched between metal surface and cavity. The cavity volume is 400 μ1.

Fig. 6
Fig. 6

Thin metal film stability design. (a) Silver film 500 Å thick on top of glass substrate, with cavity O-ring pressed against the surface. The damage ring is caused by removal of part of the Ag surface which adheres to the rubber O-ring; (b) same as in (a) with a 30 Å Cr layer under the Ag film; (c) surface damage is eliminated by depositing circular metal spots on the glass substrate having diameter smaller than the O-ring diameter, thus avoiding contact between metal surface and O-ring.

Fig. 7
Fig. 7

Differential reflectance, obtained by subtracting the measured value at time t from the corresponding value at t = 0, vs time for a Cr-Ag bilayer exposed to KLH, BGG and DNP-BSA protein solutions all at 1 mg/ml. The laser beam incidence angle was fixed close to the resonance value for the metal in contact with a clean saline solution. The rinsing induced desorption is indicated as a drop in reflectance for the KLH and DNP-BSA cases.

Fig. 8
Fig. 8

(a) Resonance curves before and after KLH deposition. The dots and continuous curves are the data and theoretical predictions, respectively. Calculated curves were obtained using the parameters given in Table I. The measurements were performed on glass slides that received more than one metal deposition, with the films chemically removed between depositions with an NH3OH solution. (b) Data and theoretical prediction for the metal-saline solution resonance curves, for a glass substrate that received a single metalization.

Fig. 9
Fig. 9

Molecular states of gastropod hemocyanin, which include Keyhole limpets, found under neutral pH conditions. The basic structure, labeled as 1/1 is a hollow cylinder having a pair of collar substructures. The 1/10 molecule is a well-defined fraction of the 1/1 molecule, which retains one molecular residue of the collar substructure. The 1/2 and 1 1/2 molecular structures are similar to the 1/1 molecule, having heights of 1/2 and 1 1/2 of the corresponding height of the 1/1 molecule, respectively. The molecular dimensions were extracted from the electron micrographs shown in Figs. 9 and 11 of Ref. 17.

Fig. 10
Fig. 10

Differential reflectance vs time for KLH coated Cr-Ag surface exposed to antiserum solution containing KLH and DNP antibodies, for varying ten-fold dilutions between 1:10–1:10,000. For this set of measurements the incidence angle was tuned close to the metal–KLH-saline solution resonance condition.

Fig. 11
Fig. 11

(a) Differential reflectance as a function of time for three different coatings exposed to a 140-fold diluted antiserum solution containing the KLH and DNP antibodies. (b) Differential reflectance vs time for a BGG coating exposed to two different antibody solutions.

Tables (3)

Tables Icon

Table I Experimental Parameters

Tables Icon

Table II Average Thickness and Standard Deviation for Different Proteins Adsorbed on Ag Surface

Tables Icon

Table III Heights for Each of the Molecular Structures Depicted In Fig. 9, and for the Case of a Cylindrical (A) or Flat Surface (B) of the Molecule Parallel to the Metal Surface

Equations (5)

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θ c = sin - 1 ( n 2 n 1 ) ,
v x = v SP ,
v x = c n 1 sin θ ,
Δ k SP = ( 2 π λ ) 1 n s 1 / 2 ( 1 - ( n s n c ) 2 ) ( n c 2 + n s 2 + ) ( n s 2 - n s 2 ) 2 2 π d λ ,
Δ θ = 1             1 n 1 cos θ SP n s 1 / 2 ( 1 - ( n s n c ) 2 ) ( n c 2 + n s 2 + ) ( n s 2 - n s 2 ) 2 2 π d λ ,

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