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A novel method of visual detection of antigens and antibodies by means of light interference
Reactive Polymers, 15 (1991) 147-151
147
Elsevier Science Publishers B.V., Amsterdam
A NOVEL METHOD OF VISUAL DETECTION OF ANTIGENS AND ANTIBODIES BY MEANS OF LIGHT INTERFERENCE TAKASHI S H I R O * , TAKEYUKI KAWAGUCHI and KAORU IWATA
Tokyo Research Center, Teo'in Ltd., 4-3-2 Asahigaoka, Hino, Tokyo 191 (Japan) (Received September 25, 1990; accepted in revised form March 20, 1991)
A novel method for detection of antigens and antibodies is proposed The detection is based on the color change due to the light interference upon the antigen-antibody reaction with an antibody (or antigen) immobilized on a substrate with a light interference layer By theoretical analysis, the application of a light-reflecting layer (colloidal gold) to the device after the reaction was found to be effective to visualization and contrast. With immobilized anti-human lgG, 1 • 10-7 g / m l of human IgG was successfully detected. Keywords: immunosensor; optical; light-interference; antigen; antibody
INTRODUCTION From the biomedical viewpoint, growing attention has been paid to methods for the detection of antigens and antibodies utilizing various techniques [1-3]. Langmuir and Schaefer proposed a method for visual detection of antigens by means of light interference [4]. The detection is based on the color change due to light interference before and after the reaction of the immobilized antibody with the antigen in an aqueous solution. The glancing angle, however, was limited. To avoid the problem, Giaever [5] and Nygren et al. [6] employed a low reflecting substrate. Although the problem of glancing angles has been over-
• To whom correspondence should be addressed,
come, these methods still have a limitation in the contrast of the interference color. In order to improve the methods in terms of both glancing angles and contrast, we analyzed the layered structure of the device proposed by Langmuir and found that the application of a light-reflecting layer (colloidal gold) to the device after antigen-antibody reaction gave an improved response. According to this method, 1 • 10-7 g / m l of human IgG could be visually detected even from the normal direction.
THEORY Figure 1 schematically shows a device composed of a light-reflecting substrate (c), a light-interference layer (b) and an immobi-
0923-1137/91/$03.50 © 1991 - Elsevier Science Publishers B.V. All rights reserved
148
Io
Ia
Io
e
Fig. 1. Illustration of the device composed of a light-refleeting substrate (c), a light-interference layer (b) and
an immobilizedantibody layer (a). lized antibody layer (a). Supposing the refractive indices of layers a and b are the same, the reflection of light from the layer b can be neglected. As a result, the light ( I a) reflecting from layer a interferes only with that (Ic) from the substrate and when the intensity and phase of I a and Ic match each other, the most effective interference occurs. The wavelength of the interfering light can be obtained according to the following equation:
plate and silicon wafer) by r.f. sputtering (400 W) in 7 • 10-1 Pa argon gas. The thickness of the light-interference layer was typically 120 nm. The gold layer of the type C device was prepared by d.c. sputtering (50 W) in 4 . 1 0 -1 Pa argon gas. Anti-human IgG was immobilized together with methyl arachidate according to the modified LB method [7,8]. The mixed monomolecular film of anti-human IgG and methyl arachidate was formed in a phosphate buffer solution (10 m M , pH 7.6) under a surface pressure of 1 m N / m . The film was then transferred onto the S i O 2 layer (110 nm) under a surface pressure of 10 m N / m by using the conventional vertical dipping method. Detection of human IgG was carried out by immersing the device with immobilized anti-human IgG in a phosphate buffer solution (10 m M , pH 7.6) of human IgG at 25 o C for 2 h. The device was then immersed in a borate buffer solution of colloidal gold for 1 h.
L = ( m + l/2))~ ( m = 0 , 1 , 2 , 3 . . . )
RESULTS AND D I S C U S S I O N
where L is the difference in the optical path length. When the device is immersed in an aqueous solution containing an antigen, the thickness of layer a increases owing to the antigen-antibody reaction resultirig in a change in the interference color,
Theoreticalanalysis
EXPERIMENTAL Human immunogloblin-G (human IgG) was supplied by the Chemo-Sero-Therapeutic Research Institute. Anti-human IgG and colloidal gold (diameter: 5 nm) were commercially available. Three model devices were prepared: Ferrotype plate/SiO2 layer (type A); silicon wafer/SiO a layer (type B); and Ferrotype plate/ SiO 2 layer/gold layer (type C). The S i O 2 used for the light-interference layer was deposited on substrates (Ferrotype
To understand the phenomena of light interference with respect to the layered structure, we performed a computer analysis. On the assumption that the refractive indices of the antibody (a) and the light interference (b) layers in Fig. 1 are the same, we simplified the model to a device composed of only an interference layer and a light-reflecting substrate. According to optical theory [9], the reflectance R can be calculated from the following equation: R = (RS+ RP)/2 2 R = I = r 1 q- rE exp(iS) To I qSr~-~2~) 8 = 4~rdE(n 2 - n l 2 sin20)1/2/X
149
where S and P denote the S- and P-polarized light; r 1 and r2 the reflection coefficients at the interface between air and the interference layer and between the interference layer and the substrate; n 1 and n 2 the refractive indices of air and the interference layer; d 2 thickness of the interference layer; ~, and 0 the wavelength and angle of incident light, respectively, Figure 2 shows the calculated reflectance spectra of a model of Ferrotype/SiO 2 (type A) as a function of incident angle. Here, refractive indices (n) and extinction indices (k) used in the calculation were measured by ellipsometry. Below 55 °, the spectra were almost flat indicating that little interference effect was observed. Above 75 ° the spectra showed a valley around 600 n m indicating development of a blue color due to an interference effect. The depth which determined the clarity of the interference color increased as the angle increased. The valley was deepest at 85 °. It should be noted that the m i n i m u m reflectance was high even at an incident angle of 85 ° indicating that the effect of interference is not enough. To match the light reflecting from the SiO 2 layer and that from the substrate, i.e., to obtain more effective interference, we either employed a low reflecting silicon wafer as the substrate or applied a light reflecting layer to the device. We 1.0 85 ° 0.8.~..... °°% °°o ~°~== ~oo om
o
=
55
ca
°A
oO~°°~...... o0° o° 75 °
°9°
qa o a
o
~eo o
~A A=
o
a~, A
Simulation by model device
°~,,~°o2S'-.. . . . "" ~ooooo"
.~6.ooo~.o=oo0OOoo°°
e =o ° 0.2
0.D
4oo
,
I
soo
,
i
6oo
,
i
~oo
Wavelength(rim)
,
i
8oo
,
9oo
Fig. 2. Calculated reflectance spectra of a type A device as a function of incident angles: F e r r o t y p e / S i O 2 (120
nm).
analyzed the spectra of them, i.e., silicon wafer/SiO 2 (type B) and F e r r o t y p e / S i O z / A u (type C). Figure 3 shows the calculated reflectance spectra for the optimized incident angles. The model device of type B showed only a flat spectrum with low reflectance. On the contrary, the model device of type C showed a sharper spectrum than that of type A indicating that the former is clearer than the latter. The calculated data on the optimum incident angle indicated that the interference color of type C is observable even from the normal direction different from that of type A. From the analysis, we conclude that type C is superior to the other types from the point of both the glancing angle and the clarity of the interference color. To simulate the color change upon the antigen-antibody reaction, we further calculated the reflectance spectra of the model device of type C with various thicknesses of the SiO 2 layer. Figure 4 shows the results. The valley shifted to the longer wavelength region u p o n an increment of only 10 nm indicating that the color change is sensitive to such a slight increment in thickness. To elucidate the color change more quantitatively, we investigated the dependence of the reflectance on the increment of the thickness of the S i O 2 layer (Fig. 5). The dependence of the devices of types A and B was slight, while that of type C was dramatic, and that over a wide range of thicknesses. These results suggest that the various increments in thickness due to the antigen-antibody reaction are detectable with high contrast.
In order to confirm the theoretical analysis, we prepared model devices of types A, B and C. Figure 6 shows photographs of them. With type A, a blue color was observed only from the highly tilted direction. With type B, a dark-blue color was discernible even from the normal direction but with difficulty. With
150 1.0
1.0
Type A
OOoo
~'
•
0.6
o
~
o
rypeC~
i "o •
& ,~ 0.4
=o g0
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o
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~ ; 600 700 Wavelength(rim)
ooOOO
c ~
& &
=~=~
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o~ 0.6
o o A %oo° 0 AA&
z~ &
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n-
o°
o
DDg Dg DD
~ j~ ~, ~' o & a a a [] a a a Type B
2r0 4~0 1'0 3'0 Increment of thickness (rim)
50
Fig. 3. Calculated reflectance spectra of devices of types A, B and C: type A, Ferrotype/SiO2 (120 nm), e = 85 o; type B, silicon wafer/SiO2 (120 nm), 0 = 0 ° ; type C, Ferrotype/SiO2 (120 nm)/Au (10 rim), 8 = 0 o.
Fig. 5. Calculated reflectance versus the thickness of the si02 layer: type A, Ferrotype/SiO2 (120 nm), 8 = 85 o; type B, silicon wafer/ SiO2 (120 nm), O=0°; type C, Ferrotype/SiO2 (140 nm)/Au (10 nm), O= 0 o.
type C, on the contrary, a bright blue color was clearly observed even from the normal direction. The results correspond well to those obtained b y the computer analysis.
light-reflecting layer. The immobilization of anti-human I g G and the procedure of detection are given in the experimental section. Figure 7 shows the photographs of the device with (type C) and without (type A) colloidal gold treatment after a n t i g e n - a n t i b o d y reac-
Detection of antigen-antibody reaction Based on the above analysis, we investigated the detection of h u m a n IgG b y utilizing a device with immobilized anti-human I gG. We used Ferrotype as the substrate, a thin film of SiO2 (110 n m thick) as the interference layer and colloidal gold with an average diameter of 5 nm as the material for the
Type A ~
==
II,
Type B
1.o
~
~~,.,,,,,,,,~ SiO2 layer (120nm) ~ ~ 0 ~ / ~ ~ /
1~
SiO2 layer (120rim) ] :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
0.6
Type
C
c
~.
SiO2 layer (120rim) 0.2
0.¢ 400
si02; 120nm
'
500
~
' 600
'
700
Wavelength(rim)
800
~
S
I
900
Fig. 4. Calculated reflectance spectra of a type C device as a function of the thickness of the SiO2 layer: Ferrotype/SiO2/Au (10 rim), 0 = 0 °.
I
~
Fig. 6. Photographs of the model devices of types A, B and C: type A, Ferrotype/SiO2 (120 nm), 0 = 85 o; type B, silicon wafer/SiO2 (120 rim), 0 = 0 o; type C, Ferrotype/SiO2 (120 nm)/Au (10 rim), 0 = 0 °.
151 TypeA
i~-. i
II1
~, ~ -,._iIF;_, I I ~
I I
layer to a L a n g m u i r type device is effective to b o t h glancing angle a n d clearness of the interference color. T h e idea was successfully applied to an actual device with immobilized a n t i - h u m a n IgG. W i t h the device, 1 . 1 0 -7 g / m l of h u m a n I g G was successfully detected.
Type C
ACKNOWLEDGMENT
~-.~I III )~< II ~ I i ! Fig. 7. Photographs of the devices of types A and C: portion I, the surface with no immobilized antihuman IgG; portion II, the surface with immobilized anti-human IgG; portion III, the surface after the antigen-antibody reaction with human-IgG (1.10 -6 g/ml). tion. W i t h type A, a clear color was observed o n l y f r o m the highly tilted direction. W i t h type C, on the contrary, we observed clear light blue on the p o r t i o n of i m m o b i l i z e d antib o d y a n d clear d a r k blue on the p o r t i o n which was treated with a solution c o n t a i n i n g h u m a n IgG. The color difference was clearly detected even f r o m the n o r m a l direction of the device. These results indicate that effective light interference occurred on the t y p e C device, as expected f r o m the c o m p u t e r analysis. The sensitivity to h u m a n I g G was estimated. T h e sensitivity was d e f i n e d as the lowest c o n c e n t r a t i o n of h u m a n I g G solution visually detectable by the device. T h e sensitivity of the device with colloidal gold t r e a t m e n t was m e a s u r e d to be 1 • 10 -7 g / m l , while t h a t of the u n t r e a t e d device was 1 ° 10 -6 g / m l . This result shows t h a t the application of a colloidal gold layer is effective for sensitivity enhancement. CONCLUSION F r o m the theoretical analysis, we f o u n d that the application of the l i g h t - r e f l e c t i n g
T h e a u t h o r s t h a n k Dr. K. C h i b a for his suggestion on the c o m p u t e r calculation.
REFERENCE 1 L. Vroman, A.L. Adams and M. Klings, Interactions among human blood proteins at interfaces, Fed. Proc., 30 (1971) 1494. 2 N. Yamamoto, Y. Nagasawa, M. Sawai, T. Sudo and H. Tsubomura, Potentiometric investigations of antigen-antibody and enzyme-enzyme inhibitor reactions using chemically modified metal electrodes, J. Immunol. Methods, 22 (1978)309. 3 R.M. Sutherland, C. Dahne, J.F. Place and A.R. Ringrose, Optical detection of antibody-antigen reactions at a glass-liquid interface, Clin. Chem., 30 (1984) 1533. 4 I. Langmuir and V.J. Schaefer, Monolayers and multilayers of chlorophyll, J. Am. Chem. Soc., 59 (1937) 2075. 5 I. Giaever, Visual detection of carcinoembryonic antigen on surfaces, J. Immunol., 116 (1976) 766. 6 B.H. Nygren, E.T. Sandstr~m, J.E. Stenberg and L.B. Stiblert, Laminate comprising multiple layers and detection and/or measurement of the concentration of a chemical substance of a biological origin, Ger. Pat. DE 3,215,484, 1983. 7 P. Frornherz, New technique for investigating lipid protein fills, Biochim. Biophys. Acta. 225 (1971) 382. 8 T. Moriizumi, M. Sriyudthsak and Y. Ogami, Sensor and LB technology, Hyomen, 25 (1981) 242. 9 M. Born and E. Wolf, Principles of Optics, Pergamon, New York, 1964.
147
Elsevier Science Publishers B.V., Amsterdam
A NOVEL METHOD OF VISUAL DETECTION OF ANTIGENS AND ANTIBODIES BY MEANS OF LIGHT INTERFERENCE TAKASHI S H I R O * , TAKEYUKI KAWAGUCHI and KAORU IWATA
Tokyo Research Center, Teo'in Ltd., 4-3-2 Asahigaoka, Hino, Tokyo 191 (Japan) (Received September 25, 1990; accepted in revised form March 20, 1991)
A novel method for detection of antigens and antibodies is proposed The detection is based on the color change due to the light interference upon the antigen-antibody reaction with an antibody (or antigen) immobilized on a substrate with a light interference layer By theoretical analysis, the application of a light-reflecting layer (colloidal gold) to the device after the reaction was found to be effective to visualization and contrast. With immobilized anti-human lgG, 1 • 10-7 g / m l of human IgG was successfully detected. Keywords: immunosensor; optical; light-interference; antigen; antibody
INTRODUCTION From the biomedical viewpoint, growing attention has been paid to methods for the detection of antigens and antibodies utilizing various techniques [1-3]. Langmuir and Schaefer proposed a method for visual detection of antigens by means of light interference [4]. The detection is based on the color change due to light interference before and after the reaction of the immobilized antibody with the antigen in an aqueous solution. The glancing angle, however, was limited. To avoid the problem, Giaever [5] and Nygren et al. [6] employed a low reflecting substrate. Although the problem of glancing angles has been over-
• To whom correspondence should be addressed,
come, these methods still have a limitation in the contrast of the interference color. In order to improve the methods in terms of both glancing angles and contrast, we analyzed the layered structure of the device proposed by Langmuir and found that the application of a light-reflecting layer (colloidal gold) to the device after antigen-antibody reaction gave an improved response. According to this method, 1 • 10-7 g / m l of human IgG could be visually detected even from the normal direction.
THEORY Figure 1 schematically shows a device composed of a light-reflecting substrate (c), a light-interference layer (b) and an immobi-
0923-1137/91/$03.50 © 1991 - Elsevier Science Publishers B.V. All rights reserved
148
Io
Ia
Io
e
Fig. 1. Illustration of the device composed of a light-refleeting substrate (c), a light-interference layer (b) and
an immobilizedantibody layer (a). lized antibody layer (a). Supposing the refractive indices of layers a and b are the same, the reflection of light from the layer b can be neglected. As a result, the light ( I a) reflecting from layer a interferes only with that (Ic) from the substrate and when the intensity and phase of I a and Ic match each other, the most effective interference occurs. The wavelength of the interfering light can be obtained according to the following equation:
plate and silicon wafer) by r.f. sputtering (400 W) in 7 • 10-1 Pa argon gas. The thickness of the light-interference layer was typically 120 nm. The gold layer of the type C device was prepared by d.c. sputtering (50 W) in 4 . 1 0 -1 Pa argon gas. Anti-human IgG was immobilized together with methyl arachidate according to the modified LB method [7,8]. The mixed monomolecular film of anti-human IgG and methyl arachidate was formed in a phosphate buffer solution (10 m M , pH 7.6) under a surface pressure of 1 m N / m . The film was then transferred onto the S i O 2 layer (110 nm) under a surface pressure of 10 m N / m by using the conventional vertical dipping method. Detection of human IgG was carried out by immersing the device with immobilized anti-human IgG in a phosphate buffer solution (10 m M , pH 7.6) of human IgG at 25 o C for 2 h. The device was then immersed in a borate buffer solution of colloidal gold for 1 h.
L = ( m + l/2))~ ( m = 0 , 1 , 2 , 3 . . . )
RESULTS AND D I S C U S S I O N
where L is the difference in the optical path length. When the device is immersed in an aqueous solution containing an antigen, the thickness of layer a increases owing to the antigen-antibody reaction resultirig in a change in the interference color,
Theoreticalanalysis
EXPERIMENTAL Human immunogloblin-G (human IgG) was supplied by the Chemo-Sero-Therapeutic Research Institute. Anti-human IgG and colloidal gold (diameter: 5 nm) were commercially available. Three model devices were prepared: Ferrotype plate/SiO2 layer (type A); silicon wafer/SiO a layer (type B); and Ferrotype plate/ SiO 2 layer/gold layer (type C). The S i O 2 used for the light-interference layer was deposited on substrates (Ferrotype
To understand the phenomena of light interference with respect to the layered structure, we performed a computer analysis. On the assumption that the refractive indices of the antibody (a) and the light interference (b) layers in Fig. 1 are the same, we simplified the model to a device composed of only an interference layer and a light-reflecting substrate. According to optical theory [9], the reflectance R can be calculated from the following equation: R = (RS+ RP)/2 2 R = I = r 1 q- rE exp(iS) To I qSr~-~2~) 8 = 4~rdE(n 2 - n l 2 sin20)1/2/X
149
where S and P denote the S- and P-polarized light; r 1 and r2 the reflection coefficients at the interface between air and the interference layer and between the interference layer and the substrate; n 1 and n 2 the refractive indices of air and the interference layer; d 2 thickness of the interference layer; ~, and 0 the wavelength and angle of incident light, respectively, Figure 2 shows the calculated reflectance spectra of a model of Ferrotype/SiO 2 (type A) as a function of incident angle. Here, refractive indices (n) and extinction indices (k) used in the calculation were measured by ellipsometry. Below 55 °, the spectra were almost flat indicating that little interference effect was observed. Above 75 ° the spectra showed a valley around 600 n m indicating development of a blue color due to an interference effect. The depth which determined the clarity of the interference color increased as the angle increased. The valley was deepest at 85 °. It should be noted that the m i n i m u m reflectance was high even at an incident angle of 85 ° indicating that the effect of interference is not enough. To match the light reflecting from the SiO 2 layer and that from the substrate, i.e., to obtain more effective interference, we either employed a low reflecting silicon wafer as the substrate or applied a light reflecting layer to the device. We 1.0 85 ° 0.8.~..... °°% °°o ~°~== ~oo om
o
=
55
ca
°A
oO~°°~...... o0° o° 75 °
°9°
qa o a
o
~eo o
~A A=
o
a~, A
Simulation by model device
°~,,~°o2S'-.. . . . "" ~ooooo"
.~6.ooo~.o=oo0OOoo°°
e =o ° 0.2
0.D
4oo
,
I
soo
,
i
6oo
,
i
~oo
Wavelength(rim)
,
i
8oo
,
9oo
Fig. 2. Calculated reflectance spectra of a type A device as a function of incident angles: F e r r o t y p e / S i O 2 (120
nm).
analyzed the spectra of them, i.e., silicon wafer/SiO 2 (type B) and F e r r o t y p e / S i O z / A u (type C). Figure 3 shows the calculated reflectance spectra for the optimized incident angles. The model device of type B showed only a flat spectrum with low reflectance. On the contrary, the model device of type C showed a sharper spectrum than that of type A indicating that the former is clearer than the latter. The calculated data on the optimum incident angle indicated that the interference color of type C is observable even from the normal direction different from that of type A. From the analysis, we conclude that type C is superior to the other types from the point of both the glancing angle and the clarity of the interference color. To simulate the color change upon the antigen-antibody reaction, we further calculated the reflectance spectra of the model device of type C with various thicknesses of the SiO 2 layer. Figure 4 shows the results. The valley shifted to the longer wavelength region u p o n an increment of only 10 nm indicating that the color change is sensitive to such a slight increment in thickness. To elucidate the color change more quantitatively, we investigated the dependence of the reflectance on the increment of the thickness of the S i O 2 layer (Fig. 5). The dependence of the devices of types A and B was slight, while that of type C was dramatic, and that over a wide range of thicknesses. These results suggest that the various increments in thickness due to the antigen-antibody reaction are detectable with high contrast.
In order to confirm the theoretical analysis, we prepared model devices of types A, B and C. Figure 6 shows photographs of them. With type A, a blue color was observed only from the highly tilted direction. With type B, a dark-blue color was discernible even from the normal direction but with difficulty. With
150 1.0
1.0
Type A
OOoo
~'
•
0.6
o
~
o
rypeC~
i "o •
& ,~ 0.4
=o g0
%a
0.2
°'Coo
o
~
A~ a
soo
--~ ~
A A
= soo
ooooooooooooo AA A~
0.4
&
~ ~e ~
&A &
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0.C
900
°
TypeC
0.2
~ ; 600 700 Wavelength(rim)
ooOOO
c ~
& &
=~=~
Type A
o~ 0.6
o o A %oo° 0 AA&
z~ &
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n-
o°
o
DDg Dg DD
~ j~ ~, ~' o & a a a [] a a a Type B
2r0 4~0 1'0 3'0 Increment of thickness (rim)
50
Fig. 3. Calculated reflectance spectra of devices of types A, B and C: type A, Ferrotype/SiO2 (120 nm), e = 85 o; type B, silicon wafer/SiO2 (120 nm), 0 = 0 ° ; type C, Ferrotype/SiO2 (120 nm)/Au (10 rim), 8 = 0 o.
Fig. 5. Calculated reflectance versus the thickness of the si02 layer: type A, Ferrotype/SiO2 (120 nm), 8 = 85 o; type B, silicon wafer/ SiO2 (120 nm), O=0°; type C, Ferrotype/SiO2 (140 nm)/Au (10 nm), O= 0 o.
type C, on the contrary, a bright blue color was clearly observed even from the normal direction. The results correspond well to those obtained b y the computer analysis.
light-reflecting layer. The immobilization of anti-human I g G and the procedure of detection are given in the experimental section. Figure 7 shows the photographs of the device with (type C) and without (type A) colloidal gold treatment after a n t i g e n - a n t i b o d y reac-
Detection of antigen-antibody reaction Based on the above analysis, we investigated the detection of h u m a n IgG b y utilizing a device with immobilized anti-human I gG. We used Ferrotype as the substrate, a thin film of SiO2 (110 n m thick) as the interference layer and colloidal gold with an average diameter of 5 nm as the material for the
Type A ~
==
II,
Type B
1.o
~
~~,.,,,,,,,,~ SiO2 layer (120nm) ~ ~ 0 ~ / ~ ~ /
1~
SiO2 layer (120rim) ] :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
0.6
Type
C
c
~.
SiO2 layer (120rim) 0.2
0.¢ 400
si02; 120nm
'
500
~
' 600
'
700
Wavelength(rim)
800
~
S
I
900
Fig. 4. Calculated reflectance spectra of a type C device as a function of the thickness of the SiO2 layer: Ferrotype/SiO2/Au (10 rim), 0 = 0 °.
I
~
Fig. 6. Photographs of the model devices of types A, B and C: type A, Ferrotype/SiO2 (120 nm), 0 = 85 o; type B, silicon wafer/SiO2 (120 rim), 0 = 0 o; type C, Ferrotype/SiO2 (120 nm)/Au (10 rim), 0 = 0 °.
151 TypeA
i~-. i
II1
~, ~ -,._iIF;_, I I ~
I I
layer to a L a n g m u i r type device is effective to b o t h glancing angle a n d clearness of the interference color. T h e idea was successfully applied to an actual device with immobilized a n t i - h u m a n IgG. W i t h the device, 1 . 1 0 -7 g / m l of h u m a n I g G was successfully detected.
Type C
ACKNOWLEDGMENT
~-.~I III )~< II ~ I i ! Fig. 7. Photographs of the devices of types A and C: portion I, the surface with no immobilized antihuman IgG; portion II, the surface with immobilized anti-human IgG; portion III, the surface after the antigen-antibody reaction with human-IgG (1.10 -6 g/ml). tion. W i t h type A, a clear color was observed o n l y f r o m the highly tilted direction. W i t h type C, on the contrary, we observed clear light blue on the p o r t i o n of i m m o b i l i z e d antib o d y a n d clear d a r k blue on the p o r t i o n which was treated with a solution c o n t a i n i n g h u m a n IgG. The color difference was clearly detected even f r o m the n o r m a l direction of the device. These results indicate that effective light interference occurred on the t y p e C device, as expected f r o m the c o m p u t e r analysis. The sensitivity to h u m a n I g G was estimated. T h e sensitivity was d e f i n e d as the lowest c o n c e n t r a t i o n of h u m a n I g G solution visually detectable by the device. T h e sensitivity of the device with colloidal gold t r e a t m e n t was m e a s u r e d to be 1 • 10 -7 g / m l , while t h a t of the u n t r e a t e d device was 1 ° 10 -6 g / m l . This result shows t h a t the application of a colloidal gold layer is effective for sensitivity enhancement. CONCLUSION F r o m the theoretical analysis, we f o u n d that the application of the l i g h t - r e f l e c t i n g
T h e a u t h o r s t h a n k Dr. K. C h i b a for his suggestion on the c o m p u t e r calculation.
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