THE USE OF THE ISOSCOPE ELLIPSOMETER IN THE STUDY OF ADSORBED PROTEINS AND BIOSPECIFIC BINDING REACTIONS

HAL Id: jpa-00223474

https://hal.archives-ouvertes.fr/jpa-00223474

Submitted on 1 Jan 1983

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

THE USE OF THE ISOSCOPE ELLIPSOMETER IN THE STUDY OF ADSORBED PROTEINS AND

BIOSPECIFIC BINDING REACTIONS

M. Stenberg, H. Nygren

To cite this version:

M. Stenberg, H. Nygren. THE USE OF THE ISOSCOPE ELLIPSOMETER IN THE STUDY OF

ADSORBED PROTEINS AND BIOSPECIFIC BINDING REACTIONS. Journal de Physique Collo-

ques, 1983, 44 (C10), pp.C10-83-C10-86. �10.1051/jphyscol:19831017�. �jpa-00223474�

JOURNAL DE PHYSIQUE

Colloque CIO, supplkment au n012, T o m e 44, dkembre 1983 page C10-83

THE USE OF THE ISOSCOPE ELLIPSOMETER IN THE STUDY OF ADSORBED PROTEINS AND BIOSPECIFIC BINDING REACTIONS

M. Stenberg and H. ~ ~ ~ r e n *

Research Laboratory o f EZectronics, ChaLmers University o f TechnoZogy, S-412 96 Gothenburg, Sweden

''~epar-tment of HistoZogy, University o f Gothenburg, Boz 33031, 5-400 33 Gothenburg, Sweden

Rdsumd

-

On ddcrit quelques techniques expdrimentales pour une visua- lisation simple et rapide, ainsi que des mesures quantitatives sur des proteines adsorbdes et des r6actions de liaison biospdcifiques, uti- lisant L'ellipsomPtre lsoscope'.

Abstract

-

Some experimental techniques are described for fast and simple visualization and quantitative measurements of adsorbed proteins and biospecific binding reactions using the Isoscoper ellipsometer.

Ellipsometry has long been used as an accurate method to study the adsorption and interaction of organic molecules at a solid-liquid interface. However, in spite of its many advantages ellipsometry has not become a routine technique in biology compared to other analytical methods. The reason could be that in experimental biology a lot of controls are necessary and many parameters must be studied simultaneously which leads to large experimental volumes which are laborious to handle with conventional ellipsometers. This implies the need for simplified ellipsometric measurements.

One simplified ellipsometric method is that of the comparison ellipsometer [l,2,4.

We have used a comparison ellipsometer ( Isoscope 125, Sagax Instrument, Sundby- berg,Sweden) for some time in surface related experimental biology. The aim of the present study was to present some experimental techniques suitable for this particular instrument.

In the Isoscope ellipsometer a test surface is compared point to point to a refe- rence surface and analyzed with respect to differencies in polarization state of reflected light. The reference and sample surfaces are placed between two fixed

crossed

polarizers. A parallel light beam is subjected to subsequent reflections at both surfaces with the plane of incidence at the reference surface perpendi- cular to the plane of incidence at the sample surface. Extinction of light is thus achieved for identical surfaces [I]

.

Visualization of a bimolecular reac- tion zone can be performed by comparison to a reference surface with constant thickness. Measurements of adsorbed mass is performed by comparison to a refe- rence surface with a calibrated wedge-like thickness profile.

For measurements on silicon the reference surface film is normal'iy made of Si02 with n=1.46. For a small thickness difference~d between sample and reference surface the residual light going through the instrument can be written [21 :

1 = 1 kd(n n d )ad 2

1' 2'0

. . . .

⁽¹⁾

where I. is a constant and the sensitivity function kd is a function of substrate '~SflscODe is a registered trademark of Sagax Instrument

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19831017

JOURNAL DE PHYSIQUE

Fig.1 Picture obtained in the Isoscope ellipso- meter showing the dissociation of fibrinogen from a hydrophilic silicon surface. Fibrinogen (Sigma) was dissolved in in 0.15 M NaCl at a concentration of 0.3 mg/ml. Silicon slides (1x3 cm) were imer- sedinto the protein solution for 30 min. and then rinsed in running phosphate-buffered saline (total volume 5 1 exchanged with a flow of 100 ml/h) for varied rinsing time. From left to right: control, 30s, 15min, Ih, 4h and 16h rinsing time. Mass

. -

evaluated according Eq.(6).

index n2, film index n l and film thickness do. For silicon as substrate kd is at most varying a factor 4 for different d values (n,=1.5) and varies very little for small do values [2]. At d = 20 nm k has decreased approximately 5% compared to at

d

d =O. Due to the synnnetrical properties of Eq. (1) the central position of the extinction can be used for an accurate measurement of the thickness. Normally both thickness and refractive index should be equal in order to get extinction. However, for thin films on silicon there is very little variation in azimuth

Y

upon reflec- tion. Equality in phase difference Abetween sample and reference surface gives after using the Drude approximation:

2 2 2

dl=( 1- l/nox) dox nl / ( nl-1)

. . . .

⁽²⁾

where nl,d, are the sample refractive index and thickness and nox,dox are the SiO refractive index and thickness. Knowing the refractive index nl the amount

2

of adsorbed mass can be calculated according:

m = dl?

. ...

⁽³⁾

where

f

is the density of the adsorbed layer. For measurements in air f can be assumed to be the bulk densityfO and the bulk value of the refractive index can be calculated according the Lorentz-Lorenz relation:

2 2

~O=(MIA) (nl-l)l(nl-2)

....

⁽⁴⁾

where M is molecular weight and A is the molecular refractivity. Combining (2),(3) and (4) gives:

m=(1/3)(2~0-(M/A))(1-l/nox)dox 2

....

^{( 5 )}

In ref. [4] somg examples of calcu a t ~ n g (M/A) are shown. For albumin we get ( .f'O=1.37 g/cm

,

(N/A)= 4.12 g/cmb, ix=1.46):

m = 0.12 d

ox

. .. .

⁽⁶⁾

with m in pg/cm3 and dox in nm. This value is representative to most proteins.

We use Eq.(6) even for submolecular layers since the Drude approximation has been found relevant in this region with bulk values of refractive index

151 .

^Protein

adsorption and desor~tion studies can be carried out by simply exposing different samples to proteins and subsequent rinsing according fig.1. Working in this way a lot of different samples with different surface treatment can be studied in parallel.

One way of making a dose-respons study on the same surface is to use the so called diffusion-in-gel (DIG)-ellipsometry [6,3]

.

With this method the reactant is allowed to diffuse laterally over a surface coated with counter reactant. In the Isoscope the thickness profile can be viewed immidiately in the eye-piece as seen in fig.2. A theory for kinetic studies with this method has been worked out 131

.

a

Fig.2 Isoscope analysis of the reaction zone ob-

a

tained by diffusion of antibodies over an antigen- -coated surface.Silicon wafers were made hydropho-

9- bic by incubation in dichlorodimethylsilane. Bo-

*% vine serum albumin was adsorbed to the surface (100 ,ug/ml in saline for 2h) and an agarose gel(l%) was poured over the antigen-coated surface. A lon- gitudinal well (3mm~20mrn) was punched out with a razor blade and filled with anti-BSA antiserum containing 2 mg/ml of specific antibodies. The

$ 8 , 1 ~ 1 1 8 diffusion was terminated by removal of the serum

-

and the gel and the plate was rinsed in water and dried.The rapid thickness increase at the advan- cing front indicates a diffusion rate limited surface reaction, followed by a concentration dependent reaction.

As seen in fig.l very small variations in thickness can be detected around extinc- tion. The sensitivity in detecting small steps in thickness can be derived as fol- lows. Due to imperfections and misalignments the intensity around extinction does not follow Eq.(l). Instead a small intensity I will be present at 5d=O giving:

0

This does not alter the position of eheextinction but rather broadens the extinction line. The rest term I corresponds to film thickness difference less than Inm [2) so Eq. (7) can be wri?ten as:

where d 51nm. The eye can resolve an intensity step somewhere between 2 and 5%

differegce in light intensity. A small thickness increase 6d can thus be detected providing the resulting relative intensity change 6111 is greater than the minimal ( 61/I)min of 2 to 5%. After differentiating Eq. (7) we get the following relation:

In fig. 3 this relation is plotted together with an experimental point obtained by looking at what thickness a thickness step with known height vanishes for the eye The thickness step was accurately measured in a conventional ellipsometer (Rudolph Research 43303-2003). It can be concluded that around extinction thickness steps below 0.1 nm can be detected.

The geometrical resolution in the instrument is in the range of 50 pm due to the slight ^v divergence in the parallel light. The symmetri &

of Eq ( 1 ) makes it possible to use a slightly 20.2.- modified technique and measure thickness of _v, very small areas. If a small area with in- v,

creased thickness is compared to a reference with varying thickness the sample may be mo- ved around to a position where the light has equal intensity within the spot as at the

surroundings. In this way the spot will be

2

positioned half-way between extinction at the

3

surroundings and extinction at the spot. By measuring the distance from this position to ^W a position where the spot is situated in the

middle of the surrounding extinction a mea- ¹

.

^l

sure of the spot film thickness will be ob- ^{0 5} tu . tained if only the thickness slope at the THICKNESS DIFFERENCE (nm) reference surface is known. Figure 4 shows

the appearance of a small antibody spot Fig.3 Plot of Eq(9) with positioned in the extinction of the surroundings. ⁽ h I/I),;, as parameter

C10-86 JOURNAL DE PHYSIQUE

Fig.4 Picture showing a reaction zone formed by culturing antibody-producing lymphocytes over an antigen-coated surface. Hydrophobic silicon wafers were coated with BSA. The spleen of mice immunized with BSA, was removed and pressed through a nylon gauge. Lymphocytes were isolated by Ficoll-paque gradient centrifugation and were then incubated in culture medium gelified with 0.5% agarose. Incuba- tion time 4h. Antigen-antibody reaction zone are formed at the surface underlaying single cells or groups of cells producing specific antibodies.The big zone is a control area where all organic material is removed by chromosulfuric acid.1n this way also the antigen layer can be measured.

Acknowledgement

We thank Jan H. Elam for performing the fibri-

nogen experiment and Cecil Czersinsky BSc. for 0.5 1 .O help with the cell cultures. SPOT DIAMETER (mm) In order to check this method thin oxides were thermally grown on silicon and a pattern of small spots with different areas were etched with standard photoresist technique. The thicknesses were measured in a conventional ellipsometer at areas where the film were not removed. A calibrated thin film reference standard with slope'l.12 n m / m were used for the test and 10 measurements were made on every spot size. The distances were measured with a vernier callipers. Fig.5 shows the result from these measurements with mean values and standard deviations inserted.

It is seen that the method works down to about 150 pm in spot diameter. For thick- nesses greater than 20 nm the method tends to give a too small value in thickness.

This can be explained by the decrease in the sensitivity function kd for thicker films. The film thickness measurements were accurate withintlnm. On a 150 pm spot this implies an accuracy in mass determination of approximately 20 pg accor- ding Eq. (6). The accuracy is limited by the parallel light giving interference effects around the edges of the spots. This darkens the spot and lowers thereading according this method.By increasing the nume-

Fig.5 Calibration measurements on small Si02 spots.

rical aperture it should be possible to in-

crease the accuracy. Around extinction with 15 less light this effect is reduced so the sen- sitivity in visualization will be somewhat

better, around 5 pg.

-

REFERENCES

1. M. Stenberg, T. Sandstrom and L. Stiblert,Mat.Sci.and Eng., 42(1980)65

2. L. Stiblert, (Thesis), Tech. Rep. No 115, Chalmers Univ. Tech,Gothenburg(l981) 3. M. Stenberg, H. Nygren, Anal. Biochem., 127(1982)183

4. P.A. Cuypers et al.,J. Biol. Chem. 258(1983)2426 5. T. Smith, J. Opt. Soc. Am., 58 No8 (1968)1069 6. H. Elwing, M. Stenberg, J. Immun. Meth., 44(1981)343

- _IJ ^&

*.A

s

The sensitivity and accuracy of the Isoscope 5 1 0 - E

9 -.

ellipsometer together with its high capacity

,

makes it well suited for biological experi-

,

^-

ments aiming at the demonstration of biolo- gically significant effects regarding amount of surface bound material during desorption, bimolecular reactions and cell synthesis of reactive compounds.