Adsorption and immunoreactivity of proteins on polystyrene and on silica. Competition with surfactants

Colloids and Surfaces B. Blomterfaces, I (1993) 75-82 0927-7765/93/$06.00 fc’ 1993 ~ Elsevter Sctence Pubhshers

75 B.V. All rights reserved.

Adsorption and immunoreactivity of proteins on polystyrene and on silica. Competition with surfactants” J.W.Th. Lichtenbelt”,

W.J.M. Heuvelsland,

Akzo Research Laboratories The Netherlands (Received

11 February

Arnhem, Department

1993; accepted

M.E. Oldenzeel, of Physical Chemistry,

22 February

R.L.J. Zsom PO-Box 9300,680O

SB Arnhem,

1993)

Abstract From the competition between surfactants and proteins for adsorptton on the same surface we can learn more about protein adsorption itself. In thts work bovine serum albumin and monoclonal tmmunoglobuhn G and the antibodies or antigens of both proteins are used. and the surfactants are nomonic or amomc The techniques and the corresponding adsorbents are: reflectometry for layers of polystyrene and of sihca. the latter with varying hydrophobictty: quast-elastic light scattering for polystyrene particles. Nomomc surfactants were more effective than amomc surfactants in removing proteins. On hydrophobic surfaces nomonic surfactants can desorb proteins at concentrations around or above thetr CMC. but anionic surfactants (only applied at relatively low concentratron) did not have any effect. On hydrophilic surfaces results are less clear cut. Pretreatment of sthca by surfactant led m no case to an inhtbttton of protein adsorption. Only a small reduction was observed Protein adsorption on hydrophilic surfaces is usually weaker; nevertheless. dtsplacement by surfactant does not occur easily, because surfactants are also adsorbed weakly on these surfaces. Sometrmes a surfactant can make a hydrophihc surface hydrophobtc and enhance subsequent protein adsorptton. Key words. Adsorptton;

Diagnostics,

Latex: Ltght scattering:

Protein;

Introduction The adsorption of protein molecules on the liquid/solid interface has been studied extensively [l-7]. One way to obtain more information on the adsorption mechanism of proteins is to test the strength of attachment by using surfactants to compete with proteins for adsorption sites [S-l 11. Proteins are often adsorbed very strongly on hydrophobic surfaces, but in some cases can be desorbed easily by surfactants under appropriate conditions [12]. One can also add the surfactant *Correspondmg author. “The prelimmary form of this paper was presented at the 7th International Conference on Colloid and Surface Science held in Compttgne. France, 7-13 July 1991, and was coordinated for pubhcatton by Professors D. Muller and D. Labarre.

Reflectometry:

Silica: Surfactant

first and investigate the effect on the adsorption of the protein. In this respect surfactants are also called surface modifiers. They can make a hydrophobic surface hydrophilic, and thus make it less attractive for proteins [13,14]. In contrast, however, in our work there are examples in which surfactants make a hydrophilic surface hydrophobic, and enhance protein adsorption. By using such techniques as reflectometry for a flat substrate and quasi-elastic light scattering (QELS) in the case of latices, these influences can be studied in detail. We used as substrate both films of pure polystyrene and polystyrene latices. As a model substrate that covers a variety of wettabilities a SiOz substrate with a gradient in hydrophobicity was used. We have chosen two different nonionic surfac-

tants: Antarox

CO 630 and Tween 20. Both surfac-

tants are often used in diagnostic We also studied the influence

tests. of two anionic

surfactants: Aerosol MA, which is often present as a surfactant in latex, and sodium dodecyl sulphate, which is known to interact with proteins directly. Cationic

surfactants

were not applied.

The proteins

used in this study were bovine serum albumin (BSA) and a monoclonal antibody to the pregnancy chorion gonadotropin hormone anti-human (ahCG). In most cases the immunological interaction of hCG with ahCG was studied and in some cases the interaction of BSA with aBSA. If ionic interactions occurred, their nature was repulsive, since all materials (proteins, surfactants and surfaces) were negatively charged or had no charge. The pH was kept constant at 7.4. Time effects have not been studied. Experimental Muteriuls

Polystyrene latices were and polymer chemistry Research. Persulphate was the emulsion polymerization. (Aerosol MA) was removed

made by the organic department of Akzo used as an initiator in Most of the emulsifier by microfiltration after

the polymerization. The particle diameters are small (about 100 nm for the bare latex), so a large relative increase could be found upon adsorption of protein. Polystyrene layers were prepared from pure polystyrene (from microtiter plates of Organon Teknika, Boxtel. The Netherlands) which was dissolved in toluene at a concentration of 1.5% and spin-coated on the silicon wafer. After drying the wafer was used for reflectometric experiments. The following proteins were used: BSA type Boseral R used for adsorption on latex and Boseral DEM for reflectometric experiments (Organon Teknika); aBSA polyclonal (rabbit) (Nordic); ahCG (anti-pregnancy IEP=5.2-6.0. purified (Organon Teknika);

by

hormone) HPLC.

monoclonal, purity 99.1?

hCG (pregnancy

hormone)

(Diosynth).

As nonionic surfactants we used: Tween 20, which is sorbitan monolaurate

poly-

ethylene oxide (poly = 20), M = 1227 g mall ‘, CMC = 0.25 g l- ’ (Atlas); Antarox CO 630, which is nonyl phenol polyethylene oxide (poly = lo), M = 662 g molt ‘, CMC = 0.04 g 1- ’ (EAF). As anionic surfactants we used: Aerosol MA, which is sodium dihexyl sulfosucciCMC=lOg 1-l M=388 g mol-‘, nate, (Cyanamid); Sodium dodecyl sulphate (SDS), M =X38 g g ll’. mol-‘, CMC=2.3 All surfactants were used as received, without further purification. Aerosol MA was obtained by evaporation from an 80% solution in an alcohol. In some experiments this solution itself was used. The phosphate buffer solution pH = 7.4 contains 0.013 mol 1-l KH,PO, (Baker PA) and 0.053 mol 1-l Na2HP0,. 12H,O (Baker PA). Phosphate buffered saline (PBS) pH =7.4 contains 0.0018 mol 1-i NaHzP0,*2H20 (Baker PA), 0.0109 mol 1-l Na,HP04*2H,0 (Baker PA) and 0.1403 mol 1-l NaCl (Merck PA). The water was purified by means of distillation (reflectometry) or by the Mini-Q water purification system (QELS). Methods RejlectometrJ

Reflectometry

is based

on the detection

of a

change in the intensity of reflected light as a consequence of the formation of an adsorption/interaction layer Cl.51 on a flat substrate with high refractive index. Figure 1 shows the set-up of the reflectometer. which is comparable with the one developed in Sweden [ 16,171. Polarized light of a He/Ne laser passes through a solution and is reflected at a silicon substrate (refractive index= 3.76). The angle of incidence is the Brewster angle for a silicon/water interface. at which the reflectance of the parallel polarized light is zero. This is

J. W.Th. Llchtenbelt et al./Colloids Surfaces B. Biointerfaces I (1993)

11

75-82

The substrate

consisted

of a silicon

wafer strip

which was oxidized for 1.5 h at 1000°C silicon wafer strip covered with a coating styrene

obtained

by spincoating

1.2 g polystyrene

dIgItal voltmeter

Fig. 1. The reflectometer for the measurement of a film on a flat substrate.

of the thickness

used to position the laser and the cuvette, which has windows perpendicular to the incident and the reflected light. When, for example, a polymer layer is coated on silicon, the intensities of both polarizations are changed compared to the reflection at pure silicon. The reflected beam is divided into p- and s-polarized light with a polarizing beam splitter, and the intensities are measured with flat silicon photodiodes. The photocurrents i, and i, are equally amplified and electronically processed to the value

Cl81 M=

si -i i, + i,

(1)

The laser can be rotated a certain

ratio

about

its axis to obtain

of the intensities

of the incident

light I, = m x I,

(2)

which leads to M

=

4 - (m x RP) R, + Cmx RJ

where R, and R, are the ratios between reflected and incident light intensities in s and p directions respectively. The value of M obtained in this way is independent of the laser beam intensity, and proportional to the adsorbed mass per unit of surface. The experiments were all carried out in a cell of glass under stirred conditions.

or of a of poly-

from a solution

in 100 ml toluene

of

at 2000 rev

min-’ using a Dynapert Precima PRS 14E spincoat unit. To provide a layer of pure polystyrene with sufficient adhesion on silicon the wafer was first silanized with 1% chlorodimethyloctyl silane in 1,1,2-trichloroethane. The wafers were dried at 50” C before use. The measuring procedure was as follows: (1) determination of the reflectometric M value with the cell filled with buffer solution; (2) addition of the first active component and determination of the adsorption; (3) flushing with buffer; (4) addition of the second component; (5) flushing with buffer, and so on. For the reflectometric measurements all proteins were diluted in the cell from concentrated solutions. After each flushing with pure buffer the adsorbed mass was once again measured several times during at least 15 min. Calculation of the adsorbed mass took place with the same value of dn/dc = 0.185 ml and surfactants, because g -’ for both proteins usually the composition of the adsorbed layer was unknown when both protein and surfactant were present. The experimental value of dn/dc may be somewhat lower for surfactants (e.g. dn/dc = 0.14 for Tween 20). As a consequence, the absolute values of adsorbed surfactants are too small. However, the masses of adsorption of the proteins are calculated

correctly.

Wettability gradient Oxidized silicon wafers with a gradient in hydrophobicity were prepared according to the procedure of Jonsson et al. [19] and Elwing and Colander [20]. The silicon wafers (Wacker Chemitronics, Germany) were provided with a SiO, layer and were hydrophilized [19,20]. They had a contact angle for water of about 15 ‘. For silicon wafers

J. W.Th

18

Lichtenbelt

for SiO, layers between

This layer thickness was reflectometric experiments.

therefore

contact angle of water ranged from 80 to about 20’ [20]. Unfortunately, it was difficult to exactly reproduce the procedure each time.

,’ z $

1’

,

2 /’ /

0

/’ OY bare latex

QELS

ahCG ---~ .

With QELS the increase in particle size upon adsorption of proteins or surfactants can be measured. A Malvern System 4700~ was used with a 15 mW He/Ne laser, at an angle of 90” and a measurement time of 5 min. The latex concentration was 0.04 g 1~ ’ in a 0.0017 mol 1~ ’ phosphate buffer, pH = 7.4, with 0.02 mol 1-l NaCl. Protein was added to the latex in the cuvette, and after 1 h the adsorbed layer thickness was measured as the increase of the particle radius. It is an important condition for this method that particles remain single. Any coagulation would also give an increase of radius, because QELS measures kinetic units (aggregates if present). Protein adsorption can be detected very well QELS, but the hydrodynamical layer thickof surfactants is usually too small to be visible. contrast, in reflectometry, which gives adsorpin mass per unit surface area with a detection

limit of about 0.03 mg rnm2, both surfactants are clearly visible.

I 119931 75-8.2

in the

By diffusion of Cl,(CH,),Si, wafers with a gradient in hydrophobicity were formed for which the

with ness In tion

B Btotnterfaces

a-

20 and 50 nm. used

Surfhcrs

10

with SiO, layers thicker than 100 nm this procedure gave irregular results. No problems were encountered

et al./Colloids

proteins

and

Aerosol

surfactant Tween

SDS MA

20

Antarox

CO 630

Fig. 2 The Influence of surfactant (concentration of 0.1 g I on the adsorptlon of ahCG onto polystyrene latex particles The protem is added first (QELS).

the adsorbed layer thickness. The other nonionic surfactant Tween 20 present at a concentration slightly below the CMC also reduces the layer thickness to some extent. The two anionic surfactants, which are, however, applied far below their CMCs, hardly changed the layer thickness. From Fig. 3 it is evident that surfactant concentration is of importance: displacement of protein (in this case ahCG) was found for Antarox CO 630 concentrations around or larger than the CMC. Displacement of adsorbed protein is also seen for Antarox CO 630 in combination with BSA in

,,I_

/

20

/

i

1

i

,i

I

Results Hydrophobic surfaces and nonionic surfactants The adsorption of ahCG combined with a fixed surfactant concentration on polystyrene has been analyzed with QELS. As Fig. 2 shows, when ahCG was added first followed by surfactant, only the application of Antarox CO 630 (nonionic) at a concentration above the CMC completely reduces

OF bare latex

-

4 ahCG

0 005 g/l Antarox - -

0 010 g/l Antarox

Antarox

CO 630

hCG

0 043 g/l Antarox

Fig. 3. The Influence of the concentration of Antarox CO 630 on the immunological actwty of ahCG adsorbed onto polystyrene latex particles (QELS)

J. W.Th

Lxhtenbelt et al./Colloids Surfaces B, Biomterfaces I (19931 75-82

reflectometric hand

experiments,

as shown

in the left-

side of Fig. 4. The reverse order

(Antarox

CO 630 followed

A very similar pattern

of addition

by BSA) reduces

79

QELS

the

measurements,

Addition

of aBSA

is observed as

is

confirms

in comparable

shown

in

Fig. 6.

the conclusions:

the

adsorption value compared with the case when no surfactant is applied. When the concentration of Tween 20 is gradually

immunological activity disappears with increasing Tween 20 concentrations. At much higher concentrations an adsorption layer reforms, which proba-

increased

bly contains mainly surfactant. Some activity using aBSA was observed which may still indicate the presence of some BSA.

in the reflectometric

experiment

using

BSA as adsorbing protein, Fig. 5 shows that the surfactant seemed to be co-adsorbed at low concentrations. At higher concentrations there is a sudden decrease of adsorbed mass, owing to a desorption and elution of BSA. Estimated 1.5

__

contact 70”

8o”

angle

60”50”

Hydrophobic

In Fig. 2 we have seen that both anionic surfactants used far below their CMCs do not displace ahCG from the polystyrene latex surface. The same is shown in Fig. 7 for a different surface material, where we see that Aerosol MA has no effect on an adsorbed BSA layer on hydrophobic silica.

of water 40”

30

surfaces and anionic surfactants

20”

Hydrophilic

‘0

02

04 Dr&uwe

- -

Antarox CO630 BSA + Antarox

06

06

1

to hydrophobic

12

14

In Fig. 4 the influence of Antarox CO 630 on the adsorption of BSA is shown. On hydrophilic SiOz some effect was found with Antarox CO 630 (above its CMC). Addition of Antarox CO 630 followed by BSA gave larger adsorption values on the hydrophilic surface. Addition in the opposite order resulted in a partial displacement of the

16

end/cm

0 01%

--

surfaces and nonionic surfactants

BSA Boseral R (0 03%) Antarox + BSA

Fig. 4. The influence of the hydrophobicity of the silica substrate on the interaction of Antarox CO 630 (concentration of 0.1 g 1~‘) and BSA (concentration of 0.3 g I-‘).

10

I

17t

11’

pure

I

BSA

-3 5

-2.5 log[qrween

.

influence

Tween

20

@ AdsorptIon

-1 5 20) g/l] BSA

-0 6 buffer

I

a 1

0

0

,cE-.

-5

-4

-3 log[C~ween

q

Flushing

wth

buffer

Fig. 5. Reflectometruz determination of the effect of mcreasmg the concentration of nonionic Tween 20 on the adsorption of BSA (from a concentration of 0.1 g 1-l) on polystyrene.

u

Tween

20

-2

-1

20) g/l] n----o

aBSA

Fig 6. The mfluence of the concentration of Tween 20 on the lmmunologlcal activity of BSA adsorbed onto polystyrene latex particles (QELS).

80

J. W.Th. Estimated 80

\

contact 70”

angle

6O”W

of water

40

Lic~htenheif et al.iColloids

made 30

20

hardly

Surfuces B: Bmnter/&a

any

04

Distance ---

06

08

1

to hydrophobic

Aerosol MAOOl% BSA Boseral D 0 03%

-

12

14

16

end/cm Aerosol + BSA - BSA + Aerosol

Fig. 7. Influence of Aerosol MA (concentratlon of 0 1 g I- I) on the adsorption of BSA (from a concentration of 0.3 g 1-l) on silica with a contact angle of water between 80 and 20’

adsorbed protein from hydrophilic surfaces. Antarox CO 630 can probably also make hydrophilic surfaces more hydrophobic and increase the protein adsorption in this way. The interaction of the other nonionic surfactant (Tween 20) was investigated using ahCG as protein. On a hydrophilic surface there is a small reduction, comparable with Antarox CO 630. As Fig. 8 indicates, only at intermediate contact angles Tween 20 decreases the adsorbed amount of ahCG. Whether Tween 20 is added before or after ahCG Estimated

contact 70”

angle

60”50”

of water

40”

0 -

II’l”‘J’11J”‘1 02 04 06 08 Distance to hydrophobic ahCG (0.0001 g/l) Tween 20 (0 3 g/i)

----

MA

(far below

its

Discussion To obtain some idea of the interactions which play a part when the surfactants and proteins are attached to a hydrophobic surface it is possible to divide the interaction into different contributions. In Table 1 a scheme is given of the change of free energy involved in protein adsorption on a hydrophobic surface, followed by displacement of protein by surfactant. Protein adsorption (Table 1, Step 1) is promoted strongly by the disappearing hydration of the surface and by structural rearrangements of the

30”

20”

I

[1] so not all this energy needs to be returned on desorption (Table 1, Step 2). For surfactant adsorption (Table 1, Step 3) hydration energies are higher than for protein,

0

Aerosol

large protein molecules such as BSA. This structural rearrangement is not completely reversible

2 80

adsorption

CMC) had an effect on the adsorption of BSA only on hydrophilic surfaces. Addition of Aerosol MA before BSA increased the BSA response in this area. Aerosol MA had probably made the hydrophilic SiO, more hydrophobic. Aerosol MA removes adsorbed BSA in the hydrophilic area, in contrast with the lack of any significant effect when the surface is hydrophobic.

I

ll’fl’l’ll’ilfl’ 02

0

the

surface and anionic surfactants

As Fig. 7 shows,

0

on

I 1993) 75-82

of ahCG. Hydrophilic

...

difference

I

1 12 14 1.6 end/cm - ahCG + Tween Tween + ahCG

Fig. 8 The influence of the hydrophobmty of the silica substrate on the InteractIon of Tween 20 and ahCG.

because surfactant

molecules

may

cover the surface more completely than proteins. The balance for displacement (Table 1, Step 2+3) is positive, which means that nonionic surfactants may displace proteins. Anionic surfactants will have less affinity for our negatively charged hydrophobic surfaces. and displacement may be questionable. Hydrophilic surfaces are dealt with in Table 2. Hydration energy changes are absent for protein molecules and for surfaces, but structural rearrangement of protein is still important [l].

J. W.Th. Lichtenbelt et al./Colloids Surfaces B Biomterfaces 1 (IY93) Table 1 Adsorptton

of protems

and surfactants

on hydrophobic

75-82

81

surfaces

Step

1 (protem Hydration Hydration Structural Coulomb Balance

of surface of protein or surfactant rearrangement of protem interaction

adsorption)

2 (protein

desorptton)

__ _

++ + +++

3 (surfactant

adsorptron)

+++ ++

+ + -

0 or -

0 or -

for dtsplacement

Table 2 Adsorptton

of protems

2+3 (balance)

+ or *

and surfactants

on hydrophthc

surfaces

Step 1 (protem Hydration Hydration Structural Coulomb Balance

of surface of protem or surfactant rearrangement of protein Interactions

adsorption)

0 0 + +

2 (protem

desorption)

0 0 _

3 (surfactant 0 fd Oor

adsorption)

2+3 (balance)

+ -

0 or i_ or -

for displacement

“Btlayer

Displacement of protein by nonionic surfactants is possible but questionable. Anionic surfactants are hardly adsorbed or not adsorbed at all on the bare surface itself so they cannot act as displacers. The conclusions are given in Table 3 and indicate that proteins adsorbed on a hydrophobic surface are displaced by nonionic surfactants and may be displaced by anionics. On a hydrophilic surface anionic surfactants are not effective; nonionics may be effective. Table 3 Dtsplacement

In general the prediction of protein adhesion based on the affinity of the protein and of the surfactant works rather well. One effect that was not taken into account and that enhances displacement is direct interaction of the protein and surfactant, which makes the protein molecule more soluble [21]. This may explain the observed displacements from hydrophilic surfaces.

Conclusions of protem

Type of surfactant

On hydrophobic surface

On hydrophilic surface

By noniomc surfactant

Yes

7

By amonic surfactant

?

No

The observations on hydrophobic surfaces are according to calculations based on adsorption free energies: nonionic surfactants can desorb proteins at concentrations around or above their CMCs but anionic surfactants (only applied at relatively low concentration) do not have any effect. On hydrophilic surfaces the results are less clearcut. In many examples there is some desorption of

82

.I. W Th Lichtenhelt

protein

when

surfactants

are added,

indicating

a

lower affinity of the protein to such surfaces. Pretreatment of both forms of SiO, by surfactant led in no case to an inhibition of protein adsorption. Only a small reduction or sometimes even an enhancement was observed. This effect of making the surface more hydrophobic is probably very much dependent on surfactant concentration because too much surfactant makes the surface hydrophilic again by bilayer formation [22].

7 8 9 10 11 12 13 14 15

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I f IYY31 75-S-7

R.D. Tilton. C R. Robertson and A.P. Cast. Langmtur, 7 (1991) 2710. A. Gardas and A Lewartowska, J. Immunol. Methods. 106 (1988) 251. J.A De Feijter. J. BenJamins and M Tamboer, Colloids Surfaces, 27 (1987) 243. M.C. Wahlgren and T. Arnebrant. J. Collotd Interface SCI., 142 (1991) 503. E Dickmson, S.R. Euston and C.M Woskett. Prog. Colloid Polym. Sci., 82 (1990) 65. R.J. Rapoza and T.A. Horbett. J. Colloid Interface SCI.. 136 (1990) 480. J.H. Lee. J Kopecek and J.D Andrade. J Boomed. Mater. Res.. 23 (1989) 351. J S Tan and P.A. Martic, J. Collotd Interface SCI.. 136 (1990) 415. W.J.M Heuvelsland and M.E. Oldenzeel. Chem. Weekb KNCV, 32 (1989) 307 S. Welm. H. Elwmg. H. Arwin and I Lundstrom. Anal. Chum. Acta, 163 ( 1984) 263. H. Arwm and I. Lundstrom, Anal. Btochem.. 145 (1985) 106. G C. Dubbeldam, Akzo Company, Eur. Patent 278.577, 1992; U.S. Patent 4,908,508. 1990. U Jonsson, B. Ivarsson, I. Lundstrom and L. Berghem, J. Colloid Interface SCI., 90 (1982) 148. H. Elwing and C.-G. Colander. Adv. Collotd Interface SCI.. 32 (1990) 317 X.H. Guo, N.M Zhao. S.H. Chen and J. Teixeird. Btopolymers. 29 (1990) 335. M.R Biihmer. Ph.D. Thesis. Agriculture Umversity of Wagenmgen. 199 I