Download Quantitative Determination of Surface Concentration of Protein with

Quantitative Determination of Surface Concentration of Protein with
Surface Plasmon Resonance Using Radiolabeled Proteins
ESA STENBERG, BJ(~RN PERSSON, HAKAN
AND C S A B A U R B A N I C Z K Y
ROOS,
Pharmacia Biosensor AB, S-751 82 Uppsala, Sweden
Received August 21, 1990; accepted November 1, 1990
A methodology to correlate the absolute surface concentration of protein to the surface plasmon
resonance (SPR) response is described. The thickness and the optical constants for each layer on the
sensor chip used were determined with different optical techniques. In a flow injection system, the steadystate SPR response was correlated to the absolute amount of radiolabeled protein adsorbed by using a
surface scintillation counter. The proteins used, ~4C-labeledhuman transferfin and chymotrypsinogen
A, as well as in vivo 35S-labeledmonoclonal antibodies, were adsorbed via electrostatic interaction to a
carboxymethylated dextran hydrogel on the sensor chip. For these proteins, surface concentrations from
2 to 50 ng mm -2 correspond linearly to the SPR response, with specificresponse in the range 0.10 +- 0.01 °
(ng mm-2) -~ , independent of protein size. The minimum detectable surface concentration of protein
is estimated to be 50 pg mm -z with this SPR instrument. Optical models have been developed to describe
how the SPR response depends on the distribution of the adsorbed protein within the hydrogel volume
at the surface. With a thin-film optical program, the theoretical SPR responses for the different models
were calculated. Comparison with experimental data shows that the protein is distributed within an
approximately 100-nm-thick dextran hydrogel layer. © 1991AcademicPress,Inc.
1. INTRODUCTION
Surface p l a s m o n r e s o n a n c e ( S P R ) is an optical p h e n o m e n o n w h i c h is sensitive to
changes in the optical p r o p e r t i e s o f the med i u m close to a m e t a l surface ( 1 ). As will be
shown, S P R is suitable for m a c r o m o l e c u l a r
i n t e r a c t i o n studies at s o l i d / l i q u i d interfaces.
T h e d e t e c t i o n system o f a S P R m o n i t o r essentially consists o f a m o n o c h r o m a t i c a n d pp o l a r i z e d (electrical v e c t o r parallel with p l a n e
o f i n c i d e n c e ) light source, a glass prism, a thin
m e t a l film in c o n t a c t with the base o f the
prism, a n d a p h o t o d e t e c t o r ( 2 ) (Fig. l a ) .
O b l i q u e l y i n c i d e n t light o n the base o f the
p r i s m will exhibit total i n t e r n a l reflection for
angles larger t h a n the critical angle. This will
c a u s e an e v a n e s c e n t field ( 3 ) to e x t e n d f r o m
the p r i s m into the m e t a l film. This e v a n e s c e n t
field can c o u p l e to a n e l e c t r o m a g n e t i c surface
wave, a surface p l a s m o n , at the m e t a l / l i q u i d
interface. C o u p l i n g is achieved at a specific
angle o f incidence, the S P R angle. A t this angle, the reflected light intensity goes t h r o u g h
a m i n i m u m d u e to the surface p l a s m o n reso n a n c e (Fig. 1b ) . T h e S P R angle p o s i t i o n dep e n d s o n the following: the optical properties
o f the prism, the metal, the m e d i u m in contact
with the m e t a l ( u s u a l l y a l i q u i d ) , the m e t a l
film thickness, a n d the wavelength o f the light
source used.
T h e S P R angle is highly sensitive to changes
in the refractive i n d e x o f a thin layer a d j a c e n t
to the m e t a l surface which is sensed b y the
e v a n e s c e n t wave. So it is a v o l u m e close to
the surface that is p r o b e d . F o r e x a m p l e , when
a p r o t e i n layer is a d s o r b e d on the metal surface
( a n d all o t h e r p a r a m e t e r s are k e p t c o n s t a n t )
an increase in the surface c o n c e n t r a t i o n occurs
a n d the S P R angle shifts to larger values (Fig.
l b ) . T h e m a g n i t u d e o f the a n g u l a r shift, defined as the S P R response, d e p e n d s on the
m e a n refractive index change d u e to the ads o r p t i o n in the p r o b e d v o l u m e .
513
0021-9797/91 $3.00
Journal of Colloid and Interface Science, Vol. 143, No. 2, May 1991
Copyright © 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
514
STENBERG ET AL.
a
b
Flow channel
Metal film
'O
I
Angle 0
'r
FIG. 1. (a) A T R configuration according to K r e t s c h m a n n and Raether (2) for excitation of surface
plasmons. (b) Intensity of reflected light as a function of incident angle obtained from the A T R configuration
shown in (a). ( - - ) Without surface layer on the metal film; (- - -) with a surface layer on the metal film.
When the surface plasmons become excited
at the metal-liquid interface, an evanescent
electromagnetic field is formed. This field decays exponentially from the metal film surface
into the interfacing medium (Fig. 2). Hence,
the impact of the adsorbate in the probed volume on the SPR response depends on its distance from the surface.
The penetration depth of the field is the distance at which the evanescent field strength
has decayed to ~37% of that at the metal/
liquid interface (3). When protein, with its
higher refractive index, adsorbs in this layer a
change in the evanescent field profile will occur
(Fig. 2). In this situation, the contribution
from the bulk solution to the SPR response
will be supressed.
Only a few scientific papers have been published where SPR has been used in protein
interaction studies (4-11 ). Kooyman et al. (8)
defined a sensitivity expression for SPR. They
showed that Maxwell's equations could provide a reasonable description for protein adsorption in SPR measurements.
Absolute amounts of protein adsorbed to
solid surfaces have been determined by different techniques such as ellipsometry (12) or by
using fluorescent or radioactive labels ( 13, 14).
J6nsson et al. (15) correlated the adsorbed
amount of radiolabeled proteins to a silica
'•
Field strength
! ! i i i i i i ! i ~
::::::::::: ::::::::~ : :
:::::::::::
Metal film
_f
Hydrogel
'~:~\~
100
200
300
400 500 600
Distance [nm]
700
800
FIG. 2. Calculated evanescent field strength, at the SPR angle, for the A T R configuration shown in Fig.
1 without ( - - ) and with (- - -) protein in the hydrogel layer. The optical properties of the prism, the metal
(gold) film, and the liquid are listed in Table I. The refractive index for the hydrogel layer with adsorbed
protein is taken as 1.44 with a thickness of 100 nm.
Journal of Colloid and Interface Science, Vol. 143,No. 2, May 1991
SURFACE PLASMON RESONANCE
surface by a combination of ellipsometry and
radiotracer measurement. Van der Scheer et
al. (16) demonstrated that using labels can
cause erroneous surface concentration determination if the experiments are not properly
designed.
J6nsson et aL showed that biospecific interaction analysis can be performed in real
time with SPR detection (17). With a flow
injection system, it was demonstrated how to
interpret antibody-antigen interaction, for
both affinity ranking and quantitative concentration determination. The sensor chip
consisted of three layers deposited in turn on
a glass surface: ( 1 ) gold, (2) aliphatic monolayer, and (3) negatively charged dextran hydrogel covalently linked to the aliphatic
monolayer as described in detail by L~ffis and
Johnsson (18). The hydrogel layer extends
from the linker layer into the solution, thus
occupying a certain volume. Apparently,
multilayer adsorption is possible in this volume. The penetration depth of SPR sensing
and the distribution of protein adsorbed in the
dextran hydrogel complicates the SPR response interpretation.
The aim of this study is to clarify the correlation between the SPR response and absolute surface concentration of protein. Absolute quantitative determinations require that
the SPR instrument is calibrated in absolute
angle units. The thickness and optical constants for the different layers of the sensor chip
are also determined with other instruments,
mostly ellipsometers.
By using in vitro 14C-labeled chymotrypsinogen A, and human transferrin as well as in
vivo 35S-labeled monoclonal antibody, the absolute surface concentration of the model proteins in the dextran hydrogel is determined by
means of a surface and a liquid scintillation
counter. When both the absolute SPR response and the surface concentration of protein are determined, thin-film models are used
to evaluate the distribution of the adsorbed
protein in the hydrogel. The detection limit
for the SPR instrument will also be discussed.
515
2. EXPERIMENTAL
2.1. S P R Instrument
The central part of the SPR instrument system is shown schematically in Fig. 3. It consists
of an illumination system, a glass prism and
a detector array with imaging optics. The sensor chip, a gold-coated glass slide (see details
below), is placed onto the prism base with the
coating facing upward. Optical contact between the prism and the glass side of the sensor
chip is achieved by a refractive index matching
fluid [bis(2-hydroxyethyl)sulfide, nD = 1, 52,
Huka, puriss ] and the flow cell is placed onto
the sensor chip.
A high-output light-emitting diode (Hitachi
HPL40RA, Hitachi Ltd) with wavelength Xp
= 760 n m and bandwidth ~ 15 nm, is used
as the light source. The divergent beam from
the LED is collimated by a spherical lens. A
cylindrical lens focuses the light onto the glassgold interface through the prism (glass quality
BSC 7, Hoya Corp., Tokyo, Japan). The illumination system produces a wedge-shaped
beam of light enabling multichannel monitoring. The measurable angular range in this
configuration is 66-72 ° .
8
Flow Channel
/
A
..... Oh,
tL_Jll
I
'"-- I
%
.~\
\/ "/~Z'~l~']
./'~
Detector
b
Outret O
~
,
FIG. 3. Schematic view of the optical unit in the SPR
instrument used in the protein adsorption experiments.
(a) Side view where the attenuated light is schematically
visualized as a dark band. (b) Top view, which schematically shows the four channels of the flowcell projected
onto the detector.
Journal of Colloid and lnterfoce Science, Vol, 143, No. 2, May 1991
516
STENBERG
The reflected beams of light from the glassgold interface are collimated by a spherical lens
and projected by a cylindrical lens onto the
photo detector array (RA 1662N, EG&G Reticon). The detector has 16 rows with 62 pixels
per row.
As shown in Fig. 3, only four rows are used
in the measurements, one row for each measuring position.
The p-polarization is achieved by a film polarizer (HN32, Polaroid) placed in front of
the detector.
The detector readings were performed with
a VME card cage computer, including a Motorola card computer (Motorola Inc.). The
signals were analog-to-digital converted and
transferred to a computer ( H P 310, HewlettPackard Corp.) for data evaluation.
The flow cell used in the SPR instrument
is constructed from silicone rubber. The sample channel size is 14.8(1) × 0.50(w)
× 0.050(h) m m with a total surface area of
7.2 + 0.1 m m 2. The adsorption process can
be measured at four positions simultaneously
(Fig. 3 ) and this feature was used to check the
homogeneity of the protein adsorbate. The
protein solution was injected into the flow cell
by means of a peristaltic pump (Pharmacia,
Uppsala, Sweden). The viton tubing (VIN
050, Labinette, Mrlnlycke) is approximately
150 m m long with an inner diameter of 0.51
mm. The total volume of the recirculating flow
system is approximately 35 tzl.
SPR is observed as a minimum in reflected
intensity of light (a dark band) as a function
of the incident angle (Figs. lb and 3). The
reflected light is projected onto the pixel row
on the detector array. The signals are amplified
and a plot of light intensity vs pixel unit (p)
is generated by the computer. The minimum
position is estimated by fitting a second-order
polynomial to the signal from five pixels closest to the pixel with the lowest intensity value.
To calibrate the detection unit, the refractive
indices of glycerol (Merck) solutions were
measured in an Abb6 refractometer (Zeiss) at
25°C. The calibration of pixel units into reJournal of Colloid and Interface ,Science, Vol. 143, No. 2, May 1991
E T AL.
fractive index units as well as angle degrees
was performed by measuring the response for
these glycerol solutions in the SPR instrument
and the SPR goniometer (see instrument description below).
2.2. Surface and Liquid
Scintillation Counter
The /3 radiation for the protein adsorbate
on the sensor chips was measured with a surface scintillation counter. The main parts of
the instrumentation were a flat carouselle, a
detector with preamplifier in a black anodized
box, an external power supply and amplifier,
and a computer equipped with a multichannel
analyzer.
There were 20 slots along the radius of the
carouselle and each slot holds one sensor chip.
The carouselle was revolved by a stepper motor (Mechanica Cortini, Forli, Italy) under
computer control and the sensor chips were
moved automatically, one by one, into the
measuring position. The detection unit consisted of a photomultiplier tube (R1450, Hamamatsu, Hamamatsu City, Japan) in a lead
housing with a scintillation film (NE 102A,
Nuclear Enterprise) 0.5 m m thick, glued onto
the flat detector surface of the tube. A copper
planchet with a circular aperture of 10 m m in
diameter was glued onto the scintillation film.
The distance from the sensor chip surface to
the photomultiplier tube surface was about
2 mm.
A conventional amplifier system (TC948,
TC145, TC241, Tennelec Inc., Oak Ridge,
TN) was connected to a multichannel analyser
(Personal Computer Analyser, the Nucleus
Inc., Oak Ridge, T N ) .
An internal ~4C-labeled reference sample
(NES-200A, Lot NES-200A-112387, Du
Pont) was included in all the surface scintillation counter measurements.
The liquid scintillation counter (Rack-beta,
1214, LKB / Wallac, Turku, Finland) was calibrated according to the supplier (19). An internal reference sample (14C-labeled palmitate
SURFACE
PLASMON
solvated in scintillation fluid) was used in all
the liquid scintillation measurements.
2.3. Chemicals
For in vivo labeling, L [ 35S] methionine (SJ
1015, Amersham, England) was used. Labeling in vitro was performed with [ ~4C]formaldehyde (CFA. 343, Amersham) and sodium
cyanoborohydride. All other chemicals were
of analytical grade and Milli Q grade water
was used.
Transferrin (T-3400) and chymotrypsinogen A (C-4879) were purchased from Sigma.
A lymphocyte cell line (clone 239, Pharmacia)
was used for the production of monoclonal
antitransferrin antibodies.
For the metabolic labeling of the antibodies,
the cells were cultivated in a medium containing L[35S] methionine according to the
supplier's recommendations. After labeling,
the cells were removed by centrifugation and
the supernatant was applied to a protein A Sepharose column (Pharmacia). The antibodies were eluted with 0.1 M sodium citrate,
pH 3.0, and collected in 1 M T r i s - H C 1 buffer,
pH 8.5. The antibodies were transferred to a
10 m M s o d i u m acetate buffer, pH 4.8, by applying 1.2 ml sample followed by 1.3 ml buffer
to a PD- 10 column (Pharmacia). Only the first
3 ml of eluant was collected, to avoid lowmolecular-weight contamination of radioactive material.
Transferrin and chymotrypsinogen were labeled in vitro by reductive methylation essentially according to Jentoft and Dearborn (20).
To a final reaction volume of 1 ml, one ampoule containing 1-3% aqueous solution of
[14C] formaldehyde ( 18.5 MBq), 2-5 mg protein dissolved in 0.1 M Hepes, pH 7.4, and
100 •1 aqueous cyanoborohydride (25 m M )
were added. After incubation at ambient temperature, 16 h, the proteins were purified on
a NAP-10 column (Pharmacia) equilibrated
with 10 m M Bistris propane buffer, p H 6.4.
Amino acid analysis was carried out at the
Biomedical Center at Uppsala Center. Fol-
RESONANCE
517
lowing acid hydrolysis of the proteins, amino
acids were separated by a standard ion-exchange procedure. Carbohydrate contents
were not determined but were set to 6% of the
molecular weight for the human transferrin
and 3% for the antibody (21 ). Chymotrypsinogen is free of carbohydrate.
2.4. Sensor Chip Fabrication
The gold (99.99% pure) film was deposited
in a sputtering system (MRC 903, Material
Research Corp.) on a 0.5-mm-thick glass wafer
(BSC 7, Hoya Corp.), 125 m m in diameter
and sliced into 12 × 12 m m 2 . The glass wafers
were first cleaned in a wafer cleaner (Titan,
FSI Corp.) before metal deposition by subsequent washing in potassium hydroxide, ammonia, and sulfuric acid solutions, respectively.
The gold layer was coated with a linker layer
consisting of a monolayer of 16-mercapto-1hexadecanol (18). This was followed by covalent coupling of a dextran (T500, Pharmacia) hydrogel. Finally carboxymethyl
groups were introduced to the dextran hydrogel (18).
2.5. Sensor Chip Characterization
The optical constants of the gold film were
determined from SPR goniometer measurements at the wavelength of 760 nm. The instrumentation used was essentially similar to
that described by Liedberg et al. except that a
high-efficiency 75-W xenon arc source coupled
to a monochromator (L-1 Illumination system, Photon Technology International Inc.,
Hamburg, West Germany) was used. The light
intensity was stabilized by using an optical
feedback system. The absolute values of p-polarized reflectance as a function of incident
angle were measured. These values were fitted
by a least-squares method to the theoretical
equations, and the refractive index and the extinction coefficient for the gold film was determined. This characterization technique has
been used by Innes and Sambles (22).
Journal of Colloid and lntarface Science, Vol. 143, No. 2, May t991
518
STENBERG ET AL.
Absolute gold film thickness was deter- of 25-50 #g/ml was circulated from the sammined from a stylus profilometer (Dektak ple vial over the carboxymethyl-modified
3030, Sloan Technology Corp., Santa Barbara, dextran hydrogel and back to the vial again at
CA) measurement, and a correlation with light a flow rate of 50/~1 min -1 . The bulk concentransmission measurements (Shimadzu UV- trations of the proteins were determined by
265, Shimadzu Corp., Kyoto, Japan) was amino acid analysis. The recirculation was
made. The surface topography of the gold layer continued for 10-30 rain until steady-state
was measured with a scanning tunneling mi- adsorption was achieved. The radioactive
croscope (Digital Instruments Inc.).
protein solution was displaced from the flow
Ellipsometry was used for optical charac- cell by introducing water, and the SPR angle
terization of the linker layer on the gold surface was recorded. The difference between this
and the dextran hydrogel on the linker layer. reading and the initial response obtained for
The instrumentation and methodology were the sensor surface without adsorbate defined
as described by Ivarsson et al. (23).
the SPR response of the adsorbed protein
Autoradiography was used as a qualitative layer. After measurement, the flow cell was
test of homogeneity of all the sensor chips after lifted from the surface and the surface was imcompleting the measurements in the SPR in- mediately flushed with nitrogen gas to remove
strument. Exposures of protein adsorbed sen- the water.
sor chips were made in a Kodak X-Omatic
The sensor chip with the adsorbed protein
casette using/3-max Hyperfilm (Amersham, was transferred to the surface scintillation
England), developed according to the sup- counter for radioactivity measurements. The
plier's instructions. When leakage from the amount of adsorbed protein was calculated
channel or nonuniform protein adsorbate was and correlated to the SPR response. The hodetected on the exposed film the correspond- mogeneity of the adsorbate on all surface
specimens was checked by autoradiography.
ing sensor chips were discarded.
Various levels of surface concentrations of
2.6. Measurement of Protein Adsorption
protein were achieved by adding sodium chloThe positively charged and radioactively la- ride to the sample solutions, so the final conbeled protein in solution was adsorbed by centration was 0 to 100 m M NaC1.
electrostatic interaction to the carboxymethTo check the relevance of the methodology
ylated dextran hydrogel of the sensor chip in presented above, an alternative method for the
the SPR instrument. A volume of 0.5-1.0 ml determination of SPR response in relation to
protein solution with the bulk concentration surface concentration was developed. Sensor
TABLE I
Specific Response, N u m b e r of Samples (n), Relative Standard Deviation (RSD), Concentration from A m i n o Acid
Analysis (c), and Specific Activity for the Labeled Proteins Used in the Adsorption Studies
Protein
[14C]Chymotrypsinogen A
[14C] Chymotrypsinogen A a
[lac]Transferrin
[laC]Transferrina
[35S]Antitransferrin
Specific
response
[° (ng mrrt-2)-l]
n
RSD
(%)
c
(#g ml-t )
Specificactivity
(Bq ng-I)
0.11
0.11
0.10
0.09
0.11
9
15
6
21
22
3.6
8.2
2.5
6.5
6.2
1160
1160
480
480
173
0.604
0.604
0.595
0.595
7.36
a Removal of protein adsorbate after dish incubation.
Journal of Colloid and Interface Science, Vol. 143,No. 2, May 1991
SURFACE PLASMON RESONANCE
chips were incubated in protein solutions contained in Petri dishes, and gently swirled for
at least 4 h. For this purpose, chymotrypsinogen A concentration was 25 #g ml -~ in 10
m M Hepes with 50 ppm Tween 20 (SurfactAmps, Pierce 28320, Pierce, IL), pH 7.5, containing 0 to 35 m M NaC1. Transferrin concentration was 25 #g ml -~ in 10 m M acetate
buffer with 50 ppm Tween 20, pH 4.76, containing 0 to 120 m M NaC1. After incubation,
the sensor chips were washed three times for
2 min in water and dried with flushing nitrogen
gas. The surface concentration of proteins was
determined with the surface scintillation
counter. The corresponding SPR response was
recorded by placing a sensor chip in the instrument, applying water, and instantaneously
registering the SPR angle. The SPR angle for
the carboxymethylated dextran hydrogel
without protein adsorbate was determined after the protein was eluted from the surface of
the sensor chip with buffer containing 1 M
NaC1.
3. RESULTS A N D DISCUSSION
3.1. Calibration
The calibration of the SPR instrument was
performed by measuring the responses for water-glycerol solutions with refractive indices
1.333-1.363. A least-squares curve fit with a
second-order polynomial proved more accurate than a linear curve fit. The result from
the SPR instrument measurements was
AnD = 9.82 X
10 -4 X p -- 2 . 8 ×
10 -6 X p 2
where AnD is nsolution -- nwater, P = pixel unit.
The same fitting procedure with the data obtained with the SPR goniometer gave
AnD
= 9.0 × 10 -3 × A0 -- 7.8 X 10 -5 × (A0) 2
where A0 is the shift in incident angle. This
resulted in a nearly linear relation between the
responses for the SPR instrument and the SPR
goniometer:
A0 ~ 0.109p.
519
This expression made it possible to compare
the experimentally measured SPR responses
with calculated values obtained from the simulation program. The SPR angle for the SPR
instrument was determined with a systematic
error of less than 5 × 10 -3 degrees.
The reproducibility for the surface scintillation counting measurements was determined
by performing ten consecutive measurements
with the NES-200A reference sample. The
mean counting efficiency obtained for this
sample was 16%, with a relative standard deviation of 0.6%.
The reproducibility for the liquid scintillation counting measurements was determined
by performing 20 consecutive measurements
with the ~4C-labeled palmitate reference sample. The relative standard deviation for these
measurements was 0.4% while the calibration
procedure had an accuracy o f + 3 % (19).
The total protein concentration in the solutions was determined by amino acid analysis
and the specific radioactivity was measured in
the liquid scintillation counter. The results are
presented in Table I.
The efficiency of the surface scintillation
counter was also determined by correlation
with data obtained from the liquid scintillation
counter. Samples with three levels of concentration of metabolically 35S-labeled monoclonal antibody were alternatively applied to
ten sensor chips and ten scintillation vials each,
and thereafter the radioactivity was measured
in the respective instrument. The relative
standard deviations for the results obtained
with the liquid and surface scintillation counter measurements were 3 and 4%, respectively.
The mean counting efficiency for the surface
scintillation counter was 21% in this case.
The higher efficiency obtained from experiments with metabolically 35S-labeled monoclonal antibody, compared with the NES200A reference sample, is due to the reflectivity of the underlying surface which influences
the counting efficiency. For example, increasing the gold layer thickness also increases the
counting efficiency.
Journal of Colloid and InterfaceScience, Vol. I43, No. 2, May 1991
520
STENBERG ET AL.
TABLE II
Data for Surface Plasmon Resonance and Surface Concentration Calculationsa
Layer
d
(nrn)
n
k
dn/dc
(ml g-X)
Glass
Gold
Linker
Liquid
Protein
Dextran
~
45
1.9
oo
-100-220
1.511
0.17
1.59
1.3289
---
0
4.93
0.09
0
0
0
----0,18
0.15
Each layer is defined by its thickness, d, refractive index, n, extinction coefficient, k, and the refractive index
increment, dn/dc, for protein and dextran.
a
These results, together with the data obtained f r o m the surface scintillation counter,
were used to calculate the surface protein concentration on the sensor chips.
3.2. Optical and Surface Characterization
The sensor chip used in this study can be
represented by a m o d e l consisting o f five separate layers (Fig. 4). Each layer is defined by
its thickness, di, refractive index, hi, and extinction coefficient, ki. To perform reliable
calculations o f the SPR response for the sensor
chip used in the experiments, it was necessary
to separately determine the thicknesses and
the optical constants o f the layers with other
methods.
The prism (or m o r e exactly the glass in the
sensor chip) and the liquid are semi-infinite
and the refractive indices, given in Table II,
were determined with a refractometer.
The absolute reflectance as a function o f
incident angle was obtained from SPR goniometer measurements on five gold-coated glass
surfaces. Using a nonlinear least-squares fitting
technique (22) the refractive index and the
extinction coefficient o f the gold film were det e r m i n e d and the results are s u m m a r i z e d in
Table II. T h e thickness, 45 rim, was determ i n e d by c o m b i n e d transmission and surface
profilometer measurements. The grain diameter for the gold films was about 2 0 - 3 0 n m
and the height difference approximately 5 n m
Journal of Colloid and Interface Science, Vol. 143, No. 2, May 1991
as determined by scanning tunneling microscope measurements, so the gold film is considered as h o m o g e n e o u s at this wavelength
(24). N o t e that the optical constants are dependent on the measuring m e t h o d s used, on
the substrate material, on the deposition technique, and on the thickness o f the metal. So
the optical constants can differ f r o m literature
data.
D u e to the extreme thickness o f the linker
layer ( ~ 2 n m ) , it was m o r e difficult to characterize than the gold film. The measurements
were m a d e at 632.8-nm wavelength with ellipsometry and we have assumed that the optical constants for the linker layer are the same
at 760 nm. In Table II, the m e a n values for
the linker layer are presented. The results agree
well with literature values ( 2 5 - 2 7 ) although
the refractive index is higher and the thickness
(n)
Liquid
(n, d) Hydrogel
(n, k, d) Linker
~
(n, k, d) Gold
~ ' * ~ r
(n)
Prism
,,
FIG. 4. Five-layer model of the SPR sensor chip with
interfacing liquid used in the SPR calculations. Each layer
in the model is described by the refractive index (n), the
extinction coefficient (k), and thickness (d).
SURFACE PLASMON
is slightly lower in our case. The obtained n
value of 1.59 is reasonable since an aliphatic
compound like polyethylene has a refractive
index of 1.56 in the crystalline phase (28).
The large k value obtained is probably due
to the rough interface between the linker and
gold layers. In practice, these layers construct
a composite layer and therefore the evaluation
should be made by using the Bruggeman effective medium approximation (24).
The dextran hydrogel is difficult to investigate since this layer contains flexible polymer
chains of nonuniform length. From electron
spectroscopy for chemical analysis, SPR, and
ellipsometry measurements the surface concentration of dextran is 1-3 ng m m -2 (18).
This means that medium to high grafting density is obtained and this is called "overlapping
coil regime" (29). In this regime, the hydrogel
can be considered as a polymer "brush." For
this type of hydrogel, de Gennes presents
equations that can be used for estimating the
upper limit of the hydrogel thickness. The surface concentration (2.5 ng mm-2), the molecular weight (500,000 Da), and the monomer size (0.65 nm) are used as input values
and from the published equations a maximum
thickness for the hydrogel of 220 nm is obtained. This figure is based on the assumption
that one end of the dextran chain is grafted
on the solid surface. In practice, the thickness
of the dextran layer must be thinner since there
can be several grafting points and they can be
distributed along the whole chain.
The preferential adsorption of either the labeled or the unlabeled form (16) can be caused
by a change in net charge of the protein due
to the label. The label can also cause changes
in charge distribution or conformation of the
protein. To avoid this, labeled and unlabeled
populations have not been mixed in the experiments. Modification with [ 14C]formaldehyde, gave nearly 100% of labeled proteins.
In the case of 35S modification, metabolically
labeled protein was used and this resulted in
a homogeneous population with high specific
activity.
521
RESONANCE
3.3. Models for the Protein-Dextran
Hydrogel
Models describing the steady-state protein
adsorption have been evaluated with a thinfilm simulation program to estimate the unknown parameters, thickness of the hydrogel,
and protein concentration as well as the distribution therein.
The concentration for the dextran layer
(Caext.... g ml -l ) is calculated from
Cae,,t~an = Fde×tran/daext~a.
[1]
where the surface concentration (•dextran, ng
m m -2) was determined experimentally and
the thickness for the dextran hydrogel (ddext~n,
nm) depends on the model. The surface concentration for the protein (Fprotein)is calculated
from a given protein concentration (Cprot~in):
I~protein = Cprotein X dprotein.
[2]
Here dprot~inis the layer thickness for protein
and is also model dependent.
In the thin-film simulation program, the ppolarized reflectance is calculated as a function
of incident angle by using Maxwell's equations
(30). In the calculations, it is assumed that
the interfaces between layers are flat and the
layers are homogeneous and isotropic. The
angular position of the reflectance minimum
is found with a binary search method. The
refractive index of the dextran layer (F/layer),
with or without protein in it, is used as input
to the program and it is calculated from
F/layer = F/liquid "q-
(dn/dc)dextran X
Cdextra n
+ (dF//dc)p~o~ein × Cprotein. [3]
The values of the refractive index increment,
dn/dc, used in this work for dextran (28) and
protein (31, 32) solutions are given in Table
II. De Feijter et al. reported that the refractive
index increment for proteins is constant up to
high concentrations (33).
The distribution of adsorbed protein within
the dextran hydrogel can be described by two
simplified models referred to as constant volume and growth models. They are evaluated
Journal of Colloid and Interface Science, Vol. 143, No. 2, May 1991
522
STENBERG
with two different thicknesses for the dextran
layer, 100 and 220 nm, respectively. These
models are described in turn.
In the constant volume model it is assumed
that in a hydrogel of an assumed thickness,
protein molecules are randomly distributed
throughout the entire hydrogel. In this model
dprotein is equal to da~xt~an. In Fig. 5a the calculated SPR response as a function of surface
concentration is given for protein-hydrogel
layers with thicknesses 100 and 220 nm. When
the same a m o u n t of protein is distributed in
a thinner hydrogel layer, a higher SPR response is obtained than with a thicker one.
This result was expected since the evanescent
wave decays exponentially into the hydrogel.
In addition, the specific response, defined as
the ratio between the SPR response and the
corresponding surface concentration, increases
with surface concentration with this model.
This fact will be emphasized in the interpretation of the experimental results below.
In the growth model, the protein layer
thickness is different from the hydrogel thickness (either 100 or 220 nm). So the hydrogel
layer (Fig. 4) is divided into two layers, one
with the dextran hydrogel and one with protein
in the dextran hydrogel. In this case, Cproteinis
constant and therefore also the refractive index
7,
E T AL.
for the layer. The protein layer thickness is
varied and increases from zero until the final
protein layer is as thick as the dextran hydrogel
layer, i.e., dprotein = ddextran- Adsorption is assumed to begin at the linker-dextran interface
and grows continuously outward. The SPR
response for this model was calculated twice,
with different concentrations assumed for the
protein layer. In the first case, the concentration was 0.60 g m1-1 , corresponding to a refractive index of 1.44, and the dextran hydrogel layer was set to be 100 n m thick. In the
second case, the concentration was 0.27 g
m1-1 , corresponding to a refractive index of
1.38, and the dextran hydrogel layer was set
to be 220 n m thick. In Fig. 5b, the calculated
SPR responses for the two cases are plotted as
a function of surface concentration of protein.
As seen, in the case with thinner hydrogel and
higher protein concentration, the SPR response is almost linearly dependent on surface
concentration and the specific response is
higher. In the case with thicker hydrogel and
lower surface concentration, the specific response decreases with increasing surface concentration. This is a consequence of the penetration depth dependence on refractive index.
In a variation of this model, the adsorption
is assumed to begin at the hydrogel-liquid in-
7.
a
b
6
6,
S-'
04
3,
ss
rr
~3
SS
r.r
ds
2,
ss
s S
1,
s S
0
-
0
,
10
-
•
20
-
i
30
-
,
40
.
•
50
-
•
60
Surface Concentration [ng mm -2 ]
10
20
30
40
50
60
Surface Concentration [ng mm -2 ]
FIG. 5. C a l c u l a t e d S P R response versus surface c o n c e n t r a t i o n for a p r i s m / g o l d / l i n k e r / p r o t e i n - h y d r o g e l /
l i q u i d system. ( a ) C o n t i n u o u s v o l u m e m o d e l has been used for two p r o t e i n - h y d r o g e l thicknesses, 100 n m
( - - ) a n d 220 n m (- - -). ( b ) G r o w t h m o d e l has been used for two protein concentrations; c = 0.6 g / r n l ,
i.e., n = 1.44 ( - - ) ; c = 0.27 g / m l , i.e., n = 1.38 (- - -).
Journal of Colloid and Interface Science, Vol. 143, No. 2, May 1991
523
SURFACE PLASMON RESONANCE
terface and grows continuously inward. All
these models were applied to experimental
data.
3.4. Experimental Measurements
The SPR responses as a function of time
were experimentally measured over a wide
range of concentrations for the three model
proteins. In Fig. 6 a typical result with in vivo
35S-labeled monoclonal antibody is shown. In
this case, the surface concentration of the antibody determined in the surface scintillation
counter was 24.1 ng m m -z. A wide range of
protein surface concentrations, from 5 to 50
ng m m -2, was detected in 20 measurements.
The in situ adsorption and dish incubation
experiments for ~4C-labeled transferrin and
chymotrypsinogen A resulted in surface concentrations of 9-37 and 2-38 ng m m -2, respectively.
The experimental results from surface
characterizations using the SPR instrument
and the surface scintillation counter are summarized in Table I. Within the limits of experimental error, these results suggest that the
specific responses are independent of protein
size or labeling methods used. It can also be
noted that the relative standard deviation is
good. The results also show a high specific activity of the metabolically labeled protein
which enables determinations of proteins
present at low surface concentration.
In Fig. 7 all the experimentally measured
SPR responses versus surface concentration of
proteins are presented. The best fit from
regression analysis shows a linear relationship,
with the specific response in the range of 0.10
_ 0.01 ° ( n g mm-2) -I. The high-molecularweight (monoclonal antibody, 150,000 Da)
and low-molecular-weight (chymotrypsinogen
A, 25,700 Da) proteins have the same specific
response; thus the dextran hydrogel does not
act as a molecular sieve for these proteins. This
can be expected, since no chemical crosslink-
40'
,
i
35'
3
30,
X
.~_
25.
20.
rrcr 15.
a_
O9
10
0
0
200
400
600
Time [s]
800
1000
1200
FIG. 6. Experimentally measured shift of the SPR response as a function of time for electrostatic adsorption
of an antitransferrin monoclonal antibody to the carboxymethylated dextran hydrogel. The baseline is defined
at 1, where water flows past the carboxymethylated dextran surface. The protein in Hepes buffer is introduced
to the flow cell and starts to adsorb at 2. Recirculation of the protein solution continues until a steady state
is reached and thereafter the protein solution is replaced by water. The SPR response is the difference
between the steady-state level at 3 and the baseline.
Journal of Colloid and Interface Science, Vol. 143,No. 2, May 1991
524
STENBERG
ET
AL.
X
X
X×
XX
X
E
g
4.
O
.~
°~
rr
rr
D..
m
O
x
2-
**
o
~x
o
o
~××~ ×
1o
8
0
0
5
10
15
20
25
30
35
40
45
50
Surface Concentration [ng mm -2 ]
FIG. 7. Experimentally obtained SPR response as a function of surface concentration of the radiolabeled
proteins studied. (© and • are 14C-labeled chymotrypsinogen, 0 and 0 are ~4C-labeled transferrin, × are
35S-antitransferrin monoclonal antibody. Open symbols are results from dish incubation.)
ing has been introduced between the dextran
chains. The results thus support the hypothesis
of highly flexible dextran chains.
The empirical data have been compared
with the theoretical results based on the optical
models. In Fig. 8, the specific response as a
function of surface concentration for the radiolabeled monoclonal antibody is shown.
The results obtained from the constant volume model, 100-nm dextran-protein layer,
and two versions of the growth model with
refractive index 1.44 in the protein-dextran
layer are shown: one where the protein adsorption starts at the linker-dextran interface
and continues outward and one where the
protein adsorption starts from the dextranliquid interface and grows inward. The interpretation is that the protein adsorbs in a 100nm-thick dextran layer distributed either homogeneously within the volume or preferentially near the linker layer.
We have also calculated the thickness of the
protein at the surface which would represent
that of a closely packed protein crystal. We
could not find actual values for solvent content
and specific volume in the literature for anti-
Journal of Colloid and Interface Science, Vol. 143, No. 2, May 1991
bodies. According to Matthews, the fractional
solvent content of 226 globular protein crystals
ranged from 30 to 78% (34). The most common value reported was 47%. From our experiments the maximum surface concentration of an antibody to the carboxymethylated
dextran is ~ 6 0 ng m m -2. Using the average
solvent content and a partial specific volume
of 0.736 ml g-I (32), the dextran-protein layer
thickness can be calculated to ~ 8 0 nm. The
extreme values of solvent content, 30 and 78%,
give thicknesses of 60 and 200 nm, respectively.
3.5. Detection Limit
The mean specific response of the proteins
was 0.10 +_ 0.01 ° ( n g / m m 2 ) -1 (Table I).
From this, an estimation of the detection limit
o f the instrument can be made. With an angular resolution of the SPR instrument at
0.005 ° , the minimum detectable surface concentration of protein was estimated to be 50
pg m m -2. When the active surface area is 1
m m 2, the sample volume is 50/zl, and the uptake efficiency is ~25%, 4 ng ml -~ can be de-
SURFACE
PLASMON
x
0.12
xx
~
x
£-.
x
xx
RESONANCE
x
x
,,x
~x xx x
~
~
<
525
_.___.
*°~
~
0.10
E
E
0.08
g
0.06-
0.04O3 0.02-
0.00
0
10
20
30
40
50
60
Surface Concentration [ng mm -2]
FIG. 8. Experimentally specificresponse as a function of surface concentration for metabolicallylabeled
35S-antitransferrin monoclonal antibody. The specific responses for three theoretical curvesare also shown.
(--) Growth model: the protein concentration in the layer is 0.6 g/ml and the layer grows from the linker
surface outward to the liquid. (---) Same as previous but the layergrowsfrom the hydrogel-liquidinterface
inward to the linker surface. (---) Continuous volume model: the protein concentration in the layer, 100
nm thick, increases uniformly.
tected. This is equivalent to, for instance, 25
pmole of a protein with a molecular weight of
150,000 Da. This has been verified in our laboratory and is lower than earlier reported (6,
8-10).
in the present SPR instrument configuration.
There was no significant difference in specific
response for the proteins tested.
The SPR response was calculated using a
thin-film simulation program with models
where the distribution of the proteins in the
dextran hydrogel was varied. By comparing
4. S U M M A R Y
experimental data with simulated SPR reA method for the quantification of protein sponses, we conclude that the dextran layer
surface concentration from surface plasmon thickness with protein adsorbate reaches a
resonance response has been developed. ~4C- mean thickness of approximately 100 nm.
labeled transferrin and chymotrypsinogen A This thickness lies between the calculated
and in vivo 35S-labeled monoclonal antibody maximum dextran thickness of 220 n m and
were adsorbed on a sensor surface to a steady- the m i n i m u m thickness for a protein crystal
state level via electrostatic interaction. The layer (80 nm) corresponding to a saturated
SPR response measured, defined as the change protein-hydrogel layer.
in surface plasmon resonance angle, was corThe mean specific response was 0.10
related to the absolute amount of protein sur- _+ 0.01 ° (ng m m - 2 ) -1 for all surface concenface concentration as determined from radio- tration values between 2 and 50 ng m m -2 for
active measurements with a surface scintilla- the proteins studied. The detection limit for a
tion apparatus. A linear correlation was found medium-sized protein is 25 pmole with a 50to be valid up to a surface concentration of 50 #1 sample volume for the present SPR instrung mm-2, which is the upper limit of detection ment.
Journal of Colloid andlnterface Science, Vol. 143, No. 2, May 1991
526
STENBERG ET AL.
ACKNOWLEDGMENTS
The authors are indebted to the following people for
their kind contribution to this work: L. Hellman at the
Institute of Immunology, Biomedical Centre at Uppsala
University, Sweden, for the metabolic labeling of antitransferrin; B. Ivarsson for ellipsometry work and also the
construction of the optical system together with U. Jrnsson; H. 13stlin for mechanical design; S. SjNander for construction of the flow cell; S. Lrffis for valuable discussions
of the surface chemistry; C. Johansson for critical reading
of the manuscript; and T. Lenke for drawing the figures,
all at Pharmacia Biosensor AB, Uppsala, Sweden.
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