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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. 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