Characterization of IgG LangmuirâBlodgett films immobilized on functionalized polymers

Talanta 52 (2000) 921–930

Characterization of IgG Langmuir–Blodgett filmsimmobilized on functionalized polymers

Claudia Preininger a,*, Hauke Clausen-Schaumann b, Arti Ahluwalia a,Danilo de Rossi a

a Centro E. Piaggio, Faculty of Engineering, Uni6ersity of Pisa, Via Diotisal6i 2, I-56100 Pisa, Italyb Institute of Applied Physics, LB-Uni6ersity Munich, Amalien St. 54, D-80799 Munich, Germany

Received 18 November 1999; received in revised form 5 May 2000; accepted 15 May 2000

Abstract

The bioactivity of anti-human IgG Langmuir–Blodgett (LB) films, the non-specific adsorption of protein and thetopography of anti-IgG LB films have been studied for application in immunosensors. The antibody (AB) LB filmswere horizontally deposited on glass and functionalized polymers, such as carboxy-poly(vinyl chloride) (PVC-COOH),chloropropyl and aminopropyl sol–gel. The LB films were characterized by means of ellipsometry, atomic forcemicroscopy (AFM) and bicinchoninic acid (BCA) protein test. The interpretation of ellipsometric data was performedusing a one-layer model. Non-specifically adsorbed protein was desorbed by washing the IgG film in 0.5 M NaCl, 2M NaCl and 1% N-cetyl-N,N,N-trimethylammoniumbromide detergent solution resulting in a 50% reduction of thefilm thickness. The mean thickness of an anti-IgG film on glass measured by ellipsometry, PVC-COOH andaminopropyl sol–gel was 992, 1191 and 2398 nm, respectively. According to the BCA test 6–8 mg antibody (AB)per slide was bound to the functionalized polymers, but only 3 mg AB per slide was adsorbed on glass. The averagedistance of anti-IgG granules as indicated by AFM measurements on PVC-COOH, chloropropyl and aminopropylsol–gel was 42920, 3493 and 2394 nm. The average distance of granular AB structures on glass, however, was150950 nm. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Antibody Langmuir–Blodgett films; Ellipsometry; Atomic force microscopy

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1. Introduction

Highly oriented antibody layers on solid sup-ports are required to attain high sensitivity andselectivity in immunosensing. Usually, methodsbased on Langmuir–Blodgett (LB) techniques areused in order to prepare such layers. Applicationsof protein LB films have been extensively de-scribed in literature [1–8], mainly for biosensor

* Corresponding author. Present address: Department ofLife Sciences/Biotechnology, Austrian Research Centers,A-2444 Seibersdorf, Austria. Tel.: +43-2254-7803527; fax:+43-2254-7803653.

E-mail address: [email protected] (C.Preininger).

0039-9140/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S0 039 -9140 (00 )00446 -X

C. Preininger et al. / Talanta 52 (2000) 921–930922

application and investigation of self-assemblingprotein systems. However, LB films are far frombeing stable, which might be due to the pooradhesion of protein layers to the substrate andtheir native instability. In order to prevent proteinfilms from washing off and to retain the activityof antibody molecules, an immobilization schemewas needed that provided the highest irreversiblesurface loading. Covalent immobilization indi-cates high surface loading and low protein loss,and thus is most desirable for sensor technology.There is generally higher activity loss from cova-lent immobilization, but this loss depends on theprotein, as well as the immobilization chemistryand the substrate [3].

Covalent immobilization can be achieved bothby surface immobilization on a solid support [9]and crosslinking the material with proteins [7].Quartz, silica, and conventional glass that hasbeen chemically modified exclusively with reagentsof the type (RO)3SiR, with R being ethyl ormethyl, and RO being aminopropyl, 3 chloro-propyl, 3 glycidyloxy, vinyl, or a long chain amineare easily reacted with a peptide to be immobi-lized. The linkage is through a free primary aminogroup or involves free SH groups [1,10] on theantibody or the antibody is covalently crosslinkedto a protein [7].

One of the most baffling problems that limitantibody-based applications is the non-specific ad-sorption of antibody and protein to the substratesurface. We have polymer-coated a solid support(glass) and have immobilized anti-IgG films ontovarious polymers using different immobilizationprocedures. Since the activity of the antibodymolecules in the film varies with its structure anddensity, the reactivity of the film depends on theimmobilization method used. We have studied thenon-specific adsorption, the antibody activity andthe surface characteristics of anti-IgG LB filmscovalently immobilized on carboxy-poly(vinylchloride) (PVC-COOH) and aminopropyl sol–geland physically adsorbed onto glass and chloro-propyl sol–gel. The evaluation of the immobiliza-tion procedures was based primarily on (a) theamount of antibody that can be loaded and (b)the antibody activity, which was defined as theability to recognize and bind its specific antigen.

In order to characterize and understand antibodyLB films immobilized on immunoassay surfacesbetter, various analytical methods, such as ellip-sometry [1], atomic force microscopy (AFM) [11]and surface plasmon resonance (SPR) [12] havebeen reported in the literature. The bioactivity ofthe anti-IgG film and the non-specific adsorptionof antigen were investigated by ellipsometry andthe bicinchoninic acid (BCA) test. The surfacetopography was described by AFM which hasbecome a powerful tool for the investigation ofprotein films [13,14] and the quantification ofspecific immunological reactions [11].

2. Experimental

2.1. Apparatus

The Langmuir–Blodgett trough was a pur-posely built double barrier Wilhelmy balance witha subphase volume of 250 ml and a surface areaof 250 cm2.

Ellipsometric measurements were carried outwith an AutoEl II automatic ellipsometer(Rudolph Research, USA) at an incident angle of70°C and a wavelength of 633 nm. The amplituderatio (C) and the phase difference (D) of the lightreflected from the polymer-coated glass slideswere measured at 12 positions of the slide andtranslated into an estimation of the film thickness.The data interpretation was performed using aone-layer model presuming the polymer to be thesubstrate. The refractive index of the polymer wasdetermined for each of the 12 slide positions andthe mean value of all refractive indices plus/minusthe standard deviation (n9s) was taken as therefractive index of the polymer-coated glass. Thethicknesses of the IgG films, which were consid-ered to be transparent [15] and optically isotropic,were calculated at a fixed refractive index of 1.49.

The AFM images were obtained with aNanoscope IIIa (Digital Instrument, Santa Bar-bara, CA) in constant force mode, at room tem-perature (20°C) in air. The cantilevers werecommercial silicon nitride cantilevers (DNP-S,Digital Instrument) with a nominal spring con-stant of 30 mN m−1. Loading forces were mini-

C. Preininger et al. / Talanta 52 (2000) 921–930 923

mized before recording the images. To minimizefriction-induced height artifacts the fast scan-axiswas oriented perpendicular to the cantilever’s axisof symmetry. Trace and retrace images were com-pared. Scan rates varied from 1 (lines per s) to 10Hz.

2.2. Reagents

Anti-human IgG (whole molecule) developed ingoat (I-9384), human IgG FITC conjugate (F-9636), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide were purchased from Sigma(Steinheim, Germany). (3-glycidyloxypropyl)tri-methoxysilane (GOPS), 3-aminopropyltriethoxy-silane (APTS), 3-chloropropyltrimethoxysilane(ClTMOS), tetramethoxysilane (TMOS) andbicinchoninic acid sodium salt were from Fluka(Buchs, Switzerland). Triton X-100 was fromMerck (Darmstadt, Germany) and carboxy-poly(vinyl chloride) (PVC-COOH, carboxyl con-tent 1.8%) was from Aldrich (Steinheim,Germany).

2.3. Cleaning of the glass slides

Glass slides (3×1 cm2) were immersed in con-centrated sulfuric acid for a day and washedseveral times with distilled water and acetonebefore use.

2.4. Preparation of the polymer layers

PVC-COOH layers were prepared by the spin-coating technique (6000 rpm, 30 s) using a solu-tion of 10% PVC-COOH in tetrahydrofuran.

Chloropropyl-modified sol–gel layers were dip-coated from a cocktail consisting of 1 mlClTMOS, 1 ml TMOS, 2.5 ml absolute ethanol(EtOH), 0.5 ml 0.1 N HCl and 50 ml TritonX-100, typically 3–5 days after cocktail prepara-tion. The silanized glass slides were dried at roomtemperature for 2 weeks before use.

The cocktail of aminopropyl-modified sol–gelwas composed of 1.3 ml APTS, 0.7 ml GOPS, 2.5ml absolute EtOH, 0.5 ml 0.1 N HCl and 50 mlTriton X-100. As sol–gel layers of pure APTSwere not transparent and not stable in buffer and

in water (even after drying in EtOH atmosphere),GOPS was added to stabilize the layer by partlycrosslinking the amino groups.

In order to evaluate the polymer layers forhomogeneous immobilization of anti-IgG, thesurface roughness of the polymer-coated slideswas measured using a profilometer. All surfaceroughnesses are rms values. The surface rough-ness of the empty glass slide was about 1.5 nm,and the roughness of the PVC-COOH coated, thechloropropyl sol–gel coated and the aminopropylsol–gel coated glass was 23, 8 and 41 nm, respec-tively. A rough surface provides a larger surfacefor immobilization than a smooth surface, butsuffers from a higher non-specific adsorption anda poor batch-to-batch reproducibility. The chloro-propyl sol–gel coated slide provides thesmoothest surface and we therefore expect thechloropropyl sol–gel coated layer to be the mostsuitable substrate for antibody immobilization.

2.5. Deposition of the films

Anti-human IgG monolayers were formed in apurposely built Langmuir trough with a subphasevolume of 250 ml and a surface area of 250 cm2.The subphase was 0.1 M phosphate buffer, pH7.4. Monolayers of anti-IgG were deposited byhorizontal lifting at 20 mN m−1 [1] onto glass,PVC-COOH and sol–gel coated glass slides.Therefore, 150 ml of a 5 mg ml−1 AB solution wasspread on the subphase to form a LB film, whichwas allowed to react with the polymer-coatedglass for 30 min. For deposition on aminopropylsol–gel a solution of 150 ml IgG (5 mg ml−1) and5 ml 0.005% glutaraldehyde (GA) (molar ratio 2:1)was spread on the subphase, usually 4 h afterpreparation of the solution. The isotherms ofanti-IgG and anti-IgG crosslinked with glu-taraldehyde are shown in Fig. 1.

2.6. Immobilization on PVC-COOH

The PVC-COOH coated glass slides wereplaced in 0.5% 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) overnight [16]. Thecarboxy group of the polymer and the carbo-diimide form a highly reactive O-acylisourea


intermediate which reacts with the amino groupof the antibody resulting in a stable amide bond.Since the anti-IgG film was deposited horizontallyon the PVC-COOH, the hydrophobic chains ofthe antibody which were lifted away from thesubphase surface were adsorbed on the hydropho-bic polymer. The covalent binding of the antibodyvia its aminoterminal end was possible only byre-organization of the antibody film. The AB filmwas both covalently bound and adsorbed on thePVC-COOH coated surface.

2.7. Immobilization on chloropropyl sol–gel

The formation of the sol–gel was due to hy-drolysis and polycondensation of alkoxysilanes.The reaction proceeds as long as ethoxy groups(which can be hydrolyzed) or hydroxy groups(which may condense) are available. The hydroly-sis and polycondensation can be accelerated orslowed by employing appropriate acidic and basiccatalysts. The anti-IgG LB film was horizontallyadsorbed onto the polymer-coated slide. Thesmooth surface of the chloropropyl sol–gel (seeSection 2.4) allows a homogeneous adsorption ofthe antibody film due to strong hydrophobicinteractions.

2.8. Immobilization on aminopropyl sol–gel

Anti-human IgG molecules crosslinked with0.005% GA were covalently bound to theaminopropyl-modified sol–gel by aldehyde

groups which were not involved in the antibodybinding. Furthermore, the antibodies were ad-sorbed through hydrophobic interactions, an ef-fect which is well known for silanized substrates.

2.9. Binding procedure

One hundred microliters of FITC-labeled hu-man IgG (100 mg ml−1) was placed on the IgGfilm. After 3 h the slides were washed in 0.1 Mphosphate buffer and 2 M NaCl solution. Thebinding of antigen to the immobilized AB wasdesignated as total binding. The attachment of theprotein due to adhesion was referred to as non-specific protein adsorption. The percentage of spe-cific binding was calculated as 100× [(totalbinding−non-specific adsorption)/total binding].Specific and non-specific binding was estimated bycalculating the film thickness before and afterwashing in ionic and detergent solutions.

2.10. BCA protein test

The protein measurement was based on bicin-choninic acid (BCA) [17] which is capable offorming an intense purple complex with Cu(I)ions in alkaline environment. The color producedin the reaction of alkaline Cu(II) with protein[reduction of Cu(II) to Cu(I)] was monitored at562 nm after 1-h incubation at 50°C.

3. Results and discussion

3.1. Antibody loading

The quantity of anti-IgG on the polymer-coated slides was determined by ellipsometry esti-mating the thickness of the LB film and by theBCA protein test. The antibody LB films werethoroughly washed in succession in buffer, 2 MNaCl and 1% N-cetyl-N,N,N-trimethylammoni-umbromide detergent solution to remove ad-sorbed IgG, not covalently bound. As shown inTable 1 thicknesses of 1191, 2398 and 992nm were calculated for anti-IgG films on PVC-COOH, aminopropyl-modified sol–gel and bareglass. No results were obtained for LB films on

Fig. 1. Surface pressure/area isotherms for anti-IgG and anti-IgG crosslinked with glutaraldehyde (GA).


Table 1Figures of merit for LB films on PVC-COOH, chloropropyl- and aminopropyl sol–gel and glass comparing the thickness d (nm) ofthe LB film, the antibody loading (mg AB per slide), the diameter and the average distance of the AB granules (nm)

Ellipsometry dLB filmSupport BCA test AFM diameter AFM average distance(nm)(mg AB per slide) (nm)(nm)

6.0PVC-COOH 10-401191 42910Chloropropyl – 7.5 2494 3493

sol–gel2398 8.0Aminopropyl 1294 2394

sol–gel3.0 94911 150950Glass 992

chloropropyl sol–gel which might be due to theporous structure of the gel or an excessive thick-ness of the anti-IgG film. The film thicknesses onPVC-COOH and on bare glass corresponded wellwith the dimensions of an IgG molecule deter-mined by X-ray crystallography (10×14×4.5nm) [18]. However, the thickness of the AB filmon aminopropyl sol–gel was significantly largerthan the size of an individual IgG molecule, whichwas likely to be a result of LB multilayers. As canbe seen from Fig. 1 the GA crosslinked films aremore condensed than the anti-IgG monolayers.This could either mean that crosslinking rendersthem more soluble in the subphase, or that theeffect of GA is to bind the anti-IgG moleculesinto a closely packed network, possibly consistingof multilayers. The latter hypothesis is supportedby the large thickness of anti-IgG films onaminopropyl sol–gel (2398 nm) and by ellipso-metric measurements at the air/water interfacewhich indicate that the film thickness at a certainpressure increases with increasing GA concentra-tion; and also by mechanical measurements whichshow that GA crosslinked films are more viscousand have higher elastic constants than IgG films(unpublished results).

The highest IgG loading determined by theBCA protein test was found on aminopropyl sol–gel (8 mg per slide). Only 3 mg anti-IgG per slidewas adsorbed on glass. The amount of anti-IgGon PVC-COOH and on chloropropyl sol–gelwas 6 and 7.5 mg per slide, respectively (seeTable 1).

3.2. Antibody acti6ity

After incubation of the anti-IgG film in FITC-labeled IgG the anti-IgG films immobilized onglass, PVC-COOH and aminopropyl sol–gel wererinsed in 2 M NaCl and their activity was exam-ined by ellipsometry. The increase in layer thick-ness (see Table 1) was assumed to be related tothe binding ability (activity) of the immobilizedantibody and was about 7 nm for PVC-COOH,10 nm for amino-modified sol–gel and 2–4 nmfor bare glass. This data compares well with theIgG dimensions of about 4.5–14 nm [17]. Thisindicates that the AB films are randomly oriented,so the thickness is an average. As for bare glassthe thickness of 2–4 nm indicates either a layer offlatly oriented IgG which might either be boundto the immobilized antibody or non-specificallyadsorbed on the film or to a very low surfacedensity of AB. The activity of the antibody filmson the polymer-coated slides was clearly higherthan that on bare glass due to the higher packingdensity of IgG molecules in the film.

The BCA test, which was performed after thereaction with labeled antigen, showed only aslight increase of protein which was about 1–1.5mg IgG. The low amount of bound antigen couldeither mean that parts of the LB-film were washedoff or that only 520% of the immobilized anti-IgG was active and able to bind the antigen. Noincrease in protein was observed for LB films onbare glass.


3.3. Non-specific protein adsorption

The effect of desorption agents on non-specifi-cally adsorbed protein on PVC-COOH, sol–gelsand glass was tested using 0.1 M phosphatebuffer, 0.5 M NaCl, 2 M NaCl and 1% N-cetyl-N,N,N-trimethylammoniumbromide. The filmthickness was determined at 12 slide positionsafter each washing (15 min). The respective plotsare shown in Fig. 2a–d. As is obvious from Fig.2a the surface of the antibody film is rough andheterogeneous, but appears smooth and uniformafter a washing in 2 M NaCl and 1% detergentsolution (Fig. 2d). The film thickness on PVC-COOH before washing was 19.8%, whereas afterwashing in 0.5 M NaCl, 2 M NaCl and 1%N-cetyl-N,N,N-trimethylammoniumbromidebuffer the thickness was 2091, 1591, 1391and 1091 nm (the washing detergent did notdecrease the film thickness further). Taking the

thickness to be directly proportional to the sur-face coverage 25% of the IgG was desorbed inbuffer, other 10% in 0.5 M NaCl, other 15% in 2M NaCl and in 1% detergent solution. As a result,desorption increases with increasing ionic strengthof the washing solution, which might be due tothe electrostatic interactions between the solutionand the immobilized antibody [19]. The mono-layer thickness of 10 nm was in good agreementwith the work of Turko et al. [5] who reported athickness of 10.8 nm.

3.4. Surface characterization by ellipsometry

The surface profiles of anti-IgG LB films de-posited on PVC-COOH and aminopropyl sol–gelusing ellipsometric data are shown in Fig. 3. Theellipsometric parameters C and D were measuredat 12 positions of the polymer-coated slide beforeand after LB film deposition. Reference measure-

Fig. 2. Desorption of non-specifically bound protein using six PVC-COOH layers. The thickness of the anti-IgG film immobilizedon PVC-COOH was measured (a) before washing; (b) after washing in 0.1 M phosphate buffer; (c) 0.5 M NaCl; and (d) 2 M NaCland 1% N-cetyl-N,N,N-trimethylammoniumbromide.


Fig. 3. Surface topography of an anti-IgG LB film on (a)PVC-COOH (slide positions 1–4), and (b) aminopropyl-modified sol–gel (slide positions 5–8).

glass and chloropropyl sol–gel and covalentlybound to aminopropyl sol–gel and carboxy-PVCare shown in Fig. 4a–d. The IgG molecules onglass (Fig. 4a) appeared to be heterogeneouslydistributed over the surface. The frequently ob-served granular structures had a diameter of 94911 nm. However, bigger anti-IgG clusters andaggregates were also present. The average distancebetween the granules was 150950 nm whichcorresponded well with a local maximum in thetwo-dimensional Fourier transform around 1/(100nm). Fig. 4b shows the AFM image of an anti-IgG LB film on chloropropyl sol–gel. The typicalsize (diameter) of the granular structures was2494 nm and the width fell between 31 and 37nm. The height of imaged anti-IgG was about892 nm. Compared with the anti-IgG film onbare glass, the structure of the anti-IgG film onchloropropyl sol–gel appeared to have a regularsurface feature. The AFM image of the anti-IgGmolecules on aminopropyl sol–gel (Fig. 4c)showed homogeneously distributed granules in anaverage distance of 2394 nm. The diameter ofthe granular structures was 1294 nm. The AFMimages obtained on aminopropyl sol–gel were themost stable and most reproducible, probably be-cause covalent binding of the IgG film led to ahigher mechanical stability and rigidity of thefilm. Fig. 4d shows the surface morphology of ananti-IgG film covalently immobilized on PVC-COOH. Apart from granular structures strand-like structures were observed. The surface was nothomogeneously covered with anti-IgG molecules,but showed some depressions of 60 nm diameter.The granular and strand-like structures had adiameter of 10–40 nm distributed in an averagedistance of 42910 nm. Considering the heteroge-neous distribution of granular and strand-likestructures on the PVC-COOH we assume thatmost of the antibody was adsorbed on the poly-mer rather than covalently immobilized.

4. Conclusion

Anti-human IgG LB films were characterizedon PVC-COOH, chloropropyl and aminopropylsol–gel and glass by means of ellipsometry, AFM

ments of PVC-COOH layers were performed afterovernight incubation in EDC. Measurements ofthe sol–gel layers were done after immersion inwater to avoid errors resulting from swelling ef-fects. Due to the formation of multilayers anduncontrolled crosslinking effects of GA the sur-face of the anti-IgG Langmuir film onaminopropyl sol–gel was not flat, but rough. Incontrast the anti-IgG film on PVC-COOHwas uniform and smooth which is obviousfrom the standard deviations ssol–gel=7.8 nm andsPVC-COOH=1.2 nm of the film thicknesses.

3.5. Surface characterization by atomic forcemicroscopy (AFM)

Typically, in constant force mode, height mea-surements by AFM may be obscured by friction-induced height artifacts. However, as stated inSection 2.1, in this study the fast scan-axis wasoriented perpendicular to the cantilever’s axis ofsymmetry, to minimize friction-induced height ar-tifacts. Also, trace and retrace images were com-pared and no differences in height contrasts couldbe detected. Furthermore, the loading forces wereminimized prior to AFM imaging and very softcantilevers were used (cantilever spring constant30 mM m−1). The particle sizes of the ABmolecules immobilized on the polymer-coatedslides, which are determined by AFM, are overes-timates of the real particle sizes by approximately30%.

The AFM images (2×2 mm) of anti-IgGmolecules which were physically adsorbed on


Fig. 4. Atomic force microscopy (AFM) images (2000×2000 nm) of anti-IgG LB films on (a) glass; (b) chloropropyl sol–gel; (c)aminopropyl sol–gel; and (d) PVC-COOH.


Fig. 4. (Continued)


and the BCA test. The results obtained by thesethree different analytical methods were in goodagreement with each other; 3 mg AB per slide wasadsorbed on glass resulting in a film thickness of992 nm. The diameter of the granular AB struc-tures was 94911 nm, which was four times largerthan that on PVC-COOH, chloropropyl andaminopropyl sol–gel. In contrast to the anti-IgGLB film on chloropropyl and aminopropyl sol–gelthe AB molecules were heterogeneously dis-tributed on the substrate surface. The averagedistance of the granules was 150950 nm. Thethickness of the AB LB film on PVC-COOH was1191.2 nm, which is in good agreement with thesize of an IgG molecule (10×14×4.5 nm) deter-mined by X-ray crystallography. AB (6 mg) wasimmobilized on the PVC-COOH-coated slide. TheAFM images showed granular and strandlikestructures of 10–40 nm diameter heterogeneouslydistributed over the surface with an average dis-tance of 42910 nm. The best and the mostreproducible results were achieved for LB films onsol–gel; 7.5 and 8 mg AB per slide were immobi-lized homogeneously on chloropropyl andaminopropyl sol–gel. Small granules of 2494and 1294 nm diameter were imaged on chloro-propyl and aminopropyl sol–gel indicating a veryclose packing of the AB molecules.

Acknowledgements

This work was supported by the Fonds zurForderung der wissenschaftlichen Forschung(FWF), Austria within project J01286-CHE. Themeasurements on the profilometer and the spin-coating of the glass slides were performed at theIROE-CNR, Firenze (Professor Sottini).

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Characterization of IgG LangmuirâBlodgett films immobilized on functionalized polymers