Materials Science and Engineering C 31 (2011) 370–376

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Materials Science and Engineering C

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Densities and orientations of antibodies on nano-textured silicon surfaces

Satyendra Kumar, Ramchander Ch, Dharitri Rath, Siddhartha Panda ⁎Department of Chemical Engineering and Centre for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur, UP 208016, India

⁎ Corresponding author. Tel.: +91 512 259 6146; faxE-mail address: spanda@iitk.ac.in (S. Panda).

0928-4931/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.msec.2010.10.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 July 2010Received in revised form 15 September 2010Accepted 18 October 2010Available online 28 October 2010

Keywords:ImmunosensorNano-textureSiliconAntibodyOrientation

High surface densities of properly oriented antibodies are desired for enhancing the sensitivity ofimmunosensors. A systematic investigation of the densities and orientations of antibodies, immobilizedwithout and with intermediate protein molecules (protein-A and streptavidin) on nano-textured siliconsurfaces (RMS roughnessb100 nm) was performed and the results were compared to those obtained fromnon-textured surfaces. The primary antibody densities and orientations (through densities of secondaryantibodies) were quantitatively measured for the different cases. Higher densities were obtained for all nano-textured surfaces compared to non-textured ones. It was observed that higher primary antibody densitieswere obtained when no intermediate proteins were used. The different nano-texturing conditions did nothave a significant effect on the densities with no intermediate proteins and with protein-A, but had an effectwith streptavidin. The densities of the properly oriented primary antibodies increased on most of the nano-textured surfaces used as compared to the non-textured samples. The effect of the texturing on the densitieswas observed for several of the surfaces studied. Design nano-texturing could be used to maximize as well astune the densities of the properly oriented antibodies on the substrate surface.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Early disease detection can be facilitated by sensitive sensors withlow detection limits for the disease biomarkers. One class of suchsensors is immunosensors which are based on the principle of specificand strong binding property of the bio-receptor molecules (anti-bodies) to their complementary target analytes (antigens). Forfabricating a highly efficient sensor, proper immobilization of thereceptormolecules on the sensor platform surface is a crucial step. It isexpected that higher surface densities would result in higher antigencapture which would enable lower detection limits. Antibodiesimmobilized on solid surfaces have reduced antigen capture efficiencyas compared to those in the solution due to random orientations andsteric hindrances and partial denaturation of protein molecules byinteraction with the solid surface [1,2]. The antibodies placed in anend-on orientation, i.e. with the Fab region away from the surface andthe Fc region on the surface, are known to have the maximumefficiency [2]. Hence, along with surface density, proper orientation ofthe antibody is necessary to obtain higher antigen capture and henceenable lower detection limits.

Antibodies can be immobilized on solid surfaces by nonspecificphysical adsorption [3] or by the formation of covalent bonds betweenfunctional groups on protein molecules (e.g., amines) and thecomplementary coupling groups (e.g., aldehydes) introduced onto

the solid surfaces [4]. However, it is a challenge to obtain the desiredend-on orientation of the antibody molecules with about only 20%proper immobilization reported [5]. Hence to overcome this problem,specific schemes have been proposed for the desired orientation ofantibodies [6,7]. These include immobilization via affinity tags [8,9],biotinylation of capture molecules and their immobilization onstreptavidin coated supports [10], immobilization of IgG on protein-G coated surfaces [11], and DNA-directed immobilization [12].Further, to get an even better orientation, genetically modifiedforms of protein-G have been used [13].

Silicon surfaces have been nano-textured to utilize the higherspecific surface area for enhancing the functionalized moleculedensity. Bessuille et al. used porous silicon (pore diameter 0.5–1 μm) and Oillic et al. deposited silicon grains (about 100 nm diameterand larger as estimated from SEM) on oxidized silicon surfaces toobtain bimolecular densities greater than those obtained on planarsurfaces [14,15].

As mentioned, there are reports of usage of intermediate proteinmolecules for better orientations on planar surfaces [6–13] and of theutilization of textured surfaces without the use of intermediateproteins for higher bimolecular densities [14,15]. However, to the bestof our knowledge we are not aware of any quantitative work on theeffect of nano-textured surfaces on the densities and orientations ofantibodies using the intermediate proteins. In the present study, asystematic investigation on the densities and orientations of anti-bodies, immobilized with and without intermediary protein mole-cules on nano-textured silicon surfaces is performed and the resultsare compared with those obtained from planar surfaces. In an earlier

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work [16], we studied the effect of silicon nano-texturing on theaminosilane densities, and in this work, we utilize these nano-textured silicon surfaces for further investigation. The importance ofthe present work lies in the fact that although a higher density of theantibodies is expected frommore textured (i.e. rougher) surfaces, thisneed not result in a higher number of recognition reactions needed forobtaining higher sensitivity. A study of the coupled effects of nano-textures of different dimensions with and without intermediateproteins on antibody orientation would help obtain the conditionsneeded for optimum device performance.

2. Materials and methods

2.1. Materials

Silicon wafers of (111) orientation, p-type, n-type with resistivity of1–20Ω cm were obtained from Wafer World, Inc. USA. Trichloroeth-ylene 99%, acetone 99%, toluene 99% and hydrogen peroxide 30% wereobtained from Qualigens Fine Chemicals, India. Absolute ethanol 99.9%and sulphuric acid 98%were obtained fromS.D.Fine-ChemLtd.Mumbai,India. Aminopropyltriethoxysilane (APTES) 99% and phosphate buffersaline (PBS buffer) pH: 7.4 were obtained from Sigma-Aldrich Inc.,Germany. Glutaraldehyde 25% was obtained from Loba Chemie,Mumbai, India. Protein-A (Staphylococcus aureus), streptavidin (Strep-tomyces avidinii), anti-BSA polyclonal IgG, anti-BSA IgG-biotin, anti-BSAIgG-HRP, anti-BSA IgG-FITC, 3-3′-diaminobenzidine tetrahydrochloride(DAB) substrate, bovine serum albumin (BSA), treated as antigen, andTween-20 were obtained from Merck (formally GeNie™), Bangalore,India. De-ionized water (conductivity 0–0.5 S/m) purified with a GlenRO+ systemwas obtained fromGlen India Ltd. and zero-grade nitrogenwas obtained from Sigma Gases & Services, India.

2.2. Experimental methods

2.2.1. Silicon processingThe siliconwaferswere cut into the desired size (5 mm×5 mm) and

were sonicated (1.5 l, 60W, 33 kHz) at an ambient temperature in de-ionized (DI) water for 2 min followed by cleaning at 30 °C intrichloroethylene, acetone, and ethanol for 5 min each followed by2 min of sonication in DI water and rinsing with profuse amounts of DIwater and blown dry with N2 gas. The cleanedwafers were divided into5 types, onenon-textured and fournano-textured (referred to as: T1, T2,T3, and T4). Nano-texturing of the sampleswas doneat 45 °C in a freshlyprepared isotropic etchant solution of HF/HNO3/H2O in a ratio of 5:1:10for 25 min and 15 min respectively for T1 and T2, and in the ratio 1:1:10for 15 min and 25min respectively for T3 and T4 [16]. After nano-texturing, each samplewas thoroughly rinsedwith ample amounts ofDIwater followed by drying with N2 gas and kept in a vacuum desiccator.

2.2.2. HydrolysisThe Si wafer samples were hydrolyzed in separate beakers in a

Piranha solution (to be handled with caution) (H2SO4:H2O2 in a ratio of3:1) for 3 h at 130±5 °C. After completion of the reaction, the waferpieces were put in hot (about 95 °C) DI water for 10 min in order toavoid any thermal stresses in the wafers. Later these Si wafer pieceswere rinsed with DI water (at about 23 °C) and blown dry with N2 gasand kept in a vacuum oven (Mahendra Scientific Instruments Mfg. Co.,Kanpur, India) at −700 mm Hg and 120 °C for 1 h to facilitate theremoval of adsorbed hydroxyl groups from the surface of the Si wafer.

2.2.3. SilanizationThe hydrolyzed samples were silanized using 3% APTES in toluene

as a solvent. After completion of the silanization reaction, the sampleswere taken out and rinsed thrice with the solvent and once withethanol in order to remove the unreacted APTES layer from thesurface and then were blown dry with N2 gas. These samples were

immediately taken to the next step of functionalization i.e., cross-linking.

2.2.4. Cross-linking2.5% (v%) glutaraldehyde in ethanol was and the silanized wafers

were immersed in the solution for 1 h at an ambient temperature.After completion of the stipulated reaction time, the wafers wererinsedwith ethanol, DI water and PBS 3 times each, dried under N2 gasand used for the immobilization of antibodies.

2.2.5. Primary antibody immobilizationAntibody immobilization on the glutaraldehyde treated Si wafers

was done in three different schemes. (a) No intermediate biomolecules(on the post glutaraldehyde treated surface): The glutaraldehydetreated silicon wafers were immersed in a 100 μl solution of PBScontaining 1 μg of antibody molecules (anti-BSA IgG) and incubatedovernight at 4 °C. (b) Immobilization of protein-A followed by IgGantibody: In this case the Siwaferswere immersed in a100 μl solutionofPBS containing 1 μg of protein-A and incubated for 2 h at an ambienttemperature. Then the antibody immobilization was done in the samemanner as above. FITC tagged antibodieswere used for the optical baseddetection, but for these, incubation for the immobilization of taggedantibody was for 1 h. (c) Immobilization of streptavidin followed byimmobilization of biotinylated antibodies: Here, the Si wafers wereimmersed in a 100 μl solution of PBS containing 1 μg of the streptavidinmedium for 2 h at an ambient temperature, and then washedthoroughly with PBS buffer 3 times followed by a single wash withPBST (phosphate buffered saline with Tween-20). These wafers werethen incubated in a 100 μl solution of PBS containing 1 μg of anti-BSAbiotinylated IgG in PBS and incubated overnight at 4 °C.

2.2.6. Antigen immobilizationsThe antibody immobilized wafers prepared in the above three

schemes were immersed in known quantity of bovine serum albumin(BSA) as an antigen and incubated at 37 °C for 1 h and washedthoroughly with PBS buffer. Then they were incubated for 30 min withthe milk protein casein (0.1 mg/ml) to block the unreacted glutaralde-hyde molecules (if any) from reacting with the secondary antibodies.

2.2.7. Secondary antibody immobilizationThe antigen immobilized silicon wafers prepared in the above

three schemeswere incubatedwith known amounts of FITC (anti-BSAIgG-FITC) or HRP (anti-BSA IgG-HRP) conjugated antibody in PBSbuffer for 1 h at an ambient temperature and washed thoroughly 3times with PBS buffer. In this work, these antibodies which wereimmobilized subsequent to the surface antigens are referred to assecondary antibodies.

2.3. Surface density measurement

2.3.1. Quantitative estimation of antibodiesThis was done by two methods, the bicinchoninic acid (BCA) assay

for the primary antibodies and the tagged horseradish peroxidase(HRP) enzyme method for the secondary antibodies. (a) BCA assay:BCA assay was performed to measure the amount of antibodyimmobilized on the modified silicon surfaces. Anti-BSA IgG wasused as a standard for primary antibody concentrations. The amountof immobilized antibody on the silicon surface was obtained bycomparing the original antibody concentration in the solution usedfor the immobilization to the remaining antibody concentration afterthe immobilization. (b) HRP enzyme tagged method: In thisexperiment, enzyme (HRP) tagged anti-BSA IgG was used as thestandard. Immobilization of the primary antibody on the surface wasfollowed by antigen binding and these antigens were subsequentlybound by the enzyme tagged secondary antibodies. For both cases, thelinear fit of the calibration curves had R2≥0.99 with known

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concentrations of anti-BSA IgG (for no intermediate biomolecule andprotein-A), anti-BSA biotinylated IgG (for streptavidin) and anti-BSAHRP tagged IgG for secondary antibody. The antibody densities weremeasured using an enzyme-linked immunosorbent assay (ELISA)reader (Thermo Scientific Multiskan® EX, Vantaa, Finland). We didnot perform the BCA test for secondary antibody (anti-BSA IgG-HRP)estimation due to experimental limitations as the enzyme tagged IgGwith BCA test could give interfering results. Experiments wereconducted in triplicate and the error bars represent one standarddeviation about the mean.

2.3.2. Semi-quantitative and qualitative estimation

2.3.2.1. Fluorescence spectrometry. Fluorescence intensitymeasurementson the fluorescein isothiocyanate (FITC) tagged antibody immobilizedsurfaces were conducted with the help of a fluorescence spectrometer(Edinburg Instruments, Livingston, UK). The excitation wavelength was475 nm and the scanning range was 500–600 nm. The average intensitywas taken from the area under the curve for the 520–540 nm range andplotted by using the Origin Pro 7.5 software to compare the averageintensity values. Experiments were conducted in triplicate and the errorbars represent one standard deviation about the mean.

2.3.2.2. Fluorescence microscopy. Fluorescence microscopy of theantibody immobilized surface images were taken in a Leica Micro-systems Fluorescence Microscope (Germany).

2.3.2.3. Atomic Force Microscopy (AFM). Surface topology images weretaken in non-contact mode in a Molecular Imaging™ AFM (AgilentTechnologies, Chandler, AZ, USA).

3. Results and discussion

3.1. Surface densities of the primary antibodies

A schematic of the primary antibodies on the non-textured andnano-textured surfaces with and without intermediate proteins isshown in Fig. 1. As reported in our earlier paper [16], the root meansquare (RMS) roughness value of the as received silicon wafers was1.8 nm (non-textured), where as for the nano-textured samples, RMS

Fig. 1. Schematic illustrations of antibody densities and orientations on non- and nano-texturNo intermediate protein, Protein-A, Streptavidin. T/N,P,S: Textured/No intermediate protein

roughness values were 17.6 nm, 24.2 nm, 42 nm, 69.2 nm for thecases referred to as T1, T2, T3 and T4 respectively, and this texturinghas been explained on the basis of a texturing model in the earlierpaper [16], where it was postulated that the presence of oxide islandswas responsible for the higher RMS roughness values for the T3 andT4 cases. Also, the wafer orientation did not have any effect on theresults.

Verification of the primary antibody immobilization was con-ducted with the help of fluorescence microscopy. Fig. 2 shows theimages of the FITC tagged IgG layer for both the planar (non-textured)and the nano-textured surfaces with protein-A and without anyintermediate protein layers, along with the control sample (i.e. noFITC tagging). The images verify that in all cases of immobilization, thefluorescence signal obtained was due to the FITC emission only. Weobserved more agglomeration of the FITC tagged antibodies for thecase of no intermediate proteins compared to the immobilizationwithProtein-A. The surface densities depend, among other factors, on theantibody dimensions. Based on AFM, the width of the antibodies (i.e.,the distance between the terminal regions of the two Fab domains)was estimated to be about 30 nm (Fig. 3) and it is in agreement withthe reported literature values [8]. As the hinge region between thetwo Fab domains is known to be flexible, it is difficult to predict theexact conformation of antibody immobilized on the surface.

Fig. 4 shows the density of the primary antibodies immobilized onboth non- and nano-textured surfaces with and without the use ofintermediate proteinmolecules. The results show that the overall amountof antibody immobilization increases on the nano-textured surfaces ascompared to the non-textured samples. On non-textured surfaces, weobtained average primary antibody densities of 6.37E+10/mm2, 5.03E+10/mm2 and 4.94E+10/mm2 with no intermediate protein (denoted asNN in the figure), protein-A (NP) and streptavidin (NS) respectively. It isobserved that the IgG density is higher for the case of without use ofintermediate protein compared to the case with an intermediate proteinand this can be attributed to the relatively more random orientation andagglomeration in the case of no intermediate protein [17]. From the BCAtest of primary antibody estimation, we observed surface densities higherthan those expected fromtheantibodydimensionswhichare likelydue toagglomerations as seen in the fluorescence micrographs (Fig. 2). Ourresults are in accordancewith the reported literature onprimary antibodydensities on non-textured surfaces [17–20]. Using a BCA test, Denczyk et

ed silicon surfaces with andwithout intermediate biomolecules. N/N,P,S: Non-textured/, Protein-A, Streptavidin.

Fig. 2. Fluorescence microscope images of primary antibodies immobilized on siliconsurfaces with no intermediate protein and with protein-A along with theircorresponding control samples for the non-textured as well as the four nano-texturedsurfaces (T1, T2, T3, and T4). The surface identities are as in Fig. 1.

Fig. 3. Y-shaped AFM images of IgGs immobilized on the non-textured silicon surface.

Fig. 4. Surface densities of primary antibodies immobilized on silicon non-textured andthe four nano-textured surfaces (T1, T2, T3, and T4) with no intermediate protein,protein-A and streptavidin, measured using the BCA method. The surface identities areas in Fig. 1. The error bars represent one standard deviation about the mean.

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al. [17] reported lesser amountof IgG immobilization throughprotein-Aascompared to no intermediate protein, on a borosilicate glass cover slip.Using ellipsometry, Wang and Jin [19] reported lower antibody densitieson silicon surfaces with protein-A compared to with no intermediateproteins. Using a fluorometer, Vijayendran and Leckband [20] reportedlower densities on silica fiber-optic probeswith streptavidin compared towith no intermediate proteins.

Higher primary antibody densities are obtained on the nano-texturedsurfaces compared to the planar surfaces. With no intermediate proteins,average densities of 9.66E+10/mm2, 1.01E+11/mm2 9.80E+10/mm2,

9.89E+10/mm2, were obtained on the surfaces T1 (denoted by T1N inFig. 4), T2 (T2N), T3 (T3N) and T4 (T4N) respectively. Thus the degree ofnano-texturingdid not affect thedensities as these valueswere almost thesame within experimental error. A similar result was obtained withprotein-A where average densities of 9.12E+10/mm2 , 9.81E+10/mm2,9.22E+10/mm2 and 9.56E+10/mm2 were obtained for the surfacesdenoted by T1 (T1P), T2 (T2P), T3 (T3P) and T4 (T4P) respectively.However the nano-texture showed an effect on the densities withstreptavidin with average densities of 5.98E+10/mm2, 8.21E+10/mm2,7.75E+10/mm2 and 9.81E+10/mm2 for the surfaces T1 (T1S), T2 (T2S),T3 (T3S) and T4 (T4S) respectively, although it is to be noted that thedensities obtainedon the surfacesdenotedbyT2 andT3were comparable.This could be attributed to the relatively higher rigidity and largerdimension of streptavidin [21] coupled with its structural aspects, i.e., thesmall groove of streptavidin for proper binding of biotin facilitates betterorientation but at the cost of density.

The above observations are supported by the semi-quantitativeresults of fluorescence spectroscopy (Fig. 5) and the fluorescencemicroscopy images (Fig. 2). Fig. 5 shows that the primary antibodydensity is slightly higher for the case of no intermediate proteincompared to the case with protein-A. Also, the amount of thefluorescence signal coming from the nano-textured samples for allcases is higher than those from the non-textured silicon surfaces. Theabove trends are qualitatively supported by the fluorescencemicroscopy images for the different conditions (Fig. 2).

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3.2. Orientations of the primary antibodies (i.e. densities of thesecondary antibodies)

As mentioned, merely enhancing the primary antibody densitymay not enhance the sensor functionality. What is critical is thedensity of the properly oriented antibodies. Verification of thesecondary antibody immobilization was conducted with the help offluorescence microscopy. Fig. 6 shows the images of the FITC taggedIgG layer for both the non-textured and the nano-textured surfaceswith protein-A, streptavidin and without any intermediate proteinlayers, alongwith the control sample (i.e. no FITC tagging). The imagesverify that in all cases of immobilization, the fluorescence signalobtained was due to the FITC emission only.

In our experiments, only the properly immobilized primaryantibodies have exposed antigen binding Fab domains to which theantigen (BSA) can be bound by the specific recognition epitopes. Theabsorbance obtained from the HRP is measured, which gives theamount of the properly oriented primary antibodies. This quantifiesthe antigen and hence the amount of the primary antibodies with thedesired end-on orientation.We have assumed here a 1:1 ratio for eachantigen attached to the secondary as well as the primary antibody.Hence the densities of the secondary antibodies can be assumed to bethose of the properly oriented primary antibodies.

Fig. 7 shows the densities of the secondary antibodies immobilizedon both non-textured and nano-textured surfaces with and withoutthe use of intermediate protein molecules. The results show that theoverall amount of secondary antibody immobilization increases onmost of the nano-textured surfaces (with T1N and T1S beingexceptions) as compared to the non-textured samples. On non-textured surfaces, we obtained average secondary antibody densitiesof 3.35E+09/mm2, 1.03E+10/mm2 and 1.35E+10/mm2 with nointermediate protein (NN), protein-A (NP) and streptavidin (NS)respectively. This trend is opposite to that observed for the primaryantibody densities and highlights the role of intermediate proteins infacilitating the proper orientation of the primary antibodies. Variousapproaches are reported in the literature for the proper orientation ofimmobilized antibodies on planar surfaces for the enhancement of theimmobilized antigen densities [22,23]. Lee et al. [23] reported thatsurface modification of the protein-A using thiol groups resulted inthe enhancement of the antigen binding to the antibody, measured bythe relative shift in the SPR angle though they did not quantify it.Danczyk et al. [17] reported antigen concentrations 2.2E+11/mm2

and 2.36E+11/mm2 for no intermediate protein and protein-A casesrespectively using the BCA test.

With no intermediate proteins, average densities of 3.94E+09/mm2, 1.30E+10/mm2 5.03E+09/mm2 and, 8.26E+09/mm2 wereobtained for the surfaces denoted by T1 (T1N), T2 (T2N), T3 (T3N) andT4 (T4N) respectively. However, unlike in the case of the primary

Fig. 5. Fluorescence spectroscopy data showing the relative fluorescence band area ofsilicon non- and nano-textured samples (T2), with no intermediate protein and withprotein-A. The surface identities are as in Fig. 1. The error bars represent one standarddeviation about the mean.

antibody densities where all the nano-textures used provided similardensities within experimental error, here the nano-texture affected thedensities of the secondary antibodies in some cases. The highest densitywas obtained with the surface denoted by T2 (T2N) followed by thesurfaces denoted by T4 (T4N) and T3 (T3N). The density obtained withthe surface denoted by T1 (T1N) was similar (within experimentalerror) to that obtained with the planar surface (NN). Also, unlike in thecase of the primary antibody densities obtained using Protein-A wheretoo all the nano-textures used in this study provided similar densitieswithin experimental error, here different densities of secondaryantibodies were obtained with different nano-textures in some cases.Secondary antibody densities with average values of 1.30E+10/mm2,1.71E+10/mm2, 1.47E+10/mm2 and 1.50E+10/mm2 were obtainedfor the surfaces denoted by T1 (T1P), T2 (T2P), T3 (T3P) and T4 (T4P)respectively. As in the case of primary antibody densities, withstreptavidin thenano-textured surfaces affected the secondary antibodydensities with average values of 1.37E+10/mm2, 2.00E+10/mm2

1.51E+10/mm2and1.74E+10/mm2obtained for the surfaces denotedby T1 (T1S), T2 (T2S), T3 (T3S) and T4 (T4S) respectively. For thetexturing conditions used here, T2 resulted in the highest densities andthe densities were in the decreasing order of T2NT4NT3NT1, denotingthe effect of nano-texturing.

As in the non-textured case, for the textured cases, use ofstreptavidin results in the highest densities followed by protein-Awith no intermediate proteins resulting in the lowest densities.Comparing the cases of the two intermediate protein molecules, thetilted orientation of the antibodies obtained with protein-A (asprotein-A has different antibody binding properties than streptavi-din) could result in more agglomeration than with the normallyoriented antibodies with streptavidin, resulting in higher densitieswith streptavidin. Future work on antibodies of different sizes (e.g.IgM, fragmented antibodies) could help elucidate the underlyingmechanisms.

The above observations for the secondary antibody densities aresupported by the semi-quantitative results offluorescence spectroscopy(Fig. 8) and the fluorescencemicroscopy images (Fig. 6). The histogramplot (Fig. 8) shows that for the non-textured surface, the mean value ofthefluorescence signal increases in the order of no intermediate protein(NN), protein-A (NP) and streptavidin (NS), although the later two arewithin experimental error. Also, the amount of fluorescence signalcoming from the nano-textured samples (T2) are higher than thosefrom the non-textured counterparts (i.e., T2NNNN, T2PNNP, T2SNNS).The above trends are qualitatively supported by the fluorescencemicroscopy images for the different conditions (Fig. 6). Our results arewell in agreement with the results reported earlier for the differentcases ofmodification for secondary antibody immobilization.Demirel etal. [24] showed theenhancementoffluorescence signalwith theAPTES–biotin–streptavidin multilayer systems as compared to the planehydroxylated silicon surface. Kim et al. [25] showed similar enhance-ment with the use of recombinant protein-G molecules reported withthe help of fluorescence microscopy and surface plasma resonancespectroscopy. Wang et al. [26] reported qualitative visualization of theenhancement in the antibody attachment by using the nano-texturedsurface as compared to the non-textured silicon surface.

Fig. 9 shows the percentage of oriented primary antibodies (ratioof secondary to primary antibody densities) for the different cases.Intermediate proteins are known to increase the orientation and heretoo higher orientations are obtained with the intermediate proteins.For almost all cases used here (with the exception of the densitiesobtained with T3S and T3P being similar within experimental limits),the highest orientations were obtained with streptavidin followed byprotein-A. A new finding here is the effect of nano-texturing onorientation. As seen in the case of no intermediate proteins, somenano-texturing conditions have enhanced the orientation. In the casesof intermediate proteins, the effect of nano-texturing seems to beenveloped by the effects of the intermediate proteins.

Fig. 6. Fluorescence microscope images of secondary antibodies immobilized on surfaces with no intermediate protein, with protein-A and with streptavidin along with theircorresponding control samples for the non-textured as well as the four textured surfaces (T1, T2, T3, and T4). The surface identities are as in Fig. 1.

Fig. 7. Surface densities of secondary antibodies immobilized on non-textured and thefour textured surfaces (T1, T2, T3, and T4) with no intermediate protein, protein-A andstreptavidin, measured using the HRP tagged method. The surface identities are as inFig. 1. The error bars represent one standard deviation about the mean.

Fig. 8. Fluorescence spectroscopy data showing the relative fluorescence band area ofnon- and nano-textured samples (T2), with no intermediate protein, with protein A andwith streptavidin. Surface identities are as in Fig. 1. The error bars represent onestandard deviation about the mean.

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Fig. 9. Comparison of the percentage of oriented antibodies for the different cases ofnon-textured and the four textured surfaces (T1, T2, T3, and T4) with no intermediateprotein, protein-A and streptavidin. The surface identities are as in Fig. 1. The error barsrepresent one standard deviation about the mean.

376 S. Kumar et al. / Materials Science and Engineering C 31 (2011) 370–376

4. Conclusion

A study of the coupled effects of silicon nano-textures of differentdimensions with intermediate proteins of different dimensions andstructures was conducted to help understand the mechanismsinvolved in obtaining antibodies of high densities with appropriateorientations for optimum device performance. The densities of theprimary antibodies and the densities of the properly orientedantibodies (i.e., the densities of the secondary antibodies) werequantitatively measured for the different cases and these weresupported by non-quantitative measurements. All nano-texturedsurfaces used provided higher densities compared to the non-textured ones. Higher primary antibody densities were obtainedwhen no intermediate proteins were used. The different nano-texturing conditions did not have a significant effect on the densitiesfor the surfaces with no intermediate proteins and with protein-A.However, with streptavidin, the densities were affected by the nano-texturing conditions. The densities of the properly oriented primaryantibodies increased on most of the nano-textured surfaces used ascompared to the non-textured samples. With streptavidin, the effectof the texturing is observed with densities being in the order of thesurfaces T2NT4NT3NT1. The mean values of the densities in the othertwo cases (i.e., with protein-A and without any intermediate protein)are in the same order, although some data are within experimentalerror. Thus design nano-texturing could be used to maximize as wellas tune the densities of the properly oriented antibodies on thesurface. Nano-textures, apart from increasing the density, can alsoenhance the orientation of the antibodies. Suitable nano-texturing

along with appropriate intermediate proteins can enhance the surfacedensity of properly oriented antibodies.

Acknowledgements

This work was supported by the Department of Science andTechnology, India. One of the authors (SK) acknowledges fellowshipfrom the Council of Scientific and Industrial Research, India. The helpof Dr. S. Sivakumar for the fluorescence spectrometer isacknowledged.

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