Langmuir 1997, 13, 3427-3433
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2. Assembly of Alternating Polyelectrolyte and Protein Multilayer Films for Immunosensing Frank Caruso,†,§ Kenichi Niikura,‡ D. Neil Furlong,*,† and Yoshio Okahata‡ CSIRO, Division of Chemicals and Polymers, Private Bag 10, South Clayton MDC, Clayton, Victoria 3169, Australia; and Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 227, Japan Received August 16, 1996. In Final Form: November 25, 1996X Alternating polyelectrolyte films constructed by the sequential adsorption of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) have been used as substrates for the immobilization of immunoglobulin G (IgG) and anti-IgG. Anti-IgG has also been immobilized in multilayer films by the alternate deposition of PSS and anti-IgG. The assembly process of the multilayer films was monitored using a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Film growth was achieved up to at least nine (5 anti-IgG and 4 PSS) layers. The utility of these films for immunosensing has been investigated via their subsequent interaction with IgG. The alternating polyelectrolyte/protein layers were constructed in order to increase the binding layer capacity (i.e. sensitivity) of the thin film with respect to IgG detection. The sensitivity, determined using IgG mass uptake data from quartz crystal microgravimetry, was found to be linearly dependent on the number of anti-IgG layers (and hence the amount of IgG incorporated) in the polyelectrolyte film when the anti-IgG layers are separated by one PSS layer. In contrast, for films where anti-IgG layers are separated by five polyelectrolyte (PSS(PAH/PSS)2) layers, only the outer anti-IgG layer is immunologically active. This is attributed to the formation of a dense polyelectrolyte film through which antibody permeation is restricted. The films evaluated have promise in that the sensitivity can be tuned by fabricating the desired number of protein layers, whilst the selectivity can be modified by selecting the desired biospecific biomolecule.
Introduction Thin organic films with a supramolecular architecture in which individual biologically active molecules (e.g. antibodies, DNA, enzymes) are macroscopically oriented and/or embedded are of special interest, particularly in the development of biological sensors. Such thin films have in the past been commonly fabricated using Langmuir-Blodgett or self-assembly techniques. The LB technique has been used for obtaining monomolecular protein layers on solid surfaces.1-4 The spontaneous adsorption of charged proteins onto solid surfaces of opposite charge (e.g. mica) is a widely used technique and also produces monomolecular protein layers.5-7 Multilayer protein films have been formed by using antibodyantigen pairs and biotin-streptavidin as coupling units between different molecular layers8,9 and by incorporating proteins into the interlayer space of cast membrane films, by use of the opposite charges on the protein and the membrane surface.10,11 This latter principle of opposite charge protein-surface adsorption has also been used by * To whom correspondence should be addressed. Fax: 61-3 9542 2515. E-mail:
[email protected]. † CSIRO. ‡ Tokyo Institute of Technology. § Current address: Max-Planck-Institute for Colloids and Interfaces, Rudower Chaussee 5, D-12489 Berlin, Germany. X Abstract published in Advance ACS Abstracts, May 15, 1997.
Lvov and co-workers to fabricate multilayer multicomponent protein films by electrostatic layer-by-layer adsorption of oppositely charged polyelectrolytes and proteins.12,13 In that work it was demonstrated that the alternate adsorption of oppositely charged polyelectrolytes and proteins is an effective and general procedure for the preparation of protein-containing organic thin films. In part 114 we have demonstrated the construction of alternating multilayer films of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS) on gold surfaces. Films were grown up to 24 layers, and the film thickness increases with the number of deposited layers. In the present study we investigate the immobilization of immunoglobulin G (IgG) and anti-IgG on an 8 nm thick (PAH/PSS)2 precursor film, as well as the fabrication of multilayer films via the alternate adsorption of PSS and anti-IgG or PSS(PAH/PSS)2 and anti-IgG. The aims of the present work are (i) to assess the advantages of a “soft” surface for the immobilization of antibodies, since in many cases where biomolecules have been directly bound to solid surfaces through adsorption, they have denatured in the process, thereby losing their biospecific activity,15,16 and (ii) to increase the capacity of the sensing thin film to bind subsequent antibodies through the fabrication of a multilayer antibody film. We have chosen immunoglobulins because they are widely used in various immunological solid phase assays17 and because of the
(1) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1991, 205, 113. (2) Ahluwalia, A.; De Rossi, D.; Monici, M.; Schirone, A. Biosens. Bioelectron. 1991, 6, 133. (3) Barraud, A.; Perrot, H.; Billard, V.; Martelet, C.; Therasse, J. Biosens. Biolectron. 1993, 8, 39. (4) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127. (5) Blomberg, E.; Claesson, P.; Froberg, J.; Tilton, R. Langmuir 1994, 10, 2325. (6) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1993, 142, 503. (7) Tilton, R.; Blomberg, E.; Claesson, P. Langmuir 1993, 9, 2102. (8) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Angermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (9) Spinke, J.; Liley, M.; Guder, H. -J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821.
(10) Fujita, A.; Senzu, H.; Kunitake, T.; Hamachi, I. Chem. Lett. 1994, 1219. (11) Hamachi, I.; Honda, T.; Noda, S.; Kunitake, T. Chem. Lett. 1991, 1121. (12) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (13) Lvov, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1994, 2323. (14) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir, preceding paper in this issue. (15) Brillhart, K. L.; Ngo, T. T. J. Immunol. Methods 1991, 144, 19. (16) Tatsuma, T.; Tsuzuki, H.; Okawa, J.; Joshida, S.; Watanabe, T. Thin Solid Films 1991, 202, 145. (17) Guesdon, J. L.; Avrameas, S. In Applied Biochemistry and Bioengineering; Wingard, L. B., Katchalski-Katzir, E., Goldstein, L., Eds.; Academic Press: New York, 1981; Vol. 3, p 207.
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interest in their use for the development of biosensors.18 Further, for the first time, the immunological activity of antibody layers constructed by alternate adsorption of oppositely charged polyelectrolytes and antibodies has been examined using a quartz crystal microbalance (QCM). The level of nonspecific binding in these films is assessed by interaction with BSA. The effect of salt (MnCl2) on the formation of polyelectrolyte layers and the amount of antiIgG subsequently immobilized is also investigated. Experimental Section Materials. Poly(allylamine hydrochloride) (PAH), Mr 50 00065 000, and poly(sodium 4-styrenesulfonate) (PSS), Mr 70 000, were purchased from Aldrich Chemical Co. and were used as received. 3-Mercaptopropionic acid (MPA) was obtained from Sigma and was used as obtained. The polyelectrolytes were adsorbed onto the substrates from 3 mg mL-1 aqueous solutions. The PAH solution was adjusted to pH 8.0 by adding NaOH. The PSS solutions contained 0, 0.01, 0.1, or 1 M MnCl2. Some PSS solutions also contained 0.01 M HCl (see below). Polyclonal sheep IgG (IgG), donkey anti-sheep IgG (anti-IgG), and monoclonal mouse IgG were all obtained from Sigma. All antibodies were used as supplied. Bovine serum albumin (BSA) was purchased from Sigma. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Aldrich Chemical Co. Protein solutions were made by diluting the stock solutions to the desired concentration with either 0.05 M HEPES buffer (pH 7.5, ionic strength (I) ) 0.1 M) or 0.05 M MES (containing 0.082 M NaCl, pH adjusted to 6.0 using NaOH; I ) 0.1 M). All experiments were performed at IgG and anti-IgG final concentrations of 50 µg mL-1 and 20 µg mL-1, respectively. (These concentrations are sufficient to give saturation coverage of the surface.19,20) BSA was made up in pure water and used at a final concentration of 50 µg mL-1. Sulfuric acid, nitric acid, acetone, and propan-2-ol were all AR grade and supplied by Rhoˆne-Poulenc. Hydrogen peroxide (AR grade) and ethanol (AR grade) were purchased from BDH. Spectroscopic grade chloroform was obtained from Merck. The water used in all experiments was obtained from a three-stage ‘Milli-Q’ purification system with a conductivity less than 1 µS cm-1. All experiments were performed at 22 ( 1 °C. Thin Film Formation. The substrates were prepared as described in a previous paper.14 The precursor film (PAH/PSS)2 was prepared on MPA-modified gold by repeating two alternate adsorption cycles of PAH and PSS. IgG and anti-IgG immobilization onto (PAH/PSS)2 was achieved by immersing the polyelectrolyte-coated substrate into the protein solution for ca. 60 min (or until no further frequency or reflectivity changes were observed in the case of QCM and SPR measurements, respectivelyssee below). The surface was then washed with pure water and nitrogen dried. Subsequent interaction with the antibody was examined by exposure to an aqueous IgG solution, followed by water rinsing and nitrogen drying. Multilayer polyelectrolyte/anti-IgG films were prepared by first assembling the precursor film (PAH/PSS)2 on the gold surface (as above). The surfaces were then alternately immersed in aqueous solutions (MES or HEPES) of anti-IgG for ca. 60 min and PSS for 1 min, with pure water rinsing and nitrogen drying between each deposition. (The PSS solutions contained 0, 0.01, or 0.1 M MnCl2; pH ≈ 6.) The anti-IgG/PSS deposition cycle was repeated until the desired number of anti-IgG layers was achieved. Multilayer films of anti-IgG layers separated by five polyelectrolyte layers (PSS(PAH/PSS)2) were also prepared. (The PSS solutions used to construct the PSS(PAH/PSS)2 layers contained 0.01 M HCl and 1 M MnCl2.) These films were assembled by alternately immersing the solid surface (with the attached precursor (PAH/PSS)2 film) into aqueous solutions of the protein and polyelectrolyte, or polycation and polyanion, with intermediate rinsing with pure water and nitrogen drying. Quartz Crystal Microbalance (QCM) Measurements. Assembly of the thin alternating polyelectrolyte/protein multilayer films was monitored using a QCM. Details of the QCM (18) Buijs, J.; Lichtenbelt, J. W. Th.; Norde, W.; Lyklema, J. Colloids Surf., B: Biointer. 1995, 5, 11.
Caruso et al. system can be found in previous publications.19,21,22 QCM measurements were performed in two modes: (i) stepwise and (ii) in-situ. In the stepwise experiments, the quartz crystal was immersed in a polyelectrolyte or protein solution for a given period, the surface was rinsed with pure water and nitrogen dried, and the in air frequency change (∆Fair) due to adsorption was measured. The in-situ experiments were only performed for protein immobilization and subsequent antibody immunoreactions. In in-situ experiments, only one side of the quartz crystal is in contact with solution. (One side of the quartz crystal is sealed with a rubber casing, maintaining it in an air environmentsthis casing is essential for frequency stability of the quartz crystal when immersed in liquids.19,21,23) The QCM crystals were first immersed in ca. 10 mL of buffer solution. After stabilization of the fundamental resonance frequency of the quartz crystal, an aliquot of the stock protein solution was injected into the HEPES or MES buffer solution. The in solution frequency changes (∆Fsolution) due to adsorption of protein on the surface of the quartz crystal were monitored as a function of time. All measurements were performed for at least 30 min, or until no further change in the frequency was recorded (this was interpreted as saturation of the surface). The crystal was then removed from solution, washed with pure water, and nitrogen dried. ∆Fair values were used to determine the amount of protein and/or polyelectrolyte adsorbed, since the relationship between ∆Fair and mass adsorbed is known (see later). This procedure was repeated for the interaction of anti-IgG with its antibody (IgG). HEPES buffer was used for second-layer (IgG) binding. Typically, the QCM crystals are stable to (1 and (2 Hz for air and solution measurements, respectively. Surface Plasmon Resonance (SPR) Measurements. Details of the SPR experimental system and measurement principle can be found elsewhere.21,24 As previously described,14 a full plasmon resonance curve (reflectivity vs internal angle) for the MPA-modified gold/air system was first measured. The precursor polyelectrolyte film (PAH/PSS)2 was then constructed,14 and the full SPR curve was measured in air. MES buffer was then injected into the SPR vessel, and a plasmon curve for gold/(PAH/PSS)2 in MES was recorded. These full SPR curves were fitted to Fresnel theory to extract the thickness and optical parameters of the gold/(PAH/PSS)2 film exposed to air or in contact with MES. An aliquot of the aqueous protein (anti-IgG) solution was then injected, and the solution was stirred. The kinetics of antiIgG adsorption were followed by fixing the detector at an external angle of incidence 2° off the SPR resonance minimum on the critical angle side of the minimum and monitoring the reflectivity as a function of time.21,24 At this point the rate of change of reflectivity (∆R) with angle is approximately linear. (The fixedangle reflectivity data were divided by the beam intensity in the absence of the sample to give the absolute reflectance.) When the reflectivity was constant (which was interpreted as saturation of the surface), a full plasmon resonance curve was again recorded. The solution was then removed, the anti-IgG layer was rinsed with pure water and dried with nitrogen, and a full plasmon resonance curve was recorded in air. The full plasmon resonance curves obtained (both in MES and air) after anti-IgG adsorption, and the parameters previously determined for the gold/(PAH/ PSS)2 film (in MES and air, respectively) were used to determine the effective thicknesses of the immobilized anti-IgG layers by fitting to Fresnel theory. After immobilization of anti-IgG, fresh MES buffer was introduced into the cell. A full plasmon resonance curve was again measured. It was found that desorption of the immobilized anti-IgG layers did not occur upon (19) Caruso, F.; Rodda, E.; Furlong, D. N. J. Colloid Interface Sci. 1996, 178, 104. (20) Geddes, N. J.; Martin, A. S.; Caruso, F.; Urquhart, R. S.; Furlong, D. N.; Sambles, J. R.; Than, K. A.; Edgar, J. A. J. Immunol. Methods 1994, 175, 149. (21) Caruso, F.; Serizawa, T.; Furlong, D. N.; Okahata, Y. Langmuir 1995, 11, 1546. (22) Caruso, F.; Rinia, H. A.; Furlong, D. N. Langmuir 1996, 12, 2145. (23) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Ebara, Y.; Okahata, Y.; Than, K. A.; Edgar, J. A. Sens. Actuators B 1994, 17, 125. (24) Caruso, F.; Vukusic, P. S.; Matsuura, K.; Urquhart, R. S.; Furlong, D. N.; Okahata, Y. Colloids Surf., A: Physicochem. Eng. Aspects 1995, 103, 147.
Polyelectrolyte and Protein Multilayer Films
Figure 1. QCM frequency-time profiles for the immobilization of (a) IgG onto a (PAH/PSS)2-coated gold electrode from 50 µg mL-1 IgG-HEPES solutions and (b) (PAH/PSS)2-coated gold quartz crystals with immobilized anti-IgG subsequent to the injection of IgG from 50 µg mL-1 IgG-HEPES solution. The arrow indicates the time at which IgG solution was injected into the HEPES solution. replacing the protein solution with buffer. Second-layer immunosensing was then evaluated by repeating the above procedure using an aqueous IgG-MES solution. The combined layer effective thicknesses (anti-IgG/IgG) were then determined by fitting the SPR curves to Fresnel theory. The SPR experiments were also repeated using HEPES buffer instead of MES.
Results and Discussion Monomolecular Protein Films. The frequency-time profile for the immobilization of IgG (50 µg mL-1 in HEPES) onto a (PAH/PSS)2-coated gold QCM electrode is shown in Figure 1 (curve a). The frequency change reaches equilibrium within 30 min, and the total frequency change in solution (∆Fsolution) is 184 ( 2 Hz. Frequency changes of 191 ( 5 Hz (duplicate experiments) were obtained for anti-IgG adsorption from 20 µg mL-1 anti-IgG-HEPES solutions onto (PAH/PSS)2-modified gold QCM electrodes (data not shown). Adsorption of IgG from 50 µg mL-1 IgG-HEPES onto a precursor 10-layer polyelectrolyte film ((PAH/PSS)5) on a quartz crystal yielded an equilibrium frequency change in solution of 220 ( 5 Hz (within 45 min), which is only slightly larger than that observed for the 4-layer (PAH/PSS)2 film. The ∆Fsolution values obtained cannot be directly transposed into mass of IgG (or antiIgG) adsorbed, since a quantitative relationship between ∆Fsolution and the corresponding mass uptake on the crystal surface is not yet available.25-30 Thus, ∆Fair data were used to determine the amount of protein (and/or polyelectrolyte) adsorbed, using the relationship 0.87 ng Hz-1.19,21 The ∆Fair values for IgG adsorption onto (PAH/ PSS)2- and (PAH/PSS)5-coated QCM electrodes are 67 ( 3 and 70 ( 5 Hz, respectively, which correspond to 58 ( 3 and 61 ( 4 ng, respectively. These values are the same, which indicates IgG adsorbs on the outermost layer only and is not able to penetrate into the film. The surface coverages (Γ) of 3.6 ( 0.2 (IgG adsorption onto (PAH/ PSS)2) and 3.8 ( 0.2 mg m-2 (IgG adsorption onto (PAH/ PSS)5) calculated for these QCM-derived mass loadings are in excellent agreement with that calculated for a close(25) Nomura, T.; Okuhura, M. Anal. Chim. Acta 1982, 142, 281. (26) Bruckenstein, S.; Shay, M. Electrochim. Acta 1985, 30, 1295. (27) Kanazawa, K. K.; Gordon, J. Anal. Chim. Acta 1985, 175, 99. (28) Yang, M.; Thompson, M.; Duncan-Hewitt, W. C. Langmuir 1993, 9, 802. (29) Hinsberg, W.; Wilson, C.; Kanazawa, K. K. J. Electrochem. Soc. 1986, 133, 1448. (30) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 3315.
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packed IgG monolayer in an end-on orientation with repelling F(ab) fragments (Γ ) 3.7 mg m-2).18 A mass change of 64 ( 3 ng (Γ ) 4.0 ( 0.2 mg m-2) was obtained for anti-IgG immobilized on (PAH/PSS)2-modified QCM electrodes. The Γ values for IgG and anti-IgG are considerably lower than those previously obtained on bare gold QCM surfaces (ca. 5-8 mg m-2), where significant protein aggregation occurs.19 The HEPES solution used for IgG adsorption is at pH 7.5, and thus the PSS outermost layer is negatively charged. Since IgG has an isoelectric point around pH 6.8,3,18 IgG also has an overall negative charge at pH 7.5, suggesting its adsorption is not driven by electrostatics. Adsorption of IgG or anti-IgG onto the precursor (PAH/ PSS)2 film from MES buffer solutions (pH 6.0) yielded the same frequency changes (within experimental error) as those obtained for adsorption from HEPES solutions. (At pH 6.0, IgG has an overall positive charge.) Given that electrostatics do not appear to control the adsorption of IgG onto negatively charged PSS, it is presumed to be predominantly caused by hydrophobic forces. Buijs et al.18 reported similar surface coverages for IgG on negatively charged polystyrene latex (PS-) at pH 6.0 and pH 7.5 and similarly concluded that IgG adsorption onto PS- is dominated by hydrophobic bonding. Further, IgG is wellknown to spontaneously adsorb from solution onto various hydrophobic surfaces, including gold19,20,24 and silanized silica.31,32 Adsorption of IgG onto PSS may also proceed via the interaction of PSS with positively charged segments of IgG. Although the exact mechanism of IgG adsorption onto PSS is unclear, IgG can be effectively immobilized on a precursor (PAH/PSS)2 thin film. Exposure of the (PAH/PSS)2-coated gold quartz crystal with immobilized IgG or anti-IgG to a 50 µg mL-1 mouse IgG-HEPES solution gave a ∆Fsolution of 10 Hz and a ∆Fair of 5 Hz. This shows that saturation coverage is obtained in the initial IgG and anti-IgG binding experiments and that the degree of nonspecific binding between immobilized anti-sheep IgG and mouse IgG is negligible. Further, exposure of these IgG and anti-IgG surfaces to an aqueous solution of 50 µg mL-1 bovine serum albumin (BSA) for 60 min resulted in 5 and 0 Hz for ∆Fsolution and ∆Fair, respectively, also confirming saturation coverage of the surface. The frequency change of the (PAH/PSS)2-coated gold quartz crystal with immobilized anti-IgG subsequent to the injection of sheep IgG from 50 µg mL-1 IgG-HEPES solution is shown in Figure 1 (curve b). Binding is considerably slower than that of anti-IgG (or IgG) on (PAH/ PSS)2-coated gold. This suggests differences in the kinetics of association between IgG and (PAH/PSS)2 compared with those of IgG and immobilized anti-IgG. In addition, the differences may be due to the orientational requirements for IgG binding to immobilized anti-IgG. IgG binding to immobilized anti-IgG produced a frequency change in the buffer of 122 ( 5 Hz and a corresponding ∆Fair of 40 Hz (mass uptake of 35 ng). Table 1 summarizes the mass changes due to IgG or anti-IgG immobilization onto (PAH/PSS)2-coated crystals and their subsequent binding with anti-IgG or IgG, respectively, along with the results for a bare gold surface previously obtained.19 Three main observations can be made: (i) Although the mass changes for anti-IgG on (PAH/PSS)2 suggest monolayer formation, this immobilized layer has a binding capacity similar to that of anti-IgG immobilized on a bare gold surface where IgG aggregation occurs.19 Since the ori(31) Chang, I. N.; Lin, J. N.; Andrade, J.; Herron, J. N. J. Colloid Interface Sci. 1995, 174, 10. (32) Lin, J. N.; Andrade, J. D.; Chang, I. N. J. Immunol. Methods 1989, 125, 67.
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Table 1. QCM Mass Changes for IgG or Anti-IgG Immobilization onto (PAH/PSS)2-Coated Gold QCM Electrodes and Subsequent Binding of Anti-IgG or IgG QCM surface
first layer (L1)
109∆m1a (g)
second layer (L2)
109∆m2a (g)
binding ratio (L2/L1)
(PAH/PSS)2 (PAH/PSS)2 goldb goldb
anti-IgG IgG anti-IgG IgG
64 ( 3 58 ( 3 83 ( 10 107 ( 16
IgG anti-IgG IgG anti-IgG
35 ( 3 91 ( 3 40 ( 2 90 ( 4
0.55 ( 0.07 1.57 ( 0.13 0.48 ( 0.08 0.84 ( 0.16
a The mass changes are the averages of repeat experiments. The errors represent the standard deviation of repeat experiments. b Data taken from previous work (ref 19).
entation of anti-IgG on the surface is important for subsequent IgG binding (see below), the similar binding ratios show that aggregates of anti-IgG immobilized on gold contain anti-IgG molecules which are favorably oriented for interaction with IgG, effectively producing the same IgG binding response as a monolayer of antiIgG on (PAH/PSS)2. (ii) IgG immobilized on (PAH/PSS)2 displays an enhanced binding ratio, L2/L1 ) 1.57, compared with IgG immobilized on gold (L2/L1 ) 0.84). This may be due to the maximum packing density of anti-IgG on immobilized IgG (both systems yield an anti-IgG mass change of ca. 90 ng) and suggests that the effective surface area of an IgG layer on (PAH/PSS)2 is similar to that on gold. (iii) The degree of second-layer binding is also greater when the immobilized layer is IgG. This can be explained by the fact that the surface orientation of immobilized anti-IgG controls the binding, with binding occurring only if the anti-IgG F(ab) receptor sites are accessible to IgG.18,20 The binding of anti-IgG from solution onto an immobilized IgG layer is unlikely to depend on any specific molecular orientation of the immobilized IgG molecules. In this case, the F(ab) receptor sites on the anti-IgG molecule are able to align appropriately in solution to effect binding with immobilized IgG.19 Nonspecific binding was evaluated by exposing the QCM crystals with adsorbed IgG or anti-IgG to BSA for 60 min prior to subsequent interaction with the biospecific antibody. (BSA is used to block nonspecific binding sites in immunosensing.) Less than 10% reduction in bound second-layer antibody was observed for both IgG/antiIgG and anti-IgG/IgG systems, indicating minimal nonspecific binding. Nonspecific binding for immobilized antisheep IgG evaluated by cross-reactivity with mouse IgG also showed little (
