Anal. Chem. 2006, 78, 8368-8373
Immobilization of Antibodies on Polyaniline Films and Its Application in a Piezoelectric Immunosensor V. V. R. Sai,† Sumeet Mahajan,† Aliasgar Q. Contractor,†,‡ and Soumyo Mukherji*,†
School of Biosciences and Bioengineering and Department of Chemistry, IIT Bombay, Mumbai, India-400076
Conducting polymers, especially polyaniline (PAni), have been extensively used in biosensor applications. A protocol for covalent immobilization of human IgG on polyaniline using glutaraldehyde as the cross-linker is described in this report and utilized in development of a piezoelectric immunosensor. Here, PAni was used as the substrate for immobilization. The electropolymerization parameters were optimized to get suitable thickness and surface morphology of the PAni for obtaining high density and uniformity of immobilized antibodies on the surface of our films. Possible reaction between PAni thin films and glutaraldehyde was explored using FT-IR characterization in grazing angle mode and XPS. The protocol has been characterized with the help of quartz crystal microbalance analysis. An antibody surface density of 4.86 ng/ mm2 was obtained. A piezoelectric biosensor developed for detection of IgG with the proposed protocol was capable of differentiating the target analyte concentrations between 500 ng/mL and 25 µg/mL with nonspecific binding of ∼10%. Over the past decade, conducting polymers, especially polyaniline (PAni) have been attracting immense interest among various conducting polymers with applications ranging from batteries to biosensors. This has been due to its extraordinary stability, simplicity of synthesis from an inexpensive monomer, and unique electrochemical properties. In the area of biosensors alone, polyaniline has been exploited in many different applications.1 Immobilization of biomolecules on a substrate is essential for the working of any biosensor. It plays an important role in the specificity, sensitivity, and reproducibility of a biosensor. Various methods for immobilizing biomolecules have been reported in the literature such as physical adsorption, entrapment, covalent binding, and LB films. Effective performance can be obtained only when certain requirements of immobilization are fulfilled.2 They are (1) retention of biological activity of biomolecules after immobilization onto the sensor surface; (2) reproducible, durable, and stable attachment with the substrate against variations in pH, * To whom correspondence should be addressed. Phone: 91-22-25767767. Fax: 91-22-25723480. E-mail:
[email protected]. † School of Biosciences and Bioengineering. ‡ Department of Chemistry. (1) Gerard, M.; Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 345-359. (2) Collings, A. F.; Caruso, F. Rep. Prog. Phys. 1997, 60, 1397-1445.
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temperature, ionic strength, and chemical nature of the microenvironment; and (3) uniform, dense, and oriented localization of the biomolecules. Among all the methods, covalent binding is known to satisfy most of these requisites. Although there are some limitations with covalent binding, such as loss of activity of biomolecules to some extent, it is the most preferred method of immobilization.2 Many researchers have explored the applications of enzymebased conducting polymer biosensors where enzymes were immobilized using electrochemical entrapment, cross-linking through glutaraldehyde and during potentiodynamic scanning as in cyclic voltammetry.3-5Apart from that, PAni has been used as a label conjugated to antibodies in competitive electrochemical immunoassay format employing impedance spectroscopic transduction techniques.6,7 However, there have been few reports of applications of PAni-based immunosensors. Antibodies have been immobilized onto polyaniline by using protein A adsorption,8 cyclic voltammetry,9 electrostatic binding by oxidation,10 and covalent bonding to the copolymerized PAni.11,12 Previously, glutaraldehyde has been used for immobilization of enzymes11,13 and antibodies on copolymerized polyaniline and protein A adsorbed polyaniline surface.8,12 Coeˆlho et al. have reported a procedure for immobilization of antigen on chemically synthesized PAni by directly using glutaraldehyde (GLu).14 In this study, PAni has been synthesized electrochemically and antibodies immobilized on it by directly using GLu. The secondary amine linkages present in the polyaniline chain or the primary amines present in the extremities of oligomers or both are (3) Sangodkar, H.; Sukeerthi, S.; Srinivasa, R. S.; Lal, R.; Contractor, A. Q. Anal. Chem. 1996, 68, 779-783. (4) Fernandes, K. F.; Lima, C. S.; Pinho, H.; Collins, C. H. Process Biochem. 2003, 38, 1379-1384. (5) Cosnier, S. Biosens. Bioelectron. 1999, 14, 443-456. (6) Sergeyeva, T. A.; Lavrik, N. V.; Piletsky, S. A.; Rachkov, A. E.; Eiskaya, A. V. Sens. Actuators, B 1996, 34, 283-288. (7) Tahir, Z. M.; Alocilja, E. C. IEEE Sens. J. 2003, 3, 345-351. (8) Liu, C. H.; Liao, K. T.; Huang, H. J. Anal. Chem. 2000, 72, 2925-2929. (9) Miao, Y. Q.; Guan, J. G. Anal. Lett. 2004, 37, 1053-1062. (10) Killard, A. J.; Zhang, S.; Zhao, H.; John, R.; Iwuoha, E. I.; Smyth, M. R. Anal. Chim. Acta 1999, 400, 109-119. (11) Lima, B. A. E.; Almeida, A. M. P.; Carvalho, L. B.; Azevedo, W. M. Braz. J. Med. Biol. Res. 2002, 35, 459-463. (12) Grennan, K.; Strachan, G.; Porter, A. J.; Killard, A. J.; Smyth, M. R. Anal. Chim. Acta 2003, 500, 287-298. (13) Silva, R. N.; Asquieri, E. R.; Fernandes, K. F. Process Biochem. 2005, 40, 1155-1159. (14) Coeˆlho, R. A. L.; Santos, G. M. P.; Azeveˆdo, P. H. S.; Jaques, G. A.; Azevedo, W. M.; Carvalho. L. B., Jr. J. Biomed. Mater. Res. 2001, 56, 257-260. 10.1021/ac060120a CCC: $33.50
© 2006 American Chemical Society Published on Web 11/10/2006
expected to react with the aldehyde groups on the cross-linker molecules to form enamine and imine bonds, respectively. The amine groups present in the IgG are expected to react with the aldehyde groups at the free end of the cross-linker molecules to form a stable imine bonds. Immobilization was studied as a function of varying concentrations of glutaraldehyde and different treatment times of polyaniline films with glutaraldehyde solutions. The immobilization of human IgG (HIgG) and its activity was examined with the help of FITC-tagged goat anti-HIgG antibodies (GaHIgG), which were specific to the Fc region of HIgG. The amount of active binding sites per nanogram of HIgG immobilized on the PAni surface for each treatment was determined using a quartz crystal microbalance (QCM). In addition, image analysis was used to quantify the fluorescence and help develop the protocol. We have also studied in detail the immobilization chemistry with the help of FT-IR spectroscopy and X-ray photoelectron spectroscopy (XPS). The characterization of each step of the protocol including quantification of antibody density and its specific activity as well as sensing of IgG were carried out by measurements using a QCM. Thus, a simple two-step, piezoelectric immunosensor for detection of IgG is reported. EXPERIMENTAL SECTION Reagents. Aniline was obtained from Merck and distilled under reduced pressure before use. It was stored in dark in a refrigerator (∼10 °C). Sulfuric acid used was MOS grade with 99.99% purity. Glutaraldehyde was obtained from Fluka. All antibodies, buffers, and bovine serum albumin (BSA) were obtained from Bangalore Genei. All reagents were of analytical grade. All solutions were prepared using Deionized (DI) water obtained from a MilliQ filtration system. Sensor Apparatus. 9-MHz, AT-cut, quartz crystals coated with gold in a circular shape (5-mm diameter) on both sides were used for mass-sensitive measurements. Changes in the resonant frequency were measured with the help of a quartz crystal microbalance (QCA 917, Seiko EG&G). Mass change due to the binding reactions has been calculated by using the Sauerbrey equation,15
∆m ) NAg∆f/f 2
(in g)
where, N, the frequency constant of quartz is 0.167 MHz·cm; A, the area of deposition is 0.196 cm2; g is the density of quartz, 2.648 g/cm3; and ∆f is the change in the resonant frequency, f. The resultant equation for mass change, ∆m, for a given change in resonant frequency of crystal is given by,
∆m ) 86571.53∆ f /f 2
(in g)
Preparation of Substrate. Gold-coated glass coverslips of size 18 × 18 mm2 were chosen as substrates for deposition of polyaniline. They were cleaned by sonication for 2 min in acetone and dried. A 500-nm-thick gold layer was deposited onto the surface of the coverslips by thermal vapor deposition under vacuum (10-6 Torr). Quartz crystals were cleaned in a solution containing chromic acid and concentrated sulfuric acid in the ratio of 9:1, washed in DI water, and dried. (15) Sauerbrey, Q. Z. Phys. 1959, 155, 206-222.
Polyaniline Deposition. The 0.1 M aniline monomer solution was prepared in 0.5 M sulfuric acid. Polyaniline was deposited on the gold-coated coverslips and quartz crystals by using a potentiostat (model 273, EG & G Princeton Applied Research). A homemade saturated calomel electrode (SCE) was used as the reference and a platinum foil used as the counter electrode. The potential was scanned from -0.2 to 0.8 V for growing polyaniline films. Immobilization Protocol. (a) Polyaniline-coated coverslips were washed in DI water and sonicated in DI water for 30 s. (b) Samples were dipped in various concentrations (1, 2, and 4%) of GLu for various incubation times (30 min, 2 h, and 4 h). Samples were subsequently washed in DI water and dried. (c) Samples were incubated in an aliquot of 0.5 mg/mL HIgG prepared in phosphate-buffered saline (PBS) for 1 h. The unbound HIgG molecules were washed away by rinsing in PBS. In order to reduce nonspecific binding (NSB) of proteins to the immobilized surface, HIgG immobilized samples were treated with 2 mg/mL BSA. After 1 h, samples were rinsed in PBS and airdried. (d) Immobilization on the PAni samples was tested by incubating with the FITC-tagged GaHIgG, which are specific to the Fc region of HIgG. After 20 min, samples were rinsed in PBS and air-dried. All experiments were carried out at room temperature. In all the experiments, two controls were used. In control-I, GLu treatment was bypassed and the sample was incubated in antibody solution directly in order to understand the true effect of GLu in the immobilization process as well as to observe the extent of physical adsorption of antibodies on the polyaniline matrix. As control-II, the samples were subjected to GLu treatment but were incubated in buffer solution containing 0.5 mg/mL rabbit IgG in order to examine the amount of NSB of GaHIgG. All the above experiments were performed on PAni coated on quartz crystals and planar gold-coated coverslips. Resonant frequency changes of quartz crystals were recorded after each step in the protocol. Fluorescence intensity due to the presence of FITC-tagged GaHIgG bound to HIgG immobilized on PAni-coated coverslips under different treatments (nine combinations obtained due to three GLu concentrations × three incubation times) was observed under a fluorescence microscope (Zeiss Axioskop-2 MAT) under 20× magnification. Fluorescence images were acquired by using a Peltier-cooled black and white digital camera. Scion Image software was used to determine the fluorescent intensities of the images. Each pixel in an image under investigation had a gray value between 0 (dark) and 255 (bright). Overall intensity of an image was calculated by subtracting a value that accounts for the background intensity from the mean gray value of an image. Quartz crystal microbalance (QCM) and fluorescence measurements obtained from nine treatments of GLu were compared using one-way classification of ANOVA. Comparisons of results obtained from (a) control-I samples with that of GLu-treated samples and (b) control-II samples with that of GLu-treated samples were performed with paired t-tests to find the effect of GLu treatment. Characterization. Surface chemical characterization was carried out using grazing-angle FT-IR and XPS. Water contact angle measurements were performed using Digidrop EWS contact angle Analytical Chemistry, Vol. 78, No. 24, December 15, 2006
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meter (GBX), to know the changes in the hydrophobicity of the surface. Study of interaction at each step of the protocol as well as utility as a biosensor was undertaken by QCM analysis. FT-IR Analysis. Mid-IR spectra of pure PAni, PAni treated with GLu, and PAni treated directly with HIgG (control-I), all of them electrodeposited on gold-coated coverslips, were obtained using Magna 550, Nicolet Instruments Corp., with a scan rate of 256 and a resolution of 4 cm-1. As the surface properties were of more interest, a grazing angle accessory was used. The angle of incidence of IR light on to the sample was chosen as 85°. XPS Analysis. XPS data for bare PAni and GLu-treated PAni were obtained using Thermo VG Scientific Multilab 2000 system equipped with a Mg K∝ X-ray source at 1253.60 eV and hemispherical electron energy analyzer having a nine-channel detector. The main chamber was maintained at a base pressure of 4 × 10-10 mbar. Full scan (1000-0 eV) was carried out with step size of 1 eV, and elemental scan for C, N, and O was carried out with step size of 0.05 eV. All spectra were obtained at 150 kV and 150 W. QCM Analysis. The physical parameters of immobilization such as surface density of antibodies, molar binding ratio, specific and nonspecific binding of GaHIgG to the immobilized HIgG, and hence activity of antibodies after immobilization was quantified using QCM analysis. The quartz crystals used in this study have a mass sensitivity of 1.1 ng/Hz.
Table 1. Number of Binding Sites/ng of Immobilized HIgG, Mean Fluorescence Values, and Their Respective Standard Deviations for Each of the Nine Different Treatments treatment
mean fluorescence value ( SD (arb units)a
number of active binding sites in 1 × 109/ng of HIgGb
1% for 1/2 h 1% for 2 h 1% for 4 h 2% for 1/2 h 2% for 2 h 2% for 4 h 4% for 1/2 h 4% for 2 h 4% for 4 h 0%
130.72 ( 22.73 116.35 ( 29.47 122.59 ( 26.77 139.74 ( 3.80 144.37 ( 22.58 147.14 ( 26.34 116.25 ( 19.67 137.10 ( 11.60 147.37 ( 26.81 111.64 ( 16.94
1.50 ( 0.075 2.05 ( 0.095 1.81 ( 0.234 2.04 ( 0.522 2.02 ( 0.213 1.66 ( 0.911 2.20 ( 0.832 2.43 ( 0.083 2.06 ( 0.289 1.02 ( 0.591
a Mean of three measurements. Fluorescence was recorded from FITC-tagged GaHIgG bound to the HIgG immobilized on PAni surface. Background noise is not subtracted from the fluorescence value expressed in the table. It is assumed that the variations in the excitation light intensity are negligible. b Number of active binding sites/ng of immobilized HIgG was obtained from the formula (specific activity) × Avogadro number × 10-9/molecular weight of HIgG, i.e., (∆FAg/∆FAb) × 6.0221415 × 1023 × 10-9/147000.
RESULTS AND DISCUSSION Optimization of Polymer Thickness. A prerequisite for uniform and proper covalent immobilization of antibodies is to have a smooth surface over the region of substrate used for the biosensor. Surface roughness causes higher NSB of biomolecules to the substrate. Experiments were performed with PAni samples that were prepared from 40, 20, 15, and 10 cycles of electropolymerization. At a higher number of cycles, the surface was found to be very rough with nonuniform clumps on the surface. PAni samples with 10 cycles were found to give the most uniform immobilization, probably due to the smoothness of the surface obtained (Figure S-1, Supporting Information). This also resulted in reduced thickness of polyaniline, which is desirable in many sensor applications. The thicknesses of PAni samples electrodeposited with 40, 20, 15, and 10 cycles measured using a profilometer (XP-2, Ambios Technologies) were 440, 105, 50, and 17 nm, respectively. Apart from the number of cycles, oxidation current density could be an appropriate measure for the amount of PAni deposited. It was found that the required thickness of PAni was obtained when the oxidation current density reached 2.5-3 µA/ mm2. Hence, all subsequent experiments were performed on polyaniline films deposited by 10 cycles of electropolymerization. Optimum Conditions for Immobilization. In order to find the optimum concentration of glutaraldehyde and incubation time, samples were treated in 1, 2, and 4% glutaraldehyde solutions prepared in DI water for 30 min, 2 h, and 4 h. Under different treatments, no significant variation in the frequency change due to the GLu mass loading on to quartz crystals was observed. The number of active binding sites/ng of immobilized HIgG on the PAni surface under each treatment were as shown in Table 1. It was found that the fluorescence obtained in all the combinations of GLu concentration and incubation time was almost similar (Table 1) (Figure S-2, Supporting Information). Using one-way 8370 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006
Figure 1. Variation in the fluorescence intensity for various HIgG concentrations. Fluorescence signal at each point is expressed as mean ( SD of three measurements. Significantly high density of immobilization was obtained with 0.5 mg/mL HIgG and above (sigmoidal fit with R2 ) 0.9979).
ANOVA, it could be said with 97.5% confidence that the amount of active binding sites/ng of immobilized HIgG as well as fluorescence intensity values obtained in all the combinations of GLu concentration and incubation time was not significantly different. Treatment of samples in a 1% concentration of GLu for 30 min of incubation was sufficient for obtaining good immobilization. Optimum concentration of HIgG antibodies needed to obtain a high density of immobilization was found by performing the experiments with the above-mentioned protocol. These experiments are carried out in triplicate with various HIgG concentrations prepared in PBS. The density of immobilization was observed by incubating each sample in 0.05 mg/mL FITC-tagged GaHIgG antibodies. Antibodies tagged with FITC were not directly immobilized onto the activated surface due to a severe problem of fluorescence quenching. The fluorescence intensity obtained for the variation in the concentration of HIgG is shown in Figure 1. The rising trend in the density of immobilized antibodies was observed until 0.5 mg/mL. No significant variation was seen for 0.5 mg/mL and above. Hence, further experiments were carried out with 0.5 mg/mL HIgG.
Figure 2. FT-IR spectra of (a) bare PAni and (b) GLu-treated PAni. Automatic baseline correction was made on both plots. The assignment of absorption peaks common to both spectra were as follows: 3390 cm-1, NH2 and NH; 1601 cm-1, CdN of quinoid (Q); 1514 cm-1, CsN of benzoid (B); 1310 cm-1, CsN of QBQ, BBQ, and QBB; 1165 cm-1, a mode of NdQdN; 825 cm-1, CsH on 1,4-ring. 1110 and 511 cm-1 might be due to CsH on 1,2,4-ring and aromatic ring deformation.
Control Immobilization. Control-I results showed that the amount of antibodies immobilized by physical adsorption was significantly less than that of covalent immobilization. Hence, the PAni surface was able to effectively reduce the adsorption of antibodies. A paired t-test performed on the fluorescence intensity values obtained from control-I and GLu-treated samples proved that the treatments were significantly different with 97.5% confidence. Control-II was found to give very minimal fluorescence, which means that the NSB of FITC-tagged GaHIgG to the surface was minimal on the PAni surface (Figure S-3, Supporting Information). In a comparison between control-II and fluorescence obtained due to specific binding, paired t-test showed a significant difference with more than 99% confidence. Surface Characterization. Contact angle measurements were carried out on PAni and a GLu-treated PAni surface. The water contact angle of the PAni surface significantly reduced to 63.23° ( 1.56° from 80.13° ( 0.87° (n ) 3) with GLu treatment, making the surface relatively hydrophilic and thus suitable for antibody immobilization. FT-IR Analysis. The FT-IR spectrum of a GLu-treated PAni surface (Figure 2) was found to be similar to that of pure PAni film, in addition, absorption peaks were also noticed at 2930, 2858, 1734.7, and 671 cm-1.16 The peaks obtained at 2858 and 1734.7 cm-1 can be assigned to CsH and carbonyl (CdO) stretching of aldehyde groups present in a low concentration on the surface, respectively. The absorption at 2930 and 671 cm-1 may be assigned to aliphatic C-H, C-C stretching.17,18 The expected reaction of GLu with PAni results in imine/enamine bonds (Cd C, CsN, and CdN), which are already present in untreated PAni.16 Hence, it might be difficult to find the proof of binding of GLu to PAni quantitatively, using FT-IR spectral analysis in grazing angle mode. XPS Analysis. XPS spectra of PAni and GLu-treated PAni were obtained as shown in Figure 3a and b. PAni spectrum showed the presence of C 1s and N 1s at 284.6 and 399.5 eV, respectively, in addition to oxygen in small quantities with O 1s at 532.7 eV. (16) Tang, J.; Jing, X.; Wang, B.; Wang, F. Synth. Met. 1988, 24, 231-238. (17) Stuart, B. Infrared spectroscopy: Fundamentals and Applications; Wiley: Chichester, 2004. (18) Socrates, G. Infrared Characteristic Group Frequencies; Wiley: Chichester, 1980.
Figure 3. Comparison of XPS spectra of (a) PAni and (b) GLutreated PAni scanned over wide range (1000-0 eV) and C 1s core level spectrum of (c) PAni and (d) GLu-treated PAni.
The XPS spectrum of GLu-treated PAni revealed the presence of oxygen in significant quantity at 532.7 eV in addition to a rise at C 1s and unaffected N 1s. Major differences were observed in C 1s and O 1s core level spectra. C/N ratio for PAni was found to be 4.16 and increased to 5.59 with GLu treatment. C 1s spectra of PAni and GLu-treated PAni were deconvoluted using a Gaussian multipeak fit as shown in Figure 3c and d. Spectra were found to show the presence of CsH, CsC and CdC, and CsN and CdN with the respective peaks at 284.6, 285.8, and 287.0 eV.19 The photoelectron count increased by 15, 2.5, and 54% at the respective peaks with GLu treatment. Thus, GLu might react with amine groups on PAni oligomers to form an imine (CdN) bond. Figure 3d also showed a peak at 288.2 eV that represents a carbonyl group (CdO).20 Significant increase in the photoelectron intensity of O 1s and the presence of CdO after GLu treatment indicates the presence of aldehyde groups on the surface. Hence, on the basis of XPS results, we conclude that GLu is binding to amine groups of PAni forming an imine bond. The chemical characterization using FT-IR and XPS and physical surface characterization using contact angle measurements and QCM mass loading (Table 2) shows the evidence for desired surface modification of PAni by GLu treatment. QCM Analysis. Polyaniline was deposited onto the gold electrodes of quartz crystals as described earlier. Antibodies were immobilized following the optimized protocol. HIgG antibodies were immobilized to determine the specific binding of GaHIgG antibodies. Control antibody (rabbit IgG)-immobilized quartz crystals were used to find NSB of GaHIgG antibodies to the substrate. Immobilized crystals were incubated in 0.05 mg/mL GaHIgG. Table 2 shows the quantified parameters of immobilization. The viscoelastic effect of quartz crystals has been taken into account during quantification of immobilization parameters. The frequency changes obtained were multiplied by a correction factor of 0.667 as suggested by Zhou et al.21 GLu treatment of the PAni matrix resulted in the binding of 20 pmol/mm2 GLu to the substrate. Molar binding ratio of GLu to PAni is not reported because PAni deposited on gold is not a single monolayer. It was found that 1.6 mM HIgG binds to 1 mol of surface-bound GLu. (19) Wagner, C. D.; Riggs, W. M.; Davis, L. E.; Moulder, J. F.; Muilenburg, G. E. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer Corp., 1978. (20) Kumar, S. N.; Gaillard, F.; Bouyssoux, G.; Sartre, A. Synth. Met. 1990, 36, 111-127.
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Table 2. Quantification of Immobilization and Sensing Parameters Using QCM Analysis treatment GLu HIgG GaHIgG RIgG+ GaHIgGb
frequency change (Hz) 53.57 ( 7.05 35.71 ( 4.70a 129.9 ( 9.10 86.60 ( 6.07a 75.7 ( 3.99 50.47 ( 2.66a 11.37 ( 3.42 7.58 ( 2.29a
mass change (ng) 58.94 39.29 142.93 95.29 83.29 55.53 11.37 8.34
ng/mm2 3.01 2.01 7.29 4.86 4.25 2.83 0.64 0.42
nmol/mm2
molar binding ratio
10-3
3.00 × 2.00 × 10-3 4.96 × 10-5 3.31 × 10-5 2.89 × 10-5 1.93 × 10-5 4.34 × 10-5 2.89 × 10-5
HIgG/GLu 0.16 × 10-2 GaHIgG/HIgG 0.58 GaHIgG/RIgG 0.15
a Viscoelastic effect of quartz crystal vibration was taken into consideration, which over estimates mass loading onto crystal by 1.5 times. These frequency changes were corrected for it by a correction factor of 2/3. b NSB was quantified by incubating the rabbit IgG-immobilized PAni-coated quartz crystals in 0.05 mg/mL GaHIgG.
The surface density of immobilized antibodies obtained in this study, 4.86 ng/mm2 (33 fmol/mm2), is found to be slightly more than the expected monolayer coverage, which is 4.1 ng/mm2 with an assumption of antibody dimensions of 14 × 10 × 5 nm2 with end-on orientation.22 It could be mainly due to relatively rough surface morphology of the PAni matrix whose rms roughness was found to be 6.7 nm (Figure S-5, Supporting Information). Molar binding ratio of GaHIgG to immobilized HIgG, for a saturated concentration of GaHIgG of 50 µg/mL, was obtained as 0.58. Molar binding ratio of GaHIgG due to NSB was obtained as 0.15, which is ∼15% of total analyte binding. Therefore, specific binding of GaHIgG to the immobilized HIgG, at the same concentration of 50 µg/mL, was found to be ∼85% of total binding. However, NSB was found to reduce to ∼10% for less than 50 µg/mL GaHIgG. Sensitivity. HIgG-treated PAni samples were incubated in different concentrations of FITC-tagged GaHIgG antibodies from 1 to 500 µg/mL for 20 min. The mean fluorescence signal and nonspecific binding for each concentration of antibody is shown in Figure 4. The HIgG immobilization obtained on the PAni surface has been found to be sensitive within the range of 2.5100 µg/mL GaHIgG, and binding sites reached saturation for subsequent concentrations. Image analysis data showed that BSA treatment is capable of minimizing nonspecific binding to less than 10% of total specific antibody binding for analyte concentrations less than 100 µg/ mL. Sensor Response. A piezoelectric immunosensor for GaHIgG has been devised based on the developed immobilization technique to demonstrate a working model. Polyaniline was deposited onto gold, and HIgG has been immobilized as described earlier. Each piezoelectric sensor was subjected to various concentrations from 0.25 to 100 µg/mL GaHIgG, in increasing order, successively. Quartz crystal sensors were incubated in antigen solution for 15 min and then washed in PBS followed by DI water and air-dried. Resonant frequency changes were measured, and thereafter, crystals were incubated in higher concentrations of GaHIgG antibody solution immediately. Figure 5 shows the sensor response as changes in the resonance frequency for changes in GaHIgG antibody concentration. Response was found to be (21) Zhou, C.; Friedt, J-M.; Angelova, A.; Choi, K-H.; Laureyn, W.; Frederix, F.; Francis, L. A.; Campitelli, A.; Engelborghs, Y. and Borghs, G. Langmuir 2004, 20, 5870-5878. (22) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Caruso, F.; Hoffmann, C. L.; Rabolt, J. F. Thin Film Solids 1995, 260, 192-199.
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sensitive in the range of 1-50 µg/mL GaHIgG antibodies. NSB in the dynamic range of interest, i.e., 1-25 µg/mL, was found to be ∼10%. The minimum detection limit was estimated to be 500 ng/mL or 3 nM IgG because the QCM response obtained was more than three times the maximum noise level. Unlike the cited techniques,8,23 our method does not require enzymatic reactions and additional reagents and, hence, makes the detection system simpler. Immobilization of antibodies on a polyaniline-coated gold surface has resulted in comparable performance in terms of
Figure 4. Fluorescence response due to specific binding (b) and NSB (O) obtained from HIgG-immobilized PAni surface for variation in the FITC-tagged GaHIgG antibody concentration (sigmoidal fit with R2 ) 0.9813 and R2 ) 0.9998, respectively). Background noise is subtracted from each point. (n ) 3)
Figure 5. Sensor response to the variation in GaHIgG concentration from 0.25 to 100 µg/mL due to specific binding (b) and NSB (O) (sigmoidal fit with R2 ) 0.9958 and R2 ) 0.9887, respectively). HIgGimmobilized quartz crystals were incubated in a known concentration of target analyte, GaHIgG, for 15 min and air-dried, and frequency changes were measured. Sensor was found to be sensitive between 1 and 50 µg/mL.
sensitivity and minimum detection limit compared to that of selfassembled monolayers formed on bare gold by thiols or sulfides bearing a terminal carboxylic acid24,25 and other alternate protocols such as use of PEI on gold.26 Thus, the use of polyaniline on gold for antibody immobilization could be an efficient alternative in developing piezoelectric and surface plasmon resonance-based biosensors. CONCLUSION AND FUTURE SCOPE In this report, we have described a general method of immobilization on PAni, which has been utilized in a piezoelectric immunobiosensor. The proposed protocol is a simple, fast, and cost-effective method of immobilization on thin films of PAni (
