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Chitosan polymer as support to IgG immobilization for piezoelectric applications
Accepted Manuscript Title: Chitosan polymer as support to IgG immobilization for piezoelectric applications Authors: Rosˆangela Ferreira Frade de Ara´ujo, Cosme Rafael Mart´ınez, Karla Patr´ıcia de Oliveira Luna, Renata Maria Costa Souza, Danyelly Bruneska, Rosa Fireman Dutra, Jos´e Luiz de Lima Filho PII: DOI: Reference:
S0169-4332(13)00354-1 http://dx.doi.org/doi:10.1016/j.apsusc.2013.02.046 APSUSC 25199
To appear in:
APSUSC
Received date: Revised date: Accepted date:
23-8-2012 31-1-2013 13-2-2013
Please cite this article as: R.F.F. Ara´ujo, C.R. Mart´ınez, K.P.O. Luna, R.M.C. Souza, D. Bruneska, R.F. Dutra, J.L.L. Filho, Chitosan polymer as support to IgG immobilization for piezoelectric applications, Applied Surface Science (2013), http://dx.doi.org/10.1016/j.apsusc.2013.02.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan polymer as support to IgG immobilization for piezoelectric applications
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Rosângela Ferreira Frade de Araújo1,4*, Cosme Rafael Martínez2, Karla Patrícia de Oliveira Luna1, Renata Maria Costa Souza1, Danyelly Bruneska1,4, Rosa Fireman Dutra3, José Luiz de Lima Filho1,4 1
Laboratório de Imunopatologia Keizo Asami – LIKA, Universidade Federal de Pernambuco – Recife – PE, Brazil
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Departamento de Química, Universidade Federal da Paraíba – João Pessoa – PB, Brazil
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Centro de Tecnologia, Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco – Recife-PE, Brazil
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Departamento de Bioquímica, Universidade Federal de Pernambuco – Recife – PE, Brazil
*Phone: +55 81 21268587;
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Fax: +55 81 21268485.
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E-mail address: [email protected]
Abstract
Immunoenzymatic assays using gold plates and QCM (Quartz Crystal microbalance) analysis were carried out in order to evaluate chitosan/IgG interaction. Two chitosan solutions (S1 and S2) were prepared with different concentrations of NaOH (0.8% - S1 and 8% - S2). Absorbances 3 fold higher were obtained when chitosan (S2) was used as support when compared with direct IgG adsorption on gold. S1 on gold showed a better stability (at 22ºC, for 72 hours) for IgG immobilization when compared with S2. However, S1 was used on QCM analysis and the IgG adsorption led to a non-Sauerbrey response, in which the mass on the electrode surface promote a proportional increase in the crystal resonant frequency. Direct IgG adsorption on gold electrode led to a 14.19% (± 2.43) increase in crystal frequency. When S1 was used as a support for IgG, a better immobilization occurred, causing a 24.34% (± 0.75) variation in crystal frequency. The structure of chitosan was shown to be efficient for IgG immobilization both in the immunoenzymatic method and in the QCM system.
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Keywords: antibody, chitosan, gold, immobilization, QCM.
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QCM: Quartz Crystal Microbalance; S1: Chitosan film prepared with 0.8% NaOH; S2: Chitosan film prepared with 8% NaOH; ELISA: Enzyme-Linked Immunoabsorbent Assay; OPD: Ortofenilenodiamine; ANOVA: Analysis of variance; HSD: Honestly Significant Difference.
1 Introduction
During the past few decades, there has been an increasing interest in using
natural polymers as immobilization matrixes for cell carriers, living organisms and enzymes [1]. Natural polymers contain functional groups that span wide ranges of polarity, electrostatic charge and hydrophobicity or ability to interact via van der Waals’ forces. Furthermore, biopolymer chemistry involves both covalent and noncovalent assembly processes [2]. Polymers present a better balance between 2
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chemical, physical and mechanical properties than metal or ceramic materials for biomedical applications [3]. Several advantages can be derived from the use of Carbohydrate polymers.
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First of all, probably because of the chemical similarities with heparin, polysaccharides show good hemocompatibility properties. They are non-toxic,
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show interaction with living cells and, with few exceptions, have low costs in comparison with others biopolymers such as collagen [4].
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Chitosan is a biodegradable natural polymer (Figure 1) with great potential for pharmaceutical and cosmetic applications and can be used like an ideal support
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for immobilization because it shows favorable characteristics such as
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biocompatibility, high charge density, hydrophicity, biodegradability, non-toxicity, excellent film-forming ability, susceptible to chemical modifications, antibacterial,
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antiviral properties and mucoadhesion [5-10]. The polymer is used in waste water
[11,12].
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treatment, drug development, obesity control, tissue engineering, e.g. bone repair
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Chitosan is a partially acetylated glycosamine biomolecule derived from
deacetylation of chitin, which is present on shells of crustaceans, insects, mushrooms and the cell wall of fungi [5,9-11]. Chitin, a linear polymer composed of nearly straight chains of (1-4) 2-acetoamido-2-deoxy-D-glucopyranose, kept together by strong interchain hydrogen bonding, is the second most abundant natural polysaccharide, after cellulose [12]. However, the term ‘chitosan’ usually is related to copolymers of 2-amino 2-deoxy-D-glucopyranose and 2-acetamido-2deoxy-D-glucopyranose where the degree of deacetylation is usually greater than 60% [13]. The greater potential applications of chitosan are related to its polycationic structure [14,15] and high percentage of nitrogen, present in the form
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of amino groups that are responsible for metal ion binding through chelation mechanisms [16]. Chitosan cross-linking with different chemicals has been used for
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immobilization of protein. The surfaces have been prepared with aminopropyltriethoxysilane [13], glutaraldehyde [8,14-16] and carbodiimide
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[17,18]. Issues include antibody capture performance and validation challenges in enzyme-linked immunosorbent assays (ELISAs), affinity chromatography
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separations, lateral flow assays, antibody-based diagnostics, antibody microarray assay formats, and biosensors [19]. Since the chemical and physical properties of
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polymers may be tailored by the chemist for particular needs, they gained
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importance in the construction of sensor devices [20]. The QCM biosensor for biological analyses has been reported increasingly in immunoassays. A linear
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relation occurs between a layer of external mass deposited in the surface of the
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crystal and the variation in its frequency of resonance [21,22]. In this paper, we tested the IgG immobilization directly on a chitosan film deposited on a gold
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surface by ELISA method and a QCM device.
2 Materials and methods
2.1 Preparation of chitosan solution 2mg/mL chitosan solutions were prepared by dissolving about 4mg chitosan (Sigma) from crab shells (minimum 85% deacetylated) in 0.8% (v/v) acetic acid (Vetec), in which it is soluble [23]. It seems that the effect of this acid on acetylation is nothing or very small [24]. 500μL of this solution were added to
375μL of 95% (v/v) ethanol (Merck) and 125μL of 0.8% (w/v) NaOH (Merck) – 4
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solution 1 (S1), pH bellow the isoelectric point of chitosan, or 125μL of 8% (w/v) NaOH – solution 2 (S2), pH above the isoelectric point of chitosan. The NaOH solution was used to neutralize the acidic residues [12]. Chitosan was rehydrated
2.2 Preparation of the supports and ELISA
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via ethanol to avoid the stiffening caused by rehydration in basic solutions [25].
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Gold plates (2mm x 3mm) were washed on piranha solution [sulphuric acid
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(Vetec), hydrogen peroxide (Labsynth), 7:3 (v/v)] and distilled water before chitosan application. Afterwards, they were placed into 2mL tubes and 50μL of
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chitosan solution (S1 or S2) was applied in order to completely cover the plates. After 15 minutes, the chitosan solution was drawn from the tubes. The plates were
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dried in nitrogen air, washed in PBS buffer pH 7.2 and dried again. Gold plates were covered with 2μg/mL human IgG solution (Sigma), kept at 22°C
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for 2 hours. After the exposure to IgG the surfaces were incubated overnight in
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blocking agent in order to prevent non-specific binding of the anti-IgG molecules. The different blocking agents trialled were 1.5% and 3% (w/v) casein, 1.5% and
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3.0% (w/v) BSA, 0.37% and 0.75% (w/v) glycine, 0.37% (w/v) glycine + 1% (w/v) NaOH and 0.75% (w/v) glycine + 1% (w/v) NaOH all prepared with PBS buffer pH 7.2. 50μL (0, 1 and 5μg/mL) of anti-human IgG peroxidase linked (Sigma) were
used within 2 hours of incubation at 22°C. Plates were washed in PBS buffer before being exposed to 200μL OPD (ortofenilenodiazina - Sigma) which was the reaction substrate. 2M sulphuric acid solution was used as stop solution and absorbances were recorded at 492nm. The final absorbances were determined by the difference between readings with and without IgG immobilized. The strategy for IgG immobilization on chitosan is shown in Figure 2. As a comparative analysis, the IgG immobilization directly on gold without chitosan was performed with the plates previously washed with 0.1 M HCl (Sigma) 5
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using 2μg/mL human IgG, the more efficient blocking solution (3% casein) and 5μg/mL anti-human IgG peroxidase linked. The IgG immobilization also was performed directly on polystyrene (aromatic hydrocarbon) micro plates (Nunk)
solutions and 5μg/mL anti-human IgG peroxidase linked.
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without and with chitosan (S1 and S2) using 2μg/mL human IgG, different blocking
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The stability of chitosan (S1 and S2) on gold was determined at 22°C after drying the plates in nitrogen air. The immobilization procedure was performed as
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described using 2μg/mL human IgG, 3% w/v casein and 5μg/mL anti-human IgG
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peroxidase linked at different times (0-72 hours) after preparing the supports. 2.3 QCM analysis
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In order to obtain results by different methods to evaluate the chitosan/IgG interaction, a QCM device also was used. An AT-cut quartz crystal of 10 MHz
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coated with two identical Au electrodes (diameter 8mm) was used and only one side
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of the quartz crystal was used for immobilization. The variation in the crystal resonant frequency was measured at 22ºC by a Frequency Analyzer GFC 8131 from
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GW intelligent counter.
Two different procedures of immobilization to attach IgG on gold electrodes
were tested. In the first, a frequency change was observed: a) after washing the electrode with corrosive solution and water, and drying in air nitrogen; b) after two applications of chitosan solution (20μL-dry-20μL-dry). With this approach, after
washing the crystals with distilled water and drying, a constant frequency was obtained; c) after incubation with 20μL of 0.5 to 3.5 μg/mL IgG where changes in frequency were also observed after washing with PBS buffer and drying (Figure 2). In the second procedure, a frequency change was observed in the following conditions: a) after incubation with 20μL of 0,1M HCl for 10 minutes, washing the electrode with distilled water and drying, and b) after incubation with 20μL of IgG 6
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solution (concentration chosen in the previous assay), in this case, changes in frequency were also observed after washing the electrode with PBS buffer and drying.
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2.4 Statistical analysis
All the assays were performed as replicates and analysis of variance
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(ANOVA) and Tukey’s HSD were performed to determine the statistical
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significance of the differences between the average values using Statistica (version
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6.1) software for statistics (Statsoft Inc., 2002).
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3 Results and Discussion
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3.1 ELISA
The IgG immobilization occurred either on S1 or on S2. The highest
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absorbances were obtained by using gold plates/chitosan solution prepared with 8% (w/v) NaOH (S2) and 3% (w/v) casein as blocking solution (Figure 3). Despite this, comparing the absorbances achieved using the other block solutions, it was not statistically different.
Only 1.5% (w/v) casein used as blocking solution on S2 was not efficient
when all the anti human IgG peroxidase linked concentrations had been led in account in the statistical analyses. By comparing between IgG immobilization on gold and on gold/chitosan (Figure 5) using 3% (w/v) casein as blocking solution, it was observed that with S1 and S2 occurred an increase of 63.52% and 71.13% in the absorbances respectively. The higher absorbance obtained by using S2 indicate that the increase in the 7
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concentration of NaOH in the preparation of the chitosan solutions contributed significantly for a better IgG immobilization. The chitosan (85% deacetylation) in an acidic environment (S1) is protonated due its isoelectric point at pH 6.3 which
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facilitates binding of the electrostatic type. These interactions depend of the charge density and size of the anionic agents [1,9]. However, in S2, the chitosan backbone
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probably did not present positive charges, so it is possible that the interaction
between the molecules of IgG and chitosan has been of the hydrophobic type.
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Yang et al [26] observed the binding capacity of IgG to protein A
immobilized on the chitosan/cellulose membrane and concluded that the binding
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capacity of human IgG on this support was four-fold higher than that using only
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cellulose for protein A immobilization. In this work, absorbances three-fold higher were found when only a chitosan solution (S2) was deposited on gold for IgG
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immobilization than that using only gold plates.
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The absorbances obtained with and without application of chitosan directly on microplates (Figure 5) were compared and the highest values were obtained
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without chitosan and with S2 considering all the used blocking solutions. Protein molecules that have been immobilized on a hydrophobic
polystyrene microplate by passive adsorption but can lose their activity and suffer considerable denaturation [25]. One more time the absorbances obtained using S2 were higher than that using S1. The increase in the concentration of NaOH caused a better interaction between chitosan and polystyrene. The figure 6 shows the highest absorbances found using 0.75% (w/v) glycine + 1% (w/v) NaOH, 1.5% (w/v) BSA and 3% (w/v) BSA blocking solutions considering the immobilization on S1 and S2. In the figure 7, the chitosan (S1 and S2) stability to IgG immobilization at 22ºC can be observed and S1 showed to be more stable then S2. In the different 8
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times after support preparation, the absorbances obtained by using S1 were not significantly different. A decrease in the absorbance occurred after 24 hours when S2 was used as
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support but after this time, the apparent decrease in the absorbances was not significant. Comparing absorbances achieved with S1 and S2 after 24 hours is clear
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to note highest absorbances using S1. However, this difference is more evident after 48 hours. With the time, it is possible have occurred loss of water on structure of
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chitosan. The dehydration of the structure and the highest concentration of NaOH in
interaction between chitosan and IgG.
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S2 can have contributed to become it more compact than S1 leading to a lesser
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Despite S1 also be dehydrated, the ability of chitosan to bind metals in acidic pH [27] can have contributed for better stability of this film. These results led
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to use S1 as support on the QCM system due the necessary steps of drying in the
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IgG immobilization on electrode surface. 3.2 QCM analysis
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The mass increase on the surface of the electrode (Table 1) occurred due
the interaction between IgG and the supports thus leading to a proportional change of the crystal resonant frequency. Gao et al [28] also found this relationship between resonant frequency and mass when they used a crystal immunosensor for detection of staphylococcal enterotoxin. When the chitosan film was deposited on the clean crystals, the crystals
resonance frequency showed an increase of 42.5% (± 0.59) being necessary two washes on electrode surface to keep its resonant frequency. This response is not as predicted by the Sauerbrey equation [12], which is a reduction in the piezoelectric crystals frequency as the mass increases. This non-Sauerbrey response had also been observed previously by Thompson et al. [21] while detecting IgG and 9
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investigating a crystal’s oscillation under liquid media and [29] using a QCM immunosensor for the detection of Listeria monocytogenes. This behavior was associated with interfacial interactions. It is known that this response is influenced
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by the viscosity of the film deposited on the crystal [30]. The direct IgG adsorption on gold electrode led to a 14.19% (± 2.43)
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variation on the crystal frequency and a 24.34% (± 0.75) variation on it frequency occurred when S1 was used as the support for IgG. The direct IgG adsorption on
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gold for piezoelectric detection was less efficient than that using chitosan due the force to guide a bigger amount of IgG to interact with chitosan to have been kept
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resisting the washing steps. The intrinsic washing steps in the IgG adsorption lead
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to a subsequent partial remotion of these molecules. When the molecules of IgG were immobilized in different concentrations (Figure 8) on electrodes surface
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covered with chitosan (S1), a significant variation in the crystal frequency occurred
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by using 2μg/mL IgG. Higher concentrations of IgG can have compromised its
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directed immobilization probably due to a steric impediment.
4 Conclusions
The chitosan backbone showed to be efficient for IgG immobilization
using a gold support, either through the immunoenzymatic method or through the QCM system in relation to immobilization without chitosan. The increase in the concentration of NaOH in chitosan solution promoted a better IgG immobilization either on polystyrene microplate or on gold. A possible hydrophobic interaction occurred between chitosan and molecules of IgG when pH was above isoelectric point of chitosan. The dehydration of the structure of chitosan with the time kept at 22°C and the highest NaOH concentration became it less favorable to keep the 10
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immobilized IgG. However, in QCM analyses a chitosan solution, prepared with lower NaOH concentration, was used to cover the gold electrode. The bigger variation in crystal resonant frequency occurred when IgG was immobilized on
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chitosan. Thus, this support showed better interaction with IgG comparing to gold
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electrode without chitosan.
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Acknowledgments
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This work was supported by Conselho Nacional de Desenvolvimento Científico e tecnológico/CNPq, Financiadora de Estudos e Projetos/FINEP and Japan International Cooperation Agency /JICA.
References
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[1] H. Huang, N. Hu, Y. Zeng, G. Zhou, Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films, Anal. Biochem. 308 (2002) 141–151.
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[2] D. Cunliffe, S. Pennadam, C. Alexander, Synthetic and biological polymers––merging the interface, Eur. Polym. J. 40 (2004) 5–25.
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[3] F.R.R. Teles, L.P. Fonseca, Applications of polymers for biomolecule immobilization in electrochemical biosensors, Mater. Sci. Eng. C Mater. Biol. Appl. 28 (2008) 1530–1543.
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[4] L. Yu, K. Dean, L. Li, Polymer blends and composites from renewable resources, Prog. Polym. Sci. 31 (2006) 576–602. [5] R.S. Juang, F.C. Wu, R.L. Tseng, Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes, Bioresour. Technol. 80 (2001) 187-193. [6] G. Vikhoreva, G. Bannikova, P. Stolbushkina, A. Panov, N. Drozd, V. Makarov, V. Varlamov, L. Gal’braikh, Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan, Carbohydr. Polym. 62 (2005) 327-332.
[7] S. Yi, C. Lee, J. Kim, D. Kyung, B. Kim, Y. Lee, Covalent immobilization of -transaminase from Vibrio fluvialis JS17 on chitosan beads, Process Biochem. 42 (2007) 895–898. [8] C.E. Orrego, N. Salgado, J.S. Valencia, G.I. Giraldo, O.H. Giraldo, C.A. Cardona, Novel chitosan membranes as support for lipases immobilization: Characterization aspects, Carbohydr. Polym. 79 (2010) 9–16. [9] M. Dasha, F. Chiellinia, R.M. Ottenbriteb, E. Chiellinia, Chitosan - A versatile semi-synthetic polymer in biomedical applications, Prog. Polym. Sci. 36 (2011) 981–1014. [10] A. Sionkowska, Current research on the blends of natural and synthetic polymers as new biomaterials: Review, Prog. Polym. Sci. 36 (2011) 1254– 1276. [11] X. Wang, J. Ma, Y. Wang, B. He, Bone repair in radii and tibias of rabbits with phosphorylated chitosan reinforced calcium phosphate cements, Biomaterials 23 (2002) 4167-4176.
11
Page 11 of 23
[12] Y. Wan, K.A.M. Creber, B. Peppley, V.T. Bui, Ionic conductivity of chitosan membranes, Polymer 44 (2003) 1057-1065. [13] J. Benesch, P. Tengvall, Blood Protein Adsorption onto Chitosan, Biomaterials 23 (2002) 2561-2568. [14] M. Hamdine, M.–C. Heuzey, A. Bégin, Effect of organic and inorganic acids on concentrated chitosan solutions and gels, Int. J. Biol. Macromol. 37 (2005) 134-142.
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[15] M.L. Arrascue, H.M. Garcia, O. Horna, E. Guibal, Gold sorption on chitosan derivatives, Hydrometallurgy 71 (2003) 191- 200.
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[16] S.A. Cetinus, E. Sßahin, D. Saraydin, Preparation of Cu(II) adsorbed chitosan beads for catalase immobilization, Food Chem. 114 (2009) 962–969.
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[17] J.K. Kim, D.S. Shin, W.J. Chung, K.H. Jang, K.N. Lee, Y.K. Kim, Y.S. Lee, Effects of polymer grafting on a glass surface for protein chip applications, Colloids Surf., B 33 (2004) 67-75.
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[18] I. Kolodziejska, B. Piotrowska, The water vapour permeability, mechanical properties and solubility of fish gelatin–chitosan films modified with transglutaminase or 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and plasticized with glycerol, Food Chem. 103 (2007) 295–300.
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[19] F. Liu, M. Dubey, H. Takahashi, D.G. Caster, D.W.Grainger, Immobilized Antibody Orientation Analysis using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-generated Patterns, Anal. Chem. 82(7) (2010) 2947–2958. [20] B. Adhikari, S. Majumdar, Polymers in sensor applications, Prog. Polym. Sci. 29 (2004) 699– 766.
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[21] X. Su, J. Zhang, Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance for human IgE quantification, Sens. Actuators 100, (2004) 309-314.
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[22] G.–Y. Shen, I. Wang, T. Deng, G.–L. Shen, R.–Q. Yu, A novel piezoelectric immunosensor for detection of carcinoembryonic antigen, Talanta 67 (2005) 217-220.
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[23] C. Muzzarelli, G. Tosi, O Francescangeli, R.A. Muzzarelli, Alkaline chitosan solutions, Carbohydr. Res. 338 (2003) 2247- 2255. [24] A. Ogino, M. Kral, M. Yamashita, M. Nagatsu, Effects of low-temperature surface-wave plasma treatment with various gases on surfaces modification of chitosan, Appl. Surf. Sci. 255 (2008) 2347 – 2352. [25] Y. Zhang, L. Li, C. Yu, T. Hei, Chitosan-coated polystyrene microplate for covalent immobilization of enzyme, Anal. Bioanal. Chem. 401 (7) (2011) 2311-2317. [26] L. Yang, W.W. Hsiao, P. Chen, Chitosan-cellulose composite membrane for affinity purification of biopolymers and immunoadsorption, J. Membrane Sci. 197 (2002) 185-197. [27] A.J. Varma, S.V. Deshpande, J.F. Kennedy, Metal complexation by chitosan and its derivatives: a review, Carbohydr. Polym. 55 (2004) 77- 93.
[28] Z. Gao, F. Chao, Z. Chao, G. Li, Detection of staphylococcal enterotoxin C2 employing a piezoelectric crystal immunosensor, Sens. Actuators 66 (2000) 193-196. [29] R.D. Vaughan, C.K. O’Sullivan, G.G. Guilbault, Development of a quartz crystal microbalance (QCM) immunosensor for the detection of Listeria monocytogenes, Enzyme Microb. Technol. 29 (2001) 635–638. [30] M. Rodahl, B. Kasemo, On the measurement of thin liquid overlayers with the quartz-crystal microbalance, Sens. Actuators A 54 (1996) 448- 456.
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Figure 1. Structure of chitin and chitosan (partially acetylated glycosamine biomolecule derived from deacetylation of chitin and related to copolymers of 2-amino 2-deoxy-D-glucopyranose and 2acetamido-2-deoxy-D-glucopyranose)
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Figure 2. Schematic design of the strategies used for IgG immobilization in ELISA (A) and QCM analyzes. 1- Gold support; 2- Chitosan film; 3- Blocking molecules; 4- IgG; 5- Anti-IgG; 6Peroxidase
Figure 3. The means of the differences between the absorbances found by the ELISA method with and without IgG (2μg/mL) immobilized on gold plates covered with chitosan using different blocking solutions and anti-IgG peroxidase linked (5μg/mL) where “C” is Casein and “G” is Glycine. The means followed by same letters not differ statistically (p<0.05) to Tukey’s HSD. The small letters make comparisons between chitosan film prepared with 0.8% NaOH - S1 or with 8% NaOH - S2 in different blocking solutions and the capital letters comparing S1 and S2 in each blocking solution Figure 4. Comparison between the absorbances obtained by ELISA method immobilizing IgG (2µg/mL) on gold plates without and with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and using 3% (w/v) casein (blocking solution) and anti-IgG peroxidase linked (5μg/mL)
Figure 5. Effects of the chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) in absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on microplate using different blocking solutions (1.5% and 3% casein, 1.5% and 3% BSA, 0.37% and 0.75% glycin, 0.37% and 0.75% glycin + 1% NaOH) and anti-IgG peroxidase linked (5μg/mL). The means of absorbances (considering all block solutions) follwed by the same letters do not differ statistically (p<0.05) to Tukey’s HSD
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Figure 6. Effects of blocking solutions in absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on microplate without and with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and anti-IgG peroxidase linked (5μg/mL) where “C” is Casein and “G” is Glycine.
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Figure 7. Absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on gold plates covered with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and using 3% (w/v) casein (blocking solution) and anti- IgG peroxidase linked (5μg/mL) at different times after the preparation of these supports. The means follwed by the same letters do not differ statistically (p<0.05) to Tukey’s HSD. The small letters make comparisons between S1 or S2 (chitosan film prepared with 0.8% NaOH and with 8% NaOH respectively) at different times and the capital letters comparing S1 and S2 in each time
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Figure 8. Variation of the quartz crystals resonant frequency after the immobilization of the IgG in different concentrations (0.5 to 3.5 µg/mL) on gold electrode covered with chitosan (prepared with 0.8% NaOH)
Table 1. Variation of quartz crystals resonant frequency after the immobilization of IgG (2μg/mL) on gold electrode without and with chitosan (prepared with 0.8% NaOH) Variation of the frequency (%) After chitosan film application _
After IgG immobilization
_
12.47
42.91
24.87
42,08
23.81
15.91
Averages variation after IgG immobilization 14.19 (± 2.43) 24,34 (± 0.75)
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-Chitosan backbone showed to be efficient for IgG immobilization using a gold support. -Highest concentration of NaOH on chitosan film favored a better IgG immobilization. -The response in QCM analysis was not as predicted by the Sauerbrey equation. -A significant variation in the crystal frequency occurred by using 2μg/mL IgG.
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S0169-4332(13)00354-1 http://dx.doi.org/doi:10.1016/j.apsusc.2013.02.046 APSUSC 25199
To appear in:
APSUSC
Received date: Revised date: Accepted date:
23-8-2012 31-1-2013 13-2-2013
Please cite this article as: R.F.F. Ara´ujo, C.R. Mart´ınez, K.P.O. Luna, R.M.C. Souza, D. Bruneska, R.F. Dutra, J.L.L. Filho, Chitosan polymer as support to IgG immobilization for piezoelectric applications, Applied Surface Science (2013), http://dx.doi.org/10.1016/j.apsusc.2013.02.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chitosan polymer as support to IgG immobilization for piezoelectric applications
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Rosângela Ferreira Frade de Araújo1,4*, Cosme Rafael Martínez2, Karla Patrícia de Oliveira Luna1, Renata Maria Costa Souza1, Danyelly Bruneska1,4, Rosa Fireman Dutra3, José Luiz de Lima Filho1,4 1
Laboratório de Imunopatologia Keizo Asami – LIKA, Universidade Federal de Pernambuco – Recife – PE, Brazil
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Departamento de Química, Universidade Federal da Paraíba – João Pessoa – PB, Brazil
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Centro de Tecnologia, Laboratório de Engenharia Biomédica, Universidade Federal de Pernambuco – Recife-PE, Brazil
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Departamento de Bioquímica, Universidade Federal de Pernambuco – Recife – PE, Brazil
*Phone: +55 81 21268587;
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Fax: +55 81 21268485.
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E-mail address: [email protected]
Abstract
Immunoenzymatic assays using gold plates and QCM (Quartz Crystal microbalance) analysis were carried out in order to evaluate chitosan/IgG interaction. Two chitosan solutions (S1 and S2) were prepared with different concentrations of NaOH (0.8% - S1 and 8% - S2). Absorbances 3 fold higher were obtained when chitosan (S2) was used as support when compared with direct IgG adsorption on gold. S1 on gold showed a better stability (at 22ºC, for 72 hours) for IgG immobilization when compared with S2. However, S1 was used on QCM analysis and the IgG adsorption led to a non-Sauerbrey response, in which the mass on the electrode surface promote a proportional increase in the crystal resonant frequency. Direct IgG adsorption on gold electrode led to a 14.19% (± 2.43) increase in crystal frequency. When S1 was used as a support for IgG, a better immobilization occurred, causing a 24.34% (± 0.75) variation in crystal frequency. The structure of chitosan was shown to be efficient for IgG immobilization both in the immunoenzymatic method and in the QCM system.
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Keywords: antibody, chitosan, gold, immobilization, QCM.
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QCM: Quartz Crystal Microbalance; S1: Chitosan film prepared with 0.8% NaOH; S2: Chitosan film prepared with 8% NaOH; ELISA: Enzyme-Linked Immunoabsorbent Assay; OPD: Ortofenilenodiamine; ANOVA: Analysis of variance; HSD: Honestly Significant Difference.
1 Introduction
During the past few decades, there has been an increasing interest in using
natural polymers as immobilization matrixes for cell carriers, living organisms and enzymes [1]. Natural polymers contain functional groups that span wide ranges of polarity, electrostatic charge and hydrophobicity or ability to interact via van der Waals’ forces. Furthermore, biopolymer chemistry involves both covalent and noncovalent assembly processes [2]. Polymers present a better balance between 2
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chemical, physical and mechanical properties than metal or ceramic materials for biomedical applications [3]. Several advantages can be derived from the use of Carbohydrate polymers.
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First of all, probably because of the chemical similarities with heparin, polysaccharides show good hemocompatibility properties. They are non-toxic,
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show interaction with living cells and, with few exceptions, have low costs in comparison with others biopolymers such as collagen [4].
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Chitosan is a biodegradable natural polymer (Figure 1) with great potential for pharmaceutical and cosmetic applications and can be used like an ideal support
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for immobilization because it shows favorable characteristics such as
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biocompatibility, high charge density, hydrophicity, biodegradability, non-toxicity, excellent film-forming ability, susceptible to chemical modifications, antibacterial,
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antiviral properties and mucoadhesion [5-10]. The polymer is used in waste water
[11,12].
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treatment, drug development, obesity control, tissue engineering, e.g. bone repair
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Chitosan is a partially acetylated glycosamine biomolecule derived from
deacetylation of chitin, which is present on shells of crustaceans, insects, mushrooms and the cell wall of fungi [5,9-11]. Chitin, a linear polymer composed of nearly straight chains of (1-4) 2-acetoamido-2-deoxy-D-glucopyranose, kept together by strong interchain hydrogen bonding, is the second most abundant natural polysaccharide, after cellulose [12]. However, the term ‘chitosan’ usually is related to copolymers of 2-amino 2-deoxy-D-glucopyranose and 2-acetamido-2deoxy-D-glucopyranose where the degree of deacetylation is usually greater than 60% [13]. The greater potential applications of chitosan are related to its polycationic structure [14,15] and high percentage of nitrogen, present in the form
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of amino groups that are responsible for metal ion binding through chelation mechanisms [16]. Chitosan cross-linking with different chemicals has been used for
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immobilization of protein. The surfaces have been prepared with aminopropyltriethoxysilane [13], glutaraldehyde [8,14-16] and carbodiimide
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[17,18]. Issues include antibody capture performance and validation challenges in enzyme-linked immunosorbent assays (ELISAs), affinity chromatography
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separations, lateral flow assays, antibody-based diagnostics, antibody microarray assay formats, and biosensors [19]. Since the chemical and physical properties of
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polymers may be tailored by the chemist for particular needs, they gained
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importance in the construction of sensor devices [20]. The QCM biosensor for biological analyses has been reported increasingly in immunoassays. A linear
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relation occurs between a layer of external mass deposited in the surface of the
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crystal and the variation in its frequency of resonance [21,22]. In this paper, we tested the IgG immobilization directly on a chitosan film deposited on a gold
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surface by ELISA method and a QCM device.
2 Materials and methods
2.1 Preparation of chitosan solution 2mg/mL chitosan solutions were prepared by dissolving about 4mg chitosan (Sigma) from crab shells (minimum 85% deacetylated) in 0.8% (v/v) acetic acid (Vetec), in which it is soluble [23]. It seems that the effect of this acid on acetylation is nothing or very small [24]. 500μL of this solution were added to
375μL of 95% (v/v) ethanol (Merck) and 125μL of 0.8% (w/v) NaOH (Merck) – 4
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solution 1 (S1), pH bellow the isoelectric point of chitosan, or 125μL of 8% (w/v) NaOH – solution 2 (S2), pH above the isoelectric point of chitosan. The NaOH solution was used to neutralize the acidic residues [12]. Chitosan was rehydrated
2.2 Preparation of the supports and ELISA
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via ethanol to avoid the stiffening caused by rehydration in basic solutions [25].
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Gold plates (2mm x 3mm) were washed on piranha solution [sulphuric acid
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(Vetec), hydrogen peroxide (Labsynth), 7:3 (v/v)] and distilled water before chitosan application. Afterwards, they were placed into 2mL tubes and 50μL of
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chitosan solution (S1 or S2) was applied in order to completely cover the plates. After 15 minutes, the chitosan solution was drawn from the tubes. The plates were
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dried in nitrogen air, washed in PBS buffer pH 7.2 and dried again. Gold plates were covered with 2μg/mL human IgG solution (Sigma), kept at 22°C
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for 2 hours. After the exposure to IgG the surfaces were incubated overnight in
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blocking agent in order to prevent non-specific binding of the anti-IgG molecules. The different blocking agents trialled were 1.5% and 3% (w/v) casein, 1.5% and
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3.0% (w/v) BSA, 0.37% and 0.75% (w/v) glycine, 0.37% (w/v) glycine + 1% (w/v) NaOH and 0.75% (w/v) glycine + 1% (w/v) NaOH all prepared with PBS buffer pH 7.2. 50μL (0, 1 and 5μg/mL) of anti-human IgG peroxidase linked (Sigma) were
used within 2 hours of incubation at 22°C. Plates were washed in PBS buffer before being exposed to 200μL OPD (ortofenilenodiazina - Sigma) which was the reaction substrate. 2M sulphuric acid solution was used as stop solution and absorbances were recorded at 492nm. The final absorbances were determined by the difference between readings with and without IgG immobilized. The strategy for IgG immobilization on chitosan is shown in Figure 2. As a comparative analysis, the IgG immobilization directly on gold without chitosan was performed with the plates previously washed with 0.1 M HCl (Sigma) 5
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using 2μg/mL human IgG, the more efficient blocking solution (3% casein) and 5μg/mL anti-human IgG peroxidase linked. The IgG immobilization also was performed directly on polystyrene (aromatic hydrocarbon) micro plates (Nunk)
solutions and 5μg/mL anti-human IgG peroxidase linked.
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without and with chitosan (S1 and S2) using 2μg/mL human IgG, different blocking
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The stability of chitosan (S1 and S2) on gold was determined at 22°C after drying the plates in nitrogen air. The immobilization procedure was performed as
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described using 2μg/mL human IgG, 3% w/v casein and 5μg/mL anti-human IgG
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peroxidase linked at different times (0-72 hours) after preparing the supports. 2.3 QCM analysis
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In order to obtain results by different methods to evaluate the chitosan/IgG interaction, a QCM device also was used. An AT-cut quartz crystal of 10 MHz
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coated with two identical Au electrodes (diameter 8mm) was used and only one side
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of the quartz crystal was used for immobilization. The variation in the crystal resonant frequency was measured at 22ºC by a Frequency Analyzer GFC 8131 from
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GW intelligent counter.
Two different procedures of immobilization to attach IgG on gold electrodes
were tested. In the first, a frequency change was observed: a) after washing the electrode with corrosive solution and water, and drying in air nitrogen; b) after two applications of chitosan solution (20μL-dry-20μL-dry). With this approach, after
washing the crystals with distilled water and drying, a constant frequency was obtained; c) after incubation with 20μL of 0.5 to 3.5 μg/mL IgG where changes in frequency were also observed after washing with PBS buffer and drying (Figure 2). In the second procedure, a frequency change was observed in the following conditions: a) after incubation with 20μL of 0,1M HCl for 10 minutes, washing the electrode with distilled water and drying, and b) after incubation with 20μL of IgG 6
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solution (concentration chosen in the previous assay), in this case, changes in frequency were also observed after washing the electrode with PBS buffer and drying.
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2.4 Statistical analysis
All the assays were performed as replicates and analysis of variance
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(ANOVA) and Tukey’s HSD were performed to determine the statistical
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significance of the differences between the average values using Statistica (version
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6.1) software for statistics (Statsoft Inc., 2002).
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3 Results and Discussion
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3.1 ELISA
The IgG immobilization occurred either on S1 or on S2. The highest
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absorbances were obtained by using gold plates/chitosan solution prepared with 8% (w/v) NaOH (S2) and 3% (w/v) casein as blocking solution (Figure 3). Despite this, comparing the absorbances achieved using the other block solutions, it was not statistically different.
Only 1.5% (w/v) casein used as blocking solution on S2 was not efficient
when all the anti human IgG peroxidase linked concentrations had been led in account in the statistical analyses. By comparing between IgG immobilization on gold and on gold/chitosan (Figure 5) using 3% (w/v) casein as blocking solution, it was observed that with S1 and S2 occurred an increase of 63.52% and 71.13% in the absorbances respectively. The higher absorbance obtained by using S2 indicate that the increase in the 7
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concentration of NaOH in the preparation of the chitosan solutions contributed significantly for a better IgG immobilization. The chitosan (85% deacetylation) in an acidic environment (S1) is protonated due its isoelectric point at pH 6.3 which
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facilitates binding of the electrostatic type. These interactions depend of the charge density and size of the anionic agents [1,9]. However, in S2, the chitosan backbone
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probably did not present positive charges, so it is possible that the interaction
between the molecules of IgG and chitosan has been of the hydrophobic type.
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Yang et al [26] observed the binding capacity of IgG to protein A
immobilized on the chitosan/cellulose membrane and concluded that the binding
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capacity of human IgG on this support was four-fold higher than that using only
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cellulose for protein A immobilization. In this work, absorbances three-fold higher were found when only a chitosan solution (S2) was deposited on gold for IgG
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immobilization than that using only gold plates.
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The absorbances obtained with and without application of chitosan directly on microplates (Figure 5) were compared and the highest values were obtained
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without chitosan and with S2 considering all the used blocking solutions. Protein molecules that have been immobilized on a hydrophobic
polystyrene microplate by passive adsorption but can lose their activity and suffer considerable denaturation [25]. One more time the absorbances obtained using S2 were higher than that using S1. The increase in the concentration of NaOH caused a better interaction between chitosan and polystyrene. The figure 6 shows the highest absorbances found using 0.75% (w/v) glycine + 1% (w/v) NaOH, 1.5% (w/v) BSA and 3% (w/v) BSA blocking solutions considering the immobilization on S1 and S2. In the figure 7, the chitosan (S1 and S2) stability to IgG immobilization at 22ºC can be observed and S1 showed to be more stable then S2. In the different 8
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times after support preparation, the absorbances obtained by using S1 were not significantly different. A decrease in the absorbance occurred after 24 hours when S2 was used as
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support but after this time, the apparent decrease in the absorbances was not significant. Comparing absorbances achieved with S1 and S2 after 24 hours is clear
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to note highest absorbances using S1. However, this difference is more evident after 48 hours. With the time, it is possible have occurred loss of water on structure of
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chitosan. The dehydration of the structure and the highest concentration of NaOH in
interaction between chitosan and IgG.
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S2 can have contributed to become it more compact than S1 leading to a lesser
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Despite S1 also be dehydrated, the ability of chitosan to bind metals in acidic pH [27] can have contributed for better stability of this film. These results led
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to use S1 as support on the QCM system due the necessary steps of drying in the
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IgG immobilization on electrode surface. 3.2 QCM analysis
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The mass increase on the surface of the electrode (Table 1) occurred due
the interaction between IgG and the supports thus leading to a proportional change of the crystal resonant frequency. Gao et al [28] also found this relationship between resonant frequency and mass when they used a crystal immunosensor for detection of staphylococcal enterotoxin. When the chitosan film was deposited on the clean crystals, the crystals
resonance frequency showed an increase of 42.5% (± 0.59) being necessary two washes on electrode surface to keep its resonant frequency. This response is not as predicted by the Sauerbrey equation [12], which is a reduction in the piezoelectric crystals frequency as the mass increases. This non-Sauerbrey response had also been observed previously by Thompson et al. [21] while detecting IgG and 9
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investigating a crystal’s oscillation under liquid media and [29] using a QCM immunosensor for the detection of Listeria monocytogenes. This behavior was associated with interfacial interactions. It is known that this response is influenced
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by the viscosity of the film deposited on the crystal [30]. The direct IgG adsorption on gold electrode led to a 14.19% (± 2.43)
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variation on the crystal frequency and a 24.34% (± 0.75) variation on it frequency occurred when S1 was used as the support for IgG. The direct IgG adsorption on
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gold for piezoelectric detection was less efficient than that using chitosan due the force to guide a bigger amount of IgG to interact with chitosan to have been kept
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resisting the washing steps. The intrinsic washing steps in the IgG adsorption lead
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to a subsequent partial remotion of these molecules. When the molecules of IgG were immobilized in different concentrations (Figure 8) on electrodes surface
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covered with chitosan (S1), a significant variation in the crystal frequency occurred
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by using 2μg/mL IgG. Higher concentrations of IgG can have compromised its
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directed immobilization probably due to a steric impediment.
4 Conclusions
The chitosan backbone showed to be efficient for IgG immobilization
using a gold support, either through the immunoenzymatic method or through the QCM system in relation to immobilization without chitosan. The increase in the concentration of NaOH in chitosan solution promoted a better IgG immobilization either on polystyrene microplate or on gold. A possible hydrophobic interaction occurred between chitosan and molecules of IgG when pH was above isoelectric point of chitosan. The dehydration of the structure of chitosan with the time kept at 22°C and the highest NaOH concentration became it less favorable to keep the 10
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immobilized IgG. However, in QCM analyses a chitosan solution, prepared with lower NaOH concentration, was used to cover the gold electrode. The bigger variation in crystal resonant frequency occurred when IgG was immobilized on
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chitosan. Thus, this support showed better interaction with IgG comparing to gold
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electrode without chitosan.
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Acknowledgments
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This work was supported by Conselho Nacional de Desenvolvimento Científico e tecnológico/CNPq, Financiadora de Estudos e Projetos/FINEP and Japan International Cooperation Agency /JICA.
References
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[1] H. Huang, N. Hu, Y. Zeng, G. Zhou, Electrochemistry and electrocatalysis with heme proteins in chitosan biopolymer films, Anal. Biochem. 308 (2002) 141–151.
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[2] D. Cunliffe, S. Pennadam, C. Alexander, Synthetic and biological polymers––merging the interface, Eur. Polym. J. 40 (2004) 5–25.
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[3] F.R.R. Teles, L.P. Fonseca, Applications of polymers for biomolecule immobilization in electrochemical biosensors, Mater. Sci. Eng. C Mater. Biol. Appl. 28 (2008) 1530–1543.
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[4] L. Yu, K. Dean, L. Li, Polymer blends and composites from renewable resources, Prog. Polym. Sci. 31 (2006) 576–602. [5] R.S. Juang, F.C. Wu, R.L. Tseng, Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes, Bioresour. Technol. 80 (2001) 187-193. [6] G. Vikhoreva, G. Bannikova, P. Stolbushkina, A. Panov, N. Drozd, V. Makarov, V. Varlamov, L. Gal’braikh, Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan, Carbohydr. Polym. 62 (2005) 327-332.
[7] S. Yi, C. Lee, J. Kim, D. Kyung, B. Kim, Y. Lee, Covalent immobilization of -transaminase from Vibrio fluvialis JS17 on chitosan beads, Process Biochem. 42 (2007) 895–898. [8] C.E. Orrego, N. Salgado, J.S. Valencia, G.I. Giraldo, O.H. Giraldo, C.A. Cardona, Novel chitosan membranes as support for lipases immobilization: Characterization aspects, Carbohydr. Polym. 79 (2010) 9–16. [9] M. Dasha, F. Chiellinia, R.M. Ottenbriteb, E. Chiellinia, Chitosan - A versatile semi-synthetic polymer in biomedical applications, Prog. Polym. Sci. 36 (2011) 981–1014. [10] A. Sionkowska, Current research on the blends of natural and synthetic polymers as new biomaterials: Review, Prog. Polym. Sci. 36 (2011) 1254– 1276. [11] X. Wang, J. Ma, Y. Wang, B. He, Bone repair in radii and tibias of rabbits with phosphorylated chitosan reinforced calcium phosphate cements, Biomaterials 23 (2002) 4167-4176.
11
Page 11 of 23
[12] Y. Wan, K.A.M. Creber, B. Peppley, V.T. Bui, Ionic conductivity of chitosan membranes, Polymer 44 (2003) 1057-1065. [13] J. Benesch, P. Tengvall, Blood Protein Adsorption onto Chitosan, Biomaterials 23 (2002) 2561-2568. [14] M. Hamdine, M.–C. Heuzey, A. Bégin, Effect of organic and inorganic acids on concentrated chitosan solutions and gels, Int. J. Biol. Macromol. 37 (2005) 134-142.
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[15] M.L. Arrascue, H.M. Garcia, O. Horna, E. Guibal, Gold sorption on chitosan derivatives, Hydrometallurgy 71 (2003) 191- 200.
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[16] S.A. Cetinus, E. Sßahin, D. Saraydin, Preparation of Cu(II) adsorbed chitosan beads for catalase immobilization, Food Chem. 114 (2009) 962–969.
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[17] J.K. Kim, D.S. Shin, W.J. Chung, K.H. Jang, K.N. Lee, Y.K. Kim, Y.S. Lee, Effects of polymer grafting on a glass surface for protein chip applications, Colloids Surf., B 33 (2004) 67-75.
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[18] I. Kolodziejska, B. Piotrowska, The water vapour permeability, mechanical properties and solubility of fish gelatin–chitosan films modified with transglutaminase or 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and plasticized with glycerol, Food Chem. 103 (2007) 295–300.
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[19] F. Liu, M. Dubey, H. Takahashi, D.G. Caster, D.W.Grainger, Immobilized Antibody Orientation Analysis using Secondary Ion Mass Spectrometry and Fluorescence Imaging of Affinity-generated Patterns, Anal. Chem. 82(7) (2010) 2947–2958. [20] B. Adhikari, S. Majumdar, Polymers in sensor applications, Prog. Polym. Sci. 29 (2004) 699– 766.
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[21] X. Su, J. Zhang, Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance for human IgE quantification, Sens. Actuators 100, (2004) 309-314.
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[22] G.–Y. Shen, I. Wang, T. Deng, G.–L. Shen, R.–Q. Yu, A novel piezoelectric immunosensor for detection of carcinoembryonic antigen, Talanta 67 (2005) 217-220.
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[23] C. Muzzarelli, G. Tosi, O Francescangeli, R.A. Muzzarelli, Alkaline chitosan solutions, Carbohydr. Res. 338 (2003) 2247- 2255. [24] A. Ogino, M. Kral, M. Yamashita, M. Nagatsu, Effects of low-temperature surface-wave plasma treatment with various gases on surfaces modification of chitosan, Appl. Surf. Sci. 255 (2008) 2347 – 2352. [25] Y. Zhang, L. Li, C. Yu, T. Hei, Chitosan-coated polystyrene microplate for covalent immobilization of enzyme, Anal. Bioanal. Chem. 401 (7) (2011) 2311-2317. [26] L. Yang, W.W. Hsiao, P. Chen, Chitosan-cellulose composite membrane for affinity purification of biopolymers and immunoadsorption, J. Membrane Sci. 197 (2002) 185-197. [27] A.J. Varma, S.V. Deshpande, J.F. Kennedy, Metal complexation by chitosan and its derivatives: a review, Carbohydr. Polym. 55 (2004) 77- 93.
[28] Z. Gao, F. Chao, Z. Chao, G. Li, Detection of staphylococcal enterotoxin C2 employing a piezoelectric crystal immunosensor, Sens. Actuators 66 (2000) 193-196. [29] R.D. Vaughan, C.K. O’Sullivan, G.G. Guilbault, Development of a quartz crystal microbalance (QCM) immunosensor for the detection of Listeria monocytogenes, Enzyme Microb. Technol. 29 (2001) 635–638. [30] M. Rodahl, B. Kasemo, On the measurement of thin liquid overlayers with the quartz-crystal microbalance, Sens. Actuators A 54 (1996) 448- 456.
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Figure 1. Structure of chitin and chitosan (partially acetylated glycosamine biomolecule derived from deacetylation of chitin and related to copolymers of 2-amino 2-deoxy-D-glucopyranose and 2acetamido-2-deoxy-D-glucopyranose)
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Figure 2. Schematic design of the strategies used for IgG immobilization in ELISA (A) and QCM analyzes. 1- Gold support; 2- Chitosan film; 3- Blocking molecules; 4- IgG; 5- Anti-IgG; 6Peroxidase
Figure 3. The means of the differences between the absorbances found by the ELISA method with and without IgG (2μg/mL) immobilized on gold plates covered with chitosan using different blocking solutions and anti-IgG peroxidase linked (5μg/mL) where “C” is Casein and “G” is Glycine. The means followed by same letters not differ statistically (p<0.05) to Tukey’s HSD. The small letters make comparisons between chitosan film prepared with 0.8% NaOH - S1 or with 8% NaOH - S2 in different blocking solutions and the capital letters comparing S1 and S2 in each blocking solution Figure 4. Comparison between the absorbances obtained by ELISA method immobilizing IgG (2µg/mL) on gold plates without and with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and using 3% (w/v) casein (blocking solution) and anti-IgG peroxidase linked (5μg/mL)
Figure 5. Effects of the chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) in absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on microplate using different blocking solutions (1.5% and 3% casein, 1.5% and 3% BSA, 0.37% and 0.75% glycin, 0.37% and 0.75% glycin + 1% NaOH) and anti-IgG peroxidase linked (5μg/mL). The means of absorbances (considering all block solutions) follwed by the same letters do not differ statistically (p<0.05) to Tukey’s HSD
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Figure 6. Effects of blocking solutions in absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on microplate without and with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and anti-IgG peroxidase linked (5μg/mL) where “C” is Casein and “G” is Glycine.
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Figure 7. Absorbances obtained by ELISA method immobilizing IgG (2μg/mL) on gold plates covered with chitosan (prepared with 0.8% NaOH - S1 and with 8% NaOH - S2) and using 3% (w/v) casein (blocking solution) and anti- IgG peroxidase linked (5μg/mL) at different times after the preparation of these supports. The means follwed by the same letters do not differ statistically (p<0.05) to Tukey’s HSD. The small letters make comparisons between S1 or S2 (chitosan film prepared with 0.8% NaOH and with 8% NaOH respectively) at different times and the capital letters comparing S1 and S2 in each time
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Figure 8. Variation of the quartz crystals resonant frequency after the immobilization of the IgG in different concentrations (0.5 to 3.5 µg/mL) on gold electrode covered with chitosan (prepared with 0.8% NaOH)
Table 1. Variation of quartz crystals resonant frequency after the immobilization of IgG (2μg/mL) on gold electrode without and with chitosan (prepared with 0.8% NaOH) Variation of the frequency (%) After chitosan film application _
After IgG immobilization
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12.47
42.91
24.87
42,08
23.81
15.91
Averages variation after IgG immobilization 14.19 (± 2.43) 24,34 (± 0.75)
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-Chitosan backbone showed to be efficient for IgG immobilization using a gold support. -Highest concentration of NaOH on chitosan film favored a better IgG immobilization. -The response in QCM analysis was not as predicted by the Sauerbrey equation. -A significant variation in the crystal frequency occurred by using 2μg/mL IgG.
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