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Bioelectrode for detection of human salivary amylase
Materials Science and Engineering C 32 (2012) 530–535
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Bioelectrode for detection of human salivary amylase Tatiane Vanessa da Silva Santos a, 1, Renata Roland Teixeira a, 1, Diego Leoni Franco b, João Marcos Madurro b, Ana Graci Brito-Madurro a, Foued Salmen Espindola a,⁎ a b
Institute of Genetics and Biochemistry, Federal University of Uberlandia, Uberlandia, MG, Brazil Institute of Chemistry, Federal University of Uberlandia, Uberlandia, MG, Brazil
a r t i c l e
i n f o
Article history: Received 17 March 2011 Received in revised form 12 September 2011 Accepted 7 December 2011 Available online 14 December 2011 Keywords: Salivary alpha-amylase 3-Hydroxyphenylacetic acid Bioelectrode Biosensor Polymeric film
a b s t r a c t The aim of this study is the development of a novel bioelectrode based on the immobilization of a specific antibody for salivary amylase onto a graphite electrode modified with poly(3-hydroxyphenylacetic) acid. For this purpose, human salivary alpha-amylase was applied to an immunoaffinity-purified anti-alpha-amylase polyclonal antibody. The bioelectrode was incubated with salivary amylase or lysozyme (interfering salivary), and the interaction between the antibody and the enzymes was analyzed through electrochemical impedance spectroscopy. The results indicated the specificity of the bioelectrode to salivary alpha-amylase. Therefore, the combination of the graphite electrode with poly(3-hydroxyphenylacetic acid) appears to be a promising strategy for antigen immobilization and other biological recognition elements, thus presenting the potential for the production of a label-free biochip. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Salivary alpha-amylase is the most abundant enzyme in saliva; it is primarily involved in the digestion of starch and acts as a receptor for the adhesion of bacterial flora, as well as assisting in the formation of dental plaque [1]. Several studies have evaluated the differences in activity and concentration of this protein to use it as a potential biomarker for diagnosis of some oral [2] and systemic diseases [3–5]. Several research groups have also evaluated different stress conditions through salivary alpha-amylase activity using spectrometric methods [6,7] and biosensors [8,9]. Immunosensors are interesting for clinical applications because they offer a number of potential advantages over conventional assay techniques; the detection is fast and the system can be automated [10]. Electrochemical impedance spectroscopy (EIS) can be used as a transducer to evaluate biomolecular interactions [11] such as the affinity of the antibody/antigen in an immunosensor device. The electropolymerization of phenols and aromatic ethers has been investigated as a means of producing modified electrodes ⁎ Corresponding author at: Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Laboratório de Bioquímica e Biologia Molecular, Av. Amazonas S/N-Bloco 2E/ 237, CEP: 38400.902, Uberlândia, Minas Gerais, Brazil. Tel.: + 55 34 3218 2477, + 55 34 9993 0670 (mobile). E-mail addresses: [email protected] (T.V. da Silva Santos), [email protected] (R.R. Teixeira), [email protected] (D.L. Franco), [email protected] (J.M. Madurro), [email protected] (A.G. Brito-Madurro), [email protected], [email protected] (F.S. Espindola). 1 These authors contributed equally to this manuscript. 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2011.12.005
[12–14]. Electropolymerization often leads to electrode passivation with non-conductive films; however, unlike chemical polymerization, electropolymerization permits higher flexibility and control of the polymerization conditions [15]. Studies have indicated that monomers containing aromatic groups directly bonded to oxygen are easier to polymerize. Such polymers exhibit high reproducibility, and the obtained films exhibit good mechanical resistance, which results in greater stability of the modified electrode [12–14,16]. Recently, the preparation of electrodes coated with poly(3-hydroxyphenylacetic acid) [poly(3-HPA)] has been described by our group [17]. Those results showed that the polymer is conductive when prepared in an acid medium and that graphite electrodes coated with these polymers were much more efficient in immobilizing biomolecules than were non-coated graphite surfaces. In the present study, we report for the first time the incorporation of a polyclonal antibody in bioelectrodes based on poly(3-hydroxyphenylacetic acid) and its use for the detection of human salivary alpha-amylase. 2. Experiments 2.1. Chemicals All reagents used were analytical grade. Ultra-high-purity water (Millipore Milli-Q system) was used in the preparation of the solutions. Buffer components (CH3COOH, CH3COONa, Tris, EDTA, EGTA, NH4HCO3) and other chemicals (Q-Sepharose resin, human salivary alpha-amylase antibody, lysozyme, trypsin, Freund's complete and incomplete adjuvant, iodoacetamide, DTT, mercaptoethanol) were purchased from Sigma-Aldrich, USA (ACS purity). Monomer solutions
T.V. da Silva Santos et al. / Materials Science and Engineering C 32 (2012) 530–535
Fig. 1. Ion-exchange chromatography for production of an enriched fraction of salivary alpha-amylase. In A, the electrophoretic profile of whole saliva (WS), diluted supernatant (S) and excluded volume (V). In B, the Western blotting showing human salivary alpha-amylase immunodetection in WS, S and V. In C, the ion-exchange chromatography fraction densitometry for HSAf production. Significant differences (p b 0.001): *WS different from S, **V different from WS and S.
of 3-hydroxyphenylacetic acid were prepared in 0.5 mol·L− 1 HClO4 solution immediately before their use. All reagents were used as received. The experiments were conducted at room temperature (25 ± 1 °C).
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(mean 26, SD± 2.5 years of age) performed oral hygiene with toothpaste 2 h before sample collection and washed their mouth three times with distilled water immediately before collection. Saliva secretion was stimulated with paraffin, and collection was carried out at 16:00 h. The saliva samples were pooled and frozen at −20 °C for 48 h for protein precipitation, and they were later thawed at room temperature and centrifuged at 12,000g for 10 min at 20 °C [19]. The saliva supernatant samples were dialyzed and fractionated by ion-exchange chromatography based on the procedure of Gouveia et al. [20] as follows: the saliva supernatant was diluted (1:1 v/v) in a 50 mM Tris– HCl buffer (pH 8.0) that contained 10 mM EGTA and 10 mM EDTA. A glass column (9× 2 cm) packed with 63 mL of Q-Sepharose resin was used. The column was stabilized with 5 volumes of 25 mM Tris–HCI at pH 8.0 that contained 5 mM EGTA and 5 mM EDTA. A sample of 120 mL of diluted saliva was added to the column. The excluded volume was collected, dialyzed against 50 mM NH4HCO3 and finally lyophilized. Fractions were submitted to SDS-PAGE 1D and Western blotting. For immunoblotting analysis, the human salivary alpha-amylase antibody (anti-HSA) was used. For the purification of anti-HSAf, rabbits were immunized with 500 μg/mL of HSAf and Freund's complete adjuvant. Two booster doses of 250 μg/mL with Freund's incomplete adjuvant were administered at 2-week intervals. Blood was collected from the ear vein 1 week before the first dose and 15 days after each immunization. Serum titer was determined through ELISA. The polyclonal antibody anti-HSAf was purified through the immunoaffinity method [21], and the specificity of the antibody was verified by Western blotting. 2.4. Peptide mass fingerprinting (PMF)
2.2. Apparatus The SDS-PAGE 1D and Western blotting were performed in a system from Hoefer Pharmacia Biotech (San Francisco, USA). The protein identification using peptide mass fingerprinting was conducted in a Bruker Reflex IV mass spectrometer (Bremen, Germany). The electropolymerizations were performed in a three-compartment electrochemical cell connected to a model 420A potentiostat from CH Instruments (Austin, USA). The working electrode was graphite (99.9995%) from Alfa Aesar (Ward Hill, USA), in disk form, with a diameter of 6.18 mm. A platinum plate and electrodes of Ag/AgCl and KCl (3 M) were used as auxiliary and reference electrodes, respectively. EIS was performed in an Autolab electrochemical system (PGSTAT302N and FRA2 module) from Eco Chemie (Utrecht, The Netherlands) using potassium ferric/ferricyanide (5 mmol·L− 1) in potassium chloride solution (0.1 mol·L− 1). The frequency range was from 100 kHz to 10 Hz using the open-circuit potential system, +0.24 V. The voltage amplitude was 10 mV.
The polypeptides immunodetected through Western blotting with anti-HSA were stained in the gel and digested for further identification of the proteins. The method employed was based on the reduction of proteins with DTT, alkylation with iodoacetamide and digestion with trypsin. The yielded fragments were submitted to microchromatography using C18 Zip Tips (Millipore, Billerica, USA), followed by matrixassisted laser desorption ionization time of flight (MALDI-TOF) in a Bruker Reflex IV mass spectrometer. Known trypsin autolysis peaks and keratin contaminants were removed. The Mascot [22] and Profound [23] software packages were used to search for the PMF-identified proteins in the NCBInr database. No restrictions were made regarding the molecular mass or the taxonomy of the proteins. The fragment mass tolerance was b0.2 Da for MH + monoisotopic data, and the “experimental molecular mass” (gel) and “theoretical molecular mass” (database) were compared to verify the identification.
2.3. Preparation of human salivary alpha-amylase (HSAf) and polyclonal antibody (anti-HSAf)
2.5. Functionalization of graphite electrode surface with poly(3-HPA) and its modification with anti-HSAf
Saliva was collected by the spitting method described by Navazesh [18] with some modifications. Briefly, 10 healthy, non-smoking subjects
Graphite electrodes were modified with polymer derived from 3hydroxyphenylacetic acid according to Oliveira et al. [17]. The
Table 1 Identification by peptide mass fingerprinting (PMF-MALDI-TOF/TOF MS) of the polypeptides immunodetected with anti-HSA. Protein identification
Organism
Protein ID
Mascot score
Macthed peptides
Coverage (%)
pI (Theor.)
Mr (Theor.) kDa
Chain A, role of the mobile loop in the mechanism of human salivary amylase Chain A, structure solution and refinement of the recombinant human salivary amylase AMY1A protein Alpha-amylase 1 precursor Alpha-amylase Amylase, alpha 1A (salivary)
–
gi|15988376
267
27
49
6.21
56.171
–
gi|14719766
234
20
41
6.21
56.484
gi|47124258 gi|40254482 gi|178585 gi|10280622
229 205 189 152
22 26 22 20
44 47 42 37
8.82 6.47 6.32 6.64
56.859 58.415 58.398 58.300
Homo Homo Homo Homo
sapiens sapiens sapiens sapiens
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Fig. 2. In A, the SDS-PAGE 1D electrophoretic profile of polypeptides from the enriched fraction of human salivary alpha-amylase (HSAf), lysozyme (L), human whole saliva (HWS), rat whole saliva (RWS), rat cardiac muscle (RCM), honey (H) and blocking solution skim milk samples in different concentrations of 5 mg/mL (B5), 10 mg/mL (B10) and 15 mg/mL (B15). In B, immunoblotting, showing immunodetection of salivary alpha-amylase polypeptides from HSAf, HWS and RWS samples.
modified electrode was pre-treated by applying a potential of −0.2 V in an acetate buffer, pH 4.7, for 60 s. Antibody anti-HSAf (20 ng/μL) was diluted in the same buffer, added to the modified electrodes, incubated for 1 h at 25 °C, immersed for 30 s in an acetate buffer at pH 4.7 and dried with N2. 2.6. Antigen–antibody interaction on the modified electrode and interference study For the detection of the target (salivary amylase), an acetate buffer solution containing mercaptoethanol was pre-heated in the presence of the target (HSAf 60 ng/μL) at 98 °C for 5 min, immersed for 30 s in an acetate buffer and dried with N2. As a negative control, a buffer solution containing mercaptoethanol pre-heated in the absence of the target was prepared. The interferent study was performed using lysozyme, an enzyme present in the human saliva, under the same conditions described for the target human salivary amylase. 3. Results and discussion 3.1. Preparation of the enriched fraction of human salivary alpha-amylase (HSAf) Fig. 1A shows the electrophoretic profile of the saliva samples before and after fractioning through ion-exchange chromatography with chelating divalent cations. The 1D SDS-PAGE analysis reveals the presence of a number of polypeptides in whole saliva (WS) as well as in the diluted supernatant saliva (S) fractions. However, the excluded volume (V) is enriched with two polypeptides with a Mr similar to that of the alphaamylase. The presence of salivary alpha-amylase in these fractions was confirmed by Western blotting (Fig. 1B) and PMF analysis (Table 1). Immunoblotting analysis showed a cross-reaction of the antibody anti-HSA with the ~58 kDa polypeptides in the WS, S and V samples. Moreover, these polypeptides were later identified through PMF analysis as amylase isoforms with Mr between 56 and 58 kDa. This result is in agreement with previous proteomic studies that showed amylase isoforms with different molecular masses and isoelectric points [24–26]. Hirtz et al. [27] have identified, from a databank, amylase native forms with 56 kDa as well as 59 kDa isoforms; post-translational modifications were the most likely cause for the variation of Mr between the 50 and 70 kDa regions, as observed on SDS-PAGE 2D [28]. Finally, densitometric analysis (Fig. 1C, p b 0.01) proved that the production of HSAf by ion-
exchange chromatography, as observed in the column excluded volume, was effective. 3.2. Purification and characterization of the polyclonal antibody The polyclonal anti-HSAf antibody purified by immunoaffinity revealed high specific interaction with alpha-amylase, as shown in the assay with different proteins. In addition to the sample of HSAf, the immunoblotting assay was performed with human lysozyme (L), human whole saliva (HWS), rat whole saliva (RWS), rat cardiac muscle (RCM), honey (H) and blocking solution skim milk samples in different concentrations of 5 mg/mL (B5), 10 mg/mL (B10) and 15 mg/mL (B15). The electrophoretic profiles from the samples used are shown in Fig. 2A. Western blotting analysis revealed that polypeptides from the blocking solution did not interact with the anti-HSAf (Fig. 2B), which shows that the antibody and the other proteins from the skim milk used in the blocking solution did not cross-react during the purification process. An anti-HSAf interaction occurred only with polypeptides of ~ Mr 58 kDa from the HSAf, HWS and RWS samples. The cross-reaction with alpha-amylase present in rat saliva is a result of the high homology (85%) between the rat and human amylase sequences, as demonstrated by ClustalW analysis (Fig. 3). This result is consistent with the high homology found among the amino acid sequences within exon 4 of genes AMY 1 and AMY 2 of human amylase when compared to nine mammal species [29]. The high specificity of the interaction of the anti-HSAf with amylase isoforms supports its potential use in research and diagnostic methods. 3.3. Electrochemical impedance spectroscopy studies of the modified electrodes One of the most significant advantages of impedance detection in biosensors is that antibody–antigen binding can be directly detected. Fig. 4 shows the complex-plane plot (known as a Nyquist plot) that represents the impedance response of a graphite electrode modified with the film, film/antibody or film/antibody–antigen. The experimental curves show one semicircle combined with a short straight line that exhibits a ~ 45° slope in the high-frequency domain, followed by a complex behavior in the low-frequency domain. The results suggest that the system is kinetically and mass-transfer controlled in the high-frequency domain. Fig. 5 shows the equivalent circuit used to fit the experimental data.
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Fig. 3. Human salivary alpha-amylase and rat alpha-amylase identity analysis by ClustalW. The (*) indicates identity between both proteins.
The model for the morphology of the electroactive layer that emerges from the results supports the existence of two regions. The internal region, which is close to the graphite surface, is described by the Rct,1 and Q1 data. The external region, where the biomolecules are applied, is described by (Q2[Rct,2W]), where Rs is the resistance of the solution, Rct is the charge-transfer resistance, Q is the capacitance and W is the Warburg impedance. Electron transfer in the internal region is expected to be easier, which results in lower Rct,1 values when compared to the Rct,2 values (Table 2). The chi-squared values (χ 2) of the Kramers–Kronig relations are on the order of 10 − 2 to 10 − 3, which reveals the data quality. The solution resistance (Rs) centered
at 5.0 is reasonably constant given the electrolyte composition (Table 2). The modified electrode with poly(3-HPA) showed significant blocking behavior with respect to electron-transfer reactions of the ferricyanide/ ferrocyanide redox pair (2427 Ω cm2), which indicates electrostatic repulsion with the negatively charged carboxylate groups of the polymer [17]. This charge repulsion causes higher charge-transfer resistance. In the presence of the immobilized antibody-containing polymeric film, a decrease of the charge-transfer resistance (566 Ω cm2) onto the electrode surface was observed. This decrease can be attributed to the interaction between the negative charge of the polymer and the positive charge of
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T.V. da Silva Santos et al. / Materials Science and Engineering C 32 (2012) 530–535 Table 2 The fitting values of the equivalent circuit elements.
Rs/ (Ω cm2) Q1/ (mF cm− 2) n1 Rct,1/ (Ω cm2) W/ (Ω cm2 s−1/2) Q2/ (mF cm− 2) n2 Rct,2/ (Ω cm2) χ2
poly(3-HPA)
Poly(3-HPA)/Ab
poly(3-HPA)/Ab-Ag
4.95 (0.04) 0.18 (0.007) 0.81 (0.003) 86.7 (0.9) 0.01650 (0.00467) 11.10 (2.08) 0.72 (0.034) 2427 (75) 9.98 × 10− 3
5.00 (0.05) 0.21 (0.005) 0.79 (0.004) 144.9 (0.0) 0.00786 (0.00049) 31.54 (8.39) 1.00 (0.085) 566 (20) 2.11 × 10− 2
3.08 (0.02) 0.22 (0.004) 0.80 (0.002) 253.6 (2.9) 0.00766 (0.00057) 35.90 (8.03) 1.00 (0.053) 1345(38) 7.39 × 10− 3
Q is the capacitance, n1, n2 is the phase angle displacement, W is the Warburg impedance and Rct is the resistance of charge transfer. The error is indicated in brackets.
Fig. 4. Nyquist diagram for EIS measurements in 5 mmol·L− 1 K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol·L− 1 KCl solution recorded from a graphite electrode modified with: poly(3-HPA) (Δ); poly(3-HPA)/anti-HSAf (○) and poly(3-HPA)/anti-HSAf-HSAf (□). Eap = + 0.24 V; amplitude = 10 mV; frequency range = 100 kHz to 10 Hz. The continuous lines represent the fitting to the equivalent circuit. Inset: amplification of the high-frequency region.
the probe, which increases the electrostatic interaction after the isoelectric point (pI) values reached 6.1–6.5, and the assay was conducted at pH=4.7. Thus, the presence of a specific interaction between the antibody and the antigen provided a resistance increase (1345 Ω cm2) that increased the separation of the interaction antigen–antibody formed with the electrode surface because of the precipitation or agglutination caused by the antigen–antibody interaction. This precipitation or agglutination resulted in a decrease of the charge transfer. Comparison of the Q1 and Q2 values provides the following sequence: poly(3-HPA)/Ab-Ag> poly(3-HPA)/Ab > poly(3-HPA). The capacitance increase indicates that the antigen is immobilized onto the polymer surface and that its elements are in contact with the antibody. This conformational model is consistent with the Rct-values. Such results are in agreement with the studies of some authors who have verified modifications in the impedimetric systems according to the biomolecule immobilizations over the electrode surface [30,31]. Wu et al. [32], using the impedimetric system for the detection of enrofloxacin, verified that the semicircle diameter illustrated in the Nyquist diagram increased when the antigen–antibody interaction occurred.
3.4. Interferent study
the performance and validation of the bioelectrode and will be carried out with saliva samples.
4. Conclusion The production of an enriched fraction of salivary alpha-amylase (HSAf) by ion-exchange chromatography was effective, being identified through PMF analysis as containing some amylase isoforms. The anti-HSAf purified by immunoaffinity revealed high specific interaction with human salivary alpha-amylase. A graphite electrode modified with poly(3-hydroxyphenylacetic acid) was effective for the immobilization of anti-HSAf. The interaction between anti-HSAf and human salivary amylase, as analyzed through electrochemical impedance spectroscopy, showed a significant modification in the Nyquist plot upon addition of the complementary target, with an increase in the charge transference resistance. An assay using a salivary interferent indicated the specificity of the bioelectrode based on poly(3-HPA) and anti-HSAf. The combination of the graphite electrode with poly(3-hydroxyphenylacetic acid) is a promising strategy for antigen immobilization and other biological recognition elements, presenting the potential for the production of a label-free biochip.
Acknowledgments This work was supported by grants from FAPEMIG to FSE and by the CNPq fellowship to TVS. We thank Mr. Abílio Borghi for an initial manuscript language review and Lucas Franco Ferreira for his technical and scientific assistance.
To investigate the specificity of the bioelectrode, we utilized lysozyme, a protein present in saliva that is secreted by the parotid, submandibular and sublingual glands [33]. Lysozyme was prepared under the same conditions as the specific target (HSAf), and the system was analyzed through EIS (Fig. 6). Fig. 6 shows that the bioelectrode discriminates the salivary amylase of another salivary compound (lysozyme) because the graphic profile is similar for the modified electrode in the presence or absence of lysozyme. These results indicate that lysozyme does not interact with the antibody (anti-HSAf), revealing the specificity of the bioelectrode. Future studies are planned to evaluate
Fig. 5. Equivalent electrical circuit used to fit the experimental impedance data. Rs: the resistance of the solution; Rct: charge transfer resistances; Q: capacitance; W: Warburg impedance.
Fig. 6. Nyquist diagram from EIS obtained in 5 mmol·L− 1 K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol·L− 1 KCl solution recorded from poly(3-HPA)/antibody (△) and poly(3-HPA)/antibody–lysozyme (○). Eap = + 0.24 V; amplitude = 10 mV; frequency range = 100 KHz to 10 Hz. The continuous lines represent the fitting to the equivalent circuit. Inset: amplification of the high-frequency region.
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Tatiane Vanessa da Silva Santos. Received her B.S. degree in Biological Sciences from the Educational Foundation of Ituiutaba in 2006 and her M.Sc. degree in Genetics and Biochemistry from the Federal University of Uberlandia in 2010 with a fully funded scholarship from the National Council for the Scientific and Technological Development. Currently works on the production and purification of antibodies for the development of immunosensors and also collaborates with research on salivary biomarkers of physical and psychological stress.
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Renata Roland Teixeira. Received her B.S. degree in Biological Sciences in 2005, her M.Sc. degree in Genetics and Biochemistry in 2007 both from the Federal University of Uberlandia. She is also technician in Clinical Pathology and Biodiagnostics from the Technical School of Health since 2004. Currently she is a fourth year Ph.D. student in Genetics and Biochemistry at the Federal University of Uberlandia. She works as a technician in the Laboratory of Biochemistry and Molecular Biology at the Federal University of Uberlandia. She works on proteomics of apiculture products and salivary biomarkers for the diagnostic of diseases. Diego Leoni Franco. Received his B.S. and M.Sc. degrees in Chemistry from the Federal University of Uberlandia in 2005 and 2007, respectively. He works on electropolymerization, microbalances, polymeric films, modified electrodes and biosensors.
João Marcos Madurro. Received his B.S. degree in Chemistry from the University of Sao Paulo in 1982, and in Industrial Chemistry from the University of Ribeirao Preto in 1984. He obtained his M.Sc. degree in Chemistry from the Federal University of Sao Carlos in 1987 and his Ph.D. degree in Organic Chemistry from the University of Sao Paulo in 1998. He also worked as a postdoctoral research associate at the University of Lisbon in 2003. His current research interests include the development of new materials and electrodes focusing on biosensors and electrooxidation of organic compounds.
Ana Graci Brito Madurro. Received her B.S. degree in chemistry in 1992, and her M.Sc. degree in Genetics and Biochemistry in 1997, both from the Federal University of Uberlandia. She obtained her Ph.D. degree in Biochemistry and Immunology from the University of Sao Paulo in 2001. She worked as a postdoctoral research associate in Biochemistry and Molecular Biology in Lisboa in 2003. She works as a professor at the Institute of Genetics and Biochemistry of the Federal University of Uberlandia and focuses her research interests in the development of biosensors for diagnosis of human diseases and physical chemistry of proteins. Foued Salmen Espindola. Received his B.S. degree in Biological Sciences in 1978 and Pharmacy and Biochemistry in 1980 from the Federal University of Minas Gerais (UFMG). In 1983, he obtained his M.Sc. degree in Biochemistry and Immunology and his Ph.D. degree in Biological Sciences in 1991 from the faculty of medicine of the University of Sao Paulo. He worked as a postdoctoral research associate at Yale University. Currently, He works as a professor at the Federal University of Uberlandia, Head of the “Rede Fitocerrado” and associate of “Pro Biotec Indústria e Comércio de Kits de Diagnóstico Para Saúde Ltda”.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Bioelectrode for detection of human salivary amylase Tatiane Vanessa da Silva Santos a, 1, Renata Roland Teixeira a, 1, Diego Leoni Franco b, João Marcos Madurro b, Ana Graci Brito-Madurro a, Foued Salmen Espindola a,⁎ a b
Institute of Genetics and Biochemistry, Federal University of Uberlandia, Uberlandia, MG, Brazil Institute of Chemistry, Federal University of Uberlandia, Uberlandia, MG, Brazil
a r t i c l e
i n f o
Article history: Received 17 March 2011 Received in revised form 12 September 2011 Accepted 7 December 2011 Available online 14 December 2011 Keywords: Salivary alpha-amylase 3-Hydroxyphenylacetic acid Bioelectrode Biosensor Polymeric film
a b s t r a c t The aim of this study is the development of a novel bioelectrode based on the immobilization of a specific antibody for salivary amylase onto a graphite electrode modified with poly(3-hydroxyphenylacetic) acid. For this purpose, human salivary alpha-amylase was applied to an immunoaffinity-purified anti-alpha-amylase polyclonal antibody. The bioelectrode was incubated with salivary amylase or lysozyme (interfering salivary), and the interaction between the antibody and the enzymes was analyzed through electrochemical impedance spectroscopy. The results indicated the specificity of the bioelectrode to salivary alpha-amylase. Therefore, the combination of the graphite electrode with poly(3-hydroxyphenylacetic acid) appears to be a promising strategy for antigen immobilization and other biological recognition elements, thus presenting the potential for the production of a label-free biochip. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Salivary alpha-amylase is the most abundant enzyme in saliva; it is primarily involved in the digestion of starch and acts as a receptor for the adhesion of bacterial flora, as well as assisting in the formation of dental plaque [1]. Several studies have evaluated the differences in activity and concentration of this protein to use it as a potential biomarker for diagnosis of some oral [2] and systemic diseases [3–5]. Several research groups have also evaluated different stress conditions through salivary alpha-amylase activity using spectrometric methods [6,7] and biosensors [8,9]. Immunosensors are interesting for clinical applications because they offer a number of potential advantages over conventional assay techniques; the detection is fast and the system can be automated [10]. Electrochemical impedance spectroscopy (EIS) can be used as a transducer to evaluate biomolecular interactions [11] such as the affinity of the antibody/antigen in an immunosensor device. The electropolymerization of phenols and aromatic ethers has been investigated as a means of producing modified electrodes ⁎ Corresponding author at: Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Laboratório de Bioquímica e Biologia Molecular, Av. Amazonas S/N-Bloco 2E/ 237, CEP: 38400.902, Uberlândia, Minas Gerais, Brazil. Tel.: + 55 34 3218 2477, + 55 34 9993 0670 (mobile). E-mail addresses: [email protected] (T.V. da Silva Santos), [email protected] (R.R. Teixeira), [email protected] (D.L. Franco), [email protected] (J.M. Madurro), [email protected] (A.G. Brito-Madurro), [email protected], [email protected] (F.S. Espindola). 1 These authors contributed equally to this manuscript. 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2011.12.005
[12–14]. Electropolymerization often leads to electrode passivation with non-conductive films; however, unlike chemical polymerization, electropolymerization permits higher flexibility and control of the polymerization conditions [15]. Studies have indicated that monomers containing aromatic groups directly bonded to oxygen are easier to polymerize. Such polymers exhibit high reproducibility, and the obtained films exhibit good mechanical resistance, which results in greater stability of the modified electrode [12–14,16]. Recently, the preparation of electrodes coated with poly(3-hydroxyphenylacetic acid) [poly(3-HPA)] has been described by our group [17]. Those results showed that the polymer is conductive when prepared in an acid medium and that graphite electrodes coated with these polymers were much more efficient in immobilizing biomolecules than were non-coated graphite surfaces. In the present study, we report for the first time the incorporation of a polyclonal antibody in bioelectrodes based on poly(3-hydroxyphenylacetic acid) and its use for the detection of human salivary alpha-amylase. 2. Experiments 2.1. Chemicals All reagents used were analytical grade. Ultra-high-purity water (Millipore Milli-Q system) was used in the preparation of the solutions. Buffer components (CH3COOH, CH3COONa, Tris, EDTA, EGTA, NH4HCO3) and other chemicals (Q-Sepharose resin, human salivary alpha-amylase antibody, lysozyme, trypsin, Freund's complete and incomplete adjuvant, iodoacetamide, DTT, mercaptoethanol) were purchased from Sigma-Aldrich, USA (ACS purity). Monomer solutions
T.V. da Silva Santos et al. / Materials Science and Engineering C 32 (2012) 530–535
Fig. 1. Ion-exchange chromatography for production of an enriched fraction of salivary alpha-amylase. In A, the electrophoretic profile of whole saliva (WS), diluted supernatant (S) and excluded volume (V). In B, the Western blotting showing human salivary alpha-amylase immunodetection in WS, S and V. In C, the ion-exchange chromatography fraction densitometry for HSAf production. Significant differences (p b 0.001): *WS different from S, **V different from WS and S.
of 3-hydroxyphenylacetic acid were prepared in 0.5 mol·L− 1 HClO4 solution immediately before their use. All reagents were used as received. The experiments were conducted at room temperature (25 ± 1 °C).
531
(mean 26, SD± 2.5 years of age) performed oral hygiene with toothpaste 2 h before sample collection and washed their mouth three times with distilled water immediately before collection. Saliva secretion was stimulated with paraffin, and collection was carried out at 16:00 h. The saliva samples were pooled and frozen at −20 °C for 48 h for protein precipitation, and they were later thawed at room temperature and centrifuged at 12,000g for 10 min at 20 °C [19]. The saliva supernatant samples were dialyzed and fractionated by ion-exchange chromatography based on the procedure of Gouveia et al. [20] as follows: the saliva supernatant was diluted (1:1 v/v) in a 50 mM Tris– HCl buffer (pH 8.0) that contained 10 mM EGTA and 10 mM EDTA. A glass column (9× 2 cm) packed with 63 mL of Q-Sepharose resin was used. The column was stabilized with 5 volumes of 25 mM Tris–HCI at pH 8.0 that contained 5 mM EGTA and 5 mM EDTA. A sample of 120 mL of diluted saliva was added to the column. The excluded volume was collected, dialyzed against 50 mM NH4HCO3 and finally lyophilized. Fractions were submitted to SDS-PAGE 1D and Western blotting. For immunoblotting analysis, the human salivary alpha-amylase antibody (anti-HSA) was used. For the purification of anti-HSAf, rabbits were immunized with 500 μg/mL of HSAf and Freund's complete adjuvant. Two booster doses of 250 μg/mL with Freund's incomplete adjuvant were administered at 2-week intervals. Blood was collected from the ear vein 1 week before the first dose and 15 days after each immunization. Serum titer was determined through ELISA. The polyclonal antibody anti-HSAf was purified through the immunoaffinity method [21], and the specificity of the antibody was verified by Western blotting. 2.4. Peptide mass fingerprinting (PMF)
2.2. Apparatus The SDS-PAGE 1D and Western blotting were performed in a system from Hoefer Pharmacia Biotech (San Francisco, USA). The protein identification using peptide mass fingerprinting was conducted in a Bruker Reflex IV mass spectrometer (Bremen, Germany). The electropolymerizations were performed in a three-compartment electrochemical cell connected to a model 420A potentiostat from CH Instruments (Austin, USA). The working electrode was graphite (99.9995%) from Alfa Aesar (Ward Hill, USA), in disk form, with a diameter of 6.18 mm. A platinum plate and electrodes of Ag/AgCl and KCl (3 M) were used as auxiliary and reference electrodes, respectively. EIS was performed in an Autolab electrochemical system (PGSTAT302N and FRA2 module) from Eco Chemie (Utrecht, The Netherlands) using potassium ferric/ferricyanide (5 mmol·L− 1) in potassium chloride solution (0.1 mol·L− 1). The frequency range was from 100 kHz to 10 Hz using the open-circuit potential system, +0.24 V. The voltage amplitude was 10 mV.
The polypeptides immunodetected through Western blotting with anti-HSA were stained in the gel and digested for further identification of the proteins. The method employed was based on the reduction of proteins with DTT, alkylation with iodoacetamide and digestion with trypsin. The yielded fragments were submitted to microchromatography using C18 Zip Tips (Millipore, Billerica, USA), followed by matrixassisted laser desorption ionization time of flight (MALDI-TOF) in a Bruker Reflex IV mass spectrometer. Known trypsin autolysis peaks and keratin contaminants were removed. The Mascot [22] and Profound [23] software packages were used to search for the PMF-identified proteins in the NCBInr database. No restrictions were made regarding the molecular mass or the taxonomy of the proteins. The fragment mass tolerance was b0.2 Da for MH + monoisotopic data, and the “experimental molecular mass” (gel) and “theoretical molecular mass” (database) were compared to verify the identification.
2.3. Preparation of human salivary alpha-amylase (HSAf) and polyclonal antibody (anti-HSAf)
2.5. Functionalization of graphite electrode surface with poly(3-HPA) and its modification with anti-HSAf
Saliva was collected by the spitting method described by Navazesh [18] with some modifications. Briefly, 10 healthy, non-smoking subjects
Graphite electrodes were modified with polymer derived from 3hydroxyphenylacetic acid according to Oliveira et al. [17]. The
Table 1 Identification by peptide mass fingerprinting (PMF-MALDI-TOF/TOF MS) of the polypeptides immunodetected with anti-HSA. Protein identification
Organism
Protein ID
Mascot score
Macthed peptides
Coverage (%)
pI (Theor.)
Mr (Theor.) kDa
Chain A, role of the mobile loop in the mechanism of human salivary amylase Chain A, structure solution and refinement of the recombinant human salivary amylase AMY1A protein Alpha-amylase 1 precursor Alpha-amylase Amylase, alpha 1A (salivary)
–
gi|15988376
267
27
49
6.21
56.171
–
gi|14719766
234
20
41
6.21
56.484
gi|47124258 gi|40254482 gi|178585 gi|10280622
229 205 189 152
22 26 22 20
44 47 42 37
8.82 6.47 6.32 6.64
56.859 58.415 58.398 58.300
Homo Homo Homo Homo
sapiens sapiens sapiens sapiens
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Fig. 2. In A, the SDS-PAGE 1D electrophoretic profile of polypeptides from the enriched fraction of human salivary alpha-amylase (HSAf), lysozyme (L), human whole saliva (HWS), rat whole saliva (RWS), rat cardiac muscle (RCM), honey (H) and blocking solution skim milk samples in different concentrations of 5 mg/mL (B5), 10 mg/mL (B10) and 15 mg/mL (B15). In B, immunoblotting, showing immunodetection of salivary alpha-amylase polypeptides from HSAf, HWS and RWS samples.
modified electrode was pre-treated by applying a potential of −0.2 V in an acetate buffer, pH 4.7, for 60 s. Antibody anti-HSAf (20 ng/μL) was diluted in the same buffer, added to the modified electrodes, incubated for 1 h at 25 °C, immersed for 30 s in an acetate buffer at pH 4.7 and dried with N2. 2.6. Antigen–antibody interaction on the modified electrode and interference study For the detection of the target (salivary amylase), an acetate buffer solution containing mercaptoethanol was pre-heated in the presence of the target (HSAf 60 ng/μL) at 98 °C for 5 min, immersed for 30 s in an acetate buffer and dried with N2. As a negative control, a buffer solution containing mercaptoethanol pre-heated in the absence of the target was prepared. The interferent study was performed using lysozyme, an enzyme present in the human saliva, under the same conditions described for the target human salivary amylase. 3. Results and discussion 3.1. Preparation of the enriched fraction of human salivary alpha-amylase (HSAf) Fig. 1A shows the electrophoretic profile of the saliva samples before and after fractioning through ion-exchange chromatography with chelating divalent cations. The 1D SDS-PAGE analysis reveals the presence of a number of polypeptides in whole saliva (WS) as well as in the diluted supernatant saliva (S) fractions. However, the excluded volume (V) is enriched with two polypeptides with a Mr similar to that of the alphaamylase. The presence of salivary alpha-amylase in these fractions was confirmed by Western blotting (Fig. 1B) and PMF analysis (Table 1). Immunoblotting analysis showed a cross-reaction of the antibody anti-HSA with the ~58 kDa polypeptides in the WS, S and V samples. Moreover, these polypeptides were later identified through PMF analysis as amylase isoforms with Mr between 56 and 58 kDa. This result is in agreement with previous proteomic studies that showed amylase isoforms with different molecular masses and isoelectric points [24–26]. Hirtz et al. [27] have identified, from a databank, amylase native forms with 56 kDa as well as 59 kDa isoforms; post-translational modifications were the most likely cause for the variation of Mr between the 50 and 70 kDa regions, as observed on SDS-PAGE 2D [28]. Finally, densitometric analysis (Fig. 1C, p b 0.01) proved that the production of HSAf by ion-
exchange chromatography, as observed in the column excluded volume, was effective. 3.2. Purification and characterization of the polyclonal antibody The polyclonal anti-HSAf antibody purified by immunoaffinity revealed high specific interaction with alpha-amylase, as shown in the assay with different proteins. In addition to the sample of HSAf, the immunoblotting assay was performed with human lysozyme (L), human whole saliva (HWS), rat whole saliva (RWS), rat cardiac muscle (RCM), honey (H) and blocking solution skim milk samples in different concentrations of 5 mg/mL (B5), 10 mg/mL (B10) and 15 mg/mL (B15). The electrophoretic profiles from the samples used are shown in Fig. 2A. Western blotting analysis revealed that polypeptides from the blocking solution did not interact with the anti-HSAf (Fig. 2B), which shows that the antibody and the other proteins from the skim milk used in the blocking solution did not cross-react during the purification process. An anti-HSAf interaction occurred only with polypeptides of ~ Mr 58 kDa from the HSAf, HWS and RWS samples. The cross-reaction with alpha-amylase present in rat saliva is a result of the high homology (85%) between the rat and human amylase sequences, as demonstrated by ClustalW analysis (Fig. 3). This result is consistent with the high homology found among the amino acid sequences within exon 4 of genes AMY 1 and AMY 2 of human amylase when compared to nine mammal species [29]. The high specificity of the interaction of the anti-HSAf with amylase isoforms supports its potential use in research and diagnostic methods. 3.3. Electrochemical impedance spectroscopy studies of the modified electrodes One of the most significant advantages of impedance detection in biosensors is that antibody–antigen binding can be directly detected. Fig. 4 shows the complex-plane plot (known as a Nyquist plot) that represents the impedance response of a graphite electrode modified with the film, film/antibody or film/antibody–antigen. The experimental curves show one semicircle combined with a short straight line that exhibits a ~ 45° slope in the high-frequency domain, followed by a complex behavior in the low-frequency domain. The results suggest that the system is kinetically and mass-transfer controlled in the high-frequency domain. Fig. 5 shows the equivalent circuit used to fit the experimental data.
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Fig. 3. Human salivary alpha-amylase and rat alpha-amylase identity analysis by ClustalW. The (*) indicates identity between both proteins.
The model for the morphology of the electroactive layer that emerges from the results supports the existence of two regions. The internal region, which is close to the graphite surface, is described by the Rct,1 and Q1 data. The external region, where the biomolecules are applied, is described by (Q2[Rct,2W]), where Rs is the resistance of the solution, Rct is the charge-transfer resistance, Q is the capacitance and W is the Warburg impedance. Electron transfer in the internal region is expected to be easier, which results in lower Rct,1 values when compared to the Rct,2 values (Table 2). The chi-squared values (χ 2) of the Kramers–Kronig relations are on the order of 10 − 2 to 10 − 3, which reveals the data quality. The solution resistance (Rs) centered
at 5.0 is reasonably constant given the electrolyte composition (Table 2). The modified electrode with poly(3-HPA) showed significant blocking behavior with respect to electron-transfer reactions of the ferricyanide/ ferrocyanide redox pair (2427 Ω cm2), which indicates electrostatic repulsion with the negatively charged carboxylate groups of the polymer [17]. This charge repulsion causes higher charge-transfer resistance. In the presence of the immobilized antibody-containing polymeric film, a decrease of the charge-transfer resistance (566 Ω cm2) onto the electrode surface was observed. This decrease can be attributed to the interaction between the negative charge of the polymer and the positive charge of
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T.V. da Silva Santos et al. / Materials Science and Engineering C 32 (2012) 530–535 Table 2 The fitting values of the equivalent circuit elements.
Rs/ (Ω cm2) Q1/ (mF cm− 2) n1 Rct,1/ (Ω cm2) W/ (Ω cm2 s−1/2) Q2/ (mF cm− 2) n2 Rct,2/ (Ω cm2) χ2
poly(3-HPA)
Poly(3-HPA)/Ab
poly(3-HPA)/Ab-Ag
4.95 (0.04) 0.18 (0.007) 0.81 (0.003) 86.7 (0.9) 0.01650 (0.00467) 11.10 (2.08) 0.72 (0.034) 2427 (75) 9.98 × 10− 3
5.00 (0.05) 0.21 (0.005) 0.79 (0.004) 144.9 (0.0) 0.00786 (0.00049) 31.54 (8.39) 1.00 (0.085) 566 (20) 2.11 × 10− 2
3.08 (0.02) 0.22 (0.004) 0.80 (0.002) 253.6 (2.9) 0.00766 (0.00057) 35.90 (8.03) 1.00 (0.053) 1345(38) 7.39 × 10− 3
Q is the capacitance, n1, n2 is the phase angle displacement, W is the Warburg impedance and Rct is the resistance of charge transfer. The error is indicated in brackets.
Fig. 4. Nyquist diagram for EIS measurements in 5 mmol·L− 1 K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol·L− 1 KCl solution recorded from a graphite electrode modified with: poly(3-HPA) (Δ); poly(3-HPA)/anti-HSAf (○) and poly(3-HPA)/anti-HSAf-HSAf (□). Eap = + 0.24 V; amplitude = 10 mV; frequency range = 100 kHz to 10 Hz. The continuous lines represent the fitting to the equivalent circuit. Inset: amplification of the high-frequency region.
the probe, which increases the electrostatic interaction after the isoelectric point (pI) values reached 6.1–6.5, and the assay was conducted at pH=4.7. Thus, the presence of a specific interaction between the antibody and the antigen provided a resistance increase (1345 Ω cm2) that increased the separation of the interaction antigen–antibody formed with the electrode surface because of the precipitation or agglutination caused by the antigen–antibody interaction. This precipitation or agglutination resulted in a decrease of the charge transfer. Comparison of the Q1 and Q2 values provides the following sequence: poly(3-HPA)/Ab-Ag> poly(3-HPA)/Ab > poly(3-HPA). The capacitance increase indicates that the antigen is immobilized onto the polymer surface and that its elements are in contact with the antibody. This conformational model is consistent with the Rct-values. Such results are in agreement with the studies of some authors who have verified modifications in the impedimetric systems according to the biomolecule immobilizations over the electrode surface [30,31]. Wu et al. [32], using the impedimetric system for the detection of enrofloxacin, verified that the semicircle diameter illustrated in the Nyquist diagram increased when the antigen–antibody interaction occurred.
3.4. Interferent study
the performance and validation of the bioelectrode and will be carried out with saliva samples.
4. Conclusion The production of an enriched fraction of salivary alpha-amylase (HSAf) by ion-exchange chromatography was effective, being identified through PMF analysis as containing some amylase isoforms. The anti-HSAf purified by immunoaffinity revealed high specific interaction with human salivary alpha-amylase. A graphite electrode modified with poly(3-hydroxyphenylacetic acid) was effective for the immobilization of anti-HSAf. The interaction between anti-HSAf and human salivary amylase, as analyzed through electrochemical impedance spectroscopy, showed a significant modification in the Nyquist plot upon addition of the complementary target, with an increase in the charge transference resistance. An assay using a salivary interferent indicated the specificity of the bioelectrode based on poly(3-HPA) and anti-HSAf. The combination of the graphite electrode with poly(3-hydroxyphenylacetic acid) is a promising strategy for antigen immobilization and other biological recognition elements, presenting the potential for the production of a label-free biochip.
Acknowledgments This work was supported by grants from FAPEMIG to FSE and by the CNPq fellowship to TVS. We thank Mr. Abílio Borghi for an initial manuscript language review and Lucas Franco Ferreira for his technical and scientific assistance.
To investigate the specificity of the bioelectrode, we utilized lysozyme, a protein present in saliva that is secreted by the parotid, submandibular and sublingual glands [33]. Lysozyme was prepared under the same conditions as the specific target (HSAf), and the system was analyzed through EIS (Fig. 6). Fig. 6 shows that the bioelectrode discriminates the salivary amylase of another salivary compound (lysozyme) because the graphic profile is similar for the modified electrode in the presence or absence of lysozyme. These results indicate that lysozyme does not interact with the antibody (anti-HSAf), revealing the specificity of the bioelectrode. Future studies are planned to evaluate
Fig. 5. Equivalent electrical circuit used to fit the experimental impedance data. Rs: the resistance of the solution; Rct: charge transfer resistances; Q: capacitance; W: Warburg impedance.
Fig. 6. Nyquist diagram from EIS obtained in 5 mmol·L− 1 K3Fe(CN)6/K4Fe(CN)6 containing 0.1 mol·L− 1 KCl solution recorded from poly(3-HPA)/antibody (△) and poly(3-HPA)/antibody–lysozyme (○). Eap = + 0.24 V; amplitude = 10 mV; frequency range = 100 KHz to 10 Hz. The continuous lines represent the fitting to the equivalent circuit. Inset: amplification of the high-frequency region.
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Tatiane Vanessa da Silva Santos. Received her B.S. degree in Biological Sciences from the Educational Foundation of Ituiutaba in 2006 and her M.Sc. degree in Genetics and Biochemistry from the Federal University of Uberlandia in 2010 with a fully funded scholarship from the National Council for the Scientific and Technological Development. Currently works on the production and purification of antibodies for the development of immunosensors and also collaborates with research on salivary biomarkers of physical and psychological stress.
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Renata Roland Teixeira. Received her B.S. degree in Biological Sciences in 2005, her M.Sc. degree in Genetics and Biochemistry in 2007 both from the Federal University of Uberlandia. She is also technician in Clinical Pathology and Biodiagnostics from the Technical School of Health since 2004. Currently she is a fourth year Ph.D. student in Genetics and Biochemistry at the Federal University of Uberlandia. She works as a technician in the Laboratory of Biochemistry and Molecular Biology at the Federal University of Uberlandia. She works on proteomics of apiculture products and salivary biomarkers for the diagnostic of diseases. Diego Leoni Franco. Received his B.S. and M.Sc. degrees in Chemistry from the Federal University of Uberlandia in 2005 and 2007, respectively. He works on electropolymerization, microbalances, polymeric films, modified electrodes and biosensors.
João Marcos Madurro. Received his B.S. degree in Chemistry from the University of Sao Paulo in 1982, and in Industrial Chemistry from the University of Ribeirao Preto in 1984. He obtained his M.Sc. degree in Chemistry from the Federal University of Sao Carlos in 1987 and his Ph.D. degree in Organic Chemistry from the University of Sao Paulo in 1998. He also worked as a postdoctoral research associate at the University of Lisbon in 2003. His current research interests include the development of new materials and electrodes focusing on biosensors and electrooxidation of organic compounds.
Ana Graci Brito Madurro. Received her B.S. degree in chemistry in 1992, and her M.Sc. degree in Genetics and Biochemistry in 1997, both from the Federal University of Uberlandia. She obtained her Ph.D. degree in Biochemistry and Immunology from the University of Sao Paulo in 2001. She worked as a postdoctoral research associate in Biochemistry and Molecular Biology in Lisboa in 2003. She works as a professor at the Institute of Genetics and Biochemistry of the Federal University of Uberlandia and focuses her research interests in the development of biosensors for diagnosis of human diseases and physical chemistry of proteins. Foued Salmen Espindola. Received his B.S. degree in Biological Sciences in 1978 and Pharmacy and Biochemistry in 1980 from the Federal University of Minas Gerais (UFMG). In 1983, he obtained his M.Sc. degree in Biochemistry and Immunology and his Ph.D. degree in Biological Sciences in 1991 from the faculty of medicine of the University of Sao Paulo. He worked as a postdoctoral research associate at Yale University. Currently, He works as a professor at the Federal University of Uberlandia, Head of the “Rede Fitocerrado” and associate of “Pro Biotec Indústria e Comércio de Kits de Diagnóstico Para Saúde Ltda”.