Immunosensor Based on Immobilization of Antigenic Peptide NS5A-1

Feb 17, 2013 - Copyright © 2013 American Chemical Society. *E-mail address: [email protected]. Tel: +55 (16) 3301 9768. Cite this:Langmuir 29, 1...
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Immunosensor Based on Immobilization of Antigenic Peptide NS5A‑1 from HCV and Silk Fibroin in Nanostructured Films Marli L. Moraes,* Lais R. Lima, Robson R. Silva, Mauricio Cavicchioli, and Sidney J. L. Ribeiro Institute of Chemistry, São Paulo State University, UNESP, CP355-Araraquara-SP, 14801-970 Brazil ABSTRACT: The peptide NS5A-1 (PPLLESWKDPDYVPPWHG), derived from hepatitis C virus (HCV) NS5A protein, was immobilized into layer-by-layer (LbL) silk fibroin (SF) films. Deposition was monitored by UV−vis absorption measurements at each bilayer deposited. The interaction SF/ peptide film induced secondary structure in NS5A-1 as indicated by fluorescence and circular dichroism (CD) measurements. Voltammetric sensor (SF/NS5A-1) properties were observed when the composite film was tested in the presence of anti-HCV. The peptide−silk fibroin interaction studied here showed new architectures for immunosensors based on antigenic peptides and SF as a suitable immobilization matrix. In this work, the structural organization of silk fibroin in thin films was investigated together with the evaluation of viability as immobilization matrix toward biomolecules, in this case, an antigenic peptide. Immobilization of antigenic peptides in nanostructured films has been promising for the development of highly specific immunosensors.26,27 The molecular recognition of peptide by antibodies leads to selectivity of assays based on immune principles without the necessity to use the complex molecules, such as protein or virus.27,28 In this way, the peptide NS5A-1 (PPLLESWKDPDYVPPWHG) derived from hepatitis C virus (HCV) NS5A protein was immobilized into layer-by-layer (LbL) films together with silk fibroin (SF). SF and SF/HS5A-1 LbL films were monitored and characterized by UV−vis, fluorescence, and circular dichroism (CD) spectroscopies and the antigen−antibody interaction was observed by electrochemistry measurements.

1. INTRODUCTION Immobilization of active biomolecules, such as enzymes, nucleic acids, and antibodies, on solid surfaces is a great challenge for biosensor application. Therefore, structural organization or activity of these biomolecules is essential for good performance of such devices.1,2 In order to improve the stability and sensitivity of the immobilized biomolecule, a variety of methods have been investigated, for example, self-assembled monolayers (SAM),3 Langmuir−Blodgett (LB)4 films, and layer-by-layer (LbL) films.5 The LbL method has been reported as suitable for biological molecule immobilization due to its versatility and the fact that the adsorption process is conducted under mild conditions.6,7 The assembly of the LbL film is based on intermolecular interactions between the materials,5,6 so a great deal of interest is dedicated to finding suitable materials to alternate with the biomolecule which can retain its specific biological function. In recent years, there has been a significant increase of interest in using natural polymers as immobilization matrix for biomolecules, such as chitin, 8 chitosan, 9−11 and silk fibroin.12−20 Silk fibroin (SF) is a biopolymer extracted from natural silk which exhibits excellent mechanical and structural properties,21 while also being biocompatible.17 SF protein produced by Bombyx mori consists of two chain polypeptides, one heavy (H) and the other light (L). Heavy chain is composed of 12 repetitive domains Gly-X (X is Ala in 65%, Ser in 23%, and Tyr in 9% of the repeats)22 linked by 11 amorphous domains characterized by tryptophan and charged residues, which are absent from the Gly-X-domains.23 Silk fibroin in the crystalline form shows a predominantly β-sheet structure. However, three structural models are known: (i) Silk I is a water-soluble structure exhibiting mainly random coiled chains, which are not well-understood. (ii) Silk II is insoluble displaying β-sheet conformation. (iii) Silk III is an unstable structure observed at the water−air interface.23−25 © 2013 American Chemical Society

2. MATERIALS AND METHODS 2.1. Materials. Synthetic peptide NS5A-1 was purchased from BioSynthesis Inc. The sequence PPLLESWKDPDYVPPWHG was purified and isolated by HPLC with purity >90% and sequence confirmed by mass-spectral analysis, as described by Bio-Synthesis Inc. The NS5A-1 solutions were prepared with purified water from Milli-Q system at concentration of 0.5 mg·L−1. The antibody specific to HCV (anti-HCV) was obtained from Santa Cruz Biotechnology Inc. Each vial contained 100 μg in 1.0 mL−1 which was diluted in phosphate buffered saline (PBS) in concentrations 1:100 to 1:10 000. Silk fibroin (SF) was extracted from the cocoons of the Bombyx mori silkworm silk supplied by Bratac S.A., Brazil. Ten grams of cocoons was boiled during 30 min in 2 L of the 0.02 mol·L−1 Na2CO3 solution in order to remove the sericin. For each 10 g of the silk yarn, 100 mL of CaCl2/CH3CH2OH/H2O (1:2:8) solution was added and Received: November 6, 2012 Revised: February 5, 2013 Published: February 17, 2013 3829

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the solution heated at 60 °C for dissolution. This solution was then dialyzed against Milli-Q water using a cellulose acetate membrane at room temperature for 48 h. After this procedure, SF was centrifuged three times at 20 000 rpm for 30 min at 5 °C to remove impurities and aggregates.29 The concentration of SF in solution was 4% in weight. For SF film preparation and CD measurements, aqueous solutions at concentration of 0.1% and 0.025% (weight/volume) were used, respectively. 2.2. Self-Assembly Films. SF and SF/NS5A-1 films were deposited onto quartz substrates previously treated with a 1:1:5 solution of NH4OH:H2O2:H2O for 10 min at 70 °C, and then with a 1:1:6 solution of HCl:H2O2:H2O for 10 min at 70 °C. The SF deposition process was carried out by immersing the substrate in the SF solution for 10 min. SF/NS5A-1 LbL film was assembled by immersing the substrate in the SF solution for 10 min and in NS5A-1 solution for 10 min. The adsorption time of 10 min in the preparation of the biomolecule layer, such as proteins and peptides, has been previously determined.30 After each step of deposition, the film was washed with Milli-Q water to remove poorly adsorbed molecules and dried gently with flowing nitrogen. By repeating this procedure, the desired number of SF or SF/NS5A-1 layers could be obtained. All the experiments were performed at room temperature. The buildup of the multilayers was monitored at each deposition step by UV−vis and fluorescence spectroscopy. UV−vis measurements were performed using a Cary 50 UV−vis spectrophotometer from Varian, Inc. 2.3. Circular Dichroism and Fluorescence Spectroscopy. Circular dichroism (CD) spectra of SF and peptide in aqueous solution were collected with a quartz cell of 1 mm optical path length. CD spectra of 10 layers SF and SF/NS5A-1 LbL films were performed directly over the films deposited on quartz substrates. In this case, the optical path is given by the film thickness. Measurements were performed on a J-815 Circular Dichroism Spectrometer (Jasco Inc., Tokyo, Japan), with the bandwidth of 1 nm, a response time of 0.5 s, and scanning speed 100 nm/min. CD spectra were obtained by averaging eight scans. Fluorescence measurements of SF and NS5A-1 were performed with excitation at 280 nm. At this wavelength, both contributions of the tyrosine (Tyr) and tryptophan (Trp) residues of the SF and NS5A-1 could be observed. Fluorescence spectra were obtained using a RF-5301PC Spectrofluorophotometer from Shimadzu Scientific Instruments, Inc., Columbia, MD. All the CD and fluorescence spectra were collected at room temperature. 2.4. Electrochemistry Measurements. One- and five-layer SF and SF/NS5A-1 LbL films were built on screen-printed carbon electrodes with Flow-Cell for screen-printed electrodes, both purchased from Dropsens (Oviedo, Spain). In this case, SF and NS5A-1 solutions were injected in the Flow-Cell for 10 min each, and in each step of deposition the film was washed with Milli-Q water. Voltammograms were obtained using a Bipotentiostat/Galvanostat μStat 400 with DropView software from DropSens (Oviedo, Spain) and PBS was used as electrolytic solution. The scan speed was 0.05 V/ s and potential range was −0.6 to 0.6 V. Before measurements, 100 μL aliquots of anti-HCV at concentrations of 1 to 0.01 μg.mL−1 (1:100 to 1:10 000) or pure PBS were added on films for 5 min, then washed with PBS.

Figure 1. UV−vis spectra of SF films containing different numbers of layers. Inset: increase in the absorption at 280 nm for a SF film as a function of the number of deposited layers.

Figure 2. UV−vis spectra of SF/NS5A-1 films containing different numbers of bilayers. Inset: increase in the absorption at 280 nm for a SF/NS5A-1 film as a function of the number of deposited bilayers.

the SF protein and NS5A-1 peptide, so the wavelength at 280 nm was used to monitor the film growth, since Tyr absorbs about 275 nm and Trp about 280 nm. 3.2. Characterization of the Silk Fibroin and NS5A-1 Films. Figure 3 shows CD spectra of silk fibroin in solution and film. SF in solution (0.025% (w/v)) exhibited a minimum at around 197 nm, which is characteristic of a random coil conformation. For SF adsorbed onto a solid substrate, the βsheet conformation was clearly formed with a minimum at about 217 nm. The conformational change can be attributed to molecular rearrangement due to the immobilization in films. The molecular chains of the SF can interact quickly and strongly with one another, and then, the chains can be rearranged in a regular array to some extent, which changed the conformation from a random coil to a β-sheet structure.31 Conformational changes could also be induced during the drying process itself. The dehydration process induces hydrogen bonding between the protein chains and consequent β-sheet arrangement in the protein after removal of the water molecules32 Yang and collaborators25 showed by CD spectra that SF had its β-sheet structure induced by ethanol. When ethanol was progressively added, the maximum degree of negative ellipticity

3. RESULTS AND DISCUSSION 3.1. Growth of the Silk Fibroin and SF/NS5A-1 LbL Films. Figures 1 and 2 show the absorption spectra for SF and SF/NS5A-1 film layers, respectively. The insets show linear increase (R2 = 0.98) of the maximum absorption at 280 nm with the number of deposited layers. This linear growth indicates that the same amount of material was probably adsorbed in each deposition step. The growth of SF and SF/ NS5A-1 films, also monitored by fluorescence spectroscopy, showed similar behavior with emission maximum at around 310 and 320 nm, respectively (results not shown here). Tyrosine and tryptophan residues are present in the primary structure of 3830

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Figure 4 shows the emission spectra for SF in solution and self-assembly films excited at 280 nm. The emission maxima for

Figure 4. Fluorescence spectra for SF in water (solid line) and tenlayer SF films (dashed line).

Figure 3. Circular dichroism (CD) spectra for SF in aqueous solution (solid line) and ten-layer SF LbL films (dashed line).

SF in solution is around 306 nm and a distinct contribution at 346 nm originated from Tyr and Trp residues, respectively. The emission maximum for SF films was observed at 310 nm and the contribution of Trp could not be observed. The red shift of about 4 nm in the emission maximum suggests that a change has occurred in the structure of SF, from random coil to βsheet, i.e., the Tyr moves from the interior of the protein to a more polar environment. Moreover, it is estimated that Tyr residues are exposed to the solvent in β-sheet structure.25,34 Peptide activity, i.e., recognition antigen−antibody, is related to its conformation at the binding site. The process of peptide− receptor recognition requires a preferred conformation or an induced fit.35 Thus, a preferential conformation is a characteristic feature required for its recognition. However, peptides have no preferred conformation in solution, as seen for NS5A-1 peptide in water. The CD spectrum of NS5A-1 peptide in water, shown in Figure 5, indicates the predominance of a random structure, with a minimum at 199 nm. The bioactive conformation of a peptide may depend on the environment;36 for example, conformational changes of peptides can be induced through its interaction with biomembranes. In this way, NS5-A-1 was immobilized on SF films to induce its bioactive conformation. The CD spectrum of NS5A-1 immobilized on LbL films onto SF displayed a minimum at 216 nm that can be attributed to the sum of the SF β-sheet and α-helix structures and NS5A-1 turns. The secondary structure of the NS5A protein is not well-defined,37 but the secondary structure predicts α-helix and NS5A-1 turns for the 323−340 nm region.38 The deconvolution of these spectra using Selcon, Contin, and CDSSTR methods39 gave 16% helical elements, 8% β structures, 29% turns, and 47% disordered elements for NS5A-1 in water, and 42% helical

at 217 nm was enhanced, which indicates the transition of SF from random coil to β-sheet. Nam and Park investigated the secondary structure of the silk fibroin solution prepared with various types of alcohol.31 β-sheet structure was formed when silk fibroin was prepared in methanol or ethanol, while the random coil is maintained when SF was prepared in 1-octanol. A mixed type of conformations with β-sheet, random-coil, and α-helix structures were observed in 1-butanol. This effect was attributed to the hydrophilic/hydrophobic character of the alcohol. The structure of cast SF films was investigated in ref 33 as well. The CD spectrum of the SF film showed two minima at around 205 and 220 nm attributed to a random coil conformation containing small amounts of β-sheet and helix structures. The β-sheet structure was only observed when poly(ethylene glycol) (PEG) was added to SF solution and a cast PEG-SF film was formed. Thus, it was necessary to introduce PEG into SF in order to obtain the β-sheet structure. SF thin films reported here showed that the β-sheet structure could be formed without using organic solvents or any materials for inducing structures. Fluorescence spectroscopy is widely used in the study of protein structure. The aromatic amino acids, Trp, Tyr, and phenylalanine (Phe), offer intrinsic fluorescent probes of protein conformation and dynamic and intermolecular interactions. Among these three amino acids, Trp is the most popular probe for investigating the protein structural change and interaction with other molecules because the fluorescence of the indole chromophore is highly sensitive to its environment.25,34 SF protein is composed of 9% Tyr and 0.25% Trp repeats with alternating hydrophobic and hydrophilic side groups along the chain. 3831

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Figure 5. Circular dichroism (CD) spectra for NS5A-1 in water (solid line) and ten-bilayer SF/NS5A-1 LbL films (dashed line). Inset: Secondary structure prediction for NS5A-1.38

Figure 6. Fluorescence spectra of the SF and NS5A-1 in water, tenlayer SF and SF/NS5A-1 LbL films, and SF+NS5A-1 in water for excitation at 280 nm.

elements, 26% β structures, 16% turns, and 16% disordered forms for SF/NS5A-1 LbL film. In this way, SF induced the organization of the peptide structure and the SF/NS5A-1 film spectrum could be attributed to the sum of the SF and NS5A-1 secondary structures. NS5A-1 is a peptide with 10 amino acid residues (PPLLESWKDPDYVPPWHG) containing two Trp and one Tyr. The fluorescence spectrum for NS5A-1 in solution shows an emission maximum at 351 nm consistent with Trp residues exposed to water. The maximum is observed at 320 nm for SF/ NS5A-1 LbL film suggesting that Trp is buried in the structure interior, as shown in Figure 6. For the mixture of SF and NS5A1 in solution, the emission spectrum shows the contribution of two bands attributed to the unstructured SF and NS5A-1. The fluorescence results corroborate with results obtained with CD. For both SF and NS5-1 in solution, random coil conformation was observed. When immobilized in LbL films, βsheet structures appeared. It is to be noted that this effect was only observed for nanostructured films, since no conformational change was observed for the mixture of SF and NS5-1 in solution. Thus, two conclusions can be drawn: (i) SF displays β-sheet organized structure when in nanostructrured films; (ii) SF film induced secondary structure for NS5A-1. Therefore, SF films can be the way for new architectures of biofilms, i.e., SF can be used as a suitable immobilization matrix of biomolecules. 3.3. Application of the SF/NS5A-1 Film as Immunosensor. SF and SF/NS5A-1 LbL films were assembled onto carbon screen-printed electrodes containing 1 and 5 layers, as shown schematically in Figure 7. Figure 8 shows the cyclic voltammograms obtained in a PBS buffer in the presence and in the absence of anti-HCV for five-layer SF (Figure 8A) and SF/ NS5A-1 (Figure 8B) LbL film. The voltammograms obtained with SF films in the presence of PBS and PBS+anti-HCV

showed insignificant changes in the profile with no increase in the current. In contrast, for the immunosensor made with SF/ NS5A-1 LbL films, the voltammogram exhibited a current increase of about 9 μA when the anti-HCV (1 μg.mL−1) antibody was added, as shown in Figure 8B. For one-layer SF and NS5A-1 films, the same results could be observed, but with a current increase of 7 μA (1 μg.mL−1). The negative current increase at low potentials can be related to reduction species generated by interaction between anti-HCV antibody and the NS5A-1 peptide. The mechanism of antigen−antibody interaction is not clear, but it is known that it involves the formation of multiple noncovalent bonds, such as van der Waals interactions, hydrogen bonds, salt bridges, and hydrophobic force, between the antigen and the amino acids of the binding site.35 The electrocatalytic effect appears as a gradual increase in the anodic peak current versus the antibody concentration, as can be seen in Figure 9. The molecular recognition of antigenic peptide−antibody is only possible when the peptide is structured,28,35 corroborating with results of CD and fluorescence spectroscopy. In previous work,28 antigenic peptide p17-1 from HIV-1 was immobilized on synthetic polymer, poly(allylamine hydrochloride) (PAH), and its secondary structure was not induced. This lack of structuring was found to be important, for no molecular recognition takes place for p17-1 toward the anti-p17 antibodies. In contrast, the LbL films with p17-1 encapsulated into liposomes, in which the peptide had its structure preserved, were suitable to produce electrochemical immunosensors. Figure 9 shows the response of one-bilayer SF and NS5A-1 immunosensor when the anti-HCV concentration was varied from 0 to 1 μg.mL−1. The current decrease showed a linearity range from 0 to 0.2 μg.mL−1 (dilution range from 0 to 1:5000), and above this concentration, there was a tendency to 3832

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Figure 7. Schematic representation of five-layer LbL film of SF/NS5A-1 (1) and SF (2) assembled onto carbon screen-printed electrodes.

Figure 8. Cyclic voltammograms for a five-layer LbL film of SF (A) and SF/NS5A-1 (B) in PBS buffer in the absence (black curve) and presence (red curve) of anti-HCV.

1:500, and 1:30−1:3000, respectively. The immunosensor described here was able to detect the antibody using dilution range from 1:100 to 1:10 000 with sensitivity of 28 μA.μg−1.mL. The sensitivity of the immunosensor was determined by the slope of linear response versus anti-HVC concentration of 0 to 0.2 μg.mL−1 (0 to 1:500).

4. CONCLUSIONS We have demonstrated the feasibility of immunosensors based on silk fibroin nanostructured films prepared by the layer-bylayer t ech nique. The antigenic peptide NS5A-1 (PPLLESWKDPDYVPPWHG), derived from hepatitis C virus (HCV) NS5A protein, was immobilized into the films in order to prepare a highly specific immunosensor. UV−vis absorption measurements indicated that the peptide was proportionally adsorbed onto SF in each bilayer deposited. Fluorescence and circular dichroism (CD) spectra indicated that the interaction of SF/peptide film induced secondary structure in NS5A-1. Highly sensitive amperometric sensor (SF/NS5A-1) properties were observed when the composite film was tested in the presence of anti-HCV. The peptide−silk fibroin interaction can lead to new architectures of

Figure 9. Analytical curves for one-bilayer SF and NS5A-1 films in the presence of different concentrations of anti-HCV.

saturation, indicating that all sites of biorecognition have been occupied. Immunosensors, such as Western Blotting, immunefluorescence, and solid-phase ELISA, are recommended by Santa Cruz Biotechnology, dilution range 1:100−1:1000, 1:50− 3833

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immunosensors based on antigenic peptide and SF as a suitable immobilization matrix.



AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. Tel: +55 (16) 3301 9768. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by FAPESP, CNPq, and NanobiotecCAPES network (Brazil). REFERENCES

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