Conference-ICSFS-14-Self Assemblage of Beta Sheeted Structures on Solid State Film for Biosensors

Based on modification of beta-sheeted protein structures in biosynthesis stage selfassemblage of 3D structures on solid state surfaces is investigated. A set of hybrid proteins containing the abeta40 peptide domain are constructed and characterized. The extended biomolecular structures are immobilised on the solid surfaces. Self assembled hybrid structures on the surface are obtained by deposition of the biomolecular conglomerates from buffer solutions. The surfaces of mica, SiO2, SnO and In2O3 are covered with the biomolecular layer. Formation of hybrid structures containing beta-sheeted fibrils is dependent on immobilisation period and origin of the solid surface. Morphology, mechanical and electrical properties are described by scanning probe microscope. Stabile fibrils of diameter about 2 nm and length of about 100-1000 nm are detected for lysozyme while Abeta40 peptide base proteins and TrxAbeta40 mutants result in specific structures. The biomolecular layers were described by analytical model based on digital representation of the surface profile data and analysis of the power spectral density function. [DOI: 10.1380/ejssnt.2009.491]


I. INTRODUCTION
Self assemblage of biomolecular structures on the surfaces of solids can be adapted for various purposes including catalysis, molecular electronics and sensors.Growth of two-and three-dimensional structures with specific functional properties offers unique approaches in modification of solid surfaces and development of unusual hybrid systems properties of which depend on processes in the constituents and in the interfaces.Understanding of technological details resulting in functioning hybrid system and description of characteristics defining parameters of the potential devices are the key part of development, improvement and exploitation of such technologies [1].
Combination of biological functions with electrical, magnetic and optical properties of solids is frequently implemented through self assembled monolayers of complex molecules [2].Various complex molecules with integrated functional groups are constructed and integrated on the surfaces of metals or semiconductors [3,4].The surface complexes are typically obtained after self arrangement of organic molecules on the solid surface with sites containing metal ions.Construction of the complex supramolecular objects are know being achievable in different manner.One of the approaches biomolecules are self assembled onto a particular substrate surface and then metal ions are introduced into the organic structure.Opposite approach starting with deposition of metal ions is also successfully used for obtaining hybrid structures (see [2,4] and references in these reports).
In this paper, we report fabrication and investigation of the hybrid structures obtained by immobilisation of already synthesised supramolecular structures on pure and modifies surfaces.Beta sheeted protein complex struc-tures were used for modification of the solid surfaces.Protein based fibrils and hybrid proteins were the biological objects integrated in the surface structures on insulating mica and SiO 2 as well as on semiconducting SnO 2 and In 2 O 3 surfaces.Layers consisting of separated objects and networks were formed and investigated.

A. Samples and Sample Preparation
A set of hybrid proteins containing the abeta40 (Aβ40) peptide domain and dimeric glucose dehydrogenase (GDH) or thioredoxin (Trx) have been constructed.Hen egg lysozyme was fibrilized for the reference experiments.

Expression and purification of fused Trx-Aβ40 protein
E. coli BL21(DE3) cells were transformed with pET3atrx-Aβ40 (donation of Dr. A. Olofsson).A single colony was used to inoculate 5 ml of NB medium containing 50 µg/ml carbenicillin.The culture was shaken at 30 • C (180 rpm) overnight and then was used to inoculate 1 l of NB medium supplemented with carbenicillin.The cells were grown at 30 • C until OD 600 reached 0.8, then cooled to 24 • C, 0.4 mM of isopropyl β-D-thiogalactoside (IPTG) was added for induction of the protein expression and the culture incubated overnight.Cells harvested by centrifugation (3000×g, 20 min, 4 • C) were washed and resuspended in 50 mM Tris-HCl, 100 mM NaCl, pH 8.5.The cells were disrupted by sonication (22 kHz) on ice and cell debris was removed by centrifugation (10,000×g, 30 min, 4 • C).The soluble fraction obtained after centrifugation was applied to HiTrap chelating column (GE Healthcare) according to the manufacturer's instructions.Fractions containing Trx-Aβ40 were pooled and dialyzed against 20 mM Tris-HCl, pH 8.5.

Construction of expression plasmid, expression and purification of GDH-Aβ40
The expression plasmid pT15abeco1 harbouring the GDH-Aβ40 fusion was constructed by amplifying a DNA fragment containing the Aβ40 gene from pET3atrx-Aβ40 DNA using PCR with these primers: forward, 5' CGCCATGGAGCTCGACGCTGAATTCCGT-CACG; reverse, 5' GCCGGATCCTCTACGTAAACAA-CACCACCAACCATC.The product was digested with Ecl136II and Eco105I restriction enzymes and ligated into the pPT15 plasmid (GDH gene Patent No WO2004099399) cut with the Eco47III and treated with calf intestinal phosphatase.The plasmid obtained into competent E. coli DH5α cells were transformed and selected on NB plates containing ampicillin.Positive clones were confirmed by DNA sequence analysis.
DH5α strain transformed with pT15abeco1 plasmid was grown in NB medium with 50 µg/ml carbenicillin at 30 • C until optical density (A 280nm ) reached 2 and protein expression was induced with 0.4 µg/ml anhydrotetracycline.The cells were maintained at 20 • C overnight and harvested by centrifugation (3000×g, 20 min, 4 • C).Harvested cells were washed and resuspended in 50 mM Tris-HCl, pH 8.0.Cells were disrupted by sonication.Cell debris was pelleted by centrifugation (10,000×g, 20 min, 4 • C).The soluble fraction was applied to a CM column (GE Healthcare) equilibrated with 50 mM Tris-HCl, pH 8.0 and fused protein was eluted with NaCl gradient (0-500 mM).Fractions containing GDH-Aβ40 were pooled and dialyzed against 20 mM ammonium carbonate (Fluka) buffer, pH 7.5 before freeze-drying.Protein expression and purification was monitored by SDS-PAGE using 15-17% acrylamide gels.After electrophoresis, the gels were stained with Coomassie Blue.

Fibril formation
10 mg/ml of Trx-Aβ40 was dissolved in PBS buffer, pH 7.5, containing 0.01% sodium azide and incubated at 37 • C for 3-5 days.To the solution of GDH-Aβ40 Aβ40 protein was added for the better fibrilisation (the final concentration were 1 mg/ml of GDH-Aβ40 and 2 mg/ml Aβ40).The other conditions were the same as for Trx-Aβ40.

Thioflavine T (ThT) binding assay
Fluorescence emission spectra of ThT, excited at 450 nm, were recorded between 460 and 600 nm on a PerkinElmer precisely LS55 luminescence spectrometer using excitation and emission bandwidths of 5 nm.Fibril formation were monitored by adding 10 µl aliquots from proteins solutions to 1 ml of a 10 µM ThT solution [7].

Fabrication of layers
The samples for the surface analysis were fabricated by deposition of biomolecular layers from colloidal solutions.Two solid substrates were used or the deposition of the layers.The basic substrate was typically standard mica substrate with very smooth surface roughness of which was characterised by variation in z-dimension less than about 1-2 nm.On these substrates a solution with biomolecules was dropped.The test substrate was placed on the wet basic substrate and the couple of substrates was kept still in laboratory atmosphere with relative humidity about 30% at T = 293 K for fixed period measured in minutes (immobilisation time).After that the test substrate was lifted from the basic substrate.Both of the substrates were rinsed in distilled water and dried in laboratory atmosphere for about 24 hours before experiments.Standard mica and Si (n-Si (100) resistance 0.5 Ω•cm with SiO 2 ) were used for non-modified substrates.Substrates with modified surfaces were prepared by deposition of SnO

Structure of the sample surfaces was visualised by SPM.
The images of the surfaces were obtained by SPM D3100 / Nanoscope IVa (Veeco, Digital Instruments).Standard contact and non-contact modes were used for description of topography and more sophisticated features of the sample surfaces [8].Combined modes were used for mapping of mechanical and electrical properties of the hybrid structures during one scan.
Topography of the surfaces was recorded by the contact and non-contact (Tapping Mode in the D3100) modes.In addition to this type of data, a phase shift between the actuating signal and oscillations of cantilever is measured during the same scan.Based on these measurements the phase lag distribution over the surfaces was obtained.
The surface topology of samples was analysed by processing digitized profiles of the scans.The analysis was based on approaches described by other authors (e.g.[9]).For this the power spectral density (PSD) function was derived from the SPM surface profile data by a built-in Nanoscope IVa function.

III. RESULTS AND DISCUSSIONS
A. Hybrid proteins

Expression of fused proteins
Trx-Aβ40 and GDH-Aβ40 were successfully expressed in E. coli cells.SDS-PAGE analysis revealed a strong inducible protein bands (18 kDa and 50 kDa for Trxfusion and GDH-fusion, respectively (data not shown)) in the cell-free extracts.A typical purification resulted in an almost homogeneous recombinant Trx-Aβ40 protein as judged by SDS-PAGE (Fig. 1, A) with a yield of 30 mg of the pure protein from 1 liter of the culture.Approximately 40 mg of the homogeneous protein from 1 liter of the bacterial culture was obtained in the case of GDH-Aβ40 (Fig. 2, B).The GDH-Aβ40 hybrid retained enzymatic activity but four times lower (approx.500 u/mg protein) comparing with the native GDH from the bacterial strain PT15.

Formation of fibrils
In order to induce the formation of the amyloid fibrils by the hybrid proteins physiological conditions have been chosen.After two days of incubation ThT was found to bind the ordered β-sheet structures in the proteins samples, resulting in the increase in its fluorescence intensity compared to the spectrum of the dye free in solution (Fig. 3).After 2 weeks of incubation gel-like consistence solution was formed in the case of GDH-Aβ40.This is characteristic indication of the fibril formation, too.
FIG. 2: Changes in fluorescence spectra of thioflavin T in the presence of the fused proteins.Thioflavin T (10 µM final concentration) was incubated with 0.01% of fused proteins for 3 min before measurement.Emission spectra of ThT were recorded (excitation at 450 nm) in the presence of TRX-Aβ40 (curve 1), GDH-Aβ40 (curve 2) and in the absence of the protein (curve 3).

B. SPM imaging of the surfaces
Self assemblage of extended biomolecular structures was dependent on the immobilisation time even for extremely stable lysozyme fibrils.Separate fibrils nd networks of fibrils were obtained only for comparatively short immobilisation time less than about 20-30 minutes under the conditions defined in section II.Typical images of distinguishable fibrils on mica surfaces are illustrated in Fig. 3. Longer immobilisation time was favourable for collection of fibrils into comparatively large agglomerates.Emerging agglomerates can be seen even in Figs.3(a) and 3(b).After comparatively long immobilisation time (about 50 minutes) the surfaces were typically covered by almost continuous layer of tightly packed ball like objects.Dimensions of these objects varied from about 100 nm to about 1 µm.
The density of the fibrils effectively depended on the origin of the solid surface.Electrically insulating surfaces of mica and SiO 2 were more favourable for self assemblage of fibril based structures while SnO and In 2 O 3 surfaces were covered with widely spread components.More detailed discussions about the solid surfaces will be presented in section C.
Self assemblage of hybrid proteins into extended structures was less noticeable compared to lysozyme fibrils.In general, the structures based on hybrid proteins were rarely found on the surfaces of tested samples and were originally constructed.Couple of distinctive supramolecular structures are illustrated in Fig. 4. In Fig. 4(a), GDH-Aβ40 based layer was scanned by the SPM while in Fig. 4(b) the SPM image displays unique structure self arranged of TRX-Aβ40.It must be noted here that granular like structures were obtained for most of the hybrid protein based samples in present study.Most of these structures were characterised by irregular arrange- ment of ball like components.The layers of immobilised GDH-Aβ40 structures contained linear objects that are tightly surrounded by accidentally distributed elements (see Fig. 4(a)).
Comparatively thick and short fibrils were detected on the surfaces after immobilisation of TRX-Aβ40 based structures.Diameter of typical fibril in Fig. 4(a) is about 7×10 nm.Diameter varies along the body of the fibril.It must also be noted here that comparatively large balls of diameter about 600 nm were also detected on these surfaces even after comparatively short immobilisation time (about 15 minutes).

C. Characteristics of the surface structures
Mechanical and electrical properties were analysed in the obtained structures.It was found that transfer of electrical charge between the substrate and the tip depends on the deformation of the hybrid structures.Specific spectra of the tunnelling current were obtained measuring dependence of the current on the force with which the tip is pressed to the surface.In present stage of study, the dependences are hardly reproducible and additional investigations are required.
Quantitative analysis of the surface images obtained by SPM scanning was based on processing of digitized profiles of the scans.In this analysis, the surfaces were described by parameters of the power spectral density (PSD) function derived from the AFM surface profile data.The analytical model of the PSD was supposed consisting of a few terms as it was shown in [9].The PSD analytical model includes the mathematical term describing the overall roughness contribution from the substrate (dominated by fractals) and pure film.PSD of the substrates with spatial frequencies f mostly follows a fractal model, which obeys the inverse power law of f [9].The PSD function of pure film can be described using the k-correlation model also called the ABC model [9].Based on this model, the PSD function is described by formula where  Most of the samples in this study were characterised by the parameters of the ABC model.Deviation from the model was detected for the samples which contained extended biomolecular structures.Typical results of the model analysis illustrated in Fig. 5.In this figure, the surface digital representation was obtained for the layer composed of immobilised lysozyme fibrils on mica substrate.Two samples are compared on Fig. 5.One sample was characterised by comparatively low density of fibrils (1) while another surface (2) was covered with comparatively high density of fibrils.A deviation from linear fall of the PSD function in this scale is obtained.The deviation is much more distinguishable for the high density of fibrils.In spite of this, the PSD function for sample with low density of fibrils was close to the ABC model calculation with the parameters A = 1.3 × 10 5 , B = 0.053, C = 1.0.The deviation of the experimental PSD function can be described by including the terms related to appearing superstructures on the surfaces [9].These structures can be related with practically regular distribution of the fibrils on the solid surface at the densities of fibrils when individ-ual components are clearly distinguished one from anther.It have to be noted here the samples that were covered by continuous layer after long immobilisation time were characterised by the PSD function compatible with the pure ABC model.
The model parameters were used for characterisation of the structures on various substrates used in this work.Since the density of fibrils was comparatively low on these surfaces the ABC model calculation practically matched the experimental PSD functions.The parameters obtained from these calculations are summarised in Table I.It can be seen that comparatively lower magnitudes of the parameters C were obtained for the samples on mica and SnO surfaces.Since the lower C magnitudes typically correspond to smoother films it can be supposed that the coverage of mica and SnO surfaces is more continuous than the layer on SiO 2 and In 2 O 3 .Considering the fact that B is equal to the correlation length it seems reasonable to conclude that the same type of fibrils (lysozyme) are self arranged in more tight structure on the surfaces of mica substrate than on other types of substrates.

IV. CONCLUSIONS
Hybrid proteins were constructed by original technology using prokaryotic system based on Escherichia coli.Specific procedures were performed for fibrilisation of the proteins in the solution.Presence of supramolecular structures in the solutions was verified by photoluminescence spectra.It was demonstrated that extended biomolecular structures can be immobilised on the solid surfaces under common conditions.Based on the SPM measurements stabile fibrils of diameter about 2 nm and length of about 1-10 µm were detected for hen egg lysozyme.Self arranged systems containing hybrid proteins preferably are spherical objects on the solid surfaces.The Trx-Aβ40 hybrid protein formed thick (diameter about 7-10 nm) and short (about 1-2 µm) objects.It is proved that morphology of self arranged combined systems and density of fibrils depends on the origin of solid state surfaces and can effectively be varied by duration of immobilization.The structures were described by the ABC model acceptable for quantitative comparison of the resulting combined structures on solid surfaces.It is also recognized that explicit explanation of the experimental facts requires additional studies.
and In 2 O 3 thin film on the Si/SiO 2 .The modifying films were grown by a dc-magnetron sputtering.
http://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) e-Journal of Surface Science and Nanotechnology A, B, C being the function parameters and the value of C is greater than 2. It was known that this model satisfactorily describes random rough surfaces over large length scales.FIG.5: Digital analysis of the SPM image obtained for lysozyme layer on mica surface.Points are experimental results for low (1) and high (2) density of fibrils.Lines are calculation according to the model (details are in the text).

TABLE I :
Features of biomolecular layer of lysozyme fibrils immobilised on solid surfaces described by parameters of the ABC model.