AFM characterization of protein net formation on a fibrous medium

AFM characterization of protein net formation on a fibrous medium

O.B.G. Assis1^*, D.C. Vieira^1,2 and R. Bernardes-Filho¹

¹Embrapa Agricultural Instrumentation, CP 741, 13560-970, São Carlos - SP,

Brazil, Fax [phone])

E-mail: [e-mail]

²PPG Interunidades Ciência e Engenharia de Materiais, Universidade de São Paulo

(USP), CP 369, 13560-970 São Carlos - SP, Brazil

(Received: November 3, 1999 ; Accepted: March 17, 2000)

Abstract - Lysozyme protein net is set on a glass fiber support using the self-assembly technique. Enzymatic film formation is followed by surface imaging via atomic force microscopy (AFM). Change in roughness as a function of deposition time is used as an indirect indicator of film formation. The objective was to form a protein film that would have no effect on the permeability of the medium, aiming at its application as a bioactive membrane or reactor suitable for bacteria and chemical interactions in aqueous media.

Keywords: thin-film, protein net, self-assembly, atomic force microscopy

INTRODUCTION

Due to its simplicity, thin-film formation by the self-assembly (SA) technique has been extensively studied in different types of biomolecules of an amphiphilic nature, which have a tendency to be adsorbed on different supports (Decher et al., 1992). The basis of the SA technique is the electrostatic attraction between oppositely charged functional interfaces and interacting molecules in an aqueous medium, which makes it an ideal process for the fast production of organic film in a controlled fashion.

A widespread organic compound suitable for structural production by the SA technique is the globular protein lysozyme (Lvov et al., 1995; Robeson & Tilton, 1996; Arai & Norde, 1990). Lysozyme, also known as muramidase, is a natural thermally stable enzyme found in colostrum, hen egg whites and human nasal mucus and tears. It is characterized by multiples and complex structures and presents a distribution of charges on the molecular surface with well defined hydrophobic and hydrophilic regions, depicted in a model constructed by Kayushina et al., 1996. To become suitable for spontaneous lysozyme bonding, the support has to undergo chemical treatment in order to create superficial negative charges, thereby attracting the positive dominantly charged portion of the protein.

In this work, an interconnected net of lysozyme is formed on a fibrous permeable medium by the SA technique and characterized by atomic force microscopy. Interest in creating an immobilized enzymatic net is due to the antimicrobial properties of the lysozyme (Bailey & Ollis, 1996; Imoto et al., 1972) and the possibility of studying changes in the support features using AFM.

MATERIALS AND METHODS

An aqueous solution of lysozyme from hen egg-white protein (Sigma) was prepared (as delivered) at a concentration of 10^-4 M (pH ~ 6.2) using ultrapure deionised water. Glass is a suitable cheap material for the SA substratum, owing to the ease with which superficial charges are generated after appropriate chemical treatment (Ferreira et al.,1994; Assis et al.,1997). Consequently, the permeable support used in this work was prepared with commercially available glass fiber with a thickness of 40-70 µm. The fibers underwent chemical hydrophilization treatment, which consisted of a series of surface cleaning procedures using ultrasonic baths and warmed acid solution washes, as described elsewhere (Borato et al., 1997; Herrmann, 1999). This is an activation treatment that assures a clean surface, enhancing the negative charges of the glass and consequently the hydrophilicity index of the support (Ferreira, 1994).

The activated material was tightly packed inside a column with an internal diameter of one and a height of one and a half inches, which was then filled with ultrapure water. The protein solution was then poured into the column where the solid surfaces were immersed for 10 minutes and then rinsed in distilled water. The fundamental process used for producing the SA protein coating was described previously by Raposo and Oliveira Jr. 1998.

Fiber samples were randomly drawn from the medium in the column, in an as-activated state and after 5, 10 and 12 minutes of surfactant immersion. They were dried and then surface scanned by atomic force microscopy (TopoMetrix Discover) in contact mode in order to evaluate superficial features.

RESULTS AND DISCUSSION

Since deposition proceeds under static conditions, enzyme film formation by electrostatic adsorption is expected to occur predominantly on the fiber surfaces immediately below the surfactant solution, i.e., on the surfaces on which deposition may be favored by gravitational action. Deposition on horizontal surfaces is claimed to be propitious for the formation of stable films where the globular protein shows a tendency to change conformation when adsorbed (Blomberg et al., 1994). However the disadvantage of such a condition is the lack of control of uniformity in the molecular protein array (Ulman, 1991). In the packed fiber medium, the longitudinal directions are randomly distributed throughout the volume, and it is not precise to refer to a horizontal deposition in this situation, but rather to the dissimilar rates of aggregation along the circular faces of the fibers.

The amount of adsorption is highly dependent on the concentration of enzyme diluted in the surfactant, as demonstrated by Wahlgren et al., 1995. Consequently the kinetics of perpendicular lysozyme assembling on glass substratum had been previously monitored by ultraviolet (UV) spectroscopy. It was observed that the absorbance increases rapidly in the initial stages of adsorption. A first-order process (a direct dependence on surfactant concentration) with a characteristic time of 5-8 minutes for a complete formation of a monolayer on flat substratum was reported by Assis et al. (1998) and Herrmann (1999). Based on these results, the 12-minute immersion period used in this work is assumed to be sufficient to assure the complete setting of an enzymatic net over all fiber surfaces. The final net structure desired is illustrated in Figure 1.

Figure 1:
Schematic representation of desired lysozyme net to be set on the fiber permeable medium. (A) Glass fiber hydrophilic support (B) After SA lysozyme deposited.

Fibers in as-hydrophilic state were observed to present a certain surface roughness and then smooth as enzyme adsorption proceeds. Such a change permits an evaluation of the lysozyme deposition over immersion time by means of qualitative information drawn from AFM scanning. As shown in Figure 2, it is possible based on the appearance of the deposited film to assess the overall reduction in roughness intensity on the scanned surface. After a 12-minute immersion, excess formation of lysozyme can be observed.

Figure 2:
AFM aspects of the scanned surfaces where changes in topographical features can be observed: in (a) as-hydrophilic fiber,(b) 5-minute surfactant immersion (c) 10-minute immersion and (d) 12-minute immersion. The decrease in superficial roughness indicates lysozyme deposition. Ridges observed after 12 minutes indicate excess of deposited enzyme.

Previous studies imaging self-assembled lysozyme suggested that the assembly proceeds by nucleation and growth, while aggregation in solution may play an important role in the deposition features (Haggerty & Lenhoff, 1993).

In Figure 3 two examples of roughness line profiles randomly taken from the same sample are shown (a) line 1 is the as-hydrophilic fiber and line 2 (thick) shows the same fiber after 10 minutes deposition. In (b) RMS roughness decreases as a function of the deposition time. It is possible to relate quantitatively surface roughness to the amount of film adsorbed. On the uncoated surface (0 minutes) it is possible to observe the reduction in roughness as the enzyme deposition proceeds, which is interpreted as the coating of fibers by a homogeneous lysozyme film, as similar as also observed by Tsukruk et al.,1997.

Figure 3:
(a) AFM Profile lines: In (1) nonimmobilized, as-hydrophilic fiber. In (2) after 10-minute surfactant immersion. In (b) it may be possible to observe the reduction in superficial RMS roughness as a function of deposition time.

The protein film thickness is of the order of a few nanometers, and the composite system provides a highly permeable flux without sacrificing transport selectivity. Bioactivated filters with this type of configuration are the subjects of further research.

ACKNOWLEDGMENT

We are grateful for the support received from FAPESP under grant number 97/08178-9.

• Arai, T. and Norde, W., (1990) Coll. Surf. No. 51:pp.1-15.
• Assis, O.B.G., Claro L.C. and Colnago, L.A. In Proc. of the 3^rd Brazilian Symposium on Enzymatic Technology - ENZITEC 97, R. Janeiro, RJ Oct. 1997 (in print).
• Assis, O.B.G., Bernardes-Filho, R. and Colnago, L.A. In Proceedings of the 14^th International Conference on Electron Microscopy. Int. of Physics Publ. London. (H.A.C. Benevides & M.J. Yacamán, eds.), Cancún, Mexico, Oct. 1998, Symposium AA, vol II,.pp.851-852
• Bailey, J.E. and Ollis, D.F. Biochemical Engineering Fundamentals. 2nd ed., McGraw-Hill International, Singapore, 1996.
• Borato, C.E., Herrmann, P.S.P., Colnago, L.A., Oliveira Jr., O.N. and Mattoso, L.H.C. Braz. Journal of Chem. Eng. No. 14[4] pp. 367-373 (1997).
• Blomberg, E., Claesson, P.M., Fröberg, J.C. and Tilton, R. D. Langmuir No.10 pp. 2325-2330 (1994).
• Decher, G., Hong, J.D., Schimitt, J., Thin Solid Films No. 210/211 pp. 831-835 (1992).
• Ferreira, M., Cheung, J.H. and Rubner, M.F. Thin Solid Films No. 244 pp. 806-809(1994).
• Ferreira, M., Ph.D. diss. IQSC -University of Săo Paulo, 1994 .
• Herrmann, P.S.P. Ph.D. Thesis IQSC -University of Săo Paulo, 1999.
• Haggerty, L. and Lenhoff, A.M. Biophys. J. 64 pp. 886-895 (1993):
• Imoto, T., Johnson, L.N., North, A.C.T., Phillips, D.C. and Rupley, J.A. in The Enzymes (P.D. Boyer. ed.), London: Academic vol 2 p. 665 (1972).
• Kayushina, R.L., Stepina, N.D., Belyaev, V.V. and Yu Khurgin, I. Crystall. Reports No. 41[1]: pp.156-161 (1996).
• Lvov, Y., Ariga, K., Ichinose, I. and Kunitake, T. J. Am. Chem. Soc., No. 177 p. 6117 (1995).
• Raposo, M. and Oliveira Jr, O.N. Braz. Journal of Physic No. 28[4] pp.392-404 (1998).
• Robeson, J.L. and Tilton, R.D. Langmuir, No.12[25] pp. 6104-6113(1996)
• Tsukruk, V.V., Bliznynk, V.N., Visser, D., Campbell, A.L., Bunning, T.J. and Adams, W.W. Macromolecules No. 30[21] pp. 6615-6625 (1997)
• Ulman, A. An introduction to ultrathin organic films from Langmuir-Blodgett to self-assembly. Academic Press, Inc., 1991.
• Wahlgren, M., Arnebrant, T. and Lundström, I. J. of Colloid and Interface Science, No. 175 p. 506 (1995).

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To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  06 July 2000
• Date of issue
  June 2000

History

• Received
  03 Nov 1999
• Accepted
  17 Mar 2000