생체복합재에서 계면전동에 관한 연구

윤은선, 용동희, 김용완*, 최청송

서강대학교, 화학공학과

*한국과학기술연구원, 생체재료연구센터

Study of Electrokinetic Properties of Biocomposite

EunSun Youn, DongHee Yong, YongWan Kim*, CheongSong Choi

Department of Chemical Engineering, Sogang University

*Biomaterials Research Center, KIST

서론

Zeta potential measurements provide for a unique method to quantify the actual state of the solid to solution interface in situ. In this study, zeta potentials for synthetic and biogenic calcium carbonate suspensions prepared in 1mM NaCl at different pH were measured. Also, in forming calcite crystals with macromolecules as an additive extracted from oyster shell, the zeta potentials were measured. These studies were performed to verify the primary role of macromolecules in controlling the formation, morphological development and crystallography of the biocomposite. Also, a new model of the electrical potential model of the composite interface region is proposed to evaluate effective zeta potentials.

INTRODUCTION

The morphology of biomaterials such as bone and biogenic calcium carbonate (oyster shell) may be influenced by many factors including zeta potentials dependent on the properties of the solid and fluid phases. The solid phase of the oyster shell is composed of an organic matrix and inorganic mineral, while the fluid phase is mainly extracellular matrix [1]. It has been noted that the difference in the electrokinetic behavior of calcium carbonate samples was ascribed to the organic matrix, which are composited within the biocomposite. To understand the controlled nucleation and growth of the biocomposite which are related to electric signals in the living system, it is necessary to investigate the zeta potential in vivo and the structure of the in vivo interface. But in many cases with the presence of adsorbed ions, ionic complexes and proteins, the zeta potential can not be defined as the electrostatic potential at the slip boundary. If the surface irregularities are not smooth, as might occur when long charged proteins are adsorbed onto the biocomposite and extended into the diffusion layer, then one should consider an effective zeta potentials. Thus, in this study, although the theoretically true geometry is complicated and changing, a simplified representation of the biocomposite is required because the zeta potential terminology is preferred because it underscores the phenomena in the interface. The investigations were directed to the influence of organic matter, and for that purpose the experiments with four type macromolecules as an additive were performed. The approach taken was that first amino acid compositions of macromolecules were measured, and then a quantitative information on the shell protein structure using Fourier self-deconvolution analysis of amide 1 band spectra obtained from Liquid ATR spectroscopy were estimated.

MATERIALS AND METHODS

Materials

Shells of Crassostrea Gigas (Choongmoo in Korea) were freshly collected, soaked in 5% NaOH, lightly scrubbed, and dried at room temperature. After drying, the shells were finely ground and separated using a mill according to their mineralogy. After complete decalcification (48h), the soluble matrices were separated. Amino acid analysis was performed using a Beckman 6300 amino acid analyzer.

Liquid ATR Measurements

The spectra were recorded using a BioRad FTS-60 with a resolution of 2 cm-1. The system was purged with dry N2 to reduce interfering water vapor IR absorption and verified no water contribution. To obtain the undisturbed proteins vibration spectra, water reference IR spectrum was subtracted from each sample solution, respectively. The subtracted spectra were analyzed by second derivatization, resolution enhanced Fourier self-deconvolution in the amide 1 region and Gaussian curve-fitting using the ORIGIN 5.0. The secondary structure contents were calculated from the area of the each Gaussian bands and their fraction of the total area in the amide 1 spectral region [2].

Zeta Potentials measurements

The powdered sample was further ground with a quartz mortar and transferred to a 500ml beaker containing 1mM NaCl electrolyte solution [3]. The measurements were started from 7.5 then adjusted towards the high pH direction using NaOH. Following pH adjustment, zeta potential of the biogenic calcium carbonate surface was measured by means of Malvern zetasizer 3000 and was analyzed by using a Helmholtz-Smolcowski equation. The suspensions in 1mM NaCl solution were prepared about 24h before use. The pH was adjusted with HCl (20mM) or NaOH (50mM) solution. Also, in order to identify the effect of macromolecules on crystal surface character, calcium carbonate crystals were grown by slow addition of 25ml of 1mM Na2CO3 to 25ml of 1mM CaCl2 solution that is suspended 12hr ago with containing macromolecules extracted from calcitic and aragonitic layer, respectively. Controls without the macromolecule additive were run in the parallel. Zeta potentials were measured as indicated above. The influence of added macromolecules was investigated by varying the concentration. The experimental concentrations were adjusted to imitate natural concentrations of similar intracrystalline protein substances in oyster shell.

RESULTS AND DISCUSSIONS

The surface charge of calcium carbonate crystals which have the same composition and structure but different origin may be important for researching adsorption processes on the crystal. Also, it is important to know if the crystal is positively or negatively charged, or if it is already covered with organic matter, because it leads the adsorption process in a completely different direction. The chemical species adsorbed are entirely different between synthetic calcite and biogenic calcite, that is, no organic substances on synthetic and intracrystalline proteins on biogenic calcite. The difference in their electrokinetic behaviors as shown in fig.1 (a) and (b) was assumed to be the consequence of the organic matter. The results indicated that the change of pH due to addition of acid and base caused the redistribution of ionic species in solution, so that in the range pH7.5 -11, the zeta potential of the synthetic calcium carbonate changed from +15mV to -35mV. The positive zeta potential was the result of the prevailing positive ionic species (Ca2+, CaHCO3+, CaOH+) at the particle-water interface. At pH > 9.5 the balance between positive and negative ionic species was shifted in favor of the negative ionic species (CO32-, HCO3-) which determined the negative surface charge of calcium carbonate. Table 1 shows the amino acid compositions of the organic matrix of the oyster shell. Under the same conditions the biogenic calcite was negatively charged and the resulting zeta potential ranged from -16mV to -28mV. No isoelectric point in myostracum composed of aragonite and in calcite layer was detected. Such a behavior was ascribed to the macromolecules composited, which prevented calcite surface from reacting with ions and solutes from the solution. This decrease of the zeta potential can be ascribed to the double layer compression due to the increase of the ionic strength.

The zeta potentials as shown in fig.1. (c) were influenced by the addition of the macromolecules extracted from each layer with the increase in amounts added. The results showed the highly specific adsorption of the macromolecules on a positively charged calcite surface at pH 9, leading to negatively charged one. It can be resulted from the reflection of carboxylate functional groups of acidic amino acids. The results show clearly that only organic matrices with a high density of functional groups are the main charge regulator. Also, it can be notable that the determining factor was not the concentration, but rather the type of organic matrix. That is, its adsorption on the mineral surface covers the characteristics of the mineral core and further reactions with other solutes are governed mainly by that organic cover. This decrease of the zeta potential can be ascribed to the double layer compression due to the increase of the ionic strength.

In conclusion, it is noted a strong influence of site-specific macromolecules and several groups of organic substances selectively adsorbed on the synthetic calcite surface. It is confirmed that the macromolecules influence the equilibrium at the solid-electrolyte interface. And it gives a new insight into the electrokinetic properties of the dispersed biocomposite systems, enabling us to describe the formation of biogenic crystal by intracrystalline proteins having well known secondary structure.

REFERENCES

[1] S. Mann, D. D. Archibald, Science, 261, (1993) 1286

[2] J. K. Kauppinen, D. J. Moffatt, Appl.Spectrosc., 35, (1981) 271

[3] R. M. Kowalchuk, S. R. Pollack, J.Biomed.Mat.Res., 27, (1993) 783

Table 1. Amino acid compositions of the proteins in the soluble and insoluble (parenthesis) matrices.

	Nonpolar (Ala,Val,Trp)	Acidic (Asp,Glu)	Basic (Lys,Arg)	Neutral (Gln,Cys)
Outer layer	14.09(46.26)	43.55(11.36)	1.49(9.61)	40.07(32.79)
Center layer	11.88(30.27)	40.83(18.65)	1.63(6.92)	45.67(44.16)
Inner layer	8.61(43.94)	46.89(11.30)	2.92(6.31)	41.57(38.44)
Myostracum	12.91(36.31)	38.31(18.69)	3.52(8.67)	45.27(36.33)

(a)

(b)                                     (c)

Fig. 1. (a) Zeta potentials of synthetic calcite and aragonite crystals vs. pH. (b) Zeta potentials of biogenic oyster shell vs. pH. (c) Zeta potentials of synthetic calcite vs. the concentration of the macromolecules extracted from each layer.