Adsorption of human serum albumin onto polypyrrole powder and polypyrrole-silica nanocomposites

ELSEVIER

Synthetic

Metals

102 (1999)

1419-1420

Adsorption of human serum albumin onto polypyrrole powder and polypyrrole-silica nanocomposites Ammar AZIOUNE,

Karine PECH Bachir SAOUDI, Mohamed M. CHEI-lIMI*, Gerard P. McCarthy’ and Steven P. Armes’

Institut de Topologie et de Dynamique des Systemes, Universite Paris7 - Denis Diderot, CNRS (UPRESA 7086), 1 rue Guy de la Brosse, 75005 Paris (FRANCE) # School of Chemistry, Physics and Enviromnental Science, University of Sussex at Fahner, Brighton BNl 9QJ (UK) Adsorption of human serum albumin (HSA) onto polypyrrole powder and polypyrrole-silica nanocomposite is investigated at pH 7.4 and RT using UV spectroscopy. the nanocomposite is more effective than the powder in adsorbing HSA (147 and 63 mg/g, respectively). XPS was used to detect characteristic features of the adsorbed HSA and to construct adsorption isotherms. These latter were in fair agreement with those obtained via UV. Polypyrrole-silica is adsorptive towards HSA at pH 7.4 by contrast with the quasi-zero adsorption previously obtained with the DNA adsorbate (Saoudi et al, J. Colloid Interface Sci. 1997). Introduction

3. X-ray Photoelectron

Polypyrrole is one of the most studied inherently conducting polymers (ICPs) as it has various interesting properties : redo& acid-base and ion-exchange properties [I]. It has also a high surface energy [2] and a high specitic surface area [3] compared to conventional polymers. These properties together with conductivity and long term stability make it very suitable for the development of novel biosensors [4], bioreactors [5] and bioadsorbents [6]. As a follow-up to our studies of DNA fragments adsorption onto polypyrrole [I and polypyrrole-silica nanmmposites [8], we wished to explore the adsorptive capacity of these two distinct forms of polypyrrole as bioadsorbents of a model protein : human serum albumin (I-ISA). We used the depletion method in conjunction with UV absorption and fluorescence to determine adsorption isotherms of HSA onto polypyrrole powder and polypyrrole-silica nanocomposites at pH 7.4 and 0.1 M phosphate but&r. XPS was used as a complementary technique for the direct character&&ion of the HSA-adsorbent interface.

Spectra were recorded using a VG Scientific ESCALAB MKI system. Mg Ku X-my source was used at a power of 200 W (2011~4 x 10 kV) and the pass energy was set at 50 eV. Charge referencing was determined by setting the aliphatic Cls component at 285.0 eV. We used experimental sensitivity factors for the quantitication of the surface composition of polypyrrole powders, HSA fibres and HSA-coated polypyrrole.

Specstroscopy (XPS)

Results and discussion HSA adsorption onto poljgynvle

powders and nanocomposites

Experimental 1. Synthesis of bulk powders and nanocomposites PpV bulk powder : pyrrole (1.00 ml, Acres) was added via syringe to 100 ml of a stirred aqueous solution of FeCl3.6HrO (9.74 g, Aldrich) at RT. The mixture was stirred for 24 h. The resulting black precipitate was vacuum-filtered and thoroughly washed with de-ionized water. The powder was then tied in a dessicator overnight and sieved (180 urn) before adsorption. Sulfate-doped PPy (PPySO4) was prepared as PPyCl but using ammonimn persulfate (3.12 g) as an oxidizing agent. Synthesis oJPPySO&lica

: see ref. [9] for 111 details.

2. Adsorption experiments. HSA (Sigma product) was diluted in 10 ml of phosphate butler 0.1 M (NaHp04 and Na&IP04 were purchased f&n Fhrka) to obtain the desired initial concentration. The solution was then stirred with lo-20 mg of adsorbent for 24 hours. The adsorbent was conditioned in the same buffer prior to HSA adsorption [A. The amount of HSA adsorbed on the surface was determined by the depletion method using UV absorbance of HSA at 280 mn in the 100-500 @ml range or by fluorescence at 337 mn for an initial conmtration in the lo-100 ug/ml~ 0379-6779/99/$ - see front matter PII: SO379-6779(98)00982-S

0 1999 Elsevier

Science

S.A.

All rights

0

loo

200 b&l

300

HSA conanbmtbn

400

so0

600

(w/mll

Fig. 1. HSA Adsqtirn onto PPyS04 (lIl,m)and PPySQ-silica b&m (0,O) and ai& (.,~)hw wxxssives washings.

(0,O)

Figure 1 displays HSA adsorption isotherms using UV absorbance spectroscopy. It is clear that the washing procedure was necessary to remove excess of physically adsorbed macromolecules. It is very important to note that adsorption onto the polypyrrole-silica nanocomposite (up to 147 mg/p) is much higher than that obtained on the bulk powder (63 mg/g). This is partly due to the high surface area of the nanocomposite but most probably reflects favourable electrostatic interactions between the negatively charged nanocomposite [lo] and positive charges of HSA. More importantly, PPy-silica is 10 times more adsorptive than polypyrrole latex particles stabilised with polyacroleine [6] towards HSA. HSA adsorption is up to 13 times more favourable on this nanocomposite than that of the polyanionic DNA [8] which undergoes repulsive interactions with the nanocomposite and requires surface functionalisation of reserved.

1420

MM.

Chehimi

Ed al. I Synthetic

the adsorbent with -NH2 or -COOH to obtain a significant adsorption up to 22 mg/g. We have also explored HSA adsorption from dilute solutions in the 1O-100 &ml concentration range using a more sensitive fluorescence spectrometer (see Figure 2).

i

0

20

40

inilial

HSA

60

60

ooncentmtion

100

Metals

IO2 (I 999)

1419-1420

XPS characterization

of the HSA-polypyrrole

inte$ace.

XPS was used to directly probe HSA at the surface of PPySO4 bulk powder. Figure 3 shows the C 1s peak of untreated PPySOd and HSA-coated PPySO4. The prominent shoulder at high binding energy (Figure 3b) is due to the peptide linkage carbon atom (NH-c=O). XPS provides thus direct evidence for the adsorption of HSA. This is also contiied by the detection of a low intensity S2p peak centred at 164 eV and due to sulfur from the cysteine moities. Combining peak intensities and sensitivity factors, we determined a surface chemical composition for various HSA-coated powders. We have related the sulfur surface content (%S) determined by XPS to the amount of HSA adsorption (Figure 4). A steady state is almost reached indicating perhaps that HSA occupies all surface sites first and then the protein-adsorbed layer thickens gradually.

Oa I

120

(pg/ml)

n

0.6

Fig2 HSAadsorption PPySO1-silica (B).

onto PPyCl(O),PPySO~

(O), PPyCl-silica

(0) and

n J

Fluorescence spectroscopy confiis, for low HSA initial concentration, that the nanocomposites are more adsorptive towards HSA than the bulk powders. In addition, there is a distinct dopant effect in the case of powders. PPyCl is more adsorptive perhaps because it is more conductive than PPySO4. This is the same situation for the nanocomposites, however for an initial HSA concentration higher than 70 ug/ml.

0.4

0.2

0.0 l 0

20

40

HSA

Fig. 4. Surface

content

of sulfur

adsorption

60

60

(me/p)

(%S) vs HSA adsorption.

It is important to note that the maximum %S is below that found for pure HSA (0.9%) thus suggesting that the underlying bulk PPy powder is still detected and that the adsorbed protein layer is less than 10 mn thick. Acknowledgements MMC thanks the Royal Society of Chemistry for partial financial support (RSC Journals Grant n”0697/023). References

3724

_... 1 .._

Binding Fig 3. Cls scans ofpolypyrrole

Energy

(eV)

(a), and HSA-coated

polypyrrole

(b)

[l] E. T. Kang, K. G. Neoh and K. L. Tan, Adv. Polym. Sci., 1993, 106, 135 [2] M. M. Chehimi, S. F Lascelles and S. P. Annes, Chromatographia, 1995,41,671 [3] T. H. Chao and J. March, J. Polym. Sci. Pal’. Chem. Ed., 1988,26,743 [4] T. Livache, A. Roget, E. Dejean, C. Barthet, G. Bidan and R. Teoule, Nucleic Acids Rex, 1994,22,2915 [5] R.V. Parthasarathy and C. R. Martin, Nature, 1994,239,298 [6] A. Smith and CKnowles, J. Appl. Pal’. Sci., 1991,43,399 [7] B. Saoudi, N. Jammul, M-L. Abel, M. M. Chehimi and G. Dodin, Synth. Met., 1997, 87, 97 [8] B. Saoudi, N. Jammul, M. M. Chehimi, G. P. McCarthy and S. P. Armes, J. Colloid Interface Sci., 1997, 192,269 [9] S. Maeda and S. P. Armes, J. Colloid Znterjkce Sci., 1993, 159,257 [lo] M. D. Butterworth, R. Corradi, J. Johal, S. F. Lascelles, S. Maeda and S.P. Armes, J. Colloid Inte@ce Sci.. 1995, 174,510