A. Goldzsteina, A. Aamouchea, F. Hombléa, M. Vouéb,∗, J. Contib, J. De Coninckb,
S. Devougec, J. Marchand-Brynaertc, E. Goormaghtigha
aLaboratoire de Structure et Fonction des Membranes Biologiques, Centre de Biologie Structurale et Bioinformatique, Université Libre de Bruxelles, Campus Plaine CP206/2, B-1050 Bruxelles, Belgium
bCentre de Recherche en Modélisation Moléculaire, Université de Mons-Hainaut, Place du Parc, 20, B-7000 Mons, Belgium
cUnité de Chimie Organique et Médicinale, Université Catholique de Louvain, Bâtiment Lavoisier, place L. Pasteur, 1, B-1348 Louvain-la-Neuve, Belgium
a r t i c l e i n f o
Article history:
Received 8 July 2008
Received in revised form 1 September 2008 Accepted 8 September 2008
Available online 24 September 2008
Keywords: Ligand–receptor interaction FTIR-ATR Alkylsilanes Haemophilia Factor VIII Molecular diagnosis a b s t r a c t
Detection of receptor–ligand interaction in complex media remains a challenging issue. We report exper-imental results demonstrating the specific detection of the coagulation factor VIII in the presence of a large excess of other proteins using the new BIA-ATR technology based on attenuated total reflection Fourier transform infrared spectroscopy. The principle of the detection is related to the ability of factor VIII molecules to bind to lipid membranes containing at least 8% phosphatidylserine. Several therapeutic concentrates of factor VIII were analyzed and the binding of the coagulation factor was monitored as a function of time. We show that a non-specific adsorption of stabilizing agents (typically, von Willebrand factor and human serum albumin) may be avoided by controlling the geometry of the ATR element. A linear response of the sensors as a function of the factor VIII concentration is described for different lipid membrane compositions.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Factor VIII (FVIII) is a large heterodimeric, multidomain plasma glycoprotein (approx. m.w.: 260 kDa), essential in the intrinsic pathway of blood coagulation. The functional absence of FVIII results in a severe X-linked bleeding disorder: haemophilia A. FVIII
acts as a cofactor in the Ca2+- and phospholipid-dependent
conver-sion of Factor X to Factor Xa by activated Factor IX (Bhopale and Nanda, 2003). Factor VIII consists of a light chain (m.w.: 80 kDa) and of a heavy chain (approx. m.w.: 180 kDa), linked by a metal bridge involving a single copper ion. In plasma, the association of FVIII with von Willebrand factor (vWF) results in a complete protection against proteolysis. Two binding sites have been iden-tified: one in the acid region at the N-terminus of the FVIII light chain and the other in the C-terminal C2 domain. This domain (in particular the amino acid residues 2303–2332) is also implied in binding to phospholipids (Bloom, 1987; Arai et al., 1989; Shima et al., 1993; Saenko et al., 1994). This domain is crucial in the bind-ing of FVIII to the membrane of platelets activated by thrombin or
∗ Corresponding author. Tel.: +32 65 373885/83; fax: +32 65 373881.
E-mail address:michel.voue@crmm.umh.ac.be(M. Voué).
other antagonists (Nesheim et al., 1988; Gilbert et al., 1991).
Accord-ing toGilbert and Drinkwater (1993a,b), the presence of at least
8% of phosphatidylserine (PS) is required for factor VIII binding to artificial phospholipid membrane fragments. The activation of the platelets induces a reorientation of the phosphatidylserine and phosphatidylethanolamine molecules from the inner to the outer membrane leaflet and leads to the formation of high affinity binding sites for FVIII (Zwaal et al., 1993).
Besides the classical detection methods based on
immuno-affinity (Western blot, ELISA, RIA) (see, e.g.Huang et al., 2001),
several biosensors have been developed to monitor the binding of FVIII to phospholipid membranes or to provide a quantitative detection of the coagulation factor. In particular, surface plasmon resonance (SPR) has been used to monitor the real-time protein
interaction (Saenko et al., 1999, 2001).Pflegerl et al. (2001)have
described a method using SPR and exploiting the covalent cou-pling of monoclonal antibodies against FVIII through the primary amine groups to the carboxymethyl dextran surface (CM5 sensor, Biacore). The SPR technique makes use of changes in the refractive index of medium near a thin gold film evaporated on a glass sub-strate. The target or biological receptors are immobilized via the interaction with an amorphous Dextran matrix or via the interac-tion with an alkanethiol-based self-assembled monolayer. In SPR,
0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.09.013
1832 A. Goldzstein et al. / Biosensors and Bioelectronics 24 (2009) 1831–1836
Fig. 1. Schematic view of the germanium triangular bar used for ATR-FTIR. The principle of FTIR sensors for FVIII detection and details of the functionalized interface.
the binding of the ligand to the receptor induces modifications of the refractive index in the immediate vicinity of the glass substrate. This yields a modification of the angle at which the minimum inten-sity of the reflected light is detected, i.e. of the resonance angle. The resulting detection measures the mass loading on the surface but does not provide any direct physico-chemical information about the “ligand–receptor” interaction. Recently (Voué et al., 2007), we have described the design of a new type of biodetection devices. These so-named BIA-ATR biosensors consist in a functionalized total attenuated reflection (ATR) germanium crystal and the detec-tion of the ligand–receptor interacdetec-tion is achieved using Fourier transform infrared (FTIR) spectroscopy (Fig. 1). The modification of the surface properties of the ATR elements by wet chemistry directly on the germanium or silicon surface avoids the evapora-tion or the sputtering of a thin metal layer, which considerably reduces the energy or intensity of the incoming actinic beam signal in the mid-infrared spectral range. The crucial steps in the surface functionalization are the activation of the Ge or Si surfaces and the anchoring of small spacer molecules. As recently pointed out by Andersson et al. (2008), it is still a challenge to achieve a versatile sensing device based on ATR-FTIR. Two approaches provide satis-factory results: the first one consists in an oxidation of the Si or Ge surface as Si/Ge–OH and the binding of alkyltrichloro- or tri-ethoxysilane molecules via an esterification reaction (Voué et al., 2007). The second one consists in the reduction of the ATR element surface as Ge–H and the subsequent binding of alkene molecules via a hydrogermylation reaction (Buriak, 2002). A major advantage of this method is that it allows not only a mass response of the lig-and/receptor system but also a positive identification of the ligand and a possibility to determine conformational changes occurring within the receptor.
In this article, we use the BIA-ATR approach with chemically modified germanium or silicon crystals to adsorb phospholipid membranes fragments and to detect the binding of FVIII molecules dissolved in a complex solution. The influence of the phospholipid composition is also investigated.
2. Experimental techniques 2.1. Reagents
1,2-Dioleoly-sn-glycero-3-phosphatidylserine (PS),
1,2-dioleoly-sn-glycero-3-phosphatidylcholine (PC), serum albumin,
octadecyltrichlorosilane (OTS) and all chemicals were purchased from Sigma–Aldrich unless specified otherwise. Recombinant FVIII lyophilisates Recombinate 250 and Refacto 250UI were respectively purchased from Baxter (Belgium) and from Wyeth Pharmaceuticals (Belgium). These chemicals were stabilized by human serum albumin (HSA). Octanate 500 was purchased from Octapharma (Switzerland). Octanate is a FVIII concentrate
from human plasma source and contains von Willebrand factor molecules to avoid coagulation factor proteolysis.
2.2. FTIR experiments
Attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectra were obtained on a Bruker IFS55 FTIR spectropho-tometer (Ettlingen, Germany) equipped with a MCT detector (broad
band 12,000–420 cm−1, liquid N2cooled, 24 h hold time) at a
res-olution of 2 cm−1with an aperture of 3.5 mm and acquired in the
double-sided, forward–backward mode. Two levels of zero filling of the interferogram prior to Fourier transform allowed encoding
the data every 1 cm−1. The spectrometer was continuously purged
with dry air (Whatman 75-62, Haverhill, MA, USA).
In one series of experiments trapezoidal silicon or
germa-nium crystals (50 mm×20 mm×2 mm) (ACM, Villiers St. Frédéric,
France) were used as internal reflection elements with an aperture
angle of 45◦ yielding 25 internal reflections. A crystal was then
placed in an ATR holder for liquid sample with an in- and out-let (Specac, Orpington, UK). A computer-controlled elevator (WOW Company, Belgium) allows the crystal to be separated in different lanes so that the baseline and sample could be recorded on the same crystal.
In another series of experiments triangular-shaped
germa-nium crystals (4.8 mm×4.8 mm×45 mm) were purchased from
Biosentech (Naninne, Belgium) and accommodated on the beam condenser from a Golden Gate Micro-ATR from Specac. A top plate with a groove fitting the crystal was used in replacement of the diamond-bearing plate (WOW Company, Belgium). With this geometry (Fig. 1), a single reflection occurs and more than 10 lanes can be used on a crystal.
The software used for data processing was written under MatLab 7.1 (Mathworks Inc., Natick, MA, USA).
2.3. Cleaning and grafting of ATR elements
The procedure was carried out as previously described and is summarized hereafter (Voué et al., 2007). The crystals were grafted with a self-assembled monolayer of octadecyltrichlorosilane (OTS) as follows. The usual procedure for silicon uses typically the Piranha
solution (i.e. a H2SO4/H2O2 mixture in ratio 7:3, v:v at 150◦C)
but this method strongly modifies the reflectance properties of germanium crystal surfaces. For germanium, the oxidized surface blackens and turns out to be rapidly unusable for grafting. To avoid these drawbacks, an alternative procedure has been proposed to
clean the germanium surface and build a stable GeO2interface. The
ATR element was immersed in HNO3(38%) during 1 min and rinsed
in MilliQ water. Afterwards, the surface was activated in a mixture
of H2O2and ethanedioic acid (10%) during 5 min. Finally, the surface
A. Goldzstein et al. / Biosensors and Bioelectronics 24 (2009) 1831–1836 1833
The procedure used to hydrophobize the ATR elements (sili-con or germanium) by OTS is derived from the work of Semal and co-workers (Semal et al., 1999; Voué et al., 1999). The activated ATR elements were immersed in a solution of OTS (0.08%, v:v) in
hexadecane:CCl4(ratio 7:3, v:v) during 16 h at 12◦C before being
rinsed in chloroform (2×5 min) under sonication to remove the
excess of unreacted silane.
2.4. Preparation of phospholipid vesicles and adsorption of phospholipid membranes
For the preparation of the liposomes, aliquots of 2 mg of lipids
were solubilised in chloroform, then dried under a N2stream for
15 min in a glass vessel and finally placed under vacuum for 12 h to remove the last traces of the solvent. The dried lipids were then
suspended in a 2 mM HEPES (N-[2-hydroxyethyl]piperazin-N
-[2-ethanesulfonic acid]) buffer at a final concentration of 0.5 mg/ml. The solution was vortexed for 1 min, then sonicated for 3–5 min (Branson probe sonifier, 30 W) in order to obtain small unilamellar vesicles (SUV). The PS and PC liposomes were used at a concentra-tion of 0.5 mg/l. The trapezoidal-shaped and the triangular-shaped
crystals were respectively incubated with 200l or 3l of liposome
solution in the infrared cell during 6–10 h at 4◦C. A FTIR spectrum
was recorded just after the injection of the liposomes in the infrared cell and kept as a reference spectrum to monitor the formation of the lipid film. The procedure allows the lipid membrane to slowly and uniformly adsorb on the OTS-grafted ATR element. At the end of the incubation, the excess of lipids was rinsed using 30 ml of buffer solution at 1 ml/min.
2.5. Binding of FVIII molecules
FVIII preparation at increasing concentration in 2 mM HEPES pH 7.2 was injected in the infrared cell. Typically, concentration ranged from 0 to 0.25 mg/ml over 120 min. The experiments were run at room temperature.
2.6. Dialysis experiments
The injectable lyophilisated Refacto 250UI FVIII was
reconsti-tuted in 200l of a 2 mM HEPES solution at pH 7.2. 15l of
this solution was spread on a dialysis membrane (Millipore V
0.025m). The amount of deposited FVIII was about 1.7g. The
dialysis medium was 200 ml MilliQ water. The FTIR spectra were recorded after 45, 90, 240, 285 and 330 min by spreading three
drops of 1l on an OTS-grafted triangular Ge crystal (without
lipids) and drying the ATR element under a N2flux.
3. Results and discussion
3.1. Lipid adsorption on the biosensor surface
Surface adsorption of the lipid can be monitored and
quanti-fied on-line from the FTIR intensity of the lipid CH2 stretching
bands near 2850 and 2920 cm−1. These two bands originating,
respectively, from thes(CH2) andas(CH2) are uncoupled from
the other vibrations (Fig. 2).Fig. 2also shows the PC and PS
spec-tra in the 1800–1000 cm−1 spectral domain. These lipids can be
easily differentiated on the basis of the presence/absence of the
peaks near 1639 and 1524 cm−1 tagged by the vertical arrows.
These are, respectively, attributed to the asymmetric and
symmet-ric deformations of the NH3+group of PS. References values for the
bands are 1629±1 cm−1and 1526±3 cm−1(Goormaghtigh et al.,
1994).
Fig. 2.FTIR spectra of phospholipid membranes on OTS surfaces (PC, plain line; PS, dashed line).
3.2. Binding specificity
In preliminary experiments, FVIII solution in HEPES buffer was prepared from Recombinate 250 (Baxter). As provided, the solu-tion containing recombinant FVIII was stabilized by human serum albumin (HSA) at a concentration of 12.5 mg/ml. Gel electrophore-sis experiments (not shown) indicated that HSA overwhelmingly dominates the protein content. A series of experiments was there-fore designed to evaluate the non-specific interactions of HSA with PS membranes. Solutions of Recombinate 250 or HSA were flown over an OTS-grafted trapezoidal silicon crystal coated with
phosphatidylserine (PS) lipids (see Section2). FTIR spectra were
recorded every minute. The bands around 1650 cm−1 (amide I)
and 1544 cm−1(amide II) arise mainly from the absorption of
pro-teins (Fig. A1, additional information). Amide I is the most intense absorption band of the polypeptides. It is positioned between 1700
and 1600 cm−1, but its exact frequency is determined by the
geom-etry of the polypeptide chain and hydrogen bonding.(C O) has a
predominant role in amide I accounting for 70–85% of the
poten-tial energy (Krimm and Bandekar, 1986). The band at 1542 cm−1is
the amide II contribution. Amide II occurs in the 1510–1580 cm−1
region. It derives mainly from the in-plane N–H bending. In the
1400–1800 cm−1spectral region (Fig. A1), the intensity of the amide
I and II absorption bands (1680 and 1550 cm−1, respectively) allows
the monitoring the protein/membrane interaction. As the protein binds onto the membrane, it concentrates in a layer close to the reflection interface where the evanescent wave is most intense
(Harrick, 1967) and contributes to the absorbance.Fig. 3reports the
time evolution of the amide I band intensity at 1660 cm−1. A similar
experiment was repeated with HSA in order to evaluate the speci-ficity of the binding. The behaviour of the FVIII and HSA solutions is obviously different. In the latter case, the intensity of the amide I band even decreases. A possible explanation of this phenomenon could be a slight instability of the coated phospholipid layers. As HSA molecules do not absorb on the PS membrane fragments, we
can conclude that the data represented by the open circles inFig. 3
correspond to the binding of the FVIII to the PS membrane. More-over, this experiment demonstrates the specificity of the device: despite the presence of a large excess of HSA in the FVIII solution only FVIII molecules bind to the sensor.
3.3. Spectral identification of bound FVIII
One of the most interesting features of the BIA-ATR approach is that it yields a signature of the binding molecule through its
1834 A. Goldzstein et al. / Biosensors and Bioelectronics 24 (2009) 1831–1836
Fig. 3.Evolution of the absorbance of the amide I band (1660 cm−1) for FVIII (open circles) and HSA (filled circles) when the solution is flown over a PS membrane.
IR spectrum. Additional experiments were carried out to confirm the chemical nature of the ligand bound to the PS membranes in the experiments reported above. In FVIII sample, the presence of other proteins (HSA, von Willebrand factor) might overlap the contribution of FVIII. In order to obtain a reference spectrum of FVIII, we used a recombinant preparation of FVIII. As the com-mercial injectable preparation contains excipients that interfere with the spectrum of the protein, we first performed a dialysis step to eliminate the excipient molecules. A detailed
examina-tion of the 1400–1800 cm−1spectral region after dialysis indicates
that the peaks are very similar to the absorption bands observed after association of FVIII molecules with the PS lipid bilayers. Fur-thermore, the comparison of the shape of the amide band in the
spectra represented inFig. A1 with that of HSA shows that the
detected proteins do not correspond to HSA (Fig. 4). The
spec-trum of streptavidin, a protein with high-sheet content (48%)
and no ␣-helix content has been added in Fig. 4 for the sake
of the comparison. The data reported in Fig. 4confirm that the
spectrum recorded with the PS-coated sensor corresponds to the binding of the coagulation factor. They stress the advantage of the BIA-ATR approach which allows the identification of a pro-tein among several others present in a mixture through the FTIR spectrum. Other studies reporting the spectra of more than 50 pro-teins have stressed the uniqueness of the IR spectrum, related to a
Fig. 4.Comparison of the FTIR spectra of proteins in the amide I–amide II bands spectral region (plain line, FVIII; dashed line, HSA; dotted line, streptavidin or SA).
large extent to the protein secondary structure (Goormaghtigh et al., 2006).
3.4. Effect of the crystal composition and geometry
The usual, commercially available trapezoidal-shaped crystals correspond to the one described in our previous publication (Voué et al., 2007). Two types of crystals (germanium and silicon) were grafted with OTS and used as optical basis for the membrane frag-ments. For each of these, PC and PS liposomes were considered. The FVIII solution, prepared from the Octanate therapeutic concentrate in 2 mM HEPES buffer solution, was progressively injected in the FTIR cell, with a concentration ranging from 0 to 0.25 mg/ml
dur-ing 120 min. Spectra were recorded every 2 min.Fig. A2(additional
information) reports the time evolution of the amide II absorption
bands (1550 cm−1) for the silicon (a) and the germanium (b)
crys-tals. For the sensors based on the silicon crystal, the binding of the FVIII is specific for the PS membranes. The control experiment with PC membranes indicates that the protein binds specifically to phos-phatidylserine phospholipids, as evidenced by the lack of growth of the amide II band. Concerning the sensors using a germanium crystal (Fig. A2b), our results show a preferential binding of the FVIII molecules on the PS membrane but the quality of the control exper-iment is not as good as expected on the basis of the results obtained on silicon. The intensity of the amide II band increases significantly due to specific interactions. Possible explanations for the non-specific interactions observed with the germanium support might be related to a weaker quality of the chemical functionalization of the surface and the larger size of the functionalized surface yield-ing, at the centimetre scale, some inhomogeneity of the lipid layer. We previously described that chemical grafting of OTS on germa-nium is not straightforward due to the chemical reactivity of the
Ge/GeO2 surface which fundamentally differs from the
reactiv-ity of the Si/SiO2one. In line with this problem, we also showed
that the hydrophobicity of the OTS-grafted Ge surfaces is slightly less than the hydrophobicity of the silanized Si surfaces. Therefore, obtaining homogeneously OTS-grafted Ge surfaces at a large scale remains a challenge and the large size of our trapezoidal crystals
(50 mm×20 mm) makes it difficult to form homogenous layers.
For these reasons, a triangular prism of germanium crystal was designed, built and tested as explained hereafter. With this new geometry (Fig. 1), we use lenses to condense the beam on a single
reflection spot of about 1 mm2.
The loss in the number of reflections (about 25 reflections for the trapezoidal crystal with respect to 1 reflection for the trian-gular prism) was compensated by the improvement of the optics quality in the FTIR cell. The Golden Gate optics (Specac) was used instead of a standard vertical ATR cell. The surface reduction of the