Download 12551_2010_28_MOESM1_ESM - Springer Static Content Server

SUPPLEMENTARY MATERIAL: Methods for Characterising Plasma Treated surfaces and their ability
to strongly attach bioactive protein layers
2.1. Electron Spin Resonance (ESR)
Electron Spin Resonance (ESR) or Electron paramagnetic resonance (EPR) measures the number of
unpaired electrons that are present in radical groups in a sample by measuring the absorption of
energy in a resonant microwave cavity operating at the electron spin precession frequency of the
electron spin in an applied magnetic field. As well as the density of electron spins, the technique is
gives information about the environment of the spin through the resonance linewidth and the g
value of the spin.
2.2. Infrared Spectroscopy
Infrared spectroscopy is a useful technique for probing the effects of energetic bombardment on a
polymer substrate and on a plasma polymer. Infrared radiation is made to interact with the region to
be characterised. Photons in this spectral range have energies capable of exciting vibrations of
chemical groups. Chemical groups present at significant concentrations in the material sampled can
be detected by the presence of absorption lines at their vibration frequencies.
For surface and near surface studies the radiation is applied by attenuated total reflection (ATR). In
this mode, a crystal is contacted to the surface and IR radiation is propagated into the crystal at an
angle beyond the Brewster angle. In this way, only the evanescent field which enters the material at
the interface with the crystal and penetrates to a depth of the order of a few microns interacts with
the surface. The sensitivity can be enhanced by using a multiple bounce ATR crystal.
Infrared spectroscopy in the attenuated total reflection (ATR) mode can also be used to detect the
presence of a surface attached protein layer. The amide groups which link the polypeptide backbone
of all proteins have three characteristic vibrations in the infrared. The corresponding absorption
lines are known as the amide A, amide I and amide II bands and they appear at wavenumbers of
3300, 1650 and 1540 cm-1 respectively (Keiderling 2002; Green et al. 1999). Because of the micron
scale penetration depth of ATR-IR, detection of a single monolayer of surface attached protein on a
polymer surface pushes the limits of signal to noise. Success requires careful background subtraction
to be applied. The shapes of the Amide absorption lines when well resolved can be used to infer
changes in the protein conformation (Green et al. 1999).
2.3. X-ray Photoelectron Spectroscopy
X ray photoelectron spectroscopy (XPS), also termed electron spectroscopy for chemical analysis
(ESCA), provides chemical information on the chemical composition of the surface and near surface
structure of a specimen. The energy spectrum of photoelectrons ejected by a soft x ray source is
examined. Only electrons ejected from atoms near the surface are useful, since the cross section for
their scattering is high and the information in the energy spectrum is degraded by scattering events.
The technique has been used to determine the chemical changes caused by plasma modification, to
examine the surface chemistry of plasma deposited polymers and to examine surface attached
layers of biomolecules. This is straightforward and very accurate if the underlying surface does not
contain all of the elements which occur abundantly in proteins (ie. carbon, nitrogen and oxygen).
The nitrogen N1s line is typically used to detect protein since the polymeric surfaces we discuss in
this review nearly always contain carbon and oxygen. In cases where nitrogen is also incorporated in
the underlying surface, changes in the shape of the C1s or N1s lines may be employed. This method
is effective if the chemical environments of the carbon or nitrogen atoms are significantly different
in the polymer surface and protein leading to a chemical shift in the elemental peaks which is
detectable in a high resolution spectral region scan. In some cases, where the proteins contain a
significant number of cystine residues, the presence of a sulphur peak can be used to detect the
addition of a protein layer (Kondyurin et al. 2009a). This method should be used in conjunction with
other techniques as the sulphur signal is typically very weak and can easily be affected by the noise.
2.4. Atomic Force Microscopy
Contact atomic force microscopy (AFM) reveals the topology of a surface at sufficiently high spatial
resolution to resolve single protein molecules, but normally not sufficient to reveal significant detail
of the orientation or conformation of the molecules. However, the extent to which the molecules
cover the surface is revealed, as shown in the work of Gan et al (Gan et al. 2007). Plasma treated
polymer surfaces show significantly higher coverage than untreated surfaces, and the coverage of a
plasma treated surface was observed to be an almost complete monolayer coverage.
2.5 Enzyme-Linked ImmunoSorbent Assay
Enzyme-Linked ImmunoSorbent Assay (ELISA) (Goldsby et al. 2003) utilises an antibody that is
specific the surface bound protein and then an enzyme labelled secondary antibody which binds to
the primary antibody to detect the presence of a protein. The surface is typically blocked by BSA or
equivalent prior to administration of the first antibody to ensure that it cannot be physisorbed to
sections of exposed surface. After incubation in the secondary antibody, the detection is carried out
by assaying for the presence of the enzyme label using for example a colorimetric assay.
Interpretation of these types of assays requires an understanding of the binding sites involved in the
two biorecognition steps. If the primary antibody binds only to one site on the surface of the
protein, the protein molecules orientated so that this site is inaccessible will not be detected.
Similarly if the site is destroyed by changes in the protein conformation the assay will not be
affective in detecting the presence of the protein when it is denatured. Some information about
protein conformation can thus be obtained by running two ELISA assays using one primary antibody
that binds many motifs on the surface attached protein and one that binds only a single
conformation sensitive site, for example a site which is hidden inside the protein in the native
conformation and is revealed upon unfolding.
2.6. Optical Methods for Monitoring Attachment
Ellipsometry is an optical technique useful for measuring and monitoring the changes in refractive
index and thickness of attached monolayers of biomolecules. The method relies on measuring the
changes in the amplitude and phase of the two principal polarisations of light after reflection from a
surface. It is useful as a non invasive and sensitive technique that can monitor changes in real time.
By measuring both the amplitude and phase changes, it is capable of discriminating changes in the
thickness of the layers as well as changes in their refractive index (Yin et al. 2009a)
Surface plasmon resonance is an optical technique that measures the attachment of biomolecules
with a high degree of sensitivity by detecting the small refractive index changes in the refractive
index of the solution. Surface plasmon resonance occurs when light polarised in the plane of
incidence is incident on a gold or silver film at the correct angle for launching the electromagnetic
excitation known as a surface plasmon. The angle required for resonance is slightly changed when
attachment of a layer of biomolecules occurs close to the film surface. The technique is non invasive
and is especially suited to the study of the kinetics of attachment and release processes of molecules
(George et al. 2008).
2.7. Quartz Crystal Microbalance and Dissipation
Quartz Crystal Microbalance and Dissipation (QCM-D) measures mass changes associated with the
attachment of a protein layer. The changes in mass are detected by frequency shifts of an oscillating
quartz crystal as the protein is adsorbed onto a surface mounted on the crystal. The mass measured
in this method includes the bound water attached to the protein, in contrast with optical methods
such as ellipsometry and SPR which detect changes in refractive index associated with the addition
of protein molecules alone and are not sensitive to bound water. Conformational changes may be
inferred in some cases by changes in the dissipation parameter which is a measure of the time it
takes for the crystal oscillations to be damped. The changes in the damping timescale are directly
related to viscoelastic properties of the surface attached layer which are often influenced by
changes in protein conformation.
2.8.Testing for Covalent Binding of proteins on surfaces
It is difficult to devise a definitive test for the presence of a covalent bond between a biomolecule
and a surface. There are two kinds of test that provide evidence for the presence of such a bond. The
first type of test attempts a direct measurement of the interaction force between the molecule and
the surface. A direct pull test can be conducted on a single molecule using a suitable tip that is
capable of applying and measuring a force on a single molecule. A covalent interaction can be
distinguished from strong physisorption through the force displacement curve. A series of steps in
this curve indicates the successive breakage of many weak interactions, while a covalent bond is
indicated by a large single detachment step. The strength of attachment of a monolayer of protein
to a surface can be investigated by adhesively attaching a flat probe to the protein monolayer and
measuring the force required to detach the probe normal to the surface (Yin et al. 2009b). This
method cannot distinguish between strong physisorption and covalent attachment and in this sense
the method provides necessary but not sufficient evidence for covalency.
The second type of test for covalency uses a surfactant to weaken physical interactions between the
molecule and the support surface. By appropriate choice of surfactant and the conditions under
which it is applied, physisorbed molecules can be removed completely, leaving only molecules
bound very strongly, by assumption with covalent bonds, to the surface. The second kind of test is
more convenient and is gaining acceptance as a test of covalency. Two common surfactants used for
this purpose are Triton –X and sodium dodecyl sulphate (SDS). Triton –X is a non ionic surfactant and
is typically less aggressive than SDS. SDS is ionic and is more effective at removing protein that is
physically adsorbed, especially when the surface is immersed in heated SDS solution of 1% or higher
concentration in water. For less stringent conditions (i.e. lower concentrations and room
temperature for example) the detergent is not capable of removing all of the physisorbed protein.
This is demonstrated by the residual binding observed on strongly hydrophobic surfaces, such as
PTFE, in a number of previous studies (Bohnert et al. 1990; Safranj et al. 1991; Kiaei et al. 1992; Chen
et al. 1993). The operation of removal by SDS appears to be assisted by denaturing the protein and
forming it into a complex which is incorporated into micelle structures (Bohnert and Horbett 1986).
The use of SDS elution as a test of covalency of binding was first reported by Danilich et al (Danilich
et al. 1992). Some authors have not made the link between the resistance to SDS elution and
covalency. For example, Kiaei et al (Kiaei et al. 1992) noted that hydrophobic surfaces of low energy
appear to bind protein in a more resistant manner and interpret their results on albumin binding in
this way. However, as we shall see here, their results can be interpreted in terms of covalent
attachment to certain types of plasma polymer surfaces and the weight of evidence now supports
the covalency hypothesis. Experience with many proteins and polymers shows that SDS resistant
binding , by inference covalent binding, is by no means confined to hydrophobic surfaces. This is a
fortunate occurrence since the preservation of protein function that is useful for applications would
be compromised by strong hydrophobic binding.
2.9.Testing for functional binding of biomolecules
Functionality assays test whether the immobilised molecules remain functional. It is important to
ensure that the immobilised molecules do not desorb during the assay. This is less problematic when
the molecules are covalently bound, but precautions must nevertheless be taken to carry out an
appropriate control. For example for assays that are carried out in solution with the surface present,
removal of the surface to test for the activity of the remaining solution is a useful control.
For enzymes, the functionality assay quantifies the degree to which the catalytic function is
preserved. For example for the enzyme horseradish peroxidise, the TMB assay (Josephy et al. 1982)
has been successfully used to examine the functionality of HRP over time and of the ability of the
modified surface to bind HRP functionally after storage (Ho et al. 2007). Other assays that have been
used are the hydrolysis of sucrose as a test for adsorbed invertase and the production of glucose by
cellobiase (Woodward 1985b).
Cell assays are another method of assessing biofunctionality of proteins which interact with cellular
membranes. Appropriate cell lines are examined for the number attached and their morphology.
Cells that are present in large numbers with morphology that indicates compatibility with the
surface after BSA blocking can be used as quantitative indicators of the function of the attached
protein (Bax et al. 2009a). Assurance that the specific interaction with the cell receptor is as
expected for protein in the native conformation can be attained by observing the effects of known
inhibitors.