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閱讀：1876 發(fā)布時(shí)間：2020-1-20A study of saliva lubrication using a compliant oral mimic
Due to ethical issues and the difficulty in obtaining biological tissues, it is important to find synthetic elastomers
that can be used as replacement test media for research purposes. An important example of this is friction testing
to understand the mechanisms behind mouthfeel attributes during food consumption (e.g. syrupy, body and
clean finish), which requires an oral mimic. In order to assess the suitability of possible materials to mimic oral
surfaces, a sliding contact is produced by loading and sliding a hemispherical silica pin against either a polydimethyl
siloxane (PDMS), agarose, or porcine tongue sample. Friction is measured and elastohydrodynamic
film thickness is calculated based on the elastic modulus of the samples, which is measured using an indentation
method. Tests were performed with both saliva and pure water as the lubricating fluid and results compared to
unlubricated conditions.
PDMS mimics the tongue well in terms of protein adhesion, with both samples showing significant reductions
in friction when lubricated with saliva versus water, whereas agarose showed no difference between saliva and
water lubricated conditions. This is attributed to PDMS's eOeSi(CH3)2- group which provides excellent adhesion
for the saliva protein molecules, in contrast with the hydrated agarose surface. The measured modulus of
the PDMS (2.2 MPa) is however significantly greater than that of tongue (3.5 kPa) and agarose (66–174 kPa).
This affects both the surface (boundary) friction, at low sliding speeds, and the entrained elastohydrodynamic
film thickness, at high speeds.
Utilising the transparent PDMS sample, we also use fluorescence microscopy to monitor the build-up and flow
of dyed-tagged saliva proteins within the contact during sliding. Results confirm the lubricous boundary film
forming nature of saliva proteins by showing a strong correlation between friction and average protein intensity
signals (cross correlation coefficient=0.87). This demonstrates a powerful method to study mouthfeel mechanisms.
1. Introduction
Due to ethical issues and the difficulty in obtaining biological tissues,
it is necessary to find synthetic elastomers that can be used as
replacement test media for research purposes. A key example of this is
friction testing to understand the mechanisms behind mouthfeel attributes
during food consumption (e.g. syrupy, body and clean finish),
which requires an oral mimic. This is important since the acceptability
of food and beverages depend critically on their mouthfeel, which results
from tribological and rheological processes (Stokes, Boehm, &
Baier, 2013). Moreover, a poor understanding of these processes
currently limits the development of healthy formulations that can replicate
foods while reducing ingredients such as fat (Dresselhuis, 2008),
(Drewnowski, 1997). When mimicking the oral mucosa for these in
vitro tribological studies of foods and beverages, consideration must be
made of the mucosal pellicle. Like the acquired enamel pellicle on teeth,
this is a subset of salivary proteins that specifically bind to oral epithelial
cells (Gibbins, Proctor, Yakubov, Wilson, & Carpenter, 2014).
Unlike the acquired enamel pellicle, the mucosal pellicle is mostly
composed of mucins and secretory IgA. This layer is driven by the interaction
of salivary mucins (muc5b and muc7) with membrane-bound
mucin (muc1) expressed on oral epithelial cells (Vijay, Inui, Dodds,
Proctor, & Carpenter, 2015). Mucins are large highly glycosylated
proteins which retain considerable amounts of water when initially
secreted (Corfield, 2015). Thus, in addition to saliva lubricating the
surface, there is also a hydrogel-like layer adjacent to the surface. All
too frequently however, saliva is omitted from in vitro tests as it was
cited as being too inconvenient to collect in sufficient quantities or
considered too complex to give consistent results.
Previously, the oral mucosa was mimicked using glass or other hard
substrates (Chen & Stokes, 2012). More recently elastic substrates have
been used which introduced soft-tribology with Hertzian mechanics. In
an important investigation by Dresselhuis et al. (Dresselhuis et al.,
2007), the surface characteristics of pig tongue were compared with
those of PDMS. Their investigation concluded that PDMS showed dissimilarities
in surface characteristics to those of a tongue surface, since
the oral mucosa and PDMS rubbers, even with a structured surface to
reproduce biological scenarios, were not interchangeable in tribological
experiments. However, this widely cited paper has a critical shortcoming
in that it used only emulsion as the lubricant and saliva interactions
were completely ignored. Other work carried out on biological
surfaces, but without the presence of saliva include studies by Adams
et al. (Adams, Briscoe, & Johnson, 2007) and Tang et al. (Tang &
Bhushan, 2010), (Tang, Bhushan, & Ge, 2010) into the lubricating
properties of human skin. Adams et al. used a smooth glass or polypropylene,
spherical tipped probe sliding against a human forearm,
while Tang et al. employed shaved porcine skin. Results were reported
for a range of lubricating conditions, but repeatability of testing was
difficult to achieve. Prinz et al. (Prinz, de Wijk, & Huntjens, 2007) did
investigate the frictional properties between two pig mucosal surfaces
lubricated with human saliva. However, scant data is presented and no
comparison is made between different component materials.
For the majority of research, crosslinked polydimethyl siloxane
(PDMS) has been chosen because of its elastic properties, easy handling
and relatively low stiffness, comparable to soft biological tissues (Cox,
Driessen, Boerboom, Bouten, & Baaijens, 2008; Khanafer, Duprey,
Schlicht, & Berguer, 2009). PDMS is utilized as one (de Vicente, Stokes,
& a Spikes, 2006), (Malone, Appelqvist, & Norton, 2003), (Tang &
Bhushan, 2010), (Tang et al., 2010) or both (Stokes, Bongaerts, &
Rossetti, 2007), (Lee & Spencer, 2005), (Bongaerts, Fourtouni, & Stokes,
2007) of the contacting surfaces in the tribological contact to maintain
low contact pressures and create the conditions for isoviscous-elastohydroynamic
lubrication (I-EHL) to occur. One key advantage of PDMS
which has contributed to its widespread use is its ease of fabrication.
Prior to crosslinking, PDMS can be cast into suitable moulds of almost
any desired shape. Other attractive features of PDMS include its physiological
inertness, availability, low unit cost, as well as its good
thermal and oxidative stability.
PDMS is a transparent silicon-based organic polymer, used to represent
biological materials in numerous tribological studies (e.g.
(Bongaerts et al., 2007) (Dresselhuis et al., 2007), (De Vicente, Spikes,
& Stokes, 2004)). It is highly compliant, with a Young's modulus
E≈0.57–3.7 MPa (depending on degree of crosslinking) (Wang,
Volinsky, & Gallant, 2014), due to its uniquely low glass transition
temperature (Tg≈−125 °C) (Lötters, Olthuis, Veltink, & Bergveld,
1999). The surface of PDMS is hydrophobic, due to its repeating eOeSi
(CH3)2- group (Adams et al., 2007) but can be made hydrophilic by
plasma cleaning. In addition to this, PDMS is being used extensively in
polymeric microfluidics (e.g. (Eddings, Johnson, & Gale, 2008)) research
and findings from this area may be usefully applied in this study.
The tribological properties of PDMS are now fairly well understood.
Vorvolaskos and Chaudhury (Vorvolakos & Chaudhury, 2003) investigated
the effect of molecular weight and test temperature on
friction in a pure sliding contact between a PDMS and metal surface.
Bongaerts et al. (Bongaerts et al., 2007) investigated the effect of surface
roughness of PDMS on the lubricating properties of biopolymers
and aqueous solutions. PDMS, like most elastomeric surfaces, is by
nature hydrophobic but an oxidation treatment can be employed to
create a hydrophilic surface. Hillborg et al. (Hillborg & Gedde, 1998),
(Hillborg, Sandelin, & Gedde, 2001) and Schneemilch et al.
(Schneemilch & Quirke, 2007) investigated the wettability of PDMS
before and after oxidisation by several techniques and studied the effect
of crosslink density on oxidation. de Vicente et al. (de Vicente, Stokes, &
Spikes, 2005) looked at the influence of surface modification of PDMS
on its aqueous lubrication properties. However, there remains some
debate over the suitability of PDMS as a model biosurface and instances
of PDMS being tested under saliva conditions are few in number.
The second soft matrix to be considered here as a potential substrate
to mimic the oral mucosa is agarose. Agarose, the agaropectin deficient
fraction of agar derived from seaweed and consisting of β-1,3 linked α-
galactose and α-1,4 linked 3,6-anhydro-αL-galactose residues
(Normand, Lootens, Amici, Plucknett, & Aymard, 2000), is used to
create a hydrogel-like matrix. The compliance of agarose varies enormously
depending on concentration, with Young's moduli ranging
from ∼1.5 kPa to ∼3 MPa (Benkherourou, Rochas, Tracqui, Tranqui, &
Guméry, 1999), (Normand et al., 2000), (Chen, Suki, & An, 2004). In
addition to this, agarose has the ability to grow cells in suspension and
has therefore been used in tissue culturing systems (Chen et al., 2004).
This combination of properties make agarose an attractive choice in
biomedical research, for example, as a cartilage mimic (Saris et al.,
2000), or as a phantom material for magnetic resonance elastography
(Muthupillai et al., 1995). It is therefore surprising that agarose has
been used in few tribological studies and seems to have been overlooked
completely as an oral mimic. Fernández Farrés studied its frictional
behaviour, but did so under glucose and glycerol lubrication
rather than saliva (Fernández Farrés & Norton, 2015). Shewan et al.
also recently studied the lubrication performance of agarose, but as
particles in suspensions rather than a substrate (Shewan & Stokes,
2015).
It can be concluded that it is important to be able to mimic the oral
mucosa surface and various materials have been studied for this purpose.
However, these have rarely been compared with actual biological
materials (probably because of the difficulty in source, preserving and
securing them) and almost never when lubricated by saliva. To address
this, the current study characterises the friction and film thickness
performance of polydimethyl siloxane (PDMS), agarose and porcine
tongue, with the aim of assessing their suitability as an oral mimic for
tribological testing. Particular attention is paid to the compliance and
protein binding behaviours of these substrates.
2. Test methods
2.1. Specimen preparation
PDMS specimens were moulded using a commercially available
Sylgard 184 kit from Dow-Corning, containing a base and curing agent
to produce a material with a Young's modulus 1.84 MPa at 23 °C.
Agarose gel was produced by dissolving powdered agarose (Sigma-
Aldrich, Poole, UK) into water at 1 or 2% w/v. To aid dissolution the
solution was heated to 90 °C then allowed to cool to a temperature
below the coil-helix transition at around 35 °C. At this point agarose
forms a gel, consisting of an infinite three-dimensional network of fibre
helices (Normand et al., 2000).
Prior to collection the subject refrained from food and drink for 1 h.
Resting whole mouth saliva (WMS) was collected from a single subject
by drooling into a pre-weighed tube, kept on ice. After collection saliva
was briefly centrifuged (3000 g for 3 min) to remove sloughed cells and
other debris.
Porcine tongue was procured and tested on the same day. Its underside
was removed to produce a parallel slab. This specimen was then
bonded onto a flat plate using cyanoacrylate adhesive and mounted in
the friction rig.
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
11
2.2. Indentation and surface roughness measurements
The elastic modulus of each sample material was measured using an
indention test performed on a Mach 1 rig (Biomomentum Inc., Laval,
Canada). This involved indenting the sample at 1 mm/s with a spherical
indenter with radius 3.175 mm, during three repeat tests, while measuring
the normal force and the vertical displacement. The normal force
was measured using 1.5 N single-axis load cell with a resolution of
75 μN and the vertical displacement was measured by the moving stage
of the rig with a resolution of 0.1 μm. A depth of penetration of 0.6mm
was used for agarose 1% w/v and 0.4mm for each of the other samples.
This was done in accordance with Van Dommelen et al.’s (van
Dommelen, van der Sande, Hrapko, & Peters, 2010) suggestion that the
sample thickness does not significantly affect the data if indentation
depths are restricted to less than 10% of the sample thickness. Nevertheless
a formulation that considers the finite thickness of the sample
was used (Hayes, Keer, Herrmann, & Mockros, 1972) to calculate
Young's moduli. Contact mechanics equations were fitted to the data to
give the Young's modulus, specifically,
χ = a
dR
2
(1)
=
− κ P ν
aGd
(1 )
4 (2)
Where d is the displacement of the indenter, R is the radius of the indenter,
a is the radius of the contact region, P the applied load, G the
shear modulus, and ν the Poisson's ratio. A schematic of the test is
provided in Fig. 1.
The reaction force and the indenter displacement are recorded by
the Mach-1 Motion Software and enter the above equations as P and d
respectively. The Poisson's ratio is assumed to be equal to 0.5 (incompressible
materials). The specimens' height h and the indenter radius
R are also known. The values of χ and κ are given in Table 2 in
(Hayes et al., 1972) for different values of a, h and ν. The radius of the
contact region a is estimated during the curve fit with equation (1).
Once the fitting algorithm converges, the Young's Modulus E is computed
from the Shear Modulus G using the equation
E = 2G(1 + ν) (3)
The roughness of each of the specimens was measured three times
(each at a different location) on the surface, using a Veeco optical
profilometer.
2.3. Protein staining measurements
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis)
was used to assess the degree of binding of different proteins
to the surface of the oral mimics. This immunoblotting technique targets
proteins in a sample with specific dyes and measures their progression
through a gel, due to an applied electric field. In this way,
different proteins in a sample with different molecular weights are separated.
Staining involved incubating for an hour at room temperature
with whole mouth saliva from a single subject. Coomassie Brilliant Blue
(CBB) was used stain of all proteins. In addition to this, Periodic Acid
Schiff's (PAS) was used to stain for highly glycosylated proteins and
specific antibody with sensitive chemiluminescent detection was used
for the saliva protein muc7. Samples were removed from the surface of
each of the oral mimic surfaces and tested in this way to investigate
which proteins were present.
2.4. Friction measurements
A contact was produced by loading and sliding a 5mm radius silica
hemisphere against the compliant disc specimen, using a UMT2
(Universal Materials Tester), manufactured by CETR, (Campbell, USA).
This equipment was operated in pin-on-disc mode, so that the PDMS
specimen rotated, while the silica hemisphere was held stationary. The
lower specimen was located on a rotating table, capable (with certain
modifications), to run at speeds from 0.01 rpm up to∼4000 rpm.
Friction force (Fx) and normal load (Fz) were measured using strain
gauges, bonded to the housing above the stationary silica hemisphere
specimen. Sensitive, low-load sensors were chosen for this purpose,
with measurement ranges of±0.65 N and±6 N for Fx and Fz respectively.
This experimental setup is shown in Fig. 2 a. Friction data
was recorded over a speed range from 0.002 to 0.35 ms−1 with an
applied load of 0.2 N.
2.5. Laser induced fluorescence measurements
The custom-built Laser Induced Fluorescence (LIF) microscope is
shown by the photograph and schematic in Fig. 2b. It comprises an LED
light source, which produces a beam that is focussed through the
transparent PDMS specimen onto the contact interface. For certain
tests, the proteins in the lubricating saliva were tagged with a dye,
fluorescein isothiocyanate, (FITC) in order for them to fluoresce when
excited by the LED. The emitted light is filtered and collected by a highspeed
EMCCD camera. For film thicknesses greater than 200 nm, the
recorded intensity of the fluorescence light emitted from the contact is
proportional to the thickness of the liquid in the interface. This means
that the images acquired by the camera represent maps showing the
distribution of proteins in the contact. Further details of the fluorescence
technique can be found in (Myant, Reddyhoff, & Spikes, 2010),
(Reddyhoff, Choo, Spikes, & Glovnea, 2010).
3. Results
3.1. Indentation and roughness results
Fig. 3 shows the force displacement curves for the four materials
during the indentation tests using the Biomomentum Mach-1 rig.
Equations (1)–(3) were applied to this data giving the Young's Modulus
values shown in Table 1. As, expected the Young's Modulus of the
porcine tongue at 3.5 kPa is lower than other measurements of biological
tissue found in the literature – e.g. human skin: 25–101 kPa
(Akhtar, Sherratt, Cruickshank, & Derby, 2011), human muscle:∼7 kPa
(McKee et al., 2011). These values were most closely mimicked by the
agarose with a modulus 66 and 174 kPa for the 1 and 2% concentrations.
The modulus of the PDMS was nearly two orders of magnitude
Fig. 1. Schematic diagram of indentation setup. higher than the biological sample.
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
12
Table 2 shows the surface roughness measurements for each of the
specimens, which are separated by approximately an order of magnitude
(PDMS < Agar < Tongue). The effect this variation has on friction,
however, is counteracted by the different stiffness which increases
in the opposite sense (e.g. the asperities on the tongue surface are
readily flattened). The range of values displayed for each measurement
refers to the standard error, which is due to the variation over surface of
the specimens, rather than any error in the measurement.
3.2. Protein staining results
When incubated for an hour at room temperature with whole mouth
saliva from a single subject, neither agarose nor PDMS bound significant
amounts protein as shown by Coomassie Brilliant Blue (CBB)
staining of all proteins as shown in Fig. 4. Small amounts of amylase,
the single most abundant protein in saliva, is the only protein apparent
(identity based on apparent molecular weight). When the same gel was
stained with Periodic Acid Schiff's (PAS), a stain for highly glycosylated
proteins, small amounts of muc5b and muc7 were visible in the agarose
gel but nothing in the sample eluted from PDMS. Immunoblotting for
muc7 using a specific antibody with sensitive chemiluminescent detection
again suggested agarose gel bound some mucins whereas PDMS
did not. Incorporating potentially muco-adhesive agents such as chitosan
and the lectin WGA (AWGL) into the agarose appeared to enhance
protein, and mucin in particular, binding to the agarose.
3.3. Friction results
In this section, Stribeck curves have plotted the speed on the x-axis
rather than the product of speed×viscosity as is customary (de Vicente
et al., 2006). This is because the viscosity of saliva, being highly non-
Newtonian, varies strongly as a function of shear rate (Rantonen &
Meurman, 1998) and is therefore not constant throughout each test.
Another obstacle in assuming a single viscosity is the inhomogeneous
and surface active nature of saliva means that it is not possible to assume
whether it is the high viscosity proteins or just water molecules
are entrained between the surfaces.
Fig. 5 shows the variation in friction with sliding speed for the
agarose-glass contact. Under unlubricated and water lubricated conditions,
this substrate exhibits lower friction, due to the agarose being a
hydrogel which releases water when compressed. When agarose was
submerged in water it exhibits Stribeck curve behaviour with higher
friction at low speeds which decreases rapidly with speed due to the
formation of an elastohydrodynamic film. However, when lubricated
with saliva, the friction behaviour is completely unchanged compared
to pure water.
Fig. 6 shows the variation in friction with sliding speed for the
PDMS-glass contact under different conditions. When the contact is
unlubricated, the coefficient of friction remains between 3 and 4, due to
the strong adhesive interaction between the surfaces. The increase
followed by a decrease in friction with sliding speed may be attributed
to the viscoelastic properties of the elastomer (the friction that arises
Fig. 2. Laser Induced Fluorescence setup, a) Photograph, b) Schematic diagram of indentation setup.
Fig. 3. Force-displacement curves for each material obtained during indentation.
Table 1
Young's modulus results for each test material in kPa.
Porine tongue Agarose (1%) Agarose (2%) PDMS
3.46 66.4 174 2270
Table 2
Surface roughness results for each test material. Examples of the corresponding
surface topographies are shown in the appendix.
Roughness (nm)
Average (Ra) RMS (Rq)
Tongue 5480 ± 667 656 ± 403
PDMS 10.1 ± 0.16 13.1 ± 0.23
Agar 1% 399 ± 91 514 ± 109
Agar 2% 325 ± 14 420 ± 18
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
13
from the deformation of the PDMS varies as a function of speed due to
its viscoelastic response).
At the lowest speed, the dry and water submerged friction values are
similar, showing that no water is present between the surfaces even
when submerged (i.e. no boundary film is formed). This is because the
speed is insufficient to separate the surfaces hydrodynamically and also
water molecules are not attracted to either the PDMS or glass surface. In
contrast, when flooded with whole mouth saliva, very low friction
(more than two orders of magnitude less than the dry case) is observed.
These observations are in agreement with those of Stokes and coworkers
(Bongaerts et al., 2007).
Fig. 7 shows the friction versus speed behaviour for the porcine
tongue sample. When lubricated with pure water, this sample shows
high boundary friction which reduces with speed due to lubricant entrainment.
In addition to this, the low speed boundary friction is significantly
reduced when lubricated with saliva when compared to
water. The shape of the dry, water and saliva lubricated curves are similar
for PDMS and tongue, however there was a significant difference
in terms of the magnitude of the friction.
3.4. Laser induced fluorescence results
An advantage of PDMS over both the tongue and the agarose samples
is that it is transparent, which enables imaging of the contact. To
demonstrate this, the LIF microscopy results in Fig. 8 show the build-up
and flow of FITC-dyed saliva proteins within the contact during sliding.
Images a-d in this figure are intensity maps of the contact showing the
distribution of proteins (these are frames taken from the videos provided
as Supplementary Material). Here, bright colours represent high
concentrations of proteins in the contact and the dark blue circular
region is the pressurised contact area. Proteins agglomerations of
varying morphologies are evident as they are entrained due the sliding
motion from the inlet at the top of the figure to the outlet at the bottom.
The figure also plots the variation in friction over time alongside a
measure of the fluorescence intensity within the contact. The latter was
obtained by counting the number of pixels within the contact with an
intensity greater than the test average (using a Matlab program).
There is a clear correlation between the coefficient of friction and
the presence of proteins within the contact zone. This is highlighted by
Fig. 4. Staining showing binding of proteins to each mimic surface. (note: the whole mouth saliva sample is labelled WMS).
Fig. 5. Friction versus sliding speed for an agarose disc pressed against a stationary silica hemisphere with a force of 0.2 N. a) linear scale, b) log scale.
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
14
the calculated cross correlation coefficient of 0.872 and the visible
occurrence of peaks (shown by ?) in one single coinciding with troughs
(shown by v) in the other signal, and vice versa.
4. Discussion
The stiffness of the tongue sample is far closer to that of the agarose
than the PDMS. This means that, for the agarose contact, the area and
pressure match more closely those found in the mouth. Moreover, if this
is considered in isolation, it suggests the boundary friction and hydrodynamic
film thickness separating the surfaces for the agarose are more
realistic. But it is important also to consider the mucosal pellicle for
lubrication of oral surfaces by saliva and to implement this we added
mucoadhesive components to agarose gels to enhance mucin binding.
In some ways this appeared successful with greater amounts of all
salivary proteins, including the two mucins (muc 5b and muc7),
binding in greater amounts to the chitosan and WGA lectin containing
agarose, shown by protein staining. However, there was little effect on
the tribology when the mucoadhesive agarose was compared to agarose
alone. Indeed, there was almost no difference between agarose lubricated
by water or saliva. This suggests that this substrate is already
being lubricated by the surface itself – probably water being expelled
from the hydrogel under the pressure of the tribo-pairing. Furthermore,
the interchangeability of the curves for water and saliva lubricated
contacts in the full film regime, where friction is dominated by viscous
drag, suggests high viscosity saliva proteins are not even being entrained
into the contact at entrainment high speed.
The behaviour of PDMS showed much stronger protein interactions.
When sliding at low speed (∼0.1 mm/s) in the boundary regime (i.e.
when there is insufficient hydrodynamic entrainment of liquid to separate
the surfaces), the coefficient of friction for PDMS when lubricated
by saliva is two orders of magnitude lower than when lubricated
with pure water (∼0.01 vs ∼2). Since saliva is made up of
99.5% water and<0.5% protein molecules, this shows the proteins are
highly effective surface active lubricating additives, which adhere to
PDMS and oral surfaces to produce a lubricous low shear strength interface.
More specifically, PDMS, like the tongue is hydrophobic
(Dresselhuis et al., 2007) and due to its charged eOeSi(CH3)2- group it
attracts proteins indiscriminately (Phillips & Cheng, 2005) (in fact the
adherence of biological proteins to PDMS is a problematic occurrence in
biological lab-on-chip systems (Phillips & Cheng, 2005)). The viscosity
difference between water and saliva (0.89 cP and ∼5 cP (Rantonen &
Meurman, 1998)) is insufficient to explain this difference.
It could also be hypothesised that the elasticity of the bulk saliva
may be responsible for the differences in the hydrodynamic/rheological
response of the PDMS compared to water. However, at such low speeds
elasticity should not play a role. Furthermore, as shown, the friction is
strongly affected by the chemistry of sample surface, which would not
be the case under full film hydrodynamic lubrication. Finally, as shown
by Davies et al., the elasticity of resting saliva, as tested here, is significantly
lower than that of acid stimulated saliva (Davies, Wantling, &
Stokes, 2009).
The shape of the dry, water and saliva lubricated curves for tongue
are most similar to those of PDMS, which supports the latter's use an
oral mimic. However, there was a significant difference in terms of the
magnitude of the friction. Under dry, unlubricated conditions, the
Fig. 6. Friction versus sliding speed for PDMS disc pressed against a stationary silica hemisphere with a force of 0.2 N. a) linear scale, b) log scale.
Fig. 7. Friction versus sliding speed for porcine tongue pressed against a stationary silica hemisphere with a force of 0.2 N. a) linear scale, b) log scale.
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
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PDMS shows a friction coefficient of around 3.5 in contrast to 1.5 for
the tongue sample. When water is replaced with saliva, the PDMS
friction reduces to ∼0.02 while the tongue sample only falls to 0.25.
This difference in friction coefficient magnitude between PDMS and
tongue, under low speed conditions, when the surfaces are in contact,
can be analysed as follows. As predicted by Schallamach (Schallamach,
1958) and Roberts (Barquins & Roberts, 2000), using Hertz theory, the
coefficient of friction under dry/boundary lubrication conditions (i.e.
when not liquid is separating the surfaces) is given by:
μ = πS (9R/16E)2/3W−1/3 (4)
where R is the reduced radius, E is the elastic modulus, S is the interfacial
shear stress and W is the load. This shows that higher friction
coefficients arise in contacts between compliant materials, since these
deform and produce a larger contact area to be sheared. Equation (4)
can be used to calculate the shear stress within the contact, S, under
boundary lubrication conditions since all other quantities are known,
which gives values of 0.53 and 3.2 kPa for tongue and PDMS respectively.
This suggests that, when lubricated by saliva, the lower friction
of the PDMS surface arises due to its higher stiffness and smaller contact
area, but per unit area the protein covered tongue is in fact more easily
sheared. Another factor is the difference in roughness between the two
samples. Under dry conditions, the lower roughness of the PDMS increases
the real contact areas and hence adhesion, whereas under
protein lubrication lower roughness aids the formation of a complete
surface film.
The highly lubricious nature of the saliva proteins and their adherence
to the PDMS surface are confirmed by the in-contact LIF results.
In addition to demonstrating the effectiveness of this technique to
study saliva protein entrainment, these results shed light on the details
of this intermittent process. More specifically, the observed highly
transient nature of the protein entrainment is similar to that demonstrated
by Fan et al. (Fan, Myant, Underwood, Cann, & Hart, 2011) who
attributed the build-up and breakdown of proteins within the contact to
the following inlet aggregation mechanism. Due to the contact geometry
and flow path of the lubricant, proteins are transported into the
contact inlet. Some of these proteins attach to the converging surfaces.
Over time additional proteins become entangled with the surface protein
branches, forming a larger protein mass in the inlet zone. A critical
point is then reached where surface friction forces and lubricant hydrodynamic
forces cause this protein mass to breakdown, allowing
large agglomerate of proteins to be dragged into the contact zone. This
can be observed in Fig. 8, highlighted on the plot with a * symbol,
where peak protein presence occurs with minima in friction coefficient.
Fig. 8. Laser Induced Fluorescence results from a sliding test of silica hemisphere loaded against PDSM disc and lubricated with FITC dyed saliva. a) Intensity maps
for unloaded contact, b) to d) Intensity maps during sliding, e) Variation of friction coefficient (blue) and fluorescence signal (orange), obtained by counting number
of pixels with intensity greater than the test average. To highlight the correlation, example peaks are labelled with ˆ and example troughs are labelled with v. The
arrows around 400 s highlight symmetrical trends in the two signals. Note: the step changes in fluorescence observed at 5 and 440 s correspond to increase and
decrease in in-contact proteins during the loading and unloading of the contact. (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
G. Carpenter et al. Food Hydrocolloids 92 (2019) 10–18
16
The difference in lubricating properties of saliva compared to water
are assumed to relate to the salivary proteins such as mucins and statherin.
Mucins contribute to the viscosity of saliva which may aid the
hydrodynamic mode of lubrication (Bongaerts et al., 2007) whereas
statherin, a small surface active protein is regarded as a boundary lubricant
(Douglas et al) (Harvey, Carpenter, Proctor, & Klein, 2011),
although it is entirely possible that other proteins also contribute to the
lubricating properties.
5. Conclusions
From a surface chemistry point of view, PDMS is suitable at replicating
the oral mucosa, since, like the tongue, it is hydrophobic
(Dresselhuis, 2008) and its charged groups, which attract proteins
(Phillips & Cheng, 2005). This resulted in PMDS showing similar friction
versus speed trends to the biological sample. Agarose on the other
hand shows only a minor difference in friction when lubricated by
saliva versus water. This is attributed to the hydrated agarose surface
weakly adhering to the saliva proteins. The friction properties of
agarose did not improve even after the agarose was treated with mucoadhesive
components to enhance mucin binding.
Although PDMS rubbers have similar hydrophobic qualities to a
tongue, PDMS has an elastic modulus two orders of magnitude larger.
Furthermore, even if the degree of cross linking is limited the modulus
of PDMS reduces only to around 570 kPa (Wang et al., 2014) versus
3.4 kPa for tongue. This is significant shortcoming, since the stiffness of
the sample affects both the boundary friction (μ α E′−2/3 (Schallamach,
1958)) and the elastohydrodynamic film thickness (h α E′0.66 (de
Vicente et al., 2005)). There is also considerable variation in roughness
between the specimens tested, with agarose matching the tongue most
closely. However, the effect this has on friction is limited due to the incontact
flattening of the rougher materials, which have lower stiffness.
An advantage of PDMS is that being transparent it allows in-contact
imaging of saliva lubrication mechanisms. This was demonstrated using
laser induced fluorescence and the resulting strong correlation (0.87)
between friction and protein intensity signals confirms the lubricous
boundary film forming ability of saliva proteins. Protein aggregation
was shown to be highly transient in nature. The application of this
technique to study the tribological interactions between saliva and
foods and beverages in order to scientifically characterise mouthfeel
attributes is the subject of ongoing research.
Acknowledgements
S.K. Baier and R.V Potineni are employed by PepsiCo, Inc. The views
expressed in this research article are those of the authors and do not
necessarily reflect the position or policy of PepsiCo, Inc. The research
was funded by PepsiCo, Inc (grant number: P55310-1).
Appendix A. Supplementary data
Supplementary data to this article can be found online at 01.049.
Appendix. Surface topography measurements
Fig. A1. Surface topographies of the three materials, measured using a Veeco optical profilometer, a) porcine tongue, b) PDMS, c) agrose.
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順應(yīng)性口腔模擬唾液潤(rùn)滑的研究
由于倫理問(wèn)題和獲取生物組織的困難，尋找可作為研究用替代試驗(yàn)介質(zhì)的合成彈性體非常重要。這方面的一個(gè)重要例子是摩擦測(cè)試，以了解在食用過(guò)程中（如糖漿、身體和清潔劑）口感屬性背后的機(jī)制，這需要一個(gè)口頭模擬。為了評(píng)估可能的材料對(duì)模擬口腔表面的適用性，通過(guò)將半球形硅膠針加載并滑動(dòng)到聚二甲基硅氧烷（PDMS）、瓊脂糖或豬舌樣品上，產(chǎn)生滑動(dòng)接觸。測(cè)量摩擦，并根據(jù)試樣的彈性模量計(jì)算彈流動(dòng)力膜厚度，該彈性模量采用壓痕法測(cè)量。以唾液和純水為潤(rùn)滑液進(jìn)行試驗(yàn)，并將結(jié)果與未潤(rùn)滑條件進(jìn)行比較。PDMS在蛋白質(zhì)粘附方面很好地模擬了舌頭，當(dāng)用唾液和水潤(rùn)滑時(shí)，兩種樣品的摩擦力都顯著降低，而瓊脂糖在唾液和水潤(rùn)滑條件下沒有差異。這歸因于PDMS的eOeSi（CH3）2-基團(tuán)，與水合瓊脂糖表面相比，它為唾液蛋白分子提供了良好的粘附性。然而，PDMS（2.2mpa）的測(cè)量模量明顯大于舌（3.5kpa）和瓊脂糖（66-174kpa）。這會(huì)影響低速滑動(dòng)時(shí)的表面（邊界）摩擦力，以及高速滑動(dòng)時(shí)的夾帶彈流膜厚度。利用透明的PDMS樣品，我們還使用熒光顯微鏡來(lái)監(jiān)測(cè)滑動(dòng)過(guò)程中接觸到的染色標(biāo)記唾液蛋白的積累和流動(dòng)。結(jié)果表明，唾液中蛋白質(zhì)的摩擦強(qiáng)度信號(hào)與平均蛋白質(zhì)強(qiáng)度信號(hào)之間存在很強(qiáng)的相關(guān)性（互相關(guān)系數(shù)=0.87），從而證實(shí)了唾液蛋白質(zhì)的潤(rùn)滑邊界膜形成性質(zhì)。這是研究口感機(jī)制的有力方法。一。由于倫理問(wèn)題和獲取生物組織的困難，有必要尋找可作為研究用替代試驗(yàn)介質(zhì)的合成彈性體。這方面的一個(gè)關(guān)鍵例子是摩擦測(cè)試，以了解在食用過(guò)程中（如糖漿、身體和清潔劑）口感屬性背后的機(jī)制，這需要口頭模擬。這一點(diǎn)很重要，因?yàn)槭称泛惋嬃系目山邮苄栽诤艽蟪潭壬先Q于其口感，而口感是摩擦學(xué)和流變學(xué)過(guò)程的結(jié)果（Stokes、Boehm和Baier，2013）。此外，對(duì)這些過(guò)程的不了解目前限制了健康配方的開發(fā)，這些配方可以復(fù)制食品，同時(shí)減少脂肪等成分（Dresselhuis，2008），（Drewnowski，1997）。在模擬口腔粘膜進(jìn)行食品和飲料的體外摩擦學(xué)研究時(shí)，必須考慮粘膜膜。與牙齒上獲得的琺瑯質(zhì)膜一樣，這是唾液蛋白的一個(gè)子集，專門與口腔上皮細(xì)胞結(jié)合（Gibbins、Proctor、Yakubov、Wilson和Carpenter，2014）。與獲得性釉質(zhì)膜不同，粘膜膜主要由黏蛋白和分泌性IgA組成。這一層是由唾液粘蛋白（muc5b和muc7）與口腔上皮細(xì)胞表達(dá)的膜結(jié)合粘蛋白（muc1）相互作用驅(qū)動(dòng)的（Vijay，Inui，Dodds，Proctor和Carpenter，2015）。粘蛋白是一種大的高度糖基化的蛋白質(zhì)，在初分泌時(shí)能保留相當(dāng)數(shù)量的水（Corfield，2015）。因此，除了唾液潤(rùn)滑表面外，在表面附近還有一個(gè)水凝膠狀層。然而，唾液在體外試驗(yàn)中經(jīng)常被忽略，因?yàn)樗徽J(rèn)為不方便收集足夠數(shù)量的唾液，或者被認(rèn)為太復(fù)雜而無(wú)法給出一致的結(jié)果。以前，口腔粘膜是用玻璃或其他硬基質(zhì)模擬的（Chen&Stokes，2012）。近年來(lái)，隨著赫茲力學(xué)的引入，彈性基底被廣泛應(yīng)用于軟摩擦學(xué)領(lǐng)域。在Dresselhuis等人的一項(xiàng)重要調(diào)查中。（Dresselhuis等人，2007），豬舌的表面特征與PDMS進(jìn)行了比較。他們的研究結(jié)論是，由于口腔粘膜和PDMS橡膠，即使表面結(jié)構(gòu)能夠再現(xiàn)生物場(chǎng)景，在摩擦學(xué)實(shí)驗(yàn)中也不能互換，PDMS在表面特征上與舌表面的表現(xiàn)不同。然而，這篇被廣泛引用的論文有一個(gè)嚴(yán)重的缺點(diǎn)，那就是它只使用乳狀液作為潤(rùn)滑劑，而忽略了唾液的相互作用。在生物表面進(jìn)行的其他工作，但是沒有唾液的存在包括亞當(dāng)斯等人的研究。（Adams、Briscoe和Johnson，2007）和Tang等人。（Tang&Bhushan，2010），（Tang，Bhushan，&Ge，2010）人類皮膚潤(rùn)滑特性研究。亞當(dāng)斯等人。使用光滑的玻璃或聚丙烯，球形探針頂著人類前臂滑動(dòng)，而Tang等人。用剃過(guò)的豬皮。結(jié)果報(bào)告了一系列潤(rùn)滑條件，但重復(fù)性的測(cè)試難以實(shí)現(xiàn)。Prinz等人。（Prinz，de Wijk和Huntjens，2007）研究了用人類唾液潤(rùn)滑的兩個(gè)豬粘膜表面之間的摩擦特性。然而，缺乏數(shù)據(jù)，沒有對(duì)不同的組分材料進(jìn)行比較。在大多數(shù)研究中，選擇交聯(lián)聚二甲基硅氧烷（PDMS）是因?yàn)槠鋸椥蕴匦?、易于處理和相?duì)較低的硬度，可與軟生物組織相比（Cox、Driessen、Boerboom、Bouten和Baaijens，2008；Khanafer、Duprey、Schlicht和Berguer，2009）。PDMS被用作一個(gè)（de Vicente，Stokes，&a Spikes，2006），（Malone，Appelqvist，&Norton，2003），（Tang&Bhushan，2010），（Tang et al.，2010）或兩者（Stokes，Bongaerts，&Rossetti，2007），（Lee&Spencer，2005），（Bongaerts，Fourtouni，&Stokes，在摩擦學(xué)接觸中保持低接觸壓力并為等粘彈性流體動(dòng)力潤(rùn)滑（I-EHL）創(chuàng)造條件。PDMS的一個(gè)關(guān)鍵優(yōu)點(diǎn)是易于制造，這也促進(jìn)了PDMS的廣泛應(yīng)用。在交聯(lián)之前，PDMS可以澆鑄成幾乎任何所需形狀的合適模具。PDMS的其他優(yōu)點(diǎn)包括其生理惰性、可用性、單位成本低以及良好的熱穩(wěn)定性和氧化穩(wěn)定性。PDMS是一種透明的硅基有機(jī)聚合物，用于在許多摩擦學(xué)研究中代表生物材料（例如（Bongaerts等人，2007年）（Dresselhuis等人，2007年），（De Vicente，Spikes和Stokes，2004年）。由于其*的低玻璃化轉(zhuǎn)變溫度（Tg≈125°C），其彈性模量E≈0.57–3.7兆帕（取決于交聯(lián)程度）（Wang、Volinsky和Gallant，2014）（Lótters、Olthuis、Veltink和Bergveld，1999）。PDMS的表面是疏水的，因?yàn)槠渲貜?fù)的eOeSi（CH3）2-基團(tuán)（Adams等人，2007），但可以通過(guò)等離子體清洗使其親水。除此之外，PDMS在聚合物微流控領(lǐng)域（如Eddings，Johnson，&Gale，2008）得到了廣泛的應(yīng)用，該領(lǐng)域的研究成果可以在本研究中得到有益的應(yīng)用。PDMS的摩擦學(xué)特性現(xiàn)在已經(jīng)相當(dāng)清楚了。Vorvolakos和Chaudhury（Vorvolakos和Chaudhury，2003）研究了分子量和試驗(yàn)溫度對(duì)PDMS與金屬表面純滑動(dòng)接觸摩擦的影響。Bongaerts等人。（Bongaerts等人，2007）研究了PDMS表面粗糙度對(duì)生物聚合物和水溶液潤(rùn)滑性能的影響。PDMS，像大多數(shù)彈性體表面，本質(zhì)上是疏水的，但是可以采用氧化處理來(lái)產(chǎn)生親水表面。希爾堡等人。（Hillborg和Gedde，1998），（Hillborg、Sandelin和Gedde，2001）和Schneemilch等人。（SnEnMeLCH和奎克，2007）通過(guò)幾種技術(shù)研究了PDMS在氧化前后的潤(rùn)濕性，研究了交聯(lián)密度對(duì)氧化的影響。de Vicente等人。（de Vicente，斯托克斯，&Spikes，2005）研究了PDMS表面改性對(duì)其水潤(rùn)滑性能的影響。然而，對(duì)于PDMS作為生物表面模型的適用性仍存在一些爭(zhēng)論，并且PDMS在唾液條件下被檢測(cè)的例子很少。第二種軟基質(zhì)被認(rèn)為是模擬口腔粘膜的潛在基質(zhì)是瓊脂糖。瓊脂糖是從海藻中提取的瓊脂中缺少瓊脂蛋白的部分，由β-1,3-連接的α-半乳糖和α-1,4-連接的3,6-脫水-αL-半乳糖殘基（Normand，Lootens，Amici，puckennet，&Aymard，2000）組成，用于制造水凝膠樣基質(zhì)。瓊脂糖的順應(yīng)性隨濃度變化很大，楊氏模量從1.5千帕到3千帕不等（Benkherourou、Rochas、Tracqui、Tranqui和Guméry，1999年），（Normand等人，2000年），（Chen、Suki和An，2004年）。此外，瓊脂糖還具有懸浮培養(yǎng)細(xì)胞的能力，因此被用于組織培養(yǎng)系統(tǒng)（Chen等人，2004）。這種特性的結(jié)合使得瓊脂糖在生物醫(yī)學(xué)研究中成為一種有吸引力的選擇，例如，作為軟骨模擬物（Saris等人，2000），或作為磁共振彈性成像的模型材料（Muthupillai等人，1995）。因此，令人驚訝的是，瓊脂糖在摩擦學(xué)研究中的應(yīng)用很少，而且似乎*被忽略作為一種口服模擬物。費(fèi)爾南德斯·法雷斯研究了其摩擦行為，但在葡萄糖和甘油潤(rùn)滑而非唾液潤(rùn)滑下進(jìn)行了研究（費(fèi)爾南德斯·法雷斯和諾頓，2015）。Shewan等人。近還研究了瓊脂糖的潤(rùn)滑性能，但它是懸浮液中的顆粒，而不是基質(zhì)（Shewan&Stokes，2015）。因此，能夠模擬口腔粘膜表面是非常重要的，為此，人們對(duì)各種材料進(jìn)行了研究。然而，它們很少與實(shí)際的生物材料進(jìn)行比較（可能是因?yàn)樗鼈冊(cè)趤?lái)源、保存和固定方面的困難），而且?guī)缀鯊膩?lái)沒有被唾液潤(rùn)滑過(guò)。為了解決這一問(wèn)題，本研究對(duì)聚二甲基硅氧烷（PDMS）、瓊脂糖和豬舌的摩擦和膜厚性能進(jìn)行了表征，目的是評(píng)估它們作為摩擦學(xué)試驗(yàn)的口腔模擬物的適用性。特別注意這些底物的順應(yīng)性和蛋白質(zhì)結(jié)合行為。2。試驗(yàn)方法2.1。樣品制備PDMS樣品使用道康寧市市面上可買到的Sylgard 184試劑盒進(jìn)行模壓，該試劑盒含有一種堿和固化劑，以在23°C下產(chǎn)生楊氏模量為1.84兆帕的材料。瓊脂糖凝膠是通過(guò)溶解粉狀瓊脂糖（Sigma-Aldrich，Poole，（UK）在1%或2%w/v的水中。為了幫助溶解，將溶液加熱至90°C，然后在35°C左右冷卻至低于螺旋轉(zhuǎn)變的溫度。此時(shí)，瓊脂糖形成凝膠，由無(wú)限的三維纖維螺旋網(wǎng)絡(luò)組成（諾曼德等人，2000）。在收集之前，受試者1小時(shí)內(nèi)不進(jìn)食和飲水。從單個(gè)受試者中收集靜止的全口唾液（WMS），方法是將其流涎到預(yù)先稱重的試管中，保存在冰上。收集后，對(duì)唾液進(jìn)行短暫離心（3000 g，持續(xù)3分鐘）以去除脫落的細(xì)胞和其他碎片。當(dāng)天采集豬舌并進(jìn)行檢測(cè)。它的下側(cè)被移除，形成一個(gè)平行的板。然后，使用氰基丙烯酸酯粘合劑將該試樣粘合到平板上，并安裝在摩擦裝置中。G、 Carpenter等人。食品水膠體92（2019）10–18 11 2.2。壓痕和表面粗糙度測(cè)量在Mach 1試驗(yàn)臺(tái)（加拿大拉瓦爾市Biomomentum公司）上進(jìn)行壓痕試驗(yàn)，測(cè)量每個(gè)樣品材料的彈性模量。這包括在三次重復(fù)試驗(yàn)期間，用半徑為3.175 mm的球形壓頭以1 mm/s的速度壓入樣品，同時(shí)測(cè)量法向力和垂直位移。采用1.5 N單軸測(cè)壓?jiǎn)卧獪y(cè)量75μm的法向力，用0.1μm分辨率的鉆臺(tái)移動(dòng)臺(tái)測(cè)量垂直位移，每個(gè)樣品的穿透深度為0.6mm，瓊脂糖為1% W/V，0.4mm為0.4mm。這是根據(jù)Van Dommelen等人（Van Dommelen、Van der Sande、Hrapko和Peters，2010）的建議完成的，即如果壓痕深度限制在樣品厚度的10%以下，樣品厚度不會(huì)對(duì)數(shù)據(jù)產(chǎn)生顯著影響。然而，考慮到樣本有限厚度的公式（Hayes、Keer、Herrmann和Mockros，1972）被用于計(jì)算楊氏模量。將接觸力學(xué)方程擬合到數(shù)據(jù)中，得到楊氏模量，具體地說(shuō)，χ=a d R 2（1）=−k P v a G d（1）4（2），其中d是壓頭的位移，R是壓頭的半徑，a是接觸區(qū)域的半徑，P是施加的載荷，G是剪切模量，v是泊松比。試驗(yàn)示意圖見圖1。用Mach-1運(yùn)動(dòng)軟件記錄反作用力和壓頭位移，分別以P和d形式輸入上述方程。假設(shè)泊松比等于0.5（不可壓縮材料）。試樣的高度h和壓頭半徑R也已知。表2（Hayes et al.，1972）中給出了a、h和ν不同值的x和k值。在用方程（1）進(jìn)行曲線擬合期間，估計(jì)接觸區(qū)域a的半徑。一旦擬合算法收斂，根據(jù)剪切模量G計(jì)算楊氏模量E，使用方程E=2G（1+v）（3）使用Veeco光學(xué)輪廓儀在表面上測(cè)量每個(gè)試樣的粗糙度三次（每個(gè)試樣在不同位置）。2.3條。蛋白質(zhì)染色測(cè)量SDS-PAGE（十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳）用于評(píng)估不同蛋白質(zhì)與口腔模擬物表面的結(jié)合程度。這種免疫印跡技術(shù)以帶有特定染料的樣品中的蛋白質(zhì)為靶點(diǎn)，并通過(guò)凝膠測(cè)量它們?cè)谕饧与妶?chǎng)作用下的進(jìn)展。就這樣，在不同分子量的樣品中分離出不同的蛋白質(zhì)。染色包括在室溫下用一名受試者的全口唾液孵育一小時(shí)?？捡R斯亮藍(lán)（CBB）染色所有蛋白。此外，采用周期性酸Schiff's（PAS）對(duì)高糖基化蛋白進(jìn)行染色，并用特異性抗體對(duì)唾液蛋白muc7進(jìn)行敏感的化學(xué)發(fā)光檢測(cè)。從每個(gè)口腔模擬表面的表面取下樣本，并以這種方式測(cè)試哪些蛋白質(zhì)存在。2.4條。摩擦測(cè)量使用CETR（美國(guó)坎貝爾）制造的UMT2（通用材料測(cè)試儀）將直徑為5mm的二氧化硅半球加載并滑動(dòng)到柔順圓盤試樣上，產(chǎn)生接觸。該設(shè)備在銷-盤模式下運(yùn)行，使PDMS試樣旋轉(zhuǎn)，而二氧化硅半球保持靜止。下試樣位于旋轉(zhuǎn)臺(tái)上，能夠（經(jīng)過(guò)某些修改）以0.01轉(zhuǎn)/分到4000轉(zhuǎn)/分的速度運(yùn)行。摩擦力（Fx）和法向載荷（Fz）是用應(yīng)變計(jì)測(cè)量的，該應(yīng)變計(jì)與固定硅半球試件上方的殼體連接。為此，選擇了靈敏的低負(fù)載傳感器，Fx和Fz的測(cè)量范圍分別為±0.65n和±6n。該實(shí)驗(yàn)裝置如圖2a所示。在0.002至0.35 ms-1的速度范圍內(nèi)，在0.2 N.2.5的負(fù)載下記錄摩擦數(shù)據(jù)。激光誘導(dǎo)熒光測(cè)量定制的激光誘導(dǎo)熒光（LIF）顯微鏡如圖2b中的照片和示意圖所示。它包括一個(gè)LED光源，該光源產(chǎn)生的光束通過(guò)透明PDMS樣品聚焦到接觸界面上。在某些測(cè)試中，潤(rùn)滑唾液中的蛋白質(zhì)用染料異硫氰酸熒光素（FITC）標(biāo)記，以便它們?cè)诒?/span>LED激發(fā)時(shí)發(fā)出熒光。發(fā)射的光被高速EMCCD攝像機(jī)過(guò)濾和收集。當(dāng)薄膜厚度大于200nm時(shí)，從接觸點(diǎn)發(fā)射的熒光光的記錄強(qiáng)度與界面中液體的厚度成正比。這意味著攝像機(jī)采集的圖像代表了顯示接觸中蛋白質(zhì)分布的地圖。熒光技術(shù)的更多細(xì)節(jié)可以在（Myant，Reddyhoff，&Spikes，2010），（Reddyhoff，Choo，Spikes，&Glovnea，2010）中找到。三。結(jié)果3.1。壓痕和粗糙度結(jié)果圖3顯示了使用Biomomentum Mach-1試驗(yàn)機(jī)進(jìn)行壓痕試驗(yàn)期間四種材料的力-位移曲線。方程（1）–（3）應(yīng)用于該數(shù)據(jù)，給出了表1所示的楊氏模量值。正如預(yù)期的那樣，3.5千帕下豬舌的楊氏模量低于文獻(xiàn)中發(fā)現(xiàn)的其他生物組織測(cè)量值，例如人類皮膚：25-101千帕（Akhtar、Sherratt、Cruickshank和Derby，2011年），人類肌肉：7千帕（McKee等人，2011年）。在1%和2%濃度下，這些數(shù)值 于模數(shù)為66和174kpa的瓊脂糖。PDMS的模量幾乎是圖1的兩個(gè)數(shù)量級(jí)?？s進(jìn)設(shè)置示意圖。高于生物樣本。G、 Carpenter等人。食品水膠體92（2019）10 - 18 12表2示出了每一個(gè)樣品的表面粗糙度測(cè)量，它們被分離大約一個(gè)數(shù)量級(jí)（PDMS<瓊脂<舌）。然而，這種變化對(duì)摩擦力的影響被相反意義上增加的不同剛度抵消（例如，舌面上的小突起容易變平）。每次測(cè)量顯示的數(shù)值范圍是指標(biāo)準(zhǔn)誤差，這是由于試樣表面的變化而不是測(cè)量中的任何誤差引起的。3.2條。蛋白質(zhì)染色結(jié)果：當(dāng)在室溫下與來(lái)自單個(gè)受試者的全口唾液孵育1小時(shí)時(shí)，所有蛋白質(zhì)的考馬斯亮藍(lán)（CBB）染色顯示，瓊脂糖和PDMS均未結(jié)合大量蛋白質(zhì)，如圖4所示。少量的淀粉酶是唾液中 的單一蛋白質(zhì)，是 明顯的蛋白質(zhì)（根據(jù)表觀分子量確定）。當(dāng)用高糖基化蛋白的周期性酸Schiff's（PAS）染色同一凝膠時(shí)，瓊脂糖凝膠中可見少量muc5b和muc7，但PDMS洗脫的樣品中沒有。用特異性抗體和敏感的化學(xué)發(fā)光檢測(cè)對(duì)muc7進(jìn)行免疫印跡，再次表明瓊脂糖凝膠結(jié)合了一些粘蛋白，而PDMS沒有。在瓊脂糖中加入潛在的粘液粘合劑，如殼聚糖和凝集素WGA（AWGL），似乎可以增強(qiáng)蛋白質(zhì)，特別是粘蛋白與瓊脂糖的結(jié)合。3.3條。摩擦結(jié)果在這一段中，StbBek曲線繪制了X軸上的速度，而不是慣用的速度×粘度的乘積（de Vicente等人，2006）。這是因?yàn)橥僖旱恼扯仁歉叨确桥ｎD的，隨著剪切速率的變化而變化很大（Rantonen&Meurman，1998），因此在每次測(cè)試中都不是恒定的。假設(shè)單一粘度的另一個(gè)障礙是唾液的非均質(zhì)性和表面活性，這意味著無(wú)法假設(shè)唾液表面夾帶的是高粘度蛋白質(zhì)還是水分子。圖5顯示了瓊脂糖玻璃接觸的摩擦隨滑動(dòng)速度的變化。在無(wú)潤(rùn)滑和水潤(rùn)滑條件下，由于瓊脂糖是一種水凝膠，壓縮時(shí)會(huì)釋放水分，因此這種基質(zhì)表現(xiàn)出較低的摩擦力。當(dāng)瓊脂糖浸沒在水中時(shí)，它表現(xiàn)出Stribeck曲線行為，低速時(shí)摩擦更大，由于形成彈性流體動(dòng)力膜，摩擦隨速度迅速減小。然而，當(dāng)用唾液潤(rùn)滑時(shí)，與純水相比，摩擦行為*沒有變化。圖6示出在不同條件下PDMS玻璃接觸的摩擦隨滑動(dòng)速度的變化。當(dāng)接觸是無(wú)潤(rùn)滑的，摩擦系數(shù)保持在3和4之間，由于表面之間的強(qiáng)粘著相互作用。隨著滑動(dòng)速度的增加，摩擦隨之減少，這可歸因于彈性體的粘彈性特性（圖2所示的摩擦）。激光誘導(dǎo)熒光裝置，a）照片，b）壓痕裝置示意圖。圖3。壓痕過(guò)程中獲得的每種材料的力-位移曲線。表1各試驗(yàn)材料的楊氏模量結(jié)果（單位：kPa）。多孔舌瓊脂糖（1%）瓊脂糖（2%）PDMS 3.46 66.4 174 2270表2每種試驗(yàn)材料的表面粗糙度結(jié)果。附錄中給出了相應(yīng)的地表地形圖示例。粗糙度（nm）平均值（Ra）RMS（Rq）舌片5480±667 656±403 PDMS 10.1±0.16 13.1±0.23瓊脂1%399±91 514±109瓊脂2%325±14 420±18 G。Carpenter等人。食品水膠體92（2019）10–18 13由于其粘彈性響應(yīng)，PDMS的變形隨速度的變化而變化）。在低速度下，干摩擦值和水下摩擦值相似，表明即使在水下，表面之間也不存在水（即沒有形成邊界膜）。這是因?yàn)樗俣炔蛔阋砸粤黧w動(dòng)力學(xué)的方式分離表面，而且水分子不被PDMS或玻璃表面所吸引。相反，當(dāng)全口唾液充滿時(shí)，可以觀察到非常低的摩擦力（比干燥情況下小兩個(gè)數(shù)量級(jí)以上）。這些觀察結(jié)果與斯托克斯和同事的觀察結(jié)果一致（Bongaerts等人，2007）。圖7顯示了豬舌樣品的摩擦與速度特性。當(dāng)用純水潤(rùn)滑時(shí)，該樣品顯示出高的邊界摩擦，由于潤(rùn)滑劑夾帶而隨著速度降低。此外，與水相比，用唾液潤(rùn)滑時(shí)，低速邊界摩擦顯著減少。PDMS和舌頭的干、水和唾液潤(rùn)滑曲線形狀相似，但摩擦大小有顯著差異。3.4條。激光誘導(dǎo)熒光結(jié)果PDMS比舌和瓊脂糖樣品的優(yōu)點(diǎn)是它是透明的，這使得接觸成像成為可能。為了證明這一點(diǎn)，圖8中的LIF顯微鏡結(jié)果顯示了FITC染色唾液蛋白在滑動(dòng)過(guò)程中在接觸面內(nèi)的積聚和流動(dòng)。圖中a-d是顯示蛋白質(zhì)分布的接觸強(qiáng)度圖（這些是從作為補(bǔ)充材料提供的視頻中獲取的幀）。在這里，明亮的顏色代表了接觸中高濃度的蛋白質(zhì)，深藍(lán)色的圓形區(qū)域是加壓接觸區(qū)域。由于從圖形頂部的入口到底部的出口的滑動(dòng)運(yùn)動(dòng)，不同形態(tài)的蛋白質(zhì)聚集明顯。該圖還繪制了摩擦隨時(shí)間的變化以及接觸內(nèi)熒光強(qiáng)度的測(cè)量值。后者是通過(guò)計(jì)算接觸點(diǎn)內(nèi)的像素?cái)?shù)，其強(qiáng)度大于測(cè)試平均值（使用Matlab程序）。摩擦系數(shù)與接觸區(qū)內(nèi)蛋白質(zhì)的存在有明顯的相關(guān)性。圖4突出顯示了這一點(diǎn)。染色顯示蛋白質(zhì)與每個(gè)模擬表面結(jié)合。（注：全口唾液樣本標(biāo)記為WMS）。圖5。瓊脂糖圓盤在0.2N力作用下對(duì)固定二氧化硅半球的摩擦力與滑動(dòng)速度a）線性標(biāo)度，b）對(duì)數(shù)標(biāo)度。G、 Carpenter等人。食品水膠體92（2019）10–18 14計(jì)算出的互相關(guān)系數(shù)為0.872，在一個(gè)信號(hào)中與另一個(gè)信號(hào)中的波谷（v）重合的可見波峰（以?表示）出現(xiàn)，反之亦然。四。舌苔的硬度與瓊脂糖的硬度比PDMS更接近。這意味著，對(duì)于瓊脂糖接觸，面積和壓力與口腔中發(fā)現(xiàn)的更接近。此外，如果將其單獨(dú)考慮，則表明分離瓊脂糖表面的邊界摩擦和流體動(dòng)力膜厚度更為真實(shí)。但是，考慮唾液對(duì)口腔表面的潤(rùn)滑作用的粘膜膜也是很重要的，為了實(shí)現(xiàn)這一點(diǎn)，我們?cè)诃傊悄z中添加了粘著成分，以增強(qiáng)粘蛋白結(jié)合。在某些方面，這似乎是成功的與更多的所有唾液蛋白質(zhì)，包括兩個(gè)粘蛋白（muc 5b和muc7），結(jié)合在殼聚糖和WGA凝集素含有瓊脂糖，顯示出蛋白質(zhì)染色。但粘著瓊脂糖與單獨(dú)瓊脂糖相比，對(duì)摩擦學(xué)性能影響不大。事實(shí)上，水或唾液潤(rùn)滑的瓊脂糖幾乎沒有區(qū)別。這表明，這種基底已經(jīng)被表面本身潤(rùn)滑了——可能是在摩擦學(xué)配對(duì)的壓力下，水從水凝膠中排出。此外，在摩擦主要由粘性阻力控制的全膜狀態(tài)下，水和唾液潤(rùn)滑接觸曲線的互換性表明，高粘度唾液蛋白甚至沒有以高速夾帶進(jìn)入接觸。PDMS的行為表現(xiàn)出更強(qiáng)的蛋白質(zhì)相互作用。當(dāng)在邊界區(qū)域（即當(dāng)液體的流體動(dòng)力夾帶不足以分離表面）以低速（∼0.1 mm/s）滑動(dòng)時(shí)，唾液潤(rùn)滑時(shí)的摩擦系數(shù)比純水潤(rùn)滑時(shí)的摩擦系數(shù)低兩個(gè)數(shù)量級(jí)（∼0.01 vs∼2）。由于唾液是由99.5%的水和<0.5%的蛋白質(zhì)分子組成的，這表明這些蛋白質(zhì)是高效的表面活性潤(rùn)滑添加劑，它們粘附在PDMS和口腔表面上，形成lubricous低剪切強(qiáng)度界面。更具體地說(shuō)，PDMS就像舌頭是疏水的一樣（Dresselhuis等人，2007），由于其帶電的eOeSi（CH3）2-基團(tuán)，它不分青紅皂白地吸引蛋白質(zhì)（Phillips&Cheng，2005）（事實(shí)上，生物蛋白質(zhì)粘附到PDMS是生物實(shí)驗(yàn)室芯片系統(tǒng)中的一個(gè)問(wèn)題（Phillips&Cheng，2005））。水和唾液之間的粘度差異（0.89 cP和∼5 cP（Rantonen和Meurman，1998））不足以解釋這種差異。也可以假設(shè)，與水相比，PDMS的流體動(dòng)力學(xué)/流變學(xué)響應(yīng)的差異可能是由唾液的彈性引起的。然而，在如此低的速度下，彈性不應(yīng)發(fā)揮作用。此外，如圖所示，摩擦受到樣品表面化學(xué)性質(zhì)的強(qiáng)烈影響，而在全膜流體動(dòng)力潤(rùn)滑條件下則不是這樣。后，如Davies等人所示，此處測(cè)試的靜止唾液的彈性明顯低于酸刺激唾液的彈性（Davies，Wangling，&Stokes，2009）。舌頭的干燥、水和唾液潤(rùn)滑曲線的形狀與PDMS 相似，這支持后者使用口腔模擬。然而，在摩擦力的大小方面存在顯著差異。在干燥、無(wú)潤(rùn)滑的條件下，如圖6所示。在0.2N的力作用下，PDMS圓盤對(duì)固定二氧化硅半球的摩擦與滑動(dòng)速度a）線性標(biāo)度，b）對(duì)數(shù)標(biāo)度。圖7。豬舌在0.2n.a）線性標(biāo)度，b）對(duì)數(shù)標(biāo)度力作用下對(duì)靜止二氧化硅半球的摩擦力與滑動(dòng)速度的關(guān)系。G、 Carpenter等人。食品水膠體92（2019）10–18 15 PDMS顯示摩擦系數(shù)約為3.5，而舌樣為1.5。當(dāng)用唾液代替水時(shí)，PDMS的摩擦力減小到0.02，而舌苔的摩擦力只有0.25。在低速條件下，當(dāng)表面接觸時(shí)，PDMS和舌片之間的摩擦系數(shù)大小差異可以分析如下。根據(jù)Schallamach（Schallamach，1958）和Roberts（Barquins&Roberts，2000）的預(yù)測(cè)，使用赫茲理論，干/邊界潤(rùn)滑條件下的摩擦系數(shù)（即，當(dāng)非液體分離表面時(shí)）由以下公式給出：μ=πS（9R/16E）2/3W−1/3（4），其中R是減小半徑，e是彈性模量，S為界面剪應(yīng)力，W為荷載。這表明，柔性材料之間的接觸會(huì)產(chǎn)生更高的摩擦系數(shù)，因?yàn)檫@些材料會(huì)變形并產(chǎn)生更大的接觸面積以進(jìn)行剪切。方程（4）可用于計(jì)算邊界潤(rùn)滑條件下接觸點(diǎn)S內(nèi)的剪應(yīng)力，因?yàn)樗衅渌慷家阎?，其中舌?/span>PDMS的值分別為0.53和3.2kpa。這表明，當(dāng)唾液潤(rùn)滑時(shí)，PDMS表面的摩擦力較低，這是因?yàn)樗挠捕容^高，接觸面積較小，但每單位面積的蛋白質(zhì)覆蓋舌頭實(shí)際上更容易剪切。另一個(gè)因素是兩個(gè)樣品之間粗糙度的差異。在干燥條件下，較低粗糙度的PDMS增加了實(shí)際接觸面積，從而增加了粘附力，而在蛋白質(zhì)潤(rùn)滑下，較低粗糙度有助于形成完整的表面膜。唾液蛋白的高潤(rùn)滑性及其與PDMS表面的粘附性已被接觸LIF結(jié)果證實(shí)。除了證明這項(xiàng)技術(shù)對(duì)研究唾液蛋白夾帶的有效性外，這些結(jié)果還揭示了這一間歇過(guò)程的細(xì)節(jié)。更具體地說(shuō)，觀察到的蛋白質(zhì)夾帶的高度瞬態(tài)性質(zhì)與Fan等人所證明的類似。（Fan，Myant，Underwood，Cann，&Hart，2011）他將接觸中蛋白質(zhì)的積累和分解歸因于以下的入口聚集機(jī)制。由于潤(rùn)滑劑的接觸幾何形狀和流動(dòng)路徑，蛋白質(zhì)被輸送到接觸入口。其中一些蛋白質(zhì)附著在會(huì)聚表面上。隨著時(shí)間的推移，更多的蛋白質(zhì)與表面的蛋白質(zhì)分支糾纏在一起，在入口區(qū)形成一個(gè)更大的蛋白質(zhì)團(tuán)。然后到達(dá)一個(gè)臨界點(diǎn)，在這個(gè)臨界點(diǎn)上，表面摩擦力和潤(rùn)滑劑流體動(dòng)力會(huì)導(dǎo)致蛋白質(zhì)團(tuán)分解，從而使蛋白質(zhì)的大團(tuán)塊被拖進(jìn)接觸區(qū)。這可以在圖8中觀察到，在帶有*符號(hào)的曲線圖中突出顯示，其中蛋白質(zhì)峰值出現(xiàn)在摩擦系數(shù)小的情況下。圖8。激光誘導(dǎo)熒光是在PDSM盤上加載二氧化硅半球并用FITC染色唾液潤(rùn)滑的滑動(dòng)試驗(yàn)的結(jié)果。a） 空載接觸的強(qiáng)度圖，b）至d）滑動(dòng)過(guò)程中的強(qiáng)度圖，e）摩擦系數(shù)（藍(lán)色）和熒光信號(hào)（橙色）的變化，通過(guò)計(jì)算強(qiáng)度大于試驗(yàn)平均值的像素?cái)?shù)獲得。為了突出相關(guān)性，示例峰值用ˆ標(biāo)記，示例波谷用v標(biāo)記。400 s左右的箭頭突出顯示兩個(gè)信號(hào)中的對(duì)稱趨勢(shì)。注：在5和440s觀察到的熒光階躍變化對(duì)應(yīng)于在接觸的加載和卸載過(guò)程中接觸蛋白的增加和減少。（為了解釋本圖圖例中對(duì)顏色的引用，請(qǐng)參閱本文的網(wǎng)絡(luò)版本。）G.Carpenter等人。食品類親水膠體92（2019）10–18 16唾液與水相比的潤(rùn)滑特性差異被認(rèn)為與唾液蛋白質(zhì)如粘蛋白和statherin有關(guān)。粘蛋白有助于唾液的粘度，這可能有助于流體動(dòng)力潤(rùn)滑模式（Bongaerts等人，2007），而statherin，一種小的表面活性蛋白被視為邊界潤(rùn)滑劑（Douglas等人）（Harvey，Carpenter，Proctor和Klein，2011），雖然*有可能其他蛋白質(zhì)也有助于潤(rùn)滑性能。5個(gè)。結(jié)論從表面化學(xué)的角度來(lái)看，PDMS適合于復(fù)制口腔粘膜，因?yàn)樗蜕囝^一樣，是疏水的（Dresselhuis，2008）及其帶電基團(tuán)，吸引蛋白質(zhì)（Phillips&Cheng，2005）。這導(dǎo)致PMDS顯示出與生物樣品相似的摩擦與速度趨勢(shì)。另一方面，當(dāng)唾液和水潤(rùn)滑時(shí)，瓊脂糖在摩擦力上的差別很小。這是由于水合瓊脂糖表面弱粘附唾液蛋白所致。瓊脂糖經(jīng)粘膠組分增強(qiáng)粘蛋白結(jié)合后，其摩擦性能沒有改善。雖然PDMS橡膠具有與舌頭相似的疏水性質(zhì)，但PDMS的彈性模量要大兩個(gè)數(shù)量級(jí)。此外，即使交聯(lián)度受到限制，PDMS的模量也僅降至約570千帕（Wang等人，2014年），而舌頭的模量為3.4千帕。這是一個(gè)明顯的缺點(diǎn)，因?yàn)闃悠返膭偠韧瑫r(shí)影響邊界摩擦（μαE′-2/3（Schallamach，1958））和彈性流體動(dòng)力膜厚度（hαE′0.66（de Vicente等人，2005））。被測(cè)樣本之間的粗糙度也有相當(dāng)大的差異，瓊脂糖與舌頭 匹配。然而，由于較粗糙材料的非接觸壓扁，這對(duì)摩擦的影響是有限的，因?yàn)檩^粗糙材料具有較低的剛度。PDMS的一個(gè)優(yōu)點(diǎn)是它是透明的，可以對(duì)唾液潤(rùn)滑機(jī)制進(jìn)行接觸成像。利用激光誘導(dǎo)熒光證實(shí)了這一點(diǎn)，由此產(chǎn)生的摩擦和蛋白質(zhì)強(qiáng)度信號(hào)之間的強(qiáng)相關(guān)性（0.87）證實(shí)了唾液蛋白質(zhì)的lubricous邊界膜形成能力。蛋白質(zhì)聚集在本質(zhì)上是高度短暫的。應(yīng)用這項(xiàng)技術(shù)來(lái)研究唾液與食品和飲料之間的摩擦學(xué)相互作用，以科學(xué)地描述口感特性是正在進(jìn)行的研究課題。致謝S.K.Baier和R.V.Potineni受雇于百事公司。本研究文章中表達(dá)的觀點(diǎn)是作者的觀點(diǎn)，并不一定反映百事公司的立場(chǎng)或政策。本研究由百事公司資助（批準(zhǔn)號(hào)：P55310-1）。附錄A.補(bǔ)充數(shù)據(jù)本文的補(bǔ)充數(shù)據(jù)可在01.049在線查詢。附錄。表面形貌測(cè)量圖A1。三種材料的表面形貌，使用Veeco光學(xué)輪廓儀測(cè)量，a）豬舌，b）PDMS，c）農(nóng)酶。參考文獻(xiàn)：Adams，M.J.，Briscoe，B.J.，和Johnson，S.a.（2007）。人體皮膚的摩擦和潤(rùn)滑。摩擦學(xué)快報(bào)，26（3），239-253。Akhtar，R.，Sherratt，M.J.，Cruickshank，J.K.和Derby，B.（2011年）。描述組織彈性特性的。材料今天，14（3），96-105。Barquins，M.和Roberts，A.D.（2000年）。橡膠摩擦隨速率和溫度的變化：一些新的觀察結(jié)果。物理與應(yīng)用物理學(xué)雜志，19（4），547-563。Benkherourou，M.，Rochas，C.，Tracqui，P.，Tranqui，L.和Guméry，P.Y.（1999年）。低濃度生物制劑表征方法的標(biāo)準(zhǔn)化：低濃度瓊脂糖凝膠的彈性特性。生物力學(xué)工程雜志，121（2），184-187。Bongaerts，J.H.H.，Fourtouni，K.，和Stokes，J.R.（2007年）。軟摩擦學(xué)：在一個(gè)兼容的PDMS-PDMS接觸潤(rùn)滑。摩擦學(xué)，40（10-12），1531-1542規(guī)范。Chen，J.和Stokes，J.R.（2012年）。流變學(xué)和摩擦學(xué)：食物質(zhì)感的兩種不同狀態(tài)。食品科學(xué)與技術(shù)趨勢(shì)，25（1），4-12。Chen，Q.，Suki，B.，&An，K.-N.（2004年）。用分?jǐn)?shù)階導(dǎo)數(shù)模型模擬瓊脂糖凝膠的動(dòng)態(tài)力學(xué)性質(zhì)。生物力學(xué)工程雜志，126（5），666-671。Corfield，A.P.（2015年）。粘蛋白：粘膜保護(hù)中與生物學(xué)相關(guān)的聚糖屏障。生物化學(xué)與生物物理學(xué)學(xué)報(bào)（BBA）-普通學(xué)科，1850（1），236–252。Cox，M.A.J.，Driessen，N.J.B.，Boerboom，R.A.，Bouten，C.V.C.和Baaijens，F.P.T.（2008年）。有限壓痕法研究各向異性平面生物軟組織的力學(xué)特性：實(shí)驗(yàn)可行性。生物力學(xué)雜志，41（2），422-429。Davies，G.A.，Wannilling，E.，和Stokes，J.R.（2009年）。飲料對(duì)唾液刺激和粘彈性的影響：與口感的關(guān)系？食品水膠體，23（8），2261-2269。De 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