Journal of Surface Engineered Materials and Advanced Technology, 2013, 3, 1-12
http://dx.doi.org/10.4236/jsemat.2013.34A1001 Published Online October 2013 (http://www.scirp.org/journal/jsemat)
1
Cross-Linked Alginate Film Pore Size Determination Using
Atomic Force Microscopy and Validation Using Diffusivity
Determinations*
Cheryl Simpliciano1, Larissa Clark1, Behrokh Asi1, Nathan Chu1, Maria Mercado1, Steven Diaz1,
Michel Goedert1#, Maryam Mobed-Miremadi1,2#
1Department of Biomedical, Chemical and Materials Engineering, San Jose State University, San Jose, USA; 2Department of Bioen-
gineering, Santa Clara University, Santa Clara, USA.
Received July 4th, 2013; revised August 6th, 2013; accepted September 1st, 2013
Copyright © 2013 Cheryl Simpliciano et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The deficit of organ donors has fueled the need for advances in tissue engineering and regenerative medicine. Microen-
capsulation in alginate immuno-isolation membranes has been used to treat many disabling metabolic disorders, namely,
phenylketonuria, kidney failure and diabetes mellitus. Systematic nutrient flux determinations are hindered by the lack
of experimental data on alginate-based membrane topography and the pore size thus preventing the full therapeutic po-
tential of the bio-membranes to be reached. In this study, samples of cross-linked alginate membranes were subjected to
the following analytical characterization: 1) pore size characterization using atomic force microscopy operated in con-
tact mode to detect and measure pore size; 2) differential scanning calorimetry to confirm biopolymer cross-linking; and
3) diffusivity measurements using spectrophotometry and fluorescence microscopy to confirm the presence of through
pores and to calculate reflection coefficients. The pore sizes for the pre-clinical standard formulation of 1.5% (w/v) me-
dium viscosity alginate cross-linked with 1.5% CaCl2 and 0.5% (w/v) alginate and chitosan cross-linked with 20%
CaCl2 are 5.2 nm ± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. An increase in the glass transition temperatures as a
function of cross-linker concentration was observed. Diffusivity values obtained from the inward diffusivity of
creatinine into macrocapsules (d = 1000 µm ± 75 µm) and the outward diffusivity of FITC dextrans from macrocap-
sules (d = 1000 µm ± 75 µm) and microcapsules (d = 40 µm ± 5 µm) were shown to correlate strongly (R2 = 0.9835)
with the ratio of solute to pore sizes, confirming the presence of through pores. Reflection coefficients approaching and
exceeding unity correlate with the lack of permeability of the membranes to MW markers that are 70 kDa and greater.
Keywords: Alginate; Atomic Force Microscopy; Pore Size; Stokes’ Radius; Diffusivity; Cross-linking; Differential
Scanning Calorimetry; Reflection Coefficient
1. Introduction
Novel therapies resulting from regenerative medicine and
tissue engineering technology may offer a new hope for
patients with injuries, metabolic disorders, cancer, and
end-stage organ failure. As an example, currently, pa-
tients with diseased and injured organs are often treated
with transplanted organs. However, there is a shortage of
donor organs that is worsening yearly as the population
ages and as the number of new cases of organ failure
increases [1]. Bio-printing, including microencapsulation
of cells, enzymes and drugs in biocompatible hydrogels,
has been researched in an organ prototyping and meta-
bolic disorders [2,3], stem cell encapsulation [4] and can-
cer [5]. This use of hydrogels can be attributed to the
ability of the hydrogel to form a biodegradable and bio-
compatible encapsulation matrix once cross-linked [6].
The most common hydrogel biopolymer used in trans-
plantation and cell therapy is alginate [2].
Alginate is a naturally-occurring, water-soluble poly-
mer comprised of (1,4)-linked β-D-mannuronic (M) and
(1,3)-α-L-guluronic (G) acid residues. Different varieties
of alginate contain varying ratios of M and G. Depending
*Declaration of interest: Authors have no declaration of interests to
report.
#Corresponding authors.

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
2
on the arrangement of the varying M, G, or MG blocks,
alginate copolymers of slightly different behaviors and
properties can be produced. Alginate can be gently
cross-linked by the addition of divalent cations [7]. The
G-block is stiffer and more extended in chain configura-
tion than the M-block due to a higher degree of hindered
rotation around the glycosidic linkages [8]. The removal
of the “M” residues, constituting a significant portion of
the alginate polymer, has increased biocompatibility by
many folds [9]. The substitution of calcium by barium as the
cross-linking divalent ion [10] and the use of chitosan/
genipin-chitosan alginate membranes [11] have resulted in
tremendous improvements in membrane strength. A signifi-
cant amount of research and development has been dedi-
cated to the reproducible molding of cross-linked alginate
membrane into microfibers [12], high-throughput micro-
capsule miniaturization [13] and transdermal patches [14].
The gelation of alginate is possible by interaction of car-
boxylate groups with divalent ions, namely, calcium [15].
The outcome of the gelation process and hence the pore size
can be modulated by using alginates of different molecular
weight and concentrations [16] and alginates comprised of
different amounts of G fractions [17], modulating the cross-
linker concentration and/or cross-linking reaction time [18]
and by combining interactions of all of these factors.
The molecular weight cutoff (MWCO) of the mem-
brane expressed in terms of Stokes’ radius, (a), is the
maximum molecular weight that is allowed through the
selective passage of the membrane pores given by Equa-
tion (1) [19]. This equation assumes that the solute of
molecular weight (MW) is a sphere with a density (ρ = 1
g·cm−3) equal to that of the solute in solid phase. The
pore sizes in the gel network of hydrogels vary from
macroporous (0.1 - 1 µm) to microporous (10 - 100 nm)
[20]. Shown in Figure 1 is a cross-section of an alginate
microcapsule captured by SEM.
1/3
A
3MW
4πN
a




(1)
The pore size of an encapsulation material is critical to
both encapsulation efficiency and release kinetics. Too
large of a pore size will allow content leakage while too
small of a pore size can hinder timely release. Alginate
pore size has been extensively researched through various
techniques, mainly through imaging and diffusivity meas-
urements. However, there is little agreement as to what the
pore sizes actually are. Tabulated results indicating the
variation in pore sizes appear in Table 1 . The reported pore
sizes apply to either alginate films or microcapsules. As
shown by results of diffusion studies, alginate pores can
range from 3.6 - 14 nm for 4% alginate [21,22] and 3 nm
and 14.5 - 17 nm for 1.5% and 3% alginate, respectively
[23]. In experiments where scanning electron micros-
Figure 1. SEM image of 0.5 % MV a lgi nate/20 % CaCl2 micro-
capsule cross section, dehydrated. Captured in low-vacuum
mode.
copy (SEM) was used, a larger range of pore sizes from 5
nm - 21 µm have been observed [7,15,24,25]. Numerous
atomic force microscopy (AFM) imaging experiments
produced pore sizes between 10 nm and 1.3 µm [10,26,27].
Pore sizes less than 10 nm and as large as 70 nm were re-
vealed using Transmission Electron Microscopy (TEM) in
experiments conducted by Leal-Egaña, Braumann, Diaz-
Cuenca, Nowicki and Bader [28]. A maximum pore size of
5.8 nm was obtained, based on fluorescent microscopy
measurements [29]. Sources of discrepancies include the
range of variables associated with the gelation technique,
the artifacts of sample preparation, and the resolution of the
measurement technique.
In the absence of precise pore size data, systematic flux
determinations are hindered by the lack of experimental
data on membrane topography, thus preventing the full
therapeutic potential of the alginate immuno-isolation
membranes to be reached. The research objectives of this
study are three-fold: 1) to measure the pore size of various
alginate formulations using AFM; 2) to confirm the occur-
rence of cross-linking using differential scanning calo-
rimetry (DSC); and 3) to correlate measured pore sizes to
diffusivity measurements. Of particular interest are the pore
sizes for the pre-clinical standard formulation of 1.5% (w/v)
alginate cross-linked with 1.5% CaCl2 [2] and the MWCO
of the miniaturized capsule membrane, 0.5% (w/v) algi-
nate/chitosan cross-linked with 20% CaCl2, characterized
by faster toxin clearance in-vit ro [30].
2. Materials and Methods
2.1. Materials
All chemicals used in this study were acquired from

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
3
Table 1. Literature review of pore size for various analytical methods.
Study Method Wet/Dry Imaging
Conditions Membrane Morphology/Type Pore Size
Wang, et al. [7] Cryo-SEM Dry Microcapsules (Calcium Chloride) 3.9 - 10.9 µm
Zimmerman, et al. [10] AFM Wet Thick film (Barium Chloride) 1.2 - 1.3 µm
Gombotz and Wee [15] SEM Dry Microcapsules (Calcium Chloride) 5 - 200 nm
Choi, et al. [21] Diffusion Wet Microfluidic scaffold (Calcium Chloride) 3.6 nm
Chan and Neufeld [22] Diffusion Wet Microcapsules (Calcium Chloride) 4 - 14 nm
Li, et al. [23] Diffusion Wet Cylinders (Calcium Chloride) 14.5 - 17 nm
Wright, et al. [24] SEM Dry Slabs (Calcium Chloride) 0.1 - 0.3 µm
Jejurikar, et al. [25] Cryo-SEM Dry
Low Viscosity Alginate Films
(Calcium Chloride and Barium Chloride) 0.5 - 21 um
Hsiong, et al. [26] AFM Dry Films (Calcium Chloride) 10 - 100 nm
Schmid, et al. [27] AFM Wet Films (Calcium Chloride) 50 - 300 nm
Leal-Egaña, et al. [28] TEM Microcapsules (Glutaraldehyde) 10 - 70 nm
Mobed-Miremadi, et al. [29] Fluorescence Microscopy Wet Artificial Cells (Calcium Chloride) <5.8 nm
Sigma-Aldrich (USA); these are: medium molecular
weight (MV) sodium-alginate (A2033), low molecular
weight (LV) sodium-alginate (A2158), low molecular
weight chitosan (44 886–9, 75% deacetylated, 3.8–6 kDa)
and fluorescein isothiocyanate dextran markers abbrevi-
ated as FITC Dextran markers (46947, FD70S, FD4). All
other reagent grade chemicals were provided by the
Chemistry store in the Faculty of Sciences at San Jose
State University: creatinine powder (MW = 113 Da) and
bovine serum albumin (BSA, MW = 66.4 kDa). The tri-
angular Pyrex-Nitride AFM probes (PNP-TR-20) were
purchased from NanoWorld (Neuchâtel, Switzerland).
Polylysine-coated slides were purchased from VWR
(Radnor, PA) (cat# 16002-116). Cellulose Ester (CE)
dialysis tubing with a molecular weight a cutoff (MWCO)
of 20 kDa was puchased from Spectrum Labs (Spec-
traPor # 131342, Rancho Dominguez, CA).
2.2. Methods
It should be noted that it was not possible to use a single
type of alginate structure for all analytical tests. While
films were used for AFM and differential scanning calo-
rimetry (DSC), spherical capsules were used for diffusiv-
ity measurements. Due to the approximate average ratio
of AFM scan area to capsule area (1:105), it has been
assumed that the sphere curvature can be neglected and
thus the pore sizes for the spherical and flat structures are
nterchangeable for the same formulation.
2.3. Atomic Force Microscopy (AFM)
2.3.1. Sample Prepa ration
AFM imaging was performed on spin-coated films pre-
pared with various alginate and cross-linker concentra-
tions and the dialysis tubing standard. Samples were cast
as films for ease of imaging. 1 mL of alginate dissolved
in saline (0.9% NaCl w/v) at a given concentration was
deposited onto a poly-L-Lysine (PLL)-coated glass slide
placed into a small petri dish. The dish was fixed to a
homemade spin coater comprised of a CPU fan attached
to a power supply shown in Figure 2. The alginate was
allowed to spin for 15 s producing a film with a relatively
homogenous thickness. Calcium chloride at a given con-
centration was added to the film in a drop-wise fashion to
induce cross-linking. The films were set to cross-link for
1 hr followed by a DI water rinse. The PLL-coated slide
was then transferred directly onto the AFM platform for
imaging.
2.3.2. Measurements
Surface imaging was performed on the various alginate
film formulations. The characterization was conducted
using an Agilent 5500 AFM equipped with a contact-
mode nose amplifier and 100 µm scanner N0524A
(Agilent, Santa Clara, CA). Calibrations were performed
using a TGZ02 standard (MikroMasch, Wilsonville, OR)
and dialysis tubing with a MWCO of 20 kDa. The in-
strument was operated in contact mode using a Pyrex-

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
4
Figure 2. Spin coater/fan s et up for AFM sample preparation .
Nitride probe with triangular cantilever (resonant fre-
quency 17 kHz, force constant 0.08 N/m, thickness 600
nm, length 200 µm, tip radius 7 - 10 nm). PicoView v1.8
(Agilent, Santa Clara, CA) and Gwyddion v2.3 (Czech
Metrology Institute, Brno, Czechoslovakia) were used as
qualitative real-time and quantitative image analysis soft-
ware, respectively. Scan speed was established by setting
a ratio of 128 pixels/line. The scan area ranged from 0.1
to 5.0 µm2 with a maximum possible range of 100 µm.
The pore size was obtained by measuring the distance
between the darkest areas of the pores as indicated by the
grayscale intensity in Gwyddion. Images were obtained
at different locations around the sample. These locations
were changed through the movement of the stage to ob-
tain an average pore size.
2.4. Differential Scanning Calorimetry (DSC)
2.4.1. Sample Prepa ration
DSC testing required samples that were no more than 5
mm in thickness and preferably relatively flat. 10 mL of
alginate was spread into glass dishes. Each sample was
immersed in a solution of CaCl2 of concentrations rang-
ing from 10% to 25%, allowed to cross-link for one hour
and turned over once to ensure uniform cross-linking. An
uncross-linked reference sample of bare alginate was also
tested as control. As described by Russo, Malinconico
and Santagata. [17], water may effectively mask the rela-
tively weak glass transition shoulder of alginate in a DSC
thermograph. In response, sheets of hydrated sample
material were desiccated in a dry nitrogen box for a mi-
nimum period of 24 hours prior to testing. Circular seg-
ments were die-cut from the desiccated sheets, weighed,
and sealed in high-purity aluminum crucibles. Specimen
masses varied between 6 and 12 mg. All films were com-
prised of an alginate concentration of 1.5% (w/v) MV
alginate. As previously stated, this is the nominal re-
ported molecular weight and concentration for cell en-
capsulation [2].
2.4.2. Measurements
All testing was performed by BAE Systems in Santa
Clara, CA, using a Mettler-Toledo DSC823e differential
scanning calorimeter (DSC). Temperature and heat flow
calibration were performed using NIST reference In, Hg
and Zn. All specimens were tested for glass transition
temperature characterization by DSC over a range of 30
to 200˚C at a rate of 10˚C/min. Dry nitrogen was used to
purge the sample chamber at a flow rate of 40 mL/min.
The glass transition region of each thermograph was eva-
luated per ASTM E1356. The midpoint temperature, or
the half-way point between upper and lower baselines,
was reported at the glass transition temperature (Tg) in
each case.
2.5. Macrocapsule Preparation
Macrocapsules (MA) were fabricated using the atomiza-
tion method [31]. A 1.5% MV sodium-alginate solution
was jetted into a 1.5% (w/v) CaCl2 bath. The air (FA)
and liquid (FL) flow rates were adjusted to 1.5 L/min and
0.5 mL/min, respectively. After jetting, capsules were
allowed to cross-link in the CaCl2 solution for 1 hr. The
calcified sodium-alginate beads were then washed with
0.9% NaCl twice.
2.6. Microcapsule Preparation
Microcapsules (MI) were fabricated using Microfab’s
Jetlab System using the methodology in reference [32].
The inkjet engine fires the 0.5% LV sodium alginate so-
lution through the print head into a 20% (w/v) CaCl2
solution. After jetting, capsules were allowed to cross-
link in the CaCl2 solution for 30 min. Then 1% (w/v)
chitosan was added into the 20% CaCl2 solution to make
the final chitosan concentration 0.5% (w/v). Capsules
were coated for an additional 30 min. Next, the capsules
were centrifuged at 8000 g for 5 min and washed with a
0.9% (w/v) NaCl solution 3 times.
2.7. Diffusivity Measurements and Modeling
2.7.1. Creatini n e
Creatinine was used as test solute to determine inward
diffusivity coefficients through MA according to previ-
ously established methodology [33]. Calibration stock
solutions ranging from 0 to 5 mg/mL were used. In this
range, there was a linear relationship between absorbance
and concentration that was subsequently used for con-
centration interpolation. A 5 mL suspension of micro-
capsules was poured into a 5 mL solution of solute at an
initial concentration C0. The objective was to measure
the amount of solute diffusing from the solution through
the empty MA membrane. Every 30 s, the supernatant
was tested for a change in solute concentration by meas-
uring the absorbance at
 = 265 nm for creatinine using
an Agilent 8453 UV-VIS spectrophotometer. Sampling

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
5
stopped when no more absorbance/concentration changes
were detected (dC/dt = 0). This concentration was taken
as the equilibrium concentration (Ceq).
2.7.2. FIT C Dextrans
FITC dextrans were used as test solutes to determine
outward diffusivity coefficients through MA and MI ac-
cording to previously established methodology [34]. Spec-
trophotometric methods were not sensitive enough to de-
tect transport across the MI membrane, therefore, fluo-
rescence microscopy was used. Calibration stock solu-
tions ranging from 0.1 to 15.1 mg/mL for each FITC-
Dextran MW standard (4, 70, and 500 kDa dissolved in
0.9% (w/v) NaCl) were prepared. In this range, for each
FITC-Dextran MW, there was a linear relationship be-
tween intensity and the concentration of the fluorescent
marker under observation, the results of which were
subsequently used for concentration interpolation. 10 µL
of MI or MA solution were incubated in 1 mL of FITC
solution for 24 h prior to imaging. The solution was cen-
trifuged at 8000 g for 5 min and washed once with a
0.9% (w/v) NaCl solution. Samples were then deposited
onto a microscope slide and observed under the trans-
mission microscope/camera (Nikon EclipseTi-S/Andor
Technology Interline CCD camera). The FITC/Acridine
Orange filter was chosen from the imaging software (NIS-
Elements v.3.2.2) filter selection feature to accommodate
the excitation and emission wavelengths of 468 and 520
nm of the FITC molecule. Images were captured every
30 s. Sampling stopped when no more intensity changes
were detected (dI/dt = 0). This concentration was taken
as the equilibrium concentration (Ceq).
2.7.3. Diffusivity Modelin g and Cal cul a tion of the
Sieving Coefficient
The analytical solution to Fick’s second law in spherical
coordinates was used to determine diffusivity coeffi-
cients from spectrophotometric and fluorescence meas-
urements according to previously published methodology
[29]. Residual sum of squares (RSS) minimization was
conducted using MATLAB 2010a. The membrane re-
flection coefficient (
 ) was calculated using Equation (2),
where
 is the ratio of the solute Stokes’ radius (α) and
the average membrane pore size (r) [19].
 
22
23
2
11 2110.163
3





  




 (3)
3. Results
3.1. Atomic Force Microscopy
As previously stated, AFM imaging was performed on
spin-coated films prepared with varying concentrations
of alginate and CaCl2 with and without a chitosan coating.
Prior to imaging, the samples were slightly hydrated by a
DI water rinse. AFM was chosen for imaging as it is an
imaging method that provides nanometer resolution and
three-dimensional surface imaging, requires minimal
sample preparation and allows imaging in ambient and
liquid conditions.
In Figure 3, the apparent variation in pore size due to
the increase in resolution is plotted as a function of scan
area by film formulation. A decrease in average pore size
is observed across all formulations with decreasing scan
area. The calculated Stokes’ radius corresponding to a
MWCO of 20 kDa for the standard dialysis tubing is 2.02
nm [19]. For an AFM scan area of 0.1 µm2, an average
pore diameter of 4.9 nm was obtained for the standard
sample. This value is the closest to the theoretical Stokes’
radius of 2.02 nm corresponding to a relative measure-
ment error of 16.7%. Hence all subsequent analyses and
comparisons will be conducted for the pore sizes ob-
tained at this setting. Pore size measurements conducted
using all scan areas are presented in Table 2. Shown in
Figures 4-8 are the corresponding 2D views for multiple
Figure 3. Variation in apparent pore size as a funct ion of sca n
area.
Figure 4. AFM images of 1.5% MV alginate and 1.5%
CaCl2. Clockwise from top left: 2.5, 1.0, 0.5 and 0.15 µm2
scan area.

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
6
Table 2. Effect of AFM scan area on apparent pore size.
Sample CaCl2 %
(w/v) Coating Pore Size Range (nm) at 0.5 µm2
Pore Size Range
(nm) at 0.25 µm2
Pore Size Range
(nm) at 0.15 µm2
Average Pore Size
(nm) at 0.1 µm2
Alginate MV 0.5% (w/v) 1.5 N/A 11 - 23 7.0 - 16 5.0 - 11 8.4 ± 3.0
Alginate MV 1.0% (w/v) 1.5 N/A 26 - 44 9.0 - 16 7.0 - 15 4.5 ± 1.1
Alginate MV 1.5% (w/v) 1.5 N/A 13 - 35 12 - 28 6.0 - 10 5.2 ± 0.9
Alginate LV 0.5% (w/v) 20 N/A 6.0 - 19 6.0 - 12 4.0 - 18 7.2 ± 2.9
Alginate LV 0.5% (w/v) 20 Chitosan 17 - 24 7.0 - 19 5.0 - 11 7.0 ± 3.1
Dialysis Tubing N/A N/A 6.0 - 25 6.0 - 14 5.0 - 13 4.9 ± 3.0
Figure 5. AFM images of 0.5% LV alginate (left) and 0.5%
LV alginate coated with chitosan (right): (top) Scan areas of
0.2 µm2 (left) and 0.15 µm2 (right); (middle) 0.25 µm2 (left)
and 0.25 µm2 (right); (bottom): 1.0 µm2 (left) and 1.0 µm2
(right).
scan areas and a 3D view for the 0.1 µm2 scan area for
which the measured pore sizes are tabulated.
For the purposes of comparing AFM images, the fol-
lowing concentrations will be discussed: 1.5% MV/1.5%
CaCl2, 0.5% LV/20% CaCl2, 0.5% LV/20% CaCl2 coated
with 0.5% chitosan, and the dialysis membrane standard.
Higher scan areas produced what appear to be surfaces
with deep, indented features, as indicated by the darkest
areas of the images. MV alginate (Figure 4) and coated
LV alginate (Figure 5) appear to have more defined sur-
face features, which are most likely attributed to tip-
sample interaction due to the relative softness of the bare
(a)
(b)
(c)
Figure 6. AFM 3D (left) and 2D (right) views of (a) 1.5%
MV alginate, 0.1 µm2 scan area; (b) 0.5% LV alginate, 0.1
µm2 scan area; and (c) 0.5% LV alginate coated with chito-
san, 0.1 µm2 scan area.
LV alginate. The layer of chitosan added to the LV algi-
nate had some effect on film morphology. For example,
chitosan-coated LV alginate displayed a structure with
fewer features that were also of a smaller size than what
was seen in the other films. This difference is due to the
extra layer coating these features, effectively reducing
the size of the pore openings.
As previously mentioned, it was observed that de-
creasing scan area also decreased the measured sizes of
the pores. Since image resolution typically decreases as

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
7
Figure 7. AFM images of dialysis tubing. Counterclockwise
from top left: 5.0, 2.5, 1.5 and 0.15 µm2 scan area.
Figure 8. AFM 3D view of dialysis tubing, 0.15 µm2 scan
area.
scan size approaches 0.1 µm2, this decrease in measured
size appears counterintuitive. However, a decrease in
scan area allows the ability to zoom into the deepest ar-
eas of the image, which represents the smallest opening
of the pores as seen in the 3D images in Figure 6.
For AFM imaging of the dialysis tubing standard (Fig-
ure 7), the standard appears to have tolerated the AFM
tip as indicated by the greater resolution and lower in-
stance of artifacts across the surface compared to the
alginate images. A 3D image of the dialysis tubing at 0.1
µm2 provided further means of visual comparison be-
tween a material of known porosity and the alginate po-
rosity (Figure 8). The known porosity of the standard
provided by the manufacturer lends feasibility to the use
of the grayscale in estimating the pore size in the alginate
material.
3.2. Differential Scanning Calorimetry
Shown in Figure 9 are sample DSC thermograms with
corresponding glass transition temperatures presented in
Table 3. Shown in the thermogram of sodium alginate
(Sample A) is endothermic decay at 112˚C due to re-
moval of absorbed moisture (or nonstructural water). A
Table 3. Effect of cross-linker concentration on transition
temperature.
Sample CaCl2 % (w/v) Coating Tg (˚C)
A Alginate MV 1.5% (w/v)0 N/A 112.81
B Alginate MV 1.5% (w/v)10 N/A 115.44
C Alginate MV 1.5% (w/v)15 N/A 115.42
D Alginate MV 1.5% (w/v)20 N/A 124.57
E Alginate MV 1.5% (w/v)25 N/A 127.77
F Alginate MV 1.5% (w/v)20 Chitosan 123.16
G Alginate MV 1.5% (w/v)25 Chitosan 135.68
gradual increase in transition temperature and delayed
endothermic shifts are observed with increasing CaCl2
concentration with an approximate step change of 9˚C
between the 15% - 20% cross-linker concentration range
(Samples C and D). Another marked increased is ob-
served for sample G characterized by highest degree of
cross-linking (25% (w/v) CaCl2) and chitosan coating.
3.3. Diffusivity Measurements
Table 4 was generated by combining the results of diffu-
sion experiments in MIs (d = 40 µm ± 5 µm) and MAs (d
= 1000 µm ± 75 µm) and AFM measurements. As shown
in Figure 10, solute diffusivity is inversely correlated to
 (R2 = 0.9835) and calculated in turn based on the AFM
measurements. Assuming that the majority of pores are
through pores, as the solute size approaches the pore size
(
 ≥ 1), the solute cannot be filtered through the mem-
brane.
Reflection coefficients equal to or exceeding unity in-
dicate the lack of membrane permeability to the specific
solute as reflected by the 103 - 104 fold reduction in dif-
fusivity values as the marker MW was increased.
4. Discussion
4.1. Atomic Force Microscopy
In terms of variability in pore measurement, the darkest
areas using grayscale intensity were used to measure pore
size. This method effectively used the smallest opening
as the pore width. Although measurements of dialysis
tubing AFM images using Gwyddion analysis tools
yielded an average pore size of 4.9 nm, the actual pore
size could not be definitively measured to less than 7 - 10
nm due to the manufacturer’s specification on the tip
radius. However, based on consistently-measured dialy-
sis tubing pores using grayscale intensity, the trend
clearly indicated that the ability to resolve the pores in-
creased with decreasing scan size. As the scan size de-

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
8
Glass T rans ition
Onset60. 19 °C
Midpoint115.42 °C
Left Limit52.04 °C
Right Limit159.39 ° C
Glas s T rans ition
Ons et55.37 °C
Midpoint112.81 ° C
L e f t Limit48.63 ° C
Right L imit144.05 ° C
Glas s T rans ition
Ons et85.75 °C
Midpoint123.16 ° C
L e f t Limit54.42 ° C
Right L imit162.46 ° C
Glass T rans ition
Onset83. 77 °C
Midpoint115.44 °C
Left Limit56.81 °C
Right Limit157.69 ° C
Glass T rans ition
Onset91. 47 °C
Midpoint124.57 °C
Left Limit62.69 °C
Right Limit162.78 ° C
Glas s T rans ition
Onse t104.03 °C
Midpoint127.70 ° C
L e f t Limit62.69 ° C
Right L imit162.78 ° C
Glas s T rans ition
Onse t114.60 °C
Midpoint135.68 ° C
L e f t Limit96.34 ° C
Right L imit162.78 ° C
Sam ple: [100807] DSC, B, 10% C a Cl2, 7 .5000 mg
Sam ple: [100807] DSC, C, 15% Ca Cl2, 11.3200 mg
Sam ple: [100807] DSC, D, 20% CaCl2, 7.28 00 mg
Sam ple: [100807] DSC, A, Bare alginate , 8.9000 mg
Sam ple: [100807] DSC, F, 20% CaCl2 w/ chitosa n, 6.180 0 m g
Sample: [100807] DSC, E, 25% CaCl2, 11.8100 m g
Sample: [100807] DSC, G, 25% Ca Cl2, w/ c hitosan, 11.8500 mg
Wg^-1
2
°C20406080100 120 140 160 180 200220 240 260280 300 320340 360 380
Figure 9. DSC thermograms with corresponding glass transition temperatures.
Table 4. Results of membrane diffusivity, pore size and reflection coefficient across different formulations and molecular
weight markers.
Membrane CaCl2 % (w/v) Coating Measurement Method MW Marker a (nm) r (nm) σ D (m2/s)
Alginate MV 1.5% (w/v) 1.5 N/A Spectrophotometry Creatinine 0.36 2.6 0.4 7.20E−13
Alginate LV 0.5% (w/v) 20 Chitosan Fluorescence Microscopy FITC dextran 4 kDa1.18 3.5 0.4 7.70E−14
Alginate MV 1.5% (w/v) 1.5 N/A Fluorescence Microscopy FITC dextran 4 kDa1.18 2.6 0.5 1.81E−14
Alginate LV 0.5% (w/v) 20 Chitosan Fluorescence Microscopy FITC dextran 70 kDa3.07 3.5 1.0 3.02E−16
Alginate MV 1.5% (w/v) 1.5 N/A Fluorescence Microscopy FITC dextran 70 kDa3.07 2.6 1.0 5.95E−17
Alginate MV 1.5% (w/v) 1.5 N/A Fluorescence Microscopy FITC dextran 500 kDa5.92 2.6 2.6 0
Alginate LV 0.5% (w/v) 20 Chitosan Fluorescence Microscopy FITC dextran 500 kDa5.92 3.5 1.0 0
creased, the measured and calculated Stokes’ radius of
the tubing began to converge, and these results lend con-
fidence into this method of measurement. However, for
the purposes of this study, the average pore size of 4.9
nm at a scan size of 0.1 µm2 is a relative measurement
and requires further investigation with a tip of smaller
radius or alternate imaging method.
SEM facilities were available for this study; however,
the equipment did not provide the desired resolution for
pore measurements, with a limit of 100 nm on the given
system. In addition, radiation generated by the SEM
electron beam is known to cause cross-linking, which
would have required further study in terms of potential
effect on the alginate/CaCl2 porosity. Transmission elec-
tron microscopy (TEM) has been used to image alginate
in previous studies [26]; however sample preparation
methods for both SEM and TEM include a number of
fixing media including glutaraldehyde [10,26,35], the
primary function of which is to provide structure by
cross-linking biological materials prior to dehydration,

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
9
Figure 10. Membrane diffusivity as a function of solute to
membrane pore size.
where changes to cross-linking in the alginate would not
be desired. If fixation were not an issue with the alginate/
CaCl2, SEM and TEM samples would then be dehydrated
after fixation using critical point drying [35] or lyophili-
zation [36] and coated with a conductive coating or im-
aged in low-vacuum SEM without a conductive coating;
however, SEM images of samples prepared using these
dehydration methods clearly indicate damage and shrink-
age of the material [26] and are not a desired sample
preparation method. Alcohol or acetone substitution
could have been used with environmental SEM (ESEM)
[35]; however, a cold stage was not available. A cold
stage allows a hydrated sample to remain at the dew
point in the SEM chamber. By varying the temperature
or pressure in the chamber, the user can cause the sample
to dehydrate in a controlled fashion so that some of the
surface moisture sublimates but without completely dry-
ing the surface, where with a wet surface, the SEM elec-
tron beam would image the liquid instead of the sample
surface. Lastly, scanning tunneling microscopy (STM)
offers Ångström resolution; however, dehydration or
fixing of the sample would have been required in order to
apply a conductive coating on the sample for STM.
Therefore, imaging in the native state using AFM with
minimal sample preparation was preferable.
Intermittent contact or tapping mode with the sample
fully immersed in liquid generally reduces the likelihood
of surface damage by the probe tip; however, in this
study, intermittent contact mode did not provide the de-
sired resolution. This problem may have been due to the
stickiness of the sample interacting with the tip [26]. It
was determined that the sample could be sufficiently
imaged by AFM using contact mode with a low stiffness
probe of 0.08 N/m to reduce damage to the surface.
Other groups were found to have performed AFM imag-
ing using a higher stiffness probe such as 0.12 N/m [27,
37]. In the case of the 0.5% LV alginate concentration,
the initial samples were too soft for AFM imaging and
required an increase in cross-linking concentration to
stiffen the material.
4.2. Calorimetry
Using ionotropic gelation by which all cross-linked sam-
ples have been fabricated, at a given initial alginate con-
centration, the degree of cross-linking can be varied by
either modulating the CaCl2 concentration or modifying
the G block content of the bio-polymer. The DSC analy-
sis that was performed revealed that increasing the
cross-linker concentration resulted in an increase in glass
transition temperature (Tg).
The increase in Tg can be attributed to the linking that
could restrict the molecular response to temperature
change as predicted by classical polymer theory [38]. As
would be expected, higher CaCl2 concentrations have a
more pronounced effect on free volume as an increase in
CaCl2 ions provide more opportunities for creating tie
points between polymer chains. Recent results of ther-
mogravimetric (TGA) analysis on alginate films con-
firmed the same trend [39]. It is known from the litera-
ture that, there are three kinds of absorbed water in hy-
drophilic polymers [40,41], free, freezing bound, and
non-freezing bound. Whereas freezing bound water in-
teracts weakly, non-freezing bound water forms hydro-
gen bond to bind with the polymeric chain. As stated in
the methodology section, since care was taken to remove
the free water by desiccation, and alginate [42] and chi-
tosan [43] decomposition occur at temperatures above
200˚C, it could be hypothesized that the shifts in transi-
tion temperatures detected are due to the elimination of
the freezing and non-freezing bound water. As for the
increase in Tg, as a result of the chitosan coating at higher
cross-linking concentrations, chitosan is classified as a
stiff and rigid polyelectrolyte. Once adsorbed onto the
bio-membrane, a more rigid and less fluid bio-membrane
characterized by higher glass transition temperatures has
been reported [44,45]. The results contradict findings of
Russo, Malinconico and Santagata. In that study, an in-
crease in the guluronic acid content of the alginate re-
sulted in a decrease in glass transition temperatures
measured by DSC. A higher G block content resulted in
swelling and lower Tg for the cross-linked hydrogel. The
authors hypothesize that, as expected, the cross-linking
points represent a hindrance for the packing of chains;
however, the chain segments between two consecutive
cross-linking points experience an increased mobility
because of the increase of the free volume due to swell-
ing.
4.3. Diffusivity Measurements
Whether using spectrophotometry or fluorescence mi-
croscopy for diffusivity determination, experiments were

Cross-Linked Alginate Film Pore Size Determination Using Atomic Force
Microscopy and Validation Using Diffusivity Determinations
10
designed to avoid the following interactions affecting
pore size measurements: 1) the capsules had reached an
equilibrium swollen state post-fabrication monitored by
microscopy; 2) the MW markers chosen for the graph do
not react with the pores; 3) the MW markers did not react
with the membrane using electrostatic and hydrophobic
interactions; 4) the solute was not present in excess at the
membrane interface to generate concentration polariza-
tion except for the 500 kDa marker to which the mem-
brane is impermeable [46]; and 5) multiple sources place
the 70 kDa marker at the MW cutoff of the membrane
[33,46] so diffusion was not hindered for creatinine or
the 4 kDa marker. Given these precautions and the diffu-
sivity measurements, it could be hypothesized that a por-
tion of the detected pores by AFM are through pores.
5. Conclusions
The surface morphology of cross-linked alginate struc-
tures was investigated through the use of DSC, AFM and
diffusivity measurements using spectrophotometry and
fluorescence microscopy. Through DSC measurements,
successful cross-linking was established by correlating
glass transition temperature and cross-linker concentra-
tion. AFM experiments performed on alginate films
yielded pore sizes for 1.5% CaCl2 and 0.5% (w/v) algi-
nate/chitosan cross-linked with 20% CaCl2 to be 5.2 nm
± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. Through
measurements of inward diffusivity and outward diffu-
sivity of MW marker, the presence of through pores in
the alginate membrane was confirmed. Decreasing diffu-
sivities and reflection coefficients approaching unity
concur with previous findings that the molecular weight
cutoff of the studied alginate bio-membranes is approxi-
mately 70 kDa.
Since it is difficult to confirm the accuracy of measur-
ing pore sizes through the grayscale intensity method, the
following improvements should be considered: 1) scan
size should start at 0.1 µm2, and finer AFM probe tips
should be investigated such as molecularly-functional-
ized tips; 2) the number of pixels/line should be in-
creased to improve resolution due to a slower scan rate; 3)
the use of liquid imaging with tapping or intermittent
contact mode should be re-evaluated, and a lower stiff-
ness probe should be used to further reduce damage to
the soft sample surface; and 4) high resolution field ef-
fect SEM (FESEM) imaging [26] to the scale of 1 - 5 nm
resolution [15] could be used in future work to quantify
differences in pore size or shape between the microcap-
sules and films if the material can be prepared for SEM
without additional damage to the material from sample
preparation.
This further understanding of alginate morphology can
potentially be helpful in determining how to fine-tune
alginate pore sizes and to carefully regulate release ki-
netics from alginate membranes.
6. Acknowledgements
The authors would like to acknowledge the Davidson’s
College of Engineering Faculty Development (“Inkjet
Bioprinting”) and Junior Professorship Grants, the C-
SUPERB Joint Venture Grant “Bio-Printing of Mam-
malian Cells”, and the C-SUPERB Faculty-Student Col-
laborative Research/New Investigator Grant Program
“Encapsulated Hybrid P450 enzyme as novel light-driven
biocatalyst” for funding this effort. Thermal characteri-
zation of the samples could not have been executed
without access to the DSC machine granted generously
by BAE Systems, San Jose, CA.
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