Journal of Membrane Science 503 (2016) 16–24

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Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

Selective transportation of charged ZnO nanoparticles and

microorganism dialysis through silicon nanoporous membranes
Priyanka Bhadra a, Sudeshna Sengupta a, Noel Prashant Ratchagar b, Balachandra Achar a,b,
Anju Chadha c,d, Enakshi Bhattacharya a,b,n
a
Center for NEMS and Nanophotonics, Indian Institute of Technology Madras, Chennai 600036, India
b
Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai 600036, India
c
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
d
National Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, India

art ic l e i nf o a b s t r a c t

Article history: It is well known that, besides the membrane pore size and the charge on the pore walls, transport of
Received 29 April 2015 biomolecues through nanoporous membranes is strongly dependent on the size as well as the charge on
Received in revised form the biomolecules. In this paper we do a systematic study to understand this transport using zinc oxide
18 November 2015
(ZnO) nanoparticles as the analogues for the biomolecules. The batch fabricated nanoporous membranes,
Accepted 26 December 2015
Available online 6 January 2016
with average pore size of 9 nm, were used for diffusion experiments on ZnO nanoparticles grouped as S1
(1–4 nm) and S2 (20–25 nm). The nanoparticles, S1 passed through the pores while the S2 did not due to
Keywords: the larger size. The charge on the S1 particles was varied, keeping the size constant by adding selective
Silicon nanoporous membrane capping agents. The membrane and the pore walls develop a net negative charge in alcohol medium
Microbial cells
which facilitate the transport of neutral nanoparticles while impeding both the positively and negatively
Zinc oxide nanoparticles
charged particles. Interestingly, both the neutral and slightly negative charged particles diffuse at more
Size and charge based separation
Pore blocking or less equal rates, while slightly positive charged material shows much less diffusion. This may be due to
the enhanced blocking of the pores resulting from the electrostatic interaction between the particles and
the pore walls. Transport of four different microorganisms of size 30 nm to 3 μm was also investigated
and were found to be blocked by the membranes.
& 2016 Elsevier B.V. All rights reserved.

1. Introduction column. These matrices consist of randomly oriented pores with

The use of membranes for separating and sorting in commer-
cial applications for isolation and purification of molecules from
various biological feed streams including the fields of pharma-
ceutical industry, food industry, and biotechnology is a well-
known process [1]. In various biomedical applications, it is ne-
cessary to separate out the naturally occurring nanoparticles such
as renal calculus, lipoproteins, and virions [2–4]. For example, in
the kidney dialysis process, the synthetic nanoporous membrane
acts as a blood filter which retains all the useful serum proteins
while allowing the waste substances like creatinine and urea to be
filtered out [5].
An existing technique in the laboratory to separate the in-
dividual fractions from a mixture of biomolecules is to use an
immobilized matrix such as a polymeric gel or a chromatographic
n rapid thermal annealing (RTA) [16,27–30]. The nanopores obtained
Corresponding author at: Center for NEMS and Nanophotonics, Indian Institute
of Technology Madras, Chennai 600036, India.
E-mail address: enakshi@ee.iitm.ac.in (E. Bhattacharya).
wide pore size distributions, which cannot be easily measured,
controlled, and manipulated. The major drawback of such a tech-
nique is the lack of engineering control and the diffusion is also a
time consuming process which greatly affects the efficiency [6].
Recently, molecular transport through synthetic nanoporous
membranes has received considerable attention [7,8]. Commer-
cially available polymeric porous membranes are used [9] because
of their versatility, but they pose a disadvantage of the molecules
taking a longer time to pass through the pore and getting stuck in
the pores [10–13]. Micro-fibrous membranes are also gaining
considerable interest in microfiltration and ultrafiltration [14,15].
In contrast, ultrathin silicon nanoporous membranes (SNMs) are
viable for the diffusion, separation and discrimination of small
organic and biomolecules [16]. SNMs are fabricated using techni-
ques like lithography [17–20], focused ion beam (FIB) drilling [21–
23], electrochemical etching [24], anodic oxidation [25,26], and
by FIB drilling, though highly ordered with uniform pore size,
cannot be batch processed. Those fabricated by lithography and

http://dx.doi.org/10.1016/j.memsci.2015.12.058
0376-7388/& 2016 Elsevier B.V. All rights reserved.
 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24
anodic oxidation are restricted by the minimum obtainable pore
size. Nanopores obtained by electrochemical etching have rough
and irregular pore walls, which can possibly trap the biomolecules
passing through the pore. The pores formed in the membrane can
be further narrowed down using vapour deposition [20–22]. An-
other technique to obtain uniform narrow pores without using
either an ion-beam or re-narrowing using vapour deposition is by
combining both surface and bulk micromachining [31]. This is
done by growing a thin film sacrificial oxide between the bulk
silicon and deposited polysilicon, and etching holes in the de-
posited polysilicon with a slight offset with the etched holes in
bulk silicon and finally etching the sacrificial oxide. The thickness
of the oxide will determine the diameter of the pore. In this paper,
we have used the process of RTA, first reported by Striemer et al.
[16], to fabricate ultrathin SNMs from layers grown by plasma
enhanced chemical vapour deposition (PECVD). This has the ad-
vantage of batch processed, smooth walled nanoporous mem-
branes as thin as the size of the biomolecules to be filtered.
Separation of biomolecules based on their size and charge is
one of the major interests in industrial and biomedical applica-
tions [16]. Martin et al. employed an array of nanochannels made
from gold nanotubule polymer membranes for ion separation [32],
detection [33], and sizing [34]. They have successfully achieved
charge dependent molecular transport and separation of en-
antiomeric drugs [35]. Stroeve et al. demonstrated that the self-
assembled functional molecules on these nanotubes can be used
for pH responsive transport of ions and protein analytes through
nanoporous membranes [36]. Ku and Stroeve demonstrated the
selective separation of two proteins with similar size by a nano-
porous membrane while varying the surface charge [37]. Cheow
et al. reported the selective transport of three differently charged
proteins across a conductive nanoporous alumina membrane un-
der varying potentials applied across the membrane [38]. Quoc
Hung et al. developed a synthetic nanoporous membrane for
charge selective transport of ionic molecules via control of surface
charge properties [39]. McGrath et al. have studied analytically and
experimentally the size based diffusion of proteins, molecules [40]
and gold nanoparticles [41] and reported that successful separa-
tion is possible only if the size range in the mixture is narrow.
McGrath et al. also used the porous nanocrystalline silicon mem-
branes as thin substrates for cell culture as these membranes offer
shorter pore length which helps in less product loss and lower
toxicity [42]. Microfluidic devices are powerful tools to study the
cell culture, but the fluid forces inside the channels can cause
unwanted cellular responses or sometimes change the direction of
the cells out of regions of observation or culture. A shear-free
microfluidic system is necessary to eliminate these complications
of fluid flow. Chung et al. have contributed the design of these
systems by introducing porous nanocrystalline silicon (pnc-Si) as
an ideal membrane for shear-free micro-fluidics which enables the
diffusion of small molecules despite attenuating fluid forces by
5 orders-of-magnitude with flow rates as high as 50 μL min
1 in
the flow chamber. Further, they have employed this shear reduc-
tion system to create an improved chemotaxis chamber for use
with shear-sensitive cells such as neutrophils and finally demon-
strated that their system helps to migrate neutrophils only by a
chemo attractant gradient [43]. In all the above mentioned papers,
selective transportation was demonstrated by varying the surface
charge on the molecules, by varying the pH of the solution, be-
tween negative, neutral and positive and probable mechanisms
were suggested.
The transport selectivity of molecules, based on the chemical
and physical properties, is observed through nanoporous mem-
brane as the pore size is sufficiently small that interactions be-
tween the pore surface and permeating molecules affect local
transport dynamics. In nanoporous membrane based
17
transportation, selectivity is typically achieved through control
over the membrane pore size relative to the size of the biomole-
cules that are being transported. Currently, researchers are fo-
cusing on the improvements in selectivity based on their charge.
The enhanced selective transportation based on biomolecular
charge, in addition to size, can also be achieved for molecules of
similar size. Hence in the present study, an attempt has been made
to identify the probable mechanisms behind the charge based
separation for similar sized nanoparticles. We have earlier re-
ported that the structure of the biomolecules remain unaltered
after passing through the nanoporous membranes [28]. There is a
need for a systematic study on the transportation of molecules,
with varying charge type as well as charge density of the mole-
cules, through ultrathin SNMs. In this work, we have added a
capping layer to vary the surface charge density of the nano-
particles. We have also investigated the onset as well as the ter-
mination of the diffusion process.
In order to investigate the dependence of the permeability of
the SNMs on the size and charge of the molecules, zinc oxide
(ZnO) nanoparticles of varying size and charge have been syn-
thesised and used as ‘analogues’ of the biomolecules in this work.
In addition, permeability of a few microorganisms such as gram
negative bacteria E.coli, the budding yeast Saccharomyces cerevi-
siae, Influenza A virus (causative agent of influenza) and Poliovirus
(causative agent of poliomyelitis) through the fabricated SNMs was
studied. Movements of microorganisms under simple diffusion
were analyzed to understand the role of the SNMs pore sizes in the
permeability process. The structural integrity of the filtered bac-
terial microorganisms was checked by observing their growth
before and after filtration.
2. Experimental details
2.1. Fabrication of SNMs
The process steps for the fabrication of the SNMs have been
reported earlier [27,28]. Briefly, an array of square windows of
dimensions 500 mm  500 mm were bulk micromachined in a
200 mm thick silicon wafer to a depth of 195 mm, using silicon di-
oxide as a mask. A stack of silicon dioxide, amorphous silicon, and
silicon dioxide of thickness 300 nm, 15 nm, and 300 nm respec-
tively were deposited on the unetched surface of the silicon wafer
using plasma enhanced chemical vapour deposition (PECVD). The
wafer was then subjected to rapid thermal annealing (RTA) at a
temperature of 850 °C with a ramp rate of 50 °C/s for 30 s in ni-
trogen ambient. Finally, the membrane was released by dry etch-
ing the silicon followed by stripping the oxide layers using 1%
buffered hydrofluoric acid. Fig. 1 shows the cross-section sche-
matic of the SNM.
2.2. Synthesis of ZnO nanoparticle
Spherical ZnO nanoparticles of various sizes and charges were
synthesised chemically to form a colloidal dispersion in butanol
medium with zinc acetate as a precursor and sodium hydroxide at
50 °C. The size of the nanoparticles was varied by varying the time,
Fig.1. Cross-section schematic of SNMs.
18 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24
solvent medium and concentration of the precursor. The nano-
particles were grouped into two sets based on their size de-
termined by transmission electron microscopy (TEM) at 1–4 nm
(S1) and 20–25 nm (S2). The surface charge was varied by adding a
capping agent, 3-aminopropyl triethoxysilane (APTES) for positive
charge [44] and citric acid for negative charge [45]. The charge
density was varied by changing the concentration of the capping
agent. Upon changing the concentration, the size of the nano-
particle did not vary, revealed by UV–vis spectra analysis (as in
Section 3.3). The charge of the nanoparticles was measured in
terms of the zeta potential, which was varied between
60 mV
and þ60 mV.
2.3. Characterisation of SNMs and ZnO nanoparticles
The diameter of the pores in the membranes and the ZnO na-
noparticles were determined by TEM. Powder diffraction mea-
surements were carried out by x-ray diffraction (XRD) with Cu Kά
radiation (λ ¼1.541 Å). The spectra were recorded in the region of
2θ between 30° and 80°. The pure zincite phase and the average
crystallite size of the sample was estimated with the help of
Scherer equation (B ¼Kλ/(2ravcos θ) using the diffraction intensity
of the (101) peak, where B is the full width at half-maximum
(FWHM) in radians of the diffraction peak obtained from the 2θ
plot, λ is the X-ray wavelength, K is a constant (0.94), θ is the Bragg
angle of the peak in radians, and rav is the average crystal radius
[46]. The particle sizes of the bare as well as the capped ZnO na-
noparticles were observed by TEM.
Absorption spectra were obtained using JASCO spectro-
photometer. A slit width of 0.5 nm and a sampling interval of
0.2 nm s
1 were used to record the spectra from 300 to 600 nm.
About 1 ml aliquot of the suspension was taken for analysis. Pure
solvent, butanol, was used as the reference. The average crystallite
size was obtained from the absorbance spectra using the effective
mass model [47,48] and taking Egbulk ¼3.2 eV, є ¼8.5, me ¼0.26,
mh ¼0.59, and Vm ¼14.8 cm3 mol
1.
2.4. Selective permeation of different size and charged ZnO nano-
particles through the SNMs
The systematic study of size- and charge- based permeation of
ZnO nanoparticles was carried out using a custom-made Teflons
cell setup as shown in Fig. 2. The setup has two reservoirs, cis and
trans, separated by a SNM. Butanol was added to both the cis and
trans tubes of the cell setup for hydrating the membrane. The
solution of nanoparticles and the blank solvent medium of 200 ml
each were added simultaneously to the cis and trans tubes re-
spectively. The solutions from both the tubes were taken out si-
multaneously after a time interval of 1 min. This procedure was
repeated for different time intervals of 5, 30, 60, and 120 min.
Fig. 2. Schematic of the setup used for separation of ZnO nanoparticles.
Since, there is a concentration difference of nanoparticles between
the cis and the trans tubes, the transport from cis to trans is by
diffusion and hence, the separation of nanoparticles can continue
till the concentration of nanoparticles on both the cis and trans
tubes are equal, i.e., 50% on both tubes. The optical density (O.D.)
was measured at 340 nm by measuring the UV-absorbance to
calculate the percentage of ZnO nanoparticles in each tube.
2.5. Permeation of different sizes microorganisms through SNMs
Permeability of live microbial cultures through SNMs was stu-
died using the same custom-made Teflons cell setup as described
in Section 2.4. The microbes were selected based on their size and
include Poliovirus (30 nm diameter), Influenza A virus (80–120 nm
diameter), E. coli (2.0 μm long and 0.5 μm in diameter) and yeast
cells (3–4 mm in diameter). Microbial diffusion study was done
under sterile conditions using multiple nanopores in a single na-
noporous membrane. Sterile isotonic saline (a solution of 0.90% w/
v of NaCl) was added to both the cis and trans tubes of the U-tube
of the cell for hydrating the membrane. Next 200 ml of fresh in-
dividual microbial culture suspended in saline was added to cis
tube and 200 ml of sterile isotonic saline was added to the trans
tube of the U-tube simultaneously. After 1 h, samples from both
the cis and trans tubes of the U-tube were collected.
Luria broth agar plates containing 10 g tryptone, 5 g yeast ex-
tract, 10 g NaCl and 15 g agar/L were prepared to identify the
presence of any live cells in the trans tube of the U-tube. Perme-
ability of the bacterial culture through the pores was checked by
spreading 30 ml of both the cis and trans samples on separate agar
plates in order to recover even a single live cell in the trans tube
and the cultures were allowed to grow for 24 h at 37 °C.
Similarly, permeability of the yeast cells through the pores was
checked by spreading 30 ml of both the cis and trans samples on
YPD (Yeast, potato extract and dextrose) agar plates and was al-
lowed to grow for 24 h at 30°. Poliovirus was suspended in 500 ml
of minimum essential medium (MEM) media, (Sigma, Manheim,
Germany) and was added to the cis tube of the setup. 500 ml of
sterile MEM media was added to the trans tube of the setup. After
1 h, samples from both tubes were collected and tested. Poliovirus
infection was tested on Vero cell line as well as by real time PCR
analysis. The Vero lineage was isolated from kidney epithelial cells
extracted from an African green monkey. Similarly, Influenza A
virus infection was tested on Madin-Darby Canine Kidney Epi-
thelial Cells (MDCK Line) as well as by real time PCR analysis. The
MDCK line was used as a model for epithelial cells. Epithelium
covers the internal organs and other surfaces of the body. In-
dependent experiments were done to verify the membrane cap-
ability for filtering microorganisms (naturally occurring mixed
culture) from unknown water sample (tap water). All experiments
were done in triplicate to check the reproducibility.
3. Results and discussion
3.1. XRD analysis of ZnO nanoparticles
All the diffraction peaks of the ZnO nanoparticles correspond to
a Wurtzite hexagonal structure and there are no additional re-
flections representing any intermediate phases except Zincite.
Wurtzite lattice parameters, such as the distance (d) between
adjacent planes in the Miller indices (hkl) (calculated from the
Bragg equation, λ ¼ 2dsin θ) were compared with standard values
of ZnO (Joint Committee on Powder Diffraction Standards (JCPDS)
No.036–1451) and showed excellent correlation. Impurities were
absent while changing the surface charges through capping. Fig. 3
demonstrates the XRD plots for samples of ZnO nanoparticles
 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24
Fig. 3. XRD analysis of ZnO nanoparticles for different zeta potentials.
having diameter of 1–4 nm (S1) with varying surface charge and
20–25 nm (S2) (size determined by TEM, mentioned in Section
3.2.).
3.2. TEM analysis
TEM measurements were done to study the shape and size of
the pores and the ZnO nanoparticles. The diameter of the pores
and the size of the ZnO nanoparticles were measured using ImageJ
software [49]. The pores were irregularly shaped, as shown in
Fig. 4, and were characterized by an average pore major diameter
of 8.2 nm, and an average pore minor diameter of 6.6 nm. The
nanoparticles were all spherical and the sizes were within the
range synthesised, which were grouped into two sets, viz 1–4 nm
(S1) and 20–25 nm (S2) in diameter. Upon capping, the nano-
particles S1 did not change their structure and size as observed
from the TEM images (Fig. 5).
3.3. UV–vis spectroscopy analysis
The electronic absorption spectrum of the ZnO samples in the
UV–vis range enables us to characterize the probable crystallite
size. Fig.6 shows the UV–vis spectra of ZnO nanoparticles syn-
thesised in both the presence and absence of capping agents. The
diameter of the ZnO nanoparticles was calculated to be 6.7 nm for
S1 which has a strong transmittance at 342 nm wavelength, while
S2 which had maximum transmittance at a wavelength of 357 nm
was calculated to be 24.8 nm. This right shift of wavelength is due
to the increase in particle size. The calculated diameter for both
Fig. 4. TEM analysis of SNMs.
19
samples S1 and S2 from UV–vis spectra correlates well with the
TEM analysis.
3.4. Analysis of systematic ZnO nanoparticle diffusion study
The O.D. was measured at the wavelengths 342 nm and 357 nm
for the original stock colloidal solution of ZnO nanoparticles hav-
ing diameter 1–4 nm and 20–25 nm. This O.D. value is used as a
reference for calculating the percentage of the ZnO nanoparticles
separated. The O.D. value for the solutions from cis and trans tube
after the experiment were measured at the same wavelength. If
the O.D. value of the stock solution is taken to be ODstock, and that
of the solution after the experiment is taken to be ODexpt, the
actual percentage of nanoparticles present in the cis or trans tube
ODexpt
is given by ODstock
×100. But, not all the ZnO nanoparticles diffuse
from the cis to the trans tube. This is because some of the nano-
particles can get stuck to the pore walls. The percentage of the ZnO
nanoparticles stuck to the membrane, referred to as the clogging
percentage, can be calculated by finding the difference between
the expected percentage of nanoparticles to be present in the trans
tube and the measured percentage. The expected percentage is
calculated by subtracting the measured percentage of the nano-
particles left behind in the cis tube from 100.
The separation of neutral ZnO nanoparticles based on size is
shown in Fig. 7a. The percentage of different size nanoparticles
present in both cis and trans tube is shown in Fig. 7b. The differ-
ences between the expected and measured percentage of the ZnO
nanoparticles present in the trans tube are shown in Fig. 7c. The
diffusion study reveals that the diffusion starts immediately
within one minute, as seen in Fig. 7a, and attains equilibrium or
stops in two hours. After two hours, the diffusion of the ZnO na-
noparticles of size 1–4 nm is completed, i.e., ∼50% of the nano-
particles are in the trans tube, while the concentration of nano-
particles of size 20–25 nm in the trans tube saturates at about 8%
in 1 h itself. In case of the 1–4 nm sized nanoparticles, the size of
the pores being larger than the size of the nanoparticles, the dif-
fusion of the nanoparticles is completed. In case of the 20–25 nm
sized nanoparticles, the size of the pore is smaller than the size of
the nanoparticles, and hence these nanoparticles are blocked. But,
statistically, there are a few pores which are larger in size than the
nanoparticles and hence, a small percentage of nanoparticles can
diffuse from the cis to the trans tube.
The separation of the ZnO nanoparticles based on their charge
is shown in Fig. 8a. The percentage of differently charged nano-
particles present in both cis and trans tubes is shown in Fig. 8b.
The differences between the expected and measured percentage of
the ZnO nanoparticles present in the trans tube are shown in
Fig. 8c. The charge based separation of the ZnO nanoparticles is
dependent on both the charge on the pore walls and the charge of
the nanoparticles. When the silicon pore wall comes in contact
20 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24
Fig. 5. TEM images of S1 particles, (a) Zeta potential 0 mV; (b) Zeta potential þ31 mV; (c) Zeta potential
55 mV and (d) TEM image of S2 particles.
Fig. 6. UV–vis spectra of ZnO nanoparticles for different zeta potentials.
with the alcoholic colloidal dispersion of the nanoparticles, a dif-
fused charge layer is formed. In open atmosphere, silicon with
native oxide forms surface hydroxyl groups in presence of alcohol,
which makes the pore walls negatively charged [50]. The iso-
electric pH (IEP) of silicon with native oxide is 2.3 [51]. The pH of
the ZnO nanoparticles in butanol medium is measured to be 11.
Since the pH of the solution is greater than the IEP of silicon, the
pore walls acquire a negative charge.
Since, the pore walls are negatively charged, the negative
charged ZnO nanoparticles will be repelled from the pores, the
positive charged nanoparticles will be attracted to the pores, and
there should be no effect on the neutral nanoparticles. The effec-
tive pore diameter for negatively charged particles becomes
smaller due to the repulsive force and the nanoparticles with a
high negative charge experience a higher repulsive force. Though
the nanoparticles experience a repulsive force, few of them can
still diffuse due to the variation in the pore size and Brownian
motion. The nanoparticles with a positive charge, on the other
hand, get attracted to the pore walls and the attractive force in-
creases with the magnitude of the positive charge. Thus, the pores
getting blocked is more in case of positively charged nanoparticles
than the negatively charged ones. This is evident from Fig. 8,
where the expected percentage of nanoparticles diffused from cis
to trans is always more than the measured value for positively
charged nanoparticles. The earlier work done by Striemer et al.
[16] and Achar et al. [28] demonstrated successful permeability of
biomolecules through SNMs which agrees with the present work.
Both groups looked at the variation in permeability due to the
interaction between the biomolecules and the pore walls either by
varying the charge on the membrane through functionalisation
[16] or by varying the pH of the solution [28]. The explanation of
the varying diffusion rate of biomolecules for a given pH, reported
earlier [28], was based on the surface charge of the biomolecules.
We have now substantiated that by selectively varying the charge
on the nanoparticles of the same size, with the pH unchanged.
3.5. Analysis of clogging of the pores in the membrane
Diffusion through the nanoporous membrane is impeded for
both negative and positively charged nanoparticles, as seen in
Fig. 8a, but the difference is in the percentage of blocking due to
the clogging of pores. The neutral nanoparticles exhibit negligible
pore clogging with the diffusion increasing monotonically with
time and this is true also of particles with a small negative surface
charge. As the negative charge density increases, the percentage of
the nanoparticles diffusing from cis to trans decreases from 46% for
slightly negative charged nanoparticle to 4% for highly negative
charged nanoparticle. It is evident that the negatively charged
 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24 21

Fig. 7. (a) Separation of the ZnO nanoparticles based on particle size; (b) percentage of different size nanoparticles present in both cis and trans tube after 2 h; (c) expected
and measured percentage of the ZnO nanoparticles present in the trans tube after 2 h.

Fig. 8. (a) Separation of the ZnO nanoparticles based on particle surface charge; (b) percentage of different charged nanoparticles present in both cis and trans tube after 2 h;
(c) expected and measured percentage of the ZnO nanoparticles present in the trans tube after 2 h.

Fig. 9. Plate showing (1) growth of E.coli bacterial culture; (2) and (3) the bacterial Fig. 10. Plate showing (1) growth of yeast culture; (2) and (3) the Yeast solution
solution after filtration using SNMs, (2) sample from cis tube, (3) sample from trans after filtration using SNMs, (2) sample from cis tube, (3) sample from trans tube,
tube and (4) sterile saline solution. (4) sterile saline solution.

nanoparticles experience a repulsive force from the pore walls
which reduces the effective pore diameter and thus impedes their
diffusion from cis to trans tube. In case of positive charged nano-
particles, the percentage of diffusion decreases from 24% for
slightly positive charged nanoparticles to 5% for highly positive
charged nanoparticles.
It is also observed that, for all individual positive charged na-
noparticles, the diffusion saturates indicating complete clogging
after a certain time interval. A similar saturation is also observed in
case of negative charged nanoparticles, but here the diffusion sa-
turates due to the repulsion of nanoparticle from the pore wall,
leading to an effectively smaller pore size. We now examine the
22 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24

Fig. 11. (a) Tap water filtration using SNMs and Plate showing (1) growth of E.coli bacterial culture; (2) and (3) the bacterial solution after filtration using SNMs (2) sample
from cis tube, (3) sample from trans tube, (4) sterile saline solution. (b) Tap water filtration using SNMs and Plate showing (1) growth of yeast culture; (2) and (3) the Yeast
solution after filtration using SNMs, (2) Sample from cis tube, (3) sample from trans tube, (4) sterile saline solution.

Fig. 12. (a) Infected Cell line after addition of cis tube media (poliovirus infection), (b) normal Cell line not infected after addition of trans tube media.

clogging percentage of the different positively charged nano-
particles within a specific time interval. For a one minute interval,
the clogging percentage decreases from 8% for slightly positive
charged nanoparticles to 4% for highly positive charged nano-
particles. The data after 5 min and 30 min time intervals reveal the
same i.e. the clogging percentage decreasing with increasing sur-
face charge density. A possible explanation for this can come from
the behaviour of the nanoparticle in that the particle diffusing in
the solution first attempts to locate the nanopore and then suc-
cessfully permeate it. In case of lower surface charge density na-
noparticle, they translocate through the nanopore one by one
without competition. The translocation of individual particle is
assuming that once a particle is gone, another one immediately
appears in the bounding volume, and the process repeats [52].
In case of higher positive surface charge density, the nano-
particles not only get attracted by the oppositely charged pore
walls, but at the same time they repel each other due to the higher
surface charge. Hence, they do not interact with the pore wall as
much as the lower surface charge density nanoparticles. Some
arbitrary values of clogging percentage results because the pore
clogging is mainly due to the non-covalent weak electrostatic in-
teraction and it is possible for the nanoparticles stuck on the pore
walls to become free. This phenomenon is also applicable for na-
noparticles with higher negative surface charge density. These
nanoparticles interact with the adjacent nanoparticles and are
repelled from each other. In this situation an additional repulsion
occurs between the higher negatively charged nanoparticles with
the pore wall. In Fig. 8a, it is seen that diffusion rate is inversely
proportional to the surface charge density for both positive and
negative charged nanoparticles. The interesting finding is in the
mechanism for nanoparticle diffusion. In case of higher negative
charged nanoparticles the diffusion rate reduced due to the higher
repulsive force whereas the diffusion rate of the higher positive
charged nanoparticles reduced due to the clogging of the nano-
particles inside the pores. In Fig. 8c, it is demonstrated that the
clogging percentage in higher positive surface charge (þ 56 mV) is
greater compared to that of higher negative surface charge
(
55 mV).
3.6. Analysis of microorganism diffusion study
Filtration study of live microbial cultures was carried out using
SNMs as mentioned in Section 2.5. None of the microbes, Poliovirus
(30 nm diameter), Influenza A virus (80–120 nm diameter), E.coli
(2.0 μm long and 0.5 μm in diameter) or Saccharomyces cerevisiae
yeast cells (3–4 mm in diameter), passed through the nanopores in
any of the SNMs as shown in Figs. 9 and 10. All the membranes
were intact after filtration. Filtration study with unknown water
 P. Bhadra et al. / Journal of Membrane Science 503 (2016) 16–24
sample (tap water) did not show any E. coli (Fig. 11a) or yeast cell
(Fig. 11b) colonies on suitable agar plates containing the trans
samples after 24 h of growth. This indicated that these membranes
are suitable for blocking bacterial and yeast samples. Spectro-
scopically, microbes can be detected only when present above a
particular concentration in the sample. However, when grown on
a suitable nutrient media, the doubling time for E. coli ranges from
20 min to 1 h and that of S. cerevisiae ranges from 1.25 to 2 h.
Hence, a single E. coli cell, which divides approximately every
30 min, can grow into a colony containing 107–108 cells in 12 h
(224 ¼1.7  107) [53] and a single live S. cerevisiae cell, which di-
vides approximately every 85–90 min, can grow into a colony
containing 5.24  105 cells in 24 h (219–5.24  105) [54]. Hence in
the present study, yeast and bacterial samples from the trans tube
were plated on nutrient agar plates and allowed to grow for 24 h
so that even a single live cell crossing the membrane can be de-
tected as it will form a colony on agar plate. Presence of Poliovirus
and Influenza A virus in the sample was tested by using Real time
PCR with Ct (cycle threshold) value which is the intersection be-
tween an amplification curve and a threshold line. It is a relative
measure of the concentration of target in the PCR reaction. The
measured Ct value in cis tube for poliovirus was 17.99 and for In-
fluenza A virus was 14.9, which showed strong positive reactions
indicative of abundant target nucleic acid in the samples whereas
the value in trans tube for both the samples revealed negative
results, which indicated that the viral particles in the samples
were most probably absent. However, in PCR reactions, a mini-
mum of 20–50 ng of template DNA is required. Poliovirus enters
the VERO cell line, replicates in six to eight hours, yielding 10,000
to 100,000 virus particles per cell, kills the host cell and spreads to
infect other cells, resulting in tissue lysis. Fig. 12a revealed the
infected VERO cell line after addition of cis tube media whereas in
the trans tube media (Fig. 12b) normal cell line was not infected,
even after 48 h of growth, which indicated that none of the intact
poliovirus particles, capable of replicating in host cells and causing
infection, diffused through the SNMs. Lysed cell particles might
have passed through the pores but they are not pathogenic as only
the whole live cells replicate and are virulent.
4. Conclusion
SNMs of average major and minor pore diameter of 8.2 nm and
6.6 nm were successfully fabricated and used for selective trans-
portation of ZnO nanoparticles of varying size and charge. The
nanoparticles fabricated were of spherical shape with size ranging
from 1–4 nm and 20–25 nm. 1–4 nm particles completely diffused
through the membrane, while the passage of larger nanoparticles
ranging from 20 to 25 nm is blocked. The pore walls being nega-
tively charged helps in separation of charged nanoparticles. The
nanoparticles that are neutral or have small negative charge ex-
perience no hindrance, while the positive and more negatively
charged nanoparticles are restricted based on their zeta potential.
The lower the zeta potential, the lesser is the percentage of
blocking of the nanoparticles. In addition, the positively charged
nanoparticles demonstrate a greater tendency to clog the pores.
Finally the nanoporous membrane, when used as a microbial filter,
was successful in removing 100% of all the live microbial con-
taminants (yeast, bacteria and virus) in the range of 30 nm to
3 μm.
Acknowledgement
The authors would like to thank Prof. M.G.Basavaraja, Depart-
ment of Chemical Engineering, IIT Madras for the use of the zeta
23
potential analyser. The measurements on the virus were carried
out in the laboratory of Dr. Asha Abraham at CMC, Vellore and we
thank her for providing the virus samples and the RTPCR data.
Sponsorship of CNNP by DeitY, Government of India is gratefully
acknowledged.
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