Antibacterial porous polymeric monolith columns with amphiphilic and polycationic character on cross-linked PMMA substrates for cell lysis applications

Article abstract of DOI:10.1039/c3ra43087a

The application of porous polymeric monolith (PPM) columns as an effective tool for bacterial cell lysis within microfluidic chips is demonstrated. By taking advantage of the large surface area and controllable pore size inherent to PPMs, we developed a double mechanism cell lysis technique. The bacterial cell wall is mechanically sheared by flowing through the porous medium of the PPM column, but it is also damaged and disintegrated by physical contact with the antibacterial polymeric biocide covering the porous surface. This leads to leakage of the intracellular contents. The stable and non-leaching antibacterial column introduced in this work alleviates the need for chemical or enzymatic lysins and their potential release of polymerase chain reaction (PCR) inhibitors. The PPM columns were obtained by the photoinitiated free radical copolymerization of n-butyl methacrylate (BuMA) and N-(tert-butyloxycarbonyl) aminoethyl methacrylate (Boc-AEMA) in the presence of a cross-linker and porogenic solvents. The porous network was synthesized directly inside a microfluidic channel fabricated in a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by laser micromachining. After removing the Boc protecting group with phosphoric acid, an amphiphilic and cationic network structure reminiscent of synthetic mimics of antimicrobial peptides (SMAMPs) was obtained. The antibacterial activity of the PPM columns was tested against Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli) cells. Cell lysis was evidenced by DNA release, which was then amplified by PCR and confirmed by gel electrophoresis, to verify that the antibacterial monolithic columns did not strongly interfere with the PCR process.

Full text of DOI:10.1039/c3ra43087a

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PAPER
Antibacterial porous polymeric monolith columns with
amphiphilic and polycationic character on cross-linked
PMMA substrates for cell lysis applications
Cite this: RSC Adv., 2013, 3, 24177
Mohamed Aly Saad Aly, Olivier Nguon, Mario Gauthier and John T. W. Yeow*
The application of porous polymeric monolith (PPM) columns as an effective tool for bacterial cell lysis
within microfluidic chips is demonstrated. By taking advantage of the large surface area and
controllable pore size inherent to PPMs, we developed a double mechanism cell lysis technique. The
bacterial cell wall is mechanically sheared by flowing through the porous medium of the PPM column,
but it is also damaged and disintegrated by physical contact with the antibacterial polymeric biocide
covering the porous surface. This leads to leakage of the intracellular contents. The stable and non-
leaching antibacterial column introduced in this work alleviates the need for chemical or enzymatic
lysins and their potential release of polymerase chain reaction (PCR) inhibitors. The PPM columns were
obtained by the photoinitiated free radical copolymerization of n-butyl methacrylate (BuMA) and
N-(tert-butyloxycarbonyl)aminoethyl methacrylate (Boc-AEMA) in the presence of a cross-linker and
porogenic solvents. The porous network was synthesized directly inside a microfluidic channel fabricated
in
a cross-linked poly(methyl methacrylate) (X-PMMA) substrate by laser micromachining. After
removing the Boc protecting group with phosphoric acid, an amphiphilic and cationic network structure
reminiscent of synthetic mimics of antimicrobial peptides (SMAMPs) was obtained. The antibacterial
activity of the PPM columns was tested against Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli)
cells. Cell lysis was evidenced by DNA release, which was then amplified by PCR and confirmed by gel
electrophoresis, to verify that the antibacterial monolithic columns did not strongly interfere with
the PCR process.
Received 19th June 2013
Accepted 8th October 2013
DOI: 10.1039/c3ra43087a
www.rsc.org/advances
bacteria and fungi. Research is ongoing to engineer these
polymers so as to imitate natural host defense peptides (HDPs)
used by the immune systems in living organisms to kill bacteria.
1
. Introduction
Porous polymeric monoliths (PPMs) were introduced over the
last decade, and are mainly used in liquid chromatography and That emerging class of antimicrobial polymers, termed
3
biological substances extraction. PPMs are formed within the ‘synthetic mimics of antimicrobial peptides’ (SMAMPs),
boundaries of a closed volume such as capillaries and micro- SMAMPs is synthesized to mimic the main features of HDPs:

uidic channels, by free radical polymerization of a mixture that cationic charge and amphiphilic character, which lead to the
includes functional monomers, a cross-linking monomer, a permeation and then the disintegration of the bacterial
free-radical initiator, and porogenic solvents. Recently PPMs
were utilized for cell lysis within microchips, as demonstrated
by Mahalanabis et al., in which the bacterial cells were lysed by
membrane. An antimicrobial surface is a form of antimicrobial
polymers killing cells by contact. Tiller et al. introduced in 2001
surfaces that killed bacteria upon contact, and termed it
4
1
mechanical shearing of the cell walls through owing in a PPM ‘contact killing’. Antibacterial polymers have been attached to
column within a microuidic channel assisted with detergent different surfaces by numerous techniques including chemical
5
,6
7,8
9
lytic conditions. The same group impregnated multi-walled graing,
layer-by-layer deposition,
and graing from.
9
carbon nanotubes (MWCNT) into the PPMs and claimed that Madkour et al. thus used surface-initiated atom transfer radical
this approach improved the lysis efficiency, albeit with chemical polymerization (ATRP) to grow an antibacterial copolymer,
2
and enzymatic pretreatment of the bacterial cells.
poly(butyl methacrylate-co-aminoethyl methacrylate), from
surfaces. These displayed a high antimicrobial activity that
killed 100% of S. aureus and E. coli in less than 5 minutes.
In this paper we report for the rst time on the preparation of
porous polymeric monoliths with antibacterial surfaces within
Antimicrobial polymers, oen called polymeric biocides, are
polymers possessing antimicrobial properties, i.e. the ability to
inhibit the growth and eventually kill microorganisms such as
University of Waterloo, 200 University Ave West, Waterloo, Ontario, Canada N2L 3G1. a microuidic chip, to form an antibacterial porous structure
E-mail: jyeow@uwaterloo.ca; Fax: +1 519 746-4791; Tel: +1 519 888-4567 ext. 32152
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belonging to the SMAMPs family. We fabricated antimicrobial Table 1 MMA and EGDMA volumes used in the preparation of the different
cross-linked PMMA (X-PMMA) substrates
PPM columns by in situ photoinitiated free radical copolymer-
ization of n-butyl methacrylate (BuMA) and N-(tert-butylox-
Cross-linker (mol%)
MMA (mL)
EGDMA (mL)
ycarbonyl)aminoethyl methacrylate (Boc-AEMA), cross-linked
with ethylene glycol dimethacrylate (EGDMA) in the presence of
porogenic solvents. In the PPM developed, bacterial cells
undergo lysis through a dual mechanism: mechanical lysis, by
forcing bacterial cells to ow into the porous medium of the
PPM column, and contact killing, when the cells come in
contact with the antibacterial surface of the column. The non-
3
5
1
1
2
145.6
142.9
136.4
130.4
125.0
4.4
7.1
13.6
19.6
25.0
0
5
0
2
leaching nature of the antibacterial structure yields a lysate crystallizing dish (190 ꢀ 100 cm ) covered with aluminum foil
ready to use for PCR. We also demonstrate the use of X-PMMA and kept under nitrogen atmosphere. The dish was immersed
ꢂ
as a substrate for the microuidic channel that covalently in a water bath at 35 C and leveled to insure a uniform
bonds with the monolithic column through the unreacted C]C substrate thickness. The solution was kept for 1.5 h under
double bonds on the surface of X-PMMA. This approach elim- nitrogen before turning off the water bath heater. As the poly-
inates the need of surface functionalization to create a bonding merization reaction was exothermic, cold water was added to
intermediate layer between the substrate and the PPM column. the water bath to avoid overheating, and then the dish was kept
under nitrogen at room temperature for 11 h. The transparent
cross-linked PMMA substrate was washed by soaking in meth-
anol for 30 min, and dried under air ow. The polymer was
2
. Experimental
2.1. Materials
easily removed from the dish with a spatula. The X-PMMA
2
Methyl methacrylate (MMA, 99%), butyl methacrylate (BuMA, substrate was then cut into 40 ꢀ 20 mm slices with 1 mm
9
9%), and ethylene glycol dimethacrylate (EGDMA, 98%) were thickness using a laser ablation machine. With a spectropho-
purchased from Sigma-Aldrich (Oakville, ON Canada), and tometer, the absorbance of the X-PMMA samples at 365 nm
ltered over alumina prior to use to remove inhibitors. wavelength was determined to be 0.238.

Dichloromethane (DCM, 99.9%, Chromasolv Plus), tetrahydro-
furan (THF, 99.9%, Chromasolv Plus) and triethylamine (TEA, 2.3. Microchip fabrication
9
9.5%) were also purchased from Sigma-Aldrich and were
Microuidic channels were micromachined on the X-PMMA
substrates with a 10.6 mm CO laser engraving system (Universal
distilled prior to use. Benzoyl peroxide (BPO, 97%), N,N-diiso-
propylethylamine (DIPEA, 99% Reagent Plus), methacryloyl
chloride (MACl, 97% purum), 1-dodecanol (98%, reagent
grade), cyclohexanol (99% Reagent Plus), 2,2-dimethoxy-2-phe-
nylacetophenone (DMPAP, 99%) and methanol (99.9% Chro-
masolv) were purchased from Sigma-Aldrich and used without
further purication. 2-Aminoethyl methacrylate hydrochloride
2
Laser Systems, VLS2.30). In order to obtain an enclosed
channel, another piece of the X-PMMA substrate in which two
holes were drilled for the inlet and outlet was chemically
bonded with the substrate hosting the microchannel. A thin
layer of BuMA was applied between the two X-PMMA layers to
enhance bonding between the two networks. Thermal bonding
was achieved by placing the top and bottom substrates under
(AEMA-HCl, 95%) was purchased from Polysciences Inc. (War-
rington, PA USA) and was used without further purication.
Fumed silica (powder, 0.2–0.3 mm avg. part size), phosphoric
acid (85%), 1,4-dioxane (anhydrous, 99.8%), triuoroacetic acid
ꢂ
pressure in a vise press and in a pre-heated oven at 130 C for
30 min. Two 30 G syringe needles were trimmed and placed over
the inlet and outlet holes. They were set with epoxy glue mixed
with ne fumed silica powder, to achieve a hard and stable
adhesive layer.
(TFA, 99%) and potassium bromide (ACS reagent, 99.0%, KBr)
were purchased from Sigma-Aldrich and used as received. iTaq
polymerase, 10ꢀ PCR buffer, and magnesium chloride were
obtained from Biorad. Primers, and dNTP mix (dATP, dCTP,
dGTP and dTTP) were obtained from Sigma-Aldrich. Ethidium
bromide (UltraPure 10 mg mL , EtBr) was purchased from Life
Technologies Inc. (Burlington, ON Canada). 100 bp DNA ladder
was purchased from BioLabs.
2.4. Antibacterial polymer synthesis and characterization
ꢁ
1
2
.4.1. Boc-EA synthesis. The synthetic procedure shown in
10
Scheme 1 was adapted from Fedotenko et al. Briey, distilled
triethylamine (TEA, 19.2 mL, 138 mmol) was added to a solution
of 2-aminoethanol (5.42 mL, 89.7 mmol) in 200 mL of dry
dichloromethane (DCM) and stirred at room temperature for
2.2. Synthesis of cross-linked PMMA
ꢂ
30 min before cooling to 0 C. A solution of di-tert-butyl dicar-
MMA and EGDMA were ltered over a thin layer of basic bonate (BOC
2
O, 20 g, 89.7 mmol) in 50 mL of dry DCM was then
aluminum oxide (alumina) and added to a dry Erlenmeyer ask. added drop-wise. The reaction mixture was stirred at room
The total reaction volume was set at 150 mL, and the amount temperature for 10 h, and quenched with a saturated aqueous
of EGDMA was adjusted to either 3, 5, 10, 15, or 20 mol% as solution of ammonium chloride (NH Cl). Liquid extraction with
4
shown in Table 1. Aer the addition of BPO (2.30 g, 9.5 mmol, 100 mL of DCM was repeated 3 times, and the combined
0.75 mol%) the mixture was degassed for 1 h under N
2
ow. The organic fractions were washed with brine. Aer drying over
solution was transferred with a canula to a dry soap-washed Na
2
SO
4
, the solvent was evaporated under reduced pressure.
2
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2.4.3. Network fabrication and deprotection. The compo-
sition used to prepare the antibacterial network was rst
investigated off-chip, by mixing 0.3 g of Boc-AEMA (30 wt%),
0.25 g of BuMA (2.5 wt%), 0.175 g of EDMA (17.5 wt%), 0.5 g of
distilled THF (50 wt%), and 5 mg of DMPAP (1 wt% with respect
to the monomers). The mixture was sonicated for 45 min, to
help dissolve the crystallized Boc-AEMA, and then stirred for
30 min to achieve complete dissolution. The mixture was used
to form a thin network on a at 20% X-PMMA substrate by
photoinitiated polymerization under UV irradiation. The
substrate with the graed lm was then washed with ethanol
and dried under nitrogen for 5 min, immersed in a beaker
containing 25 mL of phosphoric acid with stirring for 3 h for
deprotection, washed with ethanol, and dried for 10 min.
2
.5. Monolith formation and characterization
A mixture consisting of Boc-AEMA (15.6 wt%), BuMA (1.3 wt
), EDMA (9.1 wt%), 1-dodecanol (52.4 wt%), cyclohexanol
21.6 wt%), and DMPAP (1 wt% with respect to the monomers)
Scheme 1 Synthesis of Boc-EA.
%
(
The crude product was puried on a silica gel column (2%
methanol/DCM) and yielded a clear liquid (9 g, 55.8 mol, 62%).
was introduced into the microchannel, and polymerization
was triggered by irradiation for 15 min. The microchannel was
1
Rf: 0.29. H NMR (300 MHz, CDCl
(
3
): d 5.268 (br s, 1H), 3.647
then ipped 180 degrees and le under the UV source for
1
3
s, 2H), 3.238 (s, 2H), 3.007 (br s, 1H), 1.409 (s, 9H). C NMR
300 MHz, CDCl ): d 167.166, d 155.629, d 135.920, d 126.776, d
9.447, d 63.823, d 39.573, d 28.230, d 18.160.
.4.2. Boc-EAMA synthesis. The synthetic procedure shown
ꢁ2
1
5 min at 365 nm UV wavelength and 200 mJ cm energy in a
(
3
cabinet containing a UV lamp (ENF-260C, Spectronics Corp.
Westbury, NY, USA) as shown in Scheme 3. The microchannel
was then ipped 180 degrees and le under the UV source for
7
2
11
in Scheme 2 was adapted from Kuroda et al. Boc-EA, DCM and
DIPEA were added to an oven-dried round-bottom ask, and the
solution was degassed with nitrogen ow for 20 min. The ask
was then cooled in a dry ice/acetone bath and MACl was added
drop-wise with vigorous stirring under nitrogen atmosphere.
Aer 15 min, the solution was brought to room temperature and
allowed to react overnight. The light purple solution was
washed successively with water, 10% (w/v) citric acid, 10% (w/v)
K CO , 9% (w/v) NaHCO , and saturated NaCl aqueous solu-
1
5 min longer. Two 30 G syringe needles were trimmed and
placed over the inlet and outlet holes. They were set with epoxy
glue mixed with ne fumed silica powder, to achieve a hard
and stable adhesive layer. Using a Pico plus syringe pump
(

Harvard apparatus, Holliston, MA USA) the microchannel was
ushed with ethanol to remove the unreacted monomers and
the porogens.
Linear PMMA was rst considered as a substrate for the
microuidic channel, but it cracked and gradually dissolved
when exposed to the monomers. To overcome this obstacle
cross-linked PMMA was used. This approach also ensures that
unreacted double bonds on the cross-linked surface improve
adhesion between the PPM column and the substrate. Attenu-
ated total reectance-Fourier transform infrared spectroscopy
2
3
3
tions. The bottom organic layer was dried over Na SO , and the
2
4
solvent was evaporated under reduced pressure. Recrystalliza-
tion from DCM/hexanes yielded clear crystals (22.4 g, 61%
1
yield). H NMR (300 MHz, CDCl3): d 6.090 (s, 1H), 5.552 (s, 1H),
4.768 (br s, 1H), 4.170 (t, 2H), 3.405 (br, 2H), 1.915 (s, 3H), 1.410
(s, 9H). 13C NMR (300 MHz, CDCl3): d 167.166, d 155.629, d
(ATR-FTIR) was used to conrm the presence of dangling C]C
135.920, d 126.776, d 79.447, d 63.823, d 39.573, d 28.230, d
double bonds on the surface of the cross-linked PMMA.
Monolith pressure tests were conducted to evaluate the bonding
strength between the monolith column and the substrate. To
that end the microuidic chip containing the PPM column was
18.160.
connected to a compressed N cylinder that was used to pump
2
ethanol through the monolithic column. The pressure was
increased in 10 PSI increments every 5 min to determine the
pressure at which the monolithic column started to debond
from the substrate.
Images of the monoliths were obtained with a Hitachi SU-70
scanning electron microscope (SEM) (Hitachi High-Technolo-
gies Corporation, Tokyo, Japan) at an acceleration voltage of
10 kV. The SEM samples were prepared by immersing the
microchannel hosting the PPM column into liquid nitrogen,
Scheme 2 Synthesis of Boc-EAMA.
and then cutting perpendicularly to the monolith-lled
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techniques. The same composition of the functional and cross-
linking monomers was used in both the nonporous network
and the porous column. E. coli and B. subtilis bacterial cells were
selected to evaluate the cell lysis ability of the antibacterial PPM
column on gram-negative and gram-positive bacterial cells,
respectively.
2
.7.1. Cell samples. E. coli and B. subtilis cells were grown
ꢂ
in lysogeny broth (LB) in an incubator at 37 C, shaking at
180 RPM and le overnight. The bacterial cells were then
washed twice with ID water and then re-suspended in phos-
phate buffered saline (PBS) before the experiments. The stock
cell concentration was adjusted with an Ultrospec 2100 pro
UV/visible spectrophotometer (Biochrom Ltd., Cambridge, UK),
to prove an optical density (OD) value of 0.6 at 600 nm (OD600
.6) for the bacteria sample. The cell solutions were then diluted
as needed in different experiments.
.7.2. Non-porous polymeric network. The antimicrobial
¼
0
2
properties of the deprotected network were evaluated by the
LIVE/DEAD Cell Viability Assay (L7012, Molecular Probe, USA),
used to directly monitor cell viability. The assay uses two uo-
rescent nucleic acid stains, SYTO 9 (green) and propidium
iodide (PI; red). The SYTO 9 stain penetrates both healthy
bacterial cells (with intact membranes) and non-healthy cells; it
therefore labels both live and dead bacteria. In contrast propi-
dium iodide penetrates only bacteria with damaged
membranes, thus reducing the SYTO 9 uorescence intensity.
Consequently, live bacteria with intact membranes uoresce
green while dead bacteria (with disintegrated membranes)

uoresce red, while the background remains virtually non-

uorescent. Images were captured on a uorescence micro-
Scheme 3 Reactions for the formation of the monolith column on 20% X-
scope (Nikon Eclipse E600FN upright) with a digital camera
Nikon Photometrics Coolsnap EZ 12-Bit Monochrome Cooled
PMMA surface, and removal of the Boc protecting group with phosphoric acid.
(
CCD and NIS-ELEMENTS IMAGING Soware) through a dual-
band lter, so that both cells with and without intact cell
membranes could be visualized simultaneously. SYTO 9 and PI
X-PMMA channel. Gold was sputtered onto the samples prior to
SEM imaging.
(0.15 mL of each) were mixed on a vortex mixer, 100 mL of
bacteria suspension were added and mixed, and the stock
mixture was incubated for 15 minutes at 37 C. A 10 mL of
2
.6. Activation of the antibacterial column via deprotection
Deprotection of the Boc-AEMA units was achieved by owing
50 mL of phosphoric acid through the PPM column before
ushing with ethanol and deionized (DI) water to remove acid
ꢂ
bacterial suspension–stains mixture was dropped over the non-
porous network and covered with a thin microscope slide. The
sandwiched layers were le in contact under the microscope,
and uorescent microscopic images were recorded aer zero
and 300 s of contact time.
2

residues. Aer purging, the PPM column was opened by cutting
vertically through the monolith-lled X-PMMA channel with an
electrical saw. The column material was then removed by
scraping and ground into a ne powder, mixed with KBr in a
1
2.7.3. Porous polymeric monolith column. Cell lysis was
conrmed via the ethidium bromide (EtBr) intercalation assay
as an indicator of DNA presence in the cell lysate. When EtBr is
exposed to ultraviolet light, it uoresces with an orange color
which intensies aer intercalation in DNA. This assay thus
relates the intensity of EtBr uorescence to the DNA concen-
tration in the cell lysate. The orescence intensity of EtBr aer
intercalation in the DNA released from the microchannel
hosting the porous and antibacterial monolithic column was
quantied with a Quanta-Master 4 spectrouorometer (Photon
Technology International, London, ON). A 0.1 mL aliquot of
bacterial cells was suspended in PBS buffer (E. coli and B. sub-
ꢂ
: 50 weight ratio, and dried in a vacuum oven at 60 C over-
night. KBr pellets were prepared for analysis by pressing the
powder in a dye at 10 kpsi for 3 min on a Carver 3851 Press
(Thomas Scientic, Swedesboro, NJ USA). To validate the
deprotection process, FT-IR spectra were acquired at room
temperature on a Bruker Tensor 27 spectrometer by averaging
64 scans recorded at a rate of 1 scan per s. The wavenumber
ꢁ1
region scanned was between 400 and 4000 cm
.
2.7. Antibacterial activity
The antibacterial properties of the non-porous network and the tilis at OD600 ¼ 0.25) and pumped through the PPM column
ꢁ1
microporous monolithic column were investigated by several before and aer deprotection at a ow rate of 1 mL min , and
2
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Table 2 Primers used in PCR experiments
Primer direction
Primer sequence
0
0
0
E. coli forward primer
E. coli reverse primer
B. subtilis forward primer
B. subtilis reverse primer
5 -AAAACGGCAAGAAAAAGCAG-3
0
5 -ACGCGTGGTTACAGTCTTGCG-3
0
0
5 -AAGAGTTTGATCATGGCTCAG-3
0
0
5 -AGGAGGTGATCCAACCGCA-3
the cell lysate was collected at the outlet. In this assay 0.02 mL
Fig. 1 Microfluidic chip fabricated by laser micromachining on an X-PMMA
aliquots of E. coli and B. subtilis lysates were each added to a substrate filled with a monolith column (right) and SEM image of the PPM column
spectrophotometer cuvette containing 0.380 mL of DI water and packing (left).
0
0
.03 mL of EtBr from a stock solution with a concentration of
ꢁ1
.4 mg L
.
Furthermore, to validate the DNA released as a result of cell microuidic channel laser-micromachined onto
a
20%
lysis and to ensure that the PPM column did not leach any X-PMMA substrate hosting the monolithic column. To facilitate
polymerase chain reaction (PCR) inhibitors, the cell lysate was the tube connections between the microuidic channel and the
used in PCR. The presence of DNA amplied by PCR was bacterial suspension reservoir, syringe needles were set over the
conrmed by the increase in uorescence intensity of ethidium inlet and outlet of the microuidic channel and bonded to
bromide, and nally by gel electrophoresis. A Bio-Rad gel elec- the surface with a mixture of epoxy glue and fumed silica. The
trophoresis apparatus served to analyze the PCR products on unreacted double bonds on the surface of the cross-linked
1
.2% agarose gel using a DC voltage of 85 V and a running time PMMA substrate allowed covalent bonding with the monolith
of 30 min. The gel was subsequently removed from the chamber material and contributed to the mechanical stability of the PPM
and was imaged with a Bio-Rad Doc XR imagining system.
column within the microuidic channel, by decreasing the
.7.4. PCR reagents and experimental setup. The PCR possibility of debonding leading to the creation of voids along
2
reaction was performed in a T100 Thermal Cycler (Bio-RD, the monolithic column. Surface characterization of the cross-
Montreal, QC Canada) in a 25 mL volume consisting of 300 nM linked PMMA sheets was performed by ATR-FTIR to conrm the
of forward primer, 300 nM of reverse primer, 200 mM of dNTPs, presence of unreacted carbon–carbon double bonds upon
3
2
.5 mM of magnesium chloride, 0.625 U of iTaq polymerase, copolymerization of MMA and EGDMA; the spectra obtained are
ꢁ1
.5 mL of 10ꢀ PCR buffer, and 5 mL of the crude lysate collected shown in Fig. 2. The absorption band at 1637 cm is charac-
1
2
at the outlet of the microuidic channel. The structure of the teristic for the C]CH
2
stretching vibration mode vicinal to an
E. coli and B. subtilis primers used in the PCR reaction is ester group. Also visible is the C]CH out-of-plane bending
provided in Table 2. The PCR tubes were rst preheated and vibration mode 16 at 949 cm . Aer normalization of the
13
14
16
2
ꢁ
1
ꢂ
ꢁ1
incubated at 95 C for 3 minutes, and then the PCR thermal spectra to the C]O stretching band 1722 cm , the C]C
cycle was programmed to run for 30 cycles with 95 C for absorbance was found to increase slowly with the cross-linker
0 seconds, 63 C for 1 minute, and 73 C for 1 minute.
ꢂ
ꢂ
ꢂ
3
content, as illustrated in the inset of Fig. 2. In contrast to native
3. Results and discussion
3.1. Monolith formation and bonding to the substrate
To form the monoliths, two functional monomers: Boc-AEMA
and BuMA, were copolymerized with a third cross-linking
monomer in the presence of a porogenic solvent mixture. The
functional monomers play a key role in the nal product, as
they introduce the antibacterial component in the structure.
The cross-linking monomer is also necessary to form a network,
by contributing to the mechanical stability of the monolith. To
form a porous network and also control the pore size, porogenic
solvents were used. Since Boc-AEMA was synthesized in the
crystallized form, the monomer mixture was sonicated and
stirred to form a homogeneous solution prior to polymeriza-
tion. In order to achieve fast polymerization and complete
monomer conversion within approximately 10 min, DMPAP was
1
/2
selected as photoinitiator due to its relatively short half-life (t
)
Fig. 2 ATR-FTIR spectra for native and cross-linked PMMA samples with cross-
15
or high decomposition rate (kd). Fig. 1 (le) provides an SEM
linker contents ranging from 3 to 20 mol%. The vibrational modes at 1637 and
image for the PPM column packing, showing some pores circled
ꢁ1
9
49 cm correspond to C]CH
2
stretching and out-of-plan bending, respectively.
ꢁ1
and (right) an overall view of the microchip consisting of the Inset: absorbance at 1637 cm for the different X-PMMA samples.
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PMMA, the samples prepared with up to 15 mol% EGDMA
presented evidence for unreacted double bonds at the surface, functional group at 1518 cm , corresponding to the –N–H
becoming even more obvious for the sample containing bending vibrational mode, decreased signicantly in the spec-
The absorption bands characteristic for the carbamate
ꢁ
1
2
0 mol% of EGDMA.
trum aer the deprotection reaction and a small broad peak
ꢁ1
To conrm and quantify bonding between the PPM column visible at 1541 cm
appeared, corresponding to the –NH
2
and the cross-linked PMMA substrate, a pressure test was used. bending mode. A weaker absorbance associated with the N–
ꢁ
1
Table 3 summarizes the pressure withstood by the column CO–O symmetrical stretching mode at 872 cm
was also
17,18
before voids appeared along the channel (i.e. between the observed.
Additionally, the peak for the CH bending mode
monolithic column and the substrate), referred to as void of the tert-butyl group at 1367 cm decreased in intensity.
pressure, at 3%, 5%, 10%, 15% and 20% cross-linker content.
3
ꢁ
1
18
The results in Table 3 are consistent with improved bonding or
anchoring of the column material as the cross-linking level of
the substrate increased.
3
.3. Antibacterial activity of the non-porous network
Bacteria viability was monitored by the double staining tech-
nique described in Section 2.7.2. The green uorescence is
gradually replaced with red uorescence in these experiments,
as shown in Fig. 4A and B, providing clear evidence for
membrane disintegration and cell lysis. In contrast to the
deprotected network, the protected network did not display any
lysis ability, as shown in Fig. 4C and D. The red and green
3.2. t-Boc group deprotection
Several activation methods were investigated to optimize the
antibacterial activity of the PPM column. A mixture of hydro-
chloric acid and dioxane (1 : 2 ratio) was rst used, but the
mixture dissolved the plastic tip of the syringe needle and
degraded the connections tubes, which made it unsatisfactory
for deprotecting the amine group in the Boc-AEMA monomer.
Triuoroacetic acid (TFA) was also investigated but it was like-
wise aggressive on the syringe, the needle, and the tubes and it
slowly eroded the PPM column; therefore this deprotection
method was likewise discarded early on. Phosphoric acid was

uorescence intensities were recorded for up to 300 s as shown
in Fig. 5. For the deprotected network, the green and red
intensities decreased and increased with time, respectively, as
illustrated in Fig. 5, reecting the fact that the cell membranes
slowly became permeable, thus allowing the penetration of
propidium iodide and reducing the uorescence intensity for
SYTO 9. This provides clear evidence that the non-porous
network became antibacterial once the t-Boc protecting group
was removed. For the protected column, in contrast, the green
and red uorescence intensities remained almost constant as
shown in Fig. 5, reecting the fact that protected network does

nally preferred to deprotect the Boc-AEMA units. Deprotection
was successfully achieved by owing phosphoric acid through
the column, as evidenced by FT-IR analysis (Fig. 3).
Table 3 Debonding pressure of the column from the substrate for different not show any antibacterial activity.
cross-linking levels
Cross-linker (mol%)
Void pressure (psi) 3.4. Antibacterial activity of the PPM column
To validate semi-quantitatively the DNA released aer lysing the
bacterial cells by owing them through the antibacterial porous
3
5
50
60
1
1
2
0
5
0
80
100
150
medium of the PPM column, ethidium bromide was used as an
Fig. 3 FT-IR spectra for the PPM before (top) and after (bottom) deprotection of
the t-Boc group. Vibrational band assignments: N–CO–O symmetrical stretching
Fig. 4 Fluorescence intensities for E. coli suspended in PBS buffer and stained
with live/dead dye in contact with (A) the deprotected antibacterial non-porous
network at time zero and (B) after 300 s, and in contact with (C) the protected
non-porous network at time zero and (D) after 300 s.
ꢁ1
ꢁ1
3
at 1518 cm , CH bending at 1367 cm , N–CO–O symmetrical stretching at
ꢁ1
8
72 cm
.
2
4182 | RSC Adv., 2013, 3, 24177–24184
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Fig. 5 Green (live) and red (dead) stain intensities with respect to contact time
before and after removing the Boc protecting group.
indicator of DNA presence. Fig. 6 illustrates the effect of the
released DNA on the EtBr uorescence intensity aer owing
E. coli and B. subtilis bacterial cells through the PPM column Fig. 7 Gel electrophoresis analysis of PCR output for unlysed (column 2) and
lysed (through PPM) (column 3) B. subtilis cells, filtered (column 4) and nonfiltered
before and aer deprotection, and also aer amplifying the
(
column 5) lysed E. coli and nonlysed E. coli (column 6). Column 1 is for a 100 bp
collected DNA with PCR. The gure clearly shows that the
uorescence intensity of EtBr increased when adding the lysate
DNA ladder.

collected aer owing the bacterial cells through the narrow
porous channel of the monolithic column, even for the packing
material in its protected form, conrming that the bacterial
cells were partly lysed through shearing and their DNA was
released. However it can also be seen that the uorescence
intensity of EtBr further increased for the cell lysate collected
aer owing the bacterial cells through the monolithic column
in its deprotected form, thus conrming that an incremental
amount of cell lysis was achieved by physical contact of the cells
with the antibacterial surface, resulting in the release of more
DNA. The uorescence intensity of EtBr further increased for
the PCR output, which conrms that PCR was not strongly
inhibited by the cell lysate eluted from the column and that the
DNA genes of E. coli and B. subtilis could be successfully
amplied. The increases observed (ca. 2- to 3-fold aer 30
amplication cycles) remain rather modest, and it is not clear
whether this is due to the release of minute amounts of PCR
inhibitors by the PPM column or to the presence of interfering
proteins in the crude cell lysate used in the amplication
process. The determination of the exact origin of these modest
increases will be the topic of a future investigation.
Moreover, the DNA released from both E. coli and B. subtilis
amplied by PCR was qualitatively validated by gel electropho-
resis. Fig. 7 shows the gel electrophoresis analysis results for
the PCR products of B. subtilis bacterial cells that were not
19

own through the porous column (column 2), B. subtilis lysate
collected at the outlet of the deprotected PPM column (column
), E. coli lysate collected at the outlet of the deprotected
monolithic column before (column 4) and aer (column 5)
ltration using a 0.2 mm lter, and an E. coli bacterial suspen-
3

sion that was not own through the porous column (column 6).
Fig. 7 shows no detectable amount of DNA at the PCR output for
the E. coli and B. subtilis samples that were not own into the
monolithic column, which conrms that the bacterial cells had
intact cell membranes before passing through the antibacterial
monolithic columns. On the other hand, DNA is clearly detected
at the PCR output for the E. coli and B. subtilis samples that were

own through the deprotected PPM columns, which conrms
that the membranes of bacterial cells were damaged and dis-
integrated by owing through the PPM column. It can also be
observed that ltration of the cells before running the PCR did
not affect the amplication procedure. This shows that the
porous column lysed the bacterial cells and ltered the cell
debris and any intact cells le in the samples.
4. Conclusions
Fig. 6 Fluorescence intensity of EtBr after adding E. coli (blue) and B. subtilis (red)
cells suspended in PBS buffer (control), the lysate of E. coli and B. subtilis
suspensions flown through the PPM column before (protected PPM) and after
deprotection (deprotected PPM), and PCR-amplified product from the depro-
tected PPM cell lysate (PCR/deprotected PPM).
It has been demonstrated that the activated antibacterial
porous columns prepared have the ability to effectively lyse E.
coli and B. subtilis bacterial cells, and to yield DNA that is ready
to use in PCR experiments without further cleaning or ltration.
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The combination of shear degradation and antibacterial prop-
erties was clearly benecial to achieve cell lysis, which was
evidenced by the release of DNA. Cell lysis was conrmed by
several techniques, namely the LIVE/DEAD Cell Viability Assay
that was used to directly monitor cell viability on the nonporous
network, the ethidium bromide intercalation assay utilizing
spectrouorometry, relating the DNA concentration to the
increase in uorescence intensity of EtBr, as well as by gel
electrophoresis to validate the PCR amplication test. The
usefulness of X-PMMA as a substrate on which the microuidic
channel could be laser-micromachined without further surface
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Acknowledgements
3
We would like to express our gratitude to Grand Challenges 13 A. K. Bej, J. L. DiCesare, L. Haff and R. M. Atlas, Appl. Environ.
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4184 | RSC Adv., 2013, 3, 24177–24184
This journal is ª The Royal Society of Chemistry 2013