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Vertical electric field induced bacterial growthinactivation on amorphous carbon electrodes

http://dx.doi.org/10.1016/j.carbon.2014.09.0480008-6223/� 2014 Elsevier Ltd. All rights reserved.

* Corresponding authors. Address: Materials Research Center, Indian Institute of Science, Bangalore, India (B. Basu).E-mail addresses: ashutos@iitk.ac.in (A. Sharma), bikram@mrc.iisc.ernet.in (B. Basu).

Shilpee Jain a,d, Ashutosh Sharma b,*, Bikramjit Basu a,c,d,*

a Department of Materials Science and Engineering, Indian Institute of Technology, Kanpur, Indiab Department of Chemical Engineering, Indian Institute of Technology, Kanpur, Indiac Laboratory for Biomaterials, Materials Research Centre, Indian Institute of Science, Bangalore, Indiad Interdisciplinary Bio-Engineering Program, Indian Institute of Science, Bangalore, India

A R T I C L E I N F O A B S T R A C T

Article history:

Received 29 April 2014

Accepted 16 September 2014

Available online 28 September 2014

The objective of the present work is to understand the vertical electric field stimulation of

the bacterial cells, when grown on amorphous carbon substrates in vitro. In particular, the

antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative

Escherichia coli are studied using MTT assay, live/dead assay and inner membrane permeabi-

lization assays. In our experiments, the carbon substrate acts as one electrode and the

counter electrode is positioned outside the culture medium, thus suppressing the current,

electrokinetic motions and chemical reactions. Guided by similar experiments conducted

in our group on neuroblastoma cells, the present experimental results further establish

the interdependence of field strength and exposure duration towards bacterial growth inac-

tivation in vitro. Importantly, significant reduction in bacterial viability was recorded at the

2.5 V/cm electric field stimulation conditions, which does not reduce the neural cell viabil-

ity to any significant extent on an identical substrate. Following electrical stimulation, the

bacterial growth is significantly inhibited for S. aureus bacterial strain in an exposure time

dependent manner. In summary, our experiments establish the effectiveness of the vertical

electric field towards bacterial growth inactivation on amorphous carbon substrates, which

is a cell type dependent phenomenon (Gram-positive vs. Gram-negative).

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The failure of various biomedical devices is often caused by

prosthetic infection mediated by bacterial cell attachment,

growth and biofilm formation [1,2]. The retention and survival

of adhered bacteria depend on the surface charge, hydropho-

bicity/hydrophilicity, chemical composition and surface

roughness of the implant [3–5]. Due to adhesion of bacterial

cells, eukaryotic cells in the host human tissue need to com-

pete with prokaryotic cells (bacterial cells) for the implanted

material surface [6,7]. Due to faster growth rate (rate of divi-

sion), the formation of a mature biofilm often precede the

neotissue formation in vivo, making the implantation sites

extremely resistant to antibiotics or host defense mecha-

nisms [8]. Thus, in order to achieve appropriate host

response, it is essential to design surfaces and processes with

dual performance that encourage eukaryotic cell functional-

ity (adhesion, proliferation, differentiation etc.) and at the

same time prevent bacterial cell attachment. Apart from

developing new biomaterial with antibacterial property, a

194 C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2

strikingly different approach of the application of external

electric field stimulation to cause bactericidal effect in vitro

has been attempted in the present work.

The direct current (DC) electric fields are present in all ani-

mals during development and regeneration [9]. The applica-

tion of DC electric fields to biological systems for recovery

of organs is widely being investigated [9–11]. The application

of external electric field to prevent bacterial cell adhesion or

survival however has been investigated rather to a limited

extent. The reversible or irreversible electrical breakdown

occurs on the application of external electric field, which

depends on the electric field intensity and exposure time

[12]. In one report, DC electric field has been used to manipu-

late bacterial cell attachment, their growth and viability [13].

It was reported that a lower current (less than 20 mA) did

not induce any significant change in the surface properties

of phenol-degrading bacteria. In contrast, an electric current

of 20 mA could increase the surface hydrophobicity and flat-

ten the cell shape. Importantly, a higher current (40 mA) could

increase the surface extracellular substances and the net neg-

ative surface electrostatic charge. Further, the manipulation

of cell behavior in the presence of electric field depends on

the size, shape, membrane thickness of the cells, strength

and duration of applied electric field [14–16]. It is reported that

the weak electric fields enhance the efficacy of antibiotics in

killing bacterial biofilm even on the surfaces that are not used

as electrodes [17]. The change in cell behavior in the presence

of external electric field also depends on the mode [alternate

current (AC) vs. direct current (DC)] of applied field. Liu et al.

[18] studied the effect of DC electric currents on the popula-

tion of a Staphylococcus epidermidis (S. epidermidis) biofilm

adhered to a catheter. They found that the population of a

S. epidermidis biofilm adhered to a catheter was reduced by

the application of 100 mA direct current.

An effective implantable biomaterial for the majority of

the applications should facilitate eukaryotic cell functionality

(proliferation, differentiation etc.) and simultaneously inhibit

bacterial growth. Recently, it was shown that the vertical

Fig. 1 – (a) Schematic of the electric field setup, (b) direction of in

indicating the bacteria and (c) electric field application protocol,

be viewed online.)

electric field can stimulate neural differentiation in terms of

enhanced neurite length on carbon electrodes at an optimum

electric field [19]. It is important to reiterate that vertical E-

field configuration represents a unique protocol, which can

avoid the flow of current and electrokinetic motions in the

liquid, like electro-osmosis and electrophoresis. Unlike the

submerged electrodes, any damage by electrode corrosion or

by current flow is also minimized in the proposed electric

field application protocol. As a logical follow up to our earlier

work, we study here the effect of vertical electric field on bac-

terial adhesion and viability. In the present work, the vertical

electric field of strength 2.5 and 10 V/cm was applied to bacte-

ria and the effect of electric field duration to bacterial cell via-

bility was studied. Here, we use the identical electrodes and

optimized electric field conditions to study the effect of elec-

tric field on bacterial adhesion and growth. The current study

is therefore aimed to investigate the vertical electric field

stimulation on the suppression of bacterial cell viability and

growth under the conditions that are conducive for the neural

cell.

2. Materials and methods

2.1. Fabrication of carbon structures

For carbon film fabrication, the Polyacrylonitrile (PAN) solu-

tion in DMF solvent was spin-coated onto a Si wafer at

3000 rpm for 30 s. In order to obtain high performance and

high quality carbon films [20,21], the polymeric structures

were first stabilized at 250 �C for 1 h. After stabilization, the

samples were pyrolyzed at 900 �C under Ar atmosphere in a

tubular high temperature furnace (Nabertherm, Germany)

with a gas flow rate of 0.2 L/min. The heating rate was kept

constant at 5 �C/min and the samples were held at 900 �Cfor 1 h, followed by cooling to room temperature.

The carbon films were characterized by an atomic force

microscope (AFM) (Agilent, PICOSPM 3000) and field emission

scanning electron microscopy (FESEM, Quanta 200, FEL

duced electric field with circular features on both electrodes

followed by MTT analysis. (A color version of this figure can

C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2 195

Germany, SUPRA, Zeiss, Germany). The tapping mode AFM

accompanying Picoscan software was used to measure the

roughness of film substrates.

2.2. Electric field setup

The carbon film supporting the cells was used as an electrode

and a stainless steel plate, positioned outside the medium,

was used as the counter electrode. As shown in Fig. 1, both

the electrodes were kept parallel to ensure a uniform inten-

sity of the applied electric field. The external DC power sup-

ply, connected to the electrodes was used to apply the

electric field. On the application of 160 V/cm and 640 V/cm,

the electric field strength screened by a cell population in

Luria Broth (LB) medium was 2.5 V/cm and 10 V/cm, respec-

tively. More details of the calculations on the actual field

strength experienced by the cells is reported elsewhere [19].

2.3. Bacteria culture

Escherichia coli (E. coli; ATCC 25922) and Staphylococcus aureus (S.

aureus; ATCC 25923), bacterial strains were grown at 37 �C and

maintained on LB-Agar plates (Sigma–Aldrich). From the

plates, single colonies were used for suspension culture,

wherein cells were grown in LB overnight at 37 �C and then

100 lL of the cell suspension was incubated in fresh media

for 3 h. After incubation, the cell suspension was diluted

and the samples were seeded with 500 lL (0.5 optical density

at 670 nm wavelength). Before seeding, the samples were ster-

ilized with 70% ethanol, followed by 2 h UV exposure.

2.3.1. Live/dead assayFor live/dead assay, E. coli or S. aureus were stained with SYTO

9 (3.34 mM) and propidium iodide (PI, 20 mM). SYTO 9 is a

membrane-permeant dye, which labels live bacteria with

green fluorescence and membrane-impermeant propidium

iodide labels membrane-compromised bacteria with red fluo-

rescence [22]. For bacteria seeding, an OD value of 0.5 was

adjusted for the bacterial suspension at wavelength of

670 nm and carbon electrodes were seeded with 500 lL of bac-

terial suspension. After 2 h of initial seeding, the samples

were exposed to electric field for different time period viz.

0.25, 0.5, 1, 2 and 4 h, followed by 15 min incubation with

1:1 ratio of SYTO 9 (3.34 mM) and propidium iodide (PI,

20 mM) in dark, as suggested in ATCC protocol (Live/dead

assay kit, L7012, Invitrogen). All the samples were character-

ized using fluorescence microscope (Nikon LV100D).

2.3.2. MTT assayAfter electric field application for different time duration at

37 �C, all samples were washed carefully and then incubated

with 0.5 mg/ml 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-

zoliumbromide) (MTT, Amresco, Life Science Research Prod-

uct & Biochemicals, USA) in 1· PBS for 1 h at room

temperature. After 1 h, the formazan crystals were dissolved

in dimethyl sulfoxide (DMSO, Sigma–Aldrich, USA) and the

optical density was recorded at 540 nm with 630 nm as a ref-

erence in order to avoid any error using an ELISA microplate

reader (BioTek, USA). For positive control, bacteria were

incubated with 70% ethanol for different time points on tissue

culture polystyrene (TCPS). The MTT test was repeated for at

least four times.

2.3.3. Inner membrane permeabilization assayThe inner membrane permeabilization (IMP) of E. coli or

S. aureus was determined using o-nitrophenyl-b-D-galactopy-

ranoside (ONPG; Sigma). For IMP, the bacteria grown to

logarithmic phase in LB medium were washed and resus-

pended in LB. An OD value of 0.5 was adjusted at wavelength

of 670 nm and bacterial suspension (500 lL) was seeded on

carbon electrodes. After 2 h of seeding, LB was replaced with

1· PBS containing 0.2% glucose and 40 lL ONPG (30 mM) to

each sample. Subsequently, the electric field was applied for

different time period viz. 0.25, 0.5, 1, 2 and 4 h. After electric

field application for different time period, the cells were

removed from the samples followed by centrifugation and

the b-galactosidase activity was measured in the supernatant

using UV–Vis spectrophotometer (UV 10, Thermo Scientific) at

420 nm wavelength. Similarly, for positive control, bacteria

were incubated with 70% ethanol and 40 lL ONPG (30 mM)

after 2 h of initial seeding on TCPS.

2.3.4. Bacteria colony forming unitAfter electric field exposure, bacteria suspension was serially

diluted and vortexed for one min in the fresh LB. The bacteria

suspension was mixed with liquid LB-agar (maintained at

45 �C) and then LB-agar plates were incubated for 24 h at

37 �C. The formed bacteria colonies were counted. Bacterial

numbers were expressed as colony forming unit (cfu) per unit

area and were calculated by multiplying dilution factor.

2.4. Statistical analysis

All the experimental data were expressed as mean ± standard

deviation (SD) and were analyzed by one way ANOVA (SPSS

16.0) for the calculation of statistically significant difference

among the experimental data (n = 4). The differences were

considered as statistically significant, when p 6 0.05.

3. Results

XRD results (not shown) confirmed that the PAN derived car-

bon films are amorphous in nature, indicating absence of long

range order, as also observed previously for the same

films [19,23]. In Fig. 2a, FESEM image of the as-synthesized

carbon film is provided along with AFM image in Fig. 2b. A clo-

ser look at Fig. 2a reveals interconnectivity of the pores,

with sizes varying in the range of 200 nm to 2 lm. The rough-

ness of the film, as analyzed using AFM, was around

�372 nm.

3.1. Bacterial response to vertical electric field stimulation

3.1.1. Effect of vertical electric field on bacterial viabilityIn order to assess the survival of bacteria on these carbon

electrodes in the presence of vertical electric field, the electric

field of strength 2.5 V/cm and 10 V/cm was used. Similar field

parameters were also used for the neural cell stimulation [19].

The electric field was applied for variable periods of 0.25 h,

0.5 h, 1 h, 2 h and 4 h continuously after 2 h of initial seeding

Fig. 2 – (a) FESEM image and (b) AFM image of porous carbon electrode. (A color version of this figure can be viewed online.)

Fig. 3 – Live/dead assay of E. coli bacteria (a) control (TCPS), (b) film (0 V/cm), (c) 2.5 V/cm electric field for 4 h and live/dead

assay of S. aureus bacteria (d) control (TCPS), (e) film (0 V/cm), (f) 2.5 V/cm electric field for 4 h. (A color version of this figure can

be viewed online.)

196 C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2

as shown in Fig. 1. After such experiments, the live/dead

staining was carried out for qualitative assessment on bacte-

rial cell viability. It was evident from Fig. 3c and f that the pop-

ulation of dead bacteria (red stained cells) increased after

2.5 V/cm electric field application for 4 h. The MTT results

were used to quantify the viable cells after electric field appli-

cation and statistically significance was assessed (see Fig. 4).

A critical assessment of the results plotted in Fig. 4 reveals

three important aspects of the influence of electric field

stimulation on bacterial growth inhibition vis-a-vis positive

control (ethanol treatment) and without any electric field

exposure (0 V/cm):

(a) At 2.5 V/cm field strength, the bacterial viability sys-

tematically and significantly decreased only after expo-

sure duration of 1 h, independent of bacterial strain.

The systematic reduction in viability at 2.5 V/cm is

however most significant (sharp decrease) for E. coli

Fig. 4 – Bacterial cell viability (MTT assay) after vertical

electric field application (a) E. coli and (b) S. aureus, data were

expressed as mean ± standard deviation (SD) and

differences were considered statistically significant, when

p 6 0.05. * Represents the significant difference w.r.t. 0 V/cm,

** represents the significant difference w.r.t to 10 V/cm for

0.25 h and # represents the significant difference w.r.t. 2.5 V/

cm for 0.25 h (The inset shows the electric field application

protocol, followed by MTT analysis; EF stands for electric

field strength, i.e. either 2.5 V/cm or 10 V/cm). For positive

control, EF is not applied but a given bacterial strain is

treated with ethanol for various time periods, as mentioned

along x-axis. (A color version of this figure can be viewed

online.)

Fig. 5 – Inner membrane permeabilization in (a) E. coli and (b)

S. aureus bacteria after application of electric field. The inset

shows the timescale for electric field application, followed

by inner membrane permeabilization analysis. Data were

expressed as mean ± standard deviation (SD) and

differences were considered statistically significant, when

p 6 0.05. * Represents the significant difference w.r.t. 0 V/cm,

** represents the significant difference w.r.t. 2.5 V/cm for

0.25 h and # represents the significant difference w.r.t to

10 V/cm for 0.25 h. (The inset shows the electric field

application protocol, followed by IMP analysis; EF stands for

electric field strength, i.e. either 2.5 V/cm or 10 V/cm). (A

color version of this figure can be viewed online.)

C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2 197

bacteria at 2 h exposure w.r.t. 0.25 h exposure. However,

the decrease in S. aureus bacterial viability with expo-

sure duration is most systematic and gradual with time

at 2.5 V/cm.

(b) As far as the field effect is concerned, the electrical field

stimulation at 10 V/cm causes a significant decrease in

viability w.r.t. 2.5 V/cm exposure independent of treat-

ment time. However, the magnitude of the decrease in

viability with treatment time at 10 V/cm for both the

bacterial strains is not as significant as that recorded

at 2.5 V/cm exposure. For example, the decrease in via-

bility is within 10–12% over treatment time of 0.25–2 h

at 10 V/cm exposure for E. coli bacteria and around

20% for S. aureus bacteria. Over the variation in similar

duration, the bacterial viability undergoes 50–60% for

both the bacterial strain at 2.5 V/cm exposure.

(c) The ethanol treatment expectedly causes far most sig-

nificant effect on bacterial viability reduction for both

bacterial strains. Although the electric field exposure

of 2.5 V/cm can never induce such significant bacterial

viability reduction even after longest exposure time of

4 h for either of the bacterial strains, no statistically sig-

nificant difference is however recorded for the viability

of S. aureus bacteria after 1, 2 or 4 h of exposure at 10 V/

cm electric field strength w.r.t. positive control. In other

words, such electrical field stimulation condition is as

effective as positive control.

3.1.2. Inner membrane permeabilization and bacterialinactivationAfter application of electric field, the degradation of bacterial

membrane was investigated by inner membrane permeabili-

zation (IMP) assay and the results are plotted in Fig. 5a and

Fig. 6 – MTT measured cell viability of E. coli and S. aureus

after incubation for 4 h in normal growth conditions, which

is preceded by the electric field treatment for various

timescales, as mentioned along X-axis. Data were

expressed as mean ± standard deviation (SD) and

differences were considered statistically significant, when

p 6 0.05. * Represents the significant difference w.r.t. 0.25 h

exposure time. (The inset shows the electric field

application protocol, followed by MTT analysis). (A color

version of this figure can be viewed online.)

198 C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2

b. It is known that b-galactosidase is produced as a result of

the hydrolysis of ONPG during permeabilization of inner

membrane and the absorbance was measured at 420 nm

[24]. The IMP continuously increased with an increase in the

electric field duration. In both the cases, the IMP was signifi-

cantly higher, when compared to the positive control (bacte-

ria incubated with ethanol) [25] or 0 V/cm samples (see

Fig. 5a and b). The membrane permeabilization after 2.5 V/

cm electric field exposure for shorter time period (0.5 h) was

1.04 times in the case of E. coli and 1.4 times in the case of

S. aureus, compared to the unexposed (0 V/cm) bacteria. Upon

the application of higher electric field (10 V/cm), the mem-

brane permeabilization increased to 1.87 times in case of E.

coli and 1.57 times in the case of S. aureus. Similar to the anal-

ysis of MTT results, a critical assessment of the results plotted

in Fig. 5 unravels two important aspects of the influence of

electric field stimulation on bacterial inner membrane

permibilization:

(a) At 2.5 V/cm electric field strength, bacterial IMP signifi-

cantly increased only after exposure duration of 0.5 h in

the case of E. coli bacteria and after 1 h, the IMP was sig-

nificantly higher when compared to 0.25 h and it was

increased further with an increase in time. However,

in the case of S. aureus, there was no systematic

increase or decrease in the IMP after electric field expo-

sure, when compared to 0.25 h electric field exposure.

(b) However, the bacterial growth conditions with 10 V/cm

electric field strength causes a significant increase in

IMP w.r.t. 2.5 V/cm electric field exposure. For E. coli bac-

teria, the IMP after 0.5 h electric field exposure was

almost 25% more than that after 0.25 h and with

further increase in exposure time, there was no signif-

icant increase in the IMP. However, in the case of S. aur-

eus bacteria, the increase in the IMP was not significant

till 1 h, but after 4 h of electric field exposure, the IMP

was almost 40% more than 0.25 h electric field

exposure.

3.2. Bacterial growth recovery after removal of electricfield

It is important to assess whether the electric field stimulation

has a long term affect on bacterial growth inactivation.

Table 1 – Summary of colony forming unit (cfu), counted on agarvertical electric field (0 V/cm or 2.5 V/cm), which was applied fo

Electric fieldduration(h)

E. coli bacteria

After 2.5 V/cmelectric fieldexposure(cfu/cm2)

0 V/c(cfu/

0.25 1.7 · 103 1 · 100.50 4 · 103 6.2 ·1.00 1 · 104 2.3 ·2.00 6 · 104 5 · 10

Therefore, we also checked the recovery of bacterial cells after

removal of external electric field after the above set of exper-

iments (field strength variation and exposure variation). Such

experimental results would indicate any impact of electric

field on normal bacterial growth behavior. In view of positive

impact of 2.5 V/cm electric field on neural cell stimulation to

neurite outgrowth, further experiments were conducted on

both bacterial strains only at 2.5 V/cm exposure. In particular,

the bacteria were incubated for 4 h at 37 �C after removal of

2.5 V/cm electric field application for different time points

and the viability of the bacteria was determined by MTT assay

(Fig. 6). It is evident that the E. coli bacteria recovered better

after removal of the electric field and they actively grow even

after electric field exposure for longer time. The viability of E.

coli bacteria after 4 h electric field application followed by 4 h

incubation (in the absence of external electric field) at 37 �Cwas 80% w.r.t. without electric field (0 V/cm). However, the

recovery of S. aureus bacteria was decreased continuously

with an increase in the electric field duration. The viability

plates, after bacteria were incubated for 24 h after removal ofr various exposure duration of 0.25, 0.5, 1.0 and 2.0 h.

S. aureus bacteria

mcm2)

After 2.5 V/cmelectric fieldexposure(cfu/cm2)

0 V/cm(cfu/cm2)

3 4.2 · 103 4 · 103

103 3.6 · 104 5.2 · 104

104 >105 >105

5 >105 >105

C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2 199

of S. aureus bacteria after 4 h electric field exposure, followed

by 4 h incubation at 37 �C was only 50% w.r.t. without the elec-

tric field. Also, a sharp decrease in viability is recorded at 0.5 h

exposure w.r.t 0.25 h exposure, independent of bacterial

strain. Thereafter, a gradual/systematic increase or decrease

in bacterial viability is recorded in exposure time dependent

manner for E. coli or S. aureus, respectively. On the basis of

the data presented in Fig. 6, it can be therefore concluded that

E. coli bacteria can recover the growth pattern after the

removal of the electric field and when grown under normal

culture conditions for 4 h. In contrast, the influence of electric

field on bacterial growth inactivation is much more signifi-

cant for S. aureus bacteria, as the bacterial viability systemat-

ically decreases after the removal of electric field and no

recovery could be recorded.

The above aspects have been further analyzed by measur-

ing the colony forming unit (in terms of cfu/cm2), as summa-

rized in Table 1. In contrast to the data presented in Fig. 6, cfu

was counted after incubation for 24 h following electric field

application at 2.5 V/cm for various time points. For compari-

son, cfu measured after incubation without any electric field

(0 V/cm) are also presented in Table 1. The increase in cfu

with the exposure time is however cell type dependent. For

E. coli bacteria, the reduction in cfu after exposure to 2.5 V/

cm electric field w.r.t control (0 V/cm) can be well realized,

when the exposure duration is 0.5 h or more. A critical look

at Table 1 also reveals that the decrease in cfu of E. coli strain

becomes more prominent with the longer exposure time, e.g.,

1.5 times reduction at 0.5 h duration, 2.3 times at 1 h duration

and one order of magnitude reduction after 2 h of exposure.

Although such difference is not measurable at longer expo-

sure duration for S. aureus strain, almost 1.5 times decrease

in cfu was recorded after 0.5 h of exposure.

4. Discussion

The failure of the implanted devices due to bacterial infection

is the major concern in the field of biomedical application. In

this work, we have studied the inactivation of bacteria in the

presence of external vertical electric field on amorphous car-

bon electrodes. These amorphous carbon electrodes were por-

ous (pore size distribution 200–2000 nm), conducting (50 S/

cm) in nature with higher charge storage capacity (0.2 mC/

cm2) and lower electrochemical impedance (3.3 kX at 1 kHz)

Fig. 7 – Schematic illustration of the induction of transmembran

shaped bacteria. The dotted lines (vertical) indicate the electric fi

[19]. Recently, we have reported that over a narrow window

of vertical electric field (62.5 V/cm), the growth of neurite

and viability of the neural cells can be enhanced on these por-

ous amorphous carbon electrodes [19].

4.1. Influence of vertical E-field stimulation on cell typedependent bacterial functionality

It is worthwhile to note the novelty and advantages of the ver-

tical electric field application employed in the present work.

During the application of electric field, the position of the

electrodes plays a major role to dictate the cellular function-

ality in vitro. In most of the electric field related cell studies,

the lateral electric field with two parallel electrodes sub-

merged in the culture medium was used. In the lateral field

application, various chemical oxidants like, hydrogen perox-

ide etc. are generated at the electrodes in the presence of oxy-

gen during the flow of electric currents [26]. These toxic

agents can kill bacteria, but at the same time are also harmful

to eukaryotic cells as well [17]. Costerton et al. [17] reported

that the biofilm can be destroyed by an antibiotic on all areas

of the active electrodes and on the surfaces of conductive ele-

ments that lie within the electric field but do not themselves

function as electrodes. They have suggested that the genera-

tion of local electrochemical species at the electrode site

enhanced the efficacy of the electric field application. How-

ever, in our study the carbon electrodes are stable and the

adopted protocol avoids the flow of current. Therefore, the

culture protocol itself does not result in the generation of

electrochemical species. In this study, we have used a unique

protocol to stimulate bacterial cells using an external vertical

electric field. The porous carbon electrode was used as one of

the electrodes seeded with bacterial cells and the other stain-

less steel electrode was placed outside the LB media. Since,

these electrodes were separated by non-conducting air in

between, there is no electronic current during the application

of external electric field application. Therefore, it is expected

that the bacterial cells only experienced the electric field

induced in between the electrodes due to charge distribution

at the electrode surfaces. However, such external electric field

can induce the transmembrane potential, membrane polari-

zation and deformation, which can influence the bacterial

cell behavior. Like eukaryotic cells, the viability (metabolic

activity) of bacterial cells also depends on the physical

e potential on the application of electric field to the elliptical

eld lines. (A color version of this figure can be viewed online.)

200 C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2

phenomena such as membrane potential [27] and the cellular

electrical equilibria may be disturbed by the application of an

external electric field [17]. It has been shown that the external

fields can affect the a-helix content and the orientation of

membrane proteins in eukaryotic cells and the electropho-

retic mobilities of bacterial membrane proteins [27,28].

Electric fields can even be used to affect the electroinsertion

of specific proteins into the membranes of living cells. These

molecular perturbations of important membrane compo-

nents may affect the organization of membranes and we

expect that these structural changes would influence the

permeability of membranes in the present case [17].

In the present case, both Gram-negative and Gram-posi-

tive bacteria were exposed to vertical electric field with field

strength of up to 10 V/cm for various time points. It was

observed that both the bacteria could be killed up to 80% on

the application of higher electric field (10 V/cm) for short time

or on the application of low field (2.5 V/cm) for longer time

(Fig. 6a and b). Interestingly, the S. aureus bacteria responded

after longer exposure duration as compared to E. coli bacteria

in the presence of vertical electric field. It might be due to the

fact that S. aureus is a Gram-positive bacteria with thicker cell

wall compared to E. coli, which is a Gram-negative bacteria

[14,29].

The irreversible permeabilization of bacterial cell mem-

brane in the presence of electric field depends on the electric

field strength [26]. As shown schematically in Fig. 7, the

potential drop at the cell membrane on the application of

electric field was generated due to difference in conductivity

of cytoplasm (1S/m) and cell membrane (10�5 S/m) [14]. The

application of uniform electric field to the bacterial cell

induced transmembrane potential [14,30] and this can be cal-

culated by Schwan’s equation [31]:

D/ ¼ 32

Er cos h ð1Þ

where D/ is the induced transmembrane potential, E is the

external applied field, r is the radius of cell and h is the angle

between direction of applied field and cell orientation axis. It

is evident from the above equation that the application of

Fig. 8 – Comparison between neuroblastoma (N2a) cell

viability and bacterial cell viability after the electric field

stimulated culture experiments at two different vertical field

strength. (The inset shows the electric field application

protocol, followed by MTT analysis). (A color version of this

figure can be viewed online.)

vertical electric field (h = 0�) of 2.5 and 10 V/cm to spherical bac-

teria with radius 1 lm will induce a moderate transmembrane

potential of�0.375 mVand 1.5 mV, respectively. It is reported in

literature that for bacterial cells, the membrane poration

occurs when the potential across the membrane exceeds about

1 V [14]. In order to rupture the bacterial membrane completely,

i.e. to attain an induced transmembrane potential of 1 V in a

spherical bacteria of one lm diameter, at least 100 V/cm of

external electric field strength is required.

We observed that the inner membrane permeabilization

was different in both the bacterial types in the presence of

electric field (Fig. 5a and b). The difference in the permeabili-

zation of the E. coli and S. aureus is due to the difference in cell

size and it is clear from the Eq. (1) that if the size of the cell is

small, the induced transmembrane potential will also be

lower. It was also found that the recovery of E. coli bacteria

was possible after removal of the electric field (see Fig. 6).

From Eq. (1), it is clear that the induced transmembrane

potential is dependent on the orientation of the bacterial cell.

In the case of spherical shaped S. aureus cell, the induced

transmembrane potential will be isotropic in nature, whereas

in the case of elliptical shaped E. coli bacteria, the induced

transmembrane potential is expected to be anisotropic in nat-

ure and will depend on the orientation of the cell (see Fig. 7).

Also, it is well known that the generation time for E. coli is

20 min, whereas for S. aureus it is 30 min [32]. This difference

in the generation time of both the bacteria could be another

plausible reason for different activity of the bacteria after

electric field exposure. Since E. coli divides faster as compared

to S. aureus, the larger population of cells perhaps has dimin-

ished the effect of electric field [26].

4.2. Viability of cells (eukaryotic vs. prokaryotic) in electricfield stimulated culture conditions

Concerning the effect of electric field influence on both

eukaryotic and prokaryotic cells, Fig. 8 presents the difference

in the Neuroblastoma (N2a) and E. coli/S. aureus viability in the

presence of external vertical electric field stimulation with

identical field strength. From Fig. 8, it is evident that the via-

bility of N2a cells was higher in the case of 2.5 V/cm electric

field for 6 h, whereas the bacterial cell viability was signifi-

cantly less, when grown for 4 h in culture under identical

electric field strength [19]. The sharp and undesired decrease

in cell viability of N2a cells at 10 V/cm therefore suggests the

field strength of 2.5 V/cm as the most desirable field condi-

tions that can be adopted or further investigated in the clini-

cal scenario from biomedical application perspective.

Fig. 9 represents the effect of external electric field on differ-

ent size and shaped biological cells. The above mentioned dif-

ferences in cell response to electric field stimulation have been

described with the underlying mechanisms in Fig. 9. Due to the

larger size and thicker membrane of N2a cells compared to bac-

terial cells, the external electric field shows the positive effect

and the viability of N2a cells was higher as compared to control

(0 V/cm) [19]. However, such field stimulation can potentially

cause significant cell viability reduction in case of bacteria. It

is very interesting to note how the similar electric field stimu-

lation can have strikingly different response to eukaryotic and

Fig. 9 – Schematic illustration of the influence of electric field stimulation on the loss of membrane integrity, leading to

reduction in cell viability for two strains: (a) S. aureus, (b) E. coli as well as (c) neural cell differentiation in vitro. The neural cell

results are discussed in Ref. [19]. The stimulation condition proceeded by initial incubation is also shown. (The cell size is not

in scale). (A color version of this figure can be viewed online.)

C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2 201

prokaryotic cells, which needs to be investigated further on

implantable biomaterial substrates.

At the closure, it is worthwhile to mention that the overall

theme of our recent research encompasses experimental and

theoretical understanding of the cell functionality modula-

tion by manipulating the substrate properties and with the

application of external fields [33]. As far as the sole influence

of substrate conductivity effect is concerned, an earlier study

from our group illustrated the role of substrate conductivity

as a guiding factor in the orientation and differentiation of

C2C12 mouse myoblast cells on moderately conducting HA-

CaTiO3 substrates in the absence of electric field stimulation

during culture [16]. In a follow-up study, our research group

also reported the influence of pulse electrical stimulation (lat-

eral electric field) on the osteogeneic cell proliferation on HA-

CaTiO3 substrates [34] and stainless steel [35] over a narrow

window of field strength. A subsequent work demonstrated

how the intermittent electrical stimulation (lateral electric

field) and/or the substrate conductivity can influence the dif-

ferentiation of human mesenchymal stem cells to neural-like

cells on electroconductive polyaniline substrates in vitro [36].

Recently, the present authors reported the influence of verti-

cal electric field stimulation on mouse neuroblastoma (N2a)

cell differentiation over a narrow window of field strength

on amorphous carbon substrates [19]. Interestingly, we dem-

onstrate here that both E. coli and S. aureus bacteria can be

deactivated in the vertical electric field stimulated culture

conditions. We establish that the amorphous carbon sub-

strates with an externally applied electric field have potential

for neural tissue engineering applications with the twin aims

of neural regeneration and bactericidal action.

5. Conclusions

The objective of the present study is to understand the effect

of the vertical electric field stimulation on bactericidal prop-

erty of amorphous carbon in vitro. We demonstrate that cul-

ture condition with the application of 2.5 V/cm vertical

electric field, which is safe for neural cells, has bactericidal

effect in exposure time and cell type dependent manner. It

is also evident that the E. coli bacteria respond more promptly

as compared to S. aureus in external electric field stimulated

culture conditions. Importantly, the bacterial inactivation is

much more significant in case of S. aureus bacteria, when

grown for 4 h after the electric field exposure. In contrast, nor-

mal growth behavior is recorded for E. coli bacteria after 2.5 V/

cm electric field exposure over 4 h in culture. Additionally, the

application of lower electric field for longer time or higher

electric field for shorter time can equally deactivate bacterial

growth to both types of bacteria. Taken together, the experi-

mental results establish the interdependence of field strength

and exposure duration towards bacterial growth inactivation

in vitro. Overall, the present study establishes the bactericidal

effect of vertically applied electric field of strength �2.5 V/cm

on amorphous carbon substrates in culture. Similar culture

conditions importantly promotes neural cell functionality

in vitro.

Acknowledgements

The authors acknowledge the support provided by Depart-

ment of Science and Technology and its Unit of Excellence

on Soft Nanofabrication at IIT Kanpur for providing various

202 C A R B O N 8 1 ( 2 0 1 5 ) 1 9 3 – 2 0 2

research facilities. The financial support from Department of

Biotechnology (DBT) is also gratefully acknowledged.

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