DNA Detection and Cell Adhesion on Plasma-Polymerized Pyrrole

Zhihong Zhang, Shunli Liu, Yu Shi, Jun Dou, Shaoming FangHenan Provincial Key Laboratory of Surface and Interface Science, Zhengzhou University of Light Industry, No. 5 Dong Feng

Rd., Zhengzhou 450002, People’s Republic of China

Received 8 August 2013; accepted 31 August 2013

Published online 1 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22408

ABSTRACT:

This study investigates the application of Plasma-

polymerized pyrrole (ppPY) as bioactive platform for

DNA immobilization and cell adhesion based on the fun-

damental properties of ppPY, such as chemical structure,

electrochemical property, and protein adsorption. Varia-

tions in electrochemical properties of the ppPY film

deposited under different plasma conditions before and

after DNA immobilization were measured using electro-

chemical impedance spectroscopy (EIS). The equilibrium

concentration of the probe DNA immobilized on the

ppPY surface was deduced by detecting the variations in

the surface charge transfer resistance (Rct) of the ppPY

films after DNA immobilization with different concentra-

tions. In addition, the detection limit of the target DNA

hybridization with probe DNA, the association constant,

Ka, and the dissociation constant were deduced from

Langmuir isotherm equations simulated using the experi-

mental data collected by EIS. Moreover, inverted micro-

scope was used to observe the cell adhesions onto the

surface of the ppPY films prepared under different plasma

conditions. Different adhesive behaviors of cells were

observed, demonstrating that ppPY films could be an

alternative biomaterial used as the sensitive layer for

DNA sensor or cell adhesion. VC 2013 Wiley Periodicals,

Inc. Biopolymers 101: 496–503, 2014.

Keywords: plasma polymerization; DNA immobiliza-

tion=hybridization; cell adhesion; electrochemical

impedance spectroscopy

This article was originally published online as an accepted pre-

print. The “Published Online” date corresponds to the preprint

version. You can request a copy of the preprint by emailing the

Biopolymers editorial office at biopolymers@wiley.com

INTRODUCTION

Plasma polymerization is often applied for the prepa-

ration of polymer films that can be used as bioactive

matrix because of its feasibility and efficiency.1,2

Several studies have reported on protein adsorp-

tion,3–5 DNA immobilization,6–10 and cell adhe-

sion11,12 onto the surface of plasma-polymerized polymers.

Plasma-polymerized pyrrole films (ppPY) have gained

increasing interest in the research of its fundamental proper-

ties13 and applications as electronics or bioactive materials14

because of their stable chemical properties, relatively high

condctivity, and multifunctionality.15–17 The dielectric prop-

erties and the molecular structure of ppPY films have also

been determined in detail.18 Although numerous studies

have reported on polypyrrole prepared by electrochemical or

chemical polymerization used as the biosensor materials,

only few studies about the biomolecule adsorption or cell

adhesion onto the surface of ppPY films have been reported.

The tissue spinal cord response in rats after implantation of

polypyrrole and polyethylene glycol obtained by plasma was

investigated by Olayo et al.19

Various techniques for biomolecule anchor onto the plasma

polymer surface have been developed, such as quartz crystal

microbalance,20–22 surface plasmon resonance,23,24 X-ray pho-

toelectron spectroscopy (XPS),25 and electrochemical techni-

ques.26,27 Given that the electrochemical impedance

spectroscopy (EIS) measurement is simple, reliable, cheap,

Correspondence to: Zhihong Zhang; e-mail: mainzhh@163.com

Contract grant sponsor: National Natural Science Foundation of China (NSFC)

Contract grant number: 51173172

VC 2013 Wiley Periodicals, Inc.

496 Biopolymers Volume 101 / Number 5

sensitive, and compatible with DNA biochip, this method is

promising for applications on the detection of biomolecules.28

EIS has been proven as one of the most powerful tools for

probing the features of surface-modified electrodes. The

immobilization or biorecognition events of biomaterials at the

electrode surfaces change the capacitance and interfacial elec-

tron transfer resistance of the electrodes.

EIS has been used for the analysis of the solution behaviors

of ppPY and the protein adsorption procedure on the ppPY

surfaces in our previous work.29 The use of ppPY and their

application as supports for DNA immobilization=hybridiza-

tion using XPS has also been reported by our group.30 We have

found that ppPY films deposited under low input power

plasma with low N content and high density of –N15 compo-

nent is optimal for DNA anchoring. However, EIS has not

been used to investigate the kinetics of the more extensively

existing DNA=DNA hybridization onto the ppPY film surface.

We are currently concerned with the kinetic parameter varia-

tions in ppPY films with hybridized double DNA strands, and

the change in cell adhesion on the ppPY surfaces. The influen-

ces of different plasma conditions on the performance of DNA

biosensor and cell adhesions have also been investigated using

EIS and inverted microscopy.

RESULTS AND DISCUSSIONIn our previous work,29,30 the chemical structure, solution

behavior, and fundamental electrochemical properties of the

ppPY films deposited under different plasma conditions were

characterized in detail. The double strand DNA can be immo-

bilized onto the surface of the ppPY films because of the elec-

trostatic interaction between the positive charge of 2N15 in

the ppPY films and the negative charge of 2PO42 of the DNA

base pairs. However, the dynamic constants, such as the

detection limitation of the target DNA, the association con-

stant, and the dissociation constant, were deduced from the

Langmuir isotherm equations in this article.31

Influence Factors of DNA

Immobilization=Hybridization onto the ppPY FilmsThe chemical and physical properties of plasma polymers,

which depend on the experimental conditions used in the

preparation process, can affect the biomolecule binding,

including the input power, the gas flow rate, and the polymer

thickness, which depend on polymerization time.29 Also, DNA

adsorbing capacity is affected by the solution properties of

DNA.32

Influence of Plasma Input PowerFigure 1 shows the EIS curves of the ppPY films deposited

under 5, 50, and 100 W before and after DNA immobiliza-

tion=hybridization. Impedance spectroscopy can be described

by a simple equivalent circuit model consisting of resistance

and capacitance elements, such as solution resistance (Rs),

charge-transfer resistance (Rct), and constant phase element

(CPE), as shown in the inset of Figure 1a. Compared with the

quartz crystal microbalance and surface plasmon resonance,33,34 the EIS technique is simple, low cost, requires no labeling

of analyte with redox active moiety, and works well in highly

ionic environment appropriate for biomolecular detection.

Plasma irradiation for 10, 5, and 2 min were carried out for 5,

50, and 100 W, respectively, to obtain ppPY films with similar

thickness, 32 6 3 nm. In all three cases, Rct of the matrix was

increased after DNA adsorption. To understand whether or

not nonspecific adsorption exists between the target DNA and

probe DNA, total mismatch target DNA (TMM-DNA) was

used to hybridize with the probe DNA. Rct of the sample

FIGURE 1 EIS of ppPY deposited at (a) 5 W, (b) 50 W and (c) 100 W for 10 min, 5 min and 2

min, respectively, before and after DNA immobilization=hybridization in phosphate buffer.

(i-ppPY, ii-after P1 immobilization, iii-after TMM hybridization, and vi-after MM0 hybridization.)

Inset: simulated circuit used to model impedance data in the presence of redox couples. Rs, electro-

lyte solution resistance; Ret, element of interfacial electron transfer resistance; CPE, constant phase

element.

ppPY for DNA Immobilization and Cell Adhesion 497

Biopolymers

deposited at 5 and 50 W was increased substantially, but was

decreased slightly for the ppPY film prepared at 100 W. The

hybridization of the complementary target DNA (MM0-DNA)

was carried out after TMM-DNA was dissociated with the

probe DNA using HCl solution. For the three films, the Rct of

the composite electrode decreased. However, the Rct variation

of the ppPY film deposited at 5 W before and after MM0

hybridization was the highest. Rct of each step and the change

in Rct (DRct) during DNA immobilization=hybridization are

summarized in Figure 2. The DRct before and after DNA

hybridization and immobilization are most obvious. As dis-

cussed in our previous work, the density of the functional

group of ppPY films, 2N15, prepared at 5 W was highest

compared with that of the ppPY films deposited at 50 and 100

W. Thus, strong interaction between the ppPY film deposited

at 5 W and DNA sequence could occur, indicating that much

more DNA chains could be adsorbed, leading to the retarda-

tion of the transfer of electrons at the interface because of the

dense cover of probe DNA chains onto the surface of the ppPY

film. In addition, the double strand DNA could be produced

after target DNA hybridization, resulting in decreased Rct

caused by the conductivity of the helix structure of the hybri-

dized DNA.35 The DRct (DRct 5 Rct, TMM 2 Rct, P1) is attrib-

uted to the nonspecific interaction between the target DNA

and probe DNA. No nonspecific interaction has been observed

for the ppPY film deposited at 100 W. Given that DNA chains

prefers to adsorb onto the ppPY film deposited at lower input

power, high nonspecific interaction could take place between

the target DNA chains and polymer film.

Influence of ppPY ThicknessPlasma polymer thickness significantly affects DNA immobili-

zation onto the polymer surface due to the penetration behav-

ior of DNA into the network interior of the plasma polymeric

hydrogel when the polymer thickness is thick enough.8,9 To

eliminate the influence of the nonspecific interaction, high

plasma input power (100 W) was used to deposit the ppPY

films with a thickness series of 6, 56, and 124 nm. DNA immo-

bilization=hybridization onto these ppPY films was then

conducted.

Figure 3 shows the Rct of each step and DRct during DNA

immobilization=hybridization. The adsorption of the probe

DNA onto the 6 nm thin polymer film led to the decrease in

Rct. However, the thick ppPY film increased the Rct. This result

may due to the fact that the cross-linking degree of polymeric

films deposited at 100 W became higher, which restricted the

penetration of DNA chains into the interior of the polymeric

network, leading to the difficulty of the polymer chain move-

ment. DNA chains can only be adsorbed on the ppPY film sur-

face to produce a covered layer, subsequently increasing the Rct

at the interface between the polymeric film and the solution.

By contrast, the thin polymer film with lower cross-linking

degree is much looser, making it easier for the polymeric

chains to extend and swell in the aqueous solution. DNA

chains could be penetrated into its interior, leading to

decreased Rct. For the three cases, all DRct (DRct 5 Rct, TMM 2

Rct, P1) are quite low; whereas the DRct (DRct 5 Rct, MM0 2 Rct,

P1) is the highest for the ppPY films with thickness of 6 nm.

Nonspecific adsorption has been clearly observed. For the

ppPY film with thickness of 124 nm, DRct (DRct 5 Rct, MM0 2

Rct, P1) is still very high compared with the lower nonspecific

adsorption. Thus, the thick polymeric film is much more sen-

sitive for DNA sensors.

Influence of the Probe DNA ConcentrationIncreasing the probe concentration leads to a decrease the Rct

of the composite electrode after the oligonucleotides bound, as

shown for the ppPY film with the thickness of 6 nm deposited

FIGURE 2 (a) Rct simulated of the composited electrodes of ppPY films deposited at 5 W, 50 W

and 100 W before and after DNA immobilization=hybridization and (b) their Rct variations.

498 Zhang et al.

Biopolymers

under 100 W in Figure 4a. When the probe concentration is

above 100 nM, the Rct is almost up to the equilibrium (See

curve (i) in Figure 4b). Thus, the probe with 100 nM is used in

the following experiment. Similar result was observed for the

films prepared under 5 (See curve iii) and 50 W (See curve ii)

in Figure 4b.

Kinetics of the Target DNA Hybridizationwith Probe DNA

To understand the influence of the plasma input power on the

hybridization behavior of complementary DNA with probe

DNA onto the ppPY films, the kinetics of the variation in Rct

during the MM0-DNA hybridization are summarized in Figure

5. By plotting the Rct values versus the corresponding reaction

time, a Langmuir-like isotherm is obtained. The hybridization

reached the equilibrium after 5 h. When rinsing by PBS, Rct

decreases fast within a short period, and then goes slowly to

the equilibrium. The association and dissociation kinetics of

the hybridized double helix can be obtained based on the

Langmuir model. For the ppPY film deposited at 5 W (Figure

5a), a straight line was obtained by plotting ln Rct=Rct, eq versus

the rinsing time. The equation is ln Rct=Rct, eq 5

0.0378923.043 3 1026 t with a regression coefficient of

0.98822 from which kd 5 3.043 3 1026 s21. On the basis of

the association process a linear relationship is observed

between lnDRct=Rct, eq and the binding time. The equation is

lnDRct=Rct, eq 5 23.72 1 1.24 3 1024 t. On the basis of the

value of kd, ka 5 121 M21s21 was also obtained, and based on

FIGURE 4 (a) EIS of P1 immobilization with different concentrations onto ppPY films (i-0 nM,

ii-1 nM, iii-10 nM, iv-25 nM, v-50 nM, vi-100nM, vii-500 nM, viii-1000 nM) and (b) the relation-

ship between Rct of the composite electrodes immobilized with P1 and the concentrations of P1

solutions.

FIGURE 3 (a) Rct simulated of the composited electrodes of ppPY films deposited at 100 W with

the thickness of 6, 56, and 124 nm before and after DNA immobilization=hybridization and (b)

their Rct variations.

ppPY for DNA Immobilization and Cell Adhesion 499

Biopolymers

the following equation: Ka 5 ka=kd, i.e., 3.98 3 107 M21, the

affinity constant for the complementary DNA=DNA helix was

deduced. The same simulation method was carried out for the

hybridization process of MM0-DNA with probe DNA immo-

bilized on the ppPY film deposited at 100 W, as shown in Fig-

ure 5b; kd 5 1.305 3 1026 s21 and ka 5 184.41 M21 were

calculated. Hence, Ka is 1.41 3 108 M21, close with that

obtained using concentration titration method. 36 Both of

which are higher than that of the ppPY film deposited at 5 W.

Sensitivity of DNA Hybridization onto the ppPY film

The sensitivity of the ppPY-based electrochemical DNA bio-

sensor probe was investigated by varying the concentration of

the complementary target DNA. According to the EIS spectra

(Figure 6), DRct (DRct 5 Rct, MM0 2 Rct, P1) with the increase

in the amount of complementary target DNA sequence. The

relationship between the value of DRct (DRct 5 Rct, MM0 2 Rct,

P1) and the negative logarithm of the concentration of the

complementary target DNA [2lg(c)] is illustrated as the inset

of Figure 6. The dynamic determination range for the comple-

mentary target DNA was from 0.5 nM to 1000 nM with the

regression equation Y 5 6.81587 1 0.67247 log X (X: concen-

tration of MM0 target DNA, M; Y: DRct) and the correlation

coefficient c 5 0.9937. Thus, the detection limit was 7.31 3

10211 M, close to the label-free DNA sensors based on other

conducting polymers. 37

Cell adhesion on the Surface of the ppPY Films

Before the investigation of cell adhesion onto the surface of

ppPY films, the antibacterial activity was detected. No bacteria

could be adsorbed on the ppPY film deposited at arbitrary

plasma conditions. Given that the -N1- groups remained in

the ppPY structure in the aqueous solution, the density of the

positive charges was pretty high. Meanwhile, the high density

of the positive charges and surface activity are favorable for cell

adhesion.

Two kinds of cells, namely, myeloma SP-2=0 cell and thy-

moma cell38,39 were chosen in the present work. The former is

a semi-adherent cell and not digested by Trypsin before being

used. The latter, however, is a full-adherent cell and digested by

Trypsin. The inverted microscope was utilized to determine

the process of cell adhesion and growth. Figures 7 and 8 sum-

marize the paragraphs of myeloma SP-2=0 cell and thymoma

cell growth on the surface of ppPY films deposited at 5, 50,

and 100 W. A large amount of myeloma SP-2=0 cells preferred

to adhere on the polymeric surface prepared at high input

FIGURE 5 Rct of of the composite electrode of ppPY deposited under (a) 5 W and for 10 min and

(b) 100 W for 1 min in 0.10 M PBS (pH 5 7.4) and MM0 target hybridized with probe DNA for

different association and dissociation times from 0 to 12 h.

FIGURE 6 Calibration curves of the sensor response to MM0 tar-

get DNA onto PPpy film deposited under 100 W and for 10 min

immobilized with P1 DNA. Inset: linear regression of DRct (DRct 5

RdsDNA 2RssDNA) vs. the natural logarithm of MM0 target concen-

tration. The vertical bars designate the standard deviations for the

means of three replicative tests.

500 Zhang et al.

Biopolymers

power (100 W) compared with that on the polymer deposited

at 5 W, as shown in Figure 7. The distribution of myeloma SP-

2=0 cells is uniform. The surface of the ppPY film deposited at

100 W was rough. In addition, the film is ready to swell in the

aqueous solution into the three-dimensional polymeric hydro-

gel. The growth factors can penetrate into the interior of the

polymer network, leading to the proliferation of cells, thereby

enhancing the adhesion interaction of myeloma SP-2=0 cell on

the polymer surface. The thymoma cell, however, seems to

adhere onto the surface of ppPY deposited under 5 W. Gener-

ally, the adhesion of myeloma SP-2=0 cell on ppPY films is

stronger than that of thymoma cell.

CONCLUSIONSThe polypyrrole-like films applied as bioactive plats for DNA

immobilization and protein adsorption were investigated in

our previous work. The influence factors that have an impor-

tant role on the chemical structures of ppPY film, such as the

thickness of polymers and input power, also subsequently

affected the immobilization behavior of DNA and cell adhesion

onto the surface.Owing to the relatively high density of the

functional group of the ppPY film surface deposited at 5 W,

more probe DNA molecules were immobilized, leading to the

increase of Rct. However, no nonspecific adsorption occurred

between the probe DNA and ppPY film deposited at 100 W.

The difference in Rct of the composite electrode after the probe

DNA anchored was decided by the swelling behavior of the

ppPY film deposited at 100 W, which depended on its cross-

linking degree. The Rct decreased because of the penetration of

the double stranded helix of hybridized DNA into the poly-

meric hydrogel. Compared with the ppPY film deposited at 5

W, higher affinity constant Ka was obtained by the concentra-

tion titration method. Similarly, the chemical and solution

property have a role on cell adhesion. Myeloma SP-2=0 cell

preferred to adhere onto the surface of the ppPY film depos-

ited at 100 W, whereas a contrasting trend was observed for

thymoma cell. In conclusion, due to the simplicity and low

detection limit of EIS, the ppPY film, which shows high sensi-

tivity and changeable affinity, offer a good promise for DNA

analysis. The ppPY films can also be used as alternative bioac-

tive materials for cell adhesion.

MATERIALS AND METHODS

MaterialsPyrrole was purchased from Aladdin Chemistry Co., dried by distilla-

tion with calcium hydride. KH2PO4 and Na2HPO4�12H2O were form

FIGURE 7 The photographs of the adhesion of myeloma SP-2=0 cell onto ppPY films deposited

at (a) 5 W, (b) 50 W, and (c) 100 W.

FIGURE 8 The Photographs of the adhesion of thymoma cell onto PpPY films deposited at

(d) 5 W, (e) 50 W and (f) 100 W.

ppPY for DNA Immobilization and Cell Adhesion 501

Biopolymers

Tianjin YongDa Chemical Reagent Development Center and Tianjin

Kermel Chemical Reagent Co., respectively. DNA oligonucleotides

were purchased from Sangon Inc. (Shanghai, China).

Plasma PolymerizationPlasma polymerization was carried out on the HQ-2 PECVD system

manufactured by the Institute of Microelectronics of the Chinese

Academy of Sciences, China. The radio frequency generator was oper-

ated at 13.56 MHz. The other details of the procedures involved were

described in our previous work. 29 The thickness of ppPY films on Si

wafers were measured by a WVASE32TM ellipsometer (J.A.Wool.LAM

CO, INC) with a He=Ne laser (k 5 6328 A) at the incident angle of

70�.

Preparation of Single DNA and Double-Strand DNAThe sequences are 5’-TTT TTT TTT TTT TTT TGT ACA TCA CAA

CTA-3’ (probe 1, P1); 5’ TAG TTG TGA TGT ACA-3’ (complemen-

tary DNA target to P1, MM0); and 5’ TTA CCC CTC CTC TCA GTA

CAT CTT-3’ (noncomplementary DNA target to P1, TMM). DNA

solutions were made using phosphate buffer solution (PBS) with a

pH of 7.4. The ppPY films deposited on the Au films, with a thickness

of 50 nm, were used as substrates. Each sample was immersed in large

amount of PBS overnight to remove the residual amount of the physi-

cally adsorbed pyrrole monomer and short chain polymer.

Electrochemical MeasurementsElectrochemical measurements were performed using a conventional

three-electrode system. The silicon substrate deposited with the ppPY

films was used as working electrode, an Ag=AgCl was used as reference

electrode, and a Pt plate was used as counter electrode. EIS was per-

formed with a CHI660D electrochemical workstation (Shanghai CH

Instrument Company, China). Electrochemical measurements were

carried out in 0.01 M PBS (pH 7.4, containing 0.1 KCl). Impedance

spectra was measured in the frequency ranging from 100 MHz to 100

KHz, in a potential of 0.23 V, versus Ag=AgCl (saturated KCl), with a

voltage amplitude of 5 mV. The spectra were analyzed with the Zview2

software, which uses a nonlinear least-squares fit to determine the

parameters of the elements in the equivalent circuit. All measurements

were carried out at room temperature.

Cell Adhesion onto ppPY FilmsMyeloma SP-2=0 and Thymoma cells were obtained from the cell lab

in the College of Life Science in Shanghai University. The coverslip

was cleaned ultrasonically in a mixed solution of HCl and ethanol

with a volume ratio of 1:9 for 20 min after rinsing with deionized

water twice for 20 min and 10 min, respectively. The cover slip was

then dried with N2. The ppPY films were deposited onto the treated

coverslips, and the cells were adsorbed onto the surface of the ppPY

films. Briefly, the as-prepared ppPY samples were sterilized by UV in a

clean bench for 30 min after being immersed into 70% ethanol for 10

min. About 3 mL of cell solution with a concentration of 105 cells=mL

were transferred on the sterilized samples in a Petri dish. The Petri

dish was then placed in the carbon dioxide incubator for 3 days. The

process of the cell growth was recorded every day. An inverted Leica

TCS-SP2 laser scanning confocal microscopy system was used to

detect the images of cell adhesion captured by the computer. The eye-

piece and objective lens magnification was 310.

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