A new approach for immobilization of oligonucleotides ontopiezoelectric quartz crystal for preparation of a nucleic acid sensor for

following hybridization

Memed Duman, Reza Saber, Erhan Piskin *

Department of Chemical Engineering and Bioengineering Division, Hacettepe University, 06532 Beytepe, Ankara, Turkey

Received 31 October 2001; received in revised form 2 September 2002; accepted 19 December 2002

Abstract

The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following

hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow�/discharge apparatus with ethylene diamine (EDA)

plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the

crystal surfaces. The thickness of the EDA�/plasma film formed was about 439/24 A. Then, the amino groups on the crystal surfaces

were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA

concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface

concentration of about 270 ng/cm2. A double strand Oligonucleotides, having one extra base on 5?-end of one of the complementary

strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions

were as follows: oligonucleotide concentration: 1 mg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature:

20 8C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that

equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be

measured within the medium.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Nucleic acid sensors; Piezoelectric quartz crystals; Glow�/discharge; Plasma polymerization; Glutaraldehyde treatment; Oligonucleotide

immobilization; Hybridization/dehybridization

1. Introduction

Sequence-specific hybridization between nucleic acids,

either in solution or immobilized on fixed support, are

widely used for the detection and analysis of genetic

material for diverse applications including identification

of genetic diseases and disorders, detection and char-

acterization of viruses, bacteria, and parasites, etc.

(Symons, 1989; Downs, 1991). In nucleic acid sensors,

hybridization can be followed by several techniques,

including electrochemical (Palecek, 1988; Moser et al.,

1997; Hianik et al., 2001), optical (Jost et al., 1991;

Piscevic et al., 1995), and piezoelectric (Yamaguchi et

al., 1993; Su and Thompson, 1995; Caruso et al., 1997,

1998; Bardea et al., 1998; Xhang et al., 1998; Zhou et al.,

2000, 2001; Patolsky et al., 2001).

Due to its piezoelectric properties, quartz crystals are

known as microbalance (QCM) which can function as

an extremely sensitive mass sensor capable of measuring

subnanogram levels of mass changes. They are also

inexpensive and are considered chemically, physically

and mechanically durable systems. For immobilization

of the probe nucleic acids (mostly single-strand oligo-

nucleotides) effectively on the piezoelectric crystal

surfaces, several methods have been proposed, each

with its own advantages and disadvantages (Montrel et

al., 1997; Yamaguchi et al., 1993; Su and Thompson,

1995; Caruso et al., 1997, 1998; Bardea et al., 1998;

Xhang et al., 1998; Wittunng-Stafshede et al., 2000;

Zhou et al., 2000, 2001; Patolsky et al., 2001; Towery et

al., 2001). Recently, a novel technique has been applied

* Corresponding author. Tel.: �/90-312-297-7473; fax: �/90-312-

299-2124.

E-mail address: piskin@hacettepe.edu.tr (E. Piskin).

Biosensors and Bioelectronics 18 (2003) 1355�/1363

www.elsevier.com/locate/bios

0956-5663/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0956-5663(03)00087-3

by us and by others for surface modification of piezo-

electric crystals, in which crystals are treated in glow�/

discharge systems (Nakanishi et al., 1996; Nakamura et

al., 1997; Kurosawa et al., 1997; Mutlu et al., 1999; Wuet al., 2000; Towery et al., 2001; Saber et al., 2002).

Several reactive or non-reactive plasmas can be created

within the glow�/discharge apparatus, which modify the

surfaces by depositing films having functional groups.

These, the so-called plasma-polymerized films covering

the crystal surfaces, are extremely thin and homogenous

coatings. They adhere strongly to the crystal and are

highly resistant to chemical and physical treatments.Moreover, sensors produced using this method are more

reproducible from sample to sample and exhibit lower

noise than sensors made using conventional immobiliza-

tion methods.

In our previous studies, we have applied ethylene

diamine (EDA) plasma-polymerization (or glow�/dis-

charge treatment) to create amino groups on the quartz

crystals (with Ag electrodes) and immobilized antibodieson these crystals using glutaraldehyde (GA) (as cross-

linker and spacer arm) to prepare immunosensors

(Mutlu et al., 1999; Saber et al., 2002). Here, we report

preparation of a QCM based nucleic acid sensor for

investigation of hybridization, by following a very

similar procedure for nucleic acid immobilization.

2. Materials and methods

2.1. Materials

EDA was supplied from Fluka (Switzerland), GA was

purchased from Aldrich (UK). The 20-mer single-

stranded oligonucleotide having the sequence 5?-GTGT TTG CCT GTT CTC AGA C-3? was used as theprobe (‘‘ligand’’) in the DNA sensor, which is denoted

here as ‘‘P1’’ in this study. The complementary (‘‘tar-

get’’) strand, which is called ‘‘P2’’, has the sequence 5?-GTC TGA GAA CAG GCA AAC A-3?, which is the

most widely recognized sequence in the DNA’s of

Turkish population. Both oligonucleotides were synthe-

sized by Life Technologies (UK). Note that the probe

(P1) contains one extra nucleotide at 5?-end, and aftercomplete hybridization of these two complementary

single-strands, the double strand contains one free

base at one end, this means that the amino groups of

this base are available for covalent binding on the

crystal surfaces. It should be emphasized that only one

free amino group at the end of the double strand is

available for binding, which is the key difference

between this study and all previous studies. Buffersolutions with different pH and ionic strength were

obtained from Life Technologies and also prepared in

our laboratory by using analytical grade chemicals.

2.2. QCM system

The quartz crystal used in this study was commer-

cially available an AT-cut 10 MHz quartz crystal of a8.7�/0.17 mm wafer which was placed between 4.7 mm

silver electrodes and mounted on a ceramic holder with

a plug (AEC, Taiwan). The oscillation electronic circuit

was a typical Collpits oscillator, which has a buffer

amplifier. 15 V DC was applied to the oscillator circuit

to drive the crystal and the frequency was measured with

a Hewlett�/Packard frequency counter (Model No: HP

53181A, 225 MHz, USA). The crystal was enclosed in acase, which was maintained in a chamber at a constant

temperature of 359/0.1 8C during the measurements to

reduce frequency fluctuations due to dust. This QCM

system gave a frequency drift of less than 1 Hz in air

after a few minutes (B/5 min) initial stabilization period.

The procedure for preparing the silver surface has been

described in the following section. The mass change

(Dm ) on the quartz crystal which is related to theresulting frequency shift Df was calculated from the

Sauerbrey equation given below (Sauerbrey, 1959).

Df ��2:26�10�6f 2Dm=A (1)

Where, Df is the change in the fundamental frequency of

the coated crystal (Hz); f , the fundamental frequency of

the quartz crystal (Hz); Dm , the mass deposited on the

crystal (g); and A is the area coated (cm2).

2.3. Modification of piezoelectric quartz crystals

2.3.1. Glow�/discharge treatment

For oligonucleotide immobilization, the quartz crys-

tal surface was treated in a glow�/discharge system

(Mutlu et al., 1999). The glow�/discharge reactor was a

Pyrex† glass tube with a diameter of 5.6 cm and a length

of 52 cm. Two copper electrodes of 7�/17 cm wereexternally placed on the reactor. One of the electrodes

was connected to a radio-frequency generator with

frequency of 13.6 MHz (Tasarim Ltd., Model: T-RF-

1200, Turkey) through a matching network unit (Ta-

sarim Ltd., Model: T-RF-1100), while the other elec-

trode was grounded. The monomer tank was connected

to the reactor through a flow meter (Gilmont, F-1200,

Size 2, USA) and a needle valve (Brooks, Model 1355,CA, USA).

EDA was used as the active monomer, and was

plasma-polymerized in the glow�/discharge reactor, to

create amine-like active groups on the quartz crystal

surface for further treatments given below. Briefly, the

glow�/discharge reactor was evacuated to 10�1 mbar.

The monomer, EDA, was allowed to flow through the

reactor at a flow rate of 35 ml/min. The quartz crystalswere exposed to the EDA plasma for 2.5 min at glow�/

discharge power of 15 W. It should be noted that these

were optimal plasma treatment conditions which were

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/13631356

obtained in our related previous studies (Mutlu et al.,

1999; Saber et al., 2002) and also after preliminary

experiments (Duman, 2001). The modifications were

followed by measuring the shifts in the resonancefrequencies of the crystals before and after the surface

modification.

2.3.2. Glutaraldehyde activation

In the second step, the primary amine groups on the

crystal surface created after the plasma-polymerization

of EDA were converted to aldehyde groups by treating

the surfaces with GA. The GA concentration and the

incubation time were changed while the treatmenttemperature was kept constant at 4 8C. For reductive

animation of the secondary amine linkage (for stabiliza-

tion of the Shiff base) we also used NaCNBH3 (about

five times of the GA concentration, molar basis). In the

first set, the glow�/discharge treated quartz crystals were

incubated with solutions containing different amounts

of GA (0.1, 0.5, 1, 2.5 and 5%) for 2 h. In the second set

of experiments, the GA concentration was kept constant(2.5% GA in a carbonate buffer, pH 9.2) and the

incubation time was changed (1, 2, 4, 8 and 16 h).

Physically adsorbed GA molecules were removed by

washing with pure water several times. Following the

washing step, the crystals were dried in air (at room

temperature for about 15 min, until a constant fre-

quency was obtained) and the frequencies of the air-

dried crystals were recorded. It should be note that aGA molecule has two aldehyde groups. At this treat-

ment step, it was assumed that one aldehyde group on

each GA is covalently bonded to amine group on the

EDA treated crystal while the other aldehyde group

stays free for the next treatment step.

2.4. Oligonucleotide immobilization

In this part of the study, the oligonucleotide immo-

bilization (e.g. covalent binding of the double strand

oligonucleotide onto the GA-modified crystal surfaces)

was investigated at different conditions. Note that these

immobilization studies were conducted where the non-

specific adsorption of the oligonucleotide was at mini-

mum. These conditions were derived based on the

experiments performed in the previous step. Here, inthe first group, only the oligonucleotide concentration

(0.1, 0.5, 1.0, 2.0 and 4.0 mg/ml) was changed and the

immobilization time was kept constant at 3 h. In the

second group, the oligonucleotide concentration was 1.0

mg/ml, while the immobilization time was changed (1, 2,

3, 4, 8, 20 h). For reductive amination of the secondary

amine linkage between the surface aldehyde groups

(coming from the immobilized GA molecules) on thesurface and the terminal amine groups of the oligonu-

cleotide molecules, we also used NaCNBH3 (about five

times of the oligonucleotide concentration, molar basis).

Incubation was conducted in a shaker at room tem-

perature. After the immobilization step, the crystals

were first washed with distilled water and then with the

buffer solution for 15 min. The crystals were dried in airat room temperature, and the frequency shifts were

recorded. It should be noted that the double strand

oligonucleotide was covalently immobilized on the

crystal surface from the amino groups of the free

nucleotide on the P1 probe.

2.5. Hybridization experiments

In order to determine the performance of the QCM asa sensor, firstly the P2 strand (not covalently bound to

the surface) was removed from the crystal surfaces

(‘‘dehybridization’’). The crystals carrying the double

strand oligonucleotide were incubated in a carbonate

buffer (pH 9.2) at 94 8C for 3�/4 min. After washing and

drying several times with buffers and pure water,

frequency shifts were measured by the QCM system. It

should be noted that we have continued the washingstep until we were confident that we removed all of

complementary strand P2 from the surface. Practically,

when we reached the frequency shift (decrease) that

corresponds to about half of the frequency shift

(increase) observed in the immobilization of the double

strand oligonucleotide, we stopped the washing and

took these crystals carrying the single strand (P1) for

further tests.The nucleic acid sensor carrying the covalently bound

single strand (P1) was incubated with the solutions

containing the complementary strand (P2) at different

concentration (0.001, 0.01, 0.05, 0.1 and 1 mg/ml) for

different times in the range of 1�/25 min. Hybridization

was evidenced by a stepwise mode. The QCM sensor

was removed from the solution after hybridization,

rinsed and dried and the change in the resonancefrequency of the crystal before and after incubation

was recorded.

In the second approach, the required amount of the

P2 oligonucleotide was dissolved in 3 ml phosphate

buffer (pH 7.4). The crystal carrying the P1 oligonucleo-

tide was mounted onto a homemade cell, which was

then dipped into the solution containing P2 molecules.

The frequency shifts were recorded continuously withthe microbalance system described below, while the

medium was magnetically stirred.

2.6. STM imaging

Scanning tunneling microscopy (STM) images of the

crystal surfaces were obtained by using a homemade

STM constructed in our laboratory (Zareie, 1995).Mechanically, it consists of two main modules, a

scanner part constructed with a piezo tube (PZT tube)

(EBL No: 3 PZT-5H, Staveley Sensors, CT) glued to

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/1363 1357

aluminum body and a magnetically driven slider. The

electrical connections to the PZT are through a special

connector and the whole system is mounted on a

vibration isolation stage.For STM measurements, the oligonucleotide immo-

bilized crystals were attached to the sample holder with

silver epoxy. Then, the STM images were taken at room

temperature and at atmospheric pressure, using a 50 mV

sample bias and a tunneling current of 850 pA. Etched

tips of Pt/Ir (80:20) wires (0.5 mm in diameter, Digital

Instruments, Santa Barbara, CA) were used. Prior to

use, the tips were washed with acetone. The brightnessand contrast was adjusted using PHOTOSHOP v3.0

(Adope System, CA, USA) to enhance the images

details.

3. Result and discussion

In this study, glow�/discharge technique was selected

as a promising method for double strand oligonucleo-

tide immobilization on the piezoelectric crystal surface.

In the conventional techniques, such as gel entrapment,dip coating, irradiation of polymers, etc., the surface

generated on the crystal becomes bulky, depressing the

quartz crystal’s sensitivity, expanding response time and

also creating difficulties in some applications. However,

the polymer coating thickness created by glow�/dis-

charge technique was about 200 A eliminating the above

deficiencies while providing maximum sensitivity.

Furthermore, the functional groups (�/NH2) on thesurface enable the double strand oligonucleotide to be

quite easily linked to the treated surface by GA

activation from one strand, which have one more

nucleotide. The glow�/discharge parameters were opti-

mized and the result and the performance of the nucleic

acid sensor are summarized in the following section.

3.1. Surface modification of quartz crystals

3.1.1. Glow�/discharge treatment

Glow�/discharge treatment has been widely used to

change the surface properties of materials without

affecting their bulk structures for diverse applications(Clark and Feast, 1978; Yasuda, 1985; Fawcett et al.,

1988; d’Agustino, 1991; Piskin, 1992). Depending upon

the glow�/discharge conditions, surface modification

occurs through etching and/or deposition. Gases (e.g.

argon, oxygen, air, nitrogen), non-polymerizable polar

gases (e.g. water, ammonia), organic vapors (e.g.

methanol, acetone) or polymerizable gases or vapors

(e.g. ethylene, fluoroethylenes) have been used to createdifferent functional groups, (e.g. carboxyl, hydroxyl,

amino) on the material surfaces by this simple surface

modification technique.

In this study, in order to create amino-like active

groups on the quartz crystal surfaces for double strand

oligonucleotide immobilization, we used EDA as the

active monomer. According to preliminary studies, weconcluded that optimal conditions for plasma polymer-

ization are, the discharge power: 15 W; treatment time:

2.5 min; and the monomer flow rate: 35 ml/min. The

average frequency shift and standard deviation of these

crystals comparing with the blank crystals (without

glow�/discharge treatment) was 1439/24 Hz. This fre-

quency shift is based on measurements of ten randomly

selected crystals from different loads is quite significant.These crystals were used in the further studies presented

below.

It should be noted that in the glow�/discharge

treatment we deposited EDA�/plasma film on both sides

of the quartz crystal. The film thickness was determined

by the frequency shift of the bare crystal induced by the

film coating. Thickness is related to the frequency shift

as given in the following equation (Lawson, 1967; Wu etal., 2000; Sugimoto et al., 2000). The thickness of the

EDA films on the quartz crystals created in our studies,

was calculated from the following equation, and found

to be 439/24 A.

Dt�0:27 Df =r (2)

Where, Dt is the thickness of the polymeric film on thecrystal surface (A); Df , the change in the fundamental

frequency of the coated crystal (Hz); and r is the density

of the film which was taken as the density of the EDA

monomer (0.895 g/cm3). The polymerization rate of

EDA, similar to aliphatic amines, is slow compared with

those well-known plasma-polymerized monomers con-

taining nitrogen (e.g. allylamine) (Yasuda and Lamaze,

1973; Bell et al., 1975; Hozumi et al., 1983; Tajima et al.,1988). It was reported that these films have the

structures containing �/NH2, �/NH�/, �/N�/N�/, and �/

CN. In our study we have used EDA plasma, which

was also selected as the active plasma in similar studies

(Nakanishi et al., 1996a,b; Muguruma and Karube,

1999; Towery et al., 2001). As reported in these studies,

it was expected that amino groups exist on the surface of

the ethylene diamine plasma-polymerized film.

3.1.2. Glutaraldehyde attachment

Usually GA is used as an agent for crosslinking of

amine groups in compounds like protein and oligonu-

cleotides (Hermanson, 1996). In our preliminary experi-

ments we observed that in order to covalently bind the

oligonucleotide molecules to the crystals (coated with

the EDA�/plasma films), best results were obtained

when the crystals were incubated in carbonate buffer

(pH 9.2) with different GA concentration for differentincubation times.

In the first group of experiments, we changed the GA

concentration and followed the changes in frequency

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/13631358

shifts. Increasing the GA concentration from 0.1 to

2.5%, the frequency shift was doubled. The maximum

frequency shift was 889/13 Hz (the average and the

standard deviation of ten crystals), corresponding amass deposition of about 270 ng/cm2 (calculated from

Eq. (1)). However, at higher GA concentration, a slight

decrease in the GA deposition was observed, which may

be due to polymerization of the GA molecules in the

aqueous medium. Therefore, a GA concentration of

2.5% was selected as the optimal concentration and

applied in the later part of the study.

In the second set, the treatments were carried out at2.5% GA concentration in the same buffer, but for

different incubation times. The frequency change after 1

h was only 12 Hz. However, after 2 h, there was a steep

increase in the mass accumulation, which reached to

889/13 Hz. Thereafter the accumulation continued to

increase by increasing the incubation time, but not very

significantly. Therefore, 2 h was selected as the optimal

time and this time was used in the later part of the study.

3.2. Immobilization of double strand oligonucleotide

An oligonucleotide, known as fourth allele specific

probe of DQa’s subgroup, which is most frequently used

in genetic analysis of Turkish population’s DNA, was

selected as a model ligand and immobilized onto the

GA-attached piezoelectric crystals. This is one of the

basic probes on the test strips for identification of theDNA in criminology laboratories. As mentioned before,

there are many different methods for immobilization of

oligonucleotide onto solid surfaces. In this study, we

have applied for the first time, a quite simple technique

for immobilization a single strand oligonucleotide

ligand defined here as ‘‘P1’’. We have used two

complementary single strand oligonucleotides, P1 (hav-

ing one extra base on 5? end) and P2. These strands werebrought together in an aqueous medium and complete

hybridization was achieved. Then, this double strand

oligonucleotide was immobilized on the GA-attached

crystal surfaces by the reaction with the amino groups of

the free base (coming from the P1 strand) and the

aldehyde groups on the crystal surface.

Oligonucleotide immobilization was investigated at

different conditions. In the first group of experiments,increasing the oligonucleotide concentration in the

medium from 0.1 to 1.0 mg/ml caused an increase in

the amount of oligonucleotide immobilized, as expected

(Fig. 1). However, we observed a noticeable decrease in

the mass deposited, when the nucleotide concentration

was further increased. Note that there are quite a high

number of aldehyde groups available for immobilization

on the GA-attached crystal surface. But, the oligonu-cleotide molecules consist of relatively long chain and

contain only one base group at one end available to

react with the surface aldehyde groups. This situation

retards complete reaction between the amines and the

surface aldehydes. Increasing the oligonucleotide con-

centration in the immobilization medium, most prob-

ably increased the steric hindrance between the

competing long chains to react with the surface aldehyde

groups. In conclusion, 1 mg/ml oligonucleotide concen-

tration was selected as an optimal concentration for

effective oligonucleotide immobilization, and these crys-

tals were used in the next immobilization step. The

average frequency shift was 126 Hz and the correspond-

ing amount of mass deposited was about 380 ng/cm2.

These values were comparable with similar studies in

which different immobilization methods have been, used

(Yamaguchi et al., 1993; Su and Thompson, 1995;

Caruso et al., 1997, 1998; Bardea et al., 1998; Xhang

et al., 1998; Zhou et al., 2000, 2001; Patolsky et al.,

2001). For instance, in their recent study, Zhou and

coworkers have immobilized a 18-mer DNA probe (a

single strand) on the crystal surfaces carrying a poly-

electrolyte layer by electrostatic adsorption, and mea-

sured a frequency shift of 21 Hz after drying in air

(immobilized amount was calculated of 19 ng) (Zhou et

al., 2000, 2001). Alternatively, they have first chemically

immobilized on the crystal surface, then further reacted

with biotylated DNA for the immobilization of the

DNA probe. The immobilization amount was calculated

from the frequency decrease (38 Hz) and found to be 27

ng. In conclusion, for the 10 MHz quartz crystals used

in this work, Sauerbrey equation predicts that a

frequency change of 1 Hz corresponds to a mass

increase of 3.019/0.03 ng on the electrode (0.69 cm2).

It is almost the same amount reported in Zhou’s work

which predicted that a frequency change of 1 Hz,

corresponds to a mass increase of 0.9029/0.01 ng on

the electrode (0.204 cm2).

In order to observe the effects of immobilization time

on the immobilization, the GA-attached crystals were

incubated with the aqueous solution of the double

strand oligonucleotide (1 mg/ml) at room temperature

Fig. 1. Effects of oligonucleotide concentration on oligonucleotide

immobilization.

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/1363 1359

for different periods of time (1�/16 h). There was a

drastic increase in the first 3 h followed by more

moderate increase in the immobilization at longer time

(Fig. 2). Therefore, we assumed that 3 h would besufficient time to have adequate amounts of oligonu-

cleotide on the crystal surface. Furthermore, 3 h should

also be a reasonable period of time for practical

applications.

3.3. Performance of nucleic acid sensor

At the last step of this study, we investigated the

performance of a nucleic acid sensor carrying the ‘‘P1’’

strand. The other strand, i.e. the so-called ‘‘P2’’ (not

covalently bound to the surface) was dehybridized(removed) by applying the procedure mentioned before.

The nucleic acid sensor carrying the covalently bound

single strand was then used in the hybridization experi-

ments, in which these crystals were incubated with the

aqueous solutions of the ‘‘P2’’ strand at different

concentrations for different time periods. As seen in

Fig. 3, hybridization was almost completed in about 5

min in most cases. This short hybridization time is much

better when compared with the data given in similar

studies (Yamaguchi et al., 1993; Su and Thompson,

1995; Caruso et al., 1997, 1998; Bardea et al., 1998;

Xhang et al., 1998; Zhou et al., 2000, 2001; Patolsky et

al., 2001). We concluded that spacer arms with longer

chain lengths (longer than GA molecules) may increase

the availability of the immobilized strain for hybridiza-

tions and thus should reduce the time necessary to reach

equilibrium. Further studies are being conducted to

validate this hypothesis.

Fig. 3 also shows that there is no significant difference

in the hybridization curves (at equilibrium) at P2

concentrations of 0.1 and 1 mg/ml. This indicates that

almost all of the immobilized oligonucleotides (the P1

strand) were hybridized with the complementary mole-

cules (the P2 strand) corresponding to a P2 concentra-

tion of 0.1 mg/ml. In order to exhibit the relation

between the P2 concentration and the extent of hybri-

dization (the frequency shifts at equilibrium, taken from

Fig. 3), we prepared the graph (a semi-logarithmic scale)

given in Fig. 4. Note that we excluded the value

corresponding the P2 concentration at 1 mg/ml and

drew the best line for the first four values. From this

figure we concluded that there is a relation between the

‘‘P2’’ concentration and the frequency shift at equili-

brium, which may be used to quantitatively determine

the respective complementary strand in aqueous solu-

tions.

Alternatively, we have also investigated a direct

interaction of P2 on the immobilized P1 from aqueous

media. We used the probe carrying the P1 oligonucleo-

tide immobilized crystal, and placed it into a solution

containing P2. Two different concentrations of P2 were

used (namely 0.5 and 1 mg/ml) at pH 7.4. Fig. 5 shows

Fig. 2. Effects of immobilization time on oligonucleotide immobiliza-

tion.

Fig. 3. Effects of oligonucleotide (P2) concentration on hybridization

onto the crystal surface.

Fig. 4. Relation with oligonucleotide (P2) concentration and extent of

hybridization.

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/13631360

the frequency shifts observed during the interaction of

the probe with the P2 in the solution. As seen here, after

about 20�/30 min of pre-stabilization time, we added the

required amount of P2 oligonucleotide to the media.

There was an increase in the frequency shift at the

beginning due to noise and also volume change. Butafter that the frequency shifts started to decrease for the

crystals having P1 on their surfaces and reached plateau

in about 1 h. The decrease rate was faster with the

medium containing more P2, as expected. There was no

frequency change for the crystal that did not contain

immobilized P1 (line (a)).

3.4. STM imaging

In order to further support the conclusions obtained

from the QCM system, we attempted to observe the

double strand oligonucleotide molecules immobilized

onto the quartz crystals by using a novel STM techni-

que. Some representative STM micrographs are given in

Fig. 6 and show that the double strand that we haveused forms a double helix with two turns. This structure

has a pitch of helical periodicity of about 2.7 nm and a

height of 0.17�/0.38 nm. The corresponding values

calculated from Watson�/Crick DNA model is about

3.4 and 2 nm, respectively. It is known that the single

turn of the Watson�/Crick DNA consists of approxi-

mately 10 bp (Watson and Crick, 1953). Since the

double-stranded DNA used in this study consists of19-mer, it should have nearly two double-helix turns, as

actually observed in Fig. 6. We attempted to measure

the surface concentration of the oligonucleotides from

the STM micrographs, but this was not possible with the

system that we have used. Therefore, we were not able to

support quantitatively the amounts of immobilizations

at different conditions.

4. Conclusion

In this study, we demonstrated a simple immobiliza-

tion technique to prepare nucleic acid sensor based on

piezoelectric crystal microbalance system. The results

showed that the QCM sensors can be fabricated by

using relatively inexpensive quartz piezoelectric crystalshaving silver electrodes. Crystals can be effectively

coated with a very thin EDA�/plasma film in a simple

glow�/discharge system at a discharge power of 15 W

and a treatment time of 2.5 min. Then the amino groups

can be converted satisfactorily to aldehyde groups by

treating the surfaces with glutaraldehyde (concentration:

2.5%) at 4 8C in about 2 h. For oligonucleotide

immobilization, the double strand DNA composed of

Fig. 5. Frequency changes in direct measurements of P2 oligonucleo-

tide in aqueous media with a probe containing the piezoelectric

crystals; (a) crystal carrying no P1, and the medium concentration of

P2 is 1.0 mg/ml; (b) crystal carrying P1 on their surfaces and the

medium concentration of P2 is 0.5 mg/ml; (c) crystal carrying P1 on

their surfaces, the medium concentration of P2 is and 1.0 mg/ml.

Fig. 6. Representative STM micrographs of oligonucleotides on quartz crystal surfaces.

M. Duman et al. / Biosensors and Bioelectronics 18 (2003) 1355�/1363 1361

a strand to be identified (P2) and its complementary

strand having one extra base at 5?-end (P1) is dissolved

in a buffer (carbonate, pH 9.2) with an ionic strength of

0.1, at a concentration of 1 mg/ml, and only incubatedwith the GA-attached crystals for about 3 h. The

reaction with the amino groups of the 5?-end of the

oligonucleotide and surface aldehyde groups allows

covalent bonding of the oligonucleotide molecules

onto crystal surfaces through only the probe strand

(P1). The P2 strand can easily be removed from the

surface and the QCM sensor is brought to its final form.

Oligonucleotide immobilization can be followed withthe QCM system by measuring frequency shifts of the

crystal, which was 1269/12 Hz under optimal condition.

This frequency shift is sufficient for sensitive measure-

ments. The response time for equilibrium in the

hybridization experiments were obtained about 5 min,

which is quite satisfactory.

We also obtained a good correlation between the

concentration of the strand to be identified and themeasured frequency shifts, which may be applied for

quantitative determination of the oligonucleotides

within aqueous media. STM images supported the

QCM results where oligonucleotide chains are attached

onto the crystal surfaces.

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