156
A Feasibility Study of a Capacitive Biosensor for Direct
Detection of DNA Hybridization
Christine Berggren,+ Per StaÊlhandske,++ Jan Brundell,++ and Gillis Johansson*+
+
++
Department of Analytical Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
AB Sangtec Medical, P.O. Box 20045, S-161 02 Bromma, Sweden
Received: September 23, 1998
Final version: November 18, 1998
Abstract
This preliminary study was performed to prove the feasibility of a direct capacitive DNA biosensor for detection of nucleic acids. Two
different methods for immobilization of the oligonucleotide probes were used. The ®rst type of sensor was composed of a gold rod with a
self-assembled monolayer of a 26-base long oligonucleotide probe, modi®ed with an SH-group at the 5 0 -end. Coverage studies showed that
only around 20% of the surface was covered, probably due to the bulky nature of the probes. Hybridization studies performed in a ¯owthrough cell showed selectivity towards a DNA sample containing single stranded fragments of cytomegalo virus (CMV) possessing a
complementary sequence. As few as 25 molecules could be detected at sample concentrations of 0.2 attomolar with an injection volume of
250 mL. Controls with fragments of double-stranded CMV and single-stranded hepatitis B virus and tyrosinase mRNA gave all lower
responses. The other type of sensor was modi®ed by covalent immobilization of a phosphorylated 8-base long oligonucleotide probe to a
self-assembled monolayer of cysteamine. This biosensor also showed selectivity against single stranded fragments of CMV and also in this
case as few as 25 molecules could be detected.
Keywords: DNA biosensors, Nucleic acids, Hybridization, Capacitance
1. Introduction
Determination of speci®c DNA sequences in biological samples
can lead to detection and identi®cation of certain infectious or
inherited diseases as well as cancers [1]. The polymerase chain
reaction (PCR) has opened up great opportunities as it ampli®es
the DNA sample and is nowadays a widely used tool [2]. There
are, however, a high risk of contamination, which might lead to
ampli®cation of undesired sequences and special handling is
therefore necessary. Furthermore a higher sensitivity would be
desirable. Biosensors have been suggested to offer a promising
alternative for faster and cheaper hybridization assays [3]. A
biosensor consists of a biological recognition element in close
proximity to a transducer, which will convert the binding event
into an electrical signal. Short single-stranded oligonucleotide
probes immobilized on an electrode surface (transducer) can be
used as highly sequence-speci®c recognition elements for detection of DNA. These probes will hybridize when coming in contact
with a denatured single-stranded DNA sample, having a complementary sequence. Several different transducers for DNA detection have been reported. An electrochemical intercalator has been
used as indicator [4 ±8]. The hybridization can be measured
directly with acoustic wave [9], surface plasmon resonance [10],
impedance [11] or piezoelectric [12] transducers.
Capacitive transducers have lately shown great promise in the
®eld of immunosensors [13 ±15] and for the detection of bioavailable heavy metal ions [16], giving both high selectivity and low
detection limits. It was therefore considered as a suitable transducer for DNA detection, which is investigated in this article.
The capacitive transducer is based on the theory of the electrical
double layer [17], which in principle can be described as a build up
of two conducting phases, one consists of a metal surface and the
other of an electrolyte solution. Modi®cation at this interface by
immobilization of a recognition element to the conducting surface
will lead to a change in capacitance, the size of which will depend
on the nature and coverage of the recognition element. Further
Electroanalysis 1999, 11, No. 3
change in capacitance is expected when analyte binds to the
surface.
In this article gold electrodes were prepared either by direct
immobilization of an SH-modi®ed, 26-base oligonucleotide
probe, or by covalent coupling of an 8-base oligonucleotide
probe to a previously self-assembled monolayer of cysteamine.
The system studied was a model system and the oligonucleotide
probes were chosen to be complementary to a sequence of the
cytomegalo virus (CMV). As controls double-stranded CMV as
well as hepatitis B virus and tyrosinase mRNA were used. Gold
can easily be modi®ed by self-assembly of thiols, sul®des or
disul®des, resulting in well ordered monolayers [18] with strong
bonds between gold and sulfur [19]. Oligonucleotides immobilized on gold surfaces have earlier been reported. Coupling
between self-assembled thioctic acid via carbodiimide chemistry
to amino groups on the bases of the oligonucleotide back bone
yielded a low quantity of immobilized DNA [20]. SH-modi®ed
oligonucleotide probes have been self-assembled onto gold surfaces [4, 5] and hybridization detected with an electrochemically
active dye. Such surfaces were also studied with AFM, where it
was found that these thiolated DNAs bound strongly to gold
surfaces [21].
2. Experimental
2.1. Chemicals
The oligonucleotides CMV-SH (26-base, HS-5 0 -GTA GGG
AAG GCT GAG TTC TTG GTA AA) and LU-CMV (8-base, 5 0 TTA GGA GA) were obtained from Cybergene AB, Huddinge,
Sweden. The DNA standard fragments of cytomegalo virus
(CMV, 179 bases, Mw 118 140), hepatit B virus (HBV, 104
bases, Mw 68 640) and tyrosinase mRNA (Tyr, 207 bases, Mw
136 620) were all obtained from Sangtec Medical, Bromma,
Sweden.
# WILEY-VCH Verlag GmbH, D-69469 Weinheim, 1999 1040±0397/99/0303±0156 $17.50‡:50=0
Direct Detection of DNA Hybridization
157
2.2. Pretreatment of the Gold Surface
A gold rod with a diameter of 3 mm was used as the working
electrode. Prior to oligonucleotide modi®cation it was polished for
5 min with alumina paste (APFIN, Struers, Uppsala, Sweden),
ultrasonicated for 10 min, polished with aluminum oxide suspension with 0.05 mm particles (APSUP, Struers, Uppsala, Sweden)
and ®nally ultrasonicated for another 10 min. Thereafter the gold
rod was mounted into a Te¯on holder and plasma cleaned for
15 min (Harrick Sci. Co., New York, PDC-3XG).
2.3. Self-Assembling of an SH-Modi®ed Oligonucleotide
Probe to a Gold Surface
After pretreatment the electrode was immediately placed in a
solution of the thiol-modi®ed oligonucleotide and kept in this
solution overnight. After modi®cation the electrode was rinsed
thoroughly with ethanol to remove any loosely bound oligonucleotide and then rinsed with binding buffer (0.9 gyL sodium
azide, 0.5 gyL Tween 20 in water). The electrode was thereafter
placed in the ¯ow cell.
2.4. Immobilization of a 5 0 -Phosphorylated
Oligonucleotide Probe to a Gold Surface
With this kind of immobilization the gold rod was placed in a
thiol solution containing 2 % (wyw) cysteamine in ethanol for
16 h, immediately after the pretreatment described above. After
reaction the electrode was thoroughly rinsed in ethanol and dried.
The coupling of the oligonucleotide was performed, in a 0.1 M
imidazole buffer, pH 6 ±7, containing 0.15 M EDC, at room
temperature for 16 h. After the reaction the electrode was rinsed
thoroughly in buffer and placed in the ¯ow-cell.
2.5. Cyclic Voltammetry
Cyclic voltammetry was recorded by a Princeton Applied 273 A
potentiostat in a batch cell. A SCE was used as a reference
electrode. The coverage measurements were performed in
0.1 M H2SO4 with a scan rate of 100 mVys. To study the insulating
properties of the self-assembled monolayers cyclic voltammogramms in 5 mM K3Fe(CN)6 were recorded at a scan rate of
10 mVys.
The measuring ¯ow cell had a dead volume of approximately
10 mL. The carrier, an aqueous solution containing 0.9 gyL sodium
azide and 0.5 gyL Tween 20 was pumped through the measuring
cell at a ¯ow rate of approximately 0.5 mLymin. This carrier was
chosen since the standard samples were supplied in it and the
concentration of sodium ions were considered to be high enough in
order to act as counter ions to neutralize the DNA backbone.
Standard samples with a volume of 250 mL were injected into the
¯ow system and the change in capacitance upon hybridization was
recorded. The standard samples were all diluted in the carrier
solution to avoid blank signals. The injection loop and the tubings
in the system were made from PEEK in order to reduce adsorption
of DNA fragments and to reduce the risk for air bubbles.
Prior to injection the fragmented DNA samples were denatured
by boiling for 10 min. The hybridization at the electrode surface
was performed at room temperature in the ¯ow system.
3. Results and Discussion
3.1. Monolayer Characterization by Cyclic Voltammetry
The surface coverage of a self-assembled monolayer on an
electrode surface can be studied by cyclic voltammetry in
0.1 M H2SO4. Figure 1 shows cyclic voltammograms measured
on a bare gold electrode, as well as on a CMV-SH modi®ed
electrode and an 1-dodecanethiol modi®ed electrode. Around
800 V (vs. SCE) the gold oxide removal peak can be seen. The
surface coverage, y, can be estimated by comparing the area of the
gold oxide removal peaks for the modi®ed electrode and the bare
gold electrode [22]. For a bare electrode y ˆ 0, and for a totally
covered surface y ˆ 1. A coverage of 20 % was indicated for the
CMV-SH covered electrode and 84 % when 1-dodecanethiol was
self-assembled on the electrode surface. This can be compared
with a value of 85 % for 1-dodecanethiol obtained by Taira et al.
[23]. The much higher surface coverage obtained for 1-dodecanethiol compared with CMV-SH is probably due to that the
oligonucleotide (CMV-SH) is bulkier and can not be as close
packed as 1-dodecanethiol. It was observed earlier [13] that the
insulting properties of a similar electrode covered with antibodies
were destroyed by low pH or high salt contents. The accuracy of
2.6. Capacitance Measurements
The measurements were performed in a three electrode system
connected to a fast potentiostat, described earlier [14]. The current
transient obtained from a potential step of 50 mV was sampled and
the capacitance was calculated according to Equation 1, by using
the ®rst 10 points in the current array:
i…t† ˆ u=Rs exp…ÿt=Rs C1 †
…1†
i…t† is the current in the circuit as a function of time, u is the applied
pulse potential, Rs is the dynamic resistance of the self-assembled
layer, C1 is the capacitance measured between the gold electrode
and the solution, and t is the time elapsed after the potentiostatic
step was applied. With this method the system is described by a
model consisting of a capacitor and a resistor in series. The
instrumentation has been thoroughly described elsewhere [14].
Fig. 1. Cyclic voltammograms for a) a bare gold electrode, b) a CMVSH self-assembled gold electrode and c) a 1-dodecanethiol self-assembled gold electrode. All scans were performed in 0.1 M H2SO4, with a
scan rate of 100 mVys.
Electroanalysis 1999, 11, No. 3
158
the method may therefore be questioned. The actual coverages
could therefore be higher than the given values.
The insulating properties of the self-assembled layer were also
studied by cyclic voltammetry in 5 mM K3Fe(CN)6, see Figure 2.
It was found that the self-assembled monolayer of CMV-SH did
not insulate the gold surface towards the redox couple present in
the solution to any large extent. This was expected since only 20 %
of the gold surface was covered in this case. Additional insulation
with 1-dodecanethiol gave some improvement, but still the
oxidationyreduction currents could be seen in the voltammogram.
The insulation was much better for the immunosensors investigated in the article of Berggren and Johansson [13]. The capacitance decreased from approximately 24 600 nFcmÿ2 for a bare
gold surface to 15 600 nFcmÿ2 for a surface with self-assembled
CMV-SH and further down to 7000 nFcmÿ2 with treatment in 1dodecanethiol. The corresponding decreases for the electrode with
the 8-base probe were 10 800 nFcmÿ2 and 6 500 nFcmÿ2 respectively.
C. Berggren et al.
Fig. 3. Detection principle for the capacitive DNA biosensor. When a
sample containing DNA with a complementary sequence to the oligonucleotide probes immobilized on the electrode surface this speci®c
DNA will hybridize on the surface and other DNAs will be eluted in the
¯ow. The hybridized DNA will thereby displace water and solvated ions
away from the electrode surface given rise to a change in capacitance.
3.2. Hybridization Studies
The speci®city of a DNA probe depends on its length, composition and binding conditions such as: pH, salt content, solvent and
temperature. The speci®city of the binding will increase with
increasing temperature and decreasing salt content. Oligonucleotide probes are chemically synthesized, with a length of up to 40
bases. To selectively detect a unique human DNA sequence it has
been reported that the DNA probe must have at least 16 bases [24].
As the hybridization speed is dependent on the probe length, with a
higher speed for shorter probes, an overlapping array of short (6 ±
20 bases) oligonucleotide strands on a silicon chip can be used for
evaluation of the sequence of a DNA sample [25].
When a speci®c DNA sample hybridize to oligonucleotides
immobilized on the surface of an electrode, water and electrolyte
molecules will be displaced by the DNA sample thereby giving a
change in capacitance. The detection principle is shown in Figure
3. Denatured CMV samples, in the concentration range 10ÿ1 ±103
moleculesymL, complementary to the single stranded 26-base
oligonucleotide self-assembled on the electrode surface gave rise
to a decrease in capacitance when injected into the ¯ow system, see
Fig. 2. Cyclic voltammogramms for a) a bare gold electrode, b) a gold
electrode modi®ed with self-assembled CMV-SH and c) the same as b)
but with additional 1-dodecanethiol treatment. All scans were performed
in aqueous 5 mM K3Fe(CN)6 solution, with a scan rate of 10 mVys.
Electroanalysis 1999, 11, No. 3
Figure 4. The capacitance value was evaluated from the accumulated changes 10 min after injection of sample. A typical response
signal for a CMV sample at the lowest concentration used in this
study, 0.2 attomolar, is shown in Figure 5. As controls doublestranded CMV, denatured HBV and denatured tyrosinase were
used. These controls all gave lower responses at the corresponding
concentration than the one achieved for denatured single-stranded
CMV.
Another type of immobilization was studied by coupling an
oligonucleotide probe, consisting of 8 bases, to a self-assembled
monolayer of cysteamine on gold. Also this probe was complementary to a sequence of the cytomegalo virus (CMV). Capacitance changes were observed when a 179 long single-stranded
DNA-fragment of CMV was injected and hybridized on the
electrode surface, see Figure 6. Controls either consisting of a
207-base single-stranded fragment from tyrosinase mRNA or of a
104-base fragment from single-stranded HBV gave very low
Fig. 4. Response curves for an electrode with self-assembled CMV-SH
(26 bases) for a) single-stranded CMV, b) HBV, c) double-stranded
CMV and d) Tyrosinase mRNA. The carrier was in all cases an aqueous
solution containing 0.9 gyL sodium-azide, 0.5 gyL Tween 20 at a ¯ow
rate of 0.5 mLymin.
Direct Detection of DNA Hybridization
159
4. Conclusions
Fig. 5. A typical response signal for an electrode with self-assembled
CMV-SH upon injection of a CMV sample at a concentration of
10ÿ1 molecules ymL. The carrier was an aqueous solution containing
0.9 gyL sodium-azide, 0.5 gyL Tween 20 at a ¯ow rate of 0.5 mLymin.
responses, which shows that the CMV-electrode has a good
selectivity. However, the use of only 8 bases for binding will
probably lead to a high non-speci®c signal when real samples are
used.
Surprisingly, the 8-base electrode seems to be the most
selective. The selectivity of the CMV-SH electrode could possibly
have been increased if the experiments had been made at a higher
temperature. The maximum temperature for the present cell
material was 45 C, however. A reason for the difference in
selectivity might be that the DNA folds over and binds to the
probe with a few bases in one part of the molecule and with a few
from another part. We do not know how many bases that have to be
paired in order to bind to the electrode under the used hybridization
conditions. Eight bases are clearly binding suf®ciently hard to pick
up a CMV fragment down to below attomolar concentrations. In
this study completely different DNAs were used as a model
system. The hybridization conditions have to be performed at
higher stringency in order to measure closely-related oligos
speci®cally.
Fig. 6. Response curves for an electrode modi®ed with 8-base oligonucleotide probes a) CMV and b) HBV and c) Tyrosinase mRNA. The
carrier was in all cases an aqueous solution containing 0.9 gyL sodiumazide and 0.5 gyL Tween 20 pumped at a ¯ow rate of 0.5 mLymin.
This article reports on a label free DNA biosensor, with
possibilities to detect speci®c DNA fragments with detection
limits down to below attomolar (10ÿ1 moleculesymL) levels.
Compared with other DNA biosensors the detection limits are
several orders of magnitude lower. The speci®city was not very
high however and measurements in real samples may therefore not
be suf®ciently selective. The reproducibility of the sensors was not
very high. The signal response can differ as much as 50 % and
some electrodes showed no response at all. It was not possible to
regenerate the electrodes without causing increases in the capacitance over that of the original electrode thus indicating damage to
the electrode. Every curve in Figures 3 and 4 was made with a new
electrode. The points within a curve represents the accumulated
capacitance change and the experiments have therefore to be run
from lower to higher concentrations. A new cell for disposable
electrodes made by sputtering gold on silicon is in preparation and
is expected to produce sensors with high yield and with good
reproduciblity thereby improving on the present results. Furthermore different immobilization methods should be studied in order
to improve on the insulating properties of the oligonucleotide
layer, as well as decreasing the nonspeci®c binding to the surface.
The speci®city can also be improved by using probes based on
peptide nucleic acids (PNA) instead of DNA probes [26].
5. Acknowledgements
This research was supported by NUTEK, Sweden and
Sangtec Medical AB, Sweden.
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