High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers
Nyholm L (2011) High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled
Extraction of DNA Oligomers. PLoS ONE 6(12): e29243. doi:10.1371/journal.pone.0029243
High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers
Aamir Razaq 0
Gustav Nystro m 0
Maria Strmme 0
Albert Mihranyan 0
Leif Nyholm 0
Nikolai Lebedev, US Naval Reseach Laboratory, United States of America
0 1 The A ngstro m Laboratory, Department of Engineering Sciences , Nanotechnology and Functional Materials , Uppsala, Sweden, 2 The A ngstro m Laboratory, Department of Materials Chemistry Uppsala , Sweden
Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg21, were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30-50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m2 g21) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)6, (dT)20, and (dT)40 DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules.
Funding: The work was financially supported by the Swedish Foundation for Strategic Research (SSF) (grant RMA08-0025), the Swedish Research Council (VR)
(grants 621-2008-3690 and 621-2009-4626) and the Higher Education Commission of Pakistan (HEC). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The application of electronically conductive polymers, e.g.
polyaniline, polythiophene and polypyrrole, in biosciences has been
developing rapidly during more than two decades, particularly in
the fields of controlled drug delivery, biomedical engineering and
diagnostics . One reason for the interest in these polymers
stems from the fact that they can extract and release ions upon their
oxidation and reduction. It is well-known  that the charge
compensation upon the oxidation of conducting polymers can be
taken care of either by anions entering the polymer or cations
leaving the polymer, or a combined movement of both anions and
cations, depending on the charge and size of the ions. In
electrochemically controlled solid-phase micro extraction ,
this effect is used to perform batch-wise extraction and release of
charged species by applying an electrical potential/current to a
conducting solid phase extraction material in contact with the
solution containing the charged species. In another approach,
conducting polymer coated particles have been used as an
electrochemically controlled stationary phase in a chromatographic
separation system . The latter technique, (i.e.
electrochemically modulated liquid chromatography EMLC), has, however, not
yet found widespread use most likely due to the relatively complex
experimental set-up and problems associated with the packing of
efficient columns (the stationary phase should be composed of
uniform (210 mm) conductive particles with a sufficiently large (e.g.
150200 m2 g21) surface area.
Compared to conventional solid phase micro extraction (SPME)
, in which a material with a fixed number of exchange sites is
employed, electrochemically controlled SPME offers higher
flexibility since the properties of the material and thus the number
of exchange sites can be externally controlled by electrochemically
controlling the charge of the material. The applicability of
electrochemically controlled SPME has, however, so far been
limited by the relative low capacities  of the available extraction
materials. For conducting polymer films this problem generally
stems from mass transport limitations appearing when attempting
to increase the capacity of the films by increasing the film thickness
[7,21,22]. It has thus been reported  that only the outermost
layer of the polymer film on a planar electrode surface was active
in the extraction of ions when using electrodes coated with
micrometer thick films of conducting polymers. It can
consequently be anticipated  that the ion exchange capacities of
conducting polymer coatings could be increased significantly if a
larger fraction of the polymer layer could be utilized. As has been
reported recently [21,23,24], this can be achieved with materials
obtained by coating thin layers of conducting polymers on high
surface area cellulose substrates. Such materials should therefore
be highly interesting for electrochemically controlled solid phase
extractions of e.g. charged biomolecules.
Interactions between conducting polymers and biomolecules,
such as dopamine , cochlear neurotrophines , the
antipsychotic drug risperidone , adenosine triposphate (ATP)
 and, in particular, DNA [4,2936] have been studied by many
groups. In the DNA case, strands have been immobilized within the
structure of the conductive polymer, e.g. polypyrrole (PPy), or on the
surface of the polymer either employing adsorption [33,3739] or
polymerization (chemically or electrochemically) in the presence of
the DNA [29,4042]. For in situ polymerization, conductive
polymer monomers substituted with DNA have also been studied
. The spontaneous adsorption of DNA on conducting polymers
such as PPy has been investigated by several groups
[30,31,33,35,3739] and it has been shown [30,32,33,35,3739]
that the adsorption of DNA on partially oxidized conducting
polymers involves a diffusion controlled replacement of the counter
anions on the polymer with DNA [30,32,37,38,44]. It was also
demonstrated that the amount of DNA adsorbed depends on the
positive charge on the polymer (i.e. the oxidation state of the
polymer) and that, at least, some of the adsorbed DNA can be
competitively released upon the addition of other anions. The release
rates were, however, found to be two orders of magnitude lower than
the adsorption rates . The latter finding is in good agreement
with results  showing that PPy films act as cation exchangers
when DNA molecules are immobilized within PPy, since the
detachment of the DNA during the reduction of oxidized PPy films is
slow enough to induce charge compensation by cation movement.
Electrochemically controlled extraction and release of DNA
has, to the best of our knowledge, not been studied previously. It is
therefore not known if the extractions can be made faster and
more efficiently when the oxidation state of a conducting polymer
(e.g. PPy) is controlled electrochemically compared to when
chemically prepared (and only partially oxidized) films are
employed. It is also not known if the extraction/release rates
and yields can be increased by using a highly porous solid phase
extraction material coated with a thin conformal layer of the
conducting polymer, as can be anticipated based on the mass
transport limitations previously found for mm thick polymer films
. Although not shown so far, the efficiency of the DNA release
step most likely depends on the thickness of the PPy film as it is
well-known that sufficiently thick PPy films doped with large
anions generally serve as cation exchangers [45,46].
In the present paper, we describe electrochemically controlled
extraction and release of DNA oligomers from aqueous solutions,
containing an excess of other ions, using a high surface area
composite material. The material, which is composed of PPy and
cellulose, was used in the form of conducting paper sheets with
exceptionally high ion exchange capacities, low internal
resistances, rapid ion exchange properties, and good stabilities
[21,23,24,47]. It is shown that DNA oligomers of varying length
(i.e. containing 6, 20 and 40 bases) can be extracted and that the
extracted amount is proportional to the oxidation charge used in
the extraction step. The possibility of releasing the previously
extracted DNA by reducing the polymer is also discussed based on
the conductivity of the composite and the length of the DNA
chains. The present composite material is shown to be a very
promising candidate for rapid batch-wise electrochemically
controlled extractions of DNA in which the composite is immersed
directly in the DNA containing solution.
Materials and Methods
2.1.1. Chemicals. The cellulose used in this study was extracted from Cladophora algae, as previously described .
Pyrrole, iron (III) chloride, hydrochloric acid, sodium chloride,
sodium tetraborate decahydrate (99.5%) and boric acid (99.5%)
were purchased from Sigma Aldrich, Germany, and were used
without further purification. The carbon fibers [C005715/1,
Grade XAS, number of filaments 6000, Filament diameter
0.007 mm] were obtained from Goodfellow, UK. Buffer tablets
(pH = 6.8), containing disodium hydrogen orthophosphate
dehydrate and potassium dihydrogen phosphate, were purchased
from Merck, Germany. The fluorophore-tagged oligonucleotides
(oligomers) of varying length (i.e. containing 6, 20 and 40 bases)
were synthesized by Biomers.net, Germany. The oligomers were
single-stranded sequences of thymine (T) with no internal
modifications and the (dT)6, (dT)20 and (dT)40 oligomers were
tagged at the 39-position with 6-FAM, TexRed and Cy3
2.1.2. PPy-cellulose composite. The PPy-cellulose
composite was prepared by oxidative chemical polymerization of
pyrrole monomers on cellulose nanofibers (2030 nm thick) in the
presence of iron (III) chloride at room temperature, as previously
described . The composite was featured with morphological and
electrical properties identical to those reported previously [23,24,47].
The composite thus had the appearance of a black flexible paper
sheet which could be cut into any shape by a pair of scissors. The
composite possessed an internal specific surface area of 80 m2/g (as
determined by BET analysis of nitrogen adsorption isotherms), an
electrical conductivity of ,1 S/cm, and a thickness of the PPy
coating on the individual cellulose fibers of ,50 nm as characterized
by transmission electron microscopy (TEM) [23,24,47].
Prior to the extractions of DNA oligomers from solutions with
varying DNA concentrations, the composite was rinsed with
0.5 M HCl to maintain a high doping level.
2.1.3. Preparation of buffer solutions. The standard
phosphate buffer solution (PBS; 6.3 mM, pH = 6.8) was prepared
according to the manufacturers instructions (Merck) by dissolving a
tablet containing disodium hydrogen orthophosphate dehydrate
(0.47 g) and potassium dihydrogen phosphate (0.47 g) in 1 L of
deionized water. The borax buffer (pH = 8) was obtained by mixing
0.77 g of boric acid and 4.76 g of sodium tetraborate decahydrate
in 250 mL of deionized water. The pH of the buffer was then
adjusted to pH 8 by drop-wise addition of 0.1 M HCl.
2.2.1. Electrochemically controlled extraction of DNA
oligomers. The electrochemically controlled extraction and
release measurements were performed in a standard
threeelectrode electrochemical cell utilizing an Autolab/GPES
instrument (ECO Chemie, The Netherlands) with the composite
as the working electrode, a coiled Pt wire (23 cm long and 0.5 mm
in diameter) as the counter electrode, and a Ag/AgCl electrode as
the reference electrode. In the galvanostatic experiments, the
electrochemical cell was fitted with a Teflon cap and a glass-tube
with a frit (Bioanalytical Systems, UK) to obtain separate anode
and cathode compartments. The latter was done to minimize the
changes in the pH within the working electrode compartment as a
result of the oxidation and reduction of water taking place on the
Pt counter electrode. A schematic diagram of the electrochemical
cell used can be found in the Figure S1.
The composite paper sheets (used as the working electrodes)
were cut into rectangular pieces with the approximate dimensions
1.160.560.1 cm3, corresponding to a weight of about 20 mg. The
composites were contacted and immersed in the electrolyte using a
Pt-wire, which was coiled around the composite to obtain a good
ohmic contact. Fresh composites were used for each measurement,
and separate cells were used for the batch-wise extraction and
release experiments, respectively.
184.108.40.206 Pre-treatment of PPy-cellulose composite. Prior to
the galvanostatic extractions, all composites were first reduced at
20.5 V for 100 s in 10 mL of 2.0 M NaCl.
220.127.116.11. Time dependent extraction of DNA. a) Extraction
of DNA oligomers in the absence of applied current: Prior to the
experiments, each composite was first soaked for 10 min in 10 mL
of the PBS buffer. The composite was then moved into 10 mL of
the extraction solution containing (dT)6 oligomers tagged with
6FAM fluorophore and was kept there for varying time intervals,
i.e. 600 to 4000 s. The stock solution of 100 mM of (dT)6 oligomers
tagged with 6-FAM used in these experiments was prepared
according to the manufacturers instructions (Biomers.net,
Germany). The extraction solution was prepared by diluting
100 mL of stock solution with 10 mL of PBS. The final
concentration of the oligomers in the solution was thus
b) Constant-current controlled extraction of DNA: Following
the pre-treatment (i.e. 100 s reduction at 20.5 V in 10 mL of
2.0 M NaCl), the composite was immersed in 10 mL of the
extraction solution containing (dT)6 oligomers tagged with 6-FAM
fluorophore. The composite was then oxidized by applying a
constant anodic current of 1.2 mA. The extraction (i.e. oxidation)
time was varied between 600 and 4000 s. The extraction solution
was prepared as described above for the extractions in the absence
of an applied current.
18.104.22.168. Cumulative constant-time galvanostatic
extraction and release of DNA oligomers. In these
experiments, the composites were placed in 10 mL of the
extraction solution containing (dT)6 oligomers tagged with 6-FAM
(after the reductive pre-treatment) and oxidized by applying a
constant current of 1.2 mA for 800 s. Prior to the release (i.e. the
reduction of the PPy), the oxidized composite was rinsed with fresh
10 mL PBS solution and subsequently with deionized water to
remove non-specifically bound DNA oligomers, i.e. DNA oligomers
retained due to the extensive internal porosity of the composite (the
so-called sponge effect). The composite was then immersed in
10 mL borax buffer solution, which originally did not contain any
DNA, and the composite was reduced by applying a constant
current of 21.6 mA for 2000 s at a temperature of 70 uC. The
larger reduction charge (1.6 mA62000 s = 3200 mC) compared to
the oxidation charge (1.2 mA6800 s = 960 mC) was employed to
ensure that the reduction of the composite was as complete as
possible since it is well-known  that the reduction of a PPy film is
significantly slower than the corresponding oxidation.
Four consecutive measurements with fresh composites were
performed for both the extraction and release in the respective
solutions. As described above, all composites were thoroughly
rinsed with PBS buffer and subsequently with deionized water
when switching between the extraction and release cells.
22.214.171.124 Cumulative constant-time galvanostatic extraction
and release of DNA mixtures. The extraction solution was in
this case prepared by mixing 50 mL of 100 mM (dT)6 6-FAM,
(dT)20 Texas Red and (dT)40 Cy3 oligomers, respectively, in
10 mL of PBS. The molecular ratio of the oligomers (dT)6: (dT)20:
(dT)40 in the extraction solution was thus 1:1:1. The release
experiments were performed in 10 mL of borax buffer which
originally did not contain any DNA oligomers. The reduced
composites were immersed in the extraction solution as described
above and oxidized by applying a constant current of 1.2 mA for
800 s. The composite was then transferred to the release cell and
was reduced by applying a constant current of 21.6 mA for
2000 s at a temperature of 70 uC.
Four consecutive measurements with fresh composites were
performed for both the extraction and release in the respective
solutions. The composites were rinsed with fresh 10 mL PBS
between the extraction and release procedures.
2.2.2. Fluorescence measurements. The changes in the
DNA concentrations of the solutions following the electrochemical
extractions and release experiments were monitored using a
spectrofluorometer (TECAN Infinite M200, Austria). Black
Corning 96-well flat plates (Corning, Lowell, MA) were used,
and the gain-value was fixed at 100 in all measurements. Three
100 mL solution samples were taken for each analysis. For the
determination of the galvanostatic extraction and release yield in
the (dT)6 tagged 6-FAM oligomer experiments, as well as of the
extraction experiments involving the mixtures of the 6-FAM,
Texas Red and Cy3 tagged oligomers, an excitation wavelength of
460 nm was used while the emission spectrum was recorded
between 505 and 560 nm. For the release experiments involving
the 6-FAM, Texas Red and Cy3 fluorophore-tagged DNA,
different emission and excitation wavelengths were used
depending on the fluorophore. The excitation wavelengths 460,
573, and 523 nm and emission spectral ranges 505560, 600650
and 556586 nm were thus used for 6-FAM, Texas Red and Cy3
fluorophores, respectively. Calibration curves were constructed for
each specific fluorophore based on the respective gain-values,
excitation wavelengths, and emission spectra.
Results and Discussion
The highly porous conductive PPy-cellulose composite material
used in this study consists of black paper sheets with a large
internal specific surface area (see Figure S2), which can be directly
immersed in the DNA oligomer buffer solution for batch-wise
extractions. Numerous studies in the past have shown that DNA
molecules, which are negatively charged polyelectrolytes, adsorb
on the surface of (partially oxidized) PPy particles [3033,35,37
39]. In particular, it has been shown that DNA oligomers can
penetrate inside PPy films thanks to the presence of an extensive
channel network . However, as the adsorption times used in
these studies generally were of the order of hours, it is, interesting
to study if electrochemically controlled extraction of DNA can
provide similar extraction yields in significantly shorter times using
the present highly porous composite material. It is likewise very
interesting to study if the previously found  low release rate of
the extracted DNA can be increased by actively driving the
reduction of the PPy with an applied cathodic current and the use
of 3050 nm thin PPy coatings.
Figure 1 shows the results of a galvanostatic experiment,
performed to extract the (dT)6 oligomers tagged with 6-FAM
fluorophore in which a constant current of 1.2 mA was applied to
oxidize the PPy-cellulose composite for varying time intervals
ranging from 600 to 4000 s. During the oxidation, the PPy chains
became positively charged, and anions entered into the film to
maintain charge neutrality within the film. As a result of the
oxidation, the potential of the PPy composite increased with time
as is seen in Figure 1a. In accordance with previous results
[47,49,50], the potential increased almost linearly with time
during the first 1800 s after which a potential plateau of around
+0.7 V developed. The latter plateau can be explained by the
onset of PPy overoxidation which has been reported  to take
place at around +0.65 V vs. Ag/AgCl. For longer oxidation times
than 1800 s, a significant decrease in the pH of the solution was
found which most likely can be explained by the protons liberated
during the overoxidation reaction. No such change in the pH was
observed for shorter oxidation times. In the subsequent
experi1800 s). The results were normalized with respect to the weight of the
composite and the error bars represent the standard deviation (n = 3).
ments, care was therefore taken to ensure that the potential always
was significantly below +0.7 V. In the light of these results, it can
thus be assumed that the charge consumed during the 1800 s
experiment (viz. 2.16 C) corresponded to the practically available
ion extraction capacity of the PPy film. Based on a composite weight
of 20 mg, this charge corresponds to an overall ion exchange
capacity (for a singly charged anion) of about 1.1 mmol g21. As the
latter is about 370 and 30 times larger than the capacities reported
by Liljegren et al.  and Deinhammer et al. , respectively, it is
clear that the present material has a much higher capacity than the
materials previously employed in conjunction with
electrochemically controlled solid phase extraction.
During the oxidation of PPy, any negatively charged species can
potentially enter as counter ions within the PPy film. Since the
experiments were carried out in the presence of an excess of buffer
ions, it is reasonable to assume that mainly the buffer anions were
involved in the charge compensation step and that the DNA
subsequently replaced some of the buffer ions in an ion exchange
process. The apparent DNA exchange capacity will therefore
depend on the concentrations and type of all other competing ions
present in the sample solution. To determine the extracted DNA
amount, the decrease in the fluorescence intensity of the DNA
containing extraction solution was measured after each extraction
step. Figure 1b shows the amount of extracted DNA (evaluated
from the fluorescence intensity decrease) as a function of time in
the absence and presence of an imposed oxidation current of
1.2 mA. In accordance with previous results [30,32,33,35,3739],
(dT)6 was extracted also in the absence of a galvanostatic oxidation
of the PPy composite. The latter can be explained by two effects,
viz. (i) the retention of liquid containing (dT)6 in the voids between
composite nanofibers of the high surface area conductive paper
materials (see below) and (ii) the fact that the PPy composite
already was partially oxidized as a result of the manufacturing
process and therefore should have a certain anion exchange
capacity. More importantly, the results in Figure 1b clearly show
that the ion exchange capacity of the PPy composite can be
increased significantly by oxidizing the PPy using a constant
current. With an oxidation time of 1800 s (i.e. an oxidation charge
of 2.16 C), the amount of extracted DNA was thus about 3.5 times
larger than in the absence of the oxidation current (the horizontal
line, at around 8.4 nmol g21, in Figure 1b represents the average
uptake level of (dT)6 in the absence of an applied constant current).
Another problem with the previously employed [30,32,33,35,37
39] zero oxidation current extraction approach was that the
uncertainties in the extracted amounts of DNA were relatively
high. This can, at least partially, be ascribed to variations in the
oxidation state of the different PPy composites used in the
For galvanostatic electrochemical extraction of (dT)6 at varying
time intervals, it is seen in Figure 1b that the amount of extracted
DNA oligomers increased during the initial 1800 s, whereas a
leveling off effect was observed for longer time intervals. The
uncertainty in the number of extracted DNA oligomers for the
2400, 3000, and 4000 s time intervals was rather high due to the
interference from the previously mentioned overoxidation of the
PPy film and the associated pH changes occurring after about
1800 s. These results support our previous conclusion that
extraction times longer than 1800 s (viz. charges higher than
2.16 C) are of little analytical value. As is evident from Figure 1c,
the extracted amount of DNA oligomers increased practically
linearly with the applied oxidation charge for extraction times up
to 1800 s. This indicates that the amount of the extracted DNA
oligomers can be controlled by tuning the extraction time, but
more importantly that the amount of DNA extracted is
proportional to the oxidation time and hence to the positive
charge of the PPy composite.
Based on solid phase microextraction theory , it is expected
that the amount of extracted (dT)6 should be proportional to its
concentration in the solution. Such a linear relationship was
indeed found based on experiments with four different
concentrations of (dT)6 as is seen in Figure 2. In addition, no significant
difference in the extracted amount was found when carrying out
the experiments with a 1 mM solution in a buffer with a
concentration two times lower than that normally used. The
latter is in good agreement with previous results for zero oxidation
current adsorption of DNA on PPy [30,32,33,35,3739]. The fact
that no significant difference in the extracted amount was seen in
the absence and presence of a 800 s long zero-current period after
having oxidized the PPy composite for 800 s using a current of
1.2 mA also indicates that the ion exchange reaction between the
DNA and the buffer anions (assumed to be present within the PPy
polymer as charge compensating anions) was relatively fast. It
should be pointed out that, adsorption times of several hours were
generally employed in previous DNA adsorption studies
[30,32,33,35,3739]. We attribute the relative rapid adsorption
behavior to the porous structure of the composite and the thin
layers of PPy on the high-surface area cellulose matrix. We have
previously presented energy filtered TEM data  indicating that
DNA can access the entire volume of the PPy coatings during the
extraction. The present results thus indicate that the amounts of
(dT)6 within the PPy film after the extraction were determined
merely by the concentration of (dT)6 in the solution and the
oxidation state of the PPy composite.
While Figure 1 and 2 clearly demonstrate the advantages of
using the present high-surface area PPy-cellulose composites for
direct batch-wise electrochemically controlled solid phase
extractions of DNA in the presence of an excess of buffer ions, the
success of this approach in biotechnological applications clearly
also depends on the possibility of subsequently releasing the
extracted DNA by reducing the PPy composite. The latter is (for
obvious reasons) not readily studied using the zero current
approach since the PPy coating always will remain partially
oxidized (i.e. positively charged) in solutions containing oxygen. It
has, nevertheless, been found  that DNA (adsorbed on
partially oxidized PPy coatings) could be released into solutions
containing a competing anion at a rate approximately two orders
of magnitude lower than that for the adsorption process. Since a
reduction of the PPy film removes the positive charge from the
polymer, it can be expected that a reduction step should facilitate
the DNA release. This is, unfortunately, not necessarily true as it is
well-known that sufficiently thick PPy films doped with large
anions generally serve as cation exchangers [21,23,24,45,46], (i.e.
that the charge compensation involves ingress of cations rather
than release of anions during the reduction of the PPy film). One
approach to minimize the latter problem thus involves the use of a
very thin PPy coating such as that of the present PPy composite.
Figure 3 summarizes the results of batch-wise electrochemical
oxidation and reduction experiments carried out with the
PPycellulose composites in a solution containing (dT)6 oligomers. To
ensure an as complete reduction as possible, a larger reduction
charge, compared to the oxidation charge, was used in these
experiments. As can be seen in Figure S3 in the Supporting
Information, this approach gave rise to hydrogen evolution when the
current no longer could be entirely supported by the reduction of
the composite. As no attempts to remove oxygen from the release
solution were made, oxygen was likewise reduced during the
release step. Figure 3a shows that there was a continuous decrease
in the fluorescence intensity of the fluorophore tagged (dT)6
oligomers in the extraction solution after each oxidation step (the
galvanostatic oxidation and reduction curves for four consecutive
cycles can be found in the Figure S3). Analogously, Figure 3b shows
that the fluorescence intensity of the released fluorophore tagged
(dT)6 oligomers increased steadily from a value close to zero
following the electrochemical reductions of the PPy-cellulose
composite. The cumulative extracted and released amounts of
(dT)6 (evaluated from the changes in the fluorescence intensity of
the extraction and release solutions) as a function of the number of
extraction and release cycles for up to four cycles is shown in
Figure 3c. Note that the amount of (dT)6 oligomers
nonspecifically bound to the composite surface and removed in the
rinsing step, has been subtracted from the uptake values presented
in Figure 3c. This subtracted amount was estimated to be on
average 1.2060.22 nmol g21 (n = 3) under the employed
conditions. Note also that the slopes of the calibration curves
(see Figure S4) for the extraction (from the high oligomer
concentration PBS solution) and release (into the low oligomer
concentration borax solution) differed significantly, and that the
sensitivity was markedly higher in the extraction solution.
Based on the results in Figure 3c, the efficiency of the extraction
and release of (dT)6 can clearly be derived for the employed
experimental conditions. In Figure 3c, it is seen that the extracted
amount after one extraction was ,14 nmol/g while the
cumulative amount of extracted oligomers after four consecutive
oxidation cycles was ,44 nmol/g. The latter value, which
corresponds to ,9% of the total amount of (dT)6 present in the
10 mL extraction solution, is significantly higher than the
previously reported values obtained with electrochemically
controlled solid phase extraction [68,11,52,53]. It should,
however, be pointed out that the extraction yield mainly depends
on the volume of the (dT)6 solution employed in the extraction and
that it would be possible to increase the present extraction
efficiency significantly by using a different experimental set-up, as
was shown by Liljegren and Nyholm . As can be expected
since a new composite was used for each extraction, the
cumulative extracted amount increased linearly with the number
As is also seen in Figure 3c, the electrochemically released
amount of (dT)6 was about 4 nmol/g after the first reduction cycle
and approximately 7 nmol/g (cumulative) after the fourth
reduction cycle. These values correspond to an approximate
release efficiency of 30% for the first cycle and 16% after four
cumulative cycles. The 30% release efficiency for the first cycle is
very encouraging as it indicates that a significant release of
extracted (dT)6 indeed is possible even for release times as short as
2000 s. In the absence of a reduction of the PPy film, it was
previously reported  that the release rate was approximately
two orders of magnitude lower than the extraction rate. The more
efficient release in the present case can most likely be ascribed to
the reduction of the PPy and the thin PPy layers, both of which
should facilitate the release of the extracted DNA. The fact that
the release efficiency decreased with increasing number of release
cycles indicates that the release of the (dT)6 oligomer was
controlled by the diffusion of the DNA oligomer into the release
solution. The efficiency of such a process will clearly decrease
when the concentration in the release solution is increasing. This
suggests that the release efficiency ultimately depends on the
distribution coefficient for the oligomer with respect to the PPy
film and the release solution.
During the reduction of the PPy film, it is reasonable to assume
that the charge compensation takes place mainly via insertion of
cations and that the released DNA oligomers subsequently diffuse
out from the (poorly conducting) reduced PPy film. It is
wellknown  that the PPy film undergoes contraction during the
reduction step (and expansion during the oxidation step) which
most likely also affects the release of the DNA oligomers. Although
release experiments were also performed using a constant potential
(rather than a constant current) or with other applied constant
currents, the best release yield was observed when employing a
constant current of 21.6 mA for 2000 s at 70 uC (i.e. with the
conditions used in Figure 3). The higher efficiency at the latter
temperature compared to at room temperature supports the
hypothesis that the release rate of the DNA oligomers was limited
by diffusion. It is possible that the release efficiency could be
further increased if a significantly larger cation than Na+ was
employed in the release solution (it has previously been found that
the pH and type of cations in the release solution affect the release
of anions from PPy films ). This concept was, however,
difficult to implement in the present work due to the need for a
release solution containing a salt with a sufficiently high solubility,
buffer capacity, and conductivity. It should, however, be stressed
that the need for a constant pH in the release experiments
stemmed merely from the fact that the fluorescence intensities of
the chosen fluorophores were pH dependent (see Figure S5).
Since the conductivity of a PPy film decreases significantly upon
the reduction of the film  it may be anticipated that the DNA
release efficiency could be increased by increasing the conductivity
of the reduced PPy films as this would facilitate a more complete
reduction of the PPy film. To test this hypothesis, some experiments
were carried out with PPy composites to which chopped carbon
fibers (with a diameter of 8 mm) had been included as an additive
during the molding of composite paper sheets. With such carbon
fiber containing composites, a release yield of 40% was obtained,
indicating that the conductivity of the reduced PPy composite
indeed is one of the factors controlling the release efficiency.
Even though a complete release of the extracted DNA is yet to
be demonstrated, the results in Figure 3 clearly show that it is
possible to release a significant fraction of the extracted DNA in a
relatively short time using galvanostatic reduction of the PPy film.
This finding is certainly very encouraging, but additional
experiments with larger DNA oligomers are clearly needed to be
able to evaluate the general applicability of the method. Figure 4a
consequently displays typical fluorescence emission spectra
obtained during galvanostatic extraction of DNA oligomers from
a solution containing equal concentrations of (dT)6, (dT)20 and
(dT)40 tagged with 6-FAM, Texas Red and Cy3 fluorophore,
respectively, after four consecutive extraction cycles. In analogy
with the (dT)6 results in Figure 3, it is seen that the fluorescence
intensities decreased for all three DNA oligomers after each
extraction cycle. Figure 4b shows the cumulative number of DNA
oligomers extracted from the solution after two and four cycles,
respectively. Although it is clearly seen that all three oligomers
were extracted during the oxidation of the PPy film, it is difficult to
draw any definitive conclusions regarding any preferential
extraction of any of the oligomers due to the uncertainties in the
data. The apparent preference for an extraction of the (dT)20
oligomer with respect to, at least, the (dT)6 oligomer is thus not
statistically significant. Significantly different results were, on the
other hand, obtained for the oligomers during the subsequent
release experiments. In Figure 4c it is clearly seen that only the
(dT)6 fraction could be released to any substantial extent (the
released amounts of the (dT)20 and (dT)40 oligomers were thus
found to be negligible even after four consecutive release cycles).
The fact that the (dT)20 and (dT)40 oligomers could be extracted at
least as well as the (dT)6 oligomers can be explained by the ion
exchange process in which the oligomers replace some of the
buffer anions as counter ions in the oxidized PPy film. The finding
that only the (dT)6 oligomer could be released during the
subsequent reduction of the oxidized PPy film suggests that the
specific interactions between the oligomers and the PPy composite
 increase with increasing oligomer size. To be able to release
the larger oligomers (and to increase the release efficiency in
general) it is thus necessary to overcome these interactions. The
latter could possibly be accomplished based on a release approach
in which the negatively charged oligomers are driven out of the
PPy composite using an external electric field. Such experiments
are currently being conducted in our laboratory and the results of
these studies will consequently be discussed in a separate
communication. The present results hence indicate that it is
possible to employ the present PPy-cellulose composite to separate
small DNA oligomers from larger ones.
It has been demonstrated that porous conducting
PPynanocellulose composites can be used as high-capacity
electrochemically controlled solid phase materials for rapid batch-wise
extraction and release of (dT)6, (dT)20, and (dT)40 DNA oligomers,
respectively. This study constitutes an attempt to design efficient
and reversible electrochemically controlled ion exchange
membranes suitable for inexpensive batch-wise solid phase extractions
of biomacromolecules. It was shown that all three oligomers could
be straightforwardly extracted from a solution containing an
excess of buffer ions and that a release efficiency of 40% could be
obtained for the smallest oligomer. The release efficiency, which to
some extent depends on the conductivity of the PPy composite, is
most likely controlled by the distribution coefficient for the
oligomers with respect to the PPy composite and the release
solution. The combination of the use of electrochemically
controlled extraction and release experiments and the present
porous PPy composite enabled higher and more well-defined ion
exchange capacities and faster extractions to be obtained when
compared to previous PPy based solid phase extraction
approaches. These results indicate that the present combination of high
surface area and thin PPy films, yielding both high capacity and
rapid access to the PPy layers, could be very promising for the
development of inexpensive and efficient electrochemically
controlled ion-exchange membranes for batch-wise extraction of
a range of different biomolecules.
Figure S1 Schematic diagram of the electrochemical
cell used for galvanostatic extraction of DNA oligomers.
Figure S3 Four consecutive galvanostatic oxidation
(open symbols) and reduction (filled symbols) steps
performed with PPy-cellulose composite samples. The
oxidation (extraction step), involved a current of 1.2 mA applied
for 800 s in 10 mL PBS buffer containing 1 m M of (dT)6
oligomers tagged with 6-FAM fluorophore. The reduction (release)
Figure S4 Calibration curves used for the extraction
(PBS, pH = 6.8, upper figure) and release (borax,
pH = 8.0, lower figure) experiments. The excitation
wavelength of 460 nm was used at gain of 100 and emission spectrum
was measured between 505 and 560 nm for different
concentrations of (dT)6 tagged 6-FAM oligomers.
Conceived and designed the experiments: AM LN MS. Performed the
experiments: AR GN. Analyzed the data: AM LN MS AR GN. Wrote the
paper: AM LN MS AR GN.
1. Geetha S , Rao CRK , Vijayan M , Trivedi DC ( 2006 ) Biosensing and drug delivery by polypyrrole Anal Chim Acta 568 : 119 - 125 .
2. Guimard NK , Gomez N , Schmidt CE ( 2007 ) Conducting polymers in biomedical engineering Prog . Polym. Sci 32 : 876 - 921 .
3. Lange U , Roznyatouskaya NV , Mirsky VM ( 2008 ) Conducting polymers in chemical sensors and arrays Anal Chim Acta 614 : 1 - 26 .
4. Wallace GG , Kane-Maguire LAP ( 2002 ) Manipulating and monitoring biomolecular interactions with conducting electroactive polymers Adv . Mater 14 : 953 - 960 .
5. Inzelt G ( 2008 ) Conducting Polymers: A New Era in Electrochemistry . New York: Springer.
6. Gbatu TP , Ceylan O , Sutton KL , Rubinson JF , Galal A , et al. ( 1999 ) Electrochemical control of solid phase micro-extraction using unique conducting polymer coated fibers Anal . Comm 36 : 203 - 205 .
7. Liljegren G , Pettersson J , Markides KE , Nyholm L ( 2002 ) Electrochemical solidphase microextraction of anions and cations using polypyrrole coatings and an integrated three-electrode device . Analyst 127 : 591 - 597 .
8. Wu JC , Mullett WM , Pawliszyn J ( 2002 ) Electrochemically controlled solidphase microextraction based on conductive polypyrrole films Anal . Chem 74 : 4855 - 4859 .
9. Ahin Y , Ercan B , Sahin M ( 2008 ) Solid-phase microextraction and ion chromatographic analysis of anions based on polypyrrole electrode J . Appl Polym. Sci 108 : 3298 - 3304 .
10. Ugur T , Yucel S , Nusret E , Yasemin U , Kadir P , et al. ( 2004 ) Preparation of sulfonated overoxidized polypyrrole film applicable as an SPME tool for cationic analytes J . Electroanal Chem 570 : 6 - 12 .
11. Tamer U , Ertas N , Udum YA , Sahin Y , Pekmez K , et al. ( 2005 ) Electrochemically controlled solid-phase microextraction (EC-SPME) based on overoxidized sulfonated polypyrrole . Talanta 67 : 245 - 251 .
12. Kaykhaii M , Dicinoski GW , Haddad PR ( 2010 ) Solid-Phase Microextraction for the Determination of Inorganic Ions: Applications and Possibilities Anal . Lett 43 : 1546 - 1555 .
13. Deinhammer RS , Shimazu K , Porter MD ( 1991 ) Ion Chromatographic Separations Using Step and Linear Voltage Wave-Forms at a ChargeControllable Polymeric Stationary Phase Anal . Chem 63 : 1889 - 1894 .
14. Nagaoka T , Fujimoto M , Nakao H , Kakuno K , Yano J , et al. ( 1993 ) Electrochemical Separation of Ionic Compounds Using an Electroconductive Stationary Phase Coated with Crown-Ether or Polyaniline Layer J. Electroanal. Chem 350 : 337 - 344 .
15. Ge HL , Wallace GG ( 1990 ) Electrochemically Controlled Liquid-Chromatography on Conducting Polymer Stationary Phases J. Liq. Chromatogr 13 : 3245 - 3260 .
16. Chriswanto H , Ge H , Wallace GG ( 1993 ) Polypyrrole-Coated Silica as a New Stationary-Phase for Liquid-Chromatography . Chromatographia 37 : 423 - 428 .
17. Ponton LM , Porter MD ( 2004 ) High-speed electrochemically modulated liquid chromatography Anal . Chem 76 : 5823 - 5828 .
18. Harnisch JA , Porter MD ( 2001 ) Electrochemically modulated liquid chromatography: an electrochemical strategy for manipulating chromatographic retention . Analyst 126 : 1841 - 1849 .
19. Ge HL , Teasdale PR , Wallace GG ( 1991 ) J. Chromatogr. 544 : 305 - 316 .
20. Pawliszyn J ( 1997 ) Solid Phase Microextraction Theory and Practice . Chichester: Wiley-VCH.
21. Mihranyan A , Nyholm L , Garcia Bennett AE , Strmme M ( 2008 ) Novel high specific surface area conducting paper material composed of polypyrrole and Cladophora cellulose J. Phys. Chem B 112 : 12249 - 12255 .
22. Deinhammer RS , Porter MD , Shimazu K ( 1995 ) Retention Characteristics of Polypyrrole as a Stationary-Phase for the Electrochemically Modulated LiquidChromatographic (EMLC) Separations of Dansyl Amino- Acids J. Electroanal. Chem 387 : 35 - 46 .
23. Razaq A , Mihranyan A , Welch K , Nyholm L , Strmme M ( 2009 ) Influence of the Type of Oxidant on Anion Exchange Properties of Fibrous Cladophora Cellulose/Polypyrrole Composites J. Phys. Chem B 113 : 426 - 433 .
24. Gelin K , Mihranyan A , Razaq A , Nyholm L , Strmme M ( 2009 ) Potential controlled anion absorption in a novel high surface area composite of Cladophora cellulose and polypyrrole Electrochim Acta 54 : 3394 - 3401 .
25. Miller LL , Zhou QX ( 1986 ) Binding and Release of Fe(Cn)64- and Cs+ from Polypyrrole Abstr . Pap. Am. Chem. Soc 192 : 33 - Coll .
26. Richardson RT , Wise AK , Thompson BC , Flynn BO , Atkinson PJ , et al. ( 2009 ) Polypyrrole-coated electrodes for the delivery of charge and neurotrophins to cochlear neurons . Biomaterials 30 : 2614 - 2624 .
27. Svirskis D , Wright BE , Travas-Sejdic J , Rodgers A , Garg S ( 2010 ) Development of a Controlled Release System for Risperidone Using Polypyrrole: Mechanistic Studies. Electroanalysis 22 : 439 - 444 .
28. Boyle A , Genies E , Fouletier M ( 1990 ) Electrochemical-Behavior of Polypyrrole Films Doped with Atp Anions J. Electroanal. Chem 279 : 179 - 186 .
29. Tam PD , Trung T , Tuan Ma , Chien ND ( 2009 ) Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection . J. Phys. Conference series 187: 012042.
30. Pande R , Ruben GC , Lim JO , Tripathy S , Marx KA ( 1998 ) DNA bound to polypyrrole films: high-resolution imaging, DNA binding kinetics and internal migration . Biomaterials 19 : 1657 - 1667 .
31. Saoudi B , Despas C , Chehimi MM , Jammul N , Delamar M , et al. ( 2000 ) Study of DNA adsorption on polypyrrole: interest of dielectric monitoring . Sens. Actuators B 62 : 35 - 42 .
32. Saoudi B , Jammul N , Abel ML , Chehimi MM , Dodin G ( 1997 ) DNA adsorption onto conducting polypyrrole Synth . Met 87 : 97 - 103 .
33. Saoudi B , Jammul N , Chehimi MM , McCarthy GP , Armes SP ( 1997 ) Adsorption of DNA onto polypyrrole-silica nanocomposites J . Colloid Interface Sci 192 : 269 - 273 .
34. Park DH , Oh JM , Shul YG , Choy JH ( 2008 ) Nanohybrid for Efficient DNA Retrieval J . Nanosci. Nanotechnol, Fe3O4@Polypyrrole Core-Shell 8 : 5014 - 5017 .
35. Gambhir A , Gerard M , Jain SK , Malhotra BD ( 2001 ) Characterization of DNA immobilized on electrochemically prepared conducting polypyrrole-polyvinyl sulfonate films Appl . Biochem. Biotechn 96 : 303 - 309 .
36. Zanuy D , Aleman C ( 2008 ) DNA-conducting polymer complexes: A computational study of the hydrogen bond between building blocks . J. Phys. Chem B 112 : 3222 - 3230 .
37. Minehan DS , Marx KA , Tripathy SK ( 1994 ) Kinetics of DNA-Binding to Electrically Conducting Polypyrrole Films . Macromolecules 27 : 777 - 783 .
38. Minehan Ds , Marx KA , Tripathy SK ( 2001 ) DNA binding to electropolymerized polypyrrole: The dependence on film characteristics J. Macromol. Sci A 38 : 1245 - 1258 .
39. Pande R , Lim JO , Marx KA , Tripathy SK , Kaplan DL ( 1993 ) Comparison of Single and Double-Stranded DNA-Binding to Polypyrrole Biomol . Mat 292 : 135 - 140 .
40. Misoska V , Price WE , Ralph SF , Wallace GG , Ogata N ( 2001 ) Synthesis, characterisation and ion transport studies on polypyrrole/deoxyribonucleic acid conducting polymer membranes . Synth Met 123 : 279 - 286 .
41. Jiang M , Wang J ( 2001 ) Recognition and detection of oligonucleotides in the presence of chromosomal DNA based on entrapment within conductingpolymer networks J . Electroanal. Chem 500 : 584 - 589 .
42. Mandal SK , Dutta P ( 2004 ) Synthesis of DNA-polypyrrole nanocapsule . J. Nanosci. Nanotechn 4 : 972 - 975 .
43. Livache T , Fouque B , Roget A , Marchand J , Bidan G , et al. ( 1998 ) Polypyrrole DNA chip on a silicon device: Example of hepatitis C virus genotyping Anal . Biochem 255 : 188 - 194 .
44. Saoudi B , Jammul N , Chehimi MM , Jaubert A-S , Arkam C , et al. ( 2004 ) XPS study of the adsorption mechanisms of DNA onto polypyrrole particles . Spectroscopy 18 : 519 - 535 .
45. Inzelt G ( 1994 ) Mechanism of charge transport in polymer-modified electrodes . In Bard AJ, ed. Electroanalytical chemistry. New York : Marcel Dekker Volume 18 : 89 - 241 .
46. Heinze J ( 2001 ) ) Electrochemistry of conducting polymers . In Lund H, Hammerich O, eds. Organic electrochemistry. New York . pp 1309 - 1340 .
47. Nystrom G , Razaq A , Strmme M , Nyholm L , Mihranyan A ( 2009 ) Ultrafast All-Polymer Paper-Based Batteries Nano Lett 9 : 3635 - 3639 .
48. Mihranyan A , Llagostera AP , Karmhag R , Strmme M , Ek R ( 2004 ) Moisture sorption by cellulose powders of varying crystallinity Inter . J. Pharm 269 : 433 - 442 .
49. Olsson H , Nystrom G , Strmme M , Sjodin M , Nyholm L ( 2011 ) Cycling stability and self-protective properties of a paper-based polypyrrole energy storage device Electrochem . Commun 13 : 869 - 871 .
50. Li YF , Qian RY ( 2000 ) Electrochemical overoxidation of conducting polypyrrole nitrate film in aqueous solutions Electrochim . Acta 45 : 1727 - 1731 .
51. Rubino S , Razaq A , Nyholm L , Strmme M , Leifer K , et al. ( 2010 ) Spatial Mapping of Elemental Distributions in Polypyrrole-Cellulose Nanofibers using Energy-Filtered Transmission Electron Microscopy J. Phys. ChemB 114 : 13644 - 13649 .
52. Liljegren G , Forsgard N , Zettersten C , Pettersson J , Svedberg M , et al. ( 2005 ) On-line electrochemically controlled solid-phase extraction interfaced to electrospray and inductively coupled plasma mass spectrometry . Analyst 130 : 1358 - 1368 .
53. Yates BJ , Temsamani KR , Ceylan O , Oztemiz S , Gbatu TP , et al. ( 2002 ) Electrochemical control of solid phase micro-extraction: conducting polymer coated film material applicable for preconcentration/analysis of neutral species . Talanta 58 : 739 - 745 .
54. Liljegren G , Nyholm L ( 2003 ) Electrochemically controlled solid-phase microextraction and preconcentration using polypyrrole coated microarray electrodes in a flow system . Analyst 128 : 232 - 236 .
55. Chainet E , Billon M ( 1998 ) In situ study of polypyrrole morphology by STM: effect of the doping state J . Electroanal. Chem 451 : 273 - 277 .