Direct electrochemical sensor for fast reagent-free DNA detection

Biosensors and Bioelectronics 21 (2005) 182–189

Direct electrochemical sensor for fast reagent-free DNA detection Elena Komarova∗ , Matt Aldissi, Anastasia Bogomolova Fractal Systems Inc., 200 9th Avenue North, Ste. 100, Safety Harbor, FL 34695, USA Received 6 July 2004; accepted 21 July 2004 Available online 25 August 2004

Abstract A novel reagentless direct electrochemical DNA sensor has been developed using ultrathin films of the conducting polymer polypyrrole doped with an oligonucleotide probe. Our goal was to develop a prototype electrochemical DNA sensor for detection of a biowarfare pathogen, variola major virus. The sensor has been optimized for higher specificity and sensitivity. It was possible to detect 1.6 fmol of complementary oligonucleotide target in 0.1 ml in seconds by using chronoamperometry. The sensitivity of the developed sensor is comparable to indirect electrochemical DNA sensors, which use electrochemical labels and reagent-intensive amplification. The developed sensing electrode is reusable, highly stable and suitable for storage in solution or in dry state. © 2004 Elsevier B.V. All rights reserved. Keywords: DNA sensor; Polypyrrole; LB film; Chronoamperometry; Electrochemical; Variola major

1. Introduction One of the fastest growing areas in DNA/RNA analysis technology is the development of DNA biosensors. Typically, the biosensor employs immobilized oligonucleotides as the recognition element and measures specific binding processes of complementary DNA or RNA at the biosensor interface. Electrochemical methods of hybridization detection present a good alternative to well-developed fluorescent hybridization detection methods. Over the past decade, enormous progress has been made towards the development of electrochemical DNA biosensors. Considerable interest has been drawn to these devices due to their promise for obtaining sequence specific information in a faster, simpler and less expensive manner. Moreover, they have a high potential for automation and miniaturization since only basic electrochemical equipment is required. Indirect methods of electrochemical hybridization detection (methods that employ electrochemical labels and hybridization markers) generally have better detection limits, but take longer and require more steps, compared to direct ∗

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0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.07.025

methods. Detection of electrical current, generated by electroactive enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), was initially applied in electrochemical immunosensing. Later it was modified for DNA sensing, using electroactive enzyme as a label. Detection of electrical current, generated by HRP, was adapted to DNA sensing with a detection limit as low as 10 fM (Huang et al., 2002). Zhang et al. could detect 3000 copies of 38-bp DNA oligonucleotide in a 10 ␮l droplet (0.5 fM) with the HRP-labeled detector probe (Zhang et al., 2003). Using an AP-labeled detector probe, to generate the electroactive label p-aminophenol, it was possible to achieve a 20 fM detection limit after ferrocenyl enhancement (Kim et al., 2003). Differentiation of the signals of two DNA targets was possible in chronopotentiometric measurements with the use of two different enzymes, alkaline phosphatase and ␤-galactosidase, with a femtomole detection limit (Wang et al., 2002). Oligonucleotides modified with transition metal complexes (ruthenium, osmium, iron, rhodium and copper) as electrochemical labels can be used to detect DNA electrochemically with cyclic voltammetry. Popular use of ferrocene-modified oligonucleotides as detector probes resulted in femtomole detection levels (Wlasoff and King, 2002). Utilizing the stem–loop structure of a ferrocene-

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tagged capture probe, which undergoes conformational change upon hybridization and converts from loop to stem distancing the ferrocene tag from the electrode, it was possible to achieve a 5 fM detection limit (Fan et al., 2003). Using dipyridophenazine complex of osmium, it was possible to achieve a 0.1 pg/ml detection limit (Maruyama et al., 2002). Nanoparticles as electroactive labels are becoming increasingly popular. Thus, detector probes, labeled with gold nanoparticles lead to measurable conductivity changes with a target DNA concentration of 500 fM (Park et al., 2002). An elegant approach, using three different nanoparticles (zinc sulfide, cadmium sulfide, and lead sulfide), has been used to differentiate the signals of three DNA targets by stripping voltammetry of heavy metals at femtomolar amounts of DNA (Wang et al., 2003a,b). Wang et al. (Wang et al., 2003a,b) reported a new strategy for amplifying electrical DNA sensing based on the use of microsphere tags loaded internally with a redox marker. This allows chronopotentiometric detection of target DNA down to the 5.1 × 10−21 mol level in connection to 20 min of hybridization and “release” of the marker in an organic medium. Direct electrochemical detection of DNA hybridization allows omitting the step of pre- or post-hybridization labeling or post-hybridization incubation with hybridization markers. Several electrochemical properties of DNA can be utilized for direct DNA sensing. For example, DNA hybridization can be monitored through the oxidation signal of guanine in the target DNA when electroinactive inosine-substituted capture probes are utilized (Wang et al., 2001; Kara et al., 2003). The perturbation of the double helix ␲-stack, introduced by a mismatched nucleotide, can be detected by measuring the attenuation of the charge transfer when double-stranded DNA’s ability to transport charge along nucleotide stacking is taken into account (Marques et al., 2003). Direct registration of DNA hybridization advanced significantly with the use of electroactive conductive polymers as electrodes. Conductive polymers consist of conjugated backbones that are easily oxidized or reduced (doped/undoped) with a concomitant increase or decrease in conductivity, with each polymer having its own redox characteristics. Many conductive polymers, such as polypyrrole and polythiophene, have been modified with oligonucleotide pendant groups that serve as complementary DNA recognition units. Cha et al. achieved a 1 nM detection limit of target DNA by monitoring DNA hybridization (carried out for 3 h) by cyclic voltammetry when a polythiophene derivative, chemically modified with oligonucleotide probe, was exploited (Cha et al., 2003). It was possible to monitor DNA hybridization in real time through a capacitance measurement, with a detection limit of 0.5 nM and a rapid response time (s), upon addition of the complementary strand, using oligonucleotide modified silicon chip (Wei et al., 2003). DNA is electroactive and can serve as a dopant due to the negative charge of phosphates, and therefore can con-

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tribute to the polymer conductivity. Oligonucleotide probes can be incorporated into growing films of a conducting polymer (Wang et al., 1999; Lassalle et al., 2001), or the surface of the polymer electrode can be derivatized with covalent attachment of oligonucleotides (Lee and Shim, 2001; Cha et al., 2003; Pham et al., 2003). Hybridization with a complementary DNA target causes changes in conductivity of the electrode, which can be measured by amperometry (Wang et al., 1999), impedance spectroscopy (Lee and Shim, 2001), cyclic voltammetry (Cha et al., 2003), differential pulse voltammetry (Pham et al., 2003) or photocurrent spectroscopy (Lassalle et al., 2001). The detection limits of most direct DNA-sensing approaches are in micromolar range; however, the latest publications on direct DNA sensing report lower detection limits. In this work, the attention was focused on the development of a direct DNA sensor based on polypyrrole doped with oligonucleotide as the sole dopant. Our goal was to develop an electrochemical DNA sensor for detection of a biowarfare pathogen variola major, a DNA virus that causes smallpox. Different techniques of preparation of the sensing polypyrrole film were investigated to optimize sensitivity and specificity of the biosensor.

2. Materials and methods Pyrrole was purchased from Aldrich Chemical Co. and distilled before polymerization. Custom oligonucleotides, specific to variola major virus, were synthesized by Invitrogen Corp. (Fig. 1B). Oligonucleotides and deionized DNase/RNase-free water were purchased from Invitrogen Corp. Fragmented pBR322 plasmid, pre-digested by MspI was obtained from New England Biolabs. Fragmented pUC19 (New England Biolabs) was prepared by digestion with BbvI restriction endonuclease. To obtain single-stranded fragmented pBR322 and pUC19, pre-digested fragmented plasmids were freshly denatured at 90 ◦ C for 2 min and placed on ice. 2.1. Electropolymerization and chronoamperometry experiments Electropolymerization and chronoamperometry experiments were carried out in a custom-made electrochemical minicell (internal volume 100 ␮l) with indium tin oxide (ITO)-coated glass (Delta Technologies Ltd.) used as a working electrode and a platinum wire as an auxiliary electrode. A mini-reference electrode (Ag/AgCl/3 M KCl, Abtech Scientific) was used in all experiments (Fig. 2A). 2.2. Cyclic voltammetry (CV) electropolymerization Polymerization was performed using a CHI-660 potentiostat (CHI Instruments) with 10 voltammetric cycles between 0 and 0.7 V, using a solution of 0.05 M of pyrrole and 0.1–1 mg/ml of oligonucleotide (15-, 20- or 30-mer) dopant

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Fig. 1. (A) Doping of polypyrrole with DNA; (B) oligonucleotides used for doping pPy films (“dopant-15, -20, -30”) and for testing as complementary targets (“target-41, target-20”); (C) cyclic voltammograms of electropolymerization of pyrrole (0.05 M) on ITO glass in the presence of dopant-30 (100 ␮g/ml).

in DNase/RNase-free water. The resulting film diameter was 3 mm. 2.3. Constant potential (CP) electrochemical polymerization Constant potential (CP) electrochemical polymerization was performed at a constant potential of 0.7 V for 1 min, using solutions of 1 mg/ml of oligonucleotide dopant and 0.05 M of pyrrole in water. 2.4. Ultrathin film preparation For stearic acid Langmuir–Blodgett (LB) film deposition, the technique described elsewhere was used (Milella et al., 1997). The only change from the published procedure was that 1 mg/ml solution of stearic acid in chloroform was spread onto a subphase of deionized water that contained no additives (FeCl3 or HCl). After proper drying, the film was exposed to vapors of pyrrole for 48 h at 4 ◦ C in the dark.

The pyrrole-saturated LB film was electrochemically polymerized under a constant potential of 0.7 V for 1 min in the presence of 100 ␮l of dopant-20 (1 mg/ml) in deionized water. 2.5. Blocking of pPy/oligonucleotide sensing films Prepared pPy/oligonucleotide films were washed with deionized water and incubated with 1 mg/ml of calf thymus DNA in detection buffer (0.1 M NaCl/0.1 M glycine in water) for 1 h at 0.15 V at room temperature. After that, the film was washed in the detection buffer and used for chronoamperometric measurements. 2.6. Direct detection of oligonucleotide target by chronoamperometric technique Chronoamperometry was performed at a potential of 0.15 V using CHI-660 potentiostat in the detection buffer (0.1 M NaCl/0.1 M glycine solution in water) unless otherwise noted. pPy/oligonucleotide film on ITO-coated glass

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Fig. 2. (A) Electrochemical minicell used for preparation of DNA-doped conductive films (internal volume 100 ␮l, bottom opening 3 mm i.d.) as well as for target DNA chronoamperometric detection; (B) schematic of direct detection of DNA hybridization. Specific complex, which is formed between target single-stranded DNA in solution and complementary oligonucleotide dopant in the pPy film, is registered electrochemically; (C) schematic of indirect detection of DNA hybridization using horseradish peroxidase-labeled probe.

electrode was used as the working electrode. The system was allowed to stabilize for few seconds to achieve a flat baseline. The injection of the target DNA (5 ␮l) was performed and the change in current was recorded.

(iii) Adding HRP substrate: 10 ␮l of 10 mM H2 O2 in phosphate buffer saline solution (PBS) is injected into the electrochemical cell with the sensing film as a working electrode, with an applied potential of 0.12 V. The change in current was recorded.

2.7. Indirect detection of oligonucleotide target using enzyme-labeled probe 3. Results and discussion Polymerization and blocking was performed as described above. The films were prepared using dopant-15. (i) Hybridizing with target: the films were incubated with different concentrations of target-41 for 30 min in 10 mM HEPES, 1 M NaCl, 1 mM EDTA buffer and thoroughly washed. (ii) Hybridizing with horseradish peroxidase (HRP)-labeled probe: the films were incubated with HRP-labeled oligonucleotide (5 ␮g/ml) in a 20 mM phosphate buffer with 0.1 M NaCl for 40 min. The potential of 0.15 V is applied only half of that time (20 min). The films were rinsed with phosphate buffer.

3.1. Preparation and testing of DNA-doped pPy films In order to prepare conductive polypyrrole (pPy) films, pyrrole is polymerized in the presence of an anionic dopant, which contributes to film conductivity. A variety of anions can dope pPy, such as Cl− , NO3 − , along with larger or polymeric anions. During electropolymerization, pPy film grows on the working electrode. To prepare conductive DNA-pPy films, we used DNA as the sole dopant during

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electrochemical polymerization of pyrrole, as was first described by Wang (Wang et al., 1999). This allowed maximum possible incorporation of oligonucleotide in the conductive polymer throughout the film thickness and full contribution of oligonucleotide’s charged phosphates to the polymer’s conductivity (Fig. 1A). The incorporated oligonucleotides do not undergo any redox changes in the potential range used for electropolymerization. Oligonucleotide dopants, used to prepare pPy/oligonucleotide films, are shown in Fig. 1B. To prepare pPy/DNA films, we have used two electrochemical polymerization techniques: cyclic voltammetry (CV) electropolymerization and constant potential (CP) electropolymerization. Polymerization was carried out in deionized water, because ionic biological buffers, such as TE (10 mM Tris–HCl, 1 mM EDTA) and HEPES (HEPES–NaOH, 1 mM EDTA), were interfering with the incorporation of the oligonucleotide dopant in the pPy film. Typical CV electropolymerization, performed by continuous voltammetric scanning between 0 and 0.7 V, at the rate of 25 mV/s, is presented in Fig. 1C. The use of the oligonucleotide dopant shows a normal polymer growth with increasing current, which corresponds to incorporation of an oligonucleotide into the pPy film (Fig. 1C). The obtained pPy/oligonucleotide thin films had semitransparent, slightly colored appearance. The films were rinsed in the working buffer (0.1 M.NaCl/0.1 M glycine) and used for electrochemical detection of the target oligonucleotide. Direct electrochemical methods of DNA hybridization detection (Fig. 2B) are simpler and faster, compared to indirect methods, utilizing electrochemical labels (Fig. 2C). However, until now reported indirect methods were more sensitive and more specific. Our goal was to optimize a direct method of electrochemical detection, which would be comparable in sensitivity and specificity to indirect (enzyme-enhanced) detection.

Among direct electrochemical methods, chronoamperometry allowed us to register fast and reproducible signals immediately upon addition of the target oligonucleotide (Fig. 3A). Other techniques (chronopotentiometry, cyclic voltammetry, impedance, differential pulse voltammetry) did not reveal any considerable changes even after prolonged incubation (up to 1 h) of pPy/oligonucleotide film with the target oligonucleotide. The speed of chronoamperometric response is the major advantage of the technique. The steep increase in current happens immediately after the injection of the target oligonucleotide in a 0.1 M NaCl/0.1 M glycine buffer into the electrochemical cell, with the pPy/oligonucleotide film serving as a working electrode (Fig. 3A). The drawback of chronoamperometry is the appearance of the strong signal of the opposite polarity upon addition of non-complementary DNA (Fig. 3B). The nature of such electrochemical responses to the addition of complementary DNA target and non-complementary DNA is not fully understood and being currently investigated. The non-specific signal suppressed the specific signal from target DNA, when a mix of target oligonucleotide and 10× excess of non-specific DNA was used. It is worth mentioning, however, that no problems arose while detecting hybridization of 15 nucleotides (using pPy films with dopant-15) in a 41-oligonucleotide target. Further optimization of direct detection was targeted at eliminating the non-specific signal, along with increasing the sensitivity of direct detection. Indirect enzyme-mediated detection, using similar pPy/oligonucleotide films, was used for comparison. Briefly, as an indirect detection method, we have used the enzyme-enhanced “sandwich” approach: the target oligonucleotide (target-41, Fig. 1B) was hybridized to dopant-15 in pPy film, which was followed by hybridization of horseradish peroxidase (HRP)-labeled probe oligonucleotide (Fig. 2C). The presence of HRP, which works as a

Fig. 3. Direct chronoamperometric DNA sensing at 0.15 V. Response of pPy/dopant-20 film, prepared by constant potential electropolymerization, to injection of (A) specific oligonucleotide (50 ng of “target-41” in 0.1 M NaCl/0.1 M glycine); (B) non-specific DNA (500 ng of linearized, denatured pUC19 in 0.1 M NaCl/0.1 M glycine), sensing volume 0.1 ml.

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3.2.1. Optimization of pPy/oligonucleotide film preparation 3.2.1.1. Altering the length of the dopant in pPy film. Oligonucleotide dopants of different lengths (Fig. 1B) were used in pPy film preparation by CV electropolymerization. Films were tested for target detection in identical conditions. Films obtained with shorter dopants (dopant15 and dopant-20) produced stronger amperometric signals compared to films containing dopant-30 probably due to higher resulting molar concentration of the dopant and thus, the active hybridization sites in the sensing film (Table 1).

Fig. 4. Indirect HRP-mediated chronoamperometric hybridization detection: using pPy/dopant-15 film electrode and 5 ng in 100 ␮l test solution of target-41 oligonucleotide.

catalyst in H2 O2 electrochemical reduction, ensured a positive signal in chronoamperometry when H2 O2 is injected (Fig. 4). 3.2. Optimization of direct electrochemical DNA detection We have tested a number of approaches to optimize specificity and sensitivity of the pPy/oligonucleotide sensing films, which are discussed below. They can be separated into (1) optimization of film preparation parameters, (2) blocking the surface of the film, and (3) altering conditions of target detection. Results of optimization approaches on film sensitivity towards target oligonucleotide detection are summarized in Table 1.

3.2.1.2. Altering the concentration of the dopant in pPy film. We investigated the effect of dopant concentration on film sensitivity towards target detection, comparing films prepared by CV electropolymerization with 100, 500, and 1000 ␮g/ml of dopants, 0.05 M of pyrrole, 10 cycles from 0 to 0.7 V at a rate of 25 mV/s. The concentration of dopant dramatically influenced film sensitivity towards target DNA detection. Using higher dopant concentrations, amperometric hybridization signals were more pronounced, and smaller concentrations of the target oligonucleotide could be detected. 3.2.1.3. Electropolymerization technique. Conductive films polymerized at constant potential (CP) showed lower noise level in DNA-sensing chronoamperometry experiments (compared to films obtained using cyclic voltammetry), which allowed sensing of lower target DNA concentrations. Further increase of dopant concentration (up to 2 mg/ml) in film preparation using CP electropolymerization did not result in a significant sensitivity increase (Table 1).

Table 1 DNA detection by chronoamperometry using complementary target oligonucleotides Concentration of target oligonucleotide Target-41 (M)

Target-20 (M)

10−7

8× 4 × 10−7 2 × 10−7 4 × 10−8 2 × 10−8 4 × 10−9 8 × 10−10

mg/ml

10−2

1.6 × 10−9 4 × 10−10 8 × 10−11 1.6 × 10−11

1× 5 × 10−3 2.5 × 10−3 5 × 10−4 2.5 × 10−4 5 × 10−5 1 × 10−5 2.5 × 10−6 5 × 10−7 1 × 10−7

Detection limit (in 0.1 ml)

1 ␮g 500 ng 250 ng 50 ng 25 ng 5 ng 1 ng 250 pg 50 pg 10 pg

Chronoamperometry signal Indirect (HRP)

Direct

Dopant-15 CP

Dopant-30 CV

– 10 nA – – – 5 nA N/D – – –

10 nA 5 nA N/D N/D N/D – – – – –

Dopant-20/15 CV

CP

30 nA 14 nA 6 nA N/D N/D N/D N/D – – –

50 nA 40 nA 32 nA 20 nA 15 nA 9 nA 6 nA N/D N/D N/D

Dopant-20 ultrathin LB film, CP – – – – – – 12 nA 9 nA 4 nA 1 nA

N/D: not detected or not tested; CV: films prepared by CV polymerization (10 cycles: 0–0.7 V, 1000 ␮g/ml of dopant and 0.05 M pyrrole); CP: films prepared by constant potential polymerization: 0.7 V for 1 min, 1000 ␮g/ml of dopant and 0.05 M pyrrole); LB: ultrathin pPy/dopant-20 films, prepared by CP in preformed LB films. The magnitude of the chronoamperometry signal for each concentration is averaged from at least 25 experiments. The standard deviation is within ±5% of the peak.

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3.2.1.4. Using ultrathin pPy/DNA films. Ultrathin films of pPy/oligonucleotide were prepared by modifying a preformed Langmuir–Blodgett (LB) film of stearic acid by exposure to pyrrole vapor and then electrochemically polymerizing it in the presence of the DNA dopant. Polymerization of pyrrole within stearic acid LB films allowed us to significantly increase sensitivity towards target oligonucleotide detection (Table 1). 3.2.2. Blocking the surface of the film with irrelevant DNA To eliminate the non-specific signal, we performed “blocking” of the pPy/DNA film with 1 mg/ml of singlestranded (freshly denatured) fragmented calf thymus DNA for 1 h at room temperature with 0.15 V applied potential. Blocking resulted in complete disappearance of non-specific signal from oligo(dT13–18 ), and in reduction of non-specific signals from oligo(dA15 ) and from single-stranded fragmented pBR322 and pUC19 in the pPy/oligonucleotide films prepared by CV or CP electropolymerization. Additional incubation with 1 mg/ml of calf thymus DNA with an applied potential of 0.15 V did not result in the complete disappearance of non-complementary peaks of poly(dA15 ) and single-stranded fragmented pBR322. When blocking was performed on ultrathin LB films, it resulted in complete elimination of the non-specific signal from single-stranded fragmented pBR322 and pUC19 (Fig. 5A). We hypothesize that improved specificity (as compared to films, prepared by CV or CP electropolymerization) was related to the change in film morphology and hydrophobicity: ultrathin films were less polar and more hydrophobic. Thus, we could successfully detect specific DNA (10 pg, target-20) in the presence of 2.5 ng (250× weight excess) of non-specific ss-DNA (pUC19, pre-digested with BbvI) (Fig. 5B). All dopant oligonucleotides (dopant-15, -20, -30), “comp-41”, and the annealed duplex of “comp-41” with “target-41” gave no noticeable signal with the blocked films. Specific signal from complementary single-stranded “target-41” oligonucleotide remained unaffected by the blocking procedure. 3.2.3. Altering electrochemical conditions of target detection 3.2.3.1. Influence of hybridization potential on specificity of DNA recognition event. We investigated whether changing the potential applied on the pPy/oligonucleotide film electrode during hybridization/sensing will allow us to increase specificity of DNA recognition. Routinely, we were performing chronoamperometry at 0.15 V, with the potential applied prior to DNA injection. We hypothesized that applying negative potential will repel weaker-bound non-specific DNA. Both specific and non-specific signals remain unchanged in the range of 0.15–0.3 V. More negative potentials caused reduced intensity of non-specific signals and increased intensity of specific signal starting at −0.4V working potential. Lowering the potential to −0.7 V resulted in the disappearance

Fig. 5. Specificity of direct chronoamperometry using ultrathin films at the detection limit: (A) absence of amperometric response to 2.5 ng of ss-pUC19 in 0.1 M NaCl/0.1 M glycine solution, injected at 40 s; (B) amperometric response to 10 pg of target-20 (concentration 16 pM) in the presence of 2.5 ng of ss-pUC19 (both in 0.1 M NaCl/0.1 M glycine solution, 0.1 ml).

of the non-specific signal and further increase of the specific signal; however, the pPy/oligonucleotide film lost stability and conductivity due to undoping. 3.2.3.2. Altering ionic strength of hybridization buffer. Altering ionic strength of hybridization buffer in the range 0.1–0.5 M NaCl did not affect the height of specific or nonspecific peaks. 3.2.4. Combining optimization approaches Using blocked ultrathin pPy/oligonucleotide films, prepared by CP electropolymerization in preformed stearic acid LB film substrate, we were able to directly detect 10 pg or 1.6 fmol of the target oligonucleotide in 0.1 ml in the presence of 250× weight excess of non-complementary ss-DNA (Fig. 5B, Table 1). High sensitivity of the ultrathin pPy/oligonucleotide films results from considerable increase of signal-to-noise ratio compared to films obtained by CV or CP electrochemical polymerization. It was possible to reliably register 1 nA signal. The achieved detection limit of

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10 pg or 1.6 fmol of target oligonucleotide in 0.1 ml by direct electrochemical DNA sensing is orders of magnitude lower than reported for other direct DNA sensors. It is competitive with the indirect electrochemical DNA sensors reported lately, which use sandwich approaches, electrochemical labels and reagent-intensive amplification, and therefore employ longer detection times and are more difficult to make. 3.3. Stability of pPy/oligonucleotide films and reproducibility of oligonucleotide target detection pPy/oligonucleotide films, prepared by CV or CP electropolymerization, were repeatedly tested during the course of 2 weeks for amperometric hybridization detection and were showing stable performance, with no decrease in detection speed or sensitivity. Films were regenerated by washing in deionized water (as reported by Park et al., 2002), which resulted in dissociation of hybridized DNA target from the complementary oligonucleotide in the film. No elevated temperatures or elevated salt concentrations were needed for film regeneration. DNA-sensing ability remained unchanged upon storage in the working buffer, and films could be constantly reused. The possibility of storing the pPy/oligonucleotide sensing films in a dry state (a) at 4 ◦ C in the dark and (b) at room temperature with light for prolonged periods of time, up to 2 months has been investigated as well. Stored in both conditions, the sensing films did not lose their sensing abilities and were 100% functional towards DNA detection when placed in the working buffer.

4. Conclusions Compared to the published research on direct DNA sensing, we have achieved a significantly lower detection limit of 1.6 fmol in 0.1 ml by using direct chronoamperometric hybridization detection on ultrathin films of DNA-doped polypyrrole. This makes this direct method competitive with indirect electrochemical DNA sensors, which use electrochemical labels and reagent-intensive amplification. Compared to that, our sensor is reagentless and detects DNA in one step. In addition, we can detect DNA in seconds, which is unsurpassed by any other detection method of comparable sensitivity. The sensing electrode is reusable, highly stable, and suitable for storage in solution or in the dry state.

Acknowledgments This work was supported by the Defense Threat Reduction Agency (Contract No. DTRA01-03-P-0174). We would like to thank Dr. Manoj Ram for his helpful advice throughout the project.

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