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Label-Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes Pinar Kara,a Kagan Kerman,a Dilsat Ozkan,a Burcu Meric,a Arzum Erdem,a Peter E. Nielsen,b Mehmet Ozsoz*a a
b
Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100, Bornova ± Izmir, Turkey; e-mail:
[email protected] Center for Biomolecular Recognition, IMBG, Department of Biochemistry B, The Panum Institute, DK 2200 Copenhagen, Denmark
Received: January 10, 2002 Final version: April 2, 2002 Abstract A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label-free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects. Keywords: DNA biosensor, DNA, Chitosan, Methylene blue, PNA, Hybridization
1. Introduction Electrochemistry offers great advantages over the existing DNA biosensors based on optical detection protocols, because electrochemical biosensors provide rapid, simple and low-cost point-of-care detection of specific nucleic acid sequences. Thus, electrochemical DNA hybridization biosensors play an important role for pharmaceutical, clinical and forensic applications [1 ± 4]. Many protocols have been proposed for electrochemical monitoring of DNA hybridization. Oligonucleotides labelled with enzymes such as horseradish peroxidase and alkaline phosphatase [5 ± 7] have been employed in hybridization detection protocols. The basis of the hybridization detection scheme of Umek et al. [8] depended on the ferrocene modified adenine containing signaling oligonucleotides. After hybridization with the target DNA, the self assembled monolayer (SAM) allowed electron transfer between ferrocene and the gold electrode surface. Wang et al. [9, 10] attached biotinylated oligonucleotides onto strepavidin coated magnetic beads. Magnetic separation greatly eliminated the nonspecific adsorption effects. Wang et al. [10] described a colloidal gold tag for eletrochemical detection and amplification of DNA hybridization. Biotinylated target strands were used in the hybridization process. These target strands bound the streptavidin Electroanalysis 2002, 14, No. 24
coated gold nanoparticles. The acid dissolution of gold tags were monitored by using disposable carbon strip electrodes. The use of inosine substituted probes and the appearance of guanine signal upon hybridization with the target eliminated the external labels and shortened the assay time [9, 11 ± 13]. The oxidation signals of guanine or adenine have been employed to detect hybridization [12]. Such use of intrinsic signals of DNA are nowadays prefered because they shortened the hybridization detection assay time. In this report, the oxidation signals of guanine and adenine were also monitored for the detection of hybridization. External labels such as anticancer drugs [14 ± 17], metal complexes [17 ± 22], organic dyes [18 ± 22] have mostly been employed in hybridization biosensors. The changes in the electrochemical response of these labels were monitored to detect hybridization. PNA is a structural DNA analogue containing an uncharged N-(2-aminoethyl) glycine-based pseudopeptide backbone, which has been reported to form Watson-Crick complementary duplexes with DNA [27]. PNA, originally synthesized as a gene-targeting antisense drug, has demonstrated remarkable hybridization properties towards complementary oligonucleotides [28]. Compared to DNA duplexes, PNA hybrids have higher thermal stability and can be formed at low ionic strengths. The neutral peptidelike backbone of PNA, provides the basis for the probe to
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1040-0397/02/2412-1685 $ 17.50+.50/0
1686 hybridize to target DNA sequences with high affinity and specificity [29]. In this report, methylene blue (MB) is used as the electroactive label. MB is an organic dye that belongs to the phenothiazine family. Erdem et al. [23] reported that MB could be used as a redox-active indicator for the electrochemical detection of mismatched bases in Hepatitis B virus DNA. The detection of hybridization was accomplished by using the specific interaction of MB with guanine. The evidence of the direct interaction of MB with guanine bases on the DNA coated CPEs was also investigated by Yang et al. [26]. Rohs et al. [30] reported a modelling study for MB binding to DNA with alternating guanine-cytosine base sequence. Enescu et al. [31] investigated the conformation of MB-guanine complex by molecular dynamics simulation. The position and orientation of MB-guanine complexes were found to be in three modes; T-shaped, nonstacked and face-to-face. Kelley et al. [32] studied the intercalation of MB into the thiol terminated SAM of dsDNA on gold electrode by chronocoulometry, cyclic voltammetry, ellipsometry and 32P labeling methods. Tani et al. [33] reported that there was a shift in the peak potentials of the square wave voltammetric signals of MB obtained from the thiol terminated oligonucleotide self assembly on gold electrode. Chitosan (Scheme 1) is a biocompatible, biodegradable and non-toxic cationic polymer [34] that forms polyelectrolyte complexes with DNA. The fact that chitosan forms stable complex with DNA provides better DNA immobilization [34]. The amino groups (shown in circles in Scheme 1) electrostatically interact and form a complex with the negatively charged phosphate backbone of DNA. Thus, chitosan and its derivatives may represent potentially safe and efficient cationic carriers for gene delivery [35, 36]. Xu et al. [37] reported a chitosan modified platinum electrode by applying 2 mL of chitosan solution (1.00% chitosan in 1.00% acetic acid solution v/v) uniformly on a freshly smoothed platinum electrode. They have detected hybridization by using polymerase chain reaction (PCR) product modified with ferrocenecarboxaldehyde at chitosan modified platinum electrode. Xu et al. [38] also reported the detection of hybridization by using aminoferrocene modified oligonucleotides immobilized on glassy carbon electrode. The aim of this study was to report a novel DNA immobilization method to achieve specific hybridization detection between PNA and DNA oligonucleotides by using intrinsic DNA and MB signals. An electrochemical DNA biosensor is described for the detection of DNA hybridization using the voltammetric signals of MB and
Scheme 1. Molecular structure of chitosan. Electroanalysis 2002, 14, No. 24
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guanine at a chitosan modified surface. The features of the protocol are discussed, and results are compared with other protocols previously reported.
2. Experimental 2.1. Apparatus Voltammetric signals were collected with an AUTOLAB PGSTAT 30 electrochemical analysis system and GPES 4.8 software package (Eco Chemie, The Netherlands). The three electrode system consisted of the carbon paste working electode (CPE), the reference electrode (Ag/AgCl) and a platinum wire as the auxiliary electrode. The convective transport was provided by a magnetic stirrer.
2.2. Chemicals Methylene blue (MB) was purchased from La Pine Scientific Company, USA. Chitosan was purchased from Sigma. Calf thymus double stranded DNA (dsDNA) and calf thymus single stranded DNA (ssDNA) were obtained from Sigma Chemical Company (Sydney, Australia). The 14-mer DNA oligonucleotides were purchased from Genset Oligos (Sydney, Australia). Their base sequences are as follows: DNA target (14-base sequence A): 3'-GGG GGG CAG AGC AT-5' PNA probe (14-base sequence B): H-(3'-) ATG CTC TGC CCC CC (-5')-Lys-NH2 The 14-base sequence A is complementary to 14-base sequence B. DNA oligonucleotide stock solutions (100 mg/ L) were prepared with TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.00) and kept frozen. PNA stock solutions (100 mg/L) were prepared with 50 mM phosphate buffer solution (pH 7.40) and kept frozen. More dilute solutions of calf thymus DNA and oligonucleotides were prepared with either 0.50 M acetate buffer (pH 4.80) or 20 mM Tris HCl buffer (pH 7.00), according to the hybridization protocol. The in-house distilled and deionized water was used in all solutions.
2.3. Electrode Preparation The modified carbon paste was prepared in two steps. The first step involved a through mixing of the desired amounts of graphite powder and mineral oil (Acheson 38, Fisher) (30/ 70% (w/w) graphite/oil). An amount of the initial paste was mixed with a desired amount of chitosan to make the chitosan modified carbon paste and hand mixed. A portion of the resulting paste was packed into the end of a 3 mm (i.d.) glass tube. Electrical contact was established via a copper wire. The surface was smoothed on a weighing paper. Bare CPE was prepared by following the procedure explained above without the incorporation of chitosan in the carbon paste matrix.
Chitosan Modified Carbon Paste Electrodes
2.4. DNA Immobilization ChiCPE was immersed into the 0.50 M acetate buffer solution (pH 4.80) containing 10 ppm probe, 10 or 2 ppm dsDNA or ssDNA while applying 0.50 V with 200 rpm stirring for 5 min. ChiCPE was then washed with blank 0.50 M acetate buffer solution (pH 4.80) for 10 seconds to remove unbound DNA. Thus, a probe modified ChiCPE was obtained.
2.5. Hybridization The probe modified ChiCPE was immersed into the 20 mM Tris HCl buffer solution (pH 7.00) containing 15 ppm target while applying 0.50 V with 200 rpm stirring for 5 min. ChiCPE was then washed with blank Tris HCl buffer solution (pH 7.00) for 10 seconds to remove nonspecifically bound target. Thus, a hybrid modified ChiCPE was obtained.
2.6. Label Binding to the Hybrid MB was accumulated onto the surface hybrid by immersing the electrode into the stirred 20 mM Tris HCl buffer (pH 7.00) containing 20 mM MB with 20 mM NaCl and 200 rpm stirring for 5 minutes without applying any potential. After accumulation of MB, the electrode was rinsed with blank 20 mM Tris HCl buffer (pH 7.00) for 10 seconds to remove nonspecifically bound MB. For the label-free protocol, no MB accumulation step was employed.
2.7. Voltammetric Transduction The oxidation signals of adenine and guanine were measured by using DPV in the blank 0.50 M acetate buffer (pH 4.80) with an amplitude of 10 mV at 20 mV/s scan rate. The reduction signal of the accumulated MB was measured by using DPV in the 20 mM Tris-HCl buffer (pH 7.00) with an amplitude of 10 mV at 20 mV/s scan rate. The raw data were also treated using the Savitzky and Golay filter (level 2) of the GPES software, followed by the moving average baseline correction with a ™peak width∫ of 0.01. Repetitive measurements were carried out by renewing the surface and repeating the above assay format.
1687 configuration is particularly useful for this task, as it allows controlled loading of chitosan. The amount of incorporated chitosan and the immediate proximity of the binding sites greatly facilitate the rapid and effective immobilization of DNA. The effect of experimental parameters including ionic strength and MB accumulation time had been explored by Erdem et al. [23] for optimum analytical performance. The effect of experimental conditions including probe immobilization time for PNA-DNA hybridization were well desribed by Wang et al. [21, 22]. Therefore, specified parameters in these reports were employed for the subsequent voltammetric measurements in this article. The effect of the chitosan content upon the immobilization of DNA was also studied (not shown). The signals of guanine and adenine increased until the incorporation of 5% chitosan. The signals rapidly decreased upon increasing the percentage of chitosan within the carbon paste more than 5%. No intrinsic signals of DNA were observed for pastes containing more than 10% (w/w) of chitosan because of the fouling of the surface. Such profiles were expected from the increased binding capacity of the electrode. 5% (w/ w) of chitosan within the carbon paste was used for further experiments. Figure 1 displays the oxidation signals of guanine (G) and adenine (A) obtained from the ssDNA modified ChiCPE (A) and ssDNA modified CPE (B). The signals of guanine and adenine were higher at the ChiCPE than the ones obtained with ordinary CPE. The affinity of chitosan towards the negatively charged backbone of DNA greatly increased the voltammetric signals. Alternatively, Figure 2 displays the oxidation signals of guanine (G) and adenine (A) obtained from the dsDNA modified ChiCPE (A) and dsDNA modified CPE (B). The guanine and adenine signals were lower than those obtained with the ssDNA modified electrodes. This event was explained as the steric inhibition of the oxidizable sites of guanine and adenine. The binding of guanine to cytosine and adenine to thymine in the double helix also decreased the oxidation signals obtained from
3. Results and Discussion The ability to immobilize DNA at CPEs is illustrated in the following sections using chitosan as a model biological agent and in the presence of MB as the hybridization label. The binding of the chitosan to phosphate backbone of DNA makes it an ideal choice for the task of DNA immobilization. The binding of DNA from its backbone particularly leaves the bases available for efficient hybridization. The CPE
Fig. 1. Differential pulse voltammograms for the oxidation signals of guanine (G) and adenine (A) at A) ssDNA-modified ChiCPE and and B) ssDNA-modified CPE in 0.50 M acetate buffer (pH 4.80). Electrode pretreatment, 1 min at 1.70 V in 0.05 M phosphate buffer (pH 7.40); 10 ppm ssDNA immobilization, 5 min at 0.50 V in 0.50 M acetate buffer (pH 4.80) with 20 mM NaCl, measurement, scanning at 5 mV/s in 0.50 M acetate buffer (pH 4.80) with an amplitude of 10 mV at 20 mV/s scan rate. Electroanalysis 2002, 14, No. 24
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Fig. 2. Differential pulse voltammograms for the oxidation signals of guanine (G) and adenine (A) at A) dsDNA-modified ChiCPE and and B) dsDNA-modified CPE in 0.50 M acetate buffer (pH 4.80). Other conditions are as in Figure 1 except dsDNA was used in the immobilization step.
these redox active bases. This phenomen was used for the detection of hybridization in a previous article by the authors [12]. A series of three repetitive measurements of the oxidation of guanine resulted in reproducible results with a relative standard deviation (RSD) of 10.12% for ssDNA modified CPE and a RSD of 10.20% for ssDNA modified ChiCPE. A series of three repetitive measurements of the oxidation of adenine resulted in reproducible results, with a RSD of 7.50% for ssDNA modified CPE and a RSD of 13.20% for ssDNA modified ChiCPE. A series of three repetitive measurements of the oxidation of guanine resulted in reproducible results with a RSD of 8.20% for dsDNA modified CPE and a RSD of 10.45% for dsDNA modified ChiCPE. A series of three repetitive measurements of the oxidation of adenine resulted in reproducible results, with a RSD of 9.54% for ssDNA modified CPE and a RSD of 11.35% for ssDNA modified ChiCPE. The detection limits, estimated from S/N 3, correspond to 2.15 ng/mL ssDNA and 3.35 ng/mL dsDNA at ChiCPE, respectively. Figure 3 displays the calibration plot of the guanine oxidation signals against PNA probe immobilization time. Figure 3a shows that the guanine signals increased until 5 min and then decreased at 7 and 10 min. The guanine signals also increased until 5 min and then almost remained constant until 10 min. The guanine signals obtained from ChiCPE was always found to be higher than those found at CPE (Fig. 3b). Five min was used as the optimum probe immobilization time. The oxidation signal of guanine (Figure 4) was obtained from the hybridization between PNA probe and DNA target. Hybrid formation on the PNA probe modified CPE with the DNA target sequence resulted in a high signal of guanine (Figure 4a). In comparison, hybridization on the PNA probe modified ChiCPE with the DNA target resulted in a lower signal of guanine (Figure 4b). The difference between the signals of the CPE and the ChiCPE was clearly observed with an increase in the PNA-DNA hybrid signal at CPE. This signal, which was found higher than the guanine signal obtained from the ChiCPE, indicated that PNA Electroanalysis 2002, 14, No. 24
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Fig. 3. Calibration plot for the oxidation signals of guanine against PNA probe immobilization time at a) Chi CPE, b) CPE. 10 ppm PNA probe immobilization at pretreated CPE, different immobilization times at 0.50 V in 0.50 M acetate buffer (pH 4.80) with 20 mM NaCl, measurement, scanning in 0.50 M acetate buffer (pH 4.80) with an amplitude of 10 mV at 20 mV/s scan rate.
Fig. 4. Differential pulse voltammograms for the oxidation signals of guanine in 0.50 M acetate buffer (pH 4.80) at a) PNADNA hybrid modified CPE, b) PNA-DNA hybrid modified ChiCPE. 10 ppm PNA probe immobilization at pretreated CPE, 5 min at 0.50 V in 0.50 M acetate buffer (pH 4.80) with 20 mM NaCl, hybridization, 5 min at 0.50 V in 15 ppm DNA target containing 20 mM Tris HCl buffer solution (pH 7.00) with 20 mM NaCl; measurement, scanning in 0.50 M acetate buffer (pH 4.80) with an amplitude of 10 mV at 20 mV/s scan rate.
probes weren×t attracted to the ChiCPE surface because PNA had a neutral peptide backbone. Instead, DNA probes had a negatively charged backbone which facilitated the binding process on the ChiCPE surface. A series of three repetitive measurements of the oxidation of guanine resulted in reproducible results with a RSD of 9.17% for PNA-DNA hybrid modified ChiCPE and with a RSD of 8.60% for PNA-DNA hybrid modified CPE. The detection limits, estimated from S/N 3, correspond to 4.82 ng/mL PNA probe and 5.75 ng/mL PNA-DNA hybrid at ChiCPE, respectively. The oxidation signals of guanine obtained from the PNA probe modified ChiCPE and CPE are displayed in Figure 5. The guanine signal of PNA probe at ChiCPE (Figure 5b) is lower than that obtained with CPE (Figure 5a). This decrease was also attributed to the lack of phophate
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Fig. 5. Differential pulse voltammograms for the oxidation signals of guanine in 0.50 M acetate buffer (pH 4.80) at a) PNA probe modified CPE, b) PNA probe modified ChiCPE. No hybridization with DNA target step was employed. Other conditions are as in Figure 3.
backbone of the PNA probes. These results clearly demonstrated that chitosan could bind DNA through its negatively charged backbone at CPE surface. Label-based DNA recognition experiments are displayed in Figure 6. The reduction signal of MB at the ssDNA modified ChiCPE (Figure 6A) was higher than that obtained with ssDNA modified CPE (Figure 6C). An obvious decrease in the voltammetric signal was observed for the indicator, while the interaction between MB and guanine residues of ssDNAwas prevented by dsDNA on the ChiCPE (Figure 6B) and CPE (Figure 6D) surfaces, because MB intercalated between the duplex formed on the CPE surface. The redox active parts of intercalated MB was enveloped by the bulky hybrid and could not interact with the guanine bases, because guanine bases were bound to complementary cytosine bases in the hybrid. The slight increase in the MB signal obtained at the ChiCPE was attributed to the strong binding of DNA molecules onto the chitosan at the surface. The highest MB reduction signal was observed at the ChiCPE surface immobilized with the ssDNA (Figure 6A), because MB could interact with the free guanine bases and accumulate at the surface [23]. A series of three repetitive measurements of the reduction of MB resulted in reproducible results with a RSD of 10.80% for ssDNA modified ChiCPE, a RSD of 9.20% for ssDNA modified CPE and a RSD of 9.73% for dsDNA modified ChiCPE, a RSD of 8.20% for dsDNA modified CPE. The detection limits, estimated from S/N 3, correspond to 2.84 ng/mL ssDNA and 5.64 ng/mL dsDNA at ChiCPE, respectively.
4. Conclusions The advantages of chitosan modified electrodes for the sequence specific DNA hybridization biosensors based on intrinsic DNA signals and the label signals have been
Fig. 6. Histograms for the reduction signals of MB in 20 mM Tris HCl buffer solution (pH 7.00) with 20 mM NaCl at A) ssDNA modified ChiCPE, B) dsDNA modified ChiCPE, C) ssDNA modified CPE, D) dsDNA modified CPE. Electrode pretreatment, 1 min at 1.70 V in 0.05 M phosphate buffer (pH 7.50) ; 2 ppm ssDNA or dsDNA immobilization, 5 min at 0.50 V in 0.50 M acetate buffer (pH 4.80) with 20 mM NaCl, MB accumulation, 5 min with 200 rpm in 0.50 M acetate buffer (pH 4.80) with 20 mM NaCl, measurement, in 0.50 M acetate buffer (pH 4.8) with an amplitude of 10 mV at 20 mV/s scan rate.
demonstrated. Incorporation of chitosan into CPE is simple, cost-effective and provides strong immobilization of DNA. The use of chitosan eliminates the need for toxic mercapto and DNA self-assembled monolayers and expensive avidin and biotinylated DNA interactions for biosensor immobilization schemes. The application of DPV at ChiCPE fulfilled the expectations for the direct detection of hybridization between the known oligonucleotides. Due to the label-free and label based detection systems ChiCPE displays significantly enhanced immobilization procedure and hybridization signals. Improving the immobilization and hybridization steps through the use of ChiCPE should futher minimize non-specific adsorption effects and maximize sensitivity and speed. Reports are in progress toward these directions.
5. Acknowledgements B. M. acknowledges scholarship for PhD students from Turkish Technical and Scientific Research Council (TUBITAK), Munir Birsel Foundation. Authors acknowledge the financial support from TUBITAK (Project number: TBAG1871) and Ege University Science and Technology Research and Application Center (Project number: 2000/BIL/031).
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Announcement It is with deep sorrow that we report the death on November 28 of Professor Ralph ™Buzz∫ Adams Ralph was a phenomenal scientist and person. He was a tremendous inspiration to all of us. Ralph pioneered of solid electrodes, in vivo voltammetry. He taught and inspired whole generations of analytical chemists and neuroscientists. His passing is a severe loss to the entire international electroanalytical community.
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