ORIGINAL PAPER
An electrochemical biosensor for direct detection of DNA
using polystyrene-g-soya oil-g-imidazole graft copolymer
Izzet Kocak1&TimurŞanal1&Baki Hazer1
Received: 12 February 2016 / Revised: 11 December 2016 / Accepted: 2 January 2017 / Published online: 11 January 2017 # Springer-Verlag Berlin Heidelberg 2017
Abstract A label-free electrochemical DNA biosensor was developed through the attachment of polystyrene-g-soya oil-g-imidazole graft copolymer (PS-PSyIm) onto modified graphene oxide (GO) electrodeposited on glassy carbon elec-trode (GC). GC/GO elecelec-trode was initially functionalised via electrochemical reduction of 4-nitrobenzene diazonium salt, followed by the electrochemical reduction of NO2to NH2.
Subsequent to the electrochemical deposition of gold nano-particles on modified surface, the attachment of the PS-PSyIm graft copolymer on the resulting electrode was achieved. The interaction of PS-PSyIm with DNA at the bare glassy carbon electrode was studied by cyclic voltammetry technique, and it was found that interaction predominantly takes place through intercalation mode. The selectivity of developed DNA biosen-sor was also explored by DPV on the basis of considering hybridisation event with non-complementary, one-base mis-matched DNA and complementary target DNA sequence. Large decrease in the peak current was found upon the addi-tion of complementary target DNA. The sensitivity of the developed DNA biosensor was also investigated, and detec-tion limit was found to be 1.20 nmol L−1.
Keywords DNA biosensor . Graphene oxide . Soya oil . Gold nanoparticles
Introduction
Since deoxyribonucleic acid is considered one of the most sig-nificant molecules in living organisms due to its vital role in cell activities, there has been a growing interest in the investigation on the interaction of small molecules with DNA in order to discover potential drug candidates for cancer treatment [1–3]. The construction of electrochemical DNA biosensor has bear great importance over the last decade as they are simple and low cost and provide rapid, highly sensitive and accurate detection of DNA sequence [4–6]. Two methods have extensively been applied to detect the hybridisation of DNA sequence. In the first method, DNA hybridisation detection is completely performed in the presence of electrochemical labels and hybridisation markers. Despite that this method provides highly precise and accurate detection, it also has a disadvantage of being time con-suming and requires several complicated steps during the manufacturing process [7]. In the second method, known as label free, electrochemical tag is not required and therefore this method offers the probability of constructing inexpensive and highly sequential electrochemical DNA biosensor [8]. The most vital element regarding the architect of EC biosensor is successful immobilisation of biomolecules to electronic trans-ducer [9]. Thus, conductive polymer has drawn a great deal of attention due to being transducer for charged species [10,11]. If biomolecules are transferred onto the conductive polymers, in-trinsically, it can directly allow us to observe the hybridisation of DNA sequence without using any redox indicator. As a con-sequence of this, a lot of effort has been paid to the development of electrochemical DNA biosensor using conductive polymer in order to monitor the hybridisation event. For instance, p o l y ( i n d o l e - 5 - c a r b o x y l i c a c i d ) [1 2] , p o l y - 2 , 6 pyridinecarboxylic acid [5], polyaniline [13] and polypyrole [10,11,14] have been used to investigate their DNA biosensor applications.
Electronic supplementary material The online version of this article (doi:10.1007/s10008-017-3504-8) contains supplementary material, which is available to authorized users.
* Izzet Kocak
izzetkocak@beun.edu.tr
1 Faculty of Science and Art, Department of Chemistry, Bulent Ecevit
There has been a growing interest in synthesising environ-mentally friendly biodegradable plastic material in order to re-duce their damaging effect on our planet. Hence, it is quite crucial to develop cheap, readily available and producible biodegradable polymers from natural products, for instance, starch, oils and polysaccharides. Soya oil, well known to be one of the most common, available and low-cost source in preparing polymers with high molecular weight, is used in our study to manufacture electrochemical DNA biosensor [15,16]. To the best of our knowledge, this is the first paper that reports the development of an electrochemical DNA biosensor using soya oil graft copol-ymer containing imidazole group.
In our work, polystyrene-g-soya oil-g-imidazole graft copol-ymer (PS-PsyIm) was successfully synthesised and their electro-chemical properties and interaction with double-stranded DNA (ds-DNA) were studied at the surface of bare glassy carbon elec-trode by cyclic voltammetry (CV). Electrochemical attachment of graphene oxide was achieved, and electrochemical reduction of 4-nitrobenzene diazonium salt was then carried out, followed by electrochemical deposition of gold nanopartices at the NH2
-modified surface obtained by EC reduction of NO2. A sensitive
electrochemical DNA biosensor using PS-PsyIm was construct-ed, as displayed in the graphical abstract.
Experimental
All reagents and solvents were of commercial origin, analytical grade and used without further purification unless otherwise stat-ed. Solutions of calf thymus DNA (CT-DNA; purchased from Sigma) in 50 mM ammonium acetate (pH 7.5) had a UV–vis abso rbanc e ratio of 1.8–1.9:1 at 260 and 280 nm (A260/A280 = 1.9), indicating that the DNAwas sufficiently free of protein. The concentration of DNAwas determined spectropho-tometrically using a molar absorptivity of 6600 M−1cm−1 (260 nm) [31]. Double-distilled water was used to prepare buffers. Stock solutions of CT-DNA were stored at 4 °C and used within 4 days, as reported in our previous paper. [2].
The 21-mer synthetic oligonucleotides were also obtained from Prizma Biological Engineering. Their base sequences are as follows:
& Immobilised probe (16-mer sequence): 5′-GCTG CCAAATACCTCC-3′ (S1)
& Target (16-mer sequence): 5′-GGAGGTATTTGGCAGC-3′ (S2)
& One-base mismatched target (16-mer sequence): 5′-GGAGGGATTTGGCAGC-3′ (S3)
& Non-complementary (16-mer sequence): 5′-CTTA GCGTCCGGATGAT-3′ (S4)
All oligonucleotides were prepared in Tris-HCl buffer (pH 8.0) and kept in a freezer. Working carbon electrodes used in
this study are glassy carbon (GC; 3 mm diameter rods, HTW, Germany) sealed in glass and connected to copper wires using melted indium (Aldrich). Finally, the conductivity of each electrode was checked with an ohm meter to ensure good electrical connection before performing electrochemical ex-periments. Prior to electrochemical measurements, the GC electrodes were polished with silicon carbide polishing paper (grade 1200) then with 1, 0.3 and 0.05μm alumina (Buehler) on polishing cloth (Buehler) and rinsed thoroughly with deionised water and eventually dried under an argon stream.
Electrochemical measurements
All electrochemical measurements were performed with an SP-50 model Biologic Science Instruments Potentiostat/ Galvanostat and controlled using EC-Lab software, a conven-tional three-electrode system which consist of a GC as a work-ing electrode, a platinum wire as a counter electrode and a silver-silver chloride (Ag/AgCl) as a reference electrode and were kept in saturated potassium chloride solution when not being used. All potentials are reported vs. the Ag/AgCl in aqueous solution. Argon was purged through the cell for 10 min before electrochemical measurement was carried out.
Synthesis of graphene oxide
The procedure applied and followed for the synthesis of graphene oxide was similar to given literature [17, 18]. Graphene oxide was synthesised by the oxidation of graphite powder using modified Hummer’s method [19]. Stirring in an ice bath, graphite (4 g) and NaNO3(2 g) were mixed in 90 mL
of H2SO4(98%). After stirring the mixture for 2 h, in order to
prevent overheating and explosion, the temperature was kept under 20 °C while adding 12 g of potassium permanganate very slowly to the suspension. For the next 6 h, the mixture was stirred at 35 °C and then 500 mL of water was added to dilute it. It was stirred continuously for 1 h. Next, the solution was kept in a reflux system at 95 °C for 15 min, after that the temperature was decreased to 30 °C. The suspension was treated with 20 mL H2O2(30%) which changes the colour
of the solution from brown to yellow in order to ensure that the reaction with KMnO4was completed. The obtained
mix-ture was washed respectively with 10% HCl and water. Lastly, after the substance was centrifuged, it was vacuum dried for 24 h at 40 °C to obtain graphene oxide.
Synthesis of PS-PsyIm
Octadeca-9,12-dienoic acid (linoleic acid) was a product of Fluka (Code 62240). Its purity was 75 wt.%, and the rest
was an unsaturated triglyceride. It was used as received. Autoxidation of linoleic acid was performed according to the procedure reported in our previous work [20]; 12.7 g of linoleic acid spread out in a Petri dish (Φ = 14 cm; oil thick-ness, 0.8 mm) was exposed to daylight in the air at room temperature. After a given autoxidation time (ca. 4 weeks), a pale yellow viscous sticky liquid layer was formed (GPC re-sults: Mn, 2971 g/mol; Mw, 3675 g/mol; MWD, 1.237). Styrene, imidazole and the other solvents were purchased from Aldrich and purified in a conventional manner before use.
Polymerisation of styrene with PLina and synthesis
of PS-g-PLina graft copolymer
A simple polymerisation method was used in the free radical polymerisation of styrene. A solution of 3.2 g of poly(linoleic acid) (PLina) and 12.0 g of styrene were kept at 95 °C for 5 h in a flask with a stopper under argon stream. The crude poly-mer was dissolved in 20 mL of chloroform and precipitated into 200 mL of methanol under continuous stirring. Polystyrene-graft-poly(linoleic acid) (PS-g-PLina) copolymer was filtered, dried at room temperature and then dried under a vacuum at 40 °C for 24 h.
Condensation reaction of the PS-g-PLina
with imidazole
PLina at 1.05 g and imidazole at 3.04 g were dissolved in 10 mL of toluene under argon. The solution was kept at 95 °C for 5 h. After the reaction was completed, the solvent was partially evaporated and the polymer was precipitated with methanol. The crude polymer was filtered and dried at room temperature for 24 h. For further purification, amphi-philic polymer was soaked into distilled water at room tem-perature for 24 h in order to remove unreacted imidazole completely. The purified polymer was filtered, then dried at room temperature for 24 h, and then dried under a vacuum at 40 °C for 24 h. The overall reaction mechanism and1H NMR spectrum of the synthesised graft copolymer are given in Fig.S1.
DNA binding studies of PS-PsyIm
Homogeneous solution studies in the presence of DNA and graft copolymer are as follows. The certain amount of ds-DNA and PS-PsyIm was immersed in phosphate buffer solu-tion (pH 7). The electrochemical measurements of the inter-action of PS-PsyIm with DNA were carried out by cyclic voltammetry method. On the other hand, surface-based
experiments were carried out as follows. The immobilisation of DNA was achieved on the GC/GO/NH2/NP/ PS-PsyIm and
GC/NH2/NP/PS-PsyIm electrodes. The immobilisation of
DNA on the corresponding electrode surface was carried out by dropping 10μL DNA and then allowing to dry at room temperature. Finally, the electrode was washed with distilled water and rinsed with PBS (pH 7.0) to remove unadsorbed DNA.
Electrochemical deposition of graphene oxide
Electrochemical deposition of GO onto GC was achieved in a solution containing 1 mg mL−1graphene oxide in phosphate buffer (pH 9.8) at a scan rate of 50 mV s−1. Electrochemical reduction of GO was carried out by cycling potential from 0.5 to−1.5 V with ten runs.Electrochemical grafting of 4-nitrobenzene
diazonium salt
Electrografting of 4-nitrobenzene on glassy carbon surfaces was carried out by electrochemical reduction in a solution containing 5.0 mM of the corresponding diazonium salt dis-solved in 0.1 M tetrabutylammonium tetrafluoroborate (TBATFB) in acetonitrile. The grafting was carried out by cycling the electrode in the range from 0.9 to−0.8 V vs. Ag/ AgCl at a scan rate of 50 mV s−1until complete blockage of the current was observed.
Electrochemical deposition of gold nanoparticles
EC deposition of gold nanoparticles was carried out at the scan rate of 50 mV s−1with 30 runs by cycling the electrode from−0.4 to 1.5 V in a solution of 0.5 mM of HAuCl4con-taining 0.01 M of Na2SO4and 0.01 M of H2SO4. The size of
the electrodeposited gold nanoparticles was determined to be 10 nm by scanning electron microscopy.
DNA-binding studies of PS-PsyIm
Homogeneous solution studies in the presence of DNA and graft copolymer is as follows. The certain amount of ds-DNA and PS-PsyIm were immersed in phosphate buffer solution (pH 7). The electrochemical measurements of the interaction of PS-PsyIm with DNA were carried out by cyclic voltamm-etry method. On the other hand, surface-based experiments were carried out as follows. The immobilisation of DNA was achieved on the GC/GO/NH2/NP/ PS-PsyIm and GC/
on the corresponding electrode surface was carried out by dropping 10μL DNA and then allowing to dry at room tem-perature. Finally, the electrode was washed with distilled wa-ter and rinsed with PBS (pH 7.0) to remove unabsorbed DNA.
Hybridisation on ss-DNA probe-modified GC
electrode
The single-stranded DNA (ss-DNA)-modified electrode was immersed in 5 mM Tris-HCl buffer (pH 7.0) containing target complementary ss-DNA for 1 h at room temperature. After hybridisation is achieved, electrodes were washed with water to eliminate physically adsorbed oligonucleotides; 0.3 M NaCl and 30 mM sodium citrate buffer solution at pH 7 (2 × SSC buffer) was used for the hybridisation of DNA. The immobilisation of probe DNA to electrode surface was also carried out in PBS (pH 7).
Hybridisation on ss-DNA probe-modified GC
electrode
The ss-DNA-modified electrode was immersed in 5 mM Tris-HCl buffer (pH 7.0) containing target complementary ss-DNA for 1 h at room temperature. After hybridisation is achieved, electrodes were washed with water to eliminate physically adsorbed oligonucleotides. General description of how to pre-pare electrochemical DNA biosensor is displayed in Scheme1
of the Electronic supplementary material.
Results and discussion
The electrodeposition of graphene oxide on glassy carbon electrode was achieved through CV in PBS (pH 9.8). Figure1 shows successive cyclic voltammogram recorded for graphene oxide at the scan rate of 50 mV s−1. As clearly seen, while there is one peak in the cathodic region of the first voltammogram, two small reversible peaks are observed as we continue cycling. Furthermore, increase in the peak current, as a consequence of repeating scans, confirms that electrodepo-sition of graphene oxide onto GC is successfully accom-plished. The first cathodic peak can be attributed to irrevers-ible electrochemical reduction of graphene oxide. Other two small peaks appearing at 0.27 and 0.043 V, exhibiting revers-ible behaviour, can also be linked with electrochemically ac-tive organic functionalities containing oxygen that are hard to be reduced by CV technique. These results are also found to be quite consistent with the literature [21]. SEM image of GCE/GO (Fig.S2) also verifies the existence of reduced GO (RGO) on GCE.
Owing to its unique properties, for example, its large sur-face area, superb electrical and thermal conductivity, mechan-ical flexibility, good electrocatalytic activity and wide poten-tial window, graphene-modified electrodes are expected to play a significant role in electrochemical sensors and biosen-sors with respect to glassy carbon electrodes [22]. In terms of charge transfer abilities, one of the experimental methods in making a comparison between graphene oxide and glassy car-bon electrode is to carry out cyclic voltammetry measurement in the presence of Fe(CN)63−/4−, known to be quite sensitive
towards surface chemistry of carbon electrodes. As shown in Fig. 2, peak current acquired at the GCE/GO is 1.5 times larger compared with GCE, suggesting that the electrochem-ical active sites onto GCE have increased subsequent to mod-ification by GO. Larger peak current might also be attributed to increase in surface area from the planar GC after electro-chemical introduction of GO. Moreover, there is a decrease in peak separation for unmodified GC from 0.21 to 0.18 V, indi-cating an enhanced electron transfer kinetics by electrodepo-sition of GO onto GC, which can be attributed to an increase in edge defects on the GC [23].
The grafting of the nitrobenzene to GCE/GO electrodes was performed by electrochemical reduction of the corre-sponding diazonium salt, at 50 mV s−1(5 mM solution of NB in ACN containing 0.1 M TBATFB). As seen in Fig.3, rather broad irreversible peaks were obtained, indicating one electron reduction of NB and the formation of the radical that couples to electrode surfaces. Complete peak disappearance on the following scans points out that the electrode surface has been completely coated by NB.
FigureS3shows the successive CV of GC/GO electrode modified with NB. There is a quite large peak of around −1.1 V. However, as we continue to cycle, this peak disap-pears, indicating the complete reduction of the nitrobenzene to phenyl amine or phenylhydroxyl amine. On the other hand, on the second scan, it is quite noticeable that voltammogram
Fig. 1 CVs of electrochemical reduction of 1.0 mg mL−1GOxin PBS
began to exhibit reversible behaviour with the redox potential of−0.115 V. These reversible peaks can also be considered an evidence on the fact that NB is not completely changed into phenyl amine. The electrochemical behaviour of nitrobenzene grafted to different types of surfaces is well described in the literature [24], and herein, our results are found to be in a good agreement with them. It is widely accepted that during the conversion of NB to phenylnitrosoamine, a two-electron, two-proton oxidation process occurs. The first irreversible peak corresponds to a six-electron, six-proton electrochemical reduction of nitrobenzene to phenyl amine through the phenylhydroxyl amine.
Once the electrografting of NB to graphene oxide-modified GC and electrochemical reduction of NO2 to NH2 were
achieved, electrochemical deposition of Au nanoparticles (GNP) was carried out at GC/GO/NH2electrode. NH3+
pre-dominantly exists under neutral pH, which allows its use in
attaching the negatively charged Au nanoparticles to the pos-itively charge surface as a result of electrostatic interaction. Figure4shows successive CVs obtained at the GC/GO/NH2
electrode at the scan rate of 50 mV s−1in the presence of 1 mM of HAuCl4(pH 9.4). The electrochemical reduction of Au(III)
takes places around−0.03 V. However, in the further scans, this peak appeared at a more positive potential. Moreover, corresponding peaks grow as the number of cycles increase, referring that attachment of the Au nanoparticles to amine-modified surface was successfully achieved [25]. SEM pic-tures in Fig.S4also provide further evidence on the existence of GNPs.
The electrochemical properties of PS-PSyIm polymer was investigated by CVat the scan rate of 50 mV s−1in PBS (pH 7) containing 1 mg/1 mL corresponding polymer and 0.1 M KCl as supporting electrolyte. All CV measurements were record-ed vs. Ag/AgCl and glassy carbon, and platinum wire actrecord-ed as working electrode and counter electrode, respectively. As shown Fig.5, PS-PSyIm polymer exhibit irreversible process and broad peaks appeared around at the peak potential of -1.2 V, which is attributed to reduction of imidazole group in PS-PsyIm polymer [26]. The effect of scan rate on electro-chemical response of PS-PSyIm polymer was also studied. As seen in Fig.5(inset), voltammogram obtained at the scan range of 5 to 600 mV s−1show linear dependency between peak current and root of scan rate, indicating mass transport controlled electron transfer kinetics of PS-PSyIm polymer [27].
As demonstrated in Fig.6a, once ds-DNA is added into the solution, a decrease in the peak current takes places and the reduction peak potential of the corresponding polymer shift towards a more positive potential by 0.18 V, it can therefore be deduced that the interaction of this polymer with ds-DNA mainly occurs through intercalative binding mode. Because it is well described in the literature that if there is a negative shift in the peak potential, interaction between DNA and re-garding compound is predominantly electrostatic as electro-static interaction makes reduction at the surface more difficult. On the other hand, the peak potential appears at the more positive value in the presence of ds-DNA, referring to an intercalation binding mode [28,29].
Similar experiment was also carried out in the presence of Ps-PsyIm copolymer and ss-DNA (not shown). Decrease in the peak current was found to be greater for ds-DNA com-pared with ss-DNA, referring to the fact that Ps-PsyIm poly-mer exhibits higher affinity to ds-DNA than ss-DNA. Moreover, it is also significant to note that positive shift in the peak potential is slightly higher (30 mV) for ds-DNA. Such difference might be attributed to the fact that interaction between polymer and ss-DNA is predominantly electrostatic, whereas electrostatic interaction and intercalation binding mode play a role, therefore ds-DNA leads to a more positive shift in the peak potential.
Fig. 3 CV recorded at 50 mV s−1for GC/GOxin 5.0 mM 4-nitrobenzene
diazonium tetrafluoroborate and 0.1 M TBATFB in ACN
Fig. 2 CVs of 5.0 mM K3Fe(CN)6containing 0.1 M KCl on bare GCE
As demonstrated in Fig.6b, peak current decreases subse-quent to the addition of ds-DNA, it thus allows to calculate the binding constant of this polymer to ds-DNA by equation giv-en below [30]. I2p¼ 1 KbjDNAj I2po−I2p þ I2 po−jDNAj ð1Þ
Where is Kbbinding constant, Ipand Ipo represent peak
current obtained in the presence and free of DNA and |DNA| is concentration of ds-DNA. Figure6b shows linear relation-ship between reduction peak current and I
2 po−I2p
ð Þ
jDNAj. The binding
constant of PS-PSyIm is calculated to be as 1.2 × 105M−1. This value seems to be quite comparable with the polymeric structures used in a DNA biosensor and containing styrene and imidazole group, such as streptavidin-coated polystyrene
latex (Kb = 0.6 × 108) [31], imidazole polyamides
(Kb = 1.3 × 105 M−1) [32] and
poly(imidazole/1,4,7,10-tetrazyclodocane)-phosphaxene) (Kb= 0.7 × 105M−1) [33].
As previously shown, imidazole containing PS-PSyIm polymer interacts with ds-DNA via intercalation mode. Hence, it would be quite interesting to study and explore if this polymer could be used as an indicator for the label-free detection of DNA hybridisation at the surface of GC/ GO/NH2/GNP through CV and DPV measurements.
Figure 7 shows CV recorded for GC/GO/NH2/GNP
elec-trode. As clearly demonstrated in tFig. 7, cathodic peak potential of GC/GO/NH2/GNP/Im appears at−0.65 V vs.
Ag/AgCl. However, subsequent to immobilisation by single-stranded probe DNA (S1), cathodic peak potential
shifts to a more positive value by 70 mV and peak current decreased from −0.0035 to −0.0027 mA. When target ss-DNA (S2) is introduced onto the corresponding electrode
to achieve hybridisation, peak potential emerges at the more positive value and peak current decreases to 0.0013 mA. The shift in the peak potential can be ex-plained by the fact that an interaction takes places be-tween amino group of PS-PSyIm and functional groups that exist onto DNA. It is well known that DNA can be recognised as an electronegative compound because of the existence of phosphate groups. Thus, it reduces electron density around PS-PSyIm polymer. As a consequence, it leads to a shift in the reduction peak potential to more positive values [12]. It also needs to be stressed that a decrease in the peak current occurring subsequently to the immobilisation of DNA can be explained by a reduced penetration of counter ions into the polymeric film.
The selectivity of PS-PSyIm polymer coated onto the elec-trochemical DNA biosensor was also investigated by DPV in phosphate buffer (pH 7). DPV measurements was carried out at the GC/GO/NH2/GNP/Im electrode immobilised with
probe ss-DNA (S1) hybridised with non-complementary
ss-DNA (S4), one-base mismatched ss-DNA (S3) and
comple-mentary target DNA (S2), respectively. As seen in Fig.8,
when the complementary ss-DNA is immobilised onto elec-trode modified with probe ss-DNA, there is a large decrease in the peak current, indicating the successful hybridisation of DNA. In the case of one-base mismatched target DNA, peak current is slightly smaller than that of non-complementary target DNA. On the other hand, when the hybridisation is performed with non-complementary ss-DNA, voltammogram is almost undistinguishable from the electrode not treated with DNA. It is therefore concluded that the selectivity perfor-mance of the electrochemical DNA biosensor seems to be good and this device can effectively be used as a label-free detection of DNA hybridisation. Same methodology was also employed for the glassy carbon electrode without any treat-ment with graphene oxide, and similar results were also ac-quired (please see Fig.S5).
Fig. 4 Successive cyclic voltammograms recorded at GC/GOx/NH2at a
scan rate of 50 mV s−1in the 0.01-M Na2SO4containing 0.01 M of
H2SO4and 0.5 mM of HAuCl4solution. Red line represents the first scan
Fig. 5 CVof the PS-PSyIm polymer in PBS buffer, pH 7 at a scan rate of 50 mV s−1. Inset shows the effect of scan rate from 5 to 600 mV s−1
Adsorption of DNA to Ps-PsyIm is a crucial step in terms of the stability of electrochemical biosensor. Therefore, what forces play a significant role in the interaction of copolymer with DNA? Since Ps-PsyIm graft copolymer bears–OH and imidazole group, there is a probability of the formation of hydrogen bonding between DNA bases and polymeric struc-tures. Furthermore, because of the hydrophobic groups such as long alkyl chains and polystirene, Ps-PsyIm can intercalate into the DNA bases. Overall, as the proportion of hydrophobic groups is superior to imidazole and–OH groups, intrinsically interaction between DNA and polymer is predominantly an intercalative binding mode, as also experimentally confirmed with cyclic voltammetry. Such a constructed electrochemical DNA biosensor exhibited considerably good stability and ro-bustness as it will be discussed in following sections.
The sensitivity of the electrochemical biosensor was eval-uated by changing the concentration of the complementary target DNA (S2) sequence after immobilisation of ss-probe
DNA. Figure 9 shows the relationship between change in the peak current and increasing concentration of complemen-tary target DNA sequence. As the concentration of the target DNA increases, the peak current linearly decreases. Since this graph can be considered a calibration curve, the detection limit of the electrochemical DNA biosensor can be calculated from the slope and limit of detection (LOD), which is found to be 1.2 nmol L−1using 3σ, where σ is standard deviation of blank solution with eight parallel measurements; comparing this limit of detection value with the ones obtained by other
Fig. 6 a Cyclic voltammogram for PS-PSyIm polymer in the absence (black) and presence of 30μM of ds-DNA (red line) at a scan rate of 50 mV s−1in phosphate buffer (pH 7). b Curve for the calculation of
binding constant of PS-PSyIm polymer to ds-DNA by CVat a scan rate of 50 mV s−1phosphate buffer (pH 7)
Fig. 8 DPVs of PS-PSyIm polymer deposited on GC/GOx/NH2/GNP
electrode immobilised with probe ss-DNA (1 × 10−7mol L−1, black) and non-complementary ss-DNA sequence (1 × 10−7mol L−1, blue) and hybridised with complementary (3.0 × 10−8mol L−1, green) and single mismatched ss-DNA sequence (3.0 × 10−8mol L−1, red). Scan rate, pulse amplitude and pulse period are 10 mV s−1, 50 mV and 50 ms, respectively
Fig. 7 Cyclic voltammograms of PS-PSyIm polymer-modified electrode recorded at a scan rate of 50 mV s−1in PBS buffer (pH 7.00; black) with probe ss-DNA (red, 1.0 × 10−7mol L−1) and after hybridisation with complementary target ss-DNA sequence (blue, 3 × 10−8mol L−1)
systems, for instance, 1 nmol L−1obtained by polythiophene, poly(indoole-5-carboxlic acid) [12], poly(Py-co—PAA) films [34] and poly-2,6-pyridinecarboxylic and polypyrolle with 0.02μmol L−1[13], 1.8 nmol L−1determined with our bio-sensor through DPVas a label-free detection of DNA seems to be quite promising for DNA sensing technology. Control ex-periment was also performed for immobilised Ps-PsyIm onto GC/NH2/GNP (Fig.S6) and limit of detection is found to be
3.5 nmol L−1, referring to the fact that electrochemical intro-duction of graphene oxide to glassy carbon electrode has con-siderably improved the sensitivity of DNA biosensor.
In order to investigate the impact of PS-Psy-Im polymer on the performance of electrochemical DNA biosensor, a set of electrochemical DNA biosensors were constructed without PS-Psy-Im and nitrophenyl diazonium, Au nanoparticles and graphene oxide. Figure 9 shows calibration curves for all DNA biosensors, and LOD values for all types of DNA bio-sensors are also illustrated in Table1. It is quite clear that biosensor prepared in the absence of graphene oxide and Au nanoparticles exhibit a rather poor performance. This is prob-ably due to the fact that both components provide rather facile electron transfer kinetics. However, it is also important to note that there is a significant difference in the analytical perfor-mance of electrode prepared without polymer. This outcome might be related to the fact that the amino-phenyl layer is strongly electrostatic (positively charged). It is possible that this amino-phenyl layer is electrostatically attracting the ss-DNA probe layer. Hence, electrode modified with nitrophenyl group exhibits a rather good performance, but its LOD value is still slightly less than the electrode prepared with polymer, which proves that PS-Psy-Im copolymer have a significant impact on the analytical performance of the designed electro-chemical DNA biosensor.
Reproducibility and stability
Reproducibility and stability are recognised as key factors in developing an applicable biosensor. It was found that DNA biosensor can be regenerated by incubation of biosensor in hot water (75 °C) for 15 min. DPV measurements proved that electrochemical DNA biosensor can be repetitively used since it exhibits only about 10% loss of original performance after ten regeneration cycles (please see Fig.S7). The stability of the biosensor was also tested by storing the electrode in the fridge. DNA biosensor nearly maintained its original perfor-mance after 30 days (please see Fig.S8).
Conclusion
The binding properties of the PS-PSyIm polymer to ds-DNA and its implementation area in the development of an electro-chemical DNA biosensor were investigated by CV and DPV. It was found that PS-PSyIm polymer intercalates into the base pairs of the ds-DNA for the solution and surface-based exper-iments. It was also shown that DNA biosensor, constructed via the immobilisation of PS-PSyIm polymer onto gold nanopar-ticles electrochemically deposited on NH2-modified graphene
oxide, permits to determine the label-free hybridisation of DNA since it is capable of distinguishing the electrochemical signal obtained for single-base mismatched DNA sequence from the one recorded for complementary ss-DNA. The ana-lytical performance of the biosensor was also explored, and the limit of detection is calculated as 1.8 nmol L−1.
Acknowledgements Timur Sanal thanks The Scientific and Technological Research Council of Turkey (TUBITAK) for scholarship. The authors also thank Bulent Ecevit University Scientific Research Project Coordination Unit for financial support (project number 201572118496-09).
References
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0,0 2,0x10-9 4,0x10-9 6,0x10-9 8,0x10-9 1,0x10-8 0,002 0,004 0,006 0,008 0,010 0,012 I / mA E / V vs (Ag / AgCl)
Fig. 9 Plot of I vs. the concentration of complementary target DNA in phosphate buffer (pH 7) for immobilised Ps-PsyIm onto GC/GO/NH2/
GNP (black line), without GO (red line), without AuNP (blue line), without PS-PSyIm polymer (pink line) and without nitrophenyl diazonium (green line)
Table 1 LOD values for various type of electrochemical DNA biosensor
Electrode LOD (nmol L−1)
GC/GO/NH2/GNP/PS-PSy-Im 1.20
GC/NH2/GNP/PS-PSy-Im 3.50
GC/GO/NH2/PS-PSy-Im 3.10
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