Thin Solid Films 518 (2009) 338–342

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Electrochemical characterization of deoxyribonucleic acid onaluminum(III)/poly(L-glutamic acid) film and its application for the detection ofphosphinothricin acetyltransferase gene-specific sequence

Na Zhou, Tao Yang, Yongchun Zhang, Chen Jiang, Kui Jiao ⁎Key Laboratory of Eco-chemical Engineering (Ministry of Education), College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,Qingdao 266042, China

⁎ Corresponding author. Tel.: +86 532 84855977; faxE-mail address: kjiao@qust.edu.cn (K. Jiao).

0040-6090/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.tsf.2009.06.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 June 2008Received in revised form 3 June 2009Accepted 4 June 2009Available online 13 June 2009

Keywords:Poly(L-glutamic acid)DNA electrochemical biosensorPAT transgeneElectrochemical impedance spectroscopy

A simple strategy of transgenic sequence-specific detection without a special amplification procedure wasdeveloped on the basis of aluminum(III)/poly(L-glutamic acid) (PLGA) film. An aluminum ion (Al(III)) thinfilm was assembled on the surface of PLGA via the electrostatic binding of Al(III) with carboxyl, namely Al(III)/PLGA. The immobilization of deoxyribonucleic acid (DNA) was carried out on this Al(III)/PLGA film by Al(III)-single strand DNA (ssDNA) interaction. Surface hybridization between the immobilized ssDNA and itscomplementary ssDNAwasmonitored byelectrochemical impedance spectroscopy (EIS) using [Fe(CN)6]3−/4− asa redox probe. Under the optimal conditions, this DNA electrochemical sensor was applied to determine thespecific gene sequence related to phosphinothricin acetyltransferase transgene (PAT) in the transgenic plants bylabel-free EIS.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

During the past decade, more and more transgenic crops havereached the marketplace [1]. However, the safety of the transgenicplants for the consumer and environment has attractedwide attention[2]. Current research suggests that the accurate, sensitive and rapiddetection of the transgenic plants look forward to the detection ofsequence-specific deoxyribonucleic acid (DNA). A variety of tech-niques have been developed for the detection of DNA hybridization,such as electrochemistry, fluorescence, radiochemistry, piezoelectro-nics, surface plasmon resonance spectroscopy, and quartz crystalmicrobalance. Among all these developed technologies, electro-chemical DNA biosensor possesses the advantages of high sensitivity,compatibility with modern microfabrication, portability and low-costfor genetic detection. The efficient immobilization of DNA chain or aspecific sequence is an important factor in the fabrication of reliableelectrochemical genosensors. The earlier reports on the immobi-lization methods include adsorption [3–6], electropolymerization [5–11], self-assembly monolayer [12] and covalent binding [13–17]. Inaddition, considerable research has also been investigated toimmobilize DNA on metal ion membrane [18–22]. Wang and Bard[19] described a method based on the positively charged aluminum

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(III) membrane for immobilization of nucleic acids on self-assembledmonolayer at Au substrates. Thompson et al. [20] prepared a simpleand direct electrochemical DNA biosensor with the help of magne-sium cations. The oligonucleotide was linked to the conductingpolymer by forming a bidentate complex between Mg2+ and an alkylphosphonic acid group on the polymer and the phosphate group ofthe DNA. Recently, our group has also reported some assays on DNAimmobilization and hybridization via metal ion membrane [21,22].

Some amino acids, such as threonine [23], L-serine [24], L-lysine[25], and L-glutamic acid (LGA) [26,27], have been reported to beelectropolymerized at the electrode surface. Ma and Sun [23]fabricated a promising electrochemical sensor via electrochemicalimmobilization of poly(L-threonine) on a glassy carbon electrode,which exhibited superior electrocatalytic activity towards bothdopamine and epinephrine. Jiang et al. [25] electropolymerized L-lysine onto a carboxylic group-functionalized single-walled carbonnanotubes (SWNTs-COOH) modified electrode and detected the DNAhybridization by using the impedance changes provoked by DNA afterhybridization.

In this study, a label-free electrochemical DNA sensor based onaluminum ion/poly-L-glutamic acid (PLGA)membranewas fabricated.The aluminum ion (Al(III)) thin membrane can be adsorbed on thesurface of PLGA owing to the abundant carboxyl groups of PLGA. Thenthe probe DNA was immobilized on the positively charged Al(III) filmsurface (shown in Fig. 1) through the electrostatic attraction betweenthe Al(III) and the negatively charged phosphate backbone of DNA toform a stable DNA biosensor. Electrochemical impedance

Fig. 1. Representation of the immobilization and hybridization of DNA on Al(III)/PLGA/CPE.

339N. Zhou et al. / Thin Solid Films 518 (2009) 338–342

spectroscopy (EIS) was adopted to monitor and characterize theprocesses of immobilization and hybridization of DNA. The specificgene sequence of phosphinothricin acetyltransferase gene (PAT),which is an important transgene in many genetically modified crops,was detected sensitively by label-free EIS with this DNA electro-chemical biosensor.

2. Experimental details

2.1. Apparatus and reagents

A CHI 660C electrochemical analyzer (Shanghai CH InstrumentCompany, China), which was in connectionwith a home-made carbonpaste modified working electrode (Ф=4 mm), a saturated calomelreference electrode (SCE) and a platinumwire auxiliary electrode, wasused for the electrochemical measurement. The pH values of allsolutions were measured by a model pHS-25 digital acidimeter(Shanghai Leici Factory, China).

Herring sperm DNA was purchased from Beijing Jingke ReagentCompany and used without further purification. Solution of DNA inTris–HCl buffer solution gave a ratio of the UV absorbance at 260 nm to280 nm, A260/A280N1.8, indicating that the DNA is sufficiently free ofprotein. Denatured single-stranded DNA (ssDNA) was produced byheating native herring sperm DNA solution in a water bath at 100 °Cfor 30 min, followed by rapidly cooling in an ice bath.

Graphite powder and sodium dodecylsulfate (SDS) were purchasedfrom Shanghai Reagent Company and used as received. LGA waspurchased from Shanghai Zhengxiang Reagent Company. Al(NO3)3 waspurchased from Tianjin Chemical Engineering Co. Ltd. Tris(hydroxy-methyl) amminomethane (Tris) was got from Sigma. All the chemicalsare of analytical grade and solutionswere preparedwith doubly distilledwater.

The 20-base oligonucleotides probe, its complementary sequenceDNA (cDNA, target DNA, namely a 20-base fragment of PAT genesequence, which was selected according to the transgenic sequence ofphosphinothricin acetyltransferase gene in some transgenic plants),single-base mismatched DNA, double-base mismatched DNA andnoncomplementary sequence DNA (ncDNA) were synthesized byBeijing SBS Gene Technology Co. Ltd. Their base sequences are thesame as the previous work [25].

All oligonucleotides stock solutions of 20-base oligomers(50 µmol/L) were prepared using Tris–HCl solution (5.0 mmol/LTris–HCl, 50.0 mmol/L NaCl, pH 7.0) and stored at 4 °C. More dilutedsolutions were obtained via diluting aliquot of the stock solution withdoubly distilled water prior to use. The hybridization solutioncontaining the target DNA was diluted with 2×SSC (saline-sodiumcitrate, 0.30 mol/L NaCl and 30 mmol/L sodium citrate tribasicdihydrate (C6H5Na3O7·2H2O).

2.2. Procedure

2.2.1. Preparation of Al(III)/poly(L-glutamic acid) modified carbon pasteelectrode

3.0 g graphite powder and 1.0 g solid paraffinwere heated at 80 °Cfor 2 h and then mixed to produce a homogenous carbon paste. Forfabrication of the electrode, the prepared carbon paste was tightlypacked into a glass tube from one end (Ф=4 mm) and a copper wirewas introduced into the other end for electrical contact. A freshelectrode surface was generated rapidly by extruding a small plug ofthe paste with a stainless steel rod that was used to pack the carbonpaste tightly and smoothing the surface onwhite paper until a smoothsurface was obtained.

The PLGA/CPE was obtained by means of electropolymerization ofthe monomer using 2.5 mmol/L LGA in a 50 mmol/L phosphate buffersolution of pH 7.0. A successive cyclic voltammetric scan (1 to20 cycles) from −1.2 V to +2.0 V at 100 mV/s was carried out. Themethod is based on the modification of glassy carbon electrode, fibercarbon electrode, or gold electrodewithmembrane of PLGA generatedby direct electrooxidation of their monomer [26,27]. The PLGA/CPEwas then soaked in a 5.0 mmol/L Al(NO3)3 for 2 h to complete thepreparation of an “activated” electrode, which was denoted as Al(III)/PLGA/CPE.

2.2.2. Immobilization and hybridization of DNAThe immobilization of the DNA probe on the electrode surface

was carried out with following procedure: The Al(III)/PLGA/CPEelectrode was immersed in 1.5 mL Tris–HCl buffer (pH 7.0) solutioncontaining 1.0 µmol/L probe DNA for 500 s by constant potentialadsorption at 0.5 V, followed by washing the electrode with 0.2% SDSsolution for 5 min and then rinsing it with doubly distilled water. TheSDS could remove the unimmobilized ssDNA. Thus, the probecaptured electrode (ssDNA/Al(III)/PLGA/CPE) was ready for use.Hybridization reaction was conducted by immersing the ssDNA/Al(III)/PLGA/CPE into a stirred hybridization solution containing thetarget DNA and holding the working potential at +0.4 V for 500 s.Then the electrode was washed with 0.2% SDS for 5 min to removethe unhybridized target DNA and this hybridization modified elec-trode was denoted as dsDNA/Al(III)/PLGA/CPE. The representation ofthe immobilization and hybridization of DNA on Al(III)/PLGA/CPEwas shown in Fig. 1.

2.2.3. Electrochemical impedance spectroscopyEIS measurements were performed with a CHI 660C Electroche-

mical Analyzer in 0.5 mmol/L K3[Fe(CN)6] and 0.5 mmol/L K4[Fe(CN)6] (1:1) solution containing 0.1 mol/L KCl. The AC voltageamplitude was 5 mV and the voltage frequency range was from100 kHz to 0.01 Hz. The applied potential was 172 mV vs. SCE. Thereported result for every electrode in this paper was the mean value ofthree parallel measurements.

3. Results and discussion

3.1. Electrochemical impedance spectroscopy of DNA/Al(III)/PLGAmodified electrode

EIS is a useful tool for analyzing the changes in interfacialproperties of modified electrodes (or semiconductor) induced bybinding of charged biomolecules on the surfaces. The advantages ofEIS are listed as follows: Firstly, the detectionmethod is noninvasive ascompared with voltammetric method. Secondly, the voltammetricmeasurement is often inhibited by the highly insulating nature of thebiomolecules, which often scaffold on the electrode surface, whereasimpedance measurement can be conducted well even on insulatingsubstrates. Thirdly, this technique uses very small amplitude voltagesignals to avoid disturbing themeasured properties [28,29]. Therefore,

Fig. 2. Nyquist diagrams for the electrochemical impedance measurements in 0.1 mol/LKCl containing 0.5 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) at (a) Al(III)/PLGA/CPE,(b) ssDNA/Al(III)/PLGA/CPE and (c) dsDNA/Al(III)/PLGA/CPE.

Fig. 4. Nyquist diagrams for ssDNA/Al(III)/PLGA/CPE electrode with different ssDNAimmobilization times: (a) 0 s; (b) 100 s; (c) 200 s; (d) 300 s; (e) 400 s; (f) 500 s; (g) 600 s.0.5mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] was used as a redox probe. Immobilization potential:0.5 V.

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impedancemeasurements have beenwidely applied for the investiga-tion of DNA hybridization [30–34].

Herein, EIS is used with [Fe(CN)6]3−/4− redox couple as the redoxprobe. Fig. 2 illustrates the impedance changes of [Fe(CN)6]3−/4−

during the stepwise electrode modification process. Generally, theimpedance spectra include a semicircle portion and a linear portion.The semicircle portion (observed at high frequencies), corresponds tothe electron-transfer limiting process. The interfacial electron-transferresistance (Ret) of the electrodes can be directly measured for theredox probe as the semicircle diameter. From curve a, the Ret at theAl(III)/PLGA/CPE surface was 646 Ω. After the ssDNA probe wasattached to the Al(III)/PLGA/CPE, the Ret value increased to 2536 Ω(curve b), illustrating the negatively charged phosphate backbone ofthe probe ssDNA prevented [Fe(CN)6]3−/4− from reaching theelectrode surface during the redox process and therefore led to alarger Ret value. After hybridization of the ssDNA probe with thecomplementary DNA, the Ret value further increased to 4515 Ω(curve c). After hybridization, the negative charges on the electrodesurface increased remarkably and the surface membranes becomethicker. These two factors led to the further increase of the Ret value.

Fig. 3. The effect of the immobilization potential on the electron-transfer resistance of0.5 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] at ssDNA/Al(III)/PLGA/CPE. The immobilizationtime: 500 s.

The impedance measurements confirmed that the ssDNA/Al(III)/PLGA/CPE could recognize and detect the target DNA.

3.2. Optimization of DNA immobilization and hybridization conditions

In order to obtain a sensitive and quantitative genomic assayprocedure, the influence of relevant experimental parameters, namelythe immobilization potential, the immobilization time, the hybridiza-tion potential and the hybridization time was investigated in detail.

The Ret value at the ssDNA/Al(III)/PLGA/CPE electrode increasedwith the positive shift of the immobilization potential and reached amaximum at the immobilization potential of +0.5 V (Fig. 3). Whenthe immobilization potential was more positive than +0.5 V, Retreached a constant level. So, +0.5 Vwas chosen as the immobilizationpotential.

The influence of the immobilization time is shown in Fig. 4. Theresults showed that with the increase of the immobilization time, theRet value of the membranes increased initially and then declined,having a maximum Ret value at 500 s. Thus, 500 s was chosen as theoptimal immobilization time.

According to the samemethod, the hybridization potential and thehybridization time were also explored, respectively. According to the

Fig. 5. Nyquist diagrams recorded at (a) ssDNA/Al(III)/PLGA/CPE, (b) dsDNA/Al(III)/PLGA/CPE (hybridized with cDNA), (c) the electrode hybridized with ncDNA, (d) theelectrode hybridized with single-base mismatched DNA, (e) the electrode hybridizedwith double-base mismatched DNA. Conditions are the same as in Fig. 2.

Fig. 6. Nyquist diagrams recorded at ssDNA/Al(III)/PLGA/CPE and after hybridizationreaction with its complementary PAT gene sequence of different concentrations (mol/L):(a) 0, (b) 1×10−11, (c) 2×10−11, (d) 4×10−11, (e) 6×10−11, (f) 8×10−11, (g) 1×10−10.Conditions are the same as in Fig. 2.

341N. Zhou et al. / Thin Solid Films 518 (2009) 338–342

experimental results, 500 s was generally used as the hybridizationtime and +0.4 V as the hybridization potential in our experiments.

3.3. Sequence-specific detection related to PAT gene by label-freeelectrochemical impedance spectroscopic method

3.3.1. Selectivity of DNA hybridization recognitionThe selectivity of DNA hybridization could be judged by the

hybridization of the probe DNA with different DNA sequences. Asshown in Fig. 5, the curve a was the Nyquist diagram of [Fe(CN)6]3−/4−

at the probe DNA modified electrode. After hybridization of the probeDNA with the complementary DNA under the optimal experimentalconditions, the Nyquist diagram of [Fe(CN)6]3−/4− was shown as thecurve b. The Ret value rose obviously. When the noncomplementarysequence was used for the hybridization, the Ret value (curve c) variednegligibly as compared with the probe DNA modified electrode. Thesingle-base mismatched sequence (curve d) and the double-basemismatched sequence (curve e) could also be recognized via comparingthe change of the Ret value of [Fe(CN)6]3−/4−. The results demonstratedthat Ret was a suitable signal for sensing the different DNA sequences.

3.3.2. Sensitivity of DNA hybridization recognitionThe sensing of DNA at the Al(III)/PLGA/CPE was investigated at

different concentrations of the target DNA. The difference (ΔRet)between the Ret value at the probe ssDNA/Al(III)/PLGA/CPE electrodeand that at the hybridization electrode (dsDNA/Al(III)/PLGA/CPE)was used as the detection signal for the determination of thesequence-specific DNA related to the PAT gene. The ΔRet had a linearrelationship with the logarithm of the PAT gene-sequence concentra-tions. The dynamic determination range for the PAT gene sequencewas from 1.0×10−11 to 1.0×10−6 mol/L with the regression equation:ΔRet (Ω)=27.18 lgC+18.75, and the correlation coefficientγ=0.9933. The detection limit was 3.03×10−12 mol/L using 3σ,where σ was the standard deviation of 11 parallel measurements ofthe blank solution.

Owing to the effective combination of the excellent electron-transfer ability of PLGA, the high bioaffinity of aluminum ion mem-brane and a label-free detection using the EIS, the sensitivity for thePAT gene-sequence determination was greatly enhanced and thedetection limit was largely lowered as compared with our previousreports based on metal ion membrane, and the results are shown inTable 1.

From Fig. 6, it can be seen that this label-free DNA biosensor alsohad still nice recognition ability to the low concentrations ofcomplementary target DNA (1.0×10−11 to 1.0×10−10 mol/L).

3.4. Precision, regeneration and stability of DNA sensor

The precision of this impedance-based DNA hybridization sensorwas examined by a parallel measurementmethod for a low concentra-tion of target DNA sequence. The relative standard deviation for sevenparallel detections of 10−9 mol/L target DNA sequence was 5.8%,

Table 1Comparison of performance of the proposed biosensorwith those of other DNAbiosensorsbased on metal ion membrane.

Method This work Ref. [21] Ref. [22]

Membranes for DNAimmobilization

Al(III)/PLGA/CPE Al(III)/CPE Mg/PDC/GCE

Detection method EIS DPV with MB asindicator

EIS

Detection limit (mol/L) 3.03×10−12 2.25×10−8 3.4×10−10

Detection range of targetDNA (mol/L)

1.0×10−11−1.0×10−6

1.0×10−7−1.0×10−4

1.0×10−9−1.0×10−5

Differential pulse voltammetry (DPV); methylene blue (MB).

which showed that the precision of this DNA hybridization sensor wasgood.

The regeneration ability for this impedance-based DNA hybridi-zation sensorwas also evaluated by denaturing the hybridized dsDNAbound on the electrode surface, and the regenerated ssDNA/Al(III)/PLGA/CPE was used repetitively. The hybridized dsDNA electrodewas immersed into boiling water for 8 min, and then cooled downrapidly with the ice salt bath, followed by rinsing the electrode withthe doubly distilled water. The Nyquist diagrams of 0.5 mmol/L [Fe(CN)6]3−/4− solution at the regenerated ssDNA/Al(III)/PLGA/CPEand the hybridized dsDNA/Al(III)/PLGA/CPE were recorded. Theresults indicated that the two Ret values and ΔRet value were respec-tively almost the same values as those obtained in the first ex-periment. Repetitive experiments showed that the DNA sensor couldbe reproduced for 3 times without losing its sensitivity. However,after 3 times regeneration the Ret values and ΔRet value reducedrapidly, probably due to moving of the immobilized ssDNA off theelectrode.

An understanding of the stability of the ssDNA/Al(III)/PLGA/CPEwas obtained from the following experiments. The electrode wasincubated in the doubly distilled water, PBS of pH 7.0, Tris–HCl buffersolution of pH 7.0 and 2×SSC solution of pH 7.2 at 30 °C for 48 h,respectively, followed by rinsing the electrode with doubly distilledwater, and hybridized with the target DNA sequence and measured byEIS according to the procedure. The results showed that the incubatedelectrode had the same behavior as the unincubated electrode. So, theDNA hybridization sensor had good stability.

4. Conclusion

In conclusion, a hybridization biosensor for electrochemicaldetection of PAT gene sequences based on Al(III)/PLGA membranewas developed. The PLGA/CPE, which is abundant in carboxylic acidgroups on its surfaces, is convenient for fabrication of aluminum ionthin membrane. Al(III)/PLGA membrane is a good biocompatibleplatform to immobilize the probe DNA via electrostatic adsorption.The conditions for DNA immobilization and hybridization wereoptimized. The sequence-specific DNA of the PAT gene was detectedby this label-free DNA hybridization biosensor. The low detection limitof 3.03×10−12 mol/L may be attributed to the excellent electron-transfer ability of PLGA, the high biocompatibility of aluminum ionand a label-free electrochemical impedance detection.

342 N. Zhou et al. / Thin Solid Films 518 (2009) 338–342

Acknowledgements

This work was supported by the National Natural Science Foundationof China (No. 20635020, No. 20805025), Doctoral Foundation of theMinistry of Education of China (No. 20060426001), Foundation ofQingdao City (No. 09-1-3-25-jch) andDoctoral Fund of QUST (0022278).

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