electrochemical biosensor for detection of bcr/abl fusion gene based on hairpin locked nucleic acids...
TRANSCRIPT
Electrochemistry Communications 11 (2009) 1650–1653
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Electrochemistry Communications
journal homepage: www.elsevier .com/locate /e lecom
Electrochemical biosensor for detection of BCR/ABL fusion gene basedon hairpin locked nucleic acids probe
Liqing Lin a, Xinhua Lin a,*, Jinghua Chen a, Wei Chen a, Miao He a, Yuanzhong Chen b,*
a Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, Chinab Fujian Institute of Hematology, The Affiliated Union Hospital of Fujian Medical University, Fuzhou 350001, China
a r t i c l e i n f o
Article history:Received 21 May 2009Received in revised form 10 June 2009Accepted 18 June 2009Available online 23 June 2009
Keywords:Electrochemical biosensorBCR/ABL fusion geneLocked nucleic acids
1388-2481/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.elecom.2009.06.015
* Corresponding authors. Tel./fax: +86 591 22862083357896 (Y. Chen).
E-mail addresses: [email protected] (X. Lin), che
a b s t r a c t
This communication reports on a novel biosensor to study the hybridization specificity by using thiolatedhairpin locked nucleic acids (LNA) as the capture probe. The LNA probe was immobilized on the gold elec-trode through sulfur–Au interaction and could selectively hybridize with its target DNA. Differentialpulse voltammetry (DPV) was used to monitor the hybridization reaction on the probe electrode. Thedecrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridiza-tion of the probe with the target DNA. The results indicated this new method has excellent specificity forsingle-base mismatch and complementary after hybridization, and a high sensitivity. This LNA probe hasbeen used for assay of fusion gene in Chronic Myelogenous Leukemia (CML) of the real sample with sat-isfactory result.
� 2009 Elsevier B.V. All rights reserved.
1. Introduction
In recent years, there has been significant progress in develop-ing nucleic acid hybridization biosensors for the rapid and accuratedetections of specific gene sequence [1–3]. As with other types ofbiosensors, high selectivity is crucial for the success of hybridiza-tion biosensors [4]. The selectivity of nucleic acid hybridization as-says depends primarily on the selection of the probe and then ofthe hybridization conditions. Thus, the design of the probe is themost important pre-analytical step. Recently, some reports de-scribed the synthesis and hybridization of a novel nucleotidetermed LNA [5,6].
LNA is a nucleic acid analogue of RNA, in which the furanosering of the ribose sugar is chemically locked by the introductionof a methylene linkage between O2 and C40. The covalent bridgeeffectively ‘locks’ the ribose in the N-type (3-endo) conformationthat is dominant in A-form DNA and RNA. This conformation en-hances base stacking and phosphate backbone pre-organizationand results in improved affinity for complementary DNA orRNA sequences, with each LNA substitution increasing the melt-ing temperatures (Tm) by as much as 3.0–9.6 �C [7,8]. LNA basescan be interspersed with DNA bases, allowing binding affinity tobe tailored for individual applications. Due to the very high affin-ity of the LNA molecules, it demonstrates that LNA probes
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16 (X. Lin), tel./fax: +86 591
[email protected] (Y. Chen).
hybridize with very high affinity to perfectly complementary tar-gets, and at the same time shows an extraordinary specificity todiscriminate the targets that differ by a single-base.
In this study, an electrochemical DNA biosensor was devel-oped for recognition of target DNA by using hairpin LNA as theprobe for hybridization with BCR/ABL fusion gene in CML. Thisnovel probe demonstrates its excellent specificity for single-basemismatch and complementary after hybridization, and this probehas been used for assay of PCR real sample with satisfactoryresult.
2. Experimental
2.1. Reagents
Methylene blue (MB) and 6-mercapto-l-hexanol (MCH) werepurchased from Sigma (USA). The oligonucleotides were synthe-sized by Shanghai Shenggong Biotechnology Co. (Shanghai, China),and it has the following sequences:
hairpin LNA probe (S1): 5’-SH-CTLG CLAG ALGT TLCA ALAA GLCCCLTT CGC AG–3’, LNA probe is a DNA–LNA chimera, but for brevityit will be called LNA probe. Complementary target (S2): 5’-GAAGGG CTT TTG AAC TCT-3’, single-base mismatch (S3): 5’-GAAGGG ATT TTG AAC TCT-3’, non-complementary (S4): 5’-ACG TAATCC CCA GCT CTC-3’.
The oligonucleotide stock solutions (100 lM) were preparedwith 20 mM Tris–HCl, 10 mMMgCl2, pH 8.0 and kept frozen. Milli-pore Milli Q water was used in all experiments. Unless otherwise
L. Lin et al. / Electrochemistry Communications 11 (2009) 1650–1653 1651
indicated, all reagents and solvents were purchased in their highestavailable purity and used without further purification.
2.2. Preparation of electrochemical DNA biosensors
The scheme for preparation of the electrochemical DNA biosen-sor is illustrated in Fig. 1. The gold electrode (AuE) was mechani-cally polished with 0.3 lm, 0.05 lm Al2O3 suspension,respectively, and washed ultrasonically with ethanol and Milli-Qwater, subsequently electrochemically cleaned in 0.5 M H2SO4 bypotential scanning between �0.35 and 1.5 V until a reproduciblecyclic voltammogram was obtained. Then it was rinsed with waterand blown dry with nitrogen. Finally the cleaned AuE was im-mersed in 1 mM S1 solution in 50 mM Tris–HCl at pH 7.0 and incu-bated 2 h. Then the modified electrode was thoroughly rinsed with0.1 M PBS to remove the weakly adsorbed S1. The probe modifiedelectrode was donated as S1/AuE. S1/AuE was then immersed in1 mM MCH for 1 h to obtain S1/MCH/AuE. Hybridization was per-formed by pipetting 5 ll target DNA onto S1/MCH/AuE surface,respectively, and proceeding for 1 h. MB was accumulated ontothe surface of hybrid-modified AuE by immersing the electrodeinto stirred 0.1 M Tris–HCl containing 20 mM MB with 50 mMNaCl for 5 min without applying any potential. After accumulationof MB, the electrode was rinsed with 0.1 M PBS to remove the non-specific MB and subjected to electrochemical measurement.
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Target-ssDNA
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Au
Au
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H 3 CMB
Fig. 1. Scheme of the procedure for t
3. Results and discussion
3.1. Spectrophotometric characterization of DNA and LNA probes
The melting temperatures (Tm) for the hybridization of LNAwith their complemental DNA were examined to confirm their po-tential for selective recognition of complementary sequences. TheTm value of the S1 (78.8 �C) was greatly higher than that of its cor-responding DNA probes (50.3 �C). Comparing with the analogousDNA–DNA hybrids, Tm value for LNA binding to DNA was in-creased 28.5 �C, which was equivalent to 4.1 �C per LNA comparedto DNA duplexes [9,10]. Tm value for LNA binding to S3 was de-creased to 38.1 �C. Therefore, the affinity and specificity of theLNA probes can be confirmed by measurement of duplex Tm.
Theoretically, the optimum hybridization temperature wasabout 20 �C lower than Tm. By monitoring the UV light absorp-tion at 260 nm using the spectrophotometer, the Tm value ofthe LNA probe (S1) binding to complementary target (S2) was78.8 �C, while for single-base mismatch (S3) was decreased to38.1 �C. Therefore, the theoretical hybridization temperaturewas 58.8 and 18.1 �C for the S2 and S3, respectively. Accordingly,60 �C was selected as hybridization temperature [11]. At thistemperature, hybridization could be occurred only for the S2
strand but not for the S3. Therefore, the hybridization specificitywas dramatically increased.
e
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Au
NCH 3
CH 3
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he fabrication of DNA biosensor.
1652 L. Lin et al. / Electrochemistry Communications 11 (2009) 1650–1653
3.2. Electrochemical responses of hairpin LNA probe
Fig. 2A shows cyclic voltammograms (CV) of 10 mM K3Fe(CN)6
using a scan rate of 100 mV/s in PBS buffer at bare AuE(a), S1/MCH/AuE(b), S1/AuE(c) and S1–S2/MCH/AuE(d). Curve a is the CVat a freshly polished bare Au electrode. Which has a pair of well-defined voltammetric peaks. Curve c is the CV at the electrodemodified by hairpin LNA probe (S1), the peak current of which de-creased dramatically and the peak separation increased obviouslyas compared with curve a. The results indicated that the self-assembly of S1 on the electrode surface generates a negativelycharged interface that repels negatively charged [Fe(CN)6]3� an-ions. This repulsion is anticipated to retard the interfacialelectron-transfer kinetics of the [Fe(CN)6]3� at the electrode inter-face. The peak-to-peak separation of the reaction at S1/MCH/AuE isalmost the same as that at the S1/AuE electrode with a less signif-icant decrease in the current, indicating that the MCH monolayerhas a negligible effect on blocking interfacial electron-transfer.MCH, as a short-chain alkyl molecule, forms a self-assembledmonolayer that contains many pinholes and [Fe(CN)6]3� is suffi-ciently small to freely penetrate through these pinholes. Almostcomplete disappearance of the current is observed after bindingof S1–S2. After hybridization, there were more negative chargeson the surface and the DNA modified layer became thicker. Thesetwo factors provide an effective barrier to electron-transfer of[Fe(CN)6]3� in solution.
0 .0 0 .2 0 .4 0 .6
-80
-40
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40
I/µA
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0 400 800 1200 1600 2000 2400
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/Ohm
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Fig. 2. (A) CV of 10 mM K3Fe(CN)6 for bare AuE (a), S1/MCH/AuE (b), S1/AuE (c) andS1–S2/MCH/AuE (d). (B)Impedance plots on bare AuE (a), S1/MCH /AuE (b), S1/AuE(c) and S1–S2/MCH /AuE (d) in 10 mM [Fe(CN)6]4�/3�.
Fig. 2B shows the results of Electrochemical Impedance Spec-troscopy at bare AuE(a), S1/MCH/AuE(b), S1/AuE(c) and S1–S2/MCH/AuE(d) in the presence of equimolar [Fe(CN)6]4�/3�, respec-tively. The Randle modified equivalent circuit was used to fit theimpedance spectroscopy and to determine electrical parametervalues for each step [12]. The bare AuE shows a very small semicir-cle domain implying very fast electron-transfer process with a dif-fusion-controlled. The self-assembly of a negatively charged LNAprobe (S1) on the electrode surface effectively reduces the responseof the [Fe(CN)6]4�/3� anions and thus leads to enhanced electron-transfer resistance. The increase in electron-transfer resistanceindicates that the LNA probe is successfully immobilized on theelectrode surface. This is reflected by the appearance of the semi-circle part of the impedance spectrum with Ret = 881 X. The addi-tion of the MCH results in a relatively small electron-transferresistance Ret (765 X). The thiolated hairpin LNA is adsorbed spe-cifically through sulfur–Au interaction and nonspecifically throughthe nitrogen-containing bases. After treatment with MCH, the non-specifically adsorbed nitrogen-containing bases are largely re-moved from the surface and only one end of the LNA probe isbound to the surface with all the relevant bases freely availablefor reaction with DNA [13,14]. Such conformation along with theless densely packed monolayer of MCH could account for the de-crease in the value of electron-transfer resistance Ret of the S1/MCH/AuE compared with the S1/AuE. After the recognition reactionof the S1/MCH/AuE with complementary target (S2) for 60 min,there is an increase of Ret (1541 X), originating from the blockingof the electrode surface with the bulky negatively charged phos-phate group. These results are in agreement with the CV data.
-0.3 0.0 0.3 0.6
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I/ µA
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C/nM
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a
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I µA =-0.4206C + 10.355
r = 0.9983
Fig. 3. (A) DPV of 20 lM MB at a S1/MCH/AuE (d) and after hybridization with theS2 (a), S3 (b) and S4 (c). (B) DPV of MB accumulated on the S1 after its hybridizationwith different concentration of S2. S2 concentration (�10�9 M): (a) 1.0; (b) 4.0; (c)7.0; (d) 10.0; (e) 13.0; (f) 16.0.
L. Lin et al. / Electrochemistry Communications 11 (2009) 1650–1653 1653
3.3. Hybridization specificity of LNA probe
S1 has high hybridization specificity because of its LNA andloop–stem structure, which can easily discriminate S2 from S3.The selectivity of this assay was investigated by using the S1 asthe capture probe to hybridize with various DNA sequences.Fig. 3A shows the DPV signal of MB[15,16] at S1/MCH/AuE(d) andafter hybridization with S2(a), S3(b) and S4(c). It is clear thatFig. 3A (d) shows the highest peak current of MB on the S1/MCH/AuE, and after hybridization with S2, the peak current of MB is dra-matically decreased. It can also easily discriminate the S2 from S3.In the presence of oligonucleotide containing a single-base mis-match, significantly increased voltammetric signal can be ob-served, which indicates that the complete hybridization is notaccomplished due to the base mismatch. So the S1 has high hybrid-ization specificity for the S2 and the S3. In addition, as expected, nosignificant difference of peak current can be observed for the S1and its hybridization with S4, since no successful hybridization oc-curs due to the sequence mismatch between the S1 and S4. The sen-sitivity was studied and results were displayed in Fig. 3B. Adetection limit is 1.2 � 10–10 M.
3.4. Detection of the PCR sample
When the S1 for BCR/ABL was immobilized and hybridized withreal PCR samples. The DPV signals of MB obtained from the S1 gavea mean average of 10.72 lA with a RSD of 5.13%. The DPV signals ofMB obtained from the hybridization of the S1 with the positive andnegative real samples gave mean average of 5.86 lA with RSD of6.74% and 9.95 lA with RSD of 8.12%, respectively. The signal ofMB for the BCR/ABL probe was much higher than that of BCR/ABL probe hybridization with positive real sample. The resultsshowed that the electrochemical DNA biosensors are in goodagreement with those obtained from the gel electrophoresis.
4. Conclusions
In this communication, electrochemical DNA biosensor basedon hairpin LNA capture probe was investigated. This new biosensor
was used to detect BCR/ABL fusion gene in CML. The results illus-trated that the LNA probe biosensor has high hybridization speci-ficity. The proposed method provided a simple, rapid tool fordetection of DNA species in CML. It might have a promising futurefor investigation of DNA hybridization and solve the actual prob-lem of the early diagnosis and prognosis monitor of CML and otherdiseases.
Acknowledgements
The authors gratefully acknowledge the financial support of theNational Natural Science Foundation of China (20675015), thefinancial support of the National High Technology and Develop-ment of China (863 Project: 2006AA02Z4Z1 and 2008AA02Z433).
References
[1] Y. Zhang, N.F. Hu, Electrochem. Commun. 9 (2007) 35.[2] A. Sassolas, B.D. Leca-Bouvier, L.J. Blum, Chem. Rev. 108 (2008) 109.[3] J.H. Chen, J. Zhang, K. Wang, L.Y. Huang, X.H. Lin, G.N. Chen, Electrochem.
Commun. 10 (2008) 1448.[4] E.L.S. Wong, P. Erohkin, J.J. Gooding, Electrochem. Commun. 6 (2004) 648.[5] J. Wang, Biosens. Bioelectron. 13 (1998) 757.[6] S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi, T. Imanishi, Tetrahedron
Lett. 39 (1998) 5401.[7] C.Y. James Yang, L. Wang, Y.R. Wu, Y.M. Kin, C.D. Medley, H. Lin, W.H. Tan,
Nucleic Acids Res. 35 (2007) 4030.[8] J. Wengel, Acc. Chem. Res. 32 (1999) 301.[9] Y. You, B.G. Moreira, M.A. Behlke, R. Owczarzy, Nucleic Acids Res. 34 (2006)
e60.[10] O. Richard, Biophys. Chem. 117 (2005) 207.[11] X.H. Lin, P. Wu, W. Chen, Y.F. Zhang, X.H. Xia, Talanta 72 (2007) 468.[12] A.E. Radi, J.L.A. Sanchez, E. Baldrich, C.K. O’Sullivan, Anal. Chem. 77 (2005)
6320.[13] R. Levicky, T.M. Herne, M.J. Tarlov, S.K. Satija, J. Am. Chem. Soc. 120 (1998)
9787.[14] V. Dharuman, Jong Hoon Hahn, Sens. Actuators, B 127 (2007) 536.[15] A. Erdem, K. Kerman, B. Meric, U.S. Akarca, M. Ozsoz, Anal. Chim. Acta. 422
(2000) 139.[16] F. Yan, A. Erdem, B. Meric, K. Kerman, M. Ozsoz, O.A. Sadik, Electrochem.
Commun. 3 (2001) 224.