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Highly nsitive microRNA quanti fication with zero background signal from silver nanoparticles
Peng Miao a ,b ,⁎,Fanyu Meng a ,b ,c ,Bidou Wang a ,Xiaoli Zhu c ,Yuguo Tang a ,b ,⁎
a CAS Key La
b of Bio-Medical Diagnostics,Suzhou Institute of Biomedical Engineering and Technology,Chine Academy of Sciences,Suzhou 215163,PR China b University of Chine Academy of Sciences,Beijing 100049,PR China
c
Laboratory of Bionsing Technology,School of Life Sciences,Shanghai University,Shanghai,200444,PR China
a b s t r a c t
a r t i c l e i n f o Article history:
Received 29November 2014
Received in revid form 11December 2014Accepted 11December 2014
Available online 18December 2014Keywords:microRNA
Silver nanoparticles
Linear sweep voltammetry Ligation
Denaturation
MicroRNAs are a class of noncoding RNAs,which play vital roles in numerous cellular process.Recent studies have con firmed their signi ficance in the theranostics of various dias.We herein fabricate an electrochemical approach for microRNA quanti fication.DNA/microRNA/DNA hybridization and electrochemical signals from silver nanoparticles (Ag
NPs)are employed in this work.DNA1immobilized on a gold electrode interacts with target microRNA,along with amino group labeled DNA2,to form the sandwich hybrid.The adjacent DNA1and DNA2are then ligated,which can keep DNA2on the electrode surface during the denaturation.Amino group modi fied at the 5′end of DNA2captures AgNPs on the electrode surface,which provide sharp stripping peaks for microRNA quanti fication.This electrochemical approach offers a simple and nsitive platform for the detection of microRNA,which shows great utility in biomedical rearch and clinical diagnosis.
©2014Elvier B.V.All rights rerved.
1.Introduction
MicroRNAs are a class of small endogenous noncoding RNAs,which act as important regulators of gene expression [1].Currently,prominent roles of microRNAs have been uncovered in various cellular process such as embryonic differentiation,cardiac hypertrophy,apoptosis,immunological respon and tumor development [2–4].Recent studies have revealed that microRNAs exert signi ficant effects on various dias (neurodegeneration,lymphoma,lung cancer,colon carcinoma,etc.)and can be ud as noninvasive biomarkers for dia diagnosis [5–7].Moreover,by modulating speci fic micro
RNA expres-sion through the delivery of inhibitors,they can also be novel therapeu-tic targets for many dias [8].Therefore,there are urgent demands for flexible microRNA quanti fication approaches with high nsitivity and lectivity [9,10].
Currently,northern blotting technique is predominantly employed for RNA analysis.Due to some features of microRNA like low content,short quences and high quence homology among family members,northern blotting is not competent for the detection of microRNA,which stimulates the development of novel bionsors and signal ampli fication strategies [11,12].For example,real-time rever transcription polymera chain reaction [13],size-coded ligation
chain reaction [14],endonuclea cleavage cycles [15]and hybridiza-tion chain reaction [16]have been employed for the nsitive detection of microRNA.Many of the methods involve the application of electro-chemical techniques due to some unique advantages of electroanalysis for microRNA (fast respon,low cost,high nsitivity,etc.)[17–19].However,labor-intensive operation procedures may be required.Herein,we have developed a simple and nsitive electrochemical method for microRNA assay,which may be more promising for practical applications.In this work,we have subtly combined DNA/microRNA/DNA hybridization and ligation/denaturation proce-dures to achieve the microRNA-induced lective capture of silver nanoparticles (AgNPs).The nanoparticles t
hen exhibit a highly charac-teristic solid-state Ag/AgCl reaction and provide sharp silver stripping peaks.A signal-on electrochemical approach for microRNA quanti fi-cation is thus developed,which can detect target microRNA as low as 10fM.This method offers a concept for a simple and integrated electrochemical device for microRNA assay and dia diagnostics.2.Experimental ction 2.1.Materials and chemicals
Sodium borohydride (NaBH 4),Tris(2-carboxyethyl)phosphine hydrochloride (TCEP),diethypyrocarbonate (DEPC),ethylenedi-aminetetraacetic acid (EDTA)and mercaptohexanol (MCH)were purchad from Sigma-Aldrich (USA).Silver nitrate was ordered from Nanjing Chemical Reagent Co.,Ltd.(Nanjing,China).T4DNA liga
Electrochemistry Communications 51(2015)89–92
⁎Corresponding authors at:CAS Key Lab of Bio-Medical Diagnostics,Suzhou Institute of Biomedical Engineering and Technology,Chine Academy of Sciences,Suzhou 215163,PR China.Tel.:+8651269588279;fax:+8651269588283.
E-mail address:miaopeng@sibet.ac (P.Miao),tangyg@sibet.ac (Y.
Tang).
dx.doi/10.1016/j.elecom.2014.12.0161388-2481/©2014Elvier B.V.All rights
rerved.
Contents lists available at ScienceDirect拼音字母表读法
Electrochemistry Communications
j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m/l o c a t e /e l e c o m
was purchad from New England Biolabs Ltd.(Beijing,China).Human rum samples were supplied by local hospital(Suzhou,China).Other reagents were of analytical grade and were ud as received.All solutions were prepared with double-distilled water,purified with a Milli-Q purification system(Branstead,USA).Two DNA probes(DNA1 and DNA2)and target microRNA were synthesized and purified by Takara Biotechnology Co.,Ltd.(Dalian,China).DNA1was labeled with phosphate group at the5′end and thiol group at the3′end.DNA2 was modified with amino group at the5′end.The quences were listed as follows.The italic and underlined parts were complementary quences,respectively.
DNA1:50‐P‐CTGATAAGCTA TTTT‐SH‐30
DNA2:50‐NH2‐TTTTTCAACATCAGT‐30
microRNA miRNA‐21
ðÞ:50‐UAGCUUAUCAG ACUGAUGUUGA‐30
2.2.Preparation of AgNPs
AgNPs were prepared by borohydride reduction of AgNO3[20]. Briefly,100mL of AgNO3(0.25mM)and trisodium citrate(0.25mM) solution was prepared,which was then mixed with3mL of NaBH4 (10mM)under vigorous stirring for30min.The formed AgNPs were left overnight and purified by three cycles of centrifugation at12 000rpm for20min.
2.3.Hybridization of DNA1/microRNA/DNA2on the electrode
The substrate gold electrode was treated with piranha solution(98% H2SO4:30%H2O2=3:1)for about5min.Then it was polished with P3000silicon carbide paper and1,0.3and0.05μm alumina slurry. After sonication in ethanol and then water for5min each,the electrode was immerd in HNO3(50%)for30min.Subquently,it was electro-chemically cleaned with0.5M H2SO4.The pretreated electrode was soaked in1μM DNA1solution(10mM Tris–HCl,1mM EDTA,10mM TCEP,0.1M NaCl,pH7.4)at room temperature for8h.Then it was incubated with1mM MCH for30min[21].microRNA solutions were prepared for the incubation of DNA1modified electrode for1h.The concentrations of microRNA were from10fM to5nM.Then the electrode was washed and fu
rther treated with1μM DNA2solution for1h to form the hybrid of DNA1/microRNA/DNA2.
2.4.DNA ligation/denaturation and AgNPs capture
The nick between DNA1and DNA2in the hybrid was linked by5 unit/mL T4DNA liga(50mM Tris–HCl,10mM MgCl2,10mM DTT, 1mM ATP,pH7.5)at22°C for1h.Afterward,the electrode was immerd in10mM Tris–HCl(pH7.4)at95°C for5min and then cooled down.The electrode was rind and incubated with10nM AgNPs for4h.Through the interaction between Ag and amino group on DNA2,AgNPs were thus localized on the electrode surface.min是什么意思
2.5.Electrochemical measurements
All electrochemical experiments were performed by using the electrochemical analyzer(CHI660D,CH Instruments,China)at room temperature.Three electrode system was employed,consisting of the Ag/AgCl reference electrode,the platinum auxiliary electrode and DNA modified electrode(gold area:3.142mm2)as the working electrode. Electrochemical impedance spectroscopy(EIS)experiments were carried out in5mM[Fe(CN)6]3−/4−with1M KNO3.The parameters were as follows:bias potential,0.231V;amplitude,5mV;frequency range,0.1–100000Hz.Linear sweep voltammetry(LSV)experiments were performed in0.1M KCl,and the scan rat
e was100mV/s.3.Results and discussion
Fig.1reprents the scheme of the electrochemical approach for microRNA quantification.DNA1with3′end thiol group is immobilized on the electrode surface via gold-sulfur chemistry[22].Afterward, the electrode is treated with the spacer thiol molecules,MCH[23]. In the prence of target microRNA,hybridization reaction occurs and DNA1/microRNA/DNA2hybrid forms,which brings DNA2clo to DNA1.The adjacent two DNA probes are then ligated,which can keep DNA2on the electrode during the denaturation.The procedure of denaturation removes microRNA and unligated DNA2,retaining a monotonous electrode surface,which exhibits zero background signal. Since DNA2is labeled with amino group,AgNPs are thus localized on the electrode surface via silver-amino chemistry[24]and can be detected by a highly characteristic solid-state Ag/AgCl reaction[25]. MicroRNA levels can be calculated accordingly.
The stepwi modification of the gold electrode is recorded by EIS. The negatively charged phosphate groups of the oligonucleotides decelerate electron transfer for[Fe(CN)6]3−/4−redox probe,which is reflected by the micircle portion in the impedance spectrum [26].As shown in Fig.2A,a large micircle is obrved in the ca of DNA1modified electrode,which becomes larger after the hybridiza-tion of DNA1/microRNA/DNA2.Since ligation/denaturation procedures keep DNA2
and relea microRNA back into the solution,the diame-ter of the micircle declines.Afterward,the further attached AgNPs exhibit unique electrical properties and the micircle becomes even smaller.The EIS experiments results confirm the stepwi modification process.
Quantification of microRNA-induced sandwich hybridization and further AgNPs adsorption is then studied by LSV[27].As depicted in Fig.2B,DNA1modified electrode cannot exhibit a significant current peak unless the subquent incubation with microRNA,DNA2and AgNPs,demonstrating the fact that incomplete DNA1/microRNA/DNA2 hybrid cannot effectively capture AgNPs on the electrode surface.Fig.2C displays the nsitivity of this method.With larger amount of microRNA,the stripping silver peak ris,which is in linear relationship with the logarithmic microRNA concentration.The detection range is from10fM to5nM,with thefitting equation of y=90.27+6.44x, where x is the logarithmic microRNA concentration,y is the peak current,R2=0.994and n=3.The detection limit is calculated to be 10fM(S/N=3).To check the lectivity of this microRNA assay, 1pM single-ba mismatch microRNAs are employed in the electro-chemical measurements and the results are compared in Fig.2D.Tiny stripping silver peaks are obrved in the ca of mismatch microRNAs, which confirms the high lectivity towards target microRNA.To fur-ther verify the practical utility of this approach,human rum samples are ud.As shown in Table1,detection of mic
roRNA is satisfactorily achieved and recoveries have substantiated thefine accuracy of this method in biological samples.
4.Conclusions
In summary,we have offered a simple and nsitive electrochemical approach for the determination of microRNA levels bad on the DNA/microRNA/DNA hybridization and electrochemical signals from AgNPs localized on the electrode surface.Highly nsitive and lective detection of target microRNA is achieved by analyzing the stripping silver peaks.Zero background signal is attributed to the ligation and denaturation procedures.Moreover,due to the advantages of conve-nient operation and high stability,one might expect the design of an integrated electrochemical device for microRNA quantification bad on the proof-of-concept approach described here.
Conflict of interest
The authors declare no competingfinancial interests.
90P.Miao et al./Electrochemistry Communications51(2015)89–92
Fig.1.Schematic illustration of the electrochemical method for microRNA quanti fi
cation.
Fig.2.(A)Nyquist diagrams of impedance spectra for (a)DNA1modi fied electrode,(b)DNA1/microRNA/DNA2modi fied electrode,(c)after ligation and denaturation and (d)after further incubation with AgNPs.(B)Linear sweep voltammograms of (a)DNA1modi fied electrode,the other curves are the cas after the incubation with (b)microRNA and then AgNPs,(c)DNA2and then AgNPs and (d)microRNA,DNA2and AgNPs subquently.(C)Linear sweep voltammograms of the AgNPs modi fied electrode induced by microRNA with the concentrations from 10fM to 5nM.Int is the calibration plot of the peak current versus the logarithmic microRNA concentration.Error bars reprent standard deviations of three independent measurements.(D)Electrochemical detection of 1pM (a)target microRNA;b and c are the cas of two mismatch microRNAs.Int reveals the corresponding peak currents.
91
P.Miao et al./Electrochemistry Communications 51(2015)89–92
Acknowledgements
This work was supported by the National Natural Science Foundation of China(grant no.31400847).
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jnkTable1
Detection of miRNA-21in rum samples from breast cancer patients.
Sample Detected(pM)Added(pM)Found(pM)Recovery(%)
Serum10.970.5 1.4397.3
1.0 1.99101.0
2.0 2.9499.0
Serum20.640.5 1.17102.6
1.0 1.71104.3
multi2.0 2.5998.1
92P.Miao et al./Electrochemistry Communications51(2015)89–92
