1、文章编号: 1007- 8827( 2014) 06- 0438- 06功能化氧化石墨烯载带荧光探针检测白血病细胞李然1， 2， 覃艳红2， 陈秀花2， 任方刚2， 张耀方2， 徐智芳2， 王宏伟2( 1 山西医科大学 口腔医学系， 山西 太原 030001;2 山西医科大学第二医院 血液病研究所， 山西 太原 030001)摘要: 分别以细胞外实验和和细胞内实验来观察功能化氧化石墨烯( GO) 对急性早幼粒细胞白血病细胞株 NB4 细胞的检测情况。通过比较不同探针浓度、 温度、 孵育时间等因素， 探索最佳条件。结果表明， 在室温下， GO 吸附 200 nmol/L 单链DNA 荧光探针，
2、并淬灭其荧光; 当与含靶分子( PML/A 融合基因) 的 NB4 细胞共孵育 1 小时后， 探针便脱离 GO， 荧光得以恢复， 从而准确检测出 NB4 细胞。关键词: 氧化石墨烯; 荧光探针; 检测; 功能化中图分类号:TQ1271+1文献标识码:A基金项目: 山西医科大学科技创新基金( 01201314) ;山西省研究生优秀创新项目( 20123060) 通讯作者: 王宏伟， 博士， 教授 E- mail:wanghw68 hotmail com作者简介: 李然， 博士， 讲师 E- mail:lraner163 comFluorescence probe analysis of leuk
3、emia cells by modified graphene oxideLI an1， 2， TAN Yan- hong2， CHEN Xiu- hua2，EN Fang- gang2， ZHANG Yao- fang2， XU Zhi- fang2， WANG Hong- wei2( 1 Department of Stomatology，Shanxi Medical University，Taiyuan030001，China;2 Department of Hematology，The Second Hospital of Shanxi Medical University，Taiyu
4、an030001，China)Abstract:Developing a simple and cost- effective strategy to diagnose and treat cancer with a single and minimum dose throughnoninvasive strategies is highly challenging Nano- sized graphene and graphene oxide ( GO)are promising for biomedical applica-tions，such as drug delivery and t
5、he photo thermal therapy of cancer An acute promyelocytic leukemia line， such as NB4 cells， wasdetected by modified GO in extracellular and intracellular experiments esults shows that GO can quench the fluorescence of the ss-DNA fluorescent probe and its fluorescence is restored after a PML/A fusion
6、 gene of NB4 cell lines is added Therefore，thefusion gene can be detected accurately with this phenomenon The best detection conditions for the fusion gene are found with assD-NA fluorescent probe concentration of 200 nmol/L in the presence of 0 04 mg/L GO at room temperature for 1 hKeywords: Graphe
7、ne oxide;Fluorescence probe;Detection;Modifiedeceived date: 2014- 08- 30; evised date: 2014- 12- 03Foundation item:Science and Technology Innovation Project of Shanxi Medical University ( 01201314) ;Graduate excellent in-nouation project in Shanxi Province ( 20123060) Corresponding author:WANG Hong-
8、 wei，Doctor，Pofessor E- mail:wanghw68 hotmail comAuthor introduction:LI an，Ph D Lecturer，E- mail:lraner163 comEnglish edition available online ScienceDirect (http: www sciencedirect comsciencejournal18725805 ) DOI: 101016/S1872- 5805( 14) 60148- 71IntroductionGraphene has excellent mechanical and ph
9、ysicalproperties in electronic and biomedical applicationsIn this regard，graphene oxide ( GO)is a subject ofparticular interest due to its richness in functionalgroups，which facilitates its surface functionalizationfor applications such as drug delivery，biosensing andbioimaging as well as its biolog
10、ical safety1- 4 In thebiological detection，GO can quench fluorophores andcan be used as a carrier to detect DNA，small mole-cules and proteins，et al5 Detection function of GO can be divided into ex-tracellular detection and intracellular detection In thestudy of extracellular level:He et al6 reported
11、 aGO- based multicolor fluorescent DNA nanoprobe thatallows a rapid sensitive and selective detection ofDNA Wu et al7 found that the length of DNAs，the pH value and organic solvents affected the detec-tion performance of GOThere were many reports related to intracellular第 29 卷第 6 期2014 年 12 月新型炭材料NE
12、W CABON MATEIALSVol 29No 6Dec 2014detection Lu et al8 proved that the functionalizedGO could protect oligonucleotides from enzymaticcleavage and efficiently deliver oligonucleotides intocells so as to achieve detection purposes Wang etal9 explored the function of GO for intracellular mo-nitoring and
13、 in situ molecular probing As a good detection tool 10- 14 ， GO has an adequateadsorption capacity for the probe ( ssDNA labeled by thefluorescent ) ， and can quench it s fluorescence In thepresence of a target molecule， the probe can be detachedfrom the GO，and the fluorescence of the probe is re-st
14、ored 15 In this paper，this property of GO will beverified by extracellular detection or intracellular detec-tion The purpose of our experiment is to use GO loadedfluorescent probe to detect PML- A fusion gene ofpunched NB4 cell lines This is a fast and simple meth-od compared with those reported2Exp
15、erimental2 1Preparation and functionalization of GOOxidants such as NaNO3，H2SO4，KMnO4andH2O2were add into graphite powder GO was got af-ter stirring，centrifuging and rinsing Then GO wasmodified with polyethylene glycol ( PEG)16 Fig 1shows the scanning probe microscopic image of GOFig 1Scanning probe
16、 microscopy images of GO2 2Design probe and target moleculeL-type PML- A fusion gene had 3 alternativesplicing isoforms Among them，the alternative spli-cing isoform E5 ( - )E6 ( - )had an important clinicalsignificance because it had the longest cycle life in-duced by all- trans retinoic acid ( ATA)
17、 and was themost sensitive in monitoring dynamic change of fusiongene17 So we designed the probe according to thealternative splicing isoform E5 ( - ) E6 ( - )of PML /A fusion gene，5 - GFP- CGACGTGACCTGC-CATTGAGCGTCG- 3 The complementary targetmolecule was ACTGGACGGTAACTC2 3Extracellular experimentD
18、ifferent concentrations of GO and target mole-cules were prepared in order to observe quenching ef-ficiency of GO and the fluorescence recovery degreewhen target molecules were present Fluorescence sig-nal was detected by a CAY Elipse VAPIAN floures-ence spectrophotometer2 4Intracellular experimentN
19、B4 Cell was fixed and punched by formalde-hyde and Saponin hormone Then the prepared cellswere incubated with GO and fluorescence probes16 Fluorescence signal was detected by a flow cytometryinstrument 3esults and discussion3 1Extracellular experimentThe four concentrations of GO were used， 0 01，0 0
20、4， 0 1 and 0 2 mg /mL in order to know whetherfluorescence would be quenched by different concen-trations of GO The fluorescent signal was detectedbythefluorescencespectrophotometer toexplorequenching efficiencyA mixture containing bufferand probewasmade， whichwascomposedof100 mmol/LNaCl， 5 mmol/LKC
21、l， 5 mmol/LMgCl2and 20 mmol/L probes with a pH value of7 4 Fig 2 shows the fluorescence emission spectra ofprobe before and after different concentrations of GOwere put into the mixtureFig 2Fluorescence emission spectra of probe ( a)before and( b) ，( c) ，( d)and ( e)after different concentrations of
22、 GO beingput into the mixture containing buffer and probe( a: 0 mg /mL GO，b: 001 mg /mL GO，c: 004 mg /mL GO，d: 01 mg /mL GO，e: 02 mg /mL GO) 934第 6 期LI an et al:Fluorescence probe analysis of leukemia cells by modified graphene oxideCurve a is the fluorescence emission spectrum forthe mixture Curve
23、b，c，d and e are the fluorescenceemissionspectraafter0 01， 0 04， 0 1and0 2 mg /mL GO，being put into the mixture It is ap-parent that the higher is the concentration of GO，thestronger is the quenching efficiency Quenching effectof 0 01 mg /mL is not good，so we eliminate thisconcentration in the follow
24、ing testFluorescence probe can be detached from the GOand the fluorescence can be restored when the targetmolecule is present Based on this principle，we de-signed the experiments to verify the degree of fluores-cence recovery of probe in presence of 0 04， 0 1 and0 2 mg /mL GO when different concentr
25、ations of tar-get molecules were addedFig 3 shows the fluorescence spectra of the mix-ture containing probes and buffer and those after 300，100， 50 and 0 nmol/L target molecules were added inpresence of 0 04 mg /mL GO It is apparent that thehigher the concentration of target molecules， the high-er t
26、he intensity of fluorescence isFig 3Fluorescence emission spectra of( a)the mixture containing probe and buffer，( b) ，( c) ，( d)and ( e)after 004 mg /ml GO was put into the mixture in presence of300， 100， 50 and 0 nmol/L target molecules，respectivelyThe fluorescence spectra of the mixture contai-nin
27、g probes and buffer are shown in Fig 4 and Fig5，respectively In both GO concentrations，the re-covery extent of fluorescence intensity is low althoughit increases with the concentration of target mole-culesFrom the above results，it is summarized that0 04 mg /mL was the best concentration of GO in the
28、experiment，below which the fluorescence cannot bequenched effectively by the GO and above which thefluorescence recovery extent is affected3 2Intracellular experiment3 2 1Concentration of probeThe concentrationofGOwasidentifiedas0 04 mg /mL The fluorescent signal was observed atthree different probe
29、 concentrations，150，200 and250 nmol/L Fig 6A presents the flow cytometric re-sult for the control group without fluorescent probeand the fluorescence positive cell percentage is 1 8%for the control groupFig 4Fluorescence emission spectra of ( a)the mixturecontaining probe and buffer， ( b) ， ( c) ， (
30、 d)and ( e)after 01 mg/mLGO being put into the mixture in presence of 300， 100， 50 and0 nmol/L target molecules，respectivelyFig5Fluorescence emission spectra of ( a)the mixturecontaining probe and buffer，( b) ，( c) ，( d)and ( e)after02 mg /mL GO being put into the mixture in presence of300， 100， 50
31、and 0 nmol/L target molecules，respectivelyFig 6 B- a B- c shows the flow cytometric re-sults for the experimental groups intervened by fluo-rescence probe with its concentrations of 150，200and 250 nmol/L and the fluorescence positive cellpercentages are 52 4%， 60 1% and 59 5%，respec-tivelyIt could b
32、e seen that fluorescence signalchange little when the probe concentration is greaterthan 200 nmol/LSo the probe concentration of200 nmol/L is an optimum concentration3 2 2TemperatureFig 7A shows the flow cytometric result for thecontrol group without fluorescent probe and the fluo-rescence positive
33、cell percentage is 1 8% Fig 7B-a， 7B- b display the flow cytometric results for the ex-perimental groups with fluorescent probe concentrationof 200 nmol/L at room temperature and 37 and thefluorescence positive cell percentages are 60 1% and044新型炭材料第 29 卷63 0%，respectively There is almost no differe
34、ncebetween the two temperatures So room temperatureis selected for an easy operationFig 6The flow cytometric results of ( A)without probe and ( B)with fluorescence probe concentrations of ( a)150，( b)200 and ( c)250 nmol/L in presence of 004 mg /mL GO to detect NB4 cells with the PML /A fusion geneF
35、ig 7The flow cytometric results of ( A)without probe and ( B)with fluorescence probe concentrations of 200 nmol/L at( a)room temperature and ( b)37 in presence of 004 mg /mL GO to detect NB4 cells with the PML /A fusion gene3 2 3Incubation timeThe experiments for incubation time was carriedout at th
36、e probe concentration of 200 nmol/L androom temperature in presence of 0 04 mg /mL GOFig 8A shows the flow cytometric result for the con-trol group without fluorescent probe and the fluores-cence positive cell percentage is 1 9% Fig 8B- a-8B-c present the flow cytometric results for the exper-imenta
37、lgroupsattheprobeconcentrationof200 nmol/Lincubatedatroomtemperaturefor30 min， 1 h and 2 h in the presence of 0 04 mg /mLGO cells after and the fluorescence positive cell per-centages are 41 7%， 52 8% and 55 4%， respective-lyIt is found that fluorescence signal for 30 min isslightly weaker than thos
38、e for 1 and 2 h There is noobvious enhancement for the fluorescence signal evenif the incubation time is longer than 1 h Finally，theshortest incubation time of 1 h is chosen as the opti-mum incubation time3 2 4Sample handle on time after preparationFluorescence signals were detected per half anhour
39、after sample preparation with the flow cytometryinstrument at the probe concentration of 200 nmol/Land room temperature in presence of 0 04 mg /mLGO The fluorescence positive cell percentage is 2%( Fig 9A)without fluorescent probe144第 6 期LI an et al:Fluorescence probe analysis of leukemia cells by m
40、odified graphene oxideFig 8The flow cytometric results of ( A)without probe and( B)incubation time for ( a)30 min，( b)1 h and ( c)2 h at the probe concentration of200 nmol/L and room temperature in presence of 004 mg /mL GO to detect NB4 cells with the PML /A fusion geneFig 9The flow cytometric resu
41、lts of ( A)without probe and ( B)with probe detected every half an hour at the probeconcentration of 200 nmol/L and room temperature in presence of 004 mg /ml GO to detect NB4 cells with the PML /A fusion gene244新型炭材料第 29 卷Fig 9 B- a 9B- c shows the flow cytometric re-sults for the experimental grou
42、ps with fluorescentprobe every half an hour Fluorescence positive cellpercentages are 55 9%， 54 2%， 53 8% and 53 5%as time goes on There is no obvious change on theresults when we detected samples whether immediate-ly or after incubation or placing a period of time4ConclusionsModified GO loaded fluo
43、rescent probe was usedto detect PML- A fusion gene of NB4 cell linesExtracellular and intracellular experiments shows thatGO loaded probes could identify the target moleculeaccurately For PML /A fusion gene of NB4 celllines，200 nmol/L is the optimum concentration ofprobes，and incubation at room temp
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