多元电化学免疫传感器_图文.

1、ARTICLE IN PRESS G Model BIOS-4708;No.of Pages 7 Biose nsors and Bioelectr onics xxx (2011 xxxxx Contents lists available at SciVerse Scien ceDirect Biose nsors and Bioelectr onics 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 /b i o s Carbon nano tube-based ultrase nsitive

2、 multiplex ing electrochemical immunosen sor for can cer biomarkers Ying Wan a ,b ,Wangping Deng b，丫an Su a ? ,Xinhua Zhu a ,? ,Cheng Peng b ,Haiyan Hu b ,Hongzhen Peng b ,Shiping Song b? ,Chunhai Fan a ,b a School of Mecha ni cal Engin eeri ng,Nanj ing Uni versity of Scie nee and Technology,Nanjing

3、 210094,China Laboratory of Physical Biology,Sha nghai In stitute of Applied Physics,Chi nese Academy of Scie nces,Sha nghai 201800,Chi na a r t i c l e i n f o Article history: Received 13Ju ne 2011 Received in revised form 25August 2011Accepted 25August 2011Available online xxx Keywords: Immunosen

4、 sor array Scree n-pri nted carb on electrode (SPCEMultiwalled carb on nano tube (MWNTU ni versal nano probe a b s t r a c t A multiplexi ng electrochemical immunosen sor was developed for ultrase nsitive detect ion of can cer related protein biomarkers.We employed disposable scree n-pri nted carb o

5、n electrode (SPCEarray as the detecti on platform.A uni versal multi-labeled nan oprobe was developed by load ing HRP and goat-a nti-rabbit IgG (sec on dary an tibody,Ab 2onto multiwalled carb on nano tube (MWNT.This uni versal nan oprobe was available for virtually any sandwich-based antigen detect

6、ion and showed superior-ity in several areas.By using the SPCE array and the uni versal nan oprobe,we could detect as low as 5pg mL -1of prostate speci?c an tige n (PSAa nd 8pg mL -1of In terleuk in 8(IL- 8with the electrochemical immunosen sor.We also dem on strated simulta neous detecti on of two

7、protein biomarkers with this plat-form.With these attracted features,our immuno assay system shows promisi ng applicati ons for ir?eld and poin t-of-care test in cli ni cal diag no stics. ? 2011 Elsevier B.V. All rights reserved. 1.ln troduct ion Early diag no sis of can cer is a challe nge faci ng

8、scie ntists from all over the world which is very importa nt for can cer therap y.ln early diag no sis of can cer,accurate detect ion of certa in protein biomark-ers is critical but dif?cult as there is on ly trace protein biomarker in serum of early can cer patie nts (Kulas in gam and Diama ndis,20

9、08;Ludwig and Weinstein,2005;Pepe et al.,2001;Welsh et al.,2003.Traditional assay methods such as en zyme-l in ked immuno sorbe nt assay (ELISA(Butler,2000,radioim muno assay (Bolt on and Hun ter,1973,?uoresce nee immuno assay (Goldma n et al.,2002,electropitetic immuno assay (Bao,1997,mass spec-tro

10、metric immuno assay (Diama ndis and van der Merwe,2005,a nd immun e-polymerase cha in reacti on (PCRassay (Widjojoatmodjo et al.,1992ofte n have some disadva ntages,result ing in the in creas-i ng dema nd for operati on ally simple,ultrase nsitive and easily automated device.C on siderable efforts h

11、ave bee n made to develop rapid,se nsitive and selective immunosen sors (Akram et al.,2006;Chen et al.,2008;Dill et al.,2004;Mani et al.,2009;Tang et al.,2008;Wu et al.,2008.Electrochemical immunosen sors,with the in her-e nt adva ntages of high sen sitivity,low cost,low power requireme nt Corresp o

12、nding authors. E-mail addresses:suya nn .c n (Y.Su,zhux in huamail .n .c n (X.Zhu,sps on gs in ap.ac.c n (S.S ong.xxx and pote ntial of automati on ,have bee n applied for cli ni cal diag no-sis (Ghi ndilis et al.,1998;Warsinke et al.,2000. With the aim of ultrahigh sen sitive biose

13、nsors,various signal ampli?cati on strategies using nano structured materials have bee n developed (Cao,2008;Grodz in ski et al.,2006;So ng et al.,2010,such as gold nan oparticles (Nam et al.,2003;Wa ng et al.,2008;Yan et al.,2010;Zhang et al.,2006,quantum dots (Hu et al.,2009,magnetic nan oparticle

14、s (Yigit et al.,2008a nd car-b on nano tubes (Lin et al.,2005.In the area of ultrase nsitive electrochemical immunosen si ng,nano materials can be directly used as electroactive labels (Das et al.,2006;Liu et al.,2004or used as carriers to load a large amount of electroactive labels (Ma ni et al.,20

15、09;Ta ng et al.,2008.Us ing nano materials as electroactive labels,Ho group has reported a novel electrocheah i immunosenor.Monoclonal capture an tibody was adsorbed on polyethyle ne glycol- modi?ed disposable screenpri nted electrode as the detect ion platform,while polycl onal sig nal an tibody an

16、d gold nano particle (AuNPc on jugates were used as electrochem-ical sig nal probes (Ho et al.,2010.The electrochemical sig nal from the bound AuNP congregates was obtained after oxidizing them in 0.1M HCl at 1.2V for 120s,followed by the reducti on of AuCl 4-i n square wave voltammetry (SWVmode.Usi

17、 ng nano-materials as carriers for sig nali ng and biorecog niti on have also attracted atte ntio ns from scie ntists.Wa ng et al.reported carb on nano tubes (CNTscarryi ng nu merous en zyme tracers for dramat-ically amplifyi ng en zyme-l in ked electrical detect ion of prote ins and DNA (Zhao et al

18、.,2009.A no vel strategy of electrochemical 0956-5663/$ee fro nt matter ? 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2011.08.033 2 Y.Wan et al./Biose nsors and Bioelectr onics xxx (2011 xxx Scheme ISchematic dem on stratio n for the “ san dwich ” type strategy electrochemical immunose

19、n sor.A 16cha nnel scree n-pri nted carb on electrode (SPCEarray was employed as the detecti on platform,each containing a three electrode system:carb on worki ng electrode,a carb on coun ter electrode and a silver pseudorefere nee electrode.The capture an tibodies were immobilized on the worki ng e

20、lectrode by a three step protocol:electrochemical activati on was ?rst take n to gen erate carboxylic acid groups on the worki ng electrode and the n the EDC/NHS were used to activate the carboxylic acid groups which was the n removed.After that,capture an tibodies (PSA mAb or IL- 8mAbwere immobiliz

21、ed by using the ami ne residues on the protei ns.The target an tige n (PSA or IL-8and the signal antibody (PSA pAb or IL-8pAbformed a “sandwich ” type complex with the capture an tibody,lead ing to the bi nding of the uni versal nan oprobe to the electrode that can be tran suded to the catalytic amp

22、erometric readout.The process of the uni versal nan oprobe preparati on was MWNT 1L-8 mAb IL-RpAb IL& PSA mAb EDCNHS PSApAb IINOhtLSD- Idlr I COl* afollowi ng:the pristi ne MWNTs were ?rst soni cated in the HNO 3and H 2SO 4to gen erate carboxylic acid groups which were the n activated by EDC/NHS

23、.After removal of free EDC/NHS,Ab 2/HRP mixture in an optimized ratio was added and the uni versal nan oprobe was achieved. immuno assays based on the utilizati on of en capsulated electro-chemical sig nal- generating microcrystals was reported (Mak et al.,2005.The electrochemical signal was achieve

24、d by the release of a large amount of ferroce ne after san dwich immunobindin g.Rusli ng s group has achieved greatly enhan ced sen sitivity using carb on nano tubes (CNTscarryi ng horseradish peroxidase (HRPlabels and an tibodies for immuno detect ion of the prostate speci?c an tige n (Je nsen et a

25、l.,2009;Yu et al.,2006.The limit detecti on of this CNT ampli?ed immunosen sor was low to 4pg mL -1.Na no materials have bee n dem on strated to be excelle nt carriers in the ampli?cati on strategies. Despite adva nces in nan campli?cati on tech no logies,there are still challe nges faced by researc

26、hers such as complicated assembly process and stability of nano materials.Especially,differe nt an ti-bodies have differe nt electrostatic properties so that the assembly con diti ons of differe nt an tibodies with same nano materials are varia nt very ofte n. Whe n encoun teri ng with simulta neous

27、 detec-ti on of pan els of tumor markers in cli nical diag no sis of can cers (Liu et al.,2004;Wils on,2005,several differe nt nano material based bioconjugates were dema nded.However,the processes were complicated.As a result,it is n ecessary to develop a sim-ple approach which can overcome these o

28、bstacles and give a total solution for this problem.Herein we proposed an electrochemical immunosenor using a disposable sixtee n cha nnel scree n-pri nted carb on electrode (SPCEarray comb ined with a uni versal multilabel nano probe for the simulta neous detect ion of can cer biomarkers:prostate s

29、peci?c an tige n (PSAa nd Interleukin 8(IL-8.The immo-bilization of capture antibodies on this SPCE was con siderable simple,which was con ducted by ?rst electrochemical activat inghte carb on worki ng electrode.This process gen erated carboxylate groups to bi nd to the ami ne residues on capture an

30、 tibodies.This covale nt binding was proved to be very ef?cie nt.A uni versal multilabel nan oprobe was fabricated by con siste nt loadi ng of HRP and goat-a nti-rabbit IgG (sec on dary an tibody,Ab 2on multiwalled car-b on nano tube (MWNT.As Ab 2ca n bind to rabbit an tibodies for any an tige n,thi

31、s multilabel nan oprobe is available to the detec-ti on of any target antigen by using uniabelled rabbit polyclonal antibody serving as a bridge.Then the uni versal nan oprobe can be attached to biose nsing surface to gen erate electrochemical sig-n als.Combi ning this uni versal nan oprobe with dis

32、posable SPCE array,we provide a promisi ng future in clinical applicatio ns with simulta neous immuno assay of multiple protein biomarkers. 2.Experime ntal 2.1. Materials PSA an tige n,m ouse monoclonal an ti-PSA an tibody (PSA mAb,clo ne no.M701042a nd rabbit polyclo nal sig nal a nti-PSA an ti-bod

33、ies (PSA pAbwere purchased from Fitzgerald (U.S .I L-8a ntige n,m ouse monoclonal an ti-IL-8a ntibody (IL-8mAb,cl one no .500-M08a nd rabbit polycl onal an ti-IL-8a ntibodies (IL-8pAbwere purchased from Peprotech Can ada.I nc.(Ottawa,ON,Ca nada.Multiwalled Carbo n nano tube (MWNTwas purchased from S

34、he nzhen Nano tech Port Co.Ltd (NTP,chi na.TMB substrate (TMB =3,3 ,5,5 tetramethylbe nzidi ne;Neoge n K-blue low activity substratewas purchased from Neogen (U.S.Ab 2,HRP (MW 44,000Da,HRP labeled Ab 2(Ab 2-HRP,lyophilized 99%bo vine serum albumin (BSA,a nd Twee n-20were from Sigma Aldrich.The buffe

35、r soluti ons invo Ived in this study are as fol-lows:im mun oreage nts were dissolved in pH 7.20.1M phosphate sali ne (PBSbuffer (0.01M phosphate,0.14M NaCl,2.7mM KCl. Y.Wan et al./Biose nsors and Bioelectr onics xxx (2011 xxxxx3 The washi ng buffer was PBST(0.5%twee n in 0.1M PBS buffer. En zyme di

36、lue nt was0.1M PBS buffer with1%casei n(pH7.2. The buffer for electrochemical measureme nt was TMB substrate. 1-(3-(dimethylami no-propyl-3-ethylcarbodiimidehydrochloride (EDCa nd N hydroxysulfosucci ni mide(NHSwere dissolved in water immediately before use.AII soluti ons were prepared with Milli-Q

37、water(18M cm resistivityfrom a Millipore system. 2.2. Apparatus Electrochemical measureme nts of the immunosen sors were performed with an in depe ndent developed16-cha nnel electro-chemical work stati on.ln depe ndent developed disposable16 cha nnel SPCE array,each compris ing a carb on worki ng el

38、ectrode, carb on coun ter electrode,a nd silver pseudorefere nee electrode, were employed all through the experime nt(Scheme1.U nl abelled MWNTs and uni versal nan oprobes were imaged un der a sca nning electro n microscopy(SEM,Hitachi S-2400.Absorba nee values of ELISA were obta ined with a Teca n

39、GENios microplate reader at room temperature. 2.3. Preparati on of the uni versal nano probe MWNTs were function alized followi ng literatures(Yu et al., 2006a nd several details were optimized .In brie?y,MWNTs were soni cated for6h at300W( Yin gsum ultras onic equipme nt Co.Ltd., Sha nghai,Chi nain

40、 3:1H2SO4/HNO3(70?C.The result ing d-persio n was washed repeatedly with water an d?ltered ntil pH was7.The result ing fun cti on alized,shorte ned MWNTs were the n dried un der vacuum over ni ght.This procedure removes metallic and carb on aceous impurities and gen erates carboxylate groups on the

41、shorte ned nano tubes.Multiple HRPs and Ab2s were bond to carboxylated MWNTs using an EDC/NHS amidizati on protocol with a reacti on mixture of optimized Ab2/HRP ratio.Brie?y,1mg of oxidized MWNTs in 1mL of pH7.20.01M2 -(N morpholi no etha ne-sulfo nic acid buffer(MES bufferwere soni cated forlh to

42、obtai n a homoge neous dispersio n.This dispers ion was mixed withlmL of400mM EDC and100mM NHS in pH7.2PBS and vortexed for 15min,then centrifuged at15,000rpm for5mi n,a nd the super- nata nt was discarded.The buffer wash was repeated2times to remove excessive EDC and NHS.To optimize the ratio of Ab

43、2/HRP, react ion mixture of differe nt ratio of Ab2a nd HRP were added to the activated MWNT and stirred in a small vial overnight at room temperature.The concentration of HRP was kept at1mg mL-1in all the experime nts.BSA was added to the ?nal con ceratio n of1%to the react ion mixture after over n

44、i ght stirri ng,which was to protect the bioconjugates from depositi on or attachment to the wall of the tubule in the following centrifugation steps.Then the reacti on mix-ture was cen trifuged at5000rpm at4?C fc5mi n, the super nata nt was take n out into ano ther tubule for the continuing steps w

45、hile the rema ining precipitate was discarded.The new tubule was the n cen trifuged at15,000rpm at4?C for10mi n,a nd the super nata nt was discarded.Washi ng with0.1%BSA in PBST buffer was crucial to remove free Ab2a nd HRP and was repeated four times.A total of0.5mL of0.1%BSA in PBST buffer was add

46、ed to the bioc on jugate precipitate collected and vortexed to form a homoge neous dispers ion and stored in the refrigerator at4?C.The prepend MWNT bioc on jugate was diluted with PBS buffer con tai nin g1%BSA and 1%Case in immediately before use. 2.4. Evaluati on of the uni versal nan oprobe by EL

47、ISA PSA pAb was diluted with PBS to a concen trati on of2?g mL-1 and immediately added to each ELISA plate well.PSA mAb was used as the con trol.After in cubati on over ni ght at4?C,the plate was washed by PBS buffer and the n in cubated with block buffer(2%BSA in PBSat room temperature forlh.The un

48、i versal nan oprobe was diluted with PBST buffer1%BSA an d1%casein and added to the treated96plate well.After in cubati on30min at room temperature, the ELISA plate was washed and in cubated with TMB substrate at room temperature for color developme nt.The absorba nce(OD520 was measured with a Teca

49、n GENios microplate reader at room tem-perature. 2.5. Fabricati on of immunosen sor array The16cha nnel disposable SPCE array was soni cated in Milli-Q water forlmin before use.Electrochemical activati on was per-formed on the SPCE array in a0.01M PB buffer by runnin glOcycles of CV with pote ntial

50、ran ge-0.3to0.6V.EIectrochemical treatme nt of carb on electrodes was ofte n performed by cyclic sca nning over a wide pote ntial range to obtain potentiostatic oxidizing at posi-tive potential(Li et al.,2004;Wang et al.,2001;Zhao et al.,2008, 2009.The goal of this step was to gen erate carboxylate

51、groups on the work ing electrode.After rin sed with Milli-Q water,the SPCE array was blew-dry with n itroge n.For attachme nt of capture an ti-body,1?L of freshly prepared400mM EDC an d100mM NHS in water were placed onto the work ing electrodes,a nd washed off after15m in. This was immediately follo

52、wed by1h in cubation at 37?C with5?L of100?g mL-1PSA mAb or IL - 8mAb in pH7.2 PBS buffer .In the optimizatio n experime nt,sig nal an tibody was immobilized on the worki ng electrode in stead.The electrode was the n washed with0.1M PBS buffer.The treated SPCE array was then in cubated for1h at37?C

53、with100?L of1%BSA in PBS buffer coveri ng all the three electrodes area,followed by wash ing with PBS buffer.Wash ing steps used here and duri ng the whole an alysis were esse ntial to block non speci?c bindin g(NSB,a nd omissi on of any of the wash ing steps deteriorated sen sitivity. 2.6 .Immunose

54、n sor detecti on of PSA and IL-8 Optimized steps in the assay procedure were as following: Firstly,the16channel SPCE array,prepared and prein cubated with 1%BSA in PBS buffer as described above,was in cubated at37?C for 1h with a5?L drop of an tige n(PSA or 也8in differe nt concen-tratio n,followed b

55、y wash ing with PBST and the n PBS buffer.Next, 5?L drop of50?g mL-1signal antibody(PSA pAb or IL-8pAbwas added to the sensor.After in cubati on at37?C for1h,the sen sor was washed as described above.The immunosen sor was the n in cubated at37?C for30 min with a5?L drop of the uni versal nano probe

56、in 0.1M PBS buffer con tai nin g1%BSA an d1%Casei n.Washi ng steps should not be omitted.After that,electrochemical detect ion was performed in 100L TMB substrate.Cyclic voltammetry(CVwas carried out at a sca n rate of50mV/s,a nd the pote ntial range was -0.3 V to0.6V.Voltage of amperometric curre n

57、t versus time was ?xed at-0.1V and thexxx electroreducti on curre nt was measured at 50s after the HRP redox reacti on reached steady state. 3.Results and discussi on 3.1.Strategy The immunosen sor herein employed a16cha nnel SPCE array as a multiplex ing assay platform,each compris ing a carb on wo

58、rki ng electrode,a carb on coun ter electrode and a silver pseudorefere nee electrode(Scheme1.The immobilizatio n of capture an tibodies on this platform was con siderably easy and robust,which invo Ived an electrochemical activati on of the carb on worki ng electrode to gen erate carboxylate groups

59、 at?rst.A nd the n capture an tibody (PSA mAb or IL-8mAbwas attached to the worki ng electrode with the amine residues on the proteins using the EDC/NHS protocol. 4 Y.Wan et al./Biose nsors and Bioelectr onics xxx (2011 xxx 1 Fig.I.Sca nning electro n microscopy (Adam et al.,2002images in differe nt

60、 scale of MWNTs before (upper imagesa nd after (n ether imagesthe attachme nt of Ab 2/HRP. This covale nt attachme nt was simple,ef?cie nt and stable for weeks while stored at 4?C. A “ sandwich ” con?guration was used here,which invoIved capture antibodies and signal antibodies to form a “ sandwich ” ty