多元电化学免疫传感器

1、ARTICLE IN PRESSG ModelBIOS-4708;No.of Pages 7Biosensors and Bioelectronics xxx (2011 xxxxxxContents lists available at SciVerse ScienceDirect Biosensors andBioelectronicsj 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 osCarbon nanotube-based ultrasensitive multiplexing

2、electrochemical immunosensor for cancer biomarkersYing Wan a ,b ,Wangping Deng b ,Yan Su a ,Xinhua Zhu a ,Cheng Peng b ,Haiyan Hu b ,Hongzhen Peng b ,Shiping Song b ,Chunhai Fan a ,ba School of Mechanical Engineering,Nanjing University of Science and Technology,Nanjing 210094,ChinabLaboratory of Phy

3、sical Biology,Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai 201800,Chinaa r t i c l ei n f oArticle history:Received 13June 2011Received in revised form 25August 2011Accepted 25August 2011Available online xxxKeywords:Immunosensor arrayScreen-printed carbon electrode (SPC

4、EMultiwalled carbon nanotube (MWNTUniversal nanoprobea b s t r a c tA multiplexing electrochemical immunosensor was developed for ultrasensitive detection of cancer related protein biomarkers.We employed disposable screen-printed carbon electrode (SPCEarray as the detection platform.A universal mult

5、i-labeled nanoprobe was developed by loading HRP and goat-anti-rabbit IgG (secondary antibody,Ab 2onto multiwalled carbon nanotube (MWNT.This universal nanoprobe was available for virtually any sandwich-based antigen detection and showed superior-ity in several areas.By using the SPCE array and the

6、universal nanoprobe,we could detect as low as 5pg mL 1of prostate specic antigen (PSAand 8pg mL 1of Interleukin 8(IL-8with the electrochemical immunosensor.We also demonstrated simultaneous detection of two protein biomarkers with this plat-form.With these attracted features,our immunoassay system s

7、hows promising applications for in-eld and point-of-care test in clinical diagnostics.© 2011 Elsevier B.V. All rights reserved.1.IntroductionEarly diagnosis of cancer is a challenge facing scientists from all over the world which is very important for cancer therapy.In early diagnosis of cancer

8、,accurate detection of certain protein biomark-ers is critical but difcult as there is only trace protein biomarker in serum of early cancer patients (Kulasingam and Diamandis,2008;Ludwig and Weinstein,2005;Pepe et al.,2001;Welsh et al.,2003.Traditional assay methods such as enzyme-linked immunosorb

9、ent assay (ELISA(Butler,2000,radioimmunoassay (Bolton and Hunter,1973,uorescence immunoassay (Goldman et al.,2002,electrophoretic immunoassay (Bao,1997,mass spec-trometric immunoassay (Diamandis and van der Merwe,2005,and immune-polymerase chain reaction (PCRassay (Widjojoatmodjo et al.,1992often ha

10、ve some disadvantages,resulting in the increas-ing demand for operationally simple,ultrasensitive and easily automated device.Considerable efforts have been made to develop rapid,sensitive and selective immunosensors (Akram et al.,2006;Chen et al.,2008;Dill et al.,2004;Mani et al.,2009;Tang et al.,2

11、008;Wu et al.,2008.Electrochemical immunosensors,with the inher-ent advantages of high sensitivity,low cost,low power requirementCorresponding authors.and potential of automation,have been applied for clinical diagno-sis (Ghindilis et al.,1998;Warsinke et al.,2000.With the aim of ultrahigh sensitive

12、 biosensors,various sig-nal amplication strategies using nanostructured materials have been developed (Cao,2008;Grodzinski et al.,2006;Song et al.,2010,such as gold nanoparticles (Nam et al.,2003;Wang et al.,2008;Yan et al.,2010;Zhang et al.,2006,quantum dots (Hu et al.,2009,magnetic nanoparticles (

13、Yigit et al.,2008and car-bon nanotubes (Lin et al.,2005.In the area of ultrasensitive electrochemical immunosensing,nanomaterials 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 (Mani et al.,2009;Tang et

14、al.,2008.Using nanomaterials as electroactive labels,Hogroup has reported a novel electrochemi-cal immunosenor.Monoclonal capture antibody was adsorbed on polyethylene glycol-modied disposable screen-printed electrode as the detection platform,while polyclonal signal antibody and gold nanoparticle (

15、AuNPconjugates were used as electrochem-ical signal probes (Ho et al.,2010.The electrochemical signal from the bound AuNP congregates was obtained after oxidizing them in 0.1M HCl at 1.2V for 120s,followed by the reduction of AuCl 4in square wave voltammetry (SWVmode.Using nano-materials as carriers

16、 for signaling and biorecognition have also attracted attentions from scientists.Wang et al.reported carbon nanotubes (CNTscarrying numerous enzyme tracers for dramat-ically amplifying enzyme-linked electrical detection of proteins and DNA (Zhao et al.,2009.A novel strategy of electrochemical2Y.Wan

17、et al./Biosensors and Bioelectronics xxx (2011 xxxxxx Scheme 1.Schematic demonstration for the “sandwich”type strategy electrochemical immunosensor.A 16channel screen-printed carbon electrode (SPCEarray was employed as the detection platform,each containing a three electrode system:carbon working el

18、ectrode,a carbon counter electrode and a silver pseudoreference electrode.The capture antibodies were immobilized on the working electrode by a three step protocol:electrochemical activation was rst taken to generate carboxylic acid groups on the working electrode and then the EDC/NHS were used to a

19、ctivate the carboxylic acid groups which was then removed.After that,capture antibodies (PSA mAb or IL-8mAbwere immobilized by using the amine residues on the proteins.The target antigen (PSA or IL-8and the signal antibody (PSA pAb or IL-8pAbformed a “sandwich”type complex with the capture antibody,

20、leading to the binding of the universal nanoprobe to the electrode that can be transuded to the catalytic amperometric readout.The process of the universal nanoprobe preparation was as following:the pristine MWNTs were rst sonicated in the HNO 3and H 2SO 4to generate carboxylic acid groups which wer

21、e then activated by EDC/NHS.After removal of free EDC/NHS,Ab 2/HRP mixture in an optimized ratio was added and the universal nanoprobe was achieved.immunoassays based on the utilization of encapsulated electro-chemical signal-generating microcrystals was reported (Mak et al.,2005.The electrochemical

22、 signal was achieved by the release of a large amount of ferrocene after sandwich immuno-binding.Ruslings group has achieved greatly enhanced sensitivity using carbon nanotubes (CNTscarrying horseradish peroxidase (HRPlabels and antibodies for immunodetection of the prostate specic antigen (Jensen e

23、t al.,2009;Yu et al.,2006.The limit detection of this CNT amplied immunosensor was low to 4pg mL 1.Nano-materials have been demonstrated to be excellent carriers in the amplication strategies.Despite advances in nano-amplication technologies,there are still challenges faced by researchers such as co

24、mplicated assembly process and stability of nanomaterials.Especially,different anti-bodies have different electrostatic properties so that the assembly conditions of different antibodies with same nanomaterials are variant very often.When encountering with simultaneous detec-tion of panels of tumor

25、markers in clinical diagnosis of cancers (Liu et al.,2004;Wilson,2005,several different nanomaterial based bioconjugates were demanded.However,the processes were complicated.As a result,it is necessary to develop a sim-ple approach which can overcome these obstacles and give a total solution for thi

26、s problem.Herein we proposed an electrochemical immunosenor using a disposable sixteen channel screen-printed carbon electrode (SPCEarray combined with a universal multilabel nanoprobe for the simultaneous detection of cancer biomarkers:prostate specic antigen (PSAand Interleukin 8(IL-8.The immo-bil

27、ization of capture antibodies on this SPCE was considerable simple,which was conducted by rst electrochemical activating the carbon working electrode.This process generated carboxylate groups to bind to the amine residues on capture antibodies.Thiscovalent binding was proved to be very efcient.A uni

28、versal mul-tilabel nanoprobe was fabricated by consistent loading of HRP and goat-anti-rabbit IgG (secondary antibody,Ab 2on multiwalled car-bon nanotube (MWNT.As Ab 2can bind to rabbit antibodies for any antigen,this multilabel nanoprobe is available to the detec-tion of any target antigen by using

29、 unlabelled rabbit polyclonal antibody serving as a bridge.Then the universal nanoprobe can be attached to biosensing surface to generate electrochemical sig-nals.Combining this universal nanoprobe with disposable SPCE array,we provide a promising future in clinical applications with simultaneous im

30、munoassay of multiple protein biomarkers.2.ExperimentalY.Wan et al./Biosensors and Bioelectronics xxx (2011 xxxxxx3The washing buffer was PBST(0.5%tween in0.1M PBS buffer. Enzyme diluent was0.1M PBS buffer with1%casein(pH7.2. The buffer for electrochemical measurement was TMB substrate. 1-(3-(dimeth

31、ylamino-propyl-3-ethylcarbodiimidehydrochloride (EDCand N hydroxysulfosuccinimide(NHSwere dissolved in water immediately before use.All solutions were prepared with Milli-Q water(18M cm resistivityfrom a Millipore system.Electrochemical measurements of the immunosensors were performed with an indepe

32、ndent developed16-channel electro-chemical work station.Independent developed disposable16 channel SPCE array,each comprising a carbon working electrode, carbon counter electrode,and silver pseudoreference electrode, were employed all through the experiment(Scheme1.Unlabelled MWNTs and universal nan

33、oprobes were imaged under a scanning electron microscopy(SEM,Hitachi S-2400.Absorbance values of ELISA were obtained with a Tecan GENios microplate reader at room temperature.PSA pAb was diluted with PBS to a concentration of2g mL1 and immediately added to each ELISA plate well.PSA mAb was used as t

34、he control.After incubation overnight at4C,the plate was washed by PBS buffer and then incubated with block buffer(2%BSA in PBSat room temperature for1h.The universal nanoprobe was diluted with PBST buffer1%BSA and1%casein and added to the treated96plate well.After incubation30min at room temperatur

35、e, the ELISA plate was washed and incubated with TMB substrate at room temperature for color development.The absorbance(OD520 was measured with a Tecan GENios microplate reader at room tem-perature.3.Results and discussionThe immunosensor herein employed a16channel SPCE array as a multiplexing assay

36、 platform,each comprising a carbon working electrode,a carbon counter electrode and a silver pseudoreference electrode(Scheme1.The immobilization of capture antibodies on this platform was considerably easy and robust,which involved an electrochemical activation of the carbon working electrode to ge

37、nerate carboxylate groups atrst.And then capture antibody (PSA mAb or IL-8mAbwas attached to the working electrode with the amine residues on the proteins using the EDC/NHS protocol.4Y.Wan et al./Biosensors and Bioelectronics xxx (2011 xxxxxx This covalent attachment was simple,efcient and stable fo

38、r weeks while stored at 4C.A “sandwich”conguration was used here,which involved capture antibodies and signal antibodies to form a “sandwich”type complex with the target antigen.After that,the universal nanoprobe which could bind to the signal antibodies was added to the platfrom.The universal nanop

39、robe containing multiple HRP labels and Ab 2on carbon nanotube was developed to enhance the sensitivity of immunosensor.HRP and Ab 2were attached to the MWNT following a literature (Yu et al.,2006and the pro-cess was optimized.The carboxylated MWNT were rst activated by EDC/NHS which was then remove

40、d and reacted with Ab 2/HRP mixture of optimized ratio.This nanoprobe was to replace con-ventional Ab 2-HRP,and showed great promise of amplication for immunoassay.To be noted,the universal nanoprobe here was not directly reacted with the target antigen and this strategy had several advantages which

41、 were illustrated by the following exper-iments.A series of experiments were carried out to conrm the attach-ment of Ab 2/HRP with MWNT.First of all,The MWNTs and bioconjugates (universal nanoprobewere imaged under a scan-ning electron microscopy (Adam et al.,2002.As was shown in Fig.1,nanotubes dis

42、persed well in both conditions and the MWNT dimmer was obviously increased after the binding of Ab 2/HRP.The increased diameter was about 20nm which conrmed the con-jugation of Ab 2/HRP with the nanotubes.Of note,the increase of the dimmer here was higher than that the previous reported (Yu et al.,2

43、006.The reason was that the amount of antibodies com-bined with MWNT was much higher in this work.The increased dimmer here should attribute to the loading of antibody on carbon nanotubes,while it was determined by the loading of HRP in the previous study.The size of antibody is much larger than tha

44、t of HRP,so only the monolayer of antibodies was visible.HRP molecules were merged in the monolayer.Because the diameter of antibody (immunoglobulin Gis about 10nm,we observed a 20nm-increase in the diameter of MWNT.A simple method to test the activity of HRP after assembly on MWNT was mixing the un

45、iversal nanoprobe with TMB substrate and observing color change.The supernatant in the last centrifuga-tion during the preparation of the universal nanoprobe was taken as the control.As was shown in Fig.2A,the supernatant was color-less and the universal nanoprobe was homogeneous blank solution.When

46、 the supernatant and the universal nanoprobe were added to TMB substrate respectively,color change was observed by naked eyes.The color of TMB mixed with the universal nanoprobe was sig-nicantly changed into dark blue while puny change was observed when mixed with the supernatant,which suggested tha

47、t HRP was attached to the MWNT effectively and free HRP was removed clearly.There were several conditions to be optimized during the prepa-ration of the universal nanoprobe.Activity and stability were interrogated to evaluate the universal nanoprobe.To enhance the activity of the universal nanoprobe

48、 and achieve the better amplication,optimization of Ab 2/HRP ratio was done.MWNTs loading with different Ab 2/HRP ratio were prepared and then electrochemical performances were carried out to evaluate their capabilities.Amperometric responses for these nanoprobes were illustrated in Fig.3,the steady

49、 state current increased whenY.Wan et al./Biosensors and Bioelectronics xxx (2011 xxxxxx5 (B-4. the ratio of Ab 2/HRP increased.The reason might be that the increased amount of Ab 2led to the reactive activity of the univer-sal nanoprobe with PSA pAb.When the ratio was higher than 1/20,however,MWNTs

50、 became unstable and deposited which might because of the electrostatic affection of the Ab 2(data not shown.Thus the 1/20was chosen as the optimal ratio.To be point out,amperometric responses for all the nanoprobes were higher than that for purchased Ab 2-HRP.To enhance the stability of the univers

51、al nanoprobe,several details should be paid attention to.First,a long time sonication of MWNTs was unnegligible before the EDC/NHS activation to ensure the dispersion of MWNTs.The better MWNT was dispersed,the more stable the universal nanoprobe was.Second,BSA was added to the reaction mixture after overnight incu