Loop-Mediated Isothermal Amplification Integrated

Loop-Mediated Isothermal Amplification Integrated on Microfluidic Chips for Point-of-Care Quantitative Detection of PathogensXueen Fang,†,‡Yingyi Liu,‡Jilie Kong,*,†and Xingyu Jiang*,‡Department of Chemistry and Institutes of Biomedical Sciences,Fudan University,Shanghai200433,P.R.China,and CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety,National Center for Nanoscience and Technology,Beijing100190,P.R.ChinaThis work shows that loop-mediated isothermal amplifica-tion(LAMP)of nucleic acid can be integrated in an eight-channel microfluidic chip for readout either by the naked eye(as a result of the insoluble byproduct pyrophosphate generating during LAMP amplification)or via absorbance measured by an optic sensor;we call this system micro-LAMP(µLAMP).It is capable of analyzing target nucleic acids quantitatively with high sensitivity and specificity. The assay is straightforward in manipulation.It requires a sample volume of0.4µL and is complete within1h. The sensitivity of the assay is comparable to standard methods,where10fg of DNA sample could be detected under isothermal conditions(63°C).A real time quan-titativeµLAMP assay using absorbance detection is pos-sible by integration of opticalfibers within the chip.Pseudorabies virus(PRV)is the main pathogen of pseudora-bies,which would infect pigs with high mortality.Effective PRV detection is very important in the surveillance and control of the acute infectious disease.Traditional methods for PRV detection includes virus isolation,immunohistological assays,and various polymerase chain reactions(PCRs),which either consume unac-ceptably long time or demand sophisticated instruments for routine and large-scale assays or point-of-care detection.Loop-mediated isothermal amplification(LAMP)is a method for the amplification of nucleic acids,which amplifies DNA/RNA under isothermal conditions(60-65°C)with high specificity and sensitivity using a set of six specially designed primers and a Bst DNA polymerase.1Without the need to accurately toggle the reaction mixture between different temperatures normally re-quired for PCR,LAMP is a powerful tool for nucleic acid amplification and it has already been used widely in pathogen detection,such as human immunodeficiency virus(HIV),2severe acute respiratory syndrome coronavirus(SARS-CoV),3hepatitis B virus(HBV),4H5avian influenza virus,5and so forth.Although LAMP is more convenient and effective than technologies based on pathogen isolation,immunoassays,and PCRs,most of the methods for monitoring the process of LAMP are performed in macroscale tubes,often requiring at least tens to hundreds of microliters of solutions in polypropylene tubes,which severely limits the throughput/miniaturization of LAMP and the incorpora-tion of LAMP into automated and integrated diagnostic systems.Recent developments in microfluidics technology have enabled applications related to lab-on-a-chip or micrototal analysis systems. They allow the manipulation of small volumes of liquids in microfabricated channels and in some cases microchannels to perform all analytical steps including sample pretreatment,reac-tion,separation,and detection on a small chip in an effective and automatic format.6-8Microfluidics has been applied in many biological assays,such as electrophoresis,9immunoassays,10-14 nucleic acid amplification analysis,15-17cell manipulations18-21and*To whom correspondence should be addressed.E-mail:xingyujiang@ (X.J.);jlkong@(J.K.).†Fudan University.‡National Center for Nanoscience and Technology.(1)Notomi,T.;Okayama,H.;Masubuchi,H.Nucleic Acids Res.2000,28,E63.(2)Curtis,K.A.;Rudolph,D.L.;Owen,S.M.J.Virol.Methods2008,151,264–270.(3)Hong,T.C.;Mai,Q.L.;Cuong,D.V.J.Clin.Microbiol.2004,4,1956–1961.(4)Cai,T.;Lou,G.Q.;Yang,J.;Xu,D.;Meng,Z.H.J.Clin.Virol.2008,41,270–276.(5)Imai,M.;Ninomiya,A.;Minekawa,H.;Notomi,T.;Ishizaki,T.;Tashiro,M.;Odagiri,T.Vaccine2006,24,6679–6682.(6)Zheng,B.;Ismagilov,R.F.Angew.Chem.,Int.Ed.2005,44,2520–2523.(7)Whitesides,G.M.Nature2006,442,368–373.(8)Wang,J.B.;Zhou,Y.;Qiu,H.W.;Huang,H.;Sun,C.H.;Xi,J.Z.;Huang,b Chip2009,9,1831–1835.(9)Manz,A.;Harrison,D.J.;Verpoorte,E.M.J.;Fettinger,J.C.;Paulus,A.;Lu¨di,H.;Wider,H.M.J.Chromatogr.1992,593,253–258.(10)Jiang,X.Y.;Ng,J.M.K.;Stroock,A.D.;Dertinger,S.K.W.;Whitesides,G.M.J.Am.Chem.Soc.2003,125,5294–5295.(11)Yang,D.Y.;Niu,X.;Liu,Y.Y.;Wang,Y.;Gu,X.;Song,L.S.;Zhao,R.;Ma,L.Y.;Shao,Y.M.;Jiang,X.Y.Adv.Mater.2008,20,4770–4775. (12)Liu,Y.Y.;Yang,D.Y.;Yu,T.;Jiang,X.Y.Electrophoresis2009,30,3269–3275.(13)Shi,M.H.;Peng,Y.Y.;Zhou,J.;Liu,B.H.;Huang,Y.P.;Kong,J.L.Biosens.Bioelectron.2007,22,2841–2847.(14)Chen,H.;Jiang,C.M.;Yu,C.;Zhang,S.;Liu,B.H.;Kong,J.L.Biosens.Bioelectron.2009,24,3399–3411.(15)Northrup,M.A.;Ching,M.T.;White,R.M.;Watson,R.T.Proc.Tranducers1993,924–926.(16)Schaerli,Y.;Wootton,R.C.;Robinson,T.;Stein,V.;Dunsby,C.;Neil,M.A.A.;French,P.M.W.;deMello,A.J.;Abell,C.;Hollfelder,F.Anal.Chem.2009,81,302–306.(17)Huang,Y.Y.;Castrataro,P.;Lee,C.C.;Quake,b Chip2007,7,24–26.(18)Sun,Y.;Liu,Y.Y.;Qu,W.S.;Jiang,X.Y.Anal.Chim.Acta2009,650,98–105.(19)Chen,Z.L.;Li,Y.;Liu,W.W.;Zhang,D.Z.;Zhao,Y.Y.;Yuan,B.;Jiang,X.Y.Angew.Chem.,Int.Ed.2009,48,8303–8305.(20)Chen,Z.;Xie,S.B.;Shen,L.;Du,Y.;He,S.I.;Li,Q.;Liang,Z.W.;Meng,X.;Li,B.;Xu,X.D.;Ma,H.W.;Huang,Y.Y.;Shao,Y.H.Analyst2008, 133,1221–1228.Anal.Chem.2010,82,3002–300610.1021/ac1000652 2010American Chemical Society 3002Analytical Chemistry,Vol.82,No.7,April1,2010Published on Web03/10/2010so forth.Among these assays,nucleic acid amplification-based microfluidics is an active research bination of LAMP and microfluidic technology will miniaturize the LAMP detection system and facilitate the realization of point-of-care(POC)patho-gen detection.In this study,we integrate the LAMP on a microfluidic chip, which we call microLAMP(µLAMP)to quantitatively detect target nucleic acids with high sensitivity,specificity,and rapidity.This device potentially enables LAMP assays to be highly portable for on-site analysis.MATERIALS AND METHODSMaterials.Pseudorabies virus(PRV)derived from cell culture was provided by the Shanghai Entry-Exit Inspection and Quar-antine Bureau(SHCIQ).Total PRV genomic DNA used as the positive model was extracted using the QlAamp DNA Blood Mini Kit(Qiagen GmbH,Germany).This virus was used as the model for the development of theµLAMP assay for the following reasons: (1)as a real world virus,this model is more complex and challenging than a synthetic sequence of nucleic acids and(2) the surveillance of PRV is particularly important in countries(e.g., China)where pork is the predominant source of meat.Microfluidic Chip Design and Fabrication.A poly(dimeth-ylsiloxane)(PDMS)master with positive surface patterns was molded against a60mm×60mm poly(methyl methacrylate) (PMMA)glass fabricated by mechanical microfabrication.The PDMS replica was produced by soft lithography as the following: 19The PDMS precursor mixture prepared at a weight ratio of base to curing agent of10:1was poured carefully on the master, placed under vacuum for∼0.5h to rid the bubbles,and cured at 80°C for2h.The cured PDMS replica was gently peeled off the master,and the conically shaped inlet/outlet was drilled manually using a knife.(This kind of inlet/outlet was necessary for the con-venient and accurate addition of the DNA sample and at the same time making the capillary force available to transport the LAMP reaction mixture into microchannel.)Finally,the replica was irreversibly sealed with a microscope glass slide by an O2plasma to form a leak-proofµLAMP microchannel.Thefinal dimension of the microchannel is1mm×0.8mm×0.6mm with a volume of∼5µL.Setup for Real-Time Quantitative Analysis.A detection length of1.2mm was used in the real-time turbidity absorbance detection system while the volume of the microchannel remained to be5µL.The optical detection unit including opticalfibers(FU-76F,Keyence Corporation,Osaka,Japan)and digitalfiber optic sensor(FS-V31M,Keyence Corporation,Osaka)were applied in our system.Thefiber optic sensor employs a high-intensity red light-emitting diode(LED)light at640nm and a phototransistor. The launching and collecting opticalfibers with a265µm diameter core and400µm diameter cladding were inserted carefully into thefiber channels that oppose each other.The reduction of optical density was used to indicate the turbidity generation of the LAMP reaction:22,23optical density)ln(I/I1)=turbiditywhere I0is the intensity of incident light and I1is the intensity of transmitted light.Serial dilutions(10-fold)of PRV DNA ranging from105to10fg/µL were used as templates to evaluate the dynamics of LAMP amplification in microfluidic chips and establish standard curves for quantitative analysis.LAMP Amplification.The LAMP reaction was performed according to our previous work with minor modification.24The whole volume of the system was5µL,which contained1×ThermoPol buffer(New England Biolabs Inc.),8.0mM MgSO4, 0.8M betaine(Sigma,Germany),1.0mM dNTPs(Invitrogen), 0.2µM each of the outer primer(F3,CGCCTTCCTGCAC-TACG;B3,AGCGGGCCGTTGAAGA),1.6µM each of inner primer(FIP,AGAGGTGCACGGGGTAGAGCGGGCACGGT-GTCCATC AA;BIP,GGACGTCAACCGGCTCGTGG CGCGGG-TACACAAACTCCT),and0.8µM each of loop primer(LF, ACGCGCCACGCCTCGTGC;LB,CGACCCCTTCAACG CCAA), 0.32U/µL of Bst polymerase(large fragment;New England Biolabs Inc.)with0.4µL of nucleic acid sample as a template. The amplification was performed at63°C in a laboratory water bath for1h.The detection result was determined directly by the naked eye or afiber optic sensor according to the turbidity of the solution during LAMP amplification,which was then confirmed by agarose gel electrophoresis and restriction digestion with the Hinc II enzyme.Integrated Microfluidic LAMP Chip Operation.A sample containing0.4µL of nucleic acid wasfirst introduced via the inlet.A reaction mixture for LAMP(prepared manually according to the system above)of4.6µL was drawn slowly into the micro-channel by capillary force.The inlet and outlet were tightly sealed by uncured PDMS to form an integral microchamber for LAMP reaction.The whole microfluidic chip was incubated at63°C for 1h using a water bath.Thefinal results were analyzed by the naked eye or optical absorbance and confirmed by agarose gel electrophoresis.The presence of0.1%Triton X-100in the reaction mixture and the hydrophilicity of the PDMS replica(as a result of O2plasma treatment)could help completelyfill the micro-chamber without trapped air.25RESULTS AND DISCUSSIONFabrication of Microchips forµLAMP.We constructed a PDMS-glass hybrid microfluidic chip with eight5µL microchan-nels(Figure1).The microfluidic chip is easy to fabricate without using any precise valves or pumps.The LAMP reaction and readout could be simultaneously performed on the microchip.We prevented typical problems associated with the failure of DNA amplification in microchannels,such as bubble generation,reagent evaporation,cross contamination,by completelyfilling and sealing the microchamber with uncured PDMS in the conically shaped inlet/outlet while taking care to prevent entrapped gas.This method precludes any of the frequently encountered problems reported by researchers designing nucleic amplification micro-channels.26These advantages ofµLAMP are most likely due to(21)Go´mez-Sjo¨berg,R.;Leyrat,A.A.;Pirone,D.M.;Chen,C.S.;Quake,S.R.Anal.Chem.2007,79,8557–8563.(22)Mori,Y.;Kitao,M.;Tomita,N.;Notomi,T.J.Biochem.Biophys.Methods2004,59,145–157.(23)Lee,S.Y.;Huang,J.G.;Chuang,T.L.;Sheu,J.C.;Chuan,Y.K.;Holl,M.;Meldrum,D.R.;Lee,C.N.;Lin,C.W.Sens.Actuators,B2008,133,493–501.(24)Fang,X.E.;Xiong,W.;Li,J.;Chen,Q.J.Virol.Methods2008,151,35–39.(25)Ramalingam,N.;San,T.C.;Kai,T.J.;Mak,M.Y.M.;Gong,H.Q.Microfluid.Nanofluid.2009,7,325–336.(26)Shin,Y.S.;Cho,K.;Lim,S.H.;Chung,S.;Park,S.J.;Chung,C.;Han,D.C.;Chang,J.K.J.Micromech.Microeng.2003,13,768–774.3003 Analytical Chemistry,Vol.82,No.7,April1,2010the fact that µLAMP does not require changes in temperature,a protocol that may bring about problems such as bubble generation in PDMS.In this respect,µLAMP is particularly compatible with PDMS.As an isothermal DNA amplification device,our µLAMP chip did not require a precise thermal cycling module.A water bath or heat block alone was sufficient for performing the µLAMP,which would be more acceptable in resource-poor settings.Sensitivity and Specificity of the µLAMP.During LAMP amplification,a large amount of byproduct,a white precipitate of magnesium pyrophosphate,appears,leading to a turbid reaction mixture,which could be directly observed by the naked eye.27We incorporated this visual detection method in the µLAMP system.Such visual detection often suffers from low sensitivity in microchan-nels because of the short optical length.Lee et al.demonstrated the necessity of at least a volume of 25µL in the microchamber for the turbidity detection.Zhang et al.presented a 10µL volume LAMP microchamber for visual determination.28In our system,we designed an optical length of 800µm for turbidity detection of µLAMP while the reaction volume was reduced to 5µL.The sensitivity of the µLAMP was evaluated by the naked eye visual analysis and standard agarose gel electrophoresis using a series of PRV DNA dilutions (10-2to 10-8)as templates (original concentration of DNA sample was 10ng/µL).We observed that the detection limit of the assay was 10fg of DNA,100-1000-fold more sensitive than the standard PCRs for PRV detection (Figure 2).24The high sensitivity of our system was possibly attributable to the merits of Bst polymerase and loop-mediated mechanism of the amplification.1Otherwise,the turbidity in the microchannels did not decrease simultaneously with reduction of the initial DNA copies,which made the naked eye detection more powerful and effective (Figure 2A).To demonstrate the specificity of µLAMP,we applied a Hinc II restriction enzyme digestion assay.24Products of a band of predict-able size of ∼108bp were resolved on the gel after the Hinc II enzyme digestion assay,demonstrating that the target region of the nucleic acid was amplified specifically (Figure 3B,lane 2).To validate the specificity of µLAMP for PRV,we used viruses not targeted by the LAMP primers,namely,foot-and-mouth disease virus (FMDV),transmissible gastroenteritis of swine virus (TGEV),and porcine parvovirus (PPV)as control experiments.The result shows that µLAMP is highly specific and does not bring about cross-reaction from nontargeted viruses (Figure 3A).Moreover,the specificity of LAMP can be confirmed by the ladderlike pattern observed in gel electrophoresis (Figure 3B,lane 1).1,27Because of the very weak turbid signal of LAMP in the traditional PCR tube,many groups have developed other detection methods in recent years,such as various DNA staining methods,fluorescent LAMP primers,30fluorescent metal indicators,31and so forth.These methods typically rely on either complex equip-ment or sophisticated chemical synthesis.We can,however,easily observe the turbidity in the microchamber with the naked eye alone,which makes µLAMP suitable for integration into complex systems designed to be in a lab-on-a-chip format without having to resort to bulky equipments required in many complex methods.We ascribe the strong turbid signal in the microchamber to its larger depth-to-width ratio (DWR of the microchamber in our µLAMP and a typical PCR tube was 1.33and 1.00,respectively).In a word,the µLAMP established in our study using the direct naked eye detection was highly sensitive,specific,and could be(27)Mori,Y.;Nagamine,K.;Tomita,N.;Notomi,T.Biochem.Biophys.Res.Commun.2001,289,150–154.(28)Hataoka,Y.;Zhang,L.H.;Mori,Y.;Tomita,N.;Notomi,T.;Baba,Y.Anal.Chem.2004,76,3689–3693.(29)Vanoirschot,J.T.J.Clin.Microbiol.1991,5–9.(30)Mori,Y.;Hirano,T.;Notomi,T.BMC Biotechnol.2006,6,3.(31)Tomita,N.;Mori,Y.;Kanda,H.;Notomi,T.Nat.Protoc.2008,3,877–882.Figure 1.Eight-channel PDMS -glass hybrid microfluidic chip for LAMP:(A)photograph and (B)schematic drawing of an eight-channel PDMS -glass hybrid microfluidicchip.Figure 2.Sensitivity of the µLAMP:(A)direct naked eye detection.Channels 1-5show the white precipitate (channels appear white),while channels 6-8do not (channels appear dark).(B)Sensitivity of the LAMP determined by standard agarose gel electrophoresis.(1-7)DNA sample located at 10-2(105fg/µL),10-3(104fg/µL),...0.10-7(0.1fg/µL)dilutions,respectively,and (8)negativecontrol.Figure 3.The specificity of the µLAMP:(A)specificity of the µLAMP determined by nontargetted viruses;(1-5)PRV,FMDV,TGEV,PPV,and negative control,respectively.(B)The specific amplification confirmed by the Hinc II enzyme;(M)DL2000DNA marker,(1)ladderlike bands of µLAMP,(2)product of a band of predictable size of ∼108bp determined by the Hinc II assay,(3)negative control.3004Analytical Chemistry,Vol.82,No.7,April 1,2010conducted together with the amplification in one step without using any detection reagents or equipment.The notable merits of the µLAMP were compared with other methods,including polymerase chain reaction (PCR),enzyme-linked immunosorbant assay (ELISA),and direct virus isolation assay,which were demonstrated in Table 1.We believe that this method has great potential for developing point-of-care devices.µLAMP for Quantitative Analysis.To further show that µLAMP can be easily expanded for more sophisticated assays,we demonstrate that the µLAMP system could also be applied for the quantitative analysis via measuring the absorbance of the reaction mixture.Absorbance assay is a flexible and robust technology commonly used in microfluidic chips.32Because of the generation of turbidity in LAMP reaction,we performed the turbidity absorbance detection by integrating optical fibers in the microfluidic chip to realize real-time monitoring of the LAMP process and its quantitative analysis.We applied a single channel optical detection module with a 1.2mm detection length to develop the quantitative µLAMP (Figure 4).We obtained values of threshold time (Tt,defined as the reaction time necessary for samples to reach sufficiently positive signals above the baseline during real-time amplification)of µLAMP by measuring MPs from different initial concentrations of DNA template had different values of Tt,which can be related to the initial DNA concentration.Tt could be obtained by monitoring absorbance,which changes in real time as a result of the accumulation of precipitates.22We used serial dilutions (10-fold)of DNA templates from 105to 10fg/µL to generate standard dynamic curves by the optical µLAMP chip system and corresponding Tt values (Figure 5A).The log linear regression plot between template concentration and Tt shows a correlation coefficient of 0.9894,making the fiber optical µLAMP chip useful for quantitative DNA analysis (Figure 5B).From the results shown in Figure 5,The LAMP amplification from the lowest sample concentration (10fg/µL DNA)initiated a positive response at 46min and then proceeded rapidly at an approximate exponential rate,reaching a maximum at 50min.Experiments with high initial DNA concentrations exhibited fast positive responses in reaching the maximum.All curves decreased slightly after the maximum time.We attribute this observation to the following reasons:(1)precipitation and aggregation of magnesium pyrophosphate and (2)adsorption of pyrophosphates on the microchannel surface.This decay in turbidity absorbance does not affect the accuracy of the quantitative analysis because of the prominence of the emergence of the Tt.Compared with other known methods for detecting viruses,µLAMP is relatively fast,and virus isolation 33and immunohisto-logical methods 34for detecting PRV are both time-consuming,requiring at least 2-3days,while PCR assays also required 2-3h to finish the amplification.35-37By contrast,in our system,LAMPs from detectable DNA samples could all be accomplished within 60min (with higher concentrations of samples requiring even less time,see Figure 5.This time was comparably short among various methods.The whole diagnostic process from the sample arrival to the final result readout could be accomplished within less than 2h.Although fluctuations could not be avoided between runs in this homemade single channel optical detection system,the emergence of new technologies,such as integrated optical waveguides in microfluidics or optofluidics,may bring us the hope(32)Myers,F.B.;Lee,b Chip 2008,8,2015–2031.(33)Pensaert,M.B.;Kluge,J.P.In Virus Infections of Porcines ;Pensaert,M.B.,Ed.;Elsevier:Amsterdam,The Netherlands,1989;pp 39-65.(34)Ducatelle,R.;Coussement,W.;Hoorens,J.Res.Vet.Sci.1982,32,294–302.(35)Osorio, F. A.In First International Symposium on the Eradication ofPseudorabies Aujeszky’s Disease Virus ;Morrison,R.B.,Ed.;Elsevier:Saint Paul,MN 1991;pp 17-32.(36)Balasch,M.J.;Segale,P.J.Vet.Microbiol.1998,60,99–106.(37)Lee,C.S.;Moon,H.J.;Yang,J.S.J.Virol.Methods 2007,139,39–43.Table 1.Merits of µLAMP Compared with Other Techniquesmethodssensitivity specificity sample timeequipmentµLAMP 10fg/µL high 0.4µL 0.5-1h water bath PCR 24103fg/µL high 2µL 1.5-2h thermocycler ELISA 29∼103fg/µL low 2µL 2-3h ELISA reader neutralization 29,32,33lowhigh∼50µL3daysbiosafetylabFigure 4.Photograph (A)and schematic illustration (B)of the quantitative analysisunit.Figure 5.Results from the optical absorbance assay:dynamic curves (A)and standard curve (B)of the real-time absorbance detection of the LAMP chip.3005Analytical Chemistry,Vol.82,No.7,April 1,2010in realizing multichannel detection,which may achieve increas-ingly accurate real-time quantitative analysis in one experiment.38,39 The combination of the turbidity-based readout of LAMP and the opticalfiber incorporation in the microfluidic chip would be an attractive area and bring us a fascinating future to achieve a POC quantitative nucleic acid analytical device which could be used to survey and combat epidemics,such as SARS,tuberculosis,or influenza A(H1N1)and so forth.CONCLUSIONSIn this study,we integrated an isothermal DNA amplification, LAMP,on a microfluidic chip and fabricated a multichannel microfluidic system for parallel detection of pathogens.The readout could either be a naked-eye determination or a compact real-time absorbance detection device.TheµLAMP presented here allows the direct analysis of a sample of0.4µL of interested DNA in less than1h with a detection limit of10fg/µL.The combination of LAMP and microfluidics will perform diagnostics in a parallel,multiple,high-throughput,and integrated format.The technology presented here will eventually facilitate the realization of POC devices that can be used anywhere,by anyone to assay for agents that are associated with epidemics.ACKNOWLEDGMENTWe are grateful for the kind help from the colleagues in our groups,particularly Wanshun Ma for his help in chip fabrication and Wenying Pan,Bo Yuan,and Yi Zhang for their assistance in image illustration.We thank Dr.Hui Chen for her helpful advice in the manuscript revision.We acknowledge the National Science Foundation of China(2Grants0945001,20890020,20890022, 2009ZX10605,and90813032),the Human Frontier Science Pro-gram,the Chinese Academy of Sciences(Grant KJCX2-YW-M15), and the Ministry of Science&Technology(Grants2007CB714502, 2009CB930001,and2009ZX10004-505)forfinancial support.Received for review January9,2010.Accepted March3, 2010.AC1000652(38)Balslev,S.;Jorgensen,A.M.;Bilenberg,B.;Mogensen,K.B.;Snakenborg,D.;Geschke,O.;Kutter,J.P.;Kristensen,b Chip2006,6,213–217.(39)Keea,J.S.;Poenarb,D.P.;Neuzil,P.;Yobas,L.Sens.Actuators,B2008,134,532–538.3006Analytical Chemistry,Vol.82,No.7,April1,2010。