Sensors and Actuators B 212(2015)440–445Contents lists available at ScienceDirectSensors and Actuators B:Chemicalj 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 /s nbVisual detection of myoglobin via G-quadruplex DNAzymefunctionalized gold nanoparticles-based colorimetric biosensorQing Wang,Xiaohan Yang,Xiaohai Yang ∗,Fang Liu,Kemin Wang ∗State Key Laboratory of Chemo/Biosensing and Chemometrics,College of Chemistry and Chemical Engineering,Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province,Hunan University,Changsha 410082,Chinaa r t i c l ei n f oArticle history:Received 19December 2014Received in revised form 7February 2015Accepted 10February 2015Available online 18February 2015Keywords:Gold nanoparticles DNAzyme Aptamer Myoglobina b s t r a c tSince myoglobin plays a major role in the diagnosis of acute myocardial infarction (AMI),monitoring of myoglobin in point-of-care is fundamental.Here,a novel colorimetric assay for myoglobin detection was developed based on hemin/G-quadruplex DNAzyme functionalized gold nanoparticles (AuNPs).In the presence of myoglobin,the anti-myoglobin antibody,which was modified on the surface of polystyrene microplate,could first capture the target myoglobin.Then the captured target could further bind to DNA1probe which contained the aptamer sequence through aptamers/myoglobin interaction.Next,as the DNA2probe modified AuNPs were introduced,DNA2probe modified AuNPs could hybridize with the captured DNA1probe.Subsequently,DNA2probe which was modified on the AuNPs could fold into a G-quadruplex structure and bind to hemin,and then catalyze the oxidation of colorless ABTS 2−to green ABTS +by H 2O 2.Consequently,the relationship between the concentration of myoglobin and the absorbance was established.Due to AuNPs amplification,the myoglobin concentration as low as 2.5nM could be detected,which was lower than clinical cutoff for myoglobin in healthy patients.This assay also showed high selectivity for myoglobin and was used for the detection of myoglobin in the human serum samples.This work may provide a simple but effective tool for early diagnosis of AMI in the world,especially in developing countries.©2015Elsevier B.V.All rights reserved.1.IntroductionSince acute myocardial infarction (AMI)remains the leading cause of death in most industrialized nations,it is important to evaluate accurately the patients who show symptoms sugges-tive of AMI [1,2].Myoglobin,although not cardiac specific,has been widely suggested as one of the best candidate markers for an early diagnosis of AMI [3].Generally,myoglobin is present in very low concentrations (0.48–5.9nM)in serum of healthy indi-viduals.When muscle tissues are damaged,myoglobin is rapidly released into the circulation and the myoglobin concentration in serum is elevated to 4.8␮M subsequently [4].Some conventional approaches have been employed to detect myoglobin,such as mass spectrometry [5],liquid chromatography [6],electrochemi-cal [7–11]and surface plasmon resonance (SPR)[12–15].Most of these methods showed high sensitivity,but these methods were time consuming and required expensive equipment,which was unable to be applied in point-of-care (POC)testing [16].Recently,∗Corresponding authors.Tel.:+8673188821566;fax:+8673188821566.E-mail addresses:yangxiaohai@ (X.Yang),kmwang@ (K.Wang).we reported a novel assay for sensitive and selective detection of myoglobin using a personal glucose meter [17].Besides glucose meter,colorimetric method offers great potential for POC testing,due to its intrinsic advantages such as cost-effective,rapid,simple,and even only utilizing naked eyes.Zhang et al.reported a colori-metric method for myoglobin detection based on the aggregation of iminodiacetic acid-functionalized gold nanoparticles (AuNPs)[18].Although this method was easy to perform,low cost and time-saving,the detection limit is relatively high.Here,a novel colorimetric method was developed for myoglobin detection based on hemin/G-quadruplex DNAzyme functional-ized AuNPs.G-quadruplex DNAzyme,which is usually formed by binding G-rich nucleic acid to hemin [19–21],can exhibit peroxidase-like activity and effectively catalyze the H 2O 2-mediated oxidation of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS)[22–24].In this assay,hemin/G-quadruplex DNAzyme complex showed inherent advan-tages of simplicity,stability and relatively low cost.Moreover,since a single Au nanoparticle was loaded with hundreds of DNA2probes which contained DNAzyme section [25,26],it could enhance the sensitivity effectively.This work may provide the new tool for early diagnosis of AMI in the world,especially in developing countries./10.1016/j.snb.2015.02.0400925-4005/©2015Elsevier B.V.All rights reserved.Q.Wang et al./Sensors and Actuators B212(2015)440–445441Fig.1.Schematic illustration of myoglobin detection using hemin/G-quadruplex DNAzyme functionalized AuNPs.2.Experimental2.1.Materials and reagentsMyoglobin(from human heart tissue)and monoclonal anti-myoglobin antibody were purchased from Abcam(USA).C reactive protein(CRP)was purchased from Biovision(USA).Bovine serum albumin(BSA),human serum albumin(HSA)and human immunoglobulin G(IgG)were purchased from Beijing Dingguo Changsheng Biotechnology Co.,Ltd.(China).Hemin and ABTS were purchased from Sigma-Aldrich(USA).DNA1probe(5 -CCC TCC TTT CCT TCG ACG TAG ATC TGC TGC GTT GTT CCG ATT TTT AGA TGG CTA TGC AA-3 )[27]and DNA2probe(5 -T17GGG TAG GGC GGG TTG GGT10TTG CAT AGC CAT CT-3 )were synthesized by Sangon Biotech.(Shanghai)Co.,Ltd.The former contained the myoglobin aptamer sequence and the latter contained the DNAzyme section. All of the chemical reagents were of analytical grade or higher. Amicon ultra centrifugalfilter(30K)was purchased from Milli-pore Corporation(USA).Ultrapure water(18.2M cm)was used throughout.2.2.Preparation and modification of AuNPsAuNPs(∼13nm)were synthesized by the trisodium citrate reduction of HAuCl4[28].DNA2probe modified AuNPs were obtained as previously reported[29].Briefly,3.6␮M concentra-tion of DNA2probe was incubated with AuNPs solution for16h at4◦C.Then,the above mixture was aged for40h under0.1M NaCl solution(pH7.0,0.01M PBS buffer).Next,such solution was centrifuged(30min,13,500×g)and the red oily precipitate was washed with0.1M NaCl solution(pH7.0,0.01M PBS buffer). After a second centrifugation,the red oily precipitate was dis-persed in0.3M NaCl solution(pH7.0,0.01M PBS buffer)and DNA2probe modified AuNPs was obtained.Absorption spectra of AuNPs and DNA2probe modified AuNPs were recorded on a UV-1601spectrophotometer(Shimadzu,Japan)at room temper-ature.The maximum UV–visible absorption peak of AuNPs and DNA2probe modified AuNPs in solution were518.0and524.0nm, respectively.According to the previous work[30],it was calcu-lated that the concentration of DNA2probe modified AuNPs was ca.2.9nM.2.3.Immobilization of antibody on the microplateAnti-myoglobin antibody was bound non-covalently to the polystyrene microplate as previously reported[17].In brief,anti-myoglobin antibody(0.5␮g/ml)was incubated the polystyrene microplate for12h at4◦C in a humid chamber.After the microplate was rinsed thoroughly,it was incubated in1%BSA for1h to decrease non-specific binding.Next,the microplate was washed repeatedly,and it was ready for use.2.4.Procedures of myoglobin detectionDifferent concentration of myoglobin solution was incubated in anti-myoglobin antibody modified microplate for60min at37◦C, followed by a thorough rinsing with10mM PBS buffer(pH7.4). Then,DNA2probe modified AuNPs and a concentration of200nM of DNA1probe were added.After60min of incubation at37◦C,the microplate was thoroughly rinsed with10mM PBS buffer.Next,0.5␮M hemin was added and incubated for60min,followed bya thorough rinsing with10mM PBS buffer.After incubation with 6mM ABTS and3mM H2O2for5min at25◦C,absorption spectra of the reaction solution were measured.For identifying the target-specificity of this assay,three pro-teins,i.e.IgG,HSA and CRP,were respectively reacted with anti-myoglobin which was immobilized on the microplate for 60min at room temperature,followed by a thorough washing with10mM PBS.Then,the DNA2probe modified AuNPs and a concentration of200nM of DNA1probe were added and incu-bated for60min,and then the microplate was rinsed repeatedly with10mM PBS.Next,0.5␮M hemin was reacted for60min, followed by a thorough rinsing with10mM PBS buffer.Finally, after6mM ABTS and3mM H2O2were reacted for5min at25◦C, the reaction solution was recoded using UV–visible absorption spectrometer.3.Results and discussion3.1.Principle of colorimetric biosensor for myoglobin detectionThe principle of this novel assay is shown in Fig.1.Anti-myoglobin antibody,which was non-covalently immobilized on microplate,wasfirst used to capture target myoglobin.As the442Q.Wang et al./Sensors and Actuators B212(2015)440–445Fig.2.Effect of(A)K+ion concentration,(B)H2O2concentration,(C)pH value,(D)temperature and(E)the incubation time on the absorbance of the solution.When one parameter changed and the others were under their optimal conditions.The optimal conditions:K+ion concentration:20mM;H2O2concentration:3mM;pH:8.0; temperature:25◦C;the incubation time:5min.The concentration of myoglobin was500nM here.The error bars represent the standard deviation of three measurements.DNA1probe which contained aptamer sequence was introduced, it could be captured on the microplate since the aptamer against myoglobin and anti-myoglobin antibody can bind to myoglobin synchronously[17].Upon the addition of DNA2probe modified AuNPs,the bound DNA1probe could further capture DNA2probe modified AuNPs due to DNA hybridization.Next,DNA2probe which modified AuNPs could fold into a G-quadruplex structure for hemin binding,and then catalyze the H2O2-mediated oxidation of ABTS to release the colored radical product(i.e.ABTS radical cation (ABTS+)),resulting in an obvious color change.This reaction pro-cess could be monitored with a UV-spectrophotometer.According to the relationship between absorption change and the myoglobin concentration,myoglobin could be detected using this simple and sensitive method.Q.Wang et al./Sensors and Actuators B212(2015)440–445443Fig.3.(A)Distinguishable color changes of different concentrations of myoglobin;from right to left,the concentration of myoglobin changed from0to1000nM.(B)Absorption spectra for different concentrations of myoglobin detection.(C)The relationship between the myoglobin concentration and the absorbance enhancement(A1–A0).A0was the absorbance at418nm in the absence of target,while A1was the absorbance at418nm in the presence of target.The insert was the calibration plot for myoglobin.The error bars represented the standard deviation of the three measurements.(D)The change of absorbance for myoglobin and other proteins.3.2.Optimization of the experimental conditionsFor improving the sensitivity,some factors,such as pH,tem-perature,the concentration of K+ion and H2O2,the incubation time between anti-myoglobin antibody and target myoglobin,were optimized.Since it was reported that K+ion can act as coordination cation to stabilize the G-quadruplex structure,the effect of the concen-tration of K+ion was investigated over the range from0to25mM. As shown in Fig.2A,the absorbance increased obviously with the concentration of K+ion and reached a plateau at the concentration of20mM.Thus,a concentration of20mM of K+ion was selected.The concentration of H2O2played an important role in this assay,so the concentration of H2O2was also optimized.As shown in Fig.2B,as the H2O2concentration increased to3mM,the absorbance reached the maximum;while a decrease was observed at higher H2O2concentrations.As the concentration of H2O2was low,the low amount of green product was obtained,resulting in the low signal intensity.As the concentration of H2O2increased, the high signal could be obtained.However,the blank response also increased,resulting in the low absorbance difference.Hence,3mM H2O2was selected as the optimum H2O2concentration here.pH value can influence not only the formation of G-quadruplex,but also the catalytic activity of DNAzyme,so the effect of pH value was also investigated.As shown in Fig.2C,the absorbance increased with the pH as the pH value ranged from4.0to8.0;while a decrease was observed as pH value increased from8.0to10.0.Therefore,pH 8.0was selected as the optimum pH environment.In view that both DNAzyme construct and its enzyme activity were influenced obviously by the reaction temperature,the tem-perature was also investigated.As shown in Fig.2D,when the temperature was either lower or higher than25◦C,the absorbance444Q.Wang et al./Sensors and Actuators B212(2015)440–445signal obviously decreased.Thus,25◦C was chosen as the reaction temperature.Since the reaction was initiated by hemin/DNAzyme complex to the mixture of ABTS and H2O2,the effect of incubation time with the mixture of ABTS and H2O2was also investigated.As shown in Fig.2E,the absorbance signal shifted obviously as the incubation time increased,and then became saturated at5min.Hence,5min was selected as the incubation time with the mixture of ABTS and H2O2.3.3.Detection of myoglobinDifferent concentrations of myoglobin were detected at the fol-lowing experimental conditions:the concentration of K+ion,the concentration of H2O2,pH,temperature,and the incubation time was20mM,3mM,8.0,25◦C and5min,respectively.As shown in Fig.3A,the color of the solution was gradually turned into green as the amount of myoglobin increased,and even20nM myoglobin could be discriminated by the naked eye.As the concentration of myoglobin increased,the amount of DNA2probe modified AuNPs which was captured also increased,resulting in the increase of DNAzyme/hemin complex.Subsequently,the green product which was produced in the solution as ABTS and H2O2were added,result-ing in an obvious color change.The UV–vis spectra of the solution in the presence of different concentrations of myoglobin are depicted in Fig.3B.Obviously,as the target myoglobin was present,there appeared a clear absorption peak at418nm.Along with the increas-ing of the concentration of myoglobin,the absorbance at418nm correspondingly increased.The absorbance enhancement(A1–A0) of the solution versus the myoglobin concentration was employed to quantitatively detect myoglobin,and an acceptable standard deviation was obtained from three repeated experiments.A1was the absorbance of the solution at418nm in the presence of myo-globin,while A0was the absorbance of the solution at418nm in the absence of myoglobin.As shown in Fig.3C,the absorbance enhance-ment increased with the increase of myoglobin concentration.Inset showed a linear dependence of the absorbance enhancement on the myoglobin quantity in the range of2.5–100nM.According to the3ırule,the detection limit of this method was estimated to be 2.5nM,which was below the clinical cutoff myoglobin concentra-tion of5.4nM for healthy patients[14].Moreover,this sensitive was comparable to or better than that of those previous work [10,11,14,16,18].Besides sensitivity,the selectivity of this assay was also investi-gated.Three proteins,i.e.IgG,HSA and CRP,were used as contrasts. As shown in Fig.3D,the presence of myoglobin led to remarkable increase of the absorbance,while very low absorbance could be detected in the presence of other three proteins.It implied that this assay showed high selectivity for myoglobin detection.The excellent selectivity of this assay may be ascribed to the high speci-ficity of the anti-myoglobin antibody/myoglobin interaction and aptamer/myoglobin interaction.This method was also used to detect myoglobin in human serum.Here,three human serum samples which was obtained from hospital were detected using Automated Chemistry Analyzer and this method,respectively.For this method,human serum sam-ples werefirstfiltered using centrifugalfiltration devices(30K) to remove macromolecules,e.g.HSA and other abundant proteins. Then the treated human serum samples were analyzed.The results obtained are presented in paring with the results measured using Automated Chemistry Analyzer,it was easy to find that the results obtained by our proposed method showed good accordance with that of commercial analyzer.The results demonstrated its potentials for practical applications in disease diagnostics.Table1Detection of myoglobin in human serum samples.Myoglobin detection usingAutomated Chemistry Analyzer(nM)Myoglobin detection usingthis assay(nM)(n=3)Sample153.549.8±3.2Sample2114.8115.7±7.3Sample3184.2176.3±9.14.ConclusionIn conclusion,a novel and sensitive colorimetric assay was developed for myoglobin detection based on AuNPs and G-quadruplex DNAzyme.This method did not require any expensive instruments(only using UV–vis spectrophotometer).Due to the AuNPs amplification,this assay showed excellent sensitivity.More-over,this simple and sensitive assay could be used for myoglobin detection in human serum samples,implying that it has great potential application in clinical biological analysis,especially in developing countries.Theoretically,this assay could potentially be used for the detection of other targets which can bind to the aptamer and antibody(or aptamer)synchronously,such as adeno-sine,thrombin and cell.For prospective development,there are at least two attempts underway.One is validation of assay performance for quantitative purposes,including accuracy,precision,etc.The other is to inte-grate the sample treatment process with this assay based on the microfluidics.AcknowledgmentsThis work was supported by the National Natural Science Foundation of China(21190040,21375034,21175035),National Basic Research Program(2011CB911002),International Science& Technology Cooperation Program of China(2010DFB30300),the Fundamental Research Funds for the Central Universities and the China Scholarship council(201308430175).References[1]WHO,Health Topics,Cardiovascular Diseases,World Health Organization,www.who.int/topics/cardiovasculardiseases/en(accessed on April,2011). 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He is a professor of chemistry in Hunan University.His research interests are in the field of optical biosensing on nanometer and single molecule level.Fang Liu is a graduated M.S.student at Hunan University.Her research interest is mainly focused on the development and application of portable sensor.Kemin Wang is a professor of chemistry and biomedical engineering of Hunan Uni-versity since1992.He obtained a Ph.D.degree in analytical chemistry at Hunan University in1987and carried on post-doctor study in Union High Institute of Tech-nology of Zurich in Switzerland from1989to1991.His research interests mainly concentrate biological analytical chemistry on nanometer and single molecule level, such as nanometer biotechnology,nanometer biomedical device,chemistry and biological sensing technology,etc.。