生物传感器英文文献

A biomimetic sensor for the detection of lead in water

Wendy Chu a,Yuanchao Zhang a,b,Da Li a,Colin J.Barrow a,Hongbin Wang c,

Wenrong Yang a,c,n

a School of Life and Environmental Sciences,Deakin University,Vic.3217,Australia

b College of Chemistry and Chemical Engineering,Ocean University of China,Qingdao266100,China

c School of Chemistry an

d Biotechnology,Yunnan Minzu University,Kunming650031,China

a r t i c l e i n f o

Article history:

Received6June2014

Received in revised form

5September2014

Accepted27September2014

Available online2October2014

Keywords:

Gold nanoparticle

Core–satellite

Self-assembly

Lead

Glutathione

Colorimetric

a b s t r a c t

The monitoring of lead(II)ions(Pb2t)in water is essential for both human health and the environment.

Herein,a simple yet innovative biosensor for Pb2tdetection is presented.The sensor is developed by the

self-assembly of gold nanoparticles(GNPs)core–satellite structure using naturally occurring tripeptide

glutathione(GSH)as linker.The addition of Pb2tcaused a red-to-blue color change and the localized

surface plasmon resonance(LSPR)band was shifted to ca.650nm.The limit of detection(LOD)is found

to be47.6nM(9.9ppb)by UV–vis spectroscopy with high selectivity against other heavy metals.This

method offers a new strategy for heavy metal detection using functionalized GNPs.

&2014Elsevier B.V.All rights reserved.

1.Introduction

Lead(Pb2t)is a toxic heavy metal which commonly contam-

inates water.Tiny amount of Pb2twill eventually accumulate into

fatal level due to its non-biodegradable nature.It damages many

organ systems,including the central nervous system,reproductive

system,liver and kidney.Children are more susceptible to the

effects of Pb2t,and there are evidences suggesting exposure to

low level of Pb2tis associated with attention de?cit hyperactivity

disorder(ADHD),lower IQ and higher risk of childhood aggression

(Jakubowski,2011;Kim et al.,2013;Liu,2011).Therefore,the

monitoring of Pb2tin water is essential for both human health

and the environment.

Many methods have been developed to detect Pb2t,for

example,inductively coupled plasma mass spectroscopy was used

to detect Pb2tin parts per trillion level(Stetzenbach et al.,1994).

Although spectroscopic techniques like ICP-AES or ICP-MS can

detect Pb2twith high sensitivity,they require large and cumber-

some instruments.While previous electrochemical approaches

had high-sensitivity,but lacked selectivity for speci?c ions.(Chai

et al.,2010;Cho et al.,2012;Guo et al.,2013).The limited

approaches can provide sensitivity below pico-molar to study

the accumulation of much diluted metal for public health concern

(Cho et al.,2012).Therefore,there is an urgently need to develop a

sensitive,simple,fast and cost effective method for Pb2t

detection.

Gold nanoparticles(GNPs)have been widely used in sensing

applications due to their unique size and shape dependent optical

properties.Localized surface plasmon resonance(LSPR),the col-

lective oscillation of surface electrons due to the excitation of

incident light,is one of the most important properties of GNPs.

The binding of analyte to GNPs causes red-shift in SPR peak can be

used for GNPs-based colorimetric sensing(Fu et al.,2012;Huang

et al.,2010;Kalluri et al.,2009;Li et al.,2010).The surface

modi?cation of GNPs is essential to provide chemical functionality.

Careful selection and design of ligands for surface modi?cation of

GNPs strongly in?uence the sensitivity and selectivity of a sensor.

It has been shown that core–satellite GNPs structure is able to

provide higher sensitivity compared with uniform size GNPs(Choi

et al.,2012).For example,DNA has been widely used as a linker in

the self-assembly of core–satellite structures(Chen et al.,2010;

Chou et al.,2014;Pal et al.,2009;Schreiber et al.,2013;Sebba and

Lazarides,2008;Sebba et al.,2008).We have recently demon-

strated the linkage of large and small GNPs coated with L-cysteine

as core–satellite by Cu2t(Weng et al.,2013).Coupled plasmonic

assembly of colloidal gold and silver has also been reported(Choi

et al.,2012;Encina and Coronado,2010).However,to our

Contents lists available at ScienceDirect

journal homepage:https://www.wendangku.net/doc/3a15566163.html,/locate/bios

Biosensors and Bioelectronics

https://www.wendangku.net/doc/3a15566163.html,/10.1016/j.bios.2014.09.077

0956-5663/&2014Elsevier B.V.All rights

reserved.

n Corresponding author at:School of Life and Environmental Sciences,Deakin

University,Vic3217,Australia.

E-mail address:wenrong.yang@https://www.wendangku.net/doc/3a15566163.html,.au(W.Yang).

Biosensors and Bioelectronics67(2015)621–624

knowledge,no core–satellite structure utilizing peptide as a linker is studied.

Here,a new strategy for Pb2tdetection based on the self-assembly of core satellite GNP structure by glutathione(GSH)is demonstrated.GSH is a naturally occurring tripeptide which can be found in both animals and plants.It has strong af?nity to the GNPs,due to the thiol group of cysteine residue,which can form self-assembled monolayer on GNPs.It also contains hydrophilic functional groups,including two carboxyl groups and one amine group,which can chelate with metal ions.Moreover,the overall

negative charge of GSH molecule provides repulsive force which prevent aggregation of GNPs after ligand exchange reaction.Some studies demonstrated that Pb2tcaused changes in the level of GSH in mammalian,?sh,spider and plant tissues(Canesi et al., 1999;Wilczek et al.,2004;Yadav,2010).Thus,it is hypothesized that GSH can chelate with Pb2tand produce detectable signals via GNPs to allow the development of biomimetic sensors for Pb2tdetection.In this study,a colorimetric detection method for Pb2tusing GSH–GNPs core–satellite structure was developed.The limit of detection(LOD)was found to be47.6nM(9.9ppb)by UV–vis spectroscopy with high selectivity against other heavy metals in standardized solutions.

2.Material and methods

2.1.Materials and apparatus

Gold(III)chloride trihydrate(HAuCl4á3H2O,99.9t%metals basis),lead(II)nitrate(Pb2NO6,ACS reagent,Z99.0%),L-glu-tathione reduced(C10H17N3O6S,Z98.0%),sodium chloride(NaCl, AR grade,Z99%),sodium citrate tribasic dihydrate(C6H5Na3O7á2H2O,ACS reagent,Z99.0%)and different metal salts were purchased from Sigma-Aldrich and used as https://www.wendangku.net/doc/3a15566163.html,li-Q water (18MΩcm)was used to prepared all aqueous solutions.

pH of GNPs solutions was measured by Oakton s pH2700pH/ mV/°C/°F Meter(Oakton Instruments,Illinois,USA).UV–vis spec-troscopy was carried out using CARY300Bio UV–vis spectrometer (Agilent Technologies,California,USA)with Hellma s Analytics 100-QX Quartz SUPRASIL s300Precision cells(light path1mm) (Hellma Analytics,Müllheim,Germany).The image of core–satel-lite structure was visualized using ZEISS Supra55VP scanning electron microscope(SEM)(ZEISS,Oberkochen,Germany).Sur-face-enhanced Raman spectroscopy(SERS)was conducted by Renishaw Invia Raman Microspectrometer(Renishaw plc,Glou-cestershire,UK)with a633nm HeNe laser(RL633HeNe,Renishaw plc,Gloucestershire,UK)and a thermo-electrical cooled CCD detector using10seconds exposure time.

2.2.Colorimetric detection of Pb2t

Approximately 2.5?10à4M GNPs were synthesized as re-ported previously(Weng et al.,2013).To prepare the Pb2tmediated GNPs core–satellite structure,13nm and45nm GNPs were mixed in1:1ratio(v/v)followed by the addition of GSH which gave the?nal concentration of peptide at about100μM. The solution was incubated for30min at room temperature.A series of aliquots of Pb2tsolution and1M NaCl solution,acting as aggregating agent,were added to500μL of GSH–GNPs solution, and incubated for30min at room temperature before performing UV–vis spectroscopy.The ratio of absorbance at650nm to525nm (A650/A525)was used to express the molar ratio of aggregated to disperse GNPs.Three independent readings were taken for each sample and the protocol used for colorimetric detection was illustrated in Fig.1.To investigate the selectivity of core–satellite structure for Pb2t,other metal ions(Ca2t,Cd2t,Co2t,Cu2t,Fe2t,Mg2t,Mn2tand Ni2t)were studied under the same https://www.wendangku.net/doc/3a15566163.html,ke water collected from Deakin University Waurn Ponds Campus,VIC,Australia was used to con?rm the practical application of the assay.

3.Results and discussion

3.1.Characterization of CORE-satellite structure

The pH of mixture of13nm and45nm GNPs(1:1v/v)was around 5.5,which is slightly below the reported pI of GSH (Cutrignelli et al.,2014).The LSPR peak of the mixture was found to be ca.525nm,which was in between the peak of13nm and 45nm GNPs.The solution remained red in the absence of Pb2t. After addition of Pb2t,the solution turned blue.A newly formed peak was observed at ca.650nm(Fig.S1),which was caused by the surface plasmon coupling between the satellites and the core. Fig.2a showed the SEM image of core–satellite structures mediated by Pb2t.

3.2.Optimization of the assay

To optimize experimental condition,the effect of different ratio of13nm to45nm GNPs,overall concentration of GSH and pH to Pb2tinduced self-assemble of core–satellite structures were tested.The self-assemblies of1:1,3:1,4:1,7:1and9:1(v/v)of 13nm to45nm GSH–GNPs solutions in the presence of Pb2twere recorded by UV–vis spectrometry(Fig.S2).The optimum ratio of 13nm GNPs to45nm GNPs was expected to be equal to the amount of13nm GNPs to fully cover the surface of45nm GNPs. An approximate particle number ratio of40:1((45/13)3)of13to 45nm GNPs can be deduced for1:1(v/v)of13to45nm GNPs solutions by the diameters of GNPs.Based on UV–vis spectrum,1:1 (v/v)of13nm GNPs to45nm GNPs solution showed the best result as the650nm peak appeared in the shortest time.The 525nm peak decreased,and the650nm peak increased with time, indicating the formation of aggregated GNPs from dispersed GNPs.

The overall concentrations of GSH of10,50,100and150μM were used to functionalize the1:1(v/v)GNPs mixture(Fig.S3).It turn out that100μM GSH is best.The experimental results indicated that excess or limited GSH might hinder the formation of Pb2tmediated core–satellite structures.

Then pH were optimized(Fig.S4).At pH3,the peak at650nm appeared before addition of Pb2t,indicating aggregation by GSH–GSH interaction,but not the GSH–Pb2tchelation.Although it has been reported that GSH–GNPs speci?cally detected Pb2tat pH8 (Beqa et al.,2011;Chai et al.,2010;Mao et al.,2011),in our system, no aggregation was recorded at this pH.We also found the self-assembly was also not favorable at more basic environment(pH9). The UV–vis spectrum showed slight increase of650nm peak at pH 6,but best result was obtained at pH5.5.This might be due to curvature effect of various diameters of GNPs.According to the pKa equilibrium of GSH(Fig.S5),in a solution at pH3.59to8.75, majority of GSH would be in the form composing of a protonated amino group and two deprotonated carboxyl

groups.

Fig.1.Schematic representation of the formation of Pb2tmediated core satellite structure.

W.Chu et al./Biosensors and Bioelectronics67(2015)621–624 622

3.3.SERS spectra of GSH –GNPs before and after Pb 2taddition SERS was conducted to further understand the nature of the self-assembly of Pb 2tmediated GSH –GNPs core –satellite struc-ture.Since the LSPR peak shifted to ca.650nm when Pb 2twas added to the GSH –GNPs core –satellite solution,and the excitation was most effective when the excitation frequency was closed to the maxima of the plasmon band,633nm excitation laser was used for the SERS study in order to produce the optimal SERS results.

Raman spectrum was failed to obtain from aqueous GSH solution,thus Raman spectrum obtained from GSH powder was used as the reference (Fig.2b).The SERS spectrum of GSH-functionalized GNPs (Fig.2b)exhibited some strong features that corresponded very well with those of the spectrum of GSH powder.The disappearance of S –H peak at 2526cm à1(Fig.2b),in particular,con ?rmed the hybridization of GNPs with GSH as Au-S bond was formed instead (Qian and Krimm,1994).The peaks at 2865to 2999cm à1found in the spectrum of GSH powder were assigned to be the symmetric and asymmetric stretching of C –H bonds,these peaks were surface-enhanced and formed a broaden peak at 2934cm à1when GNPs were functionalized with GSH.The two peaks enhanced greatly at 1475and 1579cm à1,which were blue-shifted from the bands found in the reference spectrum at 1442and 1450cm à1,were assigned to be the bending of C –H bonds.While the peak at 1359cm à1,which was blue-shifted from the peak at 1337cm à1of the spectrum of GSH powder was assigned to be the stretching of C –O bond.

The state of aggregation of nanoparticles plays an important role in the enhancement of SERS (Moskovits,2005;Sztainbuch,2006).After the addition of Pb 2t,the Raman spectroscopy was enhanced greatly (Fig.2b),indicating the induction of the self-assembly of core –satellite structure by Pb 2t,which resulted in the formation of larger aggregates.Most of the peaks disappeared suggesting a lack of the previously existed zwitterion species (i.e.COO àand C –NH 3t).Pb 2tions were bound to GSH and induced the self-assembly of core –satellite structure.3.4.Kinetics

The formation of core –satellite structure caused changes in LSPR.UV –vis spectrum of different concentration of Pb 2tadded to 1:1(v/v)13nm to 45nm GNPs with an overall GSH concentration

of 100μM at pH 5.5were recorded.The kinetics were investigated to gain an insight into the process as we observed that the newly formed peak appeared faster when higher concentration of Pb 2twas added.Data from the UV –vis spectrum were used to plot the kinetics (Fig.S6).The kinetic curve for negative control was ?at,and higher Pb 2tconcentration increased the aggregation rate.The kinetic curves reached plateau after 30min.Thus,to obtain a stable measurement,the incubation time for colorimetric detec-tion was set at 30min.The kinetic pattern was found to be very similar to the prior report (Weng et al.,2013).3.5.Colorimetric detection of Pb 2t

To investigate the ability of the established core –satellite structure on Pb 2tdetection,a calibration curve was obtained by recording the absorbance ratio when different concentrations of Pb 2tions were added to the core –satellite samples.Each of the samples was incubated for 30min before conducting UV –vis spectroscopy,and three independent measurements were carried out for each samples to provide statistical signi ?cance.The shifting in LSPR band is due to the coupled plasmon resonance of GNPs when the interparticle distance changes.The peaks at 650nm and 525nm were related to the quantity of aggregated and dispersed GNPs respectively.Thus A 650/A 525was used as the indicator in this study.A good linear relationship between A 650/A 525and concen-tration of Pb 2twas observed from 2to 14m M (R 2?0.9934)(Fig.3a).The LOD was determined to be 47.6nM (9.9ppb)by 3s rule,which was just lower than the WHO standard limit (10ppb)(World Health Organization,2008).

Several commonly existing heavy metals were used to test the selectivity of the assay.It was observed that 20m M Ca 2t,Cd 2t,Co 2t,Cu 2t,Fe 2t,Mg 2t,Mn 2tand Ni 2thad no obvious effect on the color of 1:1(v/v)13nm and 45nm GSH –GNPs solution.While the solution changed from red to blue,due to the addition of 20μM Pb 2t,indicating the system was highly selective toward Pb 2t.The changes in values of A 650/A 525upon the addition of 20m M metal ions were also plotted (Fig.3b),showing the high selectivity for Pb 2t.It is proposed that the con ?guration of core –satellite structure limited the species of ions to chelate with GSH,and the rate of aggregation induced by other metal ions was relatively slow compared to Pb 2t.However,further investigation is needed for the detailed mechanism of selectivity in the future

work.

Fig.2.(a)SEM image of Pb 2tmediated core –satellite structure.(b)Raman spectra using 633nm excitation laser.(a)GSH powder (black);(b)GSH –GNPs before Pb 2taddition (red);(c)GSH –GNPs after Pb 2taddition (blue).(For interpretation of the references to color in this ?gure legend,the reader is referred to the web version of this article.)

W.Chu et al./Biosensors and Bioelectronics 67(2015)621–624623

3.6.Analysis of real samples

In real-life application,the concentration of unknown contami-nants in sample may be signi ?cantly greater than that of Pb 2t,thus causing interference with the test results,which is a critical problem to the application of many sensors developed.Untreated lake water collected from Deakin University Waurn Ponds Campus,Vic.,Australia was spiked with known concentrations of Pb 2tto mimic the contamination of water to con ?rm the practical application of this assay.The detected Pb 2tconcentration in lake water samples was shown in Table S1.The high recovery sug-gested that the core –satellite structure was not free from the matrix effect of the samples and may be selective towards other metal ions which were not tested in this study.Further investiga-tion and modi ?cation were needed for the practical application of this method.

4.Conclusions

In this paper,we described a simple yet innovative colorimetric detection method for Pb 2tbased on the self-assembly of GSH –GNPs core –satellite structures.The experimental results showed that the core –satellite structure can detect as low as 47.6nM Pb 2twith high selectivity against other heavy metals.It is suggested such core –satellite structures through nature peptides could be utilized for development of highly sensitive and sensitive colori-metric detection methods for toxic heavy metals.

Acknowledgement

WY would like to thank Australian Research Council for the ?nancial support (DPDP130101714).

Appendix A.Supplementary material

Supplementary data associated with this article can be found in the online version at https://www.wendangku.net/doc/3a15566163.html,/10.1016/j.bios.2014.09.077.

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Fig.3.(a)Fitted linear plot of A 650/A 525versus concentration of Pb 2t;(b)A 650/A 525upon the addition of 20m M other metal ions.The error bars represent the standard deviations based on three independent measurements.

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C++ [1] Gordon Hogenson. C++/Cli The Visual C++ Language For .Net [M]. Wiley India Pvt. Ltd., 2007. [2] Motor Industry Software Reliability Association. MISRA-C: 2004: guidelines for the use of the C language in critical systems.[M]. MIRA, 2008. [3] Jeff Cogswell, John Paul Mueller. C++ All-In-One Desk Reference For Dummies [M]. Wiley publishing.Inc 2009. [4] Stephen R. Davis. C++ for Dummies [M]. wiley publishing.Inc 2008. [5] Harvey Dietel, Paul Deitel. C: How to Program [M]. Pearson Education,Inc 2010. [6] Bruce Eckel. Thinking in C++[M]. Prentice Hall, 2000. [7] Herbert Schildt. C++: a beginner's guide Beginner's Guides[M]. McGraw-Hill Professional, 2003. [8] Mark Lee. C++ Programming for the Absolute Beginner For the Absolute Beginner[M]. Course Technology, 2009. MIS参考文献 [9] Kenneth C. Laudon, Jane P. Laudon . Management Information Systems: Managing the Digital Firm[M]. Publisher Prentice Hall, 2007. [10] Raymond McLeod, George P. Schell. Management information systems[M]. Pearson/Prentice Hall, 2007. [11] James A. O'Brien, George M. Marakas. Management Information Systems[M]. McGraw-Hill/Irwin, 2008.

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Title ：Magnetic motor shell stamping process and die design Author：yu Department of Materials "Magnetic motor shell stamping process and die design" literature review Abstract摘要 By read these references and documents, in-depth understanding of the contemporary mold of advanced manufacturing technology and metal forming technology, a number of instances of mold design and the understanding and learning, to further study the method of stamping die design, die design and thus have a directionalguidance.As used in this design and drawing die punching die and so on, through the design of the book related to mold in-depth study, this drawing die and the punching die and so the design methods have shape.These references and documents, the design of low-cost high-accuracy die with directional guidance. Keywords： Mold advanced manufacturing technology Mold Manufacturing Trends Drawing Punching CAE Die Materials Prices Preface前言 As China's economic integration with the world economy, the rapid development of basic industries, mold manufacturing industry is also developing fast.In the current economic situation, people pay more attention to efficiency, product quality, cost, and new product development capabilities.The innovation and development of mold manufacturing concern. 1.1 The history of the development of mold Archaeological discoveries in China, as early as 2,000 years ago, China has been used to make bronze stamping dies to prove that in ancient China stamping die stamping and achievements to the world's leading.In 1953, the Changchun First Automobile Works in China for the first time established a stamping plants, the plant began manufacturing cars in 1958, cover mold.60 years of the 20th century began producing fine blanking dies.In walked a long path of development temperature, the present, China has formed more than 300 billion yuan (not including Hong Kong, Macao and Taiwan statistics) the production capacity of various types of stamping dies. 1.2 Development Status and Trends Die Since reform and opening, with the rapid development of the national economy, the market demand of mold growing.In recent years, the mold industry has been the growth rate of about 15% of the rapid development of industrial enterprises in the ownership of the mold components also changed dramatically, in addition to professional mold factory outside of state-owned, collective, joint ventures, wholly-owned and private has been a rapid development.

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