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Biosensors and Bioelectronics
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highly selective and colorimetric assay of lysine by molecular-driven goldanorods assembly
ian Wanga, Pu Zhanga, Chun Mei Lia, Yuan Fang Lia, Cheng Zhi Huangb,∗
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR ChinaCollege of Pharmaceutical Sciences, Southwest University, Chongqing 400715, PR China
r t i c l e i n f o
rticle history:eceived 15 November 2011eceived in revised form 20 January 2012ccepted 2 February 2012vailable online 9 February 2012
eywords:
a b s t r a c t
In this contribution, a simple, rapid, colorimeteric and selective assay for lysine was achieved by acontrollable end-to-end assembly of gold nanorods (AuNRs) in the presence of Eu3+ and lysine. Thisone-pot end-to-end assembly of 11-mercaptoundecanoic acid (MUA) modified AuNRs was occurred inBritton–Robinson buffer of pH 6.0, which involves the coordination binding between Eu3+ and COO−
groups as well as the electrostatic interaction of the COO− groups of MUA with the NH3+ group of
lysine. As monitored by absorption spectra, scanning electron microscopic (SEM) images and dynamic
old nanorods (AuNRs)u3+
mino acidself-assembly
light scattering (DLS) measurement, the end-to-end chain assembly results in large red-shift in the lon-gitudinal plasmon resonance absorption (LPRA), giving red-to-blue color change of AuNRs. Importantly,it was found that the red-shift of LPRA is linearly proportional to the concentrations of lysine in the rangeof 5.0 × 10−6–1.0 × 10−3 M with the limit of detection (LOD) being 1.6 × 10−6 M (3�/k). This red-shift ofLPRA is highly selective, making it possible to develop a rapid, selective and visual assay for lysine in food
samples.
. Introduction
Assembling and ordering nanomaterials into designed patternsre of considerable significance both in fundamental research andractical application, which is due to the fact that the proper-ies of nanomaterials depend not only on the size and shape, butlso on the spatial arrangement and the degree of order amonghe collective building blocks (Nie et al., 2010). For example, goldanorods (AuNRs), a kind of anisotropic one-dimensional nano-aterials, have attracted great attention (Parab et al., 2010; Wang
t al., 2010c, 2011; Zhu et al., 2011) due to the charming plasmonesonance absorption (PRA) properties (Link and El-Sayed, 1999a).nterestingly, AuNRs can be designed into various patterns includ-ng rings (Khanal and Zubarev, 2007), hexagons (Kumar et al., 2007),nd-to-end linear chains (Chang et al., 2005; Huang et al., 2010;udeep et al., 2005; Sun et al., 2008; Wang et al., 2010d; Zhen et al.,009), or side-by-side motif (He et al., 2008; Pan et al., 2007; Sunt al., 2008; Wang et al., 2010a), which are accompanied with cou-ling of the plasmon band of AuNRs (Sun et al., 2008). However,ew of the assemblies have been applied in biosensing (Huang et al.,
010; Sudeep et al., 2005; Wang et al., 2010b).
Especially, the end-to-end assembly of AuNRs has been exten-ively studied due to the chemically active sites mainly belong to
the tips of nanorods (Chang et al., 2005; Hu et al., 2005; Huanget al., 2010; Sudeep et al., 2005; Sun et al., 2008). Therefore, thespecific recognition based end-to-end AuNRs assemblies can befabricated by anchoring ligand with thiol-groups onto the tips ofAuNRs (Wang et al., 2010d; Zhen et al., 2009). The simplest assem-bly of AuNRs can be fabricated just by the target molecules (Josephet al., 2006; Sudeep et al., 2005; Sun et al., 2008; Thomas et al.,2004), which requires dithiol (Joseph et al., 2006) or the inter-molecular interaction (such as hydrogen bonding or electrostaticinteraction) between the same kind of molecules (Joseph et al.,2006; Sudeep et al., 2005; Sun et al., 2008; Thomas et al., 2004). Themost common assemblies of AuNRs, been reported, are based onthe specific ligand–target recognition such as biotin–streptavidin(Caswell et al., 2003), antibody–antigen (Chang et al., 2005),aptamer–protein (Zhen et al., 2009) or ligand–metal (Nakashimaet al., 2007; Wang et al., 2010d). The above driving forces ortemplates, although have been successfully employed for AuNRsassembly, suffer from the request for special structure and bindingsites (Joseph et al., 2006) or cumbersome synthesis and modifi-cation using costly reagents (Chang et al., 2005). Besides, someof them just drive AuNRs to the desirable arrangement withoutfurther application because of the limited spectra or color change(Wang et al., 2010d; Zhen et al., 2009).
In previous reports, investigations of AuNRs assembly concern-ing amino acids have mainly focused on cysteine, which is basedon the fact that cysteine (a kind of unique thiol-containing aminoacid) can form Au S covalent bonds with gold (Hu et al., 2005;
uang et al., 2010; Sudeep et al., 2005; Sun et al., 2008). However,ue to lack of thiol-group, it is considerably more challenging totudy the assemblies involving other essential amino acids such asysine, which is a vital free-form amino acid as a key component ofnimal and human diets (Zhou et al., 2011). By now, the analysisf lysine mainly relies on the time-consuming high performanceiquid chromatography (HPLC), which makes it highly desirable toevelop a rapid, simple and colorimetric assay for lysine (Zhou et al.,011). It has been reported that lysine can induce the aggregationf 11-mercaptoundecaonoic acid (MUA) modified gold nanoparti-les (AuNPs) with the cooperative binding of Eu3+ (Enriquez et al.,008). Nevertheless, the aggregation was not tunable due to theandom binding sites of MUA on AuNPs surface. In this contribution,n virtue of the preferential modification of MUA on AuNRs ends (Yund Irudayaraj, 2007), a controllable end-to-end assembly of AuNRsas achieved in the presence of Eu3+ and lysine, which resulted in a
ed-shift in the longitudinal plasmon resonance absorption (LPRA)nd color change of AuNRs. More importantly, it was found thathe LPRA change was highly selective to lysine, which may poten-ially be used for selective and visual lysine biorecognition in foodamples. It is expected that assembly based on this design will findider applications in nanotechnology and biosensors.
. Experimental
.1. Chemicals and materials
MUA was obtained from Sigma–Aldrich (Milwaukee, WI, USA)nd was dissolved in ethanol. Cetyltrimethylammonium bro-ide (CTAB) and hydrogen tetrachloroaurate(III) tetrahydrate
HAuCl4·4H2O) were purchased from Sinopharm Group Chemicaleagent Co., Ltd. (Shanghai, China). AgNO3 was obtained from Bei-
ing Chemical Company (Beijing, China). Eu(NO3)3 was supplied byigma–Aldrich (Milwaukee, WI, USA). NaBH4 and l-ascorbic acidl-AA) were commercially available from Huanwei Fine Chemi-al Co., Ltd., (Tianjin, China) and Chuandong Chemical Group Co.,td. (Chongqing, China), respectively. Organic amines and aminocids were purchased form Sigma–Aldrich (Milwaukee, WI, USA)nd Shanghai Kangda Factory of Amino Acid (Shanghai, China),espectively.
.2. Experimental instrumentation
A Shimadsu UV-3600 spectrophotometer (Tokyo, Japan) wasmployed to record the PRA spectra. A Nano-zs zetasizer (Malvern,ngland) was used to estimate dynamic light scattering (DLS) ofuNRs. The images and photos of AuNRs were obtained with aitachi S-4800 scanning electron microscope (SEM, Tokyo, Japan)nd an E-510 Olympus camera (Tokyo, Japan), respectively. The pHalues of solutions were measured with a Cyber Scan pH/Ion 510igital pH meter (Eutech, USA).
.3. The preparation and modification of AuNRs
AuNRs were prepared according to the seed-mediated methodJiang et al., 2006; Wang et al., 2008). First, gold seeds were pre-ared by the reduction of HAuCl4·4H2O (2.5 × 10−4 M) with ice-coldaBH4 (9.0 × 10−4 M) in the presence of CTAB (7.5 × 10−2 M). Theixture, which rapidly developed into a light-brown color dur-
ng mixing, was aged for 2–24 h at 25 ◦C before synthesis of NRs.ext, 0.15 mL 0.01 M AgNO3 was added to the growth solution
5.00 mL 0.002 M HAuCl4·4H2O, 11.88 mL 0.2 M CTAB and 7.70 mL2O). After the addition of 0.16 mL 0.1 M l-AA, the solution color
mmediately changed to colorless. Finally, 0.11 mL 2 h aged Au seedolution was put into the above mixture and mixed vigorously,
ectronics 34 (2012) 197– 201
which induced a gradual color change to red. The resulted mix-ture was left undisturbed overnight for further growth. Accordingto Orendorff and Murphy (2006), the concentration of as-preparedAuNRs was about 0.80 nM.
The chemical modification of AuNRs with MUA was achieved asfollows (Yu and Irudayaraj, 2007): 5.0 mL of 20 mM MUA solution inethanol was added to 50 mL of the as-prepared AuNRs. After gentlemagnetic stirring for 24 h at room temperature, the AuNRs werecollected by centrifugation at 10,000 rpm for 20 min to removeexcess MUA and were resuspended in 50 mL 5 mM CTAB solution.
2.4. Experimental measurements
Into a 1.5 mL-plastic vial, 0.3 mL MUA-modified AuNRs and0.15 mL 1.0 × 10−4 M Eu3+ were added, followed by the addition of0.10 mL Britton–Robinson buffer (pH 6.0) and an appropriate vol-ume of lysine. After vortexing, the mixture was diluted to 1.0 mLwith doubly distilled water and mixed thoroughly, and then stoodfor 20 min before measurements with a UV-3600 spectrophotome-ter, which was scanned from 450.0 nm to 1000.0 nm.
For SEM imaging, the samples were dropped onto the siliconfilm and dried, then washed with water to remove CTAB in thesamples. After drying again at room temperature, the samples weretransferred for SEM measurements at a voltage of 30.0 kV and aworking current of 10.0 �A.
2.5. The pretreatment of food samples
We chose three kinds of food samples including milk, breadand cookies (Chongqing, China) to check the lysine content. Beforedetection, these samples were grinded and weighted, followed bydiluting with 50 mL H2O and were put at 37 ◦C for 24 h. Then, thesamples were filtrated with 0.45 �m filter paper to get the clearsolution. Here, we used water to extract lysine in food samplesbecause lysine owns a high solubility in water.
3. Results and discussion
3.1. Strategy of AuNRs assembly
An outline of AuNRs assembly principle is present in Scheme 1.Under the weakly acidic conditions (pH 6.0), MUA is negativelycharged with COO− group and lysine is positively charged withNH3
+. By the formation of Au S covalent bond (Yu and Irudayaraj,2007), AuNRs are modified with MUA molecules with a random dis-tribution in this case. When both lysine and Eu3+ are introduced,MUA modified AuNRs are assembled in the end-to-end manner,which is attributed to the cooperative binding between Eu3+ andCOO− groups (Enriquez et al., 2008) and the electrostatic interac-tion of COO− groups of MUA with NH3
+ of lysine (Jordan and Corn,1997), resulting in the red-to-blue color change due to the plasmoncoupling. The end-to-end chain assembly is achieved only whenboth lysine and Eu3+ are present, this is, if either lysine or Eu3+
is absent, the MUA modified AuNRs (MUA-AuNRs) are randomlydistributed.
3.2. Characteristics of AuNRs assembly
As shown in Fig. 1, the as-prepared AuNRs were in red colorwith the transverse and longitudinal PRA bands of the as-preparedAuNRs at 522 nm and 760 nm, corresponding to the electron oscil-
lation along the short and long axis (Link et al., 1999b; Yu et al.,1997), respectively. Because of a CTAB bilayer packing along thelongitudinal axis of AuNRs (Nikoobakht and El-Sayed, 2001; Smithand Korgel, 2008), the MUA molecule preferentially binds at the
J. Wang et al. / Biosensors and Bioelectronics 34 (2012) 197– 201 199
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Scheme 1. Schematic illustration of the present strategy for assembling A
nds of AuNRs through the formation of Au S covalent bond, lead-ng to a slight red shift of the LPRA (Curve B in Fig. 1) compared to thes-prepared AuNRs (Curve A). As curve C in Fig. 1 shows, the intro-uction of lysine alone induces hardly any LPRA shift, suggestingo assembly is achieved. However, a further 24 nm red shift (curve
in Fig. 1) was observed when Eu3+ alone was added, which is dueo complexation of the carboxylate groups in MUA on AuNRs endsith Eu3+ (Enriquez et al., 2008) to lead a tiny assembly. Further-ore, a large LPRA shift can be observed (curve E in Fig. 1) when
oth lysine and Eu3+ were introduced into the MUA-AuNRs solu-ion, suggesting that end-to-end chain assembly of AuNRs occurred.n accordance with the LPRA change, the color of AuNR solutionthe insert picture in Fig. 1) changed from red to blue when bothysine and Eu3+ were present. Using as-prepared AuNRs as a con-rol experiment, no wavelength shift can be observed when Eu3+,ysine or both Eu3+ and lysine were introduced (Fig. S1 in Support-ng information). These results indicate that the negatively chargedarboxylate groups of MUA on AuNRs end play a very important rolen the assembly, most likely interacting with both lysine and Eu3+
o link AuNRs together.The role of lysine, an amino acid with two amino groups, is
ssumed to be as a cross linker via an electrostatic interactionetween the two positively charged amino groups and the neg-tively charged carboxylate groups of MUA molecules on AuNRsnds. However, the electrostatic interaction is not strong enougho induce the assembly of AuNRs, which needs some reagents tossist to assemble AuNRs. It is reported that Eu3+ can complex with
he carboxylate groups of lysine (Hemminki et al., 1995; Li et al.,004). In this work, Eu3+ can cooperatively bind with the carboxy-
ate groups both of lysine and MUA to help lysine assemble AuNRs.
ig. 1. Normalized PRA spectra and picture of AuNRs assembly. (A) As-prepareduNRs; (B) MUA-AuNRs; (C) MUA-AuNRs + lysine; (D) MUA-AuNRs + Eu3+; (E) MUA-uNRs + lysine + Eu3+. cAuNRs, 0.24 nM; clysine, 1.0 × 10−3 M; cEu3+ , 1.5 × 10−5 M; pH.0.
in an end-to-end mode mediated by Eu3+ and lysine recognition system.
Thus, when both lysine and Eu3+ are present in the system, the obvi-ous red-shift of LPRA can be readily observed, illuminating that theAuNRs assemble in the end-to-end mode.
The successful end-to-end assembly of AuNRs was confirmed bySEM images (Fig. 2) and DLS (Fig. 3) measurement analysis. The SEMimage in Fig. 2A shows that the as-prepared AuNRs have an aver-age aspect ratio of ∼3.3 while highly dispersed in water withoutaggregation due to the positively charged CTAB bilayer (Nikoobakhtand El-Sayed, 2001; Smith and Korgel, 2008). However, owing tothe hydrogen bonding between the carboxylate groups of MUAs(Thomas et al., 2004; Yu and Irudayaraj, 2007), the modificationwith MUA makes AuNRs slightly assemble (Fig. 2B). Compared withMUA-AuNRs, the introduction of either lysine or Eu3+ has littleeffect on the distribution of MUA-AuNRs (Fig. 2C and D). Interest-ingly, when both lysine and Eu3+ are present in the MUA-AuNRssolution, the remarkable end-to-end assembly can be observed(Fig. 2E and F), which is consistent with the plasmon resonanceabsorption spectra change.
Similar to the SEM images, the DLS study (Fig. 3) also indicatesa size change in the presence of both lysine and Eu3+. The meanhydration size of as-prepared AuNRs increased from 43.82 nm to58.77 nm after the modification with MUA. When only lysine orEu3+ was introduced, the mean hydration size of MUA-AuNRs wasless than 77 nm. But if both lysine and Eu3+ were present, the meanhydration size sharply increased to more than 600 nm, providinganother confirmation that the assembly was achieved by the addi-tion of both lysine and Eu3+. The as-prepared AuNRs without MUAmodification were used to do a control experiment (Fig. S2 in Sup-porting information), which showed that the mean sizes were lessthan 60 nm even both lysine and Eu3+ were introduced, suggestingthat MUA molecule links AuNRs together with lysine and Eu3+.
3.3. Selectivity of lysine binding
As the LPRA and the inserted picture in Fig. 4 display, amongall amino acids, only lysine can induce the remarkable LPRAshift and red-to-blue color change, indicating the importance ofEu3+ complexation as well as the electrostatic interaction of thepositively charged ε-amino side chain and the negatively chargedcarboxylate groups of MUA molecules on the ends of AuNRs inthe assembly of AuNRs. Moreover, other positively charged aminoacids such as arginine, histidine and serine (molecule structuresshown in Scheme S1 in Supporting information) have a slightinfluence on the assembly of MUA-AuNRs (Patel and Menon,
2009), which can be attributed to the fact that the NH2 , NH ,OH can be protonated under weakly acidic conditions to linkMUA modified AuNRs together through the complexation as wellas the electrostatic interaction. However, cysteine did not affect the
200 J. Wang et al. / Biosensors and Bioelectronics 34 (2012) 197– 201
Fig. 2. SEM images of end-to-end AuNRs assembly. (A) As-prepared AuNRs; (B) MUA-AuNR(F) is the local enlarged view of the corresponding square area in image (E). cAuNRs, 0.24 n
linearly regressed as � = 803.1 + 0.974 c (10−3 M, r, 0.9917) with the−6
uNRs + lysine + Eu3+. cAuNRs, 0.24 nM; clysine, 1.0 × 10−3 M; cEu3+ , 1.5 × 10−5 M; pH.0.
ssembly of AuNRs because binding sites on AuNRs were occupied
y MUA molecules. Furthermore, we also checked the interfere ofther organic amines with more than one NH2 or NH groups,nd the results showed that neglectable effect for lysine detection
ig. 4. The selectivity for lysine detection, the inserted picture is the correspondingolor change of AuNRs. cAuNRs, 0.24 nM; cEu3+ , 1.5 × 10−5 M; camino acids, 1.0 × 10−3 M;H 6.0.
(Fig. S3 in Supporting information), suggesting that the structureof molecule also plays a very important role in AuNRs assembly.
It is found that the end-to-end assembly of AuNRs also can beinduced by other lanthanide ions such as La3+, Ce3+, Tb3+, Sm3+
and Nd3+, which are less obvious than that Eu3+ (Fig. S4 in Sup-porting information). The varying tendencies for assembly withoutlysine presence can be attributed to the differences in the coordina-tion capabilities of lanthanide ions and carboxylic group (Hui et al.,2006). Considering the structure of amino acids and coordinationcapabilities of lanthanide ions, lysine may interact with Eu3+ sostrong that it can remarkably induce the assembly of MUA-AuNRs.
3.4. Analytical application of the assembly
On account of the high selectivity for lysine to assemble AuNRswith color change, it is expected that this assembly can be applied toselective and colormetric detection of lysine. It was found that thewavelength (�) of LPRA was linearly related to the concentrations oflysine in the range of in the range of 5.0 × 10−6–1.0 × 10−3 M (Fig. 5).The relationship between � of LPRA and lysine concentration can be
limit of detection (LOD) being 1.6 × 10 M (3�/k). Besides, in theinserted picture in Fig. 5, the color of samples showed a red to bluechange with increasing the concentration of lysine, and the LOD
Fig. 5. The relationship between the concentrations of lysine and the variation ofabsorption wavelength, the inserted picture is the corresponding color change ofAuNRs. cAuNRs, 0.24 nM; cEu3+ , 1.5 × 10−5 M; pH 6.0.
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J. Wang et al. / Biosensors and
f this method with naked eyes was 250 �M. Thus, the selectiveinding of lysine can be potentially be used for visual sensing of
ysine.It would be valuable to detect lysine in the health research
ecause since lysine is a kind of essential amino acid in dietAppuhamy et al., 2011; Rutherfurd and Moughan, 2007). To moni-or the analytical application of the newly developed proposal, theysine concentrations in food samples (bread, cookies and milk)
ere tested by adding lysine of different concentrations (stan-ard addition method). Experimental results were described inable S1 (see Supplementary material). These data showed that theecovery (between 93.6% and 106.8%) and relative standard devi-tion values (between 2.28% and 5.58%) were acceptable, whichrovided a promising alternative tool for determining lysine inood samples. The result showed that lysine in milk was morehan cookie and bread, which maybe can attributed to the differentutritional requirements and the decomposability of lysine duringread and cookie baking (Rosenberg and Rohdenburg, 1951).
.5. The experimental conditions
pH played a vital role in assembling AuNRs (Fig. S5 in Support-ng information). The two amino groups of lysine are positivelyharged, as the pKa values of the amine and the amine substituentroups in lysine are 9.08 and 10.82, respectively. Furthermore, thearboxylic acid group of MUA molecule has a pKa of 5.7 (Enriquezt al., 2008). In order to let MUA to be negatively charged to bindith the positively charged lysine and coordinate with Eu3+, theH should be higher than 5.7. However, Eu3+ can complexate withhe carboxylate groups of MUA with increasing pH, leading to thend-to-end assembly of AuNRs without lysine. To minimize thessembly caused by Eu3+ alone and get the greatest wavelengthhange, pH 6.0 was chosen for the assembly of AuNRs.
The concentration of Eu3+ was also taken into considerationshown in Fig. S6). With increasing Eu3+ concentration, the LPRAharply shifts to longer wavelength in the presence of lysine. Whenhe Eu3+ concentration exceeds 1.25 × 10−5 M, the LPRA shows thereatest red-shift. However, if lysine is absent, the slight red-shiftf LPRA also can be observed due to complexation of Eu3+ by thearboxylate groups. Therefore, 1.5 × 10−5 M Eu3+ was chosen foruNRs assembly.
The dynamic assembly process (Fig. S7 in Supporting informa-ion) was detected by scanning the PRA spectra at intervals of 2 minmmediately after the addition of lysine. During 20 min, the LPRAeak shifted to longer wavelength with the wider band and theecreased intensity, suggesting the formation of the end-to-endssembly (Huang et al., 2010). With longer assembly time, the LPRAeak almost kept invariable, suggesting the assembly can be fin-
shed in a very short time, which can be applied to rapid biosensing.
. Conclusions
In summary, the assembly of AuNRs related with amino acidsainly focus on cysteine due to the formation of Au S covalent
ond between cysteine and AuNRs. In this study, AuNRs were suc-essfully used to develop new biosensor for the colorimetric andelective quantitative detection of lysine by an end-to-end assem-ly of AuNRs. This method was simple and rapid, which can benished in 20 min with the red-shift of LPRA and red-to-blue color
hange. Furthermore, among the natural amino acids, only lysinean bring about the greatest red-shift of the LPRA, providing a selec-ive assay for lysine analysis, which has been successfully appliedo detecting lyine content in food samples. This methodology is
ectronics 34 (2012) 197– 201 201
easy to perform and cost-effective, which can be developed as apotential candidate for a wider applications in biosensing.
Acknowledgments
All authors are grateful to the valuable comments of the review-ers and the editors, and the financial support of the National NaturalScience Foundation of China (no. 21035005), China NationalCenter for Biotechnology Development (2010ZX09401 -306-1-4) and Chongqing Science and Technology Commission (CSTC.2010AA2015).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2012.02.001.
