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Biosensors and Bioelectronics 26 (2011) 4798– 4803

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

jou rn al h om epa ge: www.elsev ier .com/ locate /b ios

n artificial enzyme-based assay: DNA detection using a peroxidase-likeopper–creatinine complex

mardeep Singh1, Srikanta Patra1, Jeong-Ah Lee, Kang Hyun Park, Haesik Yang ∗

epartment of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea

r t i c l e i n f o

rticle history:eceived 4 April 2011eceived in revised form 29 May 2011ccepted 10 June 2011vailable online 17 June 2011

eywords:rtificial enzymeNA sensoropper complexold nanoparticleeroxidase

a b s t r a c t

We report an artificial enzyme-based DNA assay using a peroxidase-like copper (Cu)–creatinine com-plex as a catalyst for 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation. The assay employs double signalamplification and a homogeneous catalytic reaction: (i) fast catalytic growth of Cu on a gold (Au) nanopar-ticle (NP) label forms a thick Cu layer (first amplification); (ii) dissolution of the Cu layer generatesmany Cu–creatinine complexes per NP (generation of homogeneous catalysts); (iii) peroxidase-likeCu–creatinine complexes rapidly convert TMB into a colored product (second amplification). To investi-gate the effect of ligand on the catalytic activities of Cu complexes, the kinetics of catalytic TMB oxidationis tested with and without using imidazole ring-containing ligands (creatinine, imidazole, and poly(l-histidine)). Both fast oxidation of TMB and slow further oxidation of the colored product are required forhigh signal-to-background ratios. Cu–creatinine complex allows relatively fast oxidation and slow fur-ther oxidation. Fast seed-mediated Cu growth on Au NP and slow Cu autonucleation (i.e., slow formation

of Cu NP in the absence of Au NP) are also required for high signal-to-background ratios. In tris–EDTA(tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid) buffer (pH 7.7) containing highconcentrations of Cu2+ (90 mM), ascorbic acid (50 mM), and Mg2+ (200 mM), Cu growth on Au NP is veryfast and autonucleation is significantly suppressed. Fast catalytic oxidation by Cu–creatinine complexalong with fast Cu growth on Au NP allows a detection limit of 0.1 pM for DNA in a simple microplateformat.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Signal amplification is essential for fast and sensitive detec-ion of biomolecules (Rosi and Mirkin, 2005; Das et al., 2006). Its readily achieved with the fast catalytic reactions of enzymesPorstmann and Kiessig, 1992; Das et al., 2007). Thus, enzyme-inked immunosorbent assay (ELISA) has been one of thetandard methods in biological, clinical, and environmental anal-sis (Goldberg and Djavadi-Ohaniance, 1993). In ELISA, however,nzymatic reactions are limited by the mass transfer of substrateolecules toward sensing surfaces because enzyme labels are

ttached to the surfaces (e.g., the well walls of microplates) (Wangt al., 2009; Nygren and Stenberg, 1989). The heterogeneous reac-ions require lengthy reaction times for high signal amplification.

n some cases, poor long-term stability and complex preparationrocedures of enzymes make it difficult to obtain highly repro-ucible assay results. This calls for a new catalytic label that can

∗ Corresponding author. Tel.: +82 51 510 3681; fax: +82 51 516 7421.E-mail address: hyang@pusan.ac.kr (H. Yang).

1 These authors contributed equally to this work.

956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2011.06.009

replace the enzyme label in ELISA. The catalytic label is required tobe highly catalytic, robust, readily preparable, and easily detachablefrom a sensing surface. Facile detachment allows catalytic reactionby the label to occur in the homogeneous, instead of heterogeneous,phase.

In recent years, synthetic materials with enzyme-like activities(artificial enzymes) have been used as catalytic labels to obtain highsignal amplification and to replace enzyme labels (Das et al., 2006;Asati et al., 2009; Gao et al., 2008; Gill et al., 2006; Higuchi et al.,2008). For example, cerium oxide nanoparticles (NPs) (Asati et al.,2009) and iron oxide NPs (Gao et al., 2007, 2008) have been used asperoxidase-like labels. However, the reaction rate per NP is lowerthan per enzyme, and the number of active sites per NP is limitedby its surface area, although NPs can have many active sites perNP. Moreover, the catalytic reactions occur in the heterogeneousphase, as in enzyme labels.

Metal complexes are found in active sites of metalloenzymesthat catalyze redox reactions, and similar synthetic metal com-

plexes have been developed to achieve fast catalytic reactions(Thomas and Ward, 2005; Mancin et al., 2009). Copper (Cu)–l-histidine complexes are the most common metal complexesobserved in metalloenzymes. The two stable oxidation states (Cu+

A. Singh et al. / Biosensors and Bioelectronics 26 (2011) 4798– 4803 4799

resen

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Scheme 1. Schematic rep

nd Cu2+) of Cu and the imidazole ring of l-histidine play crucialoles in their high catalytic activities (Battaini et al., 2006). For thiseason, synthetic Cu–poly(l-histidine) complexes have been widelymployed as artificial enzymes (Ikeno et al., 2007; Weng et al.,005) and the peroxidase activity of Cu–creatinine complexes haseen applied to creatinine detection (Pugia et al., 2000). Neverthe-

ess, to date, there has been no report of Cu complexes used for highignal amplification in detection of biomolecules, although Cu ionsave been used (Zhang et al., 2008).

Herein is presented an artificial enzyme-based assay using dou-le signal amplification and a homogeneous catalytic reactionScheme 1). Fast catalytic growth of Cu on a gold (Au) NP labelorms a thick Cu layer (first amplification), dissolution of the Cuayer generates many Cu complexes per NP (generation of homoge-eous catalysts), and peroxidase-like Cu complexes rapidly convert,3′,5,5′-tetramethylbenzidine (TMB) into colored oxidation prod-cts (second amplification).

. Materials and methods

.1. Chemicals

Neutravidin-coated microplates were obtained from R&Dystems (Minneapolis, MN, USA). TMB, H2O2, CuSO4, cre-tinine, imidazole, poly(l-histidine) (MW = 5000–25,000),thylenediaminetetraacetic acid (EDTA), EDTA disodium salt,ris(hydroxymethyl)aminomethane (Tris), casein blocking bufferere purchased from Sigma–Aldrich. Sodium dodecyl sulfate

SDS), horseradish peroxidase (HRP), and sodium chloride were

btained from Fluka. A solution of citrate-stabilized Au NP (10 nm,.01% HAuCl4) and streptavidin-conjugated HRP were purchasedrom Sigma. All buffer reagents and other inorganic chemicals wereupplied by Sigma–Aldrich, unless otherwise stated. All chemicals

tation of DNA detection.

were used as received. All aqueous solutions were prepared indoubly distilled water.

Phosphate buffered saline (PBS) buffer (pH 7.4) consisted of0.01 M sodium phosphate, 0.138 M NaCl, and 2.7 mM KCl. PBSBbuffer contained all of the ingredients of the PBS buffer plus1% (w/v) BSA. Washing buffer was prepared by adding 0.1% SDSto PBS buffer. Hybridization buffer (pH 7.4) was composed of20 mM Tris, 17.5 mM EDTA disodium salt, 150 mM NaCl, and 0.05%Tween 20. Tris–EDTA buffer (pH 7.7) consisted of 100 mM Trisand 25.3 mM EDTA. Acetate buffers (50 mM) were prepared withsodium acetate and acetic acid. For the preparation of growth solu-tion, a Tris–EDTA buffer containing 200 mM MgSO4 and 180 mMCuSO4 and a Tris–EDTA buffer containing 200 mM MgSO4 and100 mM ascorbic acid were mixed at a ratio of 1:1 just before use.The resulting buffer contained 200 mM MgSO4, 90 mM CuSO4, and50 mM ascorbic acid. A solution of 10 mM TMB was prepared bydissolving TMB in dimethylsufoxide (DMSO), and the solution wasdiluted to a concentration of 2 mM with acetate buffer.

HPLC-purified DNAs were obtained from Genotech (Daejeon,Korea). The DNA assay was designed for the detection of singlenucleotide polymorphism for the encoding residue 1038 of exon 11of the BRCA1 gene (Dunning et al., 1997). The DNAs had the follow-ing sequences: biotinylated capture probe, biotin-3′-(CH2)9-TCGACC GAA GAA ATT T-5′; target DNA, 3′-TAA TCT CTT TTA CAA AAATTT CTT CGG TCG A-5′; thiolated detection probe, 3′-TTG TAA AAGAGA TTA-A20-5′-(CH2)6-SH; and noncomplementary DNA, 3′-CATCTA TAT CAT TAT TCG ACT AAT TCT ACC T-5′. The concentrationsof DNAs (target, detection probe, capture probe, and noncomple-mentary DNA) refer to those of strands. Au NPs were conjugated

with the thiolated detection probe containing an A20 spacer byfollowing our previous procedure, and the amount of detectionprobe immobilized on single Au NP was 14 pmol/cm2 (Das et al.,2009).

4 Bioelectronics 26 (2011) 4798– 4803

2d

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Fig. 1. (a) Time-course spectra for TMB oxidation obtained at 652 nm at 25 ◦C inan acetate buffer solution (pH 5.9, 50 mM) containing 2% (v/v) DMSO, one ligand,

800 A. Singh et al. / Biosensors and

.2. Preparation of DNA-sensing surface and procedure of DNAetection

To immobilize a capture-probe DNA, 100 �L of PBS buffer con-aining 100 nM biotinylated capture-probe DNA was added to aell of neutravidin-coated microplate and incubated for 2 h at

5 ◦C. After the solution was discarded, 300 �L of washing bufferas added, incubated for 5 min, and discarded. This washing stepas repeated 3 times. 280 �L of blocking buffer was then added to

he well and incubated for 3 h at 25 ◦C. This blocking step was fol-owed by the washing step. To obtain hybridization of target DNAo the immobilized capture probe, 100 �L of hybridization bufferontaining a different concentration of target DNA was added tohe well and incubated for 2 h at 25 ◦C with mild shaking (a shakingpeed of 60–70 rpm). After washing with washing buffer, 100 �L of.45 nM detection-probe-conjugated Au NP was added and incu-ated for 1 h to achieve the hybridization of a detection probe tohe target. After the second hybridization, the well was washed 3imes with washing buffer, followed by being washed twice withris–EDTA buffer containing 200 mM MgSO4 for 2 min. The addi-ional washing step with MgSO4 helped to remove non-hybridizedetection-probe-conjugated Au NP and to reduce the adsorptionf Cu2+. To obtain catalytic Cu growth on Au NP, 100 �L of freshlyrepared growth solution was added to the well and incubated at5 ◦C for 5 min. After the well was washed 3 times with Tris–EDTAuffer containing MgSO4, 200 �L of a 0.5 M HCl solution was addedo the well and incubated at 25 ◦C for 20 min with mild shakingn order to dissolve the grown Cu layer. After 25 �L of the Cu2+-ontaining solution was taken and added to a new microwell, (i)75 �L of a 1.0 M sodium acetate solution containing 1.42 mM cre-tinine, (ii) 25 �L of a 1.0 M sodium acetate solution containing 1%2O2, and (iii) 25 �L of a 1.0 M sodium acetate containing 20% (v/v)MSO and 2 mM TMB was added to the microwell, mixed well withipette, and incubated for 5 min. The pH of the mixed solution was.0, and the resulting concentrations of creatinine, H2O2, and TMBere 1 mM, 0.1%, and 0.2 mM, respectively. To stop the reaction

nd convert all TMB+ to TMB2+ (Josephy et al., 1982), 50 �L of a M H2SO4 solution was added to the microwell and mixed wellith pipette. The absorbance of the TMB2+ solution was measured

t 450 nm.UV/visible absorption spectra were obtained with a spectropho-

ometer (UV-1650 PC, Shimadzu, Japan), and the absorbanceoptical density (OD)) in DNA detection was obtained with a

icroplate reader (VERSAmax, Molecular Devices, USA).

. Results and discussion

.1. TMB oxidation using Cu complexes

TMB is the most common chromogenic substrate of peroxidasenzymes such as horseradish peroxidase. Its one- or two-electronxidation develops a blue- or yellow-colored product, respectively,ith a high extinction coefficient (Josephy et al., 1982). To inves-

igate the effects of the ligand on the catalytic activities of Cuomplexes, the kinetics of catalytic TMB oxidation was tested withnd without using imidazole ring-containing ligands (creatinine,midazole, and poly(l-histidine)) (Scheme 1). Fig. 1a shows time-ourse data for TMB oxidation, recorded at 652 nm in an acetateuffer solution (pH 5.9) containing Cu2+ and one of the ligands.hen imidazole was used, the absorbance increased rapidly and

hen decreased with time (curve (i) of Fig. 1a). The absorbancencrease is due to generation of one-electron-oxidized TMB (TMB+),

hich is in equilibrium with a charge-transfer complex of TMBnd two-electron-oxidized TMB (TMB2+) (Josephy et al., 1982). Asor poly(l-histidine) and creatinine, the absorbance increased fastnd then decreased slowly (curves (ii) and (iii) of Fig. 1a). When

0.10 mM CuSO4, and 0.1% H2O2. Ligands were (i) 1.0 mM imidazole; (ii) 0.1 mg/mLpoly(l-histidine); (iii) 1.0 mM creatinine; (iv) 1.0 mM EDTA. (b) Absorption spectraobtained after a 20-min time-course experiment.

EDTA (a strongly chelating ligand) was used, the absorbance didnot change after a small, initial increase (curve (iv) of Fig. 1a).In the absence of ligand, the absorbance increased slowly (curve(v) of Fig. 1a). These results clearly show that the imidazole ring-containing ligands provide the Cu complexes with high catalyticactivities (Pugia et al., 2000; Chan and Kesner, 1980; Ueda andHanaki, 1984).

The absorbance decrease with time is due to further oxida-tion of TMB+ (Macías-Ruvalcaba and Evans, 2007). Fenton-likereactions (Aguiar and Ferraz, 2007) that occur in the presenceof Cu2+ and H2O2 could cause further oxidation of TMB+, result-ing in TMB2+ generation (Pugia et al., 2000), its decomposition(Macías-Ruvalcaba and Evans, 2007), and/or its polymerization.The absorption peaks at 652 and 380 nm in Fig. 1b are related toTMB+ (Pugia et al., 2000). If the absorbance decrease at 652 nmwere only due to the oxidation of TMB+ to TMB2+, the absorp-tion peak at 450 nm, related to TMB2+, would increase. However,the peak at 450 nm did not increase for an initial period of time,while the peak at 652 nm decreased. This result indicates thatthe absorbance decrease might be related to TMB+ decompositionand/or its oxidative polymerization. Interestingly, in the case ofthe Cu–imidazole complex, the absorption spectrum became moreand more monotonic and broadband over time (curve (i) of Fig. 1band Fig. S1a in Supplementary Data). This spectrum feature wasalso observed within hours when Cu–creatinine complex was used(Fig. S1b in Supplementary Data), and was similar to that observedin melanin (polymerized dopamine) (D’Ischia et al., 2009). Thisimplies that polymerized compounds might be formed by furtheroxidation of TMB+. Whatever the mechanism of the further oxida-tion is, its minimization was essential to obtain a high absorbance

signal. Unfortunately, the Cu–imidazole complex caused fast fur-ther oxidation of TMB+, although it allowed fast oxidation of TMBto TMB+ (curve (i) of Fig. 1a). As for the Cu–creatinine complex,further oxidation was relatively slow and TMB oxidation very fast

Bioelectronics 26 (2011) 4798– 4803 4801

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Fig. 2. Time-course spectra observed at 586 nm at 25 ◦C in a Tris–EDTA buffersolution (pH 7.7, 100 mM Tris, 25.3 mM EDTA) containing 50 mM ascorbic acidand 90 mM CuSO4 plus (i) none; (ii) 10 pM biotinylated capture probe; (iii) 10 pMbiotinylated capture probe and 10 pM target DNA; (iv) 10 pM detection-probe-

A. Singh et al. / Biosensors and

curve (iii) in Fig. 1a). In the case of the Cu-poly(l-histidine) com-lex, the absorbance change was similar to that observed in theu–creatinine complex. Because creatinine is more cost-effectivehan poly(l-histidine), the Cu–creatinine complex was selected tochieve fast catalytic oxidation of TMB to TMB+, along with slowurther oxidation of TMB+.

Further oxidation could be minimized, if a low concentration of2O2 was used. Under these conditions, however, catalytic gener-tion of TMB+ was very slow. Accordingly, a H2O2 concentration of.1% was used despite further oxidation. Generally, mixed solutionsf water and polar organic solvent are used to dissolve TMB becausets solubility is low in aqueous solutions. In this study, a mixed solu-ion of water and DMSO was used. 2% (v/v) DMSO showed betteresults than other conditions in terms of both fast TMB oxidationnd slow further oxidation (see Fig. S2 in Supplementary Data).

Catalytic TMB oxidation depends on solution pH. In order tobtain low background levels, direct TMB oxidation by H2O2 in thebsence of the Cu complex should be minimized. This direct oxi-ation also depended on the pH. Fig. S3a in Supplementary Datahows time-course spectra for TMB oxidation under different pHonditions in the presence of the Cu–creatinine complex, whereasig. S3b in Supplementary Data shows time-course spectra inhe absence of the complex. The absorbance rise with time inig. S3b in Supplementary Data is related to direct TMB oxida-ion. A pH of 5.9 was better for fast TMB oxidation and slow directxidation than other pH values.

.2. Cu growth on Au NP

Selective Cu growth on the Au NP label is essential for highignal-to-background ratios. Fast seed-mediated Cu growth on Aullows high signal amplification, while slow Cu autonucleation (i.e.,low formation of Cu NP in the absence of Au NP) allows lowackground levels. To date, there have been some reports on theeed-mediated Cu growth on Au NP (Jiang et al., 2008; Mao et al.,007; Wei et al., 2008). However, it was difficult to obtain bothast, reproducible growth and slow autonucleation by using pre-ious methods. Autonucleation was considerable in most aqueousolutions. For example, the absorbance change due to Cu autonu-leation by ascorbic acid was substantial in acetate buffer (pH 5.9)data not shown). Moreover, Cu ion readily precipitated in manyuffer solutions such as phosphate buffer solutions because of lowolubility-product (Ksp) values of Cu–buffer anion complexes. Verymportantly, however, autonucleation was significantly suppressed

hen Tris–EDTA buffer (pH 7.7) was used. In curve (i) of Fig. 2, thebsorbance obtained in Tris–EDTA buffer in the absence of Au NPnd DNA did not change with time. Moreover, this buffer allowedery fast Cu growth on Au NP, under conditions of high concentra-ions of Cu2+ (90 mM) and ascorbic acid (50 mM). In curve (iv) ofig. 2, the high increase in the absorbance at 586 nm was due toast Cu growth (Jiang et al., 2008). As for EDTA, a concentration of5 mM was optimal for fast Cu growth and slow autonucleation. To

ower the binding of positively charged Cu2+ to negatively chargedNA during Cu growth, a high concentration of Mg2+ (200 mM) wassed for competitive binding of Mg2+ to DNA.

Rotaru et al. (2010) reported that Cu growth occurs on double-tranded DNA in MOPS (3-(N-morpholino)propanesulfonic acid)uffer solutions (pH 7.5) but does not on single-stranded DNA.n Tris–EDTA buffer solutions, the change in the absorbance wasot much for 300 s in the presence of single-stranded DNA (curveii) of Fig. 2) but substantial in the presence of double-strandedNA (curve (iii) of Fig. 1b). These results indicate that Cu growth

as substantial in the presence of double-stranded DNA. However,

he degree of Cu growth in the presence of double-stranded DNAas much less than that in the presence of Au NP (curve (iv) of

ig. 2).

conjugated Au NP. Blank solution was a Tris–EDTA buffer solution containing 90 mMCuSO4. The solution of curve iii was used after 1-h hybridization.

3.3. DNA detection

DNA detection was performed in a sandwich format (Scheme 1):(i) a biotinylated capture probe was immobilized on a neutravidin-modified microwell; (ii) a target DNA was hybridized to the captureprobe; (iii) a detection-probe-conjugated Au NP was hybridized tothe target; (iv) Cu growth was performed for 5 min; (v) the grownCu layer was dissolved in an acidic solution (pH 0.3); (vi) the dis-solved Cu2+ was transferred to acetate buffer containing creatinine,TMB, and H2O2; (vii) the reaction of TMB oxidation proceeded in thesolution (pH 6.0) for 5 min; (viii) the reaction was stopped by addingan acidic solution and all generated TMB+ was converted to TMB2+

(Josephy et al., 1982); and (ix) finally, the absorbance of TMB2+

was measured at 450 nm. Generally, nonspecific binding of DNA-conjugated Au NPs to solid surfaces is higher than that of DNAs. Toreduce nonspecific binding, a neutravidin-coated microwell and acasein-based blocking buffer were used. For reproducible results, itwas necessary to fully remove washing solutions from microwellsduring the washing process.

Before obtaining DNA concentration-dependent data, controlexperiments were performed. First, Cu growth and TMB oxidation(detection steps (iv)–(ix)) were performed with a neutravidin-coated microwell without using capture probe, target DNA, anddetection-probe-conjugated Au NP (i.e., without detection steps(i)–(iii)). The measured absorbance (OD) (bar (i) of Fig. 3) was alittle higher than the OD of TMB solution (0.05; the dashed line ofFig. 3), indicating negligible Cu growth onto the microwell and/orCu2+ binding to the microwell. When DNA detection was carriedout at a target concentration of zero (bar (ii) of Fig. 3) and 1 nM(bar (iii) of Fig. 3) without treating detection-probe-conjugated AuNP (i.e., without detection step (iii)), the OD at 1 nM was a littlehigher, due to the Cu growth on the double-stranded DNA (captureprobe and target). When the detection was performed with treat-ing the Au NP (i.e., with all detection steps), the OD at 1 nM wasmuch higher than at zero (bars (iv) and (vi) of Fig. 3). When DNAdetection was carried out at a noncomplementary-DNA concentra-tion of 1 nM, the OD (bar (v) of Fig. 3) was similar to that at a targetconcentration of zero (bar (iv) of Fig. 3), indicating that nonspecificadsorption of noncomplementary DNA is negligible.

When a scanning electron microscopic (SEM) image wasobtained at a target concentration of zero, some large Cu NPs were

observed (Fig. 4a), resulting from nonspecific binding of the DNA-conjugated Au NPs. However, as the concentration of target wasincreased, the surface concentration of Cu NPs was significantly

4802 A. Singh et al. / Biosensors and Bioelectronics 26 (2011) 4798– 4803

(i)

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0.0

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0.2

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0.2

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Ft

robe-conjugated Au NP. The concentrations of target DNA were (ii, iv) 0 and (iii, vi) nM, and the concentration of noncomplementary DNA was (v) 1 nM.

ncreased (Fig. 4b and c). The typical size of Cu NP was ca. 400 nmFig. 4d). Considering that the size of Au NP was 10 nm, the largeize of Cu NP indicates that the Cu growth was extremely fast during-min incubation.

Fig. 5a shows the dependence of the measured OD on targetNA. For comparison, the OD was also measured without usingreatinine (Fig. 5b). The ODs obtained in the presence of creati-ine were higher than that in the absence of the ligand, as theu–creatinine complex allowed much faster TMB oxidation thanu2+ alone. Moreover, the change in OD according to the change in

oncentration was much higher in the case of Cu–creatinine com-lex. The calculated detection limit was ca. 0.1 pM in the case ofu–creatinine complex and a little higher than 0.1 pM in the casef Cu2+. For the comparison with enzyme-based DNA detection,

ig. 4. SEM images obtained after carrying out Cu growth with the DNA-sensing microwrations of target DNA were (a) 0, (b) 1 pM, and (c, d) 1 nM. (d) magnified view of (c).

target concentration of zero. The dashed line corresponds to three times the standarddeviation (SD) at a concentration of zero. The error bar represents the SD of at leastfour measurements.

we carried out HRP-based DNA detection. The measured detectionlimit was ca. 1 nM (see Fig. S4 in Supplementary Data), although alonger incubation time of 30 min was used for catalytic reaction.These results show that both the fast Cu growth and the fast TMB

ells treated with target DNA and detection-probe-conjugated Au NP. The concen-

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A. Singh et al. / Biosensors and

xidation by Cu–creatinine complex allow higher signal amplifica-ion and a lower detection limit.

. Conclusion

We developed an artificial enzyme-based assay that allowsensitive DNA detection in an ELISA-like microplate format.ast catalytic growth of Cu on Au NP, fast generation of manyu–creatinine complexes from the grown Cu layer, and fast cat-lytic oxidation of TMB to TMB+ by Cu–creatinine complex inomogeneous phase offered high signal levels. Slow Cu autonucle-tion in Tris–EDTA buffer (pH 7.7) and slow direct TMB oxidationy H2O2 in acetate buffer (pH 5.9) provide low background levels.he sensitive, simple spectrophotometric detection in the commonicroplate format makes the presented method appealing for a

eneral-purpose assay.

cknowledgements

This research was supported by the Public Welfare & Safetyesearch Program (2010-0020772) and the Basic Science Researchrogram (2009-0072062 and 2009-0085182) through the Nationalesearch Foundation of Korea (NRF). This study was also financiallyupported by Pusan National University in the program, Post-Doc.010.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.bios.2011.06.009.

eferences

guiar, A., Ferraz, A., 2007. Chemosphere 66, 947–954.

ctronics 26 (2011) 4798– 4803 4803

Asati, A., Santra, S., Kaittanis, C., Nath, S., Perez, J.M., 2009. Angew. Chem. Int. Ed. 48,2308–2312.

Battaini, G., Granata, A., Monzani, E., Gullotti, M., Casella, L., 2006. Adv. Inorg. Chem.58, 185–233.

Chan, P.C., Kesner, L., 1980. Biol. Trace Elem. Res. 2, 159–174.Das, J., Aziz, M.A., Yang, H., 2006. J. Am. Ceram. Soc. 128, 16022–16023.Das, J., Jo, K., Lee, J.W., Yang, H., 2007. Anal. Chem. 79, 2790–2796.Das, J., Huh, C.-H., Kwon, K., Park, S., Jon, S., Kim, K., Yang, H., 2009. Langmuir 25,

235–241.Dunning, A.M., Chiano, M., Smith, N.R., Dearden, J., Gore, M., Oakes, S., Wilson, C.,

Stratton, M., Peto, J., Easton, D., Clayton, D., Ponder, B.A., 1997. Hum. Mol. Genet.6, 285–289.

D’Ischia, M., Napolitano, A., Pezzella, A., Meredith, P., Sarna, T., 2009. Angew. Chem.Int. Ed. 48, 3914–3921.

Gao, L., Zhuang, J., Nie, L., Zhang, J., Zhang, Y., Gu, N., Wang, T., Feng, J., Yang, D.,Perrett, S., Yan, X., 2007. Nat. Nanotechnol. 2, 577–583.

Gao, L., Wu, J., Lyle, S., Zehr, K., Cao, L., Gao, D., 2008. J. Phys. Chem. C 112,17357–17361.

Gill, R., Polsky, R., Willner, I., 2006. Small 2, 1037–1041.Goldberg, M.E., Djavadi-Ohaniance, L., 1993. Curr. Opin. Immunol. 5, 278–281.Higuchi, A., Siao, Y.-D., Yang, S.-T., Hsieh, P.-V., Fukushima, H., Chang, Y., Ruaan, R.-C.,

Chen, W.-Y., 2008. Anal. Chem. 80, 6580–6586.Ikeno, S., Asakawa, H., Haruyama, T., 2007. Anal. Chem. 79, 5540–5546.Jiang, Z., Liao, X., Deng, A., Liang, A., Li, J., Pan, H., Li, J., Wang, S., Huang, Y., 2008.

Anal. Chem. 80, 8681–8687.Josephy, P.D., Eling, T., Mason, R.P., 1982. J. Biol. Chem. 257, 3669–3675.Macías-Ruvalcaba, N.A., Evans, D.H., 2007. J. Phys. Chem. C 111, 5805–5811.Mancin, F., Scrimin, P., Tecilla, P., Tonellato, U., 2009. Coord. Chem. Rev. 253,

2150–2160.Mao, X., Jiang, J., Luo, Y., Shen, G., Yu, R., 2007. Talanta 73, 420–424.Nygren, H., Stenberg, M., 1989. Immunology 66, 321–327.Porstmann, T., Kiessig, S.T., 1992. J. Immunol. Methods 150, 5–21.Pugia, M.J., Lott, J.A., Wallace, J.F., Cast, T.K., Bierbaum, L.D., 2000. Clin. Biochem. 33,

63–70.Rosi, N.L., Mirkin, C.A., 2005. Chem. Rev. 105, 1547–1562.Rotaru, A., Dutta, S., Jentzsch, E., Gothelf, K., Mokhir, A., 2010. Angew. Chem. Int. Ed.

49, 5665–5667.Thomas, C.M., Ward, T.R., 2005. Chem. Soc. Rev. 34, 337–346.Ueda, J.-I., Hanaki, A., 1984. Bull. Chem. Soc. Jpn. 57, 3511–3516.

Wang, G., Driskell, J.D., Porter, M.D., Lipert, R.J., 2009. Anal. Chem. 81, 6175–

6185.Wei, X., Liang, A., Zhang, S.-S., Jiang, Z.–L., 2008. Anal. Biochem. 380, 223–228.Weng, Y.C., Fan, F.-R.F., Bard, A.J., 2005. J. Am. Ceram. Soc. 127, 17576–17577.Zhang, S., Zhong, H., Ding, C., 2008. Anal. Chem. 80, 7206–7212.