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Analytica Chimica Acta 770 (2013) 147– 152

Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho me page: www.elsev ier .com/ locate /aca

esign of a sensitive aptasensor based on magneticicrobeads-assisted strand displacement amplification and target

ecycling

ing Li ∗, Xiaoting Ji, Weiling Song, Yingshu Guoey Laboratory of Biochemical Analysis, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science andechnology, Qingdao 266042, PR China

i g h l i g h t s

An aptasensor is fabricated by stranddisplacement amplification and tar-get recycling.The reaction efficiency is greatlyimproved by cross-circular amplifi-cation.Sensitive detection of other targetscan be achieved by using differentaptamers.

g r a p h i c a l a b s t r a c t

The ATP recognition probe (S1) was immobilized on the surface of MB-AuNPs and hybridized with itspartly complementary sequence (S2) to form a duplex. When the target ATP was introduced, S1 changedits conformation to combine with ATP (step Ia) and S2 was released (step Ib) to trigger the MB-assistedstrand displacement amplification (step II). In cycle 1, S2 hybridized with the single-stranded toeholddomain of S3 and the single-stranded DNA (S4) was dissociated from the MB-AuNPs by branch migration,enabling S3 to possess a newly exposed single-stranded toehold. Then the biotin-tagged single-strandedfuel (S6) was hybridized with the newly exposed toehold, releasing S5 and S2. At the same time, thedouble-stranded product and the biotin-tagged MB complexes were formed. The released S5 wouldfurther participate in the target recycling amplification reaction (cycle 2). S2 was used as catalyst toimplement another circulation of the MB-assisted strand displacement amplification. In cycle 1, thereleased S4 could hybridize with the recognition probe (S1) by DNA displacement reaction, making moreS2 to be dissociated to participate in cycle 1. The second amplification process (cycle 2) was initiatedby S5, which could hybridize with the extension part in S1/ATP complex and be used as the primer ofa polymerization reaction. In the presence of Klenow polymerase and dNTPs, the primer extended andformed a duplex with the ATP recognition probe. Thus the target ATP was displaced, which could bind withanother recognition probe and trigger new amplification cycles. Finally, a large amount of biotin-taggedMB complexes were produced. As the fuel (S6) was modified with biotin, the S3/S6 double-strandedproduct on MB-AuNPs could incorporate with streptavidin-HRP to perform the enzyme-amplification CLdetection.

r t i c l e i n f o a b s t r a c t

rticle history:eceived 17 October 2012eceived in revised form 21 January 2013ccepted 22 January 2013vailable online 4 February 2013

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancercells was developed based on aptamer–target interaction, magnetic microbeads (MBs)-assisted stranddisplacement amplification and target recycling. Here we described a new recognition probe possessingtwo parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on thesurface of MBs and hybridized with its complementary sequence to form a duplex. When combined with

∗ Corresponding author. Tel.: +86 532 84022946; fax: +86 532 84022750.E-mail address: liying79307@163.com (Y. Li).

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2013.01.044

148 Y. Li et al. / Analytica Chimica Acta 770 (2013) 147– 152

Keywords:Adenosine triphosphateAptamerStrand displacement amplificationTarget recyclingChemiluminescence

ATP, the probe changed its conformation, revealing the extension part in single-strand form, which fur-ther served as a toehold for subsequent target recycling. The released complementary sequence of theprobe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with targetrecycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation ofchemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1 × 10−10 M. Moreover, sample assays of ATP in RamosBurkitt’s lymphoma B cells were performed, which confirmed the reliability and practicality of the pro-tocol.

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. Introduction

The development of sensitive, rapid and simple methods foretecting and quantifying biological substances is increasinglyequired in bioanalysis and clinical diagnostics. For improving theensitivity, various amplification strategies have been proposeduch as enzyme catalysis, nanoparticle amplification, and so on.hese strategies were used to detect nucleic acids [1–4], protein andmall molecules [5–10]. Within these efforts, isothermal cyclingmplification is particularly used to address the limitations of com-licated procedures and time-consumption in some methods suchs polymerase chain reaction (PCR) [11–14].

Based on DNA polymerase and hairpin probes, strand-isplacement polymerization has emerged as a new amplificationechnique [15,16]. In the presence of a specific nucleic acidequence, which is complementary to the loop of the hairpin probe,he hairpin structure undergoes conformational change and thetem of the hairpin probe is opened. Then the opened probe annealsith the primer and triggers the polymerization reaction to form a

ully complementary DNA of the probe and the target is displacedo recognize another probe, initiating the next round of polymer-zation reaction. Zhang et al. introduced an enzyme-free strandisplacement amplification method based on toehold exchangesing a single-stranded input DNA as catalyst [17–19]. The products

nclude single-stranded outputs and double-stranded by-products.s the same single-stranded input DNA can participate in multipletrand displacement reaction cycles, many outputs and double-tranded by-products can be released and used for signal detection.hese reactions can proceed at constant temperature, so there iso need of complicated manipulation and the time required forycling amplification is less than that required for thermal cyclingechniques.

In this article, adenosine triphosphate (ATP) is served as a rep-esentative model target for testing new analytical techniques. ATPs a multifunctional nucleotide used as an energy carrier in theells of all known organisms, and it has been found that ATP isn indicator for cell viability and cell injury [20]. The discoveryf a specific aptamer for ATP promoted the development of ATPensors. Based on the high affinity and specificity of aptamer tohe target ATP, a variety of colorimetric [21], fluorescent [22,23],nd electrochemical methods [24–28] have been developed. Thesetrategies, however, suffer from intrinsic limitations of highackground or complicated manipulation, limiting their practicalpplication.

By combining MB-assisted strand displacement amplificationith target recycling, a new strategy of cross-circular amplificationas been introduced for highly sensitive and selective detection ofTP. The amplification process is triggered by the target moleculesnd proceeds at a constant temperature. Importantly, cross-circularmplification could be achieved because the product of one ampli-

cation reaction can be used as the reactant of another reaction.onsequently, the amplification efficiency is greatly improved and

large amount of biotin tagged MB complexes are formed tonhance the signal of chemiluminescence detection. The proposed

© 2013 Elsevier B.V. All rights reserved.

method holds great promise for ATP determination because of thesensitivity and simplicity.

2. Experimental

2.1. Materials and apparatus

All oligonucleotides used in the present study were synthesizedand purified by Shanghai Sangon Biological Engineering Technol-ogy & Services Co. Ltd. (China), and the sequences were listedin Table S1 (see supplementary materials). Klenow fragment ofEscherichia coli DNA polymerase I and the mixture of four dNTPswere purchased from TaKaRa Bio Inc. Magnetic microbeads coatedwith thiol groups (SH-MBs, particle size: 3.0–4.0 �m) and carboxylgroups (COOH-MBs, particle size: 3.0–4.0 �m) were purchasedfrom Tianjin BaseLine ChroTechResearch Center (China).

Adenosine triphosphate (ATP), guanosine triphosphate (GTP),cytidine triphosphate (CTP), uridine triphosphate (UTP), hydro-gen tetrachloroaurate(III) tetrahydrate (HAuCl4·4H2O), tri(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), luminol andp-iodophenol (PIP) were purchased from Sigma–Aldrich (USA).Streptavidin-HRP was obtained from Boster Biotechnology Co.Ltd. (China). Analytical grade H2O2 was purchased from ShanghaiChemical Reagent Company (Shanghai, China). Other chemicalsemployed were of analytical reagent grade and were used withoutfurther purification. Double-distilled water was used throughoutthe experiment. The CL measurements were performed with a FI-CL instrument (MPI-F, Remex Analytical Instrument Co. Ltd., Xi’an,China), including a model IFIS-D flow injection system, a modelRFL-1 luminometer and a computer.

2.2. Preparation of ATP extracts from cancer cells

The Ramos Burkitt’s lymphoma cells were cultured in RPMI 1640medium supplemented with 10% fetal calf serum and 100 IU mL−1

penicillin-streptomycin at 37 ◦C in a humidified atmosphere (95%air and 5% CO2). Cells were collected in the exponential phaseof growth and determined using a hemocytometer prior to eachexperiment. Then, a suspension of Ramos cells was dispersed inRPMI cell media buffer, centrifuged at 3500 rpm for 5 min andwashed with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) threetimes and resuspended in 250 �L of doubly distilled water. Finally,the cells were disrupted by sonication for 20 min at 0 ◦C and thelysate was centrifuged at 18 000 rpm for 20 min at 4 ◦C to removethe homogenate of cell debris.

2.3. Modification of magnetic microbeads with gold nanoparticles

The colloidal solution of gold nanoparticles (AuNPs) was syn-thesized by the citrate reduction method with a slight modification

[29]. Gold nanoparticles functionalized magnetic microbeads (MB-AuNPs) were obtained by capping the synthesized AuNPs on thesurface of SH-MBs through Au–S bonds, as it has been reportedpreviously [30].

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Y. Li et al. / Analytica Chi

.4. Preparation of the MB-immobilized three-stranded substrate

Firstly, the surface of MB-AuNPs was modified with captureNA (S3) through Au–S bonds. 100 �L of 1.0 × 10−7 M S3 was acti-ated with TCEP (10 mM) for 1 h and then was added to 100 �L ofB-AuNPs. After shaking gently for 16 h at room temperature, theB-AuNPs/S3 conjugates were “aged” in the solution (0.3 M NaCl,

0 mM Tris–acetate, pH 8.2) for another 48 h. Following removal ofhe supernatant, the resulted precipitate was washed with 300 �Lf 10 mM PBS (pH 7.4) containing 0.01 M NaCl, recentrifuged, andhen redispersed in the same buffer solution. Then, the mixture of4 and S5 (100 �L, 10−7 M) was added and hybridized with theapture DNA (S3) for 1 h at 37 ◦C. The formed MB-immobilizedhree-stranded substrate was washed three times with 300 �L of0 mM Tris-buffered saline (pH 7.4) and redispersed in the sameuffer solution for further use.

.5. ATP assay

The recognition probe was immobilized on the surface ofB-AuNPs through Au–S bonds and hybridized with its partly com-

lementary oligonucleotide for 1 h to form a duplex. Subsequently,he ATP solution with different concentration was added to makehe recognition probe change its structure for binding with ATP.hen 50 �L of MB-AuNPs/three-stranded substrate complex, 10 �Lf dNTPs (1 mM), 10 �L of Klenow buffer and 3.5 �L of Klenow poly-erase were added to perform the amplification reaction. After

ncubating at 37 ◦C for 2 h, the mixture was heated at 80 ◦C for0 min to inactivate the Klenow polymerase. The biotin tagged MBomplexes were obtained after removing the supernatant by mag-etic separation and then reacted with streptavidin-HRP at 37 ◦C

or 30 min to form HRP-modified MB complexes, which would beurther used for CL detection.

.6. CL detection

The HRP-modified MB complex was washed with 300 �L of PBS10 mM, pH 7.4) containing 0.01 M NaCl and redispersed in 1 mLf the same buffer solution. 5.0 × 10−4 M luminol and 5.0 × 10−3 M

Scheme 1. Schematic diagram for the detection of ATP based on magnetic m

cta 770 (2013) 147– 152 149

PIP were mixed together with the volume ratio of 1:1, after beingmixed with 5.0 × 10−3 M H2O2, the HRP-modified MB complexeswere pumped and reacted with the above mixture in the flow cell.The emitted chemiluminescence was collected using a photomul-tiplier tube with a voltage of −350 V, and the signals were recordedusing a computer.

3. Results and discussion

3.1. The principle of the proposed strategy for ATP detection

Our strategy for detection of ATP on the basis of MB-assisted strand displacement amplification and target recyclingwas depicted in Scheme 1. Firstly, the ATP recognition probe (S1)immobilized on the surface of MB-AuNPs (MB-1) was hybridizedwith its partly complementary sequence (S2) to form a duplex.When the target ATP was introduced, S1 changed its conformationto combine with ATP (step Ia) and S2 was released (step Ib) to triggerthe MB-assisted strand displacement amplification (step II). In cycle1, S2 hybridized with the single-stranded toehold domain of S3 andthe single-stranded DNA (S4) was dissociated from the MB-AuNPs(MB-2) by branch migration [17], enabling S3 to possess a newlyexposed single-stranded toehold. Then the biotin-tagged single-stranded fuel (S6) was hybridized with the newly exposed toehold,releasing S5 and S2. At the same time, the double-stranded productand the biotin-tagged MB complexes were formed. The released S5would further participate in the target recycling amplification reac-tion (cycle 2). S2 was used as catalyst to implement another cycleof the MB-assisted strand displacement amplification. In cycle 1,the released S4 could hybridize with the recognition probe (S1) byDNA displacement reaction [31], making more S2 to be dissociatedto participate in cycle 1.

The second amplification process (cycle 2) was initiated by S5,which could hybridize with the extension part in S1/ATP complexand be used as a primer of polymerization reaction. In the presence

of Klenow polymerase and dNTPs, the primer (S5) extended alongwith the ATP recognition probe (S1) and formed double strandedproduct bound to MB-1. The double stranded by-products couldnot participate in the amplification reaction, but the target ATP

icrobeads-assisted strand displacement amplification and target cycle.

150 Y. Li et al. / Analytica Chimica Acta 770 (2013) 147– 152

Fig. 1. FI-CL signals of (a) luminol-H2O2-PIP; (b) cycle 1 without S2; (c) cycle 1wi

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ith S2. Experimental conditions: luminol, 5.0 × 10−4 M; H2O2, 7.5 × 10−3 M; p-odophenol, 5.0 × 10−3 M. The concentration of the catalyst (S2) was 1.0 × 10−7 M.

as displaced, which could bind with another recognition probend trigger new amplification cycles. Finally, a large amount ofiotin-tagged MB complexes were produced. As the fuel (S6) wasodified with biotin, the S3/S6 double-stranded product on MB-uNPs could incorporate with streptavidin-HRP to perform thenzyme-amplification CL detection.

An essential issue in the design of the proposed strategy ishat cross-circular amplification could be achieved by combininghe MB-assisted strand displacement amplification with the tar-et recycling and the two cycles can accelerate each other. Forxample, the products of cycle 1, S4 and S5 participate in the DNAisplacement reaction and the target recycling individually, whilehe double-stranded product ingeniously serves as the carrier toncorporate with streptavidin-HRP for the CL detection.

.2. Verification of MB-assisted strand displacementmplification (cycle 1)

To verify the performance of the MB-assisted strand displace-ent amplification (cycle 1), we investigated the CL spectra of the

ycle 1 in the absence and presence of the “catalyst” (S2) and theesults were shown in Fig. 1. When the three-stranded substrate,ingle-stranded fuel and catalyst (S2) were all introduced, the CLignal was very high (Fig. 1, curve c), indicating that there were aarge amount of biotin-tagged MB complexes produced. But the CLignal was greatly decreased (Fig. 1, curve b) without catalyst (S2) ashe uncatalyzed reaction is very slow and little biotin-tagged MBomplexes was produced during the cycle time (2 h). The resultshowed that the MB-assisted strand displacement amplificationeaction could be performed successfully in the presence of S2.

.3. Control experiments

Further control experiments were performed and the resultsere shown in Figure S1. In the absence of Klenow polymerase

r dNTPs (Figure S1, columns b and c), the polymerization reactionould not be performed, let alone the target recycling, so the CLntensity was very low. However, the CL intensity (Figure S1, col-mn d) was significantly increased when Klenow polymerase and

NTPs were all introduced to the reaction system, which indicatedhat the MB-assisted strand displacement amplification and targetecycling occurred smoothly.

Fig. 2. The calibration curve of relative FI-CL peak height versus the concentrationof ATP from 1.0 × 10−9 to 8.0 × 10−8 M. Inset is the amplification of the linear rangefrom 1.0 × 10−9 to 1.0 × 10−8 M for ATP determination.

3.4. Optimization of the experimental conditions

3.4.1. Influence of the amount of Klenow polymeraseTo investigate the effect of different amount of Klenow poly-

merase on ATP detection, we did the optimization experiment using1.0 × 10−8 M ATP. The blank was treated in the same way for thedetection without ATP. As shown in Figure S2, when the volume ofpolymerase increased from 0.5 to 3.5 �L, the CL intensity increasedgradually. But after that, the CL intensity decreased slightly. There-fore, 3.5 �L of Klenow polymerase was considered to be optimumamount used in the amplification reaction.

3.4.2. Optimization of the reaction temperatureFigure S3 showed the influence of the temperature on the CL

signal produced by 1.0 × 10−8 M ATP. As it could be seen, a maxi-mal intensity was obtained when the reaction temperature of thesystem was 37 ◦C. So we employed 37 ◦C as the optimal experi-mental temperature, which was in accordance with the fact thatenzymatic reactions are usually operated at 37 ◦C by virtue of thebest bioactivity of enzymes.

3.4.3. Optimization of the reaction timeAs a cross-circular amplification system, two cycle modes are

coexisted to amplify signals, so the reactions will not end until thesubstrate and fuel are used up and the reaction time is an importantinfluencing factor. Figure S4 showed the changes of the CL signalgenerated by performing the experiment at different time intervals.The results showed that the CL intensity increased rapidly with theincrease of reaction time to 2 h, and a plateau effect was reachedafter this time. Therefore, the reaction time was controlled at 2 hthroughout the experiment.

3.5. Sensitivity of the assay

According to the protocol shown in Scheme 1, ATP was detectedunder the proposed experimental conditions. As shown in Fig. 2,the relative FI-CL intensity of luminol-H2O2-PIP system catalyzedby HRP-modified MB complexes increased with the increase of theATP concentration over two orders of magnitude, from 1.0 × 10−9 Mto 1.0 × 10−7 M. In addition, the relative CL intensity had a good

1.0 × 10−9 M to 1.0 × 10−8 M. The linear regression equation wascalculated as �I = 0.03747 + 0.00889C (R = 0.992) where �I was therelative CL intensity and C was the concentration of ATP (10−9 M).

Y. Li et al. / Analytica Chimica Acta 770 (2013) 147– 152 151

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Fig. 4. The FI-CL intensities after the addition of ATP or other nucleosidesdetected by strand-displacement recycling amplification. The concentration of ATP:1.0 × 10−8 M. The concentration of CTP, GTP and UTP: 1.0 × 10−6 M. Experimentalconditions: luminol, 5.0 × 10−4 M; H2O2, 7.5 × 10−3 M; p-iodophenol, 5.0 × 10−3 M.

Table 1Analysis of ATP in Ramos cell.

Samplea This method (�M) RSD HPLC RSD(%, n = 3) (�M) (%, n = 3)

1 4.15 4.2 4.38 2.82 4.08 3.4 4.25 3.2

ig. 3. The calibration curve of relative FI-CL peak height versus the concentrationf ATP from 6.0 × 10−8 to 6.0 × 10−7 M.

he detection limit as low as 6.1 × 10−10 M at 3� for ATP wasbtained. A relative standard deviation (RSD) was calculated toe 4.3% by serially measuring 2.0 × 10−9 M ATP for 11 repetitiveeterminations, indicating a high accuracy and reproducibility ofhe assay.

In order to verify the amplification effect of the two cycles, theTP detection experiment was performed in the absence of poly-erase, and the result was shown in Fig. 3. In the absence of Klenow

olymerase, S5 released during cycle 1 could not extend to form auplex with the ATP recognition probe and the target ATP couldot be displaced, so the target recycling (cycle 2) would not occur.s shown in Fig. 3, the detection range of ATP concentration was

rom 6.0 × 10−8 M to 6.0 × 10−7 M, which indicated that the ampli-cation efficiency was greatly improved incorporating MB-assistedtrand displacement amplification with target recycling.

.6. Specificity study

As the signaling of the CL detection is essentially based on thearget binding-induced dissociation of the complementary DNAaused by the conformational change of the ATP recognition probe,he sensor demonstrated here is specific for the target sensing. Bysing cytosine triphosphate (CTP), guanosine triphosphate (GTP)nd uridine triphosphate (UTP) as typical ATP analogs, the selectiv-ty of the method was investigated. As shown in Fig. 4, the FI-CLignal generated by ATP could obviously be distinguished fromhose produced by CTP, GTP or UTP. When mixed with CTP, GTPnd UTP, the FI-CL signal had no significant change compared withhat of ATP, which indicated that these small molecules could notnterfere with ATP analysis in the proposed protocol. With goodensitivity and selectivity, the present method provides a versatileethod for aptamer-based small molecules assay.

.7. Determination of ATP in cancer cell extract

In order to demonstrate the validation of the proposed methodor practical samples, analysis of cellular ATP from Ramos cellsas implemented here. After treatment with 100-fold dilution,

he extracted solutions were subjected to FI-CL detection and theesults were listed in Table 1. It was showed that the concentra-ion of ATP in Ramos cell lysate was 4.21 × 10−6 M (average ± S.D.,

= 3) per 105 cells. For comparison, HPLC was conducted for thenalysis of the same sample (the details for HPLC detection wereescribed in Supplementary Materials). The comparative result

ndicated that the data obtained by HPLC method was consistent

3 4.40 3.7 4.31 3.5

a Each value is the average of three measurements for 105 mL−1 Ramos cells.

with the proposed method basically. So this method could be usedto monitor the content of ATP in cell extracts without the interfer-ence of other substance.

4. Conclusions

In conclusion, a novel signal amplification method for ATP detec-tion is demonstrated in the present study based on magneticmicrobeads-assisted strand displacement amplification and tar-get recycling. This proposed method possesses several significantadvantages. Firstly, the employment of magnetic beads can signifi-cantly simplify the separation procedures of the assay, which makesthe manipulation very easy. Secondly, the cross-circular amplifica-tion can be achieved and the sensitivity of the amplification strategyis high enough to detect trace amount of target molecules. Thirdly,as the aptamers can be easily selected for diverse ranges of bio-logical targets with high affinity and specificity, the design can beconveniently used to detect other small molecule targets. In conclu-sion, with its sensitivity, specificity, and simplicity, it is reasonableto believe that this strategy holds great promise for basic researchand in vitro diagnostics.

Acknowledgments

This work was supported by the National Natural Science Foun-dation of China (21025523), the National Basic Research Programof China (2010CB732404), and the Program for Changjiang Scholarsand Innovative Research Team in University (PCSIRT).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.aca.2013.01.044.

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