Fluorescence detection of adenosine triphosphate through an aptamer–molecular beacon multiple probe

Analytical Biochemistry 424 (2012) 8–11

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Fluorescence detection of adenosine triphosphate through an aptamer–molecular beacon multiple probe Xiaodan Zeng a,b, Xiaoling Zhang a,⇑, Wen Yang a,⇑, Hongying Jia c, Yamin Li a a

Key Laboratory of Cluster Science of Ministry of Education, School of Chemistry, Beijing Institute of Technology, Beijing 100081, People’s Republic of China Center of Analysis and Measurement, Jilin Institute of Chemical Technology, Jilin 132022, People’s Republic of China c Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 18 November 2011 Received in revised form 21 January 2012 Accepted 23 January 2012 Available online 1 February 2012 Keywords: Aptamer Molecular beacon Adenosine triphosphate Fluorescence probe

a b s t r a c t An aptamer–molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity. Ó 2012 Elsevier Inc. All rights reserved.

Aptamer and molecular beacon (MB)1 are considered as promising recognition elements for biosensor applications. They have been selected for a broad range of targets, including metal ions (e.g., K+ [1], Hg2+ [2], Pb2+ [3]), small organic molecules (e.g., adenosine [4,5], amino acids [6,7], ochratoxin A [8,9]), DNA [10,11], and proteins (e.g., thrombin [12,13]). Aptamers are nucleic acids with specific recognition properties to targets such as low-molecular-weight substrates or macromolecules. They are selected from a rich library composed of 1016 nucleic acids by using the SELEX (systematic evolution of ligands by exponential enrichment) procedure, amplified by polymerase chain reaction (PCR), and then sequenced to yield a defined recognition unit [14]. Because of the high affinity and selectivity for their targets, aptamers have been broadly used in scientific and biochemical fields [15–18]. MB is a short hairpin oligonucleotide probe containing a stem and loop structure. The loop part of MB is complementary to a specific oligonucleotides, and the stem is composed of short complementary arms. More important, a fluorophore and a quencher are ⇑ Corresponding authors. Fax: +86 10 88875298. E-mail addresses: [email protected] (X. Zhang), [email protected] (W. Yang). 1 Abbreviations used: MB, molecular beacon; PCR, polymerase chain reaction; ATP, adenosine triphosphate; SS, short strand; GTP, guanosine triphosphate; CTP, cytidine triphosphate; UTP, uridine triphosphate. 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2012.01.021

linked to each end of the stem. The fluorophore and quencher are close to each other in the absence of target, resulting in energy release and low fluorescence signal. However, when MB binds to a sequence, it undergoes a conformation change separating the fluorophore and quencher, and then it will produce a fluorescence signal. Due to their high signal-to-background ratio and molecular recognition selectivity, MBs have been broadly applied in numerous hybridization assays [19–23]. Adenosine triphosphate (ATP) plays an important role in living organisms [24]. It is involved in sensory transduction, and its concentration is an important guideline in disease diagnosis such as cell viability and cell injury [25]. So, the determination of ATP is essential for diagnosis and biochemical study. Developing a method with high sensitivity, selectivity, and simplicity for ATP assay could greatly facilitate the diagnoses and cures of related diseases. There are many methods used for determination of ATP, including electrochemiluminescence [26,27], fluorescence [28], high-performance liquid chromatography (HPLC) [29,30], and electrochemistry [31]. But all of these methods are composed of complicated and time-consuming operations. Therefore, it is still a challenge for researchers to find new ways that could improve the simplicity, selectivity, and sensitivity of ATP detection. In our research, we provide a multiple fluorescence probe that uses the recognition priority and binding between ATP aptamer and beacon to target ATP assay. The probe is composed of three

Aptamer–MB fluorescent probe for ATP assay / X. Zeng et al. / Anal. Biochem. 424 (2012) 8–11

parts: an aptamer, an MB, and a nucleic acid (short strand, SS) that complements the aptamer and MB. The strategy is shown in Scheme 1. When there is no ATP added, the aptamer will bind with SS, which is easier than MB. Little fluorescence intensity is observed for the fluorophore quenched by the quencher of MB. When ATP is mixed with the solution containing these three parts, the aptamer will be occupied first accompanied by the binding of MB with SS. Significant fluorescence intensity will be observed. Based on the relation of the fluorescence intensity and ATP concentration, a method for ATP assay is established. Our method shows simplicity, high sensitivity, and high selectivity, and it proved to be a good method to reduce the background.

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together equivalently and diluted to the proper concentration with Tris–HCL buffer solution.

Performance of ATP detection The ATP was provided at a concentration of 10–4 M. The fluorescence measurements were carried out with the addition of a series of concentrations of ATP.

Results and discussion Spectral feature of interaction between designed probe and ATP

Materials and methods Materials and reagents Oligonucleotide-containing ATP aptamer (50 -Acc tgg ggg agt att gcg gag gga ggt-30 ) [32], the SS (50 -Ctcc ctcc gcaa tact cccc-30 ), and MB (50 -TAMRA-Ccta gggg agta ttgc ggag tagg-DABCYL-30 ) were synthesized and purified by Shanghai Sangon Biotechnology (Shanghai, China). ATP, guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) were purchased from Amresco (USA). All other chemicals were of analytical reagent grade and were used as received without further purification. Solutions were prepared with deionized water processed with a Milli-Q ultra-high-purity water system (Millipore, USA). Apparatus

As shown in Fig. 1, in the absence of ATP, the solution has very weak fluorescence emission because the fluorescence of TAMRA gets quenched by DABCYL. With the addition of ATP, the interaction between the ATP and the aptamer induces the binding of MB with SS, and DABCYL is forced away from the TAMRA group accompanied by fluorescence getting restored significantly. It was found that the quenching efficiency was approximately 94% and the background was low, suggesting that the design of our strategy was successful and might be applied for the detection of ATP. These measurements are all performed at room temperature, suggesting that this method may be a good probe for practical application because room temperature measurement is a more efficient way of eliminating background fluorescence [20].

ATP assay

For ATP assays, the fluorescence intensity was measured. All fluorescence measurements were carried out on a Cary Eclipse Fluorescence Spectrometer (Varian, USA). The intensity was recorded at 578 nm with an excitation wavelength of 521 nm, and slits were set at 10 nm for the excitation and emission. Preparation of probe To prepare the probe, each part was dissolved in Tris–HCl (pH 7.4) containing 5 mM MgCl2, 850 mM NaCl, and 16 mM KCl (final concentration of 100 lM). Then these three parts were mixed

The ATP was detected and quantified according to the procedure described above. Fig. 2 shows the fluorescence emission spectra of the probe at different ATP concentrations. The fluorescence intensity at 578 nm is enhanced gradually with increasing concentrations of ATP. The linear relationship (Fig. 3) is constructed between the fluorescence enhancement and target concentration in the range of 10 to 500 nM (r = 0.9945), and the detection limit is estimated to be 0.1 nM with a relative standard deviation (RSD) of 2.44%. The detection is lower than the values reported for many fluorescent aptasensors for ATP such as carbon nanotubes (2 lM) [33] and nanoparticles (28 nM) [34].

Scheme 1. Mechanism of probe for ATP assay.

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Aptamer–MB fluorescent probe for ATP assay / X. Zeng et al. / Anal. Biochem. 424 (2012) 8–11 Table 1 Results for determination of bovine serum albumin in synthetic samples. ATP in sample (nM) 50 100 300

Foreign substances (nM) +

GTP, CTP, UTP, Na GTP, CTP, UTP, K+ GTP, CTP, UTP, Fe3+

Found (nM)

Recovery (%)

52.9 103.2 295.2

105.8 103.2 95.84

Fig.1. Fluorescence spectra of solutions (from bottom to top): MB (200 nM), MB–SS (200 nM)–aptamer (200 nM), ATP (10 ìM)/aptamer–SS–MB, and SS–aptamer.

Fig.4. Selectivity of probe to ATP, GTP, UTP, and CTP (500 nM). Bars represent relative fluorescence intensities at 578 nm.

Fig.2. Influence of concentration of ATP on fluorescence intensity (final concentrations of ATP: 10, 50, 100, 200, 350, 375, 430, 460, 480, and 500 nM).

Fig.5. Interference analysis of GTP, UTP, and CTP (500 nM). Bars represent relative fluorescence intensities at 578 nm.

Fig.3. Fluorescence intensity at 578 nm plotted versus concentration of ATP (final concentrations of ATP: 10, 50, 100, 200, 350, 375, 430, 460, 480, and 500 nM).

Specificity of probe On the basis of the fact that ATP induced a fluorescence increase of the probe, we tested whether this system could detect ATP with

high selectivity. Changes of this probe were monitored in the presence of GTP (500 nM), CTP (500 nM), and UTP (500 nM), and the relative fluorescence intensities were plotted against the concentration of ATP (500 nM) (Fig. 4). The intensities of GTP, CTP, and UTP were very weak due to their weak binding affinity for ATP aptamer. The results showed high selectivity toward ATP over the others. In addition, the interference of these three analogues was investigated. In the presence of GTP, CTP, and UTP, ATP was added to the solution. As can be seen in Fig. 5, these analogues could not interact with the probe, and thus they will not interfere with the assay. All of the results imply that this probe can recognize the target with high selectivity, and this probe was able to detect ATP among its analogues.

Aptamer–MB fluorescent probe for ATP assay / X. Zeng et al. / Anal. Biochem. 424 (2012) 8–11

Analysis of synthetic samples We also applied this probe to detect ATP in synthetic samples containing some foreign substance to examine the accuracy and applicability of this assay. Some known concentrations of ATP were added to the synthetic samples, and the ATP concentration (found concentration in Table 1) is determined following the method mentioned above. The experimental results are listed Table 1. As can be seen, the recoveries of ATP in synthetic samples are 95.84% to 105.8%. The recoveries obtained proved that our probe is an excellent one for the measurement of ATP. It may become a promising probe for ATP assays. Conclusions A multiple probe containing aptamer and MB with high selectivity and sensitivity was introduced for ATP assay. This probe uses the advantage of high affinity of aptamer and low background of MB to develop a method for detecting ATP. At optimal conditions, 0.1 nm ATP could be detected. Compared with traditional ATP assays, this method was proved to be fast, highly sensitive, and highly selective without further treatment. Especially, the interference from GTP, CTP, and UTP was very minor, which is an important advantage when used in biological samples. More important, the design is based simply on recognition of aptamer and DNA hybridization, and therefore it can be applied to other targets by replacing the aptamers. Acknowledgments This work was supported by the National Natural Science Foundation of China (20975012) and the 111 Project (B07012). References [1] C. Shi, H.X. Gu, C.P. Ma, An aptamer-based fluorescent biosensor for potassium ion detection using a pyrene-labeled molecular beacon, Anal. Biochem. 400 (2010) 99–102. [2] Y.W. Lin, C.W. Liu, H.T. Chang, Fluorescence detection of mercury(II) and lead(II) ions using aptamer/reporter conjugates, Talanta 84 (2011) 324–329. [3] X.B. Zhang, Z.D. Wang, H. Xing, Y. Xiang, Y. Lu, Catalytic and molecular beacons for amplified detection of metal ions and organic molecules with high sensitivity, Anal. Chem. 82 (2010) 5005–5011. [4] Z.F. Liu, J. Ge, X.S. Zhao, Quantitative detection of adenosine in urine using silver enhancement of aptamer – gold nanoparticle aggregation and progressive dilution, Chem. Commun. 47 (2011) 4956–4958. [5] X. Zhu, Y.S. Zhang, W.Q. Yang, Q.D. Liu, Z.Y. Lin, B. Qiu, G.N. Chen, Highly sensitive electrochemiluminescent biosensor for adenosine based on structure-switching of aptamer, Anal. Chim. Acta 684 (2011) 121–125. [6] X.J. Yang, T. Bing, H.C. Mei, C.L. Fang, Z.H. Cao, D.H. Shangguan, Characterization and application of a DNA aptamer binding to L-tryptophan, Analyst 136 (2011) 577–585. [7] L.F. Chen, Q.H. Cai, F. Luo, X. Chen, X. Zhu, B. Qiu, Z.Y. Lin, G.N. Chen, A sensitive aptasensor for adenosine based on the quenching of Ru(bpy)32+-doped silica nanoparticle ECL by ferrocene, Chem. Commun. 46 (2010) 7751–7753. [8] N. Duan, S.J. Wu, Z.P. Wang, An aptamer-based fluorescence assay for ochratoxin A, Chin. J. Anal. Chem. 39 (2011) 300–304. [9] L. Bonel, J.C. Vidal, P. Duato, J.R. Castillo, An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer, Biosens. Bioelectron. 26 (2011) 3254–3259. [10] X. Mao, H. Xu, Q.X. Zeng, L.W. Zeng, G.D. Liu, Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis, Chem. Commun. 10 (2009) 3065–3067. [11] R. Varghese, H.A. Wagenknecht, Red–white–blue emission switching molecular beacons: ratiometric multicolour DNA hybridization probes, Org. Biomol. Chem. 8 (2010) 526–528.

11

[12] J. Zhang, P.P. Chen, X.Y. Wu, J.H. Chen, L.J. Xu, G.N. Chen, F. Fu, A signal-on electrochemiluminescence aptamer biosensor for the detection of ultratrace thrombin based on junction-probe, Biosens. Bioelectron. 26 (2011) 2645– 2650. [13] C.W. Chi, Y.H. Lao, Y.S. Li, L.C. Chen, A quantum dot – aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection, Biosens. Bioelectron. 26 (2011) 3346–3352. [14] B. Shlyahovsky, D. Li, Y. Weizmann, R. Nowarski, M. Kotler, I. Willner, Spotlighting of cocaine by an autonomous aptamer-based machine, J. Am. Chem. Soc. 129 (2007) 3814–3815. [15] S.Y. Yan, R. Huang, Y.Y. Zhou, M. Zhang, M.G. Deng, X.L. Wang, X.C. Weng, X. Zhou, Aptamer-based turn-on fluorescent four-branched quaternary ammonium pyrazine probe for selective thrombin detection, Chem. Commun. 47 (2011) 1273–1275. [16] A.E. Abelow, O. Schepelina, R.J. White, A. Vallée-Bélisle, K.W. Plaxco, I. Zharov, Biomimetic glass nanopores employing aptamer gates responsive to a small molecule, Chem. Commun. 46 (2010) 7984–7986. [17] X.L. Yan, Z.J. Cao, C.W. Lau, J.Z. Lu, DNA aptamer folding on magnetic beads for sequential detection of adenosine and cocaine by substrate-resolved chemiluminescence technology, Analyst 135 (2010) 2400–2407. [18] Z.L. Jiang, L.P. Zhou, A.H. Liang, Resonance scattering detection of trace melamine using aptamer-modified nanosilver probe as catalyst without separation of its aggregations, Chem. Commun. 47 (2011) 3162–3164. [19] L. Wang, C.Y. Yang, C.D. Medley, S.A. Benner, W.H. Tan, Locked nucleic acid molecular beacons, J. Am. Chem. Soc. 127 (2005) 15664–15665. [20] J.S. Li, W.Y. Zhou, X.Y. Ouyang, H. Yu, R.H. Yang, W.H. Tan, J.L. Yuan, Design of a room-temperature phosphorescence-based molecular beacon for highly sensitive detection of nucleic acids in biological fluids, Anal. Chem. 83 (2011) 1356–1362. [21] Z.W. Tang, P. Liu, C.B. Ma, X.Y. Yang, K.M. Wang, W.H. Tan, X.Y. Lv, Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase, Anal. Chem. 83 (2011) 2505–2510. [22] R.M. Kong, X.B. Zhang, L.L. Zhang, Y. Huang, D.Q. Lu, W.H. Tan, G.L. Shen, R.Q. Yu, Molecular beacon-based junction probes for efficient detection of nucleic acids via a true target-triggered enzymatic recycling amplification, Anal. Chem. 83 (2011) 14–17. [23] N. Dave, J.W. Liu, Fast molecular beacon hybridization in organic solvents with improved target specificity, J. Phys. Chem. B 114 (2010) 15694–15699. [24] J.J. Zhou, H.P. Huang, J. Xuan, J.R. Zhang, J.J. Zhu, Quantum dots electrochemical aptasensor based on three-dimensionally ordered macroporous gold film for the detection of ATP, Biosens. Bioelectron. 26 (2010) 834–840. [25] C.B. Ma, X.H. Yang, K.M. Wang, Z.W. Tang, W. Li, W.H. Tan, X.Y. Lv, A novel kinase-based ATP assay using molecular beacon, Anal. Biochem. 372 (2008) 131–133. [26] H.P. Huang, Y.L. Tan, J.J. Shi, G.X. Liang, J.J. Zhu, DNA aptasensor for the detection of ATP based on quantum dots electrochemiluminescence, Nanoscale 2 (2010) 606–612. [27] W. Yao, L. Wang, H.Y. Wang, X.L. Zhang, L. Li, Biosensor for ATP detection, Biosens. Bioelectron. 24 (2009) 3269–3274. [28] A.J. Moro, P.J. Cywinski, S. Körsten, G.J. Mohr, An ATP fluorescent chemosensor based on a Zn(II)-complexed dipicolylamine receptor coupled with a naphthalimide chromophore, Chem. Commun. 46 (2010) 1085–1087. [29] E.J.C.M. Coolen, I.C.W. Arts, E.L.R. Swennen, A. Bast, M.A.C. Stuart, P.C. Dagnelie, Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP–HPLC and UV detection, J. Chromatogr. B 864 (2008) 43–51. [30] X.F. Xue, F. Wang, J.H. Zhou, F. Chen, Y. Li, J. Zhao, Online cleanup of accelerated solvent extractions for determination of adenosine 50 -triphosphate (ATP), adenosine 50 -diphosphate (ADP), and adenosine 50 -monophosphate (AMP) in royal jelly using high-performance liquid chromatography, J. Agric. Food Chem. 57 (2009) 4500–4505. [31] Y.H. Wang, X.X. He, K.M. Wang, X.Q. Ni, A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA, Biosens. Bioelectron. 25 (2010) 2101–2106. [32] Z.W. Tang, P. Mallikaratchy, R.H. Yang, Y.M. Kim, Z. Zhu, H. Wang, W.H. Tan, Aptamer switch probe based on intramolecular displacement, J. Am. Chem. Soc. 130 (2008) 11268–11269. [33] Y. He, Z.G. Wang, H.W. Tang, D.W. Pang, Low background signal platform for the detection of ATP: when a molecular aptamer beacon meets graphene oxide, Biosens. Bioelectron. 29 (2011) 76–81. [34] Z.X. Zhou, Y. Du, S.J. Dong, Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor, Anal. Chem. 83 (2011) 5122–5127.