Ultrasensitive detection of lead (II) based on fluorescent aptamer-functionalized carbon nanotubes

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Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/etap

Ultrasensitive detection of lead (II) based on fluorescent aptamer-functionalized carbon nanotubes Seyed Mohammad Taghdisi a,1 , Somayeh Sarreshtehdar Emrani b,1 , Kaveh Tabrizian c , Mohammad Ramezani d , Khalil Abnous e,∗ , Ahmad Sarreshtehdar Emrani f,∗∗ a

Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b School of Dentistry, Mashhad University of Medical Sciences, Mashhad, Iran c Department of Toxicology, School of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran d Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran f Cardiovascular Research Center, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran

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Article history:

Lead contamination is a serious environmental problem with toxic effects in human. Here,

Received 9 February 2014

we developed a simple and sensitive sensing method employing ATTO 647N/aptamer-SWNT

Received in revised form

ensemble for detection of Pb2+ . This method is based on the super quenching capability

13 April 2014

of single-walled carbon nanotubes (SWNTs), high affinity of the aptamer toward Pb2+ and

Accepted 18 April 2014

different propensities of ATTO 647N-aptamer and ATTO 647N-aptamer/Pb2+ complex for

Available online 26 April 2014

adsorption on SWNTs. In the absence of Pb2+ , the fluorescence of ATTO 647N-aptamer is efficiently quenched by SWNTs. Upon addition of Pb2+ , the aptamer binds to its target, lead-

Keywords:

ing to the formation of a G-quadruplex/Pb2+ complex and does not interact with SWNTs

ATTO 647N

and ATTO 647N-aptamer starts fluorescing. This sensor exhibited a high selectivity toward

Aptamer

Pb2+ and a limit of detection (LOD) as low as 0.42 nM was obtained. Also this sensor could be

Sensor

applied for detection of Pb2+ ions in tap water and biological sample like serum with high

Lead

sensitivity. © 2014 Elsevier B.V. All rights reserved.

SWNT

1.

Introduction

Lead (Pb) is one of the most hazardous heavy metals contaminant in the environment with high persistency (Kaur et al.,

∗

2013; Zhang et al., 2013). Based on Environmental Protection Agency (EPA) and the international World Health Organization (WHO) regulations, maximum acceptable Pb2+ level in drinkingwater, are 50 ␮g L−1 and 10 ␮g L−1 , respectively (Gupta

Corresponding author. Tel.: +98 915 157 4437. Corresponding author. Tel.: +98 511 882 3255. E-mail addresses: [email protected], kh [email protected] (K. Abnous), ahmad [email protected] (A.S. Emrani). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.etap.2014.04.020 1382-6689/© 2014 Elsevier B.V. All rights reserved. ∗∗

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1236–1242

and Rastogi, 2008; Zhang et al., 2013). According to the Center of Disease Control and Prevention (CDCP), the maximum acceptable level of lead in blood is 10 ␮g/dL (Center of Disease Control and Prevention, 1991; Gilbert and Weiss, 2006). Lead is mainly absorbed via the gastrointestinal and respiratory systems (Abdel Moneim et al., 2011) and could induce toxic effects in liver, brain, kidneys and reproductive system (Abdel Moneim et al., 2011; Reshma Anjum et al., 2011). Sensitive and fast techniques for detection of Pb2+ in water are in great demand. Current methods for detection of lead ions are inductively coupled plasma atomic emission spectroscopy (ICP-AES), ICP-mass spectrometry (ICP-MS) and graphite furnace atomic absorption spectrometry (GFAAS) (Aydin and Soylak, 2010; He et al., 2008; Kuang et al., 2013). These techniques are time-consuming, labor-intensive and require expensive instruments (Kuang et al., 2013). Aptamers are among the most general sensing platforms (Ouyang et al., 2011). Aptamers are short single-stranded oligonucleotides derived from random sequence nucleic acid libraries, generated for specific targets. Aptamers are selected through an in vitro process called SELEX (systematic evolution of ligands by exponential enrichment) (Stead et al., 2010; Zhang and Zhang, 2012). Aptamers selectively bind to various targets ranging from small molecules to whole cells (Davis et al., 1998; Stoltenburg et al., 2007). Aptamers have distinct advantages over antibodies, including ease of production, small size, easy modification, stability in heat and low cost (Li et al., 2012; Medley et al., 2011; Taghdisi et al., 2011). In addition, aptamers have high affinity and selectivity and no or low immunogenicity and toxicity (Medley et al., 2011; Taghdisi et al., 2011). Because of these unique characteristics, aptamers have been broadly applied for construction of biosensors (Li et al., 2012; Zhu et al., 2011). Among the various signal transduction protocols, fluorescence is widely used for design of aptamer-based detections, because of its high sensitivity, simplicity and easy application (Kim et al., 2011; Ouyang et al., 2011; Zheng et al., 2012). Organic dye molecules are commonly used as both the fluorophore and the quencher in sensors based on fluorescence resonance energy transfer (FRET) (Li et al., 2013b). However, organic dye molecules suffer from photo-bleaching, low quantum yield and limitations of sensitivity when applied with biological samples (Li et al., 2013a; Zhen et al., 2010). Using ATTO 647N as a fluorescent dye could surpass these problems (Kolmakov et al., 2010a; Lesoine et al., 2012).

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Low efficiency of FRET between the conventional fluorophore and quencher causes a high background signal and decreases the sensor sensitivity. FRET sensor is expensive, because it needs both fluorophore and quencher dyes. In addition, when one part of the sensor is aptamer, it makes sensor vulnerable to digestion by nuclease when used in vivo. Application of carbon nanotubes (CNTs) as quencher could prevent these problems (Zhen et al., 2010). CNTs are molecular-scale tubes of graphitic carbon (Foldvari and Bagonluri, 2008; Taghdisi et al., 2011). CNTs are categorized structurally as multi-walled carbon nanotubes (MWNTs) and single-walled carbon nanotubes (SWNTs) (Foldvari and Bagonluri, 2008; Sgobba and Guldi, 2009). Due to their unique chemical and physical properties, CNTs have been attracting attention in various fields, such as biosensing, chemistry and drug delivery (Ouyang et al., 2011; Raffa et al., 2010; Zhu et al., 2010). Single stranded DNA (ssDNA) binds noncovalently to SWNTs by means of ␲-stacking interactions between nucleotide bases and SWNT sidewalls (Ouyang et al., 2011; Zhu et al., 2010). However, when aptamer interacts with its target, the complex loses this property (Ouyang et al., 2011). SWNTs are known as excellent nonquenchers by acting as an energy acceptor (Zhen et al., 2010; Zhu et al., 2010). Moreover, SWNTs could protect aptamer from nuclease digestion and facilitate application of designed aptasensor in biological fluids (Zhen et al., 2010; Zhu et al., 2010). Therefore, in this project a fluorescent sensor based on ATTO 647N/aptamer-SWNT ensemble was developed for Pb2+ detection. In this sensor, ATTO 647N acts as the energy donor and SWNT acts as the energy acceptor. A ssDNA aptamer, which specifically binds to Pb2+ (Li et al., 2013b, 2010), serves as the molecular recognition probe.

2.

Experimental

2.1.

Materials

The fluorescence labeled Pb2+ aptamer, 5 -ATTO 647NGGGTGGGTGGGTGGGT-3 and 5 -FAM-GGGTGGGTGGGTGGGT-3 were purchased from Microsynth (Switzerland). Cu(NO3 )2 , Pb(NO3 )2 , AgNO3 , Fe(NO3 )3 ·9H2 O, Ni(NO3 )2 ·6H2 O and NaH2 PO4 were provided by Sigma–Aldrich (USA). Commercial carboxyl-functionalized SWNTs was purchased from

Fig. 1 – Schematic description of Pb2+ fluorescence assay of based on aptamer-wrapped SWNT.

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Cheap Tubes Inc. (USA) to make sure CNTs were well soluble in water.

spectrophotometer (Cecil Company, UK) (Kam et al., 2005; Taghdisi et al., 2011).

2.2.

2.3. Effect of SWNT concentration on the fluorescence emission intensity of ATTO 647N-aptamer

Preparation of SWNT

The SWNT suspension (2 g L−1 ) was sonicated in deionized water for three 30 min cycles. The larger SWNT aggregates were permitted to precipitate for 30 min between each sonication cycle to make a well dispersed solution of SWNT. Dispersed SWNTs mixture from the third cycle was used as a stock solution for the next experiments (Rodrigues et al., 2013). The concentration of SWNT stock solution was determined by its absorbance at 808 nm using a CECIL 9000

Increasing concentrations of SWNT (0–900 ␮g/ml) were added to a fix concentration of ATTO 647N-aptamer (300 nM) in 10 mM phosphate buffer saline (PBS) solution (containing 10 mM Na2 HPO4 /NaH2 PO4 , 0.25 M NaCl and pH 7). The mixture was incubated at room temperature for 30 min and then centrifuged at 5000 rpm for 5 min. The pellet comprising aggregates and bundles of nanotubes at the bottom of centrifuge tube was discarded. The supernatant was collected.

Fig. 2 – Factors involved in fluorescence emission intensity. (a) Relative fluorescence intensity of ATTO 647N-aptamer in 10 mM PBS solution in the presence of various concentrations of SWNT, 300 nM ATTO 647N-aptamer, 0.25 M NaCl, and pH = 7. F0 and F are the fluorescence intensities at 665 nm before and after addition of various concentrations of SWNT, respectively. (b) Relative fluorescence intensity of ATTO 647N-aptamer in 10 mM PBS solution in the presence of various concentrations of NaCl, 300 nM ATTO 647N-aptamer, 100 nM Pb2+ , 150 ␮g/ml SWNT and pH = 7. F0 and F are the fluorescence intensities at 665 nm before and after addition of Pb2+ , respectively. (C) Effects of pH on the fluorescence intensity of ATTO 647N-aptamer without (d) and with Pb2+ . (e) Fluorescence recovery efficiency of ATTO 647N/aptamer-SWNT ensemble as a function of incubation time. F0 and F are the fluorescence intensities at 665 nm before and after addition of Pb2+ , respectively.

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Fluorescence intensity of obtained solution was measured using a microplate reader (TECAN Infinite 200 PRO, Austria) [Ex = 600 nm, Em = 665 nm].

2.4. Effect of NaCl concentration on the Pb2+ detection in the ATTO 647N/aptamer-SWNT sensing assay 100 nM Pb2+ was added to the mixture of 150 ␮g/ml SWNTs and 300 nM ATTO 647N-aptamer in 10 mM PBS (pH 7) with different concentrations of NaCl (0, 0.1, 0.15, 0.25 and 0.5 M). The mixture was incubated at room temperature for 30 min and then centrifuged at 5000 rpm for 5 min. Supernatants were collected and fluorescence intensities were measured as mentioned before.

2.5. Effect of pH on the fluorescence emission intensity of ATTO 647-aptamer 300 nM ATTO 647N-aptamer was added to 150 ␮g/ml SWNTs in 10 mM PBS (0.25 M NaCl) with different pH values (5–10). The mixture was centrifuged at 5000 rpm for 5 min. Supernatants were collected and fluorescence intensities were measured as describes before. The same experiment was performed in the presence of 100 nM Pb2+ and pH range from 6 to 9.

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2.6. Incubation time effect on the fluorescence recovery efficiency 100 nM Pb2+ was added to the mixture of 150 ␮g/ml SWNTs and 300 nM ATTO 647N-aptamer in 10 mM PBS (0.25 M NaCl and pH 7). The mixtures were incubated at room temperature from 0 to 60 min. After centrifugation at 5000 rpm for 5 min, supernatants were collected and fluorescence intensities were measured as mentioned before.

2.7.

Sensitivity and selectivity

Increasing concentrations of Pb2+ (0–1000 nM) were added to the assay solution contained 150 ␮g/ml SWNTs, 300 nM ATTO 647N-aptamer in 10 mM PBS (0.25 M NaCl and pH 7). Mixtures were incubated for 30 min at room temperature. After centrifugation, fluorescence of collected supernatants was monitored like previous experiments. The same experiment was performed for FAM-aptamer. The fluorescence intensities were measured using the microplate reader [Ex = 490 nm, Em = 535 nm]. The sensitivity of ATTO 647N/aptamer-SWNT ensemble was examined for Pb2+ detection in tap water. 150 ␮l of tap water was mixed with 150 ␮l of the PBS solution containing ATTO 647N-aptamer and SWNTs. The final concentrations in solution were adjusted to 300 nM ATTO 647N-aptamer, 150 ␮g/ml SWNTs, 0.25 M NaCl and pH 7. Then, increasing

Fig. 3 – (a) Relative fluorescence intensity of ATTO 647N/aptamer-SWNT ensemble as a function of Pb2+ concentration. (b) Pb2+ standard curve. F0 and F are the fluorescence intensities at 665 nm before and after addition of various concentrations of Pb2+ , respectively. (c) Efficiency of fluorescence in the presence of various metal ions.

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concentrations of Pb2+ (0–1000 nM) were added to the solution and incubated for 30 min. Mixtures were centrifuged at 5000 rpm for 5 min. Fluorescence intensities of collected supernatants were monitored as previous experiments. The selectivity of ATTO 647N/aptamer-SWNT ensemble was assessed in presence of 150 nM Ag+ , Fe3+ , Na+ , Ni2+ , Pb2+ and Cu2+ .

2.8.

Biological sample analysis

150 ␮l of rat serum was mixed with PBS solution and the pH was adjusted to 7.4. Pb2+ concentration was measured as previous experiments.

3.

Results and discussion

3.1.

Sensing scheme

The present fluorescent sensor is based on noncovalent binding of aptamer to SWNT, the ability of SWNT as an efficient quencher and principle of fluorescence energy transfer. As shown in Fig. 1, aptamer binds to SWNT noncovalently through ␲–␲ stacking interaction (Zhu et al., 2010). So that ATTO 647N comes to close proximity with the surface of

Fig. 4 – (a) Relative fluorescence intensity of FAM/aptamer-SWNT ensemble as a function of Pb2+ concentration. (b) Pb2+ Standard curve. F0 and F are the fluorescence intensities at 535 nm before and after addition of various concentrations of Pb2+ , respectively.

SWNT, leading to fluorescent energy transfer and quenching. Addition of Pb2+ ions induces a conformational change and formation of a G-quadruplex aptamer/Pb2+ complex. So that ATTO 647N-aptamer leaves the sidewall of SWNT (Li et al., 2013b) and fluorescence is turned on.

3.2.

Factors involved in fluorescence emission intensity

To determine the optimum concentration of SWNT for ATTO 647N quenching, increasing concentrations of SWNT were added to the PBS solution containing 300 nM ATTO 647Naptamer. The results showed that final concentration of 150 ␮g/ml SWNT could almost quench ATTO 647N (Fig. 2a). At concentrations of SWNT more than 150 ␮g/ml, all ATTO 647Naptamers were immobilized on the surface of SWNT via ␲–␲ interactions between SWNTs and aptamers. Ionic strength has an important impact on binding of aptamers to their targets (Li et al., 2013a). Higher ionic strengths, may weaken binding of aptamer to its target because of shielding effect of Na+ ions. Moreover in a low ionic strength solution, DNA hybridization gets difficult (Li et al., 2013b). Low fluorescence intensities at low NaCl concentrations (0.05, 0.1 and 0.15 M), indicates that aptamer could not

Fig. 5 – (a) Relative fluorescence intensity of ATTO 647N/aptamer-SWNT ensemble upon the addition of various concentrations of Pb2+ in tap water. (b) Standard curve of Pb2+ in tap water. F0 and F are the fluorescence intensities at 665 nm before and after addition of various concentrations of Pb2+ , respectively.

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in Fig. 3a. Our data showed that fluorescence intensities increased linearly and reached to plateau at concentration of 50 nM. Standard curve was plotted between 0 and 50 nM Pb2+ . The limit of detection (LOD), which was described as 3 times standard deviation/slope by the International Union of Pure and Applied Chemistry (IUPAC) standard, was determined to be 0.42 nM. We assumed that high sensitivity of our sensor is because of high affinity of aptamer toward its target, unique properties of ATTO 647N and unique properties of SWNT as a strong quencher and low background signal. LOD for FAM/aptamer-SWNT ensemble was determined to be 5.03 nM (Fig. 4). ATTO 647N/aptamer-SWNT ensemble was about 12 folds more sensitive than FAM/aptamer-SWNT ensemble, because of ATTO 647N unique properties such as high photostability and excellent fluorescence quantum yield (Kolmakov et al., 2010a; Lesoine et al., 2012). Selectivity is an important factor for a practical sensor. Fig. 3c shows the fluorescence response of ATTO 647N/aptamer-SWNT ensemble assay against varying metal ions including Ag+ , Fe3+ , Na+ , Ni2+ , Pb2+ and Cu2+ . No significant fluorescence intensity change was observed in the presence of other metal ions except for Pb2+ . These data indicated that designed sensor exhibited excellent selectivity toward Pb2+ .

3.4. Fig. 6 – (a) Relative fluorescence intensity of ATTO 647N/aptamer-SWNT ensemble upon the addition of various concentrations of Pb2+ in serum. (b) Standard curve of Pb2+ in serum. F0 and F are the fluorescence intensities at 665 nm before and after addition of various concentrations of Pb2+ , respectively.

efficiently bind to its target (Fig. 2b). The optimum concentration of NaCl was determined to be 0.25 M. It has been noted that pH may affect the fluorescence quenching features of fluorophores through protonatation of functional groups (Ouyang et al., 2011). In this study our data showed that in a range of pH from 5.5 to 9, SWNT could effectively quench ATTO 647N and H+ has no significant interference in the interaction between ATTO 647N-aptamer and SWNT (Fig. 2c). Moreover, in the presence of Pb2+ , the fluorescence of ATTO 647N could be effectively restored at pH range from 5.5 to 8.5 (Fig. 2d). The fluorescence recovery kinetics was analyzed by monitoring the fluorescence intensities as a function of time in the presence of 100 nM Pb2+ . Fig. 2e shows the fluorescence intensity increased rapidly with increasing the incubation time and the maximum fluorescence intensity at 30 min. This indicates the conformational change of aptamer was completed after this time and the assay time for Pb2+ detection is 30 min.

Preliminary application

Developed sensor was used to measure Pb2+ concentration in tap water and rat serum. No Pb2+ was detected in our sample buffer as well as sample serum. Known concentrations of Pb2+ ions were added into tap water and serum and LODs were determined to be 1.98 and 4.24 nM, respectively (Figs. 5 and 6). These LODs were much lower than the permissible levels for Pb2+ in drinking water and blood as regulated by WHO, USEPA and CDCD. Our data showed that our sensor could be successfully used for detection of Pb2+ in water and serum.

4.

Conclusion

In this study, we have designed a fluorescent sensor based on ATTO 647N/aptamer-SWNT ensemble for detection of Pb2+. This sensor takes advantages of super quenching capability of SWNT, high affinity of aptamer toward its target and unique properties of ATTO 647N as a fluorophore. This method is simple, highly sensitive and relatively fast (only 30 min). This sensor exhibits a limit of detection as low as 0.42 nM and excellent selectivity toward Pb2+ . Furthermore, the sensor could detect Pb2+ in tap water and serum with the LOD of 1.98 and 4.2 nM, respectively. It is expected this sensing platform/mechanism could be extended for detection of other metal ions and biomolecules.

Pb2+ analysis using ATTO 647N/aptamer-SWNT 3.3. complex

Conflict of interest Fluorescence intensities of ATTO 647N/aptamer-SWNT ensemble at different concentrations of Pb2+ are plotted

The authors declare that there are no conflicts of interest.

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Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgment Functional support of this study was provided by Mashhad University of Medical Sciences.

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