Detection of silver(I) ions based on the controlled self-assembly of a perylene fluorescence probe

Analytical Biochemistry 430 (2012) 48–52

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Detection of silver(I) ions based on the controlled self-assembly of a perylene fluorescence probe Yue Yang a,b,c, Wenying Li a,c, Hong Qi d,⇑, Qingfeng Zhang a, Jian Chen a, Yan Wang a,c, Bin Wang a,c, Shujie Wang b,⇑, Cong Yu a,⇑ a

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, People’s Republic of China Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China d Tumor Hospital of Jilin Province, Changchun 130061, People’s Republic of China b c

a r t i c l e

i n f o

Article history: Received 7 April 2012 Received in revised form 23 July 2012 Accepted 24 July 2012 Available online 1 August 2012 Keywords: Nucleic acid Silver(I) ion Label free Fluorescence probe Self-assembly Perylene

a b s t r a c t In the current work, we report a label-free fluorescence turn-on approach for the sensitive and selective sensing of Ag+. A cationic perylene derivative, compound A, was used as the fluorescence probe. Compound A monomer is strongly fluorescent, and the fluorescence can be efficiently quenched through self-aggregation (self-assembly). A cytosine (C)-rich oligonucleotide, oligo-C, was employed. In the absence of Ag+, oligo-C induced strong compound A aggregation due to electrostatic interactions in aqueous media, and very weak fluorescence signal was detected. However, in the presence of Ag+, the specific interactions between oligo-C and Ag+ induced hairpin structure formation of oligo-C through C–Ag+–C bonding interactions. Oligo-C binding to compound A aggregates was weakened; therefore, compound A monomer could be released and detected. The intensity of the fluorescence signal was directly related to the amount of Ag+ added to the assay solution. Our method is highly sensitive—a limit of detection of 5 nM was obtained—and also very selective. Ag+ detection in complex sample mixtures was also demonstrated. Ó 2012 Elsevier Inc. All rights reserved.

Silver is closely connected with people’s daily lives and has been widely used in the electrical, photographic imaging, and pharmaceutical industries [1]. Thousands of tons of silver waste have been directly released into the environment annually [2]. Traditional methods for Ag(I) sensing include atomic absorption spectroscopy, atomic emission spectroscopy, and inductively coupled plasma–mass spectroscopy [3–7]. However, these methods are heavily instrument dependent, with complicated assay procedures; therefore, they are expensive, time-consuming, and inconvenient. Many colorimetric, electrochemical, and fluorescent methods have been developed during recent years for silver(I) detection [8–12]. However, many of these methods require covalent labeling with a fluorophore, require the use of various nanomaterials, and are technically quite demanding and expensive. The specific interactions between nucleic acid bases and metal ions have currently attracted increasing attention due to their potential applications in biosensing [13]. It has been demonstrated that certain metal ions could covalently interact with specific nucleic acid bases to form metal-mediated base pairs, and induce ⇑ Corresponding authors. Fax: +86 431 85872638 (H. Qi), fax: +86 431 85095253 (S. Wang), fax: +86 431 8526 2710 (C. Yu). E-mail addresses: [email protected] (H. Qi), [email protected] (S. Wang), [email protected] (C. Yu). 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.07.024

conformation changes of the random coil single-stranded DNA into a hairpin structure or facilitate/stabilize the formation of DNA duplex structures [14–17]. For example, thymine (T)1 base was found to bond especially to Hg2+ and form T–Hg2+–T base pair in a duplex DNA, providing a way to sense Hg2+ with high sensitivity and specificity with T-rich single-stranded oligonucleotides [18–26]. Similarly, cytosine (C) base can interact with Ag+ to form stable C–Ag+– C base pair. Based on this observation, several methods for the detection of Ag+ have been developed [27–34]. However, these methods usually require the oligonucleotide to be labeled with a fluorescent dye (and sometime a nanomaterial/quencher as well) at its 30 or 50 end, which is technically demanding and expensive. Thus, the development of a label-free, simple, inexpensive, sensitive, and selective method for Ag+ sensing is highly desirable. Here we report a new label-free method for the highly sensitive and selective detection of Ag+. A water-soluble cationic fluorescent perylene probe, compound A (Fig. 1), and a cytosine-rich oligonucleotides, oligo-C, were employed. Compound A has a strong tendency to self-aggregate in an aqueous buffer solution via the intermolecular p–p stacking interactions. Oligo-C, a polyanion,

1 Abbreviations used: T, thymine; C, cytosine; UV–vis, ultraviolet–visible; TOC, total organic carbon.

Detection of silver(I) ions / Y. Yang et al. / Anal. Biochem. 430 (2012) 48–52

49

Assay procedures

Fig.1. Structure of compound A.

could induce aggregation of compound A and resulted in strong compound A monomer fluorescence quenching. However, in the presence of Ag+, the interactions between the cytosine bases and Ag+ (formation of the C–Ag+–C base pair) resulted in conformation changes of oligo-C from a random coil structure into a hairpin structure. As a result, the ability of oligo-C to induce aggregation of compound A was weakened, compound A monomer was released, and increased fluorescence intensity was detected, providing a facile, simple, sensitive, and selective means for Ag+ quantification [35–39].

Materials and methods

Oligo C (20 nM) and Ag+ ions of a specific concentration were mixed in an aqueous buffer solution (5 mM Mops and 20 mM NaNO3, pH 7.0). The sample solution was heated to 90 °C and incubated for 5 min, followed by gradually cooling down to room temperature (total sample volume = 247.5 ll). Then 50 nM compound A was added (total sample volume = 250 ll) and the fluorescence spectra were recorded. Sample analysis Lake water samples were collected from the South Lake of Changchun, Jilin Province, China. The samples were centrifuged two times at 10,000 rpm for 2 min, and the supernatant (25 ll) was collected and added to the assay solution for the quantitative analysis (total sample volume = 250 ll). In addition, known quantities of Ag+ were added to the lake water sample solutions, and the concentrations of Ag+ were determined by the above-mentioned assay procedures.

Materials

Results and discussion

The oligonucleotide (oligo-C, 50 -CCT CCT CCC TCC TTT TCC ACC CAC CAC C-30 ) was synthesized and ultra-PAGE purified by Sangon Biotechnology (Shanghai, China). Silver nitrate was obtained from Shanghai Chemical Reagent (Shanghai, China). Compound A was synthesized as described previously [35]. All other chemicals were of analytical grade and used as received. All stock and buffer solutions were prepared using water purified with a Milli-Q A10 filtration system (Millipore, Billerica, MA, USA). The oligonucleotide stock solutions were stored at 4 °C before use.

Sensing strategy

Instrumentation The oligonucleotide was quantified via ultraviolet–visible (UV– vis) absorption at 260 nm (Cary 50 Bio, Varian, USA). Emission spectra were obtained using a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon, USA). The excitation wavelength was 495 nm. Excitation and emission bandwidths were both 5 nm for all measurements. Each data point in the figures and table was a mean value of three repetitive measurements, and the error bars represent the standard deviations. Total organic carbon content was determined with a TOC analyzer (Shimadzu, Japan). Unless otherwise specified, all spectra were taken at an ambient temperature of 22 °C in 5 mM Mops buffer and 20 mM NaNO3 at pH 7.0.

The design strategy of our method is depicted in Scheme 1. First, oligo-C was mixed with Ag+ of a specific concentration, and the sample solution was heated to 90 °C, incubated for 5 min, and then slowly cooled down to ambient temperature. During such a process and in the presence of Ag+, Ag+ interacted with the cytosine bases and induced the conformation changes of the singlestranded oligo-C to form a hairpin structure because of the formation of the C–Ag+–C base pair. Compound A was subsequently added to the sample mixture. Oligo-C contains many negatively charged phosphate functional groups, making it a polyanion. In addition, because compound A contains two positive charges, oligo-C would induce compound A aggregation through strong electrostatic interactions. Depending on the amount of Ag+ added to the assay solution, certain portions of oligo-C would take the hairpin structure. As a result, its ability to induce compound A aggregation was weakened. With more Ag+ added, more hairpin oligo-C formed and more compound A existed in the free monomeric form. In addition, because compound A aggregate is not fluorescent, compound A monomer is highly fluorescent; the changes in the free monomer concentration would cause strong solution compound A fluorescence intensity changes, forming the basis for the selective Ag+ quantification.

Scheme 1. Schematic representation of the assay strategy for the selective sensing of Ag+.

50

Detection of silver(I) ions / Y. Yang et al. / Anal. Biochem. 430 (2012) 48–52

Fig.2. Effect of the buffer solution pH on the emission intensity changes of 50 nM compound A at 545 nm (Mes for pHs 5.5 and 6.0; Mops for pHs 6.5, 7.0, 7.5, and 8.0). Two concentrations of Ag+ (100 and 1000 nM) were studied. The error bars represent the standard deviations of three repetitive measurements.

Fig.3. Effect of the solution NaNO3 concentration on 50 nM compound A fluorescence intensity changes at 545 nm. Two different concentrations of Ag+ were tested (500 and 1000 nM). The error bars represent the standard deviations of three repetitive measurements.

Optimization of assay conditions We found that oligo-C must be incubated with Ag+ at an elevated temperature for a certain period of time to obtain the optimal results. It seems that the heating process destroyed the various possible secondary structures of oligo-C and facilitated the Ag+ cytosine bonding interactions. Other assay conditions were optimized to enhance the performance of the Ag+ detection. It was found that the solution pH value had a strong influence on the performance of the assay. Fig. 2 shows that the fluorescence intensity ratio of signal to background (F/F0) increased very slowly from pH 5.5 to 6.5. When the pH reached 7.0, the maximum value was obtained. The F/F0 ratio decreased again when the solution pH value increased further. It appears that the acidic conditions were not very suitable for the formation of the C–Ag+–C base pair because of the possible protonation of the cytosine base [40]. In addition, at a higher buffer pH,

Fig.4. (A) Changes in emission spectrum of 50 nM compound A in the presence of different concentrations of Ag+. (B) Plot of the fluorescence intensity changes at 545 nm against the Ag+ concentration. The error bars represent the standard deviations of three repetitive measurements. Inset: Expanded low-Ag+ concentration region of the calibration curve.

the performance also decreased, possibly because compound A has a greater tendency to self-aggregate at a higher buffer pH. Nucleic acid is a polyanion; for a hairpin (or duplex) structure to form, the strong negative charge electrostatic repulsive interactions need to be overcome. It is well known that one way to do this is to increase the solution ionic strength. The addition of the amount and type of salts has a strong influence on the secondary structures of the nucleic acid, including the correct folding of the molecular beacon hairpin structures [41]. In addition, adding high concentrations of salt would also reduce the degree of the induced aggregation of compound A. Our results show that 20 mM NaNO3 was the optimal salt concentration used in the current investigation (Fig. 3). Sensitivity of assay Our current method is highly sensitive for Ag+ detection, Fig. 4 shows that with the increase of the solution Ag+ concentration, a gradual increase of the compound A monomer fluorescence intensity was observed. At approximately 1000 nM Ag+ concentration, a

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Detection of silver(I) ions / Y. Yang et al. / Anal. Biochem. 430 (2012) 48–52 Table 1 Silver(I) recovery in lake water samples.

a

Fig.5. Selectivity of the assay over other metal ions. The changes in emission spectrum of 50 nM compound A in the presence of 800 nM Ag+ and other interference ions were analyzed. Assay procedures were the same as those described for Fig. 4.

Lake water sample

Silver(I) spiked (nM)

Silver(I) determineda (nM)

Recovery (%)

1 2 3 4

100 200 500 800

109.7 ± 11 207.2 ± 15 483.3 ± 21 784.1 ± 39

109.7 103.6 96.7 98.0

Values are averages of three measurements ± standard deviations.

relationship was obtained. The linear regression equation is F = 0.135C + 43.20 (correlation coefficient R2 = 0.99), where F is the fluorescence intensity at 545 nm and C is the concentration of Ag+ (in nM). The limit of detection of the current method is estimated to be 5 nM (3r/slope), which is comparable to some of the most sensitive methods for Ag+ quantification reported to date [27,29] and meets the sensitivity requirement of Ag+ detection for drinking water (460 nM) defined by the U.S. Environmental Protection Agency. Specificity of assay The selectivity of the assay over other metal ions was investigated. The potential interference ions studied include Mg2+, K+, Zn2+, Co2+, Mn2+, Cd2+, Ca2+, Ni2+, Cu2+, and Pb2+. Whereas 800 nM Ag+ gave significant compound A fluorescence enhancement, the other ions studied did not show noticeable compound A fluorescence enhancement. In addition, 800 nM Ag+ was also mixed with 800 nM of the interference ions, and little interference on the fluorescence recovery was observed (Fig. 5). The results clearly show that our assay method is highly selective for Ag+, and the selectivity apparently originates from the highly specific bonding interactions between the Ag+ ion and the nucleic acid cytosine base. Detection in complex sample mixtures The feasibility of our method to be used in complex sample mixtures (real samples) was also studied. Water samples were taken from the South Lake of Changchun. The total organic carbon (TOC) content of the lake water sample was determined to be 27.83 mg/L, which is a typical TOC value of the city lake water samples in China [42]. The samples were centrifuged briefly to get rid of any insoluble materials, and the supernatants were used for the quantitative analysis. Known concentrations of Ag+ were added, and a new calibration curve was obtained (Fig. 6). The linear regression equation is F = 0.139C + 39.60 (R2 = 0.99), where F is the fluorescence intensity at 545 nm and C is the concentration of Ag+ (in nM). By using this new calibration curve, the Ag+ recovery values were determined and the average percentage of recovery was determined to be 102% (Table 1). The results clearly show that our method can be used for the detection of Ag+ in complex sample mixtures (the lake water). In addition, the assay can clearly distinguish the lake water samples spiked with Ag+ ions of specific concentrations. Conclusions

Fig.6. (A) Emission spectra of 50 nM compound A in the presence of different concentrations of Ag+ spiked in lake water samples. (B) Plot of the fluorescence intensity changes of panel A at 545 nm against the Ag+ concentration. The error bars represent the standard deviations of three repetitive measurements.

saturation point was reached where further increases of the sample solution Ag+ concentration caused little increase in fluorescence intensity. At lower Ag+ concentrations (0–800 nM), a linear

A highly sensitive and selective method for the quantification of Ag+ has been developed. A cytosine-rich oligonucleotides, oligo-C, and a positively charged perylene probe, compound A, were used as the sensing elements. When Ag+ was added to the aqueous solution, oligo-C could specifically interact with Ag+ and fold into a hairpin structure via C–Ag+–C base pairs. Compound A was then added to the assay solution, and the interactions between oligo-C and compound A were weakened due to the conformation changes

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of oligo-C (formation of the hairpin structure). Decreased degree of induced aggregation and increased concentration of free compound A monomer resulted, and a turn-on emission signal was detected. With no Ag+ added, oligo-C could induce aggregation of compound A very efficiently and resulted in strong fluorescence quenching. Our method is highly sensitive—a limit of detection of 5 nM was obtained—and also highly selective against the common potential interference ions. In addition, because the method is label free, it is simple, fast, inexpensive, and convenient. Acknowledgments This work was supported by the ‘‘100 Talents’’ program of the Chinese Academy of Sciences, the National Natural Science Foundation of China (21075119, 91027036), the National Basic Research Program of China (973 Program, 2011CB911002), the Pillar Program of Changchun Municipal Bureau of Science and Technology (2011225), and the Open Project of State Key Laboratory of Supramolecular Structure and Materials (SKLSSM201215).

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