- Email: [email protected]
Colorimetric detection of hydrogen sulfide based on terbium-G-quadruplex-hemin DNAzyme
Accepted Manuscript Title: Colorimetric detection of hydrogen sulfide based on terbium-G-quadruplex-hemin DNAzyme Author: Gonge Tang Cailan Zhao Jie Gao Hongliang Tan PII: DOI: Reference:
S0925-4005(16)31024-3 http://dx.doi.org/doi:10.1016/j.snb.2016.06.162 SNB 20488
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
11-4-2016 27-6-2016 29-6-2016
Please cite this article as: Gonge Tang, Cailan Zhao, Jie Gao, Hongliang Tan, Colorimetric detection of hydrogen sulfide based on terbiumG-quadruplex-hemin DNAzyme, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.162 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Colorimetric detection of hydrogen sulfide based on terbium-G-quadruplex-hemin DNAzyme
Gonge Tang, Cailan Zhao, Jie Gao, Hongliang Tan*
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, P R China *Corresponding author: E-mail: [email protected]
Highlights
Terbium-G-quadruplex-hemin complex shows high peroxidase-like activity.
A simple colorimetric method for H2S detection was developed.
The colorimetric displays excellent sensitivity and selectivity to H2S.
Tb/G4-hemin DNAzyme can be cyclically used for colorimetric detection of H2S.
1
Abstract It is of great importance to simply and sensitively detect hydrogen sulfide (H2S) because of its role in various physiological processes as well as its inherent toxicity. In this work, a colorimetric method for H2S detection was developed by employing terbium-G-quadruplex-hemin (Tb/G4-hemin) DNAzyme as a peroxidase mimic, which can catalyze H2O2-mediated oxidation of 2,2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to produce radical cation (ABTS·+). Compared with the G4-hemin DNAzyme promoted by K+ and Na+, Tb/G4-hemin DNAzyme exhibits a higher catalytic activity. In the presence of Ag+, the peroxidase-like activity of Tb/G4-hemin DNAzyme can be inhibited significantly owing to the disruption of G-quadruplex structure. However, the addition of H2S can effectively suppress such negative behavior by competitive binding with Ag+, leading to the recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme, which can be reflected by an increase in absorbance signal of ABTS·+. The absorbance of ABTS·+ was enhanced linearly with increasing H2S concentration from 20 nM to 2 μM. The detection limit for H2S is 13 nM, which is much lower than most of previous methods. Moreover, the proposed method possesses the features of simple preparation, easy reproducibility and good biocompatibility. Given the fine performances and striking properties, we believe that the Tb/G4-hemin DNAzyme would have a great promise for analytical applications.
Keywords: Hydrogen sulfide; Colorimetric detection; G-quadruplex; Terbium ion; DNAzyme
2
1. Introduction Hydrogen sulfide (H2S) is well known for its characteristic smell of rotten eggs. It has been recognized as a highly toxic environmental pollutant arising from industrial activities, such as petroleum refining coal gasification and natural gas processing, for many decades [1]. However, recent studies suggest that H2S may also have important roles as a redox-active small molecule in cellular signaling pathways [2]. The endogenous H2S is mainly produced from L-cysteine by the enzymatic reactions involving cystathionine γ-lyase and cystathionine β-synthase [3]. The typical concentration of H2S in blood has been reported to be in the range of 10–100 μM [4, 5]. As a signal molecule, H2S appears to be involved in various physiological processes, including relaxation of vascular smooth muscles [6], mediation of neurotransmission [7], inhibition of insulin signaling [8], and regulation of inflammation [9]. An abnormal level of H2S has also been found to be closely related to the symptoms of many diseases, such as Alzheimer’s disease, Down syndrome and diabetes [10-12]. H2S is now accepted as the third gaseous transmitter besides nitric oxide (NO) and carbon monoxide (CO). Therefore, it is of great importance to simply and sensitively detect H2S due to its role in various physiological processes as well as its inherent toxicity.
To date, numerous methods have been developed for the detection of H2S, such as gas chromatography [13], inductively coupled plasma-atomic emission [14], electrochemistry [15], and fluorescence spectrometry [16, 17]. However, these methods usually require expensive and sophisticated instruments and/or complicated sample preprocesses, and are time-consuming. Compared with traditional methods, fluorescent methods can offer improved sensitivity and convenience, but laborious procedures and expensive reactants and reagents are often required for the preparation of fluorescent probes [18-21]. In some cases, organic dyes suffer from poor aqueous solubility and photobleaching. Colorimetric detection of H2S would be a more desirable method due to its low cost, simplicity and practicality. More importantly, the performance of colorimetric assays can be easily and real-time monitored by naked eyes and does not require any sophisticated instruments. In fact, several colorimetric sensors based on functionalized gold nanoparticles (AuNPs) have recently been presented for the detection of H2S in water and 3
biological samples [22-24]. These colorimetric sensors showed high sensitive and specific for H2S detection, but time-consuming fabrication procedures and poor stability of functionalized AuNPs and distinct interference from thiolates limits their further applications [23]. Therefore, rational design and development of new colorimetric sensors for simple and sensitive detection of H2S is highly demanded.
G-quadruplex-based DNAzyme is a kind of nucleic acid enzymes with peroxidase-like activity, which is formed from the assembly of guanine (G)-rich DNA sequence, hemin and metal ions. As a peroxidase mimic, G-quadruplex-hemin DNAzyme is able to catalyze the H2O2-mediated oxidation of 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to produce radical cation (ABTS·+) and accompanied by a detectable color change [25]. In contrast to nanomaterials-based peroxidase mimics, G-quadruplex-hemin DNAzymes are often water-soluble and do not involve in complicated preparation processes. Moreover, such DNAzyme possesses the unique properties of structural diversity, good biocompatibility, easy labeling, and excellent reproducibility [26, 27]. Based on these facts, G-quadruplex-hemin DNAzyme have already been used to develop various colorimetric sensors for the detection of protein [28], DNA [29], metal ions [30], and small molecules [31], showing their great potential in the analytical field. Nevertheless, most of these G-quadruplex-hemin DNAzyme were formed by the promotion of alkali cations, such as Na+ and K+, very few examples of other metal ions were reported, especially lanthanide ions. Recently, Zhang et al. found that, like common K+ and Na+, the introduction of terbium ions (Tb3+) is also able to promote G-rich DNA sequence to fold into proper
G-quadruplex
structure,
which
can
further
bind
with
hemin
to
form
Tb-G-quadruplex-hemin (Tb/G4-hemin) DNAzyme with peroxidase-like activity [27]. In spite of this, the application of Tb/G4-hemin DNAzyme as a peroxidase mimic for colorimetric sensing remains further explored.
In this work, we attempt to develop a simple and sensitive colorimetric sensor for the detection of H2S by employing Tb/G4-hemin DNAzyme as a peroxidase mimic. It has been demonstrated that Ag+ can coordinate with guanine bases and disrupt the structure of G-quadruplex promoted by K+ [32]. Inspired by this finding, we here propose that Ag+ may also destroy the structure of 4
Tb/G4-hemin DNAzyme, resulting in the inhibition of its peroxdase-like activity (Scheme 1). However, upon the addition of H2S, the coordinated Ag+ could be removed from G-quadruplex through competitive binding with H2S due to its strong affinity to Ag+ [16]. In this case, the structure of Tb/G4-hemin DNAzyme would be re-formed, which leads to the recovery of peroxidase-like activity. Therefore, Tb/G4-hemin DNAzyme-based colorimetric detection of H2S is expected.
Scheme 1
2. Material and methods 2.1 Chemicals All chemicals were of reagent grade and used without further purification. Terbium nitrate hexahydrate (Tb(NO3)3·6H2O, 99.99%) were purchased from Baotou Rewin Rare Earth Metal Materials Co. (Baotou, China). Metal salts, glucose and amino acids (Glycine (Gly), Serine (Ser), Alanine (Ala), Methionine (Met), and Cysteine (Cys)) were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). Glutathione (GSH, reduced) and Na2S·9H2O were obtained from Aladdin (Shanghai, China). The G-rich ssDNA was obtained from Sangon Biotech. Co. Ltd. (Shanghai, China). The ssDNA sequence is as follows: 5’-GGGTTAGGGTTAGGGTTAGGG-3’. Ultrapure water (18 MΩ, Millpore, USA) was used throughout this work.
2.2 Preparation of Tb/G4-hemin DNAzyme Briefly, 100 μL of DNA solution (50 μM) dissolved in Tris-HAc buffer (10 mM，pH 7.4) was firstly heated to 95 °C for 10 min to obtain completely ssDNA. After cooling down to room temperature, 100 μL of Tb(NO3)3 aqueous soliton (500 μM) was added to above DNA solution. This mixture was incubated at room temperature for over 1 h to form Tb/G4. Then, 170 μL of hemin (100 μM) was added for reacting another 30 min to form Tb/G4-hemin DNAzyme. Finally, Tris-HAc buffer (10 mM，pH 7.4) was added to above solution till its volume to reach 1 mL. The final concentrations of ssDNA, Tb3+ and hemin are 0.5, 5 and 1.7 μM, respectively. The MOS-450 spectrometer (Bio-Logic, Grenoble, France) was used to measure the circular dichroism (CD) spectrum of Tb/G4-hemin DNAzyme. 5
To test the peroxidase-like activity of Tb/G4-hemin DNAzyme, 10 μL of Tb/G4-hemin DNAzyme solution was added to a certain amount of Tris-HAc buffer (10 mM，pH 7.4) contained ABTS (3.5 mM) and H2O2 (10 mM) and mixed well. The final volume of the reaction solution is 100 μL. After incubating for 20 min at 37 °C, the absorption spectrum of this reaction solution was recorded by using Lambda 35 spectrophotometer (PerkinElmer, UK).
2.3 Effects of Ag+ on the catalytic activity of Tb/G4-hemin DNAzyme To investigate the effect of Ag+ on the catalytic activity of Tb/G4-hemin DNAzyme, 5 μL of AgNO3 aqueous solution (100 μM) was firstly added to 10 μL of Tb/G4-hemin DNAzyme solution and mixed well. After reacting for 10 min, 10 μL of ABTS (35 mM) and 10 μL of H2O2 (10 mM) were added to above mixture and Tris-HAc buffer (10 mM，pH 7.4) was used to make up to the final reaction volume to 100 μL. The reaction was carried out for 20 min at 37 °C. Then, the absorption spectrum of this reaction solution was recorded.
2.4 Colorimetric detection of H2S In this work, Na2S was used as the hydrogen sulfide donor because the pKs of H2S (pK1 = 6.96; pK2 = 12.90) predict that H2S and HS- are the predominant sulfide species in aqueous solution whether H2S, NaHS or Na2S is used [33]. To detect H2S in aqueous solution, 5 μL of AgNO3 aqueous solution (100 μM) was firstly reacted with 10 μL of Tb/G4-hemin DNAzyme solution in 50 μL of Tris-HAc buffer (10 mM，pH 7.4) for 10 min at room temperature. Then, Na2S aqueous solutions with final concentrations from 0 to 5 μM were added to above solution for reacting another 10 min at room temperature. Finally, 10 μL of ABTS (35 mM) and 10 μL of H2O2 (10 mM) were added to above mixture and Tris-HAc buffer (10 mM，pH 7.4) was used to kept the final reaction volume at 100 μL. The reaction was lasted 20 min at 37 °C before measuring the absorption spectra of these reaction solutions. The absorbance at 420 nm was used for quantitative analysis. Same procedure was used to for selectivity experiment of Tb/G4-hemin DNAzyme-Ag+ for H2S, in which H2S was replaced by other anions with same final concentrations.
2.5 Detection of H2S in serum samples The levels of H2S in serum samples were determined by using the standard addition method. The 6
serum samples were obtained from Jiangxi Normal University Hospital. The original serum samples were firstly diluted for 50 folds and then treated by H2O2 for 1 h at 37 °C to oxidize the thiol groups to disulfide bonds. Excess H2O2 was decomposed by heating for 30 min at 60 °C. The spiked serum samples were prepared by adding Na2S standard solution to the treated serum samples with final concentrations from 0 to 2 μM. The H2S detection procedure is the same as sensitivity test: 5 μL of AgNO3 aqueous solution (100 μM) was firstly reacted with 10 μL of Tb/G4-hemin DNAzyme solution for 10 min at room temperature and then the spiked serum samples were added. After the Tris-HAc buffer (10 mM，pH 7.4) contained ABTS (3.5 mM) and H2O2 (10 mM) was added and reacted for 20 min at 37 °C, their absorption spectra were recorded.
3. Results and discussion 3.1 Peroxidase-like activity of Tb/G4-hemin DNAzyme The peroxidase-like activity of Tb/G4-hemin DNAzyme was tested by the oxidation of ABTS in the presence of H2O2. As shown in Fig. 1A, ABTS itself is colorless and display no absorbance, even in the presence of H2O2. However, in the presence of pure hemin or the mixture of G-rich DNA and hemin, a typical absorption spectrum of ABTS·+ can be observed from the ABTS-H2O2 solution, indicating the occurrence of oxidation reaction of ABTS. Compared with pure hemin, the ABTS-H2O2 solution contained the mixture of G-rich DNA and hemin exhibited a 2.5-fold higher absorbance of ABTS·+. The results suggest that catalytic activity of the mixture of G-rich DNA and hemin is higher than the intrinsic peroxidase-like activity of pure hemin, which may be arisen from the binding of hemin with a part of G-rich DNA, leading to the formation of DNAzyme with low peroxidase-like activity [34]. Nevertheless, with the incubation of the mixture of G-rich DNA and hemin with Tb3+, a further significant increase (about 2.3 fold) in the absorbance of the ABTS-H2O2 solution was observed and accompanied by a deep green color. This indicates that the addition of Tb3+ can induce G-rich DNA folding into G-quadruplex structure, which is required to bind hemin to form Tb/G4-hemin DNAzyme with high peroxidase-like activity.
Figure 1
From Fig. S1, it can be seen that the absorbance of ABTS·+ enhanced with the increase of Tb3+ 7
concentration and reached to a plateau when Tb3+ concentration was more than 5 μM. The result reflects that the peroxidase-like activity of Tb/G4-hemin DNAzyme is primarily dependent on the Tb3+ concentration. To obtain the best sensitivity, 5 μM Tb3+ was chosen in the subsequent experiments. In addition, the peroxidase-like activity of Tb/G4-hemin DNAzyme is also pH dependent (Fig. S2). The highest catalytic activity of Tb/G4-hemin DNAzyme was obtained at pH 7.4. By contrast, in strong acidic or basic medium, Tb/G4-hemin DNAzyme displayed a significantly decrease in the peroxidase-like activity. This could be attributed to the protonation of the G-rich DNA under strong acidic condition and the formation of terbium hydroxide under strong basic condition, which will weaken the interactions between G-rich DNA and Tb3+, resulting in the dissociation of the Tb/G4 structure. For comparison, we tested the catalytic activities of G4-hemin DNAzyme promoted by Tb3+, K+ and Na+ under the optimized conditions, respectively. As shown in Fig. 1B, the Tb/G4-hemin DNAzyme displayed a higher peroxidase-like activity than the G4-hemin DNAzyme promoted by K+ and Na+, which is consistent with previous report [27]. Even the concentration of K+ and Na+ were increased to 1 mM, the lower catalytic activities of the G4-hemin DNAzyme promoted by K+ and Na+ were still not change. This may be attributed to the high selectivity of the G-rich DNA sequence to Tb3+, allowing Tb3+ to be much more effective than K+ and Na+ in term of promoting the proper folding of the G-rich DNA sequence [34].
3.2 Colorimetric assay of H2S Figure 2a showed the absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme under different conditions. Because of its high peroxidase-like activity, a strong absorbance can be observed from ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme. After the addition of Ag+, the absorbance of ABTS-H2O2 solution was decreased almost to background and the solution color changed from green to colorless. However, upon the further addition of H2S, the disappeared absorption spectrum of ABTS-H2O2 solution was found to be recorded again, which is associated with the production of a light green color that is detectable by naked eyes. The results imply that the addition of Ag+ could inhibit the peroxidase-like activity of Tb/G4-hemin DNAzyme, whereas the presence of H2S leads to the suppression of the negative effect of Ag+. Notably, no changes in the absorption spectrum of ABTS-H2O2 solution were found 8
in the presence of H2S alone, revealing that the ABTS-H2O2 is insensitive to H2S. Accordingly, the H2S-induced recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme is a result of the interaction of Ag+ and H2S.
Figure 2
It is well known that the peroxidase-like activity of G4-hemin complex highly relies on its G-quadruplex structure [35]. So, to discern the influence mechanism of Ag+ on the peroxidase-like activity of Tb/G4-hemin DNAzyme, the CD spectra of Tb/G4-hemin DNAzyme in the absence and presence of Ag+ were measured. As can be seen from Fig.2b, there are two positive peaks near 260 and 300 nm in the CD spectrum of Tb/G4-hemin DNAzyme alone, which can be assigned to the coexistence of parallel and antiparallel G-quadruplex structures [27, 36]. However, no CD peaks were observed after the addition of Ag+, reflecting the destruction of G-quadruplex structures. It has been reported that Ag+ can disrupt the structure of G-quadruplex by coordinating with the N7 and C6O groups of guanine base that are involved in the formation of G-quadruplex. The complexation of hemin with G-qudraplex does not have an influence on the interaction between Ag+ and G-quadruplex. Thus, the loss of peroxidase-like activity of Tb/G4-hemin DNAzyme in the presence of Ag+ would be attributed to the disruption of G-quadruplex structure caused by the coordination of Ag+ with guanine. By contrast, upon addition with H2S, the CD spectrum of Tb/G4-hemin DNAzyme in the presence of Ag+ becomes quite similar to that of its original state, indicating that the G-quadruplex structure is formed again. This is due to that Ag+ has a stronger affinity to H2S than that of guanine and can react with H2S to form Ag2S precipitate [16, 37]. Therefore, a simple colorimetric method for H2S detection may be developed on the basis of these findings.
Figure 3
The effects of other metal ions on the peroxidase-like activity of Tb/G4-hemin DNAzyme were then investigated. From Fig. 3a, it can be found that only Ag+ can cause significant decrease in the absorbance of ABTS·+, and no obvious changes were observed in the presence of other metal ions, 9
even at a high concentration of 1 mM. This indicates that the recognition of Tb/G4-hemin DNAzyme to Ag+ is highly specific. With the enhancement of Ag+ concentration, gradually decreased peroxidase-like activity of Tb/G4-hemin DNAzyme can be observed (Fig. 3b). When the Ag+ concentration reached to 5 μM, the peroxidase-like activity of Tb/G4-hemin DNAzyme has fallen to its original levels of 7 %. In order to avoid the interfering of excess Ag+, 5 μM Ag+ was used in the following experiments.
3.3 Sensitivity for H2S detection To evaluate the detection sensitivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S, the absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ were measured after adding different H2S concentrations (Fig. 4a). With the increase of H2S concentration from 0 to 2 μM, the absorbance of ABTS-H2O2 solution was enhanced gradually. When the H2S concentration is more than 2 μM, the absorbance of ABTS-H2O2 solution reached to a constant value. This could be due to that Ag+ can not be transformed into Ag2S even if H2S had been added to a saturation level. Figure 4b outlined the relationship between the absorbance of ABTS-H2O2 solution at 420 nm and the concentration of H2S. A good linear relationship (R = 0.9907) was observed in the range of 20 nM to 2 μM. On the basis of a signal to noise ratio of 3:1, the detection limit for H2S was estimated to be 13 nM. This detection limit is much lower than the maximum level of H2S in drinking water permitted by the World Health Organization (500 ppb, approximately 15 μM) [22] and the level of H2S in healthy human blood (10 - 100 μM) [4, 5], suggesting that this method is sufficient for detection of H2S in drinking water and human blood. Compared with previous reported methods for H2S detection (Table S1), our proposed method not only have much lower detection limit, but also offers the advantages of simple preparation, easy reproducibility and good biocompatibility. In addition, the Tb/G4-hemin DNAzyme displayed excellent stability. Almost no changes in the catalytic activity of Tb/G4-hemin DNAzyme were observed after the DNAzyme were stored for 30 days (Fig. S3). These results indicate that Tb/G4-hemin DNAzyme possesses great potential as a peroxidase mimic for colorimetric sensing of H2S. Figure 4
10
3.4 Selectivity for H2S detection To examine the selectivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S detection, we investigated the effects of various anions on the absorbance of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+. The anions employed in our selective experiments included ClO-, SO32-, CO32-, C2O42-, Ac-, SO42-, NO3-, Cl-, F-, and Br-. As shown in Fig. 5, only H2S can produce a significant enhancement in the absorbance of ABTS-H2O2 solution. However, under identical conditions, the absorbance changes of ABTS-H2O2 solution upon the addition of other anions are negligible. Even the concentrations of these anions was presented at 100-fold higher than that of H2S, the absorbance of ABTS-H2O2 solution still has not an obvious change. The results imply that these anions are not able to recover the peroxidase-like activity of Tb/G4-hemin DNAzyme. By measuring the absorption spectra of the sensor solution in the presence of H2S and other anions, moreover, we found that the absorbance of the sensor solution is almost same as the absorbance in the presence of H2S alone (data not shown). The results indicate that the presence of these anions does not interfere with the detection of H2S. Because the solubility product constant of Ag2S (Ksp = 6.3 ×10−50) is about at least 30 orders of magnitude lower than that of Ag+ with other anions [37], the high selectivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S detection could be ascribed to the ultrastong binding ability of sulfide with Ag+.
Figure 5
3.5 Reversibility On basis of the ultrastrong binding ability of sulfide with Ag+, which leads to the formation of Ag2S precipitates, we further explored the recycle use of Tb/G4-hemin DNAzyme as a peroxidase mimic for colorimetric detection of H2S. To this end, the ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme was firstly incubated with Ag+ for 10 min and then treated with H2S. When the absorbance of ABTS-H2O2 solution almost returned to its original levels, another portion of Ag+ was added. The cycles were repeated three times. From Fig. 6, it can be seen that the Tb/G4-hemin DNAzyme-Ag+ system exhibits obviously repeated switching behavior upon alternating addition of Ag+ and H2S (Fig. 6). Even the Tb/G4-hemin DNAzyme was cyclically treated for two rounds, the H2S-induce the absorbance of ABTS-H2O2 solution could still be 11
observed, although with a slightly lower signal intensity. The results suggest that Tb/G4-hemin DNAzyme can be cyclically used at least three times without significant loss of catalytic activity, endowing the Tb/G4-hemin DNAzyme-Ag+ system with the capability of reversible sensing.
Figure 6
3.6 Detection of H2S in serum samples To investigate the potential application of the Tb/G4-hemin DNAzyme-Ag+ system in real samples, the levels of H2S in spiked serum samples were tested by employing a standard addition method. Due to the fact that serum might contain some substances that would affect the catalytic activity of Tb/G4-hemin DNAzyme by coordinating with Tb3+ or Ag+, such as glucose, amino acids and GSH, the absorbance responses of the sensing system to these substrates were firstly studied. From Fig. S4, we can find that no remarkable absorbance responses were observed upon the addition of these interfering substrates except Cys and GSH, which may be due to the binding thiols group of Cys and GSH with Ag+. However, the interference of Cys and GSH can be eliminated by the addition of H2O2, which oxidizes the thiol groups to disulfide bonds. So, the analyzed samples in this work were prepared by adding different amounts of Na2S to 50-fold diluted serum solution that was treated by H2O2. Table 1 showed the results of Tb/G4-hemin DNAzyme-Ag+ system for the detection of H2S in serum samples. The recoveries are between 98.0 and 104.2 % and the relative standard deviations (RSD, n = 5) are all less than 2.91 %. The results imply that the Tb/G4-hemin DNAzyme-Ag+ system can be used for the detection of H2S with good recovery and precision.
Table 1
Conclusions In summary, we have developed a simple and sensitive colorimetric method for H2S detection by employing Tb/G4-hemin DNAzyme as a peroxidase mimic. The peroxidase-like activity was reduced significantly in the presence of Ag+ because of the disruption of G-quadruplex, whereas the addition of H2S can suppress such negative behavior by competitive binding with Ag+, leading 12
to the recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme. As a sensor for H2S detection, Tb/G4-hemin DNAzyme-Ag+ system not only exhibits much lower detection limit than most of previous methods, but also possesses the features of simple preparation, easy reproducibility and good biocompatibility. Due to the ultrastrong binding ability of H2S and Ag+, the alternating addition of Ag+ and H2S does not show an influence on the catalytic activity of Tb/G4-hemin DNAzyme, which endows the proposed sensor with the capability of reversible sensing. The successful application of the Tb/G4-hemin DNAzyme-Ag+ system in serum samples suggests that the proposed colorimetric sensor could be a potential biosensor platform for biological samples.
Acknowledgements This work was supported by the Natural Science Foundation of China (No. 21305054), Start-up Funding of Jiangxi Normal University, Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133604120002), and Natural Science Foundation of Jiangxi Province (No. 20151BAB203020).
References [1] R J Reiffenstein, a. W C Hulbert, S.H. Roth, Toxicology of hydrogen sulfide, Annu. Rev. Pharmacool. Toxicol., 32 (1992) 109-134. [2] L. Li, P. Rose, P.K. Moore, Hydrogen sulfide and cell signaling, Annu. Rev. Pharmacool. Toxicol., 51 (2011) 169-187. [3] H. Kimura, Hydrogen sulfide: its production, release and functions, Amino Acids, 41 (2010) 113-121. [4] W. Xuan, C. Sheng, Y. Cao, W. He, W. Wang, Fluorescent probes for the detection of hydrogen sulfide in biological systems, Angew. Chem. Int. Ed., 51 (2012) 2282-2284. [5] R.r. Hyšpler, A. Tichá, M. Indrová, Z. Zadák, L. Hyšplerová, J. Gasparič, J. Churáček, A simple, optimized method for the determination of sulphide in whole blood by GC-MS as a marker of bowel fermentation processes, J. Chromatogr. B, 770 (2002) 255-259. [6] G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A.K. Mustafa, W. Mu, S. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine γ-lyase, Science, 322 (2008) 587-590. [7] K. Abe, H. Kimura, The possible role of hydrogen sulfide as an endogenous neuromodulator, J. Neurosci,, 16 (1996) 1066-1071. [8] Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, L-cysteine inhibits insulin release from the 13
pancreatic β-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter, Diabetes, 55 (2006) 1391-1397. [9] J.L. Wallace, G. Caliendo, V. Santagada, G. Cirino, S. Fiorucci, Gastrointestinal safety and anti-inflammatory effects of a hydrogen sulfide-releasing diclofenac derivative in the rat, Gastroenterology, 132 (2007) 261-271. [10] D. Giuliani, A. Ottani, D. Zaffe, M. Galantucci, F. Strinati, R. Lodi, S. Guarini, Hydrogen sulfide slows down progression of experimental Alzheimer’s disease by targeting multiple pathophysiological mechanisms, Neurobiol. Learn. Mem., 104 (2013) 82-91. [11] P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi, B. Chadefaux-Vekemans, Endogenous hydrogen sulfide overproduction in Down syndrome, Am. J. Med. Genet. A, 116A (2003) 310-311. [12] W. Yang, G. Yang, X. Jia, L. Wu, R. Wang, Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms, J. Physiol., 569 (2005) 519-531. [13] A.R. Blanchette, A.D. Cooper, Determination of hydrogen sulfide and methyl mercaptan in mouth air at the parts-per-billion level by gas chromatography, Anal. Chem., 48 (1976) 729-731. [14] M. Colon, J.L. Todolí, M. Hidalgo, M. Iglesias, Development of novel and sensitive methods for the determination of sulfide in aqueous samples by hydrogen sulfide generation-inductively coupled plasma-atomic emission spectroscopy, Anal. Chim. Acta, 609 (2008) 160-168. [15] N.S. Lawrence, R.P. Deo, J. Wang, Electrochemical determination of hydrogen sulfide at carbon nanotube modified electrodes, Anal. Chim. Acta, 517 (2004) 131-137. [16] B. Liu, Y. Chen, Responsive lanthanide coordination polymer for hydrogen sulfide, Anal. Chem., 85 (2013) 11020-11025. [17] Q. Yu, K.Y. Zhang, H. Liang, Q. Zhao, T. Yang, S. Liu, C. Zhang, Z. Shi, W. Xu, W. Huang, Dual-emissive nanohybrid for ratiometric luminescence and lifetime imaging of intracellular hydrogen sulfide, ACS Appl. Mater. Interfaces, 7 (2015) 5462-5470. [18] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc., 133 (2011) 10078-10080. [19] T. Liu, Z. Xu, D.R. Spring, J. Cui, A Lysosome-targetable fluorescent probe for imaging hydrogen sulfide in living cells, Org. Lett., 15 (2013) 2310-2313. [20] V.S. Lin, A.R. Lippert, C.J. Chang, Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production, Proc. Natl. Acad. Sci., 110 (2013) 7131-7135. [21] K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, Development of a highly selective fluorescence probe for hydrogen sulfide, J. Am. Chem. Soc., 133 (2011) 18003-18005. [22] Z. Yuan, F. Lu, M. Peng, C.-W. Wang, Y.-T. Tseng, Y. Du, N. Cai, C.-W. Lien, H.-T. Chang, Y. He, E.S. Yeung, Selective colorimetric detection of hydrogen sulfide based on primary amine-active ester cross-linking of gold nanoparticles, Anal. Chem., 87 (2015) 7267-7273. 14
[23] X. Zhang, W. Zhou, Z. Yuan, C. Lu, Colorimetric detection of biological hydrogen sulfide using fluorosurfactant functionalized gold nanorods, Analyst, 140 (2015) 7443-7450. [24] Z. Zhang, Z. Chen, S. Wang, C. Qu, L. Chen, On-site visual detection of hydrogen sulfide in air based on enhancing the stability of gold nanoparticles, ACS Appl. Mater. Interfaces, 6 (2014) 6300-6307. [25] B. Ruttkay-Nedecky, J. Kudr, L. Nejdl, D. Maskova, R. Kizek, V. Adam, G-Quadruplexes as Sensing Probes, Molecules, 18 (2013) 14760-14779. [26] M. Deng, D. Zhang, Y. Zhou, X. Zhou, Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme, J. Am. Chem. Soc., 130 (2008) 13095-13102. [27] J. Zhang, Q. Gao, P. Chen, J. Chen, G. Chen, F. Fu, A novel Tb 3+-promoted G-quadruplex-hemin DNAzyme for the development of label-free visual biosensors, Biosens. Bioelectron., 26 (2011) 4053-4057. [28] T. Li, E. Wang, S. Dong, G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin, Chem. Commun., 44 (2008) 3654-3656. [29] Y. Xiao, V. Pavlov, T. Niazov, A. Dishon, M. Kotler, I. Willner, Catalytic beacons for the detection of DNA and telomerase activity, J. Am. Chem. Soc., 126 (2004) 7430-7431. [30] T. Li, S. Dong, E. Wang, Label-free colorimetric detection of aqueous mercury ion (Hg2+) using Hg2+-modulated G-quadruplex-based DNAzymes, Anal. Chem., 81 (2009) 2144-2149. [31] H. Bo, C. Wang, Q. Gao, H. Qi, C. Zhang, Selective, colorimetric assay of glucose in urine using G-quadruplex-based DNAzymes and 10-acetyl-3,7-dihydroxy phenoxazine, Talanta, 108 (2013) 131-135. [32] X.-H. Zhou, D.-M. Kong, H.-X. Shen, Ag+ and cysteine quantitation based on G-quadruplex-hemin DNAzymes disruption by Ag+, Anal. Chem., 82 (2009) 789-793. [33] S.K. Das, C.S. Lim, S.Y. Yang, J.H. Han, B.R. Cho, A small molecule two-photon probe for hydrogen sulfide in live tissues, Chem. Commun., 48 (2012) 8395-8397. [34] T. Li, E. Wang, S. Dong, G-Quadruplex-based DNAzyme as a sensing platform for ultrasensitive colorimetric potassium detection, Chem. Commun., 0 (2009) 580-582. [35] J. Kosman, B. Juskowiak, Peroxidase-mimicking DNAzymes for biosensing applications: A review, Anal. Chim. Acta, 707 (2011) 7-17. [36] T. Li, S. Dong, E. Wang, A lead(II)-driven DNA molecular device for turn-on fluorescence detection of lead(II) ion with high selectivity and sensitivity, J. Am. Chem. Soc., 132 (2010) 13156-13157. [37] J. Speight, Lange's Handbook of Chemistry, 16th Edition ed., McGraw-Hill Education: New York, 2005.
15
Biographies Gonge Tang received her BS degree from Yichun University in 2014. She is currently a graduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Cailan Zhao is currently an undergraduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Jie Gao received her BS degree from Anyang Institute of Technology in 2015. She is currently a graduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Hongliang Tan received his Ph.D degree in Biomedical Engineering from Southeast University. He is currently an associate professor at Jiangxi Normal University. His research interests involve the design and development of fluorescent and colorimetric sensors for bioanalysis.
16
Figure captions
Figure 1. (A) Absorption spectra of ABTS-H2O2 solution at different conditions: the mixture of 0.5 μM G-rich DNA and 1.7 μM hemin (a), Tb/G4-hemin DNAzyme (b), 1.7 μM hemin (c), and blank control (d). Inset is the corresponding photographs of these four samples. (B) Time-dependent absorbance of ABTS-H2O2 solution in the presence of G4-hemin DNAzyme promoted by Tb3+, K+ and Na+. Figure 2. (a) Absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme alone and after the addition of 5 μM Ag+, 2 μM H2S, and the mixture of 5 μM Ag+ and 2 μM H2S, respectively. Inset is the corresponding photographs of these samples. (b) CD spectra of Tb/G4-hemin DNAzyme in the absence and presence of 5 μM Ag+ and the mixture of 5 μM Ag+ and 2 μM H2S. Figure 3. Effects of various metal ions with a concentration of 5 μM (a) and Ag+ with different concentrations (b) on the absorbance of ABTS-H2O2 solution in presence of Tb/G4-hemin DNAzyme. Figure 4. Absorbance spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ after adding of H2S with different concentrations (a) and the linear calibration plots of absorbance of ABTS·+ at 420 nm against H2S concentrations (b). Figure 5. Effects of various anions (2 μM) on the absorbance of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ at 420 nm. Inset is the corresponding photographs of these samples. Figure 6. Reversible absorption changes of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme upon cyclic treatment with Ag+ and Cys. Scheme 1. Schematic illustration of Tb/G4-hemin DNAzyme as a peroxidase mimic for the detection of H2S.
17
Figure 1
18
Figure 2
19
Figure 3
20
Figure 4
21
Figure 5
22
Figure 6
23
Scheme 1
24
Table 1. Recovery of H2S added to serum samples. Spiked (μM)
Detected (μM)
Recovery (%)
RSD (n=5, %)
0.5
0.49 ± 0.01
98.0
2.33
1.5
1.51 ± 0.02
100.6
0.77
2
2.08 ± 0.07
104.2
2.91
25
S0925-4005(16)31024-3 http://dx.doi.org/doi:10.1016/j.snb.2016.06.162 SNB 20488
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
11-4-2016 27-6-2016 29-6-2016
Please cite this article as: Gonge Tang, Cailan Zhao, Jie Gao, Hongliang Tan, Colorimetric detection of hydrogen sulfide based on terbiumG-quadruplex-hemin DNAzyme, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.162 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Colorimetric detection of hydrogen sulfide based on terbium-G-quadruplex-hemin DNAzyme
Gonge Tang, Cailan Zhao, Jie Gao, Hongliang Tan*
Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology, Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, P R China *Corresponding author: E-mail: [email protected]
Highlights
Terbium-G-quadruplex-hemin complex shows high peroxidase-like activity.
A simple colorimetric method for H2S detection was developed.
The colorimetric displays excellent sensitivity and selectivity to H2S.
Tb/G4-hemin DNAzyme can be cyclically used for colorimetric detection of H2S.
1
Abstract It is of great importance to simply and sensitively detect hydrogen sulfide (H2S) because of its role in various physiological processes as well as its inherent toxicity. In this work, a colorimetric method for H2S detection was developed by employing terbium-G-quadruplex-hemin (Tb/G4-hemin) DNAzyme as a peroxidase mimic, which can catalyze H2O2-mediated oxidation of 2,2’-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to produce radical cation (ABTS·+). Compared with the G4-hemin DNAzyme promoted by K+ and Na+, Tb/G4-hemin DNAzyme exhibits a higher catalytic activity. In the presence of Ag+, the peroxidase-like activity of Tb/G4-hemin DNAzyme can be inhibited significantly owing to the disruption of G-quadruplex structure. However, the addition of H2S can effectively suppress such negative behavior by competitive binding with Ag+, leading to the recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme, which can be reflected by an increase in absorbance signal of ABTS·+. The absorbance of ABTS·+ was enhanced linearly with increasing H2S concentration from 20 nM to 2 μM. The detection limit for H2S is 13 nM, which is much lower than most of previous methods. Moreover, the proposed method possesses the features of simple preparation, easy reproducibility and good biocompatibility. Given the fine performances and striking properties, we believe that the Tb/G4-hemin DNAzyme would have a great promise for analytical applications.
Keywords: Hydrogen sulfide; Colorimetric detection; G-quadruplex; Terbium ion; DNAzyme
2
1. Introduction Hydrogen sulfide (H2S) is well known for its characteristic smell of rotten eggs. It has been recognized as a highly toxic environmental pollutant arising from industrial activities, such as petroleum refining coal gasification and natural gas processing, for many decades [1]. However, recent studies suggest that H2S may also have important roles as a redox-active small molecule in cellular signaling pathways [2]. The endogenous H2S is mainly produced from L-cysteine by the enzymatic reactions involving cystathionine γ-lyase and cystathionine β-synthase [3]. The typical concentration of H2S in blood has been reported to be in the range of 10–100 μM [4, 5]. As a signal molecule, H2S appears to be involved in various physiological processes, including relaxation of vascular smooth muscles [6], mediation of neurotransmission [7], inhibition of insulin signaling [8], and regulation of inflammation [9]. An abnormal level of H2S has also been found to be closely related to the symptoms of many diseases, such as Alzheimer’s disease, Down syndrome and diabetes [10-12]. H2S is now accepted as the third gaseous transmitter besides nitric oxide (NO) and carbon monoxide (CO). Therefore, it is of great importance to simply and sensitively detect H2S due to its role in various physiological processes as well as its inherent toxicity.
To date, numerous methods have been developed for the detection of H2S, such as gas chromatography [13], inductively coupled plasma-atomic emission [14], electrochemistry [15], and fluorescence spectrometry [16, 17]. However, these methods usually require expensive and sophisticated instruments and/or complicated sample preprocesses, and are time-consuming. Compared with traditional methods, fluorescent methods can offer improved sensitivity and convenience, but laborious procedures and expensive reactants and reagents are often required for the preparation of fluorescent probes [18-21]. In some cases, organic dyes suffer from poor aqueous solubility and photobleaching. Colorimetric detection of H2S would be a more desirable method due to its low cost, simplicity and practicality. More importantly, the performance of colorimetric assays can be easily and real-time monitored by naked eyes and does not require any sophisticated instruments. In fact, several colorimetric sensors based on functionalized gold nanoparticles (AuNPs) have recently been presented for the detection of H2S in water and 3
biological samples [22-24]. These colorimetric sensors showed high sensitive and specific for H2S detection, but time-consuming fabrication procedures and poor stability of functionalized AuNPs and distinct interference from thiolates limits their further applications [23]. Therefore, rational design and development of new colorimetric sensors for simple and sensitive detection of H2S is highly demanded.
G-quadruplex-based DNAzyme is a kind of nucleic acid enzymes with peroxidase-like activity, which is formed from the assembly of guanine (G)-rich DNA sequence, hemin and metal ions. As a peroxidase mimic, G-quadruplex-hemin DNAzyme is able to catalyze the H2O2-mediated oxidation of 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to produce radical cation (ABTS·+) and accompanied by a detectable color change [25]. In contrast to nanomaterials-based peroxidase mimics, G-quadruplex-hemin DNAzymes are often water-soluble and do not involve in complicated preparation processes. Moreover, such DNAzyme possesses the unique properties of structural diversity, good biocompatibility, easy labeling, and excellent reproducibility [26, 27]. Based on these facts, G-quadruplex-hemin DNAzyme have already been used to develop various colorimetric sensors for the detection of protein [28], DNA [29], metal ions [30], and small molecules [31], showing their great potential in the analytical field. Nevertheless, most of these G-quadruplex-hemin DNAzyme were formed by the promotion of alkali cations, such as Na+ and K+, very few examples of other metal ions were reported, especially lanthanide ions. Recently, Zhang et al. found that, like common K+ and Na+, the introduction of terbium ions (Tb3+) is also able to promote G-rich DNA sequence to fold into proper
G-quadruplex
structure,
which
can
further
bind
with
hemin
to
form
Tb-G-quadruplex-hemin (Tb/G4-hemin) DNAzyme with peroxidase-like activity [27]. In spite of this, the application of Tb/G4-hemin DNAzyme as a peroxidase mimic for colorimetric sensing remains further explored.
In this work, we attempt to develop a simple and sensitive colorimetric sensor for the detection of H2S by employing Tb/G4-hemin DNAzyme as a peroxidase mimic. It has been demonstrated that Ag+ can coordinate with guanine bases and disrupt the structure of G-quadruplex promoted by K+ [32]. Inspired by this finding, we here propose that Ag+ may also destroy the structure of 4
Tb/G4-hemin DNAzyme, resulting in the inhibition of its peroxdase-like activity (Scheme 1). However, upon the addition of H2S, the coordinated Ag+ could be removed from G-quadruplex through competitive binding with H2S due to its strong affinity to Ag+ [16]. In this case, the structure of Tb/G4-hemin DNAzyme would be re-formed, which leads to the recovery of peroxidase-like activity. Therefore, Tb/G4-hemin DNAzyme-based colorimetric detection of H2S is expected.
Scheme 1
2. Material and methods 2.1 Chemicals All chemicals were of reagent grade and used without further purification. Terbium nitrate hexahydrate (Tb(NO3)3·6H2O, 99.99%) were purchased from Baotou Rewin Rare Earth Metal Materials Co. (Baotou, China). Metal salts, glucose and amino acids (Glycine (Gly), Serine (Ser), Alanine (Ala), Methionine (Met), and Cysteine (Cys)) were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). Glutathione (GSH, reduced) and Na2S·9H2O were obtained from Aladdin (Shanghai, China). The G-rich ssDNA was obtained from Sangon Biotech. Co. Ltd. (Shanghai, China). The ssDNA sequence is as follows: 5’-GGGTTAGGGTTAGGGTTAGGG-3’. Ultrapure water (18 MΩ, Millpore, USA) was used throughout this work.
2.2 Preparation of Tb/G4-hemin DNAzyme Briefly, 100 μL of DNA solution (50 μM) dissolved in Tris-HAc buffer (10 mM，pH 7.4) was firstly heated to 95 °C for 10 min to obtain completely ssDNA. After cooling down to room temperature, 100 μL of Tb(NO3)3 aqueous soliton (500 μM) was added to above DNA solution. This mixture was incubated at room temperature for over 1 h to form Tb/G4. Then, 170 μL of hemin (100 μM) was added for reacting another 30 min to form Tb/G4-hemin DNAzyme. Finally, Tris-HAc buffer (10 mM，pH 7.4) was added to above solution till its volume to reach 1 mL. The final concentrations of ssDNA, Tb3+ and hemin are 0.5, 5 and 1.7 μM, respectively. The MOS-450 spectrometer (Bio-Logic, Grenoble, France) was used to measure the circular dichroism (CD) spectrum of Tb/G4-hemin DNAzyme. 5
To test the peroxidase-like activity of Tb/G4-hemin DNAzyme, 10 μL of Tb/G4-hemin DNAzyme solution was added to a certain amount of Tris-HAc buffer (10 mM，pH 7.4) contained ABTS (3.5 mM) and H2O2 (10 mM) and mixed well. The final volume of the reaction solution is 100 μL. After incubating for 20 min at 37 °C, the absorption spectrum of this reaction solution was recorded by using Lambda 35 spectrophotometer (PerkinElmer, UK).
2.3 Effects of Ag+ on the catalytic activity of Tb/G4-hemin DNAzyme To investigate the effect of Ag+ on the catalytic activity of Tb/G4-hemin DNAzyme, 5 μL of AgNO3 aqueous solution (100 μM) was firstly added to 10 μL of Tb/G4-hemin DNAzyme solution and mixed well. After reacting for 10 min, 10 μL of ABTS (35 mM) and 10 μL of H2O2 (10 mM) were added to above mixture and Tris-HAc buffer (10 mM，pH 7.4) was used to make up to the final reaction volume to 100 μL. The reaction was carried out for 20 min at 37 °C. Then, the absorption spectrum of this reaction solution was recorded.
2.4 Colorimetric detection of H2S In this work, Na2S was used as the hydrogen sulfide donor because the pKs of H2S (pK1 = 6.96; pK2 = 12.90) predict that H2S and HS- are the predominant sulfide species in aqueous solution whether H2S, NaHS or Na2S is used [33]. To detect H2S in aqueous solution, 5 μL of AgNO3 aqueous solution (100 μM) was firstly reacted with 10 μL of Tb/G4-hemin DNAzyme solution in 50 μL of Tris-HAc buffer (10 mM，pH 7.4) for 10 min at room temperature. Then, Na2S aqueous solutions with final concentrations from 0 to 5 μM were added to above solution for reacting another 10 min at room temperature. Finally, 10 μL of ABTS (35 mM) and 10 μL of H2O2 (10 mM) were added to above mixture and Tris-HAc buffer (10 mM，pH 7.4) was used to kept the final reaction volume at 100 μL. The reaction was lasted 20 min at 37 °C before measuring the absorption spectra of these reaction solutions. The absorbance at 420 nm was used for quantitative analysis. Same procedure was used to for selectivity experiment of Tb/G4-hemin DNAzyme-Ag+ for H2S, in which H2S was replaced by other anions with same final concentrations.
2.5 Detection of H2S in serum samples The levels of H2S in serum samples were determined by using the standard addition method. The 6
serum samples were obtained from Jiangxi Normal University Hospital. The original serum samples were firstly diluted for 50 folds and then treated by H2O2 for 1 h at 37 °C to oxidize the thiol groups to disulfide bonds. Excess H2O2 was decomposed by heating for 30 min at 60 °C. The spiked serum samples were prepared by adding Na2S standard solution to the treated serum samples with final concentrations from 0 to 2 μM. The H2S detection procedure is the same as sensitivity test: 5 μL of AgNO3 aqueous solution (100 μM) was firstly reacted with 10 μL of Tb/G4-hemin DNAzyme solution for 10 min at room temperature and then the spiked serum samples were added. After the Tris-HAc buffer (10 mM，pH 7.4) contained ABTS (3.5 mM) and H2O2 (10 mM) was added and reacted for 20 min at 37 °C, their absorption spectra were recorded.
3. Results and discussion 3.1 Peroxidase-like activity of Tb/G4-hemin DNAzyme The peroxidase-like activity of Tb/G4-hemin DNAzyme was tested by the oxidation of ABTS in the presence of H2O2. As shown in Fig. 1A, ABTS itself is colorless and display no absorbance, even in the presence of H2O2. However, in the presence of pure hemin or the mixture of G-rich DNA and hemin, a typical absorption spectrum of ABTS·+ can be observed from the ABTS-H2O2 solution, indicating the occurrence of oxidation reaction of ABTS. Compared with pure hemin, the ABTS-H2O2 solution contained the mixture of G-rich DNA and hemin exhibited a 2.5-fold higher absorbance of ABTS·+. The results suggest that catalytic activity of the mixture of G-rich DNA and hemin is higher than the intrinsic peroxidase-like activity of pure hemin, which may be arisen from the binding of hemin with a part of G-rich DNA, leading to the formation of DNAzyme with low peroxidase-like activity [34]. Nevertheless, with the incubation of the mixture of G-rich DNA and hemin with Tb3+, a further significant increase (about 2.3 fold) in the absorbance of the ABTS-H2O2 solution was observed and accompanied by a deep green color. This indicates that the addition of Tb3+ can induce G-rich DNA folding into G-quadruplex structure, which is required to bind hemin to form Tb/G4-hemin DNAzyme with high peroxidase-like activity.
Figure 1
From Fig. S1, it can be seen that the absorbance of ABTS·+ enhanced with the increase of Tb3+ 7
concentration and reached to a plateau when Tb3+ concentration was more than 5 μM. The result reflects that the peroxidase-like activity of Tb/G4-hemin DNAzyme is primarily dependent on the Tb3+ concentration. To obtain the best sensitivity, 5 μM Tb3+ was chosen in the subsequent experiments. In addition, the peroxidase-like activity of Tb/G4-hemin DNAzyme is also pH dependent (Fig. S2). The highest catalytic activity of Tb/G4-hemin DNAzyme was obtained at pH 7.4. By contrast, in strong acidic or basic medium, Tb/G4-hemin DNAzyme displayed a significantly decrease in the peroxidase-like activity. This could be attributed to the protonation of the G-rich DNA under strong acidic condition and the formation of terbium hydroxide under strong basic condition, which will weaken the interactions between G-rich DNA and Tb3+, resulting in the dissociation of the Tb/G4 structure. For comparison, we tested the catalytic activities of G4-hemin DNAzyme promoted by Tb3+, K+ and Na+ under the optimized conditions, respectively. As shown in Fig. 1B, the Tb/G4-hemin DNAzyme displayed a higher peroxidase-like activity than the G4-hemin DNAzyme promoted by K+ and Na+, which is consistent with previous report [27]. Even the concentration of K+ and Na+ were increased to 1 mM, the lower catalytic activities of the G4-hemin DNAzyme promoted by K+ and Na+ were still not change. This may be attributed to the high selectivity of the G-rich DNA sequence to Tb3+, allowing Tb3+ to be much more effective than K+ and Na+ in term of promoting the proper folding of the G-rich DNA sequence [34].
3.2 Colorimetric assay of H2S Figure 2a showed the absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme under different conditions. Because of its high peroxidase-like activity, a strong absorbance can be observed from ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme. After the addition of Ag+, the absorbance of ABTS-H2O2 solution was decreased almost to background and the solution color changed from green to colorless. However, upon the further addition of H2S, the disappeared absorption spectrum of ABTS-H2O2 solution was found to be recorded again, which is associated with the production of a light green color that is detectable by naked eyes. The results imply that the addition of Ag+ could inhibit the peroxidase-like activity of Tb/G4-hemin DNAzyme, whereas the presence of H2S leads to the suppression of the negative effect of Ag+. Notably, no changes in the absorption spectrum of ABTS-H2O2 solution were found 8
in the presence of H2S alone, revealing that the ABTS-H2O2 is insensitive to H2S. Accordingly, the H2S-induced recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme is a result of the interaction of Ag+ and H2S.
Figure 2
It is well known that the peroxidase-like activity of G4-hemin complex highly relies on its G-quadruplex structure [35]. So, to discern the influence mechanism of Ag+ on the peroxidase-like activity of Tb/G4-hemin DNAzyme, the CD spectra of Tb/G4-hemin DNAzyme in the absence and presence of Ag+ were measured. As can be seen from Fig.2b, there are two positive peaks near 260 and 300 nm in the CD spectrum of Tb/G4-hemin DNAzyme alone, which can be assigned to the coexistence of parallel and antiparallel G-quadruplex structures [27, 36]. However, no CD peaks were observed after the addition of Ag+, reflecting the destruction of G-quadruplex structures. It has been reported that Ag+ can disrupt the structure of G-quadruplex by coordinating with the N7 and C6O groups of guanine base that are involved in the formation of G-quadruplex. The complexation of hemin with G-qudraplex does not have an influence on the interaction between Ag+ and G-quadruplex. Thus, the loss of peroxidase-like activity of Tb/G4-hemin DNAzyme in the presence of Ag+ would be attributed to the disruption of G-quadruplex structure caused by the coordination of Ag+ with guanine. By contrast, upon addition with H2S, the CD spectrum of Tb/G4-hemin DNAzyme in the presence of Ag+ becomes quite similar to that of its original state, indicating that the G-quadruplex structure is formed again. This is due to that Ag+ has a stronger affinity to H2S than that of guanine and can react with H2S to form Ag2S precipitate [16, 37]. Therefore, a simple colorimetric method for H2S detection may be developed on the basis of these findings.
Figure 3
The effects of other metal ions on the peroxidase-like activity of Tb/G4-hemin DNAzyme were then investigated. From Fig. 3a, it can be found that only Ag+ can cause significant decrease in the absorbance of ABTS·+, and no obvious changes were observed in the presence of other metal ions, 9
even at a high concentration of 1 mM. This indicates that the recognition of Tb/G4-hemin DNAzyme to Ag+ is highly specific. With the enhancement of Ag+ concentration, gradually decreased peroxidase-like activity of Tb/G4-hemin DNAzyme can be observed (Fig. 3b). When the Ag+ concentration reached to 5 μM, the peroxidase-like activity of Tb/G4-hemin DNAzyme has fallen to its original levels of 7 %. In order to avoid the interfering of excess Ag+, 5 μM Ag+ was used in the following experiments.
3.3 Sensitivity for H2S detection To evaluate the detection sensitivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S, the absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ were measured after adding different H2S concentrations (Fig. 4a). With the increase of H2S concentration from 0 to 2 μM, the absorbance of ABTS-H2O2 solution was enhanced gradually. When the H2S concentration is more than 2 μM, the absorbance of ABTS-H2O2 solution reached to a constant value. This could be due to that Ag+ can not be transformed into Ag2S even if H2S had been added to a saturation level. Figure 4b outlined the relationship between the absorbance of ABTS-H2O2 solution at 420 nm and the concentration of H2S. A good linear relationship (R = 0.9907) was observed in the range of 20 nM to 2 μM. On the basis of a signal to noise ratio of 3:1, the detection limit for H2S was estimated to be 13 nM. This detection limit is much lower than the maximum level of H2S in drinking water permitted by the World Health Organization (500 ppb, approximately 15 μM) [22] and the level of H2S in healthy human blood (10 - 100 μM) [4, 5], suggesting that this method is sufficient for detection of H2S in drinking water and human blood. Compared with previous reported methods for H2S detection (Table S1), our proposed method not only have much lower detection limit, but also offers the advantages of simple preparation, easy reproducibility and good biocompatibility. In addition, the Tb/G4-hemin DNAzyme displayed excellent stability. Almost no changes in the catalytic activity of Tb/G4-hemin DNAzyme were observed after the DNAzyme were stored for 30 days (Fig. S3). These results indicate that Tb/G4-hemin DNAzyme possesses great potential as a peroxidase mimic for colorimetric sensing of H2S. Figure 4
10
3.4 Selectivity for H2S detection To examine the selectivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S detection, we investigated the effects of various anions on the absorbance of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+. The anions employed in our selective experiments included ClO-, SO32-, CO32-, C2O42-, Ac-, SO42-, NO3-, Cl-, F-, and Br-. As shown in Fig. 5, only H2S can produce a significant enhancement in the absorbance of ABTS-H2O2 solution. However, under identical conditions, the absorbance changes of ABTS-H2O2 solution upon the addition of other anions are negligible. Even the concentrations of these anions was presented at 100-fold higher than that of H2S, the absorbance of ABTS-H2O2 solution still has not an obvious change. The results imply that these anions are not able to recover the peroxidase-like activity of Tb/G4-hemin DNAzyme. By measuring the absorption spectra of the sensor solution in the presence of H2S and other anions, moreover, we found that the absorbance of the sensor solution is almost same as the absorbance in the presence of H2S alone (data not shown). The results indicate that the presence of these anions does not interfere with the detection of H2S. Because the solubility product constant of Ag2S (Ksp = 6.3 ×10−50) is about at least 30 orders of magnitude lower than that of Ag+ with other anions [37], the high selectivity of Tb/G4-hemin DNAzyme-Ag+ system for H2S detection could be ascribed to the ultrastong binding ability of sulfide with Ag+.
Figure 5
3.5 Reversibility On basis of the ultrastrong binding ability of sulfide with Ag+, which leads to the formation of Ag2S precipitates, we further explored the recycle use of Tb/G4-hemin DNAzyme as a peroxidase mimic for colorimetric detection of H2S. To this end, the ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme was firstly incubated with Ag+ for 10 min and then treated with H2S. When the absorbance of ABTS-H2O2 solution almost returned to its original levels, another portion of Ag+ was added. The cycles were repeated three times. From Fig. 6, it can be seen that the Tb/G4-hemin DNAzyme-Ag+ system exhibits obviously repeated switching behavior upon alternating addition of Ag+ and H2S (Fig. 6). Even the Tb/G4-hemin DNAzyme was cyclically treated for two rounds, the H2S-induce the absorbance of ABTS-H2O2 solution could still be 11
observed, although with a slightly lower signal intensity. The results suggest that Tb/G4-hemin DNAzyme can be cyclically used at least three times without significant loss of catalytic activity, endowing the Tb/G4-hemin DNAzyme-Ag+ system with the capability of reversible sensing.
Figure 6
3.6 Detection of H2S in serum samples To investigate the potential application of the Tb/G4-hemin DNAzyme-Ag+ system in real samples, the levels of H2S in spiked serum samples were tested by employing a standard addition method. Due to the fact that serum might contain some substances that would affect the catalytic activity of Tb/G4-hemin DNAzyme by coordinating with Tb3+ or Ag+, such as glucose, amino acids and GSH, the absorbance responses of the sensing system to these substrates were firstly studied. From Fig. S4, we can find that no remarkable absorbance responses were observed upon the addition of these interfering substrates except Cys and GSH, which may be due to the binding thiols group of Cys and GSH with Ag+. However, the interference of Cys and GSH can be eliminated by the addition of H2O2, which oxidizes the thiol groups to disulfide bonds. So, the analyzed samples in this work were prepared by adding different amounts of Na2S to 50-fold diluted serum solution that was treated by H2O2. Table 1 showed the results of Tb/G4-hemin DNAzyme-Ag+ system for the detection of H2S in serum samples. The recoveries are between 98.0 and 104.2 % and the relative standard deviations (RSD, n = 5) are all less than 2.91 %. The results imply that the Tb/G4-hemin DNAzyme-Ag+ system can be used for the detection of H2S with good recovery and precision.
Table 1
Conclusions In summary, we have developed a simple and sensitive colorimetric method for H2S detection by employing Tb/G4-hemin DNAzyme as a peroxidase mimic. The peroxidase-like activity was reduced significantly in the presence of Ag+ because of the disruption of G-quadruplex, whereas the addition of H2S can suppress such negative behavior by competitive binding with Ag+, leading 12
to the recovery of the peroxidase-like activity of Tb/G4-hemin DNAzyme. As a sensor for H2S detection, Tb/G4-hemin DNAzyme-Ag+ system not only exhibits much lower detection limit than most of previous methods, but also possesses the features of simple preparation, easy reproducibility and good biocompatibility. Due to the ultrastrong binding ability of H2S and Ag+, the alternating addition of Ag+ and H2S does not show an influence on the catalytic activity of Tb/G4-hemin DNAzyme, which endows the proposed sensor with the capability of reversible sensing. The successful application of the Tb/G4-hemin DNAzyme-Ag+ system in serum samples suggests that the proposed colorimetric sensor could be a potential biosensor platform for biological samples.
Acknowledgements This work was supported by the Natural Science Foundation of China (No. 21305054), Start-up Funding of Jiangxi Normal University, Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133604120002), and Natural Science Foundation of Jiangxi Province (No. 20151BAB203020).
References [1] R J Reiffenstein, a. W C Hulbert, S.H. Roth, Toxicology of hydrogen sulfide, Annu. Rev. Pharmacool. Toxicol., 32 (1992) 109-134. [2] L. Li, P. Rose, P.K. Moore, Hydrogen sulfide and cell signaling, Annu. Rev. Pharmacool. Toxicol., 51 (2011) 169-187. [3] H. Kimura, Hydrogen sulfide: its production, release and functions, Amino Acids, 41 (2010) 113-121. [4] W. Xuan, C. Sheng, Y. Cao, W. He, W. Wang, Fluorescent probes for the detection of hydrogen sulfide in biological systems, Angew. Chem. Int. Ed., 51 (2012) 2282-2284. [5] R.r. Hyšpler, A. Tichá, M. Indrová, Z. Zadák, L. Hyšplerová, J. Gasparič, J. Churáček, A simple, optimized method for the determination of sulphide in whole blood by GC-MS as a marker of bowel fermentation processes, J. Chromatogr. B, 770 (2002) 255-259. [6] G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A.K. Mustafa, W. Mu, S. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine γ-lyase, Science, 322 (2008) 587-590. [7] K. Abe, H. Kimura, The possible role of hydrogen sulfide as an endogenous neuromodulator, J. Neurosci,, 16 (1996) 1066-1071. [8] Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, L-cysteine inhibits insulin release from the 13
pancreatic β-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter, Diabetes, 55 (2006) 1391-1397. [9] J.L. Wallace, G. Caliendo, V. Santagada, G. Cirino, S. Fiorucci, Gastrointestinal safety and anti-inflammatory effects of a hydrogen sulfide-releasing diclofenac derivative in the rat, Gastroenterology, 132 (2007) 261-271. [10] D. Giuliani, A. Ottani, D. Zaffe, M. Galantucci, F. Strinati, R. Lodi, S. Guarini, Hydrogen sulfide slows down progression of experimental Alzheimer’s disease by targeting multiple pathophysiological mechanisms, Neurobiol. Learn. Mem., 104 (2013) 82-91. [11] P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi, B. Chadefaux-Vekemans, Endogenous hydrogen sulfide overproduction in Down syndrome, Am. J. Med. Genet. A, 116A (2003) 310-311. [12] W. Yang, G. Yang, X. Jia, L. Wu, R. Wang, Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms, J. Physiol., 569 (2005) 519-531. [13] A.R. Blanchette, A.D. Cooper, Determination of hydrogen sulfide and methyl mercaptan in mouth air at the parts-per-billion level by gas chromatography, Anal. Chem., 48 (1976) 729-731. [14] M. Colon, J.L. Todolí, M. Hidalgo, M. Iglesias, Development of novel and sensitive methods for the determination of sulfide in aqueous samples by hydrogen sulfide generation-inductively coupled plasma-atomic emission spectroscopy, Anal. Chim. Acta, 609 (2008) 160-168. [15] N.S. Lawrence, R.P. Deo, J. Wang, Electrochemical determination of hydrogen sulfide at carbon nanotube modified electrodes, Anal. Chim. Acta, 517 (2004) 131-137. [16] B. Liu, Y. Chen, Responsive lanthanide coordination polymer for hydrogen sulfide, Anal. Chem., 85 (2013) 11020-11025. [17] Q. Yu, K.Y. Zhang, H. Liang, Q. Zhao, T. Yang, S. Liu, C. Zhang, Z. Shi, W. Xu, W. Huang, Dual-emissive nanohybrid for ratiometric luminescence and lifetime imaging of intracellular hydrogen sulfide, ACS Appl. Mater. Interfaces, 7 (2015) 5462-5470. [18] A.R. Lippert, E.J. New, C.J. Chang, Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells, J. Am. Chem. Soc., 133 (2011) 10078-10080. [19] T. Liu, Z. Xu, D.R. Spring, J. Cui, A Lysosome-targetable fluorescent probe for imaging hydrogen sulfide in living cells, Org. Lett., 15 (2013) 2310-2313. [20] V.S. Lin, A.R. Lippert, C.J. Chang, Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production, Proc. Natl. Acad. Sci., 110 (2013) 7131-7135. [21] K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, Development of a highly selective fluorescence probe for hydrogen sulfide, J. Am. Chem. Soc., 133 (2011) 18003-18005. [22] Z. Yuan, F. Lu, M. Peng, C.-W. Wang, Y.-T. Tseng, Y. Du, N. Cai, C.-W. Lien, H.-T. Chang, Y. He, E.S. Yeung, Selective colorimetric detection of hydrogen sulfide based on primary amine-active ester cross-linking of gold nanoparticles, Anal. Chem., 87 (2015) 7267-7273. 14
[23] X. Zhang, W. Zhou, Z. Yuan, C. Lu, Colorimetric detection of biological hydrogen sulfide using fluorosurfactant functionalized gold nanorods, Analyst, 140 (2015) 7443-7450. [24] Z. Zhang, Z. Chen, S. Wang, C. Qu, L. Chen, On-site visual detection of hydrogen sulfide in air based on enhancing the stability of gold nanoparticles, ACS Appl. Mater. Interfaces, 6 (2014) 6300-6307. [25] B. Ruttkay-Nedecky, J. Kudr, L. Nejdl, D. Maskova, R. Kizek, V. Adam, G-Quadruplexes as Sensing Probes, Molecules, 18 (2013) 14760-14779. [26] M. Deng, D. Zhang, Y. Zhou, X. Zhou, Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme, J. Am. Chem. Soc., 130 (2008) 13095-13102. [27] J. Zhang, Q. Gao, P. Chen, J. Chen, G. Chen, F. Fu, A novel Tb 3+-promoted G-quadruplex-hemin DNAzyme for the development of label-free visual biosensors, Biosens. Bioelectron., 26 (2011) 4053-4057. [28] T. Li, E. Wang, S. Dong, G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin, Chem. Commun., 44 (2008) 3654-3656. [29] Y. Xiao, V. Pavlov, T. Niazov, A. Dishon, M. Kotler, I. Willner, Catalytic beacons for the detection of DNA and telomerase activity, J. Am. Chem. Soc., 126 (2004) 7430-7431. [30] T. Li, S. Dong, E. Wang, Label-free colorimetric detection of aqueous mercury ion (Hg2+) using Hg2+-modulated G-quadruplex-based DNAzymes, Anal. Chem., 81 (2009) 2144-2149. [31] H. Bo, C. Wang, Q. Gao, H. Qi, C. Zhang, Selective, colorimetric assay of glucose in urine using G-quadruplex-based DNAzymes and 10-acetyl-3,7-dihydroxy phenoxazine, Talanta, 108 (2013) 131-135. [32] X.-H. Zhou, D.-M. Kong, H.-X. Shen, Ag+ and cysteine quantitation based on G-quadruplex-hemin DNAzymes disruption by Ag+, Anal. Chem., 82 (2009) 789-793. [33] S.K. Das, C.S. Lim, S.Y. Yang, J.H. Han, B.R. Cho, A small molecule two-photon probe for hydrogen sulfide in live tissues, Chem. Commun., 48 (2012) 8395-8397. [34] T. Li, E. Wang, S. Dong, G-Quadruplex-based DNAzyme as a sensing platform for ultrasensitive colorimetric potassium detection, Chem. Commun., 0 (2009) 580-582. [35] J. Kosman, B. Juskowiak, Peroxidase-mimicking DNAzymes for biosensing applications: A review, Anal. Chim. Acta, 707 (2011) 7-17. [36] T. Li, S. Dong, E. Wang, A lead(II)-driven DNA molecular device for turn-on fluorescence detection of lead(II) ion with high selectivity and sensitivity, J. Am. Chem. Soc., 132 (2010) 13156-13157. [37] J. Speight, Lange's Handbook of Chemistry, 16th Edition ed., McGraw-Hill Education: New York, 2005.
15
Biographies Gonge Tang received her BS degree from Yichun University in 2014. She is currently a graduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Cailan Zhao is currently an undergraduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Jie Gao received her BS degree from Anyang Institute of Technology in 2015. She is currently a graduate student in the college of chemistry and chemical engineering at Jiangxi Normal University. Hongliang Tan received his Ph.D degree in Biomedical Engineering from Southeast University. He is currently an associate professor at Jiangxi Normal University. His research interests involve the design and development of fluorescent and colorimetric sensors for bioanalysis.
16
Figure captions
Figure 1. (A) Absorption spectra of ABTS-H2O2 solution at different conditions: the mixture of 0.5 μM G-rich DNA and 1.7 μM hemin (a), Tb/G4-hemin DNAzyme (b), 1.7 μM hemin (c), and blank control (d). Inset is the corresponding photographs of these four samples. (B) Time-dependent absorbance of ABTS-H2O2 solution in the presence of G4-hemin DNAzyme promoted by Tb3+, K+ and Na+. Figure 2. (a) Absorption spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme alone and after the addition of 5 μM Ag+, 2 μM H2S, and the mixture of 5 μM Ag+ and 2 μM H2S, respectively. Inset is the corresponding photographs of these samples. (b) CD spectra of Tb/G4-hemin DNAzyme in the absence and presence of 5 μM Ag+ and the mixture of 5 μM Ag+ and 2 μM H2S. Figure 3. Effects of various metal ions with a concentration of 5 μM (a) and Ag+ with different concentrations (b) on the absorbance of ABTS-H2O2 solution in presence of Tb/G4-hemin DNAzyme. Figure 4. Absorbance spectra of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ after adding of H2S with different concentrations (a) and the linear calibration plots of absorbance of ABTS·+ at 420 nm against H2S concentrations (b). Figure 5. Effects of various anions (2 μM) on the absorbance of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme and Ag+ at 420 nm. Inset is the corresponding photographs of these samples. Figure 6. Reversible absorption changes of ABTS-H2O2 solution in the presence of Tb/G4-hemin DNAzyme upon cyclic treatment with Ag+ and Cys. Scheme 1. Schematic illustration of Tb/G4-hemin DNAzyme as a peroxidase mimic for the detection of H2S.
17
Figure 1
18
Figure 2
19
Figure 3
20
Figure 4
21
Figure 5
22
Figure 6
23
Scheme 1
24
Table 1. Recovery of H2S added to serum samples. Spiked (μM)
Detected (μM)
Recovery (%)
RSD (n=5, %)
0.5
0.49 ± 0.01
98.0
2.33
1.5
1.51 ± 0.02
100.6
0.77
2
2.08 ± 0.07
104.2
2.91
25