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Thymine-based molecular beacon for sensing adenosine based on the inhibition of S-adenosylhomocysteine hydrolase activity
Biosensors and Bioelectronics 61 (2014) 404–409
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Thymine-based molecular beacon for sensing adenosine based on the inhibition of S-adenosylhomocysteine hydrolase activity Chih-Chun Nieh a, Wei-Lung Tseng a,b,n a b
Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan
art ic l e i nf o
a b s t r a c t
Article history: Received 12 March 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 21 May 2014
This study presents a thymine (T)-based molecular beacon (MB) used for probing S-adenosylhomocysteine hydrolase (SAHH)-catalyzed hydrolysis of S-adenosylhomocysteine (SAH) and for sensing adenosine based on the inhibition of SAHH activity. The designed MB (T8-MB-T8) contained a 15-mer loop and a stem that consisted of a pair of 8-mer T bases, a fluorophore unit at the 50 -end, and a quencher unit at the 30 -end. In the presence of Hg2 þ , a change in the conformation of T8-MB-T8 placed the fluorophore unit and the quencher in proximity to each other and caused collisional quenching of fluorescence between them. The Hg2 þ -induced fluorescence quenching of T8-MB-T8 occurred because the Hg2 þ induced T–T mismatches to form stable T–Hg2 þ –T coordination in the MB stem. SAHH catalyzed the hydrolysis of SAH to produce homocysteine. The generated homocysteine enabled the Hg2 þ to be removed from a hairpin-shaped T8-MB-T8 through the formation of a strong Hg2 þ –S bond, leading to the restoration of its fluorescence. The T8-MB-T8 Hg2 þ probe showed a limit of detection for SAHH of 4 units L 1 (approximately 0.24 nM) and was reusable for detecting the SAHH/SAH system. Because adenosine was an effective SAHH activity inhibitor, the T8-MB-T8 Hg2 þ probe combining the SAHH and SAH systems was used for sensitive and selective detection of adenosine in urine without the interference of other adenosine analogs. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular beacon S-adenosylhomocysteine Adenosine S-adenosylhomocysteine hydrolase Mercury ions
1. Introduction Researchers have proposed a non-Watson–Crick base pairingbased molecular beacon (MB) based on metal ion/small moleculemediated based pairs in the stem, including thymine–Hg2 þ –thymine (T–Hg2 þ –T) coordination (Lin et al., 2008; Wang et al., 2009; Yang et al., 2009), cytosine–Ag þ –cytosine (C–Ag þ –C) binding (Yang et al., 2010), adenosine2–coralyne–adenosine2 (A2–coralyne–A2) interaction (Lin and Tseng, 2012), and K þ –G-quadruplex complexes (Bourdoncle et al., 2006). These types of MBs not only discriminate perfectly matched DNA from single-base mismatched DNA at room temperature without requiring precise temperature control, but also prevent nonspecific binding of the MB to single-stranded DNAbinding proteins and endogenous nuclease degradation of the MB (Lin et al., 2008; Yang et al., 2009, 2010). In addition to the detection of target DNA, non-Waston–Crick base pairing-based MBs have been applied to the development of turn-on/off fluorescent sensors for detecting various analytes. A previous study presented an MB that
n Corresponding author at: Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan. Fax: þ886 7 3684046. E-mail address: [email protected] (W.-L. Tseng).
http://dx.doi.org/10.1016/j.bios.2014.05.031 0956-5663/& 2014 Elsevier B.V. All rights reserved.
consisted of a fluorophore moiety at the 30 -end, a quencher moiety at the 50 -ends, and a T-rich sequence in the stem. The presence of Hg2 þ induced these T–T mismatches to form stable T–Hg2 þ –T coordination in the stem, resulting in fluorescence quenching (Ono and Togashi, 2004). Thus, this MB achieved a switch-off fluorescence in response to Hg2 þ . Similarly, an MB stem containing a C- and A-rich sequence was used for selective detection of Ag þ and coralyne, respectively (Lin and Tseng, 2012; Ono et al., 2008). A T–Hg2 þ –Tbased MB was implemented to detect glutathione and homocysteine based on thiol-induced removal of Hg2 þ from T–Hg2 þ –T coordination, because biothiols can efficiently combine with Hg2 þ through the formation of a strong Hg2 þ –S bond (Li et al., 2013; Stobiecka et al., 2012; Xu and Hepel, 2011; Zhou et al., 2014). A C–Ag þ –C-based MB was designed to have an adenosine analog-binding aptamer in the loop and a short DNA sequence in the stem (Wang et al., 2010). The addition of adenosine triphosphate activated the fluorescence of this probe. Recently, another study devised an A2–coralyne–A2based MB for sensitive and selective detection of heparin based on the fact that electrostatic attraction between heparin and coralyne is much stronger than coordination between the MB stem and coralyne (Kuo and Tseng, 2013). Although the abovementioned MBs were shown to be useful in detecting a wide variety of analytes, no study has thus far reported the use of non-Waston–Crick base
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pairing-based MBs for investigating enzyme activity and sensing adenosine. S-adenosylhomocysteine hydrolase (SAHH) activity involves immunodeficiency disease and genetic disorder methionine metabolism (Baric et al., 2004), and its inhibitors medicate parasitic diseases, such as malaria and leishmaniasis (Bujnicki et al., 2003). Current methods used to detect SAHH activity include UV–vis absorption (Palmer and Abeles, 1979), Ellman's reagent (LozadaRamirez et al., 2006), gold nanoparticle-based sensors (Lin et al., 2010), and luciferase-based assays (Burgos et al., 2012). Although these methods provide high selectivity toward SAHH activity, they provide only moderate sensitivity with a detection limit in the micromolar range. Adenosine is the degraded product of intracellular adenosine triphosphate and is involved in the regulation of renal function, the release of rennin, and the control of coronary blood flow and cardiacarrhythmias (Vallon et al., 2006). Another source of adenosine generation originates from SAHH-mediated hydrolysis of S-adenosylhomocysteine (SAH), which is revisable to enable adenosine to act as a potent SAHH inhibitor (De Clercq, 1998). Typical methods for sensing adenosine have included include radioimmunoassay (Sato et al., 1982), voltammetry (Brajter-Toth et al., 2000; Swamy and Venton, 2007), high-performance liquid chromatography with ultraviolet absorption/mass spectrometry (Gayden et al., 1991; Ito et al., 2000; Krstulovic et al., 1977), and aptamer-based sensors (Liu and Lu 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). However, most of these methods have high equipment cost, sophisticated operation, poor selectivity, long analysis time, and/or safety risks. Responding to these disadvantages, a rapid, convenient, sensitive and selective MB probe for monitoring SAHH activity and sensing adenosine was developed in this study. An MB probe (5'T8 CCA GAT ACT CAC CGG T8-3') contains a pair of 8-mer T bases in the stem. The presence of Hg2 þ induced the coordination of T Hg2 þ T in the stem, switching off the MB fluorescence. Moreover, SAHH-catalyzed hydrolysis of SAH produced homocysteine, activating the fluorescence of a T Hg2 þ T-based MB through the competitive binding between thiol and the MB stem to Hg2 þ . This paper reports the use of a T Hg2 þ T-based MB to detect adenosine in urine to demonstrate its practicality.
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MO). All DNA samples were synthesized from Neogene Biomedicals Corporation (Taipei, Taiwan). Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA, USA). 2.2. Sensing of SAHH activity and its inhibition All samples were prepared in 20 mM phosphate buffer (pH 7.4). A T Hg2 þ T-based MB was prepared by mixing MB (10 nM, 50 μL) with Hg2 þ (10 1000 nM, 50 μL) at ambient temperature for 0 10 min. The reaction of SAH (0 50 μM, 100 μL) and SAHH (0 125 units/L, 100 μL) proceeded at 37 1C for 5 25 min. The mixed solution (200 μL) was incubated with a solution of a THg2 þ T-based MB (100 μL) at ambient temperature for 5 min. After the resulting solutions were adjusted to 500 μL with 20 mM phosphate buffer (pH 7.4), their fluorescence spectra were recorded by operating the fluorescence spectrophotometer (F-7000; Hitachi, Tokyo, Japan) at an excitation wavelength of 535 nm. In the study of SAHH inhibition, we initially mixed SAH (50 μM, 100 μL) and adenosine (0 333 μM, 100 μL). The resulting solution was incubated with SAHH (100 units L 1, 100 μL) at 37 1C for 20 min. The following steps were the same as those used in the study of SAHH activity. For detecting acetylcholinesterase activity and its inhibition, acetylcholinesterase (0–0.2 units mL 1) reacted with acetylthiocholine (0 2 μM, 100 μL) at 37 1C for 20 min. The following steps were the same as those used in the study of SAHH activity. 2.3. Analysis of adenosine in urine and serum Urine and serum samples were collected from a healthy adult male with the age of 23 years. A series of samples (100 μL) were spiked with standard solutions of adenosine (100 μL, 03 μM). A MonoSpin PBA column (GL. Science, Tokyo) was used to extract cis-hydroxyl group-containing compounds in urine or serum samples under the conditions provided by the manufacturer. The extracts (200 μL) containing 0.1 M HCl was neutralized with NaOH. The following steps were the same as those used in the study of SAHH inhibition. The concentration of creatinine in urine was measured by a TBA-200 FR automatic analyzer (Toshiba, Tokyo, Japan).
2. Experimental section
3. Results and discussion
2.1. Chemicals
3.1. Principle of operation
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride (NaCl), HgCl2, SAH, SAHH (rabbit erythrocytes; 50,000 units/L; MW 240,000), acetylthiocholine, acetylcholinesterase (Electrophorus electricus; 1256 units/mg protein), adenosine, adenosine monophosphate (AMP). Adenosine diphosphate (ADP), and adenosine triphosphate (ATP), homocysteine, H3PO4, NaH2PO4, Na2HPO4, and Na3PO4 were obtained from Sigma-Aldrich (St. Louis,
Fig. 1 illustrates the use of a DNA MB probe (T8-MB-T8) for monitoring SAHH activity and its inhibition. The T8-MB-T8 probe contains a 15-mer loop, a stem of a pair of 8-mer T bases, a reporter of 6-carboxy-20 ,40 ,40 ,50 ,7,70 -hexachlorofluorescein succinimidyl ester (HEX) at the 50 -end, and a quencher of 4-([4-(dimethylamino) phenyl]azo)-benzoic acid (DABCYL) at the 30 -end. In the absence of Hg2 þ , a solution of T8-MB-T8 exhibits weak fluorescence because
Fig. 1. Turn-on fluorescence sensing of the SAHH/SAH system based on the product homocysteine-induced removal of Hg2 þ from the stem of a T–Hg2 þ –T-based MB.
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HEX is separated far from DABCYL. The presence of Hg2 þ triggers these T–T mismatches to form stable T–Hg2 þ –T coordination in the MB stem, resulting in collision quenching between HEX and DABCYL. When SAHH catalyzes the hydrolysis of SAH, the produced homocysteine removes Hg2 þ from a T–Hg2 þ –T-based MB through the formation of strong Hg2 þ –S bond. Consequently, HEX and DABCYL units are distantly separated, leading to the restoration of HEX fluorescence. The original fluorescence signal of a T–Hg2 þ –Tbased MB can be regenerated by adding Hg2 þ to a solution containing T8-MB-T8 and the Hg2 þ (homocysteine)2 complex. Because adenosine is an efficient SAHH inhibitor, it can suppress the SAHH/SAH system-induced turn-on fluorescence of a hairpinshaped MB. 3.2. Sensing of SAHH activity To test our hypothesis, we implemented Hg2 þ to drive the quenching of T8-MB-T8 fluorescence and compared carboxyfluorescein (FAM) as a signal reporter with HEX. Because Hg2 þ complexes with aminothiols(Oram et al., 1996) and SAHH catalyzes the hydrolysis
of SAH to homocysteine and adenosine, the fluorescence of T8-MB-T8–Hg2þ complexes was monitored in the presence of different concentrations of homocysteine. The fluorescence intensity at 520 nm of the T8-MB-T8 Hg2þ probe, modified with FAM, gradually increased with increasing homocysteine concentrations (Fig. S1, Supplementary material). A similar result was observed when HEX was used in place of FAM (Fig. S2, Supplementary material). The limits of detection (LODs) at a signal-to-noise ratio of 3 for homocysteine obtained from FAM- and HEX-modified MB were estimated to be 60 and 10 nM, indicating that the use of HEX as a signal reporter can provide relatively high sensitivity toward homocysteine. Additionally, upon the addition of homocysteine to the T8-MB-T8 Hg2 þ probe, the relative standard deviations of the signal intensity of FAM and HEX were 3% and 1%, respectively. The T8-MB-T8 Hg2þ probe modified with HEX was chosen in terms of sensitivity and reproducibility. Homocysteine-induced fluorescence turn-on of the T8-MBT8 Hg2þ probe is suited to detect thiol product-related enzyme reactions, such as SAHH-induced hydrolysis of SAH, (Lin et al., 2010) glutathione reductase-catalyzed cleavage of glutathione disulfide, (Monostori et al., 2009) homocysteine thiolactonase-induced
Fig. 2. (A) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (a) before and (b–d) after addition of (b) 5 μM SAH, (c) 100 units/L SAHH, and (d) a mixture of 5 μM SAH and 100 units/L SAHH. Inset: time course measurement of fluorescence intensity (560 nm) of the T8-MB-T8 Hg2 þ probe (10 nM) upon the addition of a mixture of 5 μM SAH and 100 units/L SAHH. (B) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (10 nM) in the presence of increasing concentration of SAHH at a fixed concentration of 5 μM SAH. The arrow indicates the signal changes as increases in SAHH concentration (12.5, 25, 37.5, 50, 62.5, 75, 100, and 125 units/L). Inset: a plot of the value of (IF IF0)/IF0 value versus the SAHH concentration. IF0 and IF correspond to the fluorescence intensity at 560 nm of the T8-MB-T8 Hg2 þ probe in the absence and presence of the SAHH/SAH system, respectively. (C) Lineweaver–Burk plot for the hydrolysis reaction of 100 units/L SAHH with 1 30 μM SAH. (D) Reversible switching of the T8-MB-T8 Hg2 þ probe (10 nM) between the ON and OFF states through the alternating addition of (A) the SAHH/SAH system and 8 μM Hg2 þ (A D) a mixture of SAH and SAHH was incubated with the T8-MB-T8 Hg2 þ probe in 20 mM phosphate (pH 7.4) at 37 1C for 5 min. The error bars represent standard deviations based on three independent measurements.
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hydrolysis of homocysteine thiolactone, (Perla-Kajan and Jakubowski, 2012) and acetylcholinesterase-triggered hydrolysis of acetylthiocholine. (Miao et al., 2010) To test this hypothesis, the T8-MB-T8 Hg2 þ probe was utilized to follow the activity of SAHH and detect its inhibitor. Compared with the fluorescence spectrum of the T8-MBT8 Hg2 þ probe, a slight increase in the fluorescence of the T8-MBT8 Hg2 þ probe was observed upon the addition of 100 units L 1 SAHH (approximately 6 nM) or 5 μM SAH (Fig. 2A). When 5 μM SAH was incubated with 100 units L 1 SAHH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min, the generated homocysteine activated the fluorescence of the T8-MB-T8 Hg2 þ probe. This fluorescence turnon assay of SAHH was completed after 5 min (inset in Fig. 2A). The T8-MB-T8 Hg2þ probe is effective for optical detection of SAHH activity. As the concentration of SAHH increased at a fixed concentration of SAH, the fluorescence of the T8-MB-T8 Hg2 þ and the (IF IF0)/IF0 value was progressively enhanced (Fig. 2B). The variables IF0 and IF represent the fluorescence intensity at 560 nm of HEX as measured by the T8-MB-T8 Hg2 þ probe in the absence and presence of the SAHH/SAH system, respectively. The linear relationship (R2 ¼ 0.9920) of the (IF IF0)/IF0 value versus the SAHH concentration was from 12.5 to 125 units L 1 (inset in Fig. 2B). This probe enabled the detection of SAHH with LOD corresponding to 4 units L 1 (approximately 0.24 nM), which is lower than the LOD values measured from UV–vis absorption, (Palmer and Abeles, 1979) Ellman0 s reagent, (Lozada-Ramirez et al., 2006) fluorosurfactant-capped gold nanoparticles, (Lin et al., 2010) and luciferase-based assay. (Burgos et al., 2012) More importantly, this high-sensitivity probe has great potential to detect SAHH activity in red blood cells. (Carlucci et al., 2003) The hydrolytic activity of SAHH was determined by varying the SAH concentrations in the presence of fixed SAHH concentration. The fluorescence of the T8-MB-T8 Hg2þ probe and the value of (IF IF0)/ IF0 was gradually increased with increasing SAH concentration (Fig. S3, Supplementary material). The LOD of SAH was estimated to be 100 nM. According to the information in Fig. S3 (Supplementary material) and the data fitting to the Michaelis–Menten equation, the Michaelis constant (Km) was determined to be 36 μM (Fig. 2C), which was in agreement with that (0.75 60 μM) observed in previous research.(Ueland, 1982) These results signify that the fluorescence turn-on of the T8-MB-T8 Hg2 þ probe originates from the SAHH/SAHgenerated homocysteine; therefore, this probe can be used to perform the quantitative assay of SAHH and SAH. Fig. 2D indicates that the alternative addition of Hg2 þ and the SAHH/SAH system allowed the fluorescence intensity at 560 nm of the T8-MB-T8 probe to alternate between ON and OFF states. This result suggests that the T8-MBT8 Hg2 þ probe can be reused to detect SAHH and SAH. The successful probing of the SAHH activity by the T8-MB-T8 Hg2þ probe suggested it can be implemented to detect other thiol product-related enzyme systems. For example, acetylcholinesterase catalyzes the hydrolysis of acetylthiocholine to produce thiocholine and acetate. As the concentration of acetylcholinesterase increased at a fixed concentration of acetylthiocholine, the fluorescence of the T8-MB-T8 Hg2 þ probe was gradually intensified as a result of an increase in the thiocholine concentration (Fig. S4, Supplementary material). A linear calibration curve ranging from 2 to 40 units L 1 was shown in the inset of Fig. S4 (Supplementary material). Acetylcholinesterase was detected at a sensitivity as low as 2 units L 1 (approximately 4 nM). 3.3. Sensing of adensoine In addition to monitoring SAHH activity, this probe can be used for selective detection of adenosine because adenosine efficiently inhibited the catalytic reaction of SAHH and SAH (Fig. 3).(Burgos et al., 2012; Lin et al., 2010) After adding a mixture of 0 333 μM and 20 μM SAH to an SAHH solution of 100 unit L 1, the fluorescence intensity at 560 nm of the T8-MB-T8 Hg2 þ probe and the value of (IF1 IF2)/IF1 gradually diminished with an increase in
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Fig. 3. Illustration of the mechanism of the T8-MB-T8 Hg2 þ probe for sensing adenosine based on adenosine-induced inhibition of SAHH activity.
adenosine concentration (Fig. 4A). IF1 and IF2 correspond to the fluorescence intensity at 560 nm of HEX, as obtained from the addition of the T8-MB-T8 Hg2 þ probe to the SAHH/SAH system in the absence and presence of adenosine, respectively. By plotting the (IF1 IF2)/IF1 value against the adenosine concentration, a linear range (R2 ¼0.9947) for quantification of adenosine was observed from 670 to 3330 nM. The LOD of adenosine was determined to be 200 nM. The sensitivity of the T8-MB-T8 Hg2 þ probe combined with the SAHH/SAH system is superior to that of aptamer-based fluorescent and colorimetric detection (Liu and Lu, 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). Additionally, Fig. 4B indicates that the proposed probe can discriminate adenosine from adenosine analogs, including AMP, ADP, and ATP. However, aptamer-based sensors are incapable of sensing adenosine in a mixture of adenosine analogs because aptamer recognizes adenine and ribose moieties, rather than phosphate moiety (Liu and Lu, 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). The feasibility of the T8-MB-T8 Hg2þ probe coupled to the SAHH/ SAH system was validated by determining the adenosine concentration in urine. Urine samples spiked with 0 3330 nM adenosine were treated using a phenylboronic acid-containing MonoSpin PBA spin column. This column selectively captured a series of cis-hydroxyl group-containing compounds through the formation of boronate ester bonds, such as saccharides, catecholamines, and adenosine analogs (Kitahara et al., 2009; Ren et al., 2009). After the obtained extracts mixed with SAH were incubated with SAHH for 20 min, a progressive decrease in the fluorescence of the T8-MB-T8 Hg2þ probe was observed, as shown in Fig. 5. A linear correlation (R2 ¼0.9947) was obtained between the (IF1 IF2)/IF1 value versus the spiked concentration of adenosine (inset in Fig. 5). The slope of the calibration curve obtained from the spiked sample differs from obtained from that obtained from the standard sample, suggesting that a standard addition method was used for quantifying adenosine in urine. The concentration of creatinine in urine samples was observed to be 5.1 mM. By applying a standard addition method, the concentration of adenosine in urine was determined to be 1.270.1 μmol/mmol creatinine (n¼3), which is consistent with a previously reported adenosine concentration in urine (0.18 4.70 μmol/mmol creatinine) (Jiang and Ma, 2009). Also, the proposed method was used for the determination of adenosine in serum. Fig. S5 (Supplementary material) shows that the quantification of adenosine was successfully achieved by combining the T8-MB-T8 Hg2 þ probe, the SAHH/SAH system, and a MonoSpin PBA spin column, suggesting that the proposed method is well suited for routine adenosine assays in clinical studies.
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Fig. 4. (A) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (10 nM) after the addition of 0 333 μM of adenosine at fixed concentrations of 5 μM SAH and 100 units/L SAHH. The arrow indicates the signal changes as increases in adenosine concentration (0, 0.67, 1.3, 2, 2.7, 3.3, 6.7, 20, 27, 33, 67, 133, 200, 266, and 333 μM). Inset: a plot of the (IF1 IF2)/IF1 value versus the adenosine concentration. IF1 and IF2 represent the fluorescence intensity at 560 nm obtained from the addition of the T8-MB-T8 Hg2 þ probe to the SAHH/SAH system without and with adenosine, respectively. (B) The (IF1 IF2)/IF1 value of the T8-MB-T8 Hg2 þ probe (10 nM) after the addition of adenosine, AMP, ADP, and ATP. (A, B) SAHH reacted with a mixture of adenosine and SAH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min. The error bars represent standard deviations based on three independent measurements.
probe combined with the SAHH/SAH system can discriminate adenosine from adenosine analogs and its selectivity toward adenosine is superior to that of an aptamer-based sensor; the LOD of adenosine was 200 nM. These findings facilitate the implementation of MB as an optical probe to screen other thiol product-related enzyme systems. Because H2O2 can rapidly oxidize thiols to disufides in the presence of iodide (Wang et al., 2013), we suggest that the homocysteine-stimulated fluorescence turn-on of the T8-MB-T8 Hg2 þ probe can be implemented as a platform for the optical detection of oxidase/substrate-generated H2O2.
Acknowledgment We would like to thank National Science Council Taiwan (NSC 100-2628-M-110-001-MY 4) for the financial support of this study. Fig. 5. Fluorescence sensing of adenosine in urine by the combination of the T– Hg2 þ –T based MB probe, the SAHH/SAH system, and the phenylboronic acidcontaining spin column. The arrow indicates the signal changes as increases in the spiked concentration of adenosine (0, 0.67, 1.3, 2, 2.7, and 3.3 nM). Inset: a plot of the (IF1 IF2)/IF1 value versus the spiked concentration of adenosine. SAHH reacted with a mixture of adenosine and SAH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min. The error bars represent standard deviations based on three independent measurements.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.05.031. References
4. Conclusions We demonstrated that the T8-MB-T8 Hg2 þ probe was sensitive, selective, and reusable to probe thiol product-related enzyme system through thiol-induced removal of Hg2 þ from the probe. This was exemplified by the analysis of SAHH-mediated hydrolysis of SAH and acetylcholinesterase-triggered hydrolysis of acetylthiocholine; the LOD of SAHH and acetylcholinesterase was 4 units L 1 (approximately 0.24 nM) and 0.6 units L 1 (approximately 1 nM). However, thiol-containing compounds, such as glutathione and cysteine, can interfere with the detection of enzyme activity in biological fluids. To solve this problem, iodoacetamide can be used for blocking thiol group through alkylation prior to the analysis of enzyme activity (Faccenda et al., 2010). Because adenosine is highly effective at inhibiting SAHH activity, the T8-MB-T8 Hg2 þ
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Thymine-based molecular beacon for sensing adenosine based on the inhibition of S-adenosylhomocysteine hydrolase activity Chih-Chun Nieh a, Wei-Lung Tseng a,b,n a b
Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan
art ic l e i nf o
a b s t r a c t
Article history: Received 12 March 2014 Received in revised form 12 May 2014 Accepted 13 May 2014 Available online 21 May 2014
This study presents a thymine (T)-based molecular beacon (MB) used for probing S-adenosylhomocysteine hydrolase (SAHH)-catalyzed hydrolysis of S-adenosylhomocysteine (SAH) and for sensing adenosine based on the inhibition of SAHH activity. The designed MB (T8-MB-T8) contained a 15-mer loop and a stem that consisted of a pair of 8-mer T bases, a fluorophore unit at the 50 -end, and a quencher unit at the 30 -end. In the presence of Hg2 þ , a change in the conformation of T8-MB-T8 placed the fluorophore unit and the quencher in proximity to each other and caused collisional quenching of fluorescence between them. The Hg2 þ -induced fluorescence quenching of T8-MB-T8 occurred because the Hg2 þ induced T–T mismatches to form stable T–Hg2 þ –T coordination in the MB stem. SAHH catalyzed the hydrolysis of SAH to produce homocysteine. The generated homocysteine enabled the Hg2 þ to be removed from a hairpin-shaped T8-MB-T8 through the formation of a strong Hg2 þ –S bond, leading to the restoration of its fluorescence. The T8-MB-T8 Hg2 þ probe showed a limit of detection for SAHH of 4 units L 1 (approximately 0.24 nM) and was reusable for detecting the SAHH/SAH system. Because adenosine was an effective SAHH activity inhibitor, the T8-MB-T8 Hg2 þ probe combining the SAHH and SAH systems was used for sensitive and selective detection of adenosine in urine without the interference of other adenosine analogs. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular beacon S-adenosylhomocysteine Adenosine S-adenosylhomocysteine hydrolase Mercury ions
1. Introduction Researchers have proposed a non-Watson–Crick base pairingbased molecular beacon (MB) based on metal ion/small moleculemediated based pairs in the stem, including thymine–Hg2 þ –thymine (T–Hg2 þ –T) coordination (Lin et al., 2008; Wang et al., 2009; Yang et al., 2009), cytosine–Ag þ –cytosine (C–Ag þ –C) binding (Yang et al., 2010), adenosine2–coralyne–adenosine2 (A2–coralyne–A2) interaction (Lin and Tseng, 2012), and K þ –G-quadruplex complexes (Bourdoncle et al., 2006). These types of MBs not only discriminate perfectly matched DNA from single-base mismatched DNA at room temperature without requiring precise temperature control, but also prevent nonspecific binding of the MB to single-stranded DNAbinding proteins and endogenous nuclease degradation of the MB (Lin et al., 2008; Yang et al., 2009, 2010). In addition to the detection of target DNA, non-Waston–Crick base pairing-based MBs have been applied to the development of turn-on/off fluorescent sensors for detecting various analytes. A previous study presented an MB that
n Corresponding author at: Department of Chemistry, National Sun Yat-sen University, 70, Lien-hai Road, Kaohsiung, Taiwan. Fax: þ886 7 3684046. E-mail address: [email protected] (W.-L. Tseng).
http://dx.doi.org/10.1016/j.bios.2014.05.031 0956-5663/& 2014 Elsevier B.V. All rights reserved.
consisted of a fluorophore moiety at the 30 -end, a quencher moiety at the 50 -ends, and a T-rich sequence in the stem. The presence of Hg2 þ induced these T–T mismatches to form stable T–Hg2 þ –T coordination in the stem, resulting in fluorescence quenching (Ono and Togashi, 2004). Thus, this MB achieved a switch-off fluorescence in response to Hg2 þ . Similarly, an MB stem containing a C- and A-rich sequence was used for selective detection of Ag þ and coralyne, respectively (Lin and Tseng, 2012; Ono et al., 2008). A T–Hg2 þ –Tbased MB was implemented to detect glutathione and homocysteine based on thiol-induced removal of Hg2 þ from T–Hg2 þ –T coordination, because biothiols can efficiently combine with Hg2 þ through the formation of a strong Hg2 þ –S bond (Li et al., 2013; Stobiecka et al., 2012; Xu and Hepel, 2011; Zhou et al., 2014). A C–Ag þ –C-based MB was designed to have an adenosine analog-binding aptamer in the loop and a short DNA sequence in the stem (Wang et al., 2010). The addition of adenosine triphosphate activated the fluorescence of this probe. Recently, another study devised an A2–coralyne–A2based MB for sensitive and selective detection of heparin based on the fact that electrostatic attraction between heparin and coralyne is much stronger than coordination between the MB stem and coralyne (Kuo and Tseng, 2013). Although the abovementioned MBs were shown to be useful in detecting a wide variety of analytes, no study has thus far reported the use of non-Waston–Crick base
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pairing-based MBs for investigating enzyme activity and sensing adenosine. S-adenosylhomocysteine hydrolase (SAHH) activity involves immunodeficiency disease and genetic disorder methionine metabolism (Baric et al., 2004), and its inhibitors medicate parasitic diseases, such as malaria and leishmaniasis (Bujnicki et al., 2003). Current methods used to detect SAHH activity include UV–vis absorption (Palmer and Abeles, 1979), Ellman's reagent (LozadaRamirez et al., 2006), gold nanoparticle-based sensors (Lin et al., 2010), and luciferase-based assays (Burgos et al., 2012). Although these methods provide high selectivity toward SAHH activity, they provide only moderate sensitivity with a detection limit in the micromolar range. Adenosine is the degraded product of intracellular adenosine triphosphate and is involved in the regulation of renal function, the release of rennin, and the control of coronary blood flow and cardiacarrhythmias (Vallon et al., 2006). Another source of adenosine generation originates from SAHH-mediated hydrolysis of S-adenosylhomocysteine (SAH), which is revisable to enable adenosine to act as a potent SAHH inhibitor (De Clercq, 1998). Typical methods for sensing adenosine have included include radioimmunoassay (Sato et al., 1982), voltammetry (Brajter-Toth et al., 2000; Swamy and Venton, 2007), high-performance liquid chromatography with ultraviolet absorption/mass spectrometry (Gayden et al., 1991; Ito et al., 2000; Krstulovic et al., 1977), and aptamer-based sensors (Liu and Lu 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). However, most of these methods have high equipment cost, sophisticated operation, poor selectivity, long analysis time, and/or safety risks. Responding to these disadvantages, a rapid, convenient, sensitive and selective MB probe for monitoring SAHH activity and sensing adenosine was developed in this study. An MB probe (5'T8 CCA GAT ACT CAC CGG T8-3') contains a pair of 8-mer T bases in the stem. The presence of Hg2 þ induced the coordination of T Hg2 þ T in the stem, switching off the MB fluorescence. Moreover, SAHH-catalyzed hydrolysis of SAH produced homocysteine, activating the fluorescence of a T Hg2 þ T-based MB through the competitive binding between thiol and the MB stem to Hg2 þ . This paper reports the use of a T Hg2 þ T-based MB to detect adenosine in urine to demonstrate its practicality.
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MO). All DNA samples were synthesized from Neogene Biomedicals Corporation (Taipei, Taiwan). Water used in all experiments was doubly distilled and purified by a Milli-Q system (Millipore, Milford, MA, USA). 2.2. Sensing of SAHH activity and its inhibition All samples were prepared in 20 mM phosphate buffer (pH 7.4). A T Hg2 þ T-based MB was prepared by mixing MB (10 nM, 50 μL) with Hg2 þ (10 1000 nM, 50 μL) at ambient temperature for 0 10 min. The reaction of SAH (0 50 μM, 100 μL) and SAHH (0 125 units/L, 100 μL) proceeded at 37 1C for 5 25 min. The mixed solution (200 μL) was incubated with a solution of a THg2 þ T-based MB (100 μL) at ambient temperature for 5 min. After the resulting solutions were adjusted to 500 μL with 20 mM phosphate buffer (pH 7.4), their fluorescence spectra were recorded by operating the fluorescence spectrophotometer (F-7000; Hitachi, Tokyo, Japan) at an excitation wavelength of 535 nm. In the study of SAHH inhibition, we initially mixed SAH (50 μM, 100 μL) and adenosine (0 333 μM, 100 μL). The resulting solution was incubated with SAHH (100 units L 1, 100 μL) at 37 1C for 20 min. The following steps were the same as those used in the study of SAHH activity. For detecting acetylcholinesterase activity and its inhibition, acetylcholinesterase (0–0.2 units mL 1) reacted with acetylthiocholine (0 2 μM, 100 μL) at 37 1C for 20 min. The following steps were the same as those used in the study of SAHH activity. 2.3. Analysis of adenosine in urine and serum Urine and serum samples were collected from a healthy adult male with the age of 23 years. A series of samples (100 μL) were spiked with standard solutions of adenosine (100 μL, 03 μM). A MonoSpin PBA column (GL. Science, Tokyo) was used to extract cis-hydroxyl group-containing compounds in urine or serum samples under the conditions provided by the manufacturer. The extracts (200 μL) containing 0.1 M HCl was neutralized with NaOH. The following steps were the same as those used in the study of SAHH inhibition. The concentration of creatinine in urine was measured by a TBA-200 FR automatic analyzer (Toshiba, Tokyo, Japan).
2. Experimental section
3. Results and discussion
2.1. Chemicals
3.1. Principle of operation
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride (NaCl), HgCl2, SAH, SAHH (rabbit erythrocytes; 50,000 units/L; MW 240,000), acetylthiocholine, acetylcholinesterase (Electrophorus electricus; 1256 units/mg protein), adenosine, adenosine monophosphate (AMP). Adenosine diphosphate (ADP), and adenosine triphosphate (ATP), homocysteine, H3PO4, NaH2PO4, Na2HPO4, and Na3PO4 were obtained from Sigma-Aldrich (St. Louis,
Fig. 1 illustrates the use of a DNA MB probe (T8-MB-T8) for monitoring SAHH activity and its inhibition. The T8-MB-T8 probe contains a 15-mer loop, a stem of a pair of 8-mer T bases, a reporter of 6-carboxy-20 ,40 ,40 ,50 ,7,70 -hexachlorofluorescein succinimidyl ester (HEX) at the 50 -end, and a quencher of 4-([4-(dimethylamino) phenyl]azo)-benzoic acid (DABCYL) at the 30 -end. In the absence of Hg2 þ , a solution of T8-MB-T8 exhibits weak fluorescence because
Fig. 1. Turn-on fluorescence sensing of the SAHH/SAH system based on the product homocysteine-induced removal of Hg2 þ from the stem of a T–Hg2 þ –T-based MB.
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HEX is separated far from DABCYL. The presence of Hg2 þ triggers these T–T mismatches to form stable T–Hg2 þ –T coordination in the MB stem, resulting in collision quenching between HEX and DABCYL. When SAHH catalyzes the hydrolysis of SAH, the produced homocysteine removes Hg2 þ from a T–Hg2 þ –T-based MB through the formation of strong Hg2 þ –S bond. Consequently, HEX and DABCYL units are distantly separated, leading to the restoration of HEX fluorescence. The original fluorescence signal of a T–Hg2 þ –Tbased MB can be regenerated by adding Hg2 þ to a solution containing T8-MB-T8 and the Hg2 þ (homocysteine)2 complex. Because adenosine is an efficient SAHH inhibitor, it can suppress the SAHH/SAH system-induced turn-on fluorescence of a hairpinshaped MB. 3.2. Sensing of SAHH activity To test our hypothesis, we implemented Hg2 þ to drive the quenching of T8-MB-T8 fluorescence and compared carboxyfluorescein (FAM) as a signal reporter with HEX. Because Hg2 þ complexes with aminothiols(Oram et al., 1996) and SAHH catalyzes the hydrolysis
of SAH to homocysteine and adenosine, the fluorescence of T8-MB-T8–Hg2þ complexes was monitored in the presence of different concentrations of homocysteine. The fluorescence intensity at 520 nm of the T8-MB-T8 Hg2þ probe, modified with FAM, gradually increased with increasing homocysteine concentrations (Fig. S1, Supplementary material). A similar result was observed when HEX was used in place of FAM (Fig. S2, Supplementary material). The limits of detection (LODs) at a signal-to-noise ratio of 3 for homocysteine obtained from FAM- and HEX-modified MB were estimated to be 60 and 10 nM, indicating that the use of HEX as a signal reporter can provide relatively high sensitivity toward homocysteine. Additionally, upon the addition of homocysteine to the T8-MB-T8 Hg2 þ probe, the relative standard deviations of the signal intensity of FAM and HEX were 3% and 1%, respectively. The T8-MB-T8 Hg2þ probe modified with HEX was chosen in terms of sensitivity and reproducibility. Homocysteine-induced fluorescence turn-on of the T8-MBT8 Hg2þ probe is suited to detect thiol product-related enzyme reactions, such as SAHH-induced hydrolysis of SAH, (Lin et al., 2010) glutathione reductase-catalyzed cleavage of glutathione disulfide, (Monostori et al., 2009) homocysteine thiolactonase-induced
Fig. 2. (A) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (a) before and (b–d) after addition of (b) 5 μM SAH, (c) 100 units/L SAHH, and (d) a mixture of 5 μM SAH and 100 units/L SAHH. Inset: time course measurement of fluorescence intensity (560 nm) of the T8-MB-T8 Hg2 þ probe (10 nM) upon the addition of a mixture of 5 μM SAH and 100 units/L SAHH. (B) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (10 nM) in the presence of increasing concentration of SAHH at a fixed concentration of 5 μM SAH. The arrow indicates the signal changes as increases in SAHH concentration (12.5, 25, 37.5, 50, 62.5, 75, 100, and 125 units/L). Inset: a plot of the value of (IF IF0)/IF0 value versus the SAHH concentration. IF0 and IF correspond to the fluorescence intensity at 560 nm of the T8-MB-T8 Hg2 þ probe in the absence and presence of the SAHH/SAH system, respectively. (C) Lineweaver–Burk plot for the hydrolysis reaction of 100 units/L SAHH with 1 30 μM SAH. (D) Reversible switching of the T8-MB-T8 Hg2 þ probe (10 nM) between the ON and OFF states through the alternating addition of (A) the SAHH/SAH system and 8 μM Hg2 þ (A D) a mixture of SAH and SAHH was incubated with the T8-MB-T8 Hg2 þ probe in 20 mM phosphate (pH 7.4) at 37 1C for 5 min. The error bars represent standard deviations based on three independent measurements.
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hydrolysis of homocysteine thiolactone, (Perla-Kajan and Jakubowski, 2012) and acetylcholinesterase-triggered hydrolysis of acetylthiocholine. (Miao et al., 2010) To test this hypothesis, the T8-MB-T8 Hg2 þ probe was utilized to follow the activity of SAHH and detect its inhibitor. Compared with the fluorescence spectrum of the T8-MBT8 Hg2 þ probe, a slight increase in the fluorescence of the T8-MBT8 Hg2 þ probe was observed upon the addition of 100 units L 1 SAHH (approximately 6 nM) or 5 μM SAH (Fig. 2A). When 5 μM SAH was incubated with 100 units L 1 SAHH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min, the generated homocysteine activated the fluorescence of the T8-MB-T8 Hg2 þ probe. This fluorescence turnon assay of SAHH was completed after 5 min (inset in Fig. 2A). The T8-MB-T8 Hg2þ probe is effective for optical detection of SAHH activity. As the concentration of SAHH increased at a fixed concentration of SAH, the fluorescence of the T8-MB-T8 Hg2 þ and the (IF IF0)/IF0 value was progressively enhanced (Fig. 2B). The variables IF0 and IF represent the fluorescence intensity at 560 nm of HEX as measured by the T8-MB-T8 Hg2 þ probe in the absence and presence of the SAHH/SAH system, respectively. The linear relationship (R2 ¼ 0.9920) of the (IF IF0)/IF0 value versus the SAHH concentration was from 12.5 to 125 units L 1 (inset in Fig. 2B). This probe enabled the detection of SAHH with LOD corresponding to 4 units L 1 (approximately 0.24 nM), which is lower than the LOD values measured from UV–vis absorption, (Palmer and Abeles, 1979) Ellman0 s reagent, (Lozada-Ramirez et al., 2006) fluorosurfactant-capped gold nanoparticles, (Lin et al., 2010) and luciferase-based assay. (Burgos et al., 2012) More importantly, this high-sensitivity probe has great potential to detect SAHH activity in red blood cells. (Carlucci et al., 2003) The hydrolytic activity of SAHH was determined by varying the SAH concentrations in the presence of fixed SAHH concentration. The fluorescence of the T8-MB-T8 Hg2þ probe and the value of (IF IF0)/ IF0 was gradually increased with increasing SAH concentration (Fig. S3, Supplementary material). The LOD of SAH was estimated to be 100 nM. According to the information in Fig. S3 (Supplementary material) and the data fitting to the Michaelis–Menten equation, the Michaelis constant (Km) was determined to be 36 μM (Fig. 2C), which was in agreement with that (0.75 60 μM) observed in previous research.(Ueland, 1982) These results signify that the fluorescence turn-on of the T8-MB-T8 Hg2 þ probe originates from the SAHH/SAHgenerated homocysteine; therefore, this probe can be used to perform the quantitative assay of SAHH and SAH. Fig. 2D indicates that the alternative addition of Hg2 þ and the SAHH/SAH system allowed the fluorescence intensity at 560 nm of the T8-MB-T8 probe to alternate between ON and OFF states. This result suggests that the T8-MBT8 Hg2 þ probe can be reused to detect SAHH and SAH. The successful probing of the SAHH activity by the T8-MB-T8 Hg2þ probe suggested it can be implemented to detect other thiol product-related enzyme systems. For example, acetylcholinesterase catalyzes the hydrolysis of acetylthiocholine to produce thiocholine and acetate. As the concentration of acetylcholinesterase increased at a fixed concentration of acetylthiocholine, the fluorescence of the T8-MB-T8 Hg2 þ probe was gradually intensified as a result of an increase in the thiocholine concentration (Fig. S4, Supplementary material). A linear calibration curve ranging from 2 to 40 units L 1 was shown in the inset of Fig. S4 (Supplementary material). Acetylcholinesterase was detected at a sensitivity as low as 2 units L 1 (approximately 4 nM). 3.3. Sensing of adensoine In addition to monitoring SAHH activity, this probe can be used for selective detection of adenosine because adenosine efficiently inhibited the catalytic reaction of SAHH and SAH (Fig. 3).(Burgos et al., 2012; Lin et al., 2010) After adding a mixture of 0 333 μM and 20 μM SAH to an SAHH solution of 100 unit L 1, the fluorescence intensity at 560 nm of the T8-MB-T8 Hg2 þ probe and the value of (IF1 IF2)/IF1 gradually diminished with an increase in
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Fig. 3. Illustration of the mechanism of the T8-MB-T8 Hg2 þ probe for sensing adenosine based on adenosine-induced inhibition of SAHH activity.
adenosine concentration (Fig. 4A). IF1 and IF2 correspond to the fluorescence intensity at 560 nm of HEX, as obtained from the addition of the T8-MB-T8 Hg2 þ probe to the SAHH/SAH system in the absence and presence of adenosine, respectively. By plotting the (IF1 IF2)/IF1 value against the adenosine concentration, a linear range (R2 ¼0.9947) for quantification of adenosine was observed from 670 to 3330 nM. The LOD of adenosine was determined to be 200 nM. The sensitivity of the T8-MB-T8 Hg2 þ probe combined with the SAHH/SAH system is superior to that of aptamer-based fluorescent and colorimetric detection (Liu and Lu, 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). Additionally, Fig. 4B indicates that the proposed probe can discriminate adenosine from adenosine analogs, including AMP, ADP, and ATP. However, aptamer-based sensors are incapable of sensing adenosine in a mixture of adenosine analogs because aptamer recognizes adenine and ribose moieties, rather than phosphate moiety (Liu and Lu, 2005, 2006a, 2006b; Song et al., 2012; Xiang et al., 2009; Xu and Lu, 2010; Zhao et al., 2007). The feasibility of the T8-MB-T8 Hg2þ probe coupled to the SAHH/ SAH system was validated by determining the adenosine concentration in urine. Urine samples spiked with 0 3330 nM adenosine were treated using a phenylboronic acid-containing MonoSpin PBA spin column. This column selectively captured a series of cis-hydroxyl group-containing compounds through the formation of boronate ester bonds, such as saccharides, catecholamines, and adenosine analogs (Kitahara et al., 2009; Ren et al., 2009). After the obtained extracts mixed with SAH were incubated with SAHH for 20 min, a progressive decrease in the fluorescence of the T8-MB-T8 Hg2þ probe was observed, as shown in Fig. 5. A linear correlation (R2 ¼0.9947) was obtained between the (IF1 IF2)/IF1 value versus the spiked concentration of adenosine (inset in Fig. 5). The slope of the calibration curve obtained from the spiked sample differs from obtained from that obtained from the standard sample, suggesting that a standard addition method was used for quantifying adenosine in urine. The concentration of creatinine in urine samples was observed to be 5.1 mM. By applying a standard addition method, the concentration of adenosine in urine was determined to be 1.270.1 μmol/mmol creatinine (n¼3), which is consistent with a previously reported adenosine concentration in urine (0.18 4.70 μmol/mmol creatinine) (Jiang and Ma, 2009). Also, the proposed method was used for the determination of adenosine in serum. Fig. S5 (Supplementary material) shows that the quantification of adenosine was successfully achieved by combining the T8-MB-T8 Hg2 þ probe, the SAHH/SAH system, and a MonoSpin PBA spin column, suggesting that the proposed method is well suited for routine adenosine assays in clinical studies.
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Fig. 4. (A) Fluorescence spectra of the T8-MB-T8 Hg2 þ probe (10 nM) after the addition of 0 333 μM of adenosine at fixed concentrations of 5 μM SAH and 100 units/L SAHH. The arrow indicates the signal changes as increases in adenosine concentration (0, 0.67, 1.3, 2, 2.7, 3.3, 6.7, 20, 27, 33, 67, 133, 200, 266, and 333 μM). Inset: a plot of the (IF1 IF2)/IF1 value versus the adenosine concentration. IF1 and IF2 represent the fluorescence intensity at 560 nm obtained from the addition of the T8-MB-T8 Hg2 þ probe to the SAHH/SAH system without and with adenosine, respectively. (B) The (IF1 IF2)/IF1 value of the T8-MB-T8 Hg2 þ probe (10 nM) after the addition of adenosine, AMP, ADP, and ATP. (A, B) SAHH reacted with a mixture of adenosine and SAH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min. The error bars represent standard deviations based on three independent measurements.
probe combined with the SAHH/SAH system can discriminate adenosine from adenosine analogs and its selectivity toward adenosine is superior to that of an aptamer-based sensor; the LOD of adenosine was 200 nM. These findings facilitate the implementation of MB as an optical probe to screen other thiol product-related enzyme systems. Because H2O2 can rapidly oxidize thiols to disufides in the presence of iodide (Wang et al., 2013), we suggest that the homocysteine-stimulated fluorescence turn-on of the T8-MB-T8 Hg2 þ probe can be implemented as a platform for the optical detection of oxidase/substrate-generated H2O2.
Acknowledgment We would like to thank National Science Council Taiwan (NSC 100-2628-M-110-001-MY 4) for the financial support of this study. Fig. 5. Fluorescence sensing of adenosine in urine by the combination of the T– Hg2 þ –T based MB probe, the SAHH/SAH system, and the phenylboronic acidcontaining spin column. The arrow indicates the signal changes as increases in the spiked concentration of adenosine (0, 0.67, 1.3, 2, 2.7, and 3.3 nM). Inset: a plot of the (IF1 IF2)/IF1 value versus the spiked concentration of adenosine. SAHH reacted with a mixture of adenosine and SAH in 20 mM phosphate (pH 7.4) at 37 1C for 20 min. The error bars represent standard deviations based on three independent measurements.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.05.031. References
4. Conclusions We demonstrated that the T8-MB-T8 Hg2 þ probe was sensitive, selective, and reusable to probe thiol product-related enzyme system through thiol-induced removal of Hg2 þ from the probe. This was exemplified by the analysis of SAHH-mediated hydrolysis of SAH and acetylcholinesterase-triggered hydrolysis of acetylthiocholine; the LOD of SAHH and acetylcholinesterase was 4 units L 1 (approximately 0.24 nM) and 0.6 units L 1 (approximately 1 nM). However, thiol-containing compounds, such as glutathione and cysteine, can interfere with the detection of enzyme activity in biological fluids. To solve this problem, iodoacetamide can be used for blocking thiol group through alkylation prior to the analysis of enzyme activity (Faccenda et al., 2010). Because adenosine is highly effective at inhibiting SAHH activity, the T8-MB-T8 Hg2 þ
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