Article pubs.acs.org/ac
Polydimethylsiloxane Permeability-Based Method for the Continuous and Specific Detection of Hydrogen Sulfide Adam Faccenda, Jingyuan Wang, and Bulent Mutus* Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4 S Supporting Information *
ABSTRACT: Hydrogen sulfide (H2S) is known to play a physiological role in processes as diverse as vasodilation, maintenance of vascular tone, neurotransmission, and immune response. The multitude of physiological functions in which H2S is involved warrants the development of useful methods for its detection. Here, we introduce a simple and continuous H2S detection method that exploits the relatively high polydimethylsiloxane (PDMS) permeability of H2S in comparison to other thiols typically encountered in the cellular milieu. In this method, 96-well inserts constructed of PDMS act as an H2S-permeable membrane, eliminating nonspecific thiol detection. This design also makes it possible to use virtually any available thiol-specific probe such as Ellman’s reagent which was used here to detect H2S once it crossed the PDMS membrane. Utilizing this method, a detection limit of 9.2 ± 1.9 ppb(m) (parts per billion (by mole) or ∼0.51 μM in 1.6 mL of buffer) free H2S (detected as solution sulfide) was achieved. In addition, the assay was used to determine KM and Vmax for natural substrates of cystathionine γlyase (CSE), the main enzyme responsible for H2S production in peripheral tissues. The KM and Vmax of CSE for cysteine were 3.79 ± 2.07 mM and 0.37 ± 0.02 nmol H2S/min, respectively. KM and Vmax for homocysteine were 6.90 ± 1.78 mM and 1.10 ± 0.19 nmol H2S/min, respectively. In addition, the assay was used to examine the potential for a direct interaction of H2S and NO. The levels of detected H2S decreased in the presence of NO under normoxia but not under anoxia indicating that H2S does not react with NO but with N2O3 likely formed in the hydrophobic environment of PDMS.
H
pK a1= 6.75
ydrogen sulfide (H2S) is a highly toxic compound; however, much like its toxic gaseous counterparts carbon monoxide (CO) and nitric oxide (NO), an exponentially growing body of evidence points to H2S being an ubiquitous cellular signaling molecule with multiple physiological roles including the regulation of blood pressure, inflammation, and neuronal function.1−3 There is also growing evidence that the NO and H2S signaling pathways interact or “crosstalk”.2,4−7 Interestingly, NO and H 2 S share many of the same physiological effects; however, they often act through discrete mechanisms. For example, NO induces vasodilation via activation of soluble guanlyl cyclase (sGC), whereas H2S imparts the same effect through activation of ATP-sensitive potassium (K+ATP) channels.8 Li et. al provide an excellent account of the mutual physiological effects of H2S and NO, as well as CO.7 In mammals, H2S is mainly formed from the metabolism of cysteine by two pyridoxal-5′-phosphate (PLP)-dependent enzymes: cystathionine-β-synthase (CBS, EC 4.2.1.22) and cystathionine-γ-lyase (CSE, EC 4.4.1.1) which are responsible for H2S production in the central nervous system and peripheral tissues, respectively. Most recently, 3-mercaptosulfurtransferase (3-MST) was discovered as a third source of H2S in the brain.3 H2S is a weak acid and freely dissociates in aqueous solution according to the following equilibrium: © 2012 American Chemical Society
pK a2 = 11.96
H 2S ⇌ H+ + HS− ⇌ H+ + S2 −
(1)
At physiological temperature and pH, the Henderson− Hasselbalch equation predicts that approximately 18.5% of the total sulfide concentration exists as H2S and 81.5% as HS−, and the relative bioactivity of each of these species is still unknown.9 Throughout this Article, the term “sulfide” is used to refer to H2S, HS−, and S2− collectively, whereas “free H2S” is used to refer specifically to the gaseous species. An understanding of the mechanisms by which H2S regulates its protein targets is of paramount importance to solving its physiological and pathological roles. Central to this quest is the development of simple analytical methods that specifically detect H2S in a direct and real-time/continuous manner. Several methods are currently used for the detection and measurement of H2S including electrochemical,10 colorimetric (Zn-acetate trap + phenylenediamine),11 gas chromatograph (GC) with either flame photometric detection (FPD), pulsed flame photometric detection (PFPD), or sulfur chemiluminescence detection (SCD)12 as well as other HPLC-based methods.13,14 Of these, all but the electrochemical methods are indirect and discontinuous and are not conducive to kinetic Received: January 11, 2012 Accepted: May 24, 2012 Published: May 24, 2012 5243
dx.doi.org/10.1021/ac3008863 | Anal. Chem. 2012, 84, 5243−5249
Analytical Chemistry
Article
Figure 1. Schematics representing the two experimental setups used to measure H2S release and a photograph of the PDMS wells. (A) In the cuvette-based method, DTNB (1.6 mL, 50 μM) was placed into the cuvette into which the PDMS well containing the H2S source (e.g., cell suspension, cell media, or Na2S) was submerged. Absorbance at 412 nm was monitored using an Agilent 8453 UV/vis spectrophotometer, and free H2S concentration was determined using the extinction coefficient for NTB2− (ε412 = 14 150 M−1 cm−1). (B) In the plate-based method, the H2S source was placed into the 96-well plate and strips of PDMS wells (C) containing 100 μL of the thiol probe (i.e., DTNB) were inserted into the plate with minimal dead time between sample addition and acquisition. Absorbance at 405 nm was then measured using a Perkin-Elmer Victor 1420 microplate reader. Free H2S concentration using the plate-based method was calculated by comparison to a standard curve using known concentrations of Na2S. The sample volume required for each method was comparable: 20−100 μL for the cuvette-based method (A) and 140 ± 20 μL for the plate-based method (B).
Sodium sulfide nonahydrate was purchased from MP Biomedicals. 5,5′-Dithiobis-(2-nitrobenzoate), cysteine hydrochloride, and pyridoxal-5′-phosphate were all purchased from Sigma Aldrich and used without further purification.
determinations. The electrochemical methods have, however, been suspected of displaying cross reactivity with other thiols causing inflation of the determined H2S concentrations.15 Of the available methods, GC16 and GC/MS17 are the most sensitive and likely the most reliable. Although detection limits using GC can extend into the ppt range, achieving them involves considerable sample preparation (e.g., preconcentration and/or scrubbing to remove oxidants). In addition, even with direct injection of the sample into the GC, there is potential for sample loss due to adsorption to the equipment.18,19 Here, we introduce a simple method based on the selective permeability of polydimethylsiloxane (PDMS) to free H2S and not to other thiols commonly found in the biological milieu. In this method, free H2S is released from solution and, in gaseous form, traverses a thin membrane of PDMS, which acts as a liquid-impermeable barrier between the H2S source and the H2S probe (DTNB in this case). This separation allows for the specific detection of free H2S as sulfide in solution. The PDMSdependent isolation of H2S allows for detection in real-time by virtually any available colorimetric or fluorimetric thiol reagent once H2S crosses the PDMS membrane. Furthermore, we demonstrate the sensitivity and selectivity of our assay, thus validating its use as a novel method to detect H2S in real-time. There are, however, drawbacks which are discussed in detail later. Taken together, these characteristics provide a notable advantage over the majority of current methods in that it is one of few methods that is void of harsh chemical treatment and specifically detects free H2S.
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METHODS Hydrogen Sulfide Measurements. Unless otherwise specified, all solutions were prepared in 0.1 M sodium phosphate buffer (pH 7.4) with 2 mM EDTA and all buffers were degassed by stirring and sonicating under vacuum for 1 h, followed by purging with argon while stirring for 2 h. Two different procedures were used to kinetically measure H2S release: a cuvette-based (Figure 1A) and a plate-based (Figure 1B) procedure. In the cuvette-based procedure, H2S measurements using Ellman’s reagent (DTNB) were acquired using an Agilent 8453 UV/vis spectrophotometer equipped with a thermostat. Here, the H2S source (20−100 μL) was placed into the PDMS well, which was then submerged with the open end exposed to air into 1.6 mL of 50 μM DTNB in a cuvette containing a magnetic stir bar (Figure 1A). For the cuvettebased procedure, H2S concentrations were calculated using the extinction coefficient of the NTB2− anion (ε412 = 14 150 M−1 cm−1).20 In the plate-based procedure (Figure 1B), 140 μL of the H2S source was placed into the wells of a 96-well plate. DTNB (100 μL, 50 μM)-filled PDMS wells (Figure 1C) were then placed into the 96-well plate overtop the H2S source. The PDMS wells were inserted into the plate with minimal dead time between sample addition and acquisition (
