Nitrosothiol Detection via Amperometric Nitric Oxide Sensor with

an amperometric nitric oxide (NO) gas sensor with thin hydrogel layer containing an ... It is further shown that the new RSNO sensors can be used to a...
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Langmuir 2006, 22, 10830-10836

S-Nitrosothiol Detection via Amperometric Nitric Oxide Sensor with Surface Modified Hydrogel Layer Containing Immobilized Organoselenium Catalyst† Wansik Cha and Mark E. Meyerhoff* Department of Chemistry, UniVersity of Michigan, 930 North UniVersity AVenue, Ann Arbor, Michigan 48109-1055 ReceiVed May 1, 2006. In Final Form: August 23, 2006 A novel electrochemical device for the direct detection of S-nitrosothiol species (RSNO) is proposed by modifying an amperometric nitric oxide (NO) gas sensor with thin hydrogel layer containing an immobilized organoselenium catalyst. The diselenide, 3,3′-dipropionicdiselenide, is covalently coupled to primary amine groups in polyethylenimine (PEI), which is further cross-linked to form a hydrogel layer on a dialysis membrane support. Such a polymer film containing the organoselenium moiety is capable of decomposing S-nitrosothiols to generate NO(g) at the distal tip of the NO sensor. Under optimized conditions, various RSNOs (e.g., nitrosocysteine (CysNO), nitrosoglutathione (GSNO), etc.) are reversibly detected at e0.1 µM levels, with sensor lifetimes of at least 10 days. The presence of reducing agents (e.g., glutathione) added to the test solution enhances the amperometric dynamic range output to ∼25 µM levels of RSNO species. Sensitivities observed for different small molecule RSNO species are nearly equivalent, in sharp contrast to the behavior observed previously for a similar RSNO sensing configuration based on an immobilized Cu(I/II) catalytic layer. It is further shown that the new RSNO sensors can be used to assess the “NO-generating” ability of fresh blood samples by effectively detecting the total level of reactive low molecular-weight RSNO species present in such samples.

S-Nitrosothiols (RSNOs) occur in vivo, and their biosynthetic routes have been suggested to include the transformation of thiols by endogenous nitric oxide (NO) or its reactive intermediates (e.g., N2O3, peroxynitrite, or nitrosyl-metal complex).1-3 It is believed that RSNOs are a carrier or reservoir of transient NO under physiological conditions since various decomposition pathways of RSNO (e.g., thermal, photolytic, and catalytic) lead to localized NO generation.1,2,4 Therefore, the physiological functions of RSNOs often mirror those of NO, such as vasculature relaxation and the inhibition of platelet adhesion and activation.4,5 Although the metabolic life cycle of RSNOs still remains to be fully elucidated, the active involvement of living cells in RSNO metabolism has been suggested.3 For example, it is reported that eukaryotic cells maintain intracellular RSNO homeostasis via certain enzyme reactions (e.g., S-nitrosoglutathione (GSNO) reductase).6 Further, the metabolism of GSNO to NO by platelets has been observed, suggesting that cell surface proteins such as disulfide isomerase and specific copper(I/II)-containing proteins participate in the potent inhibitory effect that RSNOs have on platelet aggregation by localizing the release of NO from GSNO.7,8 Specific enzymatic degradation of low-molecular-weight RSNOs †

Part of the Electrochemistry special issue. * To whom correspondence should be addressed. E-mail: mmeyerho@ umich.edu. (1) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898-1902. (2) Stamler, J. S.; Jaraki, O.; Osborne, J. A.; Simon, D. I.; Keaney, J.; Vita, J.; Singel, D. J.; Valeri, C. R.; Loscalzo, J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7674-7677. (3) Zhang, Y.; Hogg, N. Free Radical Biol. Med. 2005, 38, 831. (4) Rassaf, T.; Kleinbongard, P.; Preik, M.; Dejam, A.; Gharini, P.; Lauer, T.; Erckenbrecht, J.; Duschin, A.; Schulz, R.; Heusch, G.; Feelisch, M.; Kelm, M. Circ. Res. 2002, 91, 470-477. (5) Radomski, M. W.; Rees, D. D.; Dutra, A. A.; Moncada, S. Br. J. Pharmacol. 1992, 107, 745-749. (6) Liu, L.; Hausladen, A.; Zeng, M.; Que, L.; Heitman, J.; Stamler, J. S. Nature 2001, 410, 490. (7) Root, P.; Sliskovic, I.; Mutus, B. Biochem. J. 2004, 382, 575-580. (8) Gordge, M. P.; Hothersall, J. S.; Neild, G. H.; Dutra, A. A. Br. J. Pharmacol. 1996, 119, 533-538.

has also been reported in the presence of glutathione peroxidase (GPx; a selenoenzyme) and g-glutamyl-transpeptidase, and both activities are known to be present in blood.9-11 These various NO generating physiological processes have inspired preparation and potential application of biomimetic catalytic species to convert endogenous RSNOs found in blood to NO. For example, polymer immobilized Cu(II)-ligand complexes have been suggested as new materials to improve the surface biocompatibility of blood-contacting devices.12-14 Due to the localized generation of NO from RSNO substrates, such artificial surfaces could ultimately mimic the surface of the endothelium that continuously releases NO, to suppress platelet adhesion and activation, and hence blocks the coagulation cascade that causes blood clots on artificial surfaces.15 Various synthetic strategies to incorporate functional or catalytic moieties on polymeric surfaces to release or generate NO have been reviewed elsewhere.15 Recently, we proposed an alternate catalytic agent to create polymeric surfaces that can generate NO when in contact with fresh blood.16 Indeed, it has been found that polymers modified with organoselenium molecules also exhibit catalytic RSNO decomposition activity.16 Cyclic redox reactions between organoselenium species (RSe) (e.g., diselenide (RSe-SeR), selenosulfide (RSe-SR) and selenol/selenolate (RSeH/-RSe-)) and RSNOs or reducing agents (thiols) are thought to be involved (9) Freedman, J. E.; Frei, B.; Welch, G. N.; J., L. J. Clin. InVest. 1995, 96, 394-400. (10) Hogg, N.; Singh, R. J.; Konorev, E.; Joseph, J.; Kalyanaraman, B. Biochem. J. 1997, 323, 477-481. (11) Hou, Y.; Guo, Z.; Li, J.; Wang, P. G. Biochem. Biophys. Res. Commun. 1996, 228, 88-93. (12) Oh, B. K.; Meyerhoff, M. E. J. Am. Chem. Soc. 2003, 125, 9552-9553. (13) Oh, B. K.; Meyerhoff, M. E. Biomaterials 2004, 25, 283-293. (14) Hwang, S.; Cha, W.; Meyerhoff, M. E. Angew. Chem., Int. Ed. 2006, 45, 2745-2748. (15) Frost, M. C.; Reynolds, M. M.; Meyerhoff, M. E. Biomaterials 2005, 26, 1685-1693. (16) Cha, W.; Meyerhoff, M. E. Biomaterials 2006, in press.

10.1021/la0612116 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/28/2006

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groups in a polyethylenimine (PEI) polymer, and the resulting derivatized polymer is further cross-linked to form a hydrogel layer on a dialysis membrane support. As illustrated schematically in Figure 1, the RSe-mediated decomposition of RSNOs is achieved within the dialysis membrane at the distal tip of the NO sensor, leading to the production of NO in this confined region. The NO generated diffuses through the gas permeable membrane of the NO sensor to a planar platinized platinum anode, where oxidation of NO takes place. Basic sensor performance and various factors influencing sensor sensitivity are examined. It is shown that sensors modified with RSe-containing polymer respond to various RSNO species at sub-µM levels and maintain amperometric response for at least 10 days. Further, it will be demonstrated that the resulting devices can be used to detect the presence and relative levels of NO generating RSNO species within fresh blood samples. Experimental Section

Figure 1. (a) Amperometric detection scheme of proposed RSNO sensor based on catalytic RSe-hydrogel layer; (b) Schematic presentation of RSePEI structure.

in this catalytic RSNO decomposition pathway (see below, eqs 1-4).16 Plasma RSNO species have been detected over a wide range of concentrations, from low nanomolar to tens of micromolar levels, due to differences in the individual species as well as differences in sampling and analytical methodologies used.17 In addition, circulating levels of RSNOs may be modulated by differences in their susceptibility to metals, thiols, and/or ascorbate content of the sample.18,19 Therefore, to eventually apply NO generating polymers to enhance the blood compatibility of biomedical devices in clinical practice, new methods are needed to estimate the fluctuation of blood RSNO levels from subject to subject.

RSe-SeR + R′SH a RSe-SR′ + RSe- + H+

(1)

RSe-SR′ + R′SH a RSe- + H+ + R′S-SR′

(2)

RSe- + H+ + R′′S-NO a 1/2RSe-SeR + R′′SH + NO (3) RSe-SeR + 2R′′S-NO a 2RSe-SR′′ + 2NO

(4)

Herein, we propose a novel method for the direct detection of RSNOs by utilizing an organoselenium catalytic species immobilized within a hydrogel layer at the surface of an amperometric NO sensor. In a previous report, we introduced a similar concept for RSNO detection by exploiting Cu(I/II) species as the surface catalytic layer.20 In the present work, a diselenide, 3,3′dipropionicdiselenide, is covalently coupled with primary amine (17) Giustarini, D.; Milzani, A.; Colombo, R.; Dalle-Donne, I.; Rossi, R. Clin. Chim. Acta 2003, 330, 85-98. (18) Pietraforte, D.; Mallozzi, C.; Scorza, G.; Minetti, M. Biochemistry 1995, 34, 7177-7185. (19) Kashiba-Iwatsuki, M.; Kitoh, K.; Kasahara, E.; Yu, H.; Nisikawa, M.; Matsuo, M.; Inoue, M. J. Biochem. (Tokyo) 1997, 122, 1208-1214. (20) Cha, W.; Lee, Y.; Oh, B. K.; Meyerhoff, M. E. Anal. Chem. 2005, 77, 3516-3524.

Materials. Polyethylenimine (PEI, avg. Mw 25k), cysteine, glutathione (GSH), N-acetyl penicillamine, penicillamine, N-acetyl cysteine, bovine serum albumin (BSA, γ-globulin and protease free, fatty acid content, 0.005%), glutaraldehyde (25 wt %), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxylsuccinimide (NHS), and sodium borohydride were obtained from Sigma-Aldrich (St. Louis, MO) and used as received. Dialysis membranes (Spectra/Por 7) were purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA). 3,3′-Dipropionicdiselenide (SeDPA) was synthesized as described in the literature.21 All chemicals were of analytical grade or better and used as received from various suppliers. Buffers, including phosphate-buffered saline (PBS), were prepared as needed in the laboratory from the appropriate reagents. Preparation of S-Nitrosothiols. Crystalline S-nitroso-N-acetyl penicillamine (SNAP) was synthesized according to a reported procedure22 using a 5 mM solution of N-acetyl-penicillamine containing 10 µM EDTA. Solutions (5 mM each) of other S-nitrosothiols (CysNO, GSNO, S-nitroso-N-acetyl cysteine (SNAC), and S-nitroso-penicillamine (SPA)) were also prepared as previously described.23 To obtain S-nitroso-albumin (AlbSNO) a modified procedure originally reported by Cha et al. was employed.16,20 Unless otherwise noted, these solutions were injected directly into PBS buffer (pH 7.4) to obtain the desired concentration of RSNOs. The concentrations and stabilities of the synthesized RSNOs were determined by using a chemiluminescence NO analyzer (NOA) (Seivers 280, Boulder, CO). Derivatization of PEI with SeDPA. The diselenide molecule was covalently coupled with PEI to create organoselenium-containing PEI (RSePEI) as described in detail elsewhere.16 Briefly, the carboxylic acid groups of SeDPA were first activated by EDC and NHS to form N-succinimide esters. A solution of PEI (40 mg/mL) in pH 5.8 2-[N-morpholino]ethanesulfonic acid sodium salt (MES) buffer was reacted with the activated SeDPA (12.5 mM), EDC, and NHS at room temperature for 2 h. The molar ratio of EDC:NHS: R-COOH was adjusted to 6:4:1 to achieve maximum coupling of diselenides on the PEI polymer. The resulting RSePEI solution was dialyzed against DI water for 1 d. The uncoupled carboxyl groups were removed by reducing the diselenide bond with sodium borohydride (10 mM), and then the final solution was exhaustively dialyzed against DI water for 3 d. Control PEI (no RSe species) was also prepared in the same way except that no diselenide was added to the solution used in the coupling reaction. The final RSePEI species was neutralized and stored in the dark at 4 °C until used. The dry polymer obtained after lyophilization was found to contain 3.6 ( 0.3 w/w% of Se by ICP-MS analysis. (21) Koch, T.; Suenson, E.; Henriksen, U.; Buchardt, O. Bioconj. Chem. 1990, 1, 296-304. (22) Field, L.; Dilts, R. V.; Ravichandran, R.; Lenhert, P. G.; Carnahan, G. E. J. Chem. Soc., Chem. Commun. 1978, 249-250. (23) Stamler, J. S.; Loscalzo, J. Anal. Chem. 1992, 64, 779-785.

10832 Langmuir, Vol. 22, No. 25, 2006 Cross-Linked RSePEI Hydrogel Formation on Dialysis Membrane. Based on the assumption that the Se content determined after the coupling reaction represents the actual coupling efficiency solely with primary amine groups on the PEI, only a small fraction (∼6%) of primary amine groups in PEI were consumed and the remaining sites can be further used to cross-link RSePEI onto a dialysis membrane through reaction with glutaraldehyde. On a patch of dialysis membrane (MWCO, 25kD; 2 cm2) previously soaked in 0.1 M Tris buffer (pH 7.5) solution with 1 wt % RSePEI and 0.1 mM EDTA for at least 2 d, 100 µL of a mixed solution of RSePEI (1 wt %) and glutaraldehyde (1 wt %) at a 1:1 ratio was added and reacted until dry. The resulting modified dialysis membrane was washed with DI water to remove any nonadhering polymer and placed into a 10 mM sodium borohydride solution to reduce imine bonds. The reduced membrane was then washed again and stored in 0.1 M phosphate buffer (pH 4.3) until use. Control dialysis membranes were also prepared in the same manner using unmodified PEI. Fabrication of Amperometric NO/RSNO Sensors. The amperometric NO gas sensors used in this work were fabricated in a manner analogous to that reported earlier.24 Briefly, a platinized Pt working electrode (Pt disk (with 250-µm o.d.)) sealed in glass wall tubing and a Ag/AgCl wire reference/counter electrode were incorporated behind a PTFE gas-permeable membrane (GPM). However, in this study, the PTFE-GPM (0.12 cm2) was treated with 0.5 µL of TeflonAF solution (1%, used as received, Dupont Fluoroproducts, Wilmington, DE) before sensor fabrication in order to enhance NO selectivity.25 To create the RSNO sensor, a dialysis membrane (DM) containing the cross-linked-RSePEI was affixed on the GPM of the NO sensor using an O-ring, allowing the RSePEItreated surface to face the GPM of the sensor. Control NO sensors were also prepared using the “control” dialysis membrane. All sensors were polarized at +0.75 V vs Ag/AgCl for at least 12 h before use, and all subsequent amperometric measurements were carried out using the same applied potential. Calibration curves toward NO and RSNO species and current recordings were obtained as described in our previous work.20,24 Detection of RSNOs with NO Sensor Modified with RSe Catalyst. PBS buffer (pH 7.4) containing 0.5 mM EDTA was used as the working buffer solution for all RSNO measurements in a 100-mL amber reaction vessel at room temperature. Other experimental conditions were as reported earlier.20 In some experiments, background NO levels in the bulk test solutions were monitored simultaneously using a control NO sensor placed into the same sample solution. The measured current levels of RSNO sensors at specific times were converted to equivalent NO concentrations based on prior NO calibration data of the RSNO sensor. For stability studies, the sensors’ amperometric responses toward both NO (calibration) and GSNO were recorded periodically. Detection of RSNOs in Blood. For direct detection of endogenous RSNO species in blood, fresh heparinized sheep blood was obtained from Extracorporeal Membrane Oxygenation (ECMO) laboratory at the University of Michigan Medical School. Detailed blood sampling procedures were as described in a prior report.20 The temperature of the sampled blood was quickly stabilized in water bath at 25 °C both before and during amperometric RSNO detection. The blood was kept in the dark and used within 30 min after removal from the animal. Two electrodes, a control NO sensor and an RSNO sensor were simultaneously used in the blood experiments. Each sensor was first calibrated with respect to its inherent response to NO in PBS buffer. Then, the sensors were placed in the same N2-saturated 70-mL of PBS (pH 7.3) solution containing EDTA and GSH (10 and 50 µM, respectively, after final blood addition) and their amperometric signals were stabilized at 25 °C in water bath. Finally, 30 mL of the fresh whole blood sample was added to the PBS solution to yield a 30% (v/v) dilution under a N2 atmosphere, and the amperometric responses of each sensor to the sample were monitored. (24) Lee, Y.; Oh, B. K.; Meyerhoff, M. E. Anal. Chem. 2004, 76, 536-544. (25) Cha, W.; Meyerhoff, M. E. Chem. Anal. (Warsaw) 2006, in press.

Cha and Meyerhoff

Results and Discussion RSNO Decomposition by RSe-Hydrogel. As shown in eq 1, the reduction of the diselenide by reducing agents (e.g., thiols or borohydride) leads to the formation of the corresponding organoselenol (oxidation states from -1 to -2), which is the catalytically active functional group, especially well-known for the reduction of peroxides and peroxynitrites.26,27 Therefore, if the RSePEI hydrogel obtained after coupling reaction contains unreacted carboxylic acid groups (-SeCH2CH2COOH) of the SeDPA, these can be liberated from the PEI backbone during the reduction reaction step with GSH as either the selenol or selenosulfide species. To prevent such fragmentation that can potentially lead to catalyst leaching, the uncoupled half of SeDPA was removed by the borohydride reduction followed by extensive dialysis. Therefore, the diselenide bonds in the resulting RSePEI polymer are comprised of partially cross-linked polymeric structures with intra- and intermolecular connection via the remaining stable diselenide species (after reoxidation by oxygen; see below). Selenols are readily oxidized and converted to diselenides by ambient oxygen. Indeed, the RSePEI polymer rich in selenol groups after the reduction step is colorless and upon exposure to atmospheric oxygen slowly oxidizes and returns to a yellow color with the characteristic absorption band of a diselenide species at ∼300 nm.28 This implies that the PEI polymer backbone is flexible enough to allow the diselenide linkages to reform from the organoselenols (RSeH) groups after the borohydride treatment, and thus most of organoselenium immobilized on the final RSePEI exists in the form of diselenides. The RSePEI species or cross-linked RSePEI hydrogel (prepared by glutaraldehyde treatment on the dialysis membrane) display close resemblance in catalytic behavior with that of LMWdiselenide molecules described elsewhere.16 Hence, the catalytic reaction mechanism to generate NO from RSNOs is thought to follow the same proposed pathway as summarized in eqs 1-4; two distinct, slow and fast, NO generation modes are shown (eq 3 and 4, respectively). In particular, even without a thiol reducing agent added, diselenides, including RSePEI, can undergo a fast reaction (eq 4) directly with RSNOs to produce NO stoichiometrically; this reaction stops as all diselenides are consumed. However, in the presence of a free thiol species as a reducing agent, both selenolate and diselenide reactions appear to participate in a catalytic cycle. The presence of thiol reducing agent shifts the equilibrium in eqs 1-3 to produce a selenolate (conjugate base and major form of selenol at neutral pH due to pKa, ∼5.5 (-SeH/-Se-), for selenocysteamine),29 which has strong reducing power,30 likely enough to decompose RSNO and create a corresponding thiol (RSH) and NO. Such an equilibrium shift toward selenol production can be enhanced within the PEI hydrogel network due to its basic nature. Indeed, the inner phase of the PEI hydrogel was found to maintain a basic environment (pH 7-9) even when the hydrogel is soaked in a low pH (4.3) buffer solution. This was confirmed by observing the color of dye molecules (pH indicator, bromocresol purple) physically embedded into the hydrogel (data not shown). On the other hand, if the pH within the hydrogel is lower than pH 7, it is expected that such a catalytic reaction becomes ineffective (26) Mugesh, G.; Singh, H. B. Chem. Soc. ReV. 2000, 29, 347-357. (27) Klotz, L.-O.; Sies, H. Toxicol. Lett. 2003, 125, 140-141. (28) Tamura, T.; Oikawa, T.; Ohtaka, A.; Fujii, N.; Esaki, N.; Soda, K. Anal. Biochem. 1993, 208, 151. (29) Arnold, A. P.; Tan, K. S.; Rabenstein, D. L. Inorg. Chem. 1986, 25, 2433-2437. (30) Jacob, C.; Giles, G. I.; Giles, N. M.; Sies, H. Angew. Chem., Int. Ed. 2003, 42, 4742-4758.

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Figure 3. Typical real-time current recordings for RSe-based RSNO sensor at low levels of three endogenous RSNO species along with varying RSNO concentrations in working buffer solutions containing 50 µM GSH.

Figure 2. Performance test of RSe-based RSNO sensor. (a) Amperometric responses of two (NO and RSNO) sensors mounted in one sample vessel monitored upon addition of GSNO in working buffer solution. Amperometric current levels of both sensors were normalized based on prior NO calibration; (b) dynamic recordings illustrating real-time reversibility of RSNO sensors in amperometric response to varying concentrations of GSNO. Sample solutions contain 0.5 mM EDTA and 50 µM GSH as a reducing agent.

as suggested by bulk solution phase studies described elsewhere.16 Those studies imply that the selenolate, not selenol, species is the key intermediate for the generation of NO via the catalytic route. Amperometric Responses to RSNO Species. Due to the high molecular-weight-cutoff (MWCO, 25 kDa) of the dialysis membrane support used to prepare the RSNO sensor, RSNO species as well as reducing agents (GSH) diffuse rapidly through this film and decompose catalytically within the RSePEI-catalyst layer to produce NO. As illustrated in Figure 1, a portion of evolved NO passes through the PTFE gas-permeable membrane of the NO sensor and is detected amperometrically on the platinized Pt electrode via oxidation (to nitrate). The resulting RSNO sensor exhibits quantitative and reversible amperometric responses upon addition of GSNO to a PBS test solution (see Figure 2). The results shown in Figure 2a demonstrate that the decomposition of RSNO occurs only on the surface of the RSNO sensor since the control NO sensor placed in the same test solution detects negligible levels of NO in the bulk sample phase. The response to GSNO and other RSNOs is fully reversible (see Figure 2b) when changing from low to high and then back down to lower concentrations of a given RSNO species. Remarkably, as expected from the organometallic nature of the RSe catalyst, the sensor’s sensitivity is not influenced by the

Figure 4. Typical calibration curves for RSNO sensor toward various RSNO species at low levels (a) and high levels (b) of RSNOs. PBS (pH 7.3) solutions containing 0.5 mM EDTA and 50 µM GSH were spiked with increasing levels of RSNOs to obtain the calibration plots.

presence of chelating agents [e.g., EDTA (data not reported)]. Therefore, relatively high concentrations of EDTA can be used in the test solution to prevent trace metal ion-induced RSNO breakdown (e.g., by trace copper or iron). Slower response times are observed for GSNO detection (∼5 min, to achieve 95% of the steady-state current following changes in RSNO levels) compared with the response time toward NO ( GSNO g SNAC ≈ AlbSNO.20 Although the contribution of the different probe design, specifically the choice of the polymer film used for catalyst immobilization (e.g., dialysis membrane vs hydrophilic polyurethane membrane for RSe- and Cu-based sensors, respectively) cannot be excluded, it is believed that such a difference in sensitivity variation is better explained by the different RSNO decomposition mechanisms for the two types of catalysts (RSe- and Cu-based) used to prepare the RSNO detection systems. The broad spectrum of sensitivities observed for the Cu-based system can be interpreted in terms of the inherent reactivities arising from the structural characteristics (e.g., N-acetylation) of the RSNOs and hence the capability to form a cyclic intermediate complex (5- or 6-membered ring structures) between the given RSNO and the Cu(I)/Cu(II) species.31 For the RSe-mediated RSNO decomposition, however, no significant variation in NO generation rate is observed even in bulk solution phase RSNO decomposition with LMW-diselenides and thiol reducing agents, as determined by using chemiluminescence detection.16 Such consistency in NO generation regardless of the nature of the RSNO species likely reflects the facile kinetics of NO generation steps as described by eqs 3 and 4 (see above). Moreover, the very similar sensitivities exhibited by the sensor toward low levels of GSNO and CysNO, both endogenous blood species, is potentially advantageous when using this new sensor for assessing the total amount endogenous LMW-RSNOs in biological fluids. Factors Influencing RSNO Sensor Sensitivity. Inherently, the amperometric signal of RSNO sensors based on an inner NO detector is dependent on the oxygen levels in the sample phase (see the Supporting Information Figures 1S and 2S). Since NO can decay rapidly by reacting with oxygen,32 the local concentration of NO produced from the catalytic surface of the RSNO sensor can decrease in the presence of high oxygen levels, thereby resulting in the reduction of the amperometric sensor signal. However, even in a medium rich in oxygen, the sensor sensitivity is still maintained at considerable levels (approximately 50% levels of those in the deoxygenated condition, see the Supporting Information Figures 1cS and 2bS). It is thought that the rapid oxidation of selenol/selenolate under such conditions leads to the increased levels of diselenide in catalytic layer,33 which can result in RSNO decomposition predominantly via rxn 4, a route presumably not sensitive to the presence of oxygen. In addition, according to the proposed RSe-mediated RSNO decomposition mechanism,16 two other factors can be identified which can greatly influence the RSNO sensor’s sensitivity; the concentration of reducing agent and the sample pH. To verify their effects, the condition of sample working buffer was varied as shown in Figures 5a,b. As shown in Figure 5a, the RSNO sensor detects GSNO even in the absence of any thiol reducing agent (GSH) added to the sample phase, and this implies the direct reaction between RSNO and the diselenide moiety, i.e., (31) Williams, D. L. H. Acc. Chem. Res. 1999, 32, 869-876. (32) Fukuto, J. M.; Cho, J. Y.; Switzer, C. H. In Nitric Oxide, Biology and Pathology; Ignarro, L. J., Ed.; Academic Press: San Diego, CA, 2000; pp 23-40. (33) Chaudiere, J.; Courtin, O.; Leclaire, J. Arch. Biochem. Biophys. 1992, 296, 328.

Cha and Meyerhoff

Figure 5. Influence of sample phase GSH concentration (a) and pH (b) on the amperometric signal of RSNO sensor in response of constant GSNO at 2.5 µM. For (a), GSH levels increased upon sequential addition of GSH; for (b), working buffers’ pH was varied in separate runs and GSH was present at a constant level of 50 µM in PBS test solution.

eq 4, above, is responsible for one path of NO generation. However, as also shown in this figure, the subsequent addition of GSH to the sample phase increases the amperometric signal, suggesting the contribution of the catalytic cycles (eqs 1-3) yielding a faster rate of conversion of GSNO in the catalytic layer. Such signal enhancement by GSH was also observed even in whole plasma and blood measurements (see the Supporting Information Figures 3S-5S). Therefore, the GSH level was fixed at 50 µM in all test conditions used to obtain data reported in this study. The limited increase of amperometric signal by adding GSH shown in Figure 5a is somewhat at odds with an alternate view that the organoselenol production steps (eqs 1 or 2) may be the rate-limiting reactions.34,35 This contradiction remains to be explored in more detail. However, we can speculate that there exist unidentified intermediate states interacting with the thiol and/or selenol, particularly in the reactions described by eqs 3 and 4, or another route leading to the consumption of selenol, e.g., an oxidation reaction with ambient oxygen.33 At this point, it is only evident that the amperometric response monitored in the presence of the reducing agent (thiol) results from both NO generation routes represented in eqs 3 and 4. Due to the relatively low pKa of selenol/selenolate (5.5 for selenocysteamine)29 compared with that of the thiol/thiolate (34) Singh, R.; Whitesides, G. M. J. Org. Chem. 1991, 56, 6931-6933. (35) Zhao, R.; Holmgren, A. J. Biol. Chem. 2002, 277, 39456-39462.

S-Nitrosothiol Detection

species (∼8.5), decreasing the pH of the reaction medium can drastically reduce the selenolate level and concomitantly induce lower catalytic NO generation compared to neutral pH conditions. However, as shown in Figure 5b, the apparent amperometric sensitivity toward GSNO as the substrate initially exhibits a relatively small dependence on pH changes in sample solution. Even at pH 4.3, the steady-state current level obtained immediately after the addition of GSNO is comparable with that observed at neutral pH. It is likely that such consistency in sensitivity against pH variation is not only due to the basic or buffer-like nature of the hydrogel layer rich in amine sites but also due to the abundant diselenide moieties in RSePEI (see above), thereby producing more NO from the chemistry depicted in eq 4, above, that is less dependent on local pH. Interestingly, as further shown in Figure 5b, the amperometric response at pH 4.3 decays much faster over time than that observed at higher pH. This strongly indicates that the RSe-hydrogel layer produces a limited amount of NO, which eventually dissipates to the bulk solution and decomposes by reacting with dissolved oxygen. We speculate that the build-up of organoselenosulfide (-RSe-SR-) or selenol species cannot be recycled at low pH to create more diselenides or selenolates, the active species for NO generation. By increasing the sample pH, this change in sensor sensitivity is found to completely recover back to the original levels observed at neutral pH. RSNO Sensor Lifetime. To ascertain the practical lifetime of the new RSNO sensors based on the cross-linked RSePEI catalytic layer, the sensitivity (nA/µM) changes in response to GSNO were monitored with time. Essentially no loss in amperometric sensitivity was observed for up to 10 d from the first use (data not shown). This suggests that the catalytic sites are covalently bound to PEI polymer chains and not capable of leaching through the dialysis membrane. Such excellent sensor stability is again in sharp contrast with the previously reported Cu-based RSNO detection system20 where copper catalytic sites (Cu(II)-complex, insoluble Cu(II) salts, or Cu0-metal) are always in an equilibrium state capable to release free copper ions into the aqueous sample phase. Direct Detection of RSNOs in Blood. To demonstrate the practical capability of the proposed RSNO sensor to detect endogenous RSNOs, both a control NO sensor and an RSemodified RSNO sensor were employed to measure NO and RSNO levels, respectively, in diluted fresh sheep blood samples. Due to the enhancing effect of reducing agent on sensor sensitivity, a working buffer containing GSH (50 µM, after final blood addition) was used to maximize the change in current response, since fresh blood is known to possess relatively low levels of potential reducing agents such as GSH (2.8 µM) and cysteine (9.7 µM).36 Indeed, it is found that even in undiluted fresh blood with added GSH, sensor sensitivity is maintained at levels comparable to those in PBS or whole plasma (see the Supporting Information Figures 4S and 5S). As illustrated in Figure 6, the RSNO sensor exhibits a significant elevation in amperometric NO response upon the injection of sheep blood into PBS (pH 7.4) at 25 °C. The difference in NO levels of two sensors, i.e., ∆R, strongly suggests that the degradation of endogenous RSNOs predominantly occurs on the RSNO sensor surface and yields a response equivalent to a change of approximately 70 nM (∆R) in effective NO levels (note: the RSNO sensor is precalibrated for its direct response to NO). The subsequent addition of GSNO to the sample of diluted sheep blood induces an additional large amperometric response for the (36) Jones, D. P.; Carlson, J. L.; Mody, V. C.; Cai, J.; Lynn, M. J.; Sternberg, P. Free Radical Biol. Med. 2000, 28, 625-635.

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Figure 6. Direct detection of endogenous RSNOs in fresh sheep blood. Two sensors (NO sensor and RSNO sensor utilizing RSehydrogel as a catalyst) were initially stabilized at 25 °C in 70 mL PBS under N2 environment. Upon injection of 30 mL of fresh sheep blood, only RSNO sensor displays significant NO response. The addition of exogenous GSNO to the given diluted blood solution also raises the amperometric current level significantly only for the RSNO sensor.

RSNO sensor, above and beyond the initial response to the endogenous RSNOs already present in the sample. In contrast, a much smaller signal change is seen on the NO sensor when the GSNO is added. These data suggest that by measuring the difference in response between NO-calibrated NO and RSNO sensors in the same sample of blood, the relative levels of low molecular weight RSNO species in a given blood sample can be easily estimated. However, the signal increase in the control NO sensor upon the addition of fresh blood is larger than expected, since it is thought that only low nM levels of free NO are present in mammalian plasma.2 We speculate that the higher background signal response is primarily due to the presence of ammonia in the blood (normally ∼30 µM, total of NH3 and NH4+)37 as an interfering substance for the NO probes used here despite the enhanced NO selectivity against ammonia achieved by employing TeflonAF treatment25 (ammonia can be oxidized at platinized platinum working electrodes38). In addition, the residual catalytic activity of trace metal ions existing in the dialysis membrane might emerge in the blood test and produce a small amount of NO in the control NO sensor, whereas such reactions are suppressed with high levels of the metal chelating agent (e.g., EDTA) present in working buffers, as shown in previous results20 (i.e., levels of calcium ions in the blood will tie up the added EDTA and prevent the EDTA from effectively removing trace Cu(II) and other metals).

Conclusions It has been demonstrated here that an organoselenium immobilized hydrogel layer at the surface of an amperometric NO sensor can be utilized as a catalyst for RSNO detection. S-Nitrosothiols that diffuse into the polymeric film can undergo catalytic decomposition to generate NO via reactions either directly with immobilized diselenide moieties or with selenolate formed in the presence of thiol reducing agents. The surface modified NO sensor exhibits fully reversible electrochemical response toward RSNOs and yields amperometric currents (37) Barsotti, R. J. J. Pediatr. 2001, 138, S11-S20. (38) Lopez de Mishima, B. A.; Lescano, D.; Holgado, T. M.; Mishima, H. T. Electrochim. Acta 1998, 43, 395-404.

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proportional to the concentrations of various LMW-RSNOs but prolonged and sluggish response toward AlbSNO. Sensitivity variation for the different RSNO species is much less than that of a Cu-based RSNO detection system reported earlier. Therefore the signal changes observed for the RSNO sensor when in contact with fresh blood better represent the total amount of LMWRSNO species that are reactive with the RSe-catalyst. Further, the present study clearly shows potential advantages of using the RSe-catalyst for RSNO detection. Most importantly, due to its organometallic nature, catalytic sites can be easily immobilized and better preserved within polymer matrix through covalent immobilization. Also, the NO generating capability is not affected by the presence of metal-chelating compounds. The sensor and measurement technique described here could be employed to predict the effectiveness of new RSe-based polymer coatings as a means to reduce thrombosis on the surface of biomedical implants. Indeed, for such materials to be useful, it will need to be demonstrated that RSNO levels in blood from patient to patient do not vary substantially, so that similar surface

Cha and Meyerhoff

levels of NO, a potent anti-platelet agent, can be generated in vivo. The sensor described should be useful for such studies. Further, if the catalyst layer of the sensor configuration can be redesigned to be in direct contact with the blood, it should be possible to obtain direct interactions of S-nitroso proteins in the sample, thus allowing a better estimation of the total RSNO species present in a given clinical sample. Efforts to prepare sensors with such a configuration are now in progress in this laboratory. Acknowledgment. This research was supported by the National Institutes of Health under Grants EB-000783 and EB004527. Supporting Information Available: Supplimentary Figures 1S-5S. This material is available free of charge via the Internet at http://pubs.acs.org. LA0612116