Analyst Accepted Manuscript
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication.
Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the Information for Authors.
Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/analyst Page 1 of 7 Journal Name Analyst Dynamic Article Links ►
1 2 3 Cite this: DOI: 10.1039/c0xx00000x 4 5 www.rsc.org/xxxxxx ARTICLE TYPE 6 7 In-vivo and continuous measurement of bisulfide in the hippocampus of 8 9 rat’s brain by on-line integrated microdialysis/droplet-based 10 11 microfluidic system
12 a a a a b b b 13 Feidan Gu , Xiaoyu Zhou , Xiaocui Zhu , Meiping Zhao* , Jie Hao , Ping Yu , and Lanqun Mao* 14 5 Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX 15 DOI: 10.1039/b000000x 16 17 An on-line and continuous approach was demonstrated for in 50 dissociated and associated forms of H S. Polarographic sensor 18 2 vivo measurement of bisulfide in rat’s brain. A modified has been developed for real time measurement of H S production 19 2 droplet-based microfluidic system was constructed which in biological samples, including cells, organ homogenates, and 20 10 allowed on-line qualification of the fluorescence responses of intact tissues.8 The gas/ion chromatography combined with 21 the gold nanoparticle-glutathione-fluorescein isothiocyanate electrochemical detection has also been applied for measurement 22 probe to the variation of bisulfide in the presence of the 9 55 of H S levels in brain. However, no approaches have been 23 cerebral microdialysate background. The on-line method 2 reported for on line and continuous monitoring of the in vivo 24 achieved a dynamic working range from 5.0 µM to 40 µM concentrations of H S in the brain tissues of living animals. A 25 15 and a detection limit of 2.5 µM. The in-vivo bisulfide 2 concentration in the hippocampus of rat’s brain was number of factors such as the instability and high volatility of 26 measured under different physiological conditions. The on- bisulfide make it difficult to obtain the actual value of H2S level 27 Manuscript line method may facilitate the study of H 2S biology by 60 in the brain tissues. To explore the functions of H S in nervous 28 2 providing a previously unattainable continuous record of H 2S systems, it is of great importance to develop more r eliable and 29 20 variation in living animals. It also provides a practical validated methods for in vivo H S detection. platform for in-vivo and continuous monitoring of other 2 30 In vivo microdialysis is a minimally invasive technique for 31 important species in cerebral systems. continuously sampling small molecules or ions from extracellular 32 10 65 fluids of various tissues. Droplet based microfluidic systems are 33 25 Introduction useful tools for measurement of the small volume samples with 34 high temporal resolution. 11 16 In our previous work, we have 35 Hydrogen sulfide (H 2S), known as a toxic gas and an synthesized a gold nanoparticle glutathione fluorescein 36 environmental hazard, has been recently identified as the third isothiocyanate (AuNP GSH FITC) probe for rapid and sensitive 37 Accepted endogenously produced gaseous transmitter, along with nitric 70 detection of bisulfide through a specific anion for molecule 38 oxide (NO) and carbon monoxide (CO).1, 2 It functions both in the ligand exchange reaction.17 By co immobilization of thiol 39 3 30 cardiovascular system and in the central nervous system (CNS). polyethylene glycol (HS PEG COOH) onto the surface of the 40 5 In the CNS, H2S is mainly produced from L cysteine (Cys) and AuNPs, the probe showed high stability in high salty solution 41 homocysteine (Hcy) by the actions of cystathionine b synthase. such as artificial cerebrospinal fluids (aCSF). Using the AuNP 42 In addition to biosynthesis, there are two forms of sulfur stores in 75 GSH FITC probe, a preliminary droplet based microfluidic 43 mammals, acid labile sulfur and bound sulfane sulfur, which fluorescent sensor was further constructed for online monitoring 44 35 release H2S under acidic conditions and reducing conditions, of bisulfide variation in the microdialysate in aCSF. 45 Analyst respectively. Bound sulfur may be a source of H2S in brain and it In this work, we attempt to further improve the integrated 46 can immediately release H2S in response to physiologic microdialysis/droplet based microfluidic chip system for in vivo 6 47 stimulation. In the CNS, H2S facilitates long term potentiation 80 measurement of the HS concentration in rat’s brain. As displayed 48 and regulates intracellular calcium concentration and pH level in in Scheme 1, a new droplet generation design was employed. In
49 40 brain cells. Abnormal generation and metabolism of H2S have addition to the inlets of the sample solution and the probe 50 been reported in the pathogenesis of ischemic stroke, Alzheimer’s solution, a third inlet was added for on line measurement quality 51 disease, Parkinson’s disease, and recurrent febrile seizure. 7 On control. The flow rates and other experimental conditions were
52 the other hand, exogenously applied H2S is demonstrated to have 85 optimized and the integrated system was successfully applied to 53 value for the treatment of febrile seizure and Parkinson’s disease. track the variation of in vivo bisulfide concentration in the rat’s
54 45 Thus, it is assumed that H2S may play a neuroprotective role at brain under different physiological conditions. To the best of our 55 physiological concentrations but may exhibit neurotoxic effects at knowledge, this is the first report on on line and continuous 56 significantly higher concentrations. measurement of in vivo concentration of bisulfide in the brain of 57 Under physiological conditions, >80% of hydrogen sulfide 90 living animals. 58 exists as bisulfide (HS ). Free H2S usually refers to the sum of 59 This journal is © The Royal Society of Chemistry [year] [journal] , [year], [vol] , 00–00 | 1 60 Analyst Page 2 of 7
1 poly(tetrafluoroethylene) (PTFE) tube (0.6 mm i.d./1.0 mm o.d.). 2 The microfluidic channels were created by using a dry film 3 photolithography fabrication technique as previously described. 17 4 50 The channels for oil phase and aqueous solution were both 80 m 5 wide, and the nozzle where the droplets were formed was 30 m 6 wide (Scheme 1A). The main channel where the droplets flew 7 through was 120 m wide. The depth was 40 m for all the 8 channels. Standard solutions of Na S in aCSF at different 9 2 55 concentrations were directly injected into the microchip via inlet 10 S to calibrate the droplet fluorescence and the bisulfide 11 concentration. 12
13 Integration of the droplet-based microfluidic chip with 14 microdialysis sampling system for continuous on-line 15 Scheme 1. (A) CAD design of the droplet formation microfluidic 60 measurement 16 5 chip; (B) Illustration of the integrated microdialysis/droplet based microfluidic chip system for in vivo measurement of the HS For the on line monitoring of bisulfide in the effluent of 17 microdialysis, the microdialysis probe was inserted into the 18 concentration in rat’s brain. standard solution of Na 2S in aCSF or the rat brain (Scheme 1B). 19 Then the microdialysis probe was perfused with aCSF driven by a 20 Experimental 65 microinjection pump through fluorinated ethylene propylene 21 (FEP) tube. The flow rates of microdialysis probe injection and 10 Reagents and Instrumentation 22 oil phase delivering were set at 1.0 L/min and 9.0 L/min, 23 Sodium sulfide nonahydrate (Na 2S⋅9H 2O), hydrogen tetrachloroaurate trihydrate (HAuCl ), sodium citrate, and other respectively. A pump was attached to the outlets of the PTFE 24 4 inorganic chemicals were purchased from Beijing Chemical tube to reduce the pressure and the flow rate was set at 19 L/min. 25 Reagent Corporation (Beijing, China). L glutathione, reduced 70 The microdialysis recovery was determined by comparing the 26 droplet fluorescent intensity of the same concentration of 15 (purity 499.5%) (GSH) and fluorescein isothiocyanate (FITC 27 Manuscript Isomer I (TLC 495%)) were purchased from Merck (Frankfurter, bisulfide in aCSF solution with and without microdialysis. For 28 Germany). Mineral oil (light oil, neat) was purchased from Sigma the in vivo measurements, brain microdialysates were sampled 29 (St. Louis, USA). Thiol polyethylene glycol (HS PEG COOH) from the rat brain with MAB6 (15 kD, 2 mm) because of the size 30 was purchased from Taiyuan Pegchem. Co., Ltd. (Taiyuan, 75 of hippocampus. 31 20 China). All chemicals were of analytical reagent grade and used 32 as received without any further purification. Animal surgery 33 Sylgard 184 prepolymer and its curing agent were purchased Animal surgery was carried out following the procedures as 18 34 from Dow Corning (Midland, USA). A film mask from Shenzhen described previously. Briefly, adult male Sprague Dawley rats 35 Microcad Photo mask Ltd. was used as the photomask. The 40 80 (3 months of age, weighing 300±50 g at the time of surgery) were 36 obtained from Experimental Animal Center of Peking University. 25 m negative dry film photoresist (FX540) was obtained from Accepted 37 DuPont Comp (Wilmington, USA). The artificial cerebrospinal The rats were housed individually in cages with free access to 38 food and water in a light cycle of 12/12 hours light/dark. The fluid (aCSF) (126 mM NaCl, 2.4 mM KCl, 1.1 mM CaCl 2, 0.85 39 animals were anesthetized with chloral hydrate (350 mg/kg i.p.) mM MgCl 2, 27.5 mM NaHCO 3, 0.5 mM Na 2SO 4, 0.5 mM 40 85 and were fixed in a stereotaxic frame with the incisor bar set at KH 2PO 4, pH 7.4) was prepared with pure water, filtered by a 41 3.3 mm below the interaural line for the flat skull position. 30 0.45 m millipore filter, and used as a perfusion solution for on 42 line measurements. A microdialysis guide cannula (BAS/MD 2250, BAS) was 43 Microdialysis probes CMA 12 (20 kD, 4 mm) and MAB6 (15 implanted into the hippocampus (4.0 mm caudal to bregma, 2.0 44 kD, 2mm) were purchased from CMA Microdialysis AB mm lateral to the midline, and 2.0 mm below the skull) in the rat 45 (Stockholm, Sweden). The droplet fluorescence intensity was 90 brain using standard stereotaxic procedures. The guide cannula Analyst was kept in place with three skull screws and dental cement. 46 35 observed and recorded by an Inverted Fluorescence Microscopy 47 (IX71, Olympus, USA) with U MWIBA module (excitation filter Stainless steel dummy blockers were inserted into the guide 48 BP460 495, emission filter BA510 550, and dichromatic filter cannula and fixed until the insertion of the microdialysis probe. 49 DM505) equipped with an EMCCD (Evolve512, Photometric, Throughout the surgery, the body temperature of the rats was 50 USA). The droplet images were analyzed by ImageJ software. 95 maintained at 37°C with a heating pad. After the surgery, the rats 51 were placed into a warm incubator individually until they recovered from anesthesia. The rats were allowed to recover for 52 40 Modification of the droplet-based microfluidic chip 53 As shown in Scheme 1, the droplet based microfluidic chip 24 h before microdialysis sampling and on line detection. 54 was fabricated by modification of a previous design.17 Liquids The surgical procedures for global two vessel occlusion (2 55 were injected into the device via four inlets: P, Q and S for the 100 VO) cerebral ischemia/reperfusion were undertaken during the 56 probe solution, the control solution and the sample solution (i.e. period of in vivo microdialysis and on line measurements. The 2 VO ischemia model was constructed with ligation of the bilateral 57 45 the effluent from microdialysis), respectively, and O for the oil 58 phase. Outlet D was connected to a 10 cm long common carotids arteries (CCAs) with a 3 0 suture to induce 59 60 2 | Journal Name , [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] Page 3 of 7 Analyst
1 permanent forebrain ischemia, with the methods reported flow rate of the oil phase was observed to be 9.0 L/min, at 2 19, 20 previously. Briefly, through a midline cervical incision, both 60 which the generated droplets were uniform and reproducible. The 3 common carotid arteries (CCA) were exposed and isolated from volume of the droplets are estimated to be 0.23±0.02 L and the 4 surrounding connective tissue. Global ischemia and reperfusion time intervals between the two neighboring droplets are 4.6±0.3 s. 5 4 5 were induced by first occluding both carotid arteries with The flow rate of the droplets is estimated to be 7.5 ×10 m/s. The 6 nontraumatic arterial clips for ischemia and then removing the bisulfide concentration was quantitated based on the fluorescence 7 clips for reperfusion. Throughout the operation, supplements of 65 intensity of the droplets recorded at the detection point with CCD 8 chloral hydrate (100 mg/kg) were given as required, and the body camera, as described previously. The physical lag time between 9 temperature of the animals was maintained at 37 °C using a the microdialysis probe and the fluorescence detect point was 10 10 heating pad. The wound was sutured immediately after the approximately 10 min. 11 ischemia procedure was completed. 12 All experimental procedures were performed in compliance 13 with the relevant laws and institutional guidelines for the care and 14 use of laboratory animals in Peking University, Beijing, China, 15 15 and the experiments were also approved by the animal ethics 16 committee of Peking University. 17 18 Results and Discussions 19 20 Modification of the droplet-based microfluidic chip for on- 21 20 line measurement of bisulfide in the microdialysate 22 23 In our previous work, we have developed an AuNP GSH FITC probe which can rapidly response to the variation of bisulfide 24 17 25 concentration in the droplets of microdialysate. The probe 25 showed high selectivity for bisulfide over other possibly 26 coexisting anions, including sulfate, sulfite, bisulfite, thiosulfate, 27 Manuscript persulfate, citrate, thiocyanate, pyrophosphate, nitrite, oxalate, 28 bicarbonate, nitrate and bromide. Because of the steric hindrance 29 of the HS PEG COOH on the surface of AuNPs, the probe also 30 30 successfully resists the interferences from relatively large co 31 existing thiol containing biological molecules, such as GSH and 32 bovine serum albumin. With the addition of proper amount of 33 glyoxal, the interferences from small thiol containing biological 70 34 molecules such as Cys and Hcy could be also be effectively 35 Fig 1. (A) The relative recoveries of microdialysis probe CMA 35 prohibited. These off line experimental results suggested that 12 (20 kD, 4 mm) at different concentrations of bisulfide in aCSF 36 under normal biological concentration conditions of these perfusion solution. (B) Optimization of the glyoxal concentration Accepted 37 possible interfering species, their influences on bisulfide 38 75 for elimination of the influences from Cys and other biothiol detection are negligible. analogues. (C) On line, continuous monitoring of the variation of 39 However, unlike ex vivo measurements, the compositions of 40 bisulfide in aCSF microdialysate by using the modified droplet 40 in vivo biofluidic samples are variable, depending on the tested 41 based microfluidic system. The inset shows the calibration curve animal and specific experimental conditions. To avoid for quantification of the bisulfide concentration. The flow rates of 42 unexpected elevation of the level of interferences under certain 43 80 the he sample solution, probe solution and control solution are 1.0 circumstances, it is desirable to be able to perform on line spiking L/min. The flow rate of the oil phase is 9.0 L/min. 44 or examination of the reaction solutions in the droplets. To this
Analyst 45 45 end, we modified the pattern of the channels for the droplets 46 Under above flow rate conditions, we determined the relative formation. As illustrated in Scheme 1, in addition to the inlets of recovery of the microdialysis probe integrated with the droplet 47 the sample solution (S) and the probe solution (P), a third inlet (Q) 85 based microfluidic system (defined as the percentage of the 48 was added to allow injection of the quality control solution to the 49 bisulfide concentration in the droplets containing the dialysate droplets. A dual channel switching valve were employed to account for the original concentration of bisulfide in the perfusion 50 50 smoothly change the composition of the control solutions without solution). Fig. 1A showed the relative recoveries of microdialysis 51 affecting the flow rate of the stream. for bisulfide when using the microdialysis probe CMA 12 (20 kD, 52 The microdialysis rate is usually between 1.0 and 5.0 L/min. 90 4 mm). It can be seen that the recoveries were all above 50% at 53 To ensure relatively high microdialysis efficiency and stable bisulfide concentrations varying from 5.0 M to 40 M in the 54 mixing ratio, we chose to fix the flow rates of the sample solution perfusion, indicating that CMA 12 microdialysis probe was well 55 55 (i.e. the perfusion rate of the microdialysis probe), the probe suitable for measurement of bisulfide in brain regions with depth 56 solution and the control solution at 1.0 L/min. Then we tested larger than 4 mm. For bisulfide concentrations higher than 80 57 different flow rates of the oil phase in order to obtain stable and 95 M, the recovery may be improved by applying slower flow 58 continuous spherical droplets in the PTFE tube. The optimum 59
60 This journal is © The Royal Society of Chemistry [year] Journal Name , [year], [vol] , 00–00 | 3 Analyst Page 4 of 7
1 rates. For other brain regions with smaller size, microdialysis among the three tested rats exhibited relatively higher responses 2 probe with shorter membrane length should be used. Therefore, 60 to the insertion of the microdialysis probe. Whether or not these 3 we further tested another microdialysis probe MAB6 (15 kD, 2 signal variations are associated with the age or weight or other 4 mm) with a smaller cut off and shorter membrane length, which conditions of the test animals merits further detailed 5 5 showed an average recovery of 25±3%. For comparison, we also investigations. 6 measured the recoveries of Cys in the concentration range from To verify that the detection system works well during the 7 50 M to 200 M under the same experimental conditions, which 65 microdialysis processes, we spiked the droplets with known 8 were found to be lower than 10%. These results suggested that amount of Na S standard solutions (10 M). As expected, the on 9 2 the microdialysis probe could effectively exclude Cys and other line system showed a large fluorescence response to the addition 10 10 thiol containing analogues and reduce their influences on the of bisulfide (Fig. 2B), indicating that the reaction between the 11 bisulfide detection. AuNP–GSH FITC probe and bisulfide were not affected by the 12 Previous off line experimental results showed that addition of 70 matrices present in the cerebral microdialyate solution. We also 13 0.1% glyoxal to the reaction solution can effectively eliminate the compared the detection results of the same cerebral microdialyate 14 influence of Cys at concentration as high as 20 M. 17 To further solution with or without the addition of 0.03% glyoxal to the 15 15 confirm that Cys and Hcy won’t influence the bisulfide detection control solution. No significant differences were observed 16 even at higher concentrations, we tested to add glyoxal to the between the fluorescence signals of bisulfide in the cerebral 17 reaction solution in the droplets to eliminate their interferences. 75 microdialyate solution, indicating that the influences of Cys and 18 Since long time co incubation of the AuNP–GSH FITC probe other thiol containing molecules on the on line detection were 19 with glyoxal may cause increase of the fluorescence background, negligible. To further rule out the false positive signals possibly 20 20 we added glyoxal to the control solution which was included in induced by other ions, more negative control experiments can be 21 the reaction solution as soon as the sample and the probe were performed by selectively masking bisulfide in the sample solution 22 mixed together. By fixing the concentration of Cys at 50 M, we 80 and monitoring whether the signals are inhibited. 23 optimized the amount of glyoxal in the control solution. As 24 shown in Fig. 1B, addition of 0.03% glyoxal in aCSF to the 25 25 droplets could reduce the signal of 50 M Cys to less than 15% 26 of the original value. As the recovery of Cys was less than 10%,
27 its concentration in the microdialysate would be far less than 50 Manuscript 28 M. Hence, addition of 0.03% glyoxal to the droplets was 29 sufficient to prohibit the interferences from Cys and other 30 30 biothiol analogues. Fig. 1C displays the on line fluorescent 31 responses to the variation of concentrations of bisulfide in the 32 perfusion solution in the presence of 0.03% glyoxal. The inset of 33 Fig. 1C shows the calibration curve on a log–log scale for 34 quantification of bisulfide based on the increase of fluorescence 35 35 intensity. The linear working range was from 5.0 M to 40 M 36 and the limit of detection (based on a signal to noise ratio of 3)
37 was calculated to be 2.5 M. Accepted 38 39 On-line monitoring of the variation of bisulfide in the brain of 40 40 awake rats by using the integrated microdialysis/microfluidic 41 chip system 42 43 For in vivo measurement, a syringe pump in withdraw mode 44 was attached to the outlet of the PTFE tube to help reduce the
45 pressure of the liquid streams in the channels of the microfluidic Analyst 46 45 chip caused by insertion of the microdialysis probe into the tissue. 47 The withdraw flow rate was set at 19.0 L/min. To measure the 48 basal level of H2S in awake rats, we anesthetized the rat with 49 ether and fixed it on a home made rat holder before it woke up. 50 Then we inserted the microdialysis probe into the guide cannula 51 50 and the bisulfide concentration in the cerebral microdialysate was Fig. 2. (A) On line fluorescence signals of the bisulfide in the 52 continuously monitored by the microfluidic droplet system. Fig. hippocampus of rat’s brain under awakened state. (B) Fluorescent 53 2A compares the on line fluorescence signals of the bisulfide in 85 responses to the on line spiking of the microdialysate with Na 2S 54 the microdialysate of three different rats. As can be seen, the standard solutions. Microdialysis probe MAB6 (15 kD, 2 mm) 55 fluorescence signal showed a significant increase when the probe was used for the in vivo microdialysis. The flow rates of the 56 55 was inserted into the brain tissue through the cannula, indicating sample solution, probe solution and control solution are all 1.0 57 a notable elevation of bisulfide concentration in response to the L/min. The flow rates of oil phase and the outflow solution are 58 slight injury of the brain tissue. Then the fluorescent signals 90 9.0 L/min and 19.0 L/min, respectively. 59 gradually declined to a stable level. Interestingly, the smallest one 60 4 | Journal Name , [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] Page 5 of 7 Analyst
1 Quantitative measurement of the bisulfide concentration in 2 rat’s brain under different physiological conditions 3 4 To measure the bisulfide level in the rat’s brain under different 5 physiological conditions, we used chloral hydrate to anesthetize 6 5 the rats. First, we compared the differences between the bisulfide 7 concentrations in the hippocampus of the same rat under 8 anesthetized state and awakened state. Fig. 3A shows the on line 9 fluorescence signals of the bisulfide in the cerebral 10 microdialysate. As can be seen, under anesthetized state, the 11 10 concentration of bisulfide didn’t show similar dramatic increase 12 as that observed in the awakened rats (see Fig. 2A). By contrast, 13 the fluorescence signals only showed a moderate increase from 14 the baseline level, indicating weak responses of the rat under 15 anesthetized state to the slight brain tissue injury caused by 16 15 insertion of the probe. After about 30 min, the rat woke up, and 17 the fluorescence signals gradually increased to a relatively high 18 concentration level, indicating production of larger amount of
19 H2S by the rat under awakened state. Based on the calibration 20 curve and the microdialysis recovery measured in the same 21 20 experiment, the in vivo concentration levels of bisulfide in the 22 hippocampus of the tested rat under anesthetized state and 23 awakened state were observed to be 7.8±4.0 M and 12.2±3.4 24 M, respectively. 25 Ischemic stroke is caused by an interruption of blood supply to 26 25 the brain (global) or part of the brain (focal) either by thrombosis
27 or embolism. Previous studies suggested that H 2S was involved in Manuscript 28 the pathogenesis of ischemic stroke. 21 We investigated the Fig. 3. Fluorescence time responses recorded for the bisulfide in 29 dynamic changes of bisulfide before and after global cerebral 60 the microdialysates continuously sampled from the hippocampus 30 ischemia by using the established on line detection system. The of the same rat under different physiological conditions. (A) The 31 30 rat was kept anesthetized with chloral hydrate throughout the basal level of bisulfide in the hippocampus under anesthetized 32 whole experiments. We started to monitor the fluorescence and awakened state. (B) Dynamic variation of the bisulfide 33 signals of bisulfide in the hippocampus right after the two concentration in the hippocampus before and during 2 VO global 34 common carotid arteries of the tested rat were exposed and 65 cerebral ischemia/reperfusion. Microdialysis probe MAB6 (15 35 isolated from surrounding connective tissues through a midline kD, 2 mm) was used for the in vivo microdialysis. The flow rates 36 35 cervical incision. After 110 min, a 10 min 2 VO ischemia was of the sample solution, probe solution and control solution are all
37 induced, followed by reperfusion. The results were shown in Fig. 1.0 L/min. The flow rates of oil phase and the outflow solution Accepted 38 3B. The average concentrations of bisulfide in the hippocampus are 9.0 L/min and 19.0 L/min, respectively. 39 of the tested rat during the first 110 min were found to be 70 40 22.1±3.5 M. These values were much higher than those shown In comparison with the existing approaches for H S detection, 41 40 in Fig. 3A which were observed under the same anesthetized 2 the established on line, in vivo approach may prevent unwanted 42 conditions, indicating that large amount of H 2S had been produced by the tested rat after it undertook the incision surgery. conversions during sample pretreatment and undesirable 43 24, 25 44 Then the bisulfide concentration was observed to decline to 75 volatilization of the target during detection process.
45 13.9±3.7 M during the 10 min 2 VO ischemia and subsequent Continuous monitoring may provide much better insight into the Analyst changes of H S concentration under different physiological 46 45 reperfusion. The reason for these variations of the H2S level in 2 47 hippocampus remain to be further investigated. conditions. The method will greatly facilitate the investigations of the broad brain functions of H S 48 Previous studies reported that the concentration of H 2S in brain 2 9, 22 80 49 was in the range from 50 to 160 M. More recently, some Conclusions 50 other studies revealed that the concentration of H 2S in brain was 6, 23 50 much lower, which was in the nanomolar range. Our study 51 In this work, we have demonstrated an effective online approach 52 showed that the bisulfide concentrations in the brain were less than 30 M under all the tested conditions, suggesting that the for in vivo, continuous measurements of cerebral bisulfide in 53 rat’s brain. The method was based on an integrated system of in 54 H2S in rat’s brain are in the range below 40 M. This agrees with the general consensus that the earlier measurements of 50 160 85 vivo microdialysis and a modified droplet based microfluidic 55 chip. The system was selective, reliable and reproducible, which 55 M were overestimated, most likely due to unintended 56 enabled continuous measurement of cerebral bisulfide under 57 conversions during tissue process. various physiological conditions. The endogenous levels of H 2S 58 were for the first time measured in the brain of living animals. 59
60 This journal is © The Royal Society of Chemistry [year] Journal Name , [year], [vol] , 00–00 | 5 Analyst Page 6 of 7
1 The results of this study not only offers a useful approach for 23. J. Furne, A. Saeed and M. D. Levitt, Am J Physiol Regul 2 Integr Comp Physiol , 2008, 295 , R1479 1485. physiological and pathological investigations associated with H2S, 3 70 24. S. K. Pandey, K. H. Kim and K. T. Tang, Trac-Trends Anal but also provides a practical platform for on line, in vivo and 4 Chem , 2012, 32 , 87 99. continuous monitoring of other important species in cerebral 25. T. S. Bailey and M. D. Pluth, J. Am. Chem. Soc. , 2013, 135 , 5 5 systems. 16697 16704. 6
7 Acknowledgments 75 8 The work was supported by National Natural Science Foundation 9 10 of China (91132717) and Beijing Natural Science Foundation 11 10 (2152014). 12 13 Notes and references 14 15 a Beijing National Laboratory for Molecular Sciences, MOE Key 16 Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, 17 15 Beijing, 100871, China. E-mail: [email protected] 18 b Beijing National Laboratory for Molecular Sciences, Key Laboratory of 19 Analytical Chemistry for Living Biosystems, Institute of Chemistry, the 20 Chinese Academy of Sciences (CAS), Beijing 100190, China. E-mail: 21 [email protected] 20 22 23 1. K. Abe and H. Kimura, J Neurosci , 1996, 16 , 1066 1071. 24 2. R. Wang, Physiol Rev , 2012, 92 , 791 896. 25 3. J. Z. Weimin Zhao, Yanjie Lu and and R. Wang, EMBO J. , 25 2001, 20 , 6008 6016. 26 4. J. W. Elrod, J. W. Calvert, J. Morrison, J. E. Doeller, D. W.
27 Kraus, L. Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Manuscript 28 Kimura, C. W. Chow and D. J. Lefer, Proc Natl Acad Sci U S A, 2007, 104 , 15560 15565. 29 30 5. B. H. Tan, P. T. H. Wong and J. S. Bian, Neurochem Int , 30 2010, 56 , 3 10. 31 6. M. Ishigami, K. Hiraki, K. Umemura, Y. Ogasawara, K. Ishii 32 and H. Kimura, Antioxid Redox Signal , 2009, 11 , 205 214. 7. L. F. Hu, M. Lu, P. T. Hon Wong and J. S. Bian, Antioxid 33 35 Redox Signal , 2011, 15 , 405 419. 34 8. J. E. Doeller, T. S. Isbell, G. Benavides, J. Koenitzer, H. Patel, 35 R. P. Patel, J. R. Lancaster, Jr., V. M. Darley Usmar and D. W. Kraus, Anal Biochem , 2005, 341 , 40 51. 36 9. L. R. Goodwin, D. Francom, F. P. Dieken, J. D. Taylor, M. W. Accepted 37 40 Warenycia, R. J. Reiffenstein and G. Dowling, J Anal Toxicol , 38 1989, 13 , 105 109. 39 10. J. J. E. G. J. Revuelto Rey, M.A. Munoz Sánchez, F. Murillo Cabezas, Medicina intensiva , 2012, 36 , 213 219. 40 11. A. Gunther, S. A. Khan, M. Thalmann, F. Trachsel and K. F. 41 45 Jensen, Lab on a Chip , 2004, 4, 278 286. 42 12. A. Gunther, M. Jhunjhunwala, M. Thalmann, M. A. Schmidt 43 and K. F. Jensen, Langmuir , 2005, 21 , 1547 1555. 13. A. Gunther and K. F. Jensen, Lab on a Chip , 2006, 6, 1487 44 1503. 45 50 14. J. G. Kralj, H. R. Sahoo and K. F. Jensen, Lab on a Chip , Analyst 46 2007, 7, 256 263. 47 15. L. W. Norton, F. Yuan and W. M. Reichert, Anal Chem , 2007, 79 , 445 452. 48 16. G. T. R. Meng Wang, Kristin Schultz, Colin Jennings, Robert 49 55 T. Kennedy, Anal Chem , 2008, 80 , 5607–5615. 50 17. X. Zhu, L. Xu, T. Wu, A. Xu, M. Zhao and S. Liu, Biosens Bioelectron , 2014, 55 , 438 445. 51 18. K. Liu, Y. Lin, P. Yu and L. Mao, Brain Res , 2009, 1253 , 161 52 168. 53 60 19. M. De Butte, T. Fortin and B. A. Pappas, Neurobiol Aging , 54 2002, 23 , 309 317. 20. I. Yonekura, N. Kawahara, H. Nakatomi, K. Furuya and T. 55 Kirino, J Cereb Blood Flow Metab , 2004, 24 , 151 158. 56 21. A. S. Tay, L. F. Hu, M. Lu, P. T. Wong and J. S. Bian, 57 65 Neuroscience , 2010, 167 , 277 286. 58 22. J. C. Savage and D. H. Gould, J Chromatogr , 1990, 526 , 540 545. 59 60 6 | Journal Name , [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] Page 7 of 7 Analyst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Graphical Abstract 24 208x98mm (300 x 300 DPI) 25 26 27 Manuscript 28 29 30 31 32 33 34 35 36
37 Accepted 38 39 40 41 42 43 44
45 Analyst 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60