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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 important species in cerebral systems. 31 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 µm 5 wide, and the nozzle where the droplets were formed was 30 µm 6 wide (Scheme 1A). The main channel where the droplets flew 7 through was 120 µm wide. The depth was 40 µm 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 µL/min and 9.0 µL/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 µL/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.