NO Microsensor Manual
Contents
1. Measurements of Nitric Oxide in Cardiovascular Research Using Electrochemi cal Microsensors J Vasc Res 43 70 85 21 TROUBLE SHOOTING Problem Possible cause Solution Problem Solution Problem Possible cause Solution Problem Possible cause Solution Problem Possible cause Solution High and drifting signal Gas bubbles present inside the sensor tip due to short circuit or electrical shock Degas water by boiling and subsequent cooling or by 10 minutes of vacuum treatment Immerse the sensor tip for 20 min in the degassed water Repeated or prolonged treatment may be necessary The sensor tip is broken Replace the NO microsensor Signal very low Inactivation of the anode surface Replace the NO microsensor Signal constantly very low and no response to NO Gas bubbles present inside the tip of the sensor causes a disruption in the electrolyte Degas water by boiling and subsequent cooling or by 10 min of vacuum treatment Immerse the sensor tip for 20 min in the degassed water Repeated or prolonged treatment may be necessary Slow response Insoluble compounds deposited at the sensor tip Consult the Cleaning section Problem Possible cause Solution Problem Possible cause Solution Slow response Especially needle sensors A gas bubble is trapped at the needle tip Remove the gas bubble by gentle movements of the sensor Unstable signal or the signal fluctuates if2. and then rinse with water this will remove most substances Alternatively it is possible to rinse with 0 1M NaOH isopropanol or detergents In some cases the deposits need physical abrasion to be removed contact Unisense for advice on this Solubility of NO in water Reference SOLUBILITY OF SELECTED GASES IN WATER by L H Gevantman CRC Handbook of Chemistry and Physics 92nd Edition 20 REFERENCES Aamand R et al 2009 Generation of nitric oxide from nitrite by carbonic anhydrase a possible link between metabolic activity and vasodilation Am J Physiol Heart Circ Physiol 297 H2068 H2074 Barak Y et al 2010 Role of nitric oxide in Salmonella typhimurium mediated cancer cell BMC Cancer 10 1 146 Ettwig et al 2010 Nitrite driven anaero bic methane oxidation by oxygenic bacteria Nature 464 543 548 Pryor W et al 2006 Free radical biology and medicine it s a gas man Am J Physiol Regu latory Integrative Comp Physiol 291 491 511 Schreiber et al 2008 Nitric Oxide Mi crosensor for High Spatial Resolution Mea surements in Biofilms and Sediments Anal Chem 80 4 1152 1158 Schreiber F et al 2009 Mechanisms of tran sient nitric oxide and nitrous oxide produc tion in a complex biofilm The ISME Journal 3 1301 1313 Schreiber F et al 2010 Denitrification in hu man dental plaque BMC Biology 8 1 24 Wadsworth R et al 2006 Physiologically Relevant
3. the set up is touched or equipment is intro duced in the medium you are measuring in Electrical disturbance of the sensor through the tip membrane Ground the set up using the blue grounding cable supplied with the picoam meter Connect the reference plug on the picoammeter blue plug with the medium you are measuring in Please see directions on page 10 of this manual If you encounter other problems and need scientific technical assistance please contact sales unisense com for online support we will answer you within one workday 23 27 dy UNISENSE UNISENSE DENMARK www unisense com infogunisense com
4. will be very high drop rapidly over the first few minutes and then drop more slowly For new sensors and sensors that have not been in use for several days it takes up to 24 hours for the signal to stabilize After shorter periods without polarization the sensor should be polarized until it has exhibited a stable signal for 10 minutes If the signal does not stabilize below 20 pA at room temperature please look in the Trouble shooting section of this manual 11 CALIBRATION Before concentration measurements can be performed a calibration should be performed The NO sensor responds linearly to NO from zero up to a maximum linear concentration Thus a two point calibration is sufficient The maximum linear concentration depends on the application the sensor is designed for The calibration standards should be within the linear range for the sensor in question and cover the expected concentration range to be measured e g zero NO 0 atm and 1 uM Using stock SOLUTION Please see the following section Stock solutions and NO donors for instruction on preparation ZERO READING Note the signal SO in water without any NO or interferents Note Be careful if you try to use gas sparged water which has contained NO NO will convert to NO in the presence of oxygen and if the water is acidic HNO is formed HNO interferes on the NO sensor and is not easily removed by sparging the water HiGH READING NO wil
5. 2 b Aresearcher implements the above principle in the following way bubbles NO gas from a pressure gas tank through two washing bottles in series containing 5 M NaOH These bottles capture impurities in the form of NO and NO gas After the washing bottles the gas is lead through a long needle that penetrates a septum in a vial The vial has a few ml distilled water inside and the NO gas bubbles through this water A second needle through the septum leads from the headspace to the exterior to allow the gas to escape The set up is kept in a fume hood Bub bling for a approx 15 minutes results in a clean saturated stock solution of NO in the vial This stock is diluted for calibra tion standards After the NO production the system is flushed for 45 minutes with Argon if NO is left in the system it will attack rubber tubing 2 Chemical NO synthesis Another alternative is NO synthesis from NaNO with the follow ing procedure Concentrated H SO is slowly added to a N flushed mixture of saturated NaNO and KI This evolves NO gas which is passed through a wash bottle with 10 M NaOH to remove traces of NO The purified NO gas is captured in a vial with a mem brane which contains a few ml of distilled water A second needle through the septum leads from the headspace to the exterior to allow the gas to escape Ref Diab et al 2005 Electrochemical nitric oxide sensor preparation a comparison of two electrochemical metho
6. E SENSORS Although the Unisense microsensors are made of glass the tip is flexible and can bend slightly and the sensors are surprisingly robust in use in coarse substrates However lateral movements ofthe sensor when the tip is in contact with a solid substrate may easily cause the tip to break Also due to the small size of the microsensor tip and to the steepness of gradients in many environments even a displacement of the sensor tip of few microns may change its environment Therefore we recommend that measurements are performed only in a stabilized set up free of moving or vibrating devices We recommend the Unisense lab stand LS18 and the Unisense micromanipulator MM 33 or double MM33 2 for laboratory use For in situ use we recommend our in situ stand 1519 and a micromanipulator or our automated in situ profiling instruments WARNING WARNING WARNING WARNING WARNING Itis extremely important to ground the NO sensor before use Otherwise the sensor is very susceptible to electrical disturbances and in some cases will be damaged Please read the information and directions in this manual and contact Unisense if you are in doubt of what to do ELECTRICAL NOISE The signal of a NO microsensor is very small 10 to 10 ampere Although both the Unisense amplifiers and microsensors in general are relatively resistant to electrical noise from the environment electrical fields may interfere with the sensor signal NO microsensors
7. Vh UNISENSE NITRIC OXIDE SENSOR USER MANUAL NITRIC OXIDE SENSOR USER MANUAL UNISENSE A S NITRIC OXIDE SENSOR USER MANUAL Copyright 2014 Unisense A S Version January 2014 TABLE OF CONTENTS SUPPORT ORDERING AND CONTACT INFORMATION OVERVIEW EE uin a E n ea RUE WORKING PRINCIPLE GETTING STARTED 5222655 bahn UNPACKING A NEW SENSOR GROUNDING THE SET UP POLARIZATION PRE POLARIZATION CALIBRATION STOCK SOLUTIONS AND NO MEASUREMENTS 5 22a MOUNTING OF THE SENSORS INTERFERENCE ADVANCED USE OF THE NO SENSORS STORAGE AND STORAGE CLEANING REFERENCES 66 dem la RE d TROUBLESHOOTING Mu un re bh br RE TER sas WARRANTY AND LIABILITY NOTICE TO PURCHASER This product is for research use only Not for use in human diagnostic or therapeutic procedures WARNING Microsensors have very pointed tips and must be handled with care to avoid personal injury and only by trained personnel Unisense A S recommends users to attend instruction courses to ensure proper use of the products WARRANTY AND LIABILITY The NO microsensor is covered by a 90 days limited warranty Microsensors are a consumable Unisense will only replace dysfunctional sensors if they have been tested according with the instructions in the manual within 14 days o
8. are more susceptible to electrical noise than e g IMPORTANT Always introduce and retract the NO microsensor axially using a micro manipulator and a stable stand when measuring in solid or semisolid substrate like sediment tissue biofilms microbial mats etc IMPORTANT To resolve the very small concentrations that are most often of interest it is very important to eliminate electrical noise as much as possible This can be done by grounding the set up if you have a PA2000 you can place an analog filter on it 17 oxygen microsensors so it is necessary to ground the set up This is done by creating an electrical connection between the Ground connection of the picoammeter and the sample to be studied tissue sediment core water etc Also unnecessary electrical mechanical equipment should be switched off and touching components of the set up during calibrations and measurements should be avoided On suspicion of sensor damage repeat calibration and consult Trouble shooting INTERFERENCE The following compounds and physical parameters are known to exhibit interference on the NO microsensor HNO 5 light temperature Keep updated on reported interferents on our web page Concentratlon nM 5 0 100 200 300 400 500 Time s The graph shows a NO sensor response to sequential addition of 12nM of NO Data points were taken every second Each of the shown points is the running ave
9. ds of electrode surface modification Bioelectrochemistry 66 105 110 15 As an alternative to NO stock solutions some researchers use the a compound that can decompose and release NO NO donor 1 Ref Richardson et al 2006 The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immu nity Molecular Microbiology 2006 61 4 927 939 Nitric oxide donating com pounds used in this study were obtained from Alexis Biochemicals San Diego CA Diethylamine NONO ate DEA NO t1 2 2 min Proline NONO ate ProliNO t1 2 1 8 s and Diethylethylenediamine NONO ate NOC 12 t1 2 100 min were dissolved to 500 mM final concentration in 0 01 N NaOH and stored at 80 C SNAP was resuspended to a final concentration of 500 mM in DMSO and aliquots were stored at 80 C The conjugate donor compounds diethylamine Sigma D 0806 proline Sigma P 0380 di ethylethylenediamine Aldrich 126942 and NAP Aldrich A19008 were diluted to same concentrations as cognate NONO ates in like solvents and stored at 80 2 Forfurther information on the SNAP method Ref Xhang et al 2000 Novel Cali bration Method for Nitric Oxide Microsensors by Stoichiometrical Generation of Nitric Oxide from SNAP Electroanalysis 12 6 425 428 MEASUREMENTS Before doing microscale measurements the seal must be broken and the protective tube removed NOTE this terminates Unisense s warranty MOUNTING OF TH
10. e NO sensor before use Otherwise the sensor is very susceptible to electrical disturbances and in some cases will be damaged Please read the information and directions in this manual and contact Unisense if you are in doubt of what to do GROUNDING THE SET UP Before connecting the NO sensor it is important to ground the set up both the measurement media and the picoammeter Grounding the set up means to stabilize its electrical potential to avoid noisy signals and or damage to the sensor Grounding is done by connecting any media liquids the sensor will touch to the ground connection of the picoammeter using the provided wire and connecting the ground plug of the picoammeter to an earth line An earth line can often be found in electrical outlets alterna tively a good electrical connection to a water pipe may be used If an earth line is not available at least keep media and picoammeter ground connected TECHNICAL DETAILS The tip membrane of a microsensor serves as an electrical barrier which minimizes the effect of electrical disturbances If however a sufficiently high electrical potential has built up across the membrane the resistance is not high enough to prevent a discharge of electricity across the membrane Often this happens when a person carrying electrical charges touches the set up The discharge across the membrane can cause a permanent WARNING Do not remove the seal and protective plastic tube before thes
11. e steps and calibration are successfully completed breakdown of the electrical resistance of the membrane and subsequently electrical noise e g 50 60 Hz noise from mains power installations can then affect the signal severely As NO sensors have particularly thin membranes they are more prone to this problem than other sensors See also the Troubleshooting section POLARIZATION The signal from the NO sensor is a small current in the picoampere range Therefore the NO sensor must be connected to a polar izing picoammeter e g a Microsensor Monometer a Microsensor Multimeter or the Unisense PA2000 NO sensors should be polarized at 1 25 V The polarization voltage is set in the amplifier For details on how to set and check the polarization consult the user manual of the picoammeter to be used If you are using a PA2000 amplifier please check the polarization voltage before connecting the sensor since incorrect polarization may destroy the sensor PRE POLARIZATION When the sensor is not in use NO will build up inside the elec trolyte This must be removed order to obtain a stable zero current as background for calibrations and measurements and thus stable operation of the sensor is possible Therefore a period of polar ization is necessary before you can use the sensor This is called the pre polarization period When the NO sensor is connected to the picoammeter and polarization of the sensor is initiated the signal
12. f receipt of the sensor s The warranty does not include repair or replacement necessitated by accident neglect misuse unauthorized repair or modification of the product In no event will Unisense A S be liable for any direct indirect consequential or incidental damages including lost profits or for any claim by any third party arising out of the use the results of use or the inability to use this product Unisense mechanical and electronic laboratory instruments must only be used under normal laboratory conditions in a dry and clean environment Unisense assumes no liability for damages on laboratory instruments due to unintended field use or exposure to dust humidity or corrosive environments REPAIR OR ADJUSTMENT Sensors and electrodes cannot be repaired Equipment that is not covered by the warranty will if possible be repaired by Unisense A S with appropriate charges paid by the customer In case of return of equipment please contact us for return authorization For further information please see the document General Terms of Sale and Delivery of Unisense A S as well as the manuals for the respective products CONGRATULATIONS WITH YOUR NEW PRODUCT SUPPORT ORDERING AND CONTACT INFORMATION The Nitric Oxide NO microsensor is a miniturized sensor for measuring the partial pressure of NO gas in the nanomolar range If you wish to order additional products or if you encounter any problems and need scientific technica
13. l assistance please do not hesitate to contact our sales and support team We will respond to your inquiry within one working day E mail sales unisense com Unisense A S Tueager 1 DK 8200 Aarhus N Denmark Tel 45 8944 9500 Fax 4 45 8944 9549 Further documentation and support is available at our website www unisense com REPLACEMENT OF SENSORS Unisense will replace sensors that have been damaged during shipment provided that The sensors were tested immediately upon receipt in accordance with the delivery note and the manual The seal is still intact Thesensors are returned to Unisense for inspection within two weeks The sensors are correctly packed for return to Unisense in accordance with the note included in the sensor box THIS MANUAL COVERS THE FOLLOWING SENSORS NO10 tip diameter 20 um NO100 tip diameter 70 110 um NO MR designed for use with microrespiration system NO N Needle type NO sensor NO R NO sensor with protective cap NO spec NO sensors with customer specified specifications RECOMMENDED METERS FOR NO SENSORS One channel meter Microsensor Monometer Multi channel meter Microsensor Multimeter OVERVIEW The Unisense nitric oxide NO microsensor is designed for WARNING research applications within physiology biotechnology environ Unisense sensors mental sciences and related areas are neither intended nor The Unisense NO microsensor can be made with approved for
14. l react spontaneously with oxygen so an NO cali bration solution should be made in an anoxic environment A continuously anoxic solution can be maintained in the following manner Get a vessel preferably made of glass with a lid with two holes in it e g Unisense CAL300 One hole is for sparging with oxygen free gas e g N gas the other hole is for inserting the NO sensor The vessel is filled 34 with neutral pH buffered water and there is thus a headspace under the lid The sensor is placed through the sensor hole in the lid with its tip a few cm inside the buffer The oxygen free gas is led through the gas sparging hole through a long pipe or needle that opens at the bottom of the vessel Bubbling oxygen free gas through the buffer solution will remove all oxygen The gas should stream fast enough to effectively flush the headspace preventing atmospheric oxygen from entering through any of the holes in the lid For the Unisense calibration chamber CAL300 5 minutes of bubbling at a rate of 0 5 12 IMPORTANT Calibration must be performed after pre polarization when the sensor signal has stabilized Always usea calibration solution with the same temperature and salinity as the sample solution L gas per minute is sufficient to drive out 9996 of the oxygen After the buffer is stripped of oxygen the gas pipe is retracted somewhat such that it opens in the headspace An small volume of an NO stock solution see Stock sol
15. rage of 10 measured data points ADVANCED USE OF THE NO SENSORS Unisense can construct NO sensors for customer requested applications at additional costs The most frequent construction options are described at our website Options include customer specified modifications response time stirring sensitivity pressure tolerance range and detection limit If your requirements for a NO sensor are not described on our website please contact sales unisense com for further options and prices EXAMPLES OF ADVANCED APPLICATIONS Respiration production rates in small samples in Unisense microrespiration cham bers MR Ch Measurements of NO under high external pressure e g in closed pressurized systems or in the deep sea Flow through cell measurements 19 STORAGE AND MAINTENANCE STORAGE Store the sensor in the protective plastic tube used for shipping The NO microsensor can be stored with the tip exposed to water or air The room in which the NO microsensor is stored should be dry and not too hot 10 30 C If the sensor is used regularly it can be stored polarized connected to a Unisense Meter CLEANING The sensor tip can be sterilized with ethanol During use different compounds may deposit on the sensor tip resulting in a less sensitive and slower sensor response Depending on the substance different solutes can remove the deposit The standard procedure is to rinse with 9696 ethanol then rinse with 0 01 M HCl
16. urements as long as the sensor is regularly calibrated Using NO Donors Some researchers prefer making calibrations with so called NO donors We will not describe a procedure here However refer ences can be found in the Stock solutions and NO donors section WARNING Bubbling of water with gas may cause the water to change temperature due to the gas temperature or evaporative cooling Monitor the temperature to find a suitable bubbling rate which does not does not change the water temperature significantly 13 STOCK SOLUTIONS AND NO DONORS There are several ways to prepare an NO stock solution 1 Using NO gas a Asealed vialis half filled with distilled water or buffer flushed with argon to remove oxygen and the headspace is finally vacuum treated NO gas from a pressure gas tank is bubbled through a washing bottle containing 1 M NaOH The bottle captures impurities in the form of N O and N O gas The gas streams long enough to flush all oxygen out After the washing bottles the gas is lead through a needle The needle is led to penetrate the septum in the vial filling the headspace with a slight over pressure of NO Subsequently the vial is shaken to equilibrate the headspace NO with the water buffer before the sur plus pressure is removed by puncturing the membrane Ref Methods in Molecular Biology Vol 100 Nitric Oxide Protocols Ed Michael A Thitheradge Humana Press Inc Totowa NJ p 23
17. use on humans asmall tip size 10 um which ensures excel lent response time and insignificant stirring sensitivity and makes it possible to make reliable and fast measurements with high spatial resolution e a larger tip size 2100 um which allows for low detection limit approx 2 3 nM For non destructive measurements the sensor should be designed to have a small tip for other applications the sensor is designed for maximum sensitivity WORKING PRINCIPLE The NO sensor is a miniaturized Clark type sensor which must be connected to a high sensitivity picoammeter The measuring anode inside the sensor tip is polarized against an internal reference electrode by the picoammeter Driven by the external partial pressure NO from the environment will penetrate the sensor tip membrane and reach the anode surface where it is oxidized The picoammeter converts the resulting oxidation current to a voltage signal The Unisense NO sensor has an extremely low consumption compared to other standard NO sensors The maximum consumption i e for the NO100 is 935 femtomol hour which corresponds to 0 196 hour in a 1ml 0 1uM NO sample GETTING STARTED UNPACKING A NEW SENSOR When receiving a new NO microsensor remove the shock absorbing grey plastic net Please do not remove the seal and protective tube before the following steps are successfully completed WARNING WARNING WARNING WARNING WARNING Itis extremely important to ground th
18. ution and NO donors section below is injected into the buffer The buffer is stirred briefly to distribute the NO evenly Stirring can be done with a a glass rod through the gas flushing hole b brief bubbling with the gas pipe or c with a stirrer magnet when the vessel is placed on a stirrer plate The continued gas flow in the headspace will keep oxygen out but will only slowly remove NO from the buffer as opposed to bubbling which will remove NO quickly The high calibration reading S1 is noted corresponding to the concentration C1 calculated from the stock concentration and the dilution ratio To convert a signal S from partial pressure to the corresponding concentrations of NO C perform a linear conversion C C1 S 150 51 50 If you use Unisense software the calibration calculations can be performed by the software Consult the software manual for details Check and repeat calibration at appropriate time intervals to ensure that all measurements can be calibrated to correct concentrations When the sensor is new you may need to calibrate more often while an older extensively used sensor may require calibration only every 24 hours or less To minimize the need for calibration it is recommended to keep the sensor polarized between measurements The membrane permeability of NO microsensors changes with time so a change in signal of up to 5096 may occur over months This does not affect the quality of the meas
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