NO - BIOSENSOR USER MANUAL
Contents
1. 0 n y N i Knob 5nid On31 Fig 8 Biochamber and transducer separated SWITCHING SPECIFICITY The sensor can switch specificity from nitrate nitrite NO to nitrite NO and vice versa by replacing the biochamber of the 27 sensor However each chamber can only be used once After a biochamber has been used the pressure has changed inside andit can not be re used Follow the procedures in the section Replacement of biochamber Lid Locking ring Tape Fig 9 Biochamber 28 SPECIFICATIONS EFFECTIVE MEASURING RANGE The effective measuring range is defined as the range of NO concentrations over which the sensor has a linear response to NO Higher temperatures result in a larger measuring range because the activity of the bacteria is positively correlated to temperature Toxic compounds in the external environment have the opposite effect as they reduce the bacterial activity MEASURING RANGE AT 20 C 0 1000 uM at maximum 3 8 salinity MEASURING RANGE AT 10 C 0 200 uM NO at maximum 3 8 salinity OUTPUT 1 25 4 nA per 100 uM net signal at 20 C Signal as a function of NO concentration is positively correlated with temperature The temperature coefficient is 2 4 per C The calibration of the sensor is dependent on the composition of ions in the external environment The net signal can vary up to 30 depending upon the ionic co2. UNISENSE NO BIOSENSOR USER MANUAL NO BIOSENSOR USER MANUAL Copyright O 2012 Unisense A S Version October 2012 BIOSENSOR USER MANUAL UNISENSE A S NO Biosensor User MANUAL Copyright 2012 Unisense A S Version October 2012 TABLE OF CONTENTS WARRANTY AND LIABILITY 0 ccc ccc cece 8 CONGRATULATIONS WITH YOUR NEW 9 SUPPORT ORDERING AND CONTACT INFORMATION 9 OVERVIEW asa Re KEI Kie did 11 GETTING STARTED sus esa samira cc sense ae 14 BEFORE USING THE BIOSENSOR 14 POLARIZATION OF THE N O TRANSDUCER 15 CALIBRATION 16 GENERAL USE OF THE BIOSENSOR asas aire cs ame een aaa aina dasa 21 MEASUREMENTS 21 CHANGING SALINITY OF THE SAMPLE SOLUTION 21 INTERFERENCE 22 ADVANCED USE OF THE BIOSENSOR 23 NITROUS OXIDE MEASUREMENTS 23 MEASUREMENTS WITH A FLOW CELL 26 STORAGE AND MAINTENANCE oossoo rra 29 STORAGE OF THE BIOSENSOR 29 STORAGE OF THE BIOCHAMBER 29 REPLACEMENT OF THE BIOCHAMBER 29 SWITCHING SPECIFICITY 32 SPECIFICATION Socio iii DO c 34 TROUBLE SHOOTIN Gita ia nas ur as oad PAG 36 APPENDIX EQUILIBRIUM N O CONCENTRATIONS 38 WARRANTY 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 car
3. required for their growth Excess cells formed by the growth of the bacteria are pushed upwards into the reservoir where there is no present As these cells are unable to respire growth will cease and the cells degenerate releasing cell components which are used as a carbon source for other living cells The bacteria are facultative aerobic microorganisms meaning that they are able to use both oxygen and NO for respiration Respiration with oxygen results in the highest energy yield and oxygen is therefore used preferentially over NO However oxygen respiration by the bacteria does not affect the sensor s response to The transducer is a Clark type sensor in which N O is reduced to nitrogen gas N on the surface of a polarized cathode As two electrons are used to reduce a N O molecule two electrons are taken from a silver silver iodide reference electrode the anode An picoammeter measures the transport of electrons between the anode and cathode and the measured current is proportional to the amount of N O reduced on the cathode surface The latter is again proportional to the concentration of diffusing into the sensor from the external environment A silicone membrane in the tip of the N O transducer electrically separates bacterial production and electrochemical detection of N O The silicone membrane allows the passage of gasses and 10 small uncharged molecules while ions are unable to pass
4. If possible measurements should therefore be planned so that the measured concentration does not vary greatly between successive individual measurements When moving the sensor from very high e g 200 uM to very low a few uM concentrations in one step the accuracy of the measurements is decreased because it takes longer for the signal to stabilize at the low concentration after exposure to very high levels of NO CHANGING SALINITY OF THE SAMPLE SOLUTION The sensor needs a period of acclimatization when shifting between sample solutions with different salinity or ionic composition for example when shifting from fresh to saline water or vice versa The length of the acclimatization period depends on the properties of the solutions that the sensor was exposed to before and after the shift Very high signals can occur during the acclimatization period as the activity of the bacteria in the biochamber is temporarily inhibited which may cause oxygen to penetrate through the biomass and interfere with the N O transducer This interference from oxygen disappears again after the bacteria have acclimatized IMPORTANT Always wipe the membrane with a wet piece of cloth or paper before use Wiping cannot damage the membrane and the paper or cloth should be rubbed against the membrane to ensure removal of any polymers on the outside of the membrane Biofilm polymers on the outside of the membrane can reduce the diffusio
5. The electrolyte of the N O transducer is therefore efficiently shielded from the environment containing the bacteria Electrical noise from the surrounding environment thus has a limited effect on the sensor s signal The bacteria performing the process inside the sensor is a pure culture and must not be contaminated with bacteria from an external source Therefore the bacteria are separated from the external environment by an ion permeable membrane which only allows the passage of small ions including NO Larger molecules and other bacterial cells cannot enter the sensor through this membrane The NO biosensor measures NO in almost all aqueous solutions mud soil and sediment fresh brackish and saline water serum urine etc However this of course excludes samples that contain substances toxic to the bacteria The sensor functions at temperatures between 10 38 C but the response time calibration and detection limits are significantly affected by temperature At temperatures below 10 C the bacterial conversion of NO to NO becomes too slow and at temperatures higher than 38 C the cells die Concentration gt gt Cathode Silicone Sheen em s K NO RE CS NO 2 Bioreaction chamber TAA 3 w bacteria NO 2 lon permeable N y NO membrane 2 High NO Low 0 5 mm Fig 2 Principle of the nitrate biosensor 11 GE
6. from the sensor Remember that both temperature and stirring during calibration must be the same as those during measurements Wait until the signal is stable This procedure can be repeated with several concentrations of NO When the calibration is completed a calibration curve can be made and the sensor is ready for use The calibration should be repeated regularly e g every two hours when starting using the sensor A guick calibration check can be IMPORTANT The sensor signal is affected by the ionic composition of the sample due to the so called Donnan effect across the membrane Exact measurements can therefore only be obtained if calibration is performed in a liquid with approximately the same ionic composition as the sample Demineralized water should thus only be used for the zero calibration value 15 performed from time to time between samples with a control measurement in a solution of known concentration 2 Using water from the sampling site Known additions calibration method By using the same liguid as the samples the ionic composition will be identical in the sample and the calibration solution a If measurements are performed in fresh water the signal for zero can be determined by placing the sensor in demineralized water measurements are performed in brackish or saline water the zero NO solution can be prepared using demineralized water with added NaCl cor
7. thermometer Prevent the membrane from drying out by holding a piece of moist tissue in front of it The sensor will need some hours to stabilise following this procedure and calibration should be repeated High and unstable zero current more than 2 nA in solutions free of oxygen and NO The N O transducer is defective Replace the N O transducer and send the defective transducer back to Unisense The N O transducer is guaranteed for one month after receipt and defective sensors will be replaced within this period The sensor is not sensitive to NO 31 Possible cause 1 Solution Possible cause 2 Solution Problem Possible cause Solution Bubbles caught on the tip of the sensor block the diffusion of NO into the biochamber Remove the bubbles It is not necessary to recalibrate the sensor Biofilm or precipitations on the membrane block the diffusion of NO into the biochamber Wipe the membrane thoroughly with a moist cloth or tissue When the sensor has a stable signal calibration should be repeated If the signal does not stabilise the biochamber should be replaced Non linear calibration higher response uM at higher concentration Contamination of the biochamber or precipitation in the biochamber medium Replace the biochamber If you encounter other problems and need scientific technical assistance please contact salesaunisense com for online support we will answer you within one wo
8. with a piece of soft cloth or tissue Heat some demineralized water to approximately 70 C in an Erlenmeyer flask and immerse the sensor into this water for 10 seconds ensuring that the sensor is immersed all the way to the white locking ring Dip the sensor into 70 alcohol for 10 seconds and place it on a clean surface that has been wiped with alcohol Be careful not to damage the tip of the N O transducer MOUNTING OF THE BIOCHAMBER ON THE N O TRANSDUCER 1 26 Take the biochamber from the refrigerator Ensure that the membrane is kept moist The two white rings hold membrane protective part and a lid respectively Fig 9 Remove one of the two pieces of tape so that a small hole is exposed to enable pressure equilibration during handling of the biochamber Unscrew the white ring in the top of the biochamber remove the lid and mount the chamber on the N O transducer as described above Test mounting of the biochamber Excess medium can subseguently be pressed out of the hole for pressure eguilibration Wipe the area around the exposed hole with alcohol and cover it carefully with the enclosed black tape 4 Place the sensor in a Unisense calibration chamber containing a calibration solution with 200 uM NO Wait for a stable signal typically 3 8 nA at 20 C which can take some hours When a stable signal is obtained the sensor is ready for calibration and use Positioning slit n y 0 a
9. TTING STARTED BEFORE USING THE BIOSENSOR The wrapping around the sensor will ensure that the membrane in the tip of the sensor does not dry out Additionally the sensor should be stored upright with the biochamber pointing downwards See Fig 3 O ring for height adjustment in calibration chamber ojqeo Tape Plastic bag Biochamber r m z 2 Locking rin g ring E Fig 3 sensor with protective wrapping plastic bag WARNING The sensor must only be used in aqueous solutions Donotexpose the sensor to toxic compounds of any sort These include detergents sulfide alcohol and solutions with a pH lower than 3 or higher than 10 Donotusethesensorin solutions with more than 3 8 salinity The temperature during storage and use must never exceed 38 C Avoid contact with pointed or sharp objects that may puncture the membrane Ensure the membrane is kept moist Follow the storage instructions carefully POLARIZATION OF THE N O TRANSDUCER The signal from the sensor is generated in picoampere Therefore it must be connected to a Unisense picoampere amplifier during measurements Biosensors or the 2 transducer part of the biosensors should be polarized with 0 80 V On the Microsensor Multimeter and Microsensor Monometer lemo biosensors are recognized as sensor for manual settings For details on how to set the polarization cons
10. cer by unscrewing the white locking ring clockwise until the biochamber is unlocked and pull the biochamber off the N O transducer The biochamber should fit tightly to the transducer 3 Rinse the N O transducer with demineralized water and put it down on a flat surface Take care not to damage the exposed tip of the transducer If the N O transducer is connected to the picoammeter the signal will be very high because the transducer is sensitive to oxygen TEST MOUNTING OF THE BIOCHAMBER Optional for users that are unsure of how to mount the chamber Enclosed with the shipment from Unisense is a dummy biochamber with no membrane media or bacteria This can be used to practice mounting of the biochamber 1 The black plastic of the N O transducer has a raised seam which fits into a positioning slit on the inside of the biochamber Fig 9 The N O transducer 15 inserted into the biochamber by fitting this seam into the positioning slit and guiding the tip of the transducer into of the biochamber 2 Thebiochamber and the transducer are then fixed together by turning the white locking ring counter clockwise until the flexible hooks lock onto the knobs on the biochamber If the transducer and 25 biochamber are assembled correctly the metal cathode can be observed through the small hole in the tip of the sensor when held up to a light STERILIZING THE N O TRANSDUCER 1 Wipe off the exposed N O transducer
11. culations The water from the sampling site can now be used as a standard with or without addition of from a stock solution If the concentration in the water from the sampling site is more than 25 uM we recommend calibrating the sensor with a prepared solution as described above 17 When the calibration is completed a linear calibration curve can be plotted and the sensor 15 ready for use Repeat the calibration procedure regularly in the beginning for example every second hour A quick calibration check can also be made with regular control measurements in a standard solution with a known NO concentration If the water from the sampling site has high biological activity it is necessary to filter it through a 0 2 um filter before using it as a calibration solution This will prevent a change in the NO concentration over time caused by the activity of microorganisms in the sample m Calibration chamber CAL300 GENERAL USE OF THE BIOSENSOR MEASUREMENTS The sensor measures online with a 90 response time of 1 2 minutes It can therefore be used to measure a change in the NO concentration of a particular solution over time or to measure the NO concentration in different samples The membrane of the sensor should be wiped daily with a piece of cloth or tissue This will prevent the growth of bacteria on the outside of the membrane which can otherwise affect the functioning of the sensor
12. e LIFE TIME OF THE BIOCHAMBER The lifetime of the biochamber mounted on a nitrous oxide transducer depends on the environment the sensor is exposed to When handled correctly it is normally several weeks The lifetime for an un mounted biochamber is more than one month if is stored 4 C DETECTION LIMITS At 10 C lt 0 3 uM At 20 C lt 0 2 uM At 30 C lt 0 2 uM 30 TROUBLE SHOOTING Problem Possible cause Solution Problem Possible cause Solution Problem Possible cause Solution Problem The signal is very high gt 2 nA and not stable in a free solution Oxygen interference Test for sensitivity to oxygen Place the sensor in a calibration solution free of oxygen and NO Oxygen can be removed by aerating with an inert gas such as N If the signal decreases below 2 nA the sensor has become oxygen sensitive Place the sensor in a stirred NO free calibration solution aerated with atmospheric air Wait until the signal stabilizes at a low value lt 2 nA and repeat calibration see Section on Calibration If the sensor is still sensitive to oxygen after 24 hours the biochamber itself has been damaged and should be replaced No or negative signal from the sensor Bubbles in the electrolyte have disconnected the electrical circuit of the N O transducer Shake the sensor while holding it upright so that bubbles within the electrolyte rise as you would shake an old style mercury
13. e 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 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 of 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 manual
14. l component the nitrous oxide transducer and a biological component the biochamber In order to optimize the lifetime of the sensor the biochamber is replaceable Nitrous oxide transducer A 0 5 a 0 n a Replaceable 5 biochamber a Bnid OWI1 Fig 1 Nitrate biosensor with replaceable biochamber Nitrate or nitrite diffuses into the biosensor from the external environment through a selective membrane Fig 2 Bacteria situated in a reaction chamber behind this membrane reduce the nitrate or nitrite to nitrous oxide N O which is detected by an electrochemical nitrous oxide transducer The amount IMPORTANT Read the technical description of the biosensor before starting of N 0 reduced on the cathode surface is proportional to the concentration of NO in the external environment The respiration of the bacteria oxidizes carbon the energy source to form CO and water and there is a simultaneous reduction of nitrate and nitrite to nitrous oxide This process is guite similar to humans respiration of carbon with oxygen In microbiology the use of nitrate and nitrite in place of oxygen for respiration is called denitrification The NO biosensor is eguipped with a reservoir containing the carbon source This carbon compound diffuses into the reaction chamber Fig 2 where the bacteria are positioned in opposing gradients of the two substrates carbon and NO
15. mposition of the sample TEMPERATURE RANGE AT MAXIMUM 3 8 SALINITY 10 38 C Sensors that are able to measure down to 5 C can be supplied RESPONSE TIME The response time is highly dependent on whether the sample is stirred The fastest response is obtained in a stirred sample In as stirred sample the 90 response time is less than 90 seconds at 20 C At higher temperatures the response time is faster while at lower temperature it is slower 29 SENSITIVITY STIRRING OF THE SAMPLE The sensitivity towards stirring depends on the temperature and salinity of the sample and can be up to 20 of the signal INTERFERENCES Nitrous oxide N O in the external environment diffuses through the reaction chamber and is detected by the N O sensor The sensitivity to N O is approximately 2 5 times the sensitivity for NO Sulfide Sulfide gas can destroy the NO sensor Do not use the sensor in solutions containing sulfide SIGNAL DRIFT When the sensor is handled correctly the signal drift is normally less than 20 per week LIFETIME OF THE TRANSDUCER The N O transducer has a guaranteed lifetime of 800 hours and an expected lifetime of 1000 1500 hours at constant polarization The lifetime is prolonged if the sensor is used for short term measurements and disconnected between periods of measurements For long term storage the sensor should therefore be disconnected from the amperemeter see section on Storage and Maintenanc
16. n of into the reaction chamber and thereby decreases the sensitivity of the sensor 19 PROCEDURE 1 Placethe sensorin a solution with the new salinity and containing nitrate Remember to stir the solution 2 Awaita stable signal and perform the proper calibration procedures If stability is not reached within 10 hours we recommend changing the biochamber See Replacement of biochamber Exposure to high concentrations of sulfide should avoided as it can severely affect the sensitivity of the biosensor Do not expose the sensor to toxic compounds of any sort These include detergents sulfide alcohol and solutions with a pH lower than 3 or higher than 10 On suspicion of sensor damage repeat calibration and consult Trouble shooting 20 IMPORTANT Small gas bubbles are easily caught on the tip of the sensor but should always be removed They may cause erroneous measurements by impeding the diffusive transport of NO into the sensor IMPORTANT Always rinse the sensor with water of the same ionic composition as the samples Do not use demineralised water ADVANCED USE OF THE BIOSENSOR MEASUREMENTS WITH A FLOW CELL It possible to measure NO or NO concentrations in very small samples down to 5 uL by using a flowcell mounted on the biosensor fig 7 The flowcell outlet tube the longest must be connected to a high quality peristaltic pump able to p
17. rations of NO 0 1000 uM at temperatures above 20 and thus it is normally sufficient to use a two point calibration e g at O and 200 uM However it is a good idea to make calibration curves with more data points in order to develop a routine and become acguainted with the use of the sensor Always make sure that the concentrations used for calibration cover the expected range of NO concentrations in your sample Consider the stirring conditions under which the sample analysis will be conducted and use the same conditions for calibration The best results are obtained in stirred liguid Stirring is best obtained using a magnetic stirrer but be aware of possible heating of the sample by the stirrer It may be necessary to use a heat insulating plate on top of the stirrer If the sample volume is very small stirring during measurements may not be possible and in this case the sensor should also be calibrated in still liguid For measuring in such small samples it is recommended to use the flow cell for the biosensor please see Advanced use of the biosensor Remove the plastic bag from the biochamber and place the sensor in the Unisense calibration chamber containing a calibration solution 200 uM NO The O ring on the shaft of the sensor should be adjusted so that the tip of the sensor is well submersed in the liguid Wait for a stable signal usually between 3 8 nA at room temperature This may take up to an hour Now the sensor i
18. responding to the same salinity as the sample to be analyzed Wait for a stable signal approximately 2 min This is the zero value b In order to find the other point for the two point calibration the sensor is now placed in a known volume of water from the sampling site Wait for a stable signal Again beware of small gas bubbles getting caught on the tip of the sensor Add to the calibration solution to obtain a known concentration e g 100 uM Now the concentration of the solution will be the background concentration of the sample plus 100 uM Watch the signal increase and stabilize The increase corresponds to the added concentration of NO and the conversion factor between the response in pA and the NO concentration in uM can be calculated Remember that the stirring velocity and temperature of the calibration solution must be identical to the samples to be analyzed The calibration procedure can be repeated with several concentrations of NO in order to obtain a better estimate of the conversion factor d Thebackground concentration in the calibration solution can be determined from the difference in signal obtained for zero in the solution multiplied by the conversion factor calculated from the calibration procedure se Fig 5 c b pA 100 uM b a pA Z pA uM Conversion factor Z Concentration x x x 100 Figure 5 Example of calibration procedure cal
19. rkday 32 APPENDIX EQUILIBRIUM N O CONCENTRATIONS Eguilibrium nitrous oxide concentrations mmol liter at ambient partial pressure of 1 atm in water as a function of temperature and salinity Sources Weiss R F Price B A Marine Chemistry 1980 8 347 359 Table 1 33 35 36 UNISENSE UNISENSE DENMARK www unisense com info unisense com
20. roduce a stable and low flow rate Using the tubes that came with the flowcell 0 1 CC M the optimal flow range is 5 100 uL minute but flow rates down to 1 uL minute can be used Biochamber Locking ring 11 Flow cell Tubes Fig 7 Biosensor equipped with a flow cell MOUNTING THE FLOW CELL 1 Mount the flow cell on the dummy biochamber that came with the flow cell The flow cell must be mounted according to Fig 7 There are 4 stubs on the flow cell that fit to 4 depressions on the biochamber The flow cell is fastened with the white locking ring Ensure that the depressions on the biochamber of the sensor are free of membrane material 2 Fill the in and outlet tubes with water to get rid of air and place a droplet of water on the surface of the flow cell to ensure that the membrane of the biosensor does not TIP Ifyou use the sensor regularly during a longer period leave it connected to the amplifier and do not turn this off This will spare you for the time consuming prepolarization period 21 dry out Use zero standard water NO free 3 Mountthe flow cell on the biochamber If the sensor has been in an upturned position during handling hold the biochamber downwards and shake the sensor to get bubbles in the electrolyte of the nitrous oxide transducer to rise to the top as you would shake an old style Mercury thermometer to reset it 4 Mountthe outlet tubes on the pump and place the end of
21. s for the respective products CONGRATULATIONS WITH YOUR NEW PRODUCT SUPPORT ORDERING AND CONTACT INFORMATION The NOx sensor is a brilliant tool for on line measurement in experiments where the resolution of time and concentrations are important If you wish to order additional products or if you encounter any problems and need scientific technical assistance please do not hesitate to contact our sales and support team We will respond to your inguiry within one working day E mail salesaunisense com Unisense A S Tueager 1 DK 8200 Aarhus N Denmark Tel 45 8944 9500 Fax 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 The sensors 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 RECOMMENDED AMPLIFIERS One channel amplifier Microsensor Monometer Multi channel amplifiers Microsensor Multimeter OVERVIEW This manual covers the use of the biosensor for nitrate nitrite NO and for nitrite NO For simplicity this will be refered to as NO in this manual The NO biosensor consists of an electrochemica
22. s ready to be calibrated The sensor s response to is affected by changes in the ionic composition of the samples being analyzed this is caused by the so called Donnan effect and the signal can differ by up to 30 from seawater to fresh water This sensor must be calibrated in a IMPORTANT Always calibrate at the same temperature salinity and stirring velocity as the measurements solution with approximately the same ionic composition as the sample Here are two ways to prepare the calibration solution using a prepared calibration solution and using water from the sampling site If you are working with samples with unknown ionic composition we recommend to use water from the sampling site and prepare solutions by the known additions method For samples from salt or brackish waters it is possible to prepare a calibration solution by adding NaCl to demineralized water to obtain the right salinity This is because NaCl will account for the majority of ions in the sample 1 Using a prepared calibration solution a Prepare a NO free solution with approximately the same ionic composition as the sample by adding NaCl Place the sensor in the solution and wait for a stable signal approximately 2 min Beware of gas bubbles that may be caught under the tip of the sensor and affect calibration Add NO to some of the above solution resulting in a known concentration and observe the increase in signal
23. te should be used and the flow rate should be kept at a fixed level The inlet tube can be extended if necessary Substances from the biochamber will diffuse into the sample that passes through the flow cell Therefore the sample which has passed the flow cell does not have the same chemical composition as the inlet sample Measurement in discrete samples Mixing of samples in the tubes does not happen and with known retention time in the tubes samples can be changed with intervals of 2 5 to 3 minutes response time of the sensor plus 1 minute Bubbles can accumulate in the flow cell and prevent correct measurements If there are indications of this erratic signals the bubbles can be removed by briefly speeding up the flow rate To ensure correct functioning and accurate measurements it is advisable to recalibrate during measurements by using standards Maintenance and storage To prevent biofilm formation in the flow cell it must be dismounted once a day in order to clean the membrane of the biosensor During long term storage remove the flow cell and follow the instructions in Storage and Maintenance 23 STORAGE AND MAINTENANCE STORAGE OF THE BIOSENSOR SHORT TERM STORAGE 2 3 DAYS During short term storage the sensor should remain polarized so that it can be used again immediately The sensor should be continuously immersed in aerobic water of the same salinity as the samples to be analyzed A simple option is to use
24. the Unisense calibration chamber and aerate the solution It is also advisable that the storage solution contains approx 100 NO LONG TERM STORAGE If the sensor will not be used for some days it should be disconnected from the amplifier During long term storage the sensor should be continuously immersed in aerobic water of the same salinity as the samples to be analyzed but without the sensor is used for freshwater analysis demineralized water can be used A simple option is to use the Unisense calibration chamber and aerate the solution When restarting the sensor repeat the start up procedures STORAGE OF THE BIOCHAMBER Biochambers should be refrigerated immediately after receipt to ensure the longest possible lifetime The biochambers can be stored for at least one month under these conditions REPLACEMENT OF THE BIOCHAMBER The biochamber should be replaced if any of the following is observed and cannot be solved by consulting Trouble shooting Non linear calibration curve Very long 90 response time gt 2 3 min Small or no response to e Stability is not regained after changing the sample solution 24 WARNING The sensor must NOT be placed in dry air as this will cause the membrane to dry out The sensor can be placed in the calibration solution between measurements REMOVAL OF THE BIOCHAMBER 1 Remove the tape on the biochamber 2 Remove the biochamber from the N O transdu
25. the inlet tube in zero standard samples must be sucked through the flow cell not pumped 5 Adjust the peristaltic pump to a flow rate of 5 100 uL minute 6 Start the pump and confirm that the sample is sucked though the flow cell by observing whether droplets are formed at the end of the outlet tube CALIBRATION AND MEASUREMENT To obtain accurate calibration and measurements it is important to ensure a stable temperature Optimal temperature control is obtained by immersing the samples flow cell and a part of the inlet tube in a temperature controlled water bath and using a low flow rate 5 20 uL minute The optimal temperature highest sensitivity fastest response is obtained at 25 30 C Calibration Await a stable signal and calibrate the sensor by placing the inlet tube in different standards To avoid intrusion of gas bubbles it is recommended to switch off the pump when changing standards Measure the time delay from switching standards to a stable signal is obtained at the chosen flow rate 22 TIP Bubbles passing through the flow cell create erratic signals In order to prevent entrapment of gas bubbles in the flow cell it is recommended to switch off the pump when changing standards or samples Measurement The biosensor eguipped with a flowcell can either be used as remote online sensor or to measure NO concentrations discrete samples Online measurement Flow rates from 5 100 uL minu
26. ult the user manual of the amplifier that you are using In the following we assume that the sensor is connected to a Unisense picoampere amplifier like the Unisense Microsensor Multimeter REMOVE THE GAS BUBBLES FROM THE ELECTROLYTE Holding the biochamber downwards shake the sensor to get bubbles inside the sensor to rise to the top as you would shake an old style Mercury thermometer to reset it Mount the sensor on a stand Do not remove the wrapping around the biochamber PRE POLARIZATION OF THE TRANSDUCER The transducer part of the biosensor must be pre polarized before measurements to ensure correct functioning of the sensor 1 Connect the sensor and adjust the pre polarization voltage to 1 3 V and adjust the pre amp range mV pA to 0 01 Wait for approximately 5 min 2 Adjust the polarization voltage to 0 8 V and the pre amp range mV pA to 0 1 Immediately after the sensor is connected the signal will be very high and fluctuate possibly out of the picoammeter s measuring range for some minutes The signal should decrease to less than 200 mV within 2 hours of connecting the sensor to the picoammeter If this is not the case consult Troubleshooting 13 CALIBRATION Please consult the relevant software manual for instruction on how to calibrate in the software The sensor must be calibrated in standard solutions before measurements can be performed The sensor responds linearly to low concent
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