ISO-HPO-100 Instruction Manual
Contents
1. 1 hours 95 0 1M PBS Slight Yes 6 8 World Precision Instruments ISO HPO 100 WARRANTY WPI World Precision Instruments Inc warrants to the original purchaser that this equipment includ ing its components and parts shall be free from defects in material and workmanship for a period of 30 days from the date of receipt WPI s obligation under this warranty shall be limited to repair or replacement at WPI s option of the equipment or defective components or parts upon receipt thereof f o b WPI Sarasota Florida U S A Return of a repaired instrument shall be f o b Sarasota The above warranty is contingent upon normal usage and does not cover products which have been modified without WPI s approval or which have been subjected to unusual physical or electrical stress or on which the original identification marks have been removed or altered The above warranty will not apply if adjustment repair or parts replacement is required because of accident neglect misuse failure of electric power air conditioning humidity control or causes other than normal and ordinary usage To the extent that any of its equipment is furnished by a manufacturer other than WPI the foregoing warranty shall be applicable only to the extent of the warranty furnished by such other manufacturer This warranty will not apply to appearance terms such as knobs handles dials or the like WPI makes no warranty of any kind express or implied or statu2. calibration should be performed at the same temperature as the experiment Calibrating the Sensor NOTE For detailed sensor calibration procedures and calibration theory refer to the free radical analyzer manual Most WPI manuals can be downloaded directly from www wpiinc com This information can also be e mailed when you contact the WPI Technical Support team at 941 371 1003 or technicalsupport wpiinc com Before attempting to use the ISO HPO 100 sensor in an experimental solution calibrate it under standard conditions to verify proper operation and performance If you have purchased several WPI sensors all sensors should be calibrated upon receipt at your facility Although WPI takes great care in packaging the sensors to avoid the possibility of damage the sensors are delicate Testing the sensors soon 4 World Precision Instruments ISO HPO 100 after arrival at your facility ensures that the sensors arrived intact The standard test is performed at room temperature or approximately 25 C H O sensors are sensitive to changes in temperature ion concentration and pH Therefore WPI recommends calibrating sensors under the experimental conditions It is especially important to use the same temperature during calibration that is used during experimentation Standard Test If you experience problems in using the ISO HPO 100 in your experiment it is a good idea to run the standard test to verify that the sensor operates properly
3. ating the Sensor on page incorrect 4 Verify the procedure used Uneven aliquots may have been Check the pipetter calibration and used aliquot placement injection spike The standard solutions may be Verify that there is stabilizer in H O unstable standard See Calibrating the Sensor on page 4 The standard solution has Prepare fresh H O standard solution expired See Calibrating the Sensor on page 4 The sensor has reached end of Replace the sensor its usable life NOTE If you have a problem issue with your ISO HPO 100 type sensor that falls outside the definitions of this troubleshooting section first perform the Standard Test exactly as describe on page 5 of this manual and contact the WPI Technical Support team at 941 371 1003 or technicalsupport wpiinc com World Precision Instruments SPECIFICATIONS ISO HPO 100 The ISO HPO 100 type sensors conform to the following specifications Outside Diameter Available Length Response Time Lowest Detection Limit Range Upper Detection LImit Range Sensitivity New Sensor Interferent Selectivity Coefficient Baseline Drift Poise Voltage Typical Quiescent Baseline Current 25 C Acceptable Baseline Range Polarization Time Recommended Polarization Solution Temperature Dependent Physiological Interference Recommended pH Range 10 100 m 4mm lt 5 sec 1nM 1mM gt 1pA nM Ascorbate 1 10 1 50 lt 2 0pA min 400mV 1000pA 150 1200pA
4. cam ISO HPO 100 Microsensor for H O Measurement INSTRUCTION MANUAL Serial No 121412 World Precision Instruments ISO HPO 100 CONTENTS ABOUT THIS MANUAL pananda ana ABAD 1 INTRODUCTION erm 2 Notes arid Wathltigs cae e coenae neg re D co ER DR RAN AKA 2 INSTRUMENT DESCRIPTION mamana maaamaa NIYANIG ete ener 3 Parts List pH Unpackltig oen kak AG NANANA NANANA eda ei NAG GN KANYANG 3 OPERATING INS TRUCTIONS maban na an NGIGING 4 Attaching the Sensor to the Microsensor Handle wi Polarizing the Sensor ao en aeree tee tr tt ace tae tiet ce m Calibratirg the SersOE niei eie AA Pad redo E HE eS 4 Standard TES kan 5 INSTRUMENT MAINTENANCE titia ceto GNG ni Re tcs 7 Storing the Sensor Cleaning the Sensor ACCESSORIES posted sic lerejy e SPECIFICATIONS it nettes E AA An lvo Bda Claims and Returns 7 zer ES Copyright 2012 by World Precision Instruments Inc All rights reserved No part of this publication may be reproduced or translated into any language in any form without prior written permission of World Precision Instruments Inc World Precision Instruments ISO HPO 100 World Precision Instruments ISO HPO 100 ABOUT THIS MANUAL The following symbols are used in this guide This symbol indicates a CAUTION Cautions warn aga
5. d from the free radical analyzer verify that the output reading is very close to zero If the output is not close to zero then adjust the output to Zero according to the directions in the user s manual of the free radical analyzer NOTE The Apollo1000 is the only WPI analyzer with a zero output level ad justment For other WPI analyzers there is no zero current adjustment If the analyzer does not output zero volts with no sensor connected then contact the WPI technical support team at 941 371 1003 or technicalsupport wpiinc com for possible return for adjustment or repair World Precision Instruments 5 10 11 ISO HPO 100 Adjust the settings of the free radical analyzer for H O Poise voltage should be set at 400mV Connect the sensor to the free radical analyzer using the microsensor cable WPI 91580 Using a suitable electrode holder like the ProGuide WPI 47520 or ProGuide Plus WPI 47530 suspend the tip of the microsensor in the 0 1M PBS solution Submerge the tip so that the liquid completely covers the sensor active area Typically 1cm or more below the surface is adequate Allow time for the background current to drop to a stable baseline value 1000pA or less with the free radical analyzer set in the 10nA range This may take 2 4 hours See Polarizing the Sensor on page 4 NOTE The sensor may be left in the PBS buffer solution overnight to polarize After the sensor has stabilized to an acceptabl
6. e baseline current typically between 150 1000pA record the baseline value This information is useful as an indicator of the general condition of the electrode as it ages If desired establish a zero reference line on the recording software or chart recorder Alternatively record the raw voltage from the sensor and subtract the baseline current from the measured test values In sequence pipette three aliquots 2uL 4uL and 8pL of Solution 2 1mM H O into the 20mL vial containing the 20mL of 0 1M PBS buffer Solution 1 and observe the output after each addition These aliquots correspond to calculated concentrations of 100nM 200nM and 400nM respectively The aliquots should be added about 30 45 seconds apart Each successive aliquot doubles the concentration of H O and a corresponding output that is linearly proportional to the concentration should be observed The nominal sensitivity of a fresh ISO HPO 100 sensor is 1pA nM or better As the electrode ages its sensitivity decreases So test the sensor before each experiment to validate its sensitivity Create a calibration curve using the observed data Plot the changes in current pA against the changes in concentration mM that are calculated The slope of the curve indicates the sensitivity of the sensor The sensor is usable as long as the baseline current is within an acceptable range 1000pA or less when the free radical analyzer is set in the 10nA range t
7. f one hand to separate the slit in the foam which contains the sensor and use the thumb and index finger of the other hand to grasp the sensor at its midsection and gently remove it from the container KEEP THE SENSOR STORAGE BOX and all the documentation in a safe place The test date and serial number of each sensor is printed on the bottom of its box Use of the sensor should begin within 30 days of receipt Returns Do not return any goods to WPI without obtaining prior approval RMA 3t required and instructions from WPI s Returns Department Goods returned unauthorized by collect freight may be refused If a return shipment is necessary use the original container if possible If the original container is not available use a suitable substitute that is rigid and of adequate size For further details please read the section entitled Claims and Returns on page 11 of this manual World Precision Instruments 3 ISO HPO 100 OPERATING INSTRUCTIONS Attaching the Sensor to the Microsensor Handle Once removed from the package plug the microsensor into a microsensor cable WPI 91580 connected to the free radical analyzer again being very careful that the sensor tip does not come into contact with anything which could damage it See page 1 The sensor should plug in easily If you encounter resistance it is probably due to misalignment of the sensor plug with the socket connector inside the microsensor cable Simply realign the senso
8. g charge Equipment which was built as a special order cannot be returned Repairs Contact our Customer Service Department for assistance in the repair of apparatus Do not return goods until instructions have been received Returned items must be securely packed to prevent fur ther damage in transit The Customer is responsible for paying shipping expenses including adequate insurance on all items returned for repairs Identification of the item s by model number name as well as complete description of the difficulties experienced should be written on the repair purchase order and on a tag attached to the item Electrodes batteries and other consumable parts are warranted for 30 days only from the date on which the customer receives these items World Precision Instruments 11 World Precision Instruments Inc USA International Trade Center 175 Sarasota Center Blvd Sarasota FL 34240 9258 Tel 941 371 1003 Fax 941 377 5428 E mail sales wpiinc com UK 1 Hunting Gate Hitchin Hertfordshire SG4 OTJ Tel 44 0 1462 424700 Fax 44 0 1462 424701 E mail wpiuk wpi europe com Germany Zossener Str 55 10961 Berlin Tel 030 6188845 Fax 030 6188670 E mail wpide wpi europe com China amp Hong Kong WPI Shanghai Trading Co Ltd Rm 20a No8 Dong Fang Rd Lu Jia Zui Financial District Shanghai PRC Tel 86 688 85517 E mail chinasales china wpiinc com Internet www wpiinc com store wpiinc com www wpi europe com www
9. he response is linear for the intended measurement range and there is adequate resolution for the purpose of the application World Precision Instruments ISO HPO 100 INSTRUMENT MAINTENANCE Storing the Sensor STANDBY When not being used for a short period of time such as overnight the microsensor should remain attached to the microsensor cable and immersed into buffer solution such as PBS Before the next experiment the sensor can be transferred from the buffer solution to the experimental solution LONG TERM If the microsensor is to be stored for a long period of time then it may be stored dry by removing it from the microsensor cable and returning it to the case in which it was shipped being very careful to avoid making contact with the sensor tip The H O microsensor is a maintenance free consumable sensor When it no longer functions remove it from the microsensor cable and dispose of it replacing it with a new one Cleaning the Sensor The sensor should be cleaned after each use If the sensor was used in a protein rich solution the tip should first be soaked in a protease solution for several minutes to remove protein build up and then rinsed with distilled water Enzymatic detergent Enzol WPI 7363 4 can also be used The sensor can be sterilized chemically using an appropriate disinfectant Cidex WPI 7364 If necessary gently dab the sensor with a Kimwipes to remove residue NOTE ALWAYS rinse with dist
10. illed water before storing the sensor dry World Precision Instruments 7 ISO HPO 100 ACCESSORIES Table 1 Accessories Part Number Description 7363 4 Enzol Enzymatic Detergent 1 gallon 7364 Cidex Disinfecting Solution 91580 Microsensor Cable 47510 ProGuide Postion Holder with Base 47520 ProGuide Position Holder 47530 ProGuide Plus Position Holder with fine adjustment 47540 ProGuide Plus with Base NOCHM 4 Four port Closed Chamber 8 World Precision Instruments ISO HPO 100 TROUBLESHOOTING Issue Possible Cause Solution Baseline current is below specified range Unstable x m Vv amp e o c 2 m N m o c 9 pa S U Sensitivity below range specified baseline The poise voltage sensor Set the poise voltage to 400mV For the setting may be incorrectly set TBR choosing the HPO sensor setting or sensor has drift selects 400mV automatically Set the range at 10nA The sensor may be nearing the Perform a 3 point calibration set using end of its usable life the standard If successful the sensor is functioning properly See Calibrating the Sensor on page 4 If the baseline hasn t stabilized Prepare fresh polarizing solution Use after 1 hour the polarizing 0 1M PBS only solution may be contaminated External electrical interferences Identify and isolate electrical may be the problem interferences The dilution factors may be See Calibr
11. inst actions that can cause damage to equipment Please read these carefully This symbol indicates a WARNING Warnings alert you to actions that can cause personal injury or pose a physical threat Please read these carefully NOTES and TIPS contain helpful information Fig 1 SO HPO 100 microsensor with cable World Precision Instruments 1 ISO HPO 100 INTRODUCTION The ISO HPO 100 is a 100 m tip diameter hydrogen peroxide micro sensor designed for use in tissues vessels and similar applications The sensor design is based on a flexible sensing electrode coated with a unique membrane that enhances hydrogen peroxide selectivity The ISO HPO 100 incorporates WPI s proprietary combination electrode technology whereby the hydrogen peroxide sensing element and separate reference electrode are encased within a single sensor design The ISO HPO 100 is also available with a hypodermic sheath ISO HPO 100H or as an L shape for use with a tissue bath or in monolayer style cell culture studies ISO HPO 100L Notes and Warnings Exercise caution when handling the H O sensor to avoid actions that could damage the sensor tip The sensor membrane and membrane coatings are extremely delicate and improper handling will lead to damage See Unpacking on page 3 n CAUTION DO NOT EXPOSE SENSOR TO ORGANIC SOLVENTS D CAUTION DO NOT POLARIZE IN DISTILLED WATER D CAUTION Carefully read the Probe Unpacking instructio
12. ns found in the sealed sensor case before handling the sensor 2 World Precision Instruments ISO HPO 100 INSTRUMENT DESCRIPTION Parts List After unpacking verify that there is no visible damage to the sensor Verify that all items are included 3 ISO HPO 100 microsensor 3 Sensor Performance Evaluation 1 Instruction Manual Unpacking Upon receipt of these sensors make a thorough inspection of the contents and check for possible damage Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing Concealed damage should be reported at once to the carrier and an inspection requested Please read the section entitled Claims and Returns on page 110f this manual Please contact WPI Customer Service if any parts are missing at 941 371 1003 or customerservice wpiinc com The ISO HPO 100 microsensor is shipped in a sealed rigid plastic hinged box with foam padding to protect it from damage during shipment To open the package carefully cut the seals on either side of the sensor box Due to the microsensor s extremely small size and intricate construction great caution should be used when handling it Avoid bringing the sensor tip into contact with solid surfaces since this may bend or break it The sensors are packaged in foam holders so that their tips are physically isolated from contact with the container To remove a microsensor from the package use the thumb and index finger o
13. r by gently rotating it until it snaps into place Current WPI free radical analyzers include the TBR4100 TBR1025 and Apollo1000 Apollo4000 is an older WPI free radical analyzer Polarizing the Sensor When a non polarized microsensor is initially connected to a monitoring instrument it may display a high sometimes off scale background current The polarization voltage applied by the instrument will cause a reduction of the background current to a stabilized baseline value over time Set the poise voltage to 400mV For the TBR4100 1025 set the Probe Select dial to HPO The amount of time required to reach a stable baseline current varies for each sensor New sensors typically take longer on the order of several hours The Performance Evaluation included with your sensor shows the baseline current when the sensor was quality tested at WPI The baseline value attainable in your lab may be slightly higher or lower depending on the temperature and composition of the test media For initial performance verification of an ISO HPO 100 in your lab WPI recommends using the Standard Test described on page 5 Once a stable baseline current is achieved usually between 10 2000pA the microsensor is ready for use NOTE The background current of the sensor will usually increase with increasing temperature of the experiment Although the sensitivity of the sensor does not change significantly within the range 20 37 C it is recommended that any
14. tory including without limitation any warranties of merchantability and or fitness for a particular purpose WPI shall not be liable for any damages whether direct indirect special or consequential arising from a failure of this product to operate in the manner desired by the user WPI shall not be liable for any damage to data or property that may be caused directly or indirectly by use of this product Claims and Returns Inspect all shipments upon receipt Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing Concealed loss or damage should be reported at once to the carrier and an inspection requested AII claims for shortage or damage must be made within ten 10 days after receipt of shipment Claims for lost shipments must be made within thirty 30 days of receipt of invoice or other notification of shipment Please save damaged or pilfered cartons until claim is settled In some instances photographic documentation may be required Some items are time sensitive WPI assumes no extended warranty or any liability for use beyond the date specified on the container Do not return any goods to us without obtaining prior approval and instructions from our Returns Department Goods returned unauthorized by collect freight may be refused Goods accepted for restocking will be exchanged or credited to your WPI account Goods returned which were ordered by customers in error are subject to a 25 restockin
15. under controlled conditions To test the ISO HPO 100 gather the following supplies Solution 1 200mL of 0 1M PBS buffer Use 1 Sigma tablet Sigma Aldrich P4417 100TAB dissolved into 200mL distilled water dH O Solution 2 20mL of 1mM H O Weigh 67 5mg Acetanilide Sigma Aldrich 397237 and dissolve it into 250mL of dH O in a volumetric flask Remove 1mL of this solution and put it in a clean container Add 231yL of 3 H O Sigma Aldrich 323381 Then add dH O to make a total volume of 250mL Store this stock solution in an amber bottle if available Alternatively wrap aluminum foil around the bottle to limit light intrusion Keep refrigerated e 20mL vial Magnetic stirring plate with a stirring bar small enough to fit in the 20mL vial e Flectrode holder for suspending the electrode within the test vial hands free Pipetters to dispense uL aliquots from the 1mM H O test solution e 2 250mL volumetric flasks or other containers suitable for mixing solutions 250mL amber bottle for storing 1mM H O solution To test the sensor 1 Measure 20mL of PBS Solution 1 and put it into a vial that will hold it 2 Drop a small stirring bar into the solution and place the vial on top of a magnetic stirring plate The RPM on the stirring plate should be set high enough to rapidly disperse aliquots of Solution 2 throughout the buffer 3 When no sensor is connected there is no sensor redox current With the sensor disconnecte
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