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T100 Instrument Handbook

1. Waste and water tray Figure 2 3 Waste and water tray and right pump compartment Biacore T100 Instrument Handbook 9 version AA Description 10 The waste tubes are attached with tube fittings to the waste cap Before starting a run make sure that the tube fittings are tightened and that the waste bottle is empty Note The waste bottle and the cap must be of the same type and size as the ones delivered with the system to avoid pressure disturbances in the liquid handling system If the outlet is lower than the top of the bottle there will be a pressure imbalance in the flow system and liquid may be siphoned out of the IFC 2 1 3 Sample compartment The sample compartment holds the autosampler and the sample injection unit see Section 2 3 2 The rack tray port on the front of the instrument is controlled from the software Loading samples and reagents The user loads the rack tray carrying the sample and reagent racks into the rack tray port before starting the run There is no access to the sample compartment during a wizard run or method run You may add samples during a manual run Figure 2 4 Loading the rack tray into the rack tray port Biacore T100 Instrument Handbook version AA Description Viewing the samples The samples are visible at all times during a run through the inspection window of the sample compartment door Illumination of the sample compartm
2. Buffer tray Figure 2 2 Left pump compartment opened Biacore T100 Instrument Handbook version AA Description Buffer tubing and selector valve The buffer tubes marked A B C and D are connected to the inputs of a buffer selector valve The selector valve determines which of the buffers is used during a run Buffer selection is controlled from the software buffer A is selected by default Attach unused buffer tubes to the holder inside the pump compartment door Buffer degasser The gas content of the running buffer is reduced to a low level by a vacuum degasser This eliminates the need to degas running buffer before use Note The buffer tubing should always be connected via the buffer degasser Do not disconnect tubes from the degasser even if you use degassed buffer 2 1 2 Waste and water tray and right pump compartment The waste and water tray on the right of the instrument holds one 2 liter bottle for waste solutions and one 500 ml bottle for distilled water One 2 liter bottle and cap with tube fittings and one 500 ml bottle and cap with gasket are provided with the system The right pump compartment houses a peristaltic pump for supply of buffer and water to the liquid supply block To open the pump compartment press on the inner edge of the door Tre I mi o Peristaltic pump ALI
3. Use ionic strength at or close to physiological levels 150 mM monovalent ions to reduce non specific binding of compounds to the sensor surface Tris based buffers can be used as an alternative for analysis but are not suitable for ligand immobilization using amine coupling 3 2 2 Setting up the liquid system WARNING The peristaltic pump may operate at any time during a run or standby Keep your hands clear of the pump if you open the right pump compartment door during operation 1 Open the right pump compartment door and check that the clamp of the peristaltic pump is properly fastened the lever should be in an upright position 2 Filla suitable bottle with running buffer Make sure that the bottle is clean before use Fit a cap with gasket on to the bottle and place it on the buffer tray Insert the tube marked A through the cap into the running buffer bottle Note Always use freshly prepared buffer solution Replace the buffer after every run or at least every 48 hours 3 If you plan to use different buffers fill up to three additional bottles with the required buffers Fit caps with gaskets on to the bottles and place them on the buffer tray Insert the tubes marked B C and D into the bottles Biacore T100 Instrument Handbook 25 version AA Operation I Figure 3 1 Bottles on the buffer tray 4 Place unused buffer tubing in the holder inside the
4. D 5 Reagent racks Reagent rack type 1 20 vials diam 11 mm 1 5 ml Eppendorf Reagent rack type 2 9 vials diam 16 mm 4 0 ml 24 vials diam 7 5 mm 0 8 ml Sample and reagent rack 9 vials diam 16 mm 4 0 ml 24 vials diam 11 mm 1 5 ml Eppendorf 45 vials diam 7 5 mm 0 8 ml Kes P C py Table D 1 Reagent rack specifications 68 Biacore T100 Instrument Handbook version AA Specifications D 6 Microplates Microplate formats 96 or 384 shallow plates conforming to SBS standard D 7 Liquid containers D 7 1 Buffer tray Running buffer 1x11 3x 250 ml screw caps with gasket D 7 2 Waste and water tray Waste 21 must be of the same type as the one delivered with the system Special screw cap with two tube fittings Water 500 ml screw cap with gasket D 8 Chemical resistance This section gives some general guidelines concerning chemical resistance for Biacore T100 components Regarding exposure to solutions not covered by these guidelines contact your Biacore representative for recommendations The flow system and sensor chip are the only parts of Biacore T100 that come into contact with solutions The guidelines in this section relate to tubing and connectors selector valves connector block IFC and sensor chip Note In most analysis situations the ligand attached to the sensor surface limits the chemical resistance of the sy
5. version AA Description 16 Figure 2 11 Reagent racks Type 1 bottom and Type 2 top The rack slides into the holder on the rack tray and clicks into position When changing racks make sure that the rack is correctly mounted and that it is pushed firmly into position If you attempt to mount the rack in the wrong orientation it will not click into position and you will not be able to insert the rack tray into the instrument Figure 2 12 Reagent rack mounted on the rack tray Always use vials with caps as supplied by Biacore It is important that the injection needle can penetrate the vial caps properly Sample and reagent rack A combined sample and reagent rack is available The sample and reagent rack is inserted into the sample compartment instead of the rack tray Biacore T100 Instrument Handbook version AA Description Figure 2 13 Sample and reagent rack 2 3 4 Liquid supply block The liquid supply block is part of the autosampler assembly It is used for washing the needle and emptying waste solutions The peristaltic pump maintains a continuous flow of running buffer and water to the liquid supply block throughout a run ensuring fresh liquids at all times Distilled water S2 Waste outlet g Running buffer S1 Figure 2 14 The liquid supply block By washing the needle in fresh buffer carry over is avoided The water is used to rinse the needle and the liquid supply blo
6. 21 version AA Description 2 6 Software 22 2 5 2 Sample compartment temperature The sample compartment is maintained at a temperature that may be set from 4 45 C max 15 C below ambient temperature The sample compartment temperature is set independently of the analysis temperature injected samples have sufficient time in the needle and IFC to equilibrate to the analysis temperature regardless of sample compartment temperature Note The system does not wait for the sample compartment temperature to stabilize The Temperature lamp shows the analysis temperature not the sample compartment temperature Condensate water may occasionally drip from the instrument during long runs at low temperatures particularly if ambient humidity is high A PC running dedicated software that handles analysis and presentation of results controls operation of the processing unit The software provides a toolbar and menu driven user interface It uses wizards to guide the user through operation steps For a description of the software and detailed instructions for use refer to the separate Biacore T100 Software Handbook Biacore T100 Instrument Handbook version AA Operation 3 Operation This chapter guides you through the basic operation of Biacore T100 It is assumed that the processing unit and the PC have been installed as described in Appendix A The chapter is divided into the following sections 1 Starting the syste
7. 384 well microplates make sure all such air bubbles are removed 3 Cover the used sample wells with recommended adhesive foil available from Biacore This prevents evaporation from the samples during analysis 4 Open the catch on the rack tray and slide the microplate onto the rack tray Place the microplate with well A1 facing towards the front of the rack tray Biacore T100 Instrument Handbook 31 version AA Operation Caution Make sure that you install the microplate with well A1 facing the front of the microplate Otherwise the sample order will be incorrect Figure 3 6 Placing the sample microplate on the rack tray 5 Close the catch and make sure that the microplate is properly seated on the rack tray Figure 3 7 Fastening the sample microplate on the rack tray 32 Biacore T100 Instrument Handbook version AA Operation Adjusting the rack tray To accommodate some brands of microplates it may be necessary to adjust the retainers on the rack tray Contact your Biacore representative for more information Adjust the retainers as follows 1 Loosen the screws underneath the rack tray and turn the retainers through 90 to accommodate the microplate 2 Tighten the screws again after the adjustment Figure 3 8 Adjusting the rack tray 3 4 3 Changing reagent racks 1 Remove the rack from the rack tray by pushing firmly at the rack cover 2 Slide the new rack into the holde
8. and Sanitize Inspect the needle and the liquid supply block Inspect the sample compartment look for signs of flooding Run System Check Section B 3 3 B 3 4 B 3 2 B 3 4 B 4 5 B 4 3 B 4 1 Biacore T100 Instrument Handbook version AA Maintenance B 2 2 User service operations In addition to the user maintenance operations you may need to perform some service operations using the available tools Service tool Description Section Superclean This is a powerful tool which can be used for extensive cleaning if the Desorb and Sanitize procedure is not sufficient to clean the flow system B 3 4 System Check Always run System Check before calling Biacore Service B 4 1 Software Problem Report Run this tool if you experience problems with Biacore T100 Software which do not have a readily apparent solution Table B 2 Tools for user service operations B 2 3 Preventive maintenance To ensure correct performance of Biacore T100 preventive maintenance should be done regularly by a qualified Biacore Service representative During the maintenance visit worn parts are replaced and all vital modules of the Biacore T100 system are tested The following components are always replaced e IFC e Opto interface e Syringe pump tips e Peristaltic pump tubing Biacore T100 Instrument Handbook version AA 45 Maintenance B 3 Use
9. available from Biacore as stock solutions To prepare running buffer dilute the stock solution with distilled and filtered water Available buffer solutions include HEPES buffered saline HBS and Phosphate buffered saline PBS HBS buffers are recommended as running buffer for most general applications HBS buffers are available with and without EDTA and surfactant PBS buffers are recommended as running buffer for small molecule assays However phosphate buffer should not be used for interaction systems that require Ca calcium phosphate will precipitate at very low Ca concentrations Degassing of buffers is not critical since Biacore T100 includes an active degasser The degasser continuously eliminates dissolved air from the running buffer see Appendix E for further information Preparing your own buffers All buffers used in Biacore T100 both as running buffer and for sample and reagent preparation should be filtered through a 0 22 pm filter Including a surfactant in the buffer can reduce non specific adsorption of proteins to the autosampler tube and the IFC channels Surfactant P20 is available from Biacore 1 Buffers in 200 ml ready to use packs are not recommended for use with Biacore T100 Biacore T100 Instrument Handbook version AA Operation You may omit surfactant if your sample is detergent sensitive However you may then want to clean the flow system more frequently see Appendix B Maintenance
10. be necessary to preserve the sensor surface during standby 3 6 2 Wash buffer tubing The maintenance tool Wash Buffer Tubing washes all buffer tubing with BIAdesorb solution to remove adsorbed material Run this tool when you change from buffers containing substances that tend to adsorb to the tubing e g detergent or BSA If you have used buffer tubes B C or D and do not plan to use them in coming runs run the maintenance tool Empty Buffer Tubing to wash and empty the buffer tubing then place unused tubes in the holder in the left pump compartment For details of buffer tubing maintenance see Appendix B 3 6 3 Shutdown If you want to shut down the instrument completely see instructions in Appendix B Biacore T100 Instrument Handbook 37 version AA Operation 38 Biacore T100 Instrument Handbook Installation A Installation A 1 General guidelines Biacore T100 will be installed by a Biacore representative The site should be prepared for the installation as described in Sections A 2 to A 4 If you need to move the instrument after installation follow the instructions in Section A S A 2 Space requirements The size of the processing unit is indicated in Figure A 1 At least 20 cm clearance is required behind and above the processing unit to allow adequate air circulation 400 mm gt lt 680 mm 580 mm 600 mm Figure A 1
11. bottle with sufficient distilled and filtered water for the standby period Fit a cap onto the bottle and place it on the buffer tray Insert the tube marked A into the water bottle Liquid consumption during standby is approximately 65 ml 24 h Empty the waste bottle Check that there is sufficient water in the bottle on the waste and water tray Choose Tools Standby Make sure that there is sufficient liquid before leaving the instrument Note Biacore recommends that you use distilled and filtered water instead of buffer to minimize salt deposits However if an immobilized sensor chip is docked and will be used later buffer may be necessary to preserve the sensor surface during standby If the instrument will be unused for a long period of time follow the instructions for shutdown in Section B 5 2 B 5 2 Shutdown To shut down the instrument completely 1 2 60 Run Desorb and Sanitize to clean the flow system Eject the rack tray carriage and remove the rack tray Biacore T100 Instrument Handbook version AA Maintenance 3 Prepare a bottle of distilled and filtered water and a bottle of Hel Tools 70 ethanol Choose Shutdown from the Tools menu Follow the sl instructions on the screen Shutdown Biacore T100 Evaluation The procedure flushes the flow system and then empties the IFC PE of liquid Total run time is about 20 minutes Sana DYa ky 4 When instructed open the right
12. buffer composition between running buffer and sample can introduce artefacts that may be difficult to interpret C 2 3 For additional help Several Biacore publications provide guidance on design and execution of experiments The Biacore Sensor Surface Handbook provides in depth consideration of the principles and practice of sensor chip preparation and regeneration The Biacore Concentration Analysis Handbook describes principles of concentration measurements with Biacore including considerations of assay design and validation The Biacore Advisor Tutorial interactive CD provides an introduction to experimental design for both new and more experienced users This publication covers basic principles sensor surface preparation experimental design and evaluation for Biacore T100 Instrument Handbook 65 version AA Troubleshooting 66 specificity multiple binding concentration kinetic and affinity studies e The Biacore Advisor Kinetics interactive CD offers a theoretical and practical guide to development and analysis of kinetic experiments including preparation of sensor surfaces pilot experiments general recommendations for experiments and data evaluation as well as discussion of common problems and troubleshooting e The Immobilization and Regeneration database on www biacore com contains protocols for specific immobilization and regeneration situations developed by scientists from Biacore as well as by Biacore
13. in the conventional transmission spectroscopy sense The light used in Biacore T100 is totally internally reflected inside the optical unit and it is the evanescent wave that penetrates the sample Consequently measurements may be made on turbid or even opaque solutions without interference from conventional light absorption or scattering by the sample E 1 2 What SPR measures In the configuration used in Biacore T100 the SPR response is a measure of the refractive index of the solution within the penetration distance of the evanescent field wave This distance is small about 300 nm in relation to the volume of sample used so that effectively SPR measures the refractive index at the surface of the sensor chip The refractive index of the solution varies with the solute content When the detecting molecule is attached to the sensor chip or when analyte binds to the detecting molecule the solute concentration at the sensor chip surface increases leading to a change in the SPR signal The response measured in Biacore T100 is related to the mass of analyte bound and is largely independent of the nature of the analyte Refractive index contributions for different solutes are additive so that the amount of detecting molecule attached and the amount of analyte bound can both be measured with the same detection principle Biacore T100 Instrument Handbook version AA Technical description E 2 Flow system Two high performance sy
14. leakage and tests pumps blank injections noise and buffer selector Total run time is about one hour NOTE This procedure should be run at 25 C with a new Sensor Chip CMS Running buffer should be HBS N Last run time 2 22 2005 11 37 AM w In the System Check dialog click Next to proceed Follow the instructions on the screen to prepare and start the system check The analysis temperature will be adjusted as part of the procedure System Check xi Select test s to run This procedure should be run at 25 C with a new Sensor Ca CM5 and with HBS N as running buffer Choose Close if you need to c sensor chip reset the temperature or change running buffer ange the A Reagent pumps and blank injection B Refractometer performance and flow cell leakage C Injections D Noise E Mix F Buffer selector lt K K K RIA A Tests if the peristaltic pump is in order and that a sample injection with buffer from the Reagent supply block is all right lt Back Close The results of the system check are displayed automatically after the run OK indicates a passed test a failed test is indicated by BAD Biacore T100 Instrument Handbook version AA Maintenance System check results Test Likely cause of failure Explanation Action A Reagent pump Air in injections 1 and 2 The clamp on the upper peristaltic pump was not Water fas
15. system at any other time choose Tools Prime to ensure that all parts of the liquid handling system are flushed with fresh buffer from tube A This procedure takes 6 7 minutes 3 3 3 Setting the temperature The temperature at the flow cell is shown in the status window of the Biacore T100 screen To change the analysis and or sample compartment temperature Choose Tools Set Temperature and enter the required Analysis temperature Set Temperature 1 x Analysis temperature 25 0 Sample compartment temperature 25 0 Help Cancel You can start a run before the temperature has stabilized However the run will be paused before a step where analysis temperature is critical A message is displayed and the instrument enters standby until temperature has stabilized You can choose to ignore this message but this is not recommended as the signal is not stable Biacore T100 Instrument Handbook 29 version AA Operation 3 4 Preparing samples and reagents 30 3 4 1 Ejecting the rack tray The removable rack tray carries one microplate and one reagent rack and is mounted on the rack tray carriage in the sample compartment The combined sample and reagent rack is mounted directly on the rack tray carriage The rack tray or the sample and reagent rack can be ejected in three situations e before a run when preparing samples and reagents e during the setup of a run e during a manual run Ejecting the rac
16. tray and keep your hands and loose items of clothing from the rack tray when it moves into the instrument A timer on the computer screen indicates when the rack tray will be automatically moved into the instrument WARNING MOVING PARTS Always shut down the instrument and switch off mains power before opening the sample compartment WARNING HEAVY OBJECT Biacore T100 weighs about 60 kg At least two people are required to lift the instrument 4 Biacore T100 Instrument Handbook version AA Introduction 1 4 4 Disposal procedures Follow applicable national and or local regulations for the disposal of chemicals and other materials Biacore T100 contains a lithium backup battery which must not be disposed of in fire The battery may only be changed by a Biacore Service engineer 1 4 5 Emergency stop routine Caution Do not leave the system in an emergency stop condition Always follow the restart procedure see Section 1 4 6 if possible to restore the processing unit into normal condition If there is time To stop a run under controlled conditions before it is complete choose Run Stop Run from the menu bar This will stop the run at the end of the current cycle In an emergency situation To stop a run immediately in an emergency situation press Ctrl Break Ctrl Pause on the keyboard Caution Do not use Ctrl Break unless you need to stop all operations immediately if there is a risk of injury dama
17. 6 EEC Electromagnetic EN 61 326 1 1997 and compatibility amendment A1 1998 and A2 2001 EMC requirements for Class B equipment 1 5 2 North America Biacore T100 Processing Unit meets the following safety standards e UL 61010A 1 and CAN CSA C 22 2 No 1010 1 Safety requirements for electrical equipment for measurement control and laboratory use 1 5 3 External equipment Any external equipment e g computer connected to the Biacore T100 Processing Unit must comply with the applicable IEC EN standards Biacore T100 Instrument Handbook version AA Description 2 1 Sample compartment door Left pump compartment door Sample compartment inspection window Rack tray port 2 Description Biacore T100 instrument Biacore T100 instrument is a processing unit with liquid handling sample handling and detection system controlled from a PC running Biacore T100 Control Software Biacore T100 instrument is used with sensor chips from Biacore A range of chemicals and reagents designed for use with Biacore T100 is also available Status lamps Sensor chip port Right pump compartment door Buffer tray Waste and water tray Figure 2 1 Biacore T100 instrument The removable sensor chip is inserted into the sensor chip port and docks into the instrument Optical detection occurs in flow cells that are formed by the sensor chip and the Inte
18. Biacore T100 Instrument Handbook Edition February 2005 Biacore T100 Instrument Handbook Edition February 2005 Version AA Copyright Biacore 2005 All rights reserved No parts of this publication may be reproduced without written permission from Biacore Biacore AB reserves the right to make changes to the design and specification of Biacore T100 and to the content of this Handbook without prior notice Minor differences may occur between illustrations in this Handbook and the appearance of the product Biacore is a registered trademark of Biacore AB Microsoft and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Biacore AB Rapsgatan 7 SE 754 50 Uppsala Sweden www biacore com Contents 1 Introduction 1 1 1 2 1 3 1 4 1 4 1 1 4 2 1 4 3 1 4 4 1 4 5 1 4 6 1 5 1 5 1 1 5 2 1 5 3 Biacore T100 system The Instrument Handbook Associated documentation Safety information Rating information Safety labels Safety instructions Disposal procedures Emergency stop routine Restart procedure Conformance with standards Europe North America External equipment 2 Description 2 1 2 1 1 21 2 2 1 3 2 1 4 2 2 2 2 1 2 2 2 2 2 3 2 3 2 3 1 2 3 2 2 3 3 2 3 4 2 3 5 2 3 6 Zid 2 3 8 2 3 9 2 3 10 2 4 2 4 1 2 5 2 5 1 2 5 2 2 6 Biacore T100 instrument Buffer tray and left pump compartment W
19. Biacore T100 Instrument Handbook version AA Introduction ATTENTION For protection against fire hazard replace only with same type and rating of fuses 2xT6 3AH 250V MAINS POWER MAINS INLET Disconnect cord before opening Autorange 100 240V 50 60Hz 6 3A Figure 1 2 Rating information on the mains input panel 1 4 2 Safety labels IMPORTANT INFORMATION Read Manual before operation Figure 1 3 Safety label This safety label is attached to the side panel of the instrument above the communication ports Biacore T100 Instrument Handbook version AA Introduction 1 4 3 Safety instructions WARNING HAZARDOUS VOLTAGES Biacore T100 Processing Unit contains mains voltage of up to 265 V ac Disconnect mains cord before replacing fuses or opening covers WARNING GENERAL PROCEDURES Always wear appropriate protective clothing during operation and maintenance of Biacore T100 Use required safety equipment when handling hazardous substances WARNING FLAMMABLE SUBSTANCES Liquids marked as flammable must not be used as running buffer Any buffer or reagent containing flammable substances must be placed in properly capped vials in the reagent rack Bear in mind that the temperature in the sample compartment can be as high as 45 C WARNING PINCH HAZARD The rack tray automatically moves into the instrument 1 minute after it has been ejected Do not place loose objects on the rack
20. Handbook version AA Description 2 4 1 Sensor chip The sensor chip is a gold coated glass slide mounted on a supporting frame The sensor chip is normally enclosed in a protective cassette For illustration purposes Figure 2 18 shows the sensor chip removed from the cassette Cassette Gold coated Frame glass slide Sensor Chip T Ta kaien C M 5 Figure 2 18 The sensor chip and the protective cassette Sensor chip types The most commonly used general purpose chip Sensor Chip CM5 has a carboxymethyl dextran matrix covalently bonded to the gold surface of the sensor chip e Other sensor surfaces are available for specialized applications see Biacore Sensor Surface Handbook Caution Biacore T100 can only be used with Series S sensor chips from Biacore 2 5 Temperature control SPR measurements are sensitive to changes in temperature It is important that a constant temperature is maintained at the sensor chip surface throughout the run 2 5 1 Analysis temperature The detection area housing the sensor chip is maintained at a precisely controlled temperature range 4 45 C max 20 C below ambient temperature Runs will not start if the temperature at the sensor surface is not stable You can choose to ignore or wait for the temperature to stabilize The Temperature lamp on the instrument front panel flashes if the analysis temperature is not stable Biacore T100 Instrument Handbook
21. Sensor Chip CMS 53 Sensor Chip lamp 12 Sensor Chip Maintenance 50 51 sensor chip port 11 serial communication 41 serial port 13 Series S Maintenance Kit 46 Service Tools 43 software installation 42 solutions parts in contact with 69 space requirements 39 specifications 67 SPR 71 standby 37 60 starting the system 23 stop run 5 superclean 56 surface plasmon resonance 71 system check 53 System lamp 12 BIACORE T100 Instrument Handbook 77 version AA Index 78 T technical specifications 67 temperature ambient 40 analysis 21 lamp 12 23 sample compartment 22 setting 29 Tools eject rack 30 shutdown 61 standby 60 system check 53 undocking the sensor chip 27 Ww waste tray 9 water tray 9 Biacore T100 Instrument Handbook version AA
22. Space requirements for Biacore T100 Biacore T100 Instrument Handbook 39 version AA Installation A 3 Mains power supply The processing unit and the PC with printer require mains power outlets with protective earth as specified in Table A 1 Mains voltage 100 240 Vac autorange 50 60 Hz Power consumption processing unit maximum 6 3 A PC and monitor see manufacturer s manual Se Table A 1 Mains power requirements A 4 Heating and ventilation For proper operation Biacore T100 requires an ambient temperature of 18 33 C and a relative humidity below 85 Make sure there is adequate circulation around the instrument A 5 Moving the instrument within the lab A 5 1 Shutting down the system Before moving the system 1 Shut down the instrument completely See Appendix B Section B S 2 Shut down the computer 3 Disconnect the serial communication cable 4 Place the instrument in a location that complies with the requirements in Sections A 2 to A 4 Note When moving to a new site contact your Biacore representative 40 Biacore T100 Instrument Handbook version AA Installation A 5 2 Connecting to mains power Caution Do not turn on the mains power switches before all connections are made 1 Connect the mains power cord delivered with the instrument to the MAINS INLET connector on the rear panel Connect the other end to a mains outlet with protective earth 2 Check th
23. To remove the sample compartment box 1 2 58 Open the sample compartment see Section B 4 3 Pull out the tubing from the water bottle and remove the bottle Remove the cap from the waste bottle and remove the bottle Pull out the tubing from the buffer bottles and remove the bottles Place the tubing ends in the holder inside the left pump compartment Remove the buffer tray and the waste and water tray Use two 2 5 mm screwdrivers to press the two catches inside the sample compartment box see Figure B 10 Pull the box out of the instrument Biacore T100 Instrument Handbook version AA Maintenance NA Figure B 10 Opening the sample compartment box Replacing the sample compartment box 1 Push the sample compartment box along two guide rails into the instrument Press until it locks into position 2 Replace the buffer tray and the waste and water tray 3 Pull down the sample compartment door and close the door by pressing it down until it snaps into position 4 Make sure that no tubing is in the way then close the door by pressing it onto the sample compartment box Biacore T100 Instrument Handbook 59 version AA Maintenance B 5 Shutting down the system B 5 1 Standby When not in use Biacore T100 should be left in standby mode The instrument maintains a low flow of liquid through the flow system The default standby period is 4 days To set the instrument in standby 1 Fill a
24. are will be pre installed on delivery If the software needs to be re installed for any reason 42 1 2 Turn on the PC Insert the Biacore Software CD in the CD drive and follow the instructions on the screen For software registration you need the Product Key which is printed on the back of the CD case Biacore software cannot be installed without a valid Product Key After restart the Biacore T100 software icons appear in the BIA Programs group in the Windows Start menu Biacore T100 Instrument Handbook version AA Maintenance B Maintenance WARNING e Always wear appropriate protective clothing during operation and maintenance e Use required safety equipment when handling hazardous substances e If the instrument is contaminated with biohazards please contact your local Biacore service representative for further information about decontamination procedures B 1 Introduction Regular maintenance of Biacore T100 is essential for reliable results It is important to keep the instrument free from contamination such as microbial growth and adsorbed proteins in the liquid handling system Regular maintenance should be performed at three levels e User maintenance operations supported by the Maintenance Tools software functions e User service operations supported by the Test Tools and Service Tools software functions e Preventive maintenance operations which should be performed on a reg
25. aste and water tray and right pump compartment Sample compartment Sensor chip port Indicators and switches Status lamps Mains power switch Computer connections Liquid handling system Overview Autosampler Microplates and racks Liquid supply block Buffer selector valve Buffer degasser Integrated u Fluidic Cartridge IFC Flow cells Sample handling Standby mode 20 Detection system Sensor chip Temperature control Analysis temperature Sample compartment temperature Software Biacore T100 Instrument Handbook version AA Contents ADDADBANAHAWNNNN e 20 21 21 21 22 22 Contents ii 3 Operation 3 1 3 2 3 2 1 3 2 2 3 3 3 3 1 3 3 2 3 3 3 3 4 3 4 1 3 4 2 3 4 3 3 4 4 3 4 5 3 5 3 6 3 6 1 3 6 2 3 6 3 Starting the system Preparing the system for a run Preparing buffers Setting up the liquid system Basic operating procedures Inserting the sensor chip Initiating the liquid handling system Setting the temperature Preparing samples and reagents Ejecting the rack tray Preparing samples Changing reagent racks Preparing reagents Installing the rack tray Starting the run Finishing the run Standby mode Wash buffer tubing Shutdown A Installation A 1 A 2 A 3 A 4 A 5 A S 1 A 2 A 5 3 A 6 General guidelines Space requirements Mains power supply Heating and ventilation Moving the instrument within the lab Shutting down the system Connect
26. at any mains voltage selectors on the PC and peripheral equipment are set correctly 3 Install the PC and peripheral equipment according to the respective instruction manuals anmam Badan adanan ADADA ODA caapanan caapanan Baan adanan ODODANAD Daa DADA ATTENTION For protection against Mains fire hazard replace DED fuses only with same type oan es and rating of fuses 2xT6 3AH 250V Mains switch La MAINS POWER 3 MAINS INLET Mains Disconnect cord inlet before opening Autorange 100 240V 50 60Hz 6 3A Figure A 2 Mains input panel at the rear of the instrument A 5 3 Connecting the instrument to the PC Connect a serial communication cable between the COM1 or IOIOIA port of the PC and the PC connector on the rear panel of the processing unit The SERVICE connector is connected to COM2 on the computer Biacore T100 Instrument Handbook 41 version AA Installation IMPORTANT INFORMATION Read Manual before operation Figure 3 3 Computer ports on the right hand side of the instrument A 6 Installing the software Biacore T100 Control Software and Biacore T100 Evaluation Software are supplied on CD The softw
27. ck Biacore T100 Instrument Handbook 17 version AA Description 2 3 5 Buffer selector valve The buffer selector valve has four input ports and three output ports two for the syringe pumps and one for the peristaltic pump One out of four buffers is selected and conveyed to all outputs The valve is controlled from the software Buffer A is selected by default The buffer can be changed during buffer scouting runs or during method based runs Note All wizards except buffer scouting use Buffer A Output ports To degasser i To degasser Input ports e M To peristaltic pump Figure 2 15 Buffer selector valve 2 3 6 Buffer degasser Two buffer lines pass through independent vacuum chambers in the degasser Gas molecules dissolved in the liquid diffuse through membrane tubing in the vacuum chamber The vacuum pump of the degasser operates as required to keep a low pressure as soon as the flow system is started Outputs to syringe pumps Inputs from buffer selector valve Figure 2 16 Buffer degasser front panel 18 Biacore T100 Instrument Handbook version AA Description 2 3 7 Integrated u Fluidic Cartridge IFC The IFC consists of a series of micro channels and membrane valves encased in a plastic housing and serves to control delivery of liquid to the sensor chip surface Samples are transferred from t
28. d software Reagent racks Microplates Liquid containers Buffer tray Waste and water tray Chemical resistance E Technical description E 1 E 1 1 E 1 2 E 2 E 2 1 E 2 2 Index Detection principle Surface plasmon resonance What SPR measures Flow system Buffer degasser Cooling unit Biacore T100 Instrument Handbook version AA Contents 67 67 67 68 68 68 69 69 69 69 71 71 72 73 74 75 iii Contents iv Biacore T100 Instrument Handbook version AA Introduction 1 Introduction 1 1 Biacore T100 system Biacore T100 from Biacore is a high performance system for real time biomolecular interaction analysis using surface plasmon resonance technology SPR The system is intended for research and development activities requiring flexibility in assay setup Biacore T100 consists of a processing unit controlled from a PC running Biacore T100 Control Software The software is wizard based for ease of use but advanced users can develop and customize methods to suit their needs Biacore T100 processing unit provides high sensitivity and a wide dynamic range The design of the flow cells is optimized for accurate reference subtraction An integrated buffer degasser eliminates precipitation of air in the flow system With an optional GxP package the Biacore T100 system can be operated in compliance with current GxP regulations Sensor chips and a range of chemicals and reagen
29. ding capacity try alternative immobilization methods or use a Biacore T100 Instrument Handbook version AA Troubleshooting capturing approach In some cases it may be sufficient to perform immobilization at a milder pH or to avoid exposure of the ligand to immobilization buffer as far as possible The immobilization wizard supports dilution of ligand into immobilization buffer immediately before injection for ligands that do not withstand extended exposure to immobilization conditions Make sure that the ligand withstands regeneration conditions without loss of activity If you cannot find suitable regeneration conditions consider using a capturing approach so that you use fresh ligand on the surface for each analysis cycle If you plan to store sensor chips with immobilized ligand for later use perform control experiments to establish that the ligand retains activity in the storage conditions Make sure you choose the correct flow path for a run If you inject samples over a flow cell where no ligand is immobilized you will not see significant binding C 2 2 Sample considerations Make sure the samples do not precipitate at the concentrations and buffer conditions used Even micro precipitation on the surface can seriously disturb the detection and is generally seen as excessive noise and irregularities in the sensorgrams Where possible samples should be prepared in running buffer or similar conditions Drastic changes in
30. door and open the tube clamp of Eject Rack the peristaltic pump to relieve the pressure on the pump tubing v Rack Illumination see Figure B 11 below euro 5 Exit from Biacore T100 Control Software by selecting File Exit Eject Chip Select Exit the software Set Temperature 6 Open the sample compartment door and clean the needle and liquid supply block if necessary Sections B 4 3 to B 4 5 Preferences More Tools 7 Remove bottles the waste cap assembly and the bottle trays Seal the loose tubing ends for instance by wrapping them in plastic bags Figure B 11 Opening the tube clamp To completely open the clamp flip the lever to the second position until it touches the compartment wall Caution Before re starting the system make sure that the tube clamp is properly fastened Biacore T100 Instrument Handbook 61 version AA Maintenance 62 Biacore T100 Instrument Handbook version AA Troubleshooting C Troubleshooting This chapter gives a brief guide to troubleshooting procedures for problems with Biacore T100 C 1 System related problems C 1 1 Instrument hygiene A fundamental requirement for trouble free operation of Biacore T100 is that the system is kept clean and is maintained regularly according to the following guidelines Always use fresh buffer and distilled water Filter both buffer and water through a 0 22 pm filter to remove particles Follow
31. e reference cell for flow cells 2 3 and 4 whereas Fc3 can be used for Fc4 only 2 3 9 Sample handling During operation samples and reagents are aspirated from the microplate or rack through the injection needle directly into the IFC The needle is fixed and the rack tray carriage moves into position as required The injection needle and IFC are washed automatically 2 3 10 Standby mode When a run is completed the instrument is automatically placed in standby mode and a continuous low flow of buffer is maintained through the flow system to prevent accumulation of buffer residues and preserve the ligand The default standby period is 4 days Liquid consumption during standby is approximately 65 ml 24 hours Buffer tube A is used in standby Note Biacore recommends that you use distilled and filtered water instead of buffer to minimize salt deposits However if an immobilized sensor chip is docked and should be used later buffer may be necessary to preserve the sensor surface during standby 2 4 Detection system The detection system consists of the sensor chip and the optical unit Molecules that bind to the sensor chip surface affect the reflection of light from a light source within the optical unit See Appendix E for a description of the SPR principle The interaction occurs on the surface of the sensor chip which forms one wall of the flow cell when the sensor chip is docked on to the IFC 20 Biacore T100 Instrument
32. e sensor chip port opens and the Insert Chip dialog is shown Insert Chip x Sensor chip ore 5 Select Chip type from the list of available types This determines the available choice of predefined immobilization methods 6 a Insert the sensor chip into the b Make sure that the sensor chip sensor chip port arrow is fully inserted pointing into instrument c Close the sensor chip port cover Press gently until it clicks into position Figure 3 3 inserting the sensor chip 28 Biacore T100 Instrument Handbook version AA Operation Tools Help Prime Shutdown Biacore T100 Evaluation Standby Stop Standby Eject Rack v Rack Illumination Insert Ghip Eject Chip Set Temperature Preferences More Tools 7 If you need to open the sensor chip port cover at this stage for example if you have inserted the wrong chip click Cancel This will close the Insert Chip dialog without docking the chip you can then choose Insert Chip again to open the port cover 8 Click Dock chip A countdown timer in the status bar shows the remaining time of the docking process A standby flow of running buffer is started automatically 3 3 2 Initiating the liquid handling system If the buffer solutions are changed check the Prime before run option in the System Preparations dialog which appears during setup of each wizard run If you want to prime the
33. ent can be switched on and off through the software command Tools Rack Illumination Note Condensation may occur transiently on the window of the sample compartment door during a temperature change WARNING Do not open the sample compartment door with the mains switch on The instrument must be off as long as the sample compartment door is open 2 1 4 Sensor chip port The cover to the sensor chip port is controlled through software commands and cannot be opened by hand Insert the sensor chip when the port has opened Close the sensor chip port by pressing on the upper edge The sensor chip is then docked into the instrument through a software command Figure 2 5 nserting a sensor chip Biacore T100 Instrument Handbook 11 version AA Description 2 2 Indicators and switches 2 2 1 Status lamps Ready green Lights when power is on and the instrument is ready for use ready System red This lamp is lit for a few seconds after the power is switched on If the lamp lights in other circumstances turn off the instrument and call your Biacore service representative system temperature Temperature yellow Lights steadily when the temperature at the flow cell is stable at the preset temperature Flashes when the temperature is not stable sensor chip Sensor chip green Lights when a sensor chip is docked and ready Run green Lights when the system is running 2 2 2 Mains po
34. er to increase the solubility of most biomolecules and salt Run the maintenance tool Desorb and Sanitize followed by Superclean if you suspect that the Desorb and Sanitize procedure is not sufficient to clean the flow system Total runtime is about 1 5 hours Sensor Chip Maintenance is required for the procedure The solutions used in the Superclean procedure may damage the surface of sensor chips used for assays Required solutions are listed in Table B 4 Proteins Small molecules deionized water at 50 C deionized water at 50 C 1 acetic acid 1 acetic acid 0 2 M sodium bicarbonate 0 2 M sodium bicarbonate 6 M guanidine HCl 50 DMSO 10 mM HCl 10 DMSO 56 Table B 4 Required solutions for the Superclean procedure B 4 3 Opening the sample compartment WARNING Always turn off the mains power switch before opening the sample compartment door The instrument must be off as long as the sample compartment door is open If you need to access the needle and the liquid supply block for cleaning 1 Choose Tools Stop Standby if the instrument is in Standby mode 2 Remove any microplate and reagent rack from the sample compartment Make sure that the rack tray is fully retracted and the sample compartment is closed 3 Turn off the MAINS POWER switch at the rear right of the instrument 4 Open the sample compartment door use a flat head scr
35. ernal reflection the light leaks an electric field intensity called an evanescent wave field across the interface into the medium of lower refractive index without actually losing net energy The amplitude of the evanescent field wave decreases exponentially with distance from the surface and the effective penetration depth is about half the wavelength of the incident light Incident Reflected light light SPR angle Sensor chip A Flow cell Reflected light intensity SPR angle Angle of reflection Figure E 12 The SPR principle Biacore T100 Instrument Handbook 71 version AA Technical description 72 At a certain combination of angle of incidence and energy wavelength the incident light excites plasmons electron charge density waves in the gold film As a result a characteristic absorption of energy via the evanescent wave field occurs and SPR is seen as a drop in the intensity of the reflected light Figure E 1 Because the evanescent wave field penetrates the solution conditions for this resonance effect are very sensitive to the refractive index of the solution within the effective penetration depth of the evanescent field Changes in solute concentration at the surface of the sensor chip cause changes in the refractive index of the solution which can be measured as changes in the SPR conditions Note The reduced intensity of reflected light is not caused by light absorption in the sample
36. ewdriver to turn the lock screw 1 8 turn counter clockwise Note The sample compartment door swings upwards when released Do not lean over the instrument when you open the sample compartment door Biacore T100 Instrument Handbook version AA Maintenance Figure B 8 Opening the sample compartment door B 4 4 Cleaning the sample compartment WARNING The injection needle is sharp and may penetrate protective latex gloves Take particular care if hazardous agents have been used 1 Remove the insulation plate at the back of the sample compartment 2 Remove spillage from the sample compartment with water or ethanol as required 3 Dry with a lint free cloth Insulation plate Figure B 9 The sample compartment door opened Biacore T100 Instrument Handbook 57 version AA Maintenance B 4 5 Cleaning the needle and the liquid supply block If salt residues have accumulated 1 Zi Wipe the needle and the liquid supply block with a wet cloth Replace the insulation plate at the back of the sample compartment Pull down the sample compartment door and close the door by pressing it down until it snaps into position Start the instrument and the software Run Tools Prime to wash the needle and the liquid supply block Required solutions Running buffer B 4 6 Removing the sample compartment box If required the sample compartment box can be removed for better access to the sample compartment
37. ge or loss of valuable material In an emergency situation 1 Press Ctrl Break on the keyboard This will immediately stop all current instrument operations including buffer flow and data collection 2 In the dialog box that appears click Yes if you want to wash the system with running buffer You should do this if possible The wash operation takes about 3 minutes Note If you suspect any error in the system exit from Biacore T100 Control Software Turn off power to the instrument with the MAINS POWER switch at the back of the unit Biacore T100 Instrument Handbook 5 version AA Introduction 1 4 6 Restart procedure To restart Biacore T100 1 Turn on mains power and check that the instrument starts normally see Section 3 1 2 Ifthe problem persists run the service tool System Check see Section B 4 1 If any error is detected call your Biacore Service representative If you need to clean the liquid handling system eject the sensor chip and insert a maintenance chip See Appendix B for further instructions 1 5 Conformance with standards 1 5 1 Europe Biacore T100 Processing Unit meets the requirements of the following directives through the referenced harmonized standards European directive Harmonized standard 73 23 EEC Low voltage devices EN 61 010 1 2001 EN 61 010 1 081 2002 Safety requirements for electrical equipment for measurement control and laboratory use 89 33
38. grated u Fluidic Cartridge IFC as part of the docking process Bottles containing buffer are placed on the buffer tray Up to four different buffers can be used A buffer selector valve determines which buffer flows over the sensor surface Biacore T100 Instrument Handbook 7 version AA Description A waste bottle and a bottle containing water are placed on the waste and water tray Reagents and samples are loaded into a reagent rack and a microplate or into a combined sample and reagent rack The reagent rack and or microplate are placed on a rack tray The rack tray or the sample and reagent rack is inserted into the rack tray port During a run samples and reagents are automatically injected into the detection unit 2 1 1 Buffer tray and left pump compartment The buffer tray on the left of the instrument holds up to four bottles for running buffer The buffer tray is designed to hold standard bottles threaded for screw caps Four bottles and caps with gaskets are provided with the system one 1 liter bottle and three 250 ml bottles The left pump compartment houses a buffer selector valve two syringe pumps for sample handling and flow system operation and a buffer degasser To open the pump compartment press on the inner edge of the door Holder for unused buffer tubes Buffer degasser Syringe pumps EIEEE Buffer selector valve o
39. he sample and reagent rack through the needle and into the IFC which connects directly with the detector flow cells Effluent from the flow cells is directed to waste Injection modes There are two injection modes for sample injection with the IFC e Single inject e Dual inject injection of one sample immediately followed by another Sample plug technique minimizes dispersion A sample plug is created by switching between sample and buffer flow in the IFC It is important that the sample is delivered to the flow cell in a well defined plug with minimum dispersion at the sample buffer boundaries To prevent diffusion between buffer and sample a number of air segments are placed between the buffer and the sample The number of air segments varies with the type of injection 2 3 8 Flow cells Four separate flow cells are formed when the sensor chip is docked against the IFC Precision cast channels in the surface of the IFC define the flow cells on the sensor chip surface IN Open valve Closed valve Figure 2 17 Simplified diagram showing sample flow through two flow cells Biacore T100 Instrument Handbook 19 version AA Description The IFC allows single or multichannel analysis in up to four flow cells The flow cells are optimized for use in pairs Fc1 Fc2 Fc3 Fc4 The dead volume between the flow cells of a pair is very small which improves the accuracy of referencing Flow cell 1 can be used as in lin
40. hould use buffer tubing A Section 3 2 2 The syringe pumps will be empty and the response will be out of range if no buffer is supplied to the flow cell e Check that there is sufficient buffer in the buffer bottle s and water in the water bottle at the start of a run C 1 4 Sample considerations e Make sure that you have sufficient sample and reagent in the vials and microplate The volumes specified in the software See the Biacore T100 Software Handbook are minimum volumes with due consideration for dead volumes in different vials and microplates Larger volumes may be needed if you have problems with injection of air in of sample or reagent injections e Use only recommended vials and microplates Appendix D e Use only recommended septa for vials and adhesive foil for microplates Sample evaporation from uncovered samples will both affect the sample concentration and reduce the volume possibly resulting in injection of air Use of unsuitable adhesive foil may block the autosampler needle with adhesive C 1 5 For further help If you are experiencing instrument related problems and are unable to identify and or correct them run System Check then contact your Biacore Service representative C 2 Assay related problems 64 C 2 1 Ligand considerations Loss of ligand activity on the surface of the sensor chip is a common source of application problems e If you cannot immobilize sufficient ligand with retained analyte bin
41. igure B 6 System Preparations dialog At any other time Run Maintenance Tools Normalize to normalize the detector response for all flow cells when changing sensor chip Note Run Normalize with the correct sensor chip docked Do not run Normalize with Sensor Chip Maintenance Biacore T100 Instrument Handbook version AA Maintenance B 4 User service operations Tools for user service operations are available under Test Tools and Service Tools in the More Tools menu Test Tools E System Check E Service Tools lf Flow System Wash z Software Problem Report ai Superclean Figure B 7 Test Tools and Service Tools B 4 1 System check This procedure performs a comprehensive check of system performance using a standard sucrose solution BIAtest solution which is provided in the Biacore Maintenance Kit type 2 Total run time is about 1 hour It is recommended to perform a system check once every month If you experience problems with the system run System Check before calling Biacore Service Use a new Sensor Chip CMS for this procedure Required solutions BIAtest solution HBS N buffer To perform a system check 1 Choose Tools More Tools and select System Check Click Start Biacore T100 Instrument Handbook 53 version AA Maintenance 3 54 I Test Tools 5 m Check Service Tools a Flow System Wash Software Problem Report Superclean This procedure checks for
42. ing to mains power Connecting the instrument to the PC Installing the software B Maintenance B 1 B 2 B 2 1 B 2 2 B 2 3 B 3 B 3 1 B 3 2 B 3 3 B 3 4 B 3 5 Introduction Maintenance summary User maintenance operations User service operations Preventive maintenance User maintenance operations Materials required Cleaning the instrument Checking for leaks Cleaning and disinfecting the flow system Normalizing the detector 23 23 24 24 25 27 27 29 29 30 30 31 33 34 35 36 37 37 37 37 39 39 39 40 40 40 40 41 41 42 43 43 44 44 45 45 46 46 47 48 50 52 Biacore T100 Instrument Handbook version AA B 4 B 4 1 B 4 2 B 4 3 B 4 4 B 4 5 B 4 6 B 5 B 5 1 B 5 2 User service operations System check Superclean Opening the sample compartment Cleaning the sample compartment Cleaning the needle and the liquid supply block Removing the sample compartment box Shutting down the system Standby Shutdown C Troubleshooting C 1 C 1 1 C 1 2 C 1 3 C 1 4 C 1 5 C 2 C 2 1 C 2 2 C 2 3 System related problems Instrument hygiene Instrument considerations Buffer considerations Sample considerations For further help Assay related problems Ligand considerations Sample considerations For additional help D Specifications D 1 D 2 D 3 D 4 D 5 D 6 D 7 D 7 1 D 7 2 D 8 General Processing unit Flow cells System controller an
43. k points Biacore T100 Instrument Handbook version AA Maintenance Tube fittings Plunger tip Pump barrel Plunger drive Figure B 4 Checking the syringe pumps for leakage Cooling unit Condensate water may drip from underneath the instrument during long runs with cooling below ambient temperature Wipe off any water from the bench This is not a leakage but a sign of normal operation of the cooling unit Biacore T100 Instrument Handbook 49 version AA Maintenance 50 B 3 4 Cleaning and disinfecting the flow system Tools for cleaning and disinfecting the flow system are available under Maintenance Tools in the More Tools menu Tro Tools x eq Maintenance Tools 2 Desorb Desorb and Sanitize Empty Buffer Tubing Normalize Wash Buffer Tubing Figure B 5 Maintenance Tools Desorb Run the maintenance tool Desorb to clean the sample tubing and IFC channels at least once a week or more often if proteins and other substances used show a tendency to adsorb to the walls of the flow system often revealed by carry over problems in assays Required solutions BIAdesorb solution 1 BIAdesorb solution 2 deionized water Sensor Chip Maintenance is required for the procedure The solutions used in the Desorb procedure may damage the surface of sensor chips used for assays For some applications a coating of protein on the tubing and IFC channel walls helps t
44. k tray before a run Click on the toolbar button or choose Tools Eject Rack to eject the rack tray carriage in order to access the rack tray The rack tray is ejected and the following dialog box displayed Eject Rack Tray xi m Rack Tray Ejected Click OK to return the rack tray to the sample compartment He Time to auto close 58 Figure 3 4 Eject Rack Tray dialog WARNING The rack tray automatically moves into the instrument 60 seconds after it has been ejected A timer in the dialog indicates when the rack tray will be automatically moved into the instrument Biacore T100 Instrument Handbook version AA Operation Removing the rack tray 1 Press the catch below the front edge of the rack tray to release the rack tray Lift the rack tray slightly and pull out of the instrument a a Release the rack tray b Pull out the rack tray Figure 3 5 Removing the rack tray from the instrument 2 When you click on OK in the Eject Rack Tray dialog the rack tray is moved into the instrument immediately Removing the sample and reagent rack Remove the sample and reagent rack using the procedure described above for the rack tray n 4 2 Preparing samples Dispense the samples into the sample microplate or the sample and reagent rack as shown in the Rack Positions window 2 Check that all samples are at the bottom of the wells It is easy to trap air bubbles at the bottom of the wells in
45. m 2 Preparing the system for a run 3 Basic operating procedures 4 Preparing samples and reagents 5 Starting the run 6 Finishing the run WARNING Wear appropriate protective clothing when preparing reagents and samples 3 1 Starting the system 1 Switch on the instrument The lamps on the front panel should light in the following sequence a All the lamps light for a few seconds and then go out b The green Ready lamp lights c The yellow Temperature lamp flashes to indicate non stabilized temperature and then is steadily lit when the temperature at the detection unit is stable The time required for temperature stabilization depends on the set temperature and ambient temperature The instrument uses the last analysis and sample compartment temperatures set before shutdown as default Temperature stabilization should not take more than an hour 2 Switch on the printer and the PC Biacore T100 Instrument Handbook 23 version AA Operation 3 Start Biacore T100 Control Software click on the Start button in the Windows taskbar and choose Biacore T100 Control Software from the BIA Programs menu 4 The software establishes connection with the instrument which takes about 30 seconds 3 2 Preparing the system for a run 24 3 2 1 Preparing buffers Using standard buffers Always keep a high standard of hygiene in the solutions used Prepare fresh buffer before each run Standard buffers are
46. nal communication cable may be connected between the SERVICE port and a second COM port on the computer for service purposes 2 3 Liquid handling system 2 3 1 Overview The liquid handling system comprises two syringe pumps one peristaltic pump the Integrated u Fluidic Cartridge IFC the injection needle and the liquid supply block The syringe pumps are used for precision delivery of samples reagents and running buffer to the sensor chip surface via the IFC The buffer is degassed before entering the syringe pumps A buffer selector valve determines which of four buffers is used during a run The peristaltic pump supplies buffer and water to the liquid supply block It also pumps waste solution from the liquid supply block to the waste bottle Four independent flow cells are formed in the IFC when a sensor chip is docked Microvalves in the IFC control the liquid flow between the needle and the flow cells Biacore T100 Instrument Handbook 13 version AA Description A simplified diagram of the liquid system showing delivery of samples and running buffer to the IFC is shown in Figure 2 8 Degasser Buffer Figure 2 8 Schematic diagram of the liquid handling system showing delivery of samples and running buffer to the IFC 2 3 2 Autosampler Samples and reagents are held in a microplate and or rack in the autosampler and are dispensed from there
47. o give reproducible results and the first few cycles after Desorb may be unreliable If you observe this behavior run Desorb when you change your protein system rather than once a week Note Do not run Desorb at analysis or sample compartment temperatures below 20 C BIAdesorb solution 1 precipitates at low temperatures Desorb and Sanitize To prevent microbial growth in the instrument keep a high standard of hygiene in the solutions used Run the maintenance tool Desorb and Sanitize at least once a month to remove adsorbed material and disinfect the system All buffer tubing is washed and buffer tubes B C and D are emptied during the procedure Biacore T100 Instrument Handbook version AA Maintenance Sensor Chip Maintenance is required for the procedure The solutions used in the Desorb and Sanitize procedure may damage the surface of sensor chips used for assays Required solutions BIAdesorb solution 1 BIAdesorb solution 2 BIAdisinfectant solution deionized water Note Do not run Desorb and Sanitize at analysis temperatures below 20 C BIAdesorb solution 1 precipitates at low temperatures Empty Buffer Tubing The maintenance tool Empty Buffer Tubing washes and empties all buffer tubing Run this tool when you have used buffer tubes B C or D and do not intend to use them any more Required solutions 70 ethanol deionized water Wash Buffer Tubing Run this tool when you change from buffers containing subs
48. ook 35 version AA Operation Installing the sample and reagent rack Insert the sample and reagent rack using the procedure described above for the rack tray Figure 3 10 Loading the sample and reagent rack into the sample compartment 3 5 Starting the run When docking of the sensor chip is ready a standby flow of running buffer is started e To start a wizard run choose Run Wizard Choose the type of run and select a template Follow the step by step instructions of the wizard e To start a method run choose Run Method Open a method or build a new method e Before you start a manual run run Tools Prime and Tools Normalize To start a manual run choose Run Manual run Define the flow path and click Start For details of the different types of run refer to Biacore T100 Software Handbook 36 Biacore T100 Instrument Handbook version AA Operation 3 6 Finishing the run 3 6 1 Standby mode When a run is completed the instrument is automatically placed in standby mode a continuous low flow of buffer using buffer tube A is maintained through the flow system to prevent accumulation of buffer residues The default standby period is 4 days Liquid consumption during standby is approximately 65 ml 24 hours Note Biacore recommends that you use distilled and filtered water instead of buffer to minimize salt deposits However if an immobilized sensor chip is docked and should be used later buffer may
49. pump compartment door 5 Place a 2 liter bottle for waste solution on the waste and water tray Fit the cap carrying the waste tubes on to the bottle Tighten the tube fittings by hand Do not use a smaller bottle for waste 6 Filla 500 ml bottle with distilled and filtered water Fit a cap with gasket and place it on the waste and water tray Insert the water tube into the water bottle Note Always use fresh water Replace before each run or at least every 48 hours Do not run the system without water 26 Biacore T100 Instrument Handbook version AA Operation Waste cap with two tube fittings Figure 3 2 Bottles on the waste and water tray 3 3 Basic operating procedures 3 3 1 Inserting the sensor chip Before Biacore T100 can be used a sensor chip must be docked in the instrument Make sure that the flow system is properly set up see Sections 3 2 1 to 3 2 2 Click on the toolbar icon or choose Insert Chip from the Tools t menu SJ If a sensor chip is already docked click on the Eject icon that is shown instead or choose the Eject Chip from the Tools menu Click Eject Chip in the dialog box that appears This will empty the flow cells and eject the sensor chip x A This will eject the sensor chip A countdown timer in the status bar shows the remaining time of the undocking process Biacore T100 Instrument Handbook 27 version AA Operation 4 Th
50. r maintenance operations B 3 1 Materials required Biacore Maintenance Kit type 2 contains the materials needed for routine user maintenance Figure B 2 Biacore Maintenance Kit type 2 Kit contents Solution Item Specification BIAdesorb solution 1 0 5 w v sodium dodecyl sulphate SDS two bottles of 95 ml BIAdesorb solution 2 50 mM glycine pH 9 5 two bottles of 95 ml BIAtest solution 15 sucrose in HBS N buffer with 3 mM EDTA one bottle of 65 ml BIAdisinfectant solution conc Sodium hypochlorite with 8 12 active chlorine three bottles of 10 ml BIAnormalizing solution 70 w w glycerol one bottle of 90 ml HBS N buffer 10X One bottle of 50 ml Table B 3 Contents of Biacore Maintenance Kit type 2 46 Biacore T100 Instrument Handbook version AA Maintenance Storage All solutions except BIAdesorb solution 1 should be stored at 4 8 C BIAdesorb solution 1 should be stored at room temperature WARNING BIAdisinfectant solution is corrosive The solution should be diluted shortly before use as described in the Instructions for Use provided with the maintenance kit In addition to the maintenance kit you will need the following accessories e Distilled and filtered water e 70 v v ethanol e Clean lint free cloths e Series S Sensor Chip CMS Caution Some maintenance procedures will destroy the ligand on a prepared sensor chip Always use the separate Sensor Chip Maintenance
51. r on the rack tray Make sure that it is correctly oriented If the rack is inserted the wrong way the rack tray will not fit into the sample compartment Biacore T100 Instrument Handbook 33 version AA Operation 3 Push the rack until it snaps into position on the rack tray Make sure that the rack is properly in place 3 4 4 Preparing reagents 1 Open the cover of the reagent rack or the sample and reagent rack 2 Cap the vials and place in the reagent rack as shown in the Rack Positions window Use only caps supplied or approved by Biacore for use in Biacore T100 3 Close the reagent rack cover and press until it snaps shut Make sure that the cover is completely closed 34 Biacore T100 Instrument Handbook version AA Operation 3 4 5 Installing the rack tray 1 Tf the rack tray port is not open when you are ready to install the rack tray eject the rack tray carriage as described in Section 3 4 1 2 Insert the rack tray Press gently until the rack tray snaps into place 3 The rack tray automatically moves into the instrument 60 seconds after it has been ejected When you click on OK in the Eject Rack Tray dialog the rack tray is moved into the instrument immediately An alert will be shown if the rack tray is not inserted properly make sure that the rack tray has snapped into place and click OK Figure 3 9 Loading the rack tray into the sample compartment Biacore T100 Instrument Handb
52. re in the vacuum chambers thereby drawing gas molecules through the membrane tubing Valves in the degasser switch pump input between the vacuum chambers and atmosphere This keeps the pump clean and increases the service life of the degasser An active carbon filter provides added protection for the vacuum pump E 2 2 Cooling unit A cooling unit based on Peltier elements is built into Biacore T100 The cooling unit is located at the bottom of the instrument behind the sample compartment The cooling unit normally requires no user maintenance Condensate water may occasionally drip from the cooling unit during long runs at low temperatures Biacore T100 Instrument Handbook version AA Index Index A adhesive foil 15 air bubbles 31 B basic operation overview 23 Biacore T100 instrument general description 7 Biacore T100 system 1 buffer selector valve 18 C changing reagent racks 33 chemical resistance 69 cleaning flow system 50 instrument 47 needle 58 sample compartment 57 computer ports 13 contamination with biohazards 43 control software installation 42 cooling unit 49 74 D degasser 18 74 Desorb 50 Desorb and Sanitize 50 detecting molecule 72 detection system 20 71 diagnostics 55 disposal procedures 5 docking the sensor chip 27 E electromagnetic compatibility European directive 6 emergency stop 5 evanescent wave 71 F flow system 73 cleaning and disinfec
53. ringe pumps and one peristaltic pump handle the distribution of buffer and water to the IFC and the liquid supply block One syringe pump maintains a low flow of buffer through the IFC The second syringe pump is used for aspiration of samples and reagents and precision delivery to the sensor chip surface via the IFC The system switches automatically between these pumps as required The peristaltic pump continuously supplies buffer and water to the liquid supply block The buffer is used for washing the injection needle in position S1 of the liquid supply block The buffer flow also helps keeping the liquid supply block clear of precipitated buffer Water is used for rinsing the needle position S2 and the liquid supply block itself Effluent from the IFC and the liquid supply block is conveyed to the waste bottle Figure E 13 shows the principle of the flow system i To waste Syringe pumps Vacuum degasser Peristaltic pump Buffer selector valve Buffer Waste Water Figure E 13 Principle of the flow system Biacore T100 Instrument Handbook 73 version AA Technical description 74 E 2 1 Buffer degasser The degasser consists of independent vacuum chambers each containing membrane tubing which is permeable to small gas molecules Each of the two buffer tubes from the selector valve is connected to a separate vacuum chamber A small vacuum pump maintains a low pressu
54. stem as a whole In general the flow system components withstand long term exposure to common aqueous buffer solutions used in biochemical laboratories Table D 2 below lists compatibility with other common substances Concentrated organic solvents as well as long term exposure to extremes of pH lt 3 and gt 11 should be avoided For solutions with short term compatibility do not use as running buffer or for injections longer than 10 minutes Solutions classed as long term compatible may be used as running buffer Biacore T100 Instrument Handbook 69 version AA Specifications 70 Solution Concentration Compatibility Acetonitrile 50 Short term Dimethyl formamide 50 Short term DMF Dimethyl sulphoxide 50 Short term DMSO 10 Long term Ethanol 70 Short term 10 Long term Ethylene glycol 100 Short term Formic acid 70 Short term Formamide 40 Long term Table D 2 Chemical resistance Biacore T100 Instrument Handbook version AA Technical description E Technical description This appendix gives a brief description of technical aspects of Biacore T100 E 1 Detection principle E 1 1 Surface plasmon resonance Biacore T100 exploits the phenomenon of surface plasmon resonance SPR to detect and measure analyte SPR is a phenomenon that occurs in thin conducting films at an interface between media of different refractive index Under conditions of total int
55. tances that tend to adsorb to the tubing e g detergent or BSA One of the buffer tubes or all buffer tubes are washed during the procedure The selection is made at the start of the procedure Required solutions BIAdesorb solution 1 BIAdesorb solution 2 deionized water Sensor Chip Maintenance is required for the procedure The solutions used in the Wash Buffer Tubing procedure may damage the surface of sensor chips used for assays Note Do not run Wash Buffer Tubing at analysis temperatures below 20 C BIAdesorb solution 1 precipitates at low temperatures Biacore T100 Instrument Handbook 51 version AA Maintenance 52 B 3 5 Normalizing the detector At the start of a run Check the Normalize detector option in the System Preparations dialog which appears before the start of each run This procedure adjusts the detector response to compensate for slight differences in individual sensor chips For best performance run this procedure once for each new chip The procedure can either be run before immobilization or before the first run using the immobilized chip Normalization injects a solution of 70 glycerol in water over the chip surface Required solution BIAnormalizing solution System Preparations x IV Prime before run IV Normalize detector M Temperature settings Analysis temperature 25 C Sample compartment temperature 25 C Help lt Back Next gt Close F
56. tened Buffer Air in single injection Tubing squeezed or not fully inserted into buffer or water Blank injection deviates from baseline Deposits in the liquid supply block B Refractometer Too low values A new chip was not used May also result in z too large spread in baseline level Fc3 Fc4 Too high or too low Wrong buffer Wrong values temperature Flow cell leakage too Bad IFC Call Biacore large Service C Injections Leaks in syringe pump Call Biacore Service Ect or other parts of flow Fc2 system Fc3 Fc4 D Noise Drifting baseline A new chip was not used Temperature not stable Call Biacore Service E Buffer selector Buffer A Buffer B Buffer C Buffer D F Mixing Mix1 Mix2 Difference Tubing in wrong bottles Check that the buffer tubes are inserted in the correct bottles Buffer selector not working Call Biacore Service Leaking syringe pump Leaks in syringe pump or other parts of flow system Salt deposits on the needle Call Biacore Service Call Biacore Service Clean the needle Section B 4 5 Check that vials are clean and unused Table B 3 System check diagnostics Biacore T100 Instrument Handbook version AA 55 Maintenance B 4 2 Superclean The Superclean procedure washes the flow system and denatures proteins to increase their solubility Warm water is used as running buff
57. that is included in the maintenance kit B 3 2 Cleaning the instrument If necessary clean the cover of the processing unit with a moist cloth Use water or a mild detergent The buffer tray and the waste and water tray can be removed for cleaning If necessary clean the waste bottle cap as follows 1 Unscrew the cap from the waste bottle 2 Loosen the tube fittings and remove the tubes from the cap 3 Rinse the cap in deionized water 4 Attach the tubes to the cap and tighten the fittings firmly Biacore T100 Instrument Handbook 47 version AA Maintenance Degasser Syringe pumps Buffer selector valve 48 B 3 3 Checking for leaks Once a week check that there are no liquid or salt deposits at the following positions see Figure B 3 e Syringe pump tube fittings inside the pump barrel at the plunger drive for details see Figure B 4 e Buffer selector valve tube fittings e Degasser tube fittings e Peristaltic pump tube fittings below the pump If you find leaks at tube fittings clean with water and tighten the connections If you find leaks in either of the syringe pumps call your Biacore Service representative AD o __ TIC Peristaltic pump Figure B 3 Leakage chec
58. the recommended maintenance procedures as described in Appendix B Do not ignore maintenance schedule reminders Section B 1 If the system is used with particularly sticky molecules or complex mixtures such as serum clean the flow system thoroughly after each run Leave the system in standby mode with freshly filtered distilled water or buffer when not in use or run the Shutdown procedure to empty the flow system Do not leave the instrument with liquid standing still in the flow system Do not take the sensor chip out of its protective cassette Dust or other particles on the sensor chip surface can seriously interfere with detection C 1 2 Instrument considerations If the PC does not seem to communicate with the instrument check that the communication cable is properly connected Section A 5 3 Make sure there is adequate ventilation around at the instrument site Section A 4 Check the accessible tube fittings on the pumps buffer selector valve and degasser regularly for leaks Section B 3 3 Biacore T100 Instrument Handbook 63 version AA Troubleshooting e Make sure the clamp on the peristaltic pump is properly closed at the start of a run Section 3 2 2 If the clamp is not closed buffer and water will not be supplied properly to the liquid supply block C 1 3 Buffer considerations e Make sure that the correct buffer tubing is inserted into the buffer bottle s If you are only using one buffer you s
59. through the injection needle The sample compartment is temperature controlled Injection needle m Liquid supply block Reagent rack Sample microplate Rack tray Rack tray carriage Figure 2 9 The autosampler 14 Biacore T100 Instrument Handbook version AA Description 2 3 3 Microplates and racks Sample microplate Biacore T100 uses standard 96 well and 384 well microplates that are mounted on the rack tray The microplate is held in position by a spring loaded catch Figure 2 10 Rack tray with 96 well microplate Biacore T100 is designed to accommodate microplates conforming to the Society of Biomolecular Screening SBS standard Microplates with deep wells are not supported However since microplate dimensions are not fully regulated in the standard compatibility can only be guaranteed for microplates supplied by Biacore For information about compatibility with other microplates please contact your local Biacore representative Adhesive foil The microplate should be covered with adhesive foil to prevent sample evaporation Note Use only foil supplied by Biacore The adhesive substance on the foil is only present between wells Align the foil with the microplate when attaching it otherwise the needle may be clogged when penetrating the foil Reagent rack The reagent rack holds reagents and other solutions Two reagent rack formats are currently supported Biacore T100 Instrument Handbook 15
60. ting 51 BIACORE T100 Instrument Handbook 75 version AA Index 76 H hazards 2 hygiene 63 I IEC EN standards 6 inserting a sensor chip 27 installing control software 42 installing the rack tray 35 instrument transport preparations 60 integrated microfluidic cartridge IFC 13 19 L leaks 48 ligand 64 liquid handling system 13 initiating 29 lithium backup battery 5 low voltage devices european directive 6 M mains inlet 41 mains power 40 mains power switch 12 maintenance accessories 47 overview 43 safety requirements 43 user 44 maintenance kit 44 46 Maintenance Kit 44 Maintenance Tools 43 microtiter plates 15 69 moving the instrument 40 N normalizing the detector 52 O online help 2 P PC connection to Biacore T100 41 plasmons 72 power requirements 40 preparing buffers 24 preparing reagents 34 preparing samples 31 preventing microbial growth 50 Biacore T100 Instrument Handbook version AA Index preventive maintenance 45 processing unit specifications 67 68 R rack cover 33 34 rack tray ejecting 30 installing 35 rating information 2 ready lamp 12 23 reagent rack 15 68 reagent supply block 17 refractive index 71 removing adsorbed proteins 50 restart Biacore T100 6 Run lamp 12 running buffer 25 69 S safety information 2 instructions 4 labels 3 sample compartment 10 sensor chip 21 docking 27 undocking 27
61. ts are available from Biacore Figure 1 1 The Biacore T100 system Biacore T100 Instrument Handbook 1 version AA Introduction 1 2 The Instrument Handbook Biacore T100 Instrument Handbook guides you through operation and maintenance of the Biacore T100 system The handbook also includes a system description safety instructions specifications and a brief description of the SPR principle 1 3 Associated documentation Biacore T100 Software Handbook describes how to use the software for creating and editing methods running experiments and evaluating results Online Help in the Biacore T100 Control Software and Evaluation Software provides detailed assistance on using the software Biacore T100 GxP Handbook describes how the Biacore T100 system is used with the optional GxP package for compliance with current GxP regulations Other documents describing the technology in more detail are available from Biacore 1 4 Safety information Biacore T100 Processing Unit contains mains voltage and handles liquids that may be hazardous Before operating or maintaining the system you must read this handbook and be aware of the hazards described below Follow the instructions provided to avoid personal injuries or damage to the equipment Do not use the equipment in any other way than described in this manual 1 4 1 Rating information Instrument ratings are printed on the mains input panel at the rear of the instrument 2
62. ular basis by your Biacore service representative Software tools for maintenance and service are accessed by choosing More Tools from the Tools menu This command opens the Tools dialog see Figure B 1 Te Tools x AQ LELGELALE lt Desorb Desorb and Sanitize Empty Buffer Tubing lt Normalize Wash Buffer Tubing Test Tools System Check Service Tools lai Flow System Wash Software Problem Report ai Superclean Figure B 1 The More Tools menu Biacore T100 Instrument Handbook 43 version AA Maintenance This chapter summarizes user maintenance and describes the use of software tools for user maintenance and service procedures If more extensive maintenance or service of the instrument is required please contact your local Biacore Service representative Make sure that a Biacore Maintenance Kit type 2 is available before starting maintenance procedures see Section B 3 1 B 2 Maintenance summary Regular checks and maintenance should be done according to the schedule below B 2 1 User maintenance operations Ssee Section B 3 for user maintenance instructions Daily after each run Monthly Table B 1 Schedule for user maintenance operations 44 Action Empty the waste bottle Inspect tube fittings and pumps check for leaks Clean the flow system Desorb Clean the instrument cover Clean and disinfect the flow system Desorb
63. users around the world If you cannot solve your assay related problems with the information provided in these publications contact your Biacore Application Support representative Biacore T100 Instrument Handbook version AA Specifications D 1 General D Specifications Automation Molecular weight detection Sample volume Sample reagent capacity Analysis time per sample D 2 Processing unit Dimensions see also Fig A 1 WxDxH Net weight Weight incl packing Mains requirements Fuses Power consumption Ambient temperature range Ambient humidity Analysis temperature Safety standard EMC Biacore T100 Instrument Handbook version AA 48 h unattended operation gt 100 Da ligand and analyte dependent 10 425 ul application dependent One 384 well or one 96 well microplate 33 reagent vials or 73 vials for samples and reagents Typically 2 15 min 600x615x690 mm 60 kg 80 kg Autorange 100 240 V ac 50 60 Hz protective earthing 2xT6 3AH Max 6 3 A 18 33 C lt 85 RH 4 45 C max 20 C below ambient temperature See Section 1 5 67 Specifications D 3 Flow cells Number of flow cells 4 Flow cell height Approximately 0 04 mm Flow cell volume approximately 0 06 ul D 4 System controller and software PC operating system Microsoft Windows 2000 or XP Professional Interfacing Possibilities for import of sample data and export of results e g to LIMS
64. wer switch The mains power switch is located on the mains input panel at the rear right of the instrument amma ADADDA DDA AD DDD anaman DDD ADADA DDAA DDD ADD ADADA ATTENTION For protection against Mains fire hazard replace an fuses only with same type oN and rating of fuses 2xT6 3AH 250V Mains switch MAINS POWER R MAINS INLET Mains Disconnect cord inlet before opening Autorange 100 240V 50 60Hz 6 3A a Figure 2 6 Mains input panel at the rear of the instrument 12 Biacore T100 Instrument Handbook version AA Description 2 2 3 Computer connections Two computer ports are located on the right hand side of the instrument The serial communication cable provided should be connected between the PC port and the COMI serial port on the computer on some PCs the COM1 port is marked IOIOIA ai aa III TI E E ED EI VOI VED EI DD EA DD DDD UTI TI RENERE RENE DW DDD EA e EP Wome IMPORTANT INFORMATION Read Manual before Operation f a 1 EDS Figure 2 7 Computer ports on the right hand side of the instrument An additio

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