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Reichert SR7000Surface Plasmon Resonance Refractometer

User GuideVersion 5

SR7000 Operating Manual

2 SPR manual.v.3-22-06

Table of Contents

Introduction ___________________________________________________________ 3

I. Getting started________________________________________________________ 3

II. Setting up___________________________________________________________ 5

Software Installation ____________________________________________________ 5

LABVIEW SPR data acquisition program installation _________________________ 5

LABVIEW Data Normalization program installation ______________________ 6

Data file Folder ______________________________________________________ 6

Reichert LABVIEW® interface _________________________________________ 6

A brief overview of the LabVIEW buttons. _______________________________ 7

Tera Term set-up ____________________________________________________ 8

SR7000 set-up_________________________________________________________ 14

Setting the prism temperature_________________________________________ 14

Fluidics set-up ________________________________________________________ 15

Valve system _______________________________________________________ 15Schematic 1 filling the sample loop.____________________________________ 16Inject Position _____________________________________________________ 17Schematic 2 injecting to the flow cell___________________________________ 17

Peristaltic Pump ____________________________________________________ 18

Testing the Fluidics__________________________________________________ 18

Mounting a gold slide on prism surface _________________________________ 19

Connecting the Flow Cell _____________________________________________ 21

Surface Plasmon Resonance Readings_____________________________________ 221. SR7000 ______________________________________________________ 222. LabVIEW Setup for a Reading____________________________________ 223. Injecting Ethanol and Checking the SR7000 Response _________________ 23

A Typical Molecular Interaction Analysis Using BSA as the Immobilized Ligand andAnti-BSA IgG as the Analyte ____________________________________________ 24

Data Analysis _________________________________________________________ 31

Importing data directly into Excel _____________________________________ 31

Normalizing Data and Preparing for Analysis____________________________ 32

System Components ____________________________________________________ 34

Optional Thermal Housing ______________________________________________ 35

Troubleshooting _______________________________________________________ 36

Copyright and Trademark information: ____________________________________ 37



Introduction

The SR7000 is a simple and flexible Surface Plasmon Refractometer. This manual is anoverview of how to get started along with some simple experiments to use to gainexperience. The sections are:

I. Getting started. II. Setting up. III. Taking a reading. IV. A standardized experiment.

I. Getting started

Several components make up the SR7000 SPR system. The packing list that shipped withthe instrument can be used to verify the presence of all the required parts. The majorcomponents are:

• The SR7000 instrument.

Fluidics include:

o A pump: A syringe pump is recommended as it will provide even flow with

low baseline noise over a wide range of flow rates. Peristaltic pumps offer the advantage of being able to recirculate

when the sample is limited or for long equilibrium experiments. Peristaltic pumps also provide easy changeover of solvents. But, peristaltic pumps increase baseline noise due to the cycling

nature of the pump mechanism.

o Tubing: PTFE and/or PEEK polymer tubing is recommended to preventsamples from adsorbing to the inside of the tubing. E.g. proteins willadhere (adsorb) to the surface of the tube and can then contaminatesubsequent experiments.

o A valve system: This is needed to change between samples, buffers,regenerating solutions and cleaning solutions. The supplied valve standholds an injection valve and a solvent select valve as well as one injectionport and one waste port.



• Software

o National Instruments LabVIEW® data acquisition and instrument controlsoftware is used for response versus time experiment set-up and tocontrol/monitor as well as the collection of data. A custom executableonly version of National Instruments LabVIEW Software is bundled withthe SR7000. We will provide the code to anyone who has appropriatelicenses for LabVIEW so that they might customize this interface to bettersuit their purpose.

o Tera Term terminal emulation software can be used to communicatebetween the PC the SR7000. It is used to download reflectivity versuspixel (angle) profiles. Transferred data is in tab delimited format. Oncetransferred, the data can be imported into another program for analysis. Atypical protocol is to import into Excel to do calculations and to graphresults.

Note that the computer should not have programs running in the background.Games, programs that automatically poll for messages, some print drivers, andlive e-mail are examples of programs that can interrupt communication andcause dropped data.

Accessories

o 12.5 x 12.5 mm gold coated glass slides. The SR7000 has been optimizedfor slide with a glass thickness of 0.95mm, a 10 Å Chromium layer and a500 Å Gold layer. The slides supplied with the instrument have a selfassembled monolayer for immobilizing e.g. proteins or DNA. Theseslides have a mixed self-assembled monolayer formed on the gold surfaceconsisting of a thiol-polyethylene glycol and a thiol-polyethylene glycolcarboxyl mixture. A ligand (protein, DNA) may be attached to thecarboxyl group by a variety of immobilization chemistries.

o The SR7000 can use any slide that will fit the prism well and has a glassthickness between 0.1 mm and 1.25mm.

Additional accessories: Tweezers, RS232 cable



II. Setting up

Remove all components from packaging. Check contents against the packing list. If anitem is missing, contact the factory for resolution.

Plug the pump and SR7000 SPR Refractometer into either a 110 V or a 220 V AC outlet.The SR7000 power supply is set for either voltage; check the pump to ensure it isappropriately set-up for the voltage. Connect the RS232 cable from the lower serial port on the SR7000 to a computer serialport (Pentium I, 32 Mbytes ROM minimum, Windows 98, 2000 or XP).

Turn on the SR7000 to allow warm-up for a suggested time of 1.5 hours. While the prismtemperature will stabilize very quickly, the internal components must come to a steadytemperature to assure a steady baseline.

Note that the angle of reflectance is a function of refractive index changes and thatrefractive index changes with temperature. Setting the SR7000 in an area where draftsand ambient temperature swings are minimized eliminates an external factor that canconfound measurement.

Software InstallationThe Reichert SPR Data Acquisition CD ROM contains:

o A custom, executable version of National Instruments LabVIEW® Software- This is the main data acquisition/instrument control program

o Tera Term© which is a terminal communication program.- This is used to run diagnostic tests and download reflectivity profiles

from the SR7000

o LabVIEW® SPR normalize program- This is used to normalize injection time and response to zero for

import into kinetic analysis programs.

o The SR7000 operating instructions.

LabVIEW SPR data acquisition program installation

The Reichert SPR Data Acquisition CD-ROM contains a custom executable only versionof National Instruments LabVIEW® Software. Open the folder named ‘SPR 2.21alpha



Dec 02’. Note that the 2.21a is the version number and that you may have a newerversion with a different number.

To install, click on the Setup.exe file and you will be guided through the setup. It issuggested that the default suggestions be accepted to avoid introducing unknowns intothe set-up. Make sure you allow the program time to install completely. Note that themain LabVIEW® screen will indicate complete before all the runtime files install. Thesefiles will automatically install after a short delay.

LabVIEW Data Normalization program installation

On the CD, open the folder labeled “SPR Normalize” and double click on “set up”.Follow the on screen instruction accepting the defaults.The LabVIEW® programs are installed in the C:\\ Program\Leica directory.

Data file FolderCreate the following file folders:

C:\Biotech\Data This data folder is where the LabVIEW® data acquisition program will store data.

Reichert LABVIEW® interface

Temperature

Pixel, angle or µRIU

Time (seconds)



The above screen shot indicates what the LabVIEW® instrument interface will look like.This picture is showing instrument response in pixels versus time (upper chart) and prismtemperature versus time (lower chart).

Note that the x-axis is always time. The y-axis on the lower chart is temperature inCelsius. The upper chart displays response data and there are three user selectablechoices for the y-axis – Resonance angle, Microrefractive index units (µRIU), and Pixelnumber. These are selected on the Chart drop down menu as shown below.

The Serial Input box provides a means to type in commands which are sent to theSR7000 when the “send string” button is clicked. This is not used routinely.Communications the computer and the SR7000 can be checked by typing ping andclicking the “Send string” button. “Ping” runs a diagnostic and returns the message:Main OK, LSA OK, and Peltier OK in the instrument output window on theLabVIEW® screen. This message indicates successful communications are establishedbetween the computer and the 3 microprocessor boards in the SR7000.

The instrument output box continually displays the data coming from the SR7000.

The experiment time (sec) box displays the time the total running time of experiment.This will be elapsed time for the current experimental run.

A brief overview of the LabVIEW buttons.The Initiate button tells the SR7000 to set the proper illumination light level. This isdone after warm-up and before calibration. The SR7000 can be initiated with air, water orrunning buffer in contact with the gold slide

The Calibrate button tells the SR7000 to perform a self-calibration. This is done after aninitiation (see Initiate button below). Calibration sets the microrefractive index units(µRIU) and angle to a zero point (pixel) using the solvent without analyte or during an airscan. This can be done using either a plain gold slide or a biosurface gold slide in theflow cell. Since this ‘zeros’ the baseline for the slide your using, this should be done eachtime a new slide is placed in the flow cell or anytime you wish to reset the µRIU orresonance angle scales to zero.

The READ button is used to collect one data point from the SR7000 and to save it to thefile you designated in the experimental setup window.

The Mode button is to adjust the screen on the SR7000 to read in µRIU, pixel or angle.This changes nothing on the LabVIEW screen on your computer. To make these choicesin LabVIEW, use the drop down menu for Chart.



The Experimental Setup button brings up this window.

This is where you will name the data file for each experimental run to be saved toC:\Biotech\Data\. Note that leaving the name as “default” causes data to be appended tothe existing default file. This also one of the places where you can set the readinginterval (the other is on the LabVIEW® window on the left just above the off button).

The Set/Reset buffer buttons clears the buffers and sends the serial format command tothe SR7000 for the next experiment. Clicking the set/reset button returns the message“SF KLZAIH” to the instrument output window. This is the correct serial format string.

The Send String button and Serial Input box are for sending instructions to the SR7000.Note that the normal operating commands are programmed into LabVIEW® andexperiments do not require any special language or commands to be sent.

The Monitor Liquid button toggles on and off the continuous reading of data to the fileyou created in the Experiment Setup Screen. When data is being saved, it is also beingsent to the PC’s screen.

The Instrument Output window will give a new string of values each time the SR7000takes a reading. This data is automatically saved to the file created for the experimentalrun or to the default file as discussed at the top of this page.

The Exit button exits the LabVIEW program.

Tera Term set-upTera Term© is freeware terminal emulation software, which is available off the web. Wesupply it without cost on our install CD as a convenience. While other terminal emulationprograms can be use, we suggest starting with Tera Term© as there are no known issues between it and the SR7000. Before using Tera Term©, it must be properly set up tocommunicate with the PC.



To install, simply find the Tera Term© set-up icon in the Tera Firm© folder and doubleclick or down load it from the internet. Follow the instructions on the screen.

Once installed click on the Tera Term© Pro Icon

Tera Term Pro.lnk

The New connection screen will appear. Click on serial and find the correct port on the drop down box.

Click on OK This screen will appear

Click on terminal in the drop down box

Terminal size depends on the screen size of the PC-Start with defaultChange the Terminal ID to VT100New-line should have CR in both the Receive and the Transmit boxes

Click on setup



Check the local echo boxClick OK

Click setupClick WindowMake sure that the default settings are as shown below

Click OK

Click on SetupClick on FontSelect terminal and size 10

Click OK

Click on Setup,



Click on Keyboard

Check the Backspace Key boxClick OKClick on SetupClick on Serial port setup

Ensure that the correct port is selectedEnsure that the other settings are as shown

Click OKClick SetupClick on TCP/IP

Host list should be emptySet up as shownClick OK



Click on SetupClick on General

Verify that the default port is set to correct serial portClick OKClick on setup and then choose Save setup…

Note that there are two serial ports on the back of the SR7000. The bottom port cancommunicate at a band rate of 19,200 only. Setting Tera Term© or the instrument via thesetup screen to communicate at some other rate will not change the rate at which data istransferred from the lower port. The upper port can be adjusted to a faster baud rate usingthe instrument’s setup screen. The upper port is used to download reflectivity profiles at115k baud rate

Communications between Tera Term and the SR7000 can be tested by typing “ping” andreturn. If communications are properly established the message: LSA OK, Main OK andPeltier OK will be returned. If this message is not returned, check that:

• The baud rates are set appropriately in Tera Term© (19200 for the bottom serialport on the SR7000 and any rate on the top serial port as long both the instrumentand Tera Term are set the same). This is set in the Tera Term Serial Port set upscreen.

• The serial port setup is correct (see above). • The COM port is correctly specified (see above.

To check, compare the screens above to the actual Tera Term© screens.

Once you have success with communication, click Setup, save setup and Save.



Tera term is now set up to communicate with the SR7000.



SR7000 set-up

Remove all components from packaging. Check that everything on the packing list isidentified and accounted for.

The SR7000 power supply is set for either voltage; check the pump to ensure it isappropriately set-up for your voltage. Plug the pump and SR7000 SPR Refractometerinto a 110 V or a 220 V AC outlet.

A 1.5 hour warm up is usually sufficient to reach internal temperature stability.

Connect the RS232 cable from the lower serial port on the SR7000 to a computer serialport (Pentium I, 32 Mbytes ROM minimum).

Setting the prism temperature

SR7000 prism temperature can be set by pressing the “Set Up” button on the instrumentcontrol panel.

The “Set Up” button accesses the Set Up screen and will appear as shown

This is where theprism temperature isset.



Once in the Set Up screen, the function buttons become arrow buttons until you exit theSet Up screen. Navigate through this screen using the right/left arrows (⇐⇒) to switchbetween set up parameters. Use Up/Down (⇑⇓) arrows to change values of a parameter.

Set the temperature parameter to the required value in degrees centigrade (E.g. 25.00)using the Up/Down (⇑⇓) and right/left (⇐⇒) arrows.

Press READ on the SR7000 to exit set up.

Fluidics set-up

Note that tubing lengths are suggested. Actual lengths depend on distance betweencomponents and distance to solvent reservoirs. It is best to minimize distance to reducedelay, to reduce unknowns due to analyte interaction with tubing, to minimize the mixingeffects in the tubing, and to reduce the volume of solutions needed

Valve systemThe valve system uses commonly available HPLC valves and tubing. A valve stand(pictured below) provides a central point for connecting the pump, sample loop, flow cell,and inlet lines (buffers/solvents).

The diagrams below show how the valves are to be plumbed. The two diagrams showhow the valves connect inputs to outputs given the two positions (load, inject) of the‘inject valve’.

SolventSelect Valve

Waste Port

Inject Port



Load Position

Schematic 1 filling the sample loop.

The inject valve is turned fully counter clockwise in the “Load” position. The pump ispushing solvent from the pump into port 4 and out of port 5 on the inject valve. The 1 mLinjection loop connecting ports 3 & 6 is bypassed. The injection port is connected to thesample loop, which is connected to the waste port. This allows a means to fill the loopwith sample until it is noted to be coming out the waste port.

The 1 mL loop is supplied as standard equipment. Higher or lower volume sample loopsmay be used.

Schematic 2 shows fluid flow when the “inject valve” is turned clockwise to the “Inject”position. Solvent from the pump and solvent select valve is now routed to push fluidfrom the sample loop to the flow cell.

Note that standardequipment loop has avolume of 1 mL FLOW

CELL

BUFFER FROM PUMP



Inject Position

Schematic 2 injecting to the flow cell

To assemble the system, simply use the preceding diagram and picture along with thefollowing detailed instructions.

Note that the connections on the inject valves are different from the instructions of themanufacturer – please setup per the diagram. The valves should be plumbed as describedin this manual to assure “Last in, first out” fluid flow from the injector sample loop.

Fluidics System Setup

1. Syringe pump set upa. Fill a 10 mL syringe with distilled, deionized, degassed and filtered water.

Purge bubbles.b. Connect the Luer tubing adaptor to the syringe.c. Place the syringe into the syringe pump. d. Set the flow rate on the syringe pump to 0.1 mL/min but do not start.e. Set the syringe diameter setting on the syringe pump to the proper value. –

see syringe pump manual.

BUFFER FROM PUMP

FLOWCELL



2. Install nuts and ferrules into solvent select valve and inject valve.a. Note that the ports are labeled 0 for the back center port on the solvent

select valve and 1 to 6 for the ports that circle the back of the valve. b. Attach a 0.020” or 0.030” I.D. tube between the syringe and the ports 1 -6

on the perimeter of the solvent select valve.3. The inject port is located just below and to the left of the injection valve.

a. Install a threaded adjustable injection port adapter into the front of theinject port (see picture). Do not over tighten.

b. Install 4” (10 cm) of 0.010” I.D. tubing from the back of the inject port toport 1 of the injection valve

4. Inject valve. a. Port 4, using 18” (46 cm.) of 0.020” I.D. tubing, connect port 4 of the

injector valve to the pump syringe. b. Ports 3 and 6, install the one end of the sample loop into port 1 and the

other into port 6. c. Port 5, using 20” (50 cm) of 0.01” I.D. tubing, connect port 5 on the

injection valve to the flow cell inlet which is the threaded hole closest tothe front of the flow cell. Leave the connection at the flow cell loose asyou will be testing for flow before connecting.

5. Waste port. Locate waste port just below and to the right of the inject valve. a. Install 2” (5 cm) of 0.02” I.D. tubing in the front of this union slightly

down and place a small beaker under it to catch overflow from filling thesample loop.

b. Install 4” (10 cm) of tubing from the back of this waste port to port 2 ofinjection valve.

Peristaltic Pump

1. Insert peristaltic tubing adapters in each end of a 15 cm length of MasterflexLS/13 Pharmed or Viton peristaltic pump tubing. Consult pump manual forinstructions on placing the Pharmed tubing into pump cartridges and mountingcartridges on pump. Do not increase pressure on tubing at this time. This shouldbe done when testing for flow. Caution: some types of tubing will flake off insideunder high roller pressure. This can plug the tubing. Use only the amount ofpressure that is necessary to establish flow. Follow steps two through five of thesyringe pump set-up.

Testing the Fluidics

Test fluidics by pumping filtered (through a 0.2 µm filter) distilled, deionized anddegassed water through the solvent select valve to the injection valve and out through theline which connects the injector to the flow cell. Flow should be regular with the Injector



valve in both the “Load” and “Inject” positions. If flow is irregular or not flowing, firstcheck for leaks at each connection. If the problem is not apparent, check for flow at eachconnection one at a time, working from the closest to the most distant connections fromthe pump. If there is flow the problem is farther downstream.

Mounting a gold slide on prism surface

Suggestion: Starting out, mount a plain gold slide, if available. This slide can be used totest the instrument response as detailed later in these instructions

Step 1.

Clean the prism surface with ethanolfollowed by water. A cotton swab helpsremove any index matching fluid from thecorners.

Dry with a lint free wipe. Blow off with N2.

Note: The prism surface is sapphire andcannot be scratched. This surface mustbe extremely clean with no visible streaksprior to mounting a new sensor slide. Astream of compressed air or nitrogen willhelp remove dust from the prism surface.

Step 2

Place 0.75 uL of 1.515 refractive indexmatching fluid on the center of the prismsurface. The center can be located by theintersection of the three notches in thesides of the prism well. This is a smalldrop as indicated in the circled area.

notches



Step 3.

Clean the Flow cell body and gasket.New gaskets may have a black residuefrom laser cutting which is easily removedby wiping with ethanol. Mount the gasketon the flow cell body

An interference fit between the gasket andchannel in the flow cell body will hold thegasket securely. The gasket can be pushedinto the grooves using a clean forceps.

Gasket dimensions (defines flow cellarea):1.5 mm wide 8.0 mm long 0.864 mm deep The flow cell volume is 2.5 uLNote that the channel is 0.432 mm deep,leaving flow cell height estimated at 0.216mm when the gasket is compressed 50%.

Step 4.

Couple the gold sensor slide to prism.Grasp the sensor slide firmly with forceps.Align the far edge of the slide with theback edge of the stainless steel bracket.Use caution to avoid touching the topactive surface of the slide.



Step 5.

Slowly lower the slide in place beingcareful not to trap air bubbles under theslide and prism surface

Step 6. Using the flow cell clamping studs andguides, gently position the flow cell bodywith gasket over the gold slide. Lower theflow cell into position until the gasketcontacts the slide. Tighten the flow celllocking nuts evenly and to the samedegree. Visual examination at eye level ofthe prism-flow cell will allow finalleveling of the flow cell. Uneventightening can result in leaks due tomisalignment.

Look for an even gap on both sides ofgasket.

Prism Plate

Flow cellStainless

SteelBracket

StainlessSteel

Bracket

Gasket

The flow cell should be parallel relative tothe prism, so the distance between themon each side of the gasket should be the

same

Connecting the Flow Cell

Connecting input and output lines to the flow cell:

1. Before connecting the line from port 5 of the inject valve to the flow cell, turn onthe pump and watch for at the outlet of this tube.

e



2. Turn off the pump and connect the outlet line from the injector valve (Port 5) tofront (inlet) port of the flow cell.

3. Turn on the pump and observe fluid inside the flow cell by looking into the back(outlet) port. Once you are satisfied there is flow, turn the pump off.

4. Connect a 0.020” waste line to the rear flow cell port. Smaller diameter tubingcan be used to prevent outgassing of the fluid in the flow cell. This is necessarywhen running at high prism temperatures

The set-up is now complete and you are ready to begin taking readings.

Surface Plasmon Resonance Readings

This section provides an overview of typical SR7000 system operation. It covers:

• System preparation and taking surface plasmon resonance readings• Using BSA- anti-BSA as an example experiment to test the system.• Data analysis

System preparation and taking surface plasmon resonancereadings

1. SR7000a. Turn on SR7000 and allow to warm up for 1.5 hoursb. Mount a plain gold slide (included with instrument)c. Fill syringe with distilled deionized water and begin flow at 0.1

mL/min

2. LabVIEW Setup for a Reading

a. Open the Reichert SPR LabVIEW Software

b. Click on “Experiment Setup” and enter the reading time interval and filename. Be sure the “save data” button is clicked “on”. Click on the“Done” button to exit the Experiment Setup Screen.

c. Click on Set/Reset Buffers. This makes certain the LabVIEW memorylocations are clear and sends a RS232 data format command to theinstrument.

The message “SF KLZAIH” will appear in the “Instrument Output”



d. Click “Initiate” to initiate the instrument. This sets the LED light level.The instrument can also be initiated by pressing the “function” button onthe front of the SR7000.

e. Click “Calibrate” to calibrate the instrument. This resets the resonanceangle and microrefractive index unit scales to 0, based on the index of theflowing solution.

f. Click monitor liquid to continuously read at the chosen interval.

g. The y-axis units may be changed to: 1. Resonance angle, 2. MicroRefractive Index Units or 3. Pixel by choosing from the “Chart” Menu atthe top of the screen.

h. Auto scaling of either axis on both charts may be turned on or off by rightmouse clicking on the chart and then choosing Auto scale on or off.

i. When auto scale is off, the range of the axis may be set by clicking the leftmouse button. Click once to edit a digit or twice to change the numberson the axis limits to set a new range limit.

j. In a normal experiment start with Auto scale Y on (default setting) andAuto scale X off (default setting 200 Seconds). Once the baseline isestablished turn auto scale Y off and set the scale range to the desiredvalue. Turn Auto scale X on or reset the upper limit from the default 200Sec. setting to a desired range.

3. Injecting Ethanol and Checking the SR7000 Response

a. Maintain the flow of running DDH2O over the sensor surface at 0.1mL/min. Wait until a stable baseline is reached.

b. Injection Valve should be in the “Load” position.

c. Fill the injection loop with air, pushing any solution out through the wasteport. This eliminates mixing at a front between the solution in the loop –in this case water from the flushing and cleaning protocol – and the newsolution being placed in the loop.

d. Note that anytime you are changing from one sample solution to another,you may wish to fill the loop with buffer or water and then purge with airseveral times to ensure that the old sample is fully purged from the loop.

e. Fill the injection loop with 100% Ethanol. The purpose of injectingethanol is to clean the system tubing and check the instrument response.



The entire loop must be filled. When the entire loop is filled solution willflow out of the waste port on the valve stand.

f. When the baseline is flat and steady turn the injector to the “Inject”position. The refractive index of Ethanol is much higher than water. Alarge positive response will be observed.

g. Note that the injector fluid path is arranged in a last in first outconfiguration. If more than 75% of the loop volume is injected, a mixingfront will be observed. When this happens and the injector is left in the“Inject” position the baseline will slowly return due to the Water/Buffermixing front.

h. Allow the injection to continue for 10 minutes. Turn the injector back toload to resume the flow of water over the sensor surface. The baseline willsharply return.

A Typical Molecular Interaction Analysis Using BSA asthe Immobilized Ligand and Anti-BSA IgG as theAnalyte

Using the SR7000 with this BSA – Anti-BSA protocol is a good test of how well thesystem is setup. Additionally, running samples of interest will follow the same protocol.

This experiment uses a mixed 10% COOH-(PEG)6-Alkanthiol/90% OH-(PEG)3-Alkanthiol self-assembled monolayer on a gold surface.



Overview:

This example illustrates the procedure used to obtain rate and equilibrium kineticinformation from a simple bimolecular protein/protein interaction. Bovine SerumAlbumin (BSA), the ligand, is amine coupled to the surface using EDC/NHSchemistry. The analyte is Anti-BSA Immunoglobulin G which is injected over thesurface at a range of concentrations from 20 ug/mL to 1 ug/mL

EDC - 1-ethyl-3-(3-dimethylamino propyl) carbodiimide, hydrochlorideNHS - N-HydroxysuccinimideThe reaction scheme for immobilizing BSA (Ligand) is shown below:

COHAlkane Thiol (PEG)6

O

+ CN

NR1

R2

EDC

C

O

O

Alkane Thiol (PEG)6

N

NHR1

R2

N

O

OH

O

NHS

N

O

O

C

O

Alkane Thiol (PEG)6

O

+CO

NH R1

NH

R2

Succinimide ester activated Alkane Thiol (PEG) 6N

O

O

C

OAlkane Thiol (PEG)6

O

+ NH2 Ligand

CNHO

Alkane Thiol (PEG)6

Ligand+ N

O

O

OH

NHS

Immobilized ligand

+



1) Start the SR7000 and allow to warm up for at least 1.5 hour.

Note that it does not hurt baseline stability to turn the instrument on the day before theexperiment.

2) Place a slide with the 10% COOH-(PEG)6-Alkanethiol/90% OH-(PEG)3-Alkanethiolmixed self-assembled monolayer in the flow cell. Detailed guidance on slidemounting can be found on pages 19 through 21.

3) Open LabVIEW.

a) Click “Experiment Setup”. Enter a filename and set the reading time at 2seconds.

b) Click on the LabVIEW screen and look to see if the communication windowshows response from the SR7000 which should have the string “SF KLZAIH”. Ifthis does not show, click set/reset buffers.

4) Click on the initiate button on the LabVIEW screen. When initiate is complete, theinstrument screen reads “Initialization Complete” and gives instructions forCalibrating.

5) Start PBST (Phosphate Buffered Saline with 0.05% TWEEN 20) buffer flowing at 0.1mL/min. Click on the calibrate button on the LabVIEW screen. Wait for themessage on the instrument to indicate successful calibration. The instrument willindicate that you can press read to take a reading. This will take read data point forwhatever is on the slide at that moment. To run continuously, use LabVIEW.

6) Set the inject valve to load (fully counter clockwise). Fill the sample loop withPBST.

7) Turn the inject valve to inject (clockwise). There should be no response since theloop is filled with running buffer. If a response is observed it indicates the sampleloop is not clean. The loop should be cleaned by flushing with 100% EtOH, air, thenbuffer.

8) Inject buffer again. The baseline should stay stable. If not, repeat steps 6 and 7 untilno response is seen.

9) Immobilizing BSA as the ligand is a two step process. First react EDC and NHS withthe Carboxyl group on the SAM to form the linkers for the BSA.

10) The protocol can be seen graphically on the plot of a typical surface preparationscenario as seen below and is described starting on page 27.



a) Sample loop should be loaded with PBST

b) The inject valve is in the load position.

c) Pump is set to 0.1 mL/min

d) Click on monitor in LabVIEW

11) Fill the sample loop with 1 mL of a mixture of 0.2 M EDC and 0.05M NHS. It is notnecessary to fill the loop with air prior to filing with EDC/NHS. The EDC/NHSsolution displaces PBST in the loop.

Note that the EDC/NHS solution must be made fresh and used immediately.

a) Turn the inject valve to inject

b) As soon as a large positive response is observed, set the pump to 0.01 mL/min

c) Let run 10 minutes

d) Return the inject valve to load

SR 7000, 10% COOH mSAM SurfaceBSA Immobilization

3 June 2002

850

900

950

1000

1050

1100

1150

1200

1250

3250 3750 4250 4750 5250 5750 6250 6750 7250

Time, Sec

Pixe

l

EDC/NHS

120 ug/mLBSA in20 mM NaOAc, pH 5.5

PBST

1.0 M EthanolaminepH 8.5

PBST

PBST PBST

24.05 Pixels of BSA Immobilizedscale = 37 micro RIU/Pixel889 micro RIU BSA immobilized

Protocol Description:



12) Purge with air and then PBST to remove all traces of EDC/NHS from the sampleloop.

13) Next fill the sample loop with 1 mL of BSA protein solution.

a) Prepare 20 ug/mL BSA in 20 mM sodium Acetate pH 5.5.

b) Fill the inject loop with 1 mL of the BSA solution.

c) With the pump at 0.1 mL/minute, turn the injection valve to inject.

d) Run until a response is seen on the LabVIEW screen, then slow the flow rate to0.01 mL/minute.

e) Let the BSA run until the desired amount is immobilized (10 to 20 minutes isrecommended). Note that the amount of BSA immobilized can be controlled bythe amount dissolved in the solution and the reaction time.

f) Switch the inject valve to load.

g) Reset the flow to 0.1 mL/min.

14) Deactivate excess Succininide ester activated groups

a) With the inject valve in the load position, inject 1 mL of 1 M Ethanolamine (pH8.5) into the sample loop

b) Turn the inject valve to inject.

c) Run until a response is seen on the LabVIEW screen, then slow the flow rate to0.01 mL/minute.

d) Let run for 10 minutes.

e) Turn the inject valve to load

f) The baseline should return to the PBST level or slightly below.

15) Removing excess or non-bound BSA

a) Inject a dissociation solution – e.g. 10 mM glycine pH 2 – for 3 minutes. Thisremoves any non-specifically bound protein.

b) Turn inject valve to inject. The dissociation solution has a lower RI, so theresponse should go down.

c) Run for 3 minutes.

d) This removes any non-specifically bound BSA.



e) The curve on the LabVIEW screen should return to the PBST baseline.

f) Keep the pump flowing while preparing the anti-BSA

16) Prepare a series of anti-BSA concentrations.

a) Clean glassware is an absolute must.

i) Rinse the glassware with the ultra pure water and then PBST being careful toremove all excess PBST and not allowing the PBST to dry into salts beforemixing protein solutions. Do a final rinse with water.

b) Prepare 1 mg/mL anti-BSA IgG stock solution in PBST

c) Add 100 µL of 1.0 mg/mL anti-BSA stock solution to 5 mL of PBST solution tomake a 20µg/mL anti-BSA solution.

d) Take 2.5 mL of the anti-BSA solution and mix with 2.5 mL of PBST. This makesa 10 µg/mL solution. Label this container “10 µg/mL”

e) Prepare 7 small containers by putting 2.5 mL of PBST into each.

f) Label the containers:

i) 5 µg/mL

ii) 2.5 µg/mL

iii) 1.25 µg/mL

iv) 0.625 µg/mL

v) 0.3125 µg/mL

vi) 0.156 µg/mL

vii) 0.078 µg/mL

g) Take 2.5 mL of the 10 µg/mL anti-BSA solution (step c above) and put into thecontainer labeled 5 µg/mL. Mix well.

h) Repeat the serial dilution by taking 2.5 mL of the 5 µg/mL and putting into thecontainer labeled 2.5 ug/mL. Mix well.

i) Repeat serially for all the remaining concentrations.

17) Each association/dissociation curve represents a different concentration of anti-BSA.

18) Use the same protocol for each concentration injection:



a) Turn the inject valve to the load position.

b) Do an air purge, PBST purge, and air purge of the sample loop.

c) Fill the sample loop with the one of the anti-BSA solutions until it comes out ofthe waste port.

d) Check pump flow rate: 0.1 mL/min.

e) Turn the inject valve to the inject position. As the anti-BSA IgG binds to thesurface immobilized BSA, the response will increase with time. Let run 7minutes.

f) Switch the inject valve to load position. Anti-BSA IgG will slowly dissociateshowing a slow decrease in response. Let run 7 minutes.

g) Fill the loop with 1 ml of 10mM glycine pH 2. It is not necessary to do an airpurge of the loop.

h) Turn inject valve to inject and let run at least 3 minutes.

i) Turn the inject valve to load

j) Base line should return to PBST level as all the anti-BSA IgG is removed.

k) Repeat from step (a) above for each concentration of BSA. The plot above showswhat you can expect to see in LabVIEW as you run the test.

Analyte Additions10% COOH mSAM Surface, BSA Ligand

3 June 2002

850

870

890

910

930

950

970

990

1010

1030

1050

7300 8300 9300 10300 11300 12300

Time, Sec

Pixe

l

Inject25 ug/mL Anti-BSA IgG

2.5 ug/mL Anti-BSA IgG

1.25 ug/mL Anti-BSA IgG

20 mM NaOH 20 mM NaOH

Start Dissociation

Regenerate Surface with20 mM NaOH

Flow PBS Buffer

PBS Buffer



Data Analysis

Where data is stored:

In the Experimental Setup window in LabVIEW the file for each experiment is named.

These files reside in c:\Biotech\data as comma delimited files. To do analysis and plotresults, the data can be imported into Excel (or a similar program) for analysis.

The data for Time vs. Response and Temperature is saved as a comma delimited text file.The data consists of 5 columns for each time point. The columns are organized in thefollowing manner:

Col. 1 Col. 2 Col. 3 Col.4 Col. 5Time Instrument

Response,Micro RIU*

InstrumentResponse,Angle, Deg

InstrumentResponse, Pixel

Temperature,ºC.

Notes: a. 1 micro RIU = 1 * 10-6 Refractive Index Units b. Each Row represents a new time point.

Importing data directly into ExcelData can then be imported into a spreadsheet program (e.g. Microsoft Excel) for furtheranalysis. To open the saved text file in Excel:

a) Open Excel

b) Choose File, Open

c) In the “Files of Type” window use the pull down menu and choose “Text Files(*.prn; *.txt; *.csv)”.

d) In the “Look In” window use the pull down menu to select the file path. e.g. tofind files store in the Biotech default file locate (C:), then in the main windowdouble click the “Biotech” folder , then double click the “Data” folder. Finallydouble click the file you wish to open. This will bring up the text import wizardin Excel.

e) In step 1 of the text import wizard be sure “delimited” is chosen. All otherparameters are default. Select Next.



Normalizing Data and Preparing for Analysis

Time vs. Response data can be normalized to zero (0) start time and zero (0) startresponse so they may be compared directly and imported into kinetic analysisprograms.

a. Open the LabVIEW Normalize Program. See interface above.b. Click the open button and open the data file located in C:\Biotech\Datac. Choose Index, Cell(Pixel) or Angled. The time vs. response chart will appear in the upper windowe. Drag the left vertical line on the chart to the start of the 1st analyte injectionf. Move the left vertical line to the end of the injection by dragging the bar in the

ruled scale to located under the top chart the end of the injectiong. Click “Add Plot”h. Repeat for each of the injectionsi. Click save sheet, name, locate and save the file. Each injection is saved in a

tab delimited file format.



Import Normalized data into Excel

a. Open Excel, choose file, openb. In the “Files of Type” window select all filesc. Click on the normalized data filed. Click next, next, finishe. Response data for each injection will appear in columnsf. A time column must be added. With the curser in cell A1 select “Insert”

“Column” g. Enter the experimental time in column A. E.g. 0,2,4,6. Highlight the first 4 or

5 time values entered. Double right mouse Click on the box in the lower rightcorner of the cell containing the highest time value entered. This will fill theentire column with the time.

Data can be graphed as shown below:

Anti BSA IgG Injections on COOH mixed SAM Surface with Amine Coupled BSA Ligand

-10

0

10

20

30

40

50

60

0 200 400 600 800 1000 1200 1400 1600 1800

time, seconds

Res

pons

e, P

ixel

5 ug/mL2.5ug/mL1.25 ug/mL0.625 ug/mL.312 ug/mL0.156 ug/mL0.078 ug/mL0.039 ug/mL0.0078 ug/mL2.5ug/mL0.625 ug/mL10 ug/mL

Data can also be imported into a variety of kinetic analysis programs. See the Reichert“SPR Data Analysis” application bulletin for further information on data handling andanalysis.



System Components

A complete SR7000 Surface Plasmon Resonance (SPR) system consists of the SPRRefractometer, a pump (either peristaltic or syringe), a solvent select valve, an injectionvalve and fluidics consisting of tubing with fittings.

A typical system components are shown below:

Catalog Number Description13207000-408 SR7000 Sample well 13207000A SR7000 SPR Instrument 1320630US Syringe Pump Assembly 1320620US Valve System Assembly 13207040 SR7000 Flow Cell

Additional components with a complete system include 5 CM sensor slides and allnecessary tubing, fluidics, connections, check valves, starting PBST buffer packs, bufferfilter, forceps, cables and software.

Please note that accessories and options will change and you should contact the factory orlook on line at www.reichertai.com for the latest versions available.



Optional Thermal HousingPictured below is the SR7000 with optional thermal housing.



Troubleshooting

A few lessons learned

Computers are powerful devices and we all load lots of stuff on them to take advantage ofthis. The problem is that many programs reside in the background and will load fileswhen you start up. If these programs then use resources occasionally they can and willcause drop outs in data as the computer takes the time to do the task is it calling.Programs like instant messaging, music players, live internet connections, mail programs,and games…have all been shown to create a drop out problem. We suggest that you havea clean start-up that has none of these sorts of thing running while taking data from theSR7000.

Warning: unfiltered or old buffers contaminated by microorganisms can easily plug the0.25 mm tubing causing irregular or no flow. Irregular flow can cause the instrumentresponse to appear “noisy”. All solutions should be prepared fresh and filtered through a0.2 µM filter. Filtering also serves to degas the solutions. Additional degassing may berequired

Buffer that have been stored refrigerated may have absorbed more air which can thenlead to out gassing when pumped into a warm flow cell. Trapped gas in the flow cell willcreate noise. If suspicious that this is happening, putting the buffer under vacuum mayout gas the solution without having to wait for it to come to the temperature theexperiment running at.



Copyright and Trademark information:

1. Source code of Tera Term Pro and its code is a copyright (C) 1994-1998 of T.Teranishi, All Rights Reserved. For more information please visithttp://hp.vector.co.jp/authors/VA002416/teraterm.html

2. LabVIEW is a registered trademark of National Instruments Corporation, for moreinformation please visit http://www.ni.com/legal/

3. Excel is a trademark of Microsoft Corporation.

4. Clamp is a copyright of Tom Morton and David Myszka. Please see theinformation on http://www.cores.utah.edu/interaction/download.asp Please also seeMorton & Myszka (1998) Methods Enz., 295, 268-294.

5. Winspall is a copyrighted program by Juergen Worm. For more information visithttp://www.mpip-mainz.mpg.de/~johanns/ak_knoll_Software.htm

6. Masterflex and L/S are registered trademarks of Cole-Parmer Instrument Co.

7. Parmed is a registered trademark of Norton Company.

8. Teflon is a registered trademark of E.I. Dupont de Nemours and Company.

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