The NEWSR7500DC System

The Next Generation ofSurface Plasmon Resonance

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Explore Critical Interactionswith Black Magic the NewSR7500DC SPR System

ProteinsNucleic Acids

LipidsCarbohydrates

Small Molecules/DrugsWhole Cells

Bacteria/VirusesPolymers

The new SR7500DC Dual Channel SurfacePlasmon Resonance (SPR) System providesthe diverse interests of academia and industry withone of the most sensitive and flexible SPRplatforms in today’s marketplace. This reliablelabel-free system is used to characterize a broadrange of molecular interactions that are importantin numerous scientific disciplines. Theseinteractions include those occurring with andbetween the major classes of biologicalmacromolecules along with those involving smallmolecules and drugs. Quantitative information onsuch interactions is critical to research efforts inpharmaceuticals, drug discovery, antibody

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Pushing the limits of detection and sensitivity in label-free interaction analysis

Extremely low noise (0.05 RIU) and low drift (0.01 RIU/min) baseline

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The binding of 4-carboxybenzenesulfonamide to immobilized carbonic anhydrase II

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screening, protein structure/function, generegulation and systems biology. This new SPRsystem is used to generate high quality data withoutstanding precision for:

• Rigorous kinetics analysis (association/onand dissociation/off rates)

• Affinity measurements ranging fromextremely weak (1 mM) to extremely strong(1 pM) interactions

• Precise determination of thermodynamicparameters ( H, S)

• Accurate concentration analysis

Extreme Low NoiseExtreme Low DriftExtreme ValueReichert’s new low noise, component-based SPRsystem provides outstanding flexibility andexceptional sensitivity when seeking high qualitydata for interactions of interest. The newSR7500DC System pushes detection limits andsensitivity to new lower limits, expanding theboundaries of traditional biomolecular interactionanalysis. The system offers superior performancewith the following key features:

• High sample capacity (up to 768 samples)• Temperature control from 10oC belowambient to 70oC

• Fast data sampling rates (up to 10 Hz)• Broad refractive index range (1.32 to 1.52)• Sophisticated, intuitive software with 21CFR part 11 controls

• Minimal maintenance requirements• Low life cycle costs• Ultimate flexibility• Extremely low noise (0.05 RIU)• Extremely low drift (0.01 RIU/min)

Reichert SPRLess Noise, Higher Quality and PrecisionThe SR7500DC System offers high precision indetermining kinetics and affinities for a variety ofbiomolecular interactions. The system generatesreal-time data that provides invaluable insight intothe dynamics of biomolecular interactions thatregulate numerous biological processes. With itsenhanced sensitivity, the SR7500DC System isideal for:

• Cost-effective biomolecular interactionanalysis

• Monitoring binding of low molecular weightcompounds (< 100 Da)

• Identifying potential drug targets andtherapeutics

• Antibody characterization

SR7500DC Dual Channel Surface Plasmon Resonance System

New from Reichert SPR

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Autolink, Reichert’s SPRSystem Software• Integrated sophisticated control of the ReichertSPR System. Autolink is intuitive, powerful, andeasy to use.

• Reichert's Autolink integrates the threecomponents of the SR7500DC SPR System –the injector, the pump, and the SPRspectrometer.

• Fully programmable, intuitive, step-by-stepcontrol of the pump, semi-automatic or fullyautomatic injectors and the SPR spectrometer.

• Drag and drop methods to set up multi-samplerun tables.

• Autolink also allows each component to becontrolled individually without constraints – theultimate flexibility for real-time methoddevelopment.

Autolink is Validated Softwarewith 21 CFR Part 11 TechnicalControlsProtecting the security and integrity of electronic records (ER) is essential for compliance. This includes ensuring the reliability andtrustworthiness of ER used to support criticaldecisions. Features in Reichert’s Autolinksoftware include:• Data Security and Integrity - Access controlalong with file encrypted checksums

• User Authorization Levels - Administrator,factory, and user levels set access rights tosoftware functions

• Record Tracking - Experiment details, events,and user identities are logged in a date andtimed stamped secure file

• Data can be exported both manually andautomatically in a variety of formats including tab dilineated text files, and Scrubber® data analysis files

The software has been developed in accordancewith an accepted development model to ensureadequate validation.

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Small Molecule AnalysisCarbonic anhydrase II (CAII) is an enzyme that catalyzes the reversible hydration of carbon dioxide toform bicarbonate with the release of a proton. CAII activity is strongly inhibited by a variety of aromaticand heterocyclic sulfonamides. In this example, carbonic anhydrase II is amine coupled to a

carboxymethyl dextran surface andthen the binding of a very lowmolecular weight inhibitor (95 Da),methanesulfonamide, is followed overa series of concentrations. The redlines show the global fit to a simplebimolecular binding model and thecalculated association rate constant,dissociation rate constant andequilibrium dissociation constantvalues for this interaction are shownin the inset. This experimentillustrates the remarkable sensitivityand performance of the SR7500DCSystem.

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Data Acquisition windowData from the left channel, right channel, and difference channelcan be viewed together or separately in real-time. Both the response (Y axis) and time (X axis) axis may be auto-scaled or setto user-defined limits. The data window can also be expandedover a defined region to allow close examination of the SPR response curves. In addition, this window continuously displaysthe status of each system component, read time interval, flowrate, temperature, file name and injection/dissociation markers.

Autolink post process windowSPR sensorgrams are extracted and aligned to the same zero starttime and zero response. The aligned sensorgrams are then savedfor analysis using Scrubber® or the curve fitting program of choice.

Programming a run table A typical run table injection sequenceMethods are dragged and dropped from the "Available Methods"section (center) to the Run Table Section. In this sequence step 1is a pump refill and step 2 is a 1 minute wait for baseline stabilization. The third step is a 2 minute 150 L sample injectionfrom vial 1A followed by a 2 minute dissociation period. The finaltwo steps are a 100 L injection of regeneration solution and a oneminute dissociation. The checked "Rinse" box in the next to laststep washes the sample loop and needle with buffer prior to upcoming sample injections. Run table sequences are easilycopied and pasted to quickly program run tables with many injections from 96 and 384 well plates or 48 vial sample trays.

Sample set editorThe window allows the labeling of each injection with a user-defined description and concentration. This sample information isthen displayed within the run table for each injection and ultimatelycarries over to the data analysis program.

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The SR7120 AutosamplerReichert’s autosampler provides the user withflexibility in terms of sample holder options, traycooling, sample aspiration speeds, and loopvolume choices. The autosampler allows for theseamless execution of an experimental protocolover 24 hrs and can precisely inject samples ofvarying compositions. Features include:

• Sample cooling to 4oC • Ability to inject from two sample trays,which can be 48 vial sample trays, 96 or384 well plates, and/or large volume 12vial formats

• Push/Pull sample aspiration enablesprecise pick-up of viscous samples andeliminates outgassing

• Accommodates a wide selection of sampleloop volumes

Specialized Collection Reichert offers a standard flow cell with eachinstrument. This flow cell is used to performmolecular interaction studies and has lowchannel volume resulting in extremely fastsolution exchange dynamics. In addition to astandard flow cell, Reichert offers specializedcells that provide the ability to couple SPRmeasurements with other valuable analyticaltechniques. They open up new avenues ofinvestigation for specialized applications.

Standard Flow CellReichert's new teflon body standard flow cellrepresents a significant improvement in flow cellperformance. This gasketless flow cell featuresvery small dead volumes, low channel volumewith extremely fast rise and decaytimes on both channels.

The flow cell uses standardPEEK™ HPLC/FPLC fittings and iscompatible with tubing sizes down to0.0025" inner diameter (65micrometers). The mountingmechanism for the flow cell hasbeen completely redesigned.

The flow cell is placed over the sensor chip andlocked into place with a mechanical lever. Thisensures the flow cell is always mounted in alevel position with consistent pressure.

Quartz Window Flow CellThe Reichert quartz window flow cell facilitatesexperiments combining SPR withphotochemistry, imaging fluorescent labeledmolecules on the sensorsurface by direct excitationand surface plasmon fieldenhanced fluorescencespectroscopy. Surfaceplasmon field enhancedfluorescence spectroscopy is an extremelysensitive and effective tool for detecting andquantifying biomolecular binding. This techniquedepends on excitation of a fluorophore near thegold sensor surface of an evanescent field.

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Sensor Chips

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Flow Cells

Resonance of p-polarized light with surfaceplasmons (oscillating electrons) in the gold layerproduces the evanescent field.

Electrochemical Flow CellReichert’s electrochemistry flow cell utilizes athree-electrode design. Electrical contact ismade between a platinum wire counterelectrode, an Ag/AgCl reference electrode, andthe working electrode, whichis a standard Reichert SPRgold slide. The electricalleads from the electrodesare connected to apotentiostat to control thepotential being applied tothe gold slide surface. You can adjust thepotential at the gold surface simultaneously withSPR data collection to carry out a variety ofexperiments including SPR/amperometry,SPR/pulse voltammetry and SPR/cyclicvoltammetry. These experiments have beencarried out to monitor polymer formation and forother novel applications.

MALDI Spectrometry Flow Cell A novel flow cell for combining SPR with massspectrometry (MS), is the matrix-assisted laserdesorption and ionization (MALDI)flow cell which carries removable,miniaturized sensing pins that canbe inserted into MALDI targetplates for mass spectrometricdetection of analytes on thesensor surface. This is especiallyimportant in a ligand fishing experimentwhere the aim is to identify the molecule(s)captured on the sensor surface. After verifyingbinding of the unknown species with SPR, theseremovable pins can be inserted into a modifiedMALDI target, where the ligand can be directlyanalyzed or subjected to a digestive treatment.Combined with the application of new hydrogelsensor surfaces, this flow cell allowsmeasurements of higher sensitivity and betterreproducibility with SPR-MS.

Extreme Sensitivity and the Ultimate inComputing PowerThe sensitivity starts with image detection. TheSR7500DC System uses two RL1210 PerkinElmer Photodiode 1024 pixel arrays that feature100,000:1 dynamic range and 0.4 picoAmpdark current. This all translates to extremesensitivity.

On board computing power is via an AlteraFPGA with a virtual softcore 32-bit processor.This equates to massively fast and furiouspipeline processing. And the processor is fieldprogrammable for future upgrades. Illumination

is via two 780 nm arrays, each with 66 LEDs.The twin LED banks illuminate an integratingsphere to provide two watts of fullyhomogenized light power. LED current iscritically controlled to provideconstant output powervia an isolatedfeedback circuit.

The SR7500DC Systemfeatures a new high speedUSB interface. Data samplingrate is 0.5 to 10 Hz. An embedded peripheralhub controls the complete system including thesyringe pump, autosampler and/or semi-automatic injection valve.

Reichert offers a wide variety of sensor chips ataffordable prices so researchers can explore moreinteractions without higher running costs. Theavailable sensor chips include:

Plain Gold Sensor ChipThe plain gold chip provides theopportunity to study surface formationand adsorption in real-time on bare Auand allows researchers to coat the chip withuser-defined chemistries.

Carboxymethyl Dextran Sensor ChipHydrogel surfaces, particularly carboxymethyldextran hydrogels, offer many advantages whenused as a SPR sensor chip surface. Carboxymethyldextran surfaces are very stable and resistant tonon-specific binding of biomolecules. The dextranlayer is in the form of a highly flexible non-cross-linked brush like structure extending 100 to 200nm from the surface. The flexible nature of thedextran contributes to theaccessibility of binding sites onan immobilized ligand.Biomolecules are easily coupledto the surface utilizing a varietyof techniques similar to those used in affinitychromatography. Large amounts of protein, up to50 ng/mm3, can be immobilized on carboxymethylhydrogel surfaces due to the 3-dimensional natureof the hydrogel layer. This is important forexperiments where a low molecular weight analytebinds to a surface immobilized ligand.

Planar Polyethylene Glycol/ CarboxylSensor ChipThis surface consists of a mixed, self-assembledmonolayer of alkanethiolates generated from thecombination of polyethylene glycol-terminatedalkanethiol (90%) and COOH-terminatedalkanethiol (10%). The terminal polyethylene glycolchains minimize non-specificbinding while the COOH groupsprovide a functional attachmentsite for immobilizing/capturing amolecule of interest. Amine coupling, utilizingEDC/NHS chemistry, is the most common ligandimmobilization approach but thiol, aldehyde, andmaleimide coupling are also possible with thissurface using the appropriate cross-linkingchemistry.

Nickel Nitrilotriacetic Acid Sensor ChipThis surface is used to capture histidine-taggedmolecules such as recombinant proteins. Thecaptured molecule is oriented well on the surfaceas the ligand is captured directly at the histidinetag via Ni2+/NTA chelation. The surface is easilyregenerated with an injection of imidazole or EDTAto remove the metal ions and the captured ligand.

Hydrophobic Planar Alkyl Sensor ChipThis surface is a self-assembled monolayer oflong-chained alkanethiol groups directly attached

It is ideal for studying membrane-a

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Figure 1: Temperature-dependent response curvesof 4-CBS binding to CAII at 20oC (blue lines) and35oC (red lines).

Figure 2: van't Hoff plot of the Thermodynamic Datafrom 4-CBS binding to CAII.

Low Immobilization LevelAnti-HSA/HSA DataIn this application, a very low amount of anti-HSA (< 100 RIU) is immobilized to acarboxylmethyl dextran surface via aminecoupling. HSA is then injected over theimmobilized antibody at different concentrationsranging from 7.5 to 0.45 nM. Eachconcentration is injected in duplicate to ensurereproducibility. The red lines show the global fitof the data to a simple bimolecular bindingmodel.

This example shows that high quality SPR datacan be obtained on the SR7500DC Systemdespite having extremely low ligandimmobilization levels. In fact, low ligandimmobilization levels are often desired toprevent crowding on the surface and thisapplication illustrates that the SR7500DCSystem can provide precise kinetic data for lowresponses with great confidence.

Kinetic TitrationIdentifying a suitable regeneration solution cansometimes be a bottleneck when carrying out atraditional SPR experiment. In this case, a kinetictitration approach is desired where analyte isinjected over the surface at increasingconcentration in a single cycle withoutregenerating the surface between eachconcentration injection. This kinetic titration

approach reduces the amount of time spent onassay development as it eliminates the need tooptimize regeneration conditions. This approachalso allows for the analysis of molecularinteractions that were previously difficult tocharacterize due to problems in finding anappropriate regeneration solution. Reichert’s

Applications

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to gold. It is ideal for studying membrane-associated interactions. In addition, vesiclesspontaneously adsorb to the surface forming asupported lipid monolayer. It is easily regeneratedwith an injection of a detergent such as CHAPS.

Streptavidin/NeutrAvidin Sensor ChipThis surface is used for the high affinity capture ofbiotinylated molecules such as proteins, peptides,and nucleic acids. The binding of streptavidin tobiotin is one of the strongest non-covalentinteractions known so the surface can beregenerated without having to recapture the ligandafter each regeneration step. Minimal biotinylationof the ligand and subsequent capture on aStreptavidin/NeutrAvidin chip results in a moreoriented arrangement of ligand molecules on thesurface as compared to the more randomarrangement from chemical immobilization such asamine coupling.

Protein A Sensor ChipThis surface is used for capturing certain antibodies.Protein A contains four high affinity binding sitescapable of interacting with the Fc region from IgG ofseveral species, including human and rabbit.Optimal binding occurs at pH 8.2, although bindingis also effective at neutral or physiological conditions(pH 7.0 to 7.6).

Applications

Typical Enthalpy Application:

Thermodynamic Investigation of anEnzyme-Inhibitor Pair

Figure 1 presents the temperature-dependentresponse curves of 4-CBS binding to CAII at 20oC(blue lines) and 35oC (red lines). The resultsindicate that temperature has a drastic effect onthe profile of the response curves. Specifically, theassociation and dissociation rate constantsincrease with temperature. To quantify the changein rates, the data at each temperature was fit to asimple bimolecular model using Scrubber®

(Biologic Software) to determine the rate constantsand the equilibrium dissociation constants.

Figure 2 presents a van’t Hoff analysis of the databy plotting ln KD versus 1/T. The data fits fairlywell to a linear regression model (r2 = 0.989),thus the thermodynamic parameter H can bedetermined directly from the non-integrated formof the van’t Hoff equation. In this case, the slope isH/R. Thus, H is determined to be -6.0

kcal/mol for this inhibitor-enzyme pair.

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Capture SPR Analysis Using Reichert Carboxymethyl Dextran (CMD500k) Sensor Chips

Figure 1 (right) presents data from a capture experiment. Initially, about 2,000 RIU of goat anti-mouse IgG Fc was amine coupled to the CMD500ksurface. For each series of injections, a constant concentration of monoclonalanti-HSA IgG (50 g/mL) is captured over the surface, and then varyingconcentrations of HSA are injected. Both anti-HSA and HSA are then removedduring each regeneration cycle.

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The binding of streptavidin to

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Concentration AnalysisSPR is a very precise and accurate method fordetermining the concentration of a biomolecule.Concentration measurements are based on theconcept that as the concentration of an analyteincreases, the rate of binding to an immobilizedbinding partner increases. Generally, a calibrationcurve is constructed by plotting responses at a time point in the sensorgram versus knownconcentrations.

Response points are generally chosen at laterstages of the binding association or dissociationcurves. The response at later stages in theassociation is typically exponentially increasing ordecreasing if a point is chosen in the dissociation

Figure 2 (below) shows the excellent reproducibility of the capture step andthe chemical stability of the CMD500k surface. Even after multiple injection-regeneration cycles, the surface is stable and gives reproducible results.

Figure 3 (below) shows the data and global kinetic fit of the HSAinjections. HSA is injected at concentrations ranging from 1.25 to 20 nM.The red lines are the fit obtained in Scrubber® for binding to a 1:1 model.The equilibrium dissociation constant (KD) obtained is 4.93 nM.

Applications

SR7500DC System can collect kinetic titration dataand analyze it to determine the binding kinetics ofthe interaction of interest. The figure (left) showskinetic titration data from the anti-HSA/HSA assay.In this example, HSA is injected at successivelyhigher concentrations ranging from 1.25 to 20 nMin a single cycle (no regeneration). The data isthen fit to a kinetic titration model (red line is the fitto the data) and the kinetic rate constants aredetermined for the interaction.

phase. These response versus concentration plotsare non-linear. These plots are generally fit to anon-linear function such as a quadratic, 3rd orderpolynomial or a 4-parameter equation. Figure 1 is acalibration plot of the 175-second response pointversus concentration. This data was fit to a 4-parameter function. Generally, choosing points inthe non-linear later stages of a sensorgramprovides better accuracy and reproducibility.

Response points chosen very early in thesensorgram - 2 to 10 seconds - represent theinitial rate of binding. The initial rate is typicallylinear. The calibration plot of concentration versusinitial rate or a time point in this region of thesensorgram is linear (see Figure 2 below).

Figure 1 shows the calibration tabfrom Reichert’s Autolink Softwareconcentration module. The plot isthe 175-second association responsepoint vs. concentration for anantibody (immombilized ligand) /antigen (injected analyte) interaction.

Figure 2 shows response at 10seconds vs. analyte concentration.

Concentration Analysis

Figure 1 Figure 2

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Fluidics KitOur SPR system is designed to use low-cost,off-the-shelf HPLC fittings and tubing, allowingfor quick and easy changeover. Reichert SPRsystems are supplied with a fluidics kit thatprovides all the connectors and tubing neededto properly plumb the system, includingspares. The primary contents of the kit includean assortment of tubing nuts and ferrules, lueradapters, a tubing cutter, a pair of forceps,and a selection of different inner diametertubing spindles and sample volume loops. The SPR system fluidics can accommodate awide range of sample compositions (from saltsolution to cell lysates), varying viscosities, and particulate matter.

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Corporate HeadquartersReichert, Inc.3362 Walden AvenueBuffalo, New York 14043USATel: +1 716-686-4500Fax: +1 716-686-4555Toll Free USA: 1-888-849-8955

ReichertSPR.come-mail: lifesciences@reichert.com

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Features in Reichert’s A

Technical Information

Transducer Principle Kretschmann prism, multiple angles from fixed light inputMeasurement Channels Two (either parallel or series fluid connection)Sample Loading Autosampler or semi-automatic injector, standard HLPC tubing and

connectors, external syringe pumpSample Capacity Any combination of up to 2 trays can be used. Choose: 12 (10 mL) or 48

(2 mL) Vials; or 96-well (high or low) or 384-well PlatesSample Storage 4°C or ambient temperatureFlow Cell Volume per Channel 0.18 LFlow Cell Surface Area per Channel 4.5 mm² (reference value)Aspect Ratio >25 (width/height)Fluid Contact Materials Teflon™, Acetal Copolymer, PEEK™, Kalrez™, ETFE (Tefzal™)Sample Volume 1 to 5000 L (depends on installed loop volume)System Fluid Volume (typically) 28 L (0.01" I.D. tubing) or 7.5 L (0.005" I.D. tubing)Temperature Range 10°C below ambient to 70°C

Measurement Sensitivity

Refractive Index Resolution < 10-7 RIU (< 0.1 RIU)Refractive Index Range 1.33 to 1.42; up to 1.52 with higher RI glass (@780nm)Analyte Concentration Range 1 mM to 1 pMMinimum Molecular Weight Detection < 100 DaltonsBaseline Noise 0.1 RIU peak-to-peak, 0.05 RIU RMS, @ 25 L/minBaseline Drift < 0.01 RIU/min

Typical Kinetic and Equilibrium Constant Ranges

Association Rate Constant 103 to 107 M-1s-1

Dissociation Rate Constant 10-1 to 10-5 s-1

Equilibrium Dissociation Constant 0.1 mM to 1 pM

Electrical

AC Power Supply, standard international voltage range w/universal adapter from 100 to 240 V & 50 to 60 Hz

Regulatory

Compliance with the applicable sections of the European EMC Directive and IEC safety requirements for laboratory electrical equipmentfor measurement and control

Product Safety

Compliance with IEC 61010-1 (Low Voltage Directive) under a Category classification EMC and Safety: CE mark certification (Class A, Type II)

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In this example, carbonic anhydrase II is amine coupled to a

c

The redl

This experiment

i

P

The data window can also be expanded

o In addition, this window continuously displays

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The aligned sensorgrams are then saved

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In this sequence step 1

i The third step is a 2 minute 150

The finalt

Run table sequences are easilyc

This sample information ist

Specifications

The autosampler allows for the

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• Sample cooling to 4o

The system offers superior performancew

Copyright © 2011 Reichert Technologies, Inc.