Biosensing Instrument Inc.

Installation and Training Tutorial

BI 2000 SPR Instrument

Please read the manual before

viewing this quick tutorial

Tutorial Sections

• Installation

• Operation

• Experimental Tips

• Clean Up

• Example Experiments

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Installation

• Unpacking

• Instrument Setup

• Software Installation

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Unpacking

• Check that the instrument was not damaged during shipping.

• Do not obstruct the ventilation slots.

• Place the instrument away from breezeways such as windows, door, AC vents, and major traffic areas.

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Instrument Setup

• Plug the power cords into the back of the SPR instrument and syringe pump

• Connect the SPR instrument to the computer via the USB communication cable

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Software Installation

• If a computer system was not purchased from BI, you will need to install the software yourself.

• Located the Data Acquisition program on the BI Software disk and install it first.

• Next install the BI SPR software package located on the disk.

• Simply, follow the onscreen instructions to complete the software installation process.

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BI Software Package

• BI- Control

• BI- Data Analysis

• BI- Kinetics Analysis

For more details, please review the BI Manual

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BI Control - Plotting Features

Measuring Tool Double-click on axis to

adjust rangesTake notes

More plot options in

drop-down menu

Quickly zoom out

Baseline correction

tool

Start data collection

Valve information

Plot Labels

Double-click on plot

name to change plot

options

Select any area to

quickly zoom in the

data

Export to BI- Data

Analysis

Real-time label-free

data acquisiton

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BI Data AnalysisMore plot options in

drop-down menu

Make local or global

notes

Data Selection Tool

Referenced Data

Selection Tool

Add multiple date

files to workplace

Overlay multiple

plots

Remove plots

Select name to

isolate data

Export to BI-KA

Selected region for

analysis or export

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BI Kinetics Analysis

Calculate Kinetics

Overlay simulation

and experimental

data Residuals Calculate Affinity

Reference

subtraction

Crop injections

Extracted kinetic

constants

Align baselines

Align injections

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Operation

• Data Acquisition

• Calibration

• Flow Cell

• Valves and Syringes

• Preparation

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Data Acquisition

• Data acquisition property options are found in the setup drop down menu.

• Higher gain settings increase sensitivity, but decrease detection range. – The Gain can be set : 1, 10, 100, and 1000.

– 10 is the default value

• Higher sapling rates have faster time resolution, but noisier data and larger files.– 10 is the default value

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Calibration

• Calibration property options are found in the setup drop down menu.

• Accurate calibration of both channels critical in obtaining accurate data and high quality background and reference subtraction.

• System calibration should be checked every several months.

• A calibration example is found later in this tutorial.

The potential may be

inverted for EC-SPR

applications

A drifting signal is a good indicator of poor calibration.

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Flow Cell (FC)• Open Position

– Lift the FC holder, rotate CCW, then gently lower.

• Closed Position– Lift the FC holder, rotate back over the detection area, then gently

lower the FC holder.

– Gently press down on the FC holder to ensure contact.

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Valves

Mode Select

InjectionChannel Select

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Mode Select ValveUse this valve to select single or serial flow modes.

– In the single flow mode (CCW): the injected sample only passes through one channel.

– In the serial flow mode(CW): the injected sample passes through both channels serially.

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CH1

CH2

CH1

CH2

Single Flow Mode Serial Flow Mode

Channel Select Valve

Use this valve to select Channel 1 or Channel 2 operation.

– Turn the handle CCW to select Channel 1.

– Turn the handle CW to select Channel2.

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CH1

CH2

Channel 1 Channel 2

CH1

CH2

Injection Valve• Use this valve to Load and Inject

samples.1. Turn the handle CCW to load samples

through the injection port.

2. Turn the handle CW to release the sample to the sensor.

3. Turn the handle CCW to stop the sample release and reload another samples.

• The default loop size is 100uL, and may be changed.

• Use the Injection Timer to time the injections

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Loading Injection Syringes

• Load the injection syringe with an additional 20-25 uL of sample for operational waste. – At least 10 uL of waste-sample should precede the test-sample to

flush out prior residue

– At least 10 uL of waste-sample should remain in the injection syringe to avoid injecting air bubbles.

• Wipe dry the injection syringe needle before inserting it into the injection valve for loading.

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Preparation• Warm Up

• Carrier Solution

• Syringe Pump

• Carrier Pre-Flow

• Sensor Chips

• Pre-Experiment Check

• Experimental Tips

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Warm up

• Turn on the instrument and laser

• Allow at least 15 minutes for warm up

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Carrier Solution Preparation

Filter and degas the carrier solutions prior to use

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Syringe filters are a

convenient way to

filter solutions.

Solutions may be degassed

with a low-pressure vacuum

for 10-15 min with gentle

intermittent shaking.

Loading Carrier Solution

• Load the filtered degassed carrier solution into the two provided carrier syringes

• Tap out any large air pockets that may have trapped in the syringes

• Connect the syringe fittings onto the syringes

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Watch for air pockets

Loading the Syringe Pump Loading the Pump

1. Press the Drive Button to slide the Drive Bar back

2. Lift and rotate the Syringe Retainer Bar away

3. Load the two syringes into the Syringe Cradle. Make sure the collars of the syringes are flush with the Syringe Cradle.

4. Secure the syringes with the Retainer Bar.

5. Press the Drive Button to slide the Drive Bar against the syringe plungers

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Programming the Pump

• Set the syringe diameter– Scroll through the table menu and select Beckman Plastic, 10cc

– A diameter of 14.48mm should automatically load

• Set a syringe volume for automatic stop, otherwise input zero.

• Flow rates should be less 150ul/min on BI-2000 models.

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Carrier Pre-FlowPre-Flow the carrier solution before loading the chip to remove residual air pockets and equilibrate the fluidic lines.

• Place the flow cell and holder in the closed position (either on a chip or the bare prism),

• Switch to single flow mode

• Run the pump at a high flow rate, ~150 uL/min, for 3-5 minutes.

• Stop the pump, and wait ~2 minutes for residual pressure to pass.

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Sensor Chips

• Prism Stage Cleaning

• Flow Cell Cleaning

• Removing Chip from Container

• Chip Loading

• Closing the Flow Cell

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Prism Stage Cleaning

• Lift and rotate the Flow Cell Holder away from the detection stage.

• Clean off the detection stage using lens paper moistened with ethanol.

• Make sure the detection stage is free of residue and debris.

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Flow Cell Cleaning• Remove the FC from the holder and wipe clean the

gasket with a paper towel moistened with ethanol.

• N2 blow dry the gasket, making sure the FC is free of residue and debris

• Remount the FC into the FC holder, make sure that the FC sits flush with the back of the holder

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Removing Chip from Container

• Remove the seal from the container of SPR chips.

• Remove the lid and retainer ring.

• Carefully remove a sensor chip with a clean pair of tweezers.

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Loading Chip• Place a 3ul drop of Matching Fluid on the center of the prism stage (~3mm wide).

• With tweezers, slowly place the chip (Gold side up) on the prism stage.

• Lower the chip at an angle to avoid trapping air bubbles.

• Use a soft tip to re-center the chip on the prism stage.

Note: Be careful not to scratch the prism surface . A tooth pick or pipette tip works well as a soft point

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Which Face is Gold Coated?Color Test:• Hold the chip with a pair of tweezers

• Look at the reflections of both sides.

• The gold coated side is more golden or shinny, whereas the glass side will show a duller yellow or bronze tint.

Scratch Test (if still not sure):• Gently scratch one of the chip’s corners.

• Scratch marks appear on the gold-coated side only

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Close the Flow Cell• Lift and rotate the FC holder over the sensor chip,

and gently lower the FC holder.

• Gently press down on the FC holder to confirm the contact.

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Pre-Experiment Checks

• Interface Quality

• Baseline Quality

• Resonance Quality

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Interface Quality• Turn on the liquid laser.

• Observe that the optical reflection in the viewing window has uniform intensity.

• If an intensity spot or dark streak is observed, an air bubble may be trapped at the sensor-prism interface. – Try pressing on one of the chips’ corners with a soft point.

– A tooth pick or pipette tip works well as a soft point

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Baseline Quality• In serial flow mode, start the pump at a high flow

rate, ~150ul/min.

• Let flow for ~3 min, then reduce to ~60um/min

• Wait for the baseline to stabilize, ~10 min depending upon previous use and clean up.

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Resonance Quality

• Examine the SPR reflection in the viewing window.

• Two nearly identical SPR dark lines ~2mm wide should be clearly visible and neighboring each other.– If they are not similar, you may have trapped an air bubble. (see

maintenance section)

• SPR experiments are ready to begin.

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Experimental Tips

Obtaining high quality binding information requirescareful consideration of the following factors :

• Instrument Calibration

• Buffer Chemistry

• Surface Coverage

• Sample Preparation

• Flow Rate and Sample Volume

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Quick Calibration Check

Accurate calibration of both channels is critical in obtaining accurate data and high quality background and reference subtraction. • Use a brand new bare gold chip.

• Run DI water as the carrier solution.

• In Serial mode, inject 1% ethanol solution (diluted in DI water).

• Adjust the calibration factors so that a 60.0 mDeg response is observed in both channels.

System calibration should be checked every several months.

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Buffer Chemistry

• Buffer pH’s much greater or lower than the sample’s isoelectric point, may result in strong charge attraction or repulsion of the sample with the surface.

• Chose a buffer concentration that is high enough to sustain protein activity but low enough to prevent saturation of detectors.

• It may be helpful to include low concentrations of buffer additives/surfactants to reduce non-specific absorption.

• Degas and filter the buffer before use

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Surface Coverage• Surface Coverage densities too low will result in too small of

an analyte binding signal and densities too high which result in too demanding of a surface leading to mass transport limited kinetics.

• Mass transport limited kinetics means that the surface is demanding more analyte molecules than is currently being supplied by the flow.

• The BI-KA Software can compensate for mass limited transport effects, but a sufficient amount of kinetic curvature should still be present for a more accurate analysis.

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Sample Preparation

• To minimize the effects of bulk refractive index change, dilute samples with the carrier solution.

• Degas samples and let them warm to room temperature before use

• Start with low concentrations near that of the expected affinity constant.

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Flow Rate and Sample Volume

• Flow rates too low result in mass limited transport kinetics.

• Flow rates too high do not allow enough exposure time for binding information to be extracted.

• Note that higher flow rates consume more carrier solution and sample volume per time, thus larger sample volumes are required at higher flow rates for equal exposure times.

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Clean Up

Cleaning up after experiments is of critical importance in maintaining the high performance of the instrument.

• Valves and Tubing

• Flow Cell

• Prism Stage

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Clean Up – Valves and Tubing

1. Clean the carrier and injection syringes with DI water

2. While in inject-single position, load the carrier syringes with DI water and let flow through the system at high flow rate (~150uL/min) for a several minutes.

3. Toggle the valves several times to flush away possible internal valve debris.

4. Loosely insert injection syringe into injection port, and inject DI water so that solution escapes from port opening.

5. Load the carrier syringes with air and manually pushing the air through the system to flush out all the liquid

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Clean Up – Flow Cell

1. Lift the cell holder and rotating it such that it remains elevated.

2. Slide the Flow Cell out of the Flow Cell Holder and wipe it clean with an ethanol moistened paper towel.

3. N2 blow dry the FC, and return it to the Flow Cell Holder.

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Clean Up - Prism Stage

1. Use a soft point (cotton swab or pipette tip) to nudge most of the chip off the prism edge.

2. Use a pair of tweezers to gently lift away the chip.

3. Wipe the remaining RIM liquid off the prism with lens paper.

4. Moisten lens paper with ethanol for final wipes.

5. Gently return FC to bare Prism stage.

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Example Experiments

• Reference Subtraction

• Calibration

• BSA/ anti-BSA Experiment

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Reference Subtraction Example

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Reference Selection tool

Solid outline

Dashed outline

Align BaselineAlign injection timeReference Subtracted Injection

Calibration Example• Turn on instrument and laser, allow ~15min for instrument to warm up.

• Load filtered-degassed DI water into carrier syringes

• Allow several minutes for DI water pre-flow.

• Open the FC and wipe clean the FC

• Load a brand new chip bare gold chip

• Close FC, start pump at high flow rate

• Wait ~5min for baseline to stabilize with fluid

• In serial flow mode, iject 1% ethanol into the system

• Look for 60mDeg response change in both channels.

• Adjust the calibration values for both channels so that a 1% ethanol injection results in 60mDeg change.

• Repeat until the system is accurately calibrated.

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BSA Experiment Example• Turn on instrument and laser, allow ~15min for instrument to warm up.

• Load filtered-degassed buffer into carrier syringes

• Allow several minutes for DI water pre-flow.

• Open the FC and wipe clean the FC

• Load a modified chip onto the prism stage

• Close, start pump at high flow rate, wait ~5min for baseline to stabilize with buffer

• Begin acquiring data

• In serial flow ~30uL/min, inject NHS/EDC

• Switch to Ch1 single flow mode and inject BSA

• Switch to serial flow mode and inject EA blocker

• Inject regeneration solution to further clean the chip surface

• Increase flow rate to 100 uL/min and set the injection timer

• Inject Antibody.

• Inject regeneration solution twice

• Repeat antibody Injection or inject a higher concentration of antibody

• Export to BI-DA and BI-KA for analysis

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