Paper summary from Homola et.al. regarding SEB measurement in milk using surface plasmon resonance biosensor. SPR sensor was custom dual-channel as a measuring chamber and reference chamber.

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Spectral Surface Plasmon Resonance
Biosensor for Detection of Staphylococcal
Enterotoxin B (SEB) in Milk
Homola et.al.
2002-int. Journal of food microbiology 75 61-69
Keyword: polychromatic light source

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What This Paper Talk About
• Measurement of SEB in lowest concentration
as possible
• To use custom dual-channel SPR sensor as a
measuring channel and reference channel
• Comparing direct assay and sandwich assay
for detecting SEB using antibody a-SEB
• Comparing SEB in pure condition and in
sample (milk)

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SEB Detection using SPR
SPR
Detection of
SEB in milk
Direct Detection Sandwich Assay

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INTRODUCTION

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Types of Biosensing Devices
Based on Different Principles
Electrochemical Sensors (Ghindilis et.al. 1998)
Piezoelectric Sensors (Chu et.al. 1995)
Electrical Impedance Sensor (Pless et.al. 1994)
Optical Sensors (Boisde and Harmer, 1996;
Gauglitz, 1996)

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Ensuring Specificity in Guided Mode/SPR
Immobilized biomolecular
recognition element
Capture of analytes
molecules by
biomolecular
recognition element
Increasing in refractive index
Measured with optical
method. Can be
correlated with
concentration of analyte
Most frequently used:
Antibodies
(Byfield and Abuknesha, 1994)

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Staphylococcal Enterotoxin B (SEB)
• One of ten major serological types of emetic
enterotoxins produced by Staphylococcus Aureus
• 26-30 kDa monomeric
• Heat-stable
• Potent gastrointestinal toxins (Bergdoll 1991,1995)
• Cause of gastroenteritis form consumption of
contaminated food (Archer and Young 1988)

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Study Aim
Method for detecting SEB
below minimum intoxication
level
As low as
nanograms per gram (food) or
nanograms per mililiter (milk)
Potent SE minimum intoxication level:
200 ng in a portion of 100 gr of food
Novel dual-channel SPR sensor
based on wavelength
modulation

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MATERIALS AND
METHODS

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Instrumentation
Traditional SPR biosensor
• Based on angular
measurement
• Rely on monochromatic
light (Liedberg et.al. 1993;
Melendez et.al. 1996)
• Position constraint to
miniaturization
Wavelength modulation SPR
• Based on wavelength
measurement
• Rely on parallel
polychromatic light beam
• Allow miniaturization and
remote analysis

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SPR Theory
Excitation of SP accompanied by
transfer optical energy into SP and its
dissipation in the metal layer, resulting
in a narrow dip in the spectrum of
reflected light.
Change in refractive index will resulting
in dip shifting.
Simulated dip shift as a function of
packing density of high refractive index
glass prism, gold layer (50 nm), biolayer
(estimating SEB molecule size around
5nm, and refractive index 1.45 for 100%
surface packing density), sample water,
wavelength 750 nm.
Complete
monolayer
of SEB

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Set-up Device
Halogen lamp
6332 Oriel
instruments
Input collimator
VIS-NIR Zeiss
SF14 Schott
glass prism
Dichroic polarizer
Polarcor 653006
Corning
Output collimator
2 graded-index
lenses SLW, NSG
SD-2000
Ocean optics
Au 50 nm
Cr 2 nm
Dual channel teflon
flow cell + Reglo Digital
peristaltic pump
Additional:
• 2-m frit filters (A-712-
02 Upchurch Scientific)
• 0.05 ml/min flow rate

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Materials
C15COOH, C11OH, NHS, and EDC obtained from Sigma
Anti SEB IgG (Lot no.72700BI from Toxin Tech)
SEB and bovine serum albumin (BSA) from Sigma
PBS (0.01 M sodium citrate, 0.01 M sodium phosphate
and 0.12 M sodium chloride pH 7.4)

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Functionalizing Chip
Washing with chloroform
and 100% ethanol
Cleaning under UV
Washing with DI water
and 100% ethanol
Soak with ~1mM mixed
thiol solution
(C11OH/C15COOH 7:3) at
room temperature
Chip remained in mixed
thiol solution for ~1 day
Chip rinsed with ethanol
Chip dried in a dry
nitrogen gas stream
2 mg/ml N-hydroxysucci
nimide and 2 mg/ml 1-
ethyl-3-(3-dimethylamino
propyl)-carbodiimide
for 1 hour
Incubated with anti-SEB
IgG (1 mg/ml IgG in pH 7.4
PBS buffer for 1 day
Washed with DI water,
dried under dry nitrogen,
and stored in humidity
controlled cultur dish 4 OC
Passivate in flowed BSA-
PBS 250 ng/ml for 10
minutes before used

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Sample Preparation
SEB in PBS
•BSA in concentration of 250 ng/ml in
PBS was added to solution
SEB in milk
•Milk was prepared as 5% w/v if dry
milk in water and spiked with different
concentrations of SEB

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Experimental Protocol
• SEB in PBS • SEB in Milk
Sensing channels were incubated in BSA-PBS solution for 10 min
Measuring Ch.
BSA-PBS with SEB
Reference Ch.
BSA-PBS no SEB
Measuring Ch.
Milk with SEB
Reference Ch.
Milk no SEB
30min
Rinse with BSA-PBS solution
For 10 minutes
Rinse with BSA-PBS solution
Up to 50 minutes
Secondary antibody 3 g a-SEB in 1 ml BSA-PBS flowed for 30 minutes
BSA-PBS rinse for 10 minutes

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RESULT

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Detection SEB in Buffer
(25 ng SEB in 1 ml BSA-PBS Solution)
Measuring Channel Reference Channel

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Signal for Different SEB Concentration
• Both signal already compensated with reference channel
• Direct SEB measurement is 0.07, 0.10, 0.44 and 0.82 (5, 12.5, 25, and 50
ng/ml)
• In sandwich assay, sensor response increasing by a factor of 10
• SEB in concentration of 0.5 ng/ml produced secondary a-SEB of 0.07 ng/ml

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Equilibrium Sensor Response
• Reproducibility was
determined to be within
13% of the equilibrium
sensor response (4 times
measurement wutg 10
ng/ml SEB concentration.
• Due to high
reproducibility of the
sensor response and
elimination of baseline
drift, detection limits are
limited mainly by noise of
sensor baseline.
Typical noise in compensated sensor
response is about 0.015 nm (standard
deviation).
Lowest detection limit: 3SD
LOD:
• 5 ng/ml for direct detection
• 0.5 ng/ml for sandwich mode

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SEB Detection in Milk
Minor contribution (<0.5%) due to the
specific binding of the toxin to the
antibodies immobilized on the sensor
surface
Equilibrium response of SEB
concentration:
0.37, 0.71, 1.24, and 2.10 nm for
2, 5, 10 and 20 ng/ml.
LOD for milk samples ~0.5 ng/ml
Disrepancies between sensor
responses may doe to differences in
sensor functionalizations
(functionalized in different batch)

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DISCUSSION

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Comparable Result
LOD
• 5 ng/ml in direct measure (pure samples)
• 0.5 ng/ml in sandwich assay (pure and milk)
• Western immunoblod and ELISA at 1 ng/ml
• Rasooly and Rasooly, 1998
• Bidiffractive Grating Sensors at 1 ng/ml
• O’Brien et.al. 2000
• Fluorescense based sensor at 4 ng/ml
• Rowe-Taitt et.al. 2000

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Conclusions
Room for Improvement
• Sensor LOD may further improved by surface
functionalization
• Lowering noise in sensor optoelectronic system
• Employ more advanced data processing methods
• Combining sensor with method for preconcentration of
analyte (preconc of magnetic beads
Major Hurdle
• Sensor response time
• Caused by simple fluidic system, which is not optimized for
anlayte-transport to sensing sirface

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Thank you for your attention!