p hotonics r esearch g roup 1 introduction to biosensors peter bienstman
TRANSCRIPT
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PHOTONICS RESEARCH GROUP 1
PHOTONICS RESEARCH GROUP
Introduction to biosensors
Peter Bienstman
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PHOTONICS RESEARCH GROUP 2
Biosensors
Detect presence and concentration of biomolecules• DNA• Proteins• Virus• Bacteria• …
Two classes:• Labeled: indirect detection• Label-free: direct detection
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PHOTONICS RESEARCH GROUP 3
Applications
Diagnostics
Drug development
Food safety
Environmental monitoring
…
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PHOTONICS RESEARCH GROUP 4
Desired characteristics
Low limit of detection (“sensitivity”)
Selective
Reproducible
Cheap
Portable
Fast
Multi-parameter
…
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PHOTONICS RESEARCH GROUP 5
Labeled optical sensor types
Many, many types
E.g.• Elisa• Au nanoparticle labels• Quantum dot labels• Bead-based assays• Padlock probes
Not an exhaustive list!
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PHOTONICS RESEARCH GROUP 6
ELISA
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PHOTONICS RESEARCH GROUP 7
Elisa tests
Enzyme-Linked Immuno Sorbent Assay
Workhorse of protein detection
Detect protein by using• fluorescent labels• labels with enzymes that start a colouring reaction on a dye substrate• …
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PHOTONICS RESEARCH GROUP 8
Example: pregnancy test
Detects hCG protein (human Chorionic Gonadotropin) in urine
Based on strip which pulls fluid through by capillary action (lateral flow immunochromatography)
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PHOTONICS RESEARCH GROUP 9
Test principle
See animations at http://www.whfreeman.com/kuby/content/anm/kb07an01.htm
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PHOTONICS RESEARCH GROUP 10
Assay zones
Fluid flows through 3 zones:
R: reaction zone: hCG picks up free antibody labeled with enzyme
T: test zone: hCG+antibody+enzyme gets bound by immobilised antibody on strip, enzyme starts colouring reaction of dye if pregnant
C: control zone: antibody picks up any remaining antibody+enzyme complexes, enzyme starts colouring if test works OK
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PHOTONICS RESEARCH GROUP 11
Test result
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PHOTONICS RESEARCH GROUP 12
AU NANOPARTICLES
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PHOTONICS RESEARCH GROUP 13
Variations of pregnancy test
Don’t use enzymes to colour a dye, but use gold nanoparticles
About 10 nm in diameter
Au is nice because it’s easy to functionalise it
Red in colour, but depends on particle size (see later)
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PHOTONICS RESEARCH GROUP 14
Au nanoparticles
Two different particles sizesIn solution
Immobilised on latex beads
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PHOTONICS RESEARCH GROUP 15
Ways to use them
As a fancy dye
Changing colour on aggregation
Combined with latex beads
…
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PHOTONICS RESEARCH GROUP 16
As fancy dyeJust use them as a dye, i.e. instead of the enzyme
If there are enough of them in the test zone, they will give a red line
Used e.g. by UltiMed pregnancy test
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PHOTONICS RESEARCH GROUP 17
Colloidal gold coated with hCG antibody
Changing colour on aggregation
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PHOTONICS RESEARCH GROUP 18
hCG present
Changing colour on aggregation
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PHOTONICS RESEARCH GROUP 19
Absorption band shifts due to aggregation and colour changes (see later)
Changing colour on aggregation
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PHOTONICS RESEARCH GROUP 20
Combined with latex beads
Au nanoparticles and latex microparticles
When pregnant, Au colours the latex bead and a size filter prevents them from washing downstream
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PHOTONICS RESEARCH GROUP 21
QUANTUM DOT LABELS
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PHOTONICS RESEARCH GROUP 22
Quantum dot labels
Alternative to metallic nanoparticles
Typically colloidally grown
PbSe, CdTe, …
Much sharper spectra, widely tuneable by size
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PHOTONICS RESEARCH GROUP 23
BEAD BASED ASSAYS
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PHOTONICS RESEARCH GROUP 24
Multiparameter assays
Pregnancy test measures only single compound
Very interesting to have more than 1 target
Multiplexed, multi-parameter assays
Two formats:• 2D arrays on chip: spatial encoding
• Free floating labeled microcarriers
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PHOTONICS RESEARCH GROUP 25
Labeled microcarriers
• Don’t flow fluid over planar substrate, but break up substrate into microcarriers which float in the fluid
• Better mixing properties too
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PHOTONICS RESEARCH GROUP 26
Read-out in flow cytometer
E.g., one laser measures label on bead, the other measures the reporter fluorophore
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PHOTONICS RESEARCH GROUP 27
Colour-encoded beads
e.g. Luminex xMAP technology, 2 fluorescent dyes in different ratios
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PHOTONICS RESEARCH GROUP 28
LABELFREE SENSORS
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PHOTONICS RESEARCH GROUP 29
Labeling
• detect a molecule by attaching a label to it
• very sensitive (10-9...10-16 mol/l)
• commercial product (Elisa, DNA arrays, ..)
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PHOTONICS RESEARCH GROUP 30
Disadvantages to labeling?
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PHOTONICS RESEARCH GROUP 31
Disadvantages to labeling
• some labels are very costly
• only measures final state, no kinetics
• label can influence properties of biomolecules
• strong interest in label-free sensors
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PHOTONICS RESEARCH GROUP 32
Label-free sensors
• detect presence of biomolecules directly
• focus here: label-free optical biosensors
• selective binding causes refractive index change
biorecognition element (ligand)
matching biomolecule (analyte)
flow with biomolecules
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PHOTONICS RESEARCH GROUP 33
Index change
How to measure the refractive index change?
• Surface plasmon sensors
• Evanescent wave sensors• Mach-Zehnder interferometer• Resonant cavities
Once again, the list is not exhaustive.
Also, there are many non-optical techniques (impedimetric, mass, …)
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PHOTONICS RESEARCH GROUP 34
SURFACE PLASMON RESONANCE SENSOR
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PHOTONICS RESEARCH GROUP 35
Plasmons
Collective wave oscillations of electrons in a metal
Fig: R. Nave, Hyperphysics
motion of electrons
propagation of wave
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PHOTONICS RESEARCH GROUP 36
Surface plasmons
Interaction between:plasmon at surface of metalelectromagnetic wave
EM wave
plasmon
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PHOTONICS RESEARCH GROUP 37
Magnitude of EM field
light intensity
position Cannot be excited directly from the outside
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PHOTONICS RESEARCH GROUP 38
Reflection experiment
reflection
angle angle
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PHOTONICS RESEARCH GROUP 39
Towards a biosensor
reflection
angleangle
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PHOTONICS RESEARCH GROUP 40
Surface plasmon resonance
• Popular for biosensing (Biacore machine)High fields near the interface are very sensitive to refractive index changesGold is very suitable for biochemistry
From source
To detector
Prism
Gold
R
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PHOTONICS RESEARCH GROUP 41
advantageso very sensitive, index differences of 10-6 possibleo functionalised Au layers off-the-shelf availableo integrated microfluidics
buto bulkyo expensiveo difficult to integrate and multiplex
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PHOTONICS RESEARCH GROUP 42
EVANESCENT WAVE SENSORS
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PHOTONICS RESEARCH GROUP 43
Evanescent wave biosensor
Densmore, 2008
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PHOTONICS RESEARCH GROUP 44
Influence of mode profile
• profile should overlap maximally with the adlayer, and not with bulk fluid (noise!)
• high index contrast is best
Low contrast High contrast
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PHOTONICS RESEARCH GROUP 45
Effective index change still needs to be translated into something measurable.
Many possibilities:
• Resonators
• Interferometers
• …
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PHOTONICS RESEARCH GROUP 46
EVANESCENT WAVE SENSORS: RESONATORS
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PHOTONICS RESEARCH GROUP 47
Ring resonators
Binding of biomolecules change of refractive index
resonance wavelength shift
P
P
1.55 μm
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PHOTONICS RESEARCH GROUP 48
Towards a better sensor
High demands on read-out system, but filters noise
wavelengthtra
nsm
issi
on
initialbiomolecules
wavelengthtra
nsm
issi
on
wavelengthtra
nsm
issi
on
wavelengthtra
nsm
issi
on
More interaction between light and molecules
Narrower dipsLarger shift
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PHOTONICS RESEARCH GROUP 49
Sensitivity vs detection limit
• Sensitivity: shift of resonance wavelength (in nm) for a given excitation, e.g.
Bulk sensitivity: nm / RIU (refractive index unit)Adlayer sensitivity: nm / nm
• Detection limit: smallest measurable excitation
ysensitivit limit Detection min
Δλmin : smallest distinguishable wavelength shift
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PHOTONICS RESEARCH GROUP 50
What determines Δλmin ?
• precision of measurement equipment
• noise in the system (thermal, mechanical, …)
• design of the sensor • e.g.: higher Q is better• often in conflict with sensitivity
• quality of data analysis• averaging• analytical curve fitting• Δλmin can get smaller than measurement resolution!
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PHOTONICS RESEARCH GROUP 51
Example: measurement setup
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PHOTONICS RESEARCH GROUP 52
Surface sensing: biotin/avidin
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 5 10 15 20 25
avidin concentration [μg/ml]
reso
nanc
e w
avel
engt
h sh
ift [
nm]
• High avidin concentrations: saturation
• Low avidin concentrations: quantitative measurements
• Detection limit: lower than 3ng/ml
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PHOTONICS RESEARCH GROUP 53
Real time measurement
0 200 400 600 800 1000 1200-5
0
5
10
15
20x 10
-5
time [sec]
ou
ptu
t [A
.U.]
avidin 50ng/ml
avidin 10ng/ml
0 50 100 150 200-2
8x 10
-5
time [sec]
ou
ptu
t [A
.U.]
zoom
avidin 50ng/ml
avidin 10ng/ml
Important when studying kinetics, e.g. drug discovery