introduction to biosensors
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Introduction to biosensors. Peter Bienstman. Biosensors. Detect presence and concentration of biomolecules DNA Proteins Virus Bacteria … Two classes: Labeled: indirect detection Label-free: direct detection. Applications. Diagnostics Drug development Food safety - PowerPoint PPT PresentationTRANSCRIPT
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Introduction to biosensors
Peter Bienstman
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Biosensors
Detect presence and concentration of biomolecules• DNA• Proteins• Virus• Bacteria• …
Two classes:• Labeled: indirect detection• Label-free: direct detection
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Applications
Diagnostics
Drug development
Food safety
Environmental monitoring
…
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Desired characteristics
Low limit of detection (“sensitivity”)
Selective
Reproducible
Cheap
Portable
Fast
Multi-parameter
…
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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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ELISA
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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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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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Test principle
See animations at http://www.whfreeman.com/kuby/content/anm/kb07an01.htm
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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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Test result
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AU NANOPARTICLES
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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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Au nanoparticles
Two different particles sizesIn solution
Immobilised on latex beads
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Ways to use them
As a fancy dye
Changing colour on aggregation
Combined with latex beads
…
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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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Colloidal gold coated with hCG antibody
Changing colour on aggregation
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hCG present
Changing colour on aggregation
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Absorption band shifts due to aggregation and colour changes (see later)
Changing colour on aggregation
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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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QUANTUM DOT LABELS
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Quantum dot labels
Alternative to metallic nanoparticles
Typically colloidally grown
PbSe, CdTe, …
Much sharper spectra, widely tuneable by size
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BEAD BASED ASSAYS
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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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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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Read-out in flow cytometer
E.g., one laser measures label on bead, the other measures the reporter fluorophore
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Colour-encoded beads
e.g. Luminex xMAP technology, 2 fluorescent dyes in different ratios
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LABELFREE SENSORS
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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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Disadvantages to labeling?
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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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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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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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SURFACE PLASMON RESONANCE SENSOR
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Plasmons
Collective wave oscillations of electrons in a metal
Fig: R. Nave, Hyperphysics
motion of electrons
propagation of wave
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Surface plasmons
Interaction between:plasmon at surface of metalelectromagnetic wave
EM wave
plasmon
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Magnitude of EM field
light intensity
position Cannot be excited directly from the outside
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Reflection experiment
reflection
angle angle
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Towards a biosensor
reflection
angleangle
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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 detectorPrism
Gold
R
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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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EVANESCENT WAVE SENSORS
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Evanescent wave biosensor
Densmore, 2008
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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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Effective index change still needs to be translated into something measurable.
Many possibilities:
• Resonators
• Interferometers
• …
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EVANESCENT WAVE SENSORS: RESONATORS
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Ring resonators
Binding of biomolecules change of refractive index
resonance wavelength shift
P
P
1.55 μm
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Towards a better sensor
High demands on read-out system, but filters noise
wavelengthtrans
mis
sion
initialbiomolecules
wavelengthtrans
mis
sion
wavelengthtrans
mis
sion
wavelengthtrans
mis
sion
More interaction between light and molecules
Narrower dipsLarger shift
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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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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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Example: measurement setup
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Surface sensing: biotin/avidin
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• High avidin concentrations: saturation• Low avidin concentrations: quantitative measurements • Detection limit: lower than 3ng/ml
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Real time measurement
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avidin 50ng/mlavidin 10ng/ml
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zoom
avidin 50ng/mlavidin 10ng/ml
Important when studying kinetics, e.g. drug discovery