Download Introduction to biosensors

PHOTONICS RESEARCH GROUP
Introduction to biosensors
Peter Bienstman
PHOTONICS RESEARCH GROUP
1
Biosensors
Detect presence and concentration of biomolecules
•
•
•
•
•
DNA
Proteins
Virus
Bacteria
…
Two classes:
• Labeled: indirect detection
• Label-free: direct detection
PHOTONICS RESEARCH GROUP
2
Applications
Diagnostics
Drug development
Food safety
Environmental monitoring
…
PHOTONICS RESEARCH GROUP
3
Desired characteristics
Low limit of detection (“sensitivity”)
Selective
Reproducible
Cheap
Portable
Fast
Multi-parameter
…
PHOTONICS RESEARCH GROUP
4
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!
PHOTONICS RESEARCH GROUP
5
ELISA
PHOTONICS RESEARCH GROUP
6
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
• …
PHOTONICS RESEARCH GROUP
7
Example: pregnancy test
Detects hCG protein (human Chorionic Gonadotropin) in urine
Based on strip which pulls fluid through by capillary action (lateral
flow immunochromatography)
PHOTONICS RESEARCH GROUP
8
Test principle
See animations at
http://www.whfreeman.com/kuby/content/anm/kb07an01.htm
PHOTONICS RESEARCH GROUP
9
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
PHOTONICS RESEARCH GROUP
10
Test result
PHOTONICS RESEARCH GROUP
11
AU NANOPARTICLES
PHOTONICS RESEARCH GROUP
12
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)
PHOTONICS RESEARCH GROUP
13
Au nanoparticles
Two different particles sizes
In solution
Immobilised on latex beads
PHOTONICS RESEARCH GROUP
14
Ways to use them
As a fancy dye
Changing colour on aggregation
Combined with latex beads
…
PHOTONICS RESEARCH GROUP
15
As fancy dye
Just 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
PHOTONICS RESEARCH GROUP
16
Changing colour on aggregation
Colloidal gold coated with hCG antibody
PHOTONICS RESEARCH GROUP
17
Changing colour on aggregation
hCG present
PHOTONICS RESEARCH GROUP
18
Changing colour on aggregation
Absorption band shifts due to aggregation and
colour changes (see later)
PHOTONICS RESEARCH GROUP
19
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
PHOTONICS RESEARCH GROUP
20
QUANTUM DOT LABELS
PHOTONICS RESEARCH GROUP
21
Quantum dot labels
Alternative to metallic nanoparticles
Typically colloidally grown
PbSe, CdTe, …
Much sharper spectra, widely tuneable by size
PHOTONICS RESEARCH GROUP
22
BEAD BASED ASSAYS
PHOTONICS RESEARCH GROUP
23
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
PHOTONICS RESEARCH GROUP
24
Labeled microcarriers
• Don’t flow fluid over planar substrate, but break up
substrate into microcarriers which float in the fluid
• Better mixing properties too
PHOTONICS RESEARCH GROUP
25
Read-out in flow cytometer
E.g., one laser measures label on bead, the other measures
the reporter fluorophore
PHOTONICS RESEARCH GROUP
26
Colour-encoded beads
e.g. Luminex xMAP technology, 2 fluorescent dyes in different ratios
PHOTONICS RESEARCH GROUP
27
LABELFREE SENSORS
PHOTONICS RESEARCH GROUP
28
Labeling
• detect a molecule by attaching a label to it
• very sensitive (10-9...10-16 mol/l)
• commercial product (Elisa, DNA arrays, ..)
PHOTONICS RESEARCH GROUP
29
Disadvantages to labeling?
PHOTONICS RESEARCH GROUP
30
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
PHOTONICS RESEARCH GROUP
31
Label-free sensors
• detect presence of biomolecules directly
• focus here: label-free optical biosensors
flow with biomolecules
matching biomolecule
(analyte)
biorecognition element
(ligand)
• selective binding causes refractive index change
PHOTONICS RESEARCH GROUP
32
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, …)
PHOTONICS RESEARCH GROUP
33
SURFACE PLASMON RESONANCE
SENSOR
PHOTONICS RESEARCH GROUP
34
Plasmons
Collective wave oscillations of electrons in a metal
motion of
electrons
propagation
of wave
Fig: R. Nave, Hyperphysics
PHOTONICS RESEARCH GROUP
35
Surface plasmons
Interaction between:
plasmon at surface of metal
electromagnetic wave
EM wave
plasmon
PHOTONICS RESEARCH GROUP
36
Magnitude of EM field
position
Cannot be excited directly
from the outside
light intensity
PHOTONICS RESEARCH GROUP
37
Reflection experiment
reflection
angle
PHOTONICS RESEARCH GROUP
angle
38
Towards a biosensor
reflection
angle
PHOTONICS RESEARCH GROUP
angle
39
Surface plasmon resonance
• Popular for biosensing (Biacore machine)
High fields near the interface are very sensitive to refractive index changes
Gold is very suitable for biochemistry
From source
To detector
Prism
R

Gold
PHOTONICS RESEARCH GROUP
40
advantages
o very sensitive, index differences of 10-6 possible
o functionalised Au layers off-the-shelf available
o integrated microfluidics
but
o bulky
o expensive
o difficult to integrate and multiplex
PHOTONICS RESEARCH GROUP
41
EVANESCENT WAVE SENSORS
PHOTONICS RESEARCH GROUP
42
Evanescent wave biosensor
Densmore, 2008
PHOTONICS RESEARCH GROUP
43
Influence of mode profile
• profile should overlap maximally with the adlayer, and not with
bulk fluid (noise!)
• high index contrast is best
Low contrast
PHOTONICS RESEARCH GROUP
High contrast
44
Effective index change still needs to be translated into something
measurable.
Many possibilities:
• Resonators
• Interferometers
•…
PHOTONICS RESEARCH GROUP
45
EVANESCENT WAVE SENSORS:
RESONATORS
PHOTONICS RESEARCH GROUP
46
Ring resonators
P
P
1.55 μm


Binding of biomolecules  change of refractive index
 resonance wavelength shift
PHOTONICS RESEARCH GROUP
47
transmission
Towards a better sensor
initial
biomolecules
wavelength
Narrower dips
transmission
transmission
Larger shift
wavelength
More interaction between
light and molecules
wavelength
High demands on read-out
system, but filters noise
PHOTONICS RESEARCH GROUP
48
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
min
Detection limit 
sensitivit y
Δλmin : smallest distinguishable wavelength shift
PHOTONICS RESEARCH GROUP
49
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!
PHOTONICS RESEARCH GROUP
50
Example: measurement setup
PHOTONICS RESEARCH GROUP
51
resonance wavelength shift [nm]
Surface sensing: biotin/avidin
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
avidin concentration [μg/ml]
•
High avidin concentrations: saturation
•
Low avidin concentrations: quantitative measurements
•
Detection limit: lower than 3ng/ml
PHOTONICS RESEARCH GROUP
52
Real time measurement
-5
20
x 10
15
-5
x 10
10
zoom
5
avidin 50ng/ml
ouptut [A.U.]
ouptut [A.U.]
8
avidin 50ng/ml
avidin 10ng/ml
avidin 10ng/ml
0
-2
0
-5
0
50
100
150
200
time [sec]
200
400
600
800
1000
1200
time [sec]
Important when studying kinetics, e.g. drug discovery
PHOTONICS RESEARCH GROUP
53